Synthesis and Evaluation of Indatraline-Based Inhibitors for Trypanothione Reductase

Abstract

The search for novel compounds of relevance to the treatment of diseases caused by trypanosomatid protozoan parasites continues. Screening of a large library of known bioactive compounds has led to several drug-like starting points for further optimisation. In this study, novel analogues of the monoamine uptake inhibitor indatraline were prepared and assessed both as inhibitors of trypanothione reductase (TryR) and against the parasite Trypanosoma brucei. Although it proved difficult to significantly increase the potency of the original compound as an inhibitor of TryR, some insight into the preferred substituent on the amine group and in the two aromatic rings of the parent indatraline was deduced. In addition, detailed mode of action studies indicated that two of the inhibitors exhibit a mixed mode of inhibition.

Introduction

Trypanosomatid protozoan parasites cause a variety of important diseases, including human African sleeping sickness, Chagas disease, and leishmaniasis. Sleeping sickness is caused by Trypanosoma brucei ssp., and is endemic in certain regions of sub-Saharan Africa, covering about 20countries with an estimated 70–80 000people infected.¹ Chagas disease is present in 19countries on the American continent and is responsible for disease in around 8–11million people.¹ The causative agent of this disease is the parasite Trypanosoma cruzi. Leishmaniasis is caused by members of the genus Leishmania and is found in 88countries, threatening 350million people.¹ In combination, these diseases contribute to approximately 95 000deaths annually, but considering their socioeconomic importance, the current scale of new drug discovery and vaccine development efforts is inadequate. In addition, the available therapies are often toxic, marginally effective, administered by injection, and expensive. In particular, there is an urgent need for new CNS-active drugs to treat late-stage sleeping sickness to replace the current therapies that are losing efficacy due to parasite resistance.¹

The trypanosomatids use a polyamine–glutathione adduct, trypanothione (1, Figure 1), as a key component of their defence system. Compound 1 is prepared through a unique biosynthetic pathway in which glutathione (2) is conjugated to spermidine.² In humans, glutathione and glutathione reductase (GR) are used to maintain the intracellular redox balance, whereas the analogous chemistry in the parasite is carried out by trypanothione reductase (TryR), which reduces trypanothione disulfide (T[S]₂) to 1. Previous genetic knockout studies have illustrated the essential role of TryR in parasite viability,³ validating it as a target for drug development in all three diseases. Importantly, comparison of TryR and human GR crystal structures reveal significant differences between their active sites,⁴ suggesting that these differences may be exploited to gain selectivity for TryR over GR.

Figure 1.

Structures of trypanothione (1), glutathione (2), and indatraline (3).

As part of a concerted campaign to discover new treatments for trypanosomatid-based diseases, we undertook a high-throughput screen for inhibitors of TryR. The Sigma-LOPAC¹²⁸⁰ collection, a library of compounds with known pharmacological activity, was screened against TryR.⁵ The thinking behind screening a library of known drugs is encapsulated in Sir James Black’s famous quote: “The most fruitful basis for the discovery of a new drug is to start with an old drug”.⁶ It was planned that hits derived from small molecules that already have desirable drug-like properties could be modified to tune their selectivity away from their original protein targets and towards TryR without too much disruption of the desirable drug-like properties.

As reported previously,⁵ assessment of initial screening hits against human GR and T. brucei cells together with in silico analysis of chemical properties revealed three new classes of TryR inhibitors that merited further development. Investigation of one of these classes, based on 1-[1-(2-benzo[b]thienyl)cyclohexyl)]piperidine (BTCP), was reported previously.⁷ Herein we describe a related investigation of a further class of compounds based on indatraline (3), a nonselective monoamine reuptake inhibitor.⁸ The molecule contains an indanamine core structure with a second aromatic unit at the 3-position (Figure 1). This CNS-active molecule was considered suitable for extensive modification and was of sufficiently low molecular weight to allow adherence to Lipinski’s rules, even following considerable alteration.⁹ We identified four possible sites (A–D, Figure 1) for modification of the core structure. Details of the parallel synthetic routes that were developed along with careful analysis of the activity of the analogues generated against TryR in vitro and T. brucei in culture are reported. Whilst it proved difficult in this chemical series to improve potency against the desired target, a new important insight into the mode of inhibition of TryR by these analogues was discovered, progressing our thinking on how to inhibit effectively this important enzyme.

Results and Discussion

Synthesis of indatraline analogues

Initial studies focused on the amino substituent in 3 (site A, Figure 1) starting from the common intermediate 3-phenylindanone (4a, Scheme 1). Compound 4a was prepared according to published methods.¹⁰ Treatment of 4a with methylamine in the presence of titanium tetrachloride followed by reduction of the resulting imine with sodium borohydride afforded indanamine 5 as the cis isomer, as reported by Bøgesø et al.^8a Access to the trans-indanamines was achieved as follows:^8a Reduction of indanone 4a with sodium borohydride gave 3-phenylindan-1-ol (6a) in high yield and high cis selectivity (97:3). A single recrystallisation was required to afford the pure cis isomer. Reaction of 6a with thionyl chloride resulted in an isomeric mixture of cis- and trans-1-chloro-3-phenylindanes (7), with a cis/trans ratio of 7:3. Crude 7 was then reacted with a series of primary and secondary alkylamines to produce the corresponding 3-phenylindan-1-amines with, as expected, a reversal of the cis/trans ratio (3:7). The pure trans isomers 8i–vi were isolated following purification by semi-preparative HPLC, and the stereochemistry was assigned by comparison with published work.^8a

Scheme 1.

Reagents and conditions: a) MeNH₂, TiCl₄, PhMe, −10 °C, 1h; b) NaBH₄, MeOH, RT, 3h (62 %); c) NaBH₄, MeOH, RT, 2h (77 %); d) SOCl₂, Tol., RT, 3h; e) NHR¹R², THF, 90 °C, 4h; f) (PhO)₂P(O)N₃, DBU, THF, RT, o/n (93 %); g) PS–PPh₃, H₂O, THF, RT, 16h (quant); h) R¹CHO, NaBH(OAc)₃ or CH₃COCl or TsCl, THF, RT, o/n. R¹ and R² are defined in Table 1 and Supporting Information.¹¹

