Tryptoline-Based Benzothiazoles Re-sensitize MRSA to β-Lactam Antibiotics

Abstract

Resistance-modifying agents (RMAs) offer a promising solution to combat bacterial antibiotic resistance. Here we report the discovery and structure-activity relationships of a new class of RMAs with a novel tryptoline-based benzothiazole scaffold. Our most potent compound in this series (4ad) re-sensitizes multiple MRSA strains to cephalosporins at low concentrations (2 μg/mL) and has low mammalian cytotoxicity with a half growth inhibitory concentration (GI₅₀) >100 μg/mL in human cervical carcinoma (HeLa) cells. In addition, the same core scaffold with different substitutions also gives good antibacterial activity against MRSA.

Graphical Abstract

1. Introduction

Bacterial antibiotic resistance is a world-wide health concern. Among the growing incidence of antibiotic resistant infectious bacteria, methicillin-resistant Staphylococcus aureus (MRSA) is the most frequently identified resistant pathogen in US hospitals and is associated with exceptionally high rates of morbidity and mortality due to hospital acquired infections (HAIs).^1–8 In fact, a recent report released by the Centers for diseases control and prevention (CDC) estimates that nearly 119,000 noninvasive MRSA infections directly caused more than 20,000 deaths in the 2017 alone.⁹ Furthermore, MRSA infections account for more deaths in the United States than HIV/AIDS and tuberculosis combined.¹⁰ These issues illustrate the necessity for continuous discovery of new antibiotics classes with novel structures and mechanisms of action. Although efforts have been in place to further new antibiotic discovery, only a few antibiotics with novel modes of action have been brought to the clinic in the last 50 years.^{11, 12} This is largely because the rate of antibiotic resistance development has surpassed the rate at which new antibiotics are being discovered and developed.

Resistance-modifying agents (RMAs) offer a promising alterative strategy to combat bacterial resistance.¹³ An RMA is a treatment alternative which does not kill or inhibit the growth of bacteria on its own, but enhances the antibiotic activity of already established antibacterial drugs.¹⁴ A notable advantage of the RMAs is that they can extend the market lifespan of known antibiotics that have already been optimized for large-scale production with well-studied toxicity profiles.¹⁵

Previously, our group demonstrated that compounds containing the tricyclic indoline and chlorobenzene fragment are capable of acting as RMAs in combination with β-lactam antibiotics against MRSA (Of1, Figure 1).^16–21 We showed that structures with bridged tetracyclic indolenine also sensitize a variety of MRSA strains to β-lactams (2, Figure 1).¹⁹ Furthermore, an aza-tricyclic indoline (3, Figure 1) was developed to optimize the physiochemical properties of Of1. while maintaining the RMA activity.²⁰ Since 3 still possesses the RMA properties of 1 while providing a site for facile modification, we used this compound as a core to test the effects of functionalization with different chemical moieties on anti-MRSA activities and mammalian cell toxicity.

Figure 1.

Tricyclic indoline and tryptoline as core structures of RMAs.

Benzimidazole,^{22, 23} benzoxazole²⁴ and benzothiazoles²⁵ are bioactive heterocyclic compounds found in many natural products and pharmaceutical agents. These moieties represent ideal sources of core scaffolds and capping fragments for the design and synthesis of targeted molecules.^26–29 We decided to examine whether functionalization of our tricyclic-indoline core with any of the aforementioned motifs might enhance RMA activity or decrease observed cytotoxicity. Herein we report the discovery of tryptoline-based benzothiazoles (4, Figure 1) as novel RMAs and antibiotics though a rigorous structure-activity relationship (SAR) studies.

2. Results and discussion

2.1. Chemisttry

Tryptoline-based benzothiazoles were either prepared by a one-step reaction using 2-chlorobenzothiazoles and tryptoline as reactants under basic conditions (4a-4k, 4p, 4r-4s, 4u-4x, 4af, Scheme 1), or by a two-step method, which included the synthesis of thiourea (5a-5c, Scheme 1) and palladium catalyzed cyclization of thiourea (4t, 4y-4z, 4ac and 4ag, Scheme 1). The synthesis of tryptoline-based benzoxazoles also followed this method (7a-7c, Scheme 1).

Scheme 1.

General method for the synthesis of tryptoline-based benzothiazoles, benzimidazoles and benzoxazoles. Reagents and conditions: (a) K₂CO₃, DMF, 12 h, 90 °C.; (b) DCM, 2 h, rt; (c) Pd(PPh₃)₄, MnO₂, CH₃CN, O₂, 80 °C, 8 h; (d) BPO, Na₂HPO₄, DMF, 12 h, rt; (e) N-methyl benzimidazole, (TMP)ZnCl•LiCl, Cu(OAc)₂, THF, 12 h, rt.

For the synthesis of tryptoline-based benzimidazoles, we used copper-catalyzed amination of benzimidazole.³⁰ The tryptoline was transformed into O-acylhydroxylamine, and then reacted with N-methyl benzimidazole using C-H zincation/copper-catalyzed electrophilic amination (6a-6c, Scheme 1).

The tryptoline cores ^{31, 32} with ester group at different positions (R⁵, R⁶ and R⁷) reacted with 2, 6-dichlorobenzo[d]thiazole affording 4l-4n (Scheme 2). Replacement of indole nitrogen proton of 4a with ethyl group gave compound 4o (Scheme 2), and compound 4q was prepared from reduction of its nitro precursor (Scheme 2).

Scheme 2.

Synthesis of 4l-4n, 4o and 4q. Reagents and conditions: (a) DMF, TEA, 110 °C, 12 h; (b) NaH, EtI, DMF, overnight, rt; (c) Fe, AcOH, 24 h, rt.

De-protection of methoxy group of 4z with BBr₃ afforded 4aa, which was further functionalized into amine 4ab (Scheme 3). By using copper catalyzed amination, 4ac was transformed into 4ad, which was coupled with amino acid Boc-Gly-OH giving compound 4ae after the removal of N-Boc group (Scheme 3).

Scheme 3.

Synthesis of 4aa-4ae. Reagents and conditions: (a) BBr₃, DCM, 12 h, −78 °C - rt,; (b) 1) N-(2-hydroxyethyl)phthalimide, PPh₃, DIAD, THF, 15 h, reflux; 2) hydrazine hydrate, EtOH, 3 h, reflux; (c) ammonium hydroxide solution, Cu₂O, NMP, 48 h, 80 °C; (d) 1) Boc-Gly-OH, DMAP, EDCI, DCM, 2 h, rt; 2) HCl, 1,4-dioxane, 4 h, rt.

Compound 4ah was also prepared by copper catalyzed amination of compound 4ag (Scheme 4). Further reductive amination of 4ah gave 4ai and 4aj. (Scheme 4).

Scheme 4.

Synthesis of 4ah-4aj. Reagents and conditions: (a) ammonium hydroxide solution, Cu₂O, NMP, 48 h, 80 °C; (b) 1) Na, (HCHO)_n, MeOH, 2 h, reflux; 2) NaBH₄, MeOH, 2 h, 0 °C to reflux; (c) 1) N-Boc-2-aminoacetaldehyde, AcOH, NaHB(OAc)₃, ClCH₂CH₂Cl, 16 h, rt; 2) HCl, 1,4-dioxane, 4 h, rt.

Various functional groups, such as amide, alcohol, amine and thioether were introduced to R⁷ position to probe the SAR and to optimize the physiochemical properties. All these chiral series prepared here were racemic mixture. As show in Scheme 5, hydrolysis of the ester 4n afforded free acid 8, and then amide coupling with dimethylamine afforded amide 9. Reduction of the ester 4n with LiAlH₄ provided the alcohol 10, and further reaction with MOM chloride afforded 11. Compound 12a-12e were prepared via the corresponding iodide intermediate. Further functionalization of the primary amine afforded compound 12d, 12f and 12g (Scheme 5).

Scheme 5.

Synthesis of 8-11 and 12a-12g. Reagents and conditions: (a) LiOH, MeOH/H₂O, 12 h, rt; (b) 1) (COCl)₂, DMF, DCM, 30 min; 2) NHMe₂, TEA, DCM, 3 h, rt; (c) LiAlH₄, THF, 12 h, 0 °C to rt; (d)MOMCl, TEA, DCM, rt; (e) 1) PPh₃, I₂, imidazole, DCM, 1 h, rt; 2) HSCH₂CH₂NHBoc, NaOH, t-BuOH, 120 °C, 3 h; 3) HCl, 1,4-dioxane, rt; (f) 1) PPh₃, I₂, imidazole, DCM, 1 h, rt; 2) NH₃H₂O, t-BuOH, 120 °C, 3 h; (g) 1) PPh₃, I₂, imidazole, DCM, 1 h, rt; 2) HNMe₂, t-BuOH, 120 °C, 2 h; (h) 1) PPh₃, I₂, imidazole, DCM, 1 h, rt; 2) H₂NCH₂CH₂NHBoc, NaOH, t-BuOH, 120 °C, 3 h; 3) HCl, 1,4-dioxane, rt; (i) CH₃I, MeOH, rt, 12 h; (j) Ethyl formimidate hydrochloride, DIEA, THF, −55 °C, 1 h; (k) 1) Bis-Boc-pyrazolocarboxamidine, DIEA, DCM, 2 h, rt; 2) HCl, 1,4-dioxane, rt.

2.2. Structure activity relationship (SAR) study

RMA activity of the analogues was tested by assessing their abilities to sensitize MRSA to the antibiotic cefazolin (a first-generation cephalosporin). The well characterized strain MRSA ATCC BAA-44 was tested as previously described.¹⁶ The minimum re-sensitizing concentration (MRC) was defined as the concentration of analogues at which no overnight growth was observed in the presence of the CLSI breakpoint for antibiotic sensitivity (8 μg/mL for cefazolin). The minimal inhibitory concentration (MIC), or the lowest concentration at which S. aureus is considered susceptible to an antibacterial, was determined by the standard broth microdilution method detailed in the CLSI handbook.¹⁶ RMA activity was compared by the ratio of MIC/MRC. The half growth inhibitory concentration (GI₅₀) of each analogue against HeLa cells was determined as previously described.¹⁶ Compounds that displayed improved RMA activity relative were then tested for toxicity against the growth of human cervical adenocarcinoma (HeLa) cells by incubating different concentrations of each compound with cells for 24 h and assessing viability at each concentration using the Cell Titer Glo mammalian viability assay (Promega). The luminescence of each sample was recorded in an Envision Multilabel Plate Reader (Perkin Elmer). Results of MICs and MRCs were confirmed by testing in triplicate. The GI₅₀ assay was performed in duplicate. Cefazolin and Cefuroxime were used as antibiotic controls and inhibited the growth of MRSA BAA-44 on average at 128 μg/mL or 256 μg/mL for both Cefazolin and Cefuroxime.

2.2.1. Initial screening of tryptoline-based structure for the RMA activity

For the initial screening, we chose chloro-substituted tryptoline as our core structure based on our previous results,²⁰ and found that tryptoline with chloro-substituted benzothiazole motif 4a showed good RMA activity (32 folds, entry 1, Table 1), while benzothiazole without Cl-substitution 4b (entry 2, Table 1) or with methyl substitution 4c (entry 3, Table 1) gave no RMA activity. When the benzothiazole motif was replaced by [1,3]thiazolo[4,5-b]pyridine 4d or [1,3]thiazolo[4,5-b]pyridine with CF₃ substitution 4e, the RMA activity was abolished (entries 4 and 5, Table 1). Interestingly, compounds with a thiourea motif (5a-5c, entries 6-8, Table 1), similar structure to benzothiazole, showed low RMA activity (2-fold), while giving good antibacterial activity instead by themselves (MIC = 2 μg/mL). Surprisingly, compounds with benzimidazole motif (6a-6c, entries 9-11, Table 1) or with benzoxazole motif (7a-7c, entries 12-14, Table 1) had no RMA activity. The SAR studies of tryptoline-based structure revealed that benzothiazole core motif has the most optimal RMA activity and the thiourea motif has good antibacterial activity and moderate RMA activity.

Table 1.

Initial screening of tryptoline-based structure for the RMA activity.

Compd	X	Y	R	MICa	MRCb
4a	S	C	Cl	>64	2
4b	S	C	H	>64	>64
4c	S	C	Me	>64	>64
4d	S	N	H	>64	>64
4e	S	N	CF3	>64	>64
5a	S	C	Cl	4	2
5b	S	C	OCF3	2	1
5c	S	C	CF3	2	1
6a	NMe	C	H	>64	>64
6b	NMe	C	Cl	>64	>64
6c	NMe	c	CF3	>64	>64
7a	O	C	H	>64	>64
7b	O	C	Cl	>64	>64
7c	O	C	CF3	>64	>64

^aAll MIC values are determined by using MRSA BAA-44 strain and reported in μg/mL;

^bA11 MRC values are determined by using MRSA BAA-44 in combination with cefazolin and reported in μg/mL.

