Structure–Activity Relationship for the First-in-Class Clinical Steroid Sulfatase Inhibitor Irosustat (STX64, BN83495)

Abstract

Structure–activity relationship studies were conducted on Irosustat (STX64, BN83495), the first steroid sulfatase (STS) inhibitor to enter diverse clinical trials for patients with advanced hormone-dependent cancer. The size of its aliphatic ring was expanded; its sulfamate group was N,N-dimethylated, relocated to another position and flanked by an adjacent methoxy group; and series of quinolin-2(1H)-one and quinoline derivatives of Irosustat were explored. The STS inhibitory activities of the synthesised compounds were assessed in a preparation of JEG-3 cells. Stepwise enlargement of the aliphatic ring from 7 to 11 members increases potency, although a further increase in ring size is detrimental. The best STS inhibitors in vitro had IC₅₀ values between 0.015 and 0.025 nm. Other modifications made to Irosustat were found to either abolish or significantly weaken its activity. An azomethine adduct of Irosustat with N,N-dimethylformamide (DMF) was isolated, and crystal structures of Irosustat and this adduct were determined. Docking studies were conducted to explore the potential interactions between compounds and the active site of STS, and suggest a sulfamoyl group transfer to formylglycine 75 during the inactivation mechanism.

Introduction

The inhibition of steroid sulfatase (STS) as a new target for endocrine therapy has attracted considerable attention over the past two decades after recognition that the STS pathway could also be a significant source of oestrogens alongside those originating from aromatase, the enzyme that aromatises androgens to oestrogens. Evidence to support this hypothesis includes: 1) a millionfold higher STS activity than aromatase activity in liver as well as normal and malignant breast tissues,[1] 2) the origin of oestrone (E1) from oestrone sulfate (E1S) in breast cancer tissue is ∼10-fold greater than that from androstenedione,[2] and 3) STS expression is an important prognostic factor in human breast carcinoma.[3, 4] Most oestrogens that originate from the aromatase pathway are converted into and stored in the body as sulfate conjugates that per se are biologically inactive. However, this reservoir of oestrogen sulfates could significantly contribute to overall oestrogenic stimulation of the growth and development of hormone-dependent tumours when STS catalyses the hydrolysis of substrates such as E1S to E1, and dehydroepiandrosterone sulfate (DHEA-S) to DHEA. The formation of DHEA via the STS pathway accounts for the production of 90 % of androstenediol (Adiol). Although structurally an androgen, Adiol possesses oestrogenic properties. It is ∼100-fold weaker than oestradiol[5–8] and has a lower affinity for the oestrogen receptor.[9] However, the 100-fold higher concentrations of Adiol in the circulation have led some to speculate that it may have oestrogenic properties equipotent to oestradiol.[10] Thus, STS is an attractive and novel target for rendering potentially more effective oestrogen deprivation through therapeutic intervention in hormone-dependent cancers such as those of the breast, endometrium, and prostate.

Considerable progress has been made since the early 1990s in the development of STS inhibitors. Many structurally (steroidal and nonsteroidal) and mechanistically (principally reversible and irreversible) diverse inhibitors have been developed. However, compounds that contain the pharmacophore for irreversible inhibition of STS, i.e., an aryl sulfamate ester, have consistently shown distinctive and potent in vitro and in vivo inhibitory activities.[11–13] One compound, the nonsteroidal inhibitor 1 (Irosustat, STX64, BN83495, Figure 1), is the first STS inhibitor to enter clinical trials for postmenopausal patients with advanced hormone-dependent breast cancer and has shown encouraging results.[14, 15] Progress has been made since the completion of this first trial.[16] Currently, 1 is undergoing phase I trials for advanced prostate cancer and phase II trials for endometrial and advanced breast cancer.

Figure 1.

Structure of 1 (Irosustat, STX64), 1 a (the parent phenol of 1), and derivatives 2–5.

On the discovery of 1 as a potent STS inhibitor, a basic study was carried out to provide a preliminary structure–activity relationship (SAR).[17] The main focus of that work was on ring contraction (from 7- down to 6- and 5-membered rings: compounds 2 and 3, Figure 1) and expansion (from 7- to 8-membered rings: 4, Figure 1) of the aliphatic ring of 1. In addition, a tricyclic oxepin derivative of 3 (compound 5, Figure 1) was synthesised and evaluated. Herein we report a more extensive SAR study for 1, further expansion of the aliphatic ring size, N,N-dimethylation of the sulfamate group, relocation of the sulfamate group to another position, introduction of a substituent adjacent to the sulfamate group, and exploration of a series of quinolin-2(1H)-one and quinoline derivatives of 1. The biological activities of the synthesised compounds were assessed in a preparation of JEG-3 cells. In addition, an azomethine adduct of 1 and N,N-dimethylformamide (DMF) is reported. The crystal structures of 1 and its azomethine adduct were determined. Docking studies were conducted to explore the potential interactions between the compounds and the active site of STS.

Results and Discussion

Chemistry

With the exception of ethyl 2-oxocyclotridecanecarboxylate, which is available commercially, the starting cyclic β-keto esters required for the synthesis of tricyclic coumarins 6 b–9 b and 11 b were prepared by treating the corresponding cycloalkyl ketone with diethyl carbonate in the presence of two equivalents of sodium hydride at room temperature.[18] The parent tricyclic coumarins were formed under Pechmann conditions by cyclising resorcinol and the corresponding ethyl 2-oxocycloalkylcarboxylates in the presence of an equimolar mixture of trifluoroacetic acid and concentrated sulfuric acid as the condensing agent (Scheme 1). The yields of the tricyclic coumarins ranged from 14 to 33 %, presumably due to severe ring strain experienced by cycloalkenyl rings, in particular cyclononene and cycloundecene, during the cyclisation of the cyclic β-keto esters with resorcinol.

Scheme 1.

Synthesis of tricyclic coumarin sulfamates (6–11). Reagents and conditions: a) 2 NaH, N₂, 15 h, RT; b) concd H₂SO₄/CF₃COOH, 3 h, 0 °C→RT; c) anhydrous DMF, NaH, N₂, H₂NSO₂Cl, 0 °C→RT.

An earlier method was used for the sulfamoylation of parent hydroxycoumarins (Scheme 1). This involved treating a solution of the phenol in anhydrous N,N-dimethylformamide (DMF) with sodium hydride followed by the addition of a freshly concentrated solution of sulfamoyl chloride in toluene, which was prepared according to the method of Woo et al.[19]

The synthesis of 12 was initially attempted by deprotonation of 1 in N,N-dimethylacetamide (DMA) with sodium hydride at 0 °C followed by N,N-dimethylation with methyl iodide (Scheme 2). However, compound 12 obtained by this route was persistently contaminated by a trace amount of 3- methoxy-8,9,10,11-tetrahydrocyclohepta[c]chromen-6(7H)-one, which is most likely the product of desulfamoylation of 1 followed by methylation of the phenol released (compound 1 a) under the reaction conditions employed. This ethereal contaminant was particularly difficult to remove, and hence a different synthetic approach was sought. Compound 12 was subsequently prepared with high purity by heating 1 a in N,N-dimethylcyclohexylamine with N,N-dimethylsulfonyl chloride (Scheme 2).

Scheme 2.

Synthesis of 12, the N,N-dimethyl derivative of 1. Reagents and conditions: a) NaH, CH₃I, 0 °C (12 obtained in this manner was contaminated by a trace amount of the 3-methoxy derivative of 1 a); b) N,N-dimethylcyclohexylamine, Me₂NSO₂Cl, 90–95 °C, 1 h.

Similar to 1, the synthesis of 13 b was achieved by a Pechmann route, although resorcinol was replaced by 4-methoxybenzene-1,3-diol (13 a) as starting material, which was prepared according to the method of Godfrey et al. (Scheme 3).[20] Sulfamoylation of a solution of 13 b in DMA gave the methoxylated tricyclic coumarin sulfamate 13.

Scheme 3.

Synthesis of compound 13. Reagents and conditions: a) concd H₂SO₄/CF₃COOH, 0 °C→RT, 60 h; b) anhydrous DMA, N₂, H₂NSO₂Cl, 0 °C→RT.

The synthesis of 2-hydroxy-8,9,10,11-tetrahydrocyclohepta[c]chromen-6(7H)-one (14 a) was carried out by allowing hydroquinone to react with methyl 2-oxo-1-cycloheptanecarboxylate under Pechmann conditions (Scheme 4). As anticipated, the isolated yield of 14 a was extremely low (3 %) due to the 2-position of hydroquinone not being electron-rich and hence activated for ring closure by a Pechmann mechanism. Nonetheless, a sufficient quantity of 14 a was isolated for further sulfamoylation to give the 2-sulfamate 14.

Scheme 4.

Synthesis of compound 14. Reagents and conditions: a) concd H₂SO₄/CF₃COOH, 0 °C→RT, 60 h; b) anhydrous DMA, N₂, H₂NSO₂Cl, 0 °C→RT.

Compound 15 is a low-yielding azomethine adduct of 1 with DMF. Only a very small amount of 15 was isolated during a very large-scale synthesis of 1 that was performed for determination of its crystal structure. With an earlier method for conducting sulfamoylation, which involves the use of sodium hydride in excess for deprotonating the phenolic parent compound 1 a in DMF prior to the addition of sulfamoyl chloride, the formation of 15 is anticipated, as we reported earlier a similar azomethine adduct between 2-nitrophenyl sulfamate and DMF.[21] It is reasoned that the presence of excess sodium hydride in the reaction mixture deprotonates the sulfamate group of 1 after its formation, and the resulting anion undergoes a nucleophilic attack on the formyl group of DMF to give compound 15 upon subsequent dehydration, as illustrated in Scheme 5.

Scheme 5.

Proposed mechanism for the formation of 15, an azomethine adduct between compound 1 and DMF.

The quinolinone derivative 16 a was prepared in good yield (73 %) by heating a mixture of 3-aminophenol and methyl 3-oxo-1-cycloheptane carboxylate (Scheme 6). Sulfamoylation of 16 a in the usual manner gave the quinolinone sulfamate 16.

Scheme 6.

Synthesis of quinoline and quinolinone derivatives of 1. Reagents and conditions: a) 150 °C, 8 h; b) anhydrous DMF, NaH, N₂, H₂NSO₂Cl, 0 °C→RT; c) NaH, DMF, 0 °C, BnBr, 90 °C; d) NaH, DMF, 0 °C, CH₃I, 80 °C; e) Pd/C (10 %), THF, H₂ (balloon); f) POCl₃, reflux; g) anhydrous DMF, NaH, anhydrous MeOH/DMF, 70 °C, 2 h; h) Pd/C (10 %), abs. EtOH, H₂ (balloon); i) 2,6-di-tert-butyl-4-methylpyridine, N₂, anhydrous CH₂Cl₂, H₂NSO₂Cl, RT; j) NaH, anhydrous DMF, 1-bromopentane or 1-bromo-3-phenylpropane, 100 °C, 1 h [X=OSO₂NH₂].

The key intermediate for synthesising the rest of the quinoline and quinolinone derivatives reported herein is compound 17, which was prepared by O-benzyl protection of 16 a (Scheme 6). After deprotonation of 17 with sodium hydride and heating the resulting anion with methyl iodide, the N-methyl derivative 18 a was obtained in high yield. Debenzylation by hydrogenation gave the phenolic quinolinone 18 b, which was sulfamoylated to give the 5-methyl quinolinone sulfamate 18.

The 3-O-benzyl-protected quinolinone 17 was converted into the 6-chloroquinoline 19 a with phosphorus oxychloride. Holding 19 a at reflux in anhydrous DMF with freshly prepared sodium methoxide gave the 6-methoxyquinoline 19 b. The 6-methylquinolinyl sulfamate 19 was obtained by first debenzylating 19 b followed by sulfamoylating the phenolic derivative 19 c.

Quinolinones 20 and 22 and quinolines 21 and 23 were prepared by a different route from their corresponding lower members 18 and 19. Holding the anion of 17 at reflux in DMF with either 1-bromopentane or 1-bromo-3-phenylpropane rendered a mixture of both the N- (20 a and 22 a) and O-alkylated (21 a and 23 a) derivatives. Interestingly, the isolated yields of quinolinones 20 a (62 %) and 22 a (55 %) were both found to be higher than their quinoline counterparts 21 a (41 %) and 23 a (42 %), suggesting that N-alkylation is slightly more favourable under the reaction conditions. In addition, both quinolinones were retained longer by silica in flash chromatography than quinolines, suggesting that 20 a and 22 a are more polar than 21 a and 23 a. Debenzylation by hydrogenation of 20 a–23 a in the usual manner gave the phenolic derivatives 20 b–23 b, which upon sulfamoylation gave the corresponding sulfamates 20–23.

The aminoquinolinone 24 a was prepared by heating a mixture of 1,3-phenylenediamine and methyl 2-oxo-1-cycloheptane carboxylate at 150 °C overnight (Scheme 7). Upon sulfamoylation of a solution of 24 a in DMF in the presence of 2,6-di-tert-butyl-4-methylpyridine (DBMP) and sulfamoyl chloride gave the sulfamido quinolinone 24.

Scheme 7.

Synthesis of compound 24. Reagents and conditions: a) 150 °C, 18 h; b) anhydrous DMF, N₂, DBMP, H₂NSO₂Cl, 0 °C→RT.

Crystal structures

A crystal of 1 with approximate dimensions of 0.25×0.10×0.08 mm was used for data collection. As shown in Figure 2 b, molecules of 1 interact via a network of intermolecular hydrogen bonds. In particular, one proton of the sulfamate NH₂ group (H1B) interacts with the carbonyl oxygen atom (O5) of the coumarin ring in a proximate molecule, whereas the other NH proton (H1A) interacts with an oxygen atom (O2) of the SO₂ group of a neighbouring sulfamate group. Additionally, there are possible intermolecular π–π interactions present (centroid_{C9-C10-C15-C16} to centroid_{C1-C2-C3-C4-C5-C6} distance=3.52 Å). As predicted in previous work by molecular modelling, the 7-membered aliphatic ring of 1 is in the chair form (Figure 2 a,b), which is similar to that of cycloheptene with the C=C moiety taking the place of one of the ring carbon atoms in the cyclohexane chair.[17]

Figure 2.

a) X-ray crystal structure of 1 (CCDC deposition code: 826524); ellipsoids are represented at 30 % probability. b) Portion of extended structure present in 1 showing the network of intermolecular hydrogen bonding. c) X-ray crystal structure of 15 (CCDC deposition code: 826525); ellipsoids are represented at 30 % probability.

