C-3- and C-4-Substituted Bicyclic Coumarin Sulfamates as Potent Steroid Sulfatase Inhibitors

Abstract

Synthetic routes to potent bicyclic nonsteroidal sulfamate-based active-site-directed inhibitors of the enzyme steroid sulfatase (STS), an emerging target in the treatment of postmenopausal hormone-dependent diseases, including breast cancer, are described. Sulfamate analogs 9–27 and 28–46 of the core in vivo active two-ring coumarin template, modified at the 4- and 3-positions, respectively, were synthesized to expand structure–activity relationships. α-Alkylacetoacetates were used to synthesize coumarin sulfamate derivatives with 3-position modifications, and the bicyclic ring of other parent coumarins was primarily constructed via the Pechmann synthesis of hydroxyl coumarins. Compounds were examined for STS inhibition in intact MCF-7 breast cancer cells and in placental microsomes. Low nanomolar potency STS inhibitors were achieved, and some were found to inhibit the enzyme in MCF-7 cells ca. 100–500 more potently than the parent 4-methylcoumarin-7-O-sulfamate 3, with the best compounds close in potency to the tricyclic clinical drug Irosustat. 3-Hexyl-4-methylcoumarin-7-O-sulfamate 29 and 3-benzyl-4-methylcoumarin-7-O-sulfamate 41 were particularly effective inhibitors with IC₅₀ values of 0.68 and 1 nM in intact MCF-7 cells and 8 and 32 nM for placental microsomal STS, respectively. They were docked into the STS active site for comparison with estrone 3-O-sulfamate and Irosustat, showing their sulfamate group close to the catalytic hydrated formylglycine residue and their pendant group lying between the hydrophobic sidechains of L103, F178, and F488. Such highly potent STS inhibitors expand the structure–activity relationship for these coumarin sulfamate-based agents that possess therapeutic potential and may be worthy of further development.

Introduction

Breast cancer is a major health threat to women of all age groups and a prime contributor to cancer deaths in women. About two-thirds of cases when first diagnosed are classified as hormone-dependent (ER⁺), in which to grow and develop the tumors need estrogens, which act via the estrogen receptor (ER).¹ Endocrine therapy administered by the oral route is an effective form of treatment for this type of cancer.² Although newer targeted agents such as mTOR and CDK4/6 inhibitors, e.g., everolimus and palbociclib, are now gaining recognition in treatment, they are expensive and are administered in conjunction with endocrine therapy.^3,4 Currently, the first-line treatment for patients with hormone-dependent breast cancer (HDBC) includes either a selective estrogen-receptor modulator, such as tamoxifen, which blocks the action of estrogens at the ER or an ER downregulator (SERD),⁵ or a “third-generation” aromatase inhibitor (AI) such as letrozole, anastrozole, and exemestane. This strategy leads to a reduction in the biosynthesis of estrogens and has been found to be superior to tamoxifen alone.⁶ Also, in one study, anastrozole has shown significant preventative activity in high-risk postmenopausal women with undiagnosed breast cancer.⁷ However, resistance will inevitably occur and blocking the action of estrogens at the ER and inhibiting the aromatase enzyme are not the only strategies available for endocrine therapy. There is now evidence that inhibition of steroid sulfatase (STS),⁸ the enzyme that converts the biologically inactive estrone sulfate to estrone, as well as dehydroepiandrosterone sulfate to dehydroepiandrosterone, may render significant estrogen deprivation in patients treated with an STS inhibitor. This strategy works in an intracrine fashion because in the postmenopausal setting tumor cells can convert the large reservoir of circulating estrone sulfates, imported through organic anion transporters, to active estrogen in situ. Moreover, STS inhibition also decreases levels of androstenediol, an estrogenic androgen,⁹ levels of which are unaffected by AI inhbition. Chronic AI treatment leads to compensatory increases in both STS and 17β-HSD1 levels.¹⁰ Moreover, the contribution of both STS and organic anion transporters to AI resistance was recently established and could be overcome by an STS inhibitor.¹¹ These ideas have led to STS inhibitors reaching phase II clinical trials for several indications in oncology including breast cancer and endometrial cancer and a phase I trial in prostate cancer.¹²

The first STS inhibitor discovered with a remarkable potency was the steroidal sulfamate ester estrone-3-O-sulfamate (EMATE, 1, Figure 1).¹³ This agent is orally active and inhibits STS in an irreversible manner. However, EMATE was subsequently shown to be highly estrogenic in rats and this undesirable property effectively precluded its further development for use in the treatment of HDBC, although the estradiol variant (E2MATE, PGL2001, 1a) has nevertheless proceeded to clinical trials in the hormone-dependent nononcology setting of endometriosis.^12a,12b,14

Figure 1.

Structures of STS inhibitors: 1 (EMATE), 1a (E2MATE), and 2 (Irosustat, STX64, 667COUMATE, BN83495).

In an attempt to search for a nonestrogenic alternative to 1 with a comparable or even superior STS inhibitory profile, many structurally diverse inhibitors that contain the pharmacophore for irreversible inhibition of STS, i.e., an aryl sulfamate ester, have been developed,^8,12,15 leading to the clinical inhibitor Irosustat 2 (Figure 1).¹⁶ Initial work focused on designing A/B ring mimics of 1 such as derivatives of indanone, tetralone, and tetrahydronaphthol,¹⁵ and this yielded a series of bicyclic coumarin sulfamates¹⁷ (3–8, Figure 2) that were promising leads, showed a significant improvement over the first lead nonsteroidal candidate 5,6,7,8-tetrahydronaphthalene 7-O-sulfamate, and, more significantly, possessed in vivo activity. A main lead was the two-ring 4-methylcoumarin-7-O-sulfamate (3) that was orally active in vivo and, like EMATE, was a highly potent time- and concentration-dependent STS inhibitor, but importantly with no rodent estrogenic activity.^18,19 The related 3,4-dimethylcoumarin-7-O-sulfamate (6) inhibited STS in MCF-7 cells with IC₅₀ = 30 nM, and a series of derived tricyclic compounds was subsequently synthesized that proved even more potent.^16,17 Further development of this series of nonsteroidal inhibitors led to the discovery of the tricyclic coumarin sulfamate (2) (Irosustat, STX64, 667COUMATE, BN83495, Figure 1) which has proven to be the most successful STS inhibitor to date.^12,16 Recently, other examples of both mono-^20,21 and two-ring sulfamate-based STS inhibitors²² have been published, but these compounds are generally still of relatively modest inhibitory activity. Irosustat was the first STS inhibitor to enter clinical trials for postmenopausal patients with advanced HDBC and has shown encouraging results.^12,23−26 Irosustat has just completed CRUK sponsored phase II trials in both early breast cancer and in advanced breast cancer in combination with an AI, with positive indications of efficacy.^27,28

Figure 2.

Structures of bicyclic coumarin sulfamates 3–8, including COUMATE 3. X = OSO₂NH₂.

However, despite the significant progress made in developing irreversible inhibitors of STS and although 1a and 2 have reached clinical trials, their mechanism of action remains unresolved. The crystal structure of human STS has been solved,^29,30 and several hypotheses have been postulated to suggest how a sulfamate-based STS inhibitor might inhibit STS irreversibly. The currently favored hypothesis is a transfer of the sulfamoyl group (or as sulfonylamine) to a hydrated or unhydrated STS active site formylglycine residue, and this leads to inactivation of the active site machinery.^12b Although multiple mechanisms have been proposed for this, e.g. ref. (12e), see ref. (12b) for a full up-to-date discussion.

Because of the unique role that a coumarin ring system plays in the design of potent STS inhibitors, we further expand here the bicyclic coumarin sulfamate series exemplified by 3–8 to give derivatives that bear various substituents at the 3- and/or 4-position(s), following on from preliminary encouraging data.¹⁷ The inhibitory activities of most new candidates against STS were evaluated in MCF-7 cells and in placental microsomes. This study, in conjunction with other studies carried out on 2,^16,17 provides a more comprehensive structure–activity relationship (SAR) for coumarin sulfamates and has also produced highly active inhibitors of picomolar potency in vitro. The activity of two of the best bicyclic compounds is supported by molecular modeling and by comparing binding poses with those of the benchmark compounds EMATE and Irosustat.

Chemistry

The compounds synthesized in this work fall into two different series: (i) those with an alkyl group of increasing carbon chain length or other functionalities at the C-4 position of the coumarin ring (A, Figure 3) and (ii) those with an alkyl group of increasing carbon chain length or other functionalities at the C-3 position and with a methyl group at the C-4 position of the coumarin ring (B, Figure 3).

Figure 3.

General structures of bicyclic coumarin sulfamates: A: 4-substituted series, B: 3,4-disubstituted series.

We employed β-keto esters as starting materials for synthesizing coumarins with a substituent at the 3-position, and this also leads to a 4-methyl substituent. Because 4-methylcoumarin 7-O-sulfamate (COUMATE) is more active as an STS inhibitor than unsubstituted coumarin 7-O-sulfamate, we retained this 4-substituted group, which imbues good activity.^18,19 Thus, to study further the SAR of COUMATE, we ideally needed to keep this 4-methyl group for comparison while we explored different substituents at the 3-position. Apart from coumarin 46a, the bicyclic ring of other parent coumarins was constructed by the Pechmann synthesis of hydroxyl coumarins. For our purposes, this route was preferred because the target structures can be prepared with relative ease by condensing resorcinol with an appropriate β-keto ester. The only synthetic hurdle to overcome is the synthesis of the various β-keto esters required because most of them are not available commercially. The 7-hydroxycoumarins synthesized are subsequently sulfamoylated with freshly prepared sulfamoyl chloride to form the corresponding coumarin sulfamates.

The alkanoyl acetate esters required as starting material for the coumarins in the 4-alkyl series (A, Figure 3) were synthesized by treating the inexpensive ethyl potassium malonate with the corresponding acid chloride in the presence of magnesium chloride (MgCl₂), triethylamine (Et₃N), and acetonitrile (CH₃CN) as the solvent (Scheme 1).³¹ This method has the advantage of being relatively safe, clean, economical, and suitable for scaling up with the product produced in high yield and purity, which is free from any unnecessary side products. Rathke and Cowan³² have shown that the combination of anhydrous MgCl₂ and Et₃N provides a system with enough basicity for metallating ethyl potassium malonate and that the reactions failed when MgCl₂ was replaced with other metal chlorides such as ZnCl₂, CuCl₂, FeCl₃, TiCl₄, LiCl, and AlCl₃.³² The number of equivalents of reagents used in the reaction determines the yield of the product obtained. For aromatic acid chlorides which have electron-withdrawing substituents such as fluoro, chloro, or nitro groups, 2.1 equiv of potassium ethyl malonate, 2.5 equiv of MgCl₂, and 2.2 equiv of Et₃N are optimal, and the yield is around 90%. On the other hand, aliphatic acid chlorides or aromatic acid chlorides containing electron-donating substituents generate side products, which are minimized by employing extra equivalent of Et₃N to obtain the alkanoyl acetate in high yield.

Scheme 1. Synthesis of Various Ethyl Alkanoyl Acetates for the Preparation of 4-Alkylcoumarin Sulfamates.

(i) SOCl₂/tetrahydrofuran (THF), reflux; (ii) (a) MeCN, MgCl₂, Et₃N, 10–25 °C, 2.5 h, (b) RCOCl, Et₃N, 0 °C, 0.5 h, room temperature (rt), 12 h; (iii) RCHO, SnCl₂, CH₂Cl₂, rt, 3 h.

β-Ketoesters were also prepared efficiently by reacting the corresponding aldehyde with ethyl diazoacetate in the presence of a catalytic amount of tin(II) chloride (SnCl₂) (Scheme 1). The mechanism of the reaction is likely to proceed via a betaine intermediate, followed by a preferential migration of the aldehyde hydrogen to the β-carbon i.e., a 1,2-hydride shift producing the required β-keto ester and N₂ as products. The two most noteworthy aspects of this method are its selectivity and the mild conditions involved. The reaction is insensitive to atmosphere, is complete between 1 and 2 h at room temperature, and can be catalyzed by various Lewis acids, such as BF₃, ZnCl₂, ZnBr₂, AlCl₃, SnCl₂, GeCl₂, and SnCl₄, but the highest yield is obtained with SnCl₂.³³ Although other common organic solvents can be used, CH₂Cl₂ is often employed because it gives the best results and can be easily removed.

The α-alkylacetoacetates required for the synthesis of coumarins in the 3-substituted-4-methyl series were prepared conveniently by treating a solution of ethyl acetoacetate in CH₂Cl₂ with the corresponding alkyl bromide in the presence of potassium carbonate (K₂CO₃) and tetrabutylammonium chloride (Bu₄NCl) (Scheme 2).³⁴

Scheme 2. Synthesis of Various α-Alkylacetoacetates for the 3-Alkyl-4-methylcoumarin Sulfamates.

(i) RBr, K₂CO₃, H₂O, Bu₄NCl, CH₂Cl₂, reflux, 40 h.

Pechmann synthesis of coumarins with a β-keto ester and resorcinol was carried out in the presence of an equimolar mixture of trifluoroacetic acid (CF₃COOH) and concentrated sulfuric acid (H₂SO₄) warmed from ice-water temperature to room temperature (Schemes 3 and 4). The use of a 1:1 mixture of conc. H₂SO₄ and conc. CF₃COOH as the condensing agent for the Pechmann synthesis of coumarins was first described by Hua et al.,³⁵ and, in our hands, such a mixture has been found to be as effective as using conc. H₂SO₄ alone, which is playing a role as a catalyst. The role of conc. CF₃COOH in this reaction is not entirely clear, although it might be acting as an organic solvent and lowering the viscosity of the reaction mixture, rendering the stirring process more efficient.

Scheme 3. Synthesis of 4-Substituted-7-O-coumarin Sulfamates.

(i) Conc. H₂SO₄/conc. CF₃COOH, 3 h, 0 °C to rt, (ii) anhydrous dimethylformamide (DMF), NaH, N₂, 0 °C and H₂NSO₂Cl (∼5 equiv), 0 °C to rt.

Scheme 4. Synthesis of 4-Methyl-3-substituted-7-O-coumarin Sulfamates.

(i) Conc. H₂SO₄/conc. CF₃COOH, 3 h, 0 °C to rt; (ii) (Bu)₄N⁺HSO₄^–, CH₂Cl₂, rt; (iii) anhydrous DMF, NaH, N₂, 0 °C and H₂NSO₂Cl (∼5 equiv), 0 °C to rt.

The sulfamoylation reaction was performed by reacting the hydroxyl coumarins with an excess of (∼5 equiv) sulfamoyl chloride after treating with 1 equiv of NaH as described previously by Woo et al.³⁶

Biological Results and Discussion

The in vitro inhibition of STS activity by most of the sulfamates synthesized in this work was measured in two assay systems: (i) a preparation of an intact monolayer of MCF-7 cells, which assesses the ability of compounds to cross the cell membrane and inhibit STS under conditions that closely resemble the tissue/physiological situation and (ii) a placental microsomes preparation where a higher concentration of substrate is employed, with which a compound has to compete for binding to the enzyme active site. For the placental microsome STS assay, a saturating substrate concentration of 20 μM was used and inhibitors were tested under initial rate conditions. The MCF-7 STS assay is meant to mimic/reflect physiological conditions (intact, living cells). Hence, a physiological concentration of 3 nM of E1S was used. The results are reported as % inhibition at various concentrations, and IC₅₀ values are determined for several compounds (Tables 1–4). Although time- and concentration-dependence studies have not been carried out to confirm the nature of inhibition for those compounds tested, it is anticipated that they act mechanistically in a similar manner to other aryl sulfamates like 1 and 2, which have been shown to be active-site-directed inhibitors

Table 1. % Inhibition of STS Activity in Intact MCF-7 Breast Cancer Cells by 4-Alkylcoumarin Sulfamates Evaluated at Various Concentrations and Their IC₅₀ Values if Determined^a,c.

no.	R1	10 μM	1 μM	0.1 μM	0.01 μM	IC50 nM
3b	CH3	93	86	43	<10	*
4	–CH2CH3	*	*	*	*	*
5	–(CH2)2CH3	*	*	*	*	*
9	–(CH2)3CH3	93	91	85	*	*
10	–(CH2)4CH3	95	92	88	52	10
11	–(CH2)5CH3	93	90	87	*	*
12	–(CH2)6CH3	99	95	93	*	*
13	–(CH2)7CH3	99	97	90	*	*
14	–(CH2)8CH3	100	98	96	90	*
15	–(CH2)9CH3	100	98	95	86	*
16	–(CH2)10CH3	*	*	*	*	*
17	–(CH2)11CH3	98	95	67	22	4.3
18	–(CH2)12CH3	*	*	96	72	3.2
19	isopropyl	*	*	*	*	*
20	isobutyl	*	*	*	*	*
21	–CH2Cl	92	82	33	6	220
22	–Ph	87	79	36	*	*
23	–CH2Ph	84	70	55	8	75
24	–(CH2)2Ph	98	95	84	39	18
25	4-ethylphenyl	84	73	24	6	350
26	–C6H11	93	80	72	37	24
27	CH2Ad	*	*	*	*	*

^aUnless stated otherwise, errors are <5% of the reported value (from triplicate experiments).

^bRef (15).

^c*: Results not determined; cf. EMATE (IC₅₀ = 65 pM).^13b

Table 4. % Inhibition of STS Activity in Placental Microsomes by 3-Substituted-4-methylcoumarin Sulfamates Evaluated at Various Concentrations and Their IC₅₀ Values if Determined^a,d.

no.	R	10 μM	1 μM	0.1 μM	0.01 μM	IC50 nM
3b	H	93	63	<10	*	*
6b	–CH3	97	88	35	*	*
7c	–CH2CH3	>99	96	57	*	*
8c	–(CH2)2CH3	>99	97	83	*	*
28	–(CH2)4CH3	100	99	94	41	12
29	–(CH2)5CH3	99	99	88	13	32
30	–(CH2)6CH3	99	97	65	23	*
31	–(CH2)7CH3	99	98	71	21	*
32	–(CH2)8CH3	98	97	85	21	320
33	–(CH2)9CH3	94	79	33	3	250
34	–(CH2)10CH3	*	*	*	*	2000
35	–(CH2)11CH3	*	*	*	*	*
36	–(CH2)12CH3	*	*	*	*	*
37	–(CH2)13CH3	*	*	*	*	10 000
38	–(CH2)14CH3	*	*	*	*	*
39	Cl	*	*	*	*	*
40	Ph	99	95	65	8	54
41	–CH2Ph	*	99	94	53	8
42	–(CH2)2Ph	100	99	91	45	33
43	–(CH2)3Ph	*	99	47	*	*
44	–CH2(C6H11)	*	99	74	*	*
45	–(CH2)2(C6H11)	*	99	37	*	*
46	4-methoxyphenyl	*	*	*	*	*

^aUnless stated otherwise, errors are <5% of the reported value (from triplicate experiments).

^bRef (15).

^cRef (17).

^d*: Results not determined.

4-Substituted Compounds

MCF-7

For 4-n-alkyl derivatives 9–15, 17, and 18, all derivatives inhibit STS activity >90% at 1 μM. Based on the % inhibition observed at 0.1 and 0.01 μM, the inhibitory activity of the compounds increases slightly as the chain length of the alkyl group increases, and it peaks at the nonyl (14, 90% at 0.01 μM) and decyl (15, 86% at 0.01 μM) derivatives. This may be attributed to the increase in lipophilicity of the compounds as their alkyl group becomes longer until steric hindrance potentially becomes a limiting factor. A similar observation was reported in studies of (p-O-sulfamoyl)-N-alkanoyl tyramines.^37,38 We and others have also noted the existence of a hydrophobic pocket at the end of the steroid binding pocket³⁹ that might also be accessed by the present compound series to improve binding, although seeking the right compromise between hydrophobicity and steric hindrance is important.⁴⁰ For other substituents at the 4-position of the coumarin ring, their inhibitory activities vary with the phenethyl derivative 24 (39% at 0.01 μM, IC₅₀ = 18 nM) and the cyclohexyl derivative 26 (37% at 0.01 μM, IC₅₀ = 24 nM) being the most active. However, both 24 and 26 are less potent as STS inhibitors than the 4-n-alkyl derivatives. One possibility is that the active site of STS, like many other enzymes with steroids as substrate in general, has limited accommodation for substituents at the C1/C11/C12 edge of the steroid scaffold. Hence, the more flexible aliphatic alkyl chains may be better tolerated by the enzyme active site than the bulkier and more rigid substituents such as phenyl, benzyl, phenethyl, 4-ethylphenyl, and cyclohexyl when these substituents are placed at the 4-position of the coumarin ring system, which mimics the A/B ring of the steroidal STS inhibitor 1.

Placental Microsomes

Apart from the two lower members 3 and 4 and the two higher members 17 and 18, other 4-n-alkyl derivatives (5–16) tested show >90% inhibition at 1 μM. However, the SAR between chain length and inhibitory activity is not clear because all derivatives evaluated show the same order of magnitude in regard to % inhibition at 0.01 μM and IC₅₀ values. Nonetheless, the n-pentyl (10, IC₅₀ = 40 nM) and n-dodecyl (17, IC₅₀ = 45 nM) derivatives appear to be the most active inhibitors in this group. For those derivatives that have bulkier substituents at the 4-position, they are significantly less potent than their n-alkyl derivatives with the exception of 23 (benzyl, IC₅₀ = 64 nM), 24 (phenethyl, IC₅₀ = 82 nM), and 26 (cyclohexyl, IC₅₀ = 42 nM), the IC₅₀ values of which are of the same order of magnitude as those of n-alkyl derivatives. As expected for a cell-based assay, the IC₅₀ values against STS obtained for compounds in Table 1 are much lower than those obtained from the cell-free placental microsomes assay (Tables 2 and 3). A similar phenomenon was observed in previous work.⁴¹

Table 2. % Inhibition of STS Activity in Placental Microsomes by 4-Alkylcoumarin Sulfamates Evaluated at Various Concentrations and Their IC₅₀ Values if Determined^a,d.

no.	R1	10 μM	1 μM	0.1 μM	0.01 μM	IC50 nM
3b	CH3	93	63	<10	*	*
4c	–CH2CH3	>99	88	35	*	*
5c	–(CH2)2CH3	96	94	42	*	*
9	–(CH2)3CH3	98	91	49	10	102
10	–(CH2)4CH3	99	97	70	22	40
11	–(CH2)5CH3	99	97	70	6	52
12	–(CH2)6CH3	99	97	64	8	90
13	–(CH2)7CH3	99	97	74	28	68
14	–(CH2)8CH3	99	97	79	18	60
15	–(CH2)9CH3	99	96	74	17	60
16	–(CH2)10CH3	97	92	56	8	73
17	–(CH2)11CH3	96	89	68	17	45
18	–(CH2)12CH3	91	80	55	7	85
19	isopropyl	94	90	86	*	*
20	isobutyl	*	97	19	*	*
21	–CH2Cl	94	59	19	10	600
22	–Ph	*	*	*	*	200
23	–CH2Ph	99	96	61	2	64
24	–(CH2)2Ph	98	93	53	8	82
25	4-ethylphenyl	85	27	*	*	2600
26	–C6H11	99	94	69	9	42
27	CH2Ad	*	96	8	*	*

^aUnless stated otherwise, errors are <5% of the reported value (from triplicate experiments).

^bRef (15).

^cRef (17).

^d*: Results not determined; cf. EMATE (IC₅₀ = 25 nM),¹⁷2 (IC₅₀ = 8 nM).¹⁷

Table 3. % Inhibition of STS Activity in Intact MCF-7 Breast Cancer Cells by 3-Substituted-4-Methylcoumarin Sulfamates Evaluated at Various Concentrations and Their IC₅₀ Values if Determined^a,c.

no.	R	10 μM	1 μM	0.1 μM	0.01 μM	IC50 nM
3b	H	93	86	43	<10	*
6b	–CH3	98	95	85	23	*
7	–CH2CH3	*	*	*	*	*
8	–(CH2)2CH3	*	*	*	*	*
28	–(CH2)4CH3	100	99	97	*	*
29	–(CH2)5CH3	100	100	99	*	0.68
30	–(CH2)6CH3	100	100	99	*	*
31	–(CH2)7CH3	100	100	99	*	*
32	–(CH2)8CH3	99	99	96	57	∼10
33	–(CH2)9CH3	97	99	97	80	<10
34	–(CH2)10CH3	*	*	*	*	*
35	–(CH2)11CH3	*	*	*	*	*
36	–(CH2)12CH3	*	*	*	*	*
37	–(CH2)13CH3	*	*	*	*	*
38	–(CH2)14CH3	*	*	*	*	*
39	Cl	87	67	29	*	600
40	Ph	*	*	75	32	25
41	–CH2Ph	97	94	91	85	1
42	–(CH2)2Ph	*	99	98	93	1.1
43	–(CH2)3Ph	100	99	87	*	*
44	–CH2(C6H11)	*	*	*	*	*
45	–(CH2)2(C6H11)	*	*	*	*	*
46	4-methoxyphenyl	*	*	*	*	*

^aUnless stated otherwise, errors are <5% of the reported value (from triplicate experiments).

