Synthesis and Evaluation of 1-(1-(Benzo[b]thiophen-2-yl)cyclohexyl)piperidine (BTCP) Analogues as Inhibitors of Trypanothione Reductase

Thirty two analogues of phencyclidine were synthesised and tested as inhibitors of trypanothione reductase (TryR), a potential drug target in trypanosome and leishmania parasites. The lead compound BTCP (1, 1-(1-benzo[b]thiophen-2-yl-cyclohexyl) piperidine) was found to be a competitive inhibitor of the enzyme (Ki=1 μm) and biologically active against bloodstream T. brucei (EC50=10 μm), but with poor selectivity against mammalian MRC5 cells (EC50=29 μm). Analogues with improved enzymatic and biological activity were obtained. The structure–activity relationships of this novel series are discussed.


Introduction
Parasites of the order Kinetoplastida are the causative agents of a number of human and animal diseases including Human African Trypanosomiasis (HAT) (caused by Trypanosoma brucei rhodesiense and T. b. gambiense), Chagas' disease (T. cruzi) and the leishmaniases (Leishmania sp.). Collectively these diseases have a large unmet disease burden, [1] with the current therapeutics used to treat them possessing severe limitations. [2] All of these trypanosomatid parasites use a trypanothione-based redox metabolism, [3] which is absent in humans. The enzymes of this redox pathway are therefore considered to be attractive targets for the development of new antitrypanosomatid drugs. [4] One component of the trypanothione-based redox pathway is trypanothione reductase (TryR), which is responsible for reducing trypanothione disulfide to the dithiol trypanothione and in doing so provides reducing equivalents to protect the parasites from oxidative damage. [3] In T. brucei it has been demonstrated that TryR activity is required for parasites to grow in culture and to be infective in a mouse disease model. [5] Therefore, TryR is a validated drug target, and there are a number of recent reports outlining the discovery and development of A C H T U N G T R E N N U N G inhibitors of this key enzyme. [6] A recently reported high-throughput screening (HTS) of known bioactive compounds against T. cruzi TryR identified a number of novel TryR inhibitors [7] including the arylcyclohexylamine BTCP [8] (1, 1-(1-benzo[b]thiophen-2-yl-cyclohexyl)-piperidine). BTCP (1) is an analogue of the anaesthetic drug PCP (2, 1-(1-phenyl-cyclohexyl)-piperidine, phenylcyclidine). However, despite the structural similarity between compounds 1 and 2, they have been shown to possess a different pharmacological selectivity. [8] BTCP (1) is a more potent dopamine uptake inhibitor and has a much lower affinity for the PCP receptor.
BTCP (1) was considered to be a promising screening hit for further development due to its low molecular weight (299), low micromolar potency against T. cruzi TryR (IC 50 = 3.7 mm), a promising ligand efficiency (0.35 kcal mol À1 L), lack of activity against the human homologue of TryR, glutathione reductase (GR), and the fact that phencyclidines are known to cross the blood-brain barrier, an essential property for the successful treatment of stage 2 HAT. BTCP (1) also has the advantage of being a druglike molecule, in contrast to some of the more potent reported TryR inhibitors, many of which are polyamine analogues [6a,d,f] designed to mimic the spermidine moiety of the enzyme substrate trypanothione. In addition, there are a number of publications relating to BTCP (1) and other phencyclidines detailing both synthetic strategies for analogue synthesis and their associated pharmacological activities. [9] Due to the limitations of the current treatments for HAT, there is a need for the identification of new compound classes displaying antitrypanosomal activity. Therefore, a systematic structure-activity relationship (SAR) analysis of BTCP (1) was undertaken to optimise activity against both TryR and the intact parasite T. brucei. The results of these investigations are reported herein.
Thirty two analogues of phencyclidine were synthesised and tested as inhibitors of trypanothione reductase (TryR), a potential drug target in trypanosome and leishmania parasites. The lead compound BTCP (1, 1-(1-benzo[b]thiophen-2-yl-cyclohexyl) piperidine) was found to be a competitive inhibitor of the enzyme (K i = 1 mm) and biologically active against bloodstream T. brucei (EC 50 = 10 mm), but with poor selectivity against mammalian MRC5 cells (EC 50 = 29 mm). Analogues with improved enzymatic and biological activity were obtained. The structureactivity relationships of this novel series are discussed.

