Discovery and Optimization of Tambjamines as a Novel Class of Antileishmanial Agents

Abstract

Leishmaniasis is a neglected tropical disease that is estimated to afflict over 12 million people. Current drugs for leishmaniasis suffer from serious deficiencies, including toxicity, high cost, modest efficacy, primarily parenteral delivery, and emergence of widespread resistance. We have discovered and developed a natural product inspired tambjamine chemotype, known to be effective against Plasmodium spp, as a novel class of antileishmanial agents. Herein, we report in-vitro, and in-vivo antileishmanial activities, detailed structure-activity relationships and metabolic/pharmacokinetic profiles of a large library of tambjamines. A number of tambjamines exhibited excellent potency against both Leishmania mexicana and Leishmania donovani parasites with good safety and metabolic profiles. Notably, tambjamine 110 offered excellent potency and provided partial protection to leishmania-infected mice at 40 and/or 60 mg/kg/10 days of oral treatment. This study presents the first account of antileishmanial activity in the tambjamine family, and paves the way for the generation of new oral antileishmanial drugs.

Graphical Abstract

INTRODUCTION

Leishmaniasis is a spectrum of diseases caused by parasitic protists of the genus Leishmania, with about 20 species responsible for human disease.¹ Infection is initiated by a bite of a parasite-carrying sand fly, and animals often serve as reservoirs for transmission of infection between animals and humans. The pathology is dependent upon the species of parasite and the host immune response and can range from self-healing but potentially disfiguring cutaneous disease, to highly disfiguring mucocutaneous leishmaniasis, and to fatal visceral leishmaniasis where the liver, spleen, and bone marrow are colonized.² This latter and most serious disease is usually caused by Leishmania donovani or Leishmania infantum. Two parasites that cause cutaneous leishmaniasis, Leishmania major and Leishmania mexicana, have been employed widely to study the basic cell and molecular biology of these protozoa but are also agents of widespread disease. The principal cells infected by the parasite are those of the reticuloendothelial system such as macrophages and dendritic cells. Within the sand fly vector, Leishmania parasites exist as various developmental forms of the elongated, flagellated, motile, extracellular promastigotes.³ When an infected sand fly bites a vertebrate host, it injects infectious metacyclic promastigotes that invade macrophages, where they are incorporated into phagolysosomes that mature into acidic parasitophorous vacuoles. Within these vacuoles, the promastigotes transform into nonmotile oval-shaped amastigotes that are adapted for survival and replication within the vacuole and that ultimately lyse the infected host cell, releasing free amastigotes that reinvade other macrophages.

Disease burden globally is estimated to involve ~12 million cases with 1 million new cases of cutaneous leishmaniasis and ~300,000 cases of visceral leishmaniasis per year.¹ Furthermore, asymptomatic leishmaniasis doubtlessly significantly increases the number of infected individuals well beyond the numbers cited above.⁴ Treatment options are poor, with current drugs such as pentavalent antimonials, pentamidine, amphotericin B, and paromomycin suffering from toxicity, high cost, and development of widespread resistance.⁵ Only one drug, miltefosine^5-7 is orally deliverable but exhibits severe toxicity and high cost. Hence, there is a pressing need for novel drugs with improved efficacy and safety profiles that can be employed to combat this burdensome but neglected disease that afflicts largely tropical and subtropical regions of the globe.

One obstacle to the discovery of novel drugs for leishmaniasis is the limited number of validated targets. Hence, phenotypic screening has played an important role the in recent discovery of drug candidates. These include nitroimidazooxazoles,⁸ the benzoxaborale DNDI-6148,⁹ and the nitroimidazooxazine DNDI-0690,^{10, 11} and other nitro compounds have activity against Leishmania parasites.^{9, 12} In addition, the Genomics Institute of the Novartis Foundation discovered the dimethyl oxazole-carboxamide derivative GNF6702 and a related compound LXE408¹³ as drug leads, and using genetic screens for resistance these compounds were determined to be inhibitors of the parasite proteasome. Cytochrome bc1 has been identified as a promiscuous drug target by phenotypic screening.¹⁴ However, the pyrollopyrimidine DNDI-6174, which targets the Qi site of Cytochrome b, was highly effective in decreasing the burden of L. infantum in the livers of infected mice.¹⁵ A screen of an ~200,000 compound library identified 15 chemotypes with demonstrated activity against L. major, one of which was tested for efficacy in a mouse model of cutaneous leishmaniasis.¹⁶ Screening of an ~600,000 compound library against L. mexicana identified 9 chemotypes with potential for drug development and two of these were shown to have partial efficacy in an animal model.¹⁷ In addition, a recent study established pyrazolopyrrolidinones as a new antileishmanial chemotype.¹⁸ Despite these notable advances, there is still a need for discovery of additional antileishmanial oral drug leads to maintain a robust pipeline in the face of inevitable failures on the way to clinical approval.

As a part of an ongoing interest in the discovery of a novel class of antiparasitic agents with novel mode(s) of action (MoA) to overcome the emerging drug resistance, recently we have discovered and developed a class of intriguing natural and synthetic products that has exhibited strong potency against the malaria parasite Plasmodium falciparum, including the prodiginines (PGs) and the structurally related tambjamines (TAs)^19-23 (Figure 1). PGs and TAs contain 3 and 2 pyrrole rings, respectively, and share 4-methoxy-2,2'-bipyrrole-5-carboxaldehyde (MBC, 1; Figure 1) as a common precursor in their biosynthetic pathways.^24-26 In addition, these natural and synthetic compounds have been extensively investigated for various therapeutic applications, including antimicrobial,^27-29 anticancer,^30-35 antitumor,^{27, 29, 36} and immunosuppressive³⁷ activities with various MoA.^38-41 Indeed, a recent study demonstrated the effectiveness of both TAs and PGs against the kinetoplastid parasites such as Trypanosoma cruzi,⁴² the causative agent of Chagas disease. To the best of our knowledge, the exact MoA are unknown for these novel TA chemotypes; however, they likely induce mitochondrial swelling and lysosomal dysfunction leading to autophagy and necrotic cell death in cancer cells.⁴³

Figure 1.

Biologically active natural PGs (2 and 3), and TAs (4 and 5), and their common biosynthetic precursor MBC (1).

To date, antileishmanial activities of these promising PG and TA compounds have not been investigated. Although Plasmodium and Leishmania parasites belong to different orders of protozoa, apicomplexa and kinetoplastida, respectively, the efficacy of PGs and TAs against the former genus suggested that they might be active against Leishmania as well. To test this possibility, we exposed intracellular amastigotes of both L. mexicana and L. donovani to various PGs and TAs and found that a subset of TA compounds had inhibitory activity for growth of the parasites with EC₅₀ values in the nanomolar (nM) range while exhibiting significantly less toxicity toward the host macrophages. Exploration of a large library of TA analogues allowed assignment of structure-activity relationships (SAR). Furthermore, TAs with potent in vitro activity were tested for in vivo activity, via oral administration in a mouse model of cutaneous leishmaniasis induced by infection with L. mexicana, and they were shown to have partial efficacy in controlling disease. In addition, we also report the in vitro metabolic and in vivo pharmacokinetic (PK) profiles of the most potent TAs. These results establish TAs as a promising chemotype for the development of novel antileishmanial oral drugs and define several early lead compounds.

RESULTS AND DISCUSSION

Chemistry.

To explore the effects of TAs on L. mexicana and L. donovani intracellular amastigotes, and understand the SARs, we relied upon TAs both previously synthesized in our studies on P. falciparum¹⁹ and a large library of novel TAs recently synthesized with various structural features (Tables 1-5). The key 2,2'-bipyrrole-5-carboxaldehyde intermediates 1 and 6–24 required in the synthesis of various A- and B-ring functionalized TAs are depicted in Figure 2. Using the reported methodology,⁴⁴ MBC (1) was prepared from commercially available starting materials in two steps, and the intermediates 6–19 were synthesized by our recently reported synthetic routes^{19, 20, 22, 45} in good to excellent yields. The synthetic routes of aryloxy- and aryl-substituted 2,2'-bipyrrole-5-carboxaldehyde analogues 20–24 are shown in Scheme 1.

Table 1.