^bRef (15).

^c*: Results not determined.

3-Substituted-4-methyl Compounds

MCF-7

A relatively smaller number of synthesized compounds in this series compared to their 4-substituted relatives were tested for their inhibitory activities. From the results available, 3-alkylated-4-methyl compounds 28–33 show >97% inhibition at 0.1 μM, whereas compounds 39–43, which have other substituents at the 3-position, inhibit STS between 29 and 98% at 0.1 μM. Of those five compounds that have IC₅₀ values determined, 29 is the most potent (0.68 nM), closely followed by 41 and 42 (1 and 1.1 nM, respectively). On comparing 23 (4-benzyl, IC₅₀ = 75 nM, Table 1) and 24 (4-phenethyl, IC₅₀ = 18 nM, Table 1) with 41 (3-benzyl-4-methyl, IC₅₀ = 1 nM) and 42 (3-phenethyl-4-methyl, IC₅₀ = 1.1 nM), there is 1 order of magnitude difference between the potency of the two pairs of compounds. This finding suggests that placing either a benzyl or a phenethyl group at the 3-position of the coumarin ring produces a more potent STS inhibitor. It is anticipated that on binding of 41 and 43 into the active site of STS, the coumarin ring of which is designed to mimic the A/B ring of 1, their substituents at the 3-position extend into the same area where the C/D ring of 1 resides.

Placental Microsomes

On the basis of the IC₅₀ values available, it appears that compounds with shorter alkyl chains at the 3-position of the coumarin ring (28, n-pentyl, IC₅₀ = 12 nM and 29, n-hexyl, IC₅₀ = 32 nM) are more potent STS inhibitors than those with longer alkyl chains (32–34, 37; IC₅₀ > 300 nM). This contrasts with those compounds in the 4-alkylated series (Table 2) the IC₅₀ values (40–102 nM) of which are tighter and fall within the same order of magnitude. This finding suggests that a long alkyl chain placed at the 4-position of the coumarin ring may interact better with the enzyme active site than its counterpart placed at the 3-position of the coumarin ring. To this effect, the sulfamate group of 4-alkylated compounds may be better positioned within the catalytic site of the enzyme for inactivation.

For compounds 40–43, the inhibitory activity observed at 0.1 μM starts from 65% for 40 (phenyl) and rises to 94% for 41 (benzyl) before it falls to 91 and 47% for 42 (phenethyl) and 43 (phenylpropyl), respectively. A similar pattern is observed when the IC₅₀ values of 40 (54 nM), 41 (8 nM), and 42 (33 nM) are compared. In regard to potency, the benzyl group is therefore the optimal substituent for this group of 3-substituted-4-methyl coumarin sulfamates. It is possible that the phenyl, phenethyl, and phenylpropyl groups interact less favorably with or are less well accommodated by the enzyme active site (Table 4).

When the benzyl group of 41 is replaced by a cyclohexylmethyl group to give 44, a reduction in potency is observed (at 0.1 μM, 94% for 41 vs 74% for 44). The same pattern, but to a greater extent, is observed when the phenethyl group is replaced by a cyclohexylethyl group as shown by the 91% inhibition of the STS observed for 42 at 0.1 μM compared to the 37% inhibition for 45 at the same concentration. This finding suggests that the more rigid and electron-rich phenyl group may interact better with the enzyme active site (such as through π-interactions with neighboring amino acids) than the more flexible aliphatic cyclohexyl group. The best inhibitors are illustrated in Figure 4, with an attempt to illustrate the mimicry of the steroidal C and D rings and also with some comparative activities shown in Table 5.

Figure 4.

Diagrammatic comparison of some potent inhibitors evaluated: 4-pentylcoumarin-7-O-sulfamate (10), 4-nonylcoumarin-7-O-sulfamate (14), 4-tridecylcoumarin-7-O-sulfamate (18), 3-hexyl-4-methylcoumarin-7-O-sulfamate (29), 3-nonyl-4-methylcoumarin-7-O-sulfamate (32), 3-benzyl-4-methylcoumarin-7-O-sulfamate (41), 3-phenethyl-4-methylcoumarin-7-O-sulfamate (42), in comparison with EMATE (1), Irosustat (2), and COUMATE (3). Solid lines denote similarity to steroid C and D rings.

Table 5. IC₅₀ Values and, Where Not Determined, % Inhibition for Potent STS Inhibitory Activity in Intact MCF-7 Breast Cancer Cells and Placental Microsomes.

		IC50 (nM)
no.	compound	intact MCF-7 cells	placental microsomes
1	EMATE	0.06513b	2517
2	Irosustat	0.242	1817
3	COUMATE	38018	63%@1 μM
10	4-n-pentyl	10	40
14	4-n-nonyl	90%@10 nM	60
18	4-n-tridecyl	3.2	85
29	3-n-hexyl-4-methyl	0.68	32
32	3-n-nonyl-4-methyl	∼10	320
41	3-benzyl-4-methyl	1.0	8
42	3-phenethyl-4-methyl	1.1	33

Molecular Modeling

Docking studies were conducted to explore potential interactions between the substituted bicyclic coumarin derivatives and the STS active site, in a similar fashion to those carried out for STX64/Irosustat and related series members.¹⁶ They show that the two most active compounds 29 and 41 are placed in a very similar fashion to the irreversible STS inhibitor Irosustat, with the sulfamoyl group in close proximity and opposite to the catalytic FGly 75 (Figure 5), suggesting that a putative sulfamoyl group transfer could also readily occur that might lead to similar irreversible inhibition (although note that no experiments were conducted to explore the reversibility/irreversibility of 29 and 41 against STS). Residue V486 on one side and residues L103 and V177 on the other sandwich the bicyclic ring system. Both compounds possibly form a hydrogen bond (N···O = ∼3.2 Å) from their chromen-2-one oxygen to the NH of G100 in the same manner as Irosustat (Figure 5). These more potent compounds have fairly small hydrophobic pendant groups attached to the 3-position of the chromen-2-one ring. These hydrophobic moieties lie between the hydrophobic sidechains of L103, F178, and F488. Those compounds with larger pendant groups may be less active due to the hydrophobic nature of the group making the compound less soluble. Alternatively, because STS is a membrane-bound protein and any substrate or inhibitor has to pass through the membrane to access the active site, it may be that larger hydrophobic tails result in the inhibitor failing to fully transit through the membrane: the hydrophobic tail stays, preferentially, embedded in the membrane.

Figure 5.

Left: docking of EMATE (cyan) and 29 (brown) into the crystal structure of human STS. Right: docking of Irosustat (cyan) and 41 (brown) into the crystal structure of human STS. In both figures, the Ca²⁺ ion is depicted as a yellow sphere and FG75 is the gem-diol form of FGly 75 aldehyde. The dotted yellow lines are potential hydrogen bonds.

On examining the data in Table 5, where the best compounds are benchmarked against the steroidal EMATE and the nonsteroidal Irosustat and, more particularly, against the known two-ring coumarin sulfamate COUMATE, it is readily apparent that highly potent compounds have been designed through the targeted 3- and 4-substitutions undertaken in this work. Some of these (29, 32, 41, and 42) have a potency approaching the clinical drug Irosustat in the more definitive intact MCF-7 cell assay, and of these, 29 is highly significant with a similar picomolar IC₅₀. Compound 41 is perhaps of the widest interest with an IC₅₀ of 1 nM but also with an inhibitory activity better than that of Irosustat in the more challenging placental microsomal STS assay. It has an attractive 3-benzyl substituent that, as for the highly active homolog 42, could potentially be substituted to further refine activity. Moreover, 41 and 42 are structurally distinct from the fully saturated C-ring surrogate of Irosustat and, as more versatile compounds, could form the basis of an attractive series for further optimization and eventual preclinical development. In any case, if we sensibly take COUMATE 3 for comparison, the best compounds are gratifyingly some 100–500 times more potent in the MCF-7 assay.

Conclusions

Synthetic routes to two-ring coumarin 7-O-sulfamate derivatives possessing 3- and 4-modified substitutions were devised, generally using an α-alkylacetoacetate strategy and the Pechman hydroxycoumain synthesis.^15,17 Compounds were shown to inhibit, often highly potently, the emerging clinical drug target STS⁸ now validated for hormone-dependent diseases¹² using an intact MCF-7 cell assay and an assay against placental microsomal STS activity. The best compounds were benchmarked for activity against the steroidal sulfamate drug EMATE,¹³ the nonsteroidal Irosustat,^12,16 and the known two-ring parent coumarin sulfamate COUMATE.^18,19 Through the targeted 3- and 4-substitution strategy undertaken, highly potent compounds were designed. In intact MCF-7 cells, compounds 29, 32, 41, and 42 had a potency approaching Irosustat with 29 having an IC₅₀ of 680 pM. 41 had a similar IC₅₀ of 1 nM but was also better than Irosustat against placental microsomal STS. With COUMATE 3 taken as the most relevant comparative structural benchmark for non-tricyclic derivatives, the best compounds were ca. 100–500 times more potent in the MCF-7 assay. Both 41 and 42 possess motifs structurally distinct from the fully saturated cyclic C-ring of Irosustat with attractive pendant 3-benzyl and 3-phenethyl substituents, respectively, that could potentially be further optimized through aromatic substitution. Compounds 29 and 41 were modeled into STS in comparison to benchmarks and dock well into the active site, placing the aryl sulfamate moiety opposite the catalytic FGly, as for Irosustat¹⁶ and with their pendant side chains occupying a hydrophobic pocket noted previously.³⁹ The expectation is that, in a similar fashion to Irosustat and EMATE, such compounds will act as irreversible inhibitors by transfer of their sulfamoyl group to the STS enzyme,^12b,13b although this has not been formally explored here. Thus, the versatile 3-benzyl-4-methyl- and 3-phenethyl-4-methyl-derivatives 41 and 42, respectively, and possibly also the 3-n-hexyl-4-methyl-derivative 29 from this study are potent STS inhibitors and could represent new leads for potential preclinical development.

Experimental Section

In Vitro Steroid Sulfatase Assay

STS inhibitory assays were performed essentially as previously described.^13b,43 The ability of the compounds synthesized to inhibit E1S was tested in vitro using MCF-7 cells and a placental microsomal preparation from a sulfatase-positive human placenta from a normal term pregnancy and compared with that of EMATE. For the placental microsome STS assay, a saturating substrate concentration of 20 μM was used and inhibitors were tested under initial rate conditions. For the MCF-7 STS assay, a physiological concentration of 3 nM E1S was used. Thus, for the placental microsome assay: [E1S] = 20 μM, [I] = 0.1 nM to 10 μM; MCF-7: [E1S] = 3 nM, [I] = 0.1 nM to 10 μM (NB for highly potent compounds, this was changed to 0.1 pM to 10 nM).

Chemicals and Analyses

All reagents were purchased commercially either from Aldrich Chemicals Co. (Gillingham, Dorset, U.K.) or Lancaster synthesis (Morecambe, Lancashire, U.K.). All organic solvents used were of general purpose or analytical grade and were obtained from Fisons Plc. (Loughborough, U.K.) and stored over 4 Å molecular sieves. Anhydrous dimethylformamide (DMF) used for all sulfamoylation reactions was purchased from Aldrich and was stored under a positive pressure of N₂ after use. Sulfamoyl chloride was prepared by adapting a method originally reported by Appel and Berger⁴⁴ and was stored as a standard solution in purified sulfur-free dry toluene.³⁶

Thin-layer chromatography (TLC) was carried out using precoated plates (Merck TLC aluminum sheets silica gel 60 F254, art. no. 5554). Product(s) and starting material were detected by treating plates with a methanolic solution of phosphomolybdic acid followed by heating or simply by viewing directly under UV light. Flash column chromatography was carried out by gradient elution (solvents used are indicated in the text) on wet-packed silica gel (Sorbsil C60). IR spectra were recorded using a PerkinElmer 782 spectrophotometer with peak positions expressed in cm^–1. ¹H and ¹³C NMR spectra were recorded using either a Jeol Delta 270 MHz or Varian Mercury VX 400 MHz spectrometer. Chemical shifts (δ) are reported in parts per million (ppm) using an internal standard of tetramethylsilane. Coupling constants (J) are quoted to the nearest 0.1 Hz. Mass spectra were acquired at the Mass Spectrometry Service Centre, Bath and FAB mass spectra used m-nitrobenzyl alcohol as matrix. Elemental analyses were carried out by the Microanalysis Service, Bath. Melting points are uncorrected and were determined using a Reichert-Jung Thermo Galen Kofler block. High-performance liquid chromatography (HPLC) was performed using a Waters 660E instrument equipped with an autosampler and photo diode array detector. A Waters Radialpak column (RP18, 8 mm × 100 mm) was used. The conditions of elution and analytical data are as indicated for each compound analyzed.

Molecular Modeling

Schrödinger software (running under Maestro 9.0) was used to build and minimize all of the ligands. The ALS75 residue in PDB crystal structure 1P49 (human placental estrone/dehydroepiandrosterone sulfatase) was mutated to the gem-diol form using the Schrödinger software editing tools. Minimization of the resulting structure, with the position of the backbone atoms fixed, allowed the atoms of the gem-diol and surrounding side chains to adopt low-energy conformations. Ligands were docked into the rigid protein using GOLD. A 10 Å sphere centered on the ALS75 sulfate was defined as the binding site. The GOLDScore fitness function was used to score the docked poses (25 for each ligand).

General Methods for the Synthesis of Ethyl 3-Oxo-alkanoates for the Preparation of 4-Alkylcoumarin Sulfamates

Method A³¹

To ethyl potassium malonate (2.1 equiv) in MeCN (100 mL/5 g of acid chloride) at 10–15 °C and under N₂ was added Et₃N (3.2 equiv), followed by MgCl₂ (2.5 equiv). The mixture was stirred at 20–25 °C for 2.5 h and then at 0 °C for 0.5 h before the corresponding acid chloride (1 equiv) was added dropwise during 25 min. The mixture was further treated with Et₃N (5 mL) and stirred overnight at 20 °C. The evaporation residue was dissolved in toluene and re-concentrated. More toluene was added, stirred, and cooled to 10–15 °C before aq HCl (1 M, 50 mL) was added cautiously while keeping the temperature <25 °C. The organic layer was washed with 1 M aq HCl (50 mL) and water. Drying, evaporation, and distillation or chromatography (CHCl₃ or CHCl₃/acetone, 10:1) gave the corresponding ethyl α-alkanoylacetate.

Method B³³

To anhydrous SnCl₂ (0.1 equiv) was added CH₂Cl₂ (∼100 mL/5 g of aldehyde), followed by ethyl diazoacetate (1.05 equiv). The reaction was initiated by adding a few drops of the corresponding aldehyde in CH₂Cl₂. When N₂ evolution began, the remaining solution of aldehyde (1 equiv) was added dropwise over 30 min. After the evolution of N₂ had stopped (∼1–3 h), the mixture was washed with brine (50 mL) and extracted twice (Et₂O). Drying, evaporation, and chromatography (CHCl₃ or CHCl₃/acetone, 10:1) or distillation gave the corresponding ethyl alkanoylacetate.

General Method for the Synthesis of Ethyl α-Alkylacetoacetates for the Preparation of 3-Alkyl-4-methylcoumarin Sulfamates³⁴

K₂CO₃ (2.4 equiv), water (50 mL), alkyl bromide (1 equiv), ethyl acetoacetate (1 equiv), CH₂Cl₂ (50 mL/5 g of alkyl bromide), and Bu₄NCl (1 or 2 equiv) were boiled under reflux for 3 days. After cooling, the separated organic layer was washed with 5 M aq HCl (30 mL). The mixture was extracted twice with Et₂O. The combined ethereal extracts were dried, filtered, and concentrated in vacuo. Chromatography (CHCl₃ or CHCl₃/acetone 10:1) or distillation gave the corresponding ethyl α-alkylacetoacetate.

General Method for the Synthesis of 3- or 4-Alkyl-7-hydroxycoumarins¹⁷

Resorcinol (1 equiv) was dissolved in the corresponding hot β-keto ester (1 equiv). The resulting syrup was cooled to 0 °C and treated dropwise with a mixture of CF₃COOH (2 equiv) and conc. H₂SO₄ (2 equiv) while keeping the temperature <10 °C. After stirring for 3 h at room temperature, the mixture was cautiously quenched with ice-water. The brightly colored gluey mass formed was stirred for further 1 h. The bright yellow/brown precipitate resulted was collected by suction filtration, washed exhaustively with water, and re-dissolved in acetone. The yellow/brown solid obtained upon evaporation was purified by flash chromatography (CHCl₃/acetone, 8:1 to 4:1 gradient) and/or recrystallized from hot absolute ethanol, acetone/hexane (4:1), or THF/hexane (2:0.5) to give the corresponding coumarin as a crystalline solid.

General Method for the Sulfamoylation Reaction¹⁵

To a solution of the compound (1 equiv) in anhydrous DMF (5 mL) at 0 °C under N₂ was added NaH (1 equiv). When the evolution of H₂ had ceased, previously prepared sulfamoyl chloride (∼3–5 equiv) was introduced. After stirring at room temperature under N₂ overnight, the mixture was quenched with ice-water. The organic fractions were extracted into ethyl acetate (∼150 mL) and washed with brine (4 × 100 mL). Drying, evaporation, chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient), and/or recrystallization with either ethyl acetate/hexane (5:2) or THF/hexane (2:1) gave the corresponding crystalline sulfamate.

Ethyl 3-Oxo-heptanoate (9a)

This was prepared by method A using ethyl potassium malonate (12.6 g, 74.0 mmol), CH₃CN (110 mL), Et₃N (11.6 g, 115 mmol), MgCl₂ (8.39 g, 88.1 mmol), and pentanoyl chloride (4.34 g, 36.0 mmol). The crude oily residue was purified by flash chromatography (CHCl₃) to give 9a as a pale yellow oil (4.65 g, 78%): R_f = 0.92 (CHCl₃/acetone, 10:1); ¹H NMR (400 MHz, CDCl₃): δ = 0.91 (t, J = 7.3 Hz, 3H, C7–H₃), 1.28 (t, J = 7.0 Hz, 3H, CH₂CH₃), 1.29–1.37 (m, 2H, CH₂), 1.54–1.62 (m, 2H, CH₂), 2.55 (t, J = 7.3 Hz, 2H, C4–H₂), 3.44 (s, 2H, C2–H₂) and 4.19 ppm (q, J = 7.3 Hz, 2H, CH₂CH₃). MS (FAB⁺): m/z (%) 173.1 (100) [M + H]⁺; MS (FAB^–): m/z (%) 171.1 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺; Anal. calcd for C₉H₁₇O₃: 173.1099, found: 173.1089.

4-Butyl-7-hydroxycoumarin (9b)

This was prepared with resorcinol (2.0 g, 18 mmol), 9a (3.13 g, 18.2 mmol), and a mixture of CF₃COOH (2.77 mL, 36.3 mmol) and conc. H₂SO₄ (1.83 mL, 36.3 mmol). The crude yellow/brown solid was recrystallized from acetone/hexane to give 9b as cream crystals (1.87 g, 47%): R_f = 0.63 (CHCl₃/acetone, 3:1); mp 135–138 °C (Lit.⁴⁵ mp 139–140 °C, ethanol); IR (KBr) ṽ = 3440, 1650 cm^–1; ¹H NMR (400 MHz, DMSO-d₆): δ = 0.92 (t, J = 7.3 Hz, 3H, CH₃), 1.34–1.43 (m, 2H, CH₂), 1.54–1.62 (m, 2H, CH₂), 2.73 (t, J = 7.6 Hz, 2H, C1′–H₂), 6.08 (s, 1H, C3–H), 6.71 (d, J = 2.4 Hz, 1H, C8–H), 6.80 (dd, J = 8.6 and 2.4 Hz, 1H, C6–H), 7.6 (d, J = 8.5 Hz, 1H, C5–H) and 10.53 ppm (s, 1H, OH); MS (FAB⁺): m/z (%) 437.2 (15) [2M + H]⁺, 219.2 (100) [M + H]⁺; MS (FAB^–): m/z (%) 435.3 (20) [2M – H]⁻, 217.2 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₃H₁₅O₃: 219.1021, found: 219.1034; Anal. calcd for C₁₃H₁₄O₃: C 71.54, H 6.47, found: C 71.40, H 6.49.

4-Butylcoumarin-7-O-sulfamate (9)

Upon sulfamoylation, 9b (700 mg, 3.21 mmol) gave a crude white solid, which was fractionated by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient). The white solid isolated was recrystallized from ethyl acetate/hexane to give 9 as white crystals (98 mg, 10%): R_f = 0.36 (CHCl₃/ethyl acetate, 4:1); mp 147–150 °C; IR (KBr) ṽ = 3400–3100, 1750, 1450–1300, 1100–1150 cm^–1; ¹H NMR (400 MHz, DMSO-d₆): δ = 0.93 (t, J = 7.3 Hz, 3H, CH₃), 1.36–1.45 (m, 2H, CH₂), 1.57–1.64 (m, 2H, CH₂), 2.82 (t, J = 7.6 Hz, 2H, C1′–H₂), 6.38 (s, 1H, C3–H), 7.29 (dd, J = 2.4 and 8.8 Hz, 1H, C6–H), 7.33 (d, J = 2.4 Hz, 1H, C8–H), 7.94 (d, J = 8.8 Hz, 1H, C5–H) and 8.24 (s, 2H, NH₂); MS (FAB⁺): m/z (%) 595.2 (70) [2M + H]⁺, 298.1 (100) [M + H]⁺, 219.1 (10) [M + H – HNSO₂]⁺; MS (FAB^–): m/z (%) 593.2 (15) [2M – H]⁻, 296.2 (100) [M – H]⁻, 217.2 (60) [M – H₂NSO₂]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₃H₁₆NO₅S: 298.0749, found: 298.0742; Anal. calcd for C₁₃H₁₅NO₅S: C 52.52, H 5.09, N 4.71%, found: C 52.00, H 5.00, N 4.61.

Ethyl 3-Oxo-octanoate (10a)

This was prepared by method A using ethyl potassium malonate (13.0 g, 74.4 mmol), CH₃CN (120 mL), Et₃N (16.2 mL, 116 mmol), MgCl₂ (8.66 g, 90.1 mmol), and hexanoyl chloride (5.31 g, 38.2 mmol). The crude oily residue was purified by flash chromatography (CHCl₃) to give 10a as a pale yellow oil (6.58 g, 93%): R_f = 0.88 (CHCl₃); ¹H NMR (400 MHz, CDCl₃): δ = 0.89 (t, J = 7.1 Hz, 3H, CH₃), 1.29 (t, J = 7.3 Hz, 3H, OCH₂CH₃), 1.31–1.37 (m, 4H, CH₂CH₂), 1.56–1.63 (m, 2H, CH₂), 2.54 (t, J = 7.3 Hz, 2H, C4–H₂), 3.43 (s, 2H, C2–H₂) and 4.19 (q, J = 7.3 Hz, 2H, OCH₂CH₃); MS (FAB⁺): m/z (%) 187.2 (100) [M + H]⁺; MS (FAB^–): m/z (%) 185.2 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺; Anal. calcd for C₁₀H₁₉O₃: 187.1334, found: 187.1342.