2.2.2. Optimization of tryptoline motif for RMA activity

Our SAR study continued with compound 4a (entry 1, Table 1). First, we kept chloro-substituted benzothiazole motif and explored the SAR with various substituted tryptolines. RMA activity was lost when the R² chloride was moved to a different position on the substituted tryptoline (R¹, R³and R⁴, 4f-4h, entries 2-4, Table 2). Other substitutions on the R² position also led to a decrease or abolition of RMA activity (F, Br and OMe, 4i-4k, entries 5-7, Table 2). Furthermore, additional substitutions on the R⁵, R⁶ or R⁸ position (CO₂Et for 4l, CO₂Me for 4m, Et for 4o, entries 8, 9 and 11, Table 2) with Cl on R² position maintained also abolished the RMA activity. However, analog with R⁷-CO₂Me has similar RMA activity to the parent compound 4a (32 folds, 4n, entry10, Table 2) indicating that modifications on this position might be tolerated. These SAR studies show that the nature of substituents and their substitution position on benzene ring of tryptoline both play crucial roles in retaining RMA activity, and chloride at R² position is essential for good RMA activity. Finally, we discovered that substitutions on indole nitrogen or R⁵ and R⁶-piperidine substitutions led to loss of the RMA activity, while the R⁷-piperidine substitution was tolerated for RMA activity.

Table 2.

Optimization of tryptoline motif for the RMA activity.

Compd	R1/R2/R3/R4	R5	R6	R7	R8	MICa	MRCb
4a	H/Cl/H/H	H	H	H	H	>64	2
4f	Cl/H/H/H	H	H	H	H	>16	16
4g	H/H/Cl/H	H	H	H	H	>64	>64
4h	H/H/H/Cl	H	H	H	H	>16	16
4i	H/F/H/H	H	H	H	H	>64	>64
4j	H/Br/H/H	H	H	H	H	4	1
4k	H/OMe/H/H	H	H	H	H	>64	>64
4l	H/Cl/H/H	CO2Et	H	H	H	>64	>64
4m	H/Cl/H/H	H	CO2Me	H	H	>64	>64
4n	H/Cl/H/H	H	H	CO2Me	H	>32	1
4o	H/Cl/H/H	H	H	H	Et	>64	>64

^aAll MIC values are determined by using MRSA BAA-44 strain and reported in μg/mL;

^bAll MRC values are determined by using MRSA BAA-44 in combination with cefazolin and reported in μg/mL.

2.2.3. Optimization of benzothiazole motif for RMA activity

After we optimized tryptoline core, we next attempted to improve the RMA activity and lower toxicity by modifying benzothiazole motif. First, we explored the chloride replacements on the R¹¹ of benzothiazole motif. While bromide analog 4p retained the RMA activity, the corresponding NH₂- and CO₂Et-substituted analogs 4q-4r had a complete loss of RMA activity. Interestingly, the CF₃-substituted analog 4s had a 4 fold increase of RMA activity relative to 4a (entry 5, Table 3), however, the GI₅₀ (HeLa) decreased from 13.2 μg/mL to 4.6 μg/mL. The OCF₃- analog 4t lost the RMA activity completely, while possessing great antibacterial activity by itself (MIC = 1 ug/mL). Moving the chloride from R¹¹ to different positions led to various degrees of RMA activities: R⁹ analog 4w maintained similar RMA activity; R¹⁰ analog 4v showed no RMA activity; R¹² analog 4u displayed reduced RMA activity and increased MIC (entries 7-9, Table 3). R⁹-CF₃ analog 4af gave similar RMA activity to R⁹-Cl analog 4w with 3 fold worse GI₅₀ (entry 16, Table 3). Next, we prepared a series of compounds with either Cl or CF₃ on R⁹ while varying R¹¹ substitutions (Cl: 4x-4ae, entries 10-17; CF₃: 4af-4aj, entries 18-22, Table 3). The R¹¹ substituents covered a wide range, including halide, hydroxyl, ether, amine and amide. While three analogs (4ad, 4af, 4ai) displayed good RMA activities, and several (4x, 4y, 4ae, 4aj) showed excellent MICs. Compound 4ad with Cl on R⁹ and NH₂ on R¹¹ stands out due to its great RMA activity and low mammalian cytotoxicity (GI₅₀ > 100 μg/mL, the highest concentration tested, entry 16, Table 3).

Table 3.

Optimization of benzothiazole motif for the RMA activity.

Compd	R9	R10	R11	R12	MICa	MRCb	GI50c
4a	H	H	Cl	H	>64	2	13.2
4p	H	H	Br	H	>64	2	5.1
4q	H	H	NH2	H	>64	>64	NTd
4r	H	H	CO2Et	H	>64	>64	NTd
4s	H	H	CF3	H	>64	0.5	4.6
4t	H	H	OCF3	H	1	1	7.3
4u	H	H	H	Cl	4	1	6.7
4v	H	Cl	H	H	>64	>64	NTd
4w	Cl	H	H	H	>64	2	27.7
4x	Cl	H	Cl	H	2	1	16.1
4y	Cl	Cl	Cl	H	2	1	11.7
4z	Cl	H	OMe	H	>32	>32	NTd
4aa	Cl	H	OH	H	>32	8	5.5
4ab	Cl	H	OCH2CH2NH2	H	8	4	NTd
4ac	Cl	H	Br	H	>32	8	22
4ad	Cl	H	NH2	H	>32	2	>100
4ae	Cl	H	NHCOCH2NH3Cl	H	2	2	14.8
4af	CF3	H	H	H	>32	1	8
4ag	CF3	H	Br	H	4	0.5	5.9
4ah	CF3	H	NH2HC1	H	16	2	32
4ai	CF3	H	NHCH3	H	>32	2	15.4
4aj	CF3	H	NH(CH2)2NH3Cl	H	1	1	7.4

^aAll MIC values are determined by using MRSA BAA-44 strain and reported in μg/mL;

^bAll MRC values are determined by using MRSA BAA-44 in combination with cefazolin and reported in μg/mL;

^cHeLa was used for determination of GI₅₀ and reported in μg/mL;

^dGI₅₀ was not test.

2.2.4. Further optimization of tryptoline motif on R⁷ for the RMA activity.

During the preliminary SAR studies of tryptoline motif core, we found that R⁷ position can tolerate the methyl ester substitution without compromising the RMA activity (4n, entry 1, Table 4). Further SAR exploration at this position was then performed. The introduction of carboxylic acid and amide significantly decreased or abolished the RMA activity (8 and 9, entries 2-3, Table 4). Surprisingly, conversion of the ester 4n into alcohol 10 and ether 11 led to antibacterial activity with good MIC (MIC = 2.0 μg/mL, entry 4-5, Table 4). Additional analogs incorporating basic amines, charged quarternary ammonium salt, amidine and guarnidine etc. (12a-12g) resulted in good to excellent MICs by themselves, therefore masking their potential RMA activities. Among them, quarternary ammonium salt analog 12d and guarnidine analog 12g showed relatively low level of mammalian cytotoxicity (GI₅₀ of 17.8 and 14.1 ug/mL respectively). While these R⁷-substituted analogs showed diminished RMA activities, their potent antibacterial activities combined with improved physicochemical properties offer promise for the discovery of novel antibacterial agents.

Table 4.

Further optimization of tryptoline motif on R⁷ for the RMA activity.

Compd	R7	MICa	MRCb	GI50c
4n	CO2Me	>32	1	6.6
8	COOH	32	16	65
9	CONMe2	>32	>32	NTd
10	CH2OH	2	2	10.8
11	CH2OCH2OMe	2	2	7.0
12a	CH2SCH2CH2NH3Cl	1	1	3.8
12b	CH2NH2	1	1	1.5
12c	CH2NMe2	2	1	5.2
12d	CH2N+Me3I−	8	4	18
12e	CH2NHCH2CH2NH2	2	2	2.4
12f	CH2NHCH=NH	4	2	3.8
12g	CH2NHC=NHNH3Cl	2	1	14.1

^aAll MIC values are determined by using MRSA BAA-44 strain and reported in μg/mL;

^bAll MRC values are determined by using MRSA BAA-44 in combination with cefazolin and reported in μg/mL;

^cHeLa was used for determination of GI₅₀ and reported in μg/mL;

^dGI₅₀ was not test.

On the basis of the findings and analysis described above, a summary of the SAR of tryptoline-based benzothiazoles in this study is illustrated in Figure 2.

Figure 2.

SAR studies summary of tryptoline-based benzothiazoles

We next explored the scope of RMA activity of 4ad in different MRSA strains, including MRSA BAA-1683³³, MRSA BAA-1764³³, MRSA NR-46411³⁴, NRS-384³⁵ and NRS-100³⁶. These strains were selected because of their diverse geographical origins, genetic background and resistance profile. We also tested RMA activity in combination with another commercial antibiotic, cefuroxime, which is a second-generation cephalosporin. MRCs in the presence of cefuroxime were determined using the same method as those for cefazolin. Our most potent compound 4ad potentiates both antibiotic cefazolin and cefuroxime in the five MRSA strains tested here (Table 5).

Table 5.

Evaluation of the MRC values of 4ad in a panel of MRSA.

Strains	MICa	MRC (cefazolin)b	MRC (cefuroxime)c
MRSA BAA-1683	>32	1	8
MRSA BAA-1764	>32	0.5	32
MRSANR-46411	>32	0.5	<0.25
NRS-384	>32	4	4
NRS-100	>32	4	4

^aAll MIC values are determined by using corresponding strain and reported in μg/mL;

^bAll MRC values are determined by using corresponding strain in combination with cefazolin and reported in μg/mL;

^cAll MRC values are determined by using corresponding strain in combination with cefuroxime and reported in μg/mL.

Furthermore, we explored the scope of the antibacterial activity of 12g in a panel of MRSA and MSSA strains including BAA-44³³ (MIC = 1 μg/mL), MRSA-252³⁷ (MIC = 2 μg/mL), B.subtilis NR-607³⁸ (MIC = 2 μg/mL), E.faecium HM-460³⁹ (MIC = 4 μg/mL), E.faecium 28977⁴⁰ (MIC = 2 μg/mL) and MSSA (MIC = 0.5 μg/mL). Analogue 12g displayed good antibacterial activities (MIC range from 0.5 μg/mL to 4 μg/mL) for all six MRSA and MSSA strains tested here, despite the resistance profiles.

3. Conclusion

In continuation of our efforts to discover new classes of RMAs scaffolds against MRSA, this paper describes tryptoline-based benzothiazoles as a novel class of RMAs. A variety of tryptoline-based benzothiazoles were synthesized and their ability to potentiate the representative β-lactam antibiotics in MRSA and their toxicity in mammalian cells were evaluated. The most potent compound 4ad has strong RMA activity and low mammalian cytotoxicity (MRC = 2 μg/mL, MIC > 32 μg/mL, with GI₅₀ >100 μg/mL). We also identified compound 12g as a novel anti-MRSA antibiotic (MRC = 1 μg/mL, MIC = 2 μg/mL, with GI₅₀ of 14.1 μg/mL). Further investigation of the mode of action of these compounds and the evaluation of their efficacy in vivo are ongoing and will be reported soon.

4. Experimental

All reagents were obtained commercially and used without further purification unless otherwise noted. MRSA strain ATCC BAA-44 was a gift from the laboratory of Daniel Feldheim. Strains BAA-1683(MRSA), BAA-1764(MRSA), NR-46411(MRSA), NRS-100(MRSA), MRSA-252, B.subtilis NR-607, E.faecium HM-460, E.faecium 28977, MSSA, NRS-384(MRSA) and HeLa cells were purchased from ATCC (http://www.atcc.org). CellTiter-Glo® luminescent cell viability assay kit was purchased from Promega Corp. Thin-layer chromatography (TLC) analysis of reaction mixtures was performed on Dynamicadsorbents silica gel F-254 TLC plates. Flash chromatography was carried out on Zeoprep 60 ECO silica gel. ¹H NMR spectra were recorded with Varian INOVA (400, 500 MHz) and Bruker spectrometers. Mass spectral and analytical data were obtained via the PE SCIEX/ABI API QSTAR Pulsar iHybrid LC/MS/MS (Applied Biosystems) operated by the Central Analytical Laboratory, University of Colorado at Boulder. All compounds were evaluated for purity by using an Agilent 1260 series HPLC system coupled with a 6120 Quadrupole mass spectrometer (column: ZORBAX Narrow Bore SB-C18 RRHT, 2.1× 50 mm, 1.8 μm, PN 827700-902) with a minimum purity standard of ≥ 90%. The system was eluted at 0.35 mL/min with a gradient of water/acetonitrile with 0.1% formic acid: 0-5 min, 5-95% acetonitrile; 5-7 min, 95% acetonitrile; 7-7.25 min, 95-5% acetonitrile; 7.25-12 min, 5% acetonitrile.

4.1. General procedure of the synthesis of tryptoline-based benzothiazoles

Method A:⁴¹ To a round bottom flask was added tryptoline (21 mg, 0.100 mmol), 2-chlorobenzothiazole (31 mg, 0.150 mmol), K₂CO₃ (70 mg, 0.500 mmol) and DMF (2.0 mL), and the reaction mixture was stirred for 12 h at 110 °C. After the reaction was then cooled to room temperature, ethyl acetate (10 mL) and H₂O (10 mL) were added into the mixture, and the aqueous layer was extracted with 2 x 10 mL ethyl acetate. The organic layer then was combined and washed with brine, and dried over Na₂SO₄. The solvents were removed under vacuum, and the residue was purified by flash chromatography on silica gel using ethyl acetate/hexane as eluent.