In Vitro Antileishmanial Activity of TAs (42–63) with Modifications at Terminal Amine.

		survival (%) at 0.1 μMa vs		EC50 (nM)a vs
compound	R1	L. mexicana amastigote	J774A.1 macrophage	L. mexicana amastigote	J774A.1 macrophage	TI
42	n-C6H13	11.1 ± 4.91	127 ± 71.0	52.5 ± 0.71	> 1000 ± 0.0	> 19
43	n-C11H23	11.9 ± 3.80	112 ± 72.7	47.5 ± 0.71	> 1000 ± 0.0	> 21
44		7.33 ± 0.96	88.9 ± 25.1	40.5 ± 4.95	> 1000 ± 0.0	> 25
45		4.84 ± 1.19	99.6 ± 59.1	32.5 ± 2.12	550 ± 630	17
46		5.30 ± 2.33	72.2 ± 25.9	48.5 ± 3.53	> 1000 ± 0.0	> 21
47		12.4 ± 1.90	66.6 ± 12.5	40.0 ± 0.0	696 ± 734	17
48		9.48 ± 1.73	74.4 ± 20.7	46.7 ± 17.8	1098 ± 1218	24
49		28.3 ± 3.0	51.1 ± 13.6	87.7 ± 41.9	2986 ± 0.0	34
50		24.6 ± 12.0	124 ± 78.6	79.5 ± 10.6	625 ± 530	7.9
51		14.3 ± 2.81	72.8 ± 23.7	73.0 ± 2.83	1041 ± 1262	14
52		5.49 ± 2.59	50.8 ± 14.8	41.0 ± 2.83	171 ± 165	4.2
53		9.85 ± 0.69	95.4 ± 31.9	47.0 ± 9.89	> 1000 ± 0.0	> 21
54		18.9 ± 10.8	89.6 ± 61.5	72.5 ± 29.0	> 1000 ± 0.0	> 14
55		26.0 ± 11.9	69.8 ± 33.6	93.0 ± 3.61	2741 ± 0.0	29
56		8.65 ± 2.03	50.2 ± 14.6	66.0 ± 4.24	> 1000 ± 0.0	> 15
57		13.6 ± 2.55	65.3 ± 6.27	91.5 ± 14.8	570 ± 610	6.2
58		11.4 ± 1.01	83.7 ± 13.1	54.0 ± 4.24	760 ± 340	14
59		69.9 ± 13.8	81.0 ± 22.9	ND	ND	ND
60		23.8 ± 9.67	67.5 ± 25.7	100 ± 7.07	> 1000 ± 0.0	> 10
61		77.6 ± 23.3	57.3 ± 12.9	ND	ND	ND
62		98.3 ± 20.7	58.7 ± 14.9	ND	ND	ND
63		9.03 ± 4.12	77.3 ± 26.2	32.0 ± 7.07	> 1000 ± 0.0	> 31
miltefosine	-	0.020 ± 0.010	39.2 ± 5.1	3460 ± 781	>100000 ± 0.0	> 29

^aData represent the mean ± standard deviation of results of at least two independent experiments; an error of 0.0 indicates identical values were obtained for each replicate; TI = Therapeutic Index ((J774A.1 EC₅₀/L. mexicana EC₅₀); ND, not determined.

Figure 2.

Key 2,2'-bipyrrole-5-carboxaldehydes 1 and 6–24 for the synthesis of A- and B-ring functionalized TAs.

Scheme 1.

Synthesis of Aryloxy and Aryl Substituted 2,2'-Bipyrrole-5-carboxaldehydes (20–24).

Synthesis of 4-Aryloxy- and 4-Aryl-2,2'-bipyrrole-5-carboxaldehydes (20–23).

Coupling of Boc-glycine (25) with Meldrum’s acid in the presence of 4-(N,N-dimethylamino)pyridine (DMAP) and isopropyl chloroformate led to the acylated Meldrums’s acid, which when refluxed in ethyl acetate furnished the 4-hydroxy-1,5-dihydro-pyrrol-2-one (26) in a 64% yield.⁴⁶ The hydroxy intermediate 26 was then treated with p-toluenesulfonyl chloride in the presence of N,N-diisopropylethylamine (DIPEA) to give the tosylated intermediate 27 in a 94% yield. According to Marchal’s procedure,⁴⁷ 27 was coupled with substituted phenols 28a–c in the presence of 1,4-diazabicyclo[2.2.2]octane (DABCO) at room temperature for 16 h to furnish the corresponding aryloxy intermediates 29a–c (Scheme 1, top panel). Conversely, a standard Suzuki cross-coupling reaction^{19, 45} between intermediate 27 and 4-(trifluoromethoxy)phenylboronic acid (33) furnished the desired intermediate 34. Upon treatment with trifluoroacetic acid (TFA), 29a–c and 34 smoothly delivered the corresponding free-NH intermediates 30a–c and 35, respectively, in excellent yields. Subjecting 30a–c and 35 to a standard Vilsmeier formylation⁴⁴ led to the bromo intermediates 31a–c and 36, which when further subjected to Suzuki cross-coupling with N-Boc-2-pyrroleboronic acid (32) followed by deprotection of the N-Boc group gave the desired 4-aryloxy-2,2'-bipyrrole-5-carboxaldehydes 20–22 and 4-aryl-2,2'-bipyrrole-5-carboxaldehyde 23, respectively, in good yields (Scheme 1, top panel).

Synthesis of 5'-Aryl-2,2'-bipyrrole-5-carboxaldehyde (24).

By employing a similar strategy to the synthesis of 20–23, compound 24 was obtained in good yield from readily available 4-bromoanisole (37) and 4-methoxy-3-pyrrolin-2-one (40) via the appropriate intermediates 39 and 41 (Scheme 1, bottom panel). The pyrroleboronic acid intermediate 39 was easily synthesized from 37 via a two-step sequence of Suzuki cross-coupling with 32 followed by treatment of lithium diisopropylamide (LDA) and trimethyl borate.

Synthesis of Tambjamine Analogues (42–124).

The acid-catalyzed condensation¹⁹ of the key 2,2'-bipyrrole-5-carboxaldehydes 1 and 6–24 with various alkyl/cycloalkyl/arylamines provided the desired TA analogues 42–48 and 50–124 (Tables 1-5) in good to excellent yields (Scheme 2, top panel). Conversely, compound 49 was synthesized in excellent yield under the neutral conditions²² (Na₂SO₄, dichloromethane, 60 °C, in a sealed tube) to avoid deprotection of the N-Boc group, from 1 and tert-butyl ((1r,4r)-4-aminocyclohexyl)carbamate (Scheme 2, bottom panel). All of the intermediates and final target compounds were structurally characterized using NMR and HRMS analyses.

Scheme 2.

Synthesis of Tambjamine Analogues 42–124.

Biological Activity.

Growth Inhibitory Activity of Intracellular Amastigotes.

To measure the growth inhibitory activity of our target TA analogues against Leishmania parasites, we infected J774A.1 murine macrophage-like cells with stationary phase L. mexicana or L. donovani promastigotes, expressing red-shifted firefly (Photinus pyralis) luciferase,⁴⁸ (RS-LUC) from an integrated rRNA site, at a multiplicity of 10:1. After 24 h, parasites that had not been phagocytosed were removed by washing, test compounds were added over a range of concentrations, and luciferase activity representing viable intracellular amastigotes was measured following 96 h incubation in minimal essential medium (MEM) plus 10% heat inactivated fetal bovine serum. Initially, all target TA analogues 42–124 were tested at 1 μM (data not shown) and 0.1 μM (Tables 1-5), and those that exhibited pronounced inhibition (typically ≥ 50% at 0.1 μM) were subsequently tested in a dose-response format to determine the EC₅₀ value as a measure of potency (Tables 1-6). Conversely, a series of PG analogues^{21, 22} were also tested in this assay at 1 and 0.1 μM, however, they have shown only moderate potency (data not shown) and were thus not further pursued. In parallel, cytotoxicity against the uninfected J774A.1 macrophages was determined in a similar format, except that viability was determined using SYBR Green II fluorescence as a measure of DNA content.⁴⁸ The therapeutic index (TI) was calculated as the ratio of the EC₅₀ value for J774A.1 macrophages over the EC₅₀ value for L. mexicana and/or L. donovani amastigotes (TI = EC₅₀(J774A.1)/EC₅₀(L. mexicana or L. donovani)). All of these results are summarized in Tables 1-6, and discussed in the following sections.

In Vitro Antileishmanial Activity and SAR of TAs (42–63).