7-Hydroxy-4-pentylcoumarin (10b)

This was prepared with resorcinol (2.0 g, 18 mmol), 10a (3.4 g, 18 mmol), and a mixture of CF₃COOH (2.8 mL, 36 mmol) and conc. H₂SO₄ (1.8 mL, 36 mmol). The crude yellow/brown solid was recrystallized from acetone/hexane to give 10b as pale yellow crystals (2.32 g, 56%): R_f = 0.86 (CHCl₃/acetone, 3:1); mp 148–150 °C (Lit.⁴⁶ mp 145–146 °C); ¹H NMR (400 MHz, DMSO-d₆): δ = 0.87 (t, J = 7.1 Hz, 3H, C5′–H₃), 1.33–1.34 (m, 4H, CH₂CH₂), 1.58–1.61 (m, 2H, CH₂), 2.72 (t, J = 7.6 Hz, 2H, C1′–H₂), 6.08 (s, 1H, C3–H), 6.71 (d, J = 2.4 Hz, 1H, C8–H), 6.80 (dd, J = 2.4 and 8.8 Hz, 1H, C6–H), 7.64 (d, J = 8.8 Hz, 1H, C5–H) and 10.53 (s, 1H, OH); MS (FAB⁺): m/z (%) 465.3 (15) [2M + H]⁺, 233.2 (100) [M + H]⁺; MS (FAB^–): m/z (%) 463.4 (10) [2M – H]⁻, 231.2 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₄H₁₇O₃: 233.1178, found: 233.1181; Anal. calcd for C₁₄H₁₆O₃: C 72.39, H, 6.94%, found: C 72.33, H, 6.96.

4-Pentylcoumarin-7-O-sulfamate (10)

Upon sulfamoylation, 10b (700 mg, 3.01 mmol) gave a crude white sold (893 mg), which was fractionated by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient). The white solid isolated was recrystallized from ethyl acetate/hexane to give 10 as white crystals (251 mg, 27%): R_f = 0.36 (CHCl₃/ethyl acetate, 4:1); mp 128–132 °C; ¹H NMR (400 MHz, DMSO-d₆): δ = 0.88 (t, J = 7.1 Hz, 3H, C5′–H₃), 1.31–1.39 (m, 4H, CH₂CH₂), 1.59–1.64 (m, 2H, CH₂), 2.81 (t, J = 7.6 Hz, 2H, C1′–H₂), 6.37 (s, 1H, C3–H), 7.28 (dd, J = 1.5 and 8.5 Hz, 1H, C6–H), 7.33 (d, J = 1.5 Hz, 1H, C8–H), 7.93 (d, J = 8.5 Hz, 1H, C5–H) and 8.23 (s, 2H, NH₂); MS (FAB⁺): m/z (%) 623.2 (70) [2M + H]⁺, 312.1 (100) [M + H]⁺, 233.1 (20) [M + H – HNSO₂]⁺; MS (FAB^–): m/z (%) 621.2 (20) [2M – H]⁻, 310.2 (100) [M – H]⁻, 231.2 (100) [M – H₂NSO₂]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₄H₁₈NO₅S: 312.0906, found: 312.0908; Anal. calcd for C₁₄H₁₇NO₅S: C 54.01, H 5.50, N 4.50, found: C 54.70, H 5.56, N 4.50.

Ethyl 3-Oxo-nonanoate (11a)

This was prepared by method A using ethyl potassium malonate (13 g, 74 mmol), CH₃CN (120 mL), Et₃N (16.2 g, 116 mmol), MgCl₂ (8.7 g, 91 mmol), and heptanoyl chloride (5.91 g, 36.2 mmol). The crude oily residue was purified by flash chromatography (CHCl₃) to give 11a as a pale yellow oil (4.51 g, 62%): R_f = 0.64 (CHCl₃); ¹H NMR (400 MHz, CDCl₃): δ = 0.88 (t, J = 7.3 Hz, 3H, C9–H₃), 1.26–1.32 (m, 9H, CH₂CH₃ and 3 × CH₂), 1.59 (m, 2H, 5-CH₂), 2.35 (t, J = 7.3 Hz, 2H, C4–H₂), 3.43 (s, 2H, C2–H₂) and 4.19 (q, J = 7.1 Hz, 2H, CH₂CH₃); MS (FAB⁺): m/z (%) 201.2 (100) [M + H]⁺; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₁H₂₁O₃: 201.1491, found: 201.1492.

4-Hexyl-7-hydroxycoumarin (11b)

This was prepared with resorcinol (2.20 g, 19.9 mmol), 11a (4.0 g, 20 mmol), and a mixture of CF₃COOH (3.1 mL, 40 mmol) and conc. H₂SO₄ (2.04 mL, 39.9 mmol). The crude orange solid obtained was recrystallized from acetone/hexane to 11b as off-white crystals (2.95 g, 60%): R_f = 0.72 (CHCl₃/acetone, 3:1); mp 124–126 °C; ¹H NMR (400 MHz, DMSO-d): δ = 0.86 (t, J = 7.1 Hz, 3H, C6′–H₃), 1.27–1.37 (m, 6H, 3 × CH₂), 1.55–1.63 (m, 2H, CH₂), 2.72 (t, J = 7.6 Hz, 2H, C1′–H₂), 6.08 (s, 1H, C3–H), 6.71 (d, J = 2.4 Hz, 1H, C8–H), 6.80 (dd, J = 2.4 and 8.8 Hz, 1H, C6–H), 7.64 (d, J = 8.8 Hz, 1H, C5–H) and 10.53 (s, 1H, OH); MS (FAB⁺): m/z (%) 493.4 (10) [2M + H]⁺, 247.2 (100) [M + H]⁺; MS (FAB^–): m/z (%) 491.3 (15) [2M – H]⁻, 245.2 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₅H₁₉O₃: 247.1334, found: 247.1334; Anal. calcd for C₁₅H₁₈O₃: C 73.15, H 7.37, found: C 73.30, H 7.40.

4-Hexylcoumarin-7-O-sulfamate (11)

Upon sulfamoylation, 11b (700 mg, 2.84 mmol) gave a crude white solid, which was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient). The white solid isolated was recrystallized from ethyl acetate/hexane to give 11 as fine white crystals (441 mg, 48%): R_f = 0.24 (CHCl₃/ethyl acetate, 4:1); mp 126–128 °C; ¹H NMR (400 MHz, DMSO-d₆): δ = 0.87 (t, J = 6.7 Hz, 3H, C6′–H₃), 1.29–1.39 (m, 6H, 3 × CH₂), 1.58–1.66 (m, 2H, CH₂), 2.81 (t, J = 7.9 Hz, 2H, C1′–H₂), 6.37 (s, 1H, C3–H), 7.29 (dd, J = 2.4 and 8.6 Hz, 1H, C6–H), 7.33 (d, J = 2.4 Hz, 1H, C8–H), 7.93 (d, J = 8.6 Hz, 1H, C5–H) and 8.24 (s, 2H, NH₂); MS (FAB⁺): m/z (%) 651.3 (10) [2M + H]⁺, 326.2 (100) [M + H]⁺, 247.2 (10), [M + H – HNSO₂]⁺; MS (FAB^–): m/z (%) 649.3 (15) [2M – H]⁻, 324.2 (100) [M – H]⁻, 245.2 (60), [M – H₂NSO₂]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₅H₂₀NO₅S: 326.1062, found: 326.1079; Anal. calcd for C₁₅H₁₉NO₅S: C 55.37, H 5.89, N 4.30, found: C 55.20, H 5.88, N 4.25.

Ethyl 3-Oxo-decanoate (12a)

This was prepared by method A using ethyl potassium malonate (10.5 g, 61.5 mmol), CH₃CN (120 mL), Et₃N (13.1 mL, 93.8 mmol), MgCl₂ (7.0 g, 73 mmol), and octanoyl chloride (5.0 mL, 29 mmol). The crude oily residue was purified by flash chromatography (CHCl₃) to give 12a as a pale yellow oil (3.86 g, 61%): R_f = 0.76 (CHCl₃); ¹H NMR (400 MHz, CDCl₃): δ = 0.88 (t, J = 7.3 Hz, 3H, C10–H₃), 1.26–1.29 (m, 11H, CH₂CH₃ and 4 × CH₂), 1.57–1.61 (m, 2H, CH₂), 2.53 (t, J = 6.8 Hz, 2H, C4–H₂), 3.43 (s, 2H, C2–H₂) and 4.19 (q, J = 7.3 Hz, 2H, CH₂CH₃); MS (FAB⁺): m/z (%) 215.2 (100) [M + H]⁺; MS (FAB^–): m/z (%) 213.2 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₂H₂₃O₃: 215.1647, found: 215.1652.

4-Heptyl-7-hydroxycoumarin (12b)

This was prepared with resorcinol (1.8 g, 16 mmol), 12a (3.5 g, 16 mmol), and a mixture of CF₃COOH (2.52 mL, 32.7 mmol) and conc. H₂SO₄ (1.67 mL, 32.7 mmol). The crude yellow solid was recrystallized from acetone/hexane to give 12b as yellow crystals (2.32 g, 55%): R_f = 0.78 (CHCl₃/acetone, 3:1); mp 106–107 °C; ¹H NMR (400 MHz, DMSO-d): δ = 0.86 (t, J = 7.1 Hz, 3H, C7′–H₃), 1.24–1.36 (m, 8H, 4 × CH₂), 1.55–1.63 (m, 2H, CH₂), 2.72 (t, J = 7.6 Hz, 2H, C1′–H₂), 6.08 (s, 1H, C3–H), 6.71 (d, J = 2.4 Hz, 1H, C8–H), 6.80 (dd, J = 2.4 and 8.8 Hz, 1H, C6–H), 7.64 (d, J = 8.8 Hz, 1H, C5–H) and 10.53 (s, 1H, OH); MS (FAB⁺): m/z (%) 261.2 (100) [M + H]⁺; MS (FAB^–): m/z (%) 519.3 (60) [2M – H]⁻, 259.2 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₆H₂₁O₃: 261.1491, found: 261.1501; Anal. calcd for C₁₆H₂₀O₃: C 73.82, H 7.74, found: C 73.60, H 7.82.

4-Heptylcoumarin-7-O-sulfamate (12)

Upon sulfamoylation, 12b (700 mg, 2.69 mmol) gave a crude white solid, which was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient). The white solid isolated was recrystallized from ethyl acetate/hexane to give 12 as fine white crystals (503 mg, 55%): R_f = 0.42 (CHCl₃/ethyl acetate, 4:1); mp 137–139 °C; ¹H NMR (400 MHz, DMSO-d₆): δ = 0.86 (t, J = 7.1 Hz, 3H, C7′–H₃), 1.27–1.39 (m, 8H, 4 × CH₂), 1.58–1.64 (m, 2H, CH₂), 2.81 (t, J = 7.3 Hz, 2H, C1′–H₂), 6.37 (s, 1H, C3–H), 7.27–7.33 (m, 2H, C6–H and C8–H), 7.39 (d, J = 8.8 Hz, 1H, C5–H) and 8.22 (s, 2H, NH₂); MS (FAB⁺): m/z (%) 679.3 (60) [2M + H]⁺, 340.1 (100) [M + H]⁺, 261.1 (10) [M + H – HNSO₂]⁺; MS (FAB^–): m/z (%) 677.1 (20) [2M – H]⁻, 338.1 (100) [M – H]⁻, 259.1 (60) [M – H₂NSO₂]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₆H₂₂NO₅S: 340.1218, found: 340.1215; Anal. calcd for C₁₆H₂₁NO₅S: C 56.62, H 6.24, N 4.13, found: C 56.90, H 6.31, N 4.15.

Ethyl 3-Oxo-undecanoate (13a)

This was prepared by method A using ethyl potassium malonate (13.0 g, 76.4 mmol), CH₃CN (120 mL), Et₃N (16.2 mL, 116 mmol), MgCl₂ (8.7 g, 91 mmol), and nonanoyl chloride (6.69 mL, 37.8 mmol). The crude oily residue was purified by flash chromatography (CHCl₃) to give 13a as a pale yellow oil (6.73 g, 78%): R_f = 0.65 (CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ = 0.88 (t, J = 7.3 Hz, 3H, C11–H₃), 1.26–1.61 (m, 15H, CH₂CH₃ and 6 × CH₂), 2.53 (t, J = 7.6 Hz, 2H, C4–H₂), 3.43 (s, 2H, C2–H₂) and 4.19 (q, J = 7.3 Hz, 2H, CH₂CH₃); MS (FAB⁺): m/z (%) 229.2 (100) [M + H]⁺; MS (FAB^–): m/z (%) 227.2 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₃H₂₅O₃: 229.1725, found: 229.1794.

7-Hydroxy-4-octylcoumarin (13b)

This was prepared with resorcinol (1.93 g, 17.5 mmol), 13a (4.0 g, 18 mmol) and a mixture of CF₃COOH (2.7 mL, 35 mmol) and conc. H₂SO₄ (1.8 mL, 35 mmol). The crude yellow solid was recrystallized from acetone/hexane to give 13b as yellow crystals (2.31 g, 48%): R_f = 0.71 (CHCl₃/acetone, 3:1); mp 90–92 °C; ¹H NMR (400 MHz, DMSO-d₆): δ = 0.85 (t, J = 7.1 Hz, 3H, C8′–H₃), 1.25–1.36 (m, 10H, 5 × CH₂), 1.55–1.62 (m, 2H, CH₂), 2.51 (t, J = 7.3 Hz, 2H, C1′–H₂), 6.08 (s, 1H, C3–H), 6.71 (d, J = 2.4 Hz, 1H, C8–H), 6.80 (dd, J = 2.4 and 8.8 Hz, 1H, C6–H), 7.64 (d, J = 8.8 Hz, 1H, C5–H) and 10.53 (s, 1H, OH); MS (FAB⁺): m/z (%) 549.5 (80) [2M + H]⁺, 275.2 (100) [M + H]⁺; MS (FAB^–): m/z (%) 547.4 (75) [2M – H]⁻, 273.2 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₇H₂₃O₃: 275.1647, found: 275.1647; Anal. calcd for C₁₇H₂₂O₃: C 74.42, H 8.08, found: C 74.70, H 8.18.

4-Octylcoumarin-7-O-sulfamate (13)

Upon sulfamoylation, 13b (400 mg, 1.46 mmol) gave a crude white solid which was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient). The white solid isolated was recrystallized from ethyl acetate/hexane to give 13 as white fine crystals (279 mg, 53%): R_f = 0.37 (CHCl₃/ethyl acetate, 4:1); mp 124–125 °C; ¹H NMR (400 MHz, DMSO-d₆): δ = 0.85 (t, J = 6.7 Hz, 3H, C8′–H₃), 1.25–1.39 (m, 10H, 5 × CH₂), 1.58–1.63 (m, 2H, CH₂), 2.81 (t, J = 7.3 Hz, 2H, C1′–H₂), 6.37 (s, 1H, C3–H), 7.28 (dd, J = 2.4 and 8.5 Hz, 1H, C6–H), 7.32 (d, J = 2.4 Hz, 1H, C8–H), 7.93 (d, J = 8.5 Hz, 1H, C5–H) and 8.24 (s, 2H, NH₂); MS (FAB⁺): m/z (%) 707.0 (80) [2M + H]⁺, 354.0 (100) [M + H]⁺, 275.0 (20) [M + H – HNSO₂]⁺; MS (FAB^–): m/z (%) 705.2 (20) [2M – H]⁻, 352.1 (100) [M – H]⁻, 273.1 (70) [M – H₂NSO₂]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₇H₂₄NO₅S: 354.1375, found: 354.1375; Anal. calcd for C₁₇H₂₃NO₅S: C 57.77, H 6.56, N 3.96, found: C 58.00, H 6.50, N 3.75.

Ethyl 3-Oxo-dodecanoate (14a)

This was prepared by method A using ethyl potassium malonate (13.0 g, 76.4 mmol), CH₃CN (120 mL), Et₃N (16.2 mL, 116 mmol), MgCl₂ (8.7 g, 91 mmol), and decanoyl chloride (7.5 mL, 36 mol). The crude oily residue was purified by flash chromatography (CHCl₃) to give 14a as a pale yellow oil (7.79 g, 89%): R_f = 0.75 (CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ = 0.82 (t, J = 5.5 Hz, 3H, C12–H₃), 1.19–1.55 (m, 17H, CH₂CH₃ and 7 × CH₂), 2.48 (t, J = 7.3 Hz, 2H, C4–H₂), 3.37 (s, 2H, C2–H₂) and 4.13 (q, J = 7.3 Hz, 2H, CH₂CH₃); MS (FAB⁺): m/z (%) 243.1 (100) [M + H]⁺; MS (FAB^–): m/z (%) 241.1 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₄H₂₇O₃: 243.1960, found: 243.1959.

7-Hydroxy-4-nonylcoumarin (14b)

This was prepared with resorcinol (1.14 g, 10.3 mmol), 14a (2.5 g, 10 mmol), and a mixture of CF₃COOH (1.6 mL, 21 mmol) and conc. H₂SO₄ (1.05 mL; 20.6 mmol). The crude yellow solid was purified by flash chromatography (CHCl₃/acetone, 8:1 to 4:1 gradient), and the pale yellow solid isolated was recrystallized from acetone/hexane to give 14b as off-white fine crystals (532 mg, 18%): R_f = 0.71 (CHCl₃/acetone, 3:1); mp 91–93 °C; ¹H NMR (400 MHz, CDCl₃) δ = 0.88 (t, J = 7.1 Hz, 3H, C9′–H₃), 1.27–1.44 (m, 12H, 6 × CH₂), 1.64–1.72 (m, 2H, CH₂), 2.73 (t, J = 7.3 Hz, 2H, C1′–H₂), 6.14 (s, 1H, C3–H), 6.88 (dd, J = 2.4 and 8.8 Hz, 1H, C6–H), 7.08 (d, J = 2.4 Hz, 1H, C8–H), 7.52 (d, J = 8.8 Hz, 1H, C5–H) and 10.54 (s, 1H, OH); MS (FAB⁺): m/z (%) 577.2 (80) [2M + H]⁺, 289.1 (100) [M + H]⁺; MS (FAB^–): m/z (%) 575.2 (20) [2M – H]⁻, 287.1 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₈H₂₅O₃: 289.1804, found: 289.1807; Anal. calcd for C₁₈H₂₄O₃: C 74.97, H 8.39, found: C 75.10, H 8.39.

4-Nonylcoumarin-7-O-sulfamate (14)

Upon sulfamoylation, 14b (400 mg, 1.39 mmol) gave a crude white solid, which was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient). The white solid isolated was recrystallized from ethyl acetate/hexane to give 14 as white fine crystals (36 mg, 7%): R_f = 0.40 (CHCl₃/ethyl acetate, 4:1); mp 101–103 °C; ¹H NMR (400 MHz, DMSO-d₆): δ = 0.85 (t, J = 7.1 Hz, 3H, C9′–H₃), 1.18–1.43 (m, 12H, 6 × CH₂), 1.57–1.65 (m, 2H, CH₂), 2.81 (t, J = 7.3 Hz, 2H, C1′–H₂), 6.37 (s, 1H, C3–H), 7.28 (dd, J = 2.4 and 8.5 Hz, 1H, C6–H), 7.33 (d, J = 2.4 Hz, 1H, C8–H), 7.93 (d, J = 8.5 Hz, 1H, C5–H) and 8.24 (s, 2H, NH₂); MS (FAB⁺): m/z (%) 735.3 (90) [2M + H]⁺, 368.1 (100) [M + H]⁺; MS (FAB^–): m/z (%) 733.1 (80) [2M – H]⁻, 366.0 (100) [M – H]⁻, 287.1 (90) [M – H₂NSO₂]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₈H₂₆NO₅S: 368.153, found: 2368.1539; Anal. calcd for C₁₈H₂₅NO₅S: C 58.84, H 6.86, N 3.81, found: C 59.05, H 6.91, N 3.74.

Ethyl 3-Oxo-tridecanoate (15a)

This was prepared by method B using CH₂Cl₂ (80 mL), ethyl diazoacetate (3.52 g, 30.8 mmol), SnCl₂ (556 mg, 2.9 mmol), and undecanal (5.0 g, 29 mmol) in CH₂Cl₂. The crude oily residue was purified by fractional distillation under reduced pressure to give 15a as a pale yellow oil (4.34 g, 58%): R_f = 0.72 (CHCl₃); bp_0.15: 135–139 °C (Lit.⁴⁷ bp_0.15: 130–135 °C); ¹H NMR (400 MHz, CDCl₃) δ = 0.88 (t, J = 6.9 Hz, 3H, C13–H₃), 1.26–1.61 (m, 19H, CH₂CH₃ and 8 × CH₂), 2.53 (t, J = 7.2 Hz, 2H, C4–H₂), 3.43 (s, 2H, C2–H₂) and 4.19 (q, J = 7.2 Hz, 2H, CH₂CH₃); MS (FAB⁺): m/z (%) 257.2 (100) [M + H]⁺; MS (FAB^–): m/z (%) 255.2 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₅H₂₉O₃: 257.2117, found: 257.2129.

4-Decyl-7-hydroxycoumarin (15b)

This was prepared with resorcinol (1.07 g, 9.76 mmol), 15a (2.5 g, 9.8 mmol), and a mixture of CF₃COOH (1.5 mL, 20 mmol) and conc. H₂SO₄ (1.0 mL, 20 mmol). The crude yellow solid was purified by flash chromatography (CHCl₃/acetone, 8:1 to 4:1 gradient), and the pale yellow solid isolated was recrystallized from acetone/hexane to give 15b as off-white fine crystals (1.66 g, 54%): R_f = 0.73 (CHCl₃/acetone, 3:1); mp 98–99 °C; ¹H NMR (400 MHz, CDCl₃) δ = 0.88 (t, J = 7.0 Hz, 3H, C10′–H₃), 1.27–1.42 (m, 14H, 7 × CH₂), 1.64–1.72 (m, 2H, CH₂), 2.73 (t, J = 7.6 Hz, 2H, C1′–H₂), 6.14 (s, 1H, C3–H), 6.89 (dd, J = 2.4 and 8.8 Hz, 1H, C6–H), 7.11 (d, J = 2.4 Hz, 1H, C8–H), 7.52 (d, J = 8.8 Hz, 1H, C5–H) and 8.19 (s, 1H, OH); MS (FAB⁺): m/z (%) 303.1 (100) [M + H]⁺; MS (FAB^–): m/z (%) 301.1 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₉H₂₇O₃: 303.1960, found: 303.1973; Anal. calcd for C₁₉H₂₆O₃: C 75.46, H 8.67, found: C 75.10, H 8.72.

4-Decylcoumarin-7-O-sulfamate (15)

Upon sulfamoylation, compound 15b (400 mg, 1.32 mmol) gave a crude white solid, which was fractionated by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient). The white solid isolated was recrystallized from ethyl acetate/hexane to give 15 as white fine needles (288 mg, 57%): R_f = 0.55 (CHCl₃/ethyl acetate, 4:1); mp 112–115 °C; ¹H NMR (400 MHz, DMSO-d₆): δ = 0.85 (t, J = 7.1 Hz, 3H, C10′–H₃), 1.16–1.38 (m, 14H, 7 × CH₂), 1.58–1.63 (m, 2H, CH₂), 2.81 (t, J = 7.6 Hz, 2H, C1′–H₂), 6.37 (s, 1H, C3–H), 7.28 (dd, J = 2.1 and 8.8 Hz, 1H, C6–H), 7.33 (d, J = 2.1 Hz, 1H, C8–H), 7.93 (d, J = 8.8 Hz, 1H, C5–H) and 8.24 (s, 2H, NH₂); MS (FAB⁺): m/z (%) 763.2 (65) [2M + H]⁺, 382.0 (100) [M + H]⁺, 303.1 (20) [M + H – HNSO₂]⁺; MS (FAB^–): m/z (%) 761.0 (80) [2M – H]⁻, 380.0 (100) [M – H]⁻, 301.1 (90) [M – H₂NSO₂]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₉H₂₈NO₅S: 382.1688, found: 382.1696; Anal. calcd for C₁₉H₂₇NO₅S: C 59.82, H 7.13, N 3.67, found: C 60.15, H 7.12. N 3.54.

Ethyl 3-Oxo-tetradecanoate (16a)

This was prepared by method B using CH₂Cl₂ (80 mL), ethyl diazoacetate (3.25 g, 28.5 mmol), SnCl₂ (514 mg, 2.7 mmol), and dodecyl aldehyde (5.0 g, 27 mmol) in CH₂Cl₂ (20 mL). The crude oily residue was purified by fractional distillation under reduced pressure to give 16a as a colorless oil (5.3 g, 72%): R_f = 0.74 (CHCl₃); bp_0.15: 122–123 °C (Lit.⁴⁸ bp_0.1: 123–125 °C); ¹H NMR (400 MHz, CDCl₃) δ = 0.88 (t, J = 7.0 Hz, 3H, C14–H₃), 1.25–1.61 (m, 21H, CH₂CH₃ and 9 × CH₂), 2.53 (t, J = 7.3 Hz, 2H, C4–H₂), 3.43 (s, 2H, C2–H₂) and 4.19 (q, J = 7.3 Hz, 2H, CH₂CH₃); MS (FAB⁺): m/z (%) 271.1 (100) [M + H]⁺; MS (FAB^–): m/z (%) 269.2 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₆H₃₁O₃: 271.2273, found: 271.2285.

