Alkylidene phosphonate esters as p-glycoprotein inducers

Abstract

The present invention relates to the alkylidene phosphonate esters of formula I wherein, R.sub.1 is selected from a group consisting of hydrogen or alkyl group; R.sub.2 is selected from a group consisting of hydrogen, hydroxy, alkyl, alkoxy, nitro, halogen, amino, N-substituted alkylamino; alkyl group is selected from a group consisting of methyl, ethyl and isopropyl; Ar is selected from a group consisting of aryl, substituted aryl, fused aryl, heteroaryl, and substituted heteroaryl. The present invention particularly relates to synthesis and p-glycoprotein induction activity of the alkylidene phosphonate esters. In addition, the invention relates to methods of using compounds for treating or preventing Alzheimer's disease. ##STR00001##

Claims

1. A compound represented by the following structural formulae: ##STR00012## ##STR00013##

2. The compound as claimed in claim 1, wherein the said compounds are useful for the treatment of Alzheimer's disease.

3. The compound as claimed in claim 1, wherein the compound is compound 1l, and the compound 1l displayed Pgp induction EC.sub.50 value of 0.90 nM.

4. A pharmaceutical composition for the treatment of Alzheimer's disease comprising; an effective amount of the compound as claimed in claim 1 optionally along with a pharmaceutically acceptable excipients, diluents.

5. A composition as claimed in claim 4, wherein the pharmaceutically acceptable excipient is selected from a group consisting of saccharides, stearates, polyvinyl pyrrolidine, dicalcium phosphate dihydrate, eudragit polymers, celluloses, polyethylene glycol, polysorbate 80, sodium lauryl sulfate, magnesium oxide, silicon dioxide, carbonates and talc.

6. The composition as claimed in claim 5, wherein the saccharides are at least one selected from the group consisting of lactose, starch and dextrose.

7. The composition as claimed in claim 5, wherein the stearates are at least one selected from the group consisting of stearic acid and magnesium stearate.

8. The composition as claimed in claim 5, wherein the carbonates are at least one selected from the group consisting of sodium carbonate and sodium bicarbonate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a diagram illustrating the synthetic scheme for preparation of phosphonate esters of formula I.

(2) FIG. 2 is a diagram illustrating the Pgp induction activity of phosphonate esters 1a-m, measured in terms of the % intracellular accumulation of rhodamine 123/total protein inside LS180 cells. The decrease in the % intracellular accumulation (compared to control) of Rh123 indicates induction of Pgp. Rifampicin (10 M) was used as a reference Pgp inducer. Statistical comparisons were made between control vs compounds by using Bonferroni test. The p value <0.5 was considered to be significant. P value *<0.5, **<0.01, ***<0.001.

(3) FIG. 3. Pgp Western-blot analysis of phosphonate ester 11. The quantitative comparison of Pgp expression is also shown.

LIST OF ABBREVIATIONS

(4) A; Amyloid-, CNS; Central nervous system, HMG-CoA; 3-hydroxy-3-methylglutaryl-coenzyme A, LDL; Low density lipoprotein, PXR; Pregnane X receptor, SRC-1; Steroid receptor co-activator-1, ANOVA: Analysis of variance; P-gp: P-glycoprotein; Rh123: Rhodamine 123; THF; Tetrahydrofuran, CCl.sub.4; Carbon tetrachloride, TiCl4; Titanium tetrachloride.

DETAILED DESCRIPTION OF THE INVENTION

(5) The present invention reports phosphonate esters represented by general structure I as potent Pgp inducer.

(6) ##STR00007##

(7) The present invention relates to new phosphonate ester compounds (synthesis shown in FIG. 1) that shows promising p-glycoprotein inducing activity. The bis-phosphonate esters 1b-m showed ability to induce p-glycoprotein as showed by decrease in the % intracellular levels of rhodamine 123 in LS-180 cells (FIG. 2). The compound 1l showed promising induction of p-glycoprotein which was further confirmed by western-blot analysis. The Western-blot results (FIG. 3) clearly indicated that new phosphonate ester 1l induces p-glycoprotein expression. The phosphonate ester 1l showed better Pgp induction activity (EC.sub.50=0.9 nM) as compared to SR12813 (1a). The compound 1l possesses 2-fold higher EC.sub.50 value than SR12813 (1a). The EC.sub.50 results are shown in Table 2. Furthermore, the phosphonate ester 1l also showed optimal solubility in water as well as in biological fluids. The solubility results are shown in Table 3.

