Therapeutic compounds

09561201 · 2017-02-07

Assignee

Fibrotech Therapeutics Pty Ltd (Wonga Park, AU)

Inventors

Cpc classification

International classification

Abstract

Substituted cinnamoyl anthranilate compounds exhibiting anti-fibrotic activity; or derivatives thereof, analogs thereof, pharmaceutically acceptable salts thereof, and metabolites thereof, with the proviso that the compound is no Tranilast.

Claims

1. A method for treating focal segmental glomerulosclerosis in a patient comprising: administering to the patient a compound of Formula (I): ##STR00212## wherein R.sub.1 and R.sub.2, which may be the same or different, are selected from the group consisting of H, NHR.sub.6, NR.sub.6R.sub.7, OR.sub.8, halogen, C.sub.1 to C.sub.10 alkyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 cycloalkylmethyl, C.sub.3 to C.sub.10 alkene, C.sub.3 to C.sub.10 alkyne, aryl, C.sub.5 to C.sub.20 alkaryl, fused C.sub.5 to C.sub.20 aryl or alkaryl, and a hydrocarbon chain containing a heterocyclic or fused ring, any of which may be optionally substituted; R.sub.3 is selected from the group consisting of H, C.sub.1 to C.sub.10 alkyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 cycloalkylmethyl, C.sub.3 to C.sub.10 alkene, C.sub.3 to C.sub.10 alkyne, aryl, C.sub.5 to C.sub.20 alkaryl, and a hydrocarbon chain containing a heterocyclic or fused ring, any of which may be optionally substituted; R.sub.4 is selected from the group consisting of H, OH, OR.sub.6, NHR.sub.6 and NR.sub.6R.sub.7; R.sub.5 is selected from the group consisting of H, NHR.sub.6, NR.sub.6R.sub.7, OR.sub.8, halogen, C.sub.1 to C.sub.10 alkyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 cycloalkylmethyl, C.sub.3 to C.sub.10 alkene, C.sub.3 to C.sub.10 alkyne, aryl, C.sub.5 to C.sub.20 alkaryl, fused C.sub.5 to C.sub.20 aryl or alkaryl, and a hydrocarbon chain containing a heterocyclic or fused ring, any of which may be optionally substituted; X.sub.1 and X.sub.2, which may be the same or different, are selected from the group consisting of a bond, C, O, N and S; X.sub.3 is C or N, wherein when X.sub.3 is N, m is 0; T is a single or double bond; m is the integer 0 or 1; n is an integer between 0 and 4; R.sub.6 and R.sub.7, which may be the same or different, are selected from the group consisting of H, C.sub.1 to C.sub.10 alkyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 cycloalkylmethyl, C.sub.3 to C.sub.10 alkene, C.sub.3 to C.sub.10 alkyne, aryl, C.sub.5 to C.sub.20 alkaryl, and a hydrocarbon chain containing a heterocyclic or fused ring, any of which may be optionally substituted; R.sub.8 is selected from the group consisting of H, C.sub.1 to C.sub.10 alkyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 cycloalkylmethyl, C.sub.3 to C.sub.10 alkene, C.sub.3 to C.sub.10 alkyne, aryl, C.sub.5 to C.sub.20 alkaryl, and a hydrocarbon chain containing a heterocyclic or fused ring, any of which may be optionally substituted; a pharmaceutically acceptable salt thereof, or metabolites thereof; with the proviso that the compound is not Tranilast.

2. The method of claim 1, wherein the compound of Formula I is of formula: ##STR00213## wherein R.sub.1 and R.sub.2, which may be the same or different, are selected from the group consisting of a C.sub.1 to C.sub.10 alkyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 cycloalkylmethyl, C.sub.3 to C.sub.10 alkyne and a hydrocarbon chain containing a heterocyclic or fused ring, any of which may be optionally substituted; X.sub.1 and X.sub.2 are the same or different and are selected from the group consisting of a bond, O, N and S; T is a single or double bond; R.sub.3 is selected from the group consisting of H, C.sub.3 to C.sub.10 alkene, C.sub.3 to C.sub.10 alkyne and a hydrocarbon chain containing a heterocyclic or fused ring, any of which may be optionally substituted; R.sub.4 is selected from the group consisting of H, OH, OR.sub.6, NHR.sub.6 and NR.sub.6R.sub.7; R.sub.5 is selected from the group consisting of H, NHR.sub.6, NR.sub.6R.sub.7, OR.sub.8, halogen, C.sub.3 to C.sub.10 alkene, and C.sub.3 to C.sub.10 alkyne, any of which may be optionally substituted; R.sub.6 and R.sub.7, which may be the same or different, are selected from the group consisting of H, C.sub.1 to C.sub.10 alkyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 cycloalkylmethyl, C.sub.3 to C.sub.10 alkene, C.sub.3 to C.sub.10 alkyne, aryl, C.sub.5 to C.sub.20 alkaryl, and a hydrocarbon chain containing a heterocyclic or fused ring, any of which may be optionally substituted; R.sub.8 is selected from the group consisting of H, C.sub.1 to C.sub.10 alkyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 cycloalkylmethyl, C.sub.3 to C.sub.10 alkene, C.sub.3 to C.sub.10 alkyne, aryl, C.sub.5 to C.sub.20 alkaryl, and a hydrocarbon chain containing a heterocyclic or fused ring, any of which may be optionally substituted; and n is an integer between 0 and 4; or a pharmaceutically acceptable salt thereof; and with the proviso that when X.sub.1 and X.sub.2 are both O or a bond, and one of R.sub.1 or R.sub.2 is a C.sub.1 to C.sub.4 alkyl, the other of R.sub.1 or R.sub.2 is a C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 cycloalkylmethyl, C.sub.3 to C.sub.10 alkyne, or a hydrocarbon chain containing a heterocyclic or fused ring, any of which are optionally substituted.

3. The method of claim 2, wherein X.sub.1 and X.sub.2 are O.

4. The method of claim 3, wherein R.sub.1 or R.sub.2 is methyl.

5. The method of claim 4, wherein R.sub.3 is H.

6. The method of claim 5, wherein R.sub.1 or R.sub.2 is an alkyne.

7. The method of claim 1, wherein: R.sub.1 and R.sub.2, which may be the same or different, are selected from the group consisting of a C.sub.1 to C.sub.10 alkyl, C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 cycloalkylmethyl, C.sub.3 to C.sub.10 alkyne and a hydrocarbon chain containing a heterocyclic or fused ring, any of which may be optionally substituted; and R.sub.5 is selected from the group consisting of H, NHR.sub.6, NR.sub.6R.sub.7, OR.sub.8, halogen, C.sub.3 to C.sub.10 alkene, and C.sub.3 to C.sub.10 alkyne, any of which may be optionally substituted; with the proviso that when X.sub.1 and X.sub.2 are both O or a bond, and one of R.sub.1 or R.sub.2 is a C.sub.1 to C.sub.4 alkyl, the other of R.sub.1 or R.sub.2 is a C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 cycloalkylmethyl, C.sub.3 to C.sub.10 alkyne, or a hydrocarbon chain containing a heterocyclic or fused ring.

8. The method of claim 1, wherein the compound of Formula I of formula: ##STR00214## wherein R.sub.9 or R.sub.10, which may be the same or different, are selected from the group consisting of H, C.sub.1 to C.sub.10 alkyl, C.sub.3 to C.sub.8 terminal or non-terminal alkyne, cyclopentyl, cyclohexyl, cyclohexylmethyl, and cyclopentylmethyl; or a pharmaceutically acceptable salt thereof; with the proviso that when one of R.sub.9 or R.sub.10 is a C.sub.1 to C.sub.4 alkyl, the other of R.sub.9 or R.sub.10 is a C.sub.3 to C.sub.10 cycloalkyl, C.sub.3 to C.sub.10 cycloalkylmethyl, C.sub.3 to C.sub.10 alkyne or a hydrocarbon chain containing a heterocyclic or fused ring, any of which are optionally substituted.

9. The method of claim 1, wherein the compound of Formula I is of formula: ##STR00215## wherein p is an integer between 1 and 10, inclusive; and R is selected from the group consisting of H and C.sub.1 to C.sub.10 alkyl; or a pharmaceutically acceptable salt thereof.

10. The method of claim 1, wherein the compound of Formula I is of formula: ##STR00216## wherein G is a cyclopentyl ring, a cyclohexyl ring or a 1,4-disubstituted 1,2,3-triazole ring; and q is an integer between 0 and 6, inclusive; or a pharmaceutically acceptable salt thereof.

11. A method for treating focal segmental glomerulosclerosis in a patient comprising: administering to the patient a compound of Formula I selected from the group consisting of: ##STR00217## ##STR00218## ##STR00219## ##STR00220## ##STR00221## and a pharmaceutically acceptable salt thereof.

12. The method of claim 1, wherein the compound of Formula I is: ##STR00222## or a pharmaceutically acceptable salt thereof.

13. The method of claim 1, wherein the compound of Formula I is selected from the group consisting of: ##STR00223## or a pharmaceutically acceptable salt thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1 & 2: In vitro effects of FT011 and tranilast on transforming growth factor- induced .sup.3H-proline incorporation in cultured rat mesangial cells (concentration of compounds in M). Values are expressed as meansem. #p<0.05 versus cells grown in control medium,

(2) *p<0.05 versus TGF- treated cells.

(3) FIG. 3: Mesangial cells stimulated with Platelet derived growth factor (PDGF) to stimulate proline formation (matrix synthesis).

(4) FIG. 4: Shows the inhibition of TGF- stimulated fibrosis (indicated by proline formation) in neonatal cardiac fibroblasts.

(5) FIG. 5: Shows the inhibition of angiotensin II-stimulated fibrosis (indicated by proline formation) in neonatal cardiac fibroblasts.

(6) FIG. 6: Inhibition of TGF- stimulated proline incorporationtranilast vs FT011 (SEM).

(7) FIG. 7: Inhibition of TGF- stimulated proline incorporationFT017 (SEM).

(8) FIG. 8: Inhibition of TGF- stimulated proline incorporationFT019 (SEM).

(9) FIG. 9: Inhibition of TGF- stimulated proline incorporationFT023 (SEM).

(10) FIG. 10: Inhibition of TGF- stimulated proline incorporationFT026 (SEM).

(11) FIG. 11: Inhibition of TGF- stimulated proline incorporationFT039 (SEM).

(12) FIG. 12: Inhibition of TGF- stimulated proline incorporationFT040 (SEM).

(13) FIG. 13: Inhibition of TGF- stimulated proline incorporationFT016 (SEM).

(14) FIG. 14: Inhibition of TGF- stimulated proline incorporationFT018 (SEM).

(15) FIG. 15: Inhibition of TGF- stimulated proline incorporationFT027 (SEM).

(16) FIG. 16: Inhibition of TGF- stimulated proline incorporationFT029 (SEM).

(17) FIG. 17: Inhibition of TGF- stimulated proline incorporationFT033 (SEM).

(18) FIG. 18: Inhibition of TGF- stimulated proline incorporationFT034.

(19) FIG. 19: Inhibition of TGF- stimulated proline incorporationFT035 (SEM).

(20) FIG. 20: Inhibition of TGF- stimulated proline incorporationFT036 (SEM).

(21) FIG. 21: Plasma levels of FT011 in Sprague Dawley rats.

(22) FIG. 22: Urinary levels of FT011.

(23) FIG. 23: Comparison of myocardial infarct (MI) size in treated and untreated MI groups.

(24) FIG. 24: Quantitation of collagenous matrix in the NIZ (non-infarct zone), expressed as the proportional area stained blue on Masson's trichrome stained sections of rat heart. *P<0.01 versus shams and .sup.#P<0.05 versus untreated MI rats.

(25) FIG. 25: Representative Masson's trichrome-stained sections from sham and MI rats treated with FT011. In sham (A) and sham rats treated with FT011 (B), very little collagen (blue staining) is present within the interstitium, while extensive myocardial interstitial fibrosis was noted in NIZ of rats post MI (C). Treatment of MI rats with FT011 (D) was associated with a marked reduction in the extent of interstitial fibrosis in NIZ. Magnification 350.

(26) FIG. 26: Quantitation of collagen I (A) and III (B) immunostaining in rat heart from sham, sham treated with FT011, MI and MI rats treated with FT011. Values are expressed as meanSEM. *P<0.05 versus shams. .sup.#P<0.05 versus MI. Magnification 350.

(27) FIG. 27: Representative sections of immunohistochemistry for type I (A-D) and type III (E-H) collagen in sham (A, E), sham treated with FT011 (B, F), MI (C, G) and MI rats treated with FT011 (D, H). In sham rats there was minimal evidence of immunostaining for type I or III collagen, while MI rats were associated with a marked increase in collagen immunostaining. Treatment with FT011 was associated with a reduction in immunostaining for types I and III collagen.

(28) FIG. 28: Quantitation of ED-1 positive macrophages in rat heart from Sham, sham+FT011, MI and MI+FT011 (NIZ) groups. Values are represented as meansem.

(29) *p<0.05 when compared to shams. .sup.#p<0.05 when compared to MI.

(30) FIG. 29: ED-1 positive macrophages from sham, sham treated with FT011, MI and MI rats treated with FT011. In sham and sham+FT011 (A-B) only an occasional macrophage were observed while MI (C) was associated with increased macrophages (brown) at NIZ. Treatment of MI rats with FT011 (D) was associated with a reduction in macrophage number. Magnification 350.

(31) FIG. 30: Representative PV loop analysis of systolic and diastolic function from sham (A), sham treated with FT011 (B), MI (C) and MI treated with FT011 (D).

(32) FIG. 31: Albumin excretion rate in control and diabetic Ren-2 rats with and without treatment with FT011.

(33) *p<0.01 when compared to control

(34) .sup.#p<0.05 when compared to diabetes

(35) Legend: Blue 4 weeks post streptozotocin (STZ), red 8 weeks post STZ, yellow 12 weeks post STZ, green 16 weeks post STZ.

(36) FIG. 32: Glomerulosclerotic Index (upper panel) and tubulointerstitial fibrosis (lower panel) in control and diabetic rats treated with and without FT011. Data are expressed as meanSEM. *p<0.01 compared with controls, p<0.01 versus untreated diabetic rat kidneys.

(37) FIG. 33: Representative photomicrograph of periodic acid Schiff (PAS)-stained sections from control, diabetic and diabetic rats treated with FT011. In control (A) and control treated with FT011 (B) rats, there is no glomerulosclerosis, while diabetic (C) is associated with a dramatic increase in glomerulosclerosis. Treatment of diabetic rats with FT011 (D) was associated with a reduction in extent of glomerulosclerosis. Magnification 350.

(38) FIG. 34: Representative trichrome-stained sections showing tubulointerstitial fibrosis in control, diabetic and diabetic rats treated with FT011. In control and control treated with FT011 (A, B) there is minimal cortical tubular fibrosis, while diabetic (C) was associated with marked increase in interstitial fibrosis (blue). Treatment of diabetic rats with FT011 was associated with a reduction in tubular fibrosis (D). Magnification 350.

EXAMPLES

Example 1

General Description of Synthetic Chemistry

(39) Two general approaches were used for the synthesis of various substituted cinnamoyl anthranilates. In the first approach via a piperidine-catalyzed Knoevenagel condensation of a carboxyacetamidobenzoic acid and a benzaldehyde derivative thereof to provide a substituted cinnamoyl anthranilate as a piperidinium salt followed by acidification and recrystallization to produce a cinnamoyl anthranilate as the free acid providing an N-cinnamoyl-4-aminobenzoic acid via the following synthesis.

(40) ##STR00028##

(41) In the second approach, converting a substituted cinnamic acid to the corresponding acid chloride and condensing with a 2-aminobenzamide, or aniline.

(42) ##STR00029##

(43) The benzaldehyde precursors required for the above reactions were either obtained from commercial sources, or were synthesized by alkylation of precursor phenolic benzaldehydes with assorted alkyl halides or alkyl tosylates (derived in turn from the corresponding alcohols). Alkylations were typically performed using potassium carbonate as base in acetone. Carboxyacetamidobenzoic acids were obtained by the condensation of various anthranilic acid derivatives with Meldrum's acid. 2-Aminobenzamides were synthesized by the reaction of primary amines with isatoic anhydride. Saturation of the internal alkene of tranilast was performed by reduction with hydrogen in the presence of palladium on carbon catalyst. Cinnamic acids were prepared by Knoevenagel condensation of benzaldehydes with malonic acid. Formation of triazole substituted derivatives was performed using copper(I) catalyzed condensation of azides and terminal alkynes and provides only the 1,4-regioisomer.

Experimental

(44) High resolution mass spectra (HRMS) were obtained using on a Finnigan hybrid linear quadrupole ion trap-Fourier transform (LTQ-FT) mass spectrometer (Thermo Electron, San Jose, Calif.) equipped with an electrospray ionization source. Proton nuclear magnetic resonance (.sup.1H NMR) and proton decoupled carbon nuclear magnetic resonance (.sup.13C NMR) spectra were obtained on Unity 400, Innova 400 and Innova 500 instruments (Melbourne, Australia) operating at 400 MHz and 500 MHz for .sup.1H and at 100 MHz and 125 MHz for .sup.13C. All signals were referenced to solvent peaks (CDCl.sub.3: 7.26 ppm for .sup.1H and 77.0 ppm for .sup.13C; DMSO-d.sub.6: 2.49 ppm for .sup.1H and 39.5 ppm for .sup.13C). Infrared (IR) spectra were obtained using a PerkinElmer Spectrum One FT-IR spectrometer with zinc selenide/diamond Universal ATR Sampling Accessory. Melting points were obtained using a Reichert-Jung hot stage apparatus and are corrected. Analytical thin layer chromatography (TLC) was conducted on 2 mm thick silica gel GF.sub.254 (Merck). Compounds were visualised with solutions of 20% w/w phosphomolybdic acid in ethanol, 20% w/w potassium permanganate in water, or under UV (365 nm). Flash chromatography was performed according to the method of Still et al. [20] with Merck Silica Gel 60. Petrol refers to the fraction boiling at 40-60 C. All other reagents were used as received.

(45) Procedure 1

(46) Anthranilic acid (1.1 eq.) was added to a solution of Meldrum's acid (1.0 eq.) in toluene. The reaction flask was fitted with a Dean-Stark apparatus and the suspension was heated to reflux for 3 h. The suspension was cooled, and the precipitate collected by filtration, washed with toluene and dried.

(47) Procedure 2

(48) Piperidine (1.1 eq.) was added to a suspension of an aldehyde (1.1 eq.) and diacid (1.0 eq.) in toluene. The reaction flask was fitted with a Dean-Stark apparatus and heated to reflux for 4 h, cooled to rt and stirred for 1 h. The resulting suspension was filtered, and the filter cake was washed with toluene to afford the piperidinium salt. The piperidinium salt was dissolved in MeOH (5 mL/g) and water (2 mL per/g) at 40 C. The solution was acidified and the resulting precipitate was collected by filtration.

(49) Procedure 3

(50) Propargyl bromide (1.1-1.5 eq.) was added to a suspension of the phenol (1.0 eq.) and potassium carbonate (2.0 eq.) in acetone. The suspension was heated to reflux for 16 h and then the suspension was filtered, using acetone to rinse the filter cake. The filtrate was concentrated under reduced pressure, and water was added to the residue and the aqueous phase was extracted with EtOAc. The organic extract was washed with water, brine, dried and concentrated.

(51) Procedure 4

(52) 4-Methylbenzenesulfonyl chloride (1.5 eq.) was added to a cooled solution of alcohol (1.0 eq.) and pyridine (2.0 eq.) in CH.sub.2Cl.sub.2 at 0 C. The solution was stirred at 0 C. for 1 h, warmed to rt and stirred for 4 h. Water was added and the aqueous phase was extracted with ether. The organic extract was washed with 1 M HCl, saturated aqueous NaHCO.sub.3, water, brine and dried. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography, to afford the methylbenzenesulfonate. The methylbenzenesulfonate (1.5 eq.) was added to a suspension of phenol (1.0 eq.), potassium carbonate (3.0 eq.) and sodium iodide (0.1 eq.) in acetonitrile. The suspension was heated to reflux for 16 h, filtered, and the filter cake rinsed with acetonitrile. The filtrate was concentrated under reduced pressure. Water was added to the residue and the aqueous phase was extracted with EtOAc, washed with water, brine, dried and concentrated.

2-[(Carboxyacetyl)amino]benzoic acid

(53) ##STR00030##

(54) Anthranilic acid (181 g, 1.32 mol) and Meldrum's acid (200 g, 1.39 mol) in toluene (1.50 L) were treated according to Procedure 1. 2-[(Carboxyacetyl)amino]benzoic acid (263 g, 89%) was obtained as a colourless solid; mp 171-173 C., lit. [21] 178-180 C.; .sub.H (500 MHz, DMSO-d.sub.6) 3.45 (br s, 2H, CH.sub.2), 7.16 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.59 (td, J.sub.4,5=J.sub.5,6=8.0, J.sub.3,5=1.5 Hz, 1H, H5), 7.97 (dd, J.sub.3,4=8.0, J.sub.3,5=1.5 Hz, 1H, H3), 8.44 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.27 (s, 1H, NH), 12.83 (br s, 1H, CO.sub.2H), 13.57 (br s, 1H, CO.sub.2H); .sub.C (125 MHz, DMSO-d.sub.6) 45.0, 117.0, 120.3, 123.1, 131.2, 134.1, 140.4, 164.9, 169.1, 169.3.

(E)-2-[[3-(3,4-Dimethoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (tranilast) (1)

(55) ##STR00031##

(56) Piperidine (0.96 mL, 9.7 mmol) was added to a suspension of 3,4-dimethoxybenzaldehyde (1.6 g, 9.7 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (1.9 g, 8.6 mmol) in toluene (5.0 mL) and treated according to Procedure 2, acidifying with 1 M HCl. (E)-2-[[3-(3,4-Dimethoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (tranilast) (2.1 g, 74%) was obtained as a yellow crystalline solid; mp 208-209 C., lit. [22] 206 C.; .sub.H (500 MHz, DMSO-d.sub.6) 3.79 (s, 3H, OCH.sub.3), 3.82 (s, 3H, OCH.sub.3), 6.79 (d, J=15.5 Hz, 1H, CHCHCO), 6.99 (d, J.sub.5,6=8.5 Hz, 1H, H5), 7.16 (t, J.sub.3,4=J.sub.4,5=7.9 Hz, 1H, H4), 7.25 (d, J.sub.5,6=8.5 Hz, 1H, H6), 7.38 (s, 1H, H2), 7.56 (d, J=15.5 Hz, 1H, CHCHCO), 7.61 (t, J.sub.4,5=J.sub.5,6=7.9 Hz, 1H, H5), 8.00 (d, J.sub.3,4=7.9 Hz, 1H, H3), 8.62 (d, J.sub.5,6=7.9 Hz, 1H, H6), 11.30 (s, 1H, NH), 13.61 (br s, 1H, CO.sub.2H).

(E)-2-[(1-Oxo-3-phenyl-2-propenyl)amino]benzoic acid (2)

(57) ##STR00032##

(58) Piperidine (0.42 mL, 4.2 mmol) was added to a suspension of benzaldehyde (0.43 mL, 4.2 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.83 g, 3.7 mmol) in toluene (5.0 mL) and treated according to Procedure 2, acidifying with 1 M HCl. (E)-2-[(1-oxo-3-phenyl-2-propenyl)amino]benzoic acid (0.95 g, 96%) was obtained as a pale yellow crystalline solid; mp 188-189 C., lit. [23] 196-197 C.; .sub.H (500 MHz, DMSO-d.sub.6) 6.88 (d, J=16.0 Hz, 1H, CHCHCO), 7.18 (t, J.sub.3,4=J.sub.4,5=8.0, 1H, H4), 7.41-7.45 (m, 3H, H3, H4, H5), 7.62 (td, J.sub.4,5=J.sub.5,6=8.0, J.sub.3,5=1.5 Hz, 1H, H5), 7.62 (d, J=16.0 Hz, 1H, CHCHCO), 7.72-7.74 (m, 2H, H2, H6), 8.00 (dd, J.sub.3,4=8.0, J.sub.3,5=1.5 Hz, 1H, H3), 8.59 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.32 (s, 1H, NH).

(E)-2-[[3-(4-Methoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (3)

(59) ##STR00033##

(60) Piperidine (0.42 mL, 4.2 mmol) was added to a suspension of 4-methoxybenzaldehyde (0.51 mL, 4.2 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.83 g, 3.7 mmol) in toluene (5.0 mL) and treated according to Procedure 2, acidifying with 1 M HCl. (E)-2-[[3-(4-Methoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (0.95 g, 86%) was obtained as a pale yellow crystalline solid; mp 194-195 C., lit. [24] 195-198 C.; .sub.H (500 MHz, DMSO-d.sub.6) 3.80 (s, 3H, OCH.sub.3), 6.72 (d, J=15.5 Hz, 1H, CHCHCO), 6.98 (d, J.sub.2,3=J.sub.5,6=9.0 Hz, 2H, H3, H5), 7.16 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.57 (d, J=15.5 Hz, 1H, CHCHCO), 7.60 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 7.68 (d, J.sub.2,3=J.sub.5,6=9.0 Hz, 2H, H2, H6), 7.99 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.60 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.28 (s, 1H, NH).