7-Hydroxy-4-undecylcoumarin (16b)

This was prepared with resorcinol (1.22 g, 11.1 mmol), 16a (3.0 g, 11 mmol), and a mixture of CF₃COOH (2.0 mL, 22 mmol) and conc. H₂SO₄ (2.0 mL, 22 mmol). The crude yellow solid was purified by flash chromatography (CHCl₃/acetone, 8:1 to 4:1 gradient), and the pale yellow solid isolated was recrystallized from acetone/hexane to give 16b as white crystals (1.92 g, 55%): R_f = 0.86 (CHCl₃/acetone, 3:1); mp 102–105 °C; ¹H NMR (400 MHz, DMSO-d₆): δ = 0.85 (t, J = 6.2 Hz, 3H, C11′–H₃), 1.23–1.36 (m, 16H, 8 × CH₂), 1.54–1.60 (m, 2H, CH₂), 2.72 (t, J = 7.6 Hz, 2H, C1′–H₂), 6.07 (s, 1H, C3–H), 6.71 (d, J = 2.1 Hz, 1H, C8–H), 6.79 (dd, J = 2.1 and 8.5 Hz, 1H, C6–H), 7.63 (d, J = 8.5 Hz, 1H, C5–H) and 10.52 (s, 1H, OH); MS (FAB⁺): m/z (%) 317.2 (100) [M + H]⁺; MS (FAB⁺): m/z (%) 631.4 (10) [2M – H]⁻, 315.3 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₂₀H₂₉O₃: 317.2117, found: 317.2121; Anal. calcd for C₂₀H₂₈O₃: C 75.91, H 8.92, found: C 75.50, H 8.97.

4-Undecylcoumarin-7-O-sulfamate (16)

Upon sulfamoylation, compound 16b (400 mg, 1.27 mmol) gave a crude white solid, which was fractionated by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient). The white solid isolated was recrystallized from ethyl acetate/hexane to give 16 as white fine fluffy crystals (88 mg, 18%): R_f = 0.54 (CHCl₃/ethyl acetate, 4:1); mp 114–116 °C; ¹H NMR (400 MHz, DMSO-d₆): δ = 0.85 (t, J = 7.0 Hz, 3H, C11′–H₃), 1.24–1.38 (m, 16H, 8 × CH₂), 1.58–1.65 (m, 2H, CH₂), 2.81 (t, J = 7.4 Hz, 2H, C1′–H₂), 6.37 (s, 1H, C3–H), 7.28 (dd, 1H, J = 2.3 and 8.9 Hz, C6–H), 7.33 (d, J = 2.3 Hz, 1H, C8–H), 7.93 (d, J = 8.6 Hz, 1H, C5–H) and 8.24 (s, 2H, NH₂); MS (FAB⁺): m/z (%) 396.1 (100) [M + H]⁺, 317.2 (20) [M + H – HNSO₂]⁻; MS (FAB⁺): m/z (%) 394.3 (100) [M – H]⁻, 315.3 (70) [M – H₂NSO₂]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₂₀H₃₀NO₅S: 396.1845, found: 396.1843; Anal. calcd for C₂₀H₂₉NO₅S: C 60.74, H 7.39, N 3.54, found: C 61.00, H 7.44, N 3.52.

Ethyl 3-Oxo-pentadecanoate (17a)

This was prepared by method B using CH₂Cl₂ (80 mL), ethyl diazoacetate (3.02 g, 26.5 mmol), SnCl₂ (478 mg, 2.5 mmol), and tridecanal (5.0 g, 25 mmol) in CH₂Cl₂ (20 mL). The crude oily residue was purified by flash chromatography (CHCl₃) to give 17a as a colorless oil, which solidified to a white soft solid on standing (6.98 g, 97%): R_f = 0.65 (CHCl₃); mp 28 °C (Lit.⁴⁷ mp < 20 °C); ¹H NMR (400 MHz, CDCl₃) δ = 0.88 (t, J = 6.4 Hz, 3H, C15–H₃), 1.25–1.64 (m, 23H, CH₂CH₃ and 10 × CH₂), 2.53 (t, J = 7.3 Hz, 2H, C4–H₂), 3.43 (s, 2H, C2–H₂) and 4.19 (q, J = 7.3 Hz, 2H, CH₂CH₃); MS (FAB⁺): m/z (%) 285.2 (100) [M + H]⁺, MS (FAB^–): m/z (%) 283.2 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₇H₃₃O₃: 285.2429, found: 285.2426.

4-Dodecyl-7-hydroxycoumarin (17b)

This was prepared with resorcinol (1.16 g, 10.5 mmol), 17a (3.0 g, 11 mmol), and a mixture of CF₃COOH (2.0 mL, 21 mmol) and conc. H₂SO₄ (1.5 mL, 21 mmol). The crude brown solid was purified by flash chromatography (CHCl₃/acetone, 8:1 to 4:1 gradient), and the yellow solid isolated was recrystallized from acetone/hexane to give 17b as fine yellow crystals (747 mg, 22%): R_f = 0.77 (CHCl₃/acetone, 3:1); mp 101–103 °C; ¹H NMR (400 MHz, DMSO-d₆): δ = 0.85 (t, J = 7.0 Hz, 3H, C12′–H₃), 1.17–1.36 (m, 18H, 9 × CH₂), 1.55–1.60 (m, 2H, CH₂), 2.72 (t, J = 7.4 Hz, 2H, C1′–H₂), 6.08 (s, 1H, C3–H), 6.71 (d, J = 2.3 Hz, 1H, C8–H), 6.79 (dd, J = 2.3 and 8.6 Hz, 1H, C6–H), 7.64 (d, J = 8.9 Hz, 1H, C5–H) and 10.54 (s, 1H, OH); MS (FAB⁺): m/z (%) 331.2 (100) [M + H]⁺; MS (FAB^–): m/z (%) 329.3 (100) [M – H]⁺; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₂₁H₃₁O₃: 330.2273, found: 331.2279; Anal. calcd for C₂₁H₃₀O₃: C 76.33, H 9.15, found C 76.80, H 8.80.

4-Dodecylcoumarin-7-O-sulfamate (17)

Upon sulfamoylation, compound 17b (400 mg; 1.21 mmol) gave a crude white solid, which was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient). The white solid isolated was recrystallized from ethyl acetate/hexane to give 17 as white fine crystals (91 mg, 18%): R_f = 0.55 (CHCl₃/ethyl acetate, 4:1); mp 104–106 °C; ¹H NMR (400 MHz, DMSO-d₆): δ = 0.85 (t, J = 7.0 Hz, 3H, C12′–H₃), 1.24–1.34 (m, 18H, 9 × CH₂), 1.58–1.63 (m, 2H, CH₂), 2.81 (t, J = 7.6 Hz, 2H, C1′–H₂), 6.37 (s, 1H, C3–H), 7.28 (dd, J = 2.3 and 8.9 Hz, 1H, C6–H), 7.33 (d, J = 2.3 Hz, 1H, C8–H), 7.93 (d, J = 8.6 Hz, 1H, C5–H) and 8.23 (s, 2H, NH₂); MS (FAB⁺): m/z (%) 410.2 (100) [M + H]⁺, 331.2 (20) [M + H – HNSO₂]⁺; MS (FAB^–): m/z (%) 817.3 (20) [2M – H]⁻, 408.3 (100) [M – H]⁻, 329.3 (70) [M – H₂NSO₂]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₂₁H₃₂NO₅S: 410.2001, found: 410.2006; Anal. calcd for C₂₁H₃₁NO₅S: C 61.59, H 7.63, N 3.42, found: C 61.90, H 7.65, N, 3.34.

Ethyl 3-Oxo-hexadecanoate (18a)

This was prepared by method B using CH₂Cl₂ (80 mL), ethyl diazoacetate (2.82 g, 24.7 mmol), SnCl₂ (446 mg, 2.35 mmol), and tetradecanal (5.0 g, 24 mmol) in CH₂Cl₂ (20 mL). The crude oily residue was purified by flash chromatography (CHCl₃) to give 18a as a colorless oil, which solidified to an off-white soft solid on standing (5.45 g, 78%): R_f = 0.71 (CHCl₃); mp 45–47 °C (Lit.⁴⁹ mp 41–42 °C); MS (FAB⁺) m/z: 299.2 [100, (M + H)⁺]; ¹H NMR (400 MHz, CDCl₃) δ = 0.88 (t, J = 6.67 Hz, 3H, C16–H₃), 1.20–1.69 (m, 25H, CH₂CH₃ and 11 × CH₂), 2.53 (t, J = 7.3 Hz, 2H, C4–H₂), 3.43 (s, 2H, C2–H₂) and 4.19 (q, J = 7.3 Hz, 2H, CH₂CH₃); MS (FAB⁺): m/z (%) 299.2 (100) [M + H]⁺; MS (FAB^–): m/z (%) 297.2 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₈H₃₅O₃: 299.2586, found: 299.2599.

7-Hydroxy-4-tridecylcoumarin (18b)

This was prepared with resorcinol (1.11 g, 10.1 mmol), 18a (3.0 g, 10 mmol), and a mixture of CF₃COOH (2.0 mL, 20 mmol) and conc. H₂SO₄ (1.5 mL, 20 mmol). The crude brown solid was purified by flash chromatography (CHCl₃/acetone, 8:1 to 4:1 gradient), and the beige solid isolated was recrystallized from acetone/hexane to give 18b as fine cream-colored crystals (757 mg, 22%): R_f = 0.72 (CHCl₃/acetone, 3:1); mp 95–97 °C; ¹H NMR (400 MHz, DMSO-d₆): δ = 0.85 (t, J = 7.0 Hz, 3H, C13′–H₃), 1.19–1.36 (m, 20H, 10 × CH₂), 1.54–1.62 (m, 2H, CH₂), 2.72 (t, J = 7.4 Hz, 2H, C1′–H₂), 6.08 (s, 1H, C3–H), 6.71 (d, J = 2.3 Hz, 1H, C8–H), 6.79 (dd, J = 2.3 and 8.9 Hz, 1H, C6–H), 7.64 (d, J = 8.6 Hz, 1H, C5–H) and 10.54 (s, 1H, OH); MS (FAB⁺): m/z (%) 345.2 (100) [M + H]⁺; MS (FAB^–): m/z (%) 343.3 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₂₂H₃₃O₃: 345.2429, found: 345.2438; Anal. calcd for C₂₂H₃₂O₃: C 76.70, H 9.36, found: C 76.85, H 9.31.

4-Tridecylcoumarin-7-O-sulfamate (18)

Upon sulfamoylation, compound 18b (350 mg, 1.01 mmol) gave a crude white solid, which was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient). The white solid isolated was recrystallized from ethyl acetate/hexane to give 18 as white crystals (101 mg, 23%): R_f = 0.53 (CHCl₃/ethyl acetate, 4:1); mp 120–121 °C; ¹H NMR (400 MHz, DMSO-d₆): δ = 0.86 (t, J = 7.0 Hz, 3H, C13′–H₃), 1.20–1.39 (m, 20H, 10 × CH₂), 1.58–1.66 (m, 2H, CH₂), 2.81 (t, J = 7.8 Hz, 2H, C1′–H₂), 6.38 (s, 1H, C3–H), 7.29 (dd, J = 2.3 and 8.6 Hz, 1H, C6–H), 7.33 (d, J = 2.3 Hz, 1H, C8–H), 7.93 (d, J = 8.9 Hz, 1H, C5–H) and 8.24 (s, 2H, NH₂); MS (FAB⁺): m/z (%) 424.3 (100) [M + H]⁺, 345.3 (25) [M + H – HNSO₂]⁺; MS (FAB^–): m/z (%) 422.3 (60) [M – H]⁻, 343.3 (100) [M – H₂NSO₂]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₂₂H₃₄NO₅S: 424.2158, found: 424.2168; Anal. calcd for C₂₂H₃₃NO₅S: C 62.38, H 7.85, N 3.31, found: C 62.60, H 7.90, N 3.46.

7-Hydroxy-4-(prop-2-yl)coumarin (19a)

This was prepared with resorcinol (1.21 g, 11.1 mmol), ethyl 4-methyl-3-oxopentanoate (1.6 g, 10 mmol), and a mixture of CF₃COOH (1.7 mL, 22 mmol) and conc. H₂SO₄ (2.2 mL, 22 mmol). The crude yellow solid was purified by recrystallization from acetone/hexane to give 19a as pale white fine crystals (420 mg, 21%): R_f = 0.42 (CHCl₃/acetone 3:1); mp 120–122 °C (Lit.⁵⁰ mp 62–64 °C—NB we are unable to explain this discrepancy) ¹H NMR (400 MHz, DMSO-d₆): δ = 1.24 (d, J = 6.7 Hz, 6H, CH(CH₃)₂), 3.28–3.35 (m, 1H, CH(CH₃)₂), 6.08 (s, 1H, C3–H), 6.72 (d, J = 2.1 Hz, 1H, C8–H), 6.81 (dd, J = 2.1 and 8.8 Hz, 1H, C6–H), 7.71 (d, J = 8.8 Hz, 1H, C5–H) and 10.54 (s, 1H, OH); MS (FAB⁺): m/z (%) 409.2 (20) [2M + H]⁺, 205.2 (100) [M + H]⁺; MS (FAB^–): m/z (%) 407.2 (20) [2M – H]⁻, 203.1 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₂H₁₃O₃: 205.0865, found: 205.0874; Anal. calcd for C₁₂H₁₂O₃: C 70.57, H 5.92, found: C 70.60, H 6.00.

4-(Prop-2-yl)coumarin-7-O-sulfamate (19)

Upon sulfamoylation, compound 19a (400 mg, 1.96 mmol) gave a crude white solid, which was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient). The white solid isolated was recrystallized from ethyl acetate/hexane to give 19 as white fine crystals (150 mg, 30%): R_f = 0.22 (CHCl₃/ethyl acetate, 4:1); mp 164–167 °C; ¹H NMR (400 MHz, DMSO-d₆): δ = 1.26 (d, J = 6.7 Hz, 6H, CH(CH₃)₂), 3.31–3.34 (m, 1H, CH(CH₃)₂), 6.35 (s, 1H, C3–H), 7.29 (dd, J = 2.4 and 8.8 Hz, 1H, C6–H), 7.33 (d, J = 2.4 Hz, 1H, C8–H), 7.99 (d, J = 8.8 Hz, 1H, C5–H) and 8.23 (s, 2H, NH₂); MS (FAB⁺): m/z (%) 567.1 (70) [2M + H]⁺, 284.1 (100) [M + H]⁺; MS (FAB^–): m/z (%) 565.2 (30) [2M – H]⁻, 282.1 (100) [M – H]⁻, 203.1 (60) [M – H₂NSO₂]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₂H₁₄NO₅S: 284.0593, found: 284.0599; Anal. calcd for C₁₂H₁₃NO₅S: C 50.88, H 4.63, N 4.94, found: C 50.80, H 4.62, N 4.97.

7-Hydroxy-4-(1′,1′-dimethylethyl)coumarin (20a)

This was prepared with resorcinol (7.0 g, 63 mmol), methyl 4,4-dimethyl-3-oxopentanoate (10.0 g, 63.2 mmol), and a mixture of CF₃COOH (10 mL, 0.2 mol) and conc. H₂SO₄ (6.5 mL, 0.2 mol). The crude brown residue was purified by flash chromatography (CHCl₃/acetone, 8:1 to 4:1 gradient), and the pale yellow solid isolated was recrystallized from THF/hexane to give 20a as pale yellow crystals (380 mg, 0.03%): R_f = 0.61 (CHCl₃/acetone, 3:1); mp 159–161 °C; ¹H NMR (400 MHz, CDCl₃) δ = 1.49 (s, 9H, C(CH₃)₃), 6.28 (s, 1H, C3–H), 6.85 (dd, J = 2.7 and 8.9 Hz, 1H, C6–H), 7.11 (d, J = 2.7 Hz, 1H, C8–H), 7.13 (s, 1H, OH) and 7.91 (d, J_6,5 = 8.9 Hz, 1H, C5–H); MS (FAB⁺): m/z (%) 219.1 (100) [M + H]⁺; MS (FAB^–): m/z (%) 217.1 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₃H₁₅O₃: 219.1021, found: 219.1029; Anal. calcd for C₁₃H₁₄O₃: C 71.54, H 6.47%, found: C 71.90, H 6.49; HPLC: MeOH/H₂O (90:10), flow rate = 2 mL min^–1, λ_max = 321 nm, t_R = 1.91 min.

4-(1′,1′-Dimethylethyl)coumarin-7-O-sulfamate (20)

Upon sulfamoylation, compound 20a (310 mg, 1.43 mmol) gave a crude white solid, which was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient). The white solid isolated was recrystallized from ethyl THF/hexane to give 20 as white fine crystals (43 mg, 10%): R_f = 0.48 (CHCl₃/ethyl acetate, 4:1); mp 187–189 °C; ¹H NMR (400 MHz, DMSO-d₆): δ = 1.45 (s, 9H, C(CH₃)₃), 6.33 (s, 1H, C3–H), 7.27 (dd, J = 2.3 and 8.9 Hz, 1H, C6–H), 7.34 (d, J = 2.3 Hz, 1H, C8–H), 8.26 (s, 2H, NH₂) and 8.28 (d, J = 8.9 Hz, 1H, C5–H); MS (FAB⁺): m/z (%) 298.0 (100) [M + H]⁺, 219.1 (15) [M + H – HNSO₂]⁺; MS (FAB^–): m/z (%) 296.0 (100) [M – H]⁻, 217.0 (40) [M – H₂NSO₂]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₃H₁₆NO₅S: 298.0749, found: 298.0746; Anal. calcd for C₁₃H₁₅O₅NS: C 52.51, H 5.08, N 4.71, found: C 52.40, H 4.91, N 4.76; HPLC: MeOH/H₂O (70:30), flow rate = 2 mL min^–1, λ_max = 273.4 and 310.1 nm, t_R = 1.6 min.

4-Chloromethyl-7-hydroxycoumarin (21a)

This was prepared with resorcinol (2.93 g, 29.6 mmol), methyl 4-chloro-3-oxo-butanoate (4.0 g, 29 mmol), and a mixture of CF₃COOH (4.1 mL, 53 mmol) and conc. H₂SO₄ (2.7 mL, 53 mmol). The crude orange solid was purified by recrystallization from acetone/hexane to give 21a as off-white fine crystals (1.31 g, 23%): R_f = 0.73 (CHCl₃/acetone, 3:1); mp 183–185 °C (Lit.⁵¹ mp 181 °C); ¹H NMR (400 MHz, DMSO-d₆): δ = 4.96 (s, 2H, CH₂), 6.42 (s, 1H, C3–H), 6.76 (d, J = 2.4 Hz, 1H, C8–H), 6.85 (dd, J = 2.4 and 8.7 Hz, 1H, C6–H), 7.69 (d, J = 8.8 Hz, 1H, C5–H) and 10.69 (s, 1H, OH); MS (FAB⁺): m/z (%) 421.2 (15) [2M + H]⁺, 211.1 (100) [M(Cl³⁵) + H]⁺; MS (FAB^–): m/z (%) 419.1 (15) [2M – H]⁻, 209.1 (100) [M(Cl³⁵) – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₀H₈³⁷ClO₃: 213.0132 and 211.0151, C₁₀H₈³⁵ClO₃: 211.0162, found: 213.0127; Anal. calcd for C₁₀H₇ClO₃: C 57.03, H 3.35%, found: C 57.00, H 3.20.

4-Chloromethylcoumarin-7-O-sulfamate (21)

Upon sulfamoylation, compound 21a (400 mg, 1.9 mmol) gave a crude yellow solid, which was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient). The white solid isolated was recrystallized from ethyl acetate/hexane to give 21 as pale green fine crystals (59 mg, 11%): R_f = 0.44 (CHCl₃/ethyl acetate, 4:1); mp 172–175 °C; ¹H NMR (400 MHz, DMSO-d₆): δ = 5.05 (s, 2H, CH₂), 6.72 (s, 1H, C3–H), 7.34 (dd, J = 2.4 and 8.8 Hz, 1H, C6–H), 7.38 (d, J = 2.4 Hz, 1H, C8–H), 7.96 (d, J = 8.8 Hz, 1H, C5–H) and 8.28 (s, 2H, NH₂); MS (FAB⁺): m/z (%) 289.9 (95) [M(Cl³⁵) + H]⁺, 210.9 (100) [M + H – HNSO₂]⁺; MS (FAB^–): m/z (%) 287.9 (100) [M(Cl³⁵) – H]⁻, 208.9 (90) [M – H₂NSO₂]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₀H₉³⁵ClNO₅S: 289.9889 and 291.9862, C₁₀H₉³⁷ClNO₅S: 291.9860, found: 289.9893; Anal. calcd for C₁₀H₈ClNO₅S: C 41.46, H 2.78, N 4.84, found: C 41.50, H 2.79, N 3.48.

7-Hydroxy-4-phenylcoumarin (22a)

This was prepared with resorcinol (2.0 g, 18 mmol), ethyl 3-oxo-3-phenylpropanoate (2.0 g, 18 mmol), and a mixture of CF₃COOH (2.8 mL, 36 mmol) and conc. H₂SO₄ (1.85 mL, 36.3 mmol). The crude orange solid was purified by recrystallization from hot absolute ethanol to give 22a as yellow crystals (1.85 g, 43%): R_f = 0.71 (CHCl₃/acetone, 3:1); mp 248–252 °C (Lit.⁵² mp 247–248 °C); ¹H NMR (400 MHz, DMSO-d₆): δ = 6.15 (s, 1H, C3–H), 6.79 (dd, J = 1.2 and 8.8 Hz, 1H, C6–H), 6.81 (d, J = 1.2 Hz, 1H, C8–H), 7.27 (d, J = 8.8 Hz, 1H, C5–H), 7.51–7.57 (m, 5H, Ph–H) and 10.67 (s, 1H, OH); MS (FAB⁺): m/z (%) 239.1 (100) [M + H]⁺; MS (FAB^–): m/z (%) 237.1 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₅H₁₁O₃: 239.0629, found: 239.0634; Anal. calcd for C₁₅H₁₀O₃: C 75.62, H 4.23. found: C 75.40, H 4.13.

4-Phenylcoumarin-7-O-sulfamate (22)

Upon sulfamoylation, compound 22a (700 mg, 2.94 mmol) gave a crude white solid, which was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient). The white solid isolated was recrystallized from ethyl acetate/hexane to give 22 as white fine fluffy crystals (304 mg, 33%): R_f = 0.60 (CHCl₃/ethyl acetate, 4:1); mp 185–190 °C; ¹H NMR (400 MHz, DMSO-d₆): δ = 6.36 (s, 1H, C3–H), 7.28 (dd, J = 2.1 and 8.7 Hz, 1H, C6–H), 7.39 (d, J = 2.1 Hz, 1H, C8–H), 7.58 (d, J = 8.7 Hz, 1H, C5–H) and 7.59–7.62 (m, 7H, Ph–H and NH₂—reduced to 5H when exchanged with D₂O); MS (FAB⁺): m/z (%) 318.1 (100) [M + H]⁺; MS (FAB^–): m/z (%) 316.2 (100) [M – H]⁻, 237.2 (65) [M – H₂NSO₂]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₅H₁₂NO₅S: 318.0357, found: 318.0379; Anal. calcd for C₁₅H₁₁NO₅S: C 56.78, H 3.49, N 4.41, found: C 56.70, H 3.53, N 4.48.

Ethyl 3-Oxo-4-phenylbutanoate (23a)

This was prepared by method B using CH₂Cl₂ (80 mL), ethyl diazoacetate (4.99 g, 43.7 mmol), SnCl₂ (790 mg, 3.73 mmol), and phenylacetaldehyde (5.0 g, 42 mmol) in CH₂Cl₂. The crude oily residue was purified by distillation under reduced pressure to give 23a as a pale yellow oil (5.27 g, 61%): R_f = 0.62 (CHCl₃); bp_0.3 185–189 °C (Lit.⁵³ bp₉ 154–156 °C—NB we are unable to explain this discrepancy) ¹H NMR (400 MHz, DMSO-d₆): δ = 2.16 (t, J = 7.3 Hz, 3H, CH₂CH₃), 3.45 (s, 2H, PhCH₂), 3.83 (s, 2H, C2–H₂), 4.17 (q, J = 7.3 Hz, 2H, CH₂CH₃) and 7.20–7.36 (m, 5H, PhH); MS (FAB⁺): m/z (%) 207.1 (100) [M + H]⁺, 91.1 (40) [PhCH₂]⁺; MS (FAB^–): m/z (%) 205.1 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₂H₁₅O₃: 207.1021, found: 207.1014.

