Uncharged isocoumarin-based inhibitors of urokinase-type plasminogen activator
© Heynekamp et al; licensee BioMed Central Ltd. 2006
Received: 24 May 2005
Accepted: 08 February 2006
Published: 08 February 2006
Urokinase-type plasminogen activator (uPA) plays a major role in extracellular proteolytic events associated with tumor cell growth, migration and angiogenesis. Consequently, uPA is an attractive target for the development of small molecule active site inhibitors. Most of the recent drug development programs aimed at nonpeptidic inhibitors targeted at uPA have focused on arginino mimetics containing amidine or guanidine functional groups attached to aromatic or heterocyclic scaffolds. There is a general problem of limited bioavailability of these charged inhibitors. In the present study, uPA inhibitors were designed on an isocoumarin scaffold containing uncharged substituents.
4-Chloro-3-alkoxyisocoumarins were synthesized in which the 3-alkoxy group contained a terminal bromine; these were compared with similar inhibitors that contained a charged terminal functional group. Additional variations included functional groups attached to the seven position of the isocoumarin scaffold. N- [3-(3-Bromopropoxy)-4-chloro-1-oxo-1H-isochromen-7-yl]benzamide was identified as an uncharged lead inhibitor of uPA, Ki = 0.034 μM. Molecular modeling of human uPA with these uncharged inhibitors suggests that the bromine occupies the same position as positively charged arginino mimetic groups.
This study demonstrates that potent uncharged inhibitors of uPA can be developed based upon the isocoumarin scaffold. A tethered bromine in the three position and an aromatic group in the seven position are important contributors to binding. Although the aim was to develop compounds that act as mechanism-based inactivators, these inhibitors are competitive reversible inhibitors.
Multiple proteases, including matrix metalloproteases (MMP-2, MMP-9 and MMP-14), cysteine proteases (cathepsin B and cathepsin L), aspartyl protease (cathepsin D) and serine proteases (plasmin, matriptase and urokinase) participate in cancer cell growth, metastasis and angiogenesis [1–4]. High expression of proteases often correlates with a poor prognosis [5, 6]. Urokinase (uPA) plays an especially important role in extracellular proteolysis that contributes to cancer cell metastasis. Many cancer cells secrete pro-uPA and its receptor uPAR; binding of pro-uPA to uPAR leads to its activation, with subsequent generation of plasmin by the uPA-catalyzed hydrolysis of extracellular plasminogen [7, 8]. The increased production of plasmin leads to degradation of extracellular matrix both by plasmin itself and by other proteases that are activated by plasmin. The surface location of bound uPA provides directionality to the degradation of matrix, thereby assisting the directional migration of cancer cells. uPA in complex with uPAR also affects other biological processes including signaling pathways that influence cell proliferation . uPA has become a major target for development of non-peptidic small molecule inhibitors as potential anti-cancer drugs [10, 11].
In the present study, we have focused on the synthesis and testing of uncharged compounds as leads for the development of uPA inhibitors with improved bioavailability. 4-Chloroisocoumarin was selected as the scaffold, in which substituted 3-alkoxy groups were introduced that contained neutral terminal functional groups or charged terminal functional groups . Additional substituents were introduced into the seven position. 4-Chloroisocoumarin scaffolds have been used in studies of serine protease inhibitors,  but with limited application to uPA . The choice of the 4-chloroisocoumarin scaffold was based upon the potential of these compounds to function as mechanism-based inactivators . In this study we demonstrate that introduction of bromine in place of a terminal charged functional group in the 3-alkoxy substituent provides uncharged uPA inhibitors with low micromolar dissociation constants. Further introduction of substituents at the seven position of these uncharged uPA inhibitors provides compounds with low nanomolar dissociation constants. These inhibitors may serve as lead compounds for the development of new uPA inhibitors. Molecular modeling with human uPA suggests that the bromine occupies the same site as the arginino mimetic functional groups.
Results and discussion
Compounds 4a-4e, which are 3-bromoalkoxy-4-chloroisocoumarins, were synthesized as shown in Figure 1. Two of the compounds, 4a and 4b, have a nitro group in position seven. These compounds have varying lengths of bromoalkoxy groups tethered in position three of the isocoumarin scaffold. 5-Nitrohomophthalic acid (1a) was prepared by regioselective nitration of homophthalic acid (1c) using fuming nitric acid . 5-Nitrohomophthalic acid (1a) and homophthalic acid (1c) were monoesterified using bromoalcohols, compounds 2c-2e, in the presence of sulfuric acid to give moderate yields of bromoesters, 3a-3e. Monoesterification at the saturated acid has been attributed to the mesomeric effect of the carboxyl with the double bond in the aryl ring . Cyclization of the esters, compounds 3a-3e, with phosphorus pentachloride in toluene gave 3-bromoalkoxy-4-chloroisocoumarins, 4a-4e, in moderate yields using a variation of a published method . Compounds 4a-4e were synthesized to test the importance of an uncharged group in the three position of 4-chloroisocoumarins and the length of the tether of the alkoxy group.