(8) A class of phosphonate esters is presented and defined by structural formula I:

(9) ##STR00008##
wherein, R.sub.1 is selected from hydrogen and alkyl. Ar is selected from aryl, substituted aryl, fused aryl (for example naphthalene and anthracene), heteroaryl, and substituted heteroaryl. R.sub.2 is selected from alkyl, alkoxy, nitro, halogen, amino, N-substituted amino. Alkyl group is selected from methyl, ethyl and isopropyl. The substituted aryl or substituted hetoroaryl groups are substituted with alkoxy group such as methoxy or ethoxy group, nitro or N,N-dimethyl amino group.

(10) Compounds of the invention derived from formula I include, but are not limited to, the following chemical structures:

(11) ##STR00009## ##STR00010## ##STR00011##
As used herein, the terms below have the meanings indicated.

(12) The term alkyl, as used herein, alone or in combination, refers to a straight-chain or branched-chain alkyl radical optionally substituted containing from 1 to 20 and including 20, preferably 1 to 10, and more preferably 1 to 6, carbon atoms. Examples of alkyl radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl, octyl, nonyl and the like.

(13) The term aryl as used herein, alone or in combination, means a carbocyclic aromatic system containing one, two or three rings wherein such rings may be attached together in a pendent manner or may be fused.

(14) The term halo, or halogen, as used herein, alone or in combination, refers to fluorine, chlorine, bromine, or iodine.

(15) The term alkoxy as used herein, alone or in combination, refers to oxygen linked alkyl moieties.

(16) The term heteroaryl, as used herein, alone or in combination, refers to 3 to 7 membered, preferably 5 to 7 membered, unsaturated heteromonocyclic rings, or fused polycyclic rings in which at least one of the fused rings is unsaturated, wherein at least one atom is selected from the group consisting of O, S, and N. The term also embraces fused polycyclic groups wherein heterocyclic radicals are fused with aryl radicals, wherein heteroaryl radicals are fused with other heteroaryl radicals, or wherein heteroaryl radicals are fused with cycloalkyl radicals. Examples of heteroaryl groups include pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl, pyranyl, furyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, thiadiazolyl, isothiazolyl, indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, quinoxalinyl, quinazolinyl, indazolyl, benzotriazolyl, benzodioxolyl, benzopyranyl, benzoxazolyl, benzoxadiazolyl, benzothiazolyl, benzothiadiazolyl, benzofuryl, benzothienyl, chromonyl, coumarinyl, benzopyranyl, tetrahydroquinolinyl, tetrazolopyridazinyl, tetrahydroi soquinolinyl, thienopyridinyl, furopyridinyl, pyrrolopyridinyl and the like. Exemplary tricyclic heterocyclic groupsincludecarbazolyl, benzidolyl, phenanthrolinyl, dibenzofuranyl, acridinyl, phenanthridinyl, xanthenyl and the like.

(17) The phrase therapeutically effective is intended to qualify the amount of active ingredients used in the treatment of a disease or disorder. This amount will achieve the goal of reducing or eliminating the disease or disorder.

(18) As used herein, reference to treatment of a patient is intended to include prophylaxis. The term patient means all mammals including humans. Examples of patients include humans, cows, dogs, cats, goats, sheep, pigs, rabbits, and rodents (e.g., rats, mice, and guinea pigs).

(19) As used herein, the compounds of the invention can be used to treat a patient (e.g. a human) that suffers from or is at a risk of suffering from a disease, disorder, condition, or symptom described herein. The compounds of the invention can be used alone or in combination with appropriate excipients or diluents. Each such treatment described above includes the step of administering to a patient in need thereof a therapeutically effective amount of the compound of the invention described herein to delay, reduce or prevent such a disease, disorder, condition, or symptom.

(20) It is understood that the foregoing examples are merely illustrative of the present invention. Certain modifications of the articles and/or methods employed may be made and still achieve the objectives of the invention. Such modifications are contemplated as within the scope of the claimed invention.

EXAMPLES

(21) Following examples are given by way of illustration and should not construed the scope of the present invention.