4-Benzyl-7-hydroxycoumarin (23b)

This was prepared with resorcinol (1.6 g, 15 mmol), 23a (3.0 g, 15 mmol), and a mixture of CF₃COOH (2.5 mL, 29 mmol) and conc. H₂SO₄ (1.5 mL, 29 mmol). The crude yellow solid was purified by recrystallization from hot absolute ethanol to give 23b as pale yellow crystals (2.19 g, 60%): R_f = 0.80 (CHCl₃/acetone, 3:1), mp 209–212 °C (Lit.⁵⁴ mp 214–215 °C); ¹H NMR (400 MHz, CDCl₃) δ = 4.38 (s, 2H, CH₂Ph), 5.98 (s, 1H, C3–H), 6.71 (d, J = 2.3 Hz, 1H, C8–H), 6.76 (dd, J = 2.3 and 8.6 Hz, 1H, C6–H), 7.23–7.36 (m, 5H, PhH), 7.67 (d, J = 8.6 Hz, 1H, C5–H) and 10.57 (s, 1H, OH); MS (FAB⁺): m/z (%) 505.1 (10) [2M + H]⁺, 253.1 (100) [M + H]⁺; MS (FAB^–): m/z (%) 503.2 (15) [2M – H]⁻, 251.2 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₆H₁₃O₃: 253.0865, found: 253.0863; Anal. calcd for C₁₆H₁₂O₃: C 76.18, H 4.79, found: C 75.60, H 4.88.

4-Benzylcoumarin-7-O-sulfamate (23)

Upon sulfamoylation, 23b (400 mg, 1.6 mmol) gave a crude white solid, which was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient). The white solid isolated was recrystallized from ethyl acetate/hexane to give 23 as white fine crystals (135 mg, 26%): R_f = 0.39 (CHCl₃/ethyl acetate, 4:1); mp 180–182 °C; ¹H NMR (400 MHz, DMSO-d₆): δ = 4.23 (s, 2H, CH₂Ph), 6.32 (s, 1H, C3–H), 7.23–7.26 (m, 2H, C8–H and C6–H), 7.26–7.37 (m, 5H, PhH), 7.92 (d, J = 8.9 Hz, 1H, C5–H) and 8.22 (s, 2H, NH₂); MS (FAB⁺): m/z (%) 663.4 (30) [2M + H]⁺, 332.1 (100) [M + H]⁺; MS (FAB^–): m/z (%) 330.2 (100) [M – H]⁻, 251.2 (50) [M – H₂NSO₂]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₆H₁₄NO₅S: 332.0593, found: 332.0590; Anal. calcd for C₁₆H₁₃NO₅S: C 58.00, H 3.95, N 4.23%, found: C 57.70, H 3.97, N, 4.21.

Ethyl 3-Oxo-5-phenylpentanoate (24a)

This was prepared by method B using CH₂Cl₂ (80 mL), ethyl diazoacetate (4.5 g, 39 mmol), SnCl₂ (700 mg, 3.73 mmol), and hydrocinnam-aldehyde (5.0 g, 37 mmol) in CH₂Cl₂. The crude oily residue was purified by flash chromatography (CHCl₃) to give 24a as a pale yellow oil (5.38 g, 66%): R_f = 0.63 (CHCl₃); ¹H NMR (400 MHz, DMSO-d₆): δ = 1.17 (t, J = 7.3 Hz, 3H, CH₂CH₃), 2.79 (t, J = 7.5 Hz, 2H, C4–H₂), 2.87 (t, J = 7.5 Hz, 2H, C5–H₂), 3.60 (s, 2H, C2–H₂), 4.08 (q, J = 7.3 Hz, 2H, CH₂CH₃) and 7.13–7.29 (m, 5H, PhH); MS (FAB⁺): m/z (%) 221.1 (100) [M + H]⁺, 91.0 (55) [PhCH₂]⁺; MS (FAB^–): m/z (%) 219.1 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₃H₁₇O₃: 221.1178, found: 221.1181.

7-Hydroxy-4-(2-phenylethyl)coumarin (24b)

This was prepared with resorcinol (1.25 g, 11.4 mmol), 24a (2.5 g, 11 mmol), and a mixture of CF₃COOH (1.75 mL, 22.7 mmol) and conc. H₂SO₄ (1.16 mL, 22.7 mmol). The crude yellow solid was purified by recrystallization from hot absolute ethanol to give 24b as pale white crystals (1.06 g, 35%): R_f = 0.65 (CHCl₃/acetone, 3:1); mp 175–177 °C (Lit.⁵⁵ mp 175–176 °C); ¹H NMR (400 MHz, CDCl₃) δ = 2.98–3.06 (m, 4H, CH₂CH₂), 6.12 (s, 1H, C3–H), 6.59 (s, 1H, OH), 6.84 (dd, J = 2.4 and 8.8 Hz, 1H, C6–H), 7.11 (d, J = 2.4 Hz, 1H, C8–H), 7.20–7.34 (m, 5H, PhH) and 7.53 (d, J = 8.8 Hz, 1H, C5–H); MS (FAB⁺): m/z (%) 533.2 (40) [2M + H]⁺, 267.1 (100) [M + H]⁺, 91.1 (20) [CH₂Ph]⁺; MS (FAB^–): m/z (%) 531.2 (30) [2M – H]⁻, 265.0 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₇H₁₅O₃: 267.1021, found: 267.1018; Anal. calcd for C₁₇H₁₄O₃: C 76.68, H 5.30, found: C 76.70, H 5.20.

4-(2-Phenylethyl)coumarin-7-O-sulfamate (24)

Upon sulfamoylation, 24b (400 mg, 1.5 mmol) gave a crude white solid, which was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient). The white solid isolated was recrystallized from THF/hexane to give 24 as white fine crystals (395 mg, 76%): R_f = 0.27 (CHCl₃/ethyl acetate, 4:1); mp 89–93 °C; ¹H NMR (400 MHz, DMSO-d₆): δ = 2.96 (t, J = 8.8 Hz, 2H, CH₂CH₂Ph), 3.14 (t, J = 8.5 Hz, 2H, CH₂Ph), 6.36 (s, 1H, C3–H), 7.22–7.34 (m, 7H, PhH, C6–H and C8–H), 8.03 (d, J = 7.9 Hz, 1H, C5–H) and 8.25 (s, 2H, NH₂); MS (FAB⁺): m/z (%) 346.0 (100) [M + H]⁺, 91.0 (50) [CH₂Ph]⁺; MS (FAB^–): m/z (%) 344.0 (100) [M – H]⁻, 265.0 (60) [M – H₂NSO₂]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₇H₁₆NO₅S: 346.0749, found: 346.0754; Anal. calcd for C₁₇H₁₅NO₅S: C 59.12, H 4.38, N 4.06, found: C 59.30, H 4.89, N 3.86.

Ethyl 3-(4-Ethylphenyl)-3-oxo-propanoate (25a)

This was prepared by method A using ethyl potassium malonate (6.07 g, 35.7 mmol), CH₃CN (120 mL), Et₃N (7.58 mL, 54.4 mmol), MgCl₂ (4.05 g, 42.5 mmol), and 4-ethylbenzoyl chloride (2.5 mL, 17 mmol). The crude oily residue was purified by distillation under reduced pressure to give 25a as a colorless oil (3.74 g, 78%): R_f = 0.64 (CH₂Cl₂); bp_0.23 131–135 °C (Lit.⁵⁶ bp_0.004 105–112 °C); ¹H NMR (400 MHz, CDCl₃) δ = 1.22–1.33 (m, 6H, CH₃CH₂Ph and CH₂CH₃), 2.70 (q, J = 7.8 Hz, 2H, CH₂Ph), 3.97 (s, 2H, C2–H₂), 4.21 (q, J = 6.8 Hz, 2H, CH₂CH₃), 7.29 (d, J = 8.3 Hz, 2H, 2 × ArH) and 7.87 (d, J = 8.3 Hz, 2H, 2 × Ar-H); MS (FAB⁺): m/z (%) 221.0 (100) [M + H]⁺; MS (FAB^–): m/z (%) 219.0 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₃H₁₇O₃: 221.1178, found: 221.1177.

4-(4-Ethylphenyl)-7-hydroxycoumarin (25b)

This was prepared with resorcinol (1.5 g, 14 mmol), 25a (3.0 g, 14 mmol), and a mixture of CF₃COOH (2.5 mL, 27 mmol) and conc. H₂SO₄ (1.5 mL, 27 mmol). The crude orange solid was purified by recrystallization from hot ethanol to give 25b as white needles (1.66 g, 46%): R_f = 0.72 (CHCl₃/acetone, 4:1); mp 176–180 °C; ¹H NMR (400 MHz, DMSO-d₆): δ = 1.23 (t, J = 7.6 Hz, 3H, CH₃CH₂), 2.69 (q, J = 7.6 Hz, 2H, CH₃CH₂), 6.13 (s, 1H, C3–H), 6.77 (m, 2H, C6–H and C8–H), 7.31 (d, J = 8.5 Hz, 1H, C5–H), 7.38–7.44 (m, 4H, PhH) and 10.64 (s, 1H, OH); MS (FAB⁺): m/z (%) 533.1 (10) [2M + H]⁺, 267.0 (100) [M + H]⁺; MS (FAB^–): m/z (%) 531.1 (20) [2M – H]⁻, 265.1 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₇H₁₅O₃: 267.1021, found: 267.1018; Anal. calcd for C₁₇H₁₄O₃: C 76.68, H 5.30, found: C 76.30, H, 5.30.

4-(4-Ethylphenyl)coumarin-7-O-sulfamate (25)

Upon sulfamoylation, compound 25b (400 mg, 1.5 mmol) gave a crude white solid, which was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient). The white solid isolated was recrystallized from THF/hexane to give 25 as white fine crystals (114 mg, 22%): R_f = 0.35 (CHCl₃/ethyl acetate, 4:1); mp 170–173 °C; ¹H NMR (400 MHz, DMSO-d₆): δ = 1.25 (t, J = 7.4 Hz, 3H, CH₃CH₂), 2.71 (q, J = 7.4 Hz, 2H, CH₃CH₂), 6.44 (s, 1H, C3–H), 7.26 (dd, J = 2.3 and 8.9 Hz, 1H, C6–H), 7.42–7.49 (m, 5H, PhH and C8–H), 7.57 (d, J = 8.6 Hz, 1H, C5–H) and 8.28 (s, 2H, NH₂); MS (FAB⁺): m/z (%) 691.0 (30) [2M + H]⁺, 346.1 (100) [M + H]⁺; MS (FAB^–): m/z (%) 689.3 (10) [2M – H]⁻, 344.2 (100) [M – H]⁻, 265.2 (60) [M – H₂NSO₂]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₇H₁₆NO₅S: 346.0749, found: 346.0749; Anal. calcd for C₁₇H₁₅NO₅S: C 59.12, H 4.38, N 4.06, found: C 59.00, H 4.36, N 4.03.

Ethyl 3-Cyclohexyl-3-oxo-propanoate (26a)

This was prepared by method B using CH₂Cl₂ (80 mL), ethyl diazoacetate (5.34 g, 46.8 mmol), SnCl₂ (85 mg, 4.5 mmol), and cyclohexanecarboxaldehyde (5.0 g, 45 mmol) in CH₂Cl₂ (20 mL). The crude oily residue was purified by fractional distillation under reduced pressure to give 26a as a pale yellow oil (5.38 g, 66%): R_f = 0.63 (CHCl₃); bp_0.3 135–139 °C (Lit.⁵³ bp₁₈ 146–150 °C); ¹H NMR (400 MHz, CDCl₃) δ = 1.28 (t, J = 7.2 Hz, 3H, CH₂CH₃), 1.39–2.49 (m, 11H, cyclohexyl-H), 3.48 (s, 2H, C2–H₂) and 4.19 (q, J = 7.2 Hz, 2H, CH₂CH₃); MS (FAB⁺): m/z (%) 199.0 (100) [M + H]⁺; MS (FAB^–): m/z (%) 197.0 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₁H₁₉O₃: 199.1334, found: 199.1341.

4-Cyclohexyl-7-hydroxycoumarin (26b)

This was prepared with resorcinol (1.67 g, 15.1 mmol), 26a (3.0 g, 15 mmol), and a mixture of CF₃COOH (2.33 mL, 30.3 mmol) and conc. H₂SO₄ (1.5 mL, 30 mmol). The crude yellow solid was purified by flash chromatography (CHCl₃/acetone, 8:1 to 4:1 gradient), and the pale yellow solid isolated was recrystallized from hot acetone to give 26b as white crystals (1.45 g, 39%): R_f = 0.80 (CHCl₃/acetone, 4:1); mp 191–192 °C (Lit.⁵⁷ mp 176–178 °C); ¹H NMR (400 MHz, DMSO-d₆): δ = 1.38–1.87 (m, 11H, cyclohexyl-H), 6.04 (s, 1H, C3–H), 6.71 (d, J = J = 2.3 Hz, 1H, C8–H), 6.81 (dd, J = 2.4 and 8.8 Hz, 1H, C6–H), 7.70 (d, J = 8.8 Hz, 1H, C5–H) and 10.56 (s, 1H, OH); MS (FAB⁺): m/z (%) 489.1 (15) [2M + H]⁺, 245.1 (100) [M + H]⁺; MS (FAB^–): m/z (%) 487.3 (15) [2M – H]⁻, 243.2 (100) [M – H]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₅H₁₇O₃: 245.1178, found: 245.1179; Anal. calcd for C₁₅H₁₆O₃: C 73.75, H 6.60, found: C 73.90, H 6.60.

4-Cyclohexylcoumarin-7-O-sulfamate (26)

Upon sulfamoylation, compound 26a (400 mg, 1.6 mmol) gave a crude white solid, which was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient). The white solid isolated was recrystallized from THF/hexane to give 26 as white fine crystals (192 mg, 36%): R_f = 0.45 (CHCl₃/ethyl acetate, 4:1); mp 187–190 °C; ¹H NMR (400 MHz, DMSO-d₆): δ = 1.23–2.06 (m, 11H, cyclohexyl-H), 6.32 (s, 1H, C3–H), 7.32 (dd, J = 2.3 and 8.6 Hz, 1H, C6–H), 7.36 (d, J = 2.3 Hz, 1H, C8–H), 8.01 (d, J = 8.9 Hz, 1H, C5–H) and 8.31 (s, 2H, NH₂); MS (FAB⁺): m/z (%) 324.1 (100) [M + H]⁺, 245.1 (15) [M + H – HNSO₂]⁺; MS (FAB): m/z (%) 323.2 (100) [M – H]⁻, 243.2 (50) [M – H₂NSO₂]⁻; HRMS-FAB⁺: m/z [M + H]⁺ calcd for C₁₅H₁₈NO₅S: 324.0906, found: 324.0902; Anal. calcd for C₁₅H₁₇NO₅S: C 55.72, H 5.30, N 4.33, found: C 55.70, H 5.28, N 4.17.

(Adamant-1-yl)acetyl Chloride (27a)

1-Adamantane acetic acid (5.0 g; 25.7 mmol) in an excess of thionyl chloride (20 mL; 77.2 mmol) and THF (5 mL) was boiled under reflux overnight under N₂. Thionyl chloride was removed under vacuum to get the crude 27a as a brown oil (5.5 g; 101% crude), which was used for the next reaction without purification. R_f: 0.91 (CHCl₃/MeOH, 8:1); (Lit.⁵⁸ bp₃ 107–109 °C); ¹H NMR (400 MHz; CDCl₃) δ_H: 1.11–1.76 (m, 15H, adamantane H) and 2.69 (s, 2H, CH₂CO); MS (FAB⁺) m/z: 213.1 [100, (M(³⁵Cl) + H)⁺]; MS (FAB^–) m/z: 211.1 [100, (M(³⁵Cl) – H)⁻]; Acc. MS m/z (FAB⁺): 213.1097, C₁₂H₁₈³⁵ClO requires 213.1081 and 215.1006, C₁₂H₁₈³⁷ClO requires 215.1012.

Ethyl 4-(Adamant-1-yl)-3-oxo-butanoate (27b)

This was prepared by method A using ethyl potassium malonate (8.4 g; 49 mmol), MeCN (150 mL), Et₃N (11 mL; 75 mmol), MgCl₂ (5.6 g; 59 mmol), and 27a (5.0 g; 24 mmol). The crude oily residue was purified by flash chromatography (CHCl₃) to give 27b as a brown oil (4.5 g; 72%). R_f: 0.77 (CHCl₃/hexane, 9:1); (Lit.⁵⁸ bp_0.07 105–106 °C); MS (FAB⁺) m/z: 265.2 [100, (M + H)⁺], 135 [85, (C₁₀H₁₅ + H)⁺]; ¹H NMR (400 MHz; CDCl₃) δ_H: 1.24–1.58 (m, 15H, adamantane H), 1.28 (t, 3H, CH₂CH₃), 2.28 (s, 2H, CH₂CO), 3.41 (s, 2H, 2-CH₂) and 4.21 (q, 2H, CH₂CH₃, J = 7.0 Hz); MS (FAB^–) m/z: 263.3 [100, (M – H)⁻]; Acc. MS (FAB⁺): 265.1697, C₁₆H₂₅O₃ requires 265.1705.

4-(1-Adamantanemethyl)-7-hydroxycoumarin (27c)

This was prepared by general method using resorcinol (834 mg; 7.6 mmol), 27b (2.0 g; 7.6 mmol), and a mixture of CF₃COOH (1.2 mL; 15 mmol) and conc. H₂SO₄ (1.5 mL; 15 mmol). The crude yellow solid was purified by recrystallization from THF/hexane to give 27c as yellow crystals (1.82 g; 77%). R_f: 0.72 (CHCl₃/acetone, 4:1); mp 211–214 °C; ¹H NMR (400 MHz; CDCl₃) δ_H: 1.52–2.09 (m, 15H, adamantane H), 1.90 (s, 2H, CH₂), 5.95 (s, 1H, C₃–H), 6.69 (d, 1H, C₈–H, J = 2.3 Hz), 6.78 (dd, 1H, C₆–H, J = 2.3, 8.6 Hz), 7.75 (d, 1H, C₅–H, J = 8.9 Hz) and 10.54 (s, 1H, OH); found C, 77.42; H, 7.15; C₂₀H₂₂O₃ requires C, 77.39; H, 7.14%; MS (FAB⁺) m/z: 311.1 [100, (M + H)⁺]; MS (FAB^–) m/z: 309.2 [100, (M – H)⁻]; Acc. MS (FAB⁺): 311.1568, C₂₀H₂₃O₃ requires 311.1569.

4-(1-Adamantanemethyl)coumarin-7-O-sulfamate (27)

Compound 27c (400 mg; 1.3 mmol) was sulfamoylated, the crude white solid was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient), and the white solid isolated was recrystallized from THF/hexane to give 27 as white fine crystals (88 mg; 18%). R_f: 0.57 (CHCl₃/ethyl acetate, 4:1); mp 218–221 °C; ¹H NMR (400 MHz; DMSO-d₆) δ_H: 1.54–1.64 (m, 15H, adamantane H), 1.91 (s, 2H, CH₂), 6.24 (s, 1H, C₃–H), 7.24–7.31 (m, 2H, C₆–H and C₈–H), 8.04 (d, 1H, C₅–H, J = 8.9 Hz) and 8.25 (s, 2H, NH₂); MS (FAB⁺) m/z: 390.0 [100, (M + H)⁺]; MS (FAB^–) m/z: 388.1 [100, (M – H)⁻]; Acc. MS (FAB⁺): 390.1301, C₂₀H₂₄NO₅S requires 390.1297; found C, 61.40; H, 5.75; N, 3.22; C₂₀H₂₃NO₅S requires C, 61.68; H, 5.95; N, 3.60%.

Ethyl 2-Acetylheptanoate (28a)

This was prepared by general method using K₂CO₃ (11.09 g; 79.2 mmol), water (50 mL), 1-bromopentane (4.09 mL; 33 mmol), ethyl 3-oxo-butanoate (4.29 mL; 33 mmol), CH₂Cl₂ (50 mL), and Bu₄NCl (∼10 g; 33 mmol). The crude oily residue (4.68 g) was purified by flash chromatography (CHCl₃) to give 28a as a pale yellow oil (4.03 g; 61%). (Lit.⁵⁹ bp₈ 101–105 °C); R_f: 0.69 (CHCl₃); ¹H NMR (400 MHz; DMSO-d₆) δ_H: 0.85 (t, 3H, CH₃, J = 7.3 Hz), 1.19 (t, 3H, CH₂CH₃, J = 7.3 Hz), 1.23–1.27 (m, 6H, 3 × CH₂), 1.68–1.72 (q, 2H, 3-CH₂, J = 6.1 Hz), 2.17 (s, 3H, CH₃CO), 3.57 (t, 1H, 2H, J = 7.3 Hz) and 4.12 (q, 2H, CH₂CH₃, J = 7.3 Hz); MS (FAB⁺) m/z: 201.2 [100, (M + H)⁺]; Acc. MS (FAB⁺): 201.1492, C₁₁H₂₁O₃ requires 201.1491.

7-Hydroxy-4-methyl-3-pentylcoumarin (28b)

This was prepared by general method using resorcinol (2.2 g; 20 mmol), 28a (4.0 g; 20 mmol), and a mixture of CF₃COOH (3.08 mL; 40 mmol) and conc. H₂SO₄ (2.04 mL; 40 mmol). The crude pale yellow solid was purified by flash chromatography (CHCl₃/acetone, 8:1 to 4:1 gradient), and the pale yellow solid isolated was recrystallized from acetone/hexane to give 28b as white crystals (2.03 g; 38%). R_f: 0.80 (CHCl₃/acetone, 3:1); mp 101–102 °C (Lit.⁶¹ mp 111–113 °C); ¹H NMR (400 MHz; DMSO-d₆) δ_H: 0.87 (t, 3H, 5′-CH₃, J = 6.8 Hz), 1.29–1.44 (m, 6H, 3 × CH₂), 2.35 (s, 3H, C₄–CH₃), 2.51 (t, 2H, 1′-CH₂, J = 7.3 Hz), 6.71 (d, 1H, C₈–H, J = 2.4 Hz), 6.78 (dd, 1H, C₆–H, J = 2.4, 8.8 Hz), 7.59 (d, 1H, C₅–H, J = 8.8 Hz) and 10.36 (s, 1H, OH); found C, 72.90; H, 7.29; MS (FAB⁺) m/z: 493.4 [15, (2M + H)⁺], 247.3 [100, (M + H)⁺]; MS (FAB^–) m/z: 491.3 [10, (2M – H)⁻], 245.2 [100, (M – H)⁻]; Acc. MS (FAB⁺): 247.1327, C₁₅H₁₉O₃ requires 247.1334; C₁₅H₁₈O₃ requires C, 73.15; H, 7.37%.

4-Methyl-3-pentylcoumarin-7-O-sulfamate (28)

Compound 28b (700 mg; 2.84 mmol) was sulfamoylated, the crude white solid was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient), and the white solid isolated was recrystallized from ethyl acetate/hexane to give 28 as white fine crystals (479 mg; 52%). R_f: 0.82 (CHCl₃/ethyl acetate, 4:1); mp 133–135 °C; ¹H NMR (400 MHz; DMSO-d₆) δ_H: 0.87 (t, 3H, 5′-CH₃, J = 6.7 Hz), 1.31–1.49 (m, 6H, 3 × CH₂), 2.43 (s, 3H, C₄–CH₃), 2.58 (t, 2H, 1′-CH₂, J = 8.2 Hz), 7.26–7.29 (m, 2H, C₆–H and C₈–H), 7.88 (d, 1H, C₅–H, J = 7.9 Hz) and 8.19 (s, 2H, NH₂); MS (FAB⁺) m/z: 326.2 [100, (M + H)⁺], 245.2 [50, (M + H – HNSO₂)⁺]; MS (FAB^–) m/z: 326.2 [100, (M – H)⁻]; Acc. MS (FAB⁺): Acc. MS (FAB⁺): 326.1076, C₁₅H₂₀NO₅S requires 326.1062; found C, 55.20; H, 5.88; N, 4.27; C₁₅H₁₉NO₅S requires C, 55.37; H, 5.89; N, 4.30%.