Compounds 5c-5e are 3-isothioureidoalkoxy-4-chloroisocoumarin salts and were synthesized as shown in Figure 1. Nucleophilic substitution of the bromine in compounds 4c-4e was achieved by refluxing these compounds with thiourea in tetrahydrofuran to give the hydrobromide salts, compounds 5c-5e, in moderate yields. The 7-nitrosubstituted isocoumarins, compounds 4a and 4b, did not give any identifiable products when reacted with thiourea in tetrahydrofuran. Compounds 5c-5e were synthesized to test the importance of a charged alkoxy group in the three position of 4-chloroisocoumarins.
The synthesis of 3-bromo-4-chloro-7-aminoisocoumarins, compounds 6a and 6b, is shown in Figure 2. These 7-aminoisocoumarins were prepared by reduction of 3-bromoalkoxy-4-chloro-7-nitroisocoumarins, compounds 4a and 4b, using hydrogen in the presence of a catalytic amount of 10% palladium on charcoal under pressure in methanol. Compounds 6a and 6b were synthesized to test the importance of an amino group in the seven position and an uncharged alkoxy group in the three position of 4-chloroisocoumarins. Nucleophilic substitution of the bromine in compounds 6a and 6b by reaction with thiourea afforded hydrobromide salts, 7a and 7b. Compounds 7a and 7b were synthesized to test the importance of a charged alkoxy group in the three position of 4-chloro-7-aminoisocoumarins.
3-Bromoalkoxy-4-chloro-7-benzamidoisocoumarins, compounds 8a and 8b, were synthesized by reaction of 6a and 6b with benzoyl chloride in the presence of triethylamine (Figure 2). Compounds 8a and 8b were synthesized to test the importance of an uncharged alkoxy group in the three position and a hydrophobic benzamide group in the seven position of 4-chloroisocoumarins. The two amides, 8a and 8b were reacted with thiourea to give the hydrobromide salts, 9a and 9b, in moderate yields. Compounds 9a and 9b were synthesized to test the importance of a charged alkoxy group in the three position and a hydrophobic benzamide group in the seven position of 4-chloroisocoumarins.
Figure 3 describes the synthesis of two 7-nitro-3-alkoxyisocoumarins, compounds 10a and 10b, which do not have a chlorine atom at position four. The synthesis of compound 10c, which is a 3-alkoxy-4-trifluoroacetylisocoumarin, is also described in Figure 3. Cyclization of compounds 3a and 3b, which have a nitro group in position seven, using trifluoroacetic anhydride gave compounds 10a and 10b, which contain a hydrogen at position four. On the other hand, cyclization of 3c which does not contain a nitro group in position seven gave compound 10c, which contains a trifluoroacetyl group in position four. This may be attributed to the fact that the intramolecular cyclization of the initially formed enol is faster when there is resonance stabilization by the electron withdrawing nitro group. When the nitro group is absent the initially formed enol reacts with trifluoroacetic anhydride giving compound 10c. Compounds 10a and 10b were synthesized to test the importance of a chlorine atom in position four and the presence of an electron withdrawing group in position seven of the isocoumarins. Compound 10c gives information on the importance of a trifluoroacetyl group in position four of the isocoumarins.
Compound 11, a 7-amino-3-alkoxyisocoumarin, was prepared by reduction of compound 10a using hydrogen in the presence of a catalytic amount of 10% palladium on charcoal under pressure in methanol (Figure 3). Compound 11 was synthesized to test the importance of a chlorine atom in position four and the presence of an electron donating group in position seven of the isocoumarins.
Structure activity relationships
On the basis of docking studies using the crystal structure of human uPA  we examined whether the 4-chloroisocoumarin scaffold containing 3-alkoxy substituents is predicted to be a good template for the design of uPA inhibitors. Our interest focused on compounds having an uncharged bromine group in place of the charged arginino mimetic group at the terminal position of the 3-alkoxy group. Specifically, we compared the experimentally determined dissociation constants with the docking orientations predicted for compounds with the isocoumarin scaffold containing a charged isothiourea group or an uncharged bromine atom in the terminal 3-alkoxy position.
