Discovery of small molecule benzothiazole and indole derivatives tackling tau 2N4R and α-synuclein fibrils

Abstract

Tau and α-synuclein aggregates are the main histopathological hallmarks present in Alzheimer’s disease (AD), Parkinson’s disease (PD), and other neurodegenerative disorders. Intraneuronal hyperphosphorylated tau accumulation is significantly connected to the degree of cognitive impairment in AD patients. In particular, the longest 2N4R tau isoform has a propensity to rapidly form oligomers and mature fibrils. On the other hand, misfolding of α-synuclein (α-syn) is the characteristic feature in PD and dementia with Lewy bodies (DLB). There is a strong crosstalk between the two prone-to-aggregation proteins as they coprecipitated in some brains of AD, PD, and DLB patients. Simultaneous targeting of both proteinaceous oligomers and aggregates is still challenging. Here, we rationally designed and synthesized benzothiazole- and indole-based compounds using the structural hybridization strategy between the benzothiazole N744 cyanine dye and the diphenyl pyrazole Anle138b that showed anti-aggregation activity towards 2N4R tau and α-syn, respectively. The anti-aggregation effect of the prepared compounds was monitored using the thioflavin-T (ThT) fluorescence assay, while transmission electron microscopy (TEM) was employed to detect fibrils upon the completion of a time-course study with the ThT assay. Moreover, the photo-induced crosslinking of unmodified protein (PICUP) assay was used to determine the formation of oligomers. Specifically, compounds 46 and 48 demonstrated the highest anti-aggregation activity by decreasing the ThT fluorescence to 4.0 and 14.8%, respectively, against α-syn. Although no noticeable effect on 2N4R tau oligomers, 46 showed promising anti-oligomer activity against α-syn. Both compounds induced a significantly high anti-aggregation effect against the two protein fibrils as visualized by TEM. Moreover, compound 48 remarkably inhibited α-syn inclusion and cell confluence using M17D cells. Collectively, compounds 46 and 48 could serve as a basic structure for further optimization to develop clinically active AD and PD disease-modifying agents.

1. Introduction

The most prevalent neurodegenerative disorders impacting millions of patients and their caregivers annually are Alzheimer’s disease (AD) and Parkinson’s disease (PD).^1,2 Classic AD symptoms include dementia, memory decline or loss, coherent speech difficulty, and general cognitive impairment.^3,4 While in PD, the symptoms are mainly motorial including bradykinesia, tremor, and postural instability, however, nonmotor signs such as depression and sleep disturbance have also been experienced in some patients.^5–7 The amyloid cascade hypothesis suggested that progressive deposition of amyloid-β (Aβ) protein is the pivotal hallmark in AD, and all the attendant pathological events such as neurofibrillary tangles (NFTs), formation of hyperphosphorylated tau protein, neuritic and glial cytopathology, and eventually dementia directly result from this deposition.^8–10 The Food and Drug Administration (FDA) has recently approved lecanemab as an anti-Aβ monoclonal antibody for a breakthrough treatment of AD after its impressive clinical results in clearing Aβ and reducing cognitive impairment.^11–13 However, infusion reactions and amyloid-related imaging abnormalities with edema or effusions (ARIA-E) are the most common adverse effects linked with lecanemab.^14,15

Recent findings emphasized that microtubule-associated protein tau (MAPT), hyperphosphorylated tau, and NFTs are more strongly linked with the degree of cognitive decline than with Aβ aggregates.¹⁶. In addition to AD, tau misfolding plays a role in the physiopathology of many neurodegenerative diseases such as frontotemporal dementia with Parkinsonism (FTDP-17), Pick’s disease, Down’s syndrome, and progressive supranuclear palsy (PSP), collectively termed tauopathies.^17,18 Accordingly, tau has gained surprisingly high attention as a therapeutic target for AD and other tauopathies in the past few decades. Tau is present in six isoforms in the human brain which are categorized by the absence or the presence of 29-amino-acid N-terminal domain (0 N, 1 N, and 2 N) and the number of microtubule-binding C-terminal repeats (3R and 4R).^19,20 Importantly, the 4R isoforms have faster aggregation rates and consequently higher propensity to produce neurodegeneration than the 3R variants.²¹ The benzothiazole cyanine dye N744 was identified as a strong tau aggregation inhibitor.²² N744 was found not only to inhibit the full-length (2N4R) tau aggregates (IC₅₀ = 300 nM) but also remarkably induce the disaggregation of preformed filaments.²³

In contrast, aberrant accumulation of intracellular proteinaceous aggregates of α-synuclein (α-syn) is the main histopathological characteristic of PD and dementia with Lewy bodies (DLB).^24,25 α-Syn is a presynaptic acidic protein that plays potential roles in the management of synaptic vesicle release and neuronal survival.²⁶ Under pathological conditions, misfolding of α-syn forms Lewy bodies (LBs) in the neurons.²⁷ Braak and other colleagues have identified the correlation between LBs and disease progression.^28,29 Recently, the diphenyl pyrazole Anle138b has been synthesized as a disease-modifying therapy for PD by inhibiting the formation of α-syn pathological aggregates (10 μM, 77 % inhibition of α-syn fibrils) in vitro as well as in vivo settings.³⁰ Fluorescence measurements of Anle138b showed that significant fluorescence change occurred upon incubation with α-syn fibrils but not with monomers.³¹

As a corollary, there is a noticeable interplay between tau and LBs of α-syn as both aggregates coprecipitate in some brains of AD, PD, and DLB patients.^32,33 Moreover, tau and α-syn synergistically facilitate the formation of each other’s aggregates in mouse models.^34–36 The green tea catechin, (−)-epi-gallocatechin-3-gallate (EGCG), demonstrated significant anti-amyloidogenic characteristics against Aβ, tau, and α-syn aggregates.³⁷ EGCG was able to reduce the fluorescence of Thioflavin-T (ThT) of approximately 90 %, limiting substantially the formation α-syn aggregates with a powerful inhibitory activity against oligomer formation.^38,39 However, due to its polyphenol groups, EGCG exhibits a reduced membrane permeability and is subjected to high metabolic transformation which limit its clinical applications.^40,41 Accordingly, there is an urgent need to design and prepare small molecules targeting both protein aggregates to reduce the progression of neurodegeneration.

Capitalization on the structures of N744 and Anle138b, benzothiazole and indole derivatives have been judiciously designed and synthesized by simple single-step reactions to reduce the 2N4R tau and α-syn aggregates. In particular, the indole derivatives with urea linker, 46 and 48, displayed the most promising results as a dual aggregation inhibitor for 2N4R tau and α-syn fibrils. Compounds 46 and 48 significantly decreased the ThT fluorescence to below 20 % against α-syn fibrils which are comparable to that of EGCG using similar assay conditions (see Supporting Information, Fig. S1). Moreover, 46 induced a noticeable inhibitory activity against α-syn oligomers, but not with the 2N4R tau oligomers. The use of transmission electron microscopy (TEM) revealed the powerful anti-aggregation properties of 46 and 48 on 2N4R tau and α-syn aggregates. In addition, 48 was highly effective in reducing α-syn inclusion using M17D cells.

2. Results and discussion

2.1. Design of the prepared compounds

Structural hybridization of two biologically active pharmacophores has been widely used as a common strategy to build up new hybrid scaffolds capable of binding to multitarget proteins.^42–45 We selected the methoxy benzothiazole pharmacophore from N744 dye to guarantee the high affinity and inhibitory activity toward the β-sheet structures of 2N4R tau aggregates. On the other hand, structural analysis of Anle138b showed that the phenyl ring substituted with the electron-withdrawing (EWG) bromine atom has the highest activity against α-syn aggregates.³⁰ Therefore, the elaborated compounds were designed by integrating the 4-methoxy benzothiazole moiety and various EWG-phenyl rings in one hybrid molecule. To further investigate the effect of the EWG groups, we also prepared the unsubstituted, electron-releasing, and 2-thiophenyl-containing counterparts (see Supporting Information, Table S1, Figures S2–S3). The two pharmacophores were separated by three different linkers: urea, amide, and sulfonamide. The most promising candidates were further optimized by replacing the benzothiazole ring with 4-,5-, and 6-substituted indole scaffolds (Fig. 1).

Fig. 1.

Design of methoxy benzothiazole and indole derivatives as dual 2N4R tau and α-syn aggregates inhibitors.

2.2. Chemistry

The designed compounds were synthesized using the commercially available 2-amino-4-methoxy benzothiazole (4-MBT). To prepare compounds with the urea linker, a quantitative amount of the appropriate phenyl isocyanate was used in dichloromethane (DCM). The amide or the sulfonamide counterparts were synthesized using the corresponding benzoyl chloride or benzene sulfonyl chloride, respectively, in the presence of anhydrous potassium carbonate and pyridine (Scheme 1). Then, the best candidates, with the 2-iodo and the 4-nitro sulfonamides and their urea analogs, were further modified by replacing the 4-MBT with 4-, 5-, or 6-amino indole (AI) using the same reaction conditions (Scheme 2). All products were obtained with moderate to quantitative yields (51–100 %).

Scheme 1.

Reagents and conditions: (a) DCM, r.t., 3–12 h, 71–100 %; (b) anhydrous K₂CO₃, pyridine, r.t., 8–10 h, 51–88 %.

Scheme 2.

Reagents and conditions: (a) DCM, r.t., 3–12 h, 79–100 %; (b) anhydrous K₂CO₃, pyridine, r.t., 3–12 h, 73–86 %.