(E)-2-[[3-(3-Methoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (4)

(61) ##STR00034##

(62) Piperidine (0.35 mL, 3.54 mmol) was added to a suspension of 3-methoxybenzaldehyde (0.43 mL, 3.5 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.70 g, 3.1 mmol) in toluene (5.0 mL) and treated according to Procedure 2, acidifying with 1 M HCl. (E)-2-[[3-(3-Methoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (0.71 g, 76%) as a yellow crystalline solid; mp 183-184 C., lit. [24] 183-185 C.; .sub.H (500 MHz, DMSO-d.sub.6) 3.80 (s, 3H, OCH.sub.3), 6.91 (d, J=15.5 Hz, 1H, CHCHCO), 6.98 (dd, J.sub.4,5=8.0, J.sub.2,4=2.0 Hz, 1H, H4), 7.18 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.23-7.36 (m, 3H, H2, H5, H6), 7.59 (d, J=15.5 Hz, 1H, CHCHCO), 7.62 (td, J.sub.4,5=J.sub.5,6=8.0, J.sub.3,5=1.5 Hz, 1H, H5), 7.99 (dd, J.sub.3,4=8.0, J.sub.3,5=1.5 Hz, 1H, H3), 8.58 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.31 (s, 1H, NH).

(E)-2-[[3-(3,4-Dihydroxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (5)

(63) ##STR00035##

(64) Piperidine (0.39 mL, 4.0 mmol) was added to a suspension of 3,4-dihydroxybenzaldehyde (0.55 g, 4.0 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.74 g, 3.3 mmol) in toluene (5.0 mL) and treated according to Procedure 2, acidifying with 1 M HCl. (E)-2-[[3-(3,4-Dihydroxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (0.82 g, 83%) was obtained as a brown crystalline solid; mp 204-206 C.; lit. [24] 204-206 C.; .sub.H (500 MHz, DMSO-d.sub.6) 6.50 (d, J=15.5 Hz, 1H, CHCHCO), 6.77 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.00 (dd, J.sub.5,6=8.0, J.sub.2,6=2.0 Hz, 1H, H6), 7.08 (d, J.sub.2,6=2.0 Hz, 1H, H2), 7.14 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.44 (d, J=15.5 Hz, 1H, CHCHCO), 7.61 (td, J.sub.4,5=J.sub.5,6=8.0, J.sub.3,5=1.5 Hz, 1H, H5), 8.00 (dd, J.sub.3,4=8.0, J.sub.3,5=1.5 Hz, 1H, H3), 8.58 (d, J.sub.5,6=8.0 Hz, 1H, H6), 9.11 (s, 1H, OH), 9.52 (s, 1H, OH), 11.25 (s, 1H, NH).

(E)-2-[[3-(4-Hydroxy-3-methoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (6)

(65) ##STR00036##

(66) Piperidine (0.50 mL, 5.1 mmol) was added to a suspension of 4-hydroxy-3-methoxybenzaldehyde (0.77 g, 5.1 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (1.0 g, 4.5 mmol) in toluene (5.0 mL) and treated according to Procedure 2, acidifying with 1 M HCl. (E)-2-[[3-(4-Hydroxy-3-methoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (1.1 g, 78%) was obtained as a yellow crystalline solid; mp 207.5-208.5 C., lit. [25] 230-233 C.; .sub.H (500 MHz, DMSO-d.sub.6) 3.83 (s, 3H, OCH.sub.3), 6.71 (d, J=15.5 Hz, 1H, CHCHCO), 6.80 (d, J.sub.5,6=8.5 Hz, 1H, H5), 7.13 (dd, J.sub.5,6=8.5, J.sub.2,6=1.5 Hz, 1H, H6), 7.15 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.34 (d, J.sub.2,6=1.5 Hz, 1H, H2), 7.52 (d, J=15.5 Hz, 1H, CHCHCO), 7.60 (td, J.sub.4,5=J.sub.5,6=8.0, J.sub.3,5=2.0 Hz, 1H, H5), 8.00 (dd, J.sub.3,4=8.0, J.sub.3,5=2.0 Hz, 1H, H3), 8.62 (d, J.sub.5,6=8.0 Hz, 1H, H6), 9.57 (s, 1H, OH), 11.27 (s, 1H, NH), 13.61 (br s, 1H, CO.sub.2H).

(E)-2-[[3-(3-Hydroxy-4-methoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (7)

(67) ##STR00037##

(68) Piperidine (0.25 mL, 2.5 mmol) was added to a suspension of 3-hydroxy-4-methoxybenzaldehyde (0.39 g, 2.5 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.50 g, 2.2 mmol) in toluene (5.0 mL) and treated according to Procedure 2, acidifying with 1 M HCl. (E)-2-[[3-(3-Hydroxy-4-methoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (0.53 g, 76%) was obtained as a yellow crystalline solid; mp 215-216 C., lit [25] 219-222 C.; .sub.H (500 MHz, DMSO-d.sub.6) 3.81 (s, 3H, OCH.sub.3), 6.59 (d, J=15.5 Hz, 1H, CHCHCO), 6.80 (d, J.sub.5,6=8.5 Hz, 1H, H5), 7.10-7.13 (m, 2H, H2, H6), 7.15 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.47 (d, J=15.5 Hz, 1H, CHCHCO), 7.60 (td, J.sub.4,5=J.sub.5,6=8.0, J.sub.3,5=1.5 Hz, 1H, H5), 7.99 (dd, J.sub.3,4=8.0, J.sub.3,5=1.5 Hz, 1H, H3), 8.58 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.25 (s, 1H, NH), 13.56 (br s, 1H, CO.sub.2H).

3-(2-Carboxyacetamido)-2-naphthoic acid

(69) ##STR00038##

(70) 3-Aminonaphthoic acid (0.60 g, 2.6 mmol) was added to a solution of Meldrum's acid (0.46 g, 3.2 mmol) in toluene (5.0 mL) and treated according to Procedure 1. 3-(2-Carboxyacetamido)-2-naphthoic acid (0.71 g, 81%) was obtained as a brown solid; mp 225-227 C.; .sub.H (400 MHz, DMSO-d.sub.6) 3.50 (br s, 2H, CH.sub.2), 7.49 (t, J.sub.6,7=J.sub.7,8=8.0 Hz, 1H, H7), 7.61 (t, J.sub.5,6=J.sub.6,7=8.0 Hz, 1H, H6), 7.88 (d, J.sub.7,8=8.0 Hz, 1H, H8), 8.02 (d, J.sub.5,6=8.0 Hz, H5), 8.67 (s, 1H, H4), 8.88 (s, 1H, H1), 11.31 (s, 1H, NH); .sub.C (100 MHz, DMSO-d.sub.6) 44.9, 117.1, 117.9, 125.7, 127.2, 128.3, 129.0, 129.2, 133.0, 135.4, 135.6, 164.7, 169.0, 169.2; .sub.max 1134, 1195, 1245, 1369, 1552, 1661, 1697, 3099 cm.sup.1.

(E)-3-[[3-(3,4-Dimethoxyphenyl)-1-oxo-2-propenyl]amino]-2-naphthoic acid (8)

(71) ##STR00039##

(72) Piperidine (0.23 mL, 2.3 mmol) was added to a suspension of 3,4-dimethoxybenzaldehyde (0.38 g, 2.3 mmol) and 3-(2-carboxyacetamido)-2-naphthoic acid (0.56 g, 2.0 mmol) in toluene (5.0 mL) and treated according to Procedure 2, acidifying with 1 M HCl. (E)-3-[[3-(3,4-Dimethoxyphenyl)-1-oxo-2-propenyl]amino]-2-naphthoic acid (0.51 g, 66%) was obtained as a yellow crystalline solid; mp 212-213 C.; .sub.H (400 MHz, DMSO-d.sub.6) 3.80 (s, 3H, OCH.sub.3), 3.84 (s, 3H, OCH.sub.3), 6.82 (d, J=15.6 Hz, 1H, CHCHCO), 6.99 (d, J.sub.5,6=8.2 Hz, 1H, H5), 7.25 (dd, J.sub.5,6=8.2, J.sub.2,6=2.0 Hz, 1H, H6), 7.38 (d, J.sub.2,6=2.0 Hz, 1H, H2), 7.49 (t, J.sub.6,7=J.sub.7,8=8.0 Hz, 1H, H7), 7.58 (d, J=15.6 Hz, 1H, CHCHCO), 7.62 (t, J.sub.5,6=J.sub.6,7=8.0 Hz, 1H, H6), 7.89 (d, J.sub.7,8=8.0 Hz, 1H, H8), 8.03 (d, J.sub.5,6=8.0 Hz, H5), 8.71 (s, 1H, H1), 9.05 (s, 1H, H4), 11.30 (s, 1H, NH); .sub.C (100 MHz, DMSO-d.sub.6) 55.6, 55.7, 110.4, 111.6, 117.1, 117.6, 120.1, 122.6, 125.6, 127.1, 127.3, 128.2, 129.1, 129.3, 133.1, 135.5, 136.3, 141.4, 149.0, 150.6, 164.2, 169.5; HRMS (ESI) Calculated for C.sub.22H.sub.19NO.sub.5 [M+H].sup.+378.1336. found 378.1345; .sub.max 797, 1022, 1134, 1233, 1512, 1665, 1693, 3048 cm.sup.1.

2-[(Carboxyacetyl)amino]-4,5-dimethoxybenzoic acid

(73) ##STR00040##

(74) 4,5-Dimethoxyanthranilic acid (0.50 g, 2.5 mmol) was added to a solution of Meldrum's acid (0.42 g, 2.9 mmol) in toluene (5.0 mL) and treated according to Procedure 1. 2-[(Carboxyacetyl)amino]-4,5-dimethoxybenzoic acid (0.70 g, 97%) was obtained as a brown solid; .sub.H (400 MHz, DMSO-d.sub.6) 3.43 (br s, 2H, CH.sub.2), 3.75 (s, 3H, OCH.sub.3), 3.79 (s, 3H, OCH.sub.3), 7.42 (s, 1H, H3), 8.24 (s, 1H, H6), 11.40 (s, 1H, NH).

(E)-2-[[3-(3,4-Dimethoxyphenyl)-1-oxo-2-propenyl]amino]-4,5-dimethoxybenzoic acid (9)

(75) ##STR00041##

(76) Piperidine (0.28 mL, 2.8 mmol) was added to a suspension of 3,4-dimethoxybenzaldehyde (0.46 g, 2.8 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.46 g, 2.5 mmol) in toluene (5.0 mL) and treated according to Procedure 2, acidifying with 1 M HCl. (E)-2-[[3-(3,4-Dimethoxyphenyl)-1-oxo-2-propenyl]amino]-4,5-dimethoxybenzoic acid (0.69 g, 72%) was obtained as a pale yellow crystalline solid; mp 236-239 C., lit. [26] 190-191 C.; .sub.H (400 MHz, DMSO-d.sub.6) 3.76 (s, 3H, OCH.sub.3), 3.79 (s, 3H, OCH.sub.3), 3.83 (s, 3H, 2OCH.sub.3), 6.76 (d, J=15.2 Hz, 1H, CHCHCO), 6.98 (d, J.sub.5,6=8.4 Hz, 1H, H5), 7.21 (d, J.sub.5,6=8.4 Hz, 1H, H6), 7.36 (s, 1H, H2), 7.44 (s, 1H, H3), 7.53 (d, J=15.2 Hz, 1H, CHCHCO), 8.45 (s, 1H, H6), 11.37 (s, 1H, NH).

2-[[3-(3,4-Dimethoxyphenyl)-1-oxopropyl]amino]benzoic acid (10)

(77) ##STR00042##

(78) Palladium on carbon (5%, 50 mg) was added to a solution of (E)-2-[[3-(3,4-dimethoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (tranilast) (0.50 g, 1.5 mmol) in THF (9.0 mL), EtOH (1.0 mL) and AcOH (1 drop). The suspension was stirred under an atmosphere of hydrogen for 16 h and filtered. The filtrate was concentrated under reduced pressure and the crude product was recrystallised from EtOAc/petrol to give 2-[[3-(3,4-dimethoxyphenyl)-1-oxopropyl]amino]benzoic acid (0.39 g, 77%) as a colourless crystalline solid; mp 137 C., lit. [24] 136-137.5 C.; .sub.H (500 MHz, DMSO-d.sub.6) 2.68 (t, J=7.5 Hz, 2H, CH.sub.2CO), 2.87 (t, J=7.5 Hz, 2H, CH.sub.2Ar), 3.68 (s, 3H, OCH.sub.3), 3.70 (s, 3H, OCH.sub.3), 6.74 (d, J.sub.5,6=8.2 Hz, 1H, H6), 6.82 (d, J.sub.5,6=8.2 Hz, 1H, H5), 6.86 (s, 1H, H2), 7.12 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.57 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 7.95 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.47 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.11 (s, 1H, NH), 13.57 (br s, 1H, CO.sub.2H).

3-Methoxy-4-propargyloxybenzaldehyde

(79) ##STR00043##

(80) Propargyl bromide (219 mL, 80% w/v, 1.48 mol) was added to a suspension of vanillin (150 g, 0.986 mol) and potassium carbonate (408 g, 2.96 mol) in acetone (1.50 L) and treated according to Procedure 3. 3-Methoxy-4-propargyloxybenzaldehyde (162 g, 86%) was obtained as yellow crystalline solid; mp 95 C.; .sub.H (400 MHz, CDCl.sub.3) 2.56 (t, J=2.5 Hz, 1H, CCH), 3.95 (s, 3H, OCH.sub.3), 4.86 (d, J=2.5 Hz, 2H, OCH.sub.2), 7.14 (d, J.sub.5,6=6.8 Hz, 1H, H5), 7.44 (d, J.sub.2,6=1.4 Hz, 1H, H2), 7.47 (dd, J.sub.5,6=6.8, J.sub.2,6=1.4 Hz, 1H, H6), 9.87 (s, 1H, CHO); .sub.C (100 MHz, CDCl.sub.3) 56.0, 56.6, 77.2, 77.4, 109.4, 112.5, 126.3, 130.9, 150.0, 152.1, 190.9; HRMS (ESI) Calculated for C.sub.11H.sub.10O.sub.3 [M+H].sup.+ 191.0703. found 191.0706; .sub.max 1006, 1130, 1259, 1586, 1677, 2119, 2845, 2932, 3266 cm.sup.1.

(E)-2-[[3-(3-Methoxy-4-propargyloxy)phenyl)-1-oxo-2-propenyl]amino]benzoic acid (11)

(81) ##STR00044##

(82) Piperidine (85.0 mL, 85.6 mmol) was added to a suspension of 3-methoxy-4-propargyloxybenzaldehyde (163 g, 85.6 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (182 g, 81.5 mmol) in toluene (1.0 L) and treated according to Procedure 2, acidifying with 50% AcOH. The crude product was recrystallised from EtOH (35 mL/g), filtered and washed with cold EtOH to afford (E)-2-[[3-(3-methoxy-4-propargyloxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (222 g, 77%) as a yellow crystalline solid; mp 191-193 C.; .sub.H (400 MHz, DMSO-d.sub.6) 3.59 (t, J=2.4 Hz, 1H, HCC), 3.84 (s, 3H, OCH.sub.3), 4.84 (d, J=2.4 Hz, 2H, OCH.sub.2), 6.81 (d, J=15.6 Hz, 1H, CHCHCO), 7.05 (d, J.sub.5,6=8.4 Hz, 1H, H5), 7.16 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.25 (d, J.sub.5,6=8.4 Hz, 1H, H6), 7.41 (s, 1H, H2), 7.56 (d, J=15.6 Hz, 1H, CHCHCO), 7.61 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 8.00 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.62 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.31 (s, 1H, NH), 13.57 (br s, 1H, COY); .sub.C (100 MHz, DMSO-d.sub.6) 55.6, 55.9, 78.6, 79.1, 110.8, 113.5, 116.6, 120.4, 120.4, 122.2, 122.7, 128.2, 131.2, 134.0, 141.0, 141.5, 148.3, 149.3, 164.1, 169.5; HRMS (ESI) calculated for C.sub.20H.sub.17NO.sub.5 [M+H].sup.+ 352.1179. found 352.1187; .sub.max 755, 1010, 1140, 1253, 1502, 1582, 1657, 3278, 3522 cm.sup.1.

(E)-3-(3,4-Dimethoxyphenyl)-2-propenoic acid

(83) ##STR00045##

(84) A solution of 3,4-dimethoxybenzaldehyde (5.0 g, 30 mmol) and malonic acid (4.7 g, 45 mmol) in a mixture of piperidine (0.5 mL) and pyridine (15 mL) was heated to 120 C. and stirred overnight. The mixture was cooled to rt and acidified with conc. HCl. The resulting precipitate was filtered and washed with water to give (E)-3-(3,4-dimethoxyphenyl)-2-propenoic acid (5.1 g, 81%) as a pale brown solid; .sub.H (400 MHz, DMSO-d.sub.6) 3.78 (s, 3H, OCH.sub.3), 3.79 (s, 3H, OCH.sub.3), 6.42 (d, J=16.0 Hz, 1H, CHCHCO.sub.2H), 6.96 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.19 (d, J.sub.5,6=8.0 Hz, 1H, H6), 7.30 (s, 1H, H2), 7.51 (d, J=16.0 Hz, 1H, CHCHCO.sub.2H).

(E)-2-[[3-(3,4-Dimethoxyphenyl)-1-oxo-2-propenyl]amino]benzamide (12)

(85) ##STR00046##

(86) A suspension of (E)-3-(3,4-dimethoxyphenyl)-2-propenoic acid (0.51 g, 2.5 mmol) in toluene (5.0 mL) was treated with thionyl chloride (0.53 mL, 7.3 mmol) and catalytic DMF (1 drop). The solution was heated to 50 C. and stirred for 1 h and the solvent was removed under reduced pressure to give the acid chloride as a yellow solid. A solution of the acid chloride (2.5 mmol) in pyridine (2.0 mL) and THF (2.0 mL) was added to a solution of 2-aminobenzamide (0.40 g, 2.9 mmol) in pyridine (1.0 mL). The suspension was stirred at rt for 16 h, cooled to 0 C. and acidified with 1 M HCl. The crude product was filtered, dried and recrystallised from acetonitrile to give (E)-2-[[3-(3,4-dimethoxyphenyl)-1-oxo-2-propenyl]amino]benzamide (0.32 g, 40%) as a pale red crystalline solid; mp 184-186 C., lit. [27] 193-194 C.; .sub.H (400 MHz, DMSO-d.sub.6) 3.79 (s, 3H, OCH.sub.3), 3.82 (s, 3H, OCH.sub.3), 6.72 (d, J=15.4 Hz, 1H, CHCHCO), 6.98 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.13 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.22 (dd, J.sub.5,6=8.0 Hz, J.sub.2,6=1.6 Hz, 1H, H6), 7.36 (d, J.sub.2,6=1.6 Hz, 1H, H2), 7.50 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 7.52 (d, J=15.4 Hz, 1H, CHCHCO), 7.73 (s, 1H, NH.sub.2), 7.80 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.30 (s, 1H, NH.sub.2), 8.57 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.79 (s, 1H, NH).

(E)-[3-(3,4-Dimethoxyphenyl)-1-oxo-2-propenyl]aminobenzene (13)

(87) ##STR00047##

(88) A suspension of (E)-3-(3,4-dimethoxyphenyl)-2-propenoic acid (0.51 g, 2.5 mmol) in CHCl.sub.3 (5.0 mL) was treated with thionyl chloride (0.53 mL, 7.3 mmol) and catalytic DMF (1 drop). The solution was heated to reflux and stirred for 16 h and the solvent was removed under reduced pressure to give the acid chloride as a yellow solid. A solution of the acid chloride (2.5 mmol) in CH.sub.2Cl.sub.2 (2.0 mL) was added to a solution of aniline (0.25 mL, 2.7 mmol) and NEt.sub.3 (0.75 mL, 5.4 mmol) in CH.sub.2Cl.sub.2 (2.0 mL). The mixture was stirred at rt for 16 h and diluted with water. The aqueous phase was extracted with EtOAc and the combined organic extracts were washed with water, brine and dried. The crude product was recrystallised from acetonitrile to give (E)-[3-(3,4-dimethoxyphenyl)-1-oxo-2-propenyl]aminobenzene (0.23 g, 33%) as a colourless crystalline solid; mp 131-133 C., lit. [28] 111 C.; .sub.H (400 MHz, DMSO-d.sub.6) 3.79 (s, 3H, OCH.sub.3), 3.81 (s, 3H, OCH.sub.3), 6.69 (d, J=15.5 Hz, 1H, CHCHCO), 7.01 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.04 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.17 (d, J.sub.5,6=8.0 Hz, 1H, H6), 7.21 (5, 1H, H2), 7.31 (t, J.sub.2,3=J.sub.3,4=8.0 Hz, 2H, H3, H5), 7.51 (d, J=16.0 Hz, 1H, CHCHCO), 7.68 (d, J.sub.2,3=J.sub.5,6=8.0 Hz, 2H, H2, H6), 10.09 (5, 1H, NH).

4-[(Carboxyacetyl)amino]benzoic acid

(89) ##STR00048##

(90) 4-Aminobenzoic acid (0.50 g, 3.6 mmol) was added to a solution of Meldrum's acid (0.63 g, 4.4 mmol) in toluene (5.0 mL) and treated according to Procedure 1. 4-[(Carboxyacetyl)amino]benzoic acid (0.74 g, 91%) was obtained as a colourless solid; .sub.H (400 MHz, DMSO-d.sub.6) 3.38 (br s, 2H, CH.sub.2), 7.68 (t, J.sub.2,3=J.sub.5,6=8.0 Hz, 1H, H2, H6), 7.89 (d, J.sub.2,3=J.sub.5,6=8.0, 1H, H3, H5), 10.44 (s, 1H, NH), 12.70 (br s, 1H, CO.sub.2H).

(E)-4-[[3-(3,4-Dimethoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (14)

(91) ##STR00049##

(92) Piperidine (0.39 mL, 4.0 mmol) was added to a suspension of 3,4-dimethoxybenzaldehyde (0.66 g, 4.0 mmol) and 4-[(carboxyacetyl)amino]benzoic acid (0.74 g, 3.3 mmol) in toluene (5.0 mL) and treated according to Procedure 2, acidifying with 1 M HCl. The crude product was recrystallised from EtOH providing (E)-4-[[3-(3,4-dimethoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (0.58 g, 53%) as a yellow crystalline solid; mp 258-259 C., lit. [24] 267-269 C.; .sub.H (400 MHz, DMSO-d.sub.6) 3.80 (s, 3H, OCH.sub.3), 3.82 (s, 3H, OCH.sub.3), 6.72 (d, J=15.6 Hz, 1H, CHCHCO), 7.01 (d, J.sub.5,6=8.2 Hz, 1H, H5), 7.20 (d, J.sub.5,6=8.2 Hz, 1H, H6), 7.22 (s, 1H, H2), 7.56 (d, J=15.6 Hz, 1H, CHCHCO), 7.80 (d, J.sub.2,3=J.sub.5,6=8.4 Hz, 2H, H3, H5), 7.90 (d, J.sub.2,3=J.sub.5,6=8.4 Hz, 1H, H2, H6), 10.43 (s, 1H, NH), 12.68 (br s, 1H, CO.sub.2H).

2-Amino-N-propargylbenzamide

(93) ##STR00050##

(94) A solution of propargylamine (1.00 mL, 14.6 mmol) in DMF (4.0 mL) was added dropwise to a solution of isatoic anhydride (1.57 g, 9.72 mmol) in DMF (8.0 mL) at 45 C. The solution was stirred at 45 C. for 16 h and diluted with water and CH.sub.2Cl.sub.2. The aqueous phase was extracted with CH.sub.2Cl.sub.2 washed with water, brine, dried and concentrated. The crude product was recrystallised from EtOAc/petrol to give 2-amino-N-propargylbenzamide (0.85 g, 51%) as a colourless solid; mp 100-101 C., lit. [29] 98-100 C.; .sub.H (400 MHz, DMSO-d.sub.6) 3.08 (t, J=2.4 Hz, 1H, CCH), 3.97 (dd, J=5.6, 2.4 Hz, 2H, CH.sub.2), 6.45 (s, 2H, NH.sub.2), 6.49 (t, J.sub.4,5=J.sub.5,6=7.8 Hz, 1H, H5), 6.68 (d, J.sub.3,4=7.8 Hz, 1H, H3), 7.13 (t, J.sub.3,4=J.sub.4,5=7.8 Hz, 1H, H4), 7.46 (d, J.sub.5,6=7.8 Hz, 1H, H6), 6.61 (t, J=5.6 Hz, 1H, NH).