Ethyl 2-Acetyloctanoate (29a)

This was prepared by general method using K₂CO₃ (11.1 g; 79.2 mmol), water (50 mL), 1-bromohexane (4.63 mL; 33 mmol), ethyl 3-oxo-butanoate (4.29 mL; 33 mmol), CH₂Cl₂ (50 mL), and Bu₄NCl (∼10 g; 33 mmol). The crude oily residue was purified by flash chromatography (CHCl₃) to give 29a as a pale yellow oil (3.05 g; 43%). (Lit.⁶⁰ bp₁₀ 127–129 °C); R_f: 0.66 (CHCl₃); ¹H NMR (400 MHz; DMSO-d₆) δ_H: 0.85 (t, 3H, CH₃, J = 6.7 Hz), 1.18 (t, 3H, CH₂CH₃, J = 7.1 Hz), 1.22–1.36 (m, 8H, 4 × CH₂), 1.71 (q, 2H, 3-CH₂, J = 6.4 Hz), 2.17 (s, 3H, CH₃CO), 3.57 (t, 1H, 2H, J = 6.7 Hz) and 4.12 (q, 2H, CH₂CH₃, J = 7.3 Hz); MS (FAB⁺) m/z: 215.2 [100, (M + H)⁺]; MS (FAB^–) m/z: 213.1 [100, (M – H)⁻]; Acc. MS (FAB⁺): 215.1600, C₁₂H₂₃O₃ requires 215.1639.

3-Hexyl-7-hydroxy-4-methylcoumarin (29b)

This was prepared by general method using resorcinol (1.28 g; 11.7 mmol), 29a (2.5 g; 12 mmol), and a mixture of CF₃COOH (1.8 mL; 23 mmol) and conc. H₂SO₄ (1.19 mL; 23.3 mmol). The crude pale white solid was recrystallized from acetone/hexane to give 29b as white fine crystals (2.12 g; 70%). R_f: 0.81 (CHCl₃/acetone, 3:1); mp 112–114 °C (Lit.⁶¹ mp 111–112 °C); MS (FAB⁺) m/z: 521.1 [100, (2M + H)⁺], 261.2 [100, (M + H)⁺]; ¹H NMR (400 MHz; DMSO-d₆) δ_H: 0.88 (t, 3H, 6′-CH₃, J = 7.1 Hz), 1.25–1.55 (m, 8H, 4 × CH₂), 2.39 (s, 3H, C₄–CH₃), 2.63 (t, 2H, 1′-CH₂, J = 7.6 Hz), 6.83 (dd, 1H, C₆–H, J = 2.4, 8.8 Hz), 6.96 (d, 1H, C₈–H, J = 2.4 Hz), 7.49 (d, 1H, C₅–H, J = 8.8 Hz) and 10.41 (s, 1H, OH); MS (FAB^–) m/z: 259.2 [100, (M – H)⁻]; Acc. MS (FAB⁺): 261.1421, C₁₆H₂₁O₃ requires 261.1412; found C, 73.90; H, 7.78; C₁₆H₂₀O₃ requires C, 73.82; H, 7.74%.

3-Hexyl-4-methylcoumarin-7-O-sulfamate (29)

Compound 29b (700 mg; 2.69 mmol) was sulfamoylated, the crude white solid was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient), and the white solid that was isolated was recrystallized from ethyl acetate/hexane to give 29 as white fine fluffy crystals (449 mg; 49%). R_f: 0.41 (CHCl₃/ethyl acetate, 4:1); mp 132–133 °C; ¹H NMR (400 MHz; DMSO-d₆) δ_H: 0.86 (t, 3H, CH₃, J = 5.5 Hz), 1.25–1.48 (m, 8H, 4 × CH₂), 2.43 (s, 3H, C₄–H₃), 2.59 (t, 2H, 1′-CH₂, J = 7.1 Hz), 7.26 (d, 1H, C₈–H, J = 2.1 Hz), 7.28 (m, 1H, C₆–H), 7.88 (d, 1H, C₅–H, J = 7.3 Hz) and 8.19 (s, 2H, NH₂); MS (FAB⁺) m/z: 679.4 [15, (2M + H)⁺], 340.2 [100, (M + H)⁺]; MS (FAB^–) m/z: 338.2 [100, (M – H)⁻], 259.2 [45, (M – H₂NSO₂)⁻]; Acc. MS (FAB⁺): 340.1230, C₁₆H₂₂NO₅S requires 340.1219; found C, 56.90; H, 6.22; N, 4.12; C₁₆H₂₁NO₅S requires C, 56.62; H, 6.24; N, 4.13%.

Ethyl 2-Acetylnonanoate (30a)

This was prepared by general method using K₂CO₃ (11.11 g; 80.39 mmol), water (50 mL), 1-bromoheptane (6 mL; 34 mmol), ethyl acetoacetate (4.27 mL; 33.5 mmol), CH₂Cl₂ (50 mL), and Bu₄NCl (∼10 g; 34 mmol). The crude oily residue was purified by flash chromatography (CHCl₃) to give 30a as a pale yellow oil (2.45 g; 33%). (Lit.⁶² bp_0.4 80–85 °C); R_f: 0.9 (CHCl₃); ¹H NMR (400 MHz; CDCl₃) δ_H: 0.85 (t, 3H, CH₃, J = 7.1 Hz), 1.18 (t, 3H, CH₂CH₃J = 7.1 Hz), 1.23–1.73 (m, 12H, 6 × CH₂), 2.17 (s, 3H, CH₃CO), 3.56 (t, 1H, 2H, J = 7.1 Hz) and 4.19 (q, 2H, CH₂CH₃, J = 7.1 Hz); MS (FAB⁺) m/z: 229.2 [100, (M + H)⁺]; MS (FAB^–) m/z: 227.2 [100, (M – H)⁻]; Acc. MS (FAB⁺): 229.1804, C₁₃H₂₅O₃ requires 229.1803.

3-Heptyl-7-hydroxy-4-methylcoumarin (30b)

This was prepared by general method using resorcinol (965 mg; 8.76 mmol), 30a (2.0 g; 8.8 mmol), and a mixture of CF₃COOH (1.55 mL; 20.2 mmol) and conc. H₂SO₄ (1.03 mL; 20.2 mmol). The crude pale yellow solid was recrystallized from acetone/hexane to give 30b as pale yellow crystals (730 mg; 30%). R_f: 0.78 (CHCl₃/acetone, 3:1); mp 96–98 °C; ¹H NMR (400 MHz; CDCl₃) δ_H: 0.88 (t, 3H, CH₃, J = 7.3 Hz), 1.26–1.53 (m, 10H, 5 × CH₂), 2.39 (s, 3H, C₄–CH₃), 2.63 (t, 2H, 1′-CH₂), 7.01 (d, 1H, C₈–H, J = 2.4 Hz), 6.85 (dd, 1H, C₆–H, J = 2.4, 8.7 Hz), 7.49 (d, 1H, C₅–H, J = 8.85 Hz) and 10.37 (s, 1H, OH); MS (FAB⁺) m/z: 549.4 [15, (2M + H)⁺], 275.2 [100, (M + H)⁺]; MS (FAB^–) m/z: 547.4 [10, (2M – H)⁻], 273.2 [100, (M – H)⁻]; Acc. MS (FAB⁺): 275.1641, C₁₇H₂₃O₃ requires 275.1647; found C, 74.08; H, 8.03; C₁₇H₂₂O₃ requires C, 74.42; H, 8.08%.

3-Heptyl-4-methylcoumarin-7-O-sulfamate (30)

Compound 64 (400 mg; 1.46 mmol) was sulfamoylated, the crude white solid was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient), and the white solid isolated was recrystallized from ethyl acetate/hexane to give 30 as white fine crystals (75 mg; 14%). R_f: 0.51 (CHCl₃/ethyl acetate 4:1); mp 138–140 °C; ¹H NMR (400 MHz; DMSO-d₆) δ_H: 0.86 (t, 3H, CH₃, J = 6.7 Hz), 1.26–1.49 (m, 10H, 5 × CH₂), 2.43 (s, 3H, C₄–CH₃), 2.58 (t, 2H, 1′-CH₂, J = 7.1 Hz), 7.25–7.29 (m, 2H, C₆–H and C₈–H), 7.88 (d, 1H, C₅–H, J = 8.8 Hz) and 8.18 (s, 2H, NH₂); MS (FAB⁺) m/z: 707.2 [40, (2M + H)⁺], 354.1 [100, (M + H)⁺]; MS (FAB^–) m/z: 705.2 [40, (2M – H)⁻], 352.1 [100, (M – H)⁻], 273.2 [90, (M – H₂NSO₂)⁻]; Acc. MS (FAB⁺): 354.1391, C₁₇H₂₄NO₅S requires 354.1375; found C, 57.80; H, 6.58; N, 3.92; C₁₇H₂₃NO₅S requires C, 57.77; H, 6.56; N, 3.96%.

Ethyl 2-Acetyldecanoate (31a)

This was prepared by general method using K₂CO₃ (11.47 g; 82.98 mmol), water (50 mL), 1-bromooctane (6.0 mL; 35 mmol), ethyl 3-oxo-butanoate (4.41 mL; 34.6 mmol), CH₂Cl₂ (50 mL), and Bu₄NCl (∼10 g; 35 mmol). The crude oily residue was purified by flash chromatography (CHCl₃/acetone) to give 31a as a pale yellow oil (3.1 g; 37%). (Lit.⁶³ bp_0.4 79–83 °C); R_f: 0.68 (CHCl₃); ¹H NMR (400 MHz; DMSO-d₆) δ_H: 0.86 (t, 3H, CH₃, J = 6.7 Hz), 1.18 (t, 3H, CH₂CH₃, J = 7.3 Hz), 1.23–1.75 (m, 12H, 6 × CH₂), 2.06 (s, 3H, CH₃CO), 2.39 (q, 2H, 3-CH₂, J = 7.3 Hz), 3.61 (t, 1H, 2H, J = 6.7 Hz) and 4.12 (q, 2H, CH₂CH₃, J = 7.1 Hz); MS (FAB⁺) m/z: 243.2 [100, (M + H)⁺]; MS (FAB^–) m/z: 241.2 [100, (M – H)⁻]; Acc. MS (FAB⁺): 243.1968, C₁₄H₂₇O₃ requires 243.1960.

7-Hydroxy-4-methyl-3-octylcoumarin (31b)

This was prepared by general method using resorcinol (910 mg; 8.26 mmol), 31a (2.0 g; 8.3 mmol), and a mixture of CF₃COOH (1.27 mL; 16.3 mmol) and conc. H₂SO₄ (0.84 mL; 16.3 mmol). The crude brown solid was recrystallized from acetone/hexane to give 31b as white crystals (405 mg; 17%). R_f: 0.72 (CHCl₃/acetone, 3:1); mp 98–100 °C; ¹H NMR (400 MHz; DMSO-d₆) δ_H: 0.88 (t, 3H, CH₃, J = 7.3 Hz), 1.27–1.46 (m, 12H, 6 × CH₂), 2.38 (s, 3H, C₄–CH₃), 2.54 (t, 2H, 1′-CH₂, J = 6.4 Hz), 6.69 (d, 1H, C₈–H, J = 2.1 Hz), 6.81 (dd, 1H, C₆–H, J = 2.1, 8.8 Hz), 7.62 (d, 1H, C₅–H, J = 8.8 Hz) and 10.52 (s, 1H, OH); MS (FAB⁺) m/z: 577.4 [40, (2M + H)⁺], 289.2 [100, (M + H)⁺]; MS (FAB^–) m/z: 575.3 [35, (2M – H)], 287.2 [100, (M – H)⁻]; Acc. MS (FAB⁺): 289.1801, C₁₈H₂₅O₃ requires 289.1801; found C, 74.90; H, 8.40; C₁₈H₂₄O₃ requires C, 74.97; H, 8.39%.

4-Methyl-3-octylcoumarin-7-O-sulfamate (31)

Compound 31b (400 mg; 1.39 mmol) was sulfamoylated, the crude white solid was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient), and the white solid isolated was recrystallized from ethyl acetate/hexane to give 31 as white fine crystals (69 mg; 15%). R_f: 0.43 (CHCl₃/ethyl acetate, 4:1); mp 135–138 °C; ¹H NMR (400 MHz; DMSO-d₆) δ_H: 0.86 (t, 3H, CH₃, J = 7.3 Hz), 1.25–1.49 (m, 12H, 6 × CH₂), 2.43 (s, 3H, C₄–CH₃), 2.58 (t, 2H, 1′-CH₂, J = 7.1 Hz), 7.26–7.29 (m, 2H, C₆–H and C₈–H), 7.88 (d, 1H, C₅–H, J = 8.8 Hz) and 8.18 (s, 2H, NH₂); found C, 58.70; H, 6.72; MS (FAB⁺) m/z: 735.2 [35, (2M + H)⁺], 368.1 [100, (M + H)⁺]; MS (FAB^–) m/z: 733.3 [40, (2M – H)⁻], 366.2 [100, (M – H)⁻], 287.2 [60, (M – H₂NSO₂)⁻]; Acc. MS (FAB⁺): 368.1529, C₁₈H₂₆NO₅S requires 368.1532; N, 3.68; C₁₈H₂₅NO₅S requires C, 58.84; H, 6.86; N, 3.81%.

Ethyl 2-Acetylundecanoate (32a)

This was prepared by general method using K₂CO₃ (7.4 g; 58 mmol), water (50 mL), 1-bromononane (5.0 mL; 24 mmol), ethyl 3-oxo-butanoate (3.1 mL; 24 mmol), CH₂Cl₂ (50 mL), and Bu₄NCl (∼10 g; 49 mmol). The crude oily residue was purified by flash chromatography (CHCl₃) to give 32a as a pale yellow oil (2.76 g; 45%). (Lit.⁶⁴ bp_4.2 191 °C); R_f: 0.65 (CHCl₃); ¹H NMR (400 MHz; CDCl₃) δ_H: 0.88 (t, 3H, CH₃, J = 6.8 Hz), 1.26–1.91 (m, 19H, CH₂CH₃ and 8 × CH₂), 2.22 (s, 3H, CH₃CO), 3.39 (t, 1H, 2H, J = 7.3 Hz) and 4.19 (q, 2H, CH₂CH₃, J = 7.3 Hz); MS (FAB⁺) m/z: 257.2 [100, (M + H)⁺]; MS (FAB^–) m/z: 255.2 [100, (M – H)⁻]; Acc. MS (FAB⁺): 257.2126, C₁₅H₂₉O₃ requires 257.2170.

7-Hydroxy-4-methyl-3-nonylcoumarin (32b)

This was prepared by general method using resorcinol (1.07 g; 9.76 mmol), 32a (2.5 g; 9.8 mmol), and a mixture of CF₃COOH (1.5 mL; 20 mmol) and conc. H₂SO₄ (1.0 mL; 20 mmol). The crude yellow solid was purified by flash chromatography (CHCl₃/acetone, 8:1 to 4:1 gradient), and the pale yellow solid isolated was recrystallized from acetone/hexane to give 32b as white crystals (797 mg; 27%). R_f: 0.69 (CHCl₃/acetone, 3:1); mp 78–80 °C; ¹H NMR (400 MHz; DMSO-d₆) δ_H: 0.87 (t, 3H, CH₃, J = 7.1 Hz), 1.26–1.55 (m, 14H, 7 × CH₂), 2.39 (s, 3H, C₄–CH₃), 2.63 (t, 2H, 1′-CH₂, J = 7.6 Hz), 6.86 (dd, 1H, C₆–H, J = 2.4, 8.8 Hz), 7.05 (d, 1H, C₈–H, J = 2.4 Hz), 7.49 (d, 1H, C₅–H, J = 8.8 Hz) and 7.55 (s, 1H, OH); found C, 75.35; H, 8.65; MS (FAB⁺) m/z: 605.3 [35, (2M + H)⁺], 303.1 [100, (M + H)⁺]; MS (FAB^–) m/z: 603.1 [40, (2M – H)⁻], 301.1 [100, (M – H)⁻]; Acc. MS (FAB⁺): 303.1964, C₁₉H₂₇O₃ requires 303.1960; C₁₉H₂₆O₃ requires C, 75.46; H, 8.67%.

4-Methyl-3-nonylcoumarin-7-O-sulfamate (32)

Compound 32b (400 mg; 1.32 mmol) was sulfamoylated, the crude white solid was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient), and the white solid isolated was recrystallized from ethyl acetate/hexane to give 32 as white fine crystals (184 mg; 37%). R_f: 0.82 (CHCl₃/ethyl acetate, 4:1); mp 125–129 °C; ¹H NMR (400 MHz; DMSO-d₆) δ_H: 0.85 (t, 3H, CH₃, J = 6.7 Hz), 1.24–1.48 (m, 14H, 7 × CH₂), 2.42 (s, 3H, C₄–CH₃), 2.58 (t, 2H, 1′-CH₂, J = 7.3 Hz), 7.25 (d, 1H, C₈–H, J = 2.4 Hz), 7.28 (dd, 1H, C₆–H, J = 2.1, 8.5 Hz), 7.88 (d, 1H, C₅–H, J = 8.5 Hz) and 8.19 (s, 2H, NH₂); MS (FAB⁺) m/z: 763.3 [45, (2M + H)⁺], 382.1 [100, (M + H)⁺]; MS (FAB^–) m/z: 761.2 [45, (2M – H)⁻], 380.1 [100, (M – H)⁻], 301.1 [75, (M – H₂NSO₂)⁻]; Acc. MS (FAB⁺): 382.1679, C₁₉H₂₈NO₅S requires 382.1688; found C, 59.50; H, 7.08; N, 3.59; C₁₉H₂₇NO₅S requires C, 59.82; H, 7.13; N, 3.67%.

Ethyl 2-Acetyldodecanoate (33a)

This was prepared by general method using K₂CO₃ (7.5 g; 54 mmol), water (50 mL), 1-bromodecane (5.0 mL; 23 mmol), ethyl 3-oxo-butanoate (2.88 mL; 22.6 mmol), CH₂Cl₂ (50 mL), and Bu₄NCl (∼10 g; 45 mmol). The crude oily residue was purified by flash chromatography (CHCl₃) to give 33a as a pale yellow oil (2.95 g; 48%). (Lit.⁶⁵ bp₂ 140–150 °C); R_f: 0.76 (CHCl₃); ¹H NMR (400 MHz; CDCl₃) δ_H: 0.88 (t, 3H, CH₃, J = 7.3 Hz), 1.25–2.00 (m, 21H, CH₂CH₃ and 9 × CH₂), 2.22 (s, 3H, CH₃CO), 3.39 (t, 1H, 2H, J = 7.3 Hz) and 4.19 (q, 2H, CH₂CH₃, J = 7.3 Hz); MS (FAB⁺) m/z: 271.2 [100, (M + H)⁺]; MS (FAB^–) m/z: 269.2 [100, (M – H)⁻]; Acc. MS (FAB⁺): 271.2275, C₁₆H₃₁O₃ requires 271.2273.

7-Hydroxy-3-decyl-4-methylcoumarin (33b)

This was prepared by general method using resorcinol (1.02 g; 9.25 mmol), 33a (2.5 g; 9.3 mmol), and a mixture of CF₃COOH (1.42 mL; 18.5 mmol) and conc. H₂SO₄ (0.94 mL; 18.5 mmol). The crude brown solid was purified by flash chromatography (CHCl₃/acetone, 8:1 to 4:1 gradient), and the pale yellow solid isolated was recrystallized from acetone/hexane to give 33b as white crystals (807 mg; 28%). R_f: 0.81 (CHCl₃/acetone, 3:1); mp 96–100 °C; ¹H NMR (400 MHz; CDCl₃) δ_H: 0.88 (t, 3H, CH₃, J = 7.3 Hz), 1.26–1.55 (m, 16H, 8 × CH₂), 2.39 (s, 3H, C₄–CH₃), 2.63 (t, 2H, 1′-CH₂, J = 7.6 Hz), 6.85 (dd, 1H, C₆–H, J = 2.4, 8.7 Hz), 7.02 (d, 1H, C₈–H, J = 2.4 Hz), 7.32 (s, 1H, OH) and 7.49 (d, 1H, C₅–H, J = 8.5 Hz); MS (FAB⁺) m/z: 633.3 [50, (2M + H)⁺], 317.1 [100, (M + H)⁺]; MS (FAB^–) m/z: 631.2 [10, (2M – H)⁻], 315.1 [100, (M – H)⁻]; Acc. MS (FAB⁺): 317.2117, C₂₀H₂₉O₃ requires 317.2117; found C, 75.65; H, 8.99 C₂₀H₂₈O₃ requires C, 75.91; H, 8.92%.

3-Decyl-4-methylcoumarin-7-O-sulfamate (33)

Compound 33b (400 mg; 1.27 mmol) was sulfamoylated, the crude white solid was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient), and the white solid isolated was recrystallized from ethyl acetate/hexane to give 33 as white fine crystals (101 mg; 27%). R_f: 0.55 (CHCl₃/ethyl acetate, 4:1); mp 118–121 °C; ¹H NMR (400 MHz; DMSO-d₆) δ_H: 0.85 (t, 3H, CH₃, J = 7.6 Hz), 1.24–1.47 (m, 16H, 8 × CH₂), 2.43 (s, 3H, C₄–CH₃), 2.58 (t, 2H, 1′-CH₂, J = 7.3 Hz), 7.25 (d, 1H, C₈–H, J = 2.4 Hz), 7.27 (m, 1H, C₆–H); 7.87 (d, 1H, C₅–H, J = 8.5 Hz) and 8.13 (s, 2H, NH₂); MS (FAB⁺) m/z: 791.3 [20, (2M + H)⁺], 396.1 [100, (M + H)⁺], 317.1 [30, (M + H – HNSO₂)⁺]; MS (FAB^–) m/z: 394.1 [100, (M – H)⁻], 315.1 [60, (M – H₂NSO₂)⁻]; Acc. MS (FAB⁺): 396.1850, C₂₀H₃₀NO₅S requires 396.1845; found C, 60.65; H, 7.42; N, 3.12 C₂₀H₂₉NO₅S requires C, 60.74; H, 7.39; N, 3.54%.

Ethyl 2-Acetyltridecanoate (34a)

This was prepared by general method using K₂CO₃ (7.1 g; 51 mmol), water (50 mL), 1-bromoundecane (5.0 mL; 21 mmol), ethyl 3-oxo-butanoate (2.71 mL; 21.3 mmol), CH₂Cl₂ (50 mL), and Bu₄NCl (∼10 g; 43 mmol). The crude oily residue was purified by flash chromatography (CHCl₃/hexane, 8:1 to 2:1 gradient) to give 34a as a pale yellow oil (3.25 g; 54%). R_f: 0.69 (CHCl₃/hexane, 2:1); (Lit.⁶⁶ bp₁ 145–150 °C); ¹H NMR (400 MHz; CDCl₃) δ_H: 0.88 (t, 3H, CH₃, J = 7.3 Hz), 1.25–1.85 (m, 23H, CH₂CH₃ and 10 × CH₂), 2.22 (s, 3H, CH₃CO), 3.39 (t, 1H, 2H, J = 7.3 Hz) and 4.15 (q, 2H, CH₂CH₃, J = 7.3 Hz); MS (FAB⁺) m/z: 285.2 [100, (M + H)⁺]; MS (FAB^–) m/z: 283.2 [100, (M – H)⁻]; Acc. MS (FAB⁺): 285.2422, C₁₇H₃₃O₃ requires 285.2429.

7-Hydroxy-4-methyl-3-undecylcoumarin (34b)

This was prepared by general method using resorcinol (968 mg; 8.8 mmol) and 34a (2.5 g; 8.8 mmol) in the presence of CF₃COOH (1.4 mL; 18 mmol) and conc. H₂SO₄ (0.9 mL; 18 mmol). The crude brown solid was purified by flash chromatography (CHCl₃/acetone, 8:1 to 4:1 gradient), and the pale brown solid isolated was recrystallized from acetone/hexane to give 34b as white crystals (722 mg; 29%). R_f: 0.88 (CHCl₃/acetone, 3:1); mp 74–76 °C; ¹H NMR (400 MHz; CDCl₃) δ_H: 0.85 (t, 3H, CH₃, J = 7.0 Hz), 1.23–1.41 (m, 18H, 9 × CH₂), 2.35 (s, 3H, C₄-CH₃), 2.51 (t, 2H, 1′-CH₂, J = 7.4 Hz), 6.67 (d, 1H, C₈–H, J = 2.3 Hz), 6.78 (dd, 1H, C₆–H, J = 2.3, 8.6 Hz), 7.59 (d, 1H, C₅–H, J = 8.9 Hz) and 10.39 (s, 1H, OH); MS (FAB⁺) m/z: 660.9 [35, (2M + H)⁺], 331.1 [100, (M + H)⁺]; MS (FAB^–) m/z: 659.0 [10, (2M – H)⁻], 329.2 [100, (M – H)⁻]; Acc. MS (FAB⁺): 331.2268, C₂₁H₃₁O₃ requires 331.2273; found C, 76.10; H, 8.96 C₂₁H₃₀O₃ requires C, 76.33; H, 9.15%.