2.3. Biophysical evaluation

2.3.1. Thioflavin-T (ThT) fluorescence assay

Thioflavin-T (ThT) is a fluorescent benzothiazole dye that upon binding to the cross β–sheet structures present in numerous prone-to-aggregate proteins. Upon binding, the dye results in a significant fluorescence quantum yield increase with a red shift in its excitation and emission spectra. The ThT assay was used not only to detect the presence of fibrils but also to measure the kinetics of the late-stage protein fibrillization process. Dense fibrils are formed at the plateau phase of the kinetic curve where equilibrium between disaggregation and aggregation occurs. Thus, the ThT assay was applied as a first-line screening step to monitor the antifibrillar activity of the newly synthesized 4-MBT compounds on α-syn protein. As expected, the unsubstituted, electron-releasing, and 2-thiophenyl-containing derivatives showed no activity against the α-syn aggregation via ThT fluorescence (Table S1). The 2-iodophenyl sulfonamide derivative 35 and the 4-nitrophenyl counterpart 42 demonstrated the highest anti-fibrillar activity as depicted by the decreasing ThT fluorescence to 5.0 and 9.5 %, respectively, compared to the 100 % control condition (Table 1). In order to identify the structural component that is crucial for biological activity, the benzothiazole moieties in 35 and 42 compounds were replaced by 4-, 5-, and 6-aminoindoles with sulfonamide or urea linkers (Table 2). Compounds 46 and 48, resulting from these structural modifications, induced comparable or slightly higher ThT fluorescence percentages compared to 35 and 42 (Fig. 2A). Importantly, compound 46 displayed superior activity against the earlier stage α-syn oligomer formation (discussed below). In addition, compound 46 showed a dose–response relationship with the α-syn anti-fibrillation assessment (Fig. 2B). Gradual increasing the compound concentration from 3.125 to 25 μM largely reduced the ThT fluorescence. Anti-fibrillary effects of compounds 46–48 is comparable to EGCG on α-syn and tau isoform 2N4R (Fig. S1).

Table 1.

The effect of the synthesized compounds containing the methoxy benzothiazole moiety (100 μM) on α-syn fibrils (2 μM) using thioflavin-T (ThT) fluorescence assay. The linker consisted of urea for compounds 1–14, amide for compounds 15–28, and sulfonamide for compounds 29–42.

Compound ID	R	α-syn ThT %	Compound ID	R	α-syn ThT %
1	2-F	≥ 200	8	4-I	141.2 ± 14.5
2	4-F	145.3 ± 8.0	9	3-CF3	≥ 200
3	2,4-F	184.1 ± 12.3	10	4-CF3	94.8 ± 5.1
4	4-Cl	172.8 ± 7.0	11	4-CN	≥ 200
5	3,5-Cl	≥ 200	12	2-NO2	64.4 ± 3.4
6	4-Br	170.2 ± 7.1	13	3-NO2	78.1 ± 12.5
7	2-I	124.8 ± 4.9	14	4-NO2	79.0 ± 12.8
15	2-F	98.7 ± 16.9	22	4-I	≥ 200
16	4-F	103.0 ± 15.8	23	3-CF3	84.7 ± 8.2
17	2,4-F	127.5 ± 18.0	24	4-CF3	115.9 ± 21.3
18	4-Cl	116.4 ± 17.0	25	4-CN	127.8 ± 21.8
19	3,5-Cl	95.4 ± 9.4	26	2-NO2	102.0 ± 7.8
20	4-Br	≥ 200	27	3-NO2	48.1 ± 8.8
21	2-I	137.1 ± 20.9	28	4-NO2	45.4 ± 2.2
29	2-F	64.9 ± 6.1	36	4-I	42.8 ± 12.3
30	4-F	70.4 ± 11.9	37	3-CF3	56.0 ± 3.5
31	2,4-F	87.9 ± 8.9	38	4-CF3	53.8 ± 7.7
32	4-Cl	40.3 ± 7.0	39	4-CN	46.1 ± 1.6
33	3,5-Cl	39.3 ± 6.7	40	2-NO2	41.0 ± 3.1
34	4-Br	43.1 ± 6.5	41	3-NO2	40.1 ± 3.9
35	2-I	5.8 ± 0.5	42	4-NO2	9.5 ± 1.8

Table 2.

The effect of the synthesized compounds containing different indole moieties (100 μM) on α-syn fibrils (2 μM) using thioflavin-T (ThT) fluorescence assay.

Linker: 1) Urea for compounds 43–48; 2) Sulfonamide for compounds 49–54.
Compound ID	AR	R1	R2	α-syn ThT %
43	4-Aminoindole	2-I	H	38.3 ± 1.9
44	5-Aminoindole	2-I	H	40.3 ± 0.5
45	6-Aminoindole	2-I	H	83.4 ± 4.6
46	4-Aminoindole	H	4-NO2	4.0 ± 0.2
47	5-Aminoindole	H	4-NO2	2.9 ± 0.1
48	6-Aminoindole	H	4-NO2	3.6 ± 1.3
49	4-Aminoindole	2-I	H	74.8 ± 8.0
50	5-Aminoindole	2-I	H	59.7 ± 6.8
51	6-Aminoindole	2-I	H	103.5 ± 13.1
52	4-Aminoindole	H	4-NO2	83.9 ± 3.1
53	5-Aminoindole	H	4-NO2	97.6 ± 20.7
54	6-Aminoindole	H	4-NO2	63.5 ± 5.3

Fig. 2.

ThT fluorescence assay. The ThT kinetic curves of compounds 46, 47, and 48 (100 μM) when tested with: A. α-syn (2 μM); B. Dose-dependent inhibition of varying concentrations (3.125, 6.25, 12.5, 25, 50, 100 μM) of compound 46 on α-syn (2 μM) fibril formation using ThT fluorescence assay. For each concentration, triplicate data were gathered at the plateau phase from five consecutive time points. The error bars represent the individual standard error of the mean (SEM) for each condition.

2.3.2. Photo-Induced cross-linking of unmodified protein (PICUP) assay

Suppression of oligomer formation is a crucial end goal to validate the effectiveness of candidates targeting protein misfolding. The photo-induced cross-linking of unmodified proteins (PICUP) assay is a rapid and efficient photochemical technique that was used to study the early events of protein oligomerization and protofibril formation thanks to its potent ability to covalently cross-link unmodified protein assemblies. Compounds that showed the best anti-fibrillary activity, as monitored by ThT assay, were subjected to the α-syn PICUP assay (Fig. 3 and Fig. S2). The cross-linked α-syn in this assay consist of oligomers that approximately correspond to trimers (band size is between 35 and 40 kDa). Unlike 35 and 42, compound 46 showed promising anti-oligomer α-syn activity at a compound-to-protein ratio of 1:0.6. The compounds were further evaluated in a dose-dependent manner at varying concentrations (50, 100 and 200 μM) while the concentration of the tau protein remained at 10 μM. However, compounds 46, 47, and 48 showed no effect on inhibiting 2N4R or 0N4R tau oligomers induced by the PICUP assay (Fig. 4A and 4B, respectively). That could be explained by the large variation in the core structures between α-syn and tau isoforms. The ThT assay and PICUP assay results definitively confirmed the ability of compound 46 to inhibit the oligomers and fibrils of α-syn protein and mature fibrils of tau isoform 2N4R.

Fig. 3.

Inhibition of the α-syn oligomer formation induced by the PICUP assay. In the PICUP experiment, α-syn (60 μM) was cross-linked with varying concentrations of compounds 46, 47, and 48 (50, 100, and 200 μM). The optical density of the high molecular-weight bands represents α-syn oligomer while the lower bands represent the protein monomers. In particular, compound 46 reduced the development of α-syn oligomer-corresponding high molecular bands between 35 and 40 kDa. The control consisted of no light exposure and no Tris(2,2′-bipyridyl)ruthenium(II) chloride (Ru(bpy)³, a cross-linking agent).

Fig. 4.

Inhibition of the oligomer formation induced by the PICUP assay on two different tau isoforms (10 μM): A. 2N4R tau; B. 0N4R tau. In the experiment, we exposed the protein to different concentrations of compounds 46, 47, and 48 (50 μM, 100 μM, and 200 μM). None of the compounds demonstrated the ability to suppress the formation of tau oligomers using both tau isoforms. The control consisted of no light exposure and no Tris(2,2′-bipyridyl)ruthenium(II) chloride (Ru (bpy)³, a cross-linking agent).

2.3.3. In vitro transmission electron microscope (TEM) analysis

The top α-syn anti-fibrillar compounds were further tested against α-syn and tau (isoform 2N4R) fibrils using a transmission electron microscope (TEM) to further assess the anti-aggregation ability. Samples were pre-incubated for a period of 68 h (α-syn) and 72 h (tau) prior TEM analyses. Control with vehicle (DMSO 0.25 %) resulted in dense mature fibrils of α-syn and tau isoform 2N4R. TEM imaging of α-syn revealed that all three compounds (46, 47, and 48) significantly reduced the protein fibril formation (Fig. 5, upper row). Similar effect was observed with treated unfolded tau isoform 2N4R. Treatment with compounds 46–48 reduced formation of large dense accumulation of tau fibrils. A few short and fine needle-like fibrils (Fig. 5D and F) and rounded structures (Fig. 5H) were visualized after treatment of tau isoform 2N4R with compounds 46–48 in contrast to the control (Fig. 5, lower row).

Fig. 5.

TEM analysis of compounds 46, 47, and 48 (100 μM) on the inhibition of mature fibrils of α-syn (2 μM, upper panel) and tau isoform 2N4R (6 μM, lower panel). The unfolded protein was incubated with: A-B. DMSO (0.25 %); C-D. compound 46; E-F. compound 47; or G-H. compound 48. Incubation time was ~ 68 h and 72 h at 37° C for α-syn and tau (2N4R), respectively. Scale bars: 200 nm, magnification: 40 K.

2.4. α-Synuclein Inclusion-Forming neuroblastoma cell experiment

To examine the effects of the three compounds (46, 47, and 48) on cell viability and formation of α-syn inclusions, M17D neuroblastoma cells that express the inclusion-prone fusion protein α-Syn-S3K::YFP were used (Fig. 6). In addition, compounds 35, 42 were also examined using similar assay conditions (Fig. S3). Cells were incubated with different doses of compounds 24 h after plating. Then transgene expression was induced by doxycycline at 48 h and the effects of the compounds on inclusion formation were tested in a dose-dependent manner at the 96 h time-point. Compound 48 had the highest effect in the reduction of inclusions as the concentration increased. A similar trend was observed for its effect on cell confluence at a concentration range of 5 μM to 40 μM. While compounds 46 and 47 demonstrated a reasonable reducing effect on inclusions at varying concentrations, neither compound had an effect on cell confluence below 20 μM concentration, and only minor effects at 20 and 40 μM.