A crystal of 15 with approximate dimensions of 0.25×0.13×0.10 mm was used for data collection. As shown in Figure 2 c, the tricyclic coumarin scaffold of 15 has a similar conformation to that observed for 1. The stereochemistry is unambiguously E at the double bond of its (dimethylamino)methylene sulfamoyl group, suggesting that steric effects might be a contributing factor in the more favourable formation of the trans geometric isomer via the route in Scheme 5, with the bulky dimethylamino and arylsulfamoyl motifs placed diametrically opposite before the antiperiplanar elimination of water. As for 1, the aliphatic ring of 15 is clearly in the chair form. Crystal structures of two other tricyclic coumarin sulfamates 6 and 7 with larger ring sizes were also obtained and have been reported elsewhere.[22]

Structure–activity relationship and molecular modelling

Altogether, ten tricyclic coumarin sulfamates are compared in this work, out of which the syntheses of six final compounds are reported for the first time. These compounds contain a core bicyclic coumarin ring system, but differ in the size of the third (aliphatic) ring. The lowest member of the series studied is 2, because having an aliphatic ring smaller than the 5-membered cyclopentenyl would be synthetically challenging due to the significant ring strain of a cyclobutene or cyclopropene. The increase in size of the third ring was carried out in a stepwise fashion from 5 to 15 members, although the 14-membered derivative was omitted, primarily due to the lack of commercial availability of cyclotetradecanone as starting material.

We evaluated the STS inhibitory activities of the tricyclic coumarin sulfamates 1–4 and 6–11 in a placental microsome preparation, and the results were reported in a previous publication.[23] For reference and comparison, these results are listed in Table 1. In this assay, 7 (10-membered third ring) proved to be the most potent STS inhibitor of the series in vitro, with an IC₅₀ value of 1 nm, although 1 (7-membered third ring), 6 (9-membered third ring), and 8 (11-membered third ring) were also potent, with IC₅₀ values ranging from 8 to 13 nm. The least potent congeners of the series were 2 (5-membered third ring) and 11 (15-membered third ring), the IC₅₀ values for which were found to be 200 nm or higher. While it is not clear why the IC₅₀ value for 4 (8-membered third ring) is not of the same order of magnitude as its immediate lower (1) and higher (6) congeners, but is instead significantly higher at 30 nm, it is apparent that the size of the third ring in this series of compounds has a marked effect on the potency of compounds against STS. Interestingly, it was found that 7 is only marginally more potent than 1 in vivo despite its IC₅₀ value in placental microsomes at 1 nm being eightfold lower than that of 1.[23] Despite its relatively weak activity in vitro (IC₅₀=370 nm, placental microsomes), 11 was found to be the most potent tricyclic coumarin sulfamate in vivo, inhibiting rat liver STS activity by 23 and 94 % when assayed 24 h after administration at respective doses of 0.1 and 1 mg kg⁻¹,[23] which may be explained, among other things, by a depot effect relating to its high log P value.

Table 1.

Inhibition of STS activity in placental microsomes (PM) and JEG-3 cells by tricyclic coumarin sulfamates 1–4 and 6–11, the N,N-dimethyl derivative of 1 (compound 12), the 2-methoxy derivative of 1 (13), 6-oxo-6,7,8,9,10,11-hexahydrocyclohepta[c]chromen-2-yl sulfamate (14), and the azomethine adduct of 1 and DMF (15)

Compd	n	PM IC50 [nm][a]	JEG-3 IC50 [nm]
2	3	200	32
3	4	70	7
1	5	8	1.5
4	6	30	0.9
6	7	2.4	0.022
7	8	1	0.025
8	9	13	0.015
9	10	60	100
10	11	75	220
11	13	370	1600
12	NA	ND	>10 000
13	NA	ND	78±13
14	NA	ND	283±66

^[a]Data from Ref. [23]. Unless stated otherwise, errors are <5 % of the reported value (from triplicate experiments); NA: not applicable; ND: not determined.

We recently replaced the placental microsome preparation with a JEG-3 cell preparation as the standard assay for screening the in vitro STS inhibitory activities of compounds. The advantage of using intact growing JEG-3 cells is that they allow testing of the compounds under conditions that closely resemble the tissue/physiological situation in which the drug must first cross the plasma membrane before it can reach the target (STS) enzyme. These human choriocarcinoma cells have abundant STS enzyme activity, are easy to grow, and are less expensive to use than purified enzyme or placental microsomes. We therefore re-tested the STS inhibitory activities of the tricyclic coumarin sulfamates in JEG-3 cells, and their IC₅₀ values are listed in Table 1. As expected for a cell-based assay, the IC₅₀ values against STS obtained for the series of compounds are much lower than those obtained from the cell-free placental microsome assay. However, the overall in vitro inhibitory profile observed is similar, with potency increasing as the size of the third aliphatic ring increases from 5 to 11 members, but then decreasing as the ring size increases further. The most potent compounds observed are 6–8, the IC₅₀ values of which are between 0.015 and 0.025 nm, whereas 11 is the weakest STS inhibitor in vitro. These results suggest that the ability of compounds to cross the cell membrane and then to interact with the active site of STS is optimal with compounds 6–8, when the aliphatic ring contains 8–10 carbon atoms. Unexpectedly, there is a dramatic decrease in potency observed when the size of the third ring increases from 11 to 12 members. There is a five orders of magnitude difference between the IC₅₀ values of 8 and 9.

To examine the possible interactions of tricyclic coumarin derivatives with amino acid residues within the active site of STS, these molecules were docked into the crystal structure of STS (PDB ID: 1P49).[24] Importantly, the poses discussed are assumed to be those that form immediately prior to the irreversible inactivation of the enzyme by sulfamoyl transfer. Although it is currently not known what residue is involved, these docking results would be predictive of inactivation of the gem-diol form of the formylglycine residue 75 (FG75) by sulfamoyl transfer. The docking results for 1, 7, and 9 are shown in Figure 3 a and those for 7 and 11 in Figure 3 b. In common with 1 and 7, as shown in Figure 3 a, the rest of the compounds in the series, apart from compound 11, bind with the sulfamate down by the catalytically crucial FG75 residue and the calcium ion. This leaves the third aliphatic ring residing in a predominantly hydrophobic pocket formed by R98, T99, L103, V177, F178, T180, G181, T484, H485, V486, F488, and F553. As the size of the third ring increases from 5 to 11 members (compounds 1–4 and 6–8), it gives a more favourable contact with these residues, with the first and second rings (the coumarin moiety) and the sulfamate occupying nearly identical positions. This may partly explain the increase in potency of these compounds in general as the third aliphatic ring increases in size. As shown in Figure 3 a, and exemplified by compounds 1 and 7, the carbonyl groups of these compounds are within hydrogen bonding distance from the backbone NH group of G100 (∼3 Å). This additional interaction may be a contributing factor that further assists the binding of these molecules to the enzyme active site. The docking pose of compound 9 (12-membered third ring) is different from that of its lower congeners. Presumably due to steric hindrance rendered by the bulk of its third ring, 9 binds with the coumarin ring rotated in the binding site (Figure 3 a). As a result, its carbonyl group is no longer positioned to form a hydrogen bond to G100. The same observations can be made for compound 10 (13-membered third ring), as it shows a docking pose similar to that of compound 9 (not shown). With compound 11, the 15-membered third ring is too large to fit in the binding site in the same orientation as it does for compounds 1–4 and 6–10. In contrast to its congeners, 11 binds upside down in the binding site (Figure 3 b) which is a much poorer binding pose. The GOLD docking scores for compounds 1–4 and 6–10 are all in the range of 52–57 which are not sufficiently different to allow any correlation to be made between their docking poses and IC₅₀ values. However, 11 has a significantly lower GOLD docking score of 38 which may reflect the much poorer IC₅₀ observed for this compound.

Figure 3.

The docking of a) 1 (orange), 7 (cyan), and 9 (pink); and b) 7 (cyan) and 11 (pink) into the crystal structure of human STS. The Ca²⁺ ion is depicted as a yellow sphere, and FG75 is the gem-diol form of FG75. Dotted line: potential hydrogen bond.

The N,N-dimethylation of 1 to give compound 12 renders the compound inactive in vitro as an STS inhibitor (Table 1). This supports previous findings that a free sulfamate group is a prerequisite for potent irreversible inhibition of STS in vitro. Hence, N-(piperidino),[25] N,N-(dibenzyl)sulfamate,[25] and N,N-dimethyl derivatives of oestrone 3-O-sulfamate (EMATE)[26] were found to be weak reversible or inactive inhibitors of STS in placental microsomes. Only N-acetylated EMATE, but not the benzoyl derivative, inhibits STS irreversibly, albeit much less potently than EMATE.[25] However, compound 12 was found to behave differently in vivo. When administered orally to nude mice, 12 inhibits liver STS activity potently at doses of 1 and 10 mg kg⁻¹.[27] Moreover, if 12 is applied topically at 1 and 10 mg kg⁻¹, it also inhibits skin as well as liver STS effectively.[27] This shows that 12 is able to be absorbed via the percutaneous route and could then inhibit STS in the liver and possibly in other tissues throughout the body. We reason that demethylation of 12 occurs enzymatically in vivo, releasing 1 which is then the agent that inhibits STS.

Keeping a free sulfamate group at the 3-position of 1 but introducing a methoxy group at the 2-position renders the resulting compound 13 a weaker STS inhibitor in JEG-3 cells (IC₅₀=78 nm for 13 versus 1.5 nm for 1, Table 1). A similar pattern was observed with 2-methoxyestrone 3-O-sulfamate (IC₅₀=30 nm), which was found to be a weaker STS inhibitor than EMATE (IC₅₀=4 nm) in a preparation of placental microsomes.[28] Having a bulkier aliphatic substituent positioned next to an aryl sulfamate has also been found to confer weaker inhibition of STS, presumably due to steric hindrance.[28]

The relocation of the sulfamate group in 1 from the 3- to the 2-position renders a significant decrease in STS inhibitory activity of the resulting compound 14 (Table 1). It is reasoned that the high inhibitory activity observed for 1 is due to its sulfamate group being in a position conjugated to the α,β-unsaturated lactone moiety of the coumarin ring. As a result, the parent phenol 1 a has a lower pK_a value and is hence a better leaving group than unsubstituted phenol. We postulate that this effect would more effectively facilitate the transfer of the sulfamoyl group of 1 to an essential amino acid residue in the STS active site and inactivate the enzyme as a result. Relocation of the sulfamate group from the 3- to the 2-position to give 14 would essentially disrupt this process, as the pK_a of the parent phenol 14 a is expected to be close to that of unsubstituted phenol. It is also possible that a sulfamate group placed at the 2-position might not be presented properly and effectively to essential amino acid residue(s) in the enzyme catalytic site responsible for its subsequent activation, resulting in less effective inactivation of the enzyme.

The coumarin moiety has been the core bicyclic template for the development of nonsteroidal STS inhibitors by our research group. Other phenols of bicyclic nonsteroidal moieties such as tetrahydronaphthalene;[26] flavones, isoflavones, flavanones;[29, 30] and chromenone and thiochromenone[31] have also been sulfamoylated and explored by us and other research groups for designing STS inhibitors with varying degrees of success. In this work, we studied the effects of replacing the coumarin ring system of 1 with either a quinolin-2(1H)-one or a quinoline moiety. Their respective N-alkylated and alkoxyl derivatives were also investigated for STS inhibitory activity. As shown in Table 2, all compounds inhibit STS weakly in JEG-3 cells. The best STS inhibitor is the unsubstituted quinolinone derivative 16 (IC₅₀=240 nm or 98 % inhibition at 10 μm), although it is 160-fold less potent than 1 (IC₅₀=1.5 nm, Table 1). This is closely followed by the quinoline derivative 19, which inhibits STS by 68 % at 10 μm, although the inhibition remains weak. These results further confirm that the coumarin ring is essential for the potent STS inhibitory activity observed for 1. This is attributed to several factors. With 16, 18, and 19 docked into the STS active site in a fashion similar to that of 7 (Figure 4), we postulate that electronic factors such as the pK_a values of parent phenols could play a significant role for the results observed. To explore this possible causative factor further, the pK_a values of 7-hydroxy-2H-chromen-2-one (25, represents 1 a, the parent phenol of 1), 7-hydroxyquinolin-2(1H)-one (26, represents 16 a, the parent phenol of 16), 7-hydroxy-1,4-dimethylquinolin-2(1H)-one (27, represents 18 b, the parent phenol of 18), and 7-methoxynaphthalen-2-ol (28, represents 19 c, the parent phenol of 19) as calculated by ACD/Labs software version 11.01 were compared (Figure 5). As shown, the pK_a value of 1 a is expected to be between 1 and 2 log units lower than those of 16 a, 18 b, and 19 c. This factor suggests that 1 a is a much better leaving group than 16 a, 18 b, and 19 c, rendering the sulfamate group of 1 a a much stronger sulfamoylating species for the inactivation of the enzyme, and hence 1 is a more potent STS inhibitor than the quinolinone and quinoline derivatives.

Table 2.

Inhibition of STS activity in JEG-3 cells by tricyclic quinolinone sulfamates 16, 18, 20, and 22, tricyclic quinoline sulfamates 19, 21, and 23, and the tricyclic quinolinone sulfamide 24

Compd	R	Inhibition [%][a,b]	IC50 [nm][b]
16	H	96	240±40
18	Me	ND	2400
19	Me	68	ND
20	(CH2)4CH3	<10	ND
21	(CH2)4CH3	<10	ND
22	(CH2)3Ph	20	ND
23	(CH2)3Ph	<10	ND
24	ND	ND	>103

^[a]Determined at 10 μm.

^[b]Unless stated otherwise, errors are <5 % of the reported value (from triplicate experiments); ND: not determined.

Figure 4.

The docking of 7 (cyan), 16 (pink), 18 (orange), and 19 (yellow) into the crystal structure of human STS. The Ca²⁺ ion is depicted as a yellow sphere, and FG75 is the gem-diol form of FG75. Dotted line: potential hydrogen bond.

Figure 5.

Calculated pK_a values of various bicyclic phenols 25–28, which represent the parent phenols of 1, 16, 18, and 19, respectively. The calculation was performed by ACD/Labs software version 11.01.

N-Methylation of 16 to give 18 (IC₅₀=2400 nm, Table 2) is detrimental to activity, as this substitution produces a 100-fold decrease in the IC₅₀ value observed for 18 against STS. For both quinolinone and quinoline series, further enlargement of the substituent from a methyl group to either an n-pentyl or a phenethyl group significantly abolishes the STS inhibitory activities of the resulting compounds. It is possible that these substituted molecules no longer bind effectively to the active site of STS due to steric hindrance caused by the bulk of the substituent.