Having prepared analogues 5 and 8i–vi, we decided to evaluate the routes used for conversion into a parallel synthesis protocol. This was viewed as challenging due to the required separation of the isomeric mixtures of 8 on a small scale. We therefore decided to adapt the original route by incorporating a modified Mitsunobu protocol to convert indanol 6a to the azide 9a (Scheme 1).¹² This reaction occurred with complete inversion of the C1stereochemistry in 6a and generated exclusively the trans isomer of 9a, removing the requirement to separate isomeric mixtures later in the route. A Staudinger reduction of azide 9a using polymer-supported triphenylphosphine generated the indanamine 10a in high yield and purity following filtration to remove the reagent. Reductive amination of 10a with a range of aldehydes in the presence of sodium triacetoxyborohydride afforded the required trans-indanamines 8vii–xiii. Initially, purification of these compounds was necessary, as over-alkylation of 10a was observed. However when an excess of 10a (1.2equivalents) was used in this reaction, none of the dialkylated product was formed, and remaining 10a was removed using a polymer-supported benzaldehyde scavenger resin. With this protocol it was possible to prepare pure trans-indanamines 8vii–xiii in moderate to high yields. Indanamine 10a was also treated with acetyl and tosyl chloride to afford 8xiv and 8xv, respectively. The stereochemistry of 8 and 9 prepared by this route was assigned based on literature precedent from Rice and co-workers.¹²

Having developed a robust route to amino-substituted analogues, we decided to investigate modification of the two aromatic rings present in 3. Rice and colleagues reported a protocol to access the indanone core via an aldol condensation of 3-methoxyacetophenone (11b, Scheme 2) with 3,4-dichlorobenzaldehyde (12a) to generate chalcone (13b).^{12, 13} In their hands, a subsequent Nazarov cyclisation in the presence of trifluoroacetic acid afforded 6-methoxyindanone (4b) in high yield. This route was particularly appealing, as it allowed rapid access to the indanone core in two steps. Furthermore, the product from the aldol reaction crystallised out of the reaction solution and required no further purification. These factors made this approach potentially adaptable to a high-throughput synthesis format. To investigate this further, a series of chalcones (compounds 13a–e) were prepared from the aldol reaction between acetophenones 11a–e and 3,4-dichlorobenzaldehyde (12a). The chalcones 13a–e were then submitted to cyclisation reactions using trifluoroacetic acid. The Nazarov reaction only occurred when a 3-methoxy substituent was present in the chalcone (for substrate 13b). In this case, it was possible to decrease the reaction time considerably by carrying out the reactions in a microwave reactor as opposed to conventional heating (10min versus 4h, respectively). Having determined the requirements for a successful Nazarov reaction, a collection of indanones (4b,f–i) was prepared by using 3-methoxyacetophenone (11b) and a variety of benzaldehydes 12b–e. The electronic nature of the substituent on the benzaldehydes did not influence the outcome of the Nazarov cyclisation, and all the chalcones afforded the desired indanones 4f–i. These were then converted into the indanamines 8xvi–xxi via method B (Scheme 1).

Scheme 2.

Reagents and conditions: a) R⁴CHO, KOH, EtOH, 0 °C, 2h; b) TFA, 120 °C, microwave, 10min; c) NaBH₄, MeOH, RT, 2h; d) (PhO)₂P(O)N₃, DBU, THF, RT, o/n; e) PS–PPh₃, H₂O, THF, RT, 16h; f) R¹CHO, NaBH(OAc)₃, THF, RT, o/n. R¹–R⁴ are defined in Table 1 and Supporting Information.¹¹

Biological analysis against TryR, GR, and T. brucei

The analogues prepared according to the methods described above were tested against T. cruzi TryR and in an assay for T. brucei cell proliferation, as described below in the Experimental Section.

The biological data for a selected group of these compounds are summarised in Table 1. The cis isomer 5 (Table 1, Entry 1) was less active against TryR than indatraline 3, Entry 2). This resulted in the synthetic efforts being focused on the generation of trans analogues. As the size and lipophilicity of the amino substituent was increased (Entries 1and 3–6), there appeared to be a relatively flat SAR (see also analogues S8xxii–S8xxvii in the Supporting Information¹¹). This suggests that this region of the molecule contributes little to the biological activity and is probably incorporated in a large open region of the active site. The slightly lower activity of the N-tBu analogue 8iii may, however, indicate some steric constraint close to the nitrogen atom. In addition, the piperidine (8v, Entry 7) and morpholine (8vi, Entry 8) analogues were prepared. Whilst the piperidine analogue 8v retained activity, the insertion of an additional oxygen atom, in the case of the morpholine analogue 8vi, caused a significant decrease in activity (see also analogue S8xxx in the Supporting Information¹¹). By replacing the methyl substituent of indatraline with a benzyl group (8vii, Entry 9), the activity was retained. This provided the opportunity to synthesise a Topliss series of analogues.¹⁴ Therefore the 4-chloro (8viii, Entry 10), 3,4-dichloro (8ix, Entry 11), 4-methyl (8x, Entry 12), and 4-methoxy (8xi, Entry 13) analogues were prepared. The incorporation of electron-donating substituents led to the highest activity, whereas activity diminished when electron-withdrawing groups were introduced. This result led to the preparation of the remaining two compounds in the electron-rich Topliss series: 4-dimethylamino (8xii, Entry 14) and 4-amino (8xiii, Entry 15). These results further highlight the benefits associated with the inclusion of an electron-donating substituent, although no further improvement in activity was observed relative to the 4-methoxy analogue 8xi. For data on additional substituted benzyl analogues, see table S1, analogues S8xxxi–S8xxxv (Supporting Information). The acetyl (8xiv, Entry 16) and tosyl (8xv, Entry 17) analogues were inactive against TryR (see also analogue S8xxxvi in the Supporting Information¹¹).

Table 1.

Biological data for selected indatraline analogues.

Entry	Compound	R1	R2	R3	R4	TryR IC50 [μm][a]	T. brucei EC50 [μm]
	Pentamidine[b]						0.0037±0.0001
1	3	Me	H	H	3,4-Cl2	8.84±0.24	1.06±0.05
2	5[c]	Me	H	H	3,4-Cl2	13.5±0.7	1.50±0.23
3	8i[b]	Et	H	H	3,4-Cl2	7.19±0.40	2.12±0.06
4	8ii	Isoamyl	H	H	3,4-Cl2	4.05±0.39	1.08±0.07
5	8iii[b]	tBu	H	H	3,4-Cl2	15.3±1.4	0.48±0.04
6	8iv	Octyl	H	H	3,4-Cl2	7.93±0.78	0.66±0.04
7	8v[b]	–CH2(CH2)3CH2–		H	3,4-Cl2	5.47±0.32	1.31±0.11
8	8vi	–(CH2)2O(CH2)2–		H	3,4-Cl2	173±56	6.62±0.77
9	8vii[b]	Benzyl	H	H	3,4-Cl2	8.04±0.91	3.53±0.35
10	8viii	4-Cl-Bn	H	H	3,4-Cl2	17.6±3.3	3.22±0.26
11	8ix	3,4-Cl2-Bn	H	H	3,4-Cl2	45.9±4.2	8.87±0.60
12	8x	4-Me-Bn	H	H	3,4-Cl2	13.0±1.6	1.29±0.04
13	8xi	4-OMe-Bn	H	H	3,4-Cl2	4.14±0.41	2.36±0.22
14	8xii	4-Me2N-Bn	H	H	3,4-Cl2	7.38±1.21	1.98±0.23
15	8xiii	4-NH2-Bn	H	H	3,4-Cl2	4.94±0.43	2.68±1.04
16	8xiv	Acetyl	H	H	3,4-Cl2	>200	3.64±0.28
17	8xv	Tosyl	H	H	3,4-Cl2	>200	4.33±0.33
18	8xvi	Isoamyl	H	6-OMe	3,4-Cl2	2.23±0.66	1.26±0.13
19	8xvii	Isoamyl	H	6-OMe	3,4-Br2	3.15±0.23	2.47±0.25
20	8xviii	Isoamyl	H	6-OMe	4-Cl	15.8±1.6	2.51±0.33
21	8xix	Isoamyl	H	6-OMe	4-Me	32.9±4.3	1.49±0.08
22	8xx	Isoamyl	H	6-OMe	3-OMe	125±17	6.99±1.55
23	8xxi	Benzyl	H	6-OMe	3,4-Cl2	3.07±0.45	2.30±0.22

^[a]Inhibition of trypanothione reductase (TryR).