Initially, a series of TAs 42–63 containing various alkyl, cycloalkyl, and aryl moieties at the right-hand-side terminal amine and OMe group at the 4-position of ring-B (as observed in natural TAs, Figure 1), were evaluated for their in vitro antileishmanial activity against L. mexicana amastigotes and cytotoxicity against J774A.1 macrophages, and the results are shown in Table 1. TAs 42 and 43, containing n-alkyl moieties at terminal amine, exhibited excellent potency against L. mexicana amastigotes with low EC₅₀ values of 52.5 and 47.5 nM, respectively. Notably, these two compounds were much less inhibitory to the growth of the J774A.1 macrophage line (EC₅₀ ≥ 1000 nM), indicating a feasible TI (Table 1). Interestingly, the replacement of n-alkyl groups of 42 and 43 at terminal amine with cycloalkyl moieties as in 44–48, slightly improved the potency (EC₅₀ = 32.5–48.5 nM vs L. mexicana). Approximately, a 2-fold antileishmanial potency loss was observed when the cyclohexyl group of 44 was replaced with N-substituted cycloalkyl moieties as in 49 and 50 (49 EC₅₀ = 87.7 nM and 50 EC₅₀ = 79.5 nM vs 44 EC₅₀ = 40.5 nM vs L. mexicana). Next, we tested a series of TAs containing various mono-substituted aryl moieties on terminal amine (51–59). All of these mono-substituted aryl TAs, with the exception of 59 (~30% inhibition at 0.1 μM vs L. mexicana), displayed excellent antileishmanial potency (51–58 EC₅₀ = 41.0–93.0 nM vs L. mexicana) that was comparable with the alkyl/cycloalkyl TAs (42–50 EC₅₀ = 32.5–87.7 nM vs L. mexicana). These findings suggested that the OCF₃ group at ortho-position of aryl moiety on terminal amine of 59 is detrimental to antileishmanial potency. Introduction of an additional Cl substitution on the aryl moiety at terminal amine as in 60 led to a two-fold loss of potency (60 EC₅₀ = 100 nM vs 53 EC₅₀ = 47.0 nM vs L. mexicana). A significant loss of potency was observed for 61 and 62 containing a combination of halogen (Cl/F) and OMe groups on the aromatic ring at terminal amine (~22% inhibition for 61, and ~2% inhibition for 62 at 0.1 μM). Interestingly, the potency was retained after the introduction of a cyclic-diether moiety on the aryl ring (63 EC₅₀ = 32.0 nM vs L. mexicana) (Table 1). Taken together, in vitro potency and SAR analyses of TAs 42–63 demonstrated that the OMe group at the 4-position of ring-B and n-alkyl, cycloalkyl, and mono-substituted aryl moieties at terminal amine is best tolerated.

In Vitro Antileishmanial Activity and SAR of TAs (64–81).

To probe the importance of OMe group at the 4-position of ring-B of TAs, a series of new TAs (64–81), in which the OMe group is replaced by various substituted aryloxy and/or aryl moieties, was generated and evaluated for their in vitro antileishmanial activity against L. mexicana amastigotes (Table 2). In vitro antileishmanial activity of TAs 64–75 containing 4-(trifluoromethoxy)phenoxy and/or 3,5-difluorophenoxy moieties at the 4-position of ring-B and n-alkyl, and cycloalkyl moieties at terminal amine, demonstrated a 2.5- to 6-fold diminished potency when compared to the corresponding OMe group containing TAs (Table 2 vs Table 1). Conversely, TAs 76–78, which contain a 4-chlorophenoxy moiety at the 4-position of ring-B and cycloalkyl moieties at terminal amine, retained the potency (76–78 EC₅₀ = 38.0–72.3 nM), suggesting that the aryloxy moiety with a Cl substitution is well tolerated on ring-B. Replacement of the 4-(trifluoromethoxy)phenoxy moiety at the 4-position of ring-B with 4-(trifluoromethoxy)phenyl moiety as in 79–81 resulted in a decrease in antileishmanial potency, suggesting that the oxygen (O) atom at the 4-position of ring-B (i.e., OAr or OMe) plays a pivotal role in the enhancement of potency. Notably, all 1-adamantyl moiety containing TAs 70 (EC₅₀ = 41.0 nM), 74 (EC₅₀ = 79.0 nM), 77 (EC₅₀ = 38.3 nM) and 80 (EC₅₀ = 90.5 nM) exhibited excellent potency regardless of the substitutions at the 4-position of ring-B. These results demonstrated that the substitutions at the 4-position of ring-B have an important role in potent antileishmanial activity, and the OMe group can be replaced by aryloxy and/or aryl moieties when 1-adamantyl moiety exists at the terminal amine of the TA scaffold.

Table 2.

In Vitro Antileishmanial Activity of TAs (64–81) with Modifications on Ring-B and Terminal Amine.

			survival (%) at 0.1 μMa vs		EC50 (nM)a vs
compound	R1	R2	L. mexicana amastigote	J774A.1 macrophage	L. mexicana amastigote	J774A.1 macrophage	TI
64		n-C6H13	46.1 ± 13.3	110 ± 66.5	303 ± 102	1104 ± 288	3.6
65		n-C11H23	58.0 ± 18.9	96.7 ± 39.4	ND	ND	ND
66			52.7 ± 9.40	81.3 ± 25.7	84.0 ± 11.3	982 ± 557	11.7
67			43.0 ± 11.2	82.8 ± 33.4	155 ± 2.12	642 ± 250	4.1
68			37.6 ± 6.85	92.9 ± 32.9	99.0 ± 15.6	982 ± 430	9.9
69			42.9 ± 9.92	97.6 ± 39.3	167 ± 27.6	717 ± 378	4.3
70			35.7 ± 7.48	80.7 ± 28.2	41.0 ± 2.82	353 ± 196	8.6
71			79.9 ± 3.06	112 ± 39.0	ND	ND	ND
72			33.2 ± 1.10	108 ± 11.2	88.7 ± 42.5	852 ± 298	9.6
73			87.2 ± 4.48	65.8 ± 10.4	ND	ND	ND
74			27.3 ± 4.39	59.3 ± 1.66	79.0 ± 29.5	680 ± 233	8.6
75			96.2 ± 1.86	55.3 ± 7.14	ND	ND	ND
76			19.8 ± 1.30	100 ± 17.0	63.7 ± 20.3	367 ± 54	5.8
77			7.16 ± 1.96	67.1 ± 18.0	38.3 ± 26.8	567 ± 273	15
78			27.5± 5.72	80.1 ± 17.1	72.3 ± 45.2	321 ± 341	4.4
79			77.9 ± 18.4	95.8 ±15.7	ND	ND	ND
80			39.0 ± 3.94	70.6 ± 16.2	90.5 ± 32.7	908 ± 112	10
81			81.6 ± 20.5	116 ± 19.5	ND	ND	ND
miltefosine	-	-	0.020 ± 0.010	39.2 ± 5.1	3460 ± 781	>100000 ± 0.0	> 29

^aData represent the mean ± standard deviation of results of at least two independent experiments; an error of 0.0 indicates identical values were obtained for each replicate; TI = Therapeutic Index (J774A.1 EC₅₀/L. mexicana EC₅₀); ND, not determined.

In Vitro Antileishmanial Activity and SAR of TAs (82–109).

Having established the importance of the OMe, and aryloxy/aryl moieties at the 4-position of ring-B and the n-alkyl, cycloalkyl and aryl moieties at terminal amine as optimal, we then investigated the effects of the alkyl groups either at the 3-position or 4-position or both the 3-and 4-positions on ring-B (Table 3). The potency was slightly diminished when OMe at the 4-position of ring-B of 45 (Table 1) was replaced by short alkyl groups (methyl and ethyl) as in 82 and 83 (82: ~75% inhibition; 83: ~60% inhibition vs 45 >95% inhibition at 0.1 μM vs L. mexicana), suggesting that the OMe group on ring-B is playing a crucial role on antileishmanial activity. The loss of potency was also observed when the alkyl groups shifted from the 4-position to the 3-position with the compounds 84–87 (Table 3), whereas the TAs containing 1-adamatyl moiety at terminal amine and short alkyl groups at the 3-position of ring-B (88 and 89) showed excellent potency with low EC₅₀ values of 48.0 nM and 37.5 nM, respectively. Again, these results are consistent and support the findings that the 1-adamantyl moiety at terminal amine is very important for potent antileishmanial activity. We next investigated whether substitutions at both the 3- and 4-positions are tolerated. Interestingly, TAs with 3,4-dialkyl substitutions on ring-B and cyclcoalkyl moieties at terminal amine (90–95 and 101–104), exhibited better potency compared with those of the corresponding 3- and 4-monoalkyl substituted TAs (Table 3). The greatest loss of potency was observed in TAs 96–100 and 105–109, in which cycloalkyl moiety is replaced by substituted aryl moieties, suggesting that the aryl functionality is detrimental to potency when ring-B is substituted with alkyl groups. Together, from the 3-, and 4-monoalkylated and 3,4-dialkylated TAs the data showed that the combination of dialkyl substitutions on ring-B and cycloalkyl moieties at terminal amine were well tolerated (Table 3).

Table 3.