(E)-2-[[3-(3,4-Dimethoxyphenyl)-1-oxo-2-propenyl]amino]-N-propargylbenzamide (15)

(95) ##STR00051##

(96) A suspension of (E)-3-(3,4-dimethoxyphenyl)-2-propenoic acid (0.85 g, 4.1 mmol) in toluene (8.5 mL) was treated with thionyl chloride (0.89 mL, 12 mmol) and catalytic DMF (1 drop). The solution was heated to reflux and stirred for 16 h and the solvent was removed under reduced pressure to give the acid chloride as a yellow solid. A solution of the acid chloride (4.1 mmol) in pyridine (6.0 mL) was added to a solution of 2-amino-N-2-propynyl-benzamide (0.74 g, 4.3 mmol) in pyridine (2.0 mL). The mixture was stirred at rt for 16 h, cooled to 0 C. and acidified with 1M HCl. The product was filtered, dried and recrystallised from acetonitrile providing (E)-[[3-(3,4-dimethoxyphenyl)-1-oxo-2-propenyl]amino]-N-propargylbenzene (1.05 g, 71%) as a colourless crystalline solid; mp 174-176 C.; .sub.H (400 MHz, DMSO-d.sub.6) 3.17 (t, J=2.4 Hz, 1H, CCH), 3.79 (s, 3H, OCH.sub.3), 3.83 (s, 3H, OCH.sub.3), 4.08 (dd, J=5.6, 2.4 Hz, 2H, CH.sub.2), 6.76 (d, J=15.6 Hz, 1H, CHCHCO), 6.98 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.16 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.23 (dd, J.sub.5,6=8.0, J.sub.2,6=1.6 Hz, 1H, H6), 7.38 (d, J.sub.2,6=1.6 Hz, 1H, H2), 7.52 (dt, J.sub.4,5=J.sub.5,6=8.0, J.sub.3,5=1.2 Hz, 1H, H5), 7.75 (dd, J.sub.3,4=8.0, J.sub.3,5=1.2 Hz, 1H, H3), 8.55 (d, J.sub.5,6=8.0 Hz, 1H, H6), 9.23 (t, J=5.6 Hz, 1H, NH), (s, 1H, NH); .sub.C (100 MHz, DMSO-d.sub.6) 28.6, 55.5, 55.6, 73.2, 80.8, 110.2, 111.5, 119.8, 120.1, 120.9, 122.7, 122.8, 127.3, 128.2, 132.2, 139.4, 141.6, 149.0, 150.6, 164.0, 168.1; HRMS (ESI) calculated for C.sub.21H.sub.20N.sub.2O.sub.4 [M+Na].sup.+ 387.1315. found 387.1316; .sub.max 1017, 1265, 1447, 1512, 1584, 1600, 1659, 3043, 3329 cm.sup.1.

5-Bromo-2-[(carboxyacetyl)amino]benzoic acid

(97) ##STR00052##

(98) 5-Bromoanthranilic acid (0.30 g, 1.4 mmol) was added to a solution of Meldrum's acid (0.24 g, 1.7 mmol) in toluene (5.0 mL) and treated according to Procedure 1. 5-Bromo-2-[(carboxyacetyl)amino]benzoic acid (0.34 mg, 81%) was obtained as a pale brown solid; .sub.H (500 MHz, DMSO-d.sub.6) 3.48 (s, 2H, CH.sub.2), 7.78 (d, J.sub.3,4=8.4 Hz, 1H, H4), 8.04 (s, 1H, H6), 8.40 (d, J.sub.3,4=8.4 Hz, 1H, H3), 11.20 (s, 1H, NH), 12.80 (br s, 1H, CO.sub.2H); .sub.C (125 MHz, DMSO-d.sub.6) 44.7, 114.5, 119.4, 122.5, 133.1, 136.4, 139.4, 164.7, 167.8, 168.9.

(E)-2-[[3-(3,4-Dimethoxyphenyl)-1-oxo-2-propenyl]amino]-5-bromobenzoic acid (16)

(99) ##STR00053##

(100) Piperidine (0.13 mL, 1.4 mmol) was added to a suspension of 3,4-dimethoxybenzaldehyde (0.22 g, 1.4 mmol) and 5-bromo-2-[(carboxyacetyl)amino]benzoic acid (0.34 g, 1.1 mmol) in toluene (4.0 mL) and treated according to Procedure 2, acidifying with 1 M HCl. (E)-2-[[3-(3,4-Dimethoxyphenyl)-1-oxo-2-propenyl]amino]-5-bromobenzoic acid (0.30 g, 66%) was obtained as a yellow crystalline solid; mp 210-213 C.; .sub.H (400 MHz, DMSO-d.sub.6) 3.79 (s, 3H, OCH.sub.3), 3.82 (s, 3H, OCH.sub.3), 6.78 (d, J=15.6 Hz, 1H, CHCHCO), 6.98 (d, J.sub.5,6=8.4 Hz, 1H, H5), 7.24 (d, J.sub.5,6=8.4 Hz, 1H, H6), 7.36 (s, 1H, H2), 7.56 (d, J=15.6 Hz, 1H, CHCHCO), 7.78 (dd, J.sub.3,4=8.4, J.sub.4,6=2.0 Hz, 1H, H4), 8.06 (d, J.sub.4,6=2.0 Hz, 1H, H6), 8.62 (d, J.sub.3,4=8.4 Hz, 1H, H3), 11.30 (s, 1H, NH), 13.61 (br s, 1H, CO.sub.2H); .sub.C (100 MHz, DMSO-d.sub.6) 28.6, 55.5, 55.6, 110.4, 111.6, 114.0, 119.5, 122.5, 122.7, 127.1, 133.1, 136.4, 140.2, 142.0, 149.0, 150.7, 164.2, 168.1; HRMS (ESI) calculated for C.sub.18H.sub.16BrNO.sub.5 [M+Na].sup.+ 428.0104. found 428.0105; .sub.max 1026, 1247, 1510, 1595, 1698, 2515, 2829, 3226, 3619 cm.sup.1.

4-Methoxy-3-propargyloxybenzaldehyde

(101) ##STR00054##

(102) Propargyl bromide (2.90 mL, 80% w/v, 19.7 mmol) was added to a suspension of vanillin (2.00 g, 13.1 mmol) and potassium carbonate (5.46 g, 39.4 mmol) in acetone (20 mL) and treated according to Procedure 3. 4-Methoxy-3-propargyloxybenzaldehyde (2.01 g, 80%) was obtained as a colourless crystalline solid; mp 66-67 C.; .sub.H (400 MHz, CDCl.sub.3) 2.54 (t, J=2.4 Hz, 1H, CCH), 3.95 (s, 3H, OCH.sub.3), 4.81 (d, J=2.4 Hz, 1H, OCH.sub.2), 7.00 (d, J.sub.5,6=8.4 Hz, 1H, H5), 7.50-7.53 (m, 2H, H2, H6), 9.85 (s, 1H, CHO); .sub.C (100 MHz, CDCl.sub.3) 56.1, 56.6, 76.4, 77.6, 110.9, 111.9, 127.3, 129.9, 147.3, 154.9, 190.6; HRMS (ESI) Calculated for C.sub.11H.sub.10O.sub.3 [M+H].sup.+ 191.0703. found 191.0704; .sub.max 1014, 1130, 1261, 1584, 1678, 2119, 2841, 2932, 3262 cm.sup.1.

(E)-2-[[3-(4-Methoxy-3-propargyloxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (17)

(103) ##STR00055##

(104) Piperidine (0.70 mL, 7.1 mmol) was added to a suspension of 4-methoxy-3-propargyloxybenzaldehyde (1.34 g, 7.06 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (1.50 g, 6.72 mmol) in toluene (5.0 mL) and treated according to Procedure 2, acidifying with 20% AcOH. The crude product was recrystallised from EtOH, filtered and washed with cooled EtOH to afford (E)-2-[[3-(3-methoxy-4-(prop-2-ynyloxy)phenyl)-1-oxo-2-propenyl]amino]benzoic acid (1.50 g, 64%) as a yellow crystalline solid; mp 183-185 C.; .sub.H (400 MHz, DMSO-d.sub.6) 3.58 (t, J=2.0 Hz, 1H, HCC), 3.81 (s, 3H, OCH.sub.3), 4.87 (d, J=2.0 Hz, 2H, OCH.sub.2), 6.75 (d, J=15.6 Hz, 1H, CHCHCO), 7.03 (d, J.sub.5,6=8.4 Hz, 1H, H5), 7.16 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.29 (d, J.sub.5,6=8.4 Hz, 1H, H6), 7.44 (s, 1H, H2), 7.54 (d, J=15.6 Hz, 1H, CHCHCO), 7.60 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 8.00 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.61 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.34 (s, 1H, NH), 13.60 (br s, 1H, CO.sub.2H); .sub.C (100 MHz, DMSO-d.sub.6) 55.6, 56.1, 78.4, 79.2, 112.0, 112.6, 116.6, 120.0, 120.3, 122.7, 123.5, 127.0, 131.1, 134.0, 141.1, 141.5, 146.6, 151.0, 164.1, 169.5; HRMS (ESI) calculated for C.sub.20H.sub.17NO.sub.5 [M+Na].sup.+ 374.0999. found 374.1002; .sub.max 750, 1029, 1135, 1217, 1506, 1582, 1667, 3270, 3520 cm.sup.1.

3-Methoxy-4-(pent-2-ynyloxy)benzaldehyde

(105) ##STR00056##

(106) 1-Bromopent-2-yne (0.67 mL, 6.6 mmol) was added to a suspension of vanillin (0.50 g, 3.3 mmol) and potassium carbonate (1.37 g, 9.85 mmol) in acetone (5.0 mL) and treated according to Procedure 3. 3-Methoxy-4-(pent-2-ynyloxy)benzaldehyde (0.60 g, 84%) was obtained as a yellow crystalline solid; mp 47-50 C.; .sub.H (400 MHz, CDCl.sub.3) 1.11 (t, J=7.6 Hz, 2H, CH.sub.2CH.sub.3), 2.20 (tq, J=7.6, 2.4 Hz, 3H, CH.sub.2CH.sub.3), 3.93 (s, 3H, OCH.sub.3), 4.83 (t, J=2.4 Hz, 2H, OCH.sub.2), 7.13 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.42 (s, 1H, H2), 7.45 (d, J.sub.5,6=8.0 Hz, H6), 9.86 (s, 1H, CHO); .sub.C (100 MHz, CDCl.sub.3) 12.5, 13.4, 56.0, 57.3, 73.1, 90.7, 109.2, 112.3, 126.4, 130.5, 149.9, 152.5, 190.9; .sub.max 997, 1136, 1263, 1508, 1586, 1682, 2230, 2298, 2845, 2932 cm.sup.1.

(E)-2-[[3-(3-Methoxy-4-(pent-2-ynyloxy)phenyl)-1-oxo-2-propenyl]amino]benzoic acid (18)

(107) ##STR00057##

(108) Piperidine (0.22 mL, 2.2 mmol) was added to a suspension of 3-methoxy-4-(pent-2-ynyl)oxybenzaldehyde (0.50 g, 2.3 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.49 g, 2.2 mmol) in toluene (5.0 mL) and treated according to Procedure 2, acidifying with 20% AcOH. (E)-2-[[3-(3-Methoxy-4-(pent-2-ynyloxy)phenyl)-1-oxo-2-propenyl]amino]benzoic acid (0.50 g, 60%) was obtained as a colourless crystalline solid; mp 185.5-186.5 C.; .sub.H (400 MHz, DMSO-d.sub.6) 1.05 (t, J=7.4 Hz, 2H, CH.sub.2CH.sub.3), 2.20 (q, J=7.4 Hz, 3H, CH.sub.2CH.sub.3), 3.84 (s, 3H, OCH.sub.3), 4.78 (s, 2H, OCH.sub.2), 6.80 (d, J=15.6 Hz, 1H, CHCHCO), 7.03 (d, J.sub.5,6=8.4 Hz, 1H, H5), 7.16 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.24 (d, J.sub.5,6=8.4 Hz, 1H, H6), 7.39 (s, 1H, H2), 7.56 (d, J=15.6 Hz, 1H, CHCHCO), 7.61 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 8.00 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.62 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.31 (s, 1H, NH), 13.53 (br s, 1H, CO.sub.2H); .sub.C (100 MHz, DMSO-d.sub.6) 11.7, 13.5, 56.6, 56.4, 74.7, 89.3, 110.7, 113.3, 116.6, 120.2, 120.3, 122.2, 122.7, 127.9, 131.1, 134.0, 141.0, 141.5, 148.5, 149.2, 164.1, 169.5; HRMS (ESI) calculated for C.sub.22H.sub.21NO.sub.5 [M+Na].sup.+ 402.1312. found 402.1317; .sub.max 747, 1001, 1253, 1508, 1583, 1661, 2980, 3246, 3523 cm.sup.1.

4-Methoxy-3-(pent-2-ynyloxy)benzaldehyde

(109) ##STR00058##

(110) 1-Bromopent-2-yne (0.67 mL, 6.6 mmol) was added to a suspension of vanillin (0.50 g, 3.3 mmol) and potassium carbonate (1.37 g, 9.85 mmol) in acetone (5.0 mL) and treated according to Procedure 3. 4-Methoxy-3-(pent-2-ynyloxy)benzaldehyde (0.69 g, 96%) was obtained as a yellow crystalline solid; mp 38-39 C.; .sub.H (400 MHz, CDCl.sub.3) 1.10 (t, J=7.6 Hz, 2H, CH.sub.2CH.sub.3), 2.20 (tq, J=7.6, 2.0 Hz, 3H, CH.sub.2CH.sub.3), 3.95 (s, 3H, OCH.sub.3), 4.79 (t, J=2.0 Hz, 2H, OCH.sub.2), 6.99 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.49 (dd, J.sub.5,6=8.0, J.sub.2,6=2.0 Hz, 1H, H6), 7.54 (d, J.sub.2,6=2.0 Hz, 1H, H2), 9.85 (s, 1H, CHO); .sub.C (100 MHz, CDCl.sub.3) 12.5, 13.5, 56.1, 57.3, 73.4, 90.5, 110.7, 111.8, 126.9, 129.9, 147.6, 154.8, 190.8; .sub.max 1007, 1130, 1261, 1508, 1583, 1683, 2230, 2290, 2841, 2976 cm.sup.1.

(E)-2-[[3-(4-Methoxy-3-(pent-2-ynyloxy)phenyl)-1-oxo-2-propenyl]amnio]benzoic acid (19)

(111) ##STR00059##

(112) Piperidine (0.24 mL, 2.5 mmol) was added to a suspension of 4-methoxy-3-(pent-2-ynyl)oxybenzaldehyde (0.54 g, 2.5 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.53 g, 2.4 mmol) in toluene (5.0 mL) and treated according to Procedure 2, acidifying with 20% AcOH. (E)-2-[[3-(4-Methoxy-3-(pent-2-ynyloxy)phenyl)-1-oxo-2-propenyl]amino]benzoic acid (0.50 g, 60%) was obtained as a yellow crystalline solid; mp 124-125 C.; .sub.H (400 MHz, DMSO-d.sub.6) 1.06 (t, J=7.4 Hz, 2H, CH.sub.2CH.sub.3), 2.23 (q, J=7.4 Hz, 3H, CH.sub.2CH.sub.3), 3.80 (s, 3H, OCH.sub.3), 4.81 (s, 2H, OCH.sub.2), 6.73 (d, J=15.6 Hz, 1H, CHCHCO), 7.01 (d, J.sub.5,6=8.4 Hz, 1H, H5), 7.16 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.28 (dd, J.sub.5,6=8.4, J.sub.2,6=1.6 Hz, 1H, H6), 7.42 (d, J.sub.2,6=1.6 Hz, 1H, H2), 7.57 (d, J=15.6 Hz, 1H, CHCHCO), 7.61 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 8.00 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.62 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.31 (s, 1H, NH), 13.59 (br s, 1H, CO.sub.2H); .sub.C (100 MHz, DMSO-d.sub.6) 11.7, 13.6, 55.6, 56.6, 74.9, 89.3, 111.9, 112.5, 116.5, 119.9, 120.3, 122.7, 123.3, 127.0, 131.1, 134.0, 141.1, 141.6, 146.8, 151.0, 164.1, 169.5; HRMS (ESI) calculated for C.sub.22H.sub.21NO.sub.5 [M+Na].sup.+ 402.1312. found 402.1317; .sub.max 753, 1015, 1257, 1506, 1584, 1659, 2920, 3246, 3520 cm.sup.1.

(E)-2-[[3-(3-Methoxy-4-((1-(2-oxo-2-(phenylamino)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-oxo-2-propenyl]amino]benzoic acid (20)

(113) ##STR00060##

(114) Sodium ascorbate (28 mg, 140 mol), tris-(benzyltriazolylmethyl)amine (15 mg, 28 mol) and copper sulfate (4.5 mg, 28 mol) were added to a solution of (E)-2-[[3-(3-methoxy-4-propargyloxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (0.50 g, 1.4 mmol) and 2-azido-N-phenylacetamide (0.25 g, 1.4 mmol) in DMSO (20 mL) and water (5.0 mL). The solution was stirred at rt for 16 h and diluted with water. The suspension was filtered and the filter cake was washed with water and dried. The crude product was recrystallised from acetonitrile to afford (E)-2-[[3-(3-methoxy-4-((1-(2-oxo-2-(phenylamino)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-oxo-2-propenyl]amino]benzoic acid (0.60 g, 80%) as a colourless solid; mp 220-222 C.; .sub.H (400 MHz, DMSO-d.sub.6) 3.82 (s, 3H, OCH.sub.3), 5.21 (s, 2H, CH.sub.2), 5.36 (s, 2H, CH.sub.2), 6.80 (d, J=15.6 Hz, 1H, CHCHCO), 7.08 (t, J=8.0 Hz, 1H, H4), 7.16 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.21-7.28 (m, 2H, H6, H5), 7.39 (s, 1H, H2), 7.31 (t, J.sub.2,3=J.sub.3,4=J.sub.4,5=J.sub.5,6=8.0 Hz, 2H, H3, H5), 7.56-7.63 (m, 4H, CHCHCO, H5, H2, H6), 8.00 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.27 (s, 1H, CCHN), 8.62 (d, J.sub.5,6=8.0 Hz, 1H, H6), 10.48 (s, 1H, NH), 11.30 (s, 1H, NH), 13.60 (br s, 1H, CO.sub.2H); .sub.C (100 MHz, DMSO-d.sub.6) 52.2, 55.6, 61.4, 110.5, 112.9, 116.6, 119.2, 120.1, 120.4, 122.6, 122.8, 123.8, 126.6, 127.6, 129.0, 131.2, 134.1, 138.5, 141.1, 141.7, 142.2, 149.1, 149.4, 164.2, 169.5; HRMS (ESI) calculated for C.sub.28H.sub.25N.sub.5O.sub.6 [M+Na].sup.+ 550.1697. found 550.1691; .sub.max 1239, 1585, 1665, 2605, 3000, 3250 cm.sup.1.

(E)-2-[[3-(4-Methoxy-3-((1-(2-oxo-2-(phenylamino)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-oxo-2-propenyl]amino]benzoic acid (21)

(115) ##STR00061##

(116) Sodium ascorbate (22 mg, 110 mol), tris-(benzyltriazolylmethyl)amine (12 mg, 23 mol) and copper sulfate (3.6 mg, 22 mol) were added to a solution of (E)-2-[[3-(4-methoxy-3-propargyloxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (0.40 g, 1.1 mmol) and 2-azido-N-phenylacetamide (0.20 g, 1.1 mmol) in DMSO (16 mL) and water (4.0 mL). The solution was stirred at rt for 16 h and diluted with water. The suspension was filtered and the filter cake was washed with water and dried. The crude product was recrystallised from AcOH to afford (E)-2-[[3-(3-methoxy-4-((1-(2-oxo-2-(phenylamino)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-oxo-2-propenyl]amino]benzoic acid (0.60 g, 80%) as a yellow solid; mp 253-255 C.; .sub.H (400 MHz, DMSO-d.sub.6) 3.79 (s, 3H, OCH.sub.3), 5.25 (s, 2H, CH.sub.2), 5.37 (s, 2H, CH.sub.2), 6.82 (d, J=15.6 Hz, 1H, CHCHCO), 7.01 (t, J.sub.5,6=8.0 Hz, 1H, H5), 7.05 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.16 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.27-7.34 (m, 3H, H2, H6, H3, H5), 7.57-7.62 (m, 4H, CHCHCO, H5, H2, H6), 8.00 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.29 (s, 1H, CCHN), 8.64 (d, J.sub.5,6=8.0 Hz, 1H, H6), 10.48 (s, 1H, NH), 11.32 (s, 1H, NH), 13.50 (br s, 1H, CO.sub.2H); .sub.C (100 MHz, DMSO-d.sub.6) 52.2, 55.5, 61.6, 111.8, 112.0, 116.5, 119.2, 120.0, 120.3, 122.7, 123.1, 123.8, 126.5, 127.2, 128.9, 131.2, 134.0, 138.4, 141.1, 141.7, 142.4, 147.7, 150.8, 164.2, 169.5; HRMS (ESI) calculated for C.sub.28H.sub.25N.sub.5O.sub.6 [M+Na].sup.+ 550.1697. found 550.1702; .sub.max 1259, 1580, 1667, 2599, 3952, 3345 cm.sup.1.

4-(But-2-ynyloxy)-3-methoxybenzaldehyde

(117) ##STR00062##

(118) 1-Bromobut-2-yne (0.36 mL, 4.0 mmol) was added to a suspension of vanillin (0.55 g, 3.6 mmol) and potassium carbonate (1.79 g, 10.9 mmol) in acetone (10 mL) and treated according to Procedure 3. 4-(But-2-ynyloxy)-3-methoxybenzaldehyde (0.70 g, 95%) was obtained as a pale yellow crystalline solid; mp 90-92 C.; .sub.H (400 MHz, CDCl.sub.3) 1.84 (t, J=2.2 Hz, 3H, CH.sub.3), 3.93 (s, 3H, OCH.sub.3), 4.81 (q, J=2.2 Hz, 2H, OCH.sub.2), 7.12 (d, J.sub.5,6=8.4 Hz, 1H, H5), 7.42 (d, J.sub.2,6=2.0 Hz, 1H, H2), 7.45 (dd, J.sub.5,6=8.4, J.sub.2,6=2.0 Hz, 1H, H6), 9.86 (s, 1H, CHO); .sub.C (100 MHz, CDCl.sub.3) 4.0, 56.2, 57.5, 73.2, 85.2, 109.4, 112.4, 126.7, 130.7, 150.1, 152.7, 191.2; .sub.max 991, 1259, 1504, 1586, 1679, 2226, 2302, 2833, 2921 cm.sup.1.

(E)-2-[[3-(4-(But-2-ynyloxy)-3-methoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (22)

(119) ##STR00063##

(120) Piperidine (0.34 mL, 3.4 mmol) was added to a suspension of 4-(but-2-ynyloxy)-3-methoxybenzaldehyde (0.70 g, 3.4 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.70 g, 3.4 mmol) in toluene (10 mL) and treated according to Procedure 2, acidifying with 20% AcOH. (E)-2-{[3-(4-(But-2-ynyloxy)-3-methoxyphenyl)-1-oxo-2-propenyl]amino}benzoic acid (0.70 g, 61%) was obtained as a yellow crystalline solid; mp 194-195 C.; .sub.H (400 MHz, DMSO-d.sub.6) 1.82 (s, 3H, CH.sub.3), 3.83 (s, 3H, OCH.sub.3), 4.77 (s, 2H, OCH.sub.2), 6.79 (d, J=15.6 Hz, 1H, CHCHCO), 7.02 (d, J.sub.5,6=8.4 Hz, 1H, H5), 7.14 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.23 (d, J.sub.5,6=8.4 Hz, 1H, H6), 7.38 (s, 1H, H2), 7.55 (d, J=15.6 Hz, 1H, CHCHCO), 7.60 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 8.00 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.61 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.33 (s, 1H, NH), 13.59 (br s, 1H, CO.sub.2H); .sub.C (100 MHz, DMSO-d.sub.6) 3.2, 55.7, 56.4, 74.6, 83.9, 110.7, 113.3, 116.8, 120.3, 120.4, 122.3, 122.8, 127.9, 131.2, 134.0, 141.1, 141.6, 148.6, 149.3, 164.2, 169.5; HRMS (ESI) calculated for C.sub.21H.sub.19NO.sub.5 [M+Na].sup.+ 388.1155. found 388.1158; .sub.max 753, 1253, 1506, 1584, 1659, 2917, 3239, 3516 cm.sup.1.