^[b]Pentamidine was included as a reference drug in biological testing.^16a

^[c]See Ref. 11.

We decided to use the isoamyl group (see 8ii, Entry 4) as the substituent for site A, whilst an exploration of the B and C rings was carried out. The inclusion of a 6-methoxy substituent in the B ring was investigated, as this was a synthetic requirement for the Nazarov reaction. Analogue 8xvi (Entry 18) was prepared as a direct comparison with 8ii (Entry 4). The increased activity of 8xvi relative to 8ii demonstrated that the presence of an electron-donating substituent in the B ring is favourable. Therefore a collection of analogues was synthesised containing the 6-methoxy substituent but with variations in substituents in the C ring. The 3,4-dibromo (8xvii, Entry 19), 4-chloro (8xviii, Entry 20), 4-methyl (8xix, Entry 21), and 3-methoxy (8xx, entry 22) substituted analogues were prepared and tested. A clear preference for electron-withdrawing substituents in the C ring was observed (see also 8xxi and additional analogues in table S1, Supporting Information).

All the analogues were also tested against T. brucei cultured in vitro. These data predominantly followed a trend similar to that of the IC₅₀ data, with decreases in the IC₅₀ values being mirrored by corresponding decreases in the EC₅₀ values. However, there were a few notable exceptions. Compounds 8iii (EC₅₀=0.48 μm, IC₅₀=15.29 μm) and 8iv (EC₅₀=0.66 μm, IC₅₀=7.93 μm) showed much improved EC₅₀ values relative to their corresponding IC₅₀ values. Furthermore, although 8xiv (EC₅₀=3.64 μm, IC₅₀>200 μm) and 8xv (EC₅₀=4.33 μm, IC₅₀>200 μm) were inactive against TryR, they showed reasonable activity against T. brucei. These results could suggest that these analogues have additional off-target effects, or are selectively concentrated/metabolically activated, or a combination of these. In addition, all compounds were tested for inhibition of human GR as described in the Experimental Section below. Compounds were tested in duplicate at a single final concentration of 25 μm. All compounds exhibited <12 % inhibition.

Three of the analogues were chosen for assessment of the mode of inhibition with respect to T[S]₂, as described in the Experimental Section. Indatraline (3), the original indatraline starting point (Figure 2a), is a linear competitive inhibitor of TryR. However, compounds 8ii (Figure 2b) and 8iii were confirmed by an F-test to show a mode of mixed inhibition. The mixed inhibition mode indicates that these inhibitors are able to bind to the substrate–enzyme complex as well as to the free enzyme, whilst the competitive mode indicates binding only to the free enzyme. However, even for the mixed mode inhibitors, the K_i′ values are approximately fivefold greater than the K_i values, suggesting that these compounds bind much more favourably to the free enzyme. This subtle shift in mode of inhibition may represent a useful direction for further development, because mixed inhibitors are less susceptible to the effects of substrate accumulation than competitive inhibitors. For these series, driving down the K_i′ value may be more effective than focussing solely on the potency (IC₅₀) of the inhibitors.

Figure 2.

Mode of inhibition by indatraline and compound 8ii. a) Indatraline: linear competitive inhibition with respect to trypanothione disulfide, K_i=5.0±0.2 μm. b) 8ii: linear mixed inhibition, K_i=3.5±0.4 μm; K_i′=16.8±3.3 μm.

Conclusions

We previously reported the screening of the Sigma-LOPAC¹²⁸⁰ collection of bioactive compounds for inhibitors of TryR and have described follow-up studies on one of the hits we obtained from this collection.^{5, 7} Herein we report our attempts to progress a second hit obtained in this screen. Indatraline 3, a CNS-active, nonselective monoamine reuptake inhibitor, provided the drug-like starting point for our studies. Using a range of synthetic routes, novel indatraline analogues were prepared in order to explore structure–activity relationships. Whilst it proved difficult to significantly increase the potency of the original compound as an inhibitor of TryR, some insight into the preferred substituent on the amine and in the two aromatic rings was deduced. In addition, detailed mode of action studies indicated that two of the inhibitors exhibit a mixed mode of inhibition. This interesting observation has led us to further refine the criteria that need to be considered when developing inhibitors of this important enzyme.

Experimental Section

Procedures for the synthesis of analogues of indatraline (3)

Unless otherwise stated, starting materials and reagents were obtained from commercial suppliers and were used without further purification. ¹H and ¹³C NMR spectra were measured on a Bruker Advance 300/400instrument. Chemical shifts are calibrated with reference to the residual proton and carbon resonances of the solvent (CDCl₃: δ_H=7.26, δ_C=77.0ppm). Low- and high-resolution mass spectrometric analyses were recorded using chemical ionisation operating in positive or negative ion mode. For assessment of purity of novel compounds that were tested, see table S2 (Supporting Information).¹¹

General procedure for the formation of amines 8 (Method A): The corresponding amine (0.5mmol) was added to a solution of 7¹¹ (100mg, 0.34mmol) in THF (3mL), and the solution was stirred at 90 °C in a sealed tube for 4h. The solvent was removed in vacuo, and the residue partitioned between H₂O (10mL) and Et₂O (10mL). The organic layer was extracted with 10 % citric acid (3×10mL), and the extract was basified with 10 % aqueous NH₄OH solution then extracted with Et₂O (3×20mL). The combined organics were dried (Na₂SO₄) and concentrated in vacuo. Purification by semi-preparative HPLC (Phenomenex Luna silica column, EtOAc/Hex 6:4 (0.1 % Et₂NH)) afforded the desired amines.