Finally, replacement of the bridging oxygen atom of the sulfamate group in 16 with an NH moiety to give a sulfamido group abolishes the activity of the resulting compound 24 as an STS inhibitor. A similar finding was observed with oestrone 3-sulfamide.[19] We postulate that, unlike the sulfamate group of 16, an enzyme-catalysed breaking of the S–N bond of the sulfamido group of 24 is unlikely to take place because, among other things, the parent amine 24 a is a very poor leaving group. As a result, it is not anticipated that 24 would be able to inactivate STS to any degree by sulfamoylating the active site, but such an approach could provide leads for reversible STS inhibitors.

Conclusions

The nonsteroidal inhibitor Irosustat, STX64 (1) is the first agent to enter clinical trials for postmenopausal patients with advanced hormone-dependent breast cancer, and has shown encouraging results. In this work, we conducted a range of SAR studies on this drug. Expansion of the size of the aliphatic ring of 1 generally provides more potent derivatives against STS in JEG-3 cells, with best activities observed if the ring is between 9 and 11 members. However, further increasing the ring size is unfavourable, as inhibitory activities were observed to drop significantly. Molecular docking studies suggest that the aliphatic ring of 1 and its derivatives sit in a hydrophobic pocket within the enzyme active site with better contacts made with the enclosing amino acid residues as the ring size increases up to 11 members. Larger derivatives 9 and 10, and in particular 11, dock less well into the active site. Positioning of the sulfamate moiety close to the catalytic FG75 may be predictive of sulfamoyl transfer to this residue in the inactivation process. N,N-Dimethylation of the sulfamate group of 1 is detrimental to in vitro activity, as compound 12 is inactive. This supports previous findings which showed that a free sulfamate group (H₂NSO₂O⁻) is a prerequisite for potent and irreversible STS inhibition. Introducing a methoxy group at the 2-position of 1 significantly decreases the activity of the resulting 13, probably as a result of steric factors. A detrimental effect to activity is also observed with relocation of the sulfamate group of 1 from the 3- to the 2-position of the molecule. We postulate that the decrease in activity of compound 14 is due to its sulfamate group not being in a conjugated position to the α,β-unsaturated lactone moiety of the coumarin ring, which affects the ability of 14 to sulfamoylate and inactivate the enzyme. An azomethine adduct between 1 and the solvent DMF used in the sulfamoylation of 1 a was isolated. Its crystal structure shows that the stereochemistry is E at the double bond of its (dimethylamino)methylene sulfamoyl group. Replacing the coumarin ring system of 1 to give a series of quinolin-2(1 H)-one and quinoline derivatives produces essentially weak inhibitors of STS. Only the lowest members of the series inhibit STS. This confirms the unique property of the coumarin system in the design of nonsteroidal STS inhibitors that are structurally related to 1.

In summary, most of the modifications made to the clinical drug 1 decrease potency in vitro. Only a moderate enlargement of its aliphatic ring results in derivatives that are more potent STS inhibitors in vitro. However, it remains to be explored whether such compounds would show significant advantages over 1 if put through pre-clinical trial development.

Experimental Section

In vitro sulfatase assay: Biological assays were performed essentially as described previously.[32] The extent of in vitro inhibition of STS activities was assessed by using intact monolayers of JEG-3 human choriocarcinoma cells. STS activity was measured with [6,7-³H]E1S (50 Ci mmol⁻¹, PerkinElmer Life Sciences) over a 1 h period.

Molecular modelling: All ligands were built and minimised using Schrödinger software running under Maestro version 9.0. The crystal structure of human placental oestrone/DHEA sulfatase (PDB ID: 1P49)[24] was used for building the gem-diol form of STS. This involved a point mutation of the ALS75 residue in the crystal structure to the gem-diol form of the structure using editing tools within the Schrödinger software. The resulting structure was then minimised with the backbone atoms fixed to allow the gem-diol and surrounding side chain atoms to adopt low-energy confirmations. GOLD was used to dock the ligands 25 times each into the rigid protein, with the binding site being defined as a 10 Å sphere around the ALS75 sulfate. The docked poses were scored using the GOLDScore fitness function.

General methods for synthesis: All chemicals were purchased from either Aldrich Chemical Co. (Gillingham, UK) or Alfa Aesar (Heysham, UK). All organic solvents of analytical reagent grade were supplied by Fisher Scientific (Loughborough, UK). Anhydrous N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), and tetrahydrofuran (THF) were purchased from Aldrich. Sulfamoyl chloride was prepared by an adaptation of the method of Appel and Berger[33] and was stored as a solution under N₂ in toluene as described by Woo et al.[19]

Thin-layer chromatography (TLC) was performed on pre-coated plates (Merck TLC aluminium sheets silica gel 60 F₂₅₄, Art. No. 5554). Product(s) and starting material were detected by viewing under UV light and/or treating with a methanolic solution of phosphomolybdic acid followed by heating. Flash column chromatography was performed using gradient elution (solvents indicated in the text) on wet-packed silica gel (Sorbsil C₆₀). IR spectra were determined with a PerkinElmer 782 infrared spectrophotometer, and peak positions are expressed in cm⁻¹. ¹H and ¹³C NMR spectra were recorded with either a Jeol Delta 270 MHz or a Varian Mercury VX 400 MHz spectrometer. Chemical shifts (δ) are reported in parts per million (ppm) relative to tetramethylsilane (TMS) as an internal standard. Coupling constants (J) are recorded to the nearest 0.1 Hz. Mass spectra were recorded at the Mass Spectrometry Service Centre, University of Bath. FAB mass spectra were measured using m-nitrobenzyl alcohol as the matrix. Elemental analyses were performed by the Microanalysis Service, University of Bath. Melting points were determined using a Reichert–Jung Thermo Galen Kofler block and are uncorrected. HPLC was undertaken using a Waters 717 instrument equipped with an autosampler and PDA detector. The column used, conditions of elution, and purity of sample are as indicated for each compound analysed.

Crystallographic data: CCDC 826524 (1) and 826525 (15) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif

Ethyl 2-oxocyclononanecarboxylate (6 a). A solution of cyclononanone (3.0 g, 21 mmol) in diethyl carbonate (20 mL) was added dropwise to a suspension of NaH (60 % dispersion in mineral oil, 1.71 g, 42.8 mmol) and diethyl carbonate (80 mL) under N₂ over a period of 30 min. When the evolution of H₂ had ceased (∼15 h), aqueous HCl (1 m, 100 mL) was added in portions, and the resulting mixture was extracted with Et₂O (3×100 mL). The combined ethereal extracts were dried (MgSO₄) and evaporated to give a yellow oily residue, which was purified by distillation under reduced pressure to give 6 a as a clear oil (4.15 g, 91 %): R_f=0.72 (CHCl₃); bp₃: 146–150 °C; (Lit. [34] bp₂: 108–110 °C); ¹H NMR (400 MHz, CDCl₃): δ=1.23 (t, J=7.2 Hz, 1.8 H, keto CH₂CH₃), 1.30 (t, J=7.2 Hz, 1.2 H, enol CH₂CH₃), 1.37–2.66 (m, 14 H), 3.62 (m, 0.6 H, keto CHC=O), 4.14 (q, J=7.2 Hz, 1.2 H, keto CH₂CH₃), 4.21 (q, J=7.2 Hz, 0.8 H, enol CH₂CH₃), and 12.76 ppm (s, 0.4 H, ex. with D₂O, enol OH); MS (FAB⁺): m/z (%): 213.0 (100) [M+H]⁺; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₁₂H₂₁O₃: 213.1491, found: 213.1499.

3-Hydroxy-8,9,10,11,12,13-hexahydrocyclonona[c]chromen-6(7H)-one (6 b). Resorcinol (1.56 g, 14.1 mmol) was first dissolved in hot 6 a (3.0 g, 14 mmol). Upon cooling to room temperature, the resulting syrup at 0 °C was treated dropwise with a mixture of CF₃COOH (2.2 mL, 28 mmol) and concd H₂SO₄ (1.5 mL, 28 mmol) while keeping the reaction temperature <10 °C. After stirring for 3 h at room temperature, the orange gluey mass was cautiously quenched with ice-water. The orange precipitate that formed was collected by suction filtration, washed exhaustively with water and air dried. A solution of the precipitate in a minimal volume of acetone was fractionated by flash chromatography (CHCl₃/acetone, 8:1 → 4:1 gradient). The main fraction collected gave a white solid which was recrystallised from THF/hexane to give 6 b as white fine crystals (909 mg, 25 %): R_f=0.82 (CHCl₃/acetone, 3:1); mp: 197–200 °C; ¹H NMR (400 MHz, CDCl₃): δ=1.29–2.51 (m, 10 H, 5×CH₂), 2.66 (t, J=5.8 Hz, 2 H, C7-CH₂), 2.93 (t, J=6.1 Hz, 2 H, C13-CH₂), 6.69 (d, J=2.4 Hz, 1 H, C4-H), 6.78 (dd, J=2.4 and 8.8 Hz, 1 H, C2-H), 7.59 (d, J=8.8 Hz, 1 H, C1-H) and 10.39 ppm (s, 1 H, OH); MS (FAB⁺): m/z (%): 259.1 (100) [M+H]⁺; MS (FAB⁻): m/z (%): 257.1 (100), [M-H]⁻; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₁₆H₁₉O₃: 259.1334, found: 259.1323; Anal. calcd for C₁₆H₁₈O₃: C 74.40, H 7.02, found: C 74.10, H 6.91; HPLC: Waters Radialpak column (RP₁₈, 8×100 mm), MeOH/H₂O (70:30), flow rate=2 mL min⁻¹, λ_max=323.2 nm, t_R=6.5 min, purity >98 %.

6-Oxo-6,7,8,9,10,11,12,13-octahydrocyclonona[c]chromen-3-yl sulfamate (6). NaH (60 % dispersion in mineral oil, 1 equiv) was added to a solution of 6 b (400 mg, 1.55 mmol) in anhydrous DMF (20 mL) at 0 °C under N₂. When the evolution of H₂ had ceased, sulfamoyl chloride (∼0.69 m in toluene,[19] ∼3–5 equiv, evaporated down to ∼1 mL prior to addition) was introduced in one portion. After stirring at room temperature under N₂ overnight, the reaction mixture was quenched with ice-water. Upon addition of EtOAc (∼100 mL), the organic fractions were washed with brine (4×100 mL). After drying (MgSO₄), filtering and evaporating the washed organic layer, a crude white solid was obtained which was purified by flash chromatography (CHCl₃/EtOAc, 8:1 → 2:1 gradient). The main fraction isolated gave a white solid which was recrystallised from THF/hexane to give 6 as fine white crystals (201 mg, 38 %): R_f=0.46 (CHCl₃/EtOAc, 4:1); mp: 167–168 °C; ¹H NMR (400 MHz, CDCl₃): δ=0.84–1.74 (m, 10 H, 5×CH₂), 1.52 (t, J=5.8 Hz, 2 H, C7-CH₂), 1.57 (t, J=6.1 Hz, 2 H, C13-CH₂), 7.26 (dd, J=2.4 and 8.8 Hz, 1 H, C2-H), 7.31 (d, J=2.1 Hz, 1 H, C4-H), 7.89 (d, J=8.8 Hz, 1 H, C1-H) and 8.20 ppm (s, 2 H, NH₂); MS (FAB⁺): m/z (%): 338.0 (100) [M+H]⁺; MS (FAB⁻): m/z (%): 336.1 (100) [M−H]⁻, 257.1 (30) [M−H₂NSO₂]⁻; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₁₆H₂₀NO₅S: 338.1062, found: 338.1061; Anal. calcd for C₁₆H₁₉NO₅S: C 56.96, H 5.68, N 4.15, found: C 56.85, H 5.58, N 4.00; HPLC: Waters Radialpak column (RP₁₈, 8×100 mm), MeOH/H₂O (70:30), flow rate=2 mL min⁻¹, λ_max=284 and 312.5 nm, t_R=3.1 min, purity >98 %.

Ethyl 2-oxocyclodecanecarboxylate (7 a). Prepared in a similar manner to 6 a using NaH (1.3 g, 32 mmol), diethyl carbonate (60 mL), and cyclodecanone (2.5 g, 16 mmol). The crude pale-yellow oily residue was purified by distillation under reduced pressure to give 7 a as a colourless oil (2.81 g, 76 %): R_f=0.81 (CHCl₃); bp_0.23: 84–87 °C (Lit. [34] bp₁: 118–120 °C); ¹H NMR (400 MHz, CDCl₃): δ=1.24 (t, J=7.0 Hz, 1.2 H, keto CH₂CH₃), 1.31 (t, J=7.0 Hz, 1.8 H, enol CH₂CH₃), 1.34–2.76 (m, 16 H), 3.82–3.85 (m, 0.7 H, keto CHC=O), 4.13 (q, J=7.0 Hz, 0.5 H, keto CH₂CH₃), 4.22 (q, J=7.0 Hz, 1.5 H, enol CH₂CH₃) and 12.98 ppm (s, 0.3 H, ex. with D₂O, enol OH); MS (FAB⁺): m/z (%): 227.0 (100) [M+H]⁺; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₁₃H₂₃O₃: 227.1647, found: 227.1644.

3-Hydroxy-7,8,9,10,11,12,13,14-octahydro-6H-cyclodeca[c]chromen-6-one (7 b). Prepared in a similar manner to 6 b using resorcinol (970 mg, 8.84 mmol), 7 a (2.0 g, 8.8 mmol), and a mixture of CF₃COOH (1.5 mL, 18 mmol) and concd H₂SO₄ (1.0 mL, 18 mmol). The crude dark-orange solid was purified by flash chromatography (CHCl₃/acetone, 8:1 → 4:1 gradient), and the white solid that was isolated was recrystallised from THF/hexane to give 7 b as white crystals (789 mg, 33 %): R_f:=0.72 (CHCl₃/acetone, 3:1); mp: 240–241 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ=0.88–2.18 (m, 12 H, 6×CH₂), 2.83 (t, J=6.7 Hz, 2 H, C7-CH₂), 3.02 (t, J=6.7 Hz, 2 H, C14-CH₂), 5.96 (s, 1 H, OH), 6.78 (dd, J=2.7 and 8.5 Hz, 1 H, C2-H), 6.83 (d, J=2.7 Hz, 1 H, C4-H) and 7.53 ppm (d, J=8.5 Hz, 1 H, C1-H); MS (FAB⁺) m/z (%): 273.1 (100) [M+H]⁺; MS (FAB⁻): m/z (%): 271.1 (100) [M−H]⁻; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₁₇H₂₁O₃: 273.1491, found: 273.1488; Anal. calcd for C₁₇H₂₀O₃: C 74.94, H 7.40, found: C 74.30, H 7.43; HPLC: Waters Radialpak column, MeOH/H₂O (80:20), flow rate=2 mL min⁻¹, λ_max=322 nm, t_R=4.5 min, purity >95 %.