3-(But-2-ynyloxy)-4-methoxybenzaldehyde

(121) ##STR00064##

(122) 1-Bromobut-2-yne (0.37 mL, 4.0 mmol) was added to a suspension of vanillin (0.56 g, 3.7 mmol) and potassium carbonate (1.82 g, 11.0 mmol) in acetone (10 mL) and treated according to Procedure 3. 3-(But-2-ynyloxy)-4-methoxybenzaldehyde (0.72 g, 96%) was obtained as a pale yellow crystalline solid; mp 81-83 C.; .sub.H (400 MHz, CDCl.sub.3) 1.84 (t, J=2.0 Hz, 3H, CH.sub.3), 3.95 (s, 3H, OCH.sub.3), 4.77 (q, J=2.0 Hz, 2H, OCH.sub.2), 6.99 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.49 (dd, J.sub.5,6=8.4, J.sub.2,6=2.0 Hz, 1H, H6), 7.51 (d, J.sub.2,6=2.0 Hz, 1H, H2), 9.86 (s, 1H, CHO); .sub.C (100 MHz, CDCl.sub.3) 3.7, 56.1, 57.1, 73.2, 84.7, 110.6, 111.4, 126.9, 129.9, 147.6, 154.8, 190.8; .sub.max 1003, 1259, 1506, 1583, 1681, 2226, 2297, 2841, 2916 cm.sup.1.

(E)-2-{[3-(3-(But-2-ynyloxy)-4-methoxyphenyl)-1-oxo-2-propenyl]amino}benzoic acid (23)

(123) ##STR00065##

(124) Piperidine (0.35 mL, 3.5 mmol) was added to a suspension of 4-methoxy-3-(but-2-ynyl)oxybenzaldehyde (0.72 g, 3.5 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.72 g, 3.2 mmol) in toluene (10 mL) and treated according to Procedure 2, acidifying with 20% AcOH. (E)-2-{[3-(3-(But-2-ynyloxy)-4-methoxyphenyl)-1-oxo-2-propenyl]amino}benzoic acid (0.81 g, 69%) was obtained as a yellow crystalline solid; mp 170-171 C.; .sub.H (400 MHz, DMSO-d.sub.6) 1.82 (t, J=2.0 Hz, 3H, CH.sub.3), 3.80 (s, 3H, OCH.sub.3), 4.80 (d, J=2.0 Hz, 2H, OCH.sub.2), 6.74 (d, J=15.6 Hz, 1H, CHCHCO), 7.00 (d, J.sub.5,6=8.4 Hz, 1H, H5), 7.16 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.27 (d, J.sub.5,6=8.4 Hz, 1H, H6), 7.40 (s, 1H, H2), 7.54 (d, J=15.6 Hz, 1H, CHCHCO), 7.61 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 8.00 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.61 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.31 (s, 1H, NH), 13.57 (br s, 1H, CO.sub.2H); .sub.C (100 MHz DMSO-d.sub.6) 3.2, 55.6, 56.5, 74.7, 83.7, 111.9, 112.3, 116.6, 120.0, 120.3, 122.7, 123.2, 127.1, 131.2, 134.1, 141.1, 141.6, 146.9, 151.0, 164.1, 169.5; HRMS (ESI) calculated for C.sub.21H.sub.19NO.sub.5 [M+Na].sup.+ 388.1155. found 388.12158; .sub.max 749, 1261, 1512, 1584, 1659, 2917, 3239, 3520 cm.sup.1.

4-Cyclopentyloxy-3-methoxybenzaldehyde

(125) ##STR00066##

(126) Bromocyclopentane (7.0 mL, 66 mmol) was added to a suspension of vanillin (5.0 g, 33 mmol) and potassium carbonate (13.6 g, 99 mmol) in EtOH (75 mL) and treated according to Procedure 3. 4-Cyclopentyloxy-3-methoxybenzaldehyde (7.1 g, 98%) was obtained as a yellow oil; .sub.H (400 MHz, CDCl.sub.3) 1.62 (m, 2H, CH.sub.2), 1.78-2.04 (m, 6H, CH.sub.2), 3.89 (s, 3H, OCH.sub.3), 4.86 (tt, J=6.0, 3.2 Hz, 1H, OCH), 6.94 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.38 (d, J.sub.2,6=2.0 Hz, 1H, H2), 7.41 (dd, J.sub.5,6=8.0, J.sub.2,6=2.0 Hz, 1H, H6), 9.82 (s, 1H, CHO); .sub.C (100 MHz, CDCl.sub.3) 24.1, 32.8, 56.0, 80.6, 109.4, 112.8, 126.6, 129.5, 150.2, 153.4, 190.9; .sub.max 977, 1260, 1504, 1580, 1680, 2869, 2956 cm.sup.1.

(E)-2-{[3-(4-Cyclopentyloxy-3-methoxyphenyl)-1-oxo-2-propenyl]amino}benzoic acid (26)

(127) ##STR00067##

(128) Piperidine (0.45 mL, 4.5 mmol) was added to a suspension of 4-cyclopentyloxy-3-methoxybenzaldehyde (1.0 g, 4.5 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.92 g, 4.1 mmol) in toluene (5.0 mL) and treated according to Procedure 2, acidifying with 1 M HCl. The crude product was recrystallised from EtOH/water providing (E)-2-{[3-(4-cyclopentyloxy-3-methoxyphenyl)-1-oxo-2-propenyl]amino}benzoic acid (1.06 g, 67%) as a pale yellow crystalline solid; mp 96-98 C.; .sub.H (400 MHz, DMSO-d.sub.6) 1.46 (m, 2H, CH.sub.2), 1.67-1.71 (m, 4H, CH.sub.2), 1.90 (m, 2H, CH.sub.2), 3.81 (s, 3H, OCH.sub.3), 4.82 (t, J=5.6 Hz, 1H, OCH), 6.76 (d, J=15.6 Hz, 1H, CHCHCO), 6.94 (d, J.sub.5,6=8.4 Hz, 1H, H5), 7.15 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.19 (dd, J.sub.5,6=8.4, J.sub.2,6=1.6 Hz, 1H, H6), 7.35 (d, J.sub.2,6=1.6 Hz, 1H, H2), 7.54 (d, J=15.6 Hz, 1H, CHCHCO), 7.60 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 8.00 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.62 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.28 (s, 1H, NH), 13.59 (br s, 1H, CO.sub.2H); .sub.C (100 MHz, DMSO-d.sub.6) 23.7, 32.3, 55.7, 79.5, 110.9, 114.0, 116.5, 119.7, 120.3, 122.5, 122.6, 127.0, 131.1, 134.0, 141.1, 141.7, 149.0, 149.6, 164.2, 169.5; HRMS (ESI) calculated for C.sub.22H.sub.23NO.sub.5 [M+Na].sup.+ 404.1468. found 404.1468; .sub.max 747, 1261, 1506, 1584, 1659, 2964, 3524 cm.sup.1.

4-Cyclohexyloxy-3-methoxybenzaldehyde

(129) ##STR00068##

(130) Bromocyclohexane (8.0 mL, 66 mmol) was added to a suspension of vanillin (5.0 g, 33 mmol), potassium carbonate (13.6 g, 99 mmol) and sodium iodide (0.49 g, 3.3 mmol) in EtOH (75 mL) and treated according to Procedure 3 for 64 h. The crude product was purified by flash chromatography with 10-15% EtOAc/petrol as eluent to give 4-cyclohexyloxy-3-methoxybenzaldehyde (2.8 g, 37%) as a pale yellow oil; .sub.H (400 MHz, CDCl.sub.3) 1.27-1.43 (m, 4H, CH.sub.2), 1.56 (m, 2H, CH.sub.2), 1.85 (m, 2H, CH.sub.2), 2.06 (m, 2H, CH.sub.2), 3.91 (s, 3H, OCH.sub.3), 4.37 (tt, J=9.4, 3.6 Hz, 1H, OCH), 6.98 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.40 (d, J.sub.2,6=2.0 Hz, 1H, H2), 7.42 (dd, J.sub.5,6=8.0, J.sub.2,6=2.0 Hz, 1H, H6), 9.83 (s, 1H, CHO); .sub.C (100 MHz, CDCl.sub.3) 23.9, 25.4, 31.6, 56.0, 76.9, 109.8, 113.2, 126.5, 129.7, 150.5, 153.0, 190.8; .sub.max 1133, 1263, 1504, 1581, 1680, 2857, 2933 cm.sup.1.

(E)-2-{[3-(4-Cyclohexyloxy-3-methoxyphenyl)-1-oxo-2-propenyl]amino}benzoic acid (27)

(131) ##STR00069##

(132) Piperidine (0.45 mL, 4.5 mmol) was added to a suspension of 4-cyclohexyloxy-3-methoxybenzaldehyde (1.06 g, 4.54 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.92 g, 4.1 mmol) in toluene (5.0 mL) and treated according to Procedure 2, acidifying with 1 M HCl. The crude product was recystallised from EtOH/water providing (E)-2-{[3-(4-cyclohexyloxy-3-methoxyphenyl)-1-oxo-2-propenyl]amino}benzoic acid (0.98 g, 60%) as a colourless crystalline solid; mp 90-92 C.; .sub.H (400 MHz, DMSO-d.sub.6) 1.25-1.52 (m, 6H, CH.sub.2), 1.70 (m, 2H, CH.sub.2), 1.89 (m, 2H, CH.sub.2), 3.82 (s, 3H, OCH.sub.3), 4.33 (m, 1H, OCH), 6.76 (d, J=15.6 Hz, 1H, CHCHCO), 6.99 (d, J.sub.5,6=8.4 Hz, 1H, H5), 7.15 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.19 (d, J.sub.5,6=8.4 Hz, 1H, H6), 7.35 (s, 1H, H2), 7.55 (d, J=15.6 Hz, 1H, CHCHCO), 7.60 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 8.00 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.62 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.29 (s, 1H, NH), 13.56 (br s, 1H, CO.sub.2H); .sub.C (100 MHz, DMSO-d.sub.6) 23.2, 25.1, 31.4, 55.7, 79.4, 111.2, 114.8, 116.6, 119.8, 120.3, 122.4, 122.6, 127.3, 131.1, 134.0, 141.1, 141.6, 148.6, 150.0, 164.2, 169.5; HRMS (ESI) calculated for C.sub.23H.sub.25NO.sub.5 [M+Na].sup.+ 418.1625. found 418.1625; .sub.max 745, 1259, 1504, 1588, 1659, 2929, 3520 cm.sup.1.

4-Cyclohexylmethoxy-3-methoxybenzaldehyde

(133) ##STR00070##

(134) Bromomethylcyclohexane (0.78 mL, 4.2 mmol) was added to a suspension of vanillin (0.43 g, 2.8 mmol) and potassium carbonate (1.17 g, 8.47 mmol) in EtOH (7.0 mL) and treated according to Procedure 3 for 64 h. 4-Cyclohexylmethoxy-3-methoxybenzaldehyde (0.65 g, 93%) was obtained as a yellow oil; .sub.H (400 MHz, CDCl.sub.3) 1.05 (m, 2H, CH.sub.2), 1.15-1.36 (m, 4H, CH.sub.2), 1.73 (m, 2H, CH.sub.2), 1.87-1.98 (m, 2H, CH.sub.2, CH), 3.88 (d, J=6.0 Hz, 2H, OCH.sub.2), 3.92 (s, 3H, OCH.sub.3), 6.95 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.40 (d, J.sub.2,6=2.0 Hz, 1H, H2), 7.42 (dd, J.sub.5,6=8.0, J.sub.2,6=2.0 Hz, 1H, H6), 9.84 (s, 1H, CHO); .sub.C (100 MHz, CDCl.sub.3) 25.6, 26.4, 29.8, 37.3, 56.1, 74.5, 109.3, 111.4, 126.8, 129.8, 149.9, 154.4, 190.9; .sub.max 1133, 1265, 1508, 1586, 1683, 2853, 2925 cm.sup.1.

(E)-2-{[3-(4-Cyclohexylmethoxy-3-methoxyphenyl)-1-oxo-2-propenyl]amino}benzoic acid (28)

(135) ##STR00071##

(136) Piperidine (0.24 mL, 2.4 mmol) was added to a suspension of 4-cyclohexylmethoxy-3-methoxybenzaldehyde (0.59 g, 2.4 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.48 g, 2.1 mmol) in toluene (5.0 mL) and treated according to Procedure 2, acidifying with 1 M HCl. The crude product was recystallised from EtOH/water providing (E)-2-{[3-(4-cyclohexylmethoxy-3-methoxyphenyl)-1-oxo-2-propenyl]amino}benzoic acid (0.45 g, 51%) as a colourless crystalline solid; mp 207-210 C.; .sub.H (400 MHz, DMSO-d.sub.6) 1.03 (m, 2H, CH.sub.2), 1.20 (m, 4H, CH.sub.2), 1.63-1.82 (m, 5H, CH.sub.2, CH), 3.79 (d, J=6.4 Hz, 1H, OCH.sub.2), 3.83 (s, 3H, OCH.sub.3), 6.76 (d, J=15.6 Hz, 1H, CHCHCO), 6.96 (d, J.sub.5,6=8.4 Hz, 1H, H5), 7.18 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.21 (d, J.sub.5,6=8.4 Hz, J.sub.2,6=1.8 Hz, 1H, H6), 7.36 (s, J.sub.2,6=1.8 Hz, 1H, H2), 7.55 (d, J=15.6 Hz, 1H, CHCHCO), 7.62 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 8.00 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.62 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.27 (s, 1H, NH), 13.58 (br s, 1H, CO.sub.2H); .sub.C (100 MHz, DMSO-d.sub.6) 25.2, 26.0, 29.2, 37.0, 55.8, 73.3, 110.7, 112.6, 116.6, 119.7, 120.3, 122.6, 127.1, 131.1, 134.0, 141.0, 141.6, 149.1, 150.2, 164.2, 169.4; HRMS (ESI) calculated for C.sub.23H.sub.25NO.sub.5 [M+Na].sup.+ 432.1781. found 432.1781; .sub.max 759, 1142, 1504, 1581, 1667, 2925, 3123 cm.sup.1.

3-Cyclopentyloxy-4-methoxybenzaldehyde

(137) ##STR00072##

(138) Bromocyclopentane (1.4 mL, 13 mmol) was added to a suspension of isovanillin (1.0 g, 6.6 mmol) and potassium carbonate (2.7 g, 10 mmol) in EtOH (15 mL) and treated according to Procedure 3. 3-Cyclopentyloxy-4-methoxybenzaldehyde (1.4 g, 97%) was obtained as a yellow oil; .sub.H (400 MHz, CDCl.sub.3) 1.63 (m, 2H, CH.sub.2), 1.79-1.93 (m, 4H, CH.sub.2), 1.99 (m, 2H, CH.sub.2), 3.93 (s, 3H, OCH.sub.3), 4.85 (tt, J=6.4, 3.2 Hz, 1H, OCH), 6.96 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.39 (d, J.sub.2,6=2.0 Hz, 1H, H2), 7.42 (dd, J.sub.5,6=8.0, J.sub.2,6=2.0 Hz, 1H, H6), 9.84 (s, 1H, CHO); .sub.C (100 MHz, CDCl.sub.3) 24.1, 32.7, 56.1, 80.5, 110.7, 112.1, 126.3, 130.0, 148.2, 155.4, 191.0; .sub.max 1001, 1132, 1261, 1431, 1508, 1584, 1683, 2956 cm.sup.1.

(E)-2-{[3-(3-Cyclopentyloxy-4-methoxyphenyl)-1-oxo-2-propenyl]amino}benzoic acid (29)

(139) ##STR00073##

(140) Piperidine (0.63 mL, 5.8 mmol) was added to a suspension of 3-cyclopentyloxy-4-methoxybenzaldehyde (1.4 g, 6.4 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (1.3 g, 5.8 mmol) in toluene (5.0 mL) and treated according to Procedure 2, acidifying with 1 M HCl. The crude product was recrystallised from EtOH/water providing (E)-2-{[3-(3-cyclopentyloxy-4-methoxyphenyl)-1-oxo-2-propenyl]amino}benzoic acid (1.4 g, 67%) as a yellow crystalline solid; mp 211-217 C.; .sub.H (400 MHz, DMSO-d.sub.6) 1.57 (m, 2H, CH.sub.2), 1.70-1.72 (m, 4H, CH.sub.2), 1.91 (m, 2H, CH.sub.2), 3.78 (s, 3H, OCH.sub.3), 4.90 (t, J=5.6 Hz, 1H, OCH), 6.75 (d, J=15.6 Hz, 1H, CHCHCO), 6.98 (d, J.sub.5,6=8.4 Hz, 1H, H5), 7.16 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.24 (d, J.sub.5,6=8.4 Hz, 1H, H6), 7.31 (s, 1H, H2), 7.55 (d, J=15.6 Hz, 1H, CHCHCO), 7.60 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 8.00 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.60 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.25 (s, 1H, NH), 13.57 (br s, 1H, CO.sub.2H); .sub.C (100 MHz, DMSO-d.sub.6) 23.6, 32.2, 55.6, 79.5, 112.0, 113.6, 116.7, 119.9, 120.4, 122.2, 122.7, 127.2, 131.1, 134.0, 141.0, 141.6, 147.1, 151.5, 164.2, 169.4; HRMS (ESI) calculated for C.sub.22H.sub.23NO.sub.5 [M+Na].sup.+ 404.1468. found 404.1468; .sub.max 751, 1254, 1504, 1583, 1661, 2948, 3516 cm.sup.1.

(E)-2-(3-(3,4-Dimethoxyphenyl)acrylamido)-N-((1-(2-oxo-2-(phenylamino)ethyl)-1H-1,2,3-triazol-4-yl)methyl)benzamide (32)

(141) ##STR00074##

(142) Sodium ascorbate (5.4 mg, 27 mol), tris-(benzyltriazolylmethyl)amine (2.9 mg, 5.5 mol) and copper sulfate (0.88 mg, 5.5 mol) were added to a solution of (E)-2-[[3-(3,4-dimethoxyphenyl)-1-oxo-2-propenyl]amino]-N-(prop-2-ynyl)benzamide (100 mg, 0.27 mmol) and 2-azido-N-phenylacetamide (48 mg, 0.27 mmol) in DMSO (4.0 mL) and water (1.0 mL). The solution was stirred at rt for 16 h and diluted with water. The suspension was filtered and the filter cake was washed with water and dried. The product was recrystallised from acetonitrile and (E)-2-(3-(3,4-dimethoxyphenyl)acrylamido)-N-((1-(2-oxo-2-(phenylamino)ethyl)-1H-1,2,3-triazol-4-yl)methyl)benzamide (127 mg, 86%) was obtained as a colourless solid; mp 189-191 C.; .sub.H (400 MHz, DMSO-d.sub.6) 3.77 (s, 3H, OCH.sub.3), 3.81 (s, 3H, OCH.sub.3), 4.59 (d, J=6.8 Hz, 2H, CH.sub.2NH), 5.30 (s, 2H, CH.sub.2N), 6.79 (d, J=15.6 Hz, 1H, CHCHCO), 6.96 (d, J.sub.5,6=8.4 Hz, 1H, H5), 7.06 (t, J.sub.34=J.sub.45=8.0 Hz, 1H, H4), 7.15 (t, J.sub.3,4=J.sub.4,5=8.4 Hz, 1H, H4), 7.23 (dd, J.sub.5,6=8.4, J.sub.2,6=1.6 Hz, 1H, H6), 7.28 (t, J.sub.23=J.sub.34=J.sub.45=J.sub.56=8.0 Hz, 2H, H3, H5), 7.37 (d, J.sub.2,6=1.6 Hz, 1H, H2), 7.50-7.56 (m, 4H, CHCHCO, H5, H2, H6), 7.78 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.08 (s, 1H, CCH), 8.57 (d, J.sub.5,6=8.0 Hz, 1H, H6), 9.37 (t, J=5.6 Hz, 1H, CH.sub.2NH), 10.43 (s, 1H, NHPh), 11.40 (s, 1H, NH); .sub.C (100 MHz, DMSO-d.sub.6) 44.9, 40.4, 52.2, 55.5, 55.6, 110.4, 111.5, 119.2, 119.9, 120.7, 120.8, 122.6, 122.7, 123.7, 124.6, 127.3, 128.1, 128.9, 131.9, 138.4, 139.2, 141.5, 144.5, 148.9, 150.6, 164.0, 164.2, 168.3; HRMS (ESI) calculated for C.sub.29H.sub.28N.sub.6O.sub.5 [M+Na].sup.+ 563.2013. found 516.2015; .sub.max 755, 1023, 1259, 1516, 1671, 3262 cm.sup.1.

4-(Hex-5-ynyloxy)-3-methoxybenzaldehyde

(143) ##STR00075##

(144) 4-Methylbenzenesulfonyl chloride (2.9 g, 15 mmol), 5-hexyn-1-ol (1.1 mL, 10 mmol) and pyridine (1.6 mL, 20 mmol) in CH.sub.2Cl.sub.2 (10 mL) were treated according to Procedure 4 giving hex-5-ynyl 4-methylbenzenesulfonate (2.1 g, 83%) as a colourless oil. Vanillin (0.84 g, 5.6 mmol) was alkylated with hex-5-ynyl 4-methylbenzenesulfonate (2.1 g, 8.3 mmol) according to Procedure 4 and the crude product was recystallised from EtOAc/petrol to provide 4-(hex-5-ynyloxy)-3-methoxybenzaldehyde (0.80 g, 62%) as a colourless crystalline solid; mp 67-68 C.; .sub.H (400 MHz, CDCl.sub.3) 1.74 (p, J=7.0 Hz, 2H, CH.sub.2), 1.97 (t, J=2.8 Hz, 1H, CCH), 2.02 (p, J=7.0 Hz, 2H, CH.sub.2), 2.30 (td, J=7.0, 2.8 Hz, 2H, CH.sub.2CCH), 3.92 (s, 3H, OCH.sub.3), 4.14 (t, J=7.0 Hz, 2H, OCH.sub.2), 6.97 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.41 (s, 1H, H2), 7.43 (d, J.sub.5,6=8.0 Hz, 1H, H6), 9.85 (s, 1H, CHO); .sub.C (100 MHz, CDCl.sub.3) 18.1, 24.9, 27.9, 56.0, 68.5, 68.8, 83.9, 109.3, 111.4, 126.7, 130.0, 149.9, 154.0, 190.9; .sub.max 1029, 1269, 1584, 1681, 2956, 3246 cm.sup.1.

(E)-2-[[3-(4-(Hex-5-ynyloxy)-3-methoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (33)

(145) ##STR00076##

(146) Piperidine (0.30 mL, 3.0 mmol) was added to a suspension of 4-(hex-5-ynyloxy)-3-methoxybenzaldehyde (0.70 g, 3.0 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.61 g, 2.7 mmol) in toluene (5.0 mL) and treated according to Procedure 2, acidifying with 1M HCl. The crude product was recrystallised from EtOH/water providing (E)-2-[[3-(4-(Hex-5-ynyloxy)-3-methoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (0.78 g, 73%) as a yellow crystalline solid; mp 148-150 C.; .sub.H (400 MHz, DMSO-d.sub.6) 1.59 (p, J=7.6 Hz, 2H, CH.sub.2), 1.81 (p, J=7.6 Hz, 2H, CH.sub.2), 2.24 (dt, J=7.6, 2.4 Hz, 2H, CH.sub.2CCH), 2.78 (t, J=2.4 Hz, 1H, CCH), 3.83 (s, 3H, OCH.sub.3), 4.01 (t, J=7.6 Hz, 2H, OCH.sub.2), 6.77 (d, J=15.6 Hz, 1H, CHCHCO), 6.98 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.16 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.22 (d, J.sub.5,6=8.0 Hz, 1H, H6), 7.37 (s, 1H, H2), 7.55 (d, J=15.6 Hz, 1H, CHCHCO), 7.61 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 8.00 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.62 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.27 (s, 1H, NH), 13.56 (br s, 1H, CO.sub.2H); .sub.C (100 MHz, DMSO-d.sub.6) 17.4, 24.6, 27.8, 55.7, 67.6, 71.4, 84.3, 110.7, 112.6, 116.6, 119.8, 120.3, 122.6, 122.7, 127.2, 131.1, 134.0, 141.1, 141.6, 149.1, 150.0, 164.2, 169.4; HRMS (ESI) calculated for C.sub.23H.sub.23NO.sub.5 [M+H].sup.+ 394.1649. found 394.1649; .sub.max 755, 1237, 1508, 1609, 1669, 2944, 3424, 3567 cm.sup.1.

3-(Hex-5-ynyloxy)-4-methoxybenzaldehyde

(147) ##STR00077##

(148) Isovanillin (0.78 g, 5.2 mmol) was alkylated using hex-5-ynyl 4-methylbenzenesulfonate (1.95 g, 7.73 mmol according to Procedure 4. The crude product was recystallised from EtOAc/petrol to provide 3-(hex-5-ynyloxy)-4-methoxybenzaldehyde (0.68 g, 57%) as a colourless crystalline solid; mp 66-67 C.; .sub.H (400 MHz, CDCl.sub.3) 1.74 (p, J=7.2 Hz, 2H, CH.sub.2), 1.96-2.0 (m, 3H, CH.sub.2, CCH), 2.30 (td, J=7.2, 2.8 Hz, 2H, CH.sub.2CCH), 3.95 (s, 3H, OCH.sub.3), 4.11 (t, J=7.2 Hz, 2H, OCH.sub.2), 6.97 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.40 (d, J.sub.2,6=1.6 Hz, 1H, H2), 7.44 (dd, J.sub.5,6=8.0 Hz, J.sub.2,6=1.6 Hz, 1H, H6), 9.84 (s, 1H, CHO); .sub.C (100 MHz, CDCl.sub.3) 18.1, 25.0, 28.0, 56.2, 68.4, 68.7, 83.9, 110.3, 110.6, 126.7, 130.1, 149.0, 154.9, 190.9; .sub.max 1018, 1263, 1582, 1679, 2933, 3238 cm.sup.1.