Trans-[3-(3,4-dichlorophenyl)indan-1-yl]-(3-methylbutyl)amine hydrochloride (8ii): Yield 72 %, white solid; mp: 190–191 °C; ¹H NMR (400 MHz, MeOD): δ=7.71 (1 H, d, J=6.2 Hz, CH_ar), 7.49 (1 H, d, J=8.3 Hz, CH_ar) 7.45–7.43 (2 H, m, CH_ar), 7.34 (1 H, d, J=1.8 Hz, CH_ar), 7.12–7.06 (2 H, m, CH_ar), 4.97–4.95 (1 H, m, CH-N), 4.76 (1 H, t, J=7.6 Hz, CH-Ar), 3.17–3.13 (2 H, m, CH₂N), 2.86–2.81 (1 H, m, CH_syn), 2.53–2.48 (1 H, m, CH_anti), 1.76–1.61 (3 H, m, CH and CH₂), 0.99 (3 H, d, J=6.7 Hz, CH₃), 0.98 (3 H, d, J=6.7 Hz, CH₃); ¹³C NMR (100 MHz, MeOD): δ=148.8 (C), 145.9 (C), 138.4 (C), 133.7 (C), 132.0 (CH ×2), 131.9 (C), 131.2 (CH), 129.3 (CH), 129.1 (CH), 127.3 (CH), 127.0 (CH), 63.3 (CH), 49.4 (CH), 45.8 (CH₂), 40.3 (CH₂), 36.1 (CH₂), 27.3 (CH), 22.7 (CH₃), 22.6 (CH₃); LRMS (CI⁺) m/z: 348.1 [M+H]⁺; HRMS (CI⁺) [M+H]⁺ m/z expected for C₂₀H₂₄N³⁵Cl³⁷Cl 350.1256, obtained 350.1246.

Trans-[3-(3,4-dichlorophenyl)indan-1-yl]octylamine (8iv): Yield 60 %, colourless oil; ¹H NMR (300 MHz, CDCl₃): δ=7.33–7.26 (2 H, m, CH_ar), 7.20–7.15 (3 H, m, CH_ar), 6.91–6.88 (2 H, m, CH_ar), 4.44 (1 H, t, J=7.6 Hz, CH-N), 4.28 (1 H, dd, J=6.8, 3.4 Hz, CH-Ar), 2.65–2.60 (2 H, m, CH₂), 2.41–2.32 (1 H, m, CH_syn), 2.21–2.12 (1 H, m, CH_anti), 1.52–1.39 (2 H, m, CH₂), 1.28–1.12 (10 H, m, CH₂×5), 0.83–0.79 (3 H, m, CH₃); ¹³C NMR (100 MHz, CDCl₃): δ=145.7 (C), 145.4 (C), 145.4 (C), 132.5 (C), 130.4 (CH), 130.3 (C), 129.9 (CH), 128.5 (CH), 127.4 (CH), 127.3 (CH), 125.3 (CH), 124.8 (CH), 124.8 (CH), 61.9 (CH), 48.5 (CH), 47.5 (CH₂), 43.3 (CH₂), 31.8 (CH₂), 29.8 (CH₂), 29.5 (CH₂), 27.4 (CH₂), 22.7 (CH₂), 14.1 (CH₃); LRMS (CI⁺) m/z: 390.2 [M+H]⁺; HRMS (CI⁺) [M+H]⁺ m/z expected for C₂₃H₃₀NCl₂ 390.1755, obtained 390.1755.

Trans-4-[3-(3,4-dichlorophenyl)indan-1-yl]morpholine (8vi): Yield 61 %, colourless oil; ¹H NMR (300 MHz, MeOD): δ=7.80–7.77 (1 H, m, CH_ar), 7.52–7.43 (3 H, m, CH_ar), 7.37 (1 H, d, J=1.8 Hz, CH_ar), 7.12 (1 H, dd, J=8.3, 1.8 Hz, CH_ar), 7.07–7.04 (1 H, m, CH_ar), 5.10 (1 H, d, J=8.0 Hz, CH-N), 4.80 (1 H, t, J=8.0 Hz, CH-Ar), 4.09–4.04 (2 H, m, CH₂), 3.94–3.82 (2 H, m, CH₂), 3.50–3.45 (1 H, m, CH), 3.37–3.35 (2 H, m, CH₂), 3.26–3.06 (2 H, m, CH₂, CH_syn), 2.56–2.45 (1 H, m, CH_anti); ¹³C NMR (75 MHz, MeOD): δ=150.3 (C), 145.8 (C), 135.6 (C), 133.7 (C), 132.6 (CH), 132.0 (CH), 131.4 (CH), 129.3 (CH), 129.2 (CH), 128.7 (CH), 127.1 (CH), 72.0 (CH), 65.2 (CH₂), 65.0 (CH₂), 50.8 (CH₂), 50.3 (CH₂), 50.1 (CH), 38.3 (CH₂); LRMS (CI⁺) m/z: 348.1 [M+H]⁺; HRMS (CI⁺) [M+H]⁺ m/z expected for C₁₉H₂₀NOCl₂ 348.0922, obtained 348.0900.

General procedure for chalcone 13a–i formation: A solution of KOH (6mmol) in H₂O (3mL) was slowly added to a solution of ketone 11 (2mmol) and aldehyde 12 (2mmol) in EtOH (6mL) at 0 °C. The reaction mixture was stirred for 2h, and the resulting precipitate was collected by filtration, washed with EtOH (3mL), and dried in vacuo. See Supporting Information for analytical data for 13a–i.

General procedure for the Nazarov reaction (4b,f–i): A solution of 13 (1mmol) in TFA (4mL) was heated in a sealed 10mL tube in a multimode CEM Discover™ microwave at 120 °C for 10min. The powermax mode was enabled, and the microwave was used at its maximum power of 300 W until the set temperature was reached. The solvent was removed in vacuo, and the residue was poured onto ice water and extracted with EtOAc (3×10mL). The combined extracts were washed with saturated NaHCO₃ solution (20mL) and brine (20mL), dried (Na₂SO₄) and reduced in vacuo. Purification through a plug of silica (Hex/EtOAc 20:1) afforded the desired indanone. See Supporting Information for analytical data for 4b,f–i.

General procedure for the formation of alcohols (6b,f–i): NaBH₄ (0.6mmol) was added to a solution of 4 (0.6mmol) in MeOH (3mL), and the reaction was stirred at room temperature for 2h. A 2 m solution of aqueous KOH (3mL) was added, and the reaction mixture was extracted with CH₂Cl₂ (3×7mL). The combined organic layers were dried (Na₂SO₄) and reduced in vacuo. Purification of the residue through a plug of silica (Hex/EtOAc 10:1) afforded the desired alcohol. See Supporting Information for analytical data for 6b,f–i.

General procedure for the formation of azides 9b,f–i: Diphenylphosphoryl azide (0.48mmol) was added to a solution of 6 (0.4mmol) in anhydrous THF (2mL) at 0 °C, and the reaction was stirred for 10min. DBU (0.48mmol) was slowly added, and the reaction mixture was stirred overnight. H₂O (3mL) was added, and the reaction mixture was extracted with CH₂Cl₂ (3×7mL). The combined organic layers were dried (Na₂SO₄) and reduced in vacuo. Purification of the residue through a plug of silica (Hex/EtOAc 40:1) afforded the desired azide. See Supporting Information for analytical data for 9b,f–i.