Method B: Synthesis of thiourea:⁴² To a round bottom flask was added tryptoline (206 mg, 1.00 mmol), isothiocyanate (1.10 mmol) and DCM (10 mL), and the reaction mixture was stirred at room temperature for 2 h. The solvents were removed under vacuum and the residue was purified by silica gel flash chromatography using ethyl acetate/hexane (1:2, v/v) as eluent. Pd-catalyzed cyclization of thiourea:⁴³ To a flame-dried 150 × 20 mm reaction tube was added thiourea (0.500 mmol), activated manganese dioxide (0.0043 g, 0.050 mmol), and tetrakistriphenylphosphine palladium(0) (17.3 mg, 0.015 mmol). Anhydrous acetonitrile (6 mL) was then added. The reaction system was then heated to reflux (80 °C) with vigorous stirring under an oxygen atmosphere for 6 h. The reaction mixture was then cooled to room temperature and filtered to remove solid manganese dioxide. The solvents were removed under vacuum and the residue was then purified via silica gel column chromatography using ethyl acetate/hexane (1:3, v/v) as eluent.

6-Chloro-2-(6-chloro-1,3-benzothiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (4a) was prepared by method A. Pale yellow solid was obtained with a yield of 38%. ¹H NMR (300 MHz, DMSO-d₆) δ 11.15 (s, 1H), 7.91 (q, J = 2.5 Hz, 1H), 7.57 – 7.41 (m, 2H), 7.41 – 7.21 (m, 2H), 7.06 (dt, J = 8.7, 2.4 Hz, 1H),4.87 (s, 2H), 3.92 (d, J = 5.6 Hz, 2H), 2.86 (d, J = 6.0 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.73, 151.24, 134.47, 132.22, 131.86, 127.55, 126.23, 125.04, 123.32, 120.96, 120.86, 119.55, 117.06, 112.63, 106.70, 46.99, 45.62, 20.28. LC-MS, Rt = 7.483 min, [M+H]⁺ = 374.0. HRMS (ESI) m/z calcd. for C₁₈H₁₄Cl₂N₃S [M+H]⁺ = 374.0280, found 374.0262.

2-(1,3-Benzothiazol-2-yl)-6-chloro-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (4b) was prepared by method A. Pale yellow solid was obtained with a yield of 48%. ¹H NMR (400 MHz, Chloroform-d) δ 7.97 (s, 1H), 7.63 – 7.59 (m, 1H), 7.56 (d, J = 7.9 Hz, 1H), 7.45 (d, J = 2.0 Hz, 1H), 7.30 (td, J = 8.3, 7.8, 1.3 Hz, 1H), 7.25 – 7.24 (m, (s, 1H), 7.14 – 7.04 (m, 1H), 4.93 (s, 2H), 3.94 (t, J = 5.7 Hz, 2H), 2.95 (t, J = 5.8 Hz, 2H). LC-MS, Rt = 7.397 min, [M+H]⁺ = 340.0. HRMS (ESI) m/z calcd. for C₁₈H₁₅ClN₃S [M+H]⁺ = 340.0670, found 340.0671.

6-Chloro-2-(6-methyl-1,3-benzothiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (4c) was prepared by method A. Yellow solid was obtained with a yield of 45%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.14 (s, 1H), 7.51 (d, J = 1.7 Hz, 1H), 7.42 (d, J = 2.0 Hz, 1H), 7.32 (dd, J = 8.4, 3.8 Hz, 2H), 7.03 (ddd, J = 13.0, 8.4, 1.9 Hz, 2H), 4.80 (s, 2H), 3.84 (t, J = 5.7 Hz, 2H), 2.80 (t, J = 5.7 Hz, 2H), 2.28 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 167.57, 150.20, 134.47, 132.51, 130.55, 130.37, 127.61, 127.11, 123.30, 121.13, 121.05, 120.82, 118.37, 118.30, 117.08, 117.00, 112.65, 106.71, 46.94, 45.57, 20.84, 20.78, 20.29. LC-MS, Rt = 7.881 min, [M+H]⁺ = 354.0. HRMS (ESI) m/z calcd. for C₁₉H₁₇ClN₃S [M+H]⁺ = 354.0827, found 354.0840.

6-Chloro-2-([1,3]thiazolo[4,5-b]pyridin-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (4d) was prepared by method A. Yellow solid was obtained with a yield of 29%. ¹H NMR (500 MHz, DMSO-d₆) δ 11.22 (s, 1H), 8.32 (dd, J = 4.8, 1.7 Hz, 1H), 8.22 (dd, J = 7.8, 1.7 Hz, 1H), 7.50 (d, J = 2.1 Hz, 1H), 7.37 (d, J = Hz, 1H), 7.11 – 6.83 (m, 2H), 4.94 (s, 2H), 3.99 (t, J = 4.4 Hz, 2H), 2.90 (t, J = 5.8 Hz, 2H). LC-MS, Rt = 5.552 min, [M+H]⁺ = 341.0. HRMS (ESI) m/z calcd. for C₁₇H₁₄ClN₄S [M+H]⁺ = 341.0623, found 341.0658.

6-Chloro-2-[6-(trifluoromethyl)[1,3]thiazolo[4,5-b]pyridin-2-yl]-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (4e) was prepared by method A. Pale yellow solid was obtained with a yield of 39%. ¹H NMR (500 MHz, Chloroform-d) δ 8.73 – 8.61 (m, 1H), 8.12 (d, J = 2.2 Hz, 1H), 7.49 – 7.43 (m, 1H), 7.47 – 7.33 (m, 1H), 7.21 (ddd, J = 12.9, 8.7, 2.0 Hz, 1H), 5.14 (d, J = 24.3 Hz, 2H), 4.04 (d, J = 9.8 Hz, 2H), 2.98 (dddd, J = 12.4, 6.4, 4.2, 1.9 Hz, 2H). LC-MS, Rt = 6.626 min, [M+H]⁺ = 408.0. HRMS (ESI) m/z calcd. for C₁₈H₁₃ClF₃N₄S [M+H]⁺ = 409.0496, found 409.0448.

6-Chloro-N-(4-chlorophenyl)-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole-2-carbothioamide (5a) was prepared by method B. Pale yellow solid was obtained with a yield of 63%. ¹H NMR (400 MHz, Chloroform-d) δ 8.05 (s, 1H), 7.40 (d, J = 2.0 Hz, 1H), 7.36 (s, 1H), 7.27 (d, J = 8.6 Hz, 2H), 7.20 (d, J = 8.5 Hz, 1H), 7.15 (d, J = 8.7 Hz, 2H), 7.10 (dd, J = 8.6, 2.0 Hz, 1H), 5.05 (s, 2H), 4.12 – 4.02 (m, 2H), 2.82 (t, J = 5.9 Hz, 2H). LC-MS, Rt = 7.246 min, [M+H]⁺ = 376.0. HRMS (ESI) m/z calcd. for C₁₈H₁₆Cl₂N₃S [M+H]⁺ = 376.0437, found 376.0462.

6-Chloro-N-[4-(trifluoromethoxy)phenyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole-2-carbothioamide (5b) was prepared by method B. Pale yellow solid was obtained with a yield of 59%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.17 (s, 1H), 9.64 (s, 1H), 7.49 (d, J = 2.1 Hz, 1H), 7.45 (s, 1H), 7.43 (s, 1H), 7.34 (d, J = 8.6 Hz, 1H), 7.34 – 7.29 (m, 1H), 7.32 – 7.27 (m, 1H), 7.05 (dd, J = 2.1 Hz, 1H), 5.18 (s, 2H), 4.22 (t, J = 5.6 Hz, 2H), 2.85 (t, J = 5.5 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 181.91, 144.74, 144.72, 140.29, 134.59, 133.08, 127.58, 126.96, 123.30, 121.45, 120.81, 120.74, 118.91, 117.08, 112.60, 109.60, 107.16, 47.19, 46.55, 40.15, 39.94, 39.73, 39.52, 39.31, 39.10, 38.89, 20.83. LC-MS, Rt = 6.354 min, [M+H]⁺ = 426.0. HRMS (ESI) m/z calcd. for C₁₉H₁₆ClF₃N₃OS [M+H]⁺ = 426.0650, found 426.0690.

6-Chloro-N-[3-(trifluoromethyl)phenyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole-2-carbothioamide (5c) was prepared by method B. Pale yellow solid was obtained with a yield of 60%. ¹H NMR (400 MHz, Chloroform-d) δ 7.86 (s, 1H), 7.59 (d, J = 8.4 Hz, 2H), 7.44 (dd, J = 4.2, 2.0 Hz, 1H), 7.36 – 7.27 (m, 3H), 7.25 – 7.24 (m, 3H), 7.16 – 7.09 (m, 1H), 5.10 (m, 2H), 4.21 – 4.03 (m, 2H), 2.89 (t, J = 6.0 Hz, 21H). LC-MS, Rt = 7.520 min, [M+H]⁺ = 410.0. HRMS (ESI) m/z calcd. for C₁₉H₁₆ClF₃N₃S [M+H]⁺ = 410.0701, found 410.0694.

4.2. General procedure for the synthesis of tryptoline-based benzimidazoles (6a-6c)

To a round bottom flask was added tryptoline (206 mg, 1.00 mmol), BPO (242 mg, 1.00 mmol), Na₂HPO₄ (215 mg, 1.50 mmol) and DMF (10 mL), and the reaction mixture was stirred for 12 h at room temperature. Ethyl acetate (50 mL) and H₂O (50 mL) were added into the mixture, and the aqueous layer was extract with 2 x 50 mL ethyl acetate. The organic layer then was combined and washed with saturated NaHCO₃ solution and then with brine, and dried over Na₂SO₄. The solvents were removed under vacuum, and the residue was purified by silica gel flash chromatography using ethyl acetate/hexane (1:2, v/v) as eluent. O-acylhydroxylamine was obtained as pale white solid with a yield of 26%. To a 10 mL tube charged with N-methylbenzimidazole (27 mg, 0.200 mmol, 1.0 equiv) was added THF (1 mL) followed by dropwise addition of (TMP)ZnCl·LiCl solution (0.200 mmol, 1.0 equiv) under N₂. The resulting mixture was stirred vigorously at room temperature for 1 h. Then a mixture of Cu(OAc)₂ (3.70 mg, 0.020 mmol, 0.10 equiv) and O-acylhydroxylamine (0.240 mmol, 1.2 equiv) in THF (1 mL) was added dropwise to the heteroarylzinc mixture under N₂. Upon complete consumption of the N-methylbenzimidazole, the reaction was quenched by dropwise addition of a saturated NH₄Cl solution (1 mL). The reaction mixture was subsequently basified with saturated Na₂CO₃ solution (5 mL) and extracted with ethyl acetate (3 × 5 mL). The combined organic layers were washed with brine (5 mL), dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure. The crude reaction mixture was purified by silica gel flash-column chromatography.

6-Chloro-2-(1-methyl-1H-benzimidazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (6a) was prepared by the general procedure. Pale yellow solid was obtained with a yield of 39%. ¹H NMR (400 MHz, Chloroform-d) δ 8.79 (s, 1H), 7.60 – 7.55 (m, 1H), 7.45 (d, J = 2.1 Hz, 1H), 7.24 (m, 1H), 7.23 – 7.16 (m, 3H), 7.07 (dd, J = 8.6, 2.1 Hz, 3H), 4.67 (s, 2H), 3.70 (s, 3H), 3.60 (t, J = 5.6 Hz, 2H), 2.98 (t, J = 5.6 Hz, 2H). LC-MS, Rt = 4.719 min, [M+H]⁺ = 353.1. HRMS (ESI) m/z calcd. for C₁₉H₁₇ClN₄ [M+H]⁺ = 337.1215, found 337.1198.

6-Chloro-2-(6-chloro-1-methyl-1H-benzimidazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (6b) was prepared by the general procedure. Pale yellow solid was obtained with a yield of 38%. ¹H NMR (500 MHz, Chloroform-d) δ 8.11 (s, 1H), 7.57 (d, J = 1.8 Hz, 1H), 7.50 (dd, J = 8.4, 2.1 Hz, 2H), 7.28 (s, 13H), 7.22 – 7.05 (m, 1H), 4.65 (s, 2H), 3.71 (s, 3H), 3.64 (t, J = 5.7 Hz, 2H), 3.01 (t, J = 5.8 Hz, 2H). LC-MS, Rt = 5.919 min, [M+H]⁺ = 371.0. HRMS (ESI) m/z calcd. for C₁₉H₁₇Cl₂N₄ [M+H]⁺ = 371.0825, found 371.0862.

6-Chloro-2-[1-methyl-6-(trifluoromethyl)-1H-benzimidazol-2-yl]-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (6c) was prepared by the general procedure. Pale yellow solid was obtained with a yield of 36%. ¹H NMR (400 MHz, Chloroform-d) δ 9.17 (d, J = 12.5 Hz, 1H), 7.83 – 7.74 (m, 1H), 7.51 – 7.37 (m, 3H), 7.32 – 7.24 (m, 1H), 7.12 (ddd, J = 8.7, 1.5, 0.6 Hz, 1H), 7.04 (ddd, J = 8.6, 2.0, 1.3 Hz, 1H), 4.77 – 4.57 (m, 2H), 3.73 (d, J = 3.9 Hz, 3H), 3.65 (dt, J = 8.4, 5.6 Hz, 2H), 3.02 – 2.90 (m, 2H). LC-MS, Rt = 6.748 min, [M+H]⁺ = 405.1. HRMS (ESI) m/z calcd. for C₂₀H₁₇ClF₃N₄ [M+H]⁺ = 405.1089, found 405.1087.

