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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Med Chem. Author manuscript; available in PMC Oct 3, 2011.
Published in final edited form as:
PMCID: PMC3184552
NIHMSID: NIHMS112949

Design and Synthesis of Small Molecule Glycerol 3-Phosphate Acyltransferase Inhibitors

Abstract

The incidence of obesity and other diseases associated with an increased triacylglycerol mass is growing rapidly, particularly in the United States. Glycerol 3-phosphate acyltransferase (GPAT) catalyzes the rate-limiting step of glycerolipid biosynthesis, the acylation of glycerol 3-phosphate with saturated long chain acyl-CoAs. In an effort to produce small molecule inhibitors of this enzyme, a series of benzoic and phosphonic acids was designed and synthesized. In vitro testing of this series has led to the identification of several compounds, in particular 2-(nonylsulfonamido)benzoic acid (15g), possessing moderate GPAT inhibitory activity in an intact mitochondrial assay.

Introduction

Obesity is currently estimated by the World Health Organization to affect at least 400 million adults worldwide. In the U.S. alone, approximately two-thirds of adults are overweight or obese.1 Various diseases are associated with obesity, including type 2 diabetes, hypertension, cardiovascular diseases, nonalcoholic fatty liver disease, and certain types of cancer. Even though there is a growing need for anti-obesity therapeutics, there are only two drugs approved for long-term use in the U.S. Orlistat functions by blocking the absorption of fat from the diet,2 and sibutramine affects the central nervous system, reducing energy intake and increasing energy use.3 Unfortunately, each of these drugs displays limited efficacy and produces undesirable side effects. Anti-obesity drugs currently in development utilize a wide variety of mechanisms, involving both central and peripheral targets. Alteration of lipid metabolism, by decreasing the de novo synthesis of triglycerides while increasing oxidation of stored fats, is a peripheral mechanism. This approach, based on weight loss effects observed with the compounds C75,4 cerulenin,5 and hGH(177-191),6 may be highly valuable in developing anti-obesity drugs.

The mitochondrial isoform of glycerol-3-phosphate acyltransferase-1 (mtGPAT) catalyzes the esterification of long chain acyl-CoAs with sn-glycerol-3-phosphate to produce lysophosphatidic acid (LPA). This reaction constitutes the first committed and rate-limiting step of glycerolipid biosynthesis.7, 8 The purported mechanism of this reaction is similar to that of a serine protease, with the primary hydroxyl group of glycerol-3-phosphate taking the place of serine in the catalytic triad.9 Next, LPA is esterified further to produce phosphatidic acid, a precursor of various lipids including triacylglycerol (TAG), the main component of animal fat. In addition to obesity, high TAG levels in the bloodstream have been linked to several diseases, notably atherosclerosis and pancreatitis.10,11

There are four known isoforms of GPAT, two microsomal isoforms located in the endoplasmic reticulum (GPAT3 and GPAT4), and two located in mitochondria (mtGPAT1 and mtGPAT2).12, 13 mtGPAT1 displays a strong preference for incorporating palmitoyl-CoA (16:0), thereby primarily producing saturated phospholipids, whereas the other three enzymes are not selective.13, 14 For recombinant mouse mtGPAT1, activity is 2-fold greater with palmitoyl-CoA than with various unsaturated chains 18 and 20 carbons in length,15 and in rat liver, kidney, and heart, mtGPAT1 incorporates palmitoyl CoA 3-10 times more effectively than other long chain saturated or unsaturated acyl-CoAs.14, 16-18 Of the four isoforms, only mtGPAT1 is affected by changes in diet or exercise. When excess calories are available from a high-carbohydrate diet, mtGPAT1 mRNA expression increases, resulting in greater mtGPAT1 activity.19-21 Mice that remain stationary for ten hours following a prolonged exercise regimen experience an increase in mtGPAT1 activity compared to mice that did not exercise at all, resulting in a significant overshoot of triacylglycerol (TAG) synthesis.22

mtGPAT1-deficient mice exhibit lower hepatic TAG levels and secrete less very low density lipoprotein (VLDL) than control mice.23 In contrast, rat hepatocytes with 2.7-fold increased mtGPAT1 activity demonstrated a significant increase in de novo synthesis of diacylglycerol.24 Overexpression of mtGPAT1 in vivo, as expected from the previous result, causes the levels of accumulated TAG and diacylglycerol (DAG) in mouse liver to rise dramatically to 12-fold and 7-fold that of normal levels.25 In addition to producing a certain amount of TAG dependent on the amount of active enzyme present, mtGPAT1 activity is essential for controlling the partitioning of fatty-acyl CoAs to β-oxidation or glycerolipid synthesis.

Both mtGPAT1 and carnitine palmitoyltransferase-1 (CPT-1), the enzyme that catalyzes the rate-limiting step of β-oxidation, are located on the outer mitochondrial membrane.26 This suggests that there is a competition between these enzymes for fatty acyl-CoA substrates. AMP-activated protein kinase (AMPK), which inactivates acetyl-CoA carboxylase (ACC) by phosphorylation, appears to acutely regulate both of these enzymes. Inactivation of ACC by AMPK prevents the buildup of malonyl-CoA, an allosteric suppressor of CPT-1, resulting in an increase in β-oxidation.27 AMPK inhibits mtGPAT1 as well, thereby decreasing the amount of TAG produced.28 The relationship between these two processes has been demonstrated in vivo. Feeding mtGPAT1-knockout mice a high-fat, high-sugar diet to induce obesity resulted in an increase in oxidation as the long-chain acyl-CoA substrates were partitioned away from the TAG synthetic pathway toward CPT-1 and β-oxidation.29 mtGPAT1 overexpression in rat hepatocytes produced an 80% reduction in fatty acid oxidation coupled to an increase in phospholipid biosynthesis.24 Overexpression in vivo resulted in a decrease in β-oxidation as well.25

The evidence suggesting that a drop in mtGPAT1 activity leads to a decrease in TAG levels as well as an increase in the amount of β-oxidation suggests that inhibition of this enzyme with a small molecule could be an effective treatment for obesity, diabetes, and other health problems associated with increased TAG synthesis. As there are no such studies of small molecule mtGPAT1 inhibitors described in the literature, we set out to design, synthesize, and test a GPAT inhibitor as a potential weight loss strategy.

Chemistry

The fundamental design of the compounds comprised structures with a negative charge at physiological pH to mimic the phosphate group of glycerol-3-phosphate and a long saturated chain to mimic the chain of palmitoyl-CoA, the substrate for which mtGPAT1 demonstrates a strong preference.14 A sulfonamide linker was chosen to represent a stable mimic of the presumed intermediate or transition state of the acylation reaction catalyzed by GPAT (Figure 1).

Figure 1
Comparison of the Proposed GPAT Transition State (A) to the Basic Inhibitor Design (B)

The putative glycerol-3-phosphate binding pocket, as determined in GPAT isolated from squash chloroplasts, consists of several conserved positively charged amino acids, namely His-139, Lys-193, His-194, Arg-235, and Arg-237 in the squash enzyme.9 This conserved pocket is believed to closely interact with the phosphate of glycerol-3-phosphate, and could play an integral role in binding a carboxylate or phosphonate in an inhibitor. The conserved catalytic histidine, which is thought to deprotonate the primary hydroxyl group involved in the acylation reaction, could interact favorably with the relatively acidic sulfonamide hydrogen (Figure 1). Furthermore, the saturated chain of the alkyl sulfonamide would serve as a palmitoyl-CoA C16 mimic, ideally occupying the hydrophobic palmitoyl-CoA binding site extending from the glycerol-3-phosphate binding site in the classical view of a bisubstrate analog.

The spatial relationship between the acyl-CoA and glycerol-3-phosphate in the mammalian GPAT active site is not known, however, so different linkers between the two moieties had to be examined. It was thought the most efficient way to do this would be to synthesize benzoic acids and phosphonic acids with saturated alkyl sulfonamides at each position on the aromatic ring. The distances between the sulfonamide and the carboxylate or phosphonate could also be altered by placing each group one or several methylene units from the ring. The most efficient synthetic pathway for the production of the benzoic acids was the coupling of a primary amine already present on a benzoic acid methyl ester to the alkyl sulfonyl chloride. Saponification of the ester to liberate the carboxylic acid was typically the final step in the synthetic sequence. In the case of the phosphonic acids, the phosphonates were installed through an Arbuzov reaction on a primary bromide or through aryl halide coupling reactions catalyzed by tetrakis(triphenylphosphine)palladium(0).30 The protected amine already present was then deprotected, coupled to the sulfonyl chloride, and the ethyl phosphonate was deprotected to yield the phosphonic acid. The compounds produced from these several routes allowed for the determination of a preliminary SAR from the GPAT inhibition assay.

The first series of compounds was derived from the variously substituted methyl methylbenzoates (Scheme 1). The meta- and para-amines were made by following a literature protocol.31 Following radical bromination of the methyl group with NBS in CH3CN, the bromide was displaced by refluxing with NaN3 in EtOH. Under Staudinger conditions, the azide was reduced to the free amine 3, which could then be coupled to 1-pentane- or 1-nonanesulfonyl chloride, prepared as described.32 Finally, the methyl ester 4 was converted to the carboxylate product 5 by reaction with potassium t-butoxide in Et2O with water present.

Scheme 1
Reaction conditions: (a) NBS, hν, CH3CN; (b) NaN3, EtOH, reflux; (d) C9H19SO2Cl or C5H11SO2Cl, pyridine, CH2Cl2, 0 °C to rt; (e) K+Ot-Bu, Et2O, H2O, 0 °C to rt.

The ortho-substituted carboxylates required a different approach than the meta- and para- compounds (Scheme 2). Indolinone 6, formed in a reaction between the ortho-bromide and ammonia gas in MeOH,33 was coupled to the alkane sulfonyl chlorides with NaH in DMF, and the resulting γ-lactam bond was readily cleaved with NaOH in THF/H2O to produce carboxylic acids 5e and 5f.

Scheme 2
Reaction conditions: (a) NH3, MeOH, reflux; (b) NaH, RSO2Cl, DMF, 0 °C to rt; (c) NaOH, THF/H2O, 0 °C to rt.

The synthesis of the alkyl phosphonates 13a-f commenced with the protection of the starting toluidines as the bis-acylated aniline 8 (Scheme 3).34 Free-radical bromination with NBS in CH3CN afforded benzyl bromide 9, which was converted to phosphonate 10 through Arbuzov reaction with triethyl phosphite. The aniline was unmasked by exposure to a refluxing acidic solution of EtOH. Following coupling of the amine with the alkane sulfonyl chloride to produce sulfonamide 12, the phosphonic acid moiety was revealed by treatment with TMSBr in CH2Cl2 followed by methanolysis.

Scheme 3
Reaction conditions: (a) NBS, hν, CH3CN; (b) P(OEt)3, reflux; (c) H2SO4, EtOH, reflux; (d) C9H19SO2Cl or C5H11SO2Cl, pyridine, CH3CN, 0 °C to rt; (e) TMSBr, CH2Cl2, rt.

Compounds 15a-i were synthesized by coupling the commercially available starting aniline with a variety of sulfonyl chlorides (Scheme 4). The resulting sulfonamides 14a-i were then converted to the final products by hydrolysis with potassium t-butoxide and water in ether. Aromatic sulfonyl chlorides were used as well as the saturated C9 chain in an attempt to mimic the CoA portion of the acyl-CoA substrate, as opposed to the alkyl chain.

Scheme 4
Reaction conditions: (a) RSO2Cl, pyridine, CH2Cl2, 0 °C to rt; (b) K+Ot-Bu, Et2O, H2O, 0 °C to rt.