General procedure for Staudinger reduction to form 10b,f–i: PS–PPh₃ (0.6mmol) was added to a solution of 9 (0.3mmol) in anhydrous THF (5mL), and the reaction was stirred for 16h. H₂O (1mL) was added, and the reaction was stirred for a further 4h. The reaction mixture was filtered and extracted with CH₂Cl₂ (3×5mL). The combined organic layers were dried (Na₂SO₄) and concentrated in vacuo to give the desired amine 10. See Supporting Information for analytical data for 10b,f–i.

General procedure for reductive amination of 10: The corresponding aldehyde (0.15mmol) was added to a solution of 10 (0.18mmol) in anhydrous THF (1mL), and the solution was stirred for 1h. Sodium triacetoxyborohydride (0.36mmol) was added, and the mixture was stirred overnight. A 2 m solution of aqueous KOH (1mL) was added, and the reaction was extracted with CH₂Cl₂ (3×1mL). The combined organics were dried (Na₂SO₄) and filtered. PS–benzaldehyde resin (0.1mmol) was added, and the mixture was agitated for 2h. The resin was removed by filtration, and the solvent was concentrated in vacuo to afford the desired amines.

Trans-[3-(3,4-dichlorophenyl)indan-1-yl]-(4-chlorobenzyl)amine (8viii): Yield 54 %, colourless oil; ¹H NMR (300 MHz, CDCl₃): δ=7.35–7.14 (9 H, m, CH_ar), 6.93–6.86 (2 H, m, CH_ar), 4.46 (1 H, t, J=7.5 Hz, CH-N), 4.32 (1 H, dd, J=6.8, 3.5 Hz, CH-Ar), 3.79 (2 H, s, CH₂Ar), 2.44–2.36 (1 H, m, CH_syn), 2.23–2.14 (1 H, m, CH_anti); ¹³C NMR (100 MHz, CDCl₃): δ=145.5 (C), 145.5 (C), 145.1 (C), 138.8 (C), 132.7 (C), 132.5 (C), 130.5 (CH), 130.3 (C), 129.9 (CH), 129.4 (CH ×2), 128.5 (CH ×2), 128.4 (CH), 127.4 (CH), 127.3 (CH), 125.3 (CH), 124.6 (CH), 61.6 (CH), 51.0 (CH₂), 48.5 (CH), 43.8 (CH₂); LRMS (CI⁺) m/z: 402.1 [M+H]⁺; HRMS (CI⁺) [M+H]⁺ m/z expected for C₂₂H₁₉NCl₃ 402.0583, obtained 402.0573.

Trans-[3-(3,4-dichlorophenyl)indan-1-yl]-(3,4-dichlorobenzyl)amine (8ix): Yield 73 %, colourless oil; ¹H NMR (300 MHz, CDCl₃): δ=7.42 (1 H, d, J=2.0 Hz, CH_ar), 7.34 (1 H, dd, J=6.4, 2.6 Hz, CH_ar), 7.31 (1 H, d, J=8.2 Hz, CH_ar), 7.28 (1 H, d, J=8.2 Hz, CH_ar), 7.22–7.18 (2 H, m, CH_ar), 7.16–7.12 (2 H, m, CH_ar), 6.94–6.91 (1 H, m, CH_ar), 6.88 (1 H, dd, J=8.2, 2.0 Hz, CH_ar), 4.46 (1 H, t, J=7.5 Hz, CH-N), 4.32 (1 H, dd, J=6.7, 3.6 Hz, CH-Ar), 3.78 (2 H, s, CH₂-Ar), 2.43–2.35 (1 H, m, CH_syn), 2.24–2.15 (1 H, m, CH_anti); ¹³C NMR (100 MHz, CDCl₃): δ=145.4 (C), 145.4 (C), 145.0 (C), 140.8 (C), 132.5 (C), 132.4 (C), 130.8 (C), 130.5 (CH), 130.3 (CH), 129.9 (CH), 129.8 (CH), 129.5 (C), 128.5 (CH), 127.4 (CH), 127.4 (CH), 127.3 (CH), 125.3 (CH), 124.6 (CH), 61.6 (CH), 50.5 (CH₂), 48.5 (CH), 43.8 (CH₂); LRMS (CI⁺) m/z: 436.0 [M+H]⁺; HRMS (CI⁺) [M+H]⁺ m/z expected for C₂₂H₁₈NCl₄ 436.0193, obtained 436.0194.

Trans-[3-(3,4-dichlorophenyl)indan-1-yl]-(4-methylbenzyl)amine (8x): Yield 77 %, colourless oil; ¹H NMR (300 MHz, CDCl₃): δ=7.35–7.34 (1 H, m, CH_ar), 7.27 (1 H, d, J=8.2 Hz, CH_ar), 7.19–7.14 (5 H, m, CH_ar), 7.08–7.06 (2 H, m, CH_ar), 6.92–6.86 (2 H, m, CH_ar), 4.46 (1 H, t, J=7.5 Hz, CH-N), 4.33 (1 H, dd, J=6.8, 3.5 Hz, CH-Ar), 3.78 (2 H, s, CH₂-Ar), 2.45–2.37 (1 H, m, CH_syn), 2.26 (3 H, s, CH₃), 2.22–2.13 (1 H, m, CH_anti); ¹³C NMR (75 MHz, CDCl₃): δ=145.6 (C), 145.5 (C), 145.5 (C), 137.2 (C), 136.6 (C), 132.4 (C), 130.4 (CH), 130.2 (C), 129.9 (CH), 129.1 (CH ×2), 128.2 (CH), 128.1 (CH ×2), 127.4 (CH), 127.2 (CH), 125.2 (CH), 124.6 (CH), 61.4 (CH), 51.5 (CH₂), 48.6 (CH), 43.8 (CH₂), 21.1 (CH₃); LRMS (CI⁺) m/z: 382.1 [M+H]⁺; HRMS (CI⁺) [M+H]⁺ m/z expected for C₂₃H₂₂NCl₂ 382.1129, obtained 382.1134.