In Vitro Antileishmanial Activity of TAs (82–109) with Modifications on Ring-B and Terminal Amine.

				survival (%) at 0.1 μMa vs		EC50 (nM)a vs
compound	R1	R2	R3	L. mexicana amastigote	J774A.1 macrophage	L. mexicana amastigote	J774A.1 macrophage	TI
82	H	Me		26.3 ± 3.29	88.4 ± 29.4	ND	ND	ND
83	H	Et		42.1 ± 3.13	88.7 ± 22.0	ND	ND	ND
84	Me	H		89.0 ± 17.6	127 ± 83.3	ND	ND	ND
85	Me	H		48.6 ± 9.03	150 ± 60.4	ND	ND	ND
86	Et	H		33.9 ± 10.5	32.2 ± 9.96	ND	ND	ND
87	Et	H		34.6 ± 5.87	35.8 ± 11.9	ND	ND	ND
88	Me	H		15.4 ± 3.98	86.9 ± 27.5	48.0 ± 4.24	1131 ± 185	24
89	Et	H		11.3 ± 2.09	50.0 ± 22.1	37.5 ± 2.12	884 ± 163	24
90	Me	Me		18.2 ± 1.57	62.8 ± 7.71	55.0 ± 1.41	908 ± 159	16
91	Me	Me		33.5 ± 7.06	43.5 ± 14.4	ND	ND	ND
92	Me	Me		14.2 ± 1.0	53.2 ± 22.2	91.7 ± 48.3	1600 ± 900	17
93	Et	Et		27.5±3.37	26.7±5.21	ND	ND	ND
94	Et	Et		17.0±2.70	29.5±9.59	ND	ND	ND
95	Et	Et		17.1 ± 5.6	87.8 ± 10.7	73.3 ± 0.0	1424 ± 0.0	19
96	Me	Me		74.0 ± 7.9	54.2 ± 7.9	ND	ND	ND
97	Me	Me		92.2 ± 18.6	88.1 ± 23.4	ND	ND	ND
98	Me	Me		102 ± 25	77.2 ± 18.1	ND	ND	ND
99	Me	Me		108 ± 32	65.9 ± 3.7	ND	ND	ND
100	Me	Me		111 ± 30	66.2 ± 20.6	ND	ND	ND
101				40.9 ± 7.6	60.3 ± 13.4	130 ± 0.0	2934 ± 0.0	23
102				24.7 ± 12.0	80.0 ± 6.71	74.0 ± 9.54	252±0.0	3.4
103				33.4 ± 13.5	75.8 ± 25.7	ND	ND	ND
104				16.1 ± 4.5	75.0 ± 16.9	ND	ND	ND
105				112 ± 23	95.1 ± 16.9	ND	ND	ND
106				103 ± 6	67.2 ± 19.5	ND	ND	ND
107				84.9 ± 10.7	92.7 ± 51.2	ND	ND	ND
108				89.8 ± 19.4	62.5 ± 19.1	ND	ND	ND
109				102 ± 24	58.2 ± 9.5	ND	ND	ND
miltefosine		-	-	0.020±0.010	39.2 ± 5.1	3460 ± 781	>100000±0.0	>29

^aData represent the mean ± standard deviation of results of at least two independent experiments; an error of 0.0 indicates identical values were obtained for each replicate; TI = Therapeutic Index (J774A.1 EC₅₀/L. mexicana EC₅₀); ND, not determined.

In Vitro Antileishmanial Activity and SAR of TAs (110–116).

After establishing the substitution pattern at the 3-, and 4-positon of ring-B, we investigated the importance of alkyl substitutions at different positions on ring-A by keeping cycloalkyl moieties at terminal amine as an active pharmacophore for all TAs 110–116 (Table 4). Interestingly, compounds with alkyl groups (ethyl and iso-butyl) at the 5'-position of ring-A and cycloheptyl and/or cyclooctyl moiety at terminal amine (110–112) showed superior potency (>96% inhibition at 0.1 μM vs L. mexicana) to that of the corresponding TAs (90 and 91) that are lacking the substitutions on ring-A (<80% inhibition at 0.1 μM vs L. mexicana; Table 3). In subsequent dose response studies, 110–112 also exhibited excellent potency with low EC₅₀ values (EC₅₀ = 31.4–44.3 nM) and enhanced TI (Table 4). Surprisingly, shifting the ethyl group of 110 from the 5'-position to 4'-position as in 113 led to a 3-fold diminished antileishmanial potency (113 EC₅₀ = 117 nM vs 110 EC₅₀ = 40.9 nM vs L. mexicana). Approximately a 5-fold less potency was observed with the compound 114, in which ring-A was substituted with methyl groups at both the 3'- and 5'-positions, as compared to the 110 (114 EC₅₀ = 182 nM vs 110 EC₅₀ = 40.9 nM vs L. mexicana). It is noteworthy that the antileishmanial potency was retained after replacing the cycloheptyl moiety by 1-adamantyl moiety at terminal amine in 115 (115 EC₅₀ = 46.5 nM vs 114 EC₅₀ = 182 nM vs L. mexicana). Conversely, the compound 116 containing tri-alkyl substitutions on ring-A and 1-adamatnyl moiety at terminal amine exhibited great potency (116 EC₅₀ = 54.0 nM vs L. mexicana). These results demonstrated that the positions of alkyl substitutions on ring-A play a crucial role in altering the potency, specifically, alkyl substitutions at the 5'-position play a crucial role in enhancement of the antileishmanial potency and metabolic stability.

Table 4.

In Vitro Antileishmanial Activity of TAs (110–116) with Modifications on Ring-A and Terminal Amine.

		survival (%) at 0.1 μMa vs		EC50 (nM)a vs
compound	structure	L.mexicana amastigote	J774A.1 macrophage	L.mexicana amastigote	J774A.1 macrophage	TI
110		3.7 ± 1.0	71.2 ± 20.5	40.9 ± 19.6	1140 ± 498	28
111		2.3 ± 0.1	74.5 ± 15.7	44.3 ± 24.7	1237 ± 130	28
112		1.3 ± 0.4	53.6 ± 14.8	31.4 ± 12.6	573 ± 63.4	18
113		23.8 ± 4.3	63.3 ± 26.0	117 ± 47.4	1328 ± 73.5	11
114		43.8 ± 7.5	56.8 ± 15.9	182 ± 80.7	ND	ND
115		12.5 ± 3.00	93.2 ± 50.4	46.5 ± 12.0	667 ± 473	14
116		10.7 ± 5.4	63.5 ± 16.2	54.0 ± 15.5	675 ± 459	12
miltefosine	-	0.020 ± 0.010	39.2 ± 5.1	3460 ± 781	>100000 ± 0.0	> 29

^aData represent the mean ± standard deviation of results of at least two independent experiments; an error of 0.0 indicates identical values were obtained for each replicate; TI = Therapeutic Index (J774A.1 EC₅₀/L. mexicana EC₅₀); ND, not determined.

In Vitro Antileishmanial Activity and SAR of TAs (117–124).

Our detailed SAR investigations around ring-A, ring-B and terminal amine of the TA scaffold led us to valuable insight into the structural features that are required for potent antileishmanial activity. Lastly, we sought to explore whether the substitution at the 5'-position on ring-A is tolerated when ring-B is substituted with the OMe group at the 4-position (as observed in natural TAs, Figure 1). To that end, we generated a series of TAs containing alkyl, aryl and benzyl substitutions at the 5'-positon on ring-A and cycloalkyl, and aryl moieties at terminal amine (117–124) and evaluated these compounds for in vitro antileishmanial activity (Table 5). Interestingly, TAs 117–123 containing cycloalkyl moiety at terminal amine substantially inhibited the growth of L. mexicana amastigotes at 0.1 μM concentration, whereas the compound 124 with an aryl moiety at terminal amine showed poorest activity (~7% inhibition at 0.1 μM vs L. mexicana). It is noteworthy that the inhibitory percentage of 117–123 is highest (Table 5) than any of the tested TA analogues in this study (Tables 1-4). In our subsequent dose-response evaluation, these compounds 117–123 showed the greatest potency (EC₅₀ = 11.3–37.1 nM vs L. mexicana) with a feasible TI (14–88). Overall, the detailed SAR of the TA analogues suggested that various substitutions at the 5'-positon on ring-A, OMe and short alkyl groups on ring-B and cycloalkyl (including 1-adamantyl) moieties at terminal amine play key roles in enhancing in vitro antileishmanial potency with reduced toxicity toward the host macrophages. These latter potent compounds were developed subsequent to initiating the in vivo experiments discussed below and reported in Figured 3 for TAs 95, 101, and 110. However, future investigations of in vivo efficacy for some of TAs 117–123 would be a priority.

Table 5.

In Vitro Antileishmanial Activity of TAs (117–124) with Modifications on Ring-A and Terminal Amine.

			survival (%) at 0.1 μMa vs		EC50 (nM)a vs
compound	R1	R2	L. mexicana amastigote	J774A.1 macrophage	L. mexicana amastigote	J774A.1 macrophage	TI
117	Et		0.3 ± 0.1	52.5 ± 16.6	37.1 ± 1.82	512 ± 0.0	14
118			−0.02 ± 0.03	77.3 ± 45.3	18.8 ± 9.70	450 ± 20.1	24
119			1.40 ± 0.20	86.6 ± 51.4	23.8 ± 15.9	717 ± 0.0	30
120			0.010 ± 0.10	84.6 ± 46.6	22.6 ± 13.9	946 ± 687	42
121			−0.11 ± 0.02	84.6 ± 47.8	16.2 ± 6.24	959 ± 0.0	59
122			−0.11 ± 0.04	84.8 ± 63.4	15.8 ± 4.38	547 ± 0.0	35
123			−0.05 ± 0.04	59.6 ± 35.6	11.3 ± 2.04	> 1000 ± 0.0	> 88
124			93.5 ± 26.6	101 ± 46	ND	ND	ND
miltefosine	-	-	0.020 ± 0.010	39.2 ± 5.1	3460 ± 781	>100000 ± 0.0	> 29

^aData represent the mean ± standard deviation of results of at least two independent experiments; an error of 0.0 indicates identical values were obtained for each replicate; TI = Therapeutic Index (J774A.1 EC₅₀/L. mexicana EC₅₀); ND, not determined. Negative values for % survival represent luminescence measurements that were somewhat below that of background and probably reflect error in measurements of very low intensities. In some instances, e.g., 117 and 119, the rank order of % survival does not correspond to that of the EC₅₀ values. The % survival for such compounds is also very low, and the EC₅₀ values are likely more accurate, as they entail multiple measurements over a range of concentrations and luminescence values.