(22) General procedure for synthesis of alkylidene phosphonate esters 1a-1m. A flame-dried 25 ml round bottom flask with magnetic stir bar was charged with TiCl.sub.4 (10 mmol) and 0.5 ml CCl.sub.4 at 0 C. Then, 5 ml of dry THF was added dropwise to the flask which resulted in formation of a bright yellow precipitate. Substituted benzaldehyde (5 mmol) and tetraalkyl methylenediphosphonate (5 mmol) were added to the reaction mixture. To this mixture was added dropwise, a solution of 0.5 ml 4-methylmorpholine in 3.0 ml dry THF. The reaction was allowed to warm to room temperature and stirred overnight. The reaction was quenched with water and extracted with EtOAc. The organic layer was washed with brine and dried over Na.sub.2SO.sub.4. Concentration in vacuo followed by column chromatography provided the corresponding alkylidene bisphosphonates 1a-1m. In examples 1-13 desired compounds were prepared by the general; procedure as discussed above, wherein the respective aldehydes and tetraalkyl methylenediphosphonates used as reactants to prepare desired compounds are listed in Table 1.

(23) TABLE-US-00001 TABLE 1 Starting materials for synthesis of alkylidene bisphosphonate products Alkylidene Substituted Tetraalkyl bisphosphonate benzaldehyde methylenediphosphonate product 3,5-Di-tert-butyl-4- Tetraethyl 1a hydroxybenzaldehyde methylenediphosphonate 2,3,5- Tetraethyl 1b Trimethoxybenzaldehyde methylenediphosphonate 3,5-Dimethoxybenzaldehyde Tetraethyl 1c methylenediphosphonate 2-Nitrobenzaldehyde Tetraethyl 1d methylenediphosphonate 3,5-Dimethoxybenzaldehyde Tetraisopropyl 1e methylenediphosphonate 3-Bromobenzaldehyde Tetraisopropyl 1f methylenediphosphonate 3-Fluoro-4- Tetraisopropyl 1g methoxybenzaldehyde methylenediphosphonate 4-N,N-dimethylamino Tetraisopropyl 1h benzaldehyde methylenediphosphonate 2,4,5- Tetraisopropyl 1i Trimethoxybenzaldehyde methylenediphosphonate 1-Naphthaldehyde Tetraethyl 1j methylenediphosphonate 1-Naphthaldehyde Tetraisopropyl 1k methylenediphosphonate Anthracene-9-carbaldehyde Tetraethyl 1l methylenediphosphonate 4-Nitrofuraldehyde Tetraisopropyl 1m methylenediphosphonate

Example 1

(24) Tetraethyl-2-(3,5-di-tert-butyl-4hydroxyphenyl)ethane-1,1-diyldiphosphonate(1a)

(25) Yellow oil; yield 60%; .sup.1H NMR (400 MHz, CDCl.sub.3, ppm): 8.33-8.14 (dd, J.sub.1=28 Hz, J.sub.2=48 Hz, 1H), 7.76 (s, 2H), 4.32-4.15 (m, 4H), 4.12-4.02 (m, 4H), 1.46-1.40 (s, 18H), 1.39 1.29 (m, 6H), 1.27-1.16(m, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3, ppm): 6 162.91, 162.88, 157.03, 135.59, 129.87, 125.72, 125.64, 125.51, 125.43, 62.46, 62.41, 62.23, 62.17, 53.42, 34.53, 30.26, 16.39, 16.33, 16.12, 16.04; .sup.31P NMR (CDCl.sub.3, 161.98 MHz; H.sub.3PO.sub.4 as reference standard): 19.58-19.26 (d, J=51.83 Hz), 13.62-13.30 (d, J=51.83 Hz); IR (CHCl.sub.3): .sub.max 3436, 2957, 2927, 2871, 1616, 1596, 1558, 1424, 1391, 1242, 1162, 1025 cm.sup.1; ESI-MS: m/z 505 [M+1].sup.+; HRMS: m/z 505.2468 calcd for C.sub.27H.sub.43O.sub.7P.sub.2+H.sup.+ (505.2478).