6-Oxo-7,8,9,10,11,12,13,14-octahydro-6H-cyclodeca[c]chromen-3-yl sulfamate (7). Compound 7 b (400 mg, 1.47 mmol) was sulfamoylated in a similar manner to 6 b and the crude white solid obtained was purified by flash chromatography (CHCl₃/EtOAc, 8:1 → 2:1 gradient). The white solid that was isolated was recrystallised from THF/hexane to give 7 as fine white crystals (235 mg, 46 %): R_f=0.71 (CHCl₃/EtOAc, 4:1); mp: 183–185 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ=1.17–3.0 (m, 14 H, 7×CH₂), 3.09 (t, J=6.4 Hz, 2 H, C14-CH₂), 7.26 (m, 1 H, C2-CH), 7.46 (d, J=1.2 Hz, 1 H, C4-H), 7.93 (d, J=8.8 Hz, 1 H, C1-H) and 8.21 ppm (s, 2 H, NH₂); MS (FAB⁺): m/z (%): 352.0 (100) [M+H]⁺; MS (FAB⁻): m/z (%): 350.1 (100) [M−H]⁻, 271.1 (100) [M−H₂NSO₂]⁻; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₁₇H₂₂NO₅S: 352.1219, found: 352.1223; Anal. calcd for C₁₇H₂₁NO₅S: C 58.10, H 6.02, N 3.99 %, found: C 58.40, H 6.28, N, 2.63; HPLC: Waters Radialpak column, MeOH/H₂O (70:30), flow rate=2 mL min⁻¹, λ_max=284 and 312.5 nm, t_R=6.3 min, purity >98 %.

Ethyl 2-oxocycloundecanecarboxylate (8 a). Prepared in a similar manner to 6 a using NaH (1.19 g, 29.7 mmol), diethyl carbonate (70 mL), and cycloundecanone (2.5 g, 15 mmol). The crude yellow oily residue was purified by distillation under reduced pressure to give 6 a as a pale-yellow oil (2.07 g, 58 %): R_f=0.31 (CH₂Cl₂); bp_0.15: 103–108 °C (Lit. [34] bp₅: 140–143 °C); ¹H NMR (400 MHz, CDCl₃) δ=1.20 (t, J=7.0 Hz, 3 H, CH₂CH₃), 1.26–2.76 (m, 19 H) and 4.10 ppm (q, J=7.3 Hz, 2 H, CH₃CH₂); MS (FAB⁺): m/z (%): 241.1 (100) [M+H]⁺; MS (FAB⁻): m/z (%): 239.0 (100) [M−H]⁻; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₁₄H₂₅O₃: 241.1804, found: 241.1806.

3-Hydroxy-8,9,10,11,12,13,14,15-octahydrocycloundeca[c]chromen-6(7H)-one (8 b). Prepared in a similar manner to 6 b using resorcinol (917 mg, 8.33 mmol), 8 a (2.0 g, 8.3 mmol) and a mixture of CF₃COOH (2.0 mL, 17 mmol) and concd H₂SO₄ (1.6 mL, 17 mmol). The crude yellow solid was purified by flash chromatography (CHCl₃/acetone, 8:1 → 4:1 gradient) and the yellow solid that was isolated was recrystallised from THF/hexane to give 8 b as fine pale-yellow crystals (344 mg, 14 %): R_f=0.76 (CHCl₃/acetone, 3:1); mp: 214–215 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ=1.26–1.68 (m, 14 H, 7×CH₂), 2.56 (t, J=7.0 Hz, 2 H, C7-CH₂), 2.84 (t, J=7.0 Hz, 2 H, C15-CH₂), 6.67 (d, J=2.1 Hz, 1 H, C4-H), 6.78 (dd, J=2.1 and 8.7 Hz, 1 H, C2-H), 7.63 (d, J=8.8 Hz, 1 H, C1-H) and 10.42 ppm (s, 1 H, OH); MS (FAB⁺): m/z (%) 287.1 (100) [M+H]⁺; MS (FAB⁻): m/z (%): 285.1 (100) [M−H]⁻; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₁₈H₂₃O₃: 287.1647, found: 287.1644; Anal. calcd for C₁₈H₂₂O₃: C 75.50, H 7.74, found: C 75.50, H 7.75; HPLC: Waters Radialpak column, MeOH/H₂O (80:20), flow rate=2 mL min⁻¹, λ_max=323.2 nm, t_R=6.5 min, purity >98 %.

6-Oxo-6,7,8,9,10,11,12,13,14,15-decahydrocycloundeca[c]chromen-3-yl sulfamate (8). Compound 8 b (300 mg, 1.05 mmol) was sulfamoylated in a similar manner to 6 b and the crude white solid obtained was purified by flash chromatography (CHCl₃/EtOAc, 8:1 → 2:1 gradient). The white solid that was isolated was recrystallised from THF/hexane to give 8 as fine white crystals (133 mg, 35 %): R_f=0.37 (CHCl₃/EtOAc, 4:1); mp: 145–148 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ=1.28–1.76 (m, 14 H, 7×CH₂), 2.64 (t, J=7.0 Hz, 2 H, C7-CH₂), 2.93 (t, J=7.0 Hz, 2 H, C15-CH₂), 7.26 (dd, J=2.1 and 8.8 Hz, 1 H, C2-H), 7.29 (d, J=2.1 Hz, 1 H, C4-H), 7.93 (d, J=8.8 Hz, 1 H, C1-H) and 8.20 ppm (s, 2 H, NH₂); MS (FAB⁺): m/z (%) 731.2 (10) [2 M+H]⁺, 366.0 (100) [M+H]⁺; MS (FAB⁻): m/z (%): 364.1 (100) [M−H]⁻, 285.2 (40) [M−H₂NSO₂]⁻; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₁₈H₂₄NO₅S: 366.1375, found: 366.1368; Anal. calcd for C₁₈H₂₃NO₅S: C 59.16, H 6.34, found: C 59.20, H 6.57; HPLC: Waters Radialpak column, MeOH/H₂O (80:20), flow rate=2 mL min⁻¹, λ_max=285.2 and 312.5 nm, t_R=3.8 min, purity >98 %.

Ethyl 2-oxocyclododecanecarboxylate (9 a). Prepared in a similar manner to 6 a using NaH (2.19 g, 54.9 mmol), diethyl carbonate (100 mL) and cyclododecanone (5.0 g, 27 mmol). The crude dark-yellow oily residue was purified by distillation under reduced pressure to give 9 a as a pale-yellow oil (5.62 g, 81 %): R_f=0.72 (CH₂Cl₂); bp_0.23: 128–132 °C (Lit. [34] bp₃: 155–157 °C); ¹H NMR (400 MHz, CDCl₃): δ=1.25 (t, J=7.0 Hz, 3 H, CH₂CH₃), 1.29–2.73 (m, 21 H) and 4.16 ppm (q, J=6.4 Hz, 2 H, CH₃CH₂); MS (FAB⁺): m/z (%): 255.1 (100) [M+H]⁺; MS (FAB⁻): m/z (%): 253.2 (100) [M−H)⁻]; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₁₅H₂₇O₃: 255.1960, found: 255.1968.

3-Hydroxy-7,8,9,10,11,12,13,14,15,16-decahydro-6H-cyclododeca[c]chromen-6-one (9 b). Prepared in a similar manner to 6 b using resorcinol (1.08 g, 9.8 mmol), 9 a (2.5 g, 9.8 mmol) and a mixture of CF₃COOH (1.5 mL, 20 mmol) and concd H₂SO₄ (1.0 mL, 20 mmol). The crude pale-yellow solid was purified by flash chromatography (CHCl₃/acetone, 8:1 → 4:1 gradient) and the pale-yellow solid that was isolated was recrystallised from THF/hexane to give 9 b as white crystals (972 mg, 33 %): R_f=0.76 (CHCl₃/acetone, 3:1); mp: 249–251 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ=1.39–2.89 (m, 16 H, 8×CH₂), 2.93 (t, J=7.3 Hz, 2 H, C7-CH₂), 3.22 (t, J=7.3 Hz, 2 H, C16-CH₂), 6.66 (d, J=2.3 Hz, 1 H, C4-H), 6.78 (dd, J=2.3 and 8.9 Hz, 1 H, C2-H), 7.63 (d, J=8.9 Hz, 1 H, C1-H) and 10.77 ppm (s, 1 H, OH); MS (FAB⁺): m/z (%): 301.1 (100) [M+H]⁺; MS (FAB⁻): m/z (%): 299.1 (100) [M−H]⁻; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₁₉H₂₅O₃: 301.1804, found: 301.1806; Anal. calcd for C₁₉H₂₄O₃: C 75.97, H 8.05, found: C 75.90, H 8.03; HPLC: Waters Radialpak column, MeOH/H₂O (90:10), flow rate=2 mL min⁻¹, λ_max=324.4 nm, t_R=4.2 min, purity >98 %.

6-Oxo-7,8,9,10,11,12,13,14,15,16-decahydro-6H-cyclododeca[c]chromen-3-yl sulfamate (9). Compound 9 b (400 mg, 1.33 mmol) was sulfamoylated in a similar manner to 6 b and the crude white solid obtained was purified by flash chromatography (CHCl₃/EtOAc, 8:1 → 2:1 gradient). The white solid that was isolated was recrystallised from THF/hexane to give 9 as fine white crystals (182 mg, 36 %): R_f=0.47 (CHCl₃/EtOAc, 4:1); mp: 173–175 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ=1.41–2.51 (m, 16 H, 8×CH₂), 2.62 (t, J=7.3 Hz, 2 H, C7-CH₂), 3.68 (t, J=7.6 Hz, 2 H, C16-CH₂), 7.26 (dd, J=2.4 and 8.5 Hz, 1 H, C2-H), 7.28 (d, J=2.4 Hz, 1 H, C4-H), 7.94 (d, J=8.5 Hz, 1 H, C1-H) and 8.20 ppm (s, 2 H, NH₂); MS (FAB⁺): m/z (%): 380.1 (100) [M+H]⁺, 301.1 (15) [M+H−HNSO₂]⁺; MS (FAB⁻): m/z (%): 378.1 (100) [M−H]⁻, 299.1 (50) [M−H₂NSO₂]⁻; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₁₉H₂₆NO₅S: 380.1532, found: 380.1541; Anal. calcd for C₁₉H₂₅NO₅S: C 60.14, H 6.64, N 3.69, found C 60.30, H 6.85, N 3.62; HPLC: Waters Radialpak column, MeOH/H₂O (90:10), flow rate=2 mL min⁻¹, λ_max=285.2 and 312.5 nm, t_R=5.3 min, purity >98 %.

Ethyl 2-oxocyclotridecanecarboxylate (10 a). Cyclic β-keto ester 10 a was obtained commercially.

3-Hydroxy-8,9,10,11,12,13,14,15,16,17-decahydrocyclotrideca[c]chromen-6(7H)-one (10 b). Prepared in a similar manner to 6 b using resorcinol (451 mg, 4.10 mmol), 10 a (1.0 g, 3.73 mmol) and a mixture of CF₃COOH (0.64 mL, 8.20 mmol) and concd H₂SO₄ (0.83 mL, 8.20 mmol). The light-beige residue (1.14 g) that was obtained was recrystallised from hot iPrOH to give 10 b as soft yellow crystals (300 mg, 25.6 %): R_f=0.51 (CHCl₃/EtOAc, 4:1); mp: 234–238 °C; ¹H NMR (400 MHz, CDCl₃) δ=1.1–1.8 (18 H), 2.39 (m, 2 H, C7-CH₂), 2.65 (m, 2 H, C17-CH₂), 6.70 (d, J=2.4 Hz, 1 H, C4-H), 6.81 (dd, J=2.4 and 8.8 Hz, 1 H, C2-H), 7.61 (d, J=8.8 Hz, 1 H, C1-H) and 10.38 ppm (s, 1 H, ex. with D₂O, OH); MS (FAB⁺): m/z (%): 315.3 (100) [M+H]⁺; MS (FAB⁻): m/z (%): 467.4 (35) [M−H+NBA]⁻, 313.4 (100) [M−H]⁻; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₂₀H₂₇O₃: 315.1960, found: 315.1975; Anal. calcd for C₂₀H₂₆O₃: C 76.39, H 8.34, found: C 76.1, H 8.41. The mother liquor of the crystals obtained above was fractionated by flash chromatography (CHCl₃/EtOAc, 8:1 → 2:1 gradient) to yield another 150 mg of 10 b as white residue.

6-Oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydrocyclotrideca[c]chromen-3-yl sulfamate (10). Compound 10 b (370 mg, 1.18 mmol) was sulfamoylated in a similar manner to 6 b and the crude light-yellow residue obtained (407 mg) on dissolving in a minimal volume of acetone was fractionated by flash chromatography (CHCl₃/acetone, 12:1 → 2:1 gradient). The second fraction that was isolated gave a white residue (140 mg, 20 %) which was recrystallised from THF/hexane to give 10 as white crystals (74 mg): R_f=0.34 (CHCl₃/acetone, 8:1); mp: 170–174 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ=1.2–1.7 (18 H), 2.47 (m, 2 H, C17-CH₂), 7.26 (dd, J=2.3 and 8.7 Hz, 1 H, C2-H), 7.30 (d, J=2.3 Hz, 1 H, C4-H), 7.92 (d, J=8.7 Hz, 1 H, C1-H) and 8.20 ppm (s, 2 H, ex. with D₂O, NH₂); MS (FAB⁺): m/z (%): 787.1 (6) [2 M+H]⁺, 394.0 (100) [M+H]⁺; MS (FAB⁻): m/z (%): 785.2 (12) [2 M−H]⁻, 392.1 (100) [M−H]⁻, 313.2 (50) [M−H₂NSO₂]⁻; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₂₂H₂₈NO₅S: 394.1668, found, 394.1712; Anal. calcd for C₂₀H₂₇NO₅S: C 61.04, H 6.92, N 3.56, found C 61.4, H 7.22, N 3.27.

Ethyl 2-oxocyclopentadecanecarboxylate (11 a). Prepared in a similar manner to 6 a using NaH (891 mg, 22.3 mmol), diethyl carbonate (70 mL) and cyclopentadecanone (2.5 g, 11.2 mmol). The crude yellow syrup obtained was purified by flash chromatography (CH₂Cl₂) to give 11 a as a pale-yellow oil (1.62 g, 49 %): R_f=0.70 (CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃): δ=1.64 (t, J=6.7 Hz, 3 H, CH₂CH₃), 1.15–1.62 (m, 25 H), 2.55 (t, J=7.0 Hz, 2 H, ring C2-CH₂) and 4.16 ppm (q, J=7.3 Hz, 2 H, CH₂CH₃); MS (FAB⁺): m/z (%): 297.2 (100) [M+H]⁺; MS (FAB⁻): m/z (%): 295.2 (100) [M−H]⁻; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₁₈H₃₃O₃: 297.2429, found: 297.2430.