Results and Discussion
Biological characterisation of BTCP In order to determine the validity of BTCP (1) as a starting point for a target-driven approach towards the identification of a lead compound for the treatment of HAT, the inhibitory activity of BTCP against T. brucei TryR had to be determined. BTCP (1) was assayed against T. brucei TryR using a HTS format based on a published nonenzymatically coupled assay [10] and found to have an IC 50 value of 3.3 mm, confirming its suitability for further investigation. There is no significant difference between the IC 50 values for 1 against T. cruzi (IC 50 = 3.7 mm) and T. brucei TryR (IC 50 = 3.3 mm), which is as expected given the high degree of sequence identity between TryR in the two species (83 % at the amino acid level). A more detailed kinetic analysis established that BTCP is a linear competitive inhibitor of TryR (with respect to trypanothione), with a K i value of 1.00 AE 0.08 mm, in good agreement with the IC 50 value determined in the HTS-format TryR assay.
BTCP (1) was assayed against bloodstream form T. brucei brucei cells in a HTS-assay format and found to have an EC 50 value of 10 mm, in close agreement with the previously published EC 50 value of 14 mm. [7] BTCP (1) was screened against MRC-5 cells in the same 96-well format as for the trypanosome assay giving an EC 50 value of 29 mm. Unfortunately, the threefold selectivity between MRC-5 and T. brucei is suboptimal, but the selectivity is sufficient to warrant further development of the compound series.

Synthesis of BTCP analogues
There are insufficient commercially available analogues of BTCP (1) to establish a comprehensive SAR. Therefore, a chemical synthesis programme was required to support the development of the hit compound. Initial synthetic studies focussed on preparing a diverse collection of BTCP analogues systematically modifying the benzo[b]thiophene group, the piperidine ring and the cyclohexyl ring (Table 1). In particular we were interested in carrying out the following modifications to probe for new interactions with the protein: changing the benzo[b]thiophene to other aromatic rings, both monocyclic and bicyclic; modifying the size of the piperidine ring and putting heteroatoms into the ring; modifying the size of the cyclohexyl ring and adding substituents to it.
Two different synthetic methodologies were employed to prepare the initial collection: first, addition of aryl lithiums to the benzotriazole adducts of enamines [11] (Scheme 1, route A); and second, the reaction of aryl Grignards with a-amino nitriles (the Bruylants reaction, [12] Scheme 1, route B). Route A was successfully employed in reactions where the aryl group was an unsubstituted monocyclic aromatic (2 & 3), or when the aryl group was a 5/6 fused bicyclic aromatic (e.g. benzo[b]thiophene, compounds 10, 13-15 & 17). The only exception to the latter observation was that when 1-methylindole was employed in the reaction only a trace amount of the target mole-  (2) from phenyllithium also proceeded in poor yield, suggesting that the Route A methodology is not suited to the synthesis of analogues where a substituted benzene ring is directly attached to the piperidylcyclohexyl moiety. This observation may explain why when 5-bromobenzo[b]thiophene was employed as the substrate for lithiation the exclusive product of the reaction was the bromine-substituted BTCP analogue 12, possibly due to the failure of the generated benzo[b]thien-5-yl-lithium species, but not the 5-bromobenzo[b]thien-2-yl-lithium species, to react. In contrast, both analogues 10 and 11 were isolated when 3-bromobenzo[b]thiophene was employed, due to reactive species formed by lithiation at the 2 position in addition to lithium halogen exchange at the 3 position. The enamine building blocks required for the route A synthesis were obtained from commercial sources, or readily prepared using published methodologies. [13] Analogues 4, 6 and 7 have previously been prepared via the Bruylants reaction (route B), therefore, they were prepared following this procedure. [14] Attempts to prepare the 3-phenylbenzene isomer of 4 using this methodology were unsuccessful. The indole-containing analogue 8 was also prepared using this procedure. Route B has previously been utilised for the preparation of the amine-containing analogue 16, [15] therefore, this route was chosen in preference to route A (Scheme 1). Additionally, the amine-containing analogue 18 was prepared using the Bruylants reaction as the requisite a-amino nitrile 22 was considered to be more synthetically accessible than the substituted enamine that would be required to use route A (Scheme 1).
In addition, analogues containing a carbonyl "spacer" between the cyclohexylpiperidine core and the aromatic functionality were prepared by reaction of aryl lithiums with alphaamino nitrile 20 (Scheme 2). [16] Further reaction of 23 with phenyllithium gave an analogue containing two aryl groups (25).