(E)-2-[[3-(3-(Hex-5-ynyloxy)-4-methoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (34)

(149) ##STR00078##

(150) Piperidine (0.26 mL, 2.6 mmol) was added to a suspension of 3-(hex-5-ynyloxy)-4-methoxybenzaldehyde (0.60 g, 2.6 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.52 g, 2.4 mmol) in toluene (5.0 mL) and treated according to Procedure 2, acidifying with 1M HCl. The crude product was recrystallised from EtOH/water providing (E)-2-[[3-(3-(hex-5-ynyloxy)-4-methoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (0.64 g, 70%) as a pale yellow crystalline solid; mp 135-137 C.; .sub.H (400 MHz, DMSO-d.sub.6) 1.62 (p, J=7.2 Hz, 2H, CH.sub.2), 1.82 (p, J=7.2 Hz, 2H, CH.sub.2), 2.25 (dt, J=7.2, 2.4 Hz, 2H, CH.sub.2CCH), 2.78 (t, J=2.4 Hz, 1H, CCH), 3.80 (s, 3H, OCH.sub.3), 4.05 (t, J=7.2 Hz, 2H, OCH.sub.2), 6.77 (d, J=15.6 Hz, 1H, CHCHCO), 6.99 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.16 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.23 (d, J.sub.5,6=8.0 Hz, 1H, H6), 7.37 (s, 1H, H2), 7.55 (d, J=15.6 Hz, 1H, CHCHCO), 7.61 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 8.00 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.61 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.27 (s, 1H, NH), 13.58 (br s, 1H, CO.sub.2H); .sub.C (100 MHz, DMSO-d.sub.6) 18.1, 25.3, 28.5, 56.3, 68.4, 72.1, 85.0, 112.3, 112.5, 117.3, 120.5, 121.0, 123.3, 127.9, 131.8, 134.7, 141.7, 142.3, 149.0, 151.5, 164.8, 170.1; HRMS (ESI) calculated for C.sub.23H.sub.23NO.sub.5 [M+H].sup.+ 394.1649. found 394.1650; .sub.max 753, 1257, 1512, 1586, 1675, 2941, 3242, 3536 cm.sup.1.

3-Methoxy-4-(pent-4-ynyloxy)benzaldehyde

(151) ##STR00079##

(152) 4-Methylbenzenesulfonyl chloride (5.7 g, 30 mmol), 4-pentyn-1-ol (1.8 mL, 20 mmol) and pyridine (3.2 mL, 40 mmol) in CH.sub.2Cl.sub.2 (20 mL) were treated according to Procedure 4 giving pent-4-ynyl 4-methylbenzenesulfonate (4.60 g, 97%) as a colourless oil. Vanillin (0.98 g, 6.4 mmol) was alkylated with pent-4-ynyl 4-methylbenzenesulfonate (2.3 g, 8.3 mmol) according to Procedure 4 and the crude product was recystallised from EtOAc/petrol to provide 3-methoxy-4-(pent-4-ynyloxy)benzaldehyde (1.25 g, 89%) as a colourless crystalline solid; mp 91-92 C.; .sub.H (400 MHz, CDCl.sub.3) 1.98 (t, J=2.8 Hz, 1H, CCH), 2.09 (p, J=7.0 Hz, 2H, CH.sub.2), 2.43 (td, J=7.0, 2.8 Hz, 2H, CH.sub.2CCH), 3.91 (s, 3H, OCH.sub.3), 4.21 (t, J=7.0 Hz, 2H, OCH.sub.2), 6.99 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.40 (s, 1H, H2), 7.43 (d, J.sub.5,6=8.0 Hz, 1H, H6), 9.84 (s, 1H, CHO); .sub.C (100 MHz, CDCl.sub.3) 15.1, 27.8, 56.0, 67.3, 69.1, 83.1, 109.3, 111.5, 126.7, 130.1, 149.9, 153.9, 190.8; .sub.max 1028, 1265, 1583, 1674, 2956, 3214 cm.sup.1.

(E)-2-[[3-(3-Methoxy-4-(pent-4-ynyloxy)phenyl)-1-oxo-2-propenyl]amino]benzoic acid (35)

(153) ##STR00080##

(154) Piperidine (0.45 mL, 4.6 mmol) was added to a suspension of 3-methoxy-4-(pent-1-ynyloxy)benzaldehyde (1.0 g, 4.6 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.93 g, 4.2 mmol) in toluene (10 mL) and treated according to Procedure 2, acidifying with 1 M HCl. The crude product was recrystallised from EtOH/water providing (E)-2-[[3-(3-methoxy-4-(pent-4-ynyloxy)phenyl)-1-oxo-2-propenyl]amino]benzoic acid (1.2 g, 75%) as a pale yellow crystalline solid; mp 166.5-167.5 C.; .sub.H (400 MHz, DMSO-d.sub.6) 1.89 (p, J=7.6 Hz, 2H, CH.sub.2), 2.32 (dt, J=7.6, 2.4 Hz, 2H, CH.sub.2CCH), 2.81 (t, J=2.4 Hz, 1H, CCH), 3.84 (s, 3H, OCH.sub.3), 4.06 (t, J=7.6 Hz, 2H, OCH.sub.2), 6.78 (d, J=15.6 Hz, 1H, CHCHCO), 6.99 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.17 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.22 (dd, J.sub.5,6=8.0, J.sub.2,6=2.0 Hz, 1H, H6), 7.37 (d, J.sub.2,6=2.0 Hz, 1H, H2), 7.55 (d, J=15.6 Hz, 1H, CHCHCO), 7.60 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 8.00 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.62 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.27 (s, 1H, NH), 13.56 (br s, 1H, CO.sub.2H); .sub.C (100 MHz, DMSO-d.sub.6) 14.5, 27.7, 55.7, 66.7, 71.7, 83.6, 110.7, 112.7, 116.6, 120.0, 120.3, 122.6, 122.7, 127.4, 131.1, 134.0, 141.1, 141.6, 149.2, 149.8, 164.1, 169.4; HRMS (ESI) calculated for C.sub.22H.sub.21NO.sub.5 [M+H].sup.+ 380.1492. found 380.1493; .sub.max 755, 1257, 1506, 1584, 1657, 2929, 3266, 3519 cm.sup.1.

4-Methoxy-3-(pent-4-ynyloxy)benzaldehyde

(155) ##STR00081##

(156) Isovanillin (0.98 g, 6.4 mmol) was alkylated with pent-4-ynyl 4-methylbenzenesulfonate (2.3 g, 8.3 mmol) according to Procedure 4. The crude product was recystallised from EtOAc/petrol to provide 4-methoxy-3-(pent-4-ynyloxy)benzaldehyde (1.16 g, 83%) as a colourless crystalline solid; mp 73-74 C.; .sub.H (400 MHz, CDCl.sub.3) 1.98 (t, J=2.4 Hz, 1H, CCH), 2.06 (p, J=7.0 Hz, 2H, CH.sub.2), 2.43 (td, J=7.0, 2.4 Hz, 2H, CH.sub.2CCH), 3.94 (s, 3H, OCH.sub.3), 4.18 (t, J=7.0 Hz, 2H, OCH.sub.2), 6.97 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.43 (s, 1H, H2), 7.45 (d, J.sub.5,6=8.0 Hz, 1H, H6), 9.84 (s, 1H, CHO); .sub.C (100 MHz, CDCl.sub.3) 15.1, 27.9, 56.1, 67.3, 69.1, 83.2, 110.6, 110.7, 126.7, 130.0, 148.9, 154.9, 190.9; .sub.max 1025, 1263, 1584, 1665, 2849, 2936, 3254 cm.sup.1.

(E)-2-[[3-(4-Methoxy-3-(pent-4-ynyloxy)phenyl)-1-oxo-2-propenyl]amino]benzoic acid (36)

(157) ##STR00082##

(158) Piperidine (0.45 mL, 4.6 mmol) was added to a suspension of 4-methoxy-3-(pent-1-ynyloxy)benzaldehyde (1.0 g, 4.6 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.93 g, 4.2 mmol) in toluene (10 mL) and treated according to Procedure 2, acidifying with 1 M HCl. The crude product was recrystallised from EtOH/water providing (E)-2-[[3-(4-methoxy-3-(pent-4-ynyloxy)phenyl)-1-oxo-2-propenyl]amino]benzoic acid (1.2 g, 77%) as a yellow crystalline solid; mp 154-156 C.; .sub.H (400 MHz, DMSO-d.sub.6) 1.91 (p, J=7.6 Hz, 2H, CH.sub.2), 2.34 (dt, J=7.6, 2.4 Hz, 2H, CH.sub.2CCH), 2.82 (t, J=2.4 Hz, 1H, CCH), 3.80 (s, 3H, OCH.sub.3), 4.10 (t, J=7.6 Hz, 2H, OCH.sub.2), 6.78 (d, J=15.6 Hz, 1H, CHCHCO), 6.99 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.15 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.25 (d, J.sub.5,6=8.0 Hz, 1H, H6), 7.38 (s, 1H, H2), 7.55 (d, J=15.6 Hz, 1H, CHCHCO), 7.60 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 8.00 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.62 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.26 (s, 1H, NH), 13.56 (br s, 1H, CO.sub.2H); .sub.C (100 MHz, DMSO-d.sub.6) 14.5, 27.8, 55.6, 66.8, 71.6, 83.8, 111.7, 111.8, 116.6, 119.9, 120.3, 122.6, 122.8, 127.2, 131.1, 134.0, 141.1, 141.6, 148.1, 150.9, 164.1, 169.4; HRMS (ESI) calculated for C.sub.22H.sub.21NO.sub.5 [M+H].sup.+ 380.1492. found 380.1490; .sub.max 754, 1257, 1510, 1584, 1659, 2944, 3250, 3512 cm.sup.1.

4-(But-3-ynyloxy)-3-methoxybenzaldehyde

(159) ##STR00083##

(160) 4-Methylbenzenesulfonyl chloride (5.7 g, 30 mmol), 3-butyn-1-ol (1.5 mL, 20 mmol) and pyridine (3.2 mL, 40 mmol) in CH.sub.2Cl.sub.2 (20 mL) were treated according to Procedure 4 giving but-3-ynyl 4-methylbenzenesulfonate (4.15 g, 93%) as a colourless oil. Vanillin (0.86 g, 5.7 mmol) was alkylated with but-3-ynyl 4-methylbenzenesulfonate (1.9 g, 8.5 mmol) according to Procedure 4 and the crude product was recystallised from EtOAc/petrol to provide 4-(but-3-ynyloxy)-3-methoxybenzaldehyde (0.39 g, 34%) as a colourless crystalline solid; mp 101-102 C.; .sub.H (400 MHz, CDCl.sub.3) 2.07 (t, J=2.8 Hz, 1H, CCH), 2.79 (td, J=7.2, 2.8 Hz, 2H, CH.sub.2CCH), 3.93 (s, 3H, OCH.sub.3), 4.25 (t, J=7.2 Hz, 2H, OCH.sub.2), 7.00 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.42 (d, J.sub.2,6=2.0 Hz, 1H, H2), 7.45 (dd, J.sub.5,6=8.0, J.sub.2,6=2.0 Hz, 1H, H6), 9.86 (s, 1H, CHO); .sub.C (100 MHz, CDCl.sub.3) 13.3, 56.1, 67.0, 70.5, 79.6, 109.6, 112.0, 126.6, 130.5, 149.9, 153.3, 190.9; .sub.max 1021, 1269, 1586, 1677, 2940, 3246 cm.sup.1.

(E)-2-[[3-(4-(But-3-ynyloxy)-3-methoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (37)

(161) ##STR00084##

(162) Piperidine (0.19 mL, 1.9 mmol) was added to a suspension of 3-methoxy-4-(but-1-ynyloxy)benzaldehyde (0.39 g, 1.7 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.39 g, 1.9 mmol) in toluene (5 mL) and treated according to Procedure 2, acidifying with 1M HCl. The crude product was recrystallised from EtOH/water providing (E)-2-[[3-(4-(but-3-ynyloxy)-3-methoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (0.48 g, 75%) as a yellow crystalline solid; mp 178-180 C.; .sub.H (400 MHz, DMSO-d.sub.6) 2.61 (dt, J=6.8, 2.4 Hz, 2H, CH.sub.2CCH), 2.86 (t, J=2.4 Hz, 1H, CCH), 3.81 (s, 3H, OCH.sub.3), 4.06 (t, J=6.8 Hz, 2H, OCH.sub.2), 6.77 (d, J=15.6 Hz, 1H, CHCHCO), 6.98 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.13 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.20 (d, J.sub.5,6=8.0 Hz, 1H, H6), 7.36 (s, 1H, H2), 7.53 (d, J=15.6 Hz, 1H, CHCHCO), 7.58 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 7.97 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.59 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.25 (s, 1H, NH), 13.56 (br s, 1H, CO.sub.2H); .sub.C (100 MHz, DMSO-d.sub.6) 19.6, 56.4, 67.2, 73.2, 82.0, 111.5, 113.6, 117.3, 120.8, 121.0, 123.2, 123.3, 128.4, 131.8, 134.7, 141.7, 142.2, 149.8, 150.1, 164.8, 170.1; HRMS (ESI) calculated for C.sub.21H.sub.19NO.sub.5 [M+H].sup.+ 366.1336. found 366.1337; .sub.max 755, 1263, 1512, 1603, 1689, 3257, 3401 cm.sup.1.

3-(But-3-ynyloxy)-4-methoxybenzaldehyde

(163) ##STR00085##

(164) Isovanillin (0.95 g, 6.2 mmol) was alkylated with but-3-ynyl 4-methylbenzenesulfonate (2.1 g, 9.4 mmol) according to Procedure 4. The crude product was recystallised from EtOAc/petrol to provide 3-(but-3-ynyloxy)-4-methoxybenzaldehyde (0.44 g, 35%) as a colourless crystalline solid; mp 63-65 C.; .sub.H (400 MHz, CDCl.sub.3) 2.06 (t, J=2.8 Hz, 1H, CCH), 2.76 (td, J=7.2, 2.8 Hz, 2H, CH.sub.2CCH), 3.96 (s, 3H, OCH.sub.3), 4.22 (t, J=7.2 Hz, 2H, OCH.sub.2), 6.99 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.43 (d, J.sub.2,6=1.4 Hz, 1H, H2), 7.45 (dd, J.sub.5,6=8.0, J.sub.2,6=1.4 Hz, 1H, H6), 9.85 (s, 1H, CHO); .sub.C (100 MHz, CDCl.sub.3) 19.4, 56.2, 67.0, 70.3, 79.8, 110.9, 111.2, 127.1, 130.1, 148.4, 154.9, 190.7; .sub.max 1015, 1124, 1231, 1586, 1675, 2821, 3305 cm.sup.1.

(E)-2-[[3-(3-(But-3-ynyloxy)-4-methoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (38)

(165) ##STR00086##

(166) Piperidine (0.17 mL, 1.7 mmol) was added to a suspension of 4-methoxy-3-(but-1-ynyloxy)benzaldehyde (0.35 g, 1.7 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.35 g, 1.6 mmol) in toluene (5 mL) and treated according to Procedure 2, acidifying with 1 M HCl. The crude product was recrystallised from EtOH/water providing (E)-2-[[3-(3-(but-3-ynyloxy)-4-methoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (0.40 g, 70%) as a colourless crystalline solid; mp 197-198 C.; .sub.H (400 MHz, DMSO-d.sub.6) 2.65 (dt, J=6.8, 2.4 Hz, 2H, CH.sub.2CCH), 2.90 (t, J=2.4 Hz, 1H, CCH), 3.80 (s, 3H, OCH.sub.3), 4.13 (t, J=6.8 Hz, 2H, OCH.sub.2), 6.80 (d, J=15.6 Hz, 1H, CHCHCO), 7.00 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.16 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.26 (d, J.sub.5,6=8.0 Hz, 1H, H6), 7.40 (s, 1H, H2), 7.55 (d, J=15.6 Hz, 1H, CHCHCO), 7.61 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 7.99 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.61 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.27 (s, 1H, NH), 13.58 (br s, 1H, CO.sub.2H); .sub.C (100 MHz, DMSO-d.sub.6) 19.0, 55.6, 66.6, 72.5, 81.4, 111.9, 111.9, 116.6, 120.0, 120.3, 122.7, 123.0, 127.3, 131.1, 134.0, 141.0, 141.5, 147.8, 150.7, 164.2, 169.4; HRMS (ESI) calculated for C.sub.21H.sub.19NO.sub.5 [M+H].sup.+ 366.1335. found 366.1337; .sub.max 753, 1263, 1512, 1581, 1671, 2833, 3250 cm.sup.1.

4-(Hex-3-ynyloxy)-3-methoxybenzaldehyde

(167) ##STR00087##

(168) 4-Methylbenzenesulfonyl chloride (5.7 g, 30 mmol), 3-hexyn-1-ol (1.5 mL, 20 mmol) and pyridine (3.2 mL, 40 mmol) in CH.sub.2Cl.sub.2 (20 mL) were treated according to Procedure 4 giving hex-3-ynyl 4-methylbenzenesulfonate (3.8 g, 75%) as a colourless oil. Vanillin (0.76 g, 5.0 mmol) was alkylated with hex-3-ynyl 4-methylbenzenesulfonate (1.9 g, 7.5 mmol) according to Procedure 4 and the crude product was recrystallised from EtOAc/petrol to provide 4-(hex-3-ynyloxy)-3-methoxybenzaldehyde (0.45 g, 39%) as a colourless crystalline solid; mp 80-81 C.; .sub.H (400 MHz, CDCl.sub.3) 1.12 (t, J=7.6 Hz, 3H, CH.sub.3), 2.17 (tq, J=7.6, 2.4 Hz, 2H, CH.sub.3CH.sub.2), 2.73 (tt, J=7.6, 2.4 Hz, 2H, OCH.sub.2CH.sub.2C), 3.92 (s, 3H, OCH.sub.3), 4.19 (t, J=7.6 Hz, 2H, OCH.sub.2), 7.00 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.41 (d, J.sub.2,6=1.6 Hz, 1H, H2), 7.44 (dd, J.sub.5,6=8.0, J.sub.2,6=1.6 Hz, 1H, H6), 9.85 (s, 1H, CHO); .sub.C (100 MHz, CDCl.sub.3) 12.4, 14.0, 19.5, 56.0, 67.6, 74.3, 84.0, 109.5, 111.8, 126.6, 130.3, 149.9, 153.5, 190.8; .sub.max 1023, 1134, 1263, 1586, 1680, 2877, 2972 cm.sup.1.

(E)-2-[[3-(4-(Hex-3-ynyloxy)-3-methoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (39)

(169) ##STR00088##

(170) Piperidine (0.17 mL, 1.7 mmol) was added to a suspension of 3-methoxy-4-(hex-3-ynyloxy)benzaldehyde (0.40 g, 1.7 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.35 g, 1.6 mmol) in toluene (5 mL) and treated according to Procedure 2, acidifying with 1 M HCl. The crude product was recrystallised from EtOH providing (E)-2-[[3-(4-(hex-3-ynyloxy)-3-methoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (0.40 g, 65%) as a colourless crystalline solid; mp 165-166 C.; .sub.H (400 MHz, DMSO-d.sub.6) 1.04 (t, J=7.6 Hz, 3H, CH.sub.3), 2.14 (q, J=7.6 Hz, 2H, CH.sub.3CH.sub.2), 2.61 (t, J=7.6 Hz, 1H, OCH.sub.2CH.sub.2C), 3.83 (s, 3H, OCH.sub.3), 4.05 (t, J=7.6 Hz, 2H, OCH.sub.2), 6.79 (d, J=15.6 Hz, 1H, CHCHCO), 6.99 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.16 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.22 (d, J.sub.5,6=8.0 Hz, 1H, H6), 7.38 (s, 1H, H2), 7.55 (d, J=15.6 Hz, 1H, CHCHCO), 7.61 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 7.99 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.61 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.27 (s, 1H, NH), 13.57 (br s, 1H, CO.sub.2H); .sub.C (100 MHz, DMSO-d.sub.6) 11.7, 14.0, 19.2, 55.7, 66.9, 76.1, 83.0, 110.9, 113.0, 116.6, 120.1, 120.3, 122.5, 122.6, 127.6, 131.1, 134.0, 141.0, 141.5, 149.1, 149.5, 164.1, 169.4; HRMS (ESI) calculated for C.sub.23H.sub.23NO.sub.5 [M+H].sup.+ 394.1649. found 394.1647; .sub.max 755, 1235, 1510, 1601, 1669, 3234, 3563 cm.sup.1.

3-(Hex-3-ynyloxy)-4-methoxybenzaldehyde

(171) ##STR00089##

(172) Isovanillin (0.76 g, 5.0 mmol) was alkylated with hex-3-ynyl 4-methylbenzenesulfonate (1.9 g, 7.53 mmol) according to Procedure 4. The crude product was recrystallised from EtOAc/petrol to provide 3-(hex-3-ynyloxy)-4-methoxybenzaldehyde (0.58 g, 50%) as a colourless crystalline solid; mp 86.5-87.5 C.; .sub.H (400 MHz, CDCl.sub.3) 1.13 (t, J=7.6 Hz, 3H, CH.sub.3), 2.17 (tq, J=7.6, 2.4 Hz, 2H, CH.sub.3CH.sub.2), 2.72 (tt, J=7.6, 2.4 Hz, 2H, OCH.sub.2CH.sub.2C), 3.95 (s, 3H, OCH.sub.3), 4.17 (t, J=7.6 Hz, 2H, OCH.sub.2), 6.98 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.44 (d, J.sub.2,6=1.6 Hz, 1H, H2), 7.47 (dd, J.sub.5,6=8.0, J.sub.2,6=1.6 Hz, 1H, H6), 9.85 (s, 1H, CHO); .sub.C (100 MHz, CDCl.sub.3) 12.4, 14.1, 19.6, 56.2, 67.6, 74.5, 83.9, 110.8, 111.0, 126.9, 130.1, 148.6, 154.8, 190.8; .sub.max 1019, 1134, 1265, 1586, 1683, 2841, 2977 cm.sup.1.

(E)-2-[[3-(3-(Hex-3-ynyloxy)-4-methoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (40)

(173) ##STR00090##

(174) Piperidine (0.21 mL, 2.2 mmol) was added to a suspension of 4-methoxy-3-(hex-3-ynyloxy)benzaldehyde (0.50 g, 2.2 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.44 g, 2.0 mmol) in toluene (5 mL) and treated according to Procedure 2, acidifying with 1 M HCl. The crude product was recrystallised from EtOH providing (E)-2-[[3-(3-(hex-3-ynyloxy)-4-methoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (0.58 g, 75%) as a yellow crystalline solid; mp 163-165 C.; .sub.H (400 MHz, DMSO-d.sub.6) 1.05 (t, J=7.6 Hz, 3H, CH.sub.3), 2.15 (q, J=7.6 Hz, 2H, CH.sub.3CH.sub.2), 2.62 (t, J=7.6 Hz, 1H, OCH.sub.2CH.sub.2C), 3.80 (s, 3H, OCH.sub.3), 4.09 (t, J=7.6 Hz, 2H, OCH.sub.2), 6.80 (d, J=15.6 Hz, 1H, CHCHCO), 6.99 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.16 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.25 (d, J.sub.5,6=8.0 Hz, 1H, H6), 7.40 (s, 1H, H2), 7.55 (d, J=15.6 Hz, 1H, CHCHCO), 7.60 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 8.00 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.61 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.26 (s, 1H, NH), 13.58 (br s, 1H, CO.sub.2H); .sub.C (100 MHz, DMSO-d.sub.6) 11.8, 14.0, 19.3, 55.6, 67.0, 76.2, 83.0, 111.9, 112.0, 116.6, 120.0, 120.3, 122.6, 123.0, 127.3, 131.1, 134.0, 141.0, 141.6, 147.9, 150.8, 164.2, 169.4; HRMS (ESI) calculated for C.sub.23H.sub.23NO.sub.5 [M+H].sup.+ 394.1649. found 394.1648; .sub.max 755, 1253, 1510, 1604, 1657, 3238, 3524 cm.sup.1.