Fig. 6.

Inhibition of α-syn inclusion formation using M17D cells. The cells expressing the inclusion-prone α-Syn-3K::YFP fusion protein (dox-inducible) were treated with 0.1 % DMSO (vehicle; “0 μM”) as well as 1.25, 2.5, 5, 10, 20 and 40 μM of compounds 46 (A), 47 (B) and 48 (C) at t = 24 h after plating. Cells were induced with doxycycline at t = 48 h. Incucyte-based analysis of punctate YFP signals relative to 0.1 % DMSO was done at t = 96 h (N = 2 independent experiments, n = 6–12 individual wells total (0 μM, n = 12; 40, 20 and 10 μM, n = 6; all other concentrations, n = 12). Plot of confluence fold changes relative to DMSO vehicle (0 μM). D) Representative IncuCyte images of reporter cells treated with vehicle vs 5 μM compound 46, 47, and 48 (t = 96 h), green channel. Arrows indicate αS-rich YFP-positive inclusions. Scale bar, 50 μm. All data are presented as fold-changes relative to DMSO control + /− standard deviation. One-way ANOVA, Dunnett’s post-hoc test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

3. Conclusion

Benzothiazole- and indole-based scaffold compounds were rationally designed and synthesized for dual inhibition of α-syn and tau (isoform 2N4R) aggregates. The elaborated compounds were prepared by hybridizing the active pharmacophore moieties of a strong tau (2N4R) aggregation inhibitor, N744 dye, and a powerful α-syn anti-aggregation Anle138b. ThT fluorescence assay revealed the benzothiazoles 35 and 42 significantly decreased the ThT fluorescence to 5.0 and 9.5 % towards α-syn fibrils. In addition, the ThT fluorescence was decreased to lower than 20 % by the indole-based urea compounds 46 and 48 towards α-syn fibrils. Compound 46 showed a noticeable inhibition of α-syn oligomer formation as assessed by the PICUP assay. The TEM confirmed the striking α-syn and tau (2N4R) anti-aggregation ability of compounds 46 and 48. Importantly, the α-syn inclusions were remarkably decreased after treatment with compound 48. Chemical refinement is now underway to further optimize the compounds to effectively abrogate the early-stage oligomer formation of both α-syn and tau isoform 2N4R.

4. Experimental procedures

4.1. General Information

All reagents and solvents were commercially available (Sigma Aldrich, St. Louis, MO; Thermo Scientific (formerly Alfa Aesar), Waltham, MA; Matrix Scientific, Columbia, SC; Ambeed, Arlington Hts, IL) and were used without further purification. Thin-layer chromatography (TLC) was used to monitor the reaction progress. Organic solutions were dried over anhydrous sodium sulfate. The solvents were evaporated on a Büchi rotavapor R-100 equipped with a Büchi V-100 vacuum controller. The nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 500 MHz spectrometer. Proton chemical shifts are reported in parts per million (ppm) with the solvent reference relative to tetramethyl silane (TMS) employed as the internal standard (CDCl3, δ 7.26; DMSO‑d6, δ 2.54). The multiplicities of NMR signals are designated as s (singlet), d (doublet), dd (double doublet), t (triplet), q (quartet), br (broad), m (multiplet, for unresolved lines). High-resolution mass spectrometry (HRMS) of the compounds was carried out at the MSU Mass Spectrometry and Metabolomics Core Facility. Uncorrected melting points (mp) were scored using an electrothermal apparatus (Barnstead International, Dubuque, Iowa, USA).

4.2. Chemistry

4.2.1. General procedure for the synthesis of compounds with the urea linker (1–14, 43–48)

4-Methoxy-2-amino benzothiazole, 4-, 5-, or 6-amino indole (100 mg, 1.0 eq), and the appropriate phenyl isocyanate (1.0 eq) were dissolved in dichloromethane (DCM, 10 mL) and stirred for 4–12 h at room temperature. The precipitate was then filtered and washed with hexane (10 mL), DCM (2 × 10 mL), and diethyl ether (10 mL). The desired products were obtained in excellent to quantitative yields.

1-(2-Fluorophenyl)-3-(4-methoxybenzo[d]thiazol-2-yl)urea (1).

Starting with 4-methoxy-2-amino benzothiazole and 2-fluoro phenyl isocyanate. Yield: 92%, white solid; ¹H NMR (500 MHz, DMSO) δ 11.01 (s, 1H), 9.13 (s, 1H), 8.12 – 8.08 (m, 1H), 7.47 (dd, J = 7.9, 1.0 Hz, 1H), 7.30 – 7.25 (m, 1H), 7.23 – 7.15 (m, 2H), 7.14 – 7.05 (m, 1H), 6.95 (dd, J = 8.0, 1.0 Hz, 1H). ¹³C NMR (126 MHz, DMSO) δ 157.9, 153.9, 151.9, 133.3, 126.8, 126.7, 125.2, 125.2, 124.5, 121.8, 115.8, 115.7, 113.9, 108.3, 56.2. Melting Point: 209.5 – 211.3 °C.

1-(4-Fluorophenyl)-3-(4-methoxybenzo[d]thiazol-2-yl)urea (2).

Starting with 4-methoxy-2-amino benzothiazole and 4-fluoro phenyl isocyanate. Yield: 87%, white solid; ¹H NMR (500 MHz, DMSO) δ 10.86 (s, 1H), 9.16 (s, 1H), 7.55 – 7.48 (m, 2H), 7.48 – 7.42 (m, 1H), 7.21 – 7.12 (m, 3H), 6.95 (dd, J = 8.1, 1.0 Hz, 1H), 3.89 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 159.4, 157.5, 151.6, 135.3, 133.1, 124.3, 121.4, 121.4, 116.0, 115.9, 113.9, 108.3, 56.2. Melting Point: 201.0 – 202.3 °C.

1-(2,4-Difluorophenyl)-3-(4-methoxybenzo[d]thiazol-2-yl)urea (3).

Starting with 4-methoxy-2-amino benzothiazole and 2,4-difluoro phenyl isocyanate. Yield: 94%, white solid; ¹H NMR (500 MHz, DMSO) δ 11.01 (s, 1H), 9.08 (s, 1H), 8.09 – 7.95 (m, 1H), 7.46 (dd, J = 8.0, 0.9 Hz, 1H), 7.38 – 7.32 (m, 1H), 7.18 (t, J = 8.0 Hz, 1H), 7.13 – 7.03 (m, 1H), 6.95 (d, J = 7.2 Hz, 1H), 3.89 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 157.3, 154.3, 151.8, 133.2, 124.5, 123.5, 123.2, 113.9, 111.8, 111.7, 108.3, 104.7, 104.5, 104.3, 56.2. Melting Point: 220.8 – 225.0 °C.

1-(4-Chlorophenyl)-3-(4-methoxybenzo[d]thiazol-2-yl)urea (4).

Starting with 4-methoxy-2-amino benzothiazole and 4-chloro phenyl isocyanate. Yield: 82%, white solid; ¹H NMR (500 MHz, DMSO) δ 10.94 (s, 1H), 9.27 (s, 1H), 7.53 (d, J = 8.8 Hz, 2H), 7.45 (dd, J = 7.9, 0.9 Hz, 1H), 7.36 (d, J = 8.8 Hz, 2H), 7.18 (t, J = 8.0 Hz, 1H), 6.95 (dd, J = 8.1, 1.0 Hz, 1H), 3.89 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 158.9, 158.5, 152.5, 151.5, 138.0, 133.0, 129.2, 127.0, 124.4, 121.8, 121.0, 113.9, 108.3, 56.2. Melting Point: 238.9 – 240.5 °C.

1-(3,5-Dichlorophenyl)-3-(4-methoxybenzo[d]thiazol-2-yl) urea (5).

Starting with 4-methoxy-2-amino benzothiazole and 3,5-dichloro phenyl isocyanate. Yield: 91%, white solid; ¹H NMR (500 MHz, DMSO) δ 11.38 (s, 1H), 9.47 (s, 1H), 7.64 – 7.57 (m, 2H), 7.44 (d, J = 8.0 Hz, 1H), 7.31 – 7.12 (m, 2H), 6.96 (d, J = 8.0 Hz, 1H), 3.90 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 152.5, 151.1, 142.3, 141.8, 134.6, 132.6, 124.5, 122.4, 117.6, 117.2, 114.0, 108.4, 56.2. Melting Point: 246.5 – 247.8 °C.

1-(4-Bromophenyl)-3-(4-methoxybenzo[d]thiazol-2-yl)urea (6).

Starting with 4-methoxy-2-amino benzothiazole and 4-bromo phenyl isocyanate. Yield: 87%, white solid; ¹H NMR (500 MHz, DMSO) δ 10.95 (s, 1H), 9.27 (s, 1H), 7.48 (s, 4H), 7.45 (dd, J = 8.0, 1.0 Hz, 1H), 7.18 (t, J = 8.0 Hz, 1H), 6.95 (dd, J = 8.1, 1.0 Hz, 1H), 3.89 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 158.4, 152.5, 151.5, 138.5, 133.0, 132.1, 124.4, 121.5, 121.4, 115.0, 113.9, 108.3, 56.2. Melting Point: 276.8 – 279.1 °C.

1-(2-Iodophenyl)-3-(4-methoxybenzo[d]thiazol-2-yl)urea (7).

Starting with 4-methoxy-2-amino benzothiazole and 2-iodo phenyl isocyanate. Yield: 71%, white solid; ¹H NMR (500 MHz, DMSO) δ 11.55 (s, 1H), 8.47 (s, 1H), 7.87 (dd, J = 7.9, 1.5 Hz, 1H), 7.82 (dd, J = 8.2, 1.5 Hz, 1H), 7.49 – 7.34 (m, 2H), 7.19 (t, J = 8.0 Hz, 1H), 6.99 – 6.87 (m, 2H), 3.90 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 158.1, 152.2, 151.9, 139.6, 139.3, 133.3, 129.3, 126.7, 124.4, 124.2, 113.9, 108.4, 92.8, 56.3. Melting Point: 224.0 – 225.2 °C.