Compounds 17a-f were designed to probe the effect of linkers of different length in the aryl sulfonamide portion of the molecule. These were produced in the same manner as 15a-i, starting with the commercially available aniline and coupling to either benzylsulfonyl chloride or phenylethylsulfonyl chloride with pyridine in methylene chloride to yield sulfonamides 16a-f (Scheme 5). The methyl esters were then converted to the carboxylic acids 17a-f with potassium t-butoxide and water in ether.

Scheme 5
Reaction conditions: (a) RSO2Cl, pyridine, CH2Cl2, 0 °C to rt; (b) K+Ot-Bu, Et2O, H2O, 0 °C to rt.

The synthesis of aryl phosphonic acids 21a-c is shown in Scheme 6. Aryl bromide 18 underwent palladium-catalyzed aryl halide coupling with diethyl phosphite to install the phosphonate functionality.30 The aniline was then deprotected by refluxing in acidic ethanol, and the free amine was coupled with commercially-available octanesulfonyl chloride to produce 20. The final compound was then obtained by deprotecting the diethyl phosphonate with TMSBr.

Scheme 6
Reaction conditions: (a) diethyl phosphite, Et3N, Pd(PPh3)4, EtOH, reflux; (b) H2SO4, EtOH, reflux; (c) C8H17SO2Cl, Et3N, CH2Cl2, 0 °C to rt; (d) TMSBr, CH2Cl2, rt.

Compounds 24a-c, based on 15g, were designed as probes to examine the effect of installing different length alkylsulfonamides on the ortho-substituted analogs (Scheme 7). It was believed that the compound with the saturated C16-chain (24c) would exhibit significantly greater inhibitory activity than 15g, as the enzyme demonstrates a marked preference for palmitoyl-CoA over other long-chain acyl-CoAs.14 Compounds 24d-f were designed to examine the role of an electronegative group at the 4-position of the benzene ring, which could possibly mimic the electron density of the secondary alcohol on glycerol-3-phosphate. All of these compounds (24a-f) were produced with the same reaction sequence used to produce 15a-f and 17a-f.

Scheme 7
Reaction conditions: (a) RSO2Cl, pyridine, CH2Cl2, 0 °C to rt; (b) K+Ot-Bu, Et2O, H2O, 0 °C to rt.

Results and Discussion

All benzoic acids and phosphonic acids produced were then evaluated for their ability to inhibit the acylation of glycerol-3-phosphate in vitro. The acylation reaction between 14C-labelled glycerol-3-phosphate and palmitoyl-CoA, initiated by adding mtGPAT, was measured in the presence of varying concentrations of the inhibitor by scintillation counting.35 Each reaction was performed in triplicate, and the IC50 values were then calculated based on the amount of inhibitor needed to produce 50% inhibition compared to the DMSO vehicle control. Results are summarized in Tables Tables11--33.

Table 1
In Vitro Anti-mtGPAT1 Activity of Sulfonamides 5a-f and 13a-f
Table 3
In Vitro Anti-mtGPAT1 Activity of Sulfonamides 21a-c and 24a-f

Data obtained from benzoic acids 5a-f indicate that in all cases, regardless of the position of the carboxylate with respect to the sulfonamide, the longer C9 alkyl chain resulted in greater inhibition than the C5 saturated chain. The most effective orientation between the acid and sulfonamide appeared to be ortho-substitution, as 5f (IC50 = 66.5 μM) is a better inhibitor than either 5b (IC50 = 129 μM) or 5d (IC50 = 83.5 μM). The assay data from phosphonic acids 13a-f also indicated that the longer C9 alkyl chain is more effective. In this series of compounds, however, there is no significant difference in activity between the different orientations of the phosphonic acid and the alkyl sulfonamide moiety. The most active compound of this class was 13d (IC50 = 62.8 μM), the meta-substituted phosphonic acid, though not by much over 13b (IC50 = 81.3 μM) and 13f (IC50 = 81.1 μM).

Compounds 15b, 15c, 15e, 15f, 15h, 15i and 17a-f were designed to examine the possibility of an appropriately-substituted benzene ring serving as a mimic for the Coenzyme A portion of the fatty acyl-CoA natural substrate. The distance between the benzene ring and the sulfonamide sulfur does not appear to have a significant effect on the inhibitory activity of these compounds, as there is effectively no difference between one methylene and two methylene linkers. It is apparent, however, that the ortho-substituted compounds containing these linker methylenes (17e-f) are slightly more effective than the other substituted benzoic acids (17a-d). For the meta- and para-compounds, inhibitory activity is greater when the benzene ring is directly attached to the sulfur, although the ortho-compounds are all similar. The addition of a para-chloride on the benzene ring does lead to increases in activity for the para- (15c), meta- (15f), and ortho-compounds (15i). Compounds 15a, 15d, and 15g were easily obtainable targets, which allowed for examination of the effect of the methylene linker between the benzene ring and the sulfonamide in 5a-f. For every substitution, these compounds were the most effective GPAT inhibitors, with the ortho-compound (15g) demonstrating the greatest activity (IC50 = 24.7 μM). Based on these results, the long alkyl chain is likely a more effective fatty-acyl CoA recognition element for the enzyme than a simple benzene ring.

In view of the increased inhibitory activity of 15g, two other compound series were prepared. The first, 21a-c, probes the effectiveness of an aryl phosphonic acid in place of the benzoic acid moiety. In vitro, the ortho-substituted acid (21c) is less active than 15g, and substitution of the phosphonic acid moiety does not appear to significantly affect activity (Table 3). The other compounds produced (24a-f) indicate the importance of chain length of the alkyl sulfonamide, as well as the effect of adding heteroatoms para- to the sulfonamide. It appears that the longer chain is very important to the activity of these compounds, as a C1-chain (24a) results in significantly less in vitro activity than the C9 chain. Compounds 24b and 24c were produced to determine if the naturally-favored C16 chain is preferred in these compounds over other chain lengths, including the C14 chain. In this case, there is no observed preference for the C16 compound over other long chains, in contrast to that observed with the natural acyl-CoA substrates.14

Conclusions

The first preliminary structure-activity relationship of mammalian GPAT inhibition has been constructed, utilizing in vitro assay data from several simple sulfonamide-containing benzoic acids and phosphonic acids. It appears that a medium to long chain alkyl sulfonamide is necessary for inhibitory activity, but there is not a significant preference for the naturally-favored palmitoyl chain (C16) over other shorter saturated chains. Replacement of the benzoic acid of 15g with an aryl phosphonic acid moiety results in decreased inhibitory activity, as does the presence of chloride, fluoride, or a hydroxyl group at the 5-position of the benzene ring. The best inhibitor obtained from this study, which possesses modest inhibitory activity in vitro, has been evaluated in relevant biological systems, with the results to be reported in the near future.

Experimental Section

General Methods

1H and 13C NMR spectra were measured on a Bruker 300 or 400 MHz NMR spectrometer. Melting points were determined on a Thomas-Hoover capillary melting point apparatus and are uncorrected. Column chromatography was carried out on silica gel 60 (Merck, 230-400 mesh ASTM). All solvents used for reactions were distilled prior to use (Et2O and THF over Na/benzophenone, CH2Cl2 and CH3CN over CaH). High resolution mass spectra were obtained at the Mass Spectrometry Laboratory, The Johns Hopkins University, Baltimore, MD. Elemental analyses (C, H, N) were performed by Atlantic Microlab, Norcross, GA and were within 0.4% of calculated values.

General Procedure for 4a-d

To a stirring solution of the appropriate amine 3a-c (1.2 mmol) in CH2Cl2 (4 mL) at 0 °C, the sulfonyl chloride (1.3 mmol) was added dropwise, followed by Et3N (1.3 mmol). The reaction mixture was allowed to warm to room temperature, where it was stirred for 2-3 h. Saturated NH4Cl solution was added to quench the reaction, and the mixture was extracted with 3 × 10 mL CH2Cl2. The combined organic layers were dried over MgSO4, concentrated in vacuo, and the products were purified by flash chromatography (20% EtOAc in hexanes).

Methyl 4-(pentylsulfonamidomethyl)benzoate 4a

1H NMR (CDCl3) δ 8.02 (d, J = 8.1 Hz, 2H), 7.42 (d, J = 8.1 Hz, 2H), 4.85 (br s, 1H), 4.35 (s, 2H), 3.92 (s, 3H), 2.93 (t, J = 8.4 Hz, 2H), 1.76 (m, 2H), 1.30 (m, 4H), 0.89 (t, J = 6.9 Hz, 3H); 13C NMR (CDCl3) δ 166.6, 142.1, 130.0, 129.8, 127.6, 53.4, 52.1, 46.7, 30.2, 23.2, 22.1, 13.6.

Methyl 4-(nonylsulfonamidomethyl)benzoate 4b

1H NMR (DMSO-d6) δ 7.93 (d, J = 8.0 Hz, 2H), 7.71 (t, J = 6.4 Hz, 1H), 7.49 (d, J = 8.0 Hz, 2H), 4.22 (d, J = 6.4 Hz, 2H), 3.84 (s, 3H), 2.90 (t, J = 8.0 Hz, 2H), 1.56 (m, 2H), 1.23 (m, 12H), 0.85 (t, J = 6.4 Hz, 3H); 13C NMR (DMSO-d6) δ 165.9, 144.2, 129.1, 128.4, 127.7, 52.0, 51.5, 45.4, 31.2, 28.6, 28.5, 28.4, 27.4, 23.0, 22.0, 13.8.

Methyl 3-(pentylsulfonamidomethyl)benzoate 4c

1H NMR (CDCl3) δ 8.01 (s, 1H), 7.98 (d, J = 7.6 Hz, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.44 (t, J = 7.8 Hz, 1H), 4.81 (t, J = 6.0 Hz, 1H), 4.35 (d, J = 6.0 Hz, 2H), 3.92 (s, 3H), 2.92 (t, J = 8.0 Hz, 2H), 1.76 (m, 2H), 1.28 (m, 4H), 0.88 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3) δ 166.6, 137.5, 132.3, 130.7, 129.1, 128.9, 128.8, 53.4, 52.2, 46.7, 30.2, 23.2, 22.1, 13.6.

Methyl 3-(nonylsulfonamidomethyl)benzoate 4d

1H NMR (CDCl3) δ 8.01 (s, 1H), 7.98 (d, J = 7.6 Hz, 1H), 7.57 (d, J = 7.6 Hz, 1H), 7.44 (t, J = 7.6 Hz, 1H), 4.80 (t, J = 6.0 Hz, 1H), 4.35 (d, J = 6.0 Hz, 2H), 3.92 (s, 3H), 2.93 (t, J = 8.0 Hz, 2H), 1.75 (m, 2H), 1.25 (m, 12H), 0.88 (t, J = 6.4 Hz, 3H); 13C NMR (CDCl3) δ 166.6, 137.5, 132.3, 130.7, 129.1, 128.9, 128.8, 53.4, 52.2, 46.7, 31.7, 29.2, 29.1, 29.0, 28.1, 23.5, 22.6, 14.0.

General Procedure for 5a-d

To a stirring suspension of potassium t-butoxide (5.88 mmol) in Et2O (15mL) cooled to 0 °C, was added water (1.4 mmol) via syringe. The slurry was stirred for 5 min, and 4a-d (0.67 mmol) was added. The mixture was stirred at room temperature until starting material disappeared by TLC analysis (20% EtOAc in hexanes). Ice water was added until 2 clear layers formed. The aqueous layer was separated and acidified with 1 M HCl. The product was then extracted with Et2O (3 × 20 mL) and evaporated in vacuo to afford 5a-d.

4-(Pentylsulfonamidomethyl)benzoic acid 5a

mp = 188-189 °C; 1H NMR (MeOD) δ 8.02 (d, J = 8.3 Hz, 2H), 7.50 (d, J = 8.3 Hz, 2H), 4.31 (s, 2H), 2.95 (t, J = 8.1 Hz, 2H), 1.73 (m, 2H), 1.33 (m, 4H), 0.91 (t, J = 6.9 Hz, 3H); 13C NMR (MeOD) δ 169.5, 145.0, 131.1, 131.0, 128.8, 53.6, 47.2, 31.4, 24.3, 23.2, 14.0; HRMS (FAB) calcd for C13H20NO4S [M + H]+, 286.11131; found, 286.1111. Anal. (C13H19NO4S) C, H, N.