3-Methoxy-4-(oct-3-ynyloxy)benzaldehyde

(175) ##STR00091##

(176) 4-Methylbenzenesulfonyl chloride (5.70 g, 30 mmol), 3-octyn-1-ol (1.52 mL, 20 mmol) and pyridine (3.24 mL, 40 mmol) in CH.sub.2Cl.sub.2 (20 mL) were treated according to Procedure 4 giving oct-3-ynyl 4-methylbenzenesulfonate (5.21 g, 93%) as a colourless oil. Vanillin (0.90 g, 5.94 mmol) was alkylated with oct-3-ynyl 4-methylbenzenesulfonate (2.50 g, 8.92 mmol) according to Procedure 4 and the crude product was recrystallised from purified by flash chromatography with 10% EtOAc/petrol as eluent to give 3-methoxy-4-(oct-3-ynyloxy)benzaldehyde (0.25 g, 16%) as a colourless crystalline solid; mp 64.5-65.5 C.; .sub.H (400 MHz, CDCl.sub.3) 0.86 (t, J=7.2 Hz, 3H, CH.sub.3), 1.32-1.44 (m, 4H, CH.sub.3CH.sub.2CH.sub.2), 2.12 (t, J=7.2 Hz, 2H, CH.sub.2), 2.69 (t, J=7.2 Hz, 2H, CH.sub.2CH.sub.2O), 3.88 (s, 3H, OCH.sub.3), 4.15 (t, J=7.2 Hz, 2H, OCH.sub.2), 6.96 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.37 (s, 1H, H2), 7.39 (d, J.sub.5,6=8.0 Hz, 1H, H6), 9.80 (s, 1H, CHO); .sub.C (100 MHz, CDCl.sub.3) 13.5, 18.3, 19.5, 21.8, 30.8, 55.9, 67.5, 74.8, 82.5, 109.4, 111.7, 126.4, 130.2, 149.7, 153.4, 190.7; .sub.max 1020, 1132, 1262, 1508, 1584, 1684, 2931 cm.sup.1.

(E)-2-[[3-(3-Methoxy-4-(oct-3-ynyloxy)phenyl)-1-oxo-2-propenyl]amino]benzoic acid (41)

(177) ##STR00092##

(178) Piperidine (95 L, 0.96 mmol) was added to a suspension of 3-methoxy-4-(oct-3-ynyloxy)benzaldehyde (0.25 g, 0.96 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.20 g, 0.90 mmol) in toluene (5 mL) and treated according to Procedure 2, acidifying with 20% AcOH. The crude product was recrystallised from EtOH/water providing (E)-2-[[3-(3-methoxy-4-(oct-3-ynyloxy)phenyl)-1-oxo-2-propenyl]amino]benzoic acid (0.30 g, 79%) as a colourless crystalline solid; mp 169-170 C.; .sub.H (400 MHz, DMSO-d.sub.6) 0.85 (t, J=7.2 Hz, 3H, CH.sub.3), 1.31-1.42 (m, 4H, CH.sub.3CH.sub.2CH.sub.2), 2.13 (t, J=7.2 Hz, 2H, CH.sub.2), 2.60 (t, J=7.2 Hz, 2H, CH.sub.2CH.sub.2O), 3.83 (s, 3H, OCH.sub.3), 4.05 (t, J=7.2 Hz, 2H, OCH.sub.2), 6.76 (d, J=15.6 Hz, 1H, CHCHCO), 7.00 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.15 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.22 (dd, J.sub.5,6=8.0, J.sub.2,6=1.6 Hz, 1H, H6), 7.37 (d, J.sub.5,6=1.6 Hz, 1H, H2), 7.55 (d, J=15.6 Hz, 1H, CHCHCO), 7.60 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 8.00 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.62 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.30 (s, 1H, NH), 13.55 (br s, 1H, CO.sub.2H); .sub.C (100 MHz, DMSO-d.sub.6) 13.4, 17.7, 19.2, 21.3, 30.4, 55.7, 67.0, 76.7, 81.6, 110.9, 113.0, 116.7, 120.1, 120.3, 122.5, 122.6, 127.6, 131.1, 133.9, 141.0, 141.5, 149.1, 149.5, 164.1, 169.4; HRMS (ESI) calculated for C.sub.25H.sub.27NO.sub.5 [M+H].sup.+ 421.1962. found 421.1962; .sub.max 757, 1143, 1220, 1514, 1601, 1652, 1690, 2939 cm.sup.1.

4-Methoxy-3-(oct-3-ynyloxy)benzaldehyde

(179) ##STR00093##

(180) Isovanillin (0.90 g, 5.9 mmol) was alkylated with oct-3-ynyl 4-methylbenzenesulfonate (2.5 g, 8.9 mmol) according to Procedure 2. The crude product was recrystallised from purified by flash chromatography with 10% EtOAc/petrol as eluent to give 4-methoxy-3-(oct-3-ynyloxy)benzaldehyde (0.52 g, 34%) as a colourless crystalline solid; mp 42-43 C.; .sub.H (400 MHz, CDCl.sub.3) 0.90 (t, J=7.2 Hz, 3H, CH.sub.3), 1.37-1.49 (m, 4H, CH.sub.3CH.sub.2CH.sub.2), 2.16 (tt, J=7.2, 2.4 Hz, 2H, CH.sub.2), 2.72 (tt, J=7.2, 2.4 Hz, 2H, CH.sub.2CH.sub.2O), 3.95 (s, 3H, OCH.sub.3), 4.17 (t, J=7.2 Hz, 2H, OCH.sub.2), 6.98 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.44 (d, J.sub.2,6=2.0 Hz, 1H, H2), 7.47 (dd, J.sub.5,6=8.0, J.sub.2,6=2.0 Hz, 1H, H6), 9.85 (s, 1H, CHO); .sub.C (100 MHz, CDCl.sub.3) 13.6, 18.4, 19.7, 21.9, 30.9, 56.2, 67.6, 75.1, 82.5, 110.8, 111.0, 126.9, 130.1, 148.6, 154.9, 190.8; .sub.max 1019, 1132, 1262, 1508, 1585, 1684, 2932 cm.sup.1.

(E)-2-[[3-(4-Methoxy-3-(oct-3-ynyloxy)phenyl)-1-oxo-2-propenyl]amino]benzoic acid (42)

(181) ##STR00094##

(182) Piperidine (190 L, 1.9 mmol) was added to a suspension of 4-methoxy-3-(oct-3-ynyloxy)benzaldehyde (0.50 g, 1.9 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.39 g, 1.7 mmol) in toluene (5 mL) and treated according to Procedure 2, acidifying with 20% AcOH. The crude product was recrystallised from EtOH/water providing (E)-2-[[3-(4-methoxy-3-(oct-3-ynyloxy)phenyl)-1-oxo-2-propenyl]amino]benzoic acid (0.60 g, 82%) as a colourless crystalline solid; mp 157-158 C.; .sub.H (400 MHz, DMSO-d.sub.6) 0.85 (t, J=7.2 Hz, 3H, CH.sub.3), 1.32-1.42 (m, 4H, CH.sub.3CH.sub.2CH.sub.2), 2.14 (t, J=7.2 Hz, 2H, CH.sub.2), 2.62 (t, J=7.2 Hz, 2H, CH.sub.2CH.sub.2O), 3.80 (s, 3H, OCH.sub.3), 4.10 (t, J=7.2 Hz, 2H, OCH.sub.2), 6.80 (d, J=15.6 Hz, 1H, CHCHCO), 7.00 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.16 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.25 (d, J.sub.5,6=8.0 Hz, 1H, H6), 7.40 (s, 1H, H2), 7.55 (d, J=15.6 Hz, 1H, CHCHCO), 7.61 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 8.00 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.62 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.27 (s, 1H, NH), 13.58 (br s, 1H, CO.sub.2H); .sub.C (100 MHz, DMSO-d.sub.6) 13.4, 17.8, 19.3, 21.3, 30.5, 55.6, 67.1, 76.9, 81.5, 111.9, 112.1, 116.6, 120.0, 120.3, 122.6, 122.9, 127.3, 131.1, 134.0, 141.0, 141.5, 147.9, 150.8, 164.2, 169.4; HRMS (ESI) calculated for C.sub.25H.sub.27NO.sub.5 [M+H].sup.+ 421.1962. found 421.1962; .sub.max 757, 1131, 1259, 1515, 1582, 1671, 2954, 3335 cm.sup.1.

4-Benzyloxy-3-methoxybenzaldehyde

(183) ##STR00095##

(184) Benzyl bromide (1.2 mL, 9.9 mmol) was added to a suspension of vanillin (1.0 g, 6.6 mmol) and potassium carbonate (2.7 g, 20 mmol) in acetone (10 mL) and treated according to Procedure 3. The crude product was recrystallised from EtOH to give 4-benzyloxy-3-methoxybenzaldehyde (1.0 g, 64%) as a colourless crystalline solid; mp 61-62 C.; .sub.H (400 MHz, CDCl.sub.3) 3.95 (s, 3H, OCH.sub.3), 5.25 (s, 2H, OCH.sub.2), 6.99 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.32-7.45 (m, 7H, H2, H6, Ph), 9.84 (s, 1H, CHO); .sub.C (100 MHz, CDCl.sub.3) 56.1, 70.8 109.3, 112.4, 126.6 127.2, 128.2, 128.7, 130.3, 136.0, 150.1, 153.6, 190.9; .sub.max 988, 1133, 1259, 1505, 1583, 1672 cm.sup.1.

(E)-2-[[3-(4-Benzyloxy-3-methoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (43)

(185) ##STR00096##

(186) Piperidine (0.20 mL, 2.1 mmol) was added to a suspension of 4-benzyloxy-3-methoxybenzaldehyde (0.50 g, 2.1 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.42 g, 1.9 mmol) in toluene (5 mL) and treated according to Procedure 2, acidifying with 20% AcOH. The crude product was recrystallised from EtOH providing (E)-2-[[3-(4-benzyloxy-3-methoxyphenyl)-1-oxo-2-propenyl]amino]benzoic (0.48 g, 63%) as a yellow crystalline solid; mp 197-199 C.; .sub.H (400 MHz, DMSO-d.sub.6) 3.84 (s, 3H, OCH.sub.3), 5.13 (s, 2H, OCH.sub.2), 6.79 (d, J=15.6 Hz, 1H, CHCHCO), 7.07 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.16 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.22 (d, J.sub.5,6=8.0 Hz, 1H, H6), 7.31-7.46 (m, 6H, H2, Ph), 7.55 (d, J=15.6 Hz, 1H, CHCHCO), 7.61 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 8.00 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.62 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.28 (s, 1H, NH), 13.60 (br s, 1H, CO.sub.2H); .sub.C (100 MHz, DMSO-d.sub.6) 55.7, 69.8, 110.7, 113.1, 116.6, 120.0, 120.3, 122.5, 122.7, 127.5, 127.8, 127.9, 128.4, 131.1, 134.0, 136.8, 141.1, 141.6, 149.3, 149.6, 164.2, 169.5; HRMS (ESI) calculated for C.sub.24H.sub.21NO.sub.5 [MH].sup. 402.1336. found 402.1342; .sub.max 697, 1133, 1233, 1516, 1599, 1673, 1697, 3035 cm.sup.1.

3-Methoxy-4-(naphth-2-ylmethoxy)benzaldehyde

(187) ##STR00097##

(188) 2-(Bromomethyl)naphthalene (1.3 g, 5.9 mmol) was added to a suspension of vanillin (0.60 g, 3.9 mmol) and potassium carbonate (1.6 g, 12 mmol) in acetone (10 mL) and treated according to Procedure 3. The crude product was recrystallised from EtOAc/petrol providing 3-methoxy-4-(naphth-2-ylmethoxy)benzaldehyde (0.87 g, 75%) as a colourless crystalline solid; mp 107-108 C.; .sub.H (400 MHz, CDCl.sub.3) 3.97 (s, 3H, OCH.sub.3), 5.41 (s, 2H, OCH.sub.2), 7.38 (m, 1H, Naphth-H), 7.44 (d, J.sub.2,6=1.6 Hz, 1H, H2), 7.48-7.50 (m, 3H, H6, Naphth-H), 7.55 (m, 1H, Naphth-H), 7.83-7.89 (m, 4H, Naphth-H), 9.84 (s, 1H, CHO); .sub.C (100 MHz, CDCl.sub.3) 56.3, 71.3 109.7, 112.8, 125.1 126.4, 126.5, 126.6, 126.8, 128.0, 128.2, 128.9, 130.6, 133.4, 133.5, 133.7, 150.4, 153.8, 191.1; .sub.max 991, 1131, 1263, 1505, 1580, 1672, 2884 cm.sup.1.

(E)-2-[[3-(3-Methoxy-4-(naphth-2-ylmethoxy)phenyl)-1-oxo-2-propenyl]amino]benzoic acid (44)

(189) ##STR00098##

(190) Piperidine (0.27 mL, 2.7 mmol) was added to a suspension of 3-methoxy-4-(naphth-2-ylmethoxy)benzaldehyde (0.80 g, 2.7 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.55 g, 2.5 mmol) in toluene (5 mL) and treated according to Procedure 2, acidifying with 20% AcOH. The crude product was recrystallised from EtOH providing (E)-2-[[3-(3-methoxy-4-(naphth-2-ylmethoxy)phenyl)-1-oxo-2-propenyl]amino]benzoic (0.74 g, 66%) as a yellow crystalline solid; mp 197-200 C.; .sub.H (400 MHz, DMSO-d.sub.6) 3.86 (s, 3H, OCH.sub.3), 5.30 (s, 2H, OCH.sub.2), 6.76 (d, J=15.6 Hz, 1H, CHCHCO), 7.00 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.15 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.22 (dd, J.sub.5,6=8.0, J.sub.2,6=1.6 Hz, 1H, H6), 7.37 (d, J.sub.2,6=1.6 Hz, 1H, H2), 7.50-7.63 (m, 5H, CHCHCO, H5, Naphth-H), 7.91-8.01 (m, 5H, H3, Naphth-H), 8.63 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.31 (s, 1H, NH), 13.59 (br s, 1H, CO.sub.2H); O.sub.C (100 MHz, DMSO-d.sub.6) 55.7, 70.1, 110.8, 113.4, 116.6, 120.1, 120.3, 122.5, 122.7, 125.9, 126.2, 126.4, 126.6, 127.6, 127.7, 127.8, 128.1, 131.2, 132.6, 132.7, 134.0, 134.5, 141.1, 141.6, 149.3, 149.6, 164.2, 169.5; HRMS (ESI) calculated for C.sub.28H.sub.23NO.sub.5 [MH].sup. 452.1493. found 452.1495; .sub.max 1135, 1260, 1511, 1584, 1668, 3055 cm.sup.1.

3-Methoxy-4-(pent-3-yloxy)benzaldehyde

(191) ##STR00099##

(192) 3-Bromopentane (1.2 mL, 9.9 mmol) was added to a suspension of vanillin (1.0 g, 6.6 mmol), potassium carbonate (2.7 g, 20 mmol) in EtOH (10 mL) and treated according to Procedure 3. The crude product was purified by flash chromatography with 10% EtOAc/petrol as eluent to give 3-methoxy-4-(pent-3-yloxy)benzaldehyde (0.69 g, 47%) as a pale yellow oil; .sub.H (400 MHz, CDCl.sub.3) 0.96 (t, J=7.2 Hz, 6H, CH.sub.3), 1.73 (m, 4H, CH.sub.2), 3.88 (s, 3H, OCH.sub.3), 4.23 (m, 1H, OCH), 6.94 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.40 (s, 1H, H2), 7.41 (d, J.sub.5,6=8.0 Hz, 1H, H6), 9.81 (s, 1H, CHO); .sub.C (100 MHz, CDCl.sub.3) 9.6, 26.1, 56.0, 81.9, 109.8, 113.1, 126.5, 129.7, 150.5, 154.0, 190.7; .sub.max 1133, 1263, 1504, 1582, 1682, 2967 cm.sup.1.

(E)-2-[[3-(3-Methoxy-4-(pent-3-yloxy)phenyl)-1-oxo-2-propenyl]amino]benzoic acid (45)

(193) ##STR00100##

(194) Piperidine (220 L, 2.2 mmol) was added to a suspension of 3-methoxy-4-(pent-3-yloxy)benzaldehyde (0.50 g, 2.2 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.46 g, 2.1 mmol) in toluene (5 mL) and treated according to Procedure 2, acidifying with 20% AcOH. The crude product was recrystallised from EtOH/water providing (E)-2-[[3-(3-methoxy-4-(pent-3-yloxy)phenyl)-1-oxo-2-propenyl]amino]benzoic acid (0.52 g, 66%) as a yellow crystalline solid; mp 82-85 C.; .sub.H (400 MHz, DMSO-d.sub.6) 0.88 (t, J=7.2 Hz, 6H, CH.sub.3), 1.60 (p, J=7.2 Hz, 4H, CH.sub.2), 3.82 (s, 3H, OCH.sub.3), 4.25 (t, J=7.2 Hz, 1H, OCH), 6.76 (d, J=15.6 Hz, 1H, CHCHCO), 6.98 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.15 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.20 (dd, J.sub.5,6=8.0, J.sub.2,6=1.6 Hz, 1H, H6), 7.36 (d, J.sub.2,6=1.6 Hz, 1H, H2), 7.55 (d, J=15.6 Hz, 1H, CHCHCO), 7.60 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 8.00 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.62 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.28 (s, 1H, NH), 13.58 (br s, 1H, CO.sub.2H); .sub.C (100 MHz, DMSO-d.sub.6) 9.3, 25.5, 55.7, 80.1, 111.2, 114.5, 116.6, 119.8, 120.3, 122.5, 122.6, 127.1, 131.1, 134.0, 141.1, 141.6, 149.6, 149.9, 164.2, 169.5; HRMS (ESI) calculated for C.sub.22H.sub.25NO.sub.5 [MH].sup. 382.1555. found 382.1649; .sub.max 749, 1139, 1259, 1505, 1584, 1650, 2934 cm.sup.1.

(E)-2-[[3-(6-Methoxypyridin-3-yl)-1-oxo-2-propenyl]amino]benzoic acid (46)

(195) ##STR00101##

(196) Piperidine (220 L, 2.2 mmol) was added to a suspension of 6-methoxy-3-pyridine carboxaldehyde (0.30 g, 2.2 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.44 g, 2.1 mmol) in toluene (5 mL) and treated according to Procedure 2, acidifying with 20% AcOH. The crude product was recrystallised from EtOH providing (E)-2-[[3-(6-methoxypyridin-3-yl)-1-oxo-2-propenyl]amino]benzoic acid (0.33 g, 56%) as a colourless crystalline solid; mp 209-211 C.; .sub.H (400 MHz, DMSO-d.sub.6) 3.85 (s, 3H, OCH.sub.3), 6.82 (d, J=15.6 Hz, 1H, CHCHCO), 6.85 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.13 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.57 (d, J=15.6 Hz, 1H, CHCHCO), 7.58 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 7.98 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.14 (dd, J.sub.5,6=8.0, J.sub.2,6=1.6 Hz, 1H, H6), 8.43 (d, J.sub.2,6=1.6 Hz, 1H, H2), 8.57 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.29 (s, 1H, NH), 13.58 (br s, 1H, CO.sub.2H); .sub.C (100 MHz, DMSO-d.sub.6) 53.5, 111.0, 116.7, 120.3, 121.4, 122.8, 124.2, 131.1, 134.0, 137.3, 138.0, 140.9, 148.4, 163.7, 164.4, 169.4; HRMS (ESI) calculated for C.sub.16H.sub.14N.sub.2O.sub.4 [MH].sup. 297.0870. found 297.0877; .sub.max 749, 1249, 1591, 1683, 3246 cm.sup.1.

3-Methoxy-4-(adaman-2-yl-2-oxoethoxy)benzaldehyde

(197) ##STR00102##

(198) 1-Adamantyl bromomethyl ketone (300 mg, 1.19 mmol) was added to a suspension of vanillin (120 mg, 0.791 mmol), potassium carbonate (329 mg, 2.38 mmol) in acetone (5 mL) and treated according to Procedure 3. The crude product was recrystallised from EtOAc/petrol to give 3-methoxy-4-(adaman-2-yl-2-oxoethoxy)benzaldehyde (0.210 g, 81%) as a pale yellow oil; .sub.H (400 MHz, CDCl.sub.3) 1.73-1.81 (m, 6H, CH.sub.2), 1.93 (d, J=2.0 Hz, 6H, CH.sub.2), 2.09 (s, 3H, CH), 3.95 (s, 3H, OCH.sub.3), 5.05 (s, 2H, OCH.sub.2), 6.70 (d, J.sub.5,6=7.4 Hz, 1H, H5), 7.38 (dd, J.sub.5,6=7.4, J.sub.2,6=1.6 Hz, 1H, H6), 7.43 (d, J.sub.2,6=1.6 Hz, 1H, H2), 9.85 (s, 1H, CHO); .sub.C (100 MHz, CDCl.sub.3) 27.9, 36.6, 38.3, 45.8, 56.3, 69.4, 109.9, 112.0, 126.4, 130.9, 150.1, 153.1, 190.1, 207.9; .sub.max 1001, 1133, 1259, 1507, 1587, 1680, 2850, 2904 cm.sup.1.

(E)-2-[[3-(3-Methoxy-4-(adaman-2-yl-2-oxoethoxy)phenyl)-1-oxo-2-propenyl]amino]benzoic acid (47)

(199) ##STR00103##

(200) Piperidine (63 L, 0.64 mmol) was added to a suspension of 3-methoxy-4-(adaman-2-yl-2-oxoethoxy)benzaldehyde (0.21 g, 0.64 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.13 g, 0.58 mmol) in toluene (5 mL) and treated according to Procedure 2, acidifying with 20% AcOH. The crude product was recrystallised from EtOH providing (E)-2-[[3-(3-methoxy-4-(adaman-2-yl-2-oxoethoxy)phenyl)-1-oxo-2-propenyl]amino]benzoic acid (0.17 g, 59%) as a colourless crystalline solid; mp 112-114 C.; .sub.H (400 MHz, DMSO-d.sub.6) 1.66-1.72 (m, 6H, CH.sub.2), 1.85-1.86 (m, 6H, CH.sub.2), 2.00 (s, 3H, CH), 3.84 (s, 3H, OCH.sub.3), 5.12 (s, 2H, OCH.sub.2), 6.74 (d, J.sub.5,6=8.0 Hz, 1H, H5), 6.77 (d, J=15.6 Hz, 1H, CHCHCO), 7.14-7.18 (m, 2H, H4, H6), 7.37 (d, J.sub.2,6=1.6 Hz, 1H, H2), 7.54 (d, J=15.6 Hz, 1H, CHCHCO), 7.60 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 8.00 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.62 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.28 (s, 1H, NH), 13.53 (br s, 1H, CO.sub.2H); .sub.C (100 MHz, DMSO-d.sub.6) 28.0, 36.6, 37.8, 45.4, 56.5, 69.5, 111.7, 113.4, 117.3, 120.7, 121.0, 123.0, 123.4, 128.2, 131.8, 134.7, 141.8, 142.3, 149.7, 150.0, 164.9, 170.2, 209.4; HRMS (ESI) calculated for C.sub.29H.sub.31 NO.sub.6 [M+Na].sup.+ 512.2044 found 512.2045; .sub.max 749, 1143, 1249, 1508, 1588, 1687, 1712, 2848, 2908, 3380 cm.sup.1.

(E)-2-[[3-(3-Methoxy-4-(2-morpholinoethoxy)phenyl)-1-oxo-2-propenyl]amino]benzoic acid (48)

(201) ##STR00104##

(202) Piperidine (75 L, 0.75 mmol) was added to a suspension of 3-methoxy-4-(adaman-2-yl-2-oxoethoxy)benzaldehyde (0.20 g, 0.75 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.15 g, 0.69 mmol) in toluene (5 mL) and treated according to Procedure 2, neutralizing with 20% AcOH. The aqueous phase was extracted with CH.sub.2Cl.sub.2, washed with water, brine, dried and concentrated. The crude product was recrystallised from MeOH providing (E)-2-[[3-(3-methoxy-4-(2-morpholinoethoxy)phenyl)-1-oxo-2-propenyl]amino]benzoic acid (65 mg, 22%) as a pale brown solid; mp 195-201 C.; .sub.H (400 MHz, DMSO-d.sub.6) 2.62 (m, 4H, CH.sub.2N), 2.83 (t, J=4.4 Hz, 4H, CH.sub.2), 3.61 (t, J=3.3 Hz, 4H, OCH.sub.2), 3.81 (s, 3H, OCH.sub.3), 4.15 (t, J=4.4 Hz, 2H, OCH.sub.2), 6.74 (d, J=15.6 Hz, 1H, CHCHCO), 7.00 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.12 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.20 (d, J.sub.5,6=8.0 Hz, 1H, H6), 7.35 (s, 1H, H2), 7.53 (d, J=15.6 Hz, 1H, CHCHCO), 7.55 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 8.00 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.62 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.81 (s, 1H, NH); .sub.C (100 MHz, DMSO-d.sub.6) 53.4, 55.7, 56.6, 65.7, 110.7, 113.0, 118.0, 120.0, 120.3, 122.4, 122.5, 127.6, 131.2, 133.3, 141.1, 141.3, 149.2, 149.6, 164.1, 168.7; HRMS (ESI) calculated for C.sub.23H.sub.26N.sub.2O.sub.6 [M+H].sup.+ 427.1864. found 427.1864; .sub.max 764, 1139, 1249, 1502, 1583, 1621, 1676, 2964 cm.sup.1.