1-(4-Iodophenyl)-3-(4-methoxybenzo[d]thiazol-2-yl)urea (8).

Starting with 4-methoxy-2-amino benzothiazole and 4-iodo phenyl isocyanate. Yield: 85%, white solid; ¹H NMR (500 MHz, DMSO) δ 10.93 (s, 1H), 9.24 (s, 1H), 7.64 (d, J = 8.7 Hz, 2H), 7.49 – 7.41 (m, 1H), 7.35 (d, J = 8.7 Hz, 2H), 7.18 (t, J = 8.0 Hz, 1H), 6.95 (d, J = 8.0 Hz, 1H), 3.89 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 158.4, 152.4, 151.5, 138.9, 138.0, 133.0, 127.2, 124.4, 121.6, 113.9, 108.3, 86.7, 56.2. Melting Point: 215.2 – 217.0 °C.

1-(4-Methoxybenzo[d]thiazol-2-yl)-3-(3-(trifluoromethyl) phenyl)urea (9).

Starting with 4-methoxy-2-amino benzothiazole and 3-trifluoromethyl phenyl isocyanate. Yield: 91%, white solid; ¹H NMR (500 MHz, DMSO) δ 11.12 (s, 1H), 9.51 (s, 1H), 8.01 (s, 1H), 7.69 (d, J = 8.2 Hz, 1H), 7.55 (t, J = 8.0 Hz, 1H), 7.45 (d, J = 7.9 Hz, 1H), 7.38 (d, J = 7.7 Hz, 1H), 7.19 (t, J = 8.0 Hz, 1H), 6.96 (d, J = 7.9 Hz, 1H), 3.89 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 166.7, 151.7, 140.0, 130.6, 130.2, 129.9, 125.7, 124.4, 123.5, 123.1, 119.7, 115.4, 113.9, 108.3, 56.2.

1-(4-Methoxybenzo[d]thiazol-2-yl)-3-(4-(trifluoromethyl) phenyl)urea (10).

Starting with 4-methoxy-2-amino benzothiazole and 4-trifluoromethyl phenyl isocyanate. Yield: 97%, white solid; ¹H NMR (500 MHz, DMSO) δ 11.03 (s, 1H), 9.53 (s, 1H), 7.72 (d, J = 8.4 Hz, 2H), 7.67 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 7.9 Hz, 1H), 7.19 (t, J = 8.0 Hz, 1H), 6.96 (d, J = 7.9 Hz, 1H), 3.90 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 142.8, 140.6, 140.0, 128.1, 126.7, 126.0, 124.5, 123.4, 121.7, 119.2, 113.9, 108.3, 56.2.

1-(4-Cyanophenyl)-3-(4-methoxybenzo[d]thiazol-2-yl)urea (11).

Starting with 4-methoxy-2-amino benzothiazole and 4-cyano phenyl isocyanate. Yield: 88%, white solid; ¹H NMR (500 MHz, DMSO) δ 11.18 (s, 1H), 9.62 (s, 1H), 7.76 (d, J = 8.8 Hz, 2H), 7.70 (d, J = 8.8 Hz, 2H), 7.49 – 7.36 (m, 1H), 7.19 (t, J = 8.0 Hz, 1H), 6.96 (dd, J = 8.0, 0.9 Hz, 1H), 3.90 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 151.3, 143.6, 133.8, 132.7, 126.8, 124.5, 119.6, 119.4, 119.3, 114.0, 108.3, 104.9, 56.2. Melting Point: 252.4 – 252.9 °C.

1-(4-Methoxybenzo[d]thiazol-2-yl)-3-(2-nitrophenyl)urea (12).

Starting with 4-methoxy-2-amino benzothiazole and 2-nitro phenyl isocyanate. Yield: 99%, canary yellow solid; ¹H NMR (500 MHz, DMSO) δ 12.17 (s, 1H), 9.92 (s, 1H), 8.28 (d, J = 8.4 Hz, 1H), 8.12 (d, J = 6.7 Hz, 1H), 7.79 – 7.69 (m, 1H), 7.47 (d, J = 7.9 Hz, 1H), 7.28 (t, J = 8.0 Hz, 1H), 7.20 (t, J = 8.0 Hz, 1H), 6.96 (d, J = 8.0 Hz, 1H), 3.90 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ ¹³C NMR (126 MHz, DMSO) δ 138.9, 135.6, 134.0, 128.9, 126.0, 124.6, 124.0, 123.6, 118.9, 115.1, 113.9, 108.4, 56.3.

1-(4-Methoxybenzo[d]thiazol-2-yl)-3-(3-nitrophenyl)urea (13).

Starting with 4-methoxy-2-amino benzothiazole and 3-nitro phenyl isocyanate. Yield: 92%, white solid; ¹H NMR (500 MHz, DMSO) δ 11.28 (s, 1H), 9.65 (s, 1H), 8.56 (s, 1H), 7.87–––7.81 (m, 2H), 7.58 (t, J = 8.2 Hz, 1H), 7.44 (d, J = 7.9 Hz, 1H), 7.18 (t, J = 8.0 Hz, 1H), 6.95 (d, J = 8.0 Hz, 1H), 3.90 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 159.0, 153.2, 151.3, 148.6, 140.5, 132.7, 130.6, 125.5, 124.5, 117.7, 113.9, 113.4, 110.3, 108.3, 56.2. Melting Point: 266.0 – 266.7 °C.

1-(4-Methoxybenzo[d]thiazol-2-yl)-3-(4-nitrophenyl)urea (14).

Starting with 4-methoxy-2-amino benzothiazole and 4-nitro phenyl isocyanate. Yield: 100%, off-white solid; ¹H NMR (500 MHz, DMSO) δ 11.33 (s, 1H), 9.84 (s, 1H), 8.20 (d, J = 8.7 Hz, 2H), 7.76 (d, J = 8.7 Hz, 2H), 7.45 (d, J = 8.0 Hz, 1H), 7.20 (t, J = 8.0 Hz, 1H), 6.97 (d, J = 8.1 Hz, 1H), 3.90 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 151.2, 145.8, 142.2, 132.7, 125.5, 124.6, 121.9, 118.8, 114.0, 113.8, 108.6, 108.4, 56.2. Melting Point: 279.1 – 280.4 °C.

1-(1H-Indol-4-yl)-3-(2-iodophenyl)urea (43).

Starting with 4-amino indole and 2-iodo phenyl isocyanate. Yield: 95%, grey solid; ¹H NMR (500 MHz, DMSO) δ 11.11 (s, 1H), 9.08 (s, 1H), 8.21 (s, 1H), 7.84 (d, J = 8.2 Hz, 1H), 7.80 (d, J = 7.4 Hz, 1H), 7.61 (d, J = 7.6 Hz, 1H), 7.37 – 7.32 (m, 1H), 7.30 (t, J = 2.8 Hz, 1H), 7.06 (d, J = 8.0 Hz, 1H), 6.99 (t, J = 7.9 Hz, 1H), 6.89 – 6.78 (m, 1H), 6.62 (s, 1H). ¹³C NMR (126 MHz, DMSO) δ 153.1, 140.5, 139.4, 137.0, 131.8, 129.0, 125.6, 124.5, 124.2, 122.0, 119.7, 108.6, 106.6, 98.6, 92.1. Melting Point: 207.0 – 208.4 °C.

1-(1H-Indol-5-yl)-3-(2-iodophenyl)urea (44).

Starting with 5-amino indole and 2-iodo phenyl isocyanate. Yield: 92%, grey solid; ¹H NMR (500 MHz, DMSO) δ 10.93 (s, 1H), 9.18 (s, 1H), 7.87 (d, J = 8.2 Hz, 1H), 7.84 – 7.54 (m, 3H), 7.43 – 7.17 (m, 3H), 7.07 (d, J = 8.8 Hz, 1H), 6.87 – 6.69 (m, 1H), 6.34 (s, 1H). ¹³C NMR (126 MHz, DMSO) δ 153.2, 140.7, 139.4, 132.8, 131.7, 129.0, 128.2, 126.3, 125.0, 123.1, 115.1, 111.8, 110.5, 101.4, 91.2. Melting Point: 206.1 – 207.9 °C.

1-(1H-Indol-6-yl)-3-(2-iodophenyl)urea (45).

Starting with 6-amino indole and 2-iodo phenyl isocyanate. Yield: 100%, light grey solid; ¹H NMR (500 MHz, DMSO) δ 10.93 (s, 1H), 9.31 (s, 1H), 7.88 (d, J = 8.2 Hz, 1H), 7.81 (t, J = 6.7 Hz, 3H), 7.42 (d, J = 8.4 Hz, 1H), 7.33 (s, 1H), 7.21 (t, J = 2.8 Hz, 1H), 6.87 (dd, J = 8.4, 1.9 Hz, 1H), 6.81 (d, J =7.5 Hz, 1H), 6.33 (t, J = 2.6 Hz, 1H). ¹³C NMR (126 MHz, DMSO) δ 153.0, 140.6, 139.4, 136.7, 134.1, 129.0, 125.2, 125.0, 123.7, 123.2, 120.5, 112.3, 101.7, 101.4, 91.4. Melting Point: 213.1 – 215.3 °C.

1-(1H-Indol-4-yl)-3-(4-nitrophenyl)urea (46).

Starting with 4-amino indole and 4-nitro phenyl isocyanate. Yield: 89%, brown solid; ¹H NMR (500 MHz, DMSO) δ 11.16 (s, 1H), 9.60 (s, 1H), 8.71 (s, 1H), 8.19 (d, J = 9.2 Hz, 2H), 7.72 (d, J = 9.2 Hz, 2H), 7.67 – 7.60 (m, 1H), 7.36 – 7.29 (m, 1H), 7.11 – 7.09 (m, 1H), 7.02 (t, J = 7.9 Hz, 1H), 6.55 (s, 1H). ¹³C NMR (126 MHz, DMSO) δ 152.4, 146.9, 141.4, 137.0, 131.2, 125.7, 124.8, 122.0, 119.7, 117.7, 108.5, 107.1, 98.2. HRMS m/z: calcd for C₁₅H₁₂N₄O₂, 296.0909; found, 296.0988, M + H⁺. Melting Point: 223.5 – 224.1 °C.