4-(Nonylsulfonamidomethyl)benzoic acid 5b

mp = 178-180 °C; 1H NMR (MeOD) δ 8.03 (d, J = 8.3 Hz, 2H), 7.51 (d, J = 8.3 Hz, 2H), 4.32 (s, 2H), 2.94 (t, J = 7.8 Hz, 2H), 1.71 (m, 2H), 1.30 (m, 12H), 0.92 (t, J = 6.9 Hz, 3H); 13C NMR (DMSO-d6) δ 167.0, 143.6, 129.5, 129.3, 127.5, 51.5, 45.4, 31.2, 28.6, 28.5, 28.4, 27.4, 23.0, 22.0, 13.9; HRMS (FAB) calcd for C17H28NO4S [M + H]+, 342.17391; found, 342.17447. Anal. (C17H27NO4S·¼(H2O)) C, H, N.

3-(Pentylsulfonamidomethyl)benzoic acid 5c

mp = 160-161 °C; 1H NMR (MeOD) δ 8.08 (s, 1H), 7.97 (d, J = 7.8 Hz, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.48 (t, J = 7.8 Hz, 1H), 4.31 (s, 2H), 2.92 (t, J = 8.1 Hz, 2H), 1.72 (m, 2H), 1.33 (m, 4H), 0.91 (t, J = 6.9 Hz, 3H); 13C NMR (MeOD) δ 169.5, 140.2, 133.5, 132.3, 130.1, 129.8, 129.7, 53.6, 47.1, 31.4, 24.3, 23.1, 14.0; HRMS (FAB) calcd for C13H18NO3S [M − OH]+, 268.10074; found, 268.09988. Anal. (C13H19NO4S) C, H, N.

3-(Nonylsulfonamidomethyl)benzoic acid 5d

mp = 150-151 °C; 1H NMR (MeOD) δ 8.08 (s, 1H), 7.97 (d, J = 7.6 Hz, 1H), 7.63 (d, J = 7.6 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 4.31 (s, 2H), 2.91 (t, J = 8.0 Hz, 2H), 1.70 (m, 2H), 1.28 (m, 12H), 0.92 (t, J = 7.2 Hz); 13C NMR (MeOD) δ 169.5, 140.2, 133.5, 132.3, 130.1, 129.9, 129.7, 53.7, 47.2, 32.9, 30.4, 30.3, 30.1, 29.2, 24.6, 23.6, 14.4; HRMS (FAB) calcd for C17H28NO4S [M + H]+, 342.17391; found, 342.17333. Anal. (C17H27NO4S) C, H, N.

General Procedure for 7a-b

1.5 mmol 6 was added to DMF (8 mL), and the solution was cooled to 0 °C. NaH (1.65 mmol) was added, followed by the sulfonyl chloride (1.8 mmol), and the mixture was stirred and allowed to warm to room temperature. Reaction progress was monitored by TLC (25% MeOH in CHCl3). When complete, saturated ammonium chloride solution was added (80 mL), the product was extracted with EtOAc (3 × 20 mL), dried over MgSO4, and evaporated in vacuo. The product was purified by flash chromatography (2% MeOH in CHCl3).

2-(Pentylsulfonyl)isoindolin-1-one 7a

1H NMR (DMSO-d6) δ 7.81 (t, J = 7.5 Hz, 1H), 7.76 (d, J = 7.5 Hz, 1H), 7.68 (d, J = 7.5 Hz, 1H), 7.59 (t, J = 7.5 Hz, 1H), 4.94 (s, 2H), 3.60 (t, J = 7.8 Hz, 2H), 1.72 (m, 2H), 1.32 (m, 4H), 0.83 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3) δ 167.0, 141.2, 133.9, 129.8, 128.8, 124.9, 123.3, 53.2, 49.6, 30.0, 22.4, 21.9, 13.5.

2-(Nonylsulfonyl)isoindolin-1-one 7b

1H NMR (DMSO-d6) δ 7.82 (t, J = 7.5 Hz, 1H), 7.77 (d, J = 7.5 Hz, 1H), 7.69 (d, J = 7.5 Hz, 1H), 7.59 (t, J = 7.5 Hz), 4.94 (s, 2H), 3.60 (t, J = 8.1 Hz, 2H), 1.71 (m, 2H), 1.36 (m, 2H), 1.21 (m, 10H), 0.83 (t, J = 6.3 Hz, 3H); 13C NMR (CDCl3) δ 167.1, 141.2, 134.0, 130.0, 128.9, 125.1, 123.4, 53.3, 49.6, 31.7, 29.1, 29.0, 28.9, 28.0, 22.9, 22.5, 14.0

General Procedure for 5e-f

7a-b (0.66 mmol) was dissolved in THF (3 mL), and the solution was cooled to 0 °C. 1 M NaOH (1 mL, 10 equiv) was then added, and the solution was stirred and warmed to room temperature. Reaction progress was monitored by TLC (1:1 EtOAc:hexanes). When starting material had completely reacted, saturated NaHCO3 (30 mL) was added, and the solution was washed with EtOAc. The aqueous phase was acidified to pH 3 with 1 M HCl, and product was extracted with EtOAc, dried over MgSO4, and evaporated in vacuo.

2-(Pentylsulfonamidomethyl)benzoic acid 5e

mp = 100 °C; 1H NMR (DMSO-d6) δ 13.0 (s, 1H), 7.87 (d, J = 8.1 Hz, 1H), 7.60 (m, 2H), 7.39 (m, 2H), 4.51 (d, J = 6.3 Hz, 2H), 2.92 (t, J = 7.8 Hz, 2H), 1.61 (m, 2H), 1.25 (m, 4H), 0.84 (t, J = 6.9 Hz, 3H); 13C NMR (MeOD) δ 170.3, 140.8, 133.6, 132.3, 131.3, 130.7, 128.8, 53.6, 46.5, 31.3, 24.3, 23.1, 14.0; HRMS (FAB) calcd for C13H20NO4S [M + H]+, 286.11131; found, 286.11103. Anal. (C13H19NO4S) C, H, N.

2-(Nonylsulfonamidomethyl)benzoic acid 5f

mp = 79-82 °C; 1H NMR (CDCl3) δ 8.04 (d, J = 7.2 Hz, 1H), 7.60 (m, 2H), 7.43 (t, J = 6.8 Hz, 1H), 4.60 (s, 2H), 2.89 (t, J = 8.0 Hz, 2H), 1.66 (m, 2H), 1.28 (m, 12H), 0.92 (t, J = 7.2 Hz, 3H); 13C NMR (DMSO-d6) δ 168.3, 139.7, 132.1, 130.4, 129.8, 129.0, 127.2, 51.7, 44.1, 31.3, 28.7, 28.6, 28.5, 27.6, 23.1, 22.1, 14.0; HRMS (FAB) calcd for C17H28NO4S [M + H]+, 342.17391; found, 342.17478. Anal. (C17H27NO4S) C, H, N.

General Procedure for 9a-c

8a-c (31.3 mmol) was dissolved in CH3CN (150mL) and NBS (31.3 mmol) was added. The solution was then heated to reflux with a 275 W Sunlamp. Reaction progress was monitored by TLC (30% EtOAc in hexanes). The solution was then cooled, evaporated in vacuo, and the mixture was purified by flash chromatography (30% EtOAc in hexanes).

N, N-(4-Bromophenyl)diacetylaniline 9a

1H NMR (CDCl3) δ 7.47 (d, J = 8.4 Hz, 2H), 7.11 (d, J = 8.4 Hz, 2H), 4.48 (s, 2H), 2.27 (s, 6H); 13C NMR (CDCl3) δ 172.6, 139.1, 138.4, 130.3, 128.9, 32.0, 26.7.

N, N-(3-Bromophenyl)diacetylaniline 9b

1H NMR (CDCl3) δ 7.44 (m, 2H), 7.20 (s, 1H), 7.08 (m, 1H), 4.48 (s, 2H), 2.28 (s, 6H); 13C NMR (CDCl3) δ 172.7, 139.7, 139.7, 130.1, 129.3, 128.6, 32.0, 26.8.

N, N-(2-Bromophenyl)diacetylaniline 9c

1H NMR (CDCl3) δ 7.56 (d, J = 6.8 Hz, 1H), 7.44 (m, 2H), 7.12 (d, J = 6.8 Hz, 1H), 4.32 (s, 2H), 2.36 (s, 6H); 13C NMR (CDCl3) δ 172.5, 137.9, 135.0, 131.5, 129.9, 129.5, 129.4, 28.4, 26.7.

General Procedure for 10a-c

9a-c (22.2 mmol) was dissolved in P(OEt)3 (25 mL, 6.6 equiv), and the solution was heated to reflux for 18 h with a reflux condenser heated to 50 °C. Reaction progress was monitored by TLC (30% EtOAc in hexanes). The reaction mixture was then cooled, and P(OEt)3 was removed in vacuo. The product was then purified by flash chromatography (2% MeOH in CHCl3).

Diethyl N, N-4-diacetylanilinephosphonate 10a

1H NMR (CDCl3) δ 7.39 (dd, J = 8.4, 2.4 Hz, 2H), 7.07 (d, J = 8.4 Hz, 2H), 4.02 (m, 4H), 3.17 (d, J = 21.6 Hz, 2H), 2.25 (s, 6H), 1.22 (t, J = 6.8 Hz, 6H); 13C NMR (CDCl3) δ 172.7, 138.1 (d, J = 3.6 Hz), 132.7 (d, J = 6.5 Hz), 131.1 (d, 6.5 Hz), 128.7 (d, 3.3 Hz), 62.1 (d, 6.6 Hz), 33.4 (d, J = 137.7 Hz), 27.7, 16.2 (d, J = 5.9 Hz).

Diethyl N, N-3-diacetylanilinephosphonate 10b

1H NMR (CDCl3) δ 7.30 (t, J = 7.6 Hz, 1H), 7.24 (d, J = 7.6 Hz, 1H), 7.03 (s, 1H), 6.94 (d, J = 8.4 Hz, 1H), 3.91 (m, 4H), 3.06 (d, J = 21.6 Hz, 2H), 2.16 (s, 6H), 1.13 (t, J = 6.8 Hz, 6H); 13C NMR (CDCl3) δ 172.4, 139.1 (d, J = 3.1 Hz), 133.4 (d, J = 8.8 Hz), 129.8 (d, J = 3.6 Hz), 129.8 (d, J = 3.7 Hz), 129.4 (d, J = 3.0 Hz), 126.8 (d, J = 3.6 Hz), 61.8 (d, J = 6.9 Hz), 33.0 (d, J = 137.4 Hz), 26.4, 16.0 (d, J = 5.9 Hz).

Diethyl N, N-2-diacetylanilinephosphonate 10c

1H NMR (CDCl3) δ 7.60 (d, J = 7.6 Hz, 1H), 7.35 (m, 2H), 7.09 (d, J = 7.6 Hz, 1H), 4.07 (m, 4H), 2.95 (d, J = 22.0 Hz, 2H), 2.31 (s, 6H), 1.27 (t, J = 7.2 Hz, 6H); 13C NMR (CDCl3) δ 172.9, 138.3 (d, J = 8.7 Hz), 131.6 (d, J = 5.1 Hz), 130.5 (d, J = 7.4 Hz), 129.4 (d, J = 2.3 Hz), 129.1 (d, J = 3.0 Hz), 128.3 (d, J = 3.0 Hz), 62.1 (d, J = 6.7 Hz), 28.7 (d, J = 140.3 Hz), 26.9, 16.2 (d, J = 5.8 Hz).