Example 2

(26) Tetraethyl 2-(2, 3, 5-trimethoxy phenyl)ethene-1,1-diyldiphosphonate (1b)

(27) Yellow oil; yield 50%; .sup.1H NMR (400 MHz, CDCl.sub.3, ppm): 8.64-8.44 (dd, J.sub.1=28 Hz, J.sub.2=48 Hz, 1H), 7.96 (s, 1H), 6.45 (s, 1H), 4.23-4.14 (m, 6H), 4.09-4.02 (m, 2H), 3.94-3.86 (s, 9H), 1.39-1.18 (m, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3, ppm): 155.98, 154.36, 152.93, 151.42, 142.36, 114.45, 95.39, 62.51, 62.47, 62.40, 62.35, 56.43, 56.34, 56.02, 16.36, 16.31, 16.17; IR (CHCl.sub.3): .sub.max 3436, 2927, 1612, 1579, 1508, 1466, 1440, 1335, 1282, 1221, 1128, 1025 cm.sup.1; MS: m/z 467.20 [M+Na].sup.+; HRMS: m/z 467.1596 calcd for C.sub.19H.sub.33O.sub.9P.sub.2+H.sup.+ (467.1594).

Example 3

(28) Tetraethyl 2-(3,5 dimethoxy phenyl)ethene-1,1-diyldiphosphonate (1c)

(29) Yellow oil; yield: 45%; .sup.1HNMR (400 MHz, CDCl.sub.3, ppm): 8.34-8.14 (dd, J.sub.1=32 Hz, J.sub.2=48 Hz, 1H), 6.99 (d, J=8 Hz, 2H), 6.51 (t, J=4 Hz, 1H), 4.25-4.16 (m, 4H), 4.08-3.97 (m, 4H), 3.81 (s, 6H), 1.43-1.33 (m, 6H), 1.22-1.15 (s, 6H); IR (CHCl.sub.3): .sub.max 3436, 2981, 2928, 1586, 1568, 1445, 1391, 1248, 1163, 1025 cm.sup.1; ESI-MS: m/z 437.00 [M+1].sup.+; HRMS: m/z 459.1308 calcd for C.sub.18H.sub.30O.sub.8P.sub.2+Na.sup.+ (459.1308).

Example 4

(30) Tetraethyl 2-(2-nitrophenyl) ethene-1,1-diyldiphosphonate (1d)

(31) Yellow oil; yield 50%; .sup.1H NMR (400 MHz, CDCl.sub.3, ppm): 8.24-8.22 (dd, J.sub.1=28 Hz, J.sub.2=48 Hz, 1H), 8.24-8.16 (m, 1H), 7.72-7.63 (m, 1H), 7.58-7.50 (m, 2H), 4.32-4.11 (m, 4H), 4.02-3.81 (m, 4H), 1.43-1.36 (m, 6H), 1.15-1.09 (m, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3, ppm): 159.04, 145.39, 133.59, 130.64, 129.74, 124.39, 62.99, 62.95, 62.48, 62.45, 15.39, 16.33, 16.12, 16.07; .sup.31P NMR (CDCl.sub.3, H.sub.3PO.sub.4, 161.98 MHz): 14.92-14.62 (d, J=48.59 Hz), 11.10-10.80 (d, J=48.59 Hz); IR (CHCl.sub.3): .sub.max 3467, 2983, 2928, 2855, 1734, 1589, 1570, 1525, 1442, 1392, 1345, 1248, 1163, 1023 cm.sup.1; ESI-MS: m/z 443.94 [M+Na].sup.+; HRMS: m/z 444.0954 calcd for C.sub.16H.sub.25NO.sub.8P.sub.2+Na.sup.+ (444.0947).

Example 5

(32) Tetraisopropyl 2-(3,5-dimethoxy phenyl)ethene-1,1-diyldiphosphonate (1e)