1-(1H-Indol-5-yl)-3-(4-nitrophenyl)urea (47).

Starting with 5-amino indole and 4-nitro phenyl isocyanate. Yield: 86%, beige-colored solid; ¹H NMR (500 MHz, DMSO) δ 10.98 (s, 1H), 9.34 (s, 1H), 8.65 (s, 1H), 8.17 (d, J = 8.1 Hz, 2H), 7.86 – 7.54 (m, 3H), 7.45 – 7.18 (m, 2H), 7.11 (d, J = 8.6 Hz, 1H), 6.37 (s, 1H). ¹³C NMR (126 MHz, DMSO) δ 152.8, 147.4, 141.2, 133.1, 131.1, 128.2, 126.4, 125.6, 117.7, 115.5, 111.8, 111.1, 101.5. HRMS m/z: calcd for C₁₅H₁₂N₄O₂, 296.0909; found, 296.0992, M + H⁺. Melting Point: 248.5 – 250.1 °C.

1-(1H-Indol-6-yl)-3-(4-nitrophenyl)urea (48).

Starting with 6-amino indole and 4-nitro phenyl isocyanate. Yield: 79%, yellow solid; ¹H NMR (500 MHz, DMSO) δ 10.96 (s, 1H), 9.36 (s, 1H), 8.80 (s, 1H), 8.18 (d, J = 9.2 Hz, 2H), 7.80 (s, 1H), 7.69 (d, J = 9.2 Hz, 2H), 7.43 (d, J = 8.4 Hz, 1H), 7.23 (t, J = 2.7 Hz, 1H), 6.90 (dd, J = 8.4, 2.0 Hz, 1H), 6.34 (s, 1H). ¹³C NMR (126 MHz, DMSO) δ 152.6, 147.2, 141.2, 136.6, 133.4, 125.6, 125.2, 124.1, 120.5, 117.7, 112.7, 102.1, 101.4. HRMS m/z: calcd for C₁₅H₁₂N₄O₂, 296.0909; found, 296.0993, M + H⁺. Melting Point: 247.3 – 248.9 °C.

4.2.2. General procedure for the synthesis of compounds with the amide or sulfonamide linker (15–42, 49–54)

The appropriate benzoyl chloride or its corresponding benzene sulfonyl chloride (1.0 eq) was added slowly to a stirred solution of 4-methoxy-2-amino benzothiazole, 4-, 5-, or 6-amino indole (100 mg, 1.0 eq) in pyridine (3 mL) at 0 °C. Potassium carbonate (1.5 eq) was added, and the reaction was allowed to stir overnight at room temperature. After completion, an aqueous solution of 1 N hydrochloric acid (7 mL) and dichloromethane (10 mL) was added to the reaction mixture and then extracted with DCM. The organic layers were then washed with saturated solutions of ammonium chloride (7 mL), sodium bicarbonate (7 mL), and brine (7 mL), respectively. Organic layers were collected, filtered over anhydrous magnesium sulfate, and concentrated in-vacuo. The crude was purified using column chromatography to obtain the desired products with moderate to good yields.

2-Fluoro-N-(4-methoxybenzo[d]thiazol-2-yl)benzamide (15).

Starting with 4-methoxy-2-amino benzothiazole and 2-fluoro benzoyl chloride. Yield: 58%, white solid; ¹H NMR (500 MHz, CDCl₃) δ 8.32 – 8.11 (m, 1H), 7.63 – 7.56 (m, 1H), 7.44 (dd, J = 8.0, 0.9 Hz, 1H), 7.35 (d, J = 7.8, 1H), 7.30 (t, J = 8.0 Hz, 1H), 7.43 – 7.21 (m, 1H), 6.92 (dd, J = 8.0, 0.9 Hz, 1H), 4.04 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 161.3, 156.7, 152.1, 138.2, 135.2, 133.6, 132.3, 125.3, 125.3, 125.1, 116.7, 116.5, 113.5, 106.8, 55.9. Melting Point: 178.4 – 180.8 °C.

4-Fluoro-N-(4-methoxybenzo[d]thiazol-2-yl)benzamide (16).

Starting with 4-methoxy-2-amino benzothiazole and 4-fluoro benzoyl chloride. Yield: 67%, white solid; ¹H NMR (500 MHz, DMSO) δ 8.29 – 8.16 (m, 2H), 7.54 (dd, J = 8.0, 0.9 Hz, 1H), 7.39 (t, J = 8.8 Hz, 2H), 7.27 (t, J = 8.0 Hz, 1H), 7.00 (dd, J = 8.0, 0.9 Hz, 1H), 3.92 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 172.5, 165.1, 157.7, 152.3, 133.3, 131.7, 131.6, 125.2, 116.3, 116.1, 113.9, 108.0, 56.1.

2,4-Difluoro-N-(4-methoxybenzo[d]thiazol-2-yl)benzamide (17).

Starting with 4-methoxy-2-amino benzothiazole and 2,4-difluoro benzoyl chloride. Yield: 75%, white solid; ¹H NMR (500 MHz, DMSO) δ 7.88 (td, J = 8.5, 6.5 Hz, 1H), 7.55 (dd, J = 8.0, 0.9 Hz, 1H), 7.50 – 7.39 (m, 1H), 7.32 – 7.21 (m, 2H), 7.01 (dd, J = 8.1, 1.0 Hz, 1H), 3.91 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 163.0, 159.8, 156.8, 152.4, 138.8, 133.3, 132.8, 125.4, 119.3, 119.2, 114.0, 112.4, 108.1, 105.5, 56.2. Melting Point: 147.6 – 149.1 °C.

4-Chloro-N-(4-methoxybenzo[d]thiazol-2-yl)benzamide (18).

Starting with 4-methoxy-2-amino benzothiazole and 4-chloro benzoyl chloride. Yield: 81%, white solid; ¹H NMR (500 MHz, DMSO) δ 8.15 (d, J = 8.6 Hz, 2H), 7.62 (d, J = 8.6 Hz, 2H), 7.54 (dd, J = 8.0, 0.9 Hz, 1H), 7.27 (t, J = 8.0 Hz, 1H), 7.01 (dd, J = 8.1, 1.0 Hz, 1H), 3.92 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 165.2, 152.4, 138.3, 133.4, 131.2, 131.1, 130.7, 129.3, 125.2, 120.8, 113.9, 108.0, 56.1.

3,5-Dichloro-N-(4-methoxybenzo[d]thiazol-2-yl)benzamide (19).

Starting with 4-methoxy-2-amino benzothiazole and 3,5-dichloro benzoyl chloride. Yield: 85%, white solid; ¹H NMR (500 MHz, DMSO) δ 8.15 (s, 2H), 7.90 (s, 1H), 7.54 (dd, J = 8.0, 0.9 Hz, 1H), 7.28 (t, J = 8.0 Hz, 1H), 7.01 (d, J = 8.1 Hz, 1H), 3.92 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 165.4, 157.7, 152.3, 135.7, 135.0, 133.2, 132.5, 128.3, 127.6, 125.4, 114.0, 108.1, 56.2.

4-Bromo-N-(4-methoxybenzo[d]thiazol-2-yl)benzamide (20).

Starting with 4-methoxy-2-amino benzothiazole and 4-bromo benzoyl chloride. Yield: 71%, white solid; ¹H NMR (500 MHz, DMSO) δ 8.07 (d, J = 8.6 Hz, 2H), 7.75 (d, J = 8.6 Hz, 2H), 7.54 (dd, J = 7.9, 1.0 Hz, 1H), 7.27 (t, J = 8.0 Hz, 1H), 7.00 (dd, J = 8.1, 1.0 Hz, 1H), 3.91 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 165.4, 157.6, 152.4, 133.4, 132.2, 131.5, 130.8, 129.3, 127.3, 125.2, 113.9, 108.0, 56.1. Melting Point: 225.5 – 226.9 °C.

2-Iodo-N-(4-methoxybenzo[d]thiazol-2-yl)benzamide (21).

Starting with 4-methoxy-2-amino benzothiazole and 2-iodo benzoyl chloride. Yield: 75%, white solid; ¹H NMR (500 MHz, DMSO) δ 7.95 (dd, J = 7.9, 1.1 Hz, 1H), 7.60 – 7.46 (m, 3H), 7.31 – 7.22 (m, 2H), 7.00 (dd, J = 8.1, 1.0 Hz, 1H), 3.90 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 168.4, 156.8, 152.5, 140.6, 139.8, 133.4, 132.4, 129.2, 128.6, 128.6, 125.3, 114.0, 108.1, 94.2, 56.2. Melting Point: 159.0 – 161.9 °C.

4-Iodo-N-(4-methoxybenzo[d]thiazol-2-yl)benzamide (22).

Starting with 4-methoxy-2-amino benzothiazole and 4-iodo benzoyl chloride. Yield: 88%, white solid; ¹H NMR (500 MHz, DMSO) δ 7.94 – 7.91 (m, 2H), 7.91 – 7.88 (m, 2H), 7.67 (d, J = 8.4 Hz, 1H), 7.54 (dd, J = 7.9, 0.9 Hz, 1H), 7.27 (t, J = 8.0 Hz, 1H), 7.00 (dd, J = 8.0, 0.9 Hz, 1H), 3.91 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 167.4, 165.7, 157.6, 152.3, 138.1, 133.3, 131.5, 130.5, 125.2, 113.9, 108.0, 101.7, 56.1. Melting Point: 210.2 – 211.0 °C.

3-Trifluoromethyl-N-(4-methoxybenzo[d]thiazol-2-yl)benzamide (23).

Starting with 4-methoxy-2-amino benzothiazole and 3-trifluoromethyl benzoyl chloride. Yield: 67%, white solid; ¹H NMR (500 MHz, CDCl₃) δ 8.23 (s, 1H), 8.09 (d, J = 8.1 Hz, 1H), 7.74 (dd, J = 7.8, 1.7 Hz, 1H), 7.51 – 7.39 (m, 2H), 7.27 (d, J = 8.0 Hz, 1H), 6.69 (d, J = 8.0 Hz, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 164.8, 159.0, 151.4, 137.4, 133.1, 131.5, 131.1, 129.2, 125.4, 125.1, 124.4, 122.3, 113.5, 106.6, 55.0. Melting Point: 166.3 – 168.0 °C.