3-Hydroxy-8,9,10,11,12,13,14,15,16,17,18,19-dodecahydrocyclopentadeca[c]chromen-6(7H)-one (11 b). Prepared in a similar manner to 6 b using resorcinol (558 mg, 5.06 mmol), 11 a (1.5 g, 5.1 mmol) and a mixture of CF₃COOH (1.0 mL, 10 mmol) and concd H₂SO₄ (1.0 mL, 10 mmol). The crude brown solid obtained was purified by flash chromatography (CHCl₃/acetone, 8:1 → 4:1 gradient) and the yellow solid that was isolated was recrystallised from THF/hexane to give 11 b as pale-yellow crystals (432 mg, 25 %): R_f=0.69 (CHCl₃/acetone, 3:1); mp: 209–211 °C; ¹H NMR (400 MHz, CDCl₃): δ=1.25–1.62 (m, 22 H, 11×CH₂), 2.57 (t, J=7.8 Hz, 2 H, C7-CH₂), 2.74 (t, J=7.0 Hz, 2 H, C19-CH₂), 6.04 (s, 1 H, OH), 6.81 (dd, J=2.7 and 8.9 Hz, 1 H, C2-H), 6.92 (d, J=2.7 Hz, 1 H, C4-H) and 7.45 ppm (d, J=8.9 Hz, 1 H, C1-H); MS (FAB⁺): m/z (%): 343.1 (100) [M+H]⁺; MS (FAB⁻): m/z (%): 341.2 (100) [M−H]⁻; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₂₂H₃₁O₃: 343.2273, found: 343.2269; Anal. calcd for C₂₂H₃₀O₃: C 77.16, H, 8.83, found C 77.12, H 8.89; HPLC: Waters Radialpak column, MeOH/H₂O (90:10), flow rate=2 mL min⁻¹, λ_max=324.4 nm, t_R=8.5 min, purity >98 %.

6-Oxo-6,7,8,9,10,11,12,13,14,15,16,17,18,19-tetradecahydrocyclopentadeca[c]chromen-3-yl sulfamate (11). Compound 11 b (350 mg, 1.02 mmol) was sulfamoylated in a similar manner to 6 b and the crude white solid obtained was purified by flash chromatography (CHCl₃/EtOAc, 8:1 → 2:1 gradient) to give a thick waxy solid that was difficult to recrystallise. Further purification by preparative TLC (CHCl₃/EtOAc, 4:1) gave a white solid (201 mg), that was recrystallised from THF/hexane to give 11 as fine white flakes (185 mg, 43 %): R_f=0.50 (CHCl₃/EtOAc, 4:1); mp: 163–166 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ=1.32–1.59 (m, 22 H, 11×CH₂), 2.51–2.81 (m, 4 H, C7-CH₂ and C19-CH₂), 7.26–7.28 (m, 2 H, C2-H and C4-H), 7.89 (d, J=7.8 Hz, 1 H, C1-H) and 8.19 ppm (s, 2 H, NH₂); MS (FAB⁺): m/z (%): 842.3 (70) [2 M+H]⁺, 422.1 (100) [M+H]⁺; MS (FAB⁻): m/z (%): 841.4 (80) [2 M−H]⁻, 420.2 (100) [M−H]⁻, 341.2 (60) [M−H₂NSO₂]⁻; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₂₂H₃₂NO₅S: 422.1999, found: 422.1994; Anal. calcd for C₂₂H₃₁NO₅S: C 62.68, H 7.41, N 3.32, found: C 62.80, H 7.56, N 3.00; HPLC: Waters Radialpak column, MeOH/H₂O (90:10), flow rate=2 mL min⁻¹, λ_max=285.2 and 313.7 nm, t_R=4.2 min, purity >98 %.

6-Oxo-6,7,8,9,10,11-hexahydrocyclohepta[c]chromen-3-yl dimethylsulfamate (12). N,N-Dimethylsulfonyl chloride (1.90 mL, 17.55 mmol) was added dropwise to a mixture of 3-hydroxy-8,9,10,11-tetrahydrocyclohepta[c]chromen-6(7H)-one (2.0 g, 8.69 mmol)[17] in N,N-dimethylcyclohexylamine (10 mL). The resulting mixture was heated at 90 °C for 1 h. The brown slurry obtained was cooled to room temperature and diluted with EtOAc (150 mL). The organic fraction was then washed sequentially with NaOH (1 m, 2×100 mL), HCl (2 m, 2×100 mL) and brine (3×50 mL); it was dried (MgSO₄) and evaporated to give a light-yellow residue (2.94 g). Recrystallisation from hot EtOAc/hexane (2.5:1) gave 12 as a light-yellow crystalline solid (2.0 g, 5.93 mmol, 68 %): mp: 159–160.5 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ=1.52 (m, 2 H), 1.61 (m, 2 H), 1.86 (m, 2 H), 2.81 (m, 2 H), 2.94 (s, 6 H, 2×N-CH₃), 3.00 (m, 2 H, C11-H₂), 7.32 (dd, J=2 and 8.9 Hz, 1 H, C2-H), 7.39 (d, J=2.73 Hz, 1 H, C4-H) and 8.01 ppm (d, J=8.9 Hz, 1 H, C1-H); MS (AP⁺): m/z (%): 338.2 (100) [M+H]⁺; Anal. calcd for C₁₆H₁₉NO₅S: C 56.96, H 5.68, N, 4.15, found: C 57.0, H 5.71, N 4.32.

4-Methoxybenzene-1,3-diol (13 a). Starting material 13 a was prepared according to the method of Godfrey et al.[20]

3-Hydroxy-2-methoxy-8,9,10,11-tetrahydrocyclohepta[c]chromen-6(7H)-one (13 b). A mixture of 13 a (1.05 g, 7.49 mmol) and methyl 2-oxo-1-cycloheptane carboxylate (1.35 g, 7.87 mmol) at 0 °C was treated dropwise with a mixture of CF₃COOH (1.2 mL, 15 mmol) and concd H₂SO₄ (1.5 mL, 15 mmol) while keeping the reaction temperature <10 °C. After stirring for 3 h at room temperature, the dark-brown mixture was cautiously quenched with ice-water followed by the addition of EtOAc (200 mL). The organic layer that separated was washed with H₂O (4×100 mL) and dried by azeotropic evaporation with iPrOH. The dark-purple residue obtained (2.0 g) was recrystallised from hot EtOAc and hexane to give 13 b as pink crystals (1.23 g, 25 %): mp: 158–159 °C. Upon fractionation of the residue retrieved from the mother liquor by flash chromatography (EtOAc/hexane, 1:4 → 4:1 gradient), the second fraction that was isolated gave a yellow residue (321 mg) that was recrystallised from hot EtOAc and hexane to give a second crop of 13 b (184 mg, total 72 %) as creamy crystals: R_f=0.46 (EtOAc/hexane, 2:1); mp: 158–159 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ=1.49 (m, 2 H), 1.60 (m, 2 H), 1.83 (m, 2 H), 2.76 (m, 2 H, C7-H₂), 2.96 (m, 2 H, C11-CH₂), 3.86 (s, 3 H, OCH₃), 6.76 (s, 1 H, C4-H), 7.25 (s, 1 H, C1-H) and 10.15 ppm (br s, 1 H, ex. with D₂O, OH); MS (FAB⁺): m/z (%) 259.1 (100) [M+H]⁺; MS (FAB⁻): m/z (%): 257.1 (100) [M−H]⁻; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₁₆H₁₉O₃: 259.1334, found: 259.1323; Anal. calcd for C₁₅H₁₆O₄: C 69.20, H 6.20, found: C 69.1, H 6.16.

2-Methoxy-6-oxo-6,7,8,9,10,11-hexahydrocyclohepta[c]chromen-3-yl sulfamate (13). Compound 13 b (500 mg, 1.92 mmol) in anhydrous DMF (10 mL) was sulfamoylated in a similar manner to 6 b. The crude pale-yellow residue obtained was purified by flash chromatography (CHCl₃/THF, 16:1 → 2:1 gradient). The second fraction isolated gave a white solid that was recrystallised from THF/hexane to give 13 as a white powder (204 mg, 31 %): mp: 193–195 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ=1.52 (m, 2 H), 1.62 (m, 2 H), 1.86 (m, 2 H), 2.83 (m, 2 H, C7-H₂), 3.04 (m, 2 H, C11-CH₂), 3.91 (s, 3 H, OCH₃), 7.37 (s, 1 H, C4-H), 7.48 (s, 1 H, C1-H) and 8.17 ppm (s, 2 H, ex. with D₂O, OSO₂NH₂); MS (ES⁺) m/z (%): 340.0 (100) [M+H]⁺; Anal. calcd for C₁₅H₁₇NO₆S: C 53.09, H 5.05, N 4.13, found C 53.1, H 5.06, N 4.01; HPLC: Sunfire C₁₈ reversed-phase column, 4.6×75 mm, 3.5 μm pore size, MeOH/H₂O (80:20), flow rate=0.8 mL min⁻¹, t_R=2.12 min, purity >98 %.

2-hydroxy-8,9,10,11-tetrahydrocyclohepta[c]chromen-6(7H)-one (14 a). Hydroquinone (3.56 g, 32.34 mmol) was dissolved in hot methyl 2-oxo-1-cycloheptanecarboxylate (5.0 g, 4.59 mL, 29.4 mmol). To this stirred brown suspension at ice-water temperature was added dropwise a mixture of CF₃COOH (5.0 mL, 64.68 mmol) and concd H₂SO₄ (6.47 mL, 64.68 mmol) at such a rate that the reaction temperature was kept <10 °C (∼30 min). The reaction mixture was then allowed to warm to room temperature and thereupon stirred for an additional 60 h before being quenched cautiously with ice-water. After stirring the suspension that formed for 1 h, the pale-cream precipitate was collected by suction filtration, washed exhaustively with H₂O and air dried. The crude product was purified by recrystallisation from acetone to give 14 a as colourless needles (0.19 g, 3 %): mp: 204–206 °C; ¹H NMR (270 MHz, [D₆]DMSO): δ=1.49 (m, 2 H), 1.69 (m, 2 H), 1.79 (m, 2 H), 2.68 (m, 2 H), 2.83 (m, 2 H), 7.16 (dd, J=2.95 and 8.9 Hz, 1 H), 7.30 (d, J=2.9 Hz, 1 H), 7.43 (d, J=8.9 Hz, 1 H), 9.88 ppm (s, 1 H, ex. with D₂O); ¹³C NMR (100 MHz, [D₆]DMSO): δ=21.65 (CH₂), 24.48 (CH₂), 26.11 (CH₂), 31.29 (CH₂), 34.01 (CH₂), 107.80 (CH), 119.32 (CH), 120.82, 122.41 (CH), 123.08, 148.92, 154.30, 168.69, 175.45 ppm; MS (FAB⁺): m/z (%): 231.1 (100) [M+H]⁺; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₁₄H₁₅O₃: 231.10212, found 231.10255; Anal. calcd for C₁₄H₁₅O₃: C 73.0, H 6.13, found: C 73.0, H 6.15.

6-oxo-6,7,8,9,10,11-hexahydrocyclohepta[c]chromen-2-yl sulfamate (14). To an ice-cooled solution of 14 a (100 mg, 0.43 mmol) in anhydrous DMA (5 mL) was added sulfamoyl chloride (0.7 m solution in toluene, 3.04 mL; the toluene was removed in vacuo [not allowing the temperature of the water bath to exceed 30 °C] prior to addition, 4.34 mmol) and the mixture stirred (under a positive flow of dry N₂) overnight. The mixture was diluted with EtOAc (25 mL), washed with H₂O (3×50 mL) and brine (50 mL) and concentrated in vacuo (not allowing the temperature of the water bath to exceed 30 °C). The product was precipitated with Et₂O/ n-hexane, washed with n-hexane, and vacuum dried to give 14 as an off-white amorphous powder (70 mg, 52 %): mp: 185–187 °C (dec); ¹H NMR (400 MHz, [D₆]DMSO): δ=1.50 (m, 2 H), 1.69 (m, 2 H), 1.81 (m, 2 H), 2.70 (m, 2 H), 2.88 (m, 2 H), 7.59 (dd, J=3.2 and 8.97 Hz, 1 H), 7.68 (d, J=8.97 Hz, 1 H), 7.86 (d, J=3.2 Hz, 1 H), 8.08 ppm (s, 2 H, ex. with D₂O); ¹³C NMR (100 MHz, [D₆]DMSO): δ=22.49 (CH₂), 25.29 (CH₂), 26.79 (CH₂), 32.05 (CH₂), 34.81 (CH₂), 118.54, 120.79, 122.58 (CH), 123.39 (CH), 128.90 (CH), 147.27, 153.73, 170.07, 175.55 ppm; MS (FAB⁺) m/z (%): 310.1 (100) [M+H]⁺; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₁₄H₁₆NSO₅: 310.0749, found: 310.0753; Anal. calcd for C₁₄H₁₆NSO₅: C 54.4, H 4.89, N 4.53, found: C 54.0, H 5.01, N 4.31; purity of sample (as calculated by ¹H NMR): 97.4 %

3-Hydroxy-8,9,10,11-tetrahydro-5H-cyclohepta[c]quinolin-6(7H)-one (16 a). A slurry of 3-aminophenol (2.0 g, 18.33 mmol) in methyl 2-oxo-1-cycloheptane carboxylate (3.12 g, 18.33 mmol) was heated at 150 °C for 8 h. After cooling, EtOAc (50 mL) was added to the crude dark-brown residue and the resulting suspension was triturated in an ultrasonic bath for 30 min followed by filtration. The precipitate that collected was washed with more EtOAc and air dried to give 16 a as pink/light-brown residue (3.05 g, 13.30 mmol, 73 %): mp: 290–300 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ=1.44 (m, 2 H), 1.55 (m, 2 H), 1.81 (m, 2 H), 2.81 (m, 2 H, C7-H₂), 2.93 (m, 2 H, C11-H₂), 6.62 (dd, J=2.1 and 8.7 Hz, 1 H, C2-H), 6.69 (d, J=2.1 Hz, 1 H, C4-H), 7.64 (d, J=8.7 Hz, 1 H, C1-H), 10.1 (s, 1 H, ex. with D₂O, OH) and 11.4 ppm (1 H, s, ex. with D₂O, NH); MS (FAB⁺) m/z (%) 230.3 (100) [M+H]⁺; MS (FAB⁻) m/z (%) 382.3 (45) [M+NBA]⁻, 228.3 (100) [M−H]⁻; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₁₄H₁₆NO₂: 230.1181, found: 230.1184. This crude product was used for the next reaction without further purification.