In Vitro Antileishmanial Activity against L. donovani of Selected TAs.

We next investigated a subset of the potent TAs (Table 6) against the L. donovani amastigotes (causes fatal visceral leishmaniasis) to identify TAs that are broadly active against these clinically important Leishmania species. Notably all of these tested TAs exhibited activity against L. donovani amastigotes with low EC₅₀ values (Table 6). Furthermore, the TI values for these TAs, compared to inhibition of growth of HepG2 cells, were quite robust ranging, for all but 55, from 26 to 300. However, the potency against L. donovani amastigotes was typically ~2- to 3-fold lower than for L. mexicana.

Table 6.

In Vitro Antileishmanial Activity against L. donovani Amastigotes and In Vitro Cytotoxicity of Selected TAs.

	in vitro activity EC50 (nM)a vs	cytotoxicity IC50 (nM)b vs
compound	L. donovani	HepG2 cells	TI
42	118 ± 16	26700	226
45	139 ± 61	10100	73
46	88.0 ± 11	6058	69
47	122 ± 35	3425	28
48	198 ± 61	5120	26
51	226 ± 106	ND	ND
55	285 ± 57	1734	6.0
74	184 ± 88	59400	323
77	181 ± 32	14529	80
80	398 ± 127	85513	215
88	100 ± 39	30000	300
89	90.5 ± 6.3	15200	168
90	79.5 ± 16	21300	268
95	ND	15933	ND
101	ND	8208	ND
102	101 ± 53	6200	61
110	ND	2994	ND
116	81.2 ± 8.7	21323	263

^aData represent the mean ± standard deviation of results of at least two independent experiments

^bIC₅₀ values (nM) are the average of at least three determinations, each carried out in triplicate (±10%); ND, not determined; TI = Therapeutic Index (HepG2 IC₅₀/L. donovani EC₅₀).

In Vitro Cytotoxicity.

The preliminary in vitro general cytotoxicity was tested for the selected TAs using human hepatic HepG2 cells.^{19, 49} Inhibition of mammalian cells only occurred at relatively very high concentrations (IC₅₀ >10000 nM against HepG2 cells) with many of these TAs (Table 6).

In Vitro Metabolic Stability.

A number of antileishmanial TAs, selected for their potency (EC₅₀ < 100 nM) and to represent a range of structurally diverse analogues, were also assessed for in vitro metabolic stability using human liver microsomes (HLM) and mouse liver microsomes (MLM) with well-established methods.^{50, 51} Notably, many of the most active TAs appear to be stable for up to 60 minutes or longer in both HLM and MLM (Table 7).

Table 7.

In Vitro Metabolic Stability of Selected TA Analogues against Liver Microsomes.

	metabolic stability (t1/2, min) vs
compound	HLM	MLM
42	17.1	10.2
45	52.1	8.20
46	18.6	12.3
47	17.3	11.4
48	13.0	15.4
49	8.45	9.71
52	30.0	28.9
54	> 60.0	45.1
55	> 60.0	46.9
56	> 60.0	30.1
57	> 60.0	42.0
58	> 60.0	15.8
60	56.2	46.7
61	> 60.0	> 60.0
62	> 60.0	19.1
63	> 60.0	15.8
70	> 60.0	> 60.0
74	> 60.0	> 60.0
77	> 60	> 60
80	> 60.0	> 60.0
88	> 60.0	17.6
90	45.1	11.2
95	23.3	37.5
101	26.7	33.0
102	> 60.0	9.71
104	> 60.0	> 60.0
105	42.1	31.2
108	> 60.0	> 60.0
109	> 60.0	> 60.0
110	> 60.0	> 60.0
111	> 60.0	> 60.0
112	> 60.0	> 60.0
113	> 60.0	11.0
116	> 60.0	29.0
117	> 60.0	7.98
118	> 60.0	> 60.0
119	11.7	10.4
120	19.7	13.8
121	25.6	14.8
122	50.8	16.5
123	> 60.0	> 60.0

HLM = human liver microsomes; MLM = mouse live microsomes.

In Vivo Antileishmanial Efficacy of TAs.

Given the promising antileishmanial potency of a number of TAs against L. mexicana and L. donovani along with a favorable TI against J774A.1 macrophage lines, and a favorable combination of good physicochemical properties, an in vivo proof of concept study in a well-established murine model of cutaneous leishmaniasis using BALB/c mice infected by footpad injection⁵² was undertaken with 3 promising and representative TAs 95, 101, and 110. These compounds were chosen because they had a combination of properties suggesting potential in vivo efficacy, including good in vitro potency, strong TI, and acceptable in vitro metabolic stability, with 110 being the most promising by these criteria. Importantly, these three compounds had also shown strong efficacy in curing Plasmodium yoelli infections in mice (unpublished results), thus ensuring that they must have sufficient in vivo metabolic stability and bioavailability to evaluate their efficacy against L. mexicana in vivo. However, it should be noted that in general there is not strong correlation between the in vitro potencies of TAs between L. mexicana and Plasmodium parasites, in which they were first investigated.¹⁹

Twenty days post-infection (pi) with L. mexicana promastigotes expressing red-shifted luciferase, animals were treated with 95, 101, and 110 daily for 10 days by oral gavage at 40–60 mg/kg/d. Lesion growth was monitored in two independent experiments measuring either the width of the footpad lesion (Figure 3A) or the luminescence signal over the infected footpad (Figure 3B). For both experiments, TAs 95, 101, and 110 provided retardation of lesion progression compared to the animals treated with vehicle alone, particularly between weeks 5–7 pi, demonstrating partial efficacy in controlling the disease. In particular, compound 110 provided greater efficacy than both 95 and 101. However, the effect was transient, as the lesion sizes or luminescence signal (not shown) reached that of vehicle-treated animals by week 10 pi. These results imply that in vitro potency does translate to in vivo oral efficacy, although the “drug-like” properties of these TAs would need to be improved to exhibit a curative effect. Several potent TAs with a feasible TI and good metabolic stability that emerged later in this study, including 118 and 123, will be tested in vivo in future studies to identify potentially more promising antileishmanial TA lead compounds.

Figure 3.

In vivo efficacy of 95, 101, and 110 in mice. (A) mice (initially 5 per treatment) infected with L. mexicana were treated with vehicle, 101 and 110 and footpad lesion thickness was measured. p Values were determined by two-tailed paired t-test; (B) Mice infected with RS-LUC-expressing L. mexicana were treated (days 20–29) with vehicle, 95, 101 and 110 and imaged at 6 weeks post-infection. The ratio of Relative Luminescence Units (RLU) for each mouse was calculated by dividing the RLU at day 45 post-infection (pi) by the RLU at day 19, just before treatment was begun. This ratio quantifies the increase in RLU between day 19 and day 45. Statistical significance was determined using ANOVA and Dunnett’s post-test. *** p<0.001, ** p<0.01, * p<0.05.

In Vivo Pharmacokinetic Analysis.

Preliminary in vivo PK analysis of lead compound 110 was conducted following a single intragastric (po) administration in mice at 40 mg/kg with blood and liver samples taken at the following time points: 0, 0.5, 1, 2, 4, 8, 24, 30, 48, 54, and 72 h.^{53, 54} Key PK parameters in both plasma and liver are summarized in Table 8. Significantly, compound 110 showed long half-life (t_1/2 = > 72 h for plasma, and > 36 h for liver) and rapid absorption in both plasma and liver (T_max = 0.0 h for plasma and 2.0 h for liver). It is noteworthy that 110 is highly concentrated in the liver versus plasma (3667 ng/mL for liver vs 35 ng/mL for plasma), a potentially significant advantage for the treatment of visceral leishmaniasis, where the liver is a major site of parasite colonization. The PK data of 110 indicates the observed oral dose efficacy might be the result of a combination of high concentration (3667 ng/mL) and long elimination half-life (t_1/2 > 36 h) of 110 in the liver (Table 8). Indeed, these favorable PK properties were one of the reasons we chose to test 110 in vivo.

Table 8.