(33) Yellow oil; yield, 50%; .sup.1H NMR (400 MHz, CDCl.sub.3, ppm): 8.24-8.05 (dd, J.sub.1=32 Hz, J.sub.2=48 Hz, 1H), 6.98 (s, 2H), 6.43 (s, 1H), 4.77-4.69 (m, 2H), 4.65-4.57 (m, 2H), 3.74 (s, 6H), 1.33-1.26 (m, 12H), 1.18-1.11 (m, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3, ppm): 160.26, 159.92, 108.50, 108.33, 103.36, 71.55, 71.50, 71.40, 71.35, 55.57, 24.15, 24.10, 24.07, 24.01, 23.97, 23.61, 23.57; .sup.31P NMR (CDCl.sub.3, H.sub.3PO.sub.4, 161.98 MHz): 15.34-15.03 (d, J=50.21 Hz), 9.81-9.50 (d, J=50.21 Hz); IR (CHCl.sub.3): .sub.max 3436, 2978, 2926, 2852, 1738, 1595, 1573, 1458, 1385, 1307, 1241, 1206, 1156, 1106, 1065 cm.sup.1; ESI-MS: m/z 493.1 [M+1].sup.+; HRMS: m/z 493.2104 calcd for C.sub.22H.sub.39O.sub.8P.sub.2+H.sup.+ (493.2114).

Example 6

(34) Tetraisopropyl 2-(3-bromo phenyl)ethene-1,1-diyldiphosphonate (1f)

(35) Yellow oil; yield 50%; .sup.1H NMR (400 MHz, CDCl.sub.3, ppm): 8.29-8.09 (dd, J.sub.1=28 Hz, J.sub.2=48 Hz, 1H), 7.97 (s, 1H), 7.66 (d, J=8 Hz, 1H), 7.50 (d, J=8 Hz, 1H), 7.27-7.23 (m, 1H), 4.85-4.77 (m, 2H), 4.74-4.66 (m, 2H), 1.41-1.36 (m, 12H), 1.26-1.18 (m, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3, ppm): 157.62, 137.08, 136.94, 133.05, 132.81, 129.44, 129.00, 121.96, 71.75, 71.69, 71.54, 71.47, 29.68, 24.11, 24.06, 24.05, 24.01, 23.97, 23.92, 23.59, 23.53; .sup.31P NMR (CDCl.sub.3, H.sub.3PO.sub.4, 161.98 MHz): 14.24-13.93 (d, J=50.21 Hz), 9.12-8.81 (d, J=50.21 Hz); IR (CHCl.sub.3): .sub.max 3436, 2978, 2930, 1581, 1385, 1242, 1105 cm.sup.1; ESI-MS: m/z 534 [M+Na].sup.+; HRMS: m/z 511.1003 calcd for C.sub.20H.sub.34BrO.sub.6P.sub.2+H.sup.+ (511.1008).

Example 7

(36) Tetraisopropyl 2-(3-fluoro4-methoxyphenyl)ethene-1,1-diyldiphosphonate (1g)

(37) Yellow oil; yield 65%; .sup.1H NMR (400 MHz, CDCl.sub.3, ppm): 8.18-7.98 (dd, J.sub.1=28 Hz, J.sub.2=48 Hz, 1H), 7.82-7.79 (m, 1H), 7.53-7.50 (d, J=12 Hz, 1H), 6.90-6.86 (m, 1H), 4.76-4.61 (m, 4H), 1.36-1.28 (m, 12H), 1.20-1.18 (m, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3, ppm): 158.28, 152.70, 150.30, 149.89, 129.36, 119.04, 118.85, 112.23, 71.54, 71.47, 71.46, 71.39, 56.20, 24.12, 24.08, 24.03, 23.99, 23.63, 23.58; .sup.31P NMR (CDCl.sub.3, H.sub.3PO.sub.4, 161.98 MHz): 15.72-15.42 (d, J=48.59 Hz), 10.14-9.83 (d, J=50.21 Hz); IR (CHCl.sub.3): .sub.max 3436, 2978, 2928, 1668, 1615, 1511, 1443, 1385, 1285, 1138, 1105, 1017 cm.sup.1; ESI-MS: m/z 481 [M+1].sup.+; HRMS: m/z 481.1908 calcd for C.sub.21H.sub.36FO.sub.7P.sub.2+H.sup.+ (481.1914).