6-Oxo-6,7,8,9,10,11-hexahydro-5H-cyclohepta[c]quinolin-3-yl sulfamate (16). NaH (60 % in mineral oil, 53 mg, 1.31 mmol) was added to a solution of 16 a (300 mg, 1.30 mmol) in anhydrous DMF (5 mL) at 0 °C, followed by a concentrated solution of sulfamoyl chloride (∼0.69 m in toluene, ∼5 equiv) in one portion 15 min later after the evolution of H₂ had ceased. The reaction mixture was stirred at room temperature under an atmosphere of N₂ overnight before diluting with EtOAc (100 mL). The resulting mixture was washed with brine (4×50 mL), dried (MgSO₄) and concentrated in vacuo to give an off-white residue that was fractionated on silica with EtOAc. The first fraction that was collected gave an off-white syrup (284 mg), which upon crystallisation from EtOAc/hexane (5:1) gave 16 as white crystals (174 mg, 564 μmol, 43 %): mp: 180–185 °C; IR (KBr) =3420, 3300, 3200–3000, 2920, 2860, 1630, 1550, 1380, 1180 cm⁻¹; ¹H NMR (270 MHz, [D₆]DMSO): δ=1.46 (m, 2 H), 1.57 (m, 2 H), 1.85 (m, 2 H), 2.87 (m, 2 H, C7-H₂), 3.02 (m, 2 H, C11-H₂), 7.08 (dd, J=2.4 and 9 Hz, 1 H, C2-H), 7.23 (d, J=2.2 Hz, 1 H, C4-H), 7.94 (d, J=8.8 Hz, 1 H, C1-H), 8.10 (s, 2 H, ex. with D₂O, OSO₂NH₂) and 11.8 ppm (s, 1 H, ex. with D₂O, NH); MS (FAB⁺): m/z (%): 309.2 (100) [M+H]⁺, 230.2 (12) [M−H₂NSO₂]⁺; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₁₄H₁₇N₂O₄S: 309.0909, found: 309.0916; Anal. calcd for C₁₄H₁₆N₂O₄S: C 54.53, H 5.23, N 9.08, found: C 54.7, H 5.27, N 8.96.

3-(Benzyloxy)-8,9,10,11-tetrahydro-5H-cyclohepta[c]quinolin-6(7H)-one (17). NaH (60 % in mineral oil, 350 mg, 8.75 mmol) was added to a solution of 16 a (2.0 g, 8.72 mmol) in DMF (100 mL) at 0 °C, followed by benzyl bromide (1.1 mL, 9.33 mmol) 15 min later after the evolution of H₂ had ceased. The reaction mixture was heated at 90 °C for 30 min and then concentrated in vacuo after cooling to room temperature. The light-beige sludge that was obtained was diluted with EtOAc (200 mL) and filtered. The precipitate that collected was washed with more EtOAc and H₂O (4×50 mL) and air dried overnight to give 17 as a white powder (2.2 g, 6.89 mmol, 79 %): R_f=0.69 (CHCl₃/acetone, 1:2), c.f. R_f=0.58 (16 a); IR (KBr) =3000–2800, 1650 cm⁻¹; ¹H NMR (400 MHz, [D₆]DMSO): δ=1.42 (m, 2 H), 1.53 (m, 2 H), 1.81 (m, 2 H), 2.80 (m, 2 H), 2.94 (m, 2 H), 5.11 (s, 2 H, OCH₂), 6.83 (dd, J=2.7 and 9 Hz, 1 H, C2-H), 6.87 (d, J=2.7 Hz, 1 H, C4-H), 7.38 (m, 5 H, Ph), 7.73 (d, J=9 Hz, 2 H, C1-H) and 11.5 ppm (s, 1 H, ex. with D₂O, NH); MS (FAB⁺): m/z (%): 320.0 (100) [M+H]⁺, 229.0 (5) [M+H−Bn]⁺, 91.0 (42) [Bn]⁺; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₂₁H₂₂NO₂: 320.1651, found: 320.1661. This crude product was used for the next reaction without further purification.

3-(Benzyloxy)-5-methyl-8,9,10,11-tetrahydro-5H-cyclohepta[c]quinolin-6(7H)-one (18 a). NaH (60 % in mineral oil, 65 mg, 1.63 mmol) was added to a solution of 17 (500 mg, 1.57 mmol) in DMF (130 mL) at 0 °C, followed by CH₃I (0.2 mL, 3.15 mmol) 15 min later after the evolution of H₂ had ceased. The reaction mixture was heated at 80 °C for 50 min and then concentrated in vacuo after cooling to room temperature. The beige residue obtained was dissolved in EtOAc (150 mL), and the resulting mixture washed with brine (3×50 mL), dried (MgSO₄), filtered and evaporated to give a yellow residue. This crude product was fractionated on silica with CHCl₃/EtOAc (8:1 → 2:1 gradient). The second fraction that was collected upon evaporation gave 18 a as a white residue (480 mg, 1.44 mmol, 92 %). An analytical sample of this residue was recrystallised from EtOAc/hexane (1:2) to give 18 a as soft fine needle-shaped crystals: mp: 119–121 °C; R_f=0.78 (CHCl₃/EtOAc, 1:2), c.f. R_f=0.58 (17); IR (KBr) =2920, 2840, 1630, 1610, 1590, 1240 cm⁻¹; ¹H NMR (400 MHz, [D₆]DMSO): δ=1.45 (m, 2 H), 1.56 (m, 2 H), 1.83 (m, 2 H), 2.90 (m, 2 H), 2.98 (m, 2 H), 3.61 (s, 3 H, NCH₃), 5.26 (s, 2 H, OCH₂), 6.96 (dd, J=2.3 and 8.9 Hz, 1 H, C2-H), 7.04 (d, J=2.7 Hz, 1 H, C4-H), 7.43 (m, 5 H, Ph) and 7.86 ppm (d, J=8.9 Hz, 1 H, C1-H); ¹³C NMR (100.4 MHz, CDCl₃) δ=26.01 (t), 26.54 (t), 26.91 (t), 28.64 (t), 30.68 (q, NCH₃), 32.59 (t), 70.62 (t, OCH₂), 100.36 (d), 109.93 (d), 115.12 (s), 126.02 (d), 127.65 (d), 128.38 (d), 128.87 (d), 130.77 (s), 136.55 (s), 140.45 (s), 148.84 (s), 159.86 (s) and 162.57 ppm (s); MS (FAB⁺): m/z (%): 334.3 (100) [M+H]⁺, 243.2 (5) [M+H−Bn]⁺, 91.1(35) [Bn]⁺; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₂₂H₂₄NO₂: 334.1807, found: 334.1798; Anal. calcd for C₂₂H₂₃NO₂: C 79.25, H 6.95, N 4.20, found: C 79.5, H 7.00, N 4.27.

3-Hydroxy-5-methyl-8,9,10,11-tetrahydro-5H-cyclohepta[c]quinolin-6(7H)-one (18 b). Compound 18 a (460 mg, 1.38 mmol) in THF (15 mL) was added to a suspension of Pd/C (10 %, 100 mg) in THF (15 mL). The reaction mixture was stirred under an atmosphere of H₂ (balloon) at room temperature, and the progress of the reaction was monitored by TLC. After the disappearance of the starting material had completed, the suspension was filtered and the charcoal retained washed with more THF. The combined filtrates were concentrated in vacuo, and the light-yellow residue obtained was recrystallised from hot THF/hexane (1:1) to give 18 b as fine pale-yellow crystals (148 mg, 608 μmol, 44 %): R_f=0.37 (CHCl₃/EtOAc, 1:2), c.f. R_f=0.63 (18 a); mp: 255–261 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ=1.45 (m, 2 H), 1.56 (m, 2 H), 1.82 (m, 2 H), 2.88 (m, 2 H), 2.97 (m, 2 H), 3.56 (s, 3 H, NCH₃), 6.73 (dd, J=2.3 and 9 Hz, 1 H, C2-H), 6.78 (d, J=2 Hz, 1 H, C4-H), 7.76 (d, J=9 Hz, 1 H, C1-H) and 10.11 ppm (br s, 1 H, ex. with D₂O, OH); MS (FAB⁺): m/z (%): 244.2 (100) [M+H]⁺; MS (FAB⁻): m/z (%): 396.3 (43) [M+NBA]⁻, 242.2 (100) [M−H]⁻; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₁₅H₁₈NO₂: 244.1338, found: 244.1333.

5-Methyl-6-oxo-6,7,8,9,10,11-hexahydro-5H-cyclohepta[c]quinolin-3-yl sulfamate (18). Compound 18 b (100 mg, 411 μmol) in anhydrous DMF (5 mL) was sulfamoylated in a similar manner to 16 a. The crude pale-yellow syrup (130 mg) obtained was fractionated on silica with EtOAc, and the first fraction that was collected gave a pale-yellow syrup which upon crystallisation from hot EtOAc/hexane (1:2) gave 18 as white crystals (45 mg, 140 μmol, 34 %): R_f=0.78 (EtOAc), c.f. R_f=0.68 (18 b); mp: 185–187 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ=1.47 (m, 2 H), 1.58 (m, 2 H), 1.85 (m, 2 H), 2.95 (m, 2 H), 3.06 (m, 2 H), 3.63 (s, 3 H, NCH₃), 7.19 (dd, J=2.2 and 9 Hz, 1 H, C2-H), 7.37 (d, J=2.2 Hz, 1 H, C4-H), 7.76 (d, J=9.2 Hz, 1 H, C1-H) and 8.11 ppm (s, 2 H, ex. with D₂O, OSO₂NH₂); MS (FAB⁺): m/z (%): 323.1 (100) [M+H]⁺, 243.1 (10) [M−HNSO₂]⁺; MS (FAB⁻): m/z (%): 321.1(100) [M−H)⁻, 242.1 (12) [M−H₂NSO₂]⁻; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₁₅H₁₉N₂O₄S: 323.1066, found: 323.1054; Anal. calcd for C₁₅H₁₈N₂O₄S: C 55.89, H 5.63, N 8.69, found: C 55.8, H 5.63, N, 8.63.

3-(Benzyloxy)-6-chloro-8,9,10,11-tetrahydro-7H-cyclohepta[c]quinoline (19 a). A suspension of 17 (1.0 g, 3.13 mmol) in POCl₃ (20 mL) was held at reflux for 2 h. After cooling to room temperature, ice-water and EtOAc (100 mL) were added to the dark-red/brown reaction mixture. The organic layer that separated was washed with H₂O (4×50 mL), dried (MgSO₄), filtered and concentrated in vacuo to give a light-yellow residue. This crude product was recrystallised from hot iPrOH to give 19 a as light-yellow crystals (840 mg, 2.49 mmol, 79 %): R_f=0.60 (EtOAc/hexane, 1:2), c.f. R_f<0.05 (17); mp: 128.5–130.5 °C; ¹H NMR (400 MHz, [D₆]DMSO) δ=1.62 (m, 4 H), 1.87 (m, 2 H), 3.16 (m, 2 H), 3.29 (m, 2 H), 5.28 (s, 2 H, OCH₂), 7.41 (m, 7 H) and 8.16 ppm (d, J=7.4 Hz, 1 H); MS (FAB⁺): m/z (%) 338.3 (100) [M+H]⁺, 91.1 (55); HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₂₁H₂₁NO³⁵Cl: 338.1312, found: 338.1308; Anal. calcd for C₂₁H₂₀NOCl: C 74.66, H 5.97, N 4.15, found: C 74.5, H 5.94, N 4.22.

3-(Benzyloxy)-6-methoxy-8,9,10,11-tetrahydro-7H-cyclohepta[c]quinoline (19 b). NaH (60 % in mineral oil, 296 mg, 7.40 mmol) was added to a mixture of anhydrous MeOH (240 mg, 7.49 mmol) and anhydrous DMF (15 mL) at ice-water temperature. After stirring for 15 min, the resulting purple–grey mixture was then transferred dropwise through a cannula to a solution of 19 a (500 mg, 1.48 mmol) in anhydrous DMF (10 mL). The brown mixture/suspension that resulted was heated at 70 °C for 2 h, cooled, and diluted with EtOAc (150 mL). The organic fraction was washed with brine (5×100 mL), dried (MgSO₄), filtered and concentrated in vacuo to give a yellow–brown residue that was fractionated on silica with EtOAc/hexane (1:8 → 1:4 gradient). The first fraction that collected upon evaporation gave 19 b as a white residue (387 mg, 1.16 mmol, 78 %): R_f=0.60 (EtOAc/hexane, 1:4), c.f. R_f=0.51 (19 a); ¹H NMR (400 MHz, [D₆]DMSO) δ=1.52 (m, 4 H), 1.60 (m, 2 H), 1.86 (m, 2 H), 2.95 (m, 2 H), 3.16 (m, 2 H), 3.95 (s, 3 H, OCH₃), 5.25 (s, 2 H, OCH₂), 7.11 (dd, J=2.6 and 9.2 Hz, 1 H, C2-H), 7.24 (d, J=2.6 Hz, 1 H, C4-H), 7.41 (m, 5 H, Ph) and 7.99 ppm (d, J=9.2 Hz, 1 H, C1-H); MS (FAB⁺): m/z (%): 334.3 (100) [M+H]⁺, 91.1 (64); HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₂₂H₂₄NO₂: 334.1807, found: 334.1801.

6-Methoxy-8,9,10,11-tetrahydro-7H-cyclohepta[c]quinolin-3-ol (19 c). A solution of 19 b (689 mg, 2.07 mmol) in absolute EtOH (70 mL) was debenzylated by hydrogenation in a manner similar to 18 a in the presence of Pd/C (10 %, 70 mg). The crude light-yellow residue that resulted (422 mg) was recrystallised from CHCl₃/hexane (5:6) to give 19 c as white crystals (253 mg, 1.04 mmol, 50 %): R_f=0.38 (CHCl₃/EtOAc, 4:1), c.f. R_f=0.79 (19 b); mp: undefined but all melted by 177 °C; IR (KBr) =3600–2500, 2910, 2840, 1620, 1240 cm⁻¹; ¹H NMR (400 MHz, [D₆]DMSO) δ=1.51 (m, 2 H), 1.58 (m, 2 H), 1.85 (m, 2 H), 2.92 (m, 2 H), 3.12 (m, 2 H), 3.93 (s, 3 H, OCH₃), 6.92 (dd, J=2.5 and 9.2 Hz, 1 H, C2-H), 7.00 (d, J=2.3 Hz, 1 H, C4-H), 7.89 (d, J=9.3 Hz, 1 H, C1-H) and 9.84 ppm (br s, 1 H, ex. with D₂O, OH); MS (FAB⁺) m/z (%) 244.3 (100) [M+H]⁺; MS (FAB⁻) m/z (%) 395.4 (23) [M−H+NBA]⁻, 242.3 (100) [M−H]⁻; HRMS- FAB⁺: m/z [M+H]⁺ calcd for C₁₅H₁₈NO₂: 244.1338, found: 244.1333; Anal. calcd for C₁₅H₁₇NO₂: C 74.05, H 7.04, N 5.76, found: C 73.8, H 7.01, N 5.82.