(E)-2-[[3-(3-Methoxy-4-(pyridin-3-ylmethoxy)phenyl)-1-oxo-2-propenyl]amino]benzoic acid (49)

(203) ##STR00105##

(204) 3-Bromomethylpyridine (0.30 mg, 1.2 mmol) was added to a suspension of vanillin (0.12 g, 0.79 mmol), potassium carbonate (0.33 g, 2.4 mmol) in acetone (5.0 mL) and treated according to Procedure 3. 3-Methoxy-4-(pyridin-3-ylmethoxy)benzaldehyde (88 mg, 46%) was obtained as a brown oil. Piperidine (36 L, 0.36 mmol) was added to a suspension of 3-methoxy-4-(pyridin-3-ylmethoxy)benzaldehyde (0.88 mg, 0.36 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (73 mg, 0.33 mmol) in toluene (5 mL) and treated according to Procedure 2, acidifying with 20% AcOH. (E)-2-[[3-(3-Methoxy-4-(pyridin-3-ylmethoxy)phenyl)-1-oxo-2-propenyl]amino]benzoic acid (58 mg, 44%) was obtained as a pure brown crystalline solid; mp 245-251 C.; .sub.H (400 MHz, DMSO-d.sub.6) 3.84 (s, 3H, OCH.sub.3), 5.18 (s, 2H, OCH.sub.2), 6.80 (d, J=15.6 Hz, 1H, CHCHCO), 7.12 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.16 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.24 (d, J.sub.5,6=8.0 Hz, 1H, H6), 7.40-7.45 (m, 2H, H2, ArH), 7.56 (d, J=15.6 Hz, 1H, CHCHCO), 7.60 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 7.87 (d, J=8.0 Hz, 1H, ArH), 8.00 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.55-8.67 (m, 3H, H6, ArH), 11.32 (s, 1H, NH); .sub.C (100 MHz, DMSO-d.sub.6) 55.7, 67.6, 110.8, 113.4, 116.7, 120.2, 120.3, 122.4, 122.7, 123.6, 127.9, 131.1, 132.4, 133.9, 135.9, 141.0, 141.5, 149.1, 149.2, 149.3, 164.2, 169.5; HRMS (ESI) calculated for C.sub.23H.sub.20N.sub.2O.sub.5 [M+H].sup.+ 404.1445 found 404.1445; .sub.max 758, 1257, 1509, 1586, 1671, 2931 cm.sup.1.

(E)-2-[[3-((3-(3,5-Dimethylisoxazol-4-yl)methoxy)-4-methoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (50)

(205) ##STR00106##

(206) Piperidine (97 L, 0.99 mmol) was added to a suspension of 3-((3,5-dimethylisoxazol-4-yl)methoxy)-4-methoxybenzaldehyde (0.26 g, 0.99 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.20 g, 0.90 mmol) in toluene (5 mL) and treated according to Procedure 2, acidifying with 20% AcOH. The crude product was recrystallised from EtOH/water providing (E)-2-[[3-((3-(3,5-dimethylisoxazol-4-yl)methoxy)-4-methoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (0.21 g, 54%) as an orange/brown crystalline solid; mp 227-229 C.; .sub.H (400 MHz, DMSO-d.sub.6) 2.21 (s, 3H, CH.sub.3), 2.38 (s, 3H, CH.sub.3), 3.82 (s, 3H, OCH.sub.3), 4.94 (s, 2H, OCH.sub.2), 6.81 (d, J=15.6 Hz, 1H, CHCHCO), 7.10 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.16 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.26 (d, J.sub.5,6=8.0 Hz, 1H, H6), 7.39 (s, 1H, H2), 7.57 (d, J=15.6 Hz, 1H, CHCHCO), 7.61 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 8.00 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.63 (d, J.sub.5,6=8.0 Hz, 1H, H6), 11.31 (s, 1H, NH), 13.62 (br s, 1H, CO.sub.2H); .sub.C (100 MHz, DMSO-d.sub.6) 10.3, 11.2, 56.4, 60.5, 111.0, 111.6, 114.9, 117.3, 121.0, 123.0, 123.3, 128.8, 131.8, 134.6, 141.7, 142.2, 149.8, 150.3, 160.3, 164.8, 168.2, 170.1; HRMS (ESI) calculated for C.sub.23H.sub.22N.sub.2O.sub.6 [M+Na].sup.+ 445.1370. found 445.1369; .sub.max 1141, 1256, 1511, 1584, 1665, 2940, 3326 cm.sup.1.

(E)-2-[[3-(3-((Diethylamino)methyl)-4-methoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (51)

(207) ##STR00107##

(208) Piperidine (97 L, 0.99 mmol) was added to a suspension of 3-((diethylamino)methyl)-4-methoxybenzaldehyde (0.22 g, 0.99 mmol) and 2-[(carboxyacetyl)amino]benzoic acid (0.20 g, 0.90 mmol) in toluene (5 mL) and treated according to Procedure 2, neutralizing with 20% AcOH. The resulting precipitate was filtered and washed with water providing (E)-2-[[3-(3-((diethylamino)methyl)-4-methoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (0.17 g, 50%) as a colourless crystalline solid; mp 202-205 C.; .sub.H (400 MHz, DMSO-d.sub.6) 1.14 (t, J=7.2 Hz, 4H, CH.sub.2CH.sub.3), 2.87 (q, J=7.2 Hz, 6H, CH.sub.2CH.sub.3), 3.79 (s, 3H, OCH.sub.3), 3.98 (s, 2H, NCH.sub.2), 6.51 (d, J=15.6 Hz, 1H, CHCHCO), 6.98 (t, J.sub.3,4=J.sub.4,5=8.0 Hz, 1H, H4), 7.03 (d, J.sub.5,6=8.0 Hz, 1H, H5), 7.34 (t, J.sub.4,5=J.sub.5,6=8.0 Hz, 1H, H5), 7.46 (d, J=15.6 Hz, 1H, CHCHCO), 7.57 (dd, J.sub.5,6=8.0, J.sub.2,6=1.6 Hz, 1H, H6), 7.76 (d, J.sub.2,6=1.6 Hz, 1H, H2), 7.98 (d, J.sub.3,4=8.0 Hz, 1H, H3), 8.54 (d, J.sub.5,6=8.0 Hz, 1H, H6), 13.34 (br s, 1H, CO.sub.2H); .sub.C (100 MHz, DMSO-d.sub.6) 9.38, 46.4, 49.8, 55.6, 111.4, 118.9, 121.2, 121.5, 122.1, 126.9, 130.2, 130.8, 130.9, 139.2, 140.7, 158.8, 163.3, 169.7; HRMS (ESI) calculated for C.sub.22H.sub.26N.sub.2O.sub.4 [M+H].sup.+ 383.1965. found 383.1964; .sub.max 823, 1264, 1366, 1500, 1579, 1609, 1674, 2943, 3478 cm.sup.1.

(209) Comparative Structures

(210) TABLE-US-00001 Formula and FT molecular no. Structure weight 001 08 embedded image C.sub.18H.sub.17NO.sub.5 327.33 002 09 C.sub.16H.sub.13NO.sub.3 267.28 003 0 C.sub.17H.sub.15NO.sub.4 297.31 004 C.sub.17H.sub.15NO.sub.4 297.31 005 C.sub.16H.sub.13NO.sub.5 299.28 006 C.sub.17H.sub.15NO.sub.5 313.30 007 C.sub.17H.sub.15NO.sub.5 313.30 008 C.sub.22H.sub.19NO.sub.5 377.39 009 embedded image C.sub.20H.sub.21NO.sub.7 387.38 010 C.sub.18H.sub.19NO.sub.5 329.35

(211) TABLE-US-00002 Formula and FT no. Structure molecular weight 011 embedded image C.sub.20H.sub.17NO.sub.5 351.35 012 C.sub.18H.sub.18N.sub.2O.sub.4 326.35 013 0 C.sub.17H.sub.17NO.sub.3 283.32 014 C.sub.18H.sub.17NO.sub.5 327.33 015 C.sub.21H.sub.20N.sub.2O.sub.4 364.39 016 C.sub.18H.sub.16BrNO.sub.5 406.23 017 C.sub.20H.sub.17NO.sub.5 353.35 018 C.sub.22H.sub.21NO.sub.5 379.41 019 embedded image C.sub.22H.sub.21NO.sub.5 379.41 020 C.sub.28H.sub.25N.sub.5O.sub.6 527.53 021 C.sub.28H.sub.25N.sub.5O.sub.6 527.53 022 C.sub.21H.sub.19NO.sub.5 365.38 023 0 C.sub.21H.sub.19NO.sub.5 365.38 026 C.sub.22H.sub.23NO.sub.5 381.42 027 C.sub.23H.sub.25NO.sub.5 395.45 028 C.sub.24H.sub.27NO.sub.5 409.47 029 embedded image C.sub.22H.sub.23NO.sub.5 381.42 032 C.sub.29H.sub.28N.sub.6O.sub.5 540.57 033 C.sub.23H.sub.23NO.sub.5 393.43 034 C.sub.23H.sub.23NO.sub.5 393.43 035 C.sub.22H.sub.21NO.sub.5 379.41 036 C.sub.22H.sub.21NO.sub.5 379.41 037 0 C.sub.21H.sub.19NO.sub.5 365.38 038 C.sub.21H.sub.19NO.sub.5 365.38 039 embedded image C.sub.23H.sub.23NO.sub.5 393.43 040 C.sub.23H.sub.23NO.sub.5 393.43 041 C.sub.25H.sub.27NO.sub.5 421.49 042 C.sub.25H.sub.27NO.sub.5 421.49 043 C.sub.24H.sub.21NO.sub.5 403.43 044 C.sub.28H.sub.23NO.sub.5 453.49 045 C.sub.22H.sub.25NO.sub.5 383.44 046 C.sub.16H.sub.14N.sub.2O.sub.4 298.29 047 0 embedded image C.sub.29H.sub.31NO.sub.6 489.56 048 C.sub.23H.sub.26N.sub.2O.sub.6 426.46 049 C.sub.23H.sub.20N.sub.2O.sub.5 404.42 050 C.sub.23H.sub.22N.sub.2O.sub.6 422.43 051 C.sub.22H.sub.26N.sub.2O.sub.4 382.45

Proposed Compounds

(212) TABLE-US-00003 embedded image 0 0 0 0 00 01 02 03 04 05 06 07 08 09 0

Example 2

Cell Culture Studies

Transforming Growth Factor- Stimulation

(213) The anti-fibrotic effects of 3-methoxy-4-propargyloxybenzaldehyde (FT011) in a renal cell line were tested by measuring proline incorporation after transforming growth factor- stimulation.

(214) A well-characterized cloned mesangial cell line (1097) isolated from Sprague-Dawley rats [30] was used between passages 30 and 40. Cells were cultured in Dulbecco's Modified Eagle's (DME) medium (Invitrogen, Grand Island, N.Y.) with heat-inactivated fetal bovine serum (FBS), 100 u/mL penicillin and 100 ug/mL streptomycin in a humidified 5% CO.sub.2 atmosphere at 37 C.

(215) To compare the effects of tranilast and FT011 on collagen production in vitro, incorporation of tritiated proline was used [40]. Mesangial cells were plated at low density into 24-well culture plates in DME/5% FBS and allowed to adhere overnight. The subconfluent cells were starved overnight in DME/0.5% FBS and 150 mM L-ascorbic acid (Sigma-Aldrich). Tranilast or FT011 was then added to the wells, followed 4 hours later by L-[2,3,4,5-.sup.3H]-proline, 0.5 Ci/well (Amersham) and TGF-1, 5 ng/ml (R & D systems). Mesangial cells were harvested 48 hours post-stimulation, washed three times with ice cold PBS, and incubated with 10% trichloroacetic acid (TCA) for 30 minutes on ice, followed by a wash in ice cold 10% TCA. Cells were then solubilised in 750 ml 1M NaOH. Scintillation counting was performed on 500 mL aliquots of solubilized cells neutralized with 500 mL of 1M HCl in 10 mL of Instagel Plus scintillant (Perkin-Elmer, Boston, Mass.).

(216) The data shown in FIGS. 1 and 2 suggest that in renal mesangial cells, both tranilast and FT011 significantly reduce proline incorporation from 30 to 100 M. The degree of proline incorporation in vitro relates to the degree on fibrosis in vivo.

Example 3

(217) Matrix synthesis may be stimulated by platelet derived growth factor (PDGF). Accordingly, mesangial cells incubated with PDGF will demonstrate proline incorporation, which is an indicator of matrix synthesis and thereby a model for fibrosis.

(218) In order to assess the effect of FT011 on PDGF stimulated matrix synthesis, mesangial cells (prepared as described in Example 2) were incubated with FT011 or tranilast in the presence of PDGF. The results of this analysis were provided in FIG. 3. As shown in FIG. 3, FT011 inhibits PDGF-stimulated matrix synthesis (shown by reduced proline incorporation) at 30 and 100 M concentrations. At 30 M concentrations, FT011 is more potent at reducing proline incorporation than tranilast.

Example 4

(219) Matrix synthesis may be stimulated by both angiotensin II or transforming growth factor beta (TGF-). Accordingly, neonatal cardiac fibroblasts incubated with angiotensin II or TGF- will demonstrate proline incorporation, which is an indicator of matrix synthesis and thereby a model for fibrosis.

(220) Neonatal SD rat cardiac fibroblasts (NCFs) were isolated from one-day-old pups with enzymatic digestion. NCFs were purified by percoll gradient and seeded with DMEM in the present of 1% antibiotic/antimycotic (AB/AM) and 10% fatal bovine serum (FBS). NCFs were then subcultured when they are about 80% confluence. The second passage of NCFs was used for the assays.

(221) NCFs were seeded at 25,000 cells/well in 12-well plates and incubated at 37 C. and 5% CO.sub.2 overnight in DMEM with 1% AB/AM and 10% FBS. Cells were then washed with DMEM and then the media replaced with DMEM/F12 with 1% AB/AM, 0.5% Bovine Serum Albumin (BSA) and Vitamin C, before being incubated at 37 C. and 5% CO.sub.2 for 24 hours.

(222) The effect of FT011 on TGF-- or angiotensin II-stimulated fibrosis in the neonatal SD rat cardiac fibroblasts was investigated. As shown in FIG. 4, FT011 inhibited TGF--stimulated fibrosis (indicated by proline incorporation) in rat neonatal cardiac fibroblasts. As shown in FIG. 3, FT011 inhibited angiotensin II-stimulated fibrosis (indicated by proline incorporation) in neonatal cardiac fibroblasts.

Example 5

FT Compounds in Renal Mesangial Cells, and Neonatal Cardiac Fibroblasts

(223) Methodology

(224) Proline Incorporation:

(225) A well-characterized cloned rat mesangial cell line [30] (gift of D Nikolic-Patterson) is cultured in DMEM with FBS, 100 U/mL penicillin, and 100 ug/mL streptomycin in a humidified 5% CO.sub.2 atmosphere at 37 C. Cells are plated into 24-well culture dishes in DMEM/10% FBS at low density and allowed to adhere overnight. Cells are used between passages 20 and 40. The subconfluent cells are starved overnight in DMEM/0.1% FBS containing 150 uM L-ascorbic acid, prior to 4 hours of pre-treatment with or without tranilast or the FT compounds, followed by the addition of 5 ng/mL rhTGF-.sub.11 (R&D Systems) and 1 uCi/mL of L-(2,3,4,5-.sup.3H)-proline. Control wells have the compounds but no TGF-.sub.1 added. Cells are incubated for a further 44 hours during which time their appearance is visually monitored. The cells are then washed three times in ice-cold PBS, twice in ice cold 10% TCA and solubilized in 750 uL 1M NaOH for 45 minutes at 37 C. or overnight at 4 C. A 500 uL aliquot is neutralized with 500 uL 1M HCl and 10 mL scintillation fluid (Instagel Plus-Perkin-Elmer) added. Counts are performed on a beta counter.

(226) To normalize the proline incorporation counts to take into account the proliferative effects of TGF-.sub.1, a BioRad protein assay is performed on a 100-150 uL aliquot of the remaining solubilized cells. The sample is neutralized with an equal amount of 1M HCl prior to the assay. The BSA standards used to construct the standard curve have the same amount of 1M NaOH and 1M HCl added as is present in the samples for assay.

(227) Proline incorporation is expressed as cpm/ug protein. In order to compare inter-assay results, the incorporation is expressed as percentage reduction of TGF stimulated proline incorporation, where TGF alone gives 0% reduction and the zero control gives 100% reduction.

(228) MTT Assay:

(229) Mesangial cells are plated at 15000 cells per well into 96-well culture dishes in DMEM/10% FBS and allowed to adhere overnight. The subconfluent cells are starved overnight in DMEM/0.1% FBS, prior to 4 hours of pre-treatment with or without tranilast or the FT compounds. Following the addition of 5 ng/mL rhTGF-.sub.1, the cells are incubated in a humidified 5% CO.sub.2 atmosphere at 37 C. for 44 hours. Control wells have the compounds but no TGF-.sub.1 added. The culture medium is removed from each well and 100 uL MTT (0.5 mg/mL) in starve medium is added to each well. The plates are incubated for a further 4 hours at 37 C. The culture medium is then removed and replaced with 100 L isopropanol and incubated at 37 C. for 20 to 30 minutes, until the blue formazan crystals have dissolved. The absorbance is measured at a wavelength of 570 nm with background subtraction of 690 nm.

(230) Compounds in Bold have Minimal Effect on Cell Appearance and Viability

(231) Suppressed MTT Result Indicates Reduced Cell Viability

(232) TABLE-US-00004 Mesangial Cell Effect on Mesangial Assay TGF- mesangial cell MTT Formula % reduction of TGF- Collagen cell Assay Analogue Mol. Wt. stimulation_proline inc. synthesis appearance TGF- 1 Tranilast ~20-50% @ 30 m =T OK, some C.sub.18H.sub.17NO.sub.5 50-70% @ 100 M stress @ 327.33 100 M 11 C.sub.20H.sub.17NO.sub.5 15% @ 3 M >T OK, some 351.35 20-50% @ 10 M stress @ 50-75% @ 30 M 100 M 60-100% @ 100 M 16 C.sub.18H.sub.16BrNO.sub.5 ~20% @ 3 M >>FT11 OK, lifting off Suppressed 406.23 ~50% @ 10 M @ 10 M @ 30 M 17 C.sub.20H.sub.17NO.sub.5 ~20-50% @ 10 m >=FT11 OK OK 353.35 ~55% @ 30 M 18 C.sub.22H.sub.21NO.sub.5 ~55% @ 3 m >>FT11 OK, lifting off OK 379.41 ~70% @ 10 M @ 30 M 19 C.sub.22H.sub.21NO.sub.5 ~80% @ 10 M >FT11 Some death with OK 379.41 100% @ 30 M increasing concentration 23 C.sub.21H.sub.19NO.sub.5 ~12% @ 3 M, >=FT11 OK, some OK 365.38 30-60% @ 10 m stress @ 70-80% @ 30 M 100 M ~80% @ 100 M 26 C.sub.22H.sub.23NO.sub.5 ~13% @ 1 M >FT11 OK, sparse OK 381.42 30% @ 3 M @ 30 M 85% @ 10 M, lifting off 93% @ 30 M @ 100 M 27 C.sub.23H.sub.25NO.sub.5 ~18% @ 1 M >FT11 Sparse @ Suppressed 395.45 40% @ 3 M 10 M @ 30 M 75-90% @ 10 M sick @ 30 M lifting off @ 100 M 29 C.sub.22H.sub.23NO.sub.5 ~8% @ 1 M >FT11 Sparse @ Suppressed 381.42 40% @ 3 M 10 & 30 M @ 30 M ~90% @ 10 M Stressed @ 30 & 100 M 33 C.sub.23H.sub.23NO.sub.5 ~15% @ 1 M >FT11 Looking bad Severely 393.437 40% @ 3 M @ 30 M Suppressed 60% @ 10 M @ 10 M 80% @ 30 M 34 C.sub.23H.sub.23NO.sub.5 ~80% @ 10 M >FT11 Looking bad Suppressed 393.437 94% @ 30 M @ 30 M @ 30 M 35 C.sub.22H.sub.21NO.sub.5 ~25% @ 3 m =>FT11 Looking bad Suppressed 379.41 50% @ 10 M @ 30 M @ 10 M 60-100% @ 30 M 36 C.sub.22H.sub.21NO.sub.5 ~50% @ 10 M =>FT11 Crystals or Severely 379.41 80% @ 30 M dead cells Suppressed @ 30 M @ 30 M 39 C.sub.23H.sub.23NO.sub.5 ~70% @ 10 M >FT11 Looking bad OK 393.43 100% @ 30 M @ 30 M 40 C.sub.23H.sub.23NO.sub.5 ~70% @ 10 M >FT11 OK, dead @ OK 393.43 100% @ 30 M 100 M
Mesangial Cells

(233) TABLE-US-00005 Derivatives for structure activity analysis (26/02/07): (NT = not tested) N.B suppressed MTT result indicates reduced cell viability Mesangial Cell Effect on Mesangial Assay TGF- mesangial cell MTT Formula % reduction of TGF Collagen cell Assay Analogue Mol. Wt. stimulation_proline inc. Synthesis appearance TGF- 2 C.sub.16H.sub.13NO.sub.3 <<T OK NT 267.28 3 C.sub.17H.sub.15NO.sub.4 <<T OK NT 297.31 4 C.sub.17H.sub.15NO.sub.4 <<T OK NT 297.31 5 C.sub.16H.sub.13NO.sub.5 May be toxic Cells looking NT 299.28 bad & sparse 6 C.sub.17H.sub.15NO.sub.5 20% @ 10 M <=T OK NT 313.30 45% @ 50 M 7 C.sub.17H.sub.15NO.sub.5 ~50% @ 10 M > or =T cells NT 313.30 80% @ 50 M stressed 8 C.sub.22H.sub.19NO.sub.5 Toxic Heavily NT 377.39 vacuolated 9 C.sub.20H.sub.21NO.sub.7 <<T NT 387.38 10 C.sub.18H.sub.19NO.sub.5 <<T NT 329.35 12 C.sub.18H.sub.18N.sub.2O.sub.4 ~13% @ 10 M <FT11 OK, cells NT 326.35 20% @ 30 M sparse @ 36% @ 100 M 100 M 13 C.sub.17H.sub.17NO.sub.3 ~20% @ 30 M <FT11 OK, cells NT 283.32 ~55% @ 100 M sparse @ 100 M 14 C.sub.18H.sub.17NO.sub.5 ~30% @ 100 M <FT11 OK NT 327.33 15 C.sub.21H.sub.20N.sub.2O.sub.4 ~24% @ 10 M <FT11 OK, cells NT 364.39 36% @ 30 M sparse @ 30 75% @ 100 M & 100 M 20 C.sub.28H.sub.25N.sub.5O.sub.6 30% @ 10 M =FT11 OK, dead @ OK 527.53 50% @ 30 M 100 M 90% @ 100 M 21 C.sub.28H.sub.25N.sub.5O.sub.6 ~20% @ 10 M =FT11 OK, necrotic Suppressed 527.53 ~65% @ 30 M @ 100 M @ 30 M 22 C.sub.21H.sub.19NO.sub.5 ~37% @ 3 M =FT11 OK, dead @ OK 365.38 56% @ 10 M 100 M ~70% @ 30 M 24 C.sub.39H.sub.42N.sub.6O.sub.8 ~44% @ 10 M =FT11 OK, dead @ NT 722.79 68% @ 30 M 100 M 25 C.sub.38H.sub.42N.sub.6O.sub.7S ~70% @ 10 M =FT11 dissolved NT 726.84 @ 30 M 28 C.sub.24H.sub.27NO.sub.5 ~40% @ 10 M <FT11 Stressed @ deadly 409.47 173% @ 30 M 30 M, 10 M Dead @ 100 M 30 C.sub.40H.sub.45N.sub.7O.sub.7 ~28% @ 10 M Toxic Dead @ NT 735.83 50-70% @ 30 M 30 M 31 C.sub.39H.sub.45N.sub.7O.sub.6S ~50% @ 10 M Toxic Dead @ NT 739.88 30 M 32 C.sub.29H.sub.28N.sub.6O.sub.5 ~26% @ 10 M Toxic Dead NT 540.57 62% @ 30 M 37 C.sub.21H.sub.19NO.sub.5 ~50% @ 10 M =FT11 Some death OK 365.38 70% @ 30 M @ 100 M 38 C.sub.21H.sub.19NO.sub.5 ~40% @ 10 M =FT11 Some death OK 365.38 65% @ 30 M @ 100 M 41 C.sub.25H.sub.27NO.sub.5 Toxic Cells damaged @ NT 421.49 all concentrations 42 C.sub.25H.sub.27NO.sub.5 Toxic Cells damaged @ NT 421.49 all concentrations 43 C.sub.24H.sub.21NO.sub.5 48% @ 10 M Sick @ NT 403.43 70% @ 30 M 30 & 100 M 44 C.sub.28H.sub.23NO.sub.5 Toxic Cells damaged @ NT 453.49 all concentrations 45 C.sub.22H.sub.25NO.sub.5 80% @ 10 M >FT11 Cells damaged NT 383.44 142% @ 30 M @ 100 M 46 C.sub.16H.sub.14NO.sub.4 <<FT11 OK NT 298.29

Example 6

Methods

(234) Neonatal Rat Cardiomyocytes and Fibroblast Cultures

(235) Neonatal SD rat cardiac myocytes (NCMs) and fibroblasts (NCFs) were isolated from one-day-old pups with enzymatic digestion as described in detail previously [20,21]. NCFs were seeded and maintained in high-glucose (25 mmol/L) Dulbecco's modified Eagle's medium (DMEM) (Invitrogen Mount Waverley, Vic, Australia) in the presence of 1% antibiotic/antimycotic (AB/AM) and 10% fetal bovine serum (FBS) (JRH biosciences, Kansas, USA). NCFs were used at passage 2 [31]. Purified NCMs were seeded (1000 cells/mm.sup.2) in 6-well plates and then maintained in serum-free DMEM (Invitrogen, NY, USA) supplemented with insulin and transferrin as described previously (4). Bromodeoxyuridine was included for the first 3 days. 50 mmol/L KCl was added to the medium to prevent spontaneous contraction characteristic of the plated NCMs [32].