4-Trifluoromethyl-N-(4-methoxybenzo[d]thiazol-2-yl)benzamide (24).

Starting with 4-methoxy-2-amino benzothiazole and 4-trifluoromethyl benzoyl chloride. Yield: 73%, white solid; ¹H NMR (500 MHz, DMSO) δ 8.31 (d, J = 8.0 Hz, 2H), 7.92 (d, J = 7.9 Hz, 2H), 7.56 (dd, J = 8.0, 0.9 Hz, 1H), 7.29 (t, J = 8.0 Hz, 1H), 7.02 (dd, J = 8.1, 1.0 Hz, 1H), 3.92 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 172.5, 165.2, 152.4, 136.2, 133.3, 132.9, 129.7, 126.1, 125.4, 123.2, 114.0, 108.0, 56.2.

4-Cyano-N-(4-methoxybenzo[d]thiazol-2-yl)benzamide (25).

Starting with 4-methoxy-2-amino benzothiazole and 4-cyano benzoyl chloride. Yield: 72%, pale yellow solid; ¹H NMR (500 MHz, DMSO) δ 8.26 (d, J = 8.0 Hz, 2H), 8.02 (d, J = 8.0 Hz, 2H), 7.55 (d, J = 7.9 Hz, 1H), 7.28 (t, J = 8.0 Hz, 1H), 7.01 (d, J = 8.0 Hz, 1H), 3.92 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 165.0, 157.7, 152.3, 136.5, 133.3, 133.1, 130.4, 129.5, 125.3, 118.6, 115.4, 114.0, 108.0, 56.2. Melting Point: 223.3 – 224.1 °C.

2-Nitro-N-(4-methoxybenzo[d]thiazol-2-yl)benzamide (26).

Starting with 4-methoxy-2-amino benzothiazole and 2-nitro benzoyl chloride. Yield: 61%, off-white solid; ¹H NMR (500 MHz, DMSO) δ 8.19 (dd, J = 8.2, 1.2 Hz, 1H), 7.90 – 7.78 (m, 3H), 7.57 (dd, J = 8.0, 1.0 Hz, 1H), 7.29 (t, J = 8.0 Hz, 1H), 7.01 (dd, J = 8.1, 1.0 Hz, 1H), 3.90 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 165.5, 156.7, 152.5, 146.9, 134.8, 133.4, 132.4, 130.6, 130.2, 125.4, 125.0, 125.0, 114.1, 108.3, 56.3. Melting Point: 233.7–235.3 °C.

3-Nitro-N-(4-methoxybenzo[d]thiazol-2-yl)benzamide (27).

Starting with 4-methoxy-2-amino benzothiazole and 3-nitro benzoyl chloride. Yield: 64%, white solid; ¹H NMR (500 MHz, DMSO) δ 8.97 (s, 1H), 8.57 – 8.41 (m, 2H), 7.82 (t, J = 7.9 Hz, 1H), 7.53 (d, J = 7.9 Hz, 1H), 7.27 (t, J = 7.9 Hz, 1H), 7.00 (d, J = 8.0 Hz, 1H), 3.92 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 164.5, 157.9, 152.2, 148.3, 135.0, 134.0, 133.3, 130.8, 127.6, 125.3, 123.7, 114.0, 110.1, 108.1, 56.2. Melting Point: 244.3 – 246.0 °C.

4-Nitro-N-(4-methoxybenzo[d]thiazol-2-yl)benzamide (28).

Starting with 4-methoxy-2-amino benzothiazole and 4-nitro benzoyl chloride. Yield: 78%, canary yellow solid; ¹H NMR (500 MHz, DMSO) δ 8.33 (s, 4H), 7.55 (d, J = 8.0 Hz, 1H), 7.28 (t, J = 7.8 Hz, 1H), 7.01 (d, J = 7.8 Hz, 1H), 3.92 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 164.8, 152.3, 150.2, 138.0, 133.3, 130.3, 129.3, 125.4, 124.1, 118.6, 114.0, 108.1, 56.2. Melting Point: 243.5 – 244.3 °C.

2-Fluoro-N-(4-methoxybenzo[d]thiazol-2-yl)benzenesulfonamide (29).

Starting with 4-methoxy-2-amino benzothiazole and 2-fluorophenyl sulfonyl chloride. Yield: 63%, brown solid; ¹H NMR (500 MHz, CDCl₃) δ 9.76 (s, 1H), 8.04 – 8.02 (m, 1H), 7.55 – 7.48 (m, 1H), 7.27 – 7.25 (m, 1H), 7.21 (t, J = 8.1 Hz, 1H), 7.17 – 7.09 (m, 2H), 6.87 (dd, J = 8.2, 0.9 Hz, 1H), 3.94 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 167.9, 160.5, 158.4, 146.0, 134.5, 129.5, 125.1, 124.1, 117.0, 116.8, 114.0, 108.3, 56.0. Melting Point: 195.3 – 197.1 °C.

4-Fluoro-N-(4-methoxybenzo[d]thiazol-2-yl)benzenesulfonamide (30).

Starting with 4-methoxy-2-amino benzothiazole and 4-fluoro phenylsulfonyl chloride. Yield: 74%, beige colored solid; ¹H NMR (500 MHz, CDCl₃) δ 8.03 – 7.94 (m, 2H), 7.18 (t, J = 8.1 Hz, 1H), 7.14 – 7.05 (m, 3H), 6.84 (d, J = 8.1 Hz, 1H), 3.91 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 167.1, 166.0, 163.9, 146.0, 137.9, 129.2, 129.2, 125.3, 125.1, 116.0, 115.8, 114.0, 108.4, 56.0. Melting Point: 166.1 – 167.4 °C.

2,4-Difluoro-N-(4-methoxybenzo[d]thiazol-2-yl)benzenesulfonamide (31).

Starting with 4-methoxy-2-amino benzothiazole and 2,4-difluorophenyl sulfonyl chloride. Yield: 51%, beige-colored solid; ¹H NMR (500 MHz, CDCl₃) δ 9.77 (s, 1H), 8.09 – 7.99 (m, 1H), 7.21 (t, J = 8.1 Hz, 1H), 7.12 (dd, J = 8.1, 0.9 Hz, 1H), 6.96 (m, 1H), 6.91 – 6.83 (m, 2H), 3.94 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 167.9, 166.5, 164.5, 161.1, 159.2, 146.0, 131.2, 125.2, 125.1, 114.0, 108.3, 105.7, 105.5, 56.0. Melting Point: 188.2 – 190.2 °C.

4-Chloro-N-(4-methoxybenzo[d]thiazol-2-yl)benzenesulfonamide (32).

Starting with 4-methoxy-2-amino benzothiazole and 4-chlorophenyl sulfonyl chloride. Yield: 64%, off-white solid; ¹H NMR (500 MHz, DMSO) δ 7.83 (d, J = 8.7 Hz, 2H), 7.60 (d, J = 8.7 Hz, 2H), 7.34 (d, J = 8.0 Hz, 1H), 7.20 (t, J = 8.1 Hz, 1H), 7.02 (d, J = 8.1 Hz, 1H), 3.86 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 167.67, 146.91, 141.53, 137.51, 129.67, 128.23, 126.44, 125.07, 114.81, 109.60, 56.56. Melting Point: 153.9 – 155.3 °C.

3,5-Dichloro-N-(4-methoxybenzo[d]thiazol-2-yl)benzenesulfonamide (33).

Starting with 4-methoxy-2-amino benzothiazole and 3.5-dichlorophenyl sulfonyl chloride. Yield: 75%, white solid; ¹H NMR (500 MHz, CDCl₃) δ 9.94 (s, 1H), 7.81 (d, J = 1.9 Hz, 2H), 7.43 (t, J = 1.9 Hz, 1H), 7.19 (d, J = 8.1 Hz, 1H), 7.10 (dd, J = 8.1, 0.9 Hz, 1H), 6.85 (d, J = 8.2 Hz, 1H), 3.92 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 167.6, 146.1, 144.6, 135.6, 132.2, 125.4, 125.2, 125.2, 124.9, 114.0, 108.5, 56.1. Melting Point: 160.7 – 162.3 °C.

4-Bromo-N-(4-methoxybenzo[d]thiazol-2-yl)benzenesulfonamide (34).

Starting with 4-methoxy-2-amino benzothiazole and 4-bromophenyl sulfonyl chloride. Yield: 79%. pale yellow solid; ¹H NMR (500 MHz, CDCl₃) δ 7.83 (d, J = 8.6 Hz, 2H), 7.58 (d, J = 8.6 Hz, 2H), 7.19 (t, J = 8.1 Hz, 1H), 7.10 (dd, J = 8.1, 0.9 Hz, 1H), 6.85 (dd, J = 8.2, 0.9 Hz, 1H), 3.92 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 167.2, 146.0, 140.8, 132.0, 128.1, 127.2, 125.3, 125.2, 125.1, 114.0, 108.4, 56.0. Melting Point: 175.6 – 177.1 °C.

2-Iodo-N-(4-methoxybenzo[d]thiazol-2-yl)benzenesulfonamide (35).

Starting with 4-methoxy-2-amino benzothiazole and 2-iodo phenylsulfonyl chloride. Yield: 75%, white solid; ¹H NMR (500 MHz, CDCl₃) δ 9.68 (s, 1H), 8.31 (dd, J = 7.9, 1.7 Hz, 1H), 8.04 (dd, J = 7.8, 1.2 Hz, 1H), 7.48 (td, J = 7.6, 1.2 Hz, 1H), 7.22 – 7.11 (m, 3H), 6.87 (dd, J = 8.1, 0.9 Hz, 1H), 3.95 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 168.0, 145.9, 143.8, 142.3, 134.6, 133.0, 130.1, 128.1, 125.5, 125.1, 114.0, 108.3, 92.8, 56.0. HRMS m/z: calcd for C₁₄H₁₁IN₂O₃S₂, 445.9256; found, 445.9351, M + H⁺. Melting Point: 224.9.0 – 226.5 °C.