Trans-[3-(3,4-dichlorophenyl)indan-1-yl]-(4-methoxybenzyl)amine (8xi): Yield 83 %, colourless oil; ¹H NMR (300 MHz, CDCl₃): δ=7.42–7.39 (1 H, m, CH_ar), 7.35 (1 H, d, J=8.3 Hz, CH_ar), 7.30–7.22 (5 H, m, CH_ar), 6.99–6.94 (2 H, m, CH_ar), 6.91–6.85 (2 H, m, CH_ar), 4.54 (1 H, t, J=7.7 Hz, CH-N), 4.41 (1 H, dd, J=6.8, 3.5 Hz, CH-Ar), 3.84 (2 H, s, CH₂-Ar), 3.80 (3 H, s, CH₃), 2.49 (1 H, ddd, J=13.1, 7.7, 3.5 Hz, CH_syn), 2.25 (1 H, dt, J=13.1, 6.8 Hz, CH_anti), 1.69 (1 H, br s, NH); ¹³C NMR (75 MHz, CDCl₃): δ=158.7 (C), 145.6 (C), 145.5 (C), 145.5 (C), 145.3 (C), 132.4 (C), 130.4 (CH), 130.2 (C), 129.9 (CH), 129.3 (CH ×2), 128.2 (CH), 127.4 (CH), 127.2 (CH), 125.2 (CH), 124.6 (CH), 113.8 (CH ×2), 61.5 (CH), 55.3 (CH₃), 51.2 (CH₂), 48.6 (CH), 43.6 (CH₂); LRMS (CI⁺) m/z: 398.1 [M+H]⁺; HRMS (CI⁺) [M+H]⁺ m/z expected for C₂₃H₂₂NOCl₂ 398.1078, obtained 398.1082.

Trans-[3-(3,4-dichlorophenyl)indan-1-yl]-(4-dimethylaminobenzyl)amine (8xii): Yield 68 %, colourless oil; ¹H NMR (300 MHz, CDCl₃): δ=7.46–7.43 (1 H, m, CH_ar), 7.38 (1 H, d, J=8.3 Hz, CH_ar), 7.32–7.24 (5 H, m, CH_ar), 7.02–6.98 (2 H, m, CH_ar), 6.76 (2 H, d, J=8.5 Hz, CH_ar), 4.58 (1 H, t, J=7.7 Hz, CH-N), 4.46 (1 H, dd, J=6.9, 3.3 Hz, CH-Ar), 3.84 (2 H, s, CH₂-Ar), 2.97 (6 H, s, CH₃ ×2), 2.53 (1 H, ddd, J=13.0, 7.7, 3.3 Hz, CH_syn), 2.28 (1 H, dt, J=13.0, 6.9 Hz, CH_anti), 2.15 (1 H, br s, NH); ¹³C NMR (75 MHz, CDCl₃): δ=149.8 (C), 145.6 (C), 145.5 (C), 145.3 (C), 132.4 (C), 130.4 (CH), 130.1 (C), 129.9 (CH), 129.1 (CH ×2), 128.1 (CH), 128.0 (C), 127.4 (CH), 127.2 (CH), 125.1 (CH), 124.6 (CH), 112.7 (CH ×2), 61.3 (CH), 51.3 (CH₂), 48.5 (CH), 43.7 (CH₂), 40.9 (CH₃ ×2); LRMS (CI⁺) m/z: 411.1 [M+H]⁺; HRMS (CI⁺) [M+H]⁺ m/z expected for C₂₄H₂₅N₂Cl₂ 411.1395, obtained 411.1385.

Trans-[3-(3,4-dichlorophenyl)indan-1-yl]-(4-aminobenzyl)amine (8xiii): Yield 76 %, colourless oil; ¹H NMR (400 MHz, CDCl₃): δ=7.41–7.39 (1 H, m, CH_ar), 7.34 (1 H, d, J=8.3 Hz, CH_ar), 7.26–7.21 (3 H, m, CH_ar), 7.15 (2 H, d, J=8.4 Hz, CH_ar), 6.98–6.95 (2 H, m, CH_ar), 6.66 (2 H, d, J=8.4 Hz, CH_ar), 4.54 (1 H, t, J=7.8 Hz, CH-N), 4.41 (1 H, dd, J=6.9, 3.3 Hz, CH-Ar), 3.78 (2 H, d, J=3.0 Hz, CH₂-Ar), 2.49 (1 H, ddd, J=13.2, 7.8, 3.3 Hz, CH_syn), 2.24 (1 H, dt, J=13.2, 6.9 Hz, CH_anti), 1.75 (3 H, br s, NH ×3); ¹³C NMR (100 MHz, CDCl₃): δ=149.6 (C), 145.6 (C), 145.5 (C), 145.3 (C), 132.4 (C), 130.4 (CH), 130.1 (C), 129.9 (CH), 129.3 (CH ×2), 128.2 (CH), 128.0 (C), 127.4 (CH), 127.2 (CH), 125.2 (CH), 124.6 (CH), 115.2 (CH ×2), 61.3 (CH), 51.3 (CH₂), 48.6 (CH), 43.7 (CH₂); LRMS (CI⁺) m/z: 383.1 [M+H]⁺; HRMS (CI⁺) [M+H]⁺ m/z expected for C₂₂H₂₁N₂Cl₂ 383.1082, obtained 383.1084.

Trans-N-[3-(3,4-dichlorophenyl)indan-1-yl]acetamide (8xiv): Acetyl chloride (15.6 μL, 0.22mmol) and DIPEA (115 μL, 0.66mmol) were added to a solution of 10a (50mg, 0.18mmol) in anhydrous THF (1mL), and the solution was stirred overnight. A saturated aqueous solution of NaHCO₃ (1mL) was added, and the reaction was extracted with CH₂Cl₂ (3×1mL). The combined organics were dried (Na₂SO₄) and concentrated in vacuo. Purification through a plug of silica (Hex/EtOAc 5:1) afforded 8xiv as a white solid (84 %); mp: 127–128 °C; ¹H NMR (300 MHz, CDCl₃): δ=7.42–7.39 (1 H, m, CH_ar), 7.35 (1 H, d, J=8.2 Hz, CH_ar), 7.32–7.28 (2 H, m, CH_ar), 7.19 (1 H, d, J=2.1 Hz, CH_ar), 7.05–7.02 (1 H, m, CH_ar), 6.92 (1 H, dd, J=8.3, 2.1 Hz, CH_ar), 5.67–5.56 (2 H, m, NH, CH-N), 4.46 (1 H, t, J=7.2 Hz, CH-Ar), 2.52–2.42 (2 H, m, CH₂), 2.03 (3 H, s, CH₃); LRMS (CI⁺) m/z: 320.1 [M+H]⁺; HRMS (CI⁺) [M+H]⁺ m/z expected for C₁₇H₁₆Cl₂NO 320.0609, obtained 320.0616.