2-(1,3-Benzoxazol-2-yl)-6-chloro-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (7a) was prepared following method A by using 2-chlorobenzoxazole. Pale yellow solid was obtained with a yield of 38%. ¹H NMR (400 MHz, Chloroform-d) δ 8.12 (s, 1H), 7.45 (d, J = 2.0 Hz, 1H), 7.36 (dt, J = 7.8, 0.7 Hz, 1H), 7.30 (d, J = 1.0 Hz, 1H), 7.24 (m, 1H), 7.17 (td, J = 7.7, 1.1 Hz, 1H), 7.11 (dd, J = 8.6, 2.1 Hz, 1H), 7.04 (td, J = 7.7, 1.3 Hz, 1H),4.91 (s, 2H), 4.06 (t, J = 5.7 Hz, 2H), 2.93 (dd, J = 6.7, 5.1 Hz, 2H). LC-MS, Rt = 7.746 min, [M+H]⁺ = 358.0. HRMS (ESI) m/z calcd. for C₁₈H₁₅ClN₃O [M+H]⁺ = 324.0899, found 324.0901.

6-Chloro-2-(6-chloro-1,3-benzoxazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (7b) was prepared following method A by using 2,6-dichloro-benzoxazole. Pale yellow solid was obtained with a yield of 39%. ¹H NMR (400 MHz, Methanol-d₄) δ 7.40 (dd, J = 2.1, 0.6 Hz, 1H), 7.38 – 7.31 (m, 1H), 7.29 (ddd, J = 7.8, 1.3, 0.6 Hz, 1H), 7.25 (d, J = 0.6 Hz, 1H), 7.17 (td, J = 7.7, 1.2 Hz, 1H), 7.10 – 6.98 (m, 2H), 4.88 (s, 1H), 4.05 (t, J = 5.8 Hz, 2H), 2.90 (t, J = 5.7 Hz, 2H). LC-MS, Rt = 7.284 min, [M+H]⁺ = 324.1. HRMS (ESI) m/z calcd. for C₁₈H₁₄Cl₂N₃O [M+H]⁺ = 358.0509, found 358.0535.

6-Chloro-2-[6-(trifluoromethyl)-1,3-benzoxazol-2-yl]-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (7c) was prepared following method A by using 2-chloro-6-(trifluoromethyl)-benzoxazole. Pale yellow solid was obtained with a yield of 35%. ¹H NMR (500 MHz, Chloroform-d) δ 8.11 (s, 1H), 7.49 – 7.40 (m, 2H), 7.25 (d, J = 8.6 Hz, 1H), 7.16 – 7.06 (m, 2H), 4.94 (s, 2H), 4.09 (t, J = 5.8 Hz, 2H), 2.91 (t, J = 5.8 Hz, 2H). LC-MS, Rt = 7.928 min, [M+H]⁺ = 392.0. HRMS (ESI) m/z calcd. for C₁₉H₁₄ClF₃N₃0 [M+H]⁺ = 392.0773, found 392.0739.

5-Chloro-2-(6-chloro-1,3-benzothiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (4f) was prepared by method A. Yellow solid was obtained with a yield of 46%. ¹H NMR (500 MHz, Chloroform-d) δ 9.29 (s, 1H), 7.61 (d, J = 2.1 Hz, 1H), 7.47 (d, J = 8.6 Hz, 1H), 7.41 (d, J = 2.1 Hz, 1H), 7.26 (dd, J = 8.5, 2.0 Hz, 1H), 7.07 (s, 1H), 7.05 (s, 1H), 4.95 (s, 2H), 3.99 – 3.90 (m, 2H), 3.37 (td, J = 5.6, 4.7, 2.8 Hz, 2H). ). LC-MS, Rt = 7.013 min, [M+H]⁺ = 374.0. HRMS (ESI) m/z calcd. for C₁₈H₁₄Cl₂N₃S [M+H]⁺ = 374.0280, found 374.0262.

7-Chloro-2-(6-chloro-1,3-benzothiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (4g) was prepared by method A. Yellow solid was obtained with a yield of 48%. ¹H NMR (400 MHz, Chloroform-d) δ 7.94 (s, 1H), 7.57 (d, J = 2.2 Hz, 1H), 7.44 (d, J = 8.6 Hz, 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.32 (dd, J = 1.9, 0.6 Hz, 1H), 7.24 (m, 1H), 7.08 (dd, J = 8.4, 1.8 Hz, 1H), 4.89 (s, 2H), 3.93 (t, J = 5.7 Hz, 2H), 2.95 (t, J = 5.7 Hz, 2H). LC-MS, Rt = 8.132 min, [M+H]⁺ = 374.0. HRMS (ESI) m/z calcd. for C₁₈H₁₄Cl₂N₃S [M+H]⁺ = 374.0280, found 374.0262.

8-Chloro-2-(6-chloro-1,3-benzothiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (4h) was prepared by method A. Yellow solid was obtained with a yield of 68%. ¹H NMR (500 MHz, Chloroform-d) δ 8.17 (s, 1H), 7.61 (d, J = 2.1 Hz, 1H), 7.49 (d, J = 8.6 Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.31 – 7.25 (m, 1H), 7.20 (dd, J = 7.7, 1.0 Hz, 1H), 7.08 (t, J = 7.8 Hz, 1H), 4.96 (s, 2H), 3.99 (t, J = 5.7 Hz, 2H), 3.14 – 2.90 (m, 2H). LC-MS, Rt = 7.006 mm, [M+H]⁺ = 374.0. HRMS (ESI) m/z calcd. for C₁₈H₁₄Cl₂N₃S [M+H]⁺ = 374.0280, found 374.0262.

2-(6-Chloro-1,3-benzothiazol-2-yl)-6-fluoro-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (4i) was prepared by method A. Yellow solid was obtained with a yield of 48%. ¹H NMR (400 MHz, Chloroform-d) δ 7.93 (s, 1H), 7.58 (d, J = 2.2 Hz, 1H), 7.44 (d, J = 8.5 Hz, 1H), 7.32 – 7.19 (m, 3H), 7.12 (dd, J = 9.3, 2.5 Hz, 1H), 6.91 (td, J = 9.1, 2.5 Hz, 1H), 4.91 (s, 2H), 3.93 (t, J = 5.7 Hz, 2H), 2.94 (t, J = 5.7 Hz, 2H). LC-MS, Rt = 7.768 min, [M+H]⁺ = 358.0. HRMS (ESI) m/z calcd. for C₁₈H₁₄ClFN₃S [M+H]⁺ = 358.0576, found 358.0535.

6-Bromo-2-(6-chloro-1,3-benzothiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (4j) was prepared by method A. Yellow solid was obtained with a yield of 38%. ¹H NMR (400 MHz, Chloroform-d) δ 7.99 (s, 1H), 7.62 – 7.60 (m, 1H), 7.58 (d, J = 2.2 Hz, 1H), 7.44 (d, J = 8.6 Hz, 1H), 7.27 – 7.22 (m, 2H), 7.19 (d, J = 8.6 Hz, 1H), 4.91 (s, 2H), 3.92 (t, J = 5.7 Hz, 2H), 2.94 (ddd, J = 5.9, 4.1, 1.7 Hz, 2H). LC-MS, Rt = 8.217 min, [M+H]⁺ = 417.0. HRMS (ESI) m/z calcd. for C₁₈H₁₄BrClN₃S [M+H]⁺ = 417.9775, found 417.9819.

2-(6-Chloro-1,3-benzothiazol-2-yl)-6-methoxy-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (4k) was prepared by method A. Yellow solid was obtained with a yield of 42%. ¹H NMR (400 MHz, Chloroform-d) δ 7.79 (s, 1H), 7.57 (d, J = 2.1 Hz, 1H),(d, J = 8.6 Hz, 1H), 7.24 (s, 2H), 6.93 (d, J = 2.5 Hz, 1H), 6.82 (dd, J = 8.8, 2.5 Hz, 1H), 4.87 (s, 2H), 3.93 (t, J = 5.7 Hz, 2H), 3.84 (s, 3H), 2.95 (t, J = 5.7 Hz, 2H). LC-MS, Rt = 7.552 min, [M+H]⁺ = 370.1. HRMS (ESI) m/z calcd. for C₁₉H₁₇ClN₃OS [M+H]⁺ = 370.0776, found 370.0780.

Ethyl 6-chloro-2-(6-chloro-1,3-benzothiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-1-carboxylate (4l) was prepared by method A with ethyl ester modified tryptoline (6-chloro-2,3,4,9-tetrahydro-1H-Pyrido [3,4-b]indole-1-carboxylic acid ethyl ester prepared according to the literature).⁴⁴ Pale yellow solid was obtained with a yield of 21%. ¹H NMR (400 MHz, Chloroform-d) δ 8.93 (s, 1H), 7.88 (d, J = 8.7 Hz, 1H), 7.80 (d, J = 2.1 Hz, 1H), 7.47 (dd, J = 2.0, 1.0 Hz, 1H), 7.39 (ddd, J = 8.8, 2.1, 0.9 Hz, 1H), 7.29 (d, J = 8.6 Hz, 1H), 7.13 (ddd, J = 8.6, 2.1, 0.9 Hz, 1H), 4.31 (qdd, J = 7.1, 4.4, 0.9 Hz, 2H), 3.48 (s, 1H), 3.25 (dtt, J = 23.8, 12.4, 5.6 Hz, 2H), 2.90 – 2.68 (m, 2H), 1.29 (td, J = 7.1, 0.9 Hz, 3H). LC-MS, Rt = 8.200 min, [M+H]⁺ = 446.0. HRMS (ESI) m/z calcd. for C₂₁H₁₈C₁₂N₃O₂S [M+H]⁺ = 446.0492, found 446.0450.

Methyl 6-chloro-2-(6-chloro-1,3-benzothiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate (4m) was prepared by method A with methyl ester modified tryptoline (6-chloro-2,3,4,9-tetrahydro-1H-Pyrido[3,4-b]indole-3-carboxylic acid ethyl ester prepared according to the literature).⁴⁵ Pale yellow solid was obtained with a yield of 20%. ¹H NMR (400 MHz, Chloroform-d) δ 8.11 (d, J = 8.8 Hz, 1H), 7.83 (d, J = 8.6 Hz, 1H), 7.81 (d, J = 2.1 Hz, 1H), 7.47 (dd, J = 4.4, 2.1 Hz, 2H), 7.28 (dd, J = 8.8, 2.1 Hz, 1H), 4.59 (d, J = 17.1 Hz, 1H), 4.51 – 4.40 (m, 1H), 4.16 – 4.05 (m, 1H), 3.88 (d, J = 4.8 Hz, 1H), 3.81 (s, 3H), 3.13 (dd, J = 15.8, 4.7 Hz, 1H), 2.98 – 2.87 (m, 1H).LCMS, Rt = 7.860 min, [M+H]⁺ = 432.0. HRMS (ESI) m/z calcd. for C₂₀H₁₆Cl₂N₃O₂S [M+H]⁺ = 432.0335, found 432.0355.

Methyl 6-chloro-2-(6-chlorobenzo[d]thiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-4-carboxylate (4n) was prepared by method A with methyl ester modified tryptoline (6-chloro-2,3,4,9-tetrahydro-1H-Pyrido[3,4-b]indole-4-carboxylic acid methyl ester prepared according to the literature).³² Pale yellow solid was obtained with a yield of 82%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.35 (s, 1H), 7.95 (t, J = 1.7 Hz, 1H), 7.55 – 7.45 (m, 2H), 7.40 (d, J = 8.6 Hz, 1H), 7.32 (dd, J = 8.6, 2.2 Hz, 1H), 7.10 (dd, J = 8.6, 2.1 Hz, 1H), 5.07 (d, J = 16.6 Hz, 1H), 4.68 (dd, J = 16.6, 1.5 Hz, 1H), 4.38 (dd, J = 13.3, 3.1 Hz, 1H), 4.21 – 4.13 (m, 1H), 3.90 (dd, J = 13.4, 4.5 Hz, 1H), 3.61 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 172.12, 168.74, 151.03, 134.57, 133.11, 131.96, 127.43, 127.40, 126.23, 125.18, 123.64, 121.13, 119.67, 117.81, 112.81, 109.58, 104.51, 52.00, 49.08, 45.16, 37.73. LC-MS, Rt = 5.619 min, [M+H]⁺ = 432.0. HRMS (ESI) m/z calcd. for C₂₀H₁₆Cl₂N₃O₂S [M+H]⁺ = 432.0335, found 432.0355.

6-Chloro-2-(6-chloro-1,3-benzothiazol-2-yl)-9-ethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (4o) To a stirred solution of 4a (37 mg, 0.100 mmol) in dry DMF (6.0 mL), NaH (50.0 mg, 60% suspension in mineral oil, 0.300 mmol) was added portionwise under nitrogen atmosphere at 0 °C. The reaction mixture was then warmed to room temperature and stirred for 30 min. After cooling to 0 °C, EtI (47 mg, 0.300 mmol) was added dropwise to the reaction mixture. The reaction mixture was warmed to room temperature and stirred overnight. Water was added and the aqueous layer was extracted with ether. The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (n-hexane/ethyl acetate = 7/2) to give the title compound with a yield of 60%. ¹H NMR (500 MHz, Chloroform-d) δ 7.61 (d, J = 2.1 Hz, 1H), 7.53 – 7.42 (m, 2H), 7.29 (d, J = 7.9 Hz, 1H), 7.25 (d, J = 8.6 Hz, 1H), 7.17 (dd, J = 8.8, 2.0 Hz, 1H), 4.94 (s, 2H), 4.14 (q, J = 7.3 Hz, 2H), 3.92 (t, J = 5.7 Hz, 2H), 2.97 (t, J = 5.8 Hz, 2H), 1.41 (t, J = 7.2 Hz, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 168.88, 151.32, 134.60, 131.82, 131.59, 127.61, 126.74, 126.70, 126.67, 126.63, 125.11, 121.79, 120.60, 120.50, 119.90, 119.81, 117.91, 117.82, 107.28, 47.97, 44.91, 38.46, 21.11, 15.62. LC-MS, Rt = 7.660 min, [M+H]⁺ = 402.1. HRMS (ESI) m/z calcd. for C₂₀H₁₈Cl₂N₃S [M+H]⁺ = 402.0593, found 402.0575.