4-Methyl-3-undecylcoumarin-7-O-sulfamate (34)

Compound 34b (200 mg; 0.71 mmol) was sulfamoylated, the crude white solid was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient), and the white solid isolated was recrystallized from ethyl acetate/hexane to give 34 as white fine crystals (11 mg; 4%). R_f: 0.49 (CHCl₃/ethyl acetate, 4:1); mp 117–119 °C; ¹H NMR (400 MHz; DMSO-d₆) δ_H: 0.87 (t, 3H, CH₃, J = 7.2 Hz), 1.22–1.45 (m, 18H, 9 × CH₂), 2.34 (s, 3H, C₄–CH₃), 2.59 (t, 2H, 1′-CH₂, J = 7.3 Hz), 6.89 (d, 1H, C₈–H, J = 2.3 Hz), 7.19 (dd, 1H, C₆–H, J = 2.3, 8.8 Hz); 7.59 (d, 1H, C₅–H, J = 8.9 Hz) and 8.18 (s, 2H, NH₂); MS (FAB⁺) m/z: 410.3 [100, (M + H)⁺]; MS (FAB^–) m/z: 408.3 [100, (M – H)⁻]; Acc. MS (FAB⁺): 410.1992, C₂₁H₃₂NO₅S requires 410.2001; found C, 61.40; H, 7.75; N, 3.16, C₂₁H₃₁NO₅S requires C, 61.59; H, 7.63; N, 3.42%.

Ethyl 2-Acetyltetradecanoate (35a)

This was prepared by general method using K₂CO₃ (7.99 g; 57.8 mmol), water (60 mL), 1-bromododecane (6.0 mL; 24 mmol), ethyl 3-oxo-butanoate (3.1 mL; 24 mmol), CH₂Cl₂ (60 mL), and Bu₄NCl (∼10 g; 48 mmol). The crude oily residue was purified by distillation under reduced pressure to give 35a as a pale yellow oil (3.65 g; 51%). R_f: 0.66 (CH₂Cl₂); bp_0.3 159–160 °C; (Lit.⁶⁷ bp_1.1 149–152 °C); ¹H NMR (400 MHz; CDCl₃) δ_H: 0.88 (t, 3H, CH₃, J = 7.0 Hz), 1.12–1.89 (m, 25H, CH₂CH₃ and 11 × CH₂), 2.22 (s, 3H, CH₃CO), 3.53 (t, 1H, 2H, J = 7.0 Hz) and 4.19 (q, 2H, CH₂CH₃, J = 7.0 Hz); MS (FAB⁺) m/z: 299.2 [100, (M + H)⁺]; MS (FAB^–) m/z: 297.2 [100, (M – H)⁻]; Acc. MS (FAB⁺): 299.2594, C₁₈H₃₅O₃ requires 299.2586.

3-Dodecyl-7-hydroxy-4-methylcoumarin (35b)

This was prepared by general method using resorcinol (553 mg; 5.02 mmol), 35a (1.5 g; 5.0 mmol), and a mixture of CF₃COOH (0.8 mL; 10 mmol) and conc. H₂SO₄ (0.6 mL; 10 mmol). The crude brown solid was purified by flash chromatography (CHCl₃/acetone, 8:1 to 4:1 gradient), and the pale yellow solid isolated was recrystallized from acetone/hexane to give 35b as off-white crystals (146 mg; 9%). R_f: 0.74 (CHCl₃/acetone, 3:1); mp 94–96 °C; ¹H NMR (400 MHz; CDCl₃) δ_H: 0.88 (t, 3H, CH₃, J = 7.0 Hz), 1.25–1.63 (m, 20H, 10 × CH₂), 2.38 (s, 3H, C₄–CH₃), 2.63 (t, 2H, 1′-CH₂, J = 7.4 Hz), 6.19 (s, 1H), 6.81 (dd, 1H, C₆–H, J = 2.3, 8.6 Hz), 6.91 (d, 1H, C₈–H, J = 2.3 Hz) and 7.48 (d, 1H, C₅–H, J = 8.9 Hz); MS (FAB⁺) m/z: 689.4 [20, (2M + H)⁺], 345.4 [100, (M + H)⁺]; MS (FAB^–) m/z: 343.3 [100, (M – H)⁻]; Acc. MS (FAB⁺): 345.2435, C₂₂H₃₃O₃ requires 345.2429; found C, 76.60; H, 9.22; C₂₂H₃₂O₃ requires C, 76.70; H, 9.36%.

3-Dodecyl-4-methylcoumarin-7-O-sulfamate (35)

Compound 35b (100 mg; 0.29 mmol) was sulfamoylated, the crude white solid was purified by preparative TLC (CHCl₃/ethyl acetate, 6:1), and the white solid isolated was recrystallized from ethyl acetate/hexane to give 35 as white fine crystals (15 mg; 12%). R_f: 0.36 (CHCl₃/ethyl acetate, 6:1); mp 157–159 °C; ¹H NMR (400 MHz; DMSO-d₆) δ_H: 0.88 (t, 3H, CH₃, J = 7.2 Hz), 1.24–1.48 (m, 20H, 10 × CH₂), 2.45 (s, 3H, C₄–CH₃), 2.61 (t, 2H, 1′-CH₂, J = 7.4 Hz), 7.23 (d, 1H, C₈–H, J = 2.3 Hz), 7.27 (dd, 1H, C₆–H, J = 2.3, 8.6 Hz); 7.89 (d, 1H, C₅–H, J = 8.9 Hz) and 8.21 (s, 2H, NH₂); MS (FAB⁺) m/z: 847.1 [15, (2M + H)⁺], 424.1 [100, (M + H)⁺], 245.1 [30, (M + H – HNSO₂)⁺]; MS (FAB^–) m/z: 422.1 [100, (M – H)⁻], 343.2 [55, (M – H₂NSO₂)⁻]; Acc. MS (FAB⁺): 424.1246, C₂₂H₃₄NO₅S requires 424.1241.

Ethyl 2-Acetylpentadecanoate (36a)

This was prepared by general method using K₂CO₃ (6.3 g; 46 mmol), water (60 mL), 1-bromotridecane (5.0 mL; 19 mmol), ethyl 3-oxo-butanoate (2.42 mL; 18.9 mmol), CH₂Cl₂ (60 mL), and Bu₄NCl (11.0 g; 37.9 mmol). The crude oily residue was purified by distillation under reduced pressure to give 36a as a pale yellow oil (2.88 g; 49%). R_f: 0.77 (CHCl₃); bp_0.3 153–155 °C; ¹H NMR (400 MHz; CDCl₃) δ_H: 0.88 (t, 3H, CH₃, J = 7.0 Hz), 1.12–1.56 (m, 27H, CH₂CH₃ and 12 × CH₂), 2.22 (s, 3H, CH₃CO), 3.53 (t, 1H, 2H, J = 6.7 Hz) and 4.21 (q, 2H, CH₂CH₃, J = 7.0 Hz); MS (FAB⁺) m/z: 313.3 [100, (M + H)⁺]; MS (FAB^–) m/z: 311.2 [100, (M – H)⁻]; Acc. MS (FAB⁺): 313.2756, C₁₉H₃₇O₃ requires 313.2743.

7-Hydroxy-4-methyl-3-tridecylcoumarin (36b)

This was prepared by general method using resorcinol (705 mg; 6.4 mmol), 36a (2.0 g; 6.4 mmol), and a mixture of CF₃COOH (1.0 mL; 13 mmol) and conc. H₂SO₄ (0.7 mL; 13 mmol). The crude brown solid was purified by flash chromatography (CHCl₃/acetone, 8:1 to 4:1 gradient), and the pale yellow solid isolated was recrystallized from acetone/hexane to give 36b as white crystals (621 mg; 27%). R_f: 0.65 (CHCl₃/acetone, 3:1); mp 71–72 °C; ¹H NMR (400 MHz; CDCl₃) δ_H: 0.88 (t, 3H, CH₃, J = 7.0 Hz), 1.26–1.84 (m, 22H, 11 × CH₂), 2.38 (s, 3H, C₄–CH₃), 2.62 (t, 2H, 1′-CH₂, J = 7.4 Hz), 5.87 (s, 1H, OH), 6.81 (dd, 1H, C₆–H, J = 2.3, 8.7 Hz), 6.86 (d, 1H, C₈–H, J = 2.3 Hz) and 7.48 (d, 1H, C₅–H, J = 8.9 Hz); MS (FAB⁺) m/z: 359.4 [100, (M + H)⁺]; MS (FAB^–) m/z: 357.3 [100, (M – H)⁻]; Acc. MS (FAB⁺): 359.2599, C₂₃H₃₅O₃ requires 359.2586; found C, 77.20; H, 10.00; C₂₃H₃₄O₃ requires C, 77.05; H, 9.56%.

4-Methyl-3-tridecylcoumarin-7-O-sulfamate (36)

Compound 36b (400 mg; 1.12 mmol) was sulfamoylated, the crude white solid was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient), and the white solid isolated was recrystallized from ethyl acetate/hexane to give 36 as white fine crystals (35 mg; 7%). R_f: 0.67 (CHCl₃/ethyl acetate, 6:1); mp 115–119 °C; MS (FAB⁺) m/z: 438.2 [100, (M + H)⁺]; ¹H NMR (400 MHz; DMSO-d₆) δ_H: 0.84 (t, 3H, CH₃, J = 7.0 Hz), 1.23–1.44 (m, 22H, 11 × CH₂), 2.42 (s, 3H, C₄–CH₃), 2.58 (t, 2H, 1′-CH₂, J = 7.8 Hz), 7.26 (d, 1H, C₈–H, J = 2.3 Hz), 7.28 (m, 1H, C₆–H), 7.88 (d, 1H, C₅–H, J = 8.6 Hz) and 8.21 (s, 2H, NH₂); MS (FAB^–) m/z: 436.2 [100, (M – H)⁻], 357.2 [30, (M – H₂NSO₂)⁻]; Acc. MS (FAB⁺): 438.2291, C₂₃H₃₆NO₅S requires 438.2272; found C, 63.60; H, 7.98; N, 3.17; C₂₃H₃₅NO₅S requires C, 63.13; H, 8.06; N, 3.20%.

Ethyl 2-Acetylhexadecanoate (37a)

This was prepared by general method using K₂CO₃ (5.6 g; 43 mmol), water (60 mL), 1-bromotetradecane (5.0 mL; 18 mmol), ethyl 3-oxo-butanoate (2.3 mL; 18 mmol), CH₂Cl₂ (60 mL), and Bu₄NCl (11.0 g; 36.1 mmol). The crude oily residue was purified by distillation under reduced pressure to give 37a as a pale yellow oil (2.32 g; 23%). R_f: 0.65 (CHCl₃); bp_0.3 158–162 °C; (Lit.⁶⁸ bp_0.5 162 °C); ¹H NMR (400 MHz; CDCl₃) δ_H: 0.88 (t, 3H, CH₃, J = 7.0 Hz), 1.20–1.81 (m, 29H, CH₂CH₃ and 13 × CH₂), 2.17 (s, 3H, CH₃CO), 3.53 (t, 1H, 2H, J = 7.0 Hz) and 4.19 (q, 2H, CH₂CH₃, J = 7.0 Hz); MS (FAB⁺) m/z: 327.2 [100, (M + H)⁺]; MS (FAB^–) m/z: 325.2 [100, (M – H)⁻]; Acc. MS (FAB⁺): 327.2900, C₂₀H₃₉O₃ requires 327.2899.

7-Hydroxy-4-methyl-3-tetradecylcoumarin (37b)

This was prepared by general method using resorcinol (337 mg; 3.1 mmol), 37a (1.0 g; 3.1 mmol), and a mixture of CF₃COOH (0.5 mL; 6.2 mmol) and conc. H₂SO₄ (0.4 mL; 6.2 mmol). The crude brown residue was purified by flash chromatography (CHCl₃/acetone, 8:1 to 4:1 gradient) to give 37b as pale yellow waxy solid (472 mg; 41%). R_f: 0.74 (CHCl₃/acetone, 3:1); mp 64–66 °C; ¹H NMR (400 MHz; CDCl₃) δ_H: 0.88 (t, 3H, CH₃, J = 7.0 Hz), 1.12–1.82 (m, 24H, 12 × CH₂), 2.38 (s, 3H, C₄–CH₃), 2.62 (t, 2H, 1′-CH₂, J = 7.4 Hz), 6.21 (s, 1H, OH), 6.79 (dd, 1H, C₆–H, J = 2.3, 8.6 Hz), 6.84 (d, 1H, C₈–H, J = 2.3 Hz) and 8.59 (d, 1H, C₅–H, J = 8.9 Hz); MS (FAB⁺) m/z: 373.1 [100, (M + H)⁺]; MS (FAB^–) m/z: 371.2 [100, (M – H)⁻]; Acc. MS (FAB⁺): 373.2754, C₂₄H₃₇O₃ requires 373.2743; found C, 77.11; H, 10.20; C₂₄H₃₆O₃ requires C, 77.38; H, 9.74%.

4-Methyl 3-tetradecylcoumarin-7-O-sulfamate (37)

Compound 37b (300 mg; 0.81 mmol) was sulfamoylated, the crude white solid was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient), and the white solid isolated was recrystallized from ethyl acetate/hexane to give 37 as white fine crystals (3 mg; 0.8%). R_f: 0.62 (CHCl₃/ethyl acetate, 4:1); mp 119–121 °C; ¹H NMR (400 MHz; DMSO-d₆) δ_H: 0.88 (t, 3H, CH₃, J = 7.0 Hz), 1.22–1.47 (m, 24H, 12 × CH₂), 2.41 (s, 3H, C₄–CH₃), 2.57 (t, 2H, 1′-CH₂, J = 7.8 Hz), 7.22 (d, 1H, C₈–H, J = 2.3 Hz), 7.26 (dd, 1H, C₆–H, J = 2.3, 8.6 Hz), 7.79 (d, 1H, C₅–H, J = 8.9 Hz) and 8.20 (s, 2H, NH₂); MS (FAB⁺) m/z: 452.3 [100, (M + H)⁺], 373.3 [10, (M + H – HNSO₂)⁺]; MS (FAB^–) m/z: 450.2 [100, (M – H)], 371.3 [40, (M – H₂NSO₂)⁻]; Acc. MS (FAB⁺): 452.2455, C₂₄H₃₈NO₅S requires 452.2471; found C, 63.74; H, 7.99; N, 3.36 C₂₄H₃₇NO₅S requires C, 63.83; H, 8.26; N, 3.10%.

Ethyl 2-Acetylpentadecanoate (38a)

This was prepared by general method using K₂CO₃ (5.7 g; 41 mmol), water (60 mL), 1-bromopentadecane (5.0 mL; 17 mmol), ethyl 3-oxo-butanoate (2.2 mL; 17 mmol), CH₂Cl₂ (60 mL), and Bu₄NCl (∼10 g; 34 mmol). The crude oily residue was purified by distillation under reduced pressure to give 38a as a pale yellow oil (1.78 g; 31%). R_f: 0.67 (CHCl₃); bp_0.4 198–202 °C; ¹H NMR (400 MHz; CDCl₃) δ_H: 0.88 (t, 3H, CH₃, J = 7.0 Hz), 1.26–1.79 (m, 29H, CH₂CH₃ and 16 × CH₂), 2.22 (s, 3H, CH₃CO), 3.53 (t, 2H, 3-H₂, J = 6.7 Hz), 3.37 (t, 1H, 2H, J = 7.0 Hz) and 4.18 (q, 2H, CH₂CH₃, J = 7.0 Hz); MS (FAB⁺) m/z: 341.2 [100, (M + H)⁺]; MS (FAB^–) m/z: 339.2 [100, (M – H)⁻]; Acc. MS (FAB⁺): 341.3058, C₂₁H₄₀O₃ requires 341.3056.

7-Hydroxy-4-methyl-3-pentadecylcoumarin (38b)

This was prepared by general method using resorcinol (486 mg; 4.41 mmol), 38a (1.5 g; 4.5 mmol), and a mixture of CF₃COOH (0.7 mL; 8.8 mmol) and conc. H₂SO₄ (0.5 mL; 8.8 mmol). The crude brown sticky solid was purified by flash chromatography (CHCl₃/acetone gradient, 8:1 to 4:1), and the off-white waxy solid isolated was recrystallized from acetone/hexane to give 38b as a white soft solid (123 mg; 0.07%). R_f: 0.86 (CHCl₃/acetone, 3:1); mp 59–61 °C; ¹H NMR (400 MHz; CDCl₃) δ_H: 0.88 (t, 3H, CH₃, J = 7.0 Hz), 1.11–1.80 (m, 26H, 13 × CH₂), 2.38 (s, 3H, C₄–CH₃), 2.53 (t, 2H, 1′-CH₂, J = 6.6 Hz), 5.75 (s, 1H, OH), 6.79 (dd, 1H, C₆–H, J = 2.3, 8.6 Hz), 6.84 (d, 1H, C₈–H, J = 2.3 Hz) and 7.48 (d, 1H, C₅–H, J = 8.6 Hz); MS (FAB⁺) m/z: 387.3 [100, (M + H)⁺]; MS (FAB^–) m/z: 385.3 [100, (M – H)⁻]; Acc. MS (FAB⁺): 387.2892, C₂₅H₃₉O₃ requires 387.2899.

4-Methyl-3-pentadecylcoumarin-7-O-sulfamate (38)

Compound 38b (90 mg; 0.23 mmol) was sulfamoylated, the crude white solid was purified by preparative TLC (CHCl₃/ethyl acetate gradient, 6:1), and the white solid isolated was recrstallized from ethyl acetate/hexane to give 38 as a white fine solid (21 mg; 19%). R_f: 0.64 (CHCl₃/ethyl acetate, 4:1); mp 114–116 °C; ¹H NMR (400 MHz; DMSO-d₆) δ_H: 0.85 (t, 3H, CH₃, J = 7.0 Hz), 1.14–1.44 (m, 26H, 13 × CH₂), 2.43 (s, 3H, C₄–CH₃), 2.58 (t, 2H, 1′-CH₂, J = 7.4 Hz), 7.25 (d, 1H, C₈–H, J = 2.3 Hz), 7.27 (dd, 1H, C₆–H, J = 2.3, 8.6 Hz), 7.87 (d, 1H, C₅–H, J = 8.6 Hz) and 8.16 (s, 2H, NH₂); MS (FAB⁺) m/z: 466.3 [100, (M + H)⁺], 387.3 [10, (M + H – HNSO₂)⁺]; MS (FAB^–) m/z: 464.2 [100, (M – H)⁻], 385.3 [40, (M – H₂NSO₂)⁻]; Acc. MS (FAB⁺): 466.2617, C₂₅H₄₀NO₅S requires 466.2626; found C, 64.00; H, 8.82; N, 3.26; C₂₅H₃₉NO₅S requires C, 64.48; H, 8.44; N, 3.01%.

3-Chloro-7-hydroxy-4-methylcoumarin (39a)

This was prepared by general method using resorcinol (2.0 g; 18 mmol), ethyl 2-chloro-3-oxo-butanoate (2.99 g; 18 mmol), and a mixture of CF₃COOH (2.27 mL; 36.3 mmol) and conc. H₂SO₄ (1.83 mL; 36.3 mmol). The crude brown solid was purified by flash chromatography (CHCl₃/acetone, 8:1 to 4:1 gradient), and the yellow solid isolated was recrystallized from acetone/hexane to give 39a as off-white crystals (692 mg; 18%). R_f: 0.72 (CHCl₃/acetone, 3:1); mp 250–253 °C (Lit.⁶⁹ mp 250 °C); ¹H NMR (400 MHz; DMSO-d₆) δ_H: 2.09 (s, 3H, CH₃), 6.76 (d, 1H, C₈–H, J = 2.4 Hz), 6.86 (dd, 1H, C₆–H, J = 2.4, 8.8 Hz), 7.69 (d, 1H, C₅–H, J = 8.8 Hz) and 10.68 (s, 1H, OH); MS (FAB⁺) m/z: 211.1 [100, (M(³⁵Cl) + H)⁺]; MS (FAB^–) m/z: 209.1 [100, (M(³⁵Cl) – H)⁻]; Acc. MS (FAB⁺) m/z: 211.0178 C₁₀H₈³⁵ClO₃ requires 211.0162 and 213.0152 C₁₀H₈³⁷ClO₃ requires 213.0132; found C, 57.30; H, 3.39; C₁₀H₇ClO₃ requires C, 57.03; H, 3.35%.

3-Chloro-4-methylcoumarin-7-O-sulfamate (39)

Compound 39a (400 mg; 1.9 mmol) was sulfamoylated, the crude white solid was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient), and the white solid isolated was recrystallized from ethyl acetate/hexane to give 39 as white fine crystals (164 mg; 30%). R_f: 0.30 (CHCl₃/ethyl acetate, 4:1); mp 182–186 °C; ¹H NMR (400 MHz; DMSO-d₆) δ_H: 2.59 (s, 3H, CH₃), 7.35 (dd, 1H, C₆–H, J = 2.1, 8.8 Hz), 7.39 (d, 1H, C₈–H, J = 2.1 Hz), 7.98 (d, 1H, C₅–H, J = 8.8 Hz) and 8.29 (s, 2H, NH₂); MS (FAB⁺) m/z: 290.0 [100, (M(³⁷Cl) + H)⁺]; MS (FAB^–) m/z: 288.1 [100, (M(³⁵Cl) – H)⁻], 209.1 [50, (M – H₂NSO₂)⁻]; Acc. MS (FAB⁺) m/z: 290.9860, C₁₀H₉³⁷ClNO₅S requires 290.9782 and 288.9814 C₁₀H₉³⁵ClNO₅S requires 291.9813; found C, 41.50; H, 2.62; N, 4.64; C₁₀H₈ClNO₅S requires C, 41.46; H, 2.78; N, 4.84%.

4-Methyl-3-phenyl-7-hydroxycoumarin (40a)

This was prepared by general method using resorcinol (2.13 g; 19.4 mmol), ethyl 3-oxo-2-phenylbutanoate (4.0 g; 19 mmol), and a mixture of CF₃COOH (3.0 mL; 39 mmol) and conc. H₂SO₄ (2.0 mL; 39 mmol). The crude yellow solid was purified by recrystallization from ethanol to give 40a as yellow needles (4.1 g; 83%). R_f: 0.59 (CHCl₃/acetone, 3:1); mp 226–228 °C; (Lit.⁷⁰ mp 226–228 °C); ¹H NMR (400 MHz; DMSO-d₆) δ_H: 2.21 (s, 3H, CH₃), 6.75 (d, 1H, C₈–H, J = 2.4 Hz), 6.84 (dd, 1H, C₆–H, J = 2.4, 8.6 Hz), 7.27–7.47 (m, 5H, Ph–H), 7.66 (d, 1H, C₅–H, J = 8.8 Hz) and 10.67 (s, 1H, OH); MS (FAB⁺) m/z: 505.1 [10, (2M + H)⁺], 253.0 [100, (M + H)⁺]; MS (FAB^–) m/z: 503.1 [10, (2M – H)⁻], 251.1 [100, (M – H)⁻]; Acc. MS (FAB⁺): 253.0798, C₁₆H₁₂O₃ requires 253.0786; found C, 76.10; H, 4.84; C₁₆H₁₂O₃ requires C, 76.18; H, 4.79%.