General Procedure for 11a-c

Concentrated H2SO4 (3 mL) was added to a stirring solution of 10a-c (9.7 mmol) in EtOH (60 mL). The solution was heated to reflux for 18 h. Reaction progress was monitored by TLC (5% MeOH in CHCl3). The solution was diluted with water (100 mL), washed with EtOAc (30 mL), and the aqueous phase was brought to pH 9 with saturated NaHCO3 solution. The product was extracted with EtOAc (3 × 30 mL), the combined organic layers were dried over MgSO4, and solvent was removed in vacuo.

Diethyl 4-aminobenzylphosphonate 11a

1H NMR (CDCl3) δ 6.91 (dd, J = 8.0, 2.4 Hz, 2H), 6.47 (d, J = 8.0 Hz, 2H), 3.86 (m, 4H), 3.73 (br s, 2H), 2.90 (d, J = 21.2 Hz, 2H), 1.10 (t, J = 7.2 Hz, 6H); 13C NMR (CDCl3) δ 145.3 (d, J = 3.6 Hz), 129.9 (d, J = 6.5 Hz), 119.7 (d, J = 9.4 Hz), 114.6 (d, J = 2.9 Hz), 61.5 (d, J = 6.6 Hz), 32.1 (d, J = 138.1 Hz), 15.8 (d, J = 5.9 Hz).

Diethyl 3-aminobenzylphosphonate 11b

1H NMR (CDCl3) δ 7.04 (t, J = 8.0 Hz, 1H), 6.64 (m, 2H), 6.53 (d, J = 7.2 Hz, 1H), 3.98 (m, 4H), 3.68 (br s, 2H), 3.03 (d, J = 21.6 Hz, 2H), 1.22 (t, J = 7.2 Hz, 6H); 13C NMR (CDCl3) δ 146.5 (d, J = 2.9 Hz), 132.3 (d, J = 8.8 Hz), 129.2 (d, J = 3.0 Hz), 119.8 (d, J = 6.6 Hz), 116.3 (d, J = 6.6 Hz), 113.6 (d, J = 3.6 Hz), 62.0 (d, J = 6.6 Hz), 33.5 (d, J = 137.0 Hz), 16.2 (d, J = 5.8 Hz).

Diethyl 2-aminobenzylphosphonate 11c

1H NMR (CDCl3) δ 7.03 (m, 2H), 6.73 (m, 2H), 4.30 (br s, 2H), 4.03 (m, 4H), 3.12 (d, J = 21.2 Hz, 2H), 1.24 (t, J = 7.2 Hz, 6H); 13C NMR (CDCl3) δ 145.9 (d, J = 4.6 Hz), 131.4 (d, J = 6.5 Hz), 128.1 (d, J = 3.6 Hz), 118.9 (d, J = 2.9 Hz), 117.1 (d, J = 9.7 Hz), 117.0 (d, J = 3.5 Hz), 62.3 (d, J = 6.6 Hz), 30.5 (d, J = 137.9 Hz), 16.2 (d, J = 6.0 Hz).

General Procedure for 12a-d

Sulfonyl chloride (4.9 mmol) was added dropwise to a solution of 11a-b (3.3 mmol) in CH3CN (13 mL) at 0 °C. Et3N (3.63 mmol) was added dropwise, and the solution was stirred and allowed to warm to room temperature. Reaction progress was monitored by TLC (10% MeOH in CHCl3). When complete (about 2 h), the reaction was quenched by adding saturated sodium bicarbonate solution. The product was extracted with EtOAc (3 × 10 mL), and the combined organic extracts were dried over MgSO4 and concentrated in vacuo. Flash chromatography (2% MeOH in CHCl3) afforded the product.

Diethyl 4-(pentylsulfonamido)benzylphosphonate 12a

1H NMR (CDCl3) δ 8.42 (br s, 1H), 7.15 (m, 4H), 4.01 (m, 4H), 3.08 (d, J = 21.2 Hz, 2H), 2.98 (t, J = 8.0 Hz, 2H), 1.76 (m, 2H), 1.28 (m, 4H), 1.25 (t, J = 6.8 Hz, 6H), 0.82 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3) δ 136.5 (d, J = 3.6 Hz), 130.5 (d, 6.6 Hz), 127.2 (d, J = 9.7 Hz), 120.4 (d, J = 3.3 Hz), 62.2 (d, J = 6.7 Hz), 51.1, 32.6 (d, J = 138.1 Hz), 30.1, 22.8, 21.9, 16.1 (d, J = 5.8 Hz), 13.5.

Diethyl 4-(nonylsulfonamido)benzylphosphonate 12b

1H NMR (CDCl3) δ 8.61 (br s, 1H), 7.12 (m, 4H), 3.99 (m, 4H), 3.06 (d, J = 21.2 Hz, 2H), 2.96 (t, J = 8.0 Hz, 2H), 1.71 (m, 2H), 1.27 (m, 12H), 1.18 (t, J = 7.2 Hz, 6H), 0.79 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3) δ 136.5 (d, J = 3.0 Hz), 130.4 (d, J = 6.7 Hz), 127.0 (d, J = 9.1 Hz), 120.2 (d, J = 2.9 Hz), 62.1 (d, J = 7.0 Hz), 51.0, 32.5 (d, J = 137.8 Hz), 31.5, 28.9, 28.8, 28.7, 27.9, 23.0, 22.3, 16.0 (d, J = 5.9 Hz), 13.7.

Diethyl 3-(pentylsulfonamido)benzylphosphonate 12c

1H NMR (CDCl3) δ 8.27 (br s, 1H), 7.19 (m, 3H), 7.01 (d, J = 6.8 Hz, 1H), 4.00 (m, 4H), 3.11 (d, J = 21.6, 2H), 3.02 (t, J = 8.0 Hz, 2H), 1.76 (m, 2H), 1.30 (m, 4H), 1.23 (t, J = 7.2 Hz, 6H), 0.81, (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3) δ 137.8 (d, J = 2.9 Hz), 132.8 (d, J = 9.2 Hz), 129.3 (d, J = 2.9 Hz), 125.7 (d, J = 6.6 Hz), 121.2 (d, J = 6.6 Hz), 118.4 (d, J = 3.5 Hz), 62.2 (d, J = 6.7 Hz), 51.2, 33.3 (d, J = 137.3 Hz), 30.1, 22.8, 21.9, 16.1 (d, J = 5.9 Hz), 13.5.

Diethyl 3-(nonylsulfonamido)benzylphosphonate 12d

1H NMR (CDCl3) δ 8.17 (br s, 1H), 7.20 (m, 3H), 7.02 (d, J = 7.2 Hz, 1H), 3.99 (m, 4H), 3.12 (d, J = 21.6 Hz, 2H), 3.03 (t, J = 8.0 Hz, 2H), 1.76 (m, 2H), 1.31 (m, 12H), 1.26 (t, J = 6.0 Hz, 6H), 0.83 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3) δ 137.8 (d, J = 3.6 Hz), 132.8 (d, J = 9.0 Hz), 129.4 (d, J = 2.9 Hz), 125.7 (d, J = 6.7 Hz), 121.3 (d, 6.5 Hz), 118.5 (d, J = 3.6 Hz), 62.3 (d, 6.9 Hz), 51.3, 33.3 (d, J = 137.4 Hz), 31.6, 29.1, 29.0, 28.9, 28.0, 23.2, 22.4, 16.2 (d, J = 5.9 Hz), 13.9.

General Procedure for 12e-f

11c (1.36 mmol) was dissolved in CH3CN (3.3 mL), then pyridine (10.8 mmol) was added. The solution was cooled to 0 °C, and sulfonyl chloride (1.63 mmol) was added slowly by syringe. The solution was allowed to warm to room temperature. Reaction progress was monitored by TLC (5% MeOH in CHCl3). When complete, the reaction was quenched by adding saturated NaHCO3 solution. The product was extracted with EtOAc (3 × 5 mL), washed with 1 N HCl, and the combined organic extracts were dried over MgSO4 and concentrated in vacuo. The product was purified by flash chromatography (2% MeOH in CHCl3).

Diethyl 2-(pentylsulfonamido)benzylphosphonate 12e

1H NMR (CDCl3) δ 8.71 (s, 1H), 7.40 (d, J = 7.6 Hz), 7.22 (m, 1H), 7.12 (m, 2H), 3.97 (m, 4H), 3.21 (d, J = 21.2 Hz, 2H), 3.11 (t, J = 8.0 Hz, 2H), 1.86 (m, 2H), 1.37 (m, 4H), 1.19 (t, J = 7.2 Hz, 6H), 0.85 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3) δ 136.2 (d, J = 5.2 Hz), 131.5 (d, J = 7.2 Hz), 128.2 (d, J = 3.7 Hz), 125.8 (d, J = 2.9 Hz), 125.5 (d, J = 9.7 Hz), 124.6 (d, J = 3.5 Hz), 62.6 (d, J = 6.9 Hz), 53.4, 30.9 (d, J = 136.6 Hz), 30.1, 23.0, 21.9, 16.0 (d, J = 5.9 Hz), 13.5.

Diethyl 2-(nonylsulfonamido)benzylphosphonate 12f

1H NMR (CDCl3) δ 8.71 (s, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.28 (m, 1H), 7.17 (m, 2H), 4.01 (m, 4H), 3.25 (d, J = 21.2 Hz, 2H), 3.17 (t, J = 8.0 Hz, 2H), 1.91 (m, 2H), 1.40 (m, 2H), 1.25 (m, 16H), 0.89 (t, J = 7.2 Hz); 13C NMR (CDCl3) δ 136.4 (d, J = 5.0 Hz), 131.7 (d, J = 6.7 Hz), 128.5 (d, J = 3.7 Hz), 126.0 (d, J = 2.8 Hz), 125.7 (d, J = 10.1 Hz), 124.9 (d, J = 3.1 Hz), 62.8 (d, J = 6.7 Hz), 53.8, 31.7, 31.2 (d, J = 136.4 Hz), 29.1, 29.1, 29.0, 28.2, 23.5, 22.5, 16.2 (d, J = 5.8 Hz), 14.0.

General Procedure for 13a-f

TMSBr (8.6 mmol) was added to a solution of 12a-f (0.277 mmol) in CH2Cl2 (2 mL), and the solution was stirred at room temperature. After 24 h, the reaction was quenched by adding MeOH (3 × 1.6 mL). The solution was concentrated in vacuo, and dissolved in saturated NaHCO3 solution (10 mL). This solution was washed with Et2O (5 mL), then acidified with 1 N HCl. The product was extracted with Et2O (3 × 5 mL), and the combined organic extracts were dried over MgSO4 and dried in vacuo.

4-(Pentylsulfonamido)benzylphosphonic acid 13a

mp = 198-200 °C; 1H NMR (MeOD) δ 7.29 (dd, J = 8.4, 2.4 Hz, 2H), 7.20 (d, J = 8.4 Hz, 2H), 3.10 (d, J = 21.6 Hz, 2H), 3.05 (t, J = 8.0 Hz, 2H), 1.78 (m, 2H), 1.34 (m, 4H), 0.90 (t, J = 7.2 Hz, 3H); 13C NMR (MeOD) δ 137.9 (d, J = 3.7 Hz), 131.8 (d, J = 6.3 Hz), 130.6 (d, J = 9.6 Hz), 121.4 (d, J = 2.9 Hz), 51.8, 35.1 (d, J = 134.6 Hz), 31.2, 24.2, 23.1, 14.0; HRMS (FAB) calcd for C12H20NO5PS [M]+, 321.07998; found, 321.07934. Anal. (C12H20NO5PS·0.6(H2O)) C, H, N.