Trans-N-[3-(3,4-dichlorophenyl)indan-1-yl]-4-methylbenzenesulfonamide (8xv): p-Toluenesulfonyl chloride (42mg, 0.22mmol) and DIPEA (115 μL, 0.66mmol) were added to a solution of 10a (50mg, 0.18mmol) in anhydrous THF (1mL), and the solution was stirred overnight. A saturated aqueous solution of NaHCO₃ was added, and the reaction was extracted with CH₂Cl₂ (3×1mL). The combined organics were dried (Na₂SO₄) and concentrated in vacuo. Purification through a plug of silica (Hex/EtOAc 5:1) afforded 8xv as a white solid (77 %); mp: 149–150 °C; ¹H NMR (300 MHz, CDCl₃): δ=7.82 (2 H, d, J=8.3 Hz, CH_ar), 7.36–7.31 (3 H, m, CH_ar), 7.26–7.23 (2 H, m, CH_ar), 7.13–7.10 (2 H, m, CH_ar), 7.00–6.97 (1 H, m, CH_ar), 6.85 (1 H, d, J=8.3, 2.1 Hz, CH_ar), 4.97–4.91 (1 H, m, CH-N), 4.65 (1 H, d, J=8.0 Hz, NH), 4.44–4.39 (1 H, m, CH-Ar), 2.46 (3 H, s, CH₃), 2.44–2.38 (1 H, m, CH_syn), 2.27–2.18 (1 H, m, CH_anti); ¹³C NMR (75 MHz, CDCl₃): δ=144.9 (C), 144.4 (C), 143.7 (C), 141.9 (C), 137.8 (C), 134.2 (C), 132.6 (C), 130.6 (CH), 129.9 (CH ×2), 129.6 (CH), 129.5 (CH), 129.3 (CH), 128.1 (CH), 127.1 (CH ×2), 125.3 (CH), 124.7 (CH), 57.7 (CH), 47.9 (CH), 44.1 (CH₂), 21.6 (CH₃); LRMS (CI⁺) m/z: 432.1 [M+H]⁺; HRMS (CI⁺) [M+H]⁺ m/z expected for C₂₂H₂₀NO₂Cl₂S 432.0592, obtained 432.0590.

Trans-[3-(3,4-dichlorophenyl)-6-methoxyindan-1-yl]-(3-methylbutyl)amine (8xvi): Yield (52 %), colourless oil; ¹H NMR (300 MHz, CDCl₃): δ=7.28 (1 H, d, J=8.3 Hz, CH_ar), 7.13 (1 H, d, J=2.2 Hz, CH_ar), 6.90–6.80 (3 H, m, CH_ar), 6.71 (1 H, dd, J=8.3, 2.2 Hz, CH_ar), 4.36 (1 H, t, J=7.3 Hz, CH-N), 4.25 (1 H, dd, J=6.8, 4.0 Hz, CH-Ar), 3.75 (3 H, s, CH₃), 2.67–2.62 (2 H, m, CH₂), 2.40–2.32 (1 H, m, CH_syn), 2.23–2.14 (1 H, m, CH_anti), 1.58 (1 H, sept, J=6.6 Hz, CH), 1.38–1.31 (2 H, m, CH₂), 0.84 (3 H, d, J=6.6 Hz, CH₃), 0.83 (3 H, d, J=6.6 Hz, CH₃); ¹³C NMR (75 MHz, CDCl₃): δ=159.5 (C), 144.3 (C), 139.1 (C), 137.4 (C), 132.7 (C), 130.9 (C), 130.6 (CH), 129.9 (CH), 127.3 (CH), 126.3 (CH), 117.9 (CH), 110.9 (CH), 60.9 (CH), 55.7 (CH₂), 48.0 (CH), 42.8 (CH₂), 39.4 (CH₂), 34.4 (CH₂), 26.2 (CH), 22.3 (CH₃), 22.1 (CH₃); LRMS (CI⁺) m/z: 378.1 [M+H]⁺; HRMS (CI⁺) [M+H]⁺ m/z expected for C₂₁H₂₆Cl₂NO 378.1391, obtained 378.1399.

Trans-[3-(3,4-dibromophenyl)-6-methoxyindan-1-yl]-(3-methylbutyl)amine (8xvii): Yield (71 %), colourless oil; ¹H NMR (300 MHz, CDCl₃): δ=7.50 (1 H, d, J=8.2 Hz, CH_ar), 7.38 (1 H, d, J=2.0 Hz, CH_ar), 6.93–6.86 (3 H, m, CH_ar), 6.79 (1 H, dd, J=8.4, 2.5 Hz, CH_ar), 4.42 (1 H, t, J=7.5 Hz, CH-N), 4.31 (1 H, dd, J=6.8, 3.6 Hz, CH-Ar), 3.82 (3 H, s, CH₃), 2.73–2.68 (2 H, m, CH₂), 2.47–2.39 (1 H, m, CH_syn), 2.29–2.21 (1 H, m, CH_anti), 1.64 (1 H, sept, J=6.6 Hz, CH), 1.45–1.37 (2 H, m, CH₂), 0.91 (3 H, d, J=6.6 Hz, CH₃), 0.90 (3 H, d, J=6.6 Hz, CH₃); LRMS (CI⁺) m/z: 468.0 [M+H]⁺; HRMS (CI⁺) [M+H]⁺ m/z expected for C₂₁H₂₆NO⁷⁹Br⁸¹Br 468.0361, obtained 468.0356.

Trans-[3-(4-chlorophenyl)-6-methoxyindan-1-yl]-(3-methylbutyl)amine (8xviii): Yield (56 %), colourless oil; ¹H NMR (300 MHz, CDCl₃): δ=7.24–7.22 (2 H, m, CH_ar), 7.05–6.86 (4 H, m, CH_ar), 6.77 (1 H, dd, J=8.3, 2.6 Hz, CH_ar), 4.45 (1 H, t, J=7.3 Hz, CH-N), 4.31 (1 H, dd, J=7.1, 3.8 Hz, CH-Ar), 3.82 (3 H, s, CH₃), 2.72–2.69 (2 H, m, CH₂), 2.47–2.38 (1 H, m, CH_syn), 2.31–2.22 (1 H, m, CH_anti), 1.62 (1 H, sept, J=6.6 Hz, CH), 1.45–1.38 (2 H, m, CH₂), 0.91 (3 H, d, J=6.6 Hz, CH₃), 0.90 (3 H, d, J=6.6 Hz, CH₃); LRMS (CI⁺) m/z: 344.2 [M+H]⁺; HRMS (CI⁺) [M+H]⁺ m/z expected for C₂₁H₂₇NOCl 344.1781, obtained 344.1784.

Trans-[3-(4-methylphenyl)-6-methoxyindan-1-yl]-(3-methylbutyl)amine (8xix): Yield (73 %), colourless oil; ¹H NMR (300 MHz, CDCl₃): δ=7.05 (2 H, d, J=8.0 Hz, CH_ar), 7.01 (2 H, d, J=8.0 Hz, CH_ar), 6.97–6.89 (2 H, m, CH_ar), 6.76 (1 H, dd, J=8.2, 2.4 Hz, CH_ar), 4.44 (1 H, t, J=7.4 Hz, CH-N), 4.32 (1 H, dd, J=6.7, 3.7 Hz, CH-Ar), 3.82 (3 H, s, CH₃), 2.74–2.70 (2 H, m, CH₂), 2.45–2.39 (1 H, m, CH_syn), 2.31–2.24 (4 H, m, CH₃, CH_anti), 1.64 (1 H, sept, J=6.6 Hz, CH), 1.44–1.39 (2 H, m, CH₂), 0.91 (3 H, d, J=6.6 Hz, CH₃), 0.90 (3 H, d, J=6.6 Hz, CH₃); LRMS (CI⁺) m/z: 324.2 [M+H]⁺; HRMS (CI⁺) [M+H]⁺ m/z expected for C₂₂H₃₀NO 324.2327, obtained 324.2332.