Example 8

(38) Tetraisopropyl 2-(4-N,N-dimethylamino phenyl)ethene-1,1-diyldiphosphonate (1h)

(39) Yellow oil; yield 60%; .sup.1H NMR (400 MHz, CDCl.sub.3, ppm): 8.24-8.05 (dd, J.sub.1=28 Hz, J.sub.2=48 Hz, 1H), 7.92-7.89 (d, J=12 Hz, 2H), 6.64 (d, J=8 Hz, 2H), 4.79-4.69 (m, 4H), 3.01 (s, 6H), 1.39-1.34 (m, 12H), 1.34-1.24 (m, 12H); .sup.13C NMR (100 MHz, CDCl.sub.3, ppm): 160.68, 152.16, 135.0, 110.73, 71.28, 71.03, 70.99, 70.95, 39.94, 24.09, 24.05, 24.03, 23.98, 23.88, 23.65, 23.61; IR (CHCl.sub.3): .sub.max 3467, 2978, 2931, 2874, 1731, 1607, 1519, 1436 1384, 1373, 1242, 1196, 1141, 1107 cm.sup.1; ESI-MS: m/z 476.1 [M+1].sup.+; HRMS: m/z 476.2317 calcd for C.sub.22H.sub.40NO.sub.6P.sub.2+H.sup.+ (476.2325).

Example 9

(40) Tetraisopropyl 2-(2,4,5-trimethoxy phenyl)ethene-1,1-diyldiphosphonate (1i)

(41) Yellow oil; yield 45%; .sup.1H NMR (400 MHz, CDCl.sub.3, ppm): 8.58-8.39 (dd, J.sub.1=32 Hz, J.sub.2=48 Hz, 1H), 7.98 (s, 1H), 6.38 (s, 1H), 4.75-4.60 (m, 4H), 3.86 (s, 3H), 3.83 (s, 3H), 3.78 (s, 3H), 1.33-1.29 (m, 12H), 1.15-1.13 (m,12H); .sup.31P NMR (CDCl.sub.3, H.sub.3PO.sub.4, 161.98 MHz): 17.11-16.77 (d, J=55.07 Hz), 11.81-11.48 (d, J=53.45 Hz); IR (CHCl.sub.3): .sub.max 3435, 2979, 2931, 1612, 1579, 1508, 1466, 1374, 1243, 1221, 1141 cm.sup.1; ESI-MS: m/z 523.1 [M+1].sup.+; HRMS: m/z 523.2222 calcd for C.sub.23H.sub.41O.sub.9P.sub.2+H.sup.+ (523.2220).

Example 10

(42) Tetraethyl 2-(naphthalen-1-yl) ethene-1,1-diyldiphosphonate (1j)

(43) Yellow oil; yield 55%; .sup.1H NMR (400 MHz, CDCl.sub.3, ppm): 8.96-8.77 (dd, J.sub.1=28 Hz, J.sub.2=48 Hz, 1H), 7.89-7.81 (m, 4H), 7.56-7.48 (m, 3H), 4.33-4.26 (m, 4H), 3.93-3.77 (m, 4H), 1.48-1.33 (m, 6H), 0.96-0.88 (m, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3, ppm): 160.14, 160.12, 132.95, 132.02, 130.54, 130.07, 128.61, 127.34, 126.77, 126.23, 125.05, 124.78, 124.72, 124.15, 62.88, 62.84,62.42, 62.36, 16.42, 16.36, 15.86, 15.80; .sup.31P NMR (CDCl.sub.3, H.sub.3PO.sub.4, 161.98 MHz): 16.16-15.83 (d, J=53.45 Hz), 11.81-11.48 (d, J=53.45 Hz); IR (CHCl.sub.3): .sub.max 3437, 2929, 2983, 2095, 1634, 1392, 1238, 1162, 1022 cm.sup.1; ESI-MS: m/z 427.0 [M+Na].sup.+; HRMS: m/z 427.1430 calcd for C.sub.20H.sub.29O.sub.6P.sub.2+H.sup.+ (427.1433).

Example 11

(44) Tetra isopropyl 2-(naphthalen-1-yl)ethene-1,1-diyldiphosphonate (1k)