4-(Nonylsulfonamido)benzylphosphonic acid 13b

mp = 201-203 °C; 1H NMR (MeOD) δ 7.29 (dd, J = 8.6, 2.4 Hz, 2H), 7.20 (d, J = 8.6 Hz, 2H), 3.09 (d, J = 21.2, 2H), 3.05 (t, J = 8.0 Hz, 2H), 1.77 (m, 2H), 1.29 (m, 12H), 0.91 (t, J = 7.2 Hz, 3H); 13C NMR (MeOD) δ 137.9 (d, J = 3.3 Hz), 131.8 (d, J = 6.5 Hz), 130.6 (d, J = 9.3 Hz), 121.4 (d, J = 3.0 Hz), 51.8, 35.1 (d, J = 134.5 Hz), 32.9, 30.3, 30.2, 30.1, 29.1, 24.5, 14.4; HRMS (FAB) calcd for C16H29NO5PS [M + H]+, 378.15041; found, 378.14945. Anal. (C16H28NO5PS) C, H, N.

3-(Pentylsulfonamido)benzylphosphonic acid 13c

mp = 127-128 °C; 1H NMR (MeOD) δ 7.27 (t, J = 8.0 Hz, 1H), 7.22 (s, 1H), 7.11 (m, 2H), 3.11 (d, J = 21.6 Hz, 2H), 3.09 (t, J = 7.8 Hz, 2H), 1.78 (m, 2H), 1.38 (m, 4H), 0.91 (t, J = 6.4 Hz, 3H); 13C NMR (MeOD) δ 139.5 (d, J = 3.3 Hz), 136.0 (d, J = 9.3 Hz), 130.3 (d, J = 3.4 Hz), 126.8 (d, J = 5.9 Hz), 122.5 (d, J = 6.5 Hz), 119.3 (d, J = 3.4 Hz), 51.9, 35.8 (d, J = 134.2 Hz), 31.2, 24.2, 23.1, 14.0; HRMS (FAB) calcd for C12H21NO5PS [M + H]+, 322.08781; found, 322.08830. Anal. (C12H20NO5PS) C, H, N.

3-(Nonylsulfonamido)benzylphosphonic acid 13d

mp = 149-150 °C; 1H NMR (MeOD) δ 7.27 (t, J = 8.0 Hz, 1H), 7.22 (s, 1H), 7.11 (m, 2H), 3.11 (d, J = 21.6 Hz, 2H), 3.08 (t, J = 7.6 Hz, 2H), 1.77 (m, 2H), 1.33 (m, 12H), 0.91 (t, J = 6.4 Hz, 3H); 13C NMR (MeOD) δ 139.5 (d, J = 3.0 Hz), 130.3 (d, J = 3.0 Hz), 126.8 (d, J = 6.1 Hz), 122.5, (d, J = 6.3 Hz), 119.3 (d, J = 3.5 Hz), 51.9, 35.8 (d, J = 134.0 Hz), 32.9, 30.3, 30.2, 30.1, 29.1, 24.5, 23.6, 14.3; HRMS (FAB) calcd for C16H29NO5PS [M + H]+, 378.15041; found, 378.14975. Anal. (C16H28NO5PS) C, H, N.

2-(Pentylsulfonamido)benzylphosphonic acid 13e

1H NMR (DMSO-d6) δ 9.73 (s, 1H), 7.38 (d, J = 8.0 Hz, 1H), 7.25 (m, 2H), 7.13 (t, J = 7.6 Hz, 1H), 3.14 (t, J = 8.0 Hz, 2H), 3.10 (d, J = 20.8 Hz, 2H), 1.71 (m, 2H), 1.32 (m, 4H), 0.85 (t, J = 7.2 Hz, 3H); 13C NMR (DMSO-d6) δ 136.2 (d, J = 5.8 Hz), 131.6 (d, J = 6.4 Hz), 127.6 (d, J = 10.0 Hz), 127.1 (d, J = 3.4 Hz), 125.0 (d, J = 2.8 Hz), 123.7 (d, J = 3.0 Hz), 52.3, 32.5 (d, J = 130.3 Hz), 29.4, 22.7, 21.3, 13.3; HRMS (FAB) calcd for C12H20NO5PS [M + H]+, 322.08781; found, 322.08660. Anal. (C12H20NO5PS·¼(H2O)) C, H, N.

2-(Nonylsulfonamido)benzylphosphonic acid 13f

mp = 104-106 °C; 1H NMR (DMSO-d6) δ 9.80 (s, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.25 (m, 2H), 7.15 (t, J = 7.6 Hz, 1H), 3.14 (t, J = 7.6 Hz, 2H), 3.09 (d, J = 21.2 Hz, 2H), 1.68 (m, 2H), 1.35 (m, 2H), 1.22 (m, 8H), 0.85 (t, J = 6.8 Hz); 13C NMR (DMSO-d6) δ 136.3 (d, J = 5.7 Hz), 131.8 (d, J = 6.5 Hz), 127.7 (d, J = 8.7 Hz), 127.3 (d, J = 3.3 Hz), 125.2 (d, J = 2.6 Hz), 123.8 (d, J = 3.0 Hz), 52.3, 32.6 (d, J = 130.5 Hz), 31.2, 28.6, 28.5, 28.4, 27.4, 23.2, 22.0, 13.9; HRMS (FAB) calcd for C16H28NO5PS [M + H]+, 378.15041; found, 378.15020. Anal. (C16H28NO5PS) C, H, N.

General Procedure for 14a-i

To a stirring solution of the aniline starting material (3.3 mmol) in CH2Cl2 (12 mL) at 0° C was added pyridine (7.5 equiv). The sulfonyl chloride (1.2 equiv) was then added slowly via syringe. The solution was stirred and allowed to warm to room temperature. Reaction progress was monitored by TLC (20% EtOAc in hexanes). When complete, the reaction was poured into saturated NaHCO3 solution (45 mL), extracted with CH2Cl2 (3 × 15 mL), and washed with 1 M HCl (50 mL). The combined organic phases were concentrated in vacuo, and recrystallization from EtOAc / hexanes afforded 14a-i.

Methyl 4-(nonylsulfonamido)benzoate 14a

1H NMR (DMSO-d6) δ 10.33 (s, 1H), 7.90 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 8.4 Hz, 2H), 3.80 (s, 3H), 3.17 (t, J = 8.0 Hz, 2H), 1.63 (m, 2H), 1.15 (m, 12H), 0.81 (t, J = 6.8 Hz, 3H); 13C NMR (DMSO-d6) δ 165.7, 143.1, 130.7, 123.8, 117.4, 51.8, 50.8, 31.1, 28.5, 28.5, 28.2, 27.0, 22.9, 22.0, 13.8.

Methyl 4-(phenylsulfonamido)benzoate 14b

1H NMR (CDCl3) δ 7.91 (d, J = 8.4 Hz, 2H), 7.85 (d, J = 8.4 Hz, 2H), 7.56 (t, J = 7.2 Hz, 1H), 7.46 (t, J = 8.0 Hz, 2H), 7.16 (d, J = 8.4 Hz, 2H), 3.87 (s, 3H); 13C NMR (DMSO-d6) δ 165.6, 142.3, 139.2, 133.2, 130.5, 129.3, 126.6, 124.4, 118.2, 51.8.

Methyl 4-(4-chlorophenylsulfonamido)benzoate 14c

1H NMR (DMSO-d6) δ 10.94 (s, 1H), 7.83 (m, 4H), 7.63 (d, J = 8.0 Hz, 2H), 7.23 (d, J = 8.0 Hz, 2H), 3.77 (s, 3H); 13C NMR (DMSO-d6) δ 165.5, 141.9, 138.1, 138.0, 130.6, 129.5, 128.5, 124.7, 118.4, 51.8.

Methyl 3-(nonylsulfonamido)benzoate 14d

1H NMR (DMSO-d6) δ 10.05 (s, 1H), 7.83 (s, 1H), 7.65 (d, J = 6.8 Hz, 1H), 7.46 (m, 2H), 3.84 (s, 3H), 3.08 (t, J = 8.0 Hz, 2H), 1.63 (m, 2H), 1.63 (m, 2H), 1.15 (m, 12H), 0.81 (t, J = 6.8 Hz, 3H); 13C NMR (DMSO-d6) δ 165.7, 138.9, 130.6, 129.7, 124.1, 123.6, 119.4, 52.1, 50.6, 31.1, 28.5, 28.5, 28.3, 27.1, 22.9, 22.0, 13.8.

Methyl 3-(phenylsulfonamido)benzoate 14e

1H NMR (DMSO-d6) δ 10.57 (s, 1H), 7.78 (d, J = 8.0 Hz, 2H), 7.74 (s, 1H), 7.54 (m, 4H), 7.38 (m, 2H), 3.79 (s, 3H); 13C NMR (DMSO-d6) δ 165.6, 139.2, 138.1, 133.0, 130.5, 129.6, 129.3, 126.6, 124.6, 124.4, 120.3, 52.2.

Methyl 3-(4-chlorophenylsulfonamido)benzoate 14f

1H NMR (DMSO-d6) δ 10.63 (s, 1H), 7.74 (m, 3H), 7.61 (m, 3H), 7.39 (m, 2H), 3.80 (s, 3H); 13C NMR (DMSO-d6) δ 165.5, 138.0, 138.0, 137.8, 130.6, 129.8, 129.5, 128.5, 124.9, 124.6, 120.5, 52.2.

Methyl 2-(nonylsulfonamido)benzoate 14g

1H NMR (DMSO-d6) δ 10.15 (s, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.62 (m, 2H), 7.20 (t, J = 7.6 Hz, 1H), 3.88 (s, 3H), 3.27 (t, J = 8.0 Hz, 2H), 1.62 (m, 2H), 1.16 (m, 12H), 0.83 (t, J = 7.2 Hz, 3H); 13C NMR (DMSO-d6) δ 167.8, 139.8, 134.7, 131.0, 123.0, 118.5, 116.1, 52.6, 51.2, 31.1, 28.4, 28.4, 28.2, 27.0, 22.8, 21.9, 13.8.

Methyl 2-(phenylsulfonamido)benzoate 14h

1H NMR (CDCl3) δ 10.67 (s, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 8.0 Hz, 2H), 7.67 (d, J = 8.4 Hz, 1H), 7.40 (m, 4H), 7.00 (t, J = 7.6 Hz, 1H), 3.82 (s, 3H); 13C NMR (CDCl3) δ 168.1, 140.0, 139.0, 134.3, 132.9, 131.0, 128.8, 126.9, 122.9, 118.9, 115.7, 52.3.

Methyl 2-(4-chlorophenylsulfonamido)benzoate 14i

1H NMR (DMSO-d6) δ 10.41 (s, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.77 (d, J = 8.8 Hz, 2H), 7.63 (d, J = 8.8 Hz, 2H), 7.57 (t, J = 8.0 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.21 (t, J = 8.0 Hz, 1H), 3.79 (s, 3H); 13C NMR (DMSO-d6) δ 167.3, 138.3, 138.0, 137.5, 134.2, 130.9, 129.4, 128.8, 124.2, 120.5, 118.6, 52.5.

General Procedure for 15a-i

To a stirring suspension of potassium t-butoxide (5.88 mmol) in Et2O (15mL) cooled to 0 °C, was added water (1.4 mmol) via syringe. The slurry was stirred for 5 min, and 14a-i (0.67 mmol) was added. The mixture was stirred at room temperature until starting material disappeared by TLC analysis (20% EtOAc in hexanes). Ice water was added until 2 clear layers formed. The aqueous layer was separated and acidified with 1 M HCl. The product was then extracted with Et2O (3 × 20 mL) and evaporated in vacuo to afford 15a-i.