Dissociation constants for inhibition of human uPA
1.8 ± 0.2
0.020 ± 0.003
2.4 ± 0.2
0.038 ± 0.005
14 ± 1
1.4 ± 0.2
9 ± 1
0.034 ± 0.002
18 ± 2
0.084 ± 0.007
0.033 ± 0.004
0.010 ± 0.008
0.055 ± 0.003
4.2 ± 0.4
0.14 ± 0.02
4.3 ± 0.3
12 ± 1
14 ± 2
9.5 ± 1
65 ± 9
The presence of a 7-nitro group also was beneficial. Compounds 4a and 4b, which are 3-bromoalkoxy-7-nitroisocoumarins, showed improved binding to uPA compared to the unsubstituted compounds. Compound 4b which has three methylene units between the bromine atom and the oxygen of the 3-alkoxy group has a Ki = 2.4 μM, which is an 4-fold improvement over the unsubstituted compound 4d.
Compounds 10a, 10b and 11 were synthesized to determine whether the chlorine atom in position four contributed to binding. 3-Bromoalkoxy-7-substituted isocoumarins without a chlorine atom in the four position show dissociation constants that are about 2 fold higher compared to their counterparts that have a chlorine atom in the four position suggesting a modest role for the chlorine atom (Table 1). Compound 10c which has trifluoroacetyl group in the four position did not show improved binding.
The isocoumarin-based inhibitors (Table 1) have the potential to function as suicide inhibitors or as substrates. However, simple reversible competitive inhibition was observed. The inhibitors were stable for several hours at neutral pH, as evidenced by no change in the spectral properties of the inhibitors. Addition of uPA for several hours at concentrations ten-fold higher than used in the kinetic studies did not produce any detectable changes in the inhibitors, suggesting that the inhibitors are not weak substrates of uPA. In addition, there was no loss of enzyme activity under these conditions, suggesting that the inhibitors are not functioning as suicide inhibitors. The observation of simple competitive inhibition is consistent with the modeling results in which the predicted orientations of the isocoumarin scaffold bound in the active site of uPA (figures 5 and 6) are not favorable for attack by serine 195.
Inhibition of uPA by uncharged inhibitor 8b represents a proof of concept that uPA inhibitors without a charged arginino mimetic group can be developed. Inhibitor 8b, which exhibits a dissociation constant in the low nanomolar range comparable to those of known arginino mimetic inhibitors, represents a lead compound for future development of uncharged inhibitors of uPA. The present study did not address the issue of specificity. Many previous studies of uPA inhibitors with arginino mimetic groups attached to various scaffold have resulted in the development of selective inhibitors of uPA. This information should be useful for developing selective uncharged inhibitors of uPA.
The x-ray crystal structure of human urokinase (pdb code 1EJN) was obtained from the protein data bank. All compounds shown in Table 1 were docked to the enzyme using Autodock 3.0 [21, 22] on a cluster of Silicon Graphics workstations consisting of Octanes and O2s. The compounds were prepared using Sybyl 7.0 (Tripos Inc., St. Louis, MO). The molecules were drawn in, assigned partial charges using the included Gasteiger-Hückel method and energy minimized using the BFGS method. Minimizations were run for 10,000 iterations and the rotatable bonds defined before docking. The protein was prepared before docking in Sybyl by removing non-native substrates and water molecules. Polar hydrogens and Kollman Uni charges were added to the protein as well. The molecules were docked in an area defined around the active site serine 195 by a cube of 60 × 60 × 60 Å.
Reagent quality solvents were used without purification. Benzoyl chloride was distilled before use. Melting points were determined on a Thomas Hoover capillary melting point apparatus and are uncorrected. NMR spectra were recorded on a Bruker AC250 NMR spectrometer in CDCl3 unless noted. Chemical shifts are in ppm (δ) relative to TMS. High resolution mass spectra were recorded on a Waters/Micromass LCT- premier. Analytical data was obtained from Galbraith laboratories, Knoxville TN. 5-Nitrohomophthalic acid was prepared as reported . Compounds 3a-3e, 4a-4e, 5c-5e, 6a, 6b, 8a, 8b 10a, 10b and 11 were prepared according to published procedures . Compounds 7a, 7b, 9a, 9b were prepared as reported .
Human urokinase (Sigma/Aldrich, St. Louis, MO) and Spectrozyme UK (American Diagnostica, Stamford, CT) were used for the kinetic studies. Enzyme activity was routinely measured in 1 ml volumes of 0.1 M Tris, pH 8.8, Spectrozyme UK (10 μM to 150 μM) and 0.64 μg (3,770 units/mg protein) human urokinase. Reactions were monitored at 405 nm, 25°C, with a Perkin/Elmer Lambda S2 UV/vis spectrophotometer. Michaelis constants and Ki values were determined from initial rate data, measured at 8 to 10 substrate concentrations, by non-linear regression analysis with SigmaPlot's Enzyme Kinetics Module™ (Chicago, IL, USA).