6-Methoxy-8,9,10,11-tetrahydro-7H-cyclohepta[c]quinolin-3-yl sulfamate (19). Sulfamoyl chloride (∼5 equiv) was added to a solution of 19 c (70 mg, 288 μmol) and 2,6-di-tert-butyl-4-methylpyridine (60 mg, 292 μmol) in anhydrous CH₂Cl₂ (10 mL) at room temperature. After stirring for 5 h under an atmosphere of N₂, the reaction mixture was concentrated in vacuo and the resulting yellow syrup was dissolved in EtOAc (50 mL). The organic fraction was washed with HCl (0.5 m, 4×25 mL), H₂O (2×50 mL), dried (MgSO₄) and concentrated in vacuo to give a light-brown residue (114 mg) that upon recrystallisation from CHCl₃/hexane (2:5) gave 19 as white crystals (32 mg, 99.3 μmol, 34 %): R_f=0.30 (EtOAc/hexane), c.f. R_f=0.36 (19 c); mp=94–97 °C; IR (KBr) =3540, 3350, 3240, 2910, 2840, 1360, 1180, 1170 cm⁻¹; ¹H NMR (400 MHz, [D₆]DMSO): δ=1.54 (m, 2 H), 1.61 (m, 2 H), 1.89 (m, 2 H), 3.00 (m, 2 H), 3.23 (m, 2 H), 3.99 (s, 3 H, OCH₃), 7.31 (dd, J=2.3 and 9 Hz, 1 H, C2-H), 7.63 (d, J=2.3 Hz, 1 H, C4-H), 8.07 (br s, 2 H, ex. with D₂O, OSO₂NH₂) and 8.18 ppm (d, J=9 Hz, 1 H, C1-H); MS (FAB⁺): m/z (%): 323.3 (8) [M+H]⁺, 309.2 (100) [M+H−CH₂]⁺, 230.2 (13) [M+H−CH₂−HNSO₂]⁺; HRMS-FAB⁺: m/z [M+H−CH₂]⁺ calcd for C₁₄H₁₇N₂O₄S: 309.0909, found: 309.0914; Anal. calcd for C₁₅H₁₈N₂O₄S: C 55.89, H 5.63, N 8.69, found: C 54.9, H 5.71, N 8.63.

3-(Benzyloxy)-5-pentyl-8,9,10,11-tetrahydro-5H-cyclohepta[c]quinolin-6(7H)-one (20 a) and 3-(benzyloxy)-6-(pentyloxy)-8,9,10,11-tetrahydro-7H-cyclohepta[c]quinoline (21 a). NaH (60 % in mineral oil, 65 mg, 1.62 mmol) was added to a solution of 17 (500 mg, 1.57 mmol) in DMF (40 mL) at room temperature cautiously followed by 1-bromopentane (0.4 mL, 3.23 mmol) 15 min later after the evolution of H₂ had ceased. The reaction mixture was heated at 100 °C for 1 h and then concentrated in vacuo after cooling to room temperature. The crude material that obtained was dissolved in EtOAc (100 mL) and the resulting mixture was washed with brine (4×50 mL), dried (MgSO₄), filtered and evaporated to give a yellow syrup which was fractionated on silica eluting first with CHCl₃/hexane (4:1), then CHCl₃ followed by CHCl₃/EtOAc (4:1 → 1:1 gradient). The first fraction that collected upon evaporation gave 21 a as a white residue (250 mg, 642 μmol, 41 %): R_f=0.73 (CHCl₃/hexane, 2:1), c.f. R_f<0.05 (17); IR (KBr) =3000–2840, 1615, 1590, 1330 cm⁻¹; ¹H NMR (400 MHz, [D₆]DMSO): δ=0.90 (t, J=7 Hz, 3 H, CH₃), 1.3–1.9 (m, 12 H), 2.95 (m, 2 H), 3.16 (m, 2 H), 4.36 (t, J=6.4 Hz, 2 H, OCH₂CH₂), 5.24 (s, 2 H, OCH₂Ph), 7.09 (dd, J=2.7 and 8.9 Hz, 1 H, C2-H), 7.20 (d, J=2.6 Hz, 1 H, C4-H), 7.42 (m, 5 H, Ph) and 7.98 ppm (d, J=9.4 Hz, 1 H, C1-H); MS (FAB+): m/z (%) 390.4 (95) [M+H]⁺, 319.3 (23) [M+H−C₅H₁₁]⁺, 91.1 (100) [Bn⁺]; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₂₆H₃₂NO₂: 390.2433, found: 390.2440. The second fraction that collected upon evaporation gave a light-yellow syrup (380 mg, 976 μmol, 62 %) which was recrystallised from hexane (∼50 mL) to give 20 a as fine needle-shaped white crystals (189 mg): R_f=0.62 (CHCl₃), c.f. R_f<0.05 (17); mp: 96–98.5 °C; IR (KBr) =3000–2840, 1630, 1610, 1590, 1230 cm⁻¹; ¹H NMR (400 MHz, [D₆]DMSO) δ=0.87 (t, J=, 7 Hz, 3 H, CH₃), 1.2–1.62 (m, 9 H), 1.65 (m, 2 H), 2.88 (m, 2 H), 2.98 (m, 2 H), 4.18 (t, J=7.4 Hz, 2 H, NCH₂), 5.29 (s, 2 H, OCH₂), 6.95 (m, 2 H, C2-H and C4-H), 7.40 (m, 5 H, Ph) and 7.86 ppm (d, J=8.9 Hz, 1 H, C1-H); ¹³C NMR (100.4 MHz, CDCl₃): δ=14.03 (q, CH₃), 22.43 (t), 25.49 (t), 26.15 (t), 26.35 (t), 26.92 (t), 28.22 (t), 29.21 (t), 32.15 (t), 43.18 (t), 70.19 (t, OCH₂), 99.94 (d), 109.40 (d), 109.53 (d), 114.88 (s), 125.67 (d), 127.03 (d), 127.93 (d), 128.48 (d), 130.45 (s), 136.26 (s), 139.18 (s), 148.09 (s), 159.33 (s) and 161.79 (s); MS (FAB⁺): m/z (%): 390.4 (100) [M+H]⁺, 298.3 (18) [M−Bn]⁺, 91.1(39) [Bn]⁺; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₂₆H₃₂NO₂: 390.2433, found: 390.2429; Anal. calcd for C₂₆H₃₁NO₂: C 80.17, H 8.02, N, 3.60, found: C 80.0, H 7.99, N 3.57.

3-Hydroxy-5-pentyl-8,9,10,11-tetrahydro-5H-cyclohepta[c]quinolin-6(7H)-one (20 b). A solution of 20 a (405 mg, 1.04 mmol) in absolute EtOH (30 mL) was debenzylated by hydrogenation in similar manner to 18 a in the presence of Pd/C (10 %, 41 mg). The crude white residue that resulted (235 mg, 785 μmol, 76 %) was recrystallised from CHCl₃/hexane (3:2) to give 20 b as white crystals (253 mg, 1.04 mmol, 50 %): R_f=0.52 (CHCl₃/EtOAc, 8:1), c.f. R_f=0.84 (20 a); mp: undefined but all melted by 145 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ=0.90 (t, J∼7 Hz, 3 H, CH₃), 1.36 (m, 4 H), 1.45 (m, 2 H), 1.58 (m, 4 H), 1.82 (m, 2 H), 2.88 (m, 2 H), 2.97 (m, 2 H), 4.13 (t, J=7.3 Hz, 2 H, NCH₂), 6.71 (dd, J=2 and 8.8 Hz, 1 H, C2-H), 6.80 (d, J=2.3 Hz, 1 H, C4-H), 7.76 (d, J=9 Hz, 1 H, C1-H), 8.32 (CHCl₃, 0.5 H) and 10.1 ppm (br s, 1 H, ex. with D₂O, OH); MS (FAB⁺): m/z (%): 300.3 (100) [M+H]⁺, 229.2 (10) [M+H−C₅H₁₁]⁺; MS (FAB⁻): m/z (%): 452.4 (40) [M+NBA]⁻, 298.3 (100) [M−H]⁻; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₁₉H₂₆NO₂: 300.1964, found: 300.1955.

6-Oxo-5-pentyl-6,7,8,9,10,11-hexahydro-5H-cyclohepta[c]quinolin-3-yl sulfamate (20). Compound 20 b (178 mg, 595 μmol) in anhydrous DMF (10 mL) was sulfamoylated in a similar manner to 16 a. The crude light-yellow residue (199 mg) that was obtained was fractionated on silica with CHCl₃/EtOAc (8:1). The fourth fraction that was collected gave a creamy residue (125 mg, 330 μmol, 56 %) which upon recrystallisation from hot CHCl₃/hexane (1:2) gave 20 as fine white crystals (97 mg): R_f=0.49 (CHCl₃/EtOAc, 4:1), c.f. R_f=0.67 (20 b); mp: 186–188 °C; IR (KBr) =3650–3000, 3000–2800, 1610, 1560, 1380 cm⁻¹; ¹H NMR (400 MHz, [D₆]DMSO) δ=0.89 (t, J=7 Hz, 3 H, CH₃), 1.37 (m, 4 H), 1.48 (m, 2 H), 1.61 (m, 4 H), 1.84 (m, 2 H), 2.95 (m, 2 H), 3.05 (m, 2 H), 4.21 (t, J=7.7 Hz, 2 H, NCH₂), 7.17 (dd, J=2.2 and 8.8 Hz, 1 H, C2-H), 7.36 (d, J=2.2 Hz, 1 H, C4-H), 8.05 (d, J=8.8 Hz, 1 H, C1-H) and 8.10 ppm (s, 2 H, ex. with D₂O, OSO₂NH₂); MS (FAB⁺): m/z (%): 379.2 (100) [M+H]⁺, 298.2 (27) [M−H₂NSO₂]⁺; MS (FAB⁻): m/z (%): 377.2 (100) [M−H]⁻, 298.2 (18) [M−H₂NSO₂]⁻; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₁₉H₂₇N₂O₄S: 379.1682, found: 379.1702. Anal. calcd for C₁₉H₂₆N₂O₄S: C 60.30, H 6.92, N 7.40, found: C 60.1, H 6.92, N 7.43.

6-(Pentyloxy)-8,9,10,11-tetrahydro-7H-cyclohepta[c]quinolin-3-ol (21 b). A solution of 21 a (311 mg, 798 μmol) in absolute EtOH (30 mL) was debenzylated by hydrogenation in similar manner to 18 a in the presence of Pd/C (10 %, 35 mg). The crude light-brown syrup that was obtained was fractionated on silica with CHCl₃ followed by CHCl₃/EtOAc (8:1 → 4:1 gradient) to give 21 b as a light-brown syrup which partially solidified to wax upon standing at room temperature for a few days (160 mg, 534 μmol, 67 %): R_f=0.49 (CHCl₃/EtOAc, 8:1), c.f. R_f=0.90 (21 a); mp: 120 °C; IR (KBr) =3700–2500, 3000–2800, 1615, 1590, 1440 cm⁻¹; ¹H NMR (400 MHz, [D₆]DMSO) δ=0.90 (t, J∼7 Hz, 3 H, CH₃), 1.39 (m, 4 H), 1.51 (m, 2 H), 1.59 (m, 2 H), 1.75 (m, 2 H), 1.86 (m, 2 H), 2.93 (m, 2 H), 3.12 (m, 2 H), 4.33 (t, J=6.6 Hz, 2 H, OCH₂), 6.91 (dd, J=2.3 and 8.9 Hz, 1 H, C2-H), 6.97 (d, J=2.3 Hz, 1 H, C4-H), 7.88 (d, J=8.9 Hz, 1 H, C1-H) and 9.81 ppm (br s, 1 H, ex. with D₂O, OH); MS (FAB⁺): m/z (%): 300.2 (100) [M+H]⁺, 230.1 (30); MS (FAB⁻) m/z (%) 452.2 (7) [M+NBA]⁻, 298.2 (100) [M−H]⁻, 228.1(17); HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₁₉H₂₆NO₂: 300.1964, found: 300.1962; Anal. calcd for C₁₉H₂₅NO₂: C 76.22, H 8.42, N 4.68, found: C 75.9, H 8.50, N, 4.66.

6-(Pentyloxy)-8,9,10,11-tetrahydro-7H-cyclohepta[c]quinolin-3-yl sulfamate (21). Compound 21 b (140 mg, 468 μmol) in anhydrous DMF (10 mL) was sulfamoylated in a similar manner to 16 a. The crude light-brown syrup (175 mg) that was obtained was fractionated on silica with EtOAc/hexane (1:3 → 1:2). The second fraction that was collected gave 21 as a yellow syrup that solidified to wax upon standing at room temperature overnight (87 mg, 230 μmol, 49 %): R_f=0.41 (EtOAc/hexane, 1:2), c.f. R_f=0.54 (21 b); mp: 103–107 °C; ¹H NMR (400 MHz, [D₆]DMSO) δ=0.92 (t, J=7 Hz, 3 H, CH₃), 1.41 (m, 4 H), 1.54 (m, 2 H), 1.61 (m, 2 H), 1.78 (m, 2 H), 1.89 (m, 2 H), 3.01 (m, 2 H), 3.22 (m, 2 H), 4.40 (t, J=6.5 Hz, 2 H, OCH₂), 7.30 (dd, J=2.3 and 8.9 Hz, 1 H, C2-H), 7.59 (d, J=2.3 Hz, 1 H, C4-H), 8.06 (s, 2 H, ex. with D₂O, OSO₂NH₂) and 8.17 ppm (d, J=9 Hz, 1 H, C1-H); MS (FAB⁺): m/z (%): 379.2 (100) [M+H]⁺, 300.2 (5) [M+H−HNSO₂]⁺; MS (FAB⁻): m/z (%): 377.1 (100) [M−H]⁻, 298.2 (11) [M−H₂NSO₂]⁻, 77.9 (52); HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₁₉H₂₇N₂O₄S: 379.1692, found: 379.1691; Anal. calcd for C₁₉H₂₆N₂O₄S: C 60.30, H 6.92, N 7.40, found: C 60.5, H 7.05, N 7.34.