Trypanothione reductase assay of BTCP analogues
Analogues 2-25 were tested for their ability to inhibit T. brucei TryR (Table 1) using the HTS assay format previously employed to assay BTCP (1). None of the aryl analogues (compounds 2-12) showed an improvement in potency over the hit compound 1. Analogues where the benzo[b]thiophene was re-placed with a monocyclic aromatic (compounds 2-4) showed a dramatic reduction in potency against TryR (IC 50 values 57 to > 100 mm), suggesting a requirement for a fused bicyclic aromatic moiety for optimal inhibitor binding. The inhibition values from analogues containing alternative fused bicyclic systems (compounds 5-10) suggest that there is a very specific requirement for a 2-benzo[b]thiophene substitution, as demonstrated by testing close isosteres such as 2-naphthyl (compound 7, IC 50 = 28 mm vs 3.3 mm) and analogues containing minor changes in inhibitor structure for example, compound 9 where the benzo[b]thiophene is replaced with a benzo[b]thiazole (IC 50 > 100 mm). Indeed, with the exception of replacing 2benzo[b]thiophene with 2-benzo[b]furan (compound 5) all of the aromatic analogues of BTCP (1) were at least one order of magnitude less potent against T. brucei TryR (IC 50 values 28 to > 100 mm). The screening results for analogues 11 and 12 demonstrate that it is not possible to substitute 2-benzo [b]thiophene at the 5 position, but that substitution at the 3-position gives analogues that retain some activity, albeit reduced. Given these results no further exploration of the aromatic moiety was conducted and all subsequent analogues would incorporate the 2-benzo[b]thiophene functionality.
Analogues 13-16 were prepared to investigate the effect of changing the piperidine ring of BTCP (1). Exchanging the piperidine for a morpholine or piperazine ring (compounds 15 & 16) results in a threefold reduction in potency (Table 1), possibly due to the attenuated basicity of the nitrogen atom, or due to the introduction of an additional polar atom (or a combination of both). The acyclic diethylamino analogue (14) is of approximately equal potency to the hit compound 1 (IC 50 = 5.0 mm vs 3.3 mm). Unfortunately, attempts to prepare more highly substituted acyclic analogues of 1 using route B (Scheme 1) proved unsuccessful. The pyrrolidine-containing analogue 13 was marginally more potent than the hit compound (1) (IC 50 = 0.91 mm vs 3.3 mm). A full kinetic analysis of analogue 13 showed it to be a linear competitive inhibitor with respect to trypanothione (K i = 0.26 AE 0.01 mm vs 1 mm for BTCP), confirming this mode of inhibition within the BTCP compound series ( Figure 1). However, this fourfold increase in potency did not warrant any additional investigation into replacing the A C H T U N G T R E N N U N G piperidine moiety.
The investigation of BTCP cyclohexyl-analogues was limited by synthetic considerations, with just three analogues (17-19) being prepared. Altering the cyclohexyl moiety by either ring contraction to a cyclopentane ring (17), or by replacement with a gem dimethyl substitution (19) gave analogues that were three or fivefold less potent, respectively. This suggests that the cyclohexane ring contributes to inhibitory activity by either hydrophobic interactions, or by controlling the orientation by which the other moieties are presented to the protein.
The amine-containing analogue 18 showed a slight improvement in potency (IC 50 = 0.93 mm vs 3.3 mm) suggesting that it may be possible to introduce a substituted nitrogen at the 4position of the cyclohexane moiety. Additionally, it may be possible to substitute a carbon atom at the 4 position.
The "spacer"-containing analogues 23-25 were all found to be inactive in the T. brucei TryR assay (IC 50 > 100 mm). Therefore, Scheme 2. Route to BTCP analogues containing a one carbon "spacer" between the piperidylcyclohexyl and aryl moieties. [16] Reagents and conditions: direct attachment of the aromatic moiety to the cyclohexyl-A C H T U N G T R E N N U N G piperidine core is probably an absolute requirement for TryR A C H T U N G T R E N N U N G inhibition within this series. The inactivity of these analogues combined with the failure to significantly increase potency by substitution of the aromatic, or piperidine moieties, meant that substitution at the 4-position of the cyclohexyl ring became the only focus of further investigations (see below).