2- [2-(2-Bromoethoxy)-2-oxoethyl]-5-nitrobenzoic acid (3a) (66% yield) Tan crystals: mp 113–115°C (lit.  90°C);1H NMR: δ 3.50 (t, 2H, J = 5.96 Hz) 4.21 (s, 2H) 4.43 (t, 2H, J = 6.06 Hz) 7.51 (d, 1H, J = 8.34 Hz) 8.39 (dd, 1H, J = 2.59 Hz, 8.39 Hz) 8.98 (d, J = 2.39 Hz);13C NMR: δ 28.40, 40.13, 64.17, 126.24, 126.45, 130.97, 133.32, 142.59, 146.84, 167.46, 169.63.
2- [2-(3-Bromopropoxy)-2-oxoethyl]-5-nitrobenzoic acid (3b) (51% yield) White crystals: mp 122–123°C; 1H NMR: δ 2.19 (m, 2H) 3.44 (t, 2H, J = 6.56 Hz) 4.18 (s, 2H) 4.28 (t, 2H, J = 5.96 Hz) 7.51 (d, 1H, J = 8.34 Hz) 8.39 (dd, 1H, J = 2.39 Hz, 8.35 Hz) 8.98 (d, 1H, J = 2.18 Hz) 9.78 (br s, 1H); 13C NMR: δ 29.18, 31.58, 40.51, 63.12, 126.84, 127.52, 129.77, 143.37, 147.47, 169.92, 170.05.
2- [2-(2-Bromoethoxy)-2-oxoethyl]benzoic acid (3c) (50% yield) Tan crystals: mp 82–83°C; 1H NMR: δ 3.50 (t, 2H, J = 6.25 Hz) 4.07 (s, 2H) 4.40 (t, 2H, J = 6.25 Hz) 7.27 (d, 1H, J = 7.75 Hz) 7.45 (t, 1H, J = 7.55 Hz) 7.54 (td, 1H, J = 1.39 Hz, 7.55 Hz) 8.14 (d, 1H, J = 7.74 Hz); 13C NMR: δ 28.50, 40.68, 64.08, 127.65, 128.32, 131.95, 132.43, 133.35, 136.40, 170.86, 172.43.
2- [2-(3-Bromopropoxy)-2-oxoethyl]benzoic acid (3d) (70% yield) White crystals: mp 79–80°C; 1H NMR: δ 2.16 (m, 2H, J = 6.31 Hz) 3.42 (t, 2H, J = 6.55 Hz) 4.05 (s, 2H) 4.24 (t, 2H, J = 5.96 Hz) 7.27 (d, 1H, J = 7.55 Hz) 7.40 (t, 1H, J = 7.65 Hz) 7.54 (td, 1H, J = 1.2, 7.35 Hz) 8.14 (dd, 1H, J = 0.99, 7.74 Hz); 13C NMR: δ 29.50, 31.80, 40.80, 62.52, 127.60, 128.35, 131.91, 132.41, 133.35, 136.86, 171.17, 172.47.
2- [2-(4-Bromobutoxy)-2-oxoethyl]benzoic acid (3e) A solution of 4-bromo-1-butanol (2e, 6.0 mL, 41.6 mmol), homophthalic acid (1c, 2.5 g, 13.8 mmol), and five drops of concentrated sulfuric acid was refluxed in benzene (50 mL) for four hours. The solution was cooled and washed with water (2 × 25 mL), brine (1 × 25 mL), and dried over magnesium sulfate. Filtration and evaporation of the solvent gave a dark oil that was triturated with hexane to afford a crude solid. Recrystallization from hexane/ethyl acetate gave 0.91 g (40%) of compound 3e as white crystals: mp 84–86°C; 1H NMR: δ 1.79 (m, 2H) 1.87 (m, 2H) 3.38 (t, 2H, J = 6.45 Hz) 4.04 (s, 2H) 4.13 (t, 2H, J = 6.15 Hz) 7.27 (d, 1H, J = 7.55 Hz) 7.39 (t, 2H, J = 7.65 Hz) 7.54 (td, 1H, J = 1.4, 7.55 Hz) 8.13 (dd, 1H, J = 1.19, 7.74 Hz); 13C NMR: δ 27.33, 29.32, 33.19, 63.85, 127.56, 128.41, 131.88, 132.42, 133.33, 136.77, 171.32, 172.58.
3-(2-Bromoethoxy)-4-chloro-7-nitro-1 H -isochromen-1-one (4a) (43% yield) Yellow crystals: mp 126–128°C (lit.  120°C); 1H NMR: δ 3.67 (t, 2H, J = 6.16 Hz) 4.74 (t, 2H, J = 6.06 Hz) 7.86 (d, 1H, J = 8.94 Hz) 8.53 (dd, 1H, J = 2.39 Hz, 8.94 Hz) 9.03 (d, 1H, J = 2.38 Hz); 13C NMR: δ 27.72, 69.63, 90.86, 117.17, 123.81, 126.32, 129.82, 142.71, 145.47, 154.77, 157.06.