3-(Benzyloxy)-5-(3-phenylpropyl)-8,9,10,11-tetrahydro-5H-cyclohepta[c]quinolin-6(7H)-one (22 a) and 3-(benzyloxy)-6-(3-phenylpropoxy)-8,9,10,11-tetrahydro-7H-cyclohepta[c]quino-line (23 a). NaH (60 % in mineral oil, 65 mg, 1.62 mmol) was added to a solution of 17 (500 mg, 1.57 mmol) in DMF (40 mL) at room temperature cautiously followed by 1-bromo-3-phenylpropane (0.25 mL, 1.65 mmol) 15 min later after the evolution of H₂ had ceased. The reaction mixture was heated at 100 °C for 1 h and then concentrated in vacuo after cooling to room temperature. The crude material that was obtained was dissolved in EtOAc (100 mL) and the resulting mixture was washed with brine (4×50 mL), dried (MgSO₄), filtered and evaporated to give a yellow syrup which was fractionated on silica eluting first with CHCl₃/hexane (2:1 → 4:1 gradient), then CHCl₃ followed by CHCl₃/EtOAc (2:1 → 1:2 gradient). The first fraction that was collected upon evaporation gave 23 a as a light-yellow syrup (287 mg, 656 μmol, 42 %): R_f=0.71 (CHCl₃/hexane, 2:1), c.f. R_f<0.1 (17); ¹H NMR (400 MHz, [D₆]DMSO): δ=1.57 (m, 4 H), 2.09 (quintet, J=7 Hz, 2 H, OCH₂CH₂CH₂Ph), 2.77 (t, 2 H, CH₂CH₂Ph), 2.98 (m, 2 H), 3.16 (m, 2 H), 4.35 (t, J=6.3 Hz, 2 H, OCH₂CH₂), 5.23 (s, 2 H, OCH₂Ph), 7.09 (dd, J=2.5 and 9 Hz, 1 H, C2-H), 7.34 (m, 11 H, C4-H and 2×Ar) and 7.98 ppm (d, J=9 Hz, 1 H, C1-H); MS (FAB⁺): m/z (%): 438.4 (20) [M+H]⁺, 319.3 (14) [M+H-CH₂CH₂CH₂Ph]⁺, 91.1 (45) [Bn]⁺, 73.1(100); HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₃₀H₃₂NO₂: 438.2433, found: 438.2438. The second fraction that was collected upon evaporation gave a clear syrup (373 mg, 852 μmol, 55 %) that was recrystallised from EtOAc/hexane (1:15, ∼32 mL) to give 22 a as light-beige rod-shaped crystals (240 mg): R_f=0.38 (CHCl₃/hexane, 2:1), c.f. R_f<0.1 (17); mp: 120–122 °C; ¹H NMR (400 MHz, [D₆]DMSO) δ=1.45 (m, 2 H), 1.56 (m, 2 H), 1.83 (m, 4 H), 2.71 (t, J=7.4 Hz, 2 H, CH₂CH₂Ph), 2.89 (m, 2 H), 2.98 (m, 2 H), 4.22 (t, J=7.6 Hz, 2 H, NCH₂), 5.17 (s, 2 H, OCH₂Ph), 6.83 (d, J=2.3 Hz, 1 H, C4-H), 6.93 (dd, J=2.3 and 8 Hz, 1 H, C2-H), 7.34 (m, 10 H, Ar) and 7.86 ppm (d, J=9 Hz, 1 H, C1-H); MS (FAB⁺): m/z (%): 438.4 (100) [M+H]⁺, 346.3 (20) [M−Bn]⁺, 91.1 (62) [Bn]⁺; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₃₀H₃₂NO₂: 438.2433, found: 438.2423. Anal. calcd for C₃₀H₃₁NO₂: C 82.35, H 7.14, N, 3.20, found: C 82.7, H 7.14, N 3.43.

3-Hydroxy-5-(3-phenylpropyl)-8,9,10,11-tetrahydro-5H-cyclohepta[c]quinolin-6(7H)-one (22 b). A solution of 22 a (300 mg, 686 μmol) in absolute EtOH (30 mL) was debenzylated by hydrogenation in similar manner to 18 a in the presence of Pd/C (10 %, 60 mg). The crude solid that resulted (205 mg, 590 μmol, 86 %) was recrystallised from toluene/hexane (8:3) to give 22 b as creamy crystals (150 mg): R_f=0.68 (CHCl₃/EtOAc, 4:1), c.f. R_f=0.91 (22 a); mp: 182–186 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ=1.45 (m, 2 H), 1.56 (m, 2 H), 1.81 (m, 2 H), 1.90 (quintet, J=7.8 Hz, 2 H, NCH₂CH₂CH₂Ph), 2.73 (t, J=7.8 Hz, 2 H, CH₂Ph), 2.88 (m, 2 H), 2.96 (m, 2 H), 4.18 (t, J=7 Hz, 2 H, NCH₂), 6.72 (dd, J=2.3 and 8.9 Hz, 1 H, C2-H), 6.78 (d, J=2 Hz, 1 H, C4-H), 7.25 (m, 5 H, Ph), 7.76 (d, J=8.9 Hz, 1 H, C1-H) and 10.1 (br s, 1 H, ex. with D₂O, OH); MS (FAB⁺): m/z (%): 348.3 (100) [M+H]⁺, 243.2 (18); MS (FAB⁻): m/z (%): 500.3(47) [M+NBA]⁻, 346.3 (100) [M−H]⁻; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₂₃H₂₆NO₂: 348.1964, found: 348.1964.

6-Oxo-5-(3-phenylpropyl)-6,7,8,9,10,11-hexahydro-5H-cyclohepta[c]quinolin-3-yl sulfamate (22). Compound 22 b (100 mg, 288 μmol) in anhydrous DMF (10 mL) was sulfamoylated in a similar manner to 16 a. The crude light-yellow residue (122 mg) obtained was fractionated on silica with CHCl₃/EtOAc (8:1). The second fraction that was collected gave a light-yellow syrup that solidified on standing overnight to give 22 as a white solid (65 mg, 152 μmol, 53 %). Recrystallisation from hot CHCl₃/hexane (5:4) gave 22 as fine white crystals (39 mg): R_f=0.23 (CHCl₃/EtOAc, 8:1), c.f. R_f=0.43 (22 b); mp: 176–179 °C; IR (KBr) =3650–3000, 3000–2800, 1610, 1560, 1380, 1190 cm⁻¹; ¹H NMR (400 MHz, [D₆]DMSO) δ=1.45 (m, 2 H), 1.56 (m, 2 H), 1.83 (m, 2 H), 1.91 (m, 2 H), 2.71 (t, J=7.7 Hz, 2 H, CH₂Ph), 2.92 (m, 2 H), 3.03 (m, 2 H), 4.24 (t, J=7.7 Hz, 2 H, CH₂N), 7.22 (m, 6 H, C2-H and Ar), 7.35 (d, J=2.2 Hz, 1 H, C4-H), 8.03 (d, J=8.8 Hz, 1 H, C1-H) and 8.08 ppm (br s, 2 H, ex. with D₂O, OSO₂NH₂); MS (FAB⁺): m/z (%): 427.2 (100) [M+H]⁺, 346.2 (25) [M−H₂NSO₂]⁺; MS (FAB⁻): m/z (%): 425.1(100) [M−H]⁻, 346.2 (19) [M−H₂NSO₂]⁻; HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₂₃H₂₇N₂O₄S: 427.1620, found 427.1695. Anal. calcd for C₂₃H₂₆N₂O₄S: C 64.77, H 6.14, N 6.57, found: C 64.2, H 6.13, N 6.65.

6-(3-Phenylpropoxy)-8,9,10,11-tetrahydro-7H-cyclohepta[c]quinolin-3-ol (23 b). A solution of 23 a (255 mg, 583 μmol) in a mixture of absolute EtOH (30 mL) and THF (10 mL) was debenzylated by hydrogenation in similar manner to 18 a in the presence of Pd/C (10 %, 51 mg). The crude yellow syrup that was obtained solidified on standing overnight to give 23 b as a yellow wax (182 mg, 524 μmol, 90 %): R_f=0.61 (CHCl₃/EtOAc, 4:1), c.f. R_f=0.88 (23 a); mp: ∼135 °C; IR (KBr) =3700–2500, 3000–2800, 1615, 1590, 1420, 1330, 1200 cm⁻¹; ¹H NMR (400 MHz, [D₆]DMSO) δ=1.57 (m, 4 H), 1.86 (m, 2 H), 2.08 (m, 2 H, CH₂CH₂CH₂Ph), 2.77 (t, J=7.5 Hz, 2 H, CH₂Ph),2.95 (m, 2 H), 3.13 (m, 2 H), 4.33 (t, J=6.4 Hz, 2 H, OCH₂), 6.91 (dd, J=2.7 and 8.9 Hz, 1 H, C2-H), 6.95 (d, J=2.3 Hz, 1 H, C4-H), 7.25 (m, 5 H, Ar), 7.88 (d, J=8.9 Hz, 1 H, C1-H) and 9.82 (br s, 1 H, ex. with D₂O, OH); MS (FAB⁺): m/z (%): 348.3 (100) [M+H]⁺, 229.3 (40) [M+H−CH₂CH₂CH₂Ph]⁺; MS (FAB⁻): m/z (%): 346.3 (100) [M−H]⁻, 275.2 (40), 181.2 (50); HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₂₃H₂₆NO₂: 348.1964, found: 348.1969.

6-(3-Phenylpropoxy)-8,9,10,11-tetrahydro-7H-cyclohepta[c]quinolin-3-yl sulfamate (23). Compound 23 b (135 mg, 389 μmol) in anhydrous DMF (10 mL) was sulfamoylated in a similar manner to 16 a. The crude light-brown syrup (175 mg) that obtained was fractionated on silica with EtOAc/hexane (1:3 → 1:2). The second fraction that was collected gave 23 as a yellow syrup that solidified to wax upon standing at room temperature overnight (87 mg, 230 μmol, 49 %): R_f=0.41 (EtOAc/hexane, 1:2), c.f. R_f=0.54 (23 b); mp: 103–107 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ=0.92 (t, J=7 Hz, 3 H, CH₃), 1.41 (m, 4 H), 1.54 (m, 2 H), 1.61 (m, 2 H), 1.78 (m, 2 H), 1.89 (m, 2 H), 3.01 (m, 2 H), 3.22 (m, 2 H), 4.40 (t, J=6.5 Hz, 2 H, OCH₂), 7.30 (dd, J=2.3 and 8.9 Hz, 1 H, C2-H), 7.59 (d, J=2.3 Hz, 1 H, C4-H), 8.06 (s, 2 H, ex. with D₂O, OSO₂NH₂) and 8.17 ppm (d, J=9 Hz, 1 H, C1-H); MS (FAB⁺): m/z (%): 379.2 (100) [M+H]⁺, 300.2 (5) [M+H−HNSO₂]⁺; MS (FAB⁻): m/z (%): 377.1 (100) [M−H]⁻, 298.2 (11) [M−H₂NSO₂]⁻, 77.9 (52); HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₁₉H₂₇N₂O₄S: 379.1692, found: 379.1691. Anal. calcd for C₁₉H₂₆N₂O₄S: C 60.30, H 6.92, N 7.40, found: C 60.5, H 7.05, N 7.34.

3-Amino-8,9,10,11-tetrahydro-5H-cyclohepta[c]quinolin-6(7H)-one (24 a). A mixture of 1,3-phenylenediamine (5.0 g, 46.22 mmol) and methyl 2-oxo-1-cycloheptane carboxylate (7.9 g, 46.22 mmol) was heated at 150 °C overnight. The yellow sludge that formed was cooled to room temperature and diluted with Et₂O to give a yellow suspension which was filtered. The beige precipitate collected (3.91 g) was recrystallised from hot iPrOH to give 24 a as a wool-like fluff (1.59 g, 6.98 mmol, 15 %): mp: 290–300 °C (dec); IR (KBr) =3460, 3360, 2920, 2850, 1650, 1620 cm⁻¹; ¹H NMR (400 MHz, [D₆]DMSO): δ=1.42 (m, 2 H), 1.54 (m, 2 H), 1.80 (m, 2 H), 2.78 (m, 2 H, C7-H₂), 2.87 (m, 2 H, C11-H₂), 5.63 (br s, ∼2 H, ex. with D₂O, NH₂), 6.37 (d, J=2.1 Hz, 1 H, C4-H₂), 6.64 (dd, J=2.1 and 8.9 Hz, 1 H, C2-H₂), 7.46 (d, J=8.8 Hz, 1 H, C1-H₂) and 11.2 (br s, 1 H, exchanged with D₂O, CONH). Anal. calcd for C₁₄H₁₆N₂O: C 73.66, H 7.06, N 12.27, found: C 73.7, H 7.21, N, 12.1.

6-Oxo-6,7,8,9,10,11-hexahydro-5H-cyclohepta[c]quinolin-3-ylsulfamide (24). To a solution of 24 a (300 mg; 1.31 mmol) and 2,6-di-tert-butyl-4-methylpyridine (270 mg, 1.31 mmol) in anhydrous DMF (20 mL) at 0 °C under N₂ was added sulfamoyl chloride (∼0.69 m in toluene,[19] ∼3–5 equiv, evaporated down to ∼1 mL prior to addition). After stirring at room temperature under N₂ overnight, the reaction mixture was diluted with EtOAc (∼150 mL) and the organic layer washed with brine (5×100 mL). After drying with MgSO₄ and filtering, the filtrate was evaporated, during which time the precipitation of 24 occurred. Collection and air drying of the precipitate gave 24 as a white powder (113 mg; 28 %): R_f=0.46 (CHCl₃/EtOAc, 4:1); mp: 183–185 °C; IR (KBr) =3700–2700, 3360, 3280, 2920, 2840, 1630, 1340, 1160 cm⁻¹; ¹H NMR (270 MHz; [D₆]DMSO): δ=1.44 (m, 2 H), 1.56 (m, 2 H), 1.83 (m, 2 H), 2.81 (m, 2 H, C7-CH₂), 2.94 (m, 2 H, C11-CH₂), 7.03 (dd, J=2.2 and 8.8 Hz, 1 H, C2-H), 7.10 (d, J=2.1 Hz, 1 H, C4-H), 7.13 (br s, 2 H, ex. with D₂O, H₂NSO₂NH), 7.73 (d, J=9 Hz, 1 H, C1-H), 9.81 (s, 1 H, ex. with D₂O, H₂NSO₂NH) and 11.52 (s, 1 H, ex. with D₂O, CONH); MS (FAB⁺): m/z (%): 308.1 (100) [M+H]⁺; MS (FAB⁻): m/z (%): 306.2 (100) [M−H]⁻, HRMS-FAB⁺: m/z [M+H]⁺ calcd for C₁₄H₁₈N₃O₃S: 308.1069, found: 308.1055; Anal. calcd for C₁₄H₁₇N₃O₃S: C 54.71, H 5.57, N 13.67, found: C 54.5, H 5.60, N 13.5.