(45) Yellow oil; yield 45%; .sup.1HNMR (400 MHz, CDCl.sub.3, ppm): 8.85-8.67 (dd, J.sub.1=28 Hz, J.sub.2=48 Hz, 1H), 7.81-7.73 (m, 4H), 7.47-7.20 (m, 3H), 4.85-4.80 (m, 2H), 4.49-4.44 (m, 2H), 1.38-1.36 (m, 12H), 0.98 (d, J=8Hz, 6H), 0.88 (d, J=4Hz, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3, ppm): 158.21, 132.53, 130.38, 129.72, 129.28, 128.12, 127.12, 126.14, 125.61, 124.59, 123.86, 71.25, 71.20, 70.76, 70.71, 23.77, 23.74, 23.58, 23.53, 23.50, 22.95, 22.91; .sup.31P NMR (CDCl.sub.3, H.sub.3PO.sub.4, 161.98 MHz): 14.03 13.69 (d, J=55.07 Hz), 9.66-9.33 (d, J=53.45 Hz); IR (CHCl.sub.3): .sub.max 3436, 2979, 2930, 2079, 1633, 1452, 1385, 1240, 1177, 1106 cm.sup.1; ESI-MS: m/z 483 [M+H].sup.+; HRMS: m/z 483.2057 calcd for C.sub.24H.sub.37O.sub.6P.sub.2+H.sup.+ (483.2059).

Example 12

(46) Tetraethyl 2-(anthracene-10-yl) ethene-1,1-diyldiphosphonate (11)

(47) Yellow oil; yield 45%; .sup.1HNMR (400 MHz, CDCl.sub.3, ppm): 8.99-8.80 (dd, J.sub.1=28 Hz, J.sub.2=48 Hz, 1H), 8.37 (s, 1H), 7.94-7.85 (m, 4H), 7.43-7.19 (m, 4H), 4.38-4.31 (m, 4H), 3.56-3.50 (m, 2H), 3.36-3.32 (m, 2H), 1.45-1.24 (m, 6H), 0.77-0.60 (m, 6H); .sup.31P NMR (CDCl.sub.3, H.sub.3PO.sub.4, 161.98 MHz): 14.95-14.61 (d, J=55.07 Hz), 10.34-9.99 (d, J=56.69 Hz); IR (CHCl.sub.3): .sub.max 3435, 2919, 1601, 1404, 1360, 1280, 1186, 1148, 1019 cm.sup.1; ESI-MS: m/z 477 [M+1].sup.+; HRMS: m/z 477.1590 calcd for C.sub.24H.sub.31O.sub.6P.sub.2+H.sup.+ (477.1590).

Example 13

(48) Tetraisoproyl 2-(4-nitrofuran-2-yl)ethene-1,1-diyldiphosphonate (1m)

(49) Yellow oil; yield 45%; .sup.1HNMR (400 MHz, CDCl.sub.3, ppm): 8.07-7.89 (dd, J.sub.1=28 Hz, J.sub.2=48 Hz, 1H), 7.85 (d, J=4 Hz, 1H), 7.35-7.34 (m, 1H), 4.83 4.76 (m, 4H), 1.40-1.32 (m, 24H); .sup.13C NMR (100 MHz, CDCl.sub.3, ppm): 149.85, 148.62, 139.02, 124.43, 117.87, 110.35, 69.74, 69.71, 69.69, 69.67, 21.65, 21.61, 21.58, 21.55, 21.51, 21.29, 21.25; .sup.31P NMR (CDCl.sub.3, H.sub.3PO.sub.4, 161.98 MHz): 12.70-12.45 (d, J=40.49 Hz), 7.62-7.37 (d, J=40.49 Hz); IR (CHCl.sub.3): max 3436, 2980, 2928, 1591, 1530, 1454, 1386, 1351, 1247, 1142, 1104 cm.sup.1; ESI-MS: m/z 468 [M+1].sup.+; HRMS: m/z 468.1539 calcd for C.sub.18H.sub.32NO.sub.9P.sub.2+H.sup.+ (468.1546).

Example 14

(50) Pgp-Induction Assay

(51) All synthesized compounds were screened for their ability to induce Pgp using rhodamine123 (Rh123) cell exclusion method. In this method, the Pgp function was evaluated in terms of rhodamine 123 (Rh123) accumulations and efflux. Briefly, the protocol used is as follows: Colorectal LS-180 cells [obtained from ECACC (European Collection of Cell Cultures) catalogue number: 87021202; passage number 52] were seeded at a density of 210.sup.4 per well of 96 well plate and were allowed to grow for next 24 h. Cells were further incubated with the test compounds, and were diluted to a final concentration of 100 nM and rifampicin (standard) to a final concentration of 10 M in complete media for 48 h. The final concentration of DMSO was kept at 0.1%. Drugs were removed and cells were incubated with HANKS buffer for 40 minutes before further incubation with HANKS buffer (containing 10 M of Rh123 as a Pgp substrate) for 90 minutes. At the end of Rh123 treatment cells were washed four times with cold PBS followed by cell lysis for 1 h by using 200 l of lysis buffer (0.1% Triton X-100 and 0.2 N NaOH). A total of 100 l of lysate was used for reading fluorescence of Rh123 at 485 nm/529 nm. Samples were normalized by dividing fluorescence of each sample with total protein present in the lysate. For EC.sub.50 determination, different concentrations of compound were used to treat LS180 cells for 48 h. The EC.sub.50 value was determined by plotting fluoroscence of Rh123 against concentration of compound.