3-(3-Bromopropoxy)-4-chloro-7-nitro-1 H -isochromen-1-one (4b) (76% yield) Pale yellow crystals: mp 131–134°C; 1H NMR: δ 2.37 (m, 2H) 3.59 (t, 2H, J = 6.26 Hz) 4.61 (t, 2H, J = 5.86 Hz) 7.81 (d, 1H, J = 8.94 Hz) 8.50 (dd, 1H, J = 2.09 Hz, 8.84 Hz) 8.99 (d, 1H, J = 1.79 Hz); 13C NMR: δ 28.49, 31.92, 68.56, 90.46, 116.93, 123.52, 126.22, 129.68, 142.77, 145.77, 155.34, 157.18.
3-(2-Bromoethoxy)-4-chloro-1 H -isochromen-1-one (4c) (30% yield) Yellow solid: mp 81–82°C; 1H NMR: δ 3.65 (t, 2H, J = 6.35 Hz) 4.64 (t, 2H, J = 6.35 Hz) 7.41 (t, 1H, J = 7.15 Hz) 7.74 (m, 2H) 8.20 (d, 1H, J = 7.75 Hz); 13C NMR: δ 28.07, 69.37, 92.09, 117.53, 122.47, 126.55, 130.06, 135.62, 137.35, 152.08, 159.01.
3-(3-Bromopropoxy)-4-chloro-1 H -isochromen-1-one (4d) (53% yield) Yellow crystals: mp 95–97°C; 1H NMR: δ 2.33 (m, 2H) 3.60 (t, 2H, J = 6.35 Hz) 4.51 (t, 2H, J = 5.76 Hz) 7.40 (t, 1H, J = 6.75 Hz) 7.72 (m, 2H) 8.19 (d, 1H, J = 7.94 Hz); 13C NMR: δ 28.88, 32.26, 68.28, 91.91, 117.58, 122.39, 126.43, 130.12, 135.62, 137.57, 152.74, 159.33.
3-(4-Bromobutoxy)-4-chloro-1 H -isochromen-1-one (4e) A solution of 3e (0.75 g, 2.3 mmol) and phosphorus pentachloride (1.23 g, 5.9 mmol) was refluxed in benzene (50 mL) for fourteen hours. The orange solution was cooled, washed with water (2 × 25 mL), saturated sodium bicarbonate (2 × 15 mL), brine (1 × 25 mL), and dried over magnesium sulfate. Filtration and evaporation of the solvent gave a yellow oil. Trituration with hexane gave 0.55 g (70%) of compound 4e as white crystals: mp 75–77°C; 1H NMR: δ 1.98 (m, 2H) 2.06 (m, 2H) 3.48 (t, 2H, J = 6.25 Hz) 4.40 (t, 2H, J = 5.96 Hz) 7.38 (td, 1H, J = 1.59 Hz, 7.50 Hz) 7.70 (m, 2H) 8.17 (d, 1H, J = 7.55 Hz); 13C NMR: δ 27.33, 29.32, 33.12, 40.80, 63.81, 127.55, 128.41. 131.86, 132.38, 133.30, 136.76, 171.26, 172.29. Exact mass calcd for C13H12BrClO3: 329.9658, observed (M+H) 330.9734.
2- [2-(4-Chloro-1-oxo-1 H -isochromen-3-yloxy)ethyl]isothiourea hydrobromide (5c) (64% yield) Yellow solid: mp 168–170°C (lit.  167–169°C); 1H NMR: (DMSO-d6) δ 3.65 (t, 2H, J = 5.66 Hz) 4.58 (t, 2H, J = 5.67 Hz) 7.53 (t, 1H, J = 7.65 Hz) 7.69 (d, 1H, J = 8.14 Hz) 7.92 (t, 1H, J = 7.05 Hz) 8.13 (d, 1H, J = 7.75 Hz) 9.15 (br s, 4H); 13C NMR: δ 29.73, 68.11, 90.48, 117.18, 121.72, 126.70, 129.48, 135.99, 136.56, 152.18, 158.35, 169.11.
2- [3-(4-Chloro-1-oxo-1 H -isochromen-3-yloxy)propyl]isothiourea hydrobromide (5d) (40% yield) Yellow solid: mp 159–163°C (lit.  165–167°C);1H NMR: (DMSO-d6) 2.21 (m, 2H) 3.40 (t, 2H, J = 7.18 Hz) 4.55 (t, 2H, J = 6.11 Hz) 7.62 (td, 1H, J = 0.95, 7.60 Hz) 7.79 (d, 1H, J = 7.70 Hz) 8.02 (td, 1H, J = 1.23, 7.70 Hz) 8.23 (dd, 1H, J = 1.25, 7.50 Hz) 10.09 (br s, 4H); 13C NMR: δ 26.66, 28.53, 68.68, 90.43, 117.13, 121.66, 126.59, 129.47, 135.96, 136.66, 152.63, 158.53, 169.36.