4-(Nonylsulfonamido)benzoic acid 15a

mp = 193-194 °C; 1H NMR (MeOD) δ 7.99 (d, J = 8.2 Hz, 2H), 7.31 (d, J = 8.2 Hz, 2H), 3.17 (t, J = 8.0 Hz, 2H), 1.78 (m, 2H), 1.40 (m, 2H), 1.28 (m, 10H), 0.89 (t, J = 7.2 Hz, 3H); 13C NMR (MeOD) δ 169.3, 144.3, 132.3, 126.7, 118.8, 52.4, 32.9, 30.2, 30.2, 30.0, 28.9, 24.4, 23.6, 14.3; HRMS (FAB) calcd for C16H25NO4S [M]+, 327.15043; found, 327.14957. Anal. (C16H25NO4S·¼(H2O)) C, H, N.

4-(Phenylsulfonamido)benzoic acid 15b

mp = 186-188 °C; 1H NMR (DMSO-d6) δ 12.72 (br s, 1H), 10.82 (br s, 1H), 7.80 (m, 4H), 7.61 (t, J = 6.8 Hz, 1H), 7.56 (t, J = 8.0 Hz, 2H), 7.20 (t, J = 7.2 Hz, 2H); 13C NMR (DMSO-d6) δ 166.6, 141.9, 142.0, 133.2, 130.7, 129.4, 126.6, 125.6, 118.2.; HRMS (FAB) calcd for C13H11NO4S [M]+, 277.04088; found, 277.04077. Anal. (C13H11NO4S) C, H, N.

4-(4-Chlorophenylsulfonamido)benzoic acid 15c

mp = 254-256 °C; 1H NMR (DMSO-d6) δ 12.76 (br s, 1H), 10.86 (br s, 1H), 7.81 (d, J = 6.4 Hz, 4H), 7.65 (d, J = 7.2 Hz, 2H), 7.18 (d, J = 6.8 Hz, 2H); 13C NMR (DMSO-d6) δ 166.6, 141.5, 138.1, 138.0, 130.7, 129.5, 128.5, 125.9, 118.4; HRMS (FAB) calcd for C13H11ClNO4S [M + H]+, 312.00973; found, 312.00859. Anal. (C13H10ClNO4S·¼(H2O)) C, H, N.

3-(Nonylsulfonamido)benzoic acid 15d

mp = 183-184 °C; 1H NMR (DMSO-d6) δ 13.03 (br s, 1H), 9.98 (s, 1H), 7.81 (s, 1H), 7.64 (m, 1H), 7.44 (m, 2H), 3.07 (t, J = 7.6 Hz, 2H), 1.65 (m, 2H), 1.21 (m, 12H), 0.83 (t, J = 7.2 Hz, 3H); 13C NMR (DMSO-d6) δ 166.8, 138.7, 131.8, 129.5, 124.3, 123.2, 119.7, 50.5, 31.1, 28.5, 28.5, 28.3, 27.1, 22.9, 22.0, 13.8; HRMS (FAB) calcd for C16H26NO4S [M + H]+, 328.15826; found, 328.15640. Anal. (C16H25NO4S) C, H, N.

3-(Phenylsulfonamido)benzoic acid 15e

mp = 203-204 °C; 1H NMR (DMSO-d6) δ 13.02 (br s, 1H), 10.51 (br s, 1H), 7.75 (d, J = 7.2 Hz, 2H), 7.68 (s, 1H), 7.56 (m, 4H), 7.34 (m, 2H); 13C NMR (DMSO-d6) δ 166.6, 139.2, 137.9, 133.0, 131.7, 129.4, 129.3, 126.5, 124.8, 124.0, 120.5; HRMS (FAB) calcd for C13H11NO4S [M]+, 277.04088; found, 277.04054. Anal. (C13H11NO4S·¼(H2O)) C, H, N.

3-(4-Chlorophenylsulfonamido)benzoic acid 15f

mp = 242-243 °C; 1H NMR (MeOD) δ 7.76 (d, J = 8.8 Hz, 4H), 7.52 (d, J = 8.4 Hz, 2H), 7.34 (m, 2H); 13C NMR (MeOD) δ 168.9, 140.2, 139.5, 139.0, 133.0, 130.3, 130.3, 129.8, 127.0, 126.4, 123.1; HRMS (FAB) calcd for C13H10ClNO4S [M]+, 311.00191; found, 311.00152. Anal. (C13H10ClNO4S) C, H, N.

2-(Nonylsulfonamido)benzoic acid 15g

mp = 122-124 °C; 1H NMR (MeOD) δ 8.11 (d, J = 8.0 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.16 (t, J = 7.6 Hz, 1H), 3.18 (t, J = 8.0 Hz, 2H), 1.71 (m, 2H), 1.24 (m, 12H), 0.88 (t, J = 7.2 Hz, 3H); 13C NMR (MeOD) δ 171.3, 142.4, 135.7, 133.1, 123.8, 118.8, 117.0, 52.3, 32.9, 30.2, 30.1, 29.9, 28.8, 24.4, 23.6, 14.4; HRMS (FAB) calcd for C16H25NO4S [M]+, 327.15043; found, 327.15044. Anal. (C16H25NO4S) C, H, N.

2-(Phenylsulfonamido)benzoic acid 15h

mp = 213-215 °C; 1H NMR (MeOD) δ 7.95 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 8.0 Hz, 2H), 7.69 (d, J = 8.4 Hz, 1H), 7.56 (t, J = 7.6 Hz, 1H), 7.49 (m, 3H), 7.09 (t, J = 7.6 Hz, 1H); 13C NMR (DMSO-d6) δ 169.7, 139.7, 138.5, 134.4, 133.5, 131.5, 129.4, 126.8, 123.3, 118.4, 116.7; HRMS (FAB) calcd for C13H11NO4S [M]+, 277.04088; found, 277.04124. Anal. (C13H11NO4S·¼(H2O)) C, H, N.

2-(4-Chlorophenylsulfonamido)benzoic acid 15i

mp = 202-203 °C; 1H NMR (DMSO-d6) δ 13.98 (br s, 1H), 11.12 (br s, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 8.8 Hz, 2H), 7.63 (d, J = 8.8 Hz, 2H), 7.54 (t, J = 7.6 Hz, 1H), 7.48 (d, J = 8.4 Hz, 1H), 7.14 (t, J = 7.2 Hz, 1H); 13C NMR (MeOD) δ 171.1, 141.3, 140.6, 139.0, 135.4, 132.8, 130.3, 129.9, 124.7, 120.6, 118.3; HRMS (FAB) calcd for C13H10ClNO4S [M]+, 311.00191; found, 311.00136. Anal. (C13H10ClNO4S) C, H, N.

General Procedure for 16a-f

To a stirring solution of the aniline starting material (3.3 mmol) in CH2Cl2 (12 mL) at 0 °C was added pyridine (7.5 equiv). The sulfonyl chloride (1.2 equiv) was then added slowly via syringe. The solution was stirred and allowed to warm to room temperature. Reaction progress was monitored by TLC (20% EtOAc in hexanes). When complete, the reaction was poured into saturated NaHCO3 (45 mL), extracted with CH2Cl2 (3 × 15 mL), and washed with 1 M HCl (50 mL). The combined organic phases were concentrated in vacuo, and the resulting solid was recrystallized from EtOAc / hexanes to afford 16a-f.

Methyl 4-(phenylmethylsulfonamido)benzoate 16a

1H NMR (DMSO-d6) δ 10.37 (s, 1H), 7.90 (d, J = 8.0 Hz, 2H), 7.33 (m, 3H), 7.24 (m, 4H), 4.58 (s, 2H), 3.82 (s, 3H); 13C NMR (DMSO-d6) δ 165.7, 143.1, 130.9, 130.6, 129.2, 128.4, 128.3, 123.6, 117.2, 57.2, 51.8.

Methyl 4-(2-phenylethylsulfonamido)benzoate 16b

1H NMR (CDCl3) δ 7.97 (d, J = 7.4 Hz, 2H), 7.29 (m, 3H), 7.13 (d, J = 7.4 Hz, 2H), 7.06 (d, J = 6.8 Hz, 2H), 6.68 (s, 1H), 3.91 (s, 3H), 3.43 (t, J = 8.0 Hz, 2H), 3.14 (t, J = 8.0 Hz, 2H); 13C NMR (CDCl3) δ 166.3, 140.9, 137.1, 131.2, 129.0, 128.3, 127.1, 126.1, 118.1, 53.0, 52.1, 29.8.

Methyl 3-(phenylmethylsulfonamido)benzoate 16c

1H NMR (DMSO-d6) δ 10.11 (s, 1H), 7.78 (s, 1H), 7.65 (d, J = 6.8 Hz, 1H), 7.44 (m, 2H), 7.33 (m, 3H), 7.26 (m, 2H), 4.50 (s, 2H), 3.85 (s, 3H); 13C NMR (DMSO-d6) δ 165.8, 138.9, 130.8, 130.6, 129.6, 129.2, 128.3, 128.2, 123.8, 123.3, 119.1, 57.1, 52.2.

Methyl 3-(2-phenylethylsulfonamido)benzoate 16d

1H NMR (CDCl3) δ 7.84 (d, J = 7.6 Hz, 1H), 7.79 (s, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.41 (t, J = 7.6 Hz, 1H), 7.26 (m, 4H), 7.14 (d, J = 7.6 Hz, 2H), 3.96 (s, 3H), 3.41 (t, J = 7.6 Hz, 2H), 3.16 (t, J = 7.6 Hz, 2H); 13C NMR (CDCl3) δ 166.4, 137.3, 137.1, 131.4, 129.7, 128.9, 128.3, 127.0, 126.0, 124.6, 121.2, 52.8, 52.4, 29.7.

Methyl 2-(phenylmethylsulfonamido)benzoate 16e

1H NMR (CDCl3) δ 10.34 (s, 1H), 7.97 (d, J = 7.6 Hz, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.46 (t, J = 7.6 Hz, 1H), 7.25 (m, 3H), 7.14 (d, J = 7.2 Hz, 2H), 7.07 (t, J = 7.6 Hz, 1H), 4.36 (s, 2H), 3.77 (s, 3H); 13C NMR (CDCl3) δ 167.6, 140.5, 134.4, 131.0, 130.2, 128.3, 128.3, 127.8, 122.4, 117.4, 114.9, 57.7, 52.0.

Methyl 2-(2-phenylethylsulfonamido)benzoate 16f

1H NMR (CDCl3) δ 10.52 (s, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.56 (t, J = 8.4 Hz, 1H), 7.25 (m, 3H), 7.11 (m, 3H), 3.95 (s, 3H), 3.45 (t, J = 8.0 Hz, 2H), 3.14 (t, J = 8.0 Hz, 2H); 13C NMR (CDCl3) δ 168.1, 140.6, 137.1, 134.7, 131.4, 128.5, 128.0, 126.7, 122.5, 117.5, 114.9, 52.9, 52.3, 29.4.

General Procedure for 17a-f

To a stirring suspension of potassium t-butoxide (5.88 mmol) in Et2O (15 mL) cooled to 0 °C, was added water (1.4 mmol) via syringe. The slurry was stirred for 5 min, and 16a-f (0.67 mmol) was added. The mixture was stirred at room temperature until starting material disappeared by TLC analysis (20% EtOAc in hexanes). Ice water was added until 2 clear layers formed. The aqueous layer was separated and acidified with 1 M HCl. The product was then extracted with Et2O (3 × 20 mL) and evaporated in vacuo to afford 17a-f.

4-(Phenylmethylsulfonamido)benzoic acid 17a

mp = 221-223 °C; 1H NMR (DMSO-d6) δ 12.72 (br s, 1H), 10.29 (s, 1H), 7.88 (d, J = 8.4 Hz, 2H), 7.33 (m, 3H), 7.24 (m, 4H), 4.56 (s, 2H); 13C NMR (DMSO-d6) δ 166.8, 142.7, 130.9, 130.8, 129.2, 128.3, 128.3, 124.8, 117.2, 57.1; HRMS (FAB) calcd for C14H14NO4S [M + H]+, 292.06435; found, 292.06397. Anal. (C14H13NO4S) C, H, N.