2-(6-Bromo-1,3-benzothiazol-2-yl)-6-chloro-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (4p) was prepared by method A using 6-bromo-2-chloro-benzothiazole as reactant. Yellow solid was obtained with a yield of 44%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.17 (s, 1H), 8.01 (d, J = 1.2 Hz, 1H), 7.46 (d, J = 2.0 Hz, 1H), 7.40 (d, J = 1.2 Hz, 2H), 7.37 (d, J = 8.6 Hz, 1H), 7.06 (dd, J = 2.1 Hz, 1H), 4.86 (s, 2H), 3.91 (t, J = 5.7 Hz, 2H), 2.85 (t, J = 5.7 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.67, 151.55, 134.48, 132.35, 132.19, 128.93, 127.55, 123.66, 123.34, 120.87, 117.06, 112.67, 112.63, 106.69, 47.00, 45.61, 20.29. LC-MS, Rt = 7.529 min, [M+H]⁺ = 417.0. HRMS (ESI) m/z calcd. for C₁₈H₁₄BrClN₃S [M+H]⁺ = 417.9753, found 417.9729.

2-(6-Chloro-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-1,3-benzothiazol-6-amine (4q) was prepared by reduction of nitro precursor, which was prepared by method A using 2-chloro-6-nitro-benzothiazole as reactant. Nitro precursor: yellow solid was obtained with a yield of 42%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.19 (s, 1H), 8.86 (d, J = 2.5 Hz, 1H), 8.63 (d, J = 2.4 Hz, 1H), 7.57 (d, J = 8.8 Hz, 1H), 7.49 (d, J = 2.1 Hz, 1H), 7.38 (d, J = 8.6 Hz, 1H), 7.07 (dd, J = 8.6, 2.1 Hz, 1H), 4.97 (s, 2H), 4.04 (m, 2H), 2.97 – 2.86 (m, 2H).LC-MS, Rt = 7.592 min, [M+H]⁺ = 385.0. To a solution of the nitro precursor (38.5 mg, 1.00 mmol) in AcOH (10 mL) at 0-10 °C was added Fe powder (280.0 mg, 5.00 mmol). The reaction mixture was stirred at room temperature for 24 h, was then concentrated under vacuum. The residue was diluted with H₂O and basified with saturated NaHCO₃ solution. The mixture was extracted with EtOAc (2 x 50 mL), dried over Na₂SO₄, and concentrated. The residue was purified by flash chromatography on silica gel using ethyl acetate/hexane (2:1, v/v) as eluent. Pale yellow solid was obtained with a yield of 75%. ¹H NMR (400 MHz, Chloroform-d) δ 8.04 (s, 1H), 7.45 (d, J = 2.0 Hz, 1H), 7.38 (d, J = 8.5 Hz, 1H),(d, J= 8.5 Hz, 1H), 7.11 (dd, J = 8.6, 2.0 Hz, 1H), 6.98 (d, J = 2.4 Hz, 1H), 6.69 (dd, J = 8.5, 2.4 Hz, 1H), 4.88 (s, 2H), 3.89 (t, J = 5.7 Hz, 2H), 2.98 – 2.90 (m, 2H). LC-MS, Rt = 5.273 min, [M+H]⁺ = 355.1. HRMS (ESI) m/z calcd. for C₁₈H₁₆ClN₄S [M+H]⁺ = 355.0779, found 355.0784.

Ethyl 2-(6-chloro-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-1,3-benzothiazole-6-carboxylate (4r) was prepared by method A using 2-chloro-6-Benzothiazolecarboxylic acid ethyl ester as reactant. Yellow solid was obtained with a yield of 42%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.15 (s, 1H), 8.41 (d, J = 1.7 Hz, 1H), 7.85 (ddd, J = 8.5, 1.9, 0.9 Hz, 1H), 7.50 (d, J = 8.4 Hz, 1H), 7.46 (d, J = 2.0 Hz, 1H), 7.34 (d, J = 8.6 Hz, 1H), 7.03 (ddd, J = 8.6, 2.2, 1.0 Hz, 1H), 4.89 (s, 2H), 4.27 (q, J = 7.1 Hz, 2H), 4.01 – 3.90 (m, 2H), 2.86 (t, J = 5.8 Hz, 2H), 1.40 – 1.15 (m, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 170.83, 165.54, 156.25, 134.50, 132.05, 130.48, 127.53, 123.34, 123.01, 122.40, 120.91, 118.01, 117.09, 112.67, 106.72, 60.51, 39.94, 39.73, 39.52, 39.31, 39.10, 20.31, 14.30. LC-MS, Rt = 6.810 min, [M+H]⁺ = 412.0. HRMS (ESI) m/z calcd. for C₂₁H₁₉ClN₃O₂S [M+H]⁺ = 412.0882, found 412.0869.

6-Chloro-2-[6-(trifluoromethyl)-1,3-benzothiazol-2-yl]-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (4s) was prepared by method A using 2-chloro-6-(trifluoromethyl)-benzothiazole as reactant. Pale yellow solid was obtained with a yield of 44%. ¹H NMR (400 MHz, Chloroform-d) δ 8.15 (s, 1H), 7.90 – 7.83 (m, 1H), 7.61 – 7.49 (m, 1H), 7.44 (d, J = 2.0 Hz, 1H), 7.21 (dd, J = 8.6, 0.6 Hz, 1H), 7.11 (dd, J = 8.6, 2.0 Hz, 1H), 4.94 (t, J = 1.6 Hz, 2H), 3.96 (t, J = 5.7 Hz, 2H), 2.95 (tt, J = 5.6, 1.5 Hz, 2H). LC-MS, Rt = 8.021 min, [M+H]⁺ = 408.1. HRMS (ESI) m/z calcd. for C₁₉H₁₄ClF₃N₃S [M+H]⁺ = 408.0544, found 408.0568.

6-Chloro-2-[6-(trifluoromethoxy)-1,3-benzothiazol-2-yl]-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (4t) was prepared by method A using 2-chloro-6-(trifluoromethoxy)-benzothiazole as reactant. Pale yellow solid was obtained with a yield of 84%. ¹H NMR (400 MHz, Chloroform-d) δ 8.06 (s, 1H), 7.54 – 7.46 (m, 2H), 7.44 (d, J = 2.0 Hz, 1H), 7.23 (d, J = 10.8 Hz, 1H), 7.20 – 7.12 (m, 1H), 7.11 (dd, J = 8.6, 2.1 Hz, 1H), 4.91 (s, 2H), 3.93 (t, J = Hz, 2H), 2.94 (t, J = 5.7 Hz, 2H). LC-MS, Rt = 7.031 min, [M+H]⁺ = 424.0. HRMS (ESI) m/z calcd. for C₁₉H₁₄ClF₃N₃OS [M+H]⁺ = 424.0493, found 424.0534.

6-Chloro-2-(7-chloro-1,3-benzothiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (4u) was prepared by method A using 2,7-dichloro-benzothiazole as reactant. Pale yellow solid was obtained with a yield of 42%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.10 (s, 1H), 7.38 (d, J = 2.0 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.29 (d, J = 8.6 Hz, 1H), 7.22 (t, J = 8.0 Hz, 1H), 7.10 – 7.02 (m, 1H), 6.98 (dd, J = 8.5, 2.1 Hz, 1H), 4.80 (s, 2H), 3.85 (t, J = 5.7 Hz, 2H), 2.79 (t, J = 5.7 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 167.72, 153.31, 134.51, 132.08, 129.85, 127.54, 127.49, 124.81, 123.37, 120.89, 117.23, 117.11, 117.03, 112.67, 106.69, 47.16, 45.61, 20.30. LC-MS, Rt = 8.288 min, [M+H]⁺ = 374.0. HRMS (ESI) m/z calcd. for C₁₈H₁₄Cl₂N₃S [M+H]⁺ = 374.0280, found 374.0262.

6-Chloro-2-(5-chloro-1,3-benzothiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (4v) was prepared by method A using 2,5-dichloro-benzothiazole as reactant. Pale yellow solid was obtained with a yield of 44%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.12 (s, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.44 (dd, J = 17.0, 2.0 Hz, 2H), 7.31 (d, J = 8.6 Hz, 1H), 7.04 (dd, J = 8.4, 2.1 Hz, 1H), 7.00 (dd, J = 8.6, 2.1 Hz, 1H), 4.82 (s, 2H), 3.87 (t, J = 5.8 Hz, 2H), 2.81 (t, J = 5.7 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 169.37, 153.59, 134.50, 132.17, 130.75, 129.02, 127.56, 123.36, 122.59, 122.53, 121.04, 120.90, 118.06, 117.99, 117.12, 117.04, 112.68, 109.60, 106.71, 47.02, 45.62, 20.29. LC-MS, Rt = 8.002 min, [M+H]⁺ = 374.0. HRMS (ESI) m/z calcd. for C₁₈H₁₄Cl₂N₃S [M+H]⁺ = 374.0280, found 374.0262.

6-Chloro-2-(4-chloro-1,3-benzothiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (4w) was prepared by method A using 2,4-dichloro-benzothiazole as reactant. Pale yellow solid was obtained with a yield of 38%. ¹H NMR (400 MHz, Chloroform-d) δ 7.97 (s, 1H), 7.50 (dd, J = 7.9, 1.1 Hz, 1H), 7.41 (d, J = 1.9 Hz, 1H), 7.31 (dd, J = 7.9, 1.1 Hz, 1H), 7.21 (dd, J = 8.6, 0.6 Hz, 1H), 7.10 (dd, J = 8.6, 2.0 Hz, 1H), 7.00 (t, J = 7.9 Hz, 1H), 4.95 (s, 2H), 3.92 (t, J = 5.7 Hz, 1H), 2.91 (t, J = 5.7 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 168.88, 149.65, 134.66, 131.85, 131.09, 127.96, 126.55, 125.58, 123.57, 122.34, 122.13, 119.47, 117.75, 112.05, 108.38, 47.97, 45.51, 20.95. LC-MS, Rt = 7.969 min, [M+H]⁺ = 374.0. HRMS (ESI) m/z calcd. for C₁₈H₁₄Cl₂N₃S [M+H]⁺ = 374.0280, found 374.0262.

6-Chloro-2-(4,6-dichloro-1,3-benzothiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (4x) was prepared by method A using 2,4,6-trichloro-benzothiazole as reactant. Pale yellow solid was obtained with a yield of 48%. ¹H NMR (500 MHz, Chloroform-d) δ 8.10 (s, 1H), 7.48 (s, 1H), 7.41 (s, 1H), 7.33 (s, 1H), 7.22 (d, J = 8.6 Hz, 1H), 7.13 (d, J = 8.7 Hz, 1H), 4.90 (s, 2H), 3.89 (d, J = 5.9 Hz, 2H), 2.90 (t, J = 5.9 Hz, 2H). LC-MS, Rt = 7.425 min, [M+H]⁺ = 408.0. HRMS (ESI) m/z calcd. for C₁₈H₁₃Cl₃N₃S [M+H]⁺ = 407.9891, found 407.9863.

6-Chloro-2-(4,5,6-trichloro-1,3-benzothiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (4y) was prepared by method B using 1,2,3-trichloro-4-isothiocyanato-benzene as reactant. Pale yellow solid was obtained with a yield of 70%. ¹H NMR (500 MHz, Chloroform-d) δ 8.00 (s, 1H), 7.62 (s, 1H), 7.46 (d, J = 2.0 Hz, 1H), 7.30 – 7.24 (m, 1H), 7.15 (dd, J = 8.6, 2.0 Hz, 1H), 4.99 (s, 2H), 3.95 (t, J = 5.7 Hz, 2H), 2.97 (t, J = 5.7 Hz, 2H). LC-MS, Rt = 7.711 min, [M+H]⁺ = 443.9. HRMS (ESI) m/z calcd. for C₁₈H₁₂Cl₄N₃S [M+H]⁺ = 441.9501, found 441.9526.

6-Chloro-2-(4-chloro-6-methoxy-1,3-benzothiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (4z) was prepared by method B using 2-chloro-1-isothiocyanato-4-methoxy-benzene as reactant. Pale yellow solid was obtained with a yield of 89%. ¹H NMR (400 MHz, Chloroform-d) δ 7.96 (s, 1H), 7.42 (d, J = 2.1 Hz, 1H), 7.22 (d, J = 8.6 Hz, 1H), 7.10 (dd, J = 8.6, 2.0 Hz, 1H), 7.06 (d, J = 2.4 Hz, 1H), 6.94 (d, J = 2.4 Hz, 1H), 4.92 (s, 2H), 3.89 (t, J = 5.7 Hz, 21H), 3.80 (s, 3H), 2.91 (t, J = 5.7 Hz, 2H). LC-MS, Rt = 8.702 min, [M+H]⁺ = 404.0. HRMS (ESI) m/z calcd. for C₁₉H₁₆Cl₂N₃OS [M+H]⁺ = 404.0386, found 404.0377.