2- [4-(4-Chloro-1-oxo-1 H -isochromen-3-yloxy)butyl]isothiourea hydrobromide (5e) A solution of 4e (0.25 g, 0.75 mmol) and thiourea (0.075 g, 0.98 mmol) in dry tetrahydrofuran (25 mL) was refluxed for forty-eight hours. The resulting pale yellow solid was filtered and washed with hot tetrahydrofuran (3 × 10 mL) to give 0.2 g (65%) of compound 5h as a pale yellow solid: mp 160–162°C; 1H NMR: (DMSO-d6) δ 1.82 (br s, 4H) 3.24 (t, 2H, J = 6.45 Hz) 4.39 (t, 2H, J = 5.75 Hz) 7.50 (t, 1H, J = 7.45 Hz) 7.65 (d, 1H, 7.45 Hz) 7.89 (t, 1H, J = 7.25 Hz) 8.09 (d, 1H, J = 7.75 Hz) 9.07 (br s, 4H); 13C NMR: δ 24.92, 27.35, 29.59, 69.96, 90.20, 116.95, 121.56, 126.48, 129.45, 135.94, 136.73, 152.80, 158.59, 169.56.
7-Amino-3-(2-bromoethoxy)-4-chloro-1 H -isochromen-1-one (6a) Compound 4a (2.2 g, 6.3 mmol) was reduced on a Parr apparatus with hydrogen over 10% palladium on charcoal (50 mg) in ethanol (25 mL) until the reaction stopped absorbing hydrogen. The solution was filtered through celite and the filtrate was evaporated. The resulting crude solid was chromatographed (dichloromethane) to give 1.55 g (78%) of compound 6a as yellow crystals: mp 134–136°C, (lit.  134–137°C); 1H NMR: δ 3.63 (t, 2H, J = 6.46 Hz) 3.95 (br s, 2H) 4.56 (t, 2H, J = 6.36 Hz) 7.10 (dd, 1H, J = 2.58 Hz, 8.54 Hz) 7.43 (d, 1H, J = 2.58 Hz) 7.54 (d, 1H, J = 8.74 Hz); 13C NMR: δ 28.18, 69.87, 93.59, 113.09, 119.21, 123.54, 124.04, 128.24, 145.63, 149.90, 159.47.
7-Amino-3-(3-bromopropoxy)-4-chloro-1 H -isochromen-1-one (6b) (75% yield) Yellow crystals: mp 106–107°C (lit. [24, 26] 98–100°C); 1H NMR (DMSO-d6) δ 2.29 (m, 2H) 3.60 (t, 2H, J = 6.36 Hz) 4.42 (t, 2H, J = 5.76 Hz) 7.09 (dd, 1H, J = 2.09 Hz, 8.44 Hz) 7.42 (d, 1H, J = 1.99 Hz) 7.51 (d, 1H, J = 8.54 Hz); 13C NMR: δ 29.11, 32.36, 68.71, 93.35, 113.11, 119.17, 123.58, 123.91, 128.42, 145.56, 150.49, 159.74.
2- [2-(7-amino-4-chloro-1-oxo-1 H -isochromen-3-yloxy)ethyl]isothiourea hydrobromide (7a) (40% yield) Pale yellow solid: mp d 150°C; 1H NMR: (DMSO-d6) δ 3.59 (br s, 2H) 4.47 (br s, 2H) 5.81 (br s, 2H) 7.21 (d, 1H, J = 8.94 Hz) 7.26 (s, 1H) 7.44 (br d, 1H) 9.11 (br s, 4H);13C NMR: δ 29.73, 68.11, 90.48, 117.18, 121.72, 126.70, 129.48, 135.99, 136.56, 152.18, 158.35, 169.11.
2- [3-(7-Amino-4-chloro-1-oxo-1 H -isochromen-3-yloxy)propyl]isothiourea hydrobromide (7b) A solution of 6b (0.25 g, 0.75 mmol), thiourea (0.071 g, 0.94 mmol) and tetrahydrofuran (25 mL) was refluxed for forty-eight hours to give a yellow precipitate. The precipitate was filtered and washed with hot tetrahydrofuran (3 × 25 mL), and recrystallized from methanol/ether to give 0.06 g (20%) of compound 7b as a pale yellow solid: mp 173°C; (lit.  160–162°C); 1H NMR: (DMSO-d6) δ 2.07 (br s, 2H) 3.30 (br s, 2H) 4.32 (br s, 2H) 7.16 (d, 1H, J = 7.94 Hz) 7.26 (s, 1H) 7.41 (d, 1H, J = 7.95 Hz) 9.05 (br s, 4H);13C NMR: δ 26.73, 28.35, 69.26, 92.87, 110.88, 118.81, 122.84, 123.11, 124.66, 148.23, 149.41, 159.06, 169.37.