Key PK Parameters of 110 in Plasma and Liver Following Single Oral Dose of 40 mg/kg Administrations in Mice.

matrix	Cmax (ng/mL)	Tmax (h)	t1/2 (h)	AUClast (ng.h/mL)	AUCinf (ng·h/mL)	AUCextrap (%)
liver	3667	2.0	> 36	22982	23043	0.262
plasma	35.0	0.0	> 72	2421	206250	98.8

C_max, maximum plasma or hepatic concentration; T_max, time to C_max; t_1/2, apparent elimination half-life; AUC_last, area under the concentration-time curve from 0 up to the last sampling time at which a quantifiable concentration is found; AUC_inf, area under the concentration-time curve from 0 up to infinity; AUC_extrap, percentage of the AUC extrapolated from the last observed time point.

In Vitro Mutagenicity.

Given the promising in vitro and in vivo antileishmanial activities of TA analogues, lead compound 110 was investigated for the potential of mutagenicity risk using the Ames assay^{55, 56} (EPBI Inc.) at concentrations up to 10 μM, with and without S9 metabolic activation, against two Salmonella typhimurium TA100 and T98 strains. Results were negative: there was no increase over the background reversion rate with 110, suggesting low risk of mutagenicity with this TA class of compounds.

CONCLUSIONS

In summary, we have discovered and developed a natural product inspired tambjamine chemotype as a novel class of antileishmanial agents. In this work, we have generated and investigated a large library of TA analogues with various structural features on ring-A, ring-B, and terminal amine of the TA scaffold, against two Leishmania parasites, cutaneous leishmaniasis causing L. mexicana, and fatal visceral leishmaniasis causing L. donovani. Several strengths of the TA chemotype are summarized here. The robust SARs identified a number of novel TA analogues with excellent in vitro potency with low nanomolar EC₅₀ values against both L. mexicana and L. donovani parasites, while demonstrating great TI against both the J774A.1 macrophages and human hepatic HepG2 cells. It is noteworthy that the TAs demonstrated superior in vitro antileishmanial activity than the control drug, miltefosine (TAs EC₅₀s < 100 nM vs miltefosine EC₅₀ = 3460 nM), and in general the low nM potency of many TAs makes them potentially superior to currently available antileishmanial therapies. Furthermore, selected lead TAs possess good in vitro metabolic stability in both human and mouse liver microsomes and in vivo PK profiles in mice with long half-life and rapid absorption in both plasma and liver. In addition, these TAs have shown moderate in vivo efficacy in mice after oral administration. Specifically, lead compound 110 provided retardation of lesion progression compared to animals treated with vehicle alone, particularly between weeks 5 and 7 pi, demonstrating partial efficacy in controlling disease. The partial in vivo efficacy indicates that this chemotype has not yet been sufficiently optimized to qualify as a late lead. Our further structural optimizations focus on the identification of novel TAs that retain or increase antileishmanial potency, including some of those listed in Table 5, while improving efficacy in animals and “drug-like” properties such as favorable PK and oral bioavailability, with mitigated toxicity.

Another objective for future studies is to address the potential targets and MoA of TAs in Leishmania parasites. Currently, targets and MoA are not known, be it for Plasmodium parasites, T. cruzi, or L. mexicana. One approach that has been employed successfully for other anti-trypanosomatid chemotypes is to generate resistant mutants followed by whole genome sequencing to identify genetic modifications.¹³ Alternatively, analogs that incorporate photoactivatable probes can be employed to identify proteins with which TAs interact.⁵⁷ Recently, proteome-wide approaches have been developed to identify proteins that interact with specific compounds, inducing a shift in thermal denaturation.⁵⁸ In addition, phenotypic alterations in TA-treated parasites might reveal important cellular pathways whereby such compounds act. While such comprehensive studies are beyond the scope of the current initial demonstration of antileishmanial potency, they provide potential strategies for elucidation of the mechanisms whereby this chemotype targets Leishmania intracellular amastigotes.

EXPERIMENTAL SECTION

General.

NMR spectra were recorded on Bruker AMX-400 and AMX-600, spectrometers at 400 and 600 MHz, respectively. NMR experiments were recorded in CDCl₃ and/or DMSO-d₆ at 25 °C. Chemical shifts are given in parts per million (ppm) downfield from internal standard tetramethylsilane (TMS). High-resolution mass spectra (HRMS) (electrospray ionization (ESI)) were recorded on a vanquish ultrahigh-performance liquid chromatography/high-performance liquid chromatography (UHPLC/HPLC) system coupled with a high-resolution (350000) Q Exactive Orbitrap mass spectrometer. Unless otherwise stated, all reagents and solvents were purchased from commercial supplies and used without further purification. Reactions that required the use of anhydrous, inert atmosphere techniques were carried out under an atmosphere of argon/nitrogen. Chromatography was executed on a CombiFlash instrument using silica gel (230–400 mesh) as stationary phase and mixtures of ethyl acetate and hexanes as eluents. Analytical HPLC analysis was performed on an Agilent 1260 Infinity II LC System using C18 column (3.0 × 50 mm) with a linear elution gradient of water/methanol (containing 10 mM ammonium acetate) ranging from 50:50 to 0:100 for 6.0 min at a flow rate of 0.75 mL/min, at 254 nm. A purity of >95% has been established for all final compounds.

Synthesis of tert-Butyl 4-Hydroxy-2-oxo-2,5-dihydro-1H-pyrrole-1-carboxylate (26).

To a stirred solution of (tert-butoxycarbonyl)glycine (25, 10 g, 57.1 mmol) in 200 mL of anhydrous dichloromethane (DCM) were added Meldrum’s acid (9.87 g, 68.6 mmol) and DMAP (17.4 g, 143 mmol) under argon atmosphere at 0 °C. A solution of isopropyl chloroformate (86 mL, 85.7 mmol, 1 N in toluene) was added dropwise, and the reaction mixture was stirred for 4 h at 0 °C. The reaction mixture was diluted with DCM (200 mL), washed with 15% KHSO₄ (2 × 100 mL), and organic layer was dried over anhydrous Na₂SO₄, and the solvent was evaporated under reduced pressure to give the acylated Meldrum’s acid. This material was then refluxed in ethyl acetate (1 L) for 1 h, and the solvent was evaporated under reduced pressure, and the product was recrystallized from ethyl acetate to give the desired product 26 (7.28 g, 64%) as a white crystalline solid. ¹H NMR (DMSO-d₆, 400 MHz) δ 12.1 (br s, 1H), 4.88 (s, 1H), 4.14 (s, 2H), 1.44 (s, 9H); HRMS (ESI) calcd for C₉H₁₃NNaO₄ (M + Na)⁺ 222.0737; found 222.0729.

Synthesis of tert-Butyl 2-Oxo-4-(tosyloxy)-2,5-dihydro-1H-pyrrole-1-carboxylate (27).

To a stirred solution of 26 (5.0 g, 25.1 mmol) in anhydrous DCM (100 mL) were added p-toluenesulfonyl chloride (4.77 g, 25.1 mmol) and DIPEA (6.48 g, 50.2 mmol). The resulting reaction mixture was stirred for 6 h at room temperature. Then the reaction mixture was washed with 5% HCl (2 × 40 mL), and brine and dried over anhydrous Na₂SO₄. The organic solvent was removed under reduced pressure and the product was chromatographed on silica gel, with ethyl acetate/hexanes as eluent, to afford 27 (8.34 g, 94%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.86 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 5.75 (s, 1H), 4.22 (d, J = 1.2 Hz, 2H), 2.50 (s, 3H), 1.52 (s, 9H); HRMS (ESI) calcd for C₁₆H₁₉NNaO₆S (M + Na)⁺ 376.0825; found 376.0831.

Synthesis of tert-Butyl 2-Oxo-4-(4-(trifluoromethoxy)phenoxy)-2,5-dihydro-1H-pyrrole-1-carboxylate (29a).

To a stirred solution of 27 (4.0 g, 11.3 mmol) in anhydrous DCM (150 mL) were added 4-(trifluoromethoxy)phenol (28a; 4.0 g, 22.7 mmol) and DABCO (2.79 g, 24.9 mmol). The resulting reaction mixture was stirred for 16 h at room temperature. Then the reaction mixture was washed with 5% HCl (2 × 75 mL), and 10% NaOH solution (150 mL) and dried over anhydrous Na₂SO₄. The organic solvent was removed under reduced pressure and the product was chromatographed on silica gel, with ethyl acetate/hexanes as eluent, to afford 29a (2.03 g, 50%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.31 (d, J = 9.1 Hz, 2H), 7.20 (d, J = 9.1 Hz, 2H), 4.98 (s, 1H), 4.43 (s, 2H), 1.57 (s, 9H); HRMS (ESI) calcd for C₁₆H₁₆F₃NNaO₅ (M + Na)⁺ 382.0873; found 382.0860.

Synthesis of Intermediates 29b and 29c.

Compounds 29b and 29c were synthesized and purified by the same procedure as described for 29a from 27, and 28b and 28c, respectively.

tert-Butyl 4-(4-Chlorophenoxy)-2-oxo-2,5-dihydro-1H-pyrrole-1-carboxylate (29b).