4-Methyl-3-phenylcoumarin-7-O-sulfamate (40)

Compound 40a (400 mg; 1.6 mmol) was sulfamoylated, the crude white solid was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient), and the white solid isolated was recrystallized from ethyl acetate/hexane to give 40 as white fine crystals (246 mg; 52%). R_f: 0.36 (CHCl₃/ethyl acetate, 4:1); mp 184–187 °C; ¹H NMR (400 MHz; DMSO-d₆) δ_H: 2.28 (s, 3H, CH₃), 7.31–7.38 (m, 2H, C₆–H and C₈–H), 7.40–7.49 (m, 5H, Ph–H), 7.95 (d, 1H, C₅–H, J = 8.8 Hz) and 8.27 (s, 2H, NH₂); MS (FAB⁺) m/z: 332.0 [100, (M + H)⁺], 253.0 [15, (M + H – HNSO₂)⁺]; MS (FAB^–) m/z: 330.1 [100, (M – H)⁻], 251.1 [60, (M – H₂NSO₂)⁻]; Acc. MS (FAB⁺): 332.0589, C₁₆H₁₄NO₅S requires 332.0593; found C, 58.00; H, 3.94; N, 4.21; C₁₆H₁₃NO₅S requires C, 58.00; H, 3.95; N, 4.23%.

3-Benzyl-4-methyl-7-hydroxycoumarin (41a)

This was prepared by general method using resorcinol (1.99 g; 18.2 mmol), ethyl 2-benzyl-3-oxo-butanoate (4.0 g; 18 mmol), and a mixture of CF₃COOH (2.8 mL; 36 mmol) and conc. H₂SO₄ (1.8 mL; 36 mmol). The crude brown solid was purified by recrystallization from ethanol to give 41a as white crystals (4.35 g; 90%). R_f: 0.79 (CHCl₃/acetone, 3:1); mp 230–232 °C (Lit.⁷¹ mp 226–227 °C); ¹H NMR (400 MHz; DMSO-d₆) δ_H: 2.39 (s, 3H, CH₃), 3.92 (s, 2H, CH₂Ph), 6.71 (d, 1H, C₈–H, J = 2.4 Hz), 6.80 (dd, 1H, C₆–H, J = 2.4, 8.8 Hz), 7.15–7.28 (m, 5H, Ph–H), 7.64 (d, 1H, C₅–H, J = 8.8 Hz) and 10.48 (s, 1H, OH); MS (FAB⁺) m/z: 532.9 [10, (2M + H)⁺], 267.1 [100, (M + H)⁺]; MS (FAB^–) m/z: 265.1 [100, (M – H)]; Acc. MS (FAB⁺): 267.1026, C₁₇H₁₅O₃ requires 267.1012; found C, 76.60; H, 5.34; C₁₇H₁₄O₃ requires C, 76.68; H, 5.30%.

3-Benzyl-4-methylcoumarin-7-O-sulfamate (41)

Compound 41a (400 mg; 1.5 mmol) was sulfamoylated, the crude white solid was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient), and the white solid isolated was recrystallized from THF/hexane to give 41 as white fine crystals (279 mg; 54%). R_f: 0.57 (CHCl₃/ethyl acetate, 4:1); mp 168–170 °C; ¹H NMR (400 MHz; DMSO-d₆) δ_H: 2.39 (s, 3H, CH₃), 3.99 (s, 2H, PhCH₂), 7.17–7.31 (m, 6H, C₆–H and Ph–H), 7.32 (d, 1H, C₈–H, J = 2.0 Hz), 7.92 (d, 1H, C₅–H, J = 8.6 Hz) and 8.22 (s, 2H, NH₂); MS (FAB⁺) m/z: 346.1 [100, (M + H)⁺]; MS (FAB^–) m/z: 344.1 [100, (M – H)⁻], 265.1 [60, (M – H₂NSO₂)⁻]; Acc. MS (FAB⁺): 346.0755, C₁₇H₁₆NO₅S requires 346.0749; found C, 59.10; H, 4.39; N, 4.04; C₁₇H₁₅NO₅S requires C, 59.12; H, 4.38; N, 4.06%.

Ethyl 2-Acetyl-4-phenylbutanoate (42a)

This was prepared by general method using K₂CO₃ (8.97 g; 64.85 mmol), water (50 mL), (2-bromoethyl)benzene (5.0 mL; 27 mmol), ethyl 3-oxo-butanoate (3.44 mL; 27 mmol), CH₂Cl₂ (50 mL), and Bu₄NCl (∼10 g; 27 mmol). The crude oily residue was purified by flash chromatography (CHCl₃) to give 42a as a pale yellow oil (2.17 g; 34%). (Lit.⁷² bp_1.4 167–168 °C); R_f: 0.78 (CHCl₃); ¹H NMR (400 MHz; CDCl₃) δ_H: 1.25 (t, 3H, CH₂CH₃, J = 6.7 Hz), 2.29 (s, 3H, CH₃), 3.02 (q, 2H, CH₂CH₂Ph, J = 7.1 Hz), 3.71 (t, 1H, CH₂, J = 7.3 Hz), 3.95 (t, 2H, CH₂Ph, J = 7.1 Hz), 4.11 (q, 2H, CH₂CH₃, J = 7.1 Hz) and 7.16–7.34 (m, 5H, Ph–H); MS (FAB⁺) m/z: 235.0 [100, (M + H)⁺]; Acc. MS (FAB⁺): 235.1335, C₁₄H₁₉O₃ requires 235.1334.

7-Hydroxy-4-methyl-3-(2-phenylethyl)coumarin (42b)

This was prepared by general method using resorcinol (705 mg; 6.4 mmol), 42a (1.5 g; 6.4 mmol), and a mixture of CF₃COOH (1 mL; 13 mmol) and conc. H₂SO₄ (0.7 mL; 13 mmol). The crude brown solid was purified by flash chromatography (CHCl₃/acetone gradient, 8:1 to 4:1), and the white solid isolated was recrystallized from acetone/hexane to give 42b as gray crystals (746 mg; 42%). R_f: 0.80 (CHCl₃/acetone, 3:1); mp 175–178 °C ¹H NMR (400 MHz; DMSO-d₆) δ_H: 2.16 (s, 3H, CH₃), 2.73 (t, 2H, CH₂CH₂Ph, J = 5.5 Hz), 2.79 (t, 2H, PhCH₂, J = 5.2 Hz), 6.69 (d, 1H, C₈–H, J = 2.4 Hz), 6.78 (dd, 1H, C₆–H, J = 2.4, 8.5 Hz), 7.16–7.29 (m, 5H, Ph–H), 7.56 (d, 1H, C₅–H, J = 8.8 Hz) and 10.42 (s, 1H, OH); MS (FAB⁺) m/z: 561.1 [10, (2M + H)⁺], 281.0 [100, (M + H)⁺]; MS (FAB^–) m/z: 279.1 [100, (M – H)]; Acc. MS (FAB⁺): 281.1182, C₁₈H₁₇O₃ requires 281.1178; found C, 77.35; H, 5.95; C₁₈H₁₆O₃ requires C, 77.12; H, 5.75%.

4-Methyl-3-(2-phenylethyl)coumarin-7-O-sulfamate (42)

Compound 42b (400 mg; 1.43 mmol) was sulfamoylated, the crude white solid was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient), and the white solid isolated was recrystallized from ethyl acetate/hexane to give 42 as white fine crystals (132 mg; 26%). R_f: 0.54 (CHCl₃/ethyl acetate, 4:1); mp 196–198 °C; ¹H NMR (400 MHz; DMSO-d₆) δ_H: 2.21 (s, 3H, CH₃), 2.76 (t, 2H, CH₂CH₂Ph, J = 7.3 Hz), 2.87 (t, 2H, PhCH₂, J = 7.9 Hz), 7.17–7.27 (m, 7H, C₆–H, C₈–H, and Ph–H), 7.83 (d, 1H, C₅–H, J = 8.8 Hz) and 8.19 (s, 2H, NH₂); MS (FAB⁺) m/z: 360.0 [100, (M + H)⁺]; MS (FAB^–) m/z: 358.0 [100, (M – H)⁻], 279.1 [40, (M – H₂NSO₂)⁻]; Acc. MS (FAB⁺): 360.0917, C₁₈H₁₈NO₅S requires 360.0906; found C, 60.00; H, 4.81; N, 3.89; C₁₈H₁₇NO₅S requires C, 60.16; H, 4.77; N, 3.90%.

Ethyl 2-Acetyl-5-phenylpentanoate (43a)

This was prepared by general method using K₂CO₃ (16.7 g; 0.12 mol), water (60 mL), 1-bromo-3-phenylpropane (10.0 mL; 50.2 mmol), ethyl 3-oxo-butanoate (6.5 mL; 50.2 mmol), CH₂Cl₂ (60 mL), and Bu₄NCl (28 g; 0.1 mol). The crude oily residue was purified by flash chromatography (CHCl₃/hexane, 10:1) to give 43a as a colorless oil (4.92 g; 39%). R_f: 0.61 (CHCl₃/hexane, 10:1); ¹H NMR (400 MHz; CDCl₃) δ_H: 1.26 (t, 3H, CH₂CH₃, J = 7.0 Hz), 2.19 (s, 3H, CH₃), 1.85–2.61 (m, 6H, CH₂), 2.64 (t, 1H, 2H, J = 7.8 Hz), 4.21 (q, 2H, CH₂CH₃, J = 7.0 Hz) and 7.15–7.29 (m, 5H, Ph–H); MS (FAB⁺) m/z: 249.1 [100, (M + H)⁺]; MS (FAB^–) m/z: 247.1 [100, (M – H)⁻]; Acc. MS (FAB⁺): 249.1491, C₁₅H₂₁O₃ requires 249.1491.

7-Hydroxy-4-methyl-3-[3-phenylpropyl]coumarin (43b)

This was prepared by general method using resorcinol (443 mg; 4.03 mmol), 43a (1.0 g; 4.0 mmol), and a mixture of CF₃COOH (0.6 mL; 8.1 mmol) and conc. H₂SO₄ (0.4 mL; 8.1 mmol). The crude orange solid was purified by flash chromatography (CHCl₃/acetone gradient, 8:1 to 4:1), and the white solid isolated was recrystallized from THF/hexane to give 43b as pale green crystals (275 mg; 22%). R_f: 0.76 (CHCl₃/acetone, 3:1); mp 189–191 °C; MS (FAB⁺) m/z: 589.2 [10, (2M + H)⁺], 295.1 [100, (M + H)⁺]; MS (FAB^–) m/z: 293.1 [100, (M – H)⁻]; Acc. MS (FAB⁺): 295.1675, C₁₉H₁₉O₃ requires 295.1680; ¹H NMR (400 MHz; DMSO-d₆) δ_H: 1.71 (quintet, 2H, CH₂CH₂CH₂Ph, J = 7.8 Hz), 2.13 (s, 3H, CH₃), 2.54 (t, 2H, CH₂CH₂CH₂Ph, J = 7.8 Hz), 2.63 (t, 2H, CH₂CH₂CH₂Ph, J = 7.4 Hz), 6.82 (d, 1H, C₈–H, J = 2.4 Hz), 7.13–7.27 (m, 6H, C₆–H and Ph–H), 7.42 (d, 1H, C₅–H, J = 8.8 Hz) and 10.25 (s, 1H, OH); found C, 77.13; H, 6.28; C₁₉H₁₈O₃ requires C, 77.53; H, 6.16%.

4-Methyl-3-[3-phenylpropyl]coumarin-7-O-sulfamate (43)

Compound 43b (230 mg; 0.78 mmol) was sulfamoylated, the crude white solid was purified by flash chromatography (CHCl₃/ethyl acetate gradient, 8:1 to 2:1), and the white solid isolated was recrystallized from THF/hexane to give 43 as white crystals (62 mg; 21%). R_f: 0.54 (CHCl₃/ethyl acetate, 4:1); mp 154–156 °C; ¹H NMR (400 MHz; DMSO-d₆) δ_H: 1.76 (pentet, 2H, CH₂CH₂CH₂Ph, J = 8.2 Hz), 2.49 (s, 3H, CH₃), 2.62 (t, 2H, CH₂CH₂CH₂Ph, J = 8.2 Hz), 2.67 (t, 2H, CH₂CH₂CH₂Ph, J = 7.4 Hz), 7.15–7.39 (m, 7H, C₈–H, C₆–H and Ph–H), 7.87 (d, 1H, C₅–H, J = 8.8 Hz) and 8.19 (s, 2H, NH₂); MS (FAB⁺) m/z: 374.0 [100, (M + H)⁺]; MS (FAB^–) m/z: 372.1 [100, (M – H)⁻], 293.1 [40, (M – H₂NSO₂)⁻]; Acc. MS (FAB⁺): 374.1060, C₁₉H₂₀NO₅S requires 374.1062; found C, 61.30; H, 4.94; N, 3.49; C₁₉H₁₉NO₅S requires C, 61.11; H, 5.13; N, 3.75%.

Ethyl 2-(Cyclohexylmethyl)-3-oxo-butanoate (44a)

This was prepared by general method using K₂CO₃ (7.5 g; 54 mmol), water (50 mL), cyclohexylmethyl bromide (4.0 mL; 23 mmol), ethyl 3-oxo-butanoate (2.9 mL; 23 mmol), CH₂Cl₂ (50 mL) and Bu₄NCl (∼10 g; 23 mol). The crude pale yellow oil was purified by flash chromatography (CHCl₃) to give 44a as a pale yellow oil (1.44 g; 28%). (Lit.⁷³ bp₁₉ 166 °C); R_f: 0.58 (CHCl₃); ¹H NMR (400 MHz; CDCl₃) δ_H: 1.08–1.34 (m, 11H, cyclohexyl-H), 1.27 (t, 3H, CH₂CH₃, J = 7.0 Hz), 1.69 (t, 2H, CH₂, J = 7.0 Hz), 2.22 (s, 3H, CH₃), 3.53 (t, 1H, 2H, J = 7.4 Hz) and 4.21 (q, 2H, CH₂CH₃, J = 7.0 Hz); MS (FAB⁺) m/z: 227.3 [100, (M + H)⁺]; MS (FAB^–) m/z: 225.3 [100, (M – H)⁻]; Acc. MS (FAB⁺): 227.1632, C₁₃H₂₃O₃ requires 227.1647.

3-Cyclohexylmethyl-7-hydroxy-4-methylcoumarin (44b)

This was prepared by general method using resorcinol (487 mg; 4.42 mmol), 44a (1.0 g; 4.4 mmol), and a mixture of CF₃COOH (0.7 mL; 8.8 mmol) and conc. H₂SO₄ (0.5 mL; 8.8 mmol). The crude orange solid was purified by flash chromatography (CHCl₃/acetone, 8:1 to 4:1 gradient), and the white solid isolated was recrystallized from THF/hexane to give 44b as fine white crystals (409 mg; 34%). R_f: 0.74 (CHCl₃/acetone, 3:1); mp 193–196 °C; ¹H NMR (400 MHz; DMSO-d₆) δ_H: 1.11–1.91 (m, 11H, cyclohexyl-H), 2.35 (s, 3H, CH₃), 2.43 (d, 2H, CH₂, J = 7.0 Hz), 6.68 (d, 1H, C₈–H, J = 2.3 Hz), 6.79 (dd, 1H, C₆–H, J = 2.3, 8.6 Hz), 7.59 (d, 1H, C₅–H, J = 8.6 Hz) and 10.21 (s, 1H, OH); MS (FAB⁺) m/z: 545.3 [15, (2M + H)⁺], 273.2 [100, (M + H)⁺]; MS (FAB^–) m/z: 271.2 [100, (M – H)⁻]; Acc. MS (FAB⁺): 273.1491, C₁₇H₂₁O₃ requires 273.1492; found C, 74.80; H, 7.47; C₁₇H₂₀O₃ requires C, 74.97; H, 7.40%.

3-Cyclohexylmethyl-4-methylcoumarin-7-O-sulfamate (44)

Compound 44b (300 mg; 1.10 mmol) was sulfamoylated, the crude white solid was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient), and the white solid isolated was recrystallized from ethyl acetate/hexane to give 44 as white crystals (46 mg; 12%). R_f: 0.55 (CHCl₃/ethyl acetate, 4:1); mp 170–171 °C; ¹H NMR (400 MHz; DMSO-d₆) δ_H: 1.22–1.31 (m, 11H, cyclohexyl-H), 2.06 (s, 3H, CH₃), 2.58 (d, 2H, CH₂, J = 7.0 Hz), 7.32–7.35 (m, 2H, C₈–H and C₆–H), 7.93 (d, 1H, C₅–H, J = 8.6 Hz) and 8.39 (s, 2H, NH₂); MS (FAB⁺) m/z: 352.0 [100, (M + H)⁺]; MS (FAB^–) m/z: 350.0 [100, (M – H)⁻], 271.1 [45, (M – H₂NSO₂)⁻]; Acc. MS (FAB⁺): 352.1214, C₁₇H₂₂NO₅S requires 352.1218; found C, 58.30; H, 5.86; N, 3.79; C₁₇H₂₁NO₅S requires C, 58.10; H, 6.02; N, 3.99%.

Ethyl 2-Acetyl-4-cyclohexylbutanoate (45a)

This was prepared by general method using K₂CO₃ (8.68 g; 62.8 mmol), water (60 mL), 1-bromo-2-cyclohexylethane (5.0 mL; 26.2 mmol), ethyl 3-oxo-butanoate (3.34 mL; 26.2 mmol), CH₂Cl₂ (50 mL), and Bu₄NCl (7.3 g; 26 mmol). The crude orange oily residue was purified by flash chromatography (CHCl₃) to give 45a as a pale yellow oil (850 mg; 14%). (Lit.⁷³ bp₁₉ 175 °C); R_f: 0.72 (CHCl₃); ¹H NMR (400 MHz; CDCl₃) δ_H: 1.19–1.66 (m, 13H, cyclohexyl-H and CH₂), 1.27 (t, 3H, CH₂CH₃, J = 7.0 Hz), 1.80–1.87 (m, 2H, CH₂), 2.22 (s, 3H, CH₃), 3.35 (t, 1H, 2H, J = 7.4 Hz) and 4.19 (q, 2H, CH₂CH₃, J = 7.0 Hz); MS (FAB⁺) m/z: 241.3 [100, (M + H)⁺]; MS (FAB^–) m/z: 239.3 [100, (M – H)⁻]; Acc. MS (FAB⁺): 241.1804, C₁₄H₂₅O₃ requires 241.1804.

3-(2-Cyclohexylethyl)-4-methyl-7-hydroxycoumarin (45b)

This was prepared by general method using resorcinol (343 mg; 3.12 mmol), 45a (750 mg; 3.12 mmol), and a mixture of CF₃COOH (0.5 mL; 6.2 mmol) and conc. H₂SO₄ (0.4 mL; 6.2 mmol). The crude orange solid was purified by flash chromatography (CHCl₃/acetone, 8:1 to 4:1 gradient), and the white solid isolated was recrystallized from THF/hexane to give 45b as white crystals (352 mg; 39%). R_f: 0.79 (CHCl₃/acetone, 3:1); mp 148–151 °C; ¹H NMR (400 MHz; DMSO-d₆) δ_H: 1.11–1.73 (m, 11H, cyclohexyl-H), 2.34 (s, 3H, CH₃), 2.51–2.53 (m, 4H, CH₂CH₂), 6.67 (d, 1H, C₈–H, J = 2.3 Hz), 6.78 (dd, 1H, C₆–H, J = 2.3, 8.6 Hz), 7.59 (d, 1H, C₅–H, J = 8.9 Hz) and 10.38 (s, 1H, OH); MS (FAB⁺) m/z: 572.9 [10, (2M + H)⁺], 287.1 [100, (M + H)⁺]; MS (FAB^–) m/z: 571.1 [10, (2M – H)⁻], 285.2 [100, (M – H)⁻]; Acc. MS (FAB⁺): 287.1639, C₁₈H₂₃O₃ requires 287.1647; found C, 75.50; H, 7.67; C₁₈H₂₂O₃ requires C, 75.50; H, 7.74%.

3-(2-Cyclohexylethyl)-4-methylcoumarin-7-O-sulfamate (45)

Compound 45b (250 mg; 0.87 mmol) was sulfamoylated, the crude white solid was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 2:1 gradient), and the white solid isolated was recrystallized from ethyl acetate/hexane to give 45 as white fine crystals (155 mg; 49%). R_f: 0.47 (CHCl₃/ethyl acetate, 4:1); mp 179–181 °C; ¹H NMR (400 MHz; DMSO-d₆) δ_H: 1.19–1.78 (m, 11H, cyclohexyl-H), 2.41 (s, 3H, CH₃), 2.49–2.59 (m, 4H, CH₂CH₂), 7.25–7.28 (m, 2H, C₈–H and C₆–H), 7.87 (d, 1H, C₅–H, J = 8.6 Hz) and 8.20 (s, 2H, NH₂); MS (FAB⁺) m/z: 366.0 [100, (M + H)⁺]; MS (FAB^–) m/z: 364.0 [100, (M – H)⁻], 285.1 [40, (M – H₂NSO₂)⁻]; Acc. MS (FAB⁺): 366.1319, C₁₈H₂₄NO₅S requires 366.1297; found C, 59.10; H, 6.31; N, 3.62; C₁₈H₂₃NO₅S requires C, 59.16; H, 6.34; N, 3.83%.

7-Hydroxy-3-(4-methoxyphenyl)-4-methylcoumarin (46a)

To a CH₂Cl₂ (100 mL) solution of 2,4-dihydroxyacetophenone (5.0 g; 33 mmol; 1 equiv), tetrabutylammonium hydrogensulphate (300 mg; 0.88 mmol; 0.03 equiv), and 20% aq K₂CO₃ (100 mL) was added 4-methoxyphenylacetyl chloride (6.7 g; 36 mmol; 1.1 equiv) CH₂Cl₂ (30 mL) dropwise over 30 min and stirred for 4 h at rt. The organic layer was separated, washed with water (3 × 200 mL), dried, and concentrated. The crude pale brown syrup was purified by flash chromatography (CHCl₃/ethyl acetate, 8:1 to 4:1 gradient), and the pale yellow solid isolated was recrystallized from THF/hexane to give 46a as white crystals (1.2 g; 13%). R_f: 0.68 (UV visible and fluorescent) (ethyl acetate/hexane, 1:1); mp 230–232 °C (Lit.⁷⁴ mp 233–234 °C); ¹H NMR (400 MHz; DMSO-d₆) δ_H: 2.60 (s, 3H, CH₃), 3.81 (s, 3H, OCH₃), 6.63 (dd, 1H, C₆–H, J = 2.3, 8.9 Hz), 6.71 (d, 1H, C₈–H, J = 2.3 Hz), 6.90 (d, 2H, Ph-2,6-H₂, J = 8.6 Hz), 7.29 (d, 2H, Ph-3,5-H₂, J = 8.6 Hz), 7.72 (d, 1H, C₅–H, J = 8.9 Hz) and 12.42 (s, 1H, OH); MS (FAB⁺) m/z: 283.2 [100, (M + H)⁺]; MS (FAB^–) m/z: 281.2 [100, (M – H)⁻]; Acc. MS (FAB⁺): 283.0898, C₁₇H₁₅O₄ requires 283.0892; found C, 71.98; H, 5.36; C₁₇H₁₄O₄ requires C, 72.33; H, 5.00%.

3-(4-Methoxyphenyl)-4-methylcoumarin-7-O-sulfamate (46)

Upon sulfamoylation, compound 46a (500 mg; 1.77 mmol) gave a crude white solid, which was fractionated by flash chromatography (CHCl₃/ethyl acetate 8:1 to 2:1 gradient). The white solid isolated was recrystallized from ethyl acetate/hexane to give 46 as white fine leaves (331 mg; 52%). R_f: 0.86 (CHCl₃/ethyl acetate, 4:1); mp 129–132 °C; ¹H NMR (400 MHz; DMSO-d₆) δ_H: 2.77 (s, 3H, CH₃), 3.75 (s, 3H, OCH₃), 6.93 (d, 2H, Ph-2,6-H₂, J = 8.6 Hz), 7.31 (d, 2H, Ph-3,5-H₂, J = 8.6 Hz), 7.35 (dd, 1H, C₆–H, J = 1.9, 8.9 Hz), 7.48 (d, 1H, C₈–H, J = 1.9 Hz), 8.21 (d, 1H, C₅–H, J = 8.9 Hz), 8.24 (s, 2H, NH₂); MS (FAB⁺) m/z: 362.1 [100, (M + H)⁺]; MS (FAB^–) m/z: 361.2 [100, (M – H)⁻]; Acc. MS (FAB⁺): 362.0705, C₁₇H₁₆NO₆S requires 362.0698; found C, 56.30; H, 4.21; N, 3.78; C₁₇H₁₅NO₆S requires C, 56.50; H, 4.18; N, 3.88%. ref. (45)

The authors declare the following competing financial interest(s): B.V.L.P. was a Director and stockholder of Sterix Ltd and received research funding that supported some aspects of this work. A.P. was a stockholder of Sterix Ltd.