Cell-based assays of BTCP analogues
A subset of the analogues prepared as part of the initial diverse BTCP analogue collection (compounds 1, 5 & 13-19) were assayed for their ability to inhibit the growth of T. brucei in culture (Table 1). With the exception of compound 16, the analogues displayed a decrease in potency between the enzyme and cellular assays of between 2-and 15-fold. Although it is not possible to draw a reliable correlation with this small subset, this level of decrease and its consistency between analogues suggests that inhibition of TryR could be the cause of the inhibition of parasite growth and that it is not the result of an off-target effect.
Additional analogues (14, 16 & 18) were subjected to the MRC-5 counter screen and their selectivity between MRC-5 cells and T. brucei was found to be~1-to > 20-fold. Although this low selectivity is disadvantageous, it may increase in analogues with improved inhibitory activity against TryR.
Synthesis and TryR assay of BTCP analogues substituted at the 4-position of the cyclohexyl ring Two strategies were employed to functionalise the 4-position of the cyclohexane moiety; first, preparation of a bipiperidinyl analogue (28), with subsequent derivatisation of the nitrogen atom, allowing the synthesis of a number of analogues with a minimal number of synthetic transformations (Scheme 3); and second, a stepwise preparation of cis and trans 38 containing a tert-butyl substitution at C4 of the cyclohexane ring (Scheme 4).
In order to prepare the bipiperidinyl 28 it was necessary to employ a suitable protecting group for the nitrogen atom. Previously it has been reported that both the benzyl and benzoyl nitrogen protecting groups are unsuitable for the preparation of substituted phencyclidines. [9a] Therefore, the Boc protecting group was employed during the Bruylants reaction giving the key protected intermediate 27 (Scheme 3). The Boc group of 27 was deprotected under acidic conditions to yield the secondary amine 28, which subsequently underwent either acylation or alkylation reactions to give the substituted analogues 29-   Table 2    www.chemmedchem.org 33. However, the alkylation reactions proved problematic leading to the formation of significant quantities of quaternary A C H T U N G T R E N N U N G ammonium salts as side products, which proved difficult to separate from the tertiary amines by column chromatography. Therefore, LiAlH 4 reduction of the amide analogues 30 and 32 was used to prepare the tertiary amine analogues 34 and 35, respectively.
Analogues 27-35 were assayed for their ability to inhibit T. brucei TryR as described above and the results are displayed in Table 2. The free amine 28 was approximately equal in activity to BTCP (1) (IC 50 = 5.1 mm vs 3.3 mm), suggesting that the increased activity of the N-methyl analogue 18 is derived from the introduction of the methyl group, not through the introduction of a hydrogen bond donor. However, analogues containing larger hydrophobic amide or alkyl substitutions (analogues 29-31 & 34) all possessed reduced inhibitory activity (IC 50 = 6.6-19 mm). Similarly the Boc protected precursor 27 proved to be completely inactive in the TryR assay (IC 50 > 100 mm). This demonstrates that the 4-position of the cyclohexane ring of BTCP (1) is not fully occluded by TryR upon inhibitor binding, but that the protein region around this position does not form favourable hydrophobic interactions. This conclusion is supported by the fact that analogues 32 and 33 containing polar substitutions were found to be approximately equipotent with BTCP (1) (IC 50 = 2.6 mm and 4.4 mm, respectively vs 3.3 mm), and of similar potency to the N-methyl analogue 18. Analogue 35 was found to be inactive in the TryR assay inconsistent with the results observed for 32 and 33. However, this lack of activity could be due to 35 being the only analogue to contain three highly basic atoms.
Analogues 28 and 30-34 were assayed against T. brucei parasites and MRC-5 cells (Table 2). With the exception of compound 32, all of the analogues showed some degree of selectivity against the parasites (> 2-fold). However, as observed with the N-methyl analogue 16, compounds 28, 30, 31 and 34 showed improved potency in the T. brucei assay over the enzyme assay. This is suggestive of either selective uptake, or an off-target effect for these analogues.
Analogues containing alkyl substitutions at C4 of the cyclohexyl ring have been previously prepared by employing either the Bruylants reaction (Scheme 1, route B), or in a stepwise sequence from tertiary benzylic alcohols (e.g. 36) (Scheme 4). It has been demonstrated that the Bruylants reaction gives only a single isomer (cis) when 4-substituted a-aminonitriles are used as the substrates for the reaction. [17] However, there was an interest in assaying both isomers of 38 as they have been shown to possess a different pharmacological selectivity [18] and could offer an insight into the optimal arrangement of the piperidine ring, aromatic group and 4-cyclohexyl substituent relative to each other for the inhibition of TryR. Therefore, in order to access both isomers, a modification of the published synthetic route outlined in Scheme 4 was employed. The two isomers, cis-and trans-38, were separated by column chromatography at the final step. It has been demonstrated that the cis isomer elutes first when the mixture is purified with silica as the stationary phase. [9b] Cis-and trans-38 were assayed for their ability to inhibit TryR under the standard assay conditions and found to have IC 50 values of > 100 mm and 3.6 mm, respectively. This demonstrates that there is an absolute requirement for the piperidine moiety to be equatorial and conversely for the aromatic moiety to be in an axial conformation in order for BTCP analogues to inhibit TryR. Additionally, these results show that substituting BTCP with a bulky tert-butyl group at the 4-position of the cyclohexane ring leads to no appreciable change in TryR inhibitory activity (3.6 mm vs 3.3 mm for 1), supporting the conclusion that the 4-position is not occluded by the protein structure upon binding of the inhibitor with TryR. Trans-38 was also screened in the cell assay and found to have an EC 50 value of 3.2 mm against T. brucei and inactive against the mammalian cell line (EC 50 > 15 mm), again comparable to 1.