4-Chloro-2-(6-chloro-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-1,3-benzothiazol-6-ol (4aa) was prepared by using BBr₃ for the de-protection of 4z according to the literature.⁴⁶ A solution of BBr₃ (3.0 mL, 3.00 mmol, 1M in CH₂Cl₂) was added to a solution of compound 4z (201.0 mg, 0.5 mmol) in anhydrous CH₂Cl₂ (4.0 mL) under N₂ at −78°C and stirred at this temperature for 1 h before warming up to room temperature and stirred for another 12 h. The reaction mixture was quenched with methanol. The solvent was removed under vacuum and the residue was extracted with CH₂Cl₂ after basified with saturated NaHCO₃ solution. The combined organic layer was washed with brine, dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by silica gel column chromatography to give the product with yield of 90%. ¹H NMR (400 MHz, Chloroform-d) δ 7.93 (s, 1H), 7.43 (d, J = 2.0 Hz, 1H), 7.23 (d, J = 6.6 Hz, 1H), 7.11 (dd, J = 8.6, 2.0 Hz, 1H), 7.03 (dd, J = 20.2, 2.4 Hz, 1H), 6.91 (dd, J = 27.1, 2.4 Hz, 1H), 4.93 (d, J = 2.7 Hz, 2H), 3.89 (td, J = 5.7, 2.4 Hz, 2H), 3.80 (s, 1H), 2.92 (t, J = 5.8 Hz, 2H). LC-MS, Rt = 7.926 min, [M+H]⁺ = 390.0. HRMS (ESI) m/z calcd. for C₁₈H₁₄Cl₂N₃OS [M+H]⁺ = 390.0230, found 390.0198.

2-((4-Chloro-2-(6-chloro-1,3,4,9-tetrahydro-2^H-pyrido [3,4-b]indol-2-yl)benzo[d]thiazol-6-yl)oxy)ethan-1-amine (4ab) was prepared according to the literature.⁴⁷ A suspension of 4-chloro-2-(6-chloro-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-1,3-benzothiazol-6-ol 4aa (82.5 mg, 0.200 mmol), N-(2-hydroxyethyl)phthalimide (57.5 mg, 0.300 mmol) and triphenylphosphine (78.5 mg, 0.300 mmol) in anhydrous THF (5 mL) was stirred under an argon atmosphere until a clear solution was obtained. Then, the solution of diisopropyl azodicarboxylate (DIAD) in THF (61 mg, 0.300 mmol) were added dropwise and the reaction mixture was refluxed for 15 h. The solvents were evaporated under reduced pressure and the residue was purified by silica gel column chromatography. To a solution of the product obtained from the previous step in absolute ethanol (5 mL) were added 0.2 mL of hydrazine hydrate and 0.25 mL of acetic acid. The reaction mixture was refluxed for 3 h. After cooling down to room temperature, the precipitates were removed by filtration and the filtrate was concentrated under reduced pressure. The residue was resuspended in dichloromethane (50 mL) and extracted with 20% NaOH solution (3 × 50 mL). The organic layers were dried over Na₂SO₄, concentrated under reduced pressure. The residue was purified by silica gel column chromatography with a yield of 50%. ¹H NMR (500 MHz, Chloroform-d) δ 8.31 (s, 1H), 7.46 (d, J = 2.0 Hz, 1H), 7.14 (dd, J = 8.5, 2.0 Hz, 1H), 7.10 (d, J = 2.4 Hz, 1H), 7.03 – 6.95 (m, 2H), 4.97 (s, 2H), 4.00 (t, J = 5.1 Hz, 2H), 3.93 (t, J = 5.7 Hz, 2H), 3.57 (m, 2H), 2.96 (t, J = 5.7 Hz, 2H), 2.91 (s, 2H). LC-MS, Rt = 6.453 min, [M+H]⁺ = 433.0. HRMS (ESI) m/z calcd. for C₂₀H₁₉Cl₂N₄OS [M+H]⁺ = 433.0652, found 433.0703.

2-(6-Bromo-4-chloro-1,3-benzothiazol-2-yl)-6-chloro-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (4ac) was prepared by method B using 4-bromo-2-chloro-1-isothiocyanato-benzene as reactant. Pale yellow solid was obtained with a yield of 95%. ¹H NMR (400 MHz, Acetone-d₆) δ 7.89 (dd, J = 1.9, 0.6 Hz, 0H), 7.48 (dd, J = 6.7, 2.0 Hz, 1H), 7.39 (d, J = 8.6 Hz, 0H), 7.07 (dd, J = 8.6, 2.1 Hz, 0H), 4.99 (d, J = 1.6 Hz, 1H), 4.03 (t, J = 6.7 Hz, 2H), 2.98 (ddd, J = 7.3, 3.7, 1.7 Hz, 1H). LC-MS, Rt = 7.565 min, [M+H]⁺ = 453.9. HRMS (ESI) m/z calcd. for C₁₈H₁₃BrCl₂N₃S [M+H]⁺ = 451.9386, found 451.9359.

4-Chloro-2-(6-chloro-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-1,3-benzothiazol-6-amine (4ad) was prepared according to the literature.⁴⁸ 10 mL reaction vessel was charged with Cu₂O (14.3 mg, 0.100 mmol), 2-[6-bromo-4-(trifluoromethyl)-1,3-benzothiazol-2-yl]-6-chloro-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole 4ac (906.0 mg, 2.00 mmol), 1.5 mL of N-methyl pyrrolidinone (NMP), 1.5 mL of ammonium hydroxide solution (28% NH₃, 20.0 mmol) and a magnetic stir bar. The vessel was sealed with a Teflon screw cap, and was stirred at 80 °C for 48 h. The reaction mixture was cooled to room temperature, quenched with water, extracted with diethyl ether and dried over Na₂SO₄. The solvents were removed under vacuum and the residue was purified by silica gel flash chromatography. Red solid was obtained as product with a yield of 56%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.08 (s, 1H), 7.38 (d, J = 2.1 Hz, 1H), 7.27 (d, J = 8.6 Hz, 1H), 6.97 (dd, J = 8.6, 2.1 Hz, 1H), 6.81 (d, J = 2.1 Hz, 1H), 6.59 (d, J = 2.1 Hz, 1H), 5.08 (s, 2H), 4.74 (s, 2H), 3.75 (t, J = 5.7 Hz, 2H), 2.76 (t, J = 5.7 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 165.12, 144.93, 140.05, 134.68, 133.04, 132.82, 127.87, 123.56, 122.53, 121.07, 117.29, 113.33, 112.85, 106.93, 104.55, 47.30, 45.70, 20.54. LC-MS, Rt = 8.066 min, [M+H]⁺ = 389.0. HRMS (ESI) m/z calcd. for C₁₈H₁₅Cl₂N₄S [M+H]⁺ = 389.0390, found 389.0378.

2-amino-N-(4-chloro-2-(6-chloro-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)benzo[d]thiazol-6-yl)acetamide hydrochloride (4ae) To a round bottom flask was added 4-chloro-2-(6-chloro-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-1,3-benzothiazol-6-amine 4ad (21.0 mg, 0.050 mmol), Boc-Gly-OH (10.5 mg, 0.06 mmol), DMAP (7.5 mg, 0.060 mmol), EDCI (9.5 mg, 0.060 mmol) and DCM (2 mL), and the reaction mixture was stirred for 2 h. The solvents were removed under vacuum and the residue was purified by silica gel flash chromatography using ethyl acetate/hexane as eluent. To a round bottom flask was added the obtained product (35 mg, 0.050 mmol) and HCl in dioxane (2.0 mL, 4 M), and the reaction mixture was stirred for 4 h. The solvents were removed under vacuum and red solid was obtained as product with a yield of 90%. ¹H NMR (500 MHz, Methanol-d₄) δ 8.11 – 7.99 (m, 1H), 7.62 (dd, J = 17.5, 2.0 Hz, 1H), 7.43 (dd, J = 3.6, 2.0 Hz, 1H), 7.30 (dd, J = 8.6, 1.7 Hz, 1H), 7.06 (dt, J = 8.6, 2.4 Hz, 1H), 4.99 (s, 2H), 4.09 (t, J = 5.8 Hz, 2H), 3.88 (s, 2H), 2.99 (t, J = 6.1 Hz, 2H). LC-MS, Rt = 6.636 min, [M+H]⁺ = 446.0. HRMS (ESI) m/z calcd. for C₂₀H₁₈Cl₂N₅OS [M+H]⁺ = 446.0604, found 446.0634.

6-Chloro-2-[4-(trifluoromethyl)-1,3-benzothiazol-2-yl]-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (4af) was prepared by method A using 2-chloro-4-(trifluoromethyl)-benzothiazole as reactant. Pale yellow solid was obtained with a yield of 41%. ¹H NMR (400 MHz, Chloroform-d) δ 7.96 (s, 1H), 7.76 (dd, J = 7.9, 1.2 Hz, 1H), 7.59 – 7.52 (m, 1H), 7.40 (d, J = 2.0 Hz, 1H), 7.20 (dd, J = 8.5, 0.6 Hz, 1H), 7.14 – 7.07 (m, 2H), 4.95 (s, 2H), 3.91 (t, J = 5.7 Hz, 2H), 2.88 (t, J = 5.7 Hz, 2H). LC-MS, Rt = 7.142 min, [M+H]⁺ = 408.0. HRMS (ESI) m/z calcd. for C₁₉H₁₄ClF₃N₃S [M+H]⁺ = 408.0544, found 408.0568.

2-[6-Bromo-4-(trifluoromethyl)-1,3-benzothiazol-2-yl]-6-chloro-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (4ag) was prepared by method B using 4-bromo-1-isothiocyanato-2-(trifluoromethyl)--benzene as reactant. Pale yellow solid was obtained with a yield of 80%. ¹H NMR (400 MHz, Acetone-d₆) δ 8.21 (s, 1H), 7.69 (s, 1H), 7.47 (s, 1H), 7.39 (dd, J = 8.6, 1.6 Hz, 1H), 7.08 (d, J = 8.6 Hz, 1H), 5.02 (s, 2H), 4.06 (t, J = 5.2 Hz, 2H), 2.99 (tt, J = 3.9, 1.9 Hz, 2H). LC-MS, Rt = 7.644 min, [M+H]⁺ = 487.9. HRMS (ESI) m/z calcd. for C₁₉H₁₃BrClF₃N₃S [M+H]⁺ = 485.9649, found 485.9641

2-(6-Chloro-1,3,4,9-tetrahydro-2H-pyrido[3,4-b] indol-2-yl)-4-(trifluoromethyl)-1,3-benzothiazol-6-aminium chloride (4ah) was prepared according to the literature.⁴⁸ 10 mL reaction vessel was charged with Cu₂O (14.3 mg, 0.100 mmol), 2-[6-bromo-4-(trifluoromethyl)-1,3-benzothiazol-2-yl]-6-chloro-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole 4ag (973.4 mg, 2.00 mmol), 1.5 mL of N-methyl pyrrolidinone (NMP), 1.5 mL of ammonium hydroxide solution (28% NH₃, 20.0 mmol) and a magnetic stir bar. The vessel was sealed with a Teflon screw cap, and was stirred at 80 °C for 48 h. Then the reaction mixture was cooled to 25 °C, quenched with water, extracted with diethyl ether and dried over anhydrous Na₂SO₄. The solvents were removed under vacuum and the residue was purified by silica gel flash chromatography. Then, to a round bottom flask was added the product obtained from above (50 mg, 0.01 mmol) and HCl in 1, 4-dioxane (1 mL, 4 M), and the reaction mixture was stirred for 1h. The solvents were removed under vacuum and red solid was obtained as product with a yield of 78%. ¹H NMR (400 MHz, Chloroform-d) δ 7.94 (s, 1H), 7.39 (d, J = 2.0 Hz, 1H), 7.18 (d, J = 8.6 Hz, 1H), 7.13 – 7.05 (m, 2H), 6.93 (d, J = 2.3 Hz, 1H), 4.88 (s, 2H), 3.85 (t, J = 5.7 Hz, 2H), 2.86 (t, J = 5.8 Hz, 2H).). LC-MS, Rt = 8.229 min, [M-Cl]⁺ = 423.0. HRMS (ESI) m/z calcd. for C₁₉H₁₅ClF₃N₄S [M-Cl]⁺ = 423.0653, found 423.0564.

2-(6-Chloro-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-N-methyl-4-(trifluoromethyl)-1,3-benzothiazol-6-amine (4ai) was prepared according to the literature.⁴⁹ Sodium metal (12.5 mg, 0.500 mmol) was added portionwise to anhydrous MeOH (2 mL) at 0 °C. After complete consumption of the sodium metal, 2-(6-chloro-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-4-(trifluoromethyl)-1,3-benzothiazol-6-aminium chloride 4ah (25.0 mg, 0.500 mmol) and paraformaldehyde (15 mg, 0.500 mmol) were added at room temperature, and the mixture was stirred for 2 h at reflux to give an imine intermediate. The mixture was then reacted with NaBH₄ (0.500 mmol) at 0 °C, and then refluxed for an additional 2 h. The reaction mixture was then cooled to room temperature, and the solvent was removed under reduced pressure. The residue was diluted with CH₂Cl₂ (5 mL), washed with water (3 mL), dried over Na₂SO₄ and concentrated to give a residue that was purified via silica gel column chromatography with a yield of 77%. ¹H NMR (400 MHz, Chloroform-d) δ 7.93 (s, 1H), 7.40 (d, J = 2.0 Hz, 1H), 7.19 (d, J = 8.5 Hz, 1H), 7.09 (dd, J = 8.6, 2.0 Hz, 1H), 6.99 (d, J = 2.4 Hz, 1H), 6.86 (d, J = 2.4 Hz, 1H), 4.90 (s, 2H), 3.86 (t, J = 5.7 Hz, 2H), 3.14 – 2.62 (m, 5H). LC-MS, Rt = 8.081 min, [M+H]⁺ = 437.1. HRMS (ESI) m/z calcd. for C₂₀H₁₇ClF₃N₄S [M+H]⁺ = 437.0810, found 437.0849.