(52) The bis-phosphonate esters 1b-m showed ability to induce p-glycoprotein as showed by decrease in the % intracellular levels of rhodamine 123 in LS-180 cells (FIG. 2). One of the phosphonate ester compound 1l showed better Pgp induction activity (EC.sub.50=0.9 nM) as compared to SR12813 (1a) (EC.sub.50=2.04 nM). The compound 11 possesses 2-fold higher EC.sub.50 value than SR12813 (1a). The EC.sub.50 results are shown in Table 2.

(53) TABLE-US-00002 TABLE 2 Pgp induction activity in terms of EC.sub.50 values of 1a and 1l Compound Pgp induction EC.sub.50 value 1a 2.04 nM 1l 0.90 nM

Example 15

(54) Western Blot Analysis

(55) Protein was measured employing Bio-Rad protein assay kit using bovine serum albumin as standard. Proteins aliquots (70 g) were resolved on SDS-PAGE and then electro transferred to PVDF membrane overnight at 4 C. at 30V. Nonspecific binding was blocked by incubation with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature. The blots were probed with anti-Pgp antibody for 4 h and washed three times with TBST. Blot was then incubated with horseradish peroxidase conjugated antimouse secondary antibody for 1 h, washed again three times with TBST and signals detected using ECL plus chemiluminescence's kit on BioRad ChemiDoc XRS system.

(56) Upon exposure to compound 1l the p-glycoprotein mediated Rh123 efflux function in LS-180 cells is significantly increased and levels of intracellular % Rh123 is decreased as shown in FIG. 1. Furthermore in addition to that, the Western-blot results (FIG. 3) of p-glycoprotein expression clearly indicate that new phosphonate ester 1l induces p-glycoprotein expression in LS-180 cells.

Example 16

(57) Determination of Thermodynamic Equilibrium Solubility

(58) The compounds were first dissolved in methanol to prepare stock solutions (100 and 1000 g/mL). Different concentrations of stock solutions were pipetted into the 96-well plates and the solvent was evaporated to ensure that solid drug was present in the beginning of the experiment. Thereafter, 200 l of the dissolution medium (water) was added to the wells and 96-well plate was shaken horizontally at 300 rpm (Eppendorf Thermoblock Adapter, North America) for 4 h at room temperature (251 C.). The plates were kept overnight for equilibration of drug in medium. Later, the plates were centrifuged at 3000 rpm for 15 min (Jouan centrifuge BR4i). Supernatant (50 l) was pipetted into UV 96-well plates (Corning 96 Well Clear Flat Bottom UV-Transparent Microplate) for analyses with plate reader (SpectraMax Plus384) at .sub.max of 350 nm. The analyses were performed in triplicate for each compound. The solubility curve of concentration (ng/mL) vs absorbance was plotted to find out saturation point and the corresponding concentration was noted.

(59) Furthermore, the phosphonate ester 1l showed optimal solubility in water as well as in other biological fluids for good in-vivo pharmacokinetics. The solubility results are shown in Table 3.

(60) TABLE-US-00003 TABLE 3 Solubility of phosphonate esters 1a and 1l in water, phosphate buffer saline (PBS), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF). Solubility in g/mL Compound Water PBS SGF SIF 1a 200 200 200 200 1l 400 >1500 >1500 >1500

ADVANTAGES OF THE INVENTION

(61) The main advantages of the present invention are:

(62) Compounds claimed in the present invention showed promising Pgp induction activity in LS-180 cells. One of the compound claimed in the present invention showed Pgp induction at low nanomolar concentrations (EC.sub.50<1 nM). Compounds of the invention showed optimal water solubility. Compounds of the invention are stable.