Conclusions
The investigations reported herein have confirmed that analogues of BTCP (1) represent a new class of TryR inhibitors, which are to our knowledge structurally distinct from inhibitors previously reported in the literature. Enzyme and cellular assays have demonstrated that analogues of this series are competitive inhibitors with respect to the natural TryR substrate, trypanothione, and that the analogues are marginally more potent against trypanosomes than mammalian cells in culture.
Synthesis and screening of a diverse analogue collection has allowed a detailed SAR to be established for all moieties of the arylcyclohexylamine pharmacophore ( Figure 2). However, although the essential structural features for maintaining the inhibitory activity of BTCP analogues have been determined, no functional group changes that significantly increase the potency against TryR have been identified.
From the rough correlation between T. brucei TryR IC 50 and T. brucei EC 50 values it is expected that TryR inhibitors in the single nanomolar range will be a requisite for adequate inhibition of parasite growth. However, given the preliminary SAR this goal is unlikely to be realised without the aid of a proteinligand structure to identify potentially beneficial binding interactions. However, no noncovalent protein-ligand structures  [19] these inhibitors are not considered druglike (e.g. MW > 500). This requirement for high molecular weight compounds to efficiently inhibit TryR may be a direct consequence of TryR possessing a large, solvent-exposed active site. [20] To date, druglike molecules have only achieved potencies in the low micromolar range, [6] unfortunately this remains true for the BTCP series.

Experimental Section
Biology TryR enzyme assay A nonenzymatically coupled assay for detecting TyrR activity was used. [10] In this assay, the activity of TyrR is coupled to the reduction of DTNB (5,5'-dithiobis-(2-nitrobenzoic acid)) to 2TNB À by dihydrotrypanothione (T[SH] 2 ). Formation of 2TNB À is measured as an increase in absorbance at 412 nm ( Figure 3). The TyrR screening assay was miniaturised and optimised to a 384-well plate format. Assessment of the assay for robustness in an automated environment yielded the following typical performance statistics: Z' = 0.84 AE 0.001; %CV (plate) = 3.65 AE 0.4; signal to background = 10 AE 0.25; clomipramine IC 50 = 12.4 AE 0.14 mm.
Potency was determined as independent duplicates for all compounds tested. Serial titrations (10 half log increments) of test compounds from 30 mm to 1 nm were created in DMSO using the Janus automated 8 channel pipettor (Perkin-Elmer). A serial titration of clomipramine was used as a positive control in each assay plate; BTCP was used as an additional control in some screening plates. Using a Platemate Plus (Thermofisher Scientific), 500 nL of each test compound was transferred into assay plates (384 clear polystyrene plates) along with standard inhibitor and DMSO in the appropriate control wells. A TryR/DTNB/TrySH mixture (37.5 mL in buffer containing 40 mm Hepes and 1 mm Na 4 EDTA, pH 7.4) was then added to each well (Platemate Plus, Thermofisher Scientific) such that final assay concentrations were 3 nm, 50 mm and 6 mm, respectively. The reaction was started by addition of 4 mL NADPH (4 mL buffer for LO controls), to yield a final assay concentration 150 mm. The reaction was incubated for 35 min at room temperature. The absorbance was then measured at 405 AE 8 nm using the Envision plate reader (Perkin-Elmer).
ActivityBase from IDBS was used for all data processing and analysis. Database querying and report creation was undertaken using SARgen version 5.4 and SARview version 6.1 from IDBS.