N1-(2-(6-chloro-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-4-(trifluoromethyl)benzo[d]thiazol-6-yl)ethane-1,2-diamine hydrochloride (4aj) was prepared according to the literature.⁵⁰ To a solution of 2-(6-Chloro-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-4-(trifluoromethyl)-1,3-benzothiazol-6-aminium chloride 4ah (49.0 mg, 0.100 mmol, 1 equiv) in dichloroethane at room temperature were added N-Boc-2-aminoacetaldehyde (19.0 mg, 0.120 mmol), AcOH (6.0 mg, 0.100 mmol) and NaHB(OAc)₃ (42.4 mg, 0.200 mmol). The resulting mixture was stirred for 16 h. The solvent was then removed in vacuum. The residue was redissolved in ethyl acetate, washed with saturated NaHCO₃ solution, dried over Na₂SO₄ and concentrated in vacuo. The intermediate was obtained after purification by silica gel flash chromatography with a yield of 60%. To a round bottom flask was added the obtained product from above (25 mg, 0.050 mmol), and HCl in 1, 4-dioxane (2 mL, 4 M). The reaction mixture was stirred at room temperature for 4 h. The solvents were then removed under vacuum and red solid was obtained as product with a yield of 90%. ¹H NMR (500 MHz, Methanol-d₄) δ 7.42 (d, J = 2.1 Hz, 1H), 7.30 (dd, J = 8.5, 6.3 Hz, 2H), 7.14 – 6.89 (m, 2H), 3.99 (s, 2H), 3.75 (dd, J = 6.1, 4.8 Hz, 1H), 3.64 – 3.57 (m, 1H), 3.47 (d, J = 6.6 Hz, 2H), 3.19 (t, J = 6.0 Hz, 2H), 2.94 (t, J = 5.8 Hz, 2H). LC-MS, Rt = 6.706 min, [M-Cl]⁺ = 466.0. HRMS (ESI) m/z calcd. for C₂₁H₂₀ClF₃N₅S [M+H]⁺ = 466.1075, found 466.1111.

6-Chloro-2-(6-chlorobenzo[d]thiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-4-carboxylic acid (8) The mixture of methyl 6-chloro-2-(6-chlorobenzo[d]thiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-4-carboxylate 4n (20 mg, 0.046 mmol) and lithium hydroxide (20 mg, 0.46 mmol) were dissolved in H₂O and MeOH (1 mL, v: v = 1:1), and stirred at room temperature overnight. The reaction was then quenched with 1 N HCl to adjust the pH to around 5, extracted with ethyl estate. The combined organic phase dried over Na₂SO₄, concentrated under reduced pressure to afford the desired acid product (20 mg, 99%). ¹H NMR (500 MHz, DMSO-d₆) δ 12.67 (s, 1H), 11.32 (s, 1H), 7.96 (d, J = 2.2 Hz, 1H), 7.53 (d, J = 2.1 Hz, 1H), 7.49 (d, J = 8.6 Hz, 1H), 7.40 (d, J = 8.6 Hz, 1H), 7.32 (dd, J = 8.6, 2.3 Hz, 1H), 7.09 (dd, J = 8.6, 2.1 Hz, 1H), 5.05 (d, J = 16.5 Hz, 1H), 4.74 – 4.63 (m, 1H), 4.34 (d, J = 13.2 Hz, 1H), 4.06 (s, 1H), 3.87 (dd, J = 13.2, 4.6 Hz, 1H). LC-MS, Rt = 8.571 min, [M+H]⁺ = 418.0. HRMS (ESI) m/z calcd. for C₁₉H₁₄Cl₂N₃O₂S [M+H]⁺ = 418.0179, found 418.0175.

6-Chloro-2-(6-chlorobenzo[d]thiazol-2-yl)-N,N-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-4-carboxamide (9) To the mixture of 6-chloro-2-(6-chlorobenzo[d]thiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-4-carboxylic acid 8 (21 mg, 0.05 mmol) in anhydrous DCM (1 mL) was added oxalyl chloride (8.6 μL, 0.1 mmol) and 3 drops DMF. After the mixture was stirred at room temperature for 2 h, dimethylamine (26 μL, 0.25 mmol) was added, and the mixture was stirred at room temperature for another 2 h. The reaction mixture was diluted with DCM, washed with water and brine, dried over Na₂SO₄ and concentrated. The residue was purified by silica gel flash chromatography (hexane / EA = 1:1 to 1:2) to afford the title compound 9 (19 mg, 85%). ¹H NMR (500 MHz, Chloroform-d) δ 9.21 (s, 1H), 7.47 (d, J = 2.1 Hz, 1H), 7.39 (d, J = 8.6 Hz, 1H), 7.20 (dd, J = 8.6, 2.2 Hz, 1H), 7.12 (d, J = 1.9 Hz, 1H), 6.91 (dd, J = 8.6, 2.0 Hz, 1H), 6.75 (d, J = 8.5 Hz, 1H), 4.60 (d, J = 16.1 Hz, 1H), 4.41 (dd, J = 7.7, 4.9 Hz, 1H), 4.28 – 4.14 (m, 2H), 3.92 (dd, J = 13.3, 7.8 Hz, 1H), 3.40 (s, 3H), 3.15 (s, 3H). LC-MS, Rt = 5.222 min, [M+H]⁺ = 445.0. HRMS (ESI) m/z calcd. for C₂₁H₁₉Cl₂N₄OS [M+H]⁺ = 445.0646, found 445.0685.

(6-Chloro-2-(6-chlorobenzo[d]thiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-4-yl)methanol (10) To the solution of methyl 6-chloro-2-(6-chlorobenzo[d]thiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-4-carboxylate 4n (43.2 mg, 0.1 mmol) in anhydrous THF (1 mL) was added LiAlH₄ (11.4 mg, 0.3 mmol) in three portions under Argon. The reaction mixture was stirred at room temperature overnight. Then quenched with 2 N NaOH solution and water. The precipitate was filtered, and the solid was washed with MeOH three times. The combined filtrate were concentrated under reduced pressure. The residue was purified by silica gel flash chromatography to afford the alcohol 10 (39.3 mg, 97%). ¹H NMR (500 MHz, Chloroform-d) δ 8.50 (s, 1H), 7.52 (d, J = 2.2 Hz, 1H), 7.50 (d, J = 2.0 Hz, 1H), 7.40 (d, J = 8.6 Hz, 1H), 7.25 – 7.17 (m, 2H), 7.11 (dd, J = 8.6, 2.0 Hz, 1H), 4.97 (d, J = 16.1 Hz, 1H), 4.67 (dd, J = 16.0, 1.6 Hz, 1H), 4.60 – 4.47 (m, 1H), 3.97 (dd, J = 11.3, 4.9 Hz, 1H), 3.62 (dd, J = 13.5, 3.7 Hz, 1H), 3.57 (dd, J = 11.3, 9.6 Hz, 1H), 3.35 – 3.25 (m, 1H). ¹³C NMR (101 MHz, Chloroform-d) δ 169.55, 150.73, 134.70, 131.55, 131.13, 127.60, 126.98, 126.84, 125.87, 122.62, 120.62, 119.67, 117.92, 112.28, 109.28, 63.31, 48.39, 46.55, 35.90. LC-MS, Rt = 9.347 min, [M+H]⁺ = 404.1. HRMS (ESI) m/z calcd. for C₁₉H₁₆Cl₂N₃OS [M+H]⁺ = 404.0386, found 404.0377.

6-Chloro-2-(6-chloro-4-((methoxymethoxy)methyl)-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)benzo[d]thiazole (11) To the mixture of (6-chloro-2-(6-chlorobenzo[d]thiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-4-yl)methanol 10 (15 mg, 0.0371 mmol) and N,N-Diisopropylethylamine (20 μL, 0.112 mmol) in anhydrous DCM (1 mL) was added MOM chloride (10 μL, 0.132 mmol). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was then concentrated and purified by silica gel flash chromatography to afford the title product 11 (5.0 mg, 30%). ¹H NMR (500 MHz, Chloroform-d) δ 8.13 (s, 1H), 7.59 (d, J = 2.1 Hz, 1H), 7.54 (d, J = 2.0 Hz, 1H), 7.45 (d, J = 8.6 Hz, 1H), 7.27 – 7.22 (m, 2H), 7.14 (dd, J = 8.6, 2.0 Hz, 1H), 5.24 (d, J = 16.3 Hz, 1H), 4.77 – 4.67 (m, 2H), 4.65 (dd, J = 16.3, 1.5 Hz, 1H), 4.23 (dd, J = 13.2, 2.6 Hz, 1H), 3.86 (dd, J = 9.9, 4.3 Hz, 1H), 3.73 (dd, J = 13.2, 3.9 Hz, 1H), 3.55 (t, J = 9.9 Hz, 1H), 3.41 (s, 3H). LC-MS, Rt = 8.249 min, [M+H]⁺ = 448.0. HRMS (ESI) m/z calcd. for C₂₁H₂₀Cl₂N₃O₂S [M+H]⁺ = 448.0643, found 448.0659.

2-(((6-Chloro-2-(6-chlorobenzo[d]thiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-4-yl)methyl)thio)ethan-1-amine hydrochloride (12a) To the solution of triphenylphosphine (58 mg, 0.22 mmol) in anhydrous DCM (2 mL) under Argon, was added iodine (28 mg, 0.22 mmol). After the mixture was stirred for 10 min, imidazole (26 mg, 0.37 mmol) was added into the mixture and stirred for another 10 min. (6-chloro-2-(6-chlorobenzo[d]thiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-4-yl)methanol 10 (41 mg, 0.1 mmol) in anhydrous DCM (1 mL) was added slowly and stirred at room temperature for 1 h. The reaction mixture was concentrated and purified by silica gel flash chromatography to afford the iodinated intermediate (36 mg, 70%). The mixture of iodinated intermediate (30 mg, 0.058 mmol) and tert-butyl (2-mercaptoethyl)carbamate (0.012 mL, 5.0 equiv) and sodium hydroxide (2.3 mg, 0.058 mmol) in tert-Butyl alcohol (1 mL) was heated to 120 °C for 4 h. The reaction mixture was concentrated and purified by silica gel flash chromatography to afford the N-Boc-12a (26 mg, 78%). ¹H NMR (500 MHz, Chloroform-d) δ 8.74 (s, 1H), 7.56 (d, J = 2.2 Hz, 1H), 7.51 (s, 1H), 7.42 (d, J = 8.7 Hz, 1H), 7.22 (dd, J = 8.6, 2.2 Hz, 1H), 7.18 (d, J = 8.6 Hz, 1H), 7.09 (dd, J = 8.6, 2.0 Hz, 1H), 5.18 (d, J = 16.2 Hz, 1H), 5.05 (s, 1H), 4.59 – 4.51 (m, 1H), 4.33 (dd, J = 13.2, 2.3 Hz, 1H), 3.66 (dd, J = 13.3, 3.8 Hz, 1H), 3.34 (dh, J = 26.6, 6.5 Hz, 2H), 3.20 (d, J = 11.0 Hz, 1H), 3.00 (dd, J = 13.3, 3.5 Hz, 1H), 2.75 (t, J = 6.8 Hz, 2H), 2.58 (dd, J = 13.2, 10.9 Hz, 1H), 1.45 (s, 9H). ¹³C NMR (101 MHz, Chloroform-d) δ 169.44, 155.99, 151.02, 134.76, 131.81, 131.11, 127.10, 126.93, 126.72, 125.75, 122.52, 120.66, 119.79, 117.65, 112.37, 110.87, 79.78, 77.36, 50.78, 45.47, 39.98, 35.07, 33.32, 33.24, 28.54. LC-MS, Rt = 9.058 min, [M+H]⁺ = 563.1. HRMS (ESI) m/z calcd. for C₂₆H₂₉Cl₂N₄O₂S₂ [M+H]⁺ = 563.1104, found 563.1185. N-Boc protected 12a was dissolved in HCl in 1,4-dioxane solution (1 mL, 4 M) and stirred at room temperature overnight. The reaction mixture was concentrated to afford the title compound 12a in quantitative yield (18 mg, 99%). ¹H NMR (500 MHz, DMSO-d₆) δ 11.50 (s, 1H), 8.15 (d, J = 6.2 Hz, 2H), 7.98 (d, J = 2.2 Hz, 1H), 7.63 (d, J = 2.1 Hz, 1H), 7.51 (d, J = 8.6 Hz, 1H), 7.39 (d, J = 8.6 Hz, 1H), 7.33 (dd, J = 8.6, 2.3 Hz, 1H), 7.08 (dd, J = 8.6, 2.1 Hz, 1H), 5.10 (d, J = 16.5 Hz, 1H), 4.75 (d, J = 16.5 Hz, 1H), 4.26 (d, J = 13.3 Hz, 1H), 3.81 (dd, J = 13.3, 3.9 Hz, 1H), 3.76 – 3.62 (m, 1H), 3.53 – 3.41 (m, 1H), 3.37 – 3.29 (m, 1H), 3.11 – 2.97 (m, 3H), 2.95 (q, J = 6.7 Hz, 1H), 2.86 (ddd, J = 13.9, 8.0, 6.3 Hz, 1H), 2.59 (dd, J = 13.4, 10.3 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 169.12, 149.33, 134.66, 132.12, 130.84, 126.83, 126.60, 125.60, 123.56, 121.35, 121.09, 119.07, 117.52, 112.88, 109.21, 60.22, 45.75, 38.65, 34.66, 32.14, 28.91. LC-MS, Rt = 2.847mm, [M-Cl]⁺ = 463.0. HRMS (ESI) m/z calcd. for C₂₁H₂₁Cl₃N₄S₂ [M-Cl]⁺ = 463.0580, found 463.0538.