Yield: 1.61 g (45%) white solid; ¹H NMR (CDCl₃, 400 MHz) δ 7.40 (d, J = 8.7 Hz, 2H), 7.10 (d, J = 8.7 Hz, 2H), 4.95 (s, 1H), 4.40 (d, J = 0.9 Hz, 2H), 1.55 (s, 9H); HRMS (ESI) calcd for C₁₅H₁₆ClNNaO₄ (M + Na)⁺ 332.0660; found 332.0650.

tert-Butyl 4-(3,5-difluorophenoxy)-2-oxo-2,5-dihydro-1H-pyrrole-1-carboxylate (29c).

Yield: 2.21 g (44%) white solid; ¹H NMR (CDCl₃, 400 MHz) δ 7.8–6.72 (m, 3H), 5.09 (s, 1H), 4.44 (s, 2H), 1.56 (s, 9H); HRMS (ESI) calcd for C₁₅H₁₅F₂NaNO₄ (M + Na)⁺ 334.0861; found 334.0850.

Synthesis of 4-(4-(Trifluoromethoxy)phenoxy)-1,5-dihydro-2H-pyrrol-2-one (30a).

To a stirred solution of 29a (2.0 g, 5.57 mmol) in anhydrous DCM (50 mL) was added dropwise TFA (2.54 g, 22.3 mmol). The resulting reaction mixture was stirred for an additional hour at room temperature. The solvent was evaporated under reduced pressure, and the crude material was then dissolved in ethyl acetate (200 mL). The organic layer was washed with 5% NaHCO₃ (100 mL) and brine and dried over anhydrous Na₂SO₄. The organic solvent was removed under reduced pressure and the solid product was washed with DCM (50 mL) to afford the pure product 30a (1.32 g, 92%) as a white solid. ¹H NMR (DMSO-d₆, 400 MHz) δ 7.67 (br s, 1H), 7.48 (d, J = 9.2 Hz, 2H), 7.42 (d, J = 9.2 Hz, 2H), 4.85 (d, J = 1.3 Hz, 1H), 4.08 (s, 2H); HRMS (ESI) calcd for C₁₁H₉F₃NO₃ (M + H)⁺ 260.0529; found 260.0520.

Synthesis of Intermediates 30b and 30c.

Compounds 30b and 30c were synthesized and purified by the same procedure as described for 30a from 29b and 29c, respectively.

4-(4-Chlorophenoxy)-1,5-dihydro-2H-pyrrol-2-one (30b).

Yield: 1.13 g (95%) white solid; ¹H NMR (DMSO-d₆, 400 MHz) δ 7.66 (br s, 1H), 7.52 (d, J = 8.6 Hz, 2H), 7.32 (d, J = 8.6 Hz, 2H), 4.84 (s, 1H), 4.06 (s, 2H); HRMS (ESI) calcd for C₁₀H₉ClNO₂ (M + H)⁺ 210.0316; found 210.0310.

4-(3,5-Difluorophenoxy)-1,5-dihydro-2H-pyrrol-2-one (30c).

Yield: 1.34 g (90%) white solid; ¹H NMR (DMSO-d₆, 400 MHz) δ 7.72 (br s, 1H), 7.72–7.13 (m, 3H), 5.06 (d, J = 0.89 Hz, 1H), 4.06 (s, 2H); HRMS (ESI) calcd for C₁₀H₈F₂NO₂ (M + H)⁺ 212.0518; found 212.0511.

Synthesis of (Z)-N-((5-Bromo-3-(4-(trifluoromethoxy)phenoxy)-2H-pyrrol-2-ylidene)methyl)-N-ethylethanamine (31a).

To a stirred solution of diethylformamide (1.52 g, 15.0 mmol) in anhydrous DCM (10 mL) was added dropwise a solution of phosphorus oxybromide (POBr₃; 3.58 g, 12.5 mmol) in DCM (10 mL) under an argon atmosphere at 0 °C. The resulting thick suspension was stirred at 0 °C for an hour to obtain the Vilsmeier complex as a white solid. Then, the solid was crushed with a spatula, DCM (50 mL) was added, and the mixture was cooled to 0 °C. A solution of 30a (1.3 g, 5.0 mmol) in DCM (10 mL) was added dropwise, and the mixture was warmed to room temperature and then heated at 50 °C for 6 h. The mixture was poured onto ice water, and the pH was adjusted to 8 by treating with 5 N NaOH solution, and then stirred for an additional 30 min. The organic layer was separated and the aqueous layer was extracted with DCM (3 × 50 mL). The combined organic extracts were washed with water and brine, and dried over anhydrous Na₂SO₄. The solvent was evaporated under reduced pressure, and the crude product was chromatographed on silica gel, with ethyl acetate/hexanes as eluent, to afford the title compound 31a as a tan solid (1.3 g, 64%). ¹H NMR (CDCl₃, 400 MHz) δ 7.23–7.16 (m, 5H), 5.64 (s, 1H), 4.24 (q, J = 7.1 Hz, 2H), 3.48 (q, J = 7.1 Hz, 2H), 1.38–1.33 (m, 6H); HRMS (ESI) calcd for C₁₆H₁₇BrF₃N₂O₂ (M + H)⁺ 405.0420; found 405.0406 and 407.0383 (+2, isotope).

Synthesis of Intermediates 31b and 31c.

Compounds 31b and 31c were synthesized and purified by the same procedure as described for 31a from 30b and 30c, respectively.

(Z)-N-((5-Bromo-3-(4-chlorophenoxy)-2H-pyrrol-2-ylidene)methyl)-N-ethylethanamine (31b).

Yield: 1.21 g (65%) pale-brown solid; ¹H NMR (CDCl₃, 400 MHz) δ 7.21 (d, J = 8.8 Hz, 2H), 7.06 (s, 1H), 7.01 (d, J = 8.8 Hz, 2H), 5.51 (s, 1H), 4.13 (q, J = 6.9 Hz, 2H), 3.38 (q, J = 7.2 Hz, 2H), 1.28–1.24 (m, 6H); HRMS (ESI) calcd for C₁₅H₁₇BrClN₂O (M + H)⁺ 355.0207; found 355.0194 and 357.0169 (+2, isotope).

(Z)-N-((5-Bromo-3-(3,5-difluorophenoxy)-2H-pyrrol-2-ylidene)methyl)-N-ethylethanamine (31c).

Yield: 1.46 g (72%) pale-brown solid; ¹H NMR (CDCl₃, 400 MHz) δ 7.07 (s, 2H), 6.69–6.50 (m, 3H), 5.78 (s, 1H), 4.22 (q, J = 7.2 Hz, 2H), 3.46 (q, J = 7.3 Hz, 2H), 1.36–1.31 (m, 6H); HRMS (ESI) calcd for C₁₅H₁₆BrF₂N₂O (M + H)⁺ 357.0409; found 357.0394 and 359.0371 (+2, isotope).

Synthesis of 4-(4-(Trifluoromethoxy)phenoxy)-1H,1'H-[2,2'-bipyrrole]-5-carbaldehyde (20).

To a degassed solution of 31a (1.3 g, 3.21 mmol) and 2-pyrroleboronic acid (32, 1.0 g, 4.82 mmol) in 10% water/1,4-dioxane (70 mL) were added Pd(PPh₃)₄ (0.186 g, 0.16 mmol) and Na₂CO₃ (0.682 g, 6.43 mmol). The reaction mixture was stirred for 4 h at 100 °C. The solvents were evaporated under reduced pressure, and the obtained crude product was then treated with 10% NaOH solution at 90 °C for 20 min. Then the crude product was extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with brine and dried over anhydrous Na₂SO₄. The solvent was evaporated under reduced pressure, and the crude product was then chromatographed on silica gel, with ethyl acetate/hexanes as eluent, to afford the title compound 20 as a pale-yellow solid (0.886 g, 82%). ¹H NMR (CDCl₃, 400 MHz) δ 11.96 (br s, 1H), 11.66 (br s, 1H), 9.39 (s, 1H), 7.30–7.25 (m, 4H), 6.98 (m, 1H), 6.62 (m, 1H), 6.29 (m, 1H), 5.99 (d, J = 2.6 Hz, 1H); HRMS (ESI) calcd for C₁₆H₁₂F₃N₂O₃ (M + H)⁺ 337.0795; found 337.0781.

Synthesis of MBC Analogues 21 and 22.

Compounds 21 and 22 were synthesized and purified by the same procedure as described for 20 from 31b and 31c, respectively.

4-(4-Chlorophenoxy)-1H,1'H-[2,2'-bipyrrole]-5-carbaldehyde (21).

Yield: 0.687 g (85%) pale-yellow solid; ¹H NMR (DMSO-d₆, 400 MHz) δ 11.8 (br s, 1H), 11.2 (br s, 1H), 9.41 (s, 1H), 7.47 (d, J = 8.8 Hz, 2H), 7.24 (d, J = 8.8 Hz, 2H), 6.89 (m, 1H), 6.80 (m, 1H), 6.16 (d, J = 1.9 Hz, 1H), 6.11 (m, 1H); HRMS (ESI) calcd for C₁₅H₁₂ClN₂O₂ (M + H)⁺ 287.0582; found 287.0574.