Cell-based assays
Trypanosomes (T. b. brucei, BSF 427 vsg221) were seeded in 96-well plates at 2000 cells per well in a volume of 200 mL of HMI-9T [21] containing 10 % FCS. MRC-5 cells were seeded at 2000 cells per well in a volume of 200 mL of DMEM containing 10 % FCS and allowed to adhere for 24 h prior to use. For compound assessment, compounds were serially diluted in 100 % DMSO through a ten-point, one in three dilution curve, in row orientation using a Janus 8 channel Varispan. This produced a working stock of 200 final concentration in the assay. Compound plates contained six test compounds and one standard compound occupying columns 1-10: row A was omitted from screening due to potential edge effect and row H contained the standard compound. Each compound working stock (1 mL) was then stamped into replicate clear 96-well polystyrene assay plates using a Platemate 2 2 (Matrix-Thermofisher) to achieve the final assay concentration at DMSO level of 0.5 %.
Assay plates (200 mL final volume per well) were incubated for 69 h at 37 8C in an atmosphere of 5 % CO 2 . Resazurin (20 mL of 500 mm) was then added to each well and the plates incubated for another 4 h. Plates were read for fluorescence at an excitation wavelength of 528 nm and an emission wavelength of 590 nm.

Mode of inhibition studies
An assay mixture consisting of TryR, NADPH and DTNB was made up in 40 mm HEPES; 1 mm EDTA (pH 7.4). Aliquots of the assay mixture (180 mL) containing three different concentrations of test compound were added to three rows of a microtitre plate, a fourth row contained only the assay mixture. The test compound concentration ranged from~0.25 to 1 times the IC 50 value. Trypanothione disulphide was serially diluted across a fifth row of the plate to produce a 12-point range from 500 mm to 5.8 mm. The assay was initiated by transferring 20 mL of trypanothione disulphide row to each of the assay rows. The final 200 mL assay contained 150 mm NADPH; 50 mm DTNB and 20 mU mL À1 TryR. The linear rate of increase in absorbance at 412 nm was determined using a Molecular Devices Thermomax plate reader. Each data set was fitted by nonlinear regression to the Michaelis-Menten equation using GraFit 5.0 (Erithacus software). The resulting individual fits were examined as Lineweaver-Burke transformations and the graphs inspected for diagnostic inhibition patterns. The entire dataset was then globally fitted to the appropriate equation (competitive, mixed or uncompetitive inhibition).

Procedures for the synthesis of BTCP analogues
Method A (compounds 3, 5, 9-15, 17): [11] nBuLi (1.6 m in hexanes, 4 eq) was added to a solution of the corresponding heteroaromatic compound (4 eq) in anhyd THF (10 mL) at À78 8C and stirred for 1 h. The resultant ArLi solution was then added via a cannula to an ice-cooled solution of the relevant benzotriazoyl adduct prepared by stirring the corresponding enamine (1 eq) and benzotriazole (1 eq) in anhyd Et 2 O (5 mL) for 1 h. The reaction was allowed to warm to RT and stirred for 16 h. Workup was initiated by the addition of aq citrate (10 % w/v, 20 mL), the layers separated and the organic layer further extracted with aq citrate (10 % w/v, 3 20 mL). The combined aqueous layers were basified to pH 10 (2 m aq NaOH), extracted into CH 2 Cl 2 (4 50 mL) and the combined CH 2       1-(1-Naphthalen-2-yl)cyclohexyl)piperidine 7: Prepared by method B1 from 2-bromonaphthalene (10 mmol, 2.07 g). The product was obtained as a white crystalline solid (191 mg, 7 %). The reported analysis is for the HCl salt. 1