(6-Chloro-2-(6-chlorobenzo[d]thiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-4-yl)methanamine (12b) The mixture of iodinated intermediate (30 mg, 0.058 mmol, prepared in the synthesis of 12a) and aqueous ammonium hydroxyide (0.5 mL) in tert-Butyl alcohol (0.5 mL) in a sealed vial was heated at 120 °C for 2 h. The reaction mixture was concentrated and purified by silica gel flash chromatography to afford the title compound 12b as a white solid (21 mg, 89%). ¹H NMR (500 MHz, Methanol-d₄) δ 7.86 (d, J = 3.3 Hz, 1H), 7.59 (dd, J = 14.7, 2.2 Hz, 1H), 7.51 (dd, J = 5.1, 2.1 Hz, 1H), 7.36 (t, J = 8.8 Hz, 1H), 7.25 (dd, J = 8.6, 5.1 Hz, 1H), 7.22 – 7.15 (m, 1H), 7.01 (ddd, J = 8.6, 4.6, 2.1 Hz, 1H), 4.90 (d, J = 11.8 Hz, 1H), 4.58 (ddd, J = 15.9, 13.5, 1.4 Hz, 1H), 4.28 (td, J = 13.2, 2.7 Hz, 1H), 3.61 (td, J = 13.4, 3.7 Hz, 1H), 3.11 (td, J = 8.0, 3.9 Hz, 1H), 2.95 (ddd, J = 13.1, 6.8, 4.8 Hz, 1H), 2.71 (ddd, J = 13.2, 8.8, 6.5 Hz, 1H).¹³C NMR (101 MHz, Methanol-d₄) δ 171.06, 152.26, 136.50, 132.80, 128.77, 127.69, 127.54, 125.99, 122.62, 121.60, 120.39, 118.38, 113.41, 110.11, 79.45, 50.19, 47.01, 45.17, 37.04. LC-MS, Rt = 7.310 min, [M+H]⁺ = 403.0. HRMS (ESI) m/z calcd. for C₁₉H₁₇Cl₂N₄S [M+H]⁺ = 403.0540, found 403.0558.

1-(6-Chloro-2-(6-chlorobenzo[d]thiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-4-yl)-N,N-Dimethylmethanamine (12c) Prepared by the same method as 12a with a yield of 30%. ¹H NMR (500 MHz, Chloroform-d) δ 8.24 (s, 1H), 7.61 (d, J = 2.1 Hz, 1H), 7.49 (d, J = 2.0 Hz, 1H), 7.45 (d, J = 8.6 Hz, 1H), 7.27 (d, J = 3.2 Hz, 1H), 7.26 – 7.22 (m, 2H), 7.14 (dd, J = 8.6, 2.0 Hz, 1H), 5.33 (d, J = 16.3 Hz, 1H), 4.64 (dd, J = 16.3, 1.5 Hz, 1H), 4.26 (dd, J = 13.1, 2.3 Hz, 1H), 3.83 – 3.62 (m, 1H), 3.24 (dt, J = 11.5, 2.7 Hz, 1H), 2.57 – 2.43 (m, 2H), 2.40 (s, 6H). ¹³C NMR (101 MHz, Chloroform-d) δ 169.69, 151.25, 134.76, 132.08, 131.42, 127.57, 126.71, 126.62, 125.68, 122.43, 120.61, 119.67, 117.88, 112.23, 110.68, 61.86, 50.69, 46.23, 45.09, 31.87. LC-MS, Rt = 7.395 min, [M+H]⁺ = 431.0. HRMS (ESI) m/z calcd. for C₂₁H₂₁Cl₂N₄S [M+H]⁺ = 431.0853, found 431.0835.

1-(6-Chloro-2-(6-chlorobenzo[d]thiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-4-yl)-N,N,N-trimethylmethanaminium iodide (12d) The mixture of l-(6-chloro-2-(6-chlorobenzo[d]thiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-4-yl)-N,N-Dimethylmethanamine 12c (5.0 mg, 0.012 mmol) in CH₃I (0.2 mL) and MeOH (0.2 mL) was stirred at room temperature overnight. The reaction mixture was concentrated and dried under high vacuum to afford the title compound 12d (6.6 mg, 99%). ¹H NMR (500 MHz, Methanol-d₄) δ 7.74 (d, J = 2.2 Hz, 1H), 7.52 (d, J = 2.0 Hz, 1H), 7.49 (d, J = 8.6 Hz, 1H), 7.35 (d, J = 8.7 Hz, 1H), 7.30 (dd, J = 8.6, 2.2 Hz, 1H), 7.12 (dd, J = 8.6, 2.0 Hz, 1H), 4.93 – 4.89 (m, 1H), 4.69 – 4.64 (m, 1H), 3.91 (d, J = 8.7 Hz, 1H), 3.74 (ddd, J = 14.0, 3.1, 1.3 Hz, 1H), 3.68 – 3.61 (m, 1H), 3.53 (dd, J = 5.5, 4.1 Hz, 1H), 3.43 (dt, J = 14.0, 1.6 Hz, 1H), 3.39 (s, 9H). ¹³C NMR (101 MHz, Methanol-d₄) δ 178.58, 166.61, 159.86, 144.10, 142.16, 140.90, 136.64, 135.94, 134.82, 133.17, 130.63, 128.88, 127.03, 122.32, 117.00, 81.68, 80.04, 75.88, 69.69, 41.62. LC-MS, Rt = 6.434 min, [M-I]⁺ = 445.2. HRMS (ESI) m/z calcd. for C₂₂H₂₃Cl₂N₄S [M+H]⁺ = 446.1094, found 446.1095

N1-((6-chloro-2-(6-chlorobenzo[d]thiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-4-yl)methyl)ethane-1,2-diamine (12e) The iodinated intermediate (15 mg, 0.029 mmol prepared in the synthesis of 12a) and ethylenediamine (0.1 mL, 50.0 equiv) in tert-Butyl alcohol (0.5 mL) in a sealed tube were heated to 120 °C for 1 h. The mixture was then concentrated and purified via silica gel flash chromatography to afford the title compound 12e (10.0 mg, 77%). ¹H NMR (500 MHz, Methanol-d₄) δ 7.68 (t, J = 2.4 Hz, 1H), 7.54 (t, J = 2.4 Hz, 1H), 7.43 (dd, J = 9.4, 3.3 Hz, 1H), 7.34 – 7.24 (m, 2H), 7.06 (dt, J = 8.5, 2.5 Hz, 1H), 5.03 (d, J = 15.7 Hz, 1H), 4.63 (d, J = 16.2 Hz, 1H), 4.41 (d, J = 13.4 Hz, 1H), 3.72 – 3.63 (m, 1H), 3.27 – 3.19 (m, 1H), 2.92 (t, J = 10.9 Hz, 1H), 2.80 (d, J = 13.0 Hz, 3H), 2.73 – 2.60 (m, 2H). LC-MS, Rt = 5.676 min, [M+H]⁺ = 446.1. HRMS (ESI) m/z calcd. for C₂₁H₂₂Cl₂N₅S [M+H]⁺ = 446.0962, found 446.1003.

N-((6-chloro-2-(6-chlorobenzo[d]thiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-4-yl)methyl)formimidamide (12f) To the mixture of (6-chloro-2-(6-chlorobenzo[d]thiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-4-yl)methanamine 12b (7.6 mg, 0.019 mmol) and DIEA (16.5 μL, 0.095 mmol) at −55 °C was added ethyl formimidate hydrochloride (3.2 mg, 0.0285 mmol) in one portion. The reaction mixture was stirred at −55 °C for 1 h. After warming up to room temperature, the mixture was concentrated and purified by silica gel flash chromatography (DCM/MeOH = 50:1 to 10:1 ) to afford title compound 12f (4.0 mg, 49%). ¹H NMR (500 MHz, Methanol-d₄) δ 7.94 (s, 1H), 7.84 (s, 1H), 7.70 (t, J = 1.8 Hz, 1H), 7.52 (dd, J = 11.5, 2.0 Hz, 1H), 7.43 (d, J = 8.7 Hz, 1H), 7.31 (d, J = 8.6 Hz, 1H), 7.27 (dd, J = 8.7, 2.2 Hz, 1H), 7.06 (dd, J = 8.6, 2.0 Hz, 1H), 5.04 (d, J = 16.1 Hz, 1H), 4.67 (d, J = 16.0 Hz, 1H), 3.75 (dd, J = 13.7, 3.1 Hz, 1H), 3.60 – 3.46 (m, 3H), 3.03 (d, J = 7.4 Hz, 1H). LC-MS, Rt = 6.737 min, [M+H]⁺ = 430.1. HRMS (ESI) m/z calcd. for C₂₀H₁₈Cl₂N₅S [M+H]⁺ = 430.0649, found 430.0692.

1-((6-chloro-2-(6-chlorobenzo[d]thiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-4-yl)methyl)guanidine hydrochloride (12g) The mixture of (6-chloro-2-(6-chlorobenzo[d]thiazol-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-4-yl)methanamine 12b (42 mg, 0.104 mmol) and N,N’-Di-Boc-1H-pyrazole-1-carboxamidine (35 mg, 0.115 mmol) in anhydrous DCM (3 mL) was stirred at room temperature for 2 h. The mixture was concentrated and purified via silica gel flash chromatography to afford the guanidinylated intermediate (42 mg, 62%). ¹H NMR (500 MHz, Chloroform-d) δ 11.45 (s, 1H), 8.74 (t, J = 5.6 Hz, 1H), 8.22 (s, 1H), 7.70 (d, J = 1.9 Hz, 1H), 7.59 (d, J = 2.1 Hz, 1H), 7.51 (d, J = 8.6 Hz, 1H), 7.26 (d, J = 2.2 Hz, 1H), 7.23 (d, J = 8.6 Hz, 1H), 7.11 (dd, J = 8.6, 2.0 Hz, 1H), 5.10 (d, J = 16.1 Hz, 1H), 4.61 (dd, J = 16.1, 1.4 Hz, 1H), 4.24 (d, J = 13.4 Hz, 1H), 3.90 (dt, J = 13.5, 5.4 Hz, 1H), 3.64 (dd, J = 13.4, 3.8 Hz, 1H), 3.55 (d, J = 4.3 Hz, 1H), 3.43 (ddd, J = 13.8, 8.4, 5.6 Hz, 1H), 1.53 (d, J = 1.4 Hz, 9H), 1.50 (s, 9H). HRMS (ESI) m/z calcd. for C₃₀H₃₅Cl₂N₆O₄S [M+H]⁺ = 645.1807, found 645.1794. Then the N-Boc intermediate (38 mg) was dissolved in HCl in 1,4-dioxane solution (1 mL, 4 M) in a sealed tube and heated to 80 °C overnight. The mixture was concentrated and dried over high vacuum to afford the title compound 12g as a white solid (28 mg, 99%). ¹H NMR (500 MHz, DMSO-d₆) δ 11.29 (s, 1H), 7.74 (d, J = 2.2 Hz, 1H), 7.72 – 7.65 (m, 1H), 7.41 (d, J = 2.0 Hz, 1H), 7.28 (d, J = 8.6 Hz, 1H), 7.16 (d, J = 8.6 Hz, 1H), 7.11 (dd, J = 8.6, 2.2 Hz, 1H), 6.85 (dd, J = 8.6, 2.1 Hz, 1H), 4.85 (d, J = 16.4 Hz, 1H), 4.47 (d, J = 16.3 Hz, 1H), 3.57 (dd, J = 13.5, 3.6 Hz, 1H), 3.50 – 3.40 (m, 1H), 3.28 – 3.15 (m, 2H), 3.10 (dt, J = 12.2, 6.8 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 169.09, 157.13, 150.37, 134.62, 132.67, 131.42, 127.15, 126.46, 125.34, 123.69, 121.14, 121.08, 119.40, 117.54, 112.83, 107.51, 49.41, 45.66, 43.91, 43.69. LC-MS, R_t = 4.888 min, [M-Cl]⁺ = 445.0. HRMS (ESI) m z calcd. for C₂₀H₁₉Cl₂N₆S [M-Cl]⁺ = 445.0758, found 445.0777.

Abbreviations

MRSA

methicillin-resistant S. aureus

RMA

resistance-modifying agent

MSSA

methicillinsensitive S. aureus

CLSI

Clinical Laboratory Standards Institutes

SAR

structure-activity relationship

MIC

minimal inhibitory concentration

MRC

minimum resensitizing concentration

GI₅₀

half growth inhibitory concentration

HeLa cells

human cervical carcinoma cells

HAIs

hospital acquired infections

CLSI

Clinical & Laboratory Standards Institute

DMSO

dimethyl sulfoxide

TFA

trifluoroacetic acid

Rt

LCMS retention time

DIEA

N,N-Diisopropylethylamine