4-Iodo-N-(4-methoxybenzo[d]thiazol-2-yl)benzenesulfonamide (36).

Starting with 4-methoxy-2-amino benzothiazole and 4-iodophenyl sulfonyl chloride. Yield: 71%, beige colored solid; ¹H NMR (500 MHz, CDCl₃) δ 7.81 (d, J = 8.6 Hz, 2H), 7.69 (d, J = 8.6 Hz, 2H), 7.19 (d, J = 8.1 Hz, 1H), 7.11 (dd, J = 8.1, 0.9 Hz, 1H), 6.86 (dd, J = 8.2, 0.9 Hz, 1H), 3.93 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 167.2, 145.9, 141.4, 138.0, 128.0, 125.3, 125.2, 125.1, 114.1, 108.4, 99.6, 56.0. Melting Point: 206.4 – 208.0 °C.

3-Trifluoromethyl-N-(4-methoxybenzo[d]thiazol-2-yl)benzenesulfonamide (37).

Starting with 4-methoxy-2-amino benzothiazole and 3-trifluoromethylphenyl sulfonyl chloride. Yield: 68%, beige-colored solid; ¹H NMR (500 MHz, CDCl₃) δ 9.86 (s, 1H), 8.23 (d, J = 1.7 Hz, 1H), 8.17 (d, J = 7.9, 1H), 7.76 (dd, J = 7.7, 1.7 Hz, 1H), 7.59 (t, J = 7.9 Hz, 1H), 7.19 (t, J = 8.1 Hz, 1H), 7.10 (dd, J = 8.0, 0.9 Hz, 1H), 6.85 (d, J = 8.1 Hz, 1H), 3.91 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 167.5, 146.1, 142.9, 129.6, 129.0, 128.9, 125.3, 125.2, 125.2, 123.6, 123.6, 114.0, 108.5, 56.0. Melting Point: 156.3 – 158.0 °C.

4-Trifluoromethyl-N-(4-methoxybenzo[d]thiazol-2-yl)benzenesulfonamide (38).

Starting with 4-methoxy-2-amino benzothiazole and 4-trifluoromethylphenyl sulfonyl chloride. Yield: 64%, beige-colored solid, ¹H NMR (500 MHz, CDCl₃) δ 9.75 (s, 1H), 8.10 (d, J = 8.1 Hz, 2H), 7.71 (d, J = 8.1, 2H), 7.21 (d, J = 8.1 Hz, 1H), 7.12 (dd, J = 8.1, 0.9 Hz, 1H), 6.86 (dd, J = 8.2, 0.9 Hz, 1H), 3.93 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 167.5, 146.1, 145.2, 134.1, 133.9, 127.0, 126.0, 125.9, 125.3, 114.1, 108.4, 56.0. Melting Point: 154.2 – 156.4 °C.

4-Cyano-N-(4-methoxybenzo[d]thiazol-2-yl)benzenesulfonamide (39).

Starting with 4-methoxy-2-amino benzothiazole and 4-cyanophenyl sulfonyl chloride. Yield: 69%, brown solid. ¹H NMR (500 MHz, DMSO) δ 8.12 – 7.92 (m, 4H), 7.35 (d, J = 7.9 Hz, 1H), 7.20 (t, J = 8.1 Hz, 1H), 7.02 (d, J = 8.2 Hz, 1H), 3.86 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 168.01, 147.11, 146.74, 133.79, 127.04, 125.15, 118.31, 115.05, 114.80, 110.96, 109.61, 56.57. Melting Point: 184.5 – 185.6 °C.

2-Nitro-N-(4-methoxybenzo[d]thiazol-2-yl)benzenesulfonamide (40).

Starting with 4-methoxy-2-amino benzothiazole and 2-nitrophenyl sulfonyl chloride. Yield: 71%; brown solid; ¹H NMR (500 MHz, CDCl₃) δ 9.88 (brs, 1H), 8.32 – 8.23 (m, 1H), 7.73 – 7.63 (m, 3H), 7.22 (t, J = 8.1 Hz, 1H), 7.15 – 7.08 (m, 1H), 6.87 (dd, J = 8.1, 1.0 Hz, 1H), 3.94 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 167.9, 147.6, 146.1, 135.3, 133.2, 132.1, 130.3, 125.5, 125.4, 125.0, 124.3, 114.0, 108.4, 56.0. Melting Point: 201.6 – 203.1 °C.

3-Nitro-N-(4-methoxybenzo[d]thiazol-2-yl)benzenesulfonamide (41).

Starting with 4-methoxy-2-amino benzothiazole and 3-nitrophenyl sulfonyl chloride. Yield: 59%, beige-colored solid; ¹H NMR (500 MHz, CDCl₃) δ 9.79 (s, 1H), 8.78 (t, J = 2.0 Hz, 1H), 8.38 – 8.33 (m, 1H), 8.32 – 8.29 (m, 1H), 7.68 (t, J = 8.0 Hz, 1H), 7.22 (t, J = 8.1 Hz, 1H), 7.13 (dd, J = 8.1, 0.9 Hz, 1H), 6.87 (dd, J = 8.1, 0.9 Hz, 1H), 3.94 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 167.7, 148.1, 146.1, 143.8, 132.2, 130.2, 126.8, 125.5, 125.2, 125.1, 121.8, 114.1, 108.5, 56.1. Melting Point: 166.3 – 167.8 °C.

4-Nitro-N-(4-methoxybenzo[d]thiazol-2-yl)benzenesulfonamide (42).

Starting with 4-methoxy-2-amino benzothiazole and 4-nitrophenyl sulfonyl chloride. Yield: 67%, pale yellow solid; ¹H NMR (500 MHz, CDCl₃) δ 8.28 (d, J = 8.9 Hz, 2H), 8.15 (d, J = 8.9 Hz, 2H), 7.22 (t, J = 8.1 Hz, 1H), 7.13 (dd, J = 8.0, 0.9 Hz, 1H), 6.87 (dd, J = 8.2, 0.9 Hz, 1H), 3.93 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 167.7, 149.8, 147.4, 146.2, 127.8, 125.5, 125.3, 125.2, 124.1, 114.1, 108.5, 56.1. HRMS m/z: calcd for C₁₄H₁₁N₃O₅S₂, 365.0140; found, 365.0229, M + H⁺. Melting Point: 170.4 – 172.1 °C.

N-(1H-Indol-4-yl)-2-iodobenzene sulfonamide (49).

Starting with 4-amino indole and 2-iodophenyl sulfonyl chloride. Yield: 73%, grey solid; ¹H NMR (500 MHz, DMSO) δ 11.09 (s, 1H), 10.25 (s, 1H), 8.06 (d, J = 7.7 Hz, 1H), 7.95 (d, J = 8.2 Hz, 1H), 7.45 (t, J = 7.5 Hz, 1H), 7.29 – 7.15 (m, 2H), 7.09 (d, J = 8.0 Hz, 1H), 6.88 (t, J = 7.8 Hz, 1H), 6.77 (d, J = 7.7 Hz, 1H), 6.74 (t, J = 2.6 Hz, 1H). ¹³C NMR (126 MHz, DMSO) δ 143.1, 142.7, 137.3, 134.1, 131.1, 129.1, 128.8, 125.0, 122.2, 121.5, 111.2, 108.9, 100.2, 93.8. Melting Point: 158.6 – 160.4 °C.

N-(1H-Indol-5-yl)-2-iodobenzene sulfonamide (50).

Starting with 5-amino indole and 2-iodophenyl sulfonyl chloride. Yield: 86%, brown solid; ¹H NMR (500 MHz, CDCl₃) δ 8.16 (s, 1H), 8.05 (dd, J = 7.9, 1.2 Hz, 1H), 7.93 (dd, J = 7.9, 1.6 Hz, 1H), 7.41 (d, J = 2.1 Hz, 1H), 7.32 – 7.27 (m, 1H), 7.26 – 7.19 (m, 2H), 7.18 (dd, J = 3.2, 2.5 Hz, 1H), 7.14 – 7.09 (m, 1H), 6.98 (dd, J = 8.6, 2.1 Hz, 1H), 6.48 – 6.43 (m, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 142.0, 141.2, 134.4, 133.5, 132.0, 128.5, 128.1, 127.7, 125.5, 119.3, 116.7, 111.5, 102.9, 92.4. Melting Point: 163.2 – 164.4 °C.

N-(1H-Indol-6-yl)-2-iodobenzene sulfonamide (51).

Starting with 6-amino indole and 2-iodophenyl sulfonyl chloride. 78 %, reddish brown solid; ¹H NMR (500 MHz, DMSO) δ 10.98 (s, 1H), 10.23 (s, 1H), 8.07 (dd, J = 7.8, 1.2 Hz, 1H), 7.97 (dd, J = 7.9, 1.6 Hz, 1H), 7.53 – 7.44 (m, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.24 – 7.17 (m, 3H), 6.82 (dd, J = 8.5, 2.0 Hz, 1H), 6.30 (s, 1H). ¹³C NMR (126 MHz, DMSO) δ 143.0, 142.4, 136.3, 134.2, 131.2, 131.2, 128.9, 125.9, 125.2, 120.8, 114.2, 104.3, 101.4, 93.7.

N-(1H-Indol-4-yl)-4-nitrobenzene sulfonamide (52).

Starting with 4-amino indole and 4-nitrophenyl sulfonyl chloride. Yield: 83%, yellow solid; ¹H NMR (500 MHz, DMSO) δ 11.11 (s, 1H), 10.35 (s, 1H), 8.29 (d, J = 8.8 Hz, 2H), 7.95 (d, J = 8.8 Hz, 2H), 7.21 – 7.13 (m, 2H), 6.95 (t, J = 7.9 Hz, 1H), 6.82 (dd, J = 7.6, 0.9 Hz, 1H), 6.49 – 6.43 (m, 1H). ¹³C NMR (126 MHz, DMSO) δ 150.1, 146.4, 137.3, 128.8, 128.4, 125.5, 124.8, 123.1, 121.6, 113.4, 109.8, 99.6. Melting Point: 185.0 – 186.3 °C.

N-(1H-Indol-5-yl)-4-nitrobenzene sulfonamide (53).