N- [3-(2-Bromoethoxy)-4-chloro-1-oxo-1 H -isochromen-7-yl]benzamide (8a) To a solution of 6a (0.75 g, 2.4 mmol) in dry tetrahydrofuran (20 mL) was added benzoyl chloride (0.35 mL, 2.8 mmol) and triethylamine (0.33 mL, 2.3 mmol). The solution was stirred at room temperature for fourteen hours after which time the triethylamine hydrochloride was filtered off and washed with hot tetrahydrofuran (2 × 10 mL). The filtrate was evaporated to give a pale yellow solid that was recrystallized from tetrahydrofuran/hexane to afford 0.60 g (75%) of compound 8a as a pale yellow solid: mp 214–216°C; 1H NMR: (DMSO-d6) δ 3.83 (t, 2H, J = 5.46 Hz) 4.65 (t, 2H, J = 5.46 Hz) 7.56 (m, 3H) 7.71 (d, 1H, J = 8.93 Hz) 7.99 (d, 2H, J = 8.15) 8.29 (dd, 1H, J = 2.39 Hz, 8.74 Hz) 8.68 (d, 1H, J = 2.18 Hz) 10.63 (s, 1H); 13C NMR: δ 30.49, 69.98, 91.08, 117.64, 119.23, 122.38, 127.52, 127.86, 128.20, 131.62, 131.90, 134.12, 137.90, 151.40, 158.35, 165.43. Exact mass calcd for C18H13BrClNO4: 420.9716, observed (M+H) 421.9788.
N- [3-(3-Bromopropoxy)-4-chloro-1-oxo-1 H -isochromen-7-yl]benzamide (8b) (82% yield) Pale yellow solid: mp 193–194°C; 1H NMR: (DMSO-d6) δ 2.28 (m, 2H) 3.66 (t, 2H, J = 6.56 Hz) 4.44 (t, 2H, J = 5.96 Hz) 7.55 (m, 3H) 7.68 (d, 1H, J = 8.74 Hz) 7.98 (d, 6.56 Hz) 8.25 (dd, 1H, J = 1.99 Hz, 8.74 Hz) 8.66 (d, 1H, J = 1.98 Hz) 10.62 (s, 1H); 13C NMR: δ 30.74, 32.09, 69.13, 91.36, 118.01, 119.74, 122.80, 127.88, 128.43, 128.67, 133.10, 132.46, 134.47, 138.15, 152.20, 158.95, 165.96. Exact mass calcd for C19H15BrClNO4: 434.9873, observed (M+H) 435.9959.
2- [2-(7-Benzamido-4-chloro-1-oxo-1 H -isochromen-3-yloxy)ethyl]isothiourea hydrobromide (9a) A solution of 8a (0.3 g, 0.71 mmol) and thiourea (0.06 g, 0.78 mmol) in dry tetrahydrofuran (25 mL) was refluxed for twelve hours. The resulting pale yellow solids were filtered and washed with hot tetrahydrofuran (3 × 10 mL) to give 0.06 g (17%) of compound 9a as a pale yellow solid: mp 173–175°C. Evaporation of the filtrate afforded 8a (0.2 g). Yield based on recovered starting material is 51%; 1H NMR: (DMSO-d6) δ 3.68 (br s, 2H) 4.60 (br s, 2H) 7.59 (m, 3H) 7.74 (d, 1H, J = 8.54 Hz) 8.03 (d, 2H, J = 6.75 Hz) 8.32 (d, 1H, J = 8.14 Hz) 8.73 (s, 1H) 9.18 (br s, 4H) 10.69 (s, 1H); 13C NMR: δ 29.85, 68.32, 91.00, 117.72, 119.47, 122.53, 127.60, 128.22, 128.33, 131.79, 132.06, 134.12, 137.96, 151.44, 158.43, 165.63, 169.18.