4-(2-Phenylethylsulfonamido)benzoic acid 17b

mp = 222-223 °C; 1H NMR (DMSO-d6) δ 12.74 (br s, 1H), 10.38 (s, 1H), 7.90 (d, J = 8.0 Hz, 2H), 7.26 (m, 2H), 7.23 (m, 2H), 7.18 (m, 3H), 3.48 (t, J = 6.4, 2H), 2.98 (t, J = 6.4 Hz, 2H); 13C NMR (DMSO-d6) δ 166.8, 142.4, 137.8, 130.8, 128.4, 128.3, 126.5, 125.2, 117.8, 51.9, 29.0; HRMS (FAB) calcd for C15H16NO4S [M + H]+, 306.08000; found, 306.07892. Anal. (C15H15NO4S) C, H, N.

3-(Phenylmethylsulfonamido)benzoic acid 17c

mp = 205-206 °C; 1H NMR (DMSO-d6) δ 13.02 (br s, 1H), 10.06 (s, 1H), 7.79 (s, 1H), 7.64 (d, J = 7.2 Hz, 1H), 7.42 (m, 2H), 7.33 (m, 3H), 7.25 (m, 2H), 4.48 (s, 2H); 13C NMR (DMSO-d6) δ 166.9, 138.7, 131.8, 130.9, 129.4, 129.3, 128.3, 128.2, 124.1, 122.9, 119.4, 57.0; HRMS (FAB) calcd for C14H14NO4S [M + H]+, 292.06435; found, 292.06448. Anal. (C14H13NO4S) C, H, N.

3-(2-Phenylethylsulfonamido)benzoic acid 17d

mp = 199-200 °C; 1H NMR (DMSO-d6) δ 13.06 (s, 1H), 10.11 (s, 1H), 7.85 (s, 1H), 7.67 (d, J = 6.8 Hz, 1H), 7.48 (m, 2H), 7.24 (m, 2H), 7.17 (m, 3H), 3.38 (t, J = 8.0 Hz, 2H), 2.99 (t, J = 8.0 Hz, 2H); 13C NMR (DMSO-d6) δ 166.8, 138.5, 137.9, 131.9, 129.6, 128.4, 128.3, 126.5, 124.6, 123.8, 120.2, 51.7, 29.0; HRMS (FAB) calcd for C15H16NO4S [M + H]+, 306.08000; found, 306.08051. Anal. (C15H15NO4S) C, H, N.

2-(Phenylmethylsulfonamido)benzoic acid 17e

mp = 216-219 °C; 1H NMR (DMSO-d6) δ 13.86 (br s, 1H), 10.68 (s, 1H), 7.99 (d, J = 7.6 Hz, 1H), 7.58 (m, 2H), 7.32 (m, 3H), 7.19 (m, 3H), 4.69 (s, 2H); 13C NMR (DMSO-d6) δ 169.6, 140.7, 134.6, 131.5, 130.7, 128.8, 128.4, 128.3, 122.4, 117.2, 115.4, 57.2; HRMS (FAB) calcd for C14H13NO4S [M]+, 291.05653; found, 291.05655. Anal. (C14H13NO4S) C, H, N.

2-(2-Phenylethylsulfonamido)benzoic acid 17f

mp = 157-159 °C; 1H NMR (DMSO-d6) δ 13.90 (br s, 1H), 10.74 (br s, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.61 (d, J = 4.4 Hz, 2H), 7.20 (m, 2H), 7.16 (m, 4H), 3.61 (t, J = 8.0 Hz, 2H), 2.98 (t, J = 8.0 Hz, 2H); 13C NMR (DMSO-d6) δ 169.7, 140.3, 137.5, 134.6, 131.6, 128.3, 128.2, 126.5, 122.6, 117.7, 115.9, 52.0, 28.9; HRMS (FAB) calcd for C15H16NO4S [M + H]+, 306.08000; found, 306.07886. Anal. (C15H15NO4S) C, H, N.

General Procedure for 19a-c

The starting bromide 18 (1.96 mmol) was added to a round-bottomed flask containing diethyl phosphite (2.35 mmol), tetrakis(triphenylphosphine)palladium (0) (0.04 mmol), Et3N (2.94 mmol), and EtOH (8 mL), and the solution was heated to reflux overnight (16 h). The solution was then diluted with 30 mL EtOAc, washed with 50 mL saturated NaHCO3 solution, 50 mL H2O, dried over MgSO4, and concentrated in vacuo. The product was then purified by flash chromatography (EtOAc).

Diethyl 4-acetamidophenylphosphonate 19a

Characterization data are in agreement with literature values.36

Diethyl 3-acetamidophenylphosphonate 19b

1H NMR (CDCl3) δ 9.91 (s, 1H), 8.29 (s, 1H), 7.89 (d, J = 15.0 Hz, 1H), 7.37 (m, 2H), 4.02 (m, 4H), 2.17 (s, 3H), 1.26 (t, J = 7.2 Hz, 6H); 13C NMR (CDCl3) δ 169.3, 139.5 (d, J = 18.4 Hz), 129.2, (d, J = 16.1 Hz), 127.5 (d, J = 185 Hz), 125.7 (d, J = 8.6 Hz), 123.7 (d, J = 3.2 Hz), 122.7 (d, J = 12.1 Hz), 62.2 (d, J = 5.3 Hz), 24.0, 16.0 (d, J = 6.5 Hz).

Diethyl 2-acetamidophenylphosphonate 19c

Characterization data are in agreement with literature values.37

Diethyl 4-(octylsulfonamido)phenylphosphonate 20a

1H NMR (CDCl3) δ 8.87 (s, 1H), 7.72 (dd, J = 13.2, 7.8 Hz, 2H), 7.37 (dd, J = 7.8, 3.6 Hz, 2H), 4.08 (m, 4H), 3.10 (t, J = 8.0 Hz, 2H), 1.79 (m, 2H), 1.29 (t, J = 7.2 Hz, 6H), 1.23 (m, 10H), 0.82 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3) δ 142.0 (d, J = 3 Hz), 133.2 (d, J = 10.9 Hz), 122.4 (d, J = 193 Hz), 118.1 (d, J = 15.4 Hz), 62.2 (d, J = 5.5 Hz), 51.9, 31.5, 28.8, 28.7, 28.0, 23.2, 22.4, 16.1 (d, J = 6.5 Hz), 13.8.

Diethyl 3-(octylsulfonamido)phenylphosphonate 20b

1H NMR (CDCl3) δ 9.53 (s, 1H), 7.89 (d, J = 15.2 Hz, 1H), 7.67 (s, 1H), 7.38 (m, 2H), 4.11 (m, 4H), 3.04 (t, J = 8.0 Hz, 2H), 1.77 (m, 2H), 1.28 (t, J = 7.2 Hz, 6H), 1.17 (m, 10H), 0.80 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3) δ 139.0 (d, J = 9.2 Hz), 129.6 (d, J = 16.2 Hz), 128.9 (d, J = 189 Hz), 125.9 (d, J = 8.3 Hz), 123.1 (d, J = 12.6 Hz), 122.2 (d, J = 3.0 Hz), 62.4 (d, J = 5.8 Hz), 51.5, 31.4, 28.7, 28.7, 28.0, 23.1, 22.3, 16.0 (d, J = 6.4 Hz), 13.8.

Diethyl 2-(octylsulfonamido)phenylphosphonate 20c

1H NMR (CDCl3) δ 9.94 (s, 1H), 7.73 (t, J = 8.0 Hz, 1H), 7.59 (m, 1H), 7.53 (m, 1H), 7.14 (dt, J = 7.2, 2.8 Hz, 1H), 4.09 (m, 4H), 3.14 (t, J = 8.0 Hz, 2H), 1.84 (m, 2H), 1.34 (t, J = 7.2 Hz, 6H), 1.26 (m, 10H), 0.86 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3) δ 142.2 (d, J = 7.4 Hz), 134.2 (d, J = 2.3 Hz), 133.1 (d, J = 5.9 Hz), 122.9 (d, J = 13.3 Hz), 118.3 (d, J = 11.5 Hz), 114.0 (d, J = 180 Hz), 62.7 (d, J = 5.2 Hz), 52.2, 31.6, 28.9, 28.8, 28.1, 23.2, 22.4, 16.1 (d, J = 6.5 Hz), 13.9.

4-(Octylsulfonamido)phenylphosphonic acid 21a

mp = 185-187 °C; 1H NMR (MeOD) δ 7.75 (dd, J = 12.8, 8.0 Hz, 2H), 7.33 (dd, J = 8.0, 3.2 Hz, 2H), 3.15 (t, J = 8.0 Hz, 2H), 1.78 (m, 2H), 1.39 (m, 2H), 1.28 (m, 8H), 0.90 (t, J = 7.2 Hz, 3H); 13C NMR (MeOD) δ 143.0 (d, J = 3.6 Hz), 133.4 (d, J = 11.0 Hz), 127.7 (d, J = 190 Hz), 119.1 (d, J = 15.2 Hz), 52.4, 32.8, 30.0, 29.9, 29.0, 24.5, 23.6, 14.3; HRMS (FAB) calcd for C14H25NO5PS [M + H]+, 350.11911; found, 350.11869. Anal. (C14H24NO5PS·0.6(H2O)) C, H, N.

3-(Octylsulfonamido)phenylphosphonic acid 21b

mp = 112-114 °C; 1H NMR (MeOD) δ 7.72 (d, J = 14.8 Hz, 1H), 7.55 (m, 1H), 7.44 (m, 2H), 3.12 (t, J = 8.0 Hz, 2H), 1.78 (m, 2H), 1.39 (m, 2H), 1.27 (m, 8H), 0.90 (t, J = 7.2 Hz, 3H); 13C NMR (MeOD) δ 139.6 (d, J = 18.2 Hz), 134.5 (d, J = 184 Hz), 130.5 (d, J = 16.1 Hz), 127.3 (d, J = 9.5 Hz), 123.8 (d, J = 3.0 Hz), 122.8 (d, J = 11.6 Hz), 52.2, 32.7, 30.0, 29.9, 29.0, 24.4, 23.5, 14.3; HRMS (FAB) calcd for C14H25NO5PS [M + H]+, 350.11911; found, 350.11879. Anal. (C14H24NO5PS) C, H, N.

2-(Octylsulfonamido)phenylphosphonic acid 21c

mp = 92-94 °C; 1H NMR (MeOD) δ 7.70 (m, 2H), 7.54 (t, J = 8.4 Hz, 1H), 7.21 (t, J = 7.5 Hz, 1H), 3.18 (t, J = 7.8 Hz, 2H), 1.77 (m, 2H), 1.23 (m, 10H), 0.89 (t, J = 7.5 Hz, 3H); 13C NMR (MeOD) δ 141.8 (d, J = 7.0 Hz), 134.3 (d, J = 2.7 Hz), 134.1 (d, J = 6.8 Hz), 120.4 (d, J = 178 Hz), 119.6 (d, J = 10.8 Hz), 52.6, 32.8, 29.9, 29.9, 29.0, 24.3, 23.5, 14.3; HRMS (FAB) calcd for C14H25NO5PS [M + H]+, 350.11911; found, 350.11826. Anal. (C14H24NO5PS) C, H, N.

General Procedure for 23a-f

To a stirring solution of the aniline starting material (3.3 mmol) in CH2Cl2 (12 mL) at 0 °C was added pyridine (7.5 equiv). The sulfonyl chloride (1.2 equiv) was then added slowly via syringe. The solution was stirred and allowed to warm to room temperature. Reaction progress was monitored by TLC (20% EtOAc in hexanes). When complete, the reaction was poured into saturated NaHCO3 solution (45 mL), extracted with CH2Cl2 (3 × 15 mL), and washed with 1 M HCl (50 mL). The combined organic phases were concentrated in vacuo, and separated by flash chromatography (20 % EtOAc in hexanes) to afford 23a-f.