Starting with 5-amino indole and 4-nitrophenyl sulfonyl chloride. Yield: 77%, yellow solid; ¹H NMR (500 MHz, DMSO) δ 11.07 (s, 1H), 10.15 (s, 1H), 8.31 (d, J = 8.9 Hz, 2H), 7.90 (d, J = 8.9 Hz, 2H), 7.30 (t, J = 2.8 Hz, 1H), 7.25 (dd, J = 5.4, 3.2 Hz, 2H), 6.80 (dd, J = 8.7, 2.0 Hz, 1H), 6.37 – 6.30 (m, 1H). ¹³C NMR (126 MHz, DMSO) δ 150.1, 145.7, 134.4, 128.8, 128.5, 128.2, 126.9, 124.9, 117.9, 115.0, 112.3, 101.7. Melting Point: 142.1 – 144.5 °C.

N-(1H-Indol-6-yl)-4-nitrobenzene sulfonamide (54).

Starting with 6-amino indole and 4-nitrophenyl sulfonyl chloride. Yield: 76%, yellow solid; ¹H NMR (500 MHz, DMSO) δ 11.01 (s, 1H), 10.27 (s, 1H), 8.32 (d, J = 8.9 Hz, 2H), 7.92 (d, J = 8.9 Hz, 2H), 7.36 (d, J = 8.4 Hz, 1H), 7.26 (t, J = 2.7 Hz, 1H), 7.19 – 7.13 (m, 1H), 6.72 (dd, J = 8.4, 1.9 Hz, 1H), 6.34 – 6.28 (m, 1H). ¹³C NMR (126 MHz, DMSO) δ 150.1, 145.6, 136.2, 130.7, 128.8, 126.3, 125.9, 124.9, 120.9, 115.1, 105.7, 101.4. Melting Point: 193.7 – 195.2 °C.

4.3. Biophysical experiments

4.3.1. Chemical and protein source

Thioflavin-T (ThT) were purchased from Alfa Aesar (Ward Hill, MA) for the α-syn ThT assays. Heparin sodium salt was purchased from Millipore-Sigma. Procuration of recombinant α-syn and tau 2N4R was from rPeptide (WatKinsville, GA). Concerning the preparation of tau 0N4R, a bacterial expression plasmid consisting of the vector pET30a carrying a cDNA encoding the human Tau 0N4R isoform was a kind gift of Dr. Benjamin Wolozin (Boston University). E. coli stock (Rosetta BL21 Ecoli (CamR) containing pET30a[0N4R tau wt] (KanR)) were grown in LB media supplemented with kanamycin (50 μg/mL) and chloramphenicol (50 μg/mL). Protein over-expression was induced by the addition of 1 mM IPTG for ~ 18 h at 37 °C, and cells were pelleted by centrifugation at 6,000 g for 15 min at 4 °C. The cells were resuspended in lysis buffer (10 mM Hepes pH 7.4, 50 mM NaCl, 1 mM MgCl₂, 1 mM PMSF, 1X PIC, 0.5 mM DTT) and lysed by sonication at 30 sec On 1 min Off at ~ 30–45 % power for ~ 3–5 min. Lysate was centrifuged at 10000 rpm for 10 min at 4 °C and supernatant was transferred with 7.8 mL (total) of 3 M NaCl. The lysate was incubated for 10 min in 80 °C water bath, then cooled for 10 min in an ice bath. The lysate was centrifuge at 10000 rpm, 4 °C for 10 min and supernatant was transferred to new tubes. The supernatant was dialyzed overnight against cation exchange buffer (50 mM MES, 1 M NaCl, 1 mM DTT, pH 6.0). The dialysate was loaded onto a HiPrep SP HP column, and proteins were eluted with a linear gradient ranging from 50 mM to 1 M NaCl. Fractions containing tau isoform 0N4R were pooled, and the resulting protein solution was dialyzed against PBS (pH 7.4) and stored at − 80 °C. Similar procedure and plasmid vector construction were used to produce tau isoform 2N4R for the PICUP assays.

4.3.2. Thioflavin t (ThT) fluorescence assays

A technique commonly used to track the kinetics of α-syn fibril formation in response to various drug candidate treatments is the ThT fluorescence assay.^46,47 The final concentration of the tested compounds was 100 μM while ThT was applied at a final concentration of 20 μM. The recombinant α-syn has been validated with proper quality control to confirm the monomeric state of the protein. α-Syn was dissolved in 20 mM Tris–HCl (pH 7.4) to a stock solution of 280 μM prior to resuspension in ThT buffer to obtain a final concentration of 2 μM (in each well). Compounds and ThT were first added to the wells. The kinetics of fibril formation begin when the α-syn is solubilized in the ThT buffer (10 mM PBS buffer (pH 7.4), supplemented with 0.5 mM SDS and 300 mM NaCl) and added to a non-treated black 96 well microplate with a transparent flat bottom. Each well was filled with a maximum volume of 150 μL buffer with one 3 mm borosilicate bead.⁴⁸ The background fluorescence signal consisted of ThT in buffer and 0.25 % DMSO without α-syn. The excitation and emission wavelengths consisted of 440 and 485 nm, respectively. Measurements were acquired with Synergy HT multi-mode microplate reader (BioTek, Winooski, VT) and taken at 37 °C every 20 min with 10 sec shaking before reading the plate. Kinetics were of 40 to 70 h duration. Samples were measured in three replicates. For each time point, arbitrary units of fluorescence were calculated from the mean values normalized against the maximum value. The percentage of fluorescence intensity at the plateau phase in Tables 1–2 was expressed as mean ± SEM. Concerning the dose–response curve depicted in Figure 2, the data were plotted using GraphPad Prism.

4.3.3. Photo-induced cross-linking of unmodified proteins (PICUP) assay

α-Syn (resourced from rPeptide), 2N4R (produced in the lab), and 0N4R (produced in the lab) tau isoforms were diluted in 10 mM phosphate buffer to achieve final concentrations of 60 μM for α-syn and 10 μM for the tau isoforms.⁴⁹ Compounds were tested at different concentration. Before light exposure, Ru(bpy) (300 μM final concentration) and ammonium persulfate (6 mM final concentration) were added to the protein solution in the presence or absence of compound to start the cross-linking process. In order to verify the progressive impact of our compounds on the suppression of protein oligomerization, controls unexposed to light or exempt of Ru(bpy) were used. The samples underwent quick light exposure using a 53 W (120 V) incandescent bulb in a homemade dark box. Light exposures consisted of 1 s and 60 s for the α-syn and tau isoforms, respectively. The ultimate capacity of each tube was 20 μL. 8.3 μL of Laemmli loading buffer containing 15% of 2-mercaptoethanol was added to the solution right away after light exposure, and the PICUP samples were then incubated at 95 °C for 10 min. On a 16% SDS-PAGE gel, the cross-linked samples were separated and Coomassie blue staining was used for protein band visualization.

4.3.4. Transmission electron microscopy

Tau isoform 2N4R (6 μM, resourced from rPeptide) mixed with DMSO (0.25 %) was incubated in a 10 mM PBS buffer (pH 7.4) for 72 h at 37 °C. α-Syn samples (at 2 μM) recovered from kinetic investigations of ThT fibril formation were also examined. A volume of 10 μL of each sample was put to a 400-mesh Formvar-carbon-coated copper grid (Electron Microscopy Sciences, Hatfield, PA) to prepare the grids for TEM analysis. The sample was incubated with the grids for 1 min before being rinsed three times with distilled water. A new solution of 1 % uranyl acetate was then applied for 1 min after being air-dried. The filter paper was used to absorb the solution, then grids were dried in the air. A transmission electron microscope (JEOL 1400 Flash, Japan) was used to analyze the grids, and micrographs were taken at a magnification of 40 k and an acceleration voltage of 100 kV.

4.4. α-Synuclein inclusion-forming neuroblastoma cell experiment

M17D-TR/αS-3 K::YFP neuroblastoma cells that are doxycycline (dox)-induced have been previously used for this assay.⁵⁰ Compounds were introduced 24 h after the cells had been plated in 96-well plates at a density of 30,000 cells per well. αS-3 K::YFP transgene expression was stimulated 24 h later by adding culture medium dox at a final concentration of 1 μg per mL. The Incucyte Zoom 2000 platform (Essen Biosciences) was used to incubate the cells while taking continuous green bright field pictures. After 48 h of induction (96 h after plating), inclusion formation or growth endpoint analysis was carried out. The Incucyte processing definition “Inclusions” was developed as follows: parameters, fixed threshold, threshold (GCU) 50; edge split on; edge sensitivity 100; cleanup; hole fill (m2): 10; adjust size (pixels); filters; area (m2): max 50; mean intensity: min 60; integrated intensity: min 2000. The following processing definition of “Cells” was used to measure cell confluence: parameters, segmentation adjustment 0.7; Cleanup, all parameters are set to 0; filters, area (m2): min 345.00. A polyclonal antibody to GAPDH (Sigma-Aldrich, St. Louis, MO, G9545; 1:5000) and an S-specific monoclonal antibody 4B12 (Thermofisher, Waltham, MA; 1:1000) were used, as previously described, for the evaluation of protein expression by SDS-PAGE and Western blotting in the LiCor system.⁵¹.

5. Associated content

Additional data pertaining to the characterization of compounds (NMR, HRMS, IR) and additional biophysical (ThT, PICUP) and cell-based results are provided in Supporting Information.

Abbreviations:

Aβ

amyloid-β

AD

Alzheimer’s disease

AI

aminoindole

DCM

dicloromethane

dox

doxycycline

EWG

electron-withdrawing group

DLB

dementia with Lewy bodies

FTDP-17

frontotemporal dementia with Parkinsonism

MAPT

microtubule-associated protein tau

4-MBT

2-amino-4-methoxy benzothiazole

NFTs

neurofibrillary tangles

PD

Parkinson’s disease

PICUP

photo-induced cross-linking of unmodified protein

PSP

progressive supranuclear palsy

RPD

relative pixel density

SEM

standard error of the mean

α-syn

α-synuclein

ThT

thioflavin-T

TEM

transmission electron microscopy

Data availability

No data was used for the research described in the article.