(236) Measurement of Neonatal Rat Cardiomyocyte Hypertrophy

(237) NCM hypertrophy studies were performed as previously described [22]. Four hours after treatment with the compounds (concentrations various from 1 to 30 M), ANG II (10.sup.7 mol/L) was used to stimulate hypertrophy. After 60 hours of stimulation, cells were harvested and hypertrophy defined as a significant increase in protein content (Bradford assay) in the absence of any significant change in DNA content (Burton assay) [33].

(238) Measurement of Collagen Synthesis and Proliferation and Cell Viability in Neonatal Rat Cardiac Fibroblasts

(239) NCFs collagen synthesis assays were performed as described previously [31]. Briefly, NCFs plated at a density of 50,000 cells/well in 12-well plate and incubated overnight. NCFs were then serum starved for 24 hours in high-glucose DMEM. The cells were then preincubated for 30 min in the presence or absence of compounds (1 to 30 M) in fresh DMEM/F12 before stimulation with 210.sup.10 mol/L of TGF-.sub.1 or 10.sup.7 mol/L of ANG II.

(240) For collagen synthesis, 1 Ci of [.sup.3H]-Proline was added to each well and incubated for further 48 hours before harvest. Cells were harvested by precipitation with 10% TCA on ice for 30 min, before solubilisation with 0.75 mL of 1 mol/L NaOH overnight at 4 C. The samples were then neutralized with 1 mol/L HCl and .sup.3H level were counted with 10 ml scintillation fluid on a beta-counter to determine [.sup.3H]-proline incorporation.

(241) For proliferation studies, NCFs were treated with 1 mCi of [.sup.3H]-thymidine added to each well 2 hours prior to harvesting. Cells were harvested by TCA precipitation as described for collagen synthesis above determining [.sup.3H]-thymidine incorporation.

(242) Results

(243) Neonatal Cardiac Fibroblasts

(244) TABLE-US-00006 Formula NCF Analogue Mol. Wt. TGF- Collagen Synthesis 1 Tranilast NT C.sub.18H.sub.17NO.sub.5 327.33 11 C.sub.20H.sub.17NO.sub.5 32% @ 30 M 351.35 87% @ 100 M 17 C.sub.20H.sub.17NO.sub.5 ~70% at 353.35 30 M 26 C.sub.22H.sub.23NO.sub.5 28% at 381.42 10 M 29 C.sub.22H.sub.23NO.sub.5 10% @ 10 M 381.42 25% @ 30 M

Example 7

FT011 Treatment Post Myocardial Infarction or Diabetic Nephropathy

(245) FT011 is Anti-Inflammatory and Anti-Fibrotic

(246) Methods

(247) Animals

(248) The animal studies were conducted with the approval of the Animal Welfare and Ethics Committee of the St Vincent's Hospital and the National Health and Medical Research Foundation of Australia. All rats received normal rat chow (Certified Rodent Diet #5002, LabDiet, USA) and drinking water ad libitum. All animals were housed in a stable environment maintained at 221 C. with a 12-hour light/dark cycle commencing at 6 am.

(249) Pilot Study (Toxicity Assessment)

(250) A total of twenty male Sprague-Dawley (SD) rats weighing 200-250 g were randomised to either FT011 (Fibrotech Therapeutics Pty Ltd, Melbourne, Australia) or Tranilast (Pharm Chemical, Shanghai Lansheng Corporation, China) at the dose of 50 mg, 100 mg, 200 mg and 400 mg/kg/day by gavage twice daily (n=2-3 per group). A control group of animals were gavaged with vehicle (1% carboxy methyl cellulose). The study was conducted for 2 weeks. Animals were bled daily at one, four, and eight hours after oral gavage to measure the plasma concentration. Serum was also collected to assess renal and liver function at the end of the study (plasma creatine and urate, ALT and bilirubin). Rats were individually housed in metabolic cages at the end of the study, habituated for 2 to 3 hours, and urine collected over 24 hours. Animals continued to have free access to tap water and standard laboratory chow during this period. The biochemistry tests performed at the department of pathology, St Vincent's hospital. Major organs including lung, heart, liver, spleen and kidney were harvested and immersed fix with 10% neutral buffered formalin and then embedded paraffin for subsequent light microscope examination.

(251) Myocardial Infarct Rats

(252) Forty male SD rats weighing 200-250 g were randomised to two groups of 20 animals that each underwent surgery. Anaesthesia was achieved with 3% isoflurane/97% oxygen in a tidal volume of 1 ml/100 g body weight, at a rate of 72 breaths/minute. Twenty rats underwent ligation of left anterior descending coronary artery (LAD) to induce anterior myocardial infarction (MI). Briefly, surgery performed under aseptic conditions on a heated table (37 C.) to maintain body warmth during the course of the procedure. The chest swabbed with chlorhexidine in 70% ethanol to disinfect the area. An incision made into the skin to the left of the sternum and the underlying muscle layers blunt dissected. A thoracotomy performed through the fourth to fifth intercostal space and the ribs held open using retractors to enable access to the heart. The pericardial sac surrounding the heart torn open and a 6-0 prolene suture used to ligate the LAD immediately. Visible blanching and hypokinesis of the anterior wall of the left ventricle and swelling of the left atrium are indicative of successful ligation. The control groups (sham+vehicle, sham+FT011) underwent a sham operation consisting of the same procedure except that the suture passed through the myocardium beneath the LAD without ligation [34].

(253) Echocardiography was performed on all animals 2 days post surgery and randomised to sham and MI groups. Animals were re-randomised at day 7 post surgery (10 animal each) to receive: vehicle or FT011 (100 mg/kg bid gavage). Every week, systolic blood pressure (SBP) was determined in preheated conscious rats via tail-cuff plethysmography using a non-invasive blood pressure (NIBP) controller and Powerlab (AD instruments, NSW, Australia). Cardiac function was assessed by echocardiography and cardiac catheterization prior to sacrificing at day 35 post surgery for all animals [34].

(254) Diabetic (mRen-2)27 Rats

(255) Forty six-week old female, heterozygous (mRen-2)27 rats (St. Vincent's Hospital Animal House, Melbourne, Australia) were assigned to receive either 55 mg/kg of STZ (Sigma, St. Louis, USA) diluted in 0.1 M citrate buffer, pH 4.5 or citrate buffer alone (non-diabetic) by tail vein injection following an overnight fast. Control and diabetic groups were then each randomised into 2 groups (n=10), receiving either treatment with: the FT 011 (100 mg/kg bid gavage, Fibrotech Therapeutics Pty Ltd, Melbourne, Australia), or no treatment for 16 weeks. Treatment commenced within 24 hours of STZ or citrate buffer injection. Each week, rats were weighed and their blood glucose levels were measured (Accu-check Advantage II Blood Glucose Monitor, Roche Diagnostics, USA) and only STZ-treated animals with blood glucose >15 mmol/L were considered diabetic. Every 4 weeks, systolic blood pressure (SBP) was determined in preheated conscious rats via tail-cuff plethysmography using a non-invasive blood pressure (NIBP) controller and Powerlab (AD instruments, NSW, Australia). Hemoglobin A1c (HbA1c) was measured by HPLC at the end of the study. Diabetic rats received a daily injection of insulin (2-4 units intraperitoneally; Humulin NPH, Eli Lilly and Co., Indianapolis, Ind.) to reduce mortality and to promote weight gain [37, 39].

(256) Heart Function

(257) Echocardiography

(258) Echocardiography, including Doppler examination, was performed using a Vivid 7 Dimension (GE Vingmed, Horten, Norway) echocardiograph with a 10 MHz phased array probe. Electrocardiographic data were acquired simultaneously. End-diastole was defined as the peak of the R wave, and end-systole was defined as the end of the T wave.

(259) Animals were anaesthetized with pentobarbitone sodium 60 mg/kg i.p. injection. Animals underwent echocardiographic interrogation in the left recumbent position. M-mode echocardiography was performed using a parasternal short axis view at the level of the papillary muscles. Left ventricular posterior (LVPW) and anterior wall thickness (LVAW) were obtained during diastole (d) and systole (s), as were the left ventricular internal diameter at end-diastole (LVIDd) and end-systole (LVIDs). From the para-sternal short axis view, the end diastolic and end systolic cross sectional blood pool areas were measured. Fractional area change (FAC) was then calculated according to the formulae:
FAC=[(end-diastolic area-end-systolic area)/end-diastolic area]*100.

(260) The apical 4-chamber view was used to assess early and late transmitral peak diastolic flow velocity (E and A waves), using pulsed wave Doppler with a sample volume of 2 mm placed at the tips of the mitral valve leaflets. All Doppler spectra were recorded for 10 cardiac cycles at a sweep speed of 200 mm/s. All parameters were assessed using an average of three beats, and calculations were made in accordance with the American Society of Echocardiography guidelines [41]. All data were acquired and analyzed by a single blinded observer using EchoPAC (GE Vingmed) offline processing.

(261) Cardiac Catheterization

(262) Post echocardiography, animals were placed on a warming pad (37 C.), intubated using a 14 gauge catheter, and ventilated using positive pressure with a tidal volume of 10% body weight at 70 breaths per minute using room air. Animals were secured in a recumbent position and the right jugular vein was cannulated with 0.9% NaCl infused at 100 L per hour. Pressure was calibrated after warming the catheter (Model SPR-838 Millar instruments, Houston, Tex.) in 0.9% NaCl at in 37 C. for 30 minutes. The right internal carotid was then identified and ligated cranially. A 2F miniaturized combined conductance catheter-micromanometer was inserted into the carotid artery to obtain aortic blood pressure, then advanced into the left ventricle until stable pressure volume (PV) loops were obtained. The abdomen was then opened and the inferior vena cava and portal vein identified. Elastic bands were placed around these vessels to allow rapid reduction in cardiac preload. All loops were obtained with the ventilator turned off for 5-10 seconds and the animal apnoeic.

(263) Using the pressure conductance data, functional parameters were then calculated (Millar analysis software PVAN 3.4). These included the slope of the end diastolic pressure volume relationship (EDPVR) and the slope of the preload recruitable stroke work relationship (PRSW), defined as the relationship between stroke work and end diastolic volume, where stroke work is the pressure-volume loop area for each beat.

(264) Renal Function

(265) Rats were individually housed in metabolic cages at 4, 8 12 and 16 weeks, habituated for 2 to 3 hours, and urine collected over 24 hours. Animals continued to have free access to tap water and standard laboratory chow during this period. After 24 hours in metabolic cages, an aliquot of urine (5 mL) was collected from the 24-hour urine sample and stored at 70 C. for subsequent analysis of albumin by radio-immunoassay, as previously performed [36]. Prior to sacrifice, the glomerular filtration rate (GFR) was determined by injecting a single shot of 99Tc-DTPA into the tail vein and sampling the blood after 43 minutes, as previously described [37].

(266) Tissue Preparation

(267) Rats were anaesthetised (Nembutal 60 mg/kg body wt i.p. Boehringer-Ingelheim, Australia). Lungs, left ventricle (LV), right ventricle (RV) and atria were separated, blotted dry once and weighed, the LV was then sectioned immediately and tissue was either frozen fresh, stored frozen in OCT or fixed in neutral buffered formalin. Kidneys were excised, decapsulated, sliced transversely, half of the kidney snap frozen for tissue RNA assay and other half immersed fix with formalin and paraffin-embedded for subsequent light microscopic evaluation.

(268) Histopathology and Immunohistochemistry

(269) Histopathological changes in kidney and heart were assessed in a masked protocol. Sections were stained with either haematoxylin and eosin (H & E), periodic acid Schiff's stain (PAS), picrosirius red and/or Masson's modified trichrome to demonstrate collagenous matrix.

(270) Infarct Size

(271) The picrosirius red stained slides of heart were examined under light microscopy and digitised, then analysed using image analysis (AIS, Analytical imaging station version 6.0, Ontario, Canada). Infarct sizes assessed morphologically and calculated as the ration of circumferences of the endocardium and the epicardium to LV average circumferences of the endocardium and the epicardium, as previously described.

(272) Glomerulosclerotic Index

(273) In 4 m kidney sections stained with PAS, 150 to 200 glomeruli from rats were examined in a masked protocol. The extent of sclerosis in each glomerulus was subjectively graded on a scale of 0 to 4, as previously described [39] with Grade 0, normal; Grade 1, sclerotic area up to 25% (minimal); Grade 2, sclerotic area 25-50% (moderate); Grade 3, sclerotic area 50-75% (moderate to severe) and Grade 4, sclerotic area 75-100% (severe). A glomerulosclerotic index (GSI) was then calculated using the Formula (4):

(274) $GSI = {.Math.}_{i = 0}^{4} Fi (i)$
where Fi is the % of glomeruli in the rat with a given score (i).
Quantitation of Matrix Deposition

(275) To examine extracellular matrix deposition in heart sections were stained with picrosirius red and the accumulation of matrix within the non-infarct zone (NIZ) was then quantified using a modification of the technique described by Lal et al. (Lal et al., 2004) with a blinded manner. Briefly, 5 random stained sections from the mid left ventricle were digitally captured and then loaded onto a Pentium III IBM computer. To isolate the NIZ from the infarct and the peri-infarct zone, the infarct and a 2 mm zone on either side of it were excluded. To assess the tubulointerstitial fibrosis in kidney sections were stained with modified Masson's trichrome. Briefly, 5 random non-overlapping fields from 10 rats per group were captured and digitised using a BX50 microscope attached to a Fujix HC5000 digital camera. Digital images were then loaded onto a Pentium III IBM computer as described as above. An area of red in heart and blue in kidney on picrosirius red and trichrome-stained section, respectively, were selected for its color range and the proportional area of tissue with this range of color was then quantified. Calculation of the proportional area stained red and blue (matrix) was then determined using image analysis (AIS, Analytical imaging Station Version 6.0, Ontario, Canada) [37, 39].

(276) Immunohistochemistry

(277) Collagen Subtypes I and III

(278) Collagen subtypes I and III were assessed in the heart using goat and mouse anti-Collagen I (Southern Biotechnology Associates, Inc. Birmingham, Ala. 35226 USA) and III antibody (Biogenex, San Ramon Cal, 94583 USA). In brief, four micron sections were placed into histosol to remove the paraffin wax, re-hydrated in graded ethanol, and immersed into tap water before being incubated for 20 minutes with normal goat serum (NGS) diluted 1:10 with 0.1 mol/L PBS, pH 7.4. Sections were incubated respective primary antibodies overnight (18 hours) at 4 C. The following day the sections were thoroughly washed in PBS (35 minute changes), incubated with 3% hydrogen peroxide for 10 minutes to block endogenous peroxide, then rinsed with PBS (25 min), and incubated with either biotinylated swine anti-goat or goat anti mouse IgG antibody (DAKO, Carpinteria Calif.), diluted 1:200 with PBS. Sections were then rinsed with PBS (25 min) followed by incubation with an avidin-biotin peroxidase complex (Vector, Burlingame, Calif.), diluted 1:200 with PBS. Following rinsing with PBS (25 min), localization of the peroxidase conjugates was achieved by using diaminobenizidine tetrahydrochloride as a chromagen, for 1-3 minutes. Sections were rinsed in tap water for 5 minutes to stop reaction and then counterstained in Mayer's haemotoxylin, differentiated in Scott's tap water, dehydrated, cleared and mounted in Depex. Sections incubated with 1:10 NGS, instead of the primary antiserum, served as the negative controls. The accumulation of immunostaining for collagen I and III were quantified using computer-assisted image analysis. Briefly, 10 random non-overlapping fields from 10 rats per group were captured and digitized as described as above. An area of brown on immunostained sections (Collagen I and III) was selected for their color ranges. To correct for variation due to shrinkage, the area of positive immunostaining (collagen/area tissue) relative to the total area (matrix+myocytes) was determined using computer-assisted image analysis (AIS, Analytical imaging Station Version 6.0, Ontario, Canada), as previously reported [40].

(279) Macrophages

(280) Four micron heart sections were placed into histosol to remove the paraffin wax, hydrated in graded ethanol and immersed into tap water before being incubated for 20 minutes with normal goat serum (NGS) diluted 1:10 with 0.1 M PBS at pH 7.4. Sections were then incubated for 18 hours at 4 C. with specific primary monoclonal rat macrophage marker (ED-1, 1:200 Serotec, Raleigh N.C., USA). Macrophage number was estimated by counting the number of macrophages in 10 fields under light microscope with 200 power per animal from each group (n=10 per group) and expressed as numbers per field [38].

(281) Statistics

(282) Data are expressed as meanssem unless otherwise stated. Statistical significance was determined by a two-way ANOVA with a Fishers post-hoc comparison. Albuminuria was skew distributed and was analysed following log transformation and presented as geometric means x/ tolerance factors. Analyses were performed using Statview II+Graphics package (Abacus Concepts, Berkeley, Calif.) on an Apple Macintosh G4 computer (Apple Computer, Inc., Cupertino, Calif.). A p-value <0.05 was regarded as statistically significant.

(283) Results

(284) FT011 Pilot Study (Toxicity Assessment)

(285) In the in vivo pilot study, there was no change in body weight amongst all animal groups (Table 1). Plasma levels of creatinine and urate, ALT and bilirubin were similar to control rats at all doses (Table 1). Following gavage of FT011, the level of FT011 measured in plasma increased in a dose dependent manner (FIG. 1). A significant level of FT011 was also measured in the urine of rats treated with FT011 (FIG. 2).

(286) TABLE-US-00007 TABLE 1 Plasma biochemistry parameters of SD rats Body Plasma Plasma weight Creatinine Urate Bilirubin Group (Gram) (umol/L) (mmol/L) ALT (mmol/L) Control 291 11 39 2.7 <0.09 42 2 1.8 0.16 Tranilast 315 19 43 0.5 <0.09 40.5 4.5 2 0 50 mg/kg/day FT011 313 24 38 2 <0.09 41 1 1 0 50 mg/kg/day Tranilast 298 21 43 3.5 <0.09 41 1 2 0 100 mg/kg/day FT011 308 22 39 0.5 <0.09 44 2 1.5 0.5 100 mg/kg/day Tranilast 293 10 45 1 <0.09 41 4.6 2.33 0.33 200 mg/kg/day FT011 301 8 37 0.6 <0.09 36 0.5 1.33 0.33 200 mg/kg/day Tranilast 314 13 49 7.8 <0.09 40 5 1.66 0.33 400 mg/kg/day FT011 264 29 36 3.9 <0.09 32 4.4 1.66 0.33 400 mg/kg/day
Post-Myocardial Infarct in Rats Treated with FT011
Animal Characteristics

(287) In rats post myocardial infarct, RV and lung:body weight ratio was increased. The increase in lung:body weight ratio, a marker of pulmonary edema secondary to left heart failure was significantly reduced with FT011 treatment (Table 2).

(288) TABLE-US-00008 TABLE 2 Animal parameters of SD rats Group BW (g) LV/BW RV/BW Lung/BW Sham 378 11 0.19 0.004 0.04 0.001 0.33 0.015 Sham + Vehicle 377 6 0.2 0.003 0.045 0.001 0.27 0.045 MI + Vehicle 424 6* 0.21 0.003 0.056 0.001* 0.39 0.016* MI + FT011 382 6 0.22 0.004 0.048 0.002 .sup.0.32 0.011.sup.# *P < 0.05 versus shams and .sup.#p < 0.05 versus MI + Vehicle.
Cardiac Structure

(289) Myocardial infarct size was similar in the treated and untreated MI groups (FIG. 23; P=0.36). In comparison with sham rats, MI rats displayed increased interstitial fibrosis as evidenced by Masson's trichrome staining (FIGS. 24 and 25) and a greater abundance of interstitial cardiac fibrillar collagenous subtype I and III within the non-infarct zone (NIZ, FIGS. 26 and 27). MI animals also showed evidence of an increase in the infiltration of macrophages in NIZ (FIGS. 28 and 29). All of these changes were attenuated by FT011 treatment, indicative of a diminution in adverse remodelling post-MI.

(290) Echocardiography

(291) Following MI, over the 5 weeks duration of the study, echocardiography demonstrated the hallmarks of adverse LV remodelling including: ventricular dilatation, as evidenced by an increase in LVIDd and LVIDs; impaired systolic and diastolic function as evidenced by a reduction in percentage of fractional area change (FAC), and an increased E:A ratio and deceleration time, respectively (Table 3). All of these changes were significantly attenuated by FT011 treatment (Table 3).

(292) In Vivo Pressure Volume Loop Analysis

(293) Pressure volume loop analysis was used to assess both load-sensitive and load-insensitive measures of systolic and diastolic function.

(294) The preload recruitable stroke work index, used to assess systolic function, was significantly reduced in the MI animals when compared to sham (p<0.05). Treatment with FT011 preserved systolic function (P<0.05) in the MI animals. (Table 3)

(295) Chamber compliance, measured by the slope of the end diastolic pressure volume relationship was increased in the MI animals when compared to sham, indicating impaired diastolic function. Treatment with FT011 restored compliance in the MI animals to levels comparable with sham (FIG. 30).

(296) TABLE-US-00009 TABLE 3 Echocardiographic and pressure volume loop parameters of SD rats Deceleration PRSW slop EDPVR slop Group FAC % LVED (ml) LVES (ml) LVIDd (cm) LVIDs (cm) E:A ratio time (ms) (mmHg/ul) (mmHg/sec) Sham + Vehicle 66.40 1.52 0.43 0.04 0.08 0.01 0.83 0.02 0.46 0.03 2.45 0.17 34.75 1.49 72.77 6.70 0.66 0.19 MI + Vehicle 27.56 1.2** 0.90 0.08.sup.# 0.48 0.04** 0.99 0.03.sup.# 0.81 0.03** 3.46 0.52 50.09 1.78.sup.# 53.49 4.66* 0.86 0.15 MI + FT011 42.69 2.3** 0.53 0.04.sup.# 0.15 0.02** 0.92 0.03 0.70 0.03* 2.00 0.26* 38.64 1.2** 65.15 3.04* 0.51 0.05.sup. *P < 0.05, .sup.#p < 0.01, **p < 0.0001 and .sup.P = 0.06 (MI + Vehicle compared to Sham + Vehicle and MI + FT011 compared to MI + Vehicle). PRSW = preload recruitable stroke work; EDPVR = end diastolic pressure volume relationship.
Diabetic (mRen-2)27 Rats Treated with FT011
Animal Characteristics

(297) Diabetic rats had reduced body weight and were all equally hyperglycaemic Table 4). Diabetic rats had increased albuminuria and FT011 significantly attenuated the rise in albuminuria (FIG. 11)

(298) TABLE-US-00010 TABLE 4 Animal characteristic of Ren-2 rats Body Weight Plasma Glucose Group (Gram) (mmol/L) GFR (ml/min) Control 294 11 5 0.2 3.77 0.23 Control + FT011 309 7 7 0.2 3.63 0.08 Diabetic 281 22 33 0.2* 5.33 0.47* Diabetic + FT011 278 12 30 1.5* 5.90 0.20* *p < 0.01 when compared to control

CONCLUSION

(299) The above results would suggest that treatment with FT011 may provide a potential in disease or conditions characterised by inflammation and/or benign or malignant neoplastic diseases.

(300) It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

REFERENCES

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