Trans-[3-(4-methoxyphenyl)-6-methoxyindan-1-yl]-(3-methylbutyl)amine (8xx): Yield (76 %), colourless oil; ¹H NMR (300 MHz, CDCl₃): δ=7.22–7.16 (1 H, m, CH_ar), 6.94–6.91 (2 H, m, CH_ar), 6.78–6.61 (4 H, m, CH_ar), 4.45 (1 H, t, J=7.4 Hz, CH-N), 4.33 (1 H, dd, J=6.7, 3.7 Hz, CH-Ar), 3.82 (3 H, s, CH₃), 3.75 (3 H, s, CH₃), 2.74–2.70 (2 H, m, CH₂), 2.46–2.39 (1 H, m, CH_syn), 2.31–2.26 (1 H, m, CH_anti), 1.64 (1 H, sept, J=6.6 Hz, CH), 1.44–1.39 (2 H, m, CH₂), 0.91 (3 H, d, J=6.6 Hz, CH₃), 0.90 (3 H, d, J=6.6 Hz, CH₃); LRMS (CI⁺) m/z: 340.2 [M+H]⁺; HRMS (CI⁺) [M+H]⁺ m/z expected for C₂₂H₃₀NO₂ 340.2277, obtained 340.2276.

Trans-benzyl-[3-(3,4-dichlorophenyl)-6-methoxyindan-1-yl]amine (8xxi): Yield (59 %), colourless oil; ¹H NMR (300 MHz, CDCl₃): δ=7.32–7.12 (7 H, m, CH_ar), 6.89–6.81 (3 H, m, CH_ar), 6.72 (1 H, dd, J=8.3, 2.5 Hz, CH_ar), 4.39 (1 H, t, J=7.3 Hz, CH-N), 4.31 (1 H, dd, J=6.8, 4.0 Hz, CH-Ar), 3.82 (2 H, s, CH₂-Ph), 3.75 (3 H, s, CH₃), 2.46–2.37 (1 H, m, CH_syn), 2.24–2.15 (1 H, m, CH_anti); ¹³C NMR (75 MHz, CDCl₃): δ=159.5 (C), 144.4 (C), 144.4 (C), 139.2 (C), 137.5 (C), 132.6 (C), 130.6 (CH), 130.5 (CH ×2), 130.1 (C), 129.9 (CH), 129.1 (CH ×2), 127.4 (CH), 126.3 (CH), 117.9 (CH), 110.8 (CH), 59.7 (CH), 55.7 (CH₃), 47.9 (CH), 47.7 (CH₂), 39.1 (CH₂); LRMS (CI⁺) m/z: 398.1 [M+H]⁺; HRMS (CI⁺) [M+H]⁺ m/z expected for C₂₃H₂₂NOCl₂ 398.1078, obtained 398.1090.

Inhibition of trypanothione reductase: Compounds were tested for inhibition of T. cruzi TryR by using a high-throughput microplate assay.¹⁵ Briefly, assays were set up in 96-well plates using a Biotek Precision 2000automated liquid handler and initiated with NADPH. The final assay mixtures (0.18mL) contained TryR (20mU mL⁻¹), 40mm HEPES (pH 7.5), 1mm EDTA, 0.15mm NADPH, 50 μm DTNB, 6 μm T[S]₂, and inhibitor (100 μm–5nm in threefold serial dilutions). The rate of TNB⁻ formation was monitored over 5min in a Spectramax 340PC plate reader (Molecular Devices) at λ=412nm. Raw data were processed with Microsoft Excel. GraFit 5.0 (Erithacus software) was used to fit the data to a three-parameter equation and output the concentration resulting in 50 % inhibition (IC₅₀ value). Compounds were tested against TryR on three separate occasions, and the IC₅₀ values were used to calculate a mean weighted to the standard error. These values are given in Table 1. The mean Z′ value throughout the IC₅₀ testing was 0.88, indicating suitable assay performance.

Inhibition of glutathione reductase: Compounds were tested for inhibition of human GR by using a modification of the TryR assay method and processed as before. The final conditions in the assay were 7 μm glutathione disulfide, 150 μm NADPH, 50 μm DTNB, and 16nm human GR.

Assessment of mode of inhibition: Three of the compounds (3, 8ii, and 8iii) were tested for mode of inhibition with respect to trypanothione. The standard TryR assay was used as before. Aliquots of the assay mixture (180 μL) containing three different concentrations of test compound were added to three rows of a microtitre plate, a fourth row contained only the assay mixture. T[S]₂ was serially diluted across a fifth row of the plate to produce a 12-point range from 500–5.8 μm. The assay was initiated by transferring 20 μL of T[S]₂ row to each of the assay rows. The final 200 μL assay contained 150 μm NADPH, 50 μm DTNB, and 20mU mL⁻¹ TryR and 50–0.58 μm T[S]₂. The final inhibitor concentration in the three rows of the plate ranged from 0.5× to 3× IC₅₀. The rate of reaction was measured as before. 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–Burk transformations, and the graphs were inspected for diagnostic inhibition patterns. The entire dataset was then globally fitted to the appropriate equation (competitive, mixed, or uncompetitive inhibition).

Inhibition of T. brucei cell proliferation: Compounds were tested for inhibition of proliferation of whole T. brucei bloodstream form cells using a microplate assay as previously described.¹⁶ Cells were cultured in modified HMI-9media containing 10 % FBS and 10 % Serum Plus (HMI-9T), in which 0.2mm 2-mercaptoethanol was replaced with 0.056mm thioglycerol.¹⁷ Briefly, test compounds (10mm in DMSO) were serially diluted in medium in 96-well plates (Greiner). Bloodstream T. brucei (S427) cells were added to give a final density of 10³ cells mL⁻¹ in 0.5 % DMSO with inhibitor ranging from 50to 0.2 μm in a total volume of 200 μL. Pentamidine (250–1nm) was included as a standard drug control on each test plate. Plates were incubated at 37 °C in a 5 % CO₂ humidified atmosphere for 72h, then 45 μm resazurin (Sigma) was added. After incubation for 4–5h, fluorescence due to formation of resorufin was measured at λ_ex=528nm, λ_em=590nm. Compounds were tested against T. brucei cells on three separate occasions, and the EC₅₀ values were used to calculate a mean weighted to the standard error. These values are given in Table 1. The mean Z′ value throughout the T. brucei testing was 0.67, indicating suitable assay performance.