2- [3-(7-Benzamido-4-chloro-1-oxo-1 H -isochromen-3-yloxy]propyl)isothiourea hydrobromide (9b) (25% yield) Pale yellow solid: mp 203–204°C;1H NMR: (DMSO-d6) δ 2.12 (m, 2H) 3.31 (br s, 2H) 4.44 (t, 2H, J = 5.66 Hz) 7.56 (m, 3H) 7.71 (d, 1H, J = 8.74 Hz) 8.00 (d, 2H, J = 6.95 Hz) 8.28 (d, 1H, J = 8.54 Hz) 8.70 (s, 1H) 9.07 (br s, 4H) 10.65 (s, 1H); 13C NMR: δ 27.21, 28.79, 69.37, 91.38, 118.13, 119.85, 122.92, 128.04, 128.60, 128.77, 132.21, 132.57, 134.61, 138.33, 152.36, 159.05, 166.02, 169.83. Exact mass calcd for C20H18ClN3O4S: 431.0707, observed (M+H) 432.0780.
3-(2-Bromoethoxy)-7-nitro-1 H -isochromen-1-one (10a) A solution of 3a (1.5 g, 4.5 mmol) and trifluoroacetic anhydride (0.64 mL, 5.0 mmol) in dichloromethane (50 mL) was stirred at room temperature for sixteen hours. The solution was evaporated, washed with water (1 × 25 mL), saturated sodium bicarbonate solution (1 × 25 mL), dried over magnesium sulfate, and evaporated to afford 1.23 g (87%) of a crude yellow solid. Recrystallization from isopropanol gave 0.66 g (47%) of compound 10a as yellow crystals: mp 95–97°C; 1H NMR: δ 3.65 (t, 2H, J = 5.96 Hz) 4.54 (t, 2H, J = 5.86 Hz) 5.77 (s, 1H) 7.42 (d, 1H, J = 8.74 Hz) 8.38 (d, 1H, J = 8.54 Hz) 8.96 (s, 1H); 13C NMR: δ 27.51, 68.87, 81.12, 117.12, 125.70, 126.17, 129.28, 144.97, 145.11, 158.81, 160.02.
3-(3-Bromopropoxy)-7-nitro-1 H -isochromen-1-one (10b) (55% yield) Yellow crystals: mp 117–118°C; 1H NMR: δ 2.37 (m, 2H) 3.59 (t, 2H, J = 6.26 Hz) 4.38 (t, 2H, J = 5.86 Hz) 5.72 (s, 1H) 7.43 (d, 1H, J = 8.74 Hz) 8.41 (dd, 1H, J = 2.48 Hz, 8.84 Hz) 9.02 (d, 1H, J = 2.19 Hz);13C NMR: δ 28.80, 31.46, 67.29, 80.02, 117.02, 125.55, 126.07, 129.15, 144.79, 145.21, 158.94, 160.89.
3-(3-Bromopropoxy)-4-trifluoroacetyl-1 H -isochromen-1-one (10c) A solution of 3d (0.60 g, 2.0 mmol) and trifluoroacetic anhydride (0.38 mL, 2.7 mmol) in dichloromethane (25 mL) was stirred at room temperature for fourteen hours. The solution was evaporated and the oil was chromatographed (chloroform) to afford 0.45 g (59%) of compound 10c as white crystals: mp 116–117°C; 1H NMR: δ 2.38 (m, 2H) 3.54 (t, 2H, J = 6.26 Hz) 4.70 (t, 2H, J = 5.96 Hz) 7.42 (m, 1H) 7.74 (m, 1H) 8.10 (d, 1H, J = 8.34 Hz) 8.22 (d, 1H, J = 7.95 Hz); 13C NMR: δ 28.24, 31.37, 68.94, 90.90, 115.81, 116.01, 123.40, 126.75, 130.26, 135.87, 136.27, 157.73, 162.08, 179.97. Anal. Calcd. for C14H10BrF3O4: C, 44.35; H, 2.66. Found: C, 44.25; H, 2.99.
7-Amino-3-(2-bromoethoxy)-1 H -isochromen-1-one (11) A solution of 10a (1.5 g, 4.7 mmol) in methanol/ethyl acetate (1:1, 25 mL) was reduced on a Parr apparatus with hydrogen and 10% palladium on charcoal. After the reaction stopped absorbing hydrogen it was filtered through celite and the celite was washed with methylene chloride (3 × 50 mL). The filtrate was evaporated to near dryness keeping the temperature below 40°C. The semisolid was recrystallized from methylene chloride/methanol to afford 1.10 g (81%) of compound 11 as yellow crystals: mp > 280°C; 1H NMR: (DMSO-d6) 3.79 (t, 2H, J = 5.17 Hz) 4.36 (t, 2H, J = 4.97 Hz) 5.50 (s, 2H) 5.82 (s, 1H) 7.03 (d, 1H, J = 8.54 Hz) 7.19 (m, 2H); 13C NMR: δ 30.30, 68.71, 80.35, 110.20, 117.91, 123.17, 125.84, 128.03, 147.16, 154.71, 160.45.
This work was supported by a grant from the National Institutes of Health, HL68598
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