Methyl 2-(methylsulfonamido)benzoate 23a

1H NMR (CDCl3) δ 10.43 (s, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.54 (t, J = 8.4 Hz, 1H), 7.11 (t, J = 7.6 Hz, 1H), 3.91 (s, 3H), 3.05 (s, 3H); 13C NMR (CDCl3) δ 168.2, 140.7, 134.8, 131.4, 122.7, 117.9, 115.3, 52.4, 39.8.

Methyl 2-(tetradecylsulfonamido)benzoate 23b

1H NMR (CDCl3) δ 10.42 (s, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.51 (t, J = 8.4 Hz, 1H), 7.08 (t, J = 7.6 Hz, 1H), 3.91 (s, 3H), 3.12 (t, J = 8.0 Hz, 2H), 1.78 (m, 2H), 1.23 (m, 22H), 0.87 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3) δ 168.2, 141.0, 134.7, 131.4, 122.4, 117.6, 114.9, 52.4, 52.0, 31.8, 29.5, 29.5, 29.5, 29.4, 29.3, 29.2, 29.0, 28.8, 27.9, 23.2, 22.5, 14.0.

Methyl 2-(hexadecylsulfonamido)benzoate 23c

1H NMR (CDCl3) δ 10.42 (s, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.54 (t, J = 8.0 Hz, 1H), 7.11 (t, J = 8.0 Hz, 1H), 3.94 (s, 3H), 3.14 (t, J = 8.0 Hz, 2H), 1.81 (m, 2H), 1.26 (m, 26H), 0.88 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3) δ 168.3, 141.0, 134.8, 131.4, 122.4, 117.7, 115.0, 52.4, 52.1, 31.8, 29.6, 29.6, 29.6, 29.6, 29.5, 29.5, 29.4, 29.3, 29.1, 28.9, 28.0, 23.3, 22.6, 14.0.

Methyl 5-chloro-2-(nonylsulfonamido)benzoate 23d

1H NMR (CDCl3) δ 10.31 (br s, 1H), 8.01 (d, J = 2.4 Hz, 1H), 7.73 (d, J = 9.0 Hz, 1H), 7.48 (dd, J = 9.0, 2.4 Hz, 1H), 3.95 (s, 1H), 3.12 (t, J = 7.8 Hz, 2H), 1.78 (m, 2H), 1.23 (m, 12H), 0.87 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3) δ 167.3, 139.6, 134.6, 131.0, 127.8, 119.2, 116.2, 52.8, 52.3, 31.7, 29.0, 28.8, 27.9, 27.9, 23.2, 22.5, 14.0.

Methyl 5-hydroxy-2-(octylsulfonamido)benzoate 23e

1H NMR (CDCl3) δ 9.91 (s, 1H), 7.62 (d, J = 9.0 Hz, 1H), 7.52 (d, J = 3.0 Hz, 1H), 7.07 (dd, J = 3.0, 9.0 Hz, 1H), 6.36 (s, 1H), 3.91 (s, 3H), 3.05 (t, J = 8.1 Hz, 2H), 1.76 (m, 2H), 1.22 (m, 10H), 0.86 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3) δ 168.0, 151.7, 133.3, 122.3, 120.7, 117.3, 117.0, 52.6, 51.7, 31.5, 28.8, 28.7, 27.9, 23.1, 22.4, 13.9.

Methyl 5-fluoro-2-(octylsulfonamido)benzoate 23f

1H NMR (CDCl3) δ 10.15 (s, 1H), 7.75 (m, 2H), 7.25 (m, 1H), 3.95 (s, 3H), 3.09 (t, J = 8.0 Hz, 2H), 1.76 (m, 2H), 1.22 (m, 10H), 0.85 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3) δ 167.2 (d, J = 2.2 Hz), 157.5 (d, J = 242 Hz), 137.1 (d, J = 2.8 Hz), 122.0 (d, J = 22.4 Hz), 120.1 (d, J = 7.4 Hz), 117.4 (d, J = 24.3 Hz), 116.4 (d, J = 6.9 Hz), 52.7, 52.1, 31.5, 28.7, 28.7, 27.9, 23.2, 22.4, 13.8.

General Procedure for 24a-f

To a stirring suspension of potassium t-butoxide (5.88 mmol) in Et2O (15 mL) cooled to 0 °C was added water (1.4 mmol) via syringe. The slurry was stirred for 5 min, and 23a-f (0.67 mmol) was added. The mixture was stirred at room temperature until starting material disappeared by TLC analysis (20% EtOAc in hexanes). Ice water was added until two clear layers formed. The aqueous layer was separated and acidified with 1 M HCl, and the product was extracted with Et2O (3 × 20 mL) and evaporated in vacuo. If necessary, the product was then recrystallized (EtOAc / hexanes) to afford pure 24a-f.

2-(Methylsulfonamido)benzoic acid 24a

mp = 187-189 °C; 1H NMR (MeOD) δ 8.11 (d, J = 8.0 Hz, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.60 (t, J = 7.2 Hz, 1H), 7.17 (t, J = 7.6 Hz, 1H), 3.08 (s, 3H); 13C NMR (MeOD) δ 171.2, 142.2, 135.7, 133.0, 123.9, 119.2, 117.3, 39.9; HRMS (FAB) calcd for C8H9NO4S [M]+, 215.02523; found, 215.02576. Anal. (C8H9NO4S) C, H, N.

2-(Tetradecylsulfonamido)benzoic acid 24b

mp = 120-122 °C; 1H NMR (MeOD) δ 8.12 (d, J = 8.0 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.17 (t, J = 7.6 Hz, 1H), 3.19 (t, J = 8.0 Hz, 2H), 1.70 (m, 2H), 1.29 (m, 22H), 0.91 (t, J = 6.8 Hz, 3H); 13C NMR (MeOD) δ 171.4, 142.5, 135.7, 133.1, 123.8, 118.8, 117.0, 52.2, 33.0, 30.7, 30.7, 30.7, 30.6, 30.5, 30.4, 30.2, 29.9, 28.8, 24.4, 23.7, 14.4; HRMS (FAB) calcd for C21H35NO4S [M]+, 397.22835; found, 397.22835. Anal. (C21H35NO4S·¼(H2O)) C, H, N.

2-(Hexadecylsulfonamido)benzoic acid 24c

mp = 126-128 °C; 1H NMR (MeOD) δ 8.12 (d, J = 7.6 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.59 (t, J = 8.0 Hz, 1H), 7.17 (t, J = 7.6 Hz, 1H), 3.19 (t, J = 7.6 Hz, 2H), 1.73 (m, 2H), 1.23 (m, 26H), 0.91 (t, J = 7.2 Hz, 3H); 13C NMR (MeOD) δ 169.8, 140.7, 134.6, 131.6, 122.4, 117.3, 115.6, 50.9, 31.2, 29.0, 29.0, 29.0, 29.0, 29.0, 28.9, 28.8, 28.7, 28.5, 28.2, 27.0, 22.8, 22.0, 13.8; HRMS (FAB) calcd for C23H40NO4S [M + H]+, 426.26781; found, 426.26825. Anal. (C23H39NO4S) C, H, N.

5-Chloro-2-(nonylsulfonamido)benzoic acid 24d

mp = 101-103 °C; 1H NMR (MeOD) δ 8.05 (d, J = 2.8 Hz, 1H), 7.74 (d, J = 9.2 Hz, 1H), 7.59 (dd, J = 9.2, 2.8 Hz, 1H), 3.21 (t, J = 8.0 Hz, 2H), 1.72 (m, 2H), 1.23 (m, 12H), 0.90 (t, J = 7.2 Hz, 3H); 13C NMR (MeOD) δ 170.1, 141.2, 135.5, 132.4, 128.9, 120.6, 118.0, 52.5, 32.9, 30.2, 30.1, 29.9, 28.8, 24.4, 23.6, 14.4; HRMS (FAB) calcd for C16H24ClNO4S [M]+, 361.11146; found, 361.11063. Anal. (C16H24ClNO4S) C, H, N.

5-Hydroxy-2-(octylsulfonamido)benzoic acid 24e

mp = 142-144 °C; 1H NMR (MeOD) δ 7.56 (d, J = 8.8 Hz, 1H), 7.51 (d, J = 2.8 Hz, 1H), 7.03 (dd, J = 8.8, 2.8 Hz, 1H), 3.07 (t, J = 8.0 Hz, 2H), 1.68 (m, 2H), 1.23 (m, 10H), 0.89 (t, J = 7.2 Hz, 3H); 13C NMR (MeOD) δ 171.0, 154.6, 134.1, 122.8, 121.9, 119.2, 118.5, 53.1, 32.8, 29.9, 29.8, 28.8, 24.3, 23.6, 14.4. Anal. (C15H23NO5S) C, H, N.

5-Fluoro-2-(octylsulfonamido)benzoic acid 24f

mp = 141-143 °C; 1H NMR (MeOD) δ 7.77 (m, 2H), 7.38 (m, 1H), 3.17 (t, J = 8.0 Hz, 2H), 1.71 (m, 2H), 1.23 (m, 10H), 0.88 (t, J = 7.2 Hz, 3H); 13C NMR (MeOD) δ 170.2 (d, J = 1.8 Hz), 159.2 (d, J = 241 Hz), 138.6 (d, J = 2.7 Hz), 122.7 (d, J = 22.7 Hz), 121.5 (d, J = 7.6 Hz), 119.0 (d, J = 6.9 Hz), 118.8 (d, J = 24.1 Hz), 52.4, 32.8, 29.9, 29.9, 28.8, 24.4, 23.6, 14.3; HRMS (FAB) calcd for C15H22FNO4S [M]+, 331.12536; found, 331.12445. Anal. (C15H22FNO4S) C, H, N.

GPAT Assay

The mtGPAT assay has been reported previously.35 A mitochondrial preparation of glycerol 3-phosphate acyltransferase was added to the incubation mixture containing 14C-labeled glycerol 3-phosphate, palmitoyl-CoA, and varying inhibitor concentrations to initiate the reaction. After ten min, the reaction was terminated by adding chloroform, methanol, and 1% perchloric acid. Five min later, more chloroform and perchloric acid were added, and the upper aqueous layer was removed. After washing three times with 1% perchloric acid, the organic layer was evaporated under nitrogen, and the amount of 14C present was counted to determine the extent of reaction inhibition. Data points were recorded in triplicate, and IC50 values were calculated based on the amount of test inhibitor necessary to produce 50% of mtGPAT activity observed in the absence of inhibitor but in the presence of DMSO vehicle control.

Table 2
In Vitro Anti-mtGPAT1 Activity of Sulfonamides 15a-i and 17a-f

Supplementary Material

1_si_001

Acknowledgments

The work was supported by the NIH 1R43DK65423 to FASgen, Inc. and donors to the Raynam Trust. Under a license agreement between FASgen, Inc. and The Johns Hopkins University, CAT is entitled to share royalty received by the University on sales of products described in this article. CAT owns FASgen, Inc. stock, which is subject to certain restrictions under university policy. The Johns Hopkins University, in accordance with its conflict of interest policies, is managing the terms of this arrangement.

Abbreviations

GPAT
glycerol 3-phosphate acyltransferase
DIO
diet-induced obese
mtGPAT
mitochondrial glycerol 3-phosphate acyltransferase
LPA
lysophosphatidic acid
TAG
triacylglycerol
VLDL
very low density lipoprotein
DAG
diacylglycerol
CPT-1
carnitine palmitoyltransferase-1
AMPK
AMP-activated protein kinase
ACC
acetyl-CoA carboxylase
FAS
fatty acid synthase

Footnotes

Supporting Information Available: Elemental analysis data for target compounds.

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