4-(3,5-Difluorophenoxy)-1H,1'H-[2,2'-bipyrrole]-5-carbaldehyde (22).

Yield: 0.501 g (62%) pale-yellow solid; ¹H NMR (DMSO-d₆, 600 MHz) δ 11.9 (br s, 1H), 11.2 (br s, 1H), 9.40 (s, 1H), 7.06 (tt, J = 9.3, 2.0 Hz, 1H), 6.98 (dd J = 8.3, 1.8 Hz, 2H), 6.91 (m, 1H), 6.81 (br s, 1H), 6.30 (d, J = 2.0 Hz, 1H), 6.13 (m, 1H); HRMS (ESI) calcd for C₁₅H₁₁F₂N₂O₂ (M + H)⁺ 289.0783; found 289.0775.

Synthesis of tert-Butyl 2-Oxo-4-(4-(trifluoromethoxy)phenyl)-2,5-dihydro-1H-pyrrole-1-carboxylate (34).

To a degassed solution of 27 (3.9 g, 11.0 mmol) and (4-(trifluoromethoxy)phenyl)boronic acid (33, 3.4 g, 16.6 mmol) in 10% water/THF (100 mL) were added Pd(dppf)Cl₂ (0.403 g, 0.55 mmol) and Cs₂CO₃ (10.8 g, 33.1 mmol). The reaction mixture was stirred for 6 h at 85 °C and then filtered through Celite. The filtrate was evaporated under reduced pressure, and the obtained crude product was then chromatographed on silica gel, with ethyl acetate/hexanes as eluent, to afford the title compound 34 as a white solid (3.0 g, 79%). ¹H NMR (CDCl₃, 400 MHz) δ 7.61 (d, J = 8.6 Hz, 2H), 7.32 (d, J = 8.6 Hz, 2H), 6.43 (s, 1H), 4.70 (s, 2H), 1.61 (s, 9H); HRMS (ESI) calcd for C₁₆H₁₆F₃NNaO₄ (M + Na)⁺ 366.0924; found 366.0913.

Synthesis of 4-(4-(Trifluoromethoxy)phenyl)-1,5-dihydro-2H-pyrrol-2-one (35).

Compound 35 was synthesized and purified by the same procedure as described for 30a from 34. Yield: 1.91 g (90%) white solid; ¹H NMR (DMSO-d₆, 400 MHz) δ 8.24 (br s, 1H), 7.80 (d, J = 8.8 Hz, 2H), 7.44 (d, J = 8.8 Hz, 2H), 6.58 (s, 1H), 4.39 (s, 2H); HRMS (ESI) calcd for C₁₁H₉F₃NO₂ (M + H)⁺ 244.0580; found 244.0572.

Synthesis of (Z)-N-((5-Bromo-3-(4-(trifluoromethoxy)phenyl)-2H-pyrrol-2-ylidene)methyl)-N-ethylethanamine (36).

Compound 36 was synthesized and purified by the same procedure as described for 31a from 35. Yield:1.71 g (63%) pale-brown solid; ¹H NMR (CDCl₃, 400 MHz) δ 7.39–7.25 (m, 4H), 6.94 (s, 1H), 6.39 (s, 1H), 4.35 (m, 2H), 3.47 (m, 2H), 1.41 (t, J = 7.1 Hz, 3H), 1.33 (t, J = 7.2 Hz, 3H); HRMS (ESI) calcd for C₁₆H₁₇BrF₃N₂O (M + H)⁺ 389.0471; found 389.0455 and 391.0431 (+2, isotope).

Synthesis of 4-(4-(Trifluoromethoxy)phenyl)-1H,1'H-[2,2'-bipyrrole]-5-carbaldehyde (23).

Compound 23 was synthesized and purified by the same procedure as described for 20 from 32 and 36. Yield: 0.800 g (63%) pale-yellow solid; ¹H NMR (DMSO-d₆, 600 MHz) δ 12.1 (br s, 1H), 11.3 (br s, 1H), 9.46 (s, 1H), 7.70 (d, J = 8.7 Hz, 2H), 7.45 (d, J = 8.7 Hz, 2H), 6.93 (d, J = 1.1 Hz, 1H), 6.80 (d, J = 1.6 Hz, 1H), 6.73 (s, 1H), 6.14 (m, 1H); HRMS (ESI) calcd for C₁₆H₁₂F₃N₂O₂ (M + H)⁺ 321.0845; found 321.0837.

Synthesis of tert-Butyl 2-(4-Methoxyphenyl)-1H-pyrrole-1-carboxylate (38).

Compound 38 was synthesized and purified by the same procedure as described for 20 from 32 and 37, with the exception of 10% NaOH solution treatment. Yield: 4.5 g (62%) clear syrup; ¹H NMR (CDCl₃, 400 MHz) δ 7.35 (m, 1H), 7.30 (d, J = 8.6 Hz, 2H), 6.92 (d, J = 8.6 Hz, 2H), 6.23 (t, J = 3.3 Hz, 1H), 6.16 (m, 1H), 3.85 (s, 3H), 1.42 (s, 9H); HRMS (ESI) calcd for C₁₆H₁₉NNaO₃ (M + Na)⁺ 296.1259; found 296.1248.

Synthesis of (1-(tert-Butoxycarbonyl)-5-(4-methoxyphenyl)-1H-pyrrol-2-yl)boronic Acid (39).

To a stirred solution of 38 (3.5 g, 12.8 mmol) in 75 mL of anhydrous THF was added dropwise lithium diisopropylamide (LDA, 2 M; 19.3 mL, 63.6 mmol) under an argon atmosphere at −78 °C. The reaction mixture was stirred for 2 h at −78 °C, then trimethyl borate (6.60 g, 64.1 mmol) was added, and the resulting solution was allowed to react at ambient temperature overnight. The reaction was quenched with saturated NH₄Cl solution, filtered through Celite, and extracted with ethyl acetate (3 × 50 mL). The combined organic phases were dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude product was further washed with hexanes, to afford the pure product 39 as a white solid (3.82 g, 94%). ¹H NMR (CDCl₃, 400 MHz) δ 7.23 (d, J = 8.8 Hz, 2H), 7.11 (d, J = 3.4 Hz, 1H), 6.91 (br s, 2H), 6.93 (d, J = 8.8 Hz, 2H), 6.22 (d, J = 3.4 Hz, 1H), 3.86 (s, 3H), 1.22 (s, 9H); HRMS (ESI) calcd for C₁₆H₂₀BNNaO₅ (M + Na)⁺ 340.1330; found 340.1313.

Synthesis of (Z)-N-((5-Bromo-3-methoxy-2H-pyrrol-2-ylidene)methyl)-N-ethylethanamine (41).

Compound 41 was synthesized and purified by the same procedure as described for 31a from 40. Yield: 7.53 g (66%) tan solid; ¹H NMR (CDCl₃, 600 MHz) δ 7.01 (s, 1H), 5.61 (s, 1H), 4.14 (q, J = 7.4 Hz, 2H), 3.78 (s, 3H), 3.41 (q, J = 7.2 Hz, 2H), 1.31 (t, J = 7.4 Hz, 3H), 1.30 (t, J = 7.2 Hz, 3H); HRMS (ESI) calcd for C₁₀H₁₆BrN₂O (M + H)⁺ 259.0441; found 259.0434 and 259.0412 (+2, isotope).

Synthesis of 4-Methoxy-5'-(4-methoxyphenyl)-1H,1'H-[2,2'-bipyrrole]-5-carbaldehyde (24).

Compound 24 was synthesized and purified by the same procedure as described for 20 from 39 and 41. Yield: 1.72 g (75%) tan solid; ¹H NMR (DMSO-d₆, 400 MHz) δ 11.4 (br s, 1H), 11.3 (br s, 1H), 9.31 (s, 1H), 7.61 (d, J = 8.8 Hz, 2H), 6.99 (d, J = 8.8 Hz, 2H), 6.77 (t, J = 2.3 Hz, 1H), 6.51 (dd, J = 2.3, 1.0 Hz, 1H), 6.40 (d, J = 2.6 Hz, 1H), 3.86 (s, 3H), 3.79 (m, 3H); HRMS (ESI) calcd for C₁₇H₁₇N₂O₃ (M + H)⁺ 297.1234; found 297.1224.

Synthesis of (Z)-1-Cycloheptyl-N-((4'-methoxy-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)-methanamine Hydrochloride (46).

To a stirred solution of 1 (0.100 g, 0.52 mmol) and cycloheptylmethanamine (0.134 g, 1.05 mmol) in anhydrous methanol (10 mL) were added a few drops of methanolic HCl (2 N, catalytic amount) under an argon atmosphere at room temperature. The resulting pale yellow colored solution was stirred at refluxing temperature for 5 h (in some cases between 2 and10 h). On completion of the reaction, methanol was evaporated under reduced pressure and the obtained crude product was then dissolved in ethyl acetate (50 mL) and washed with 2 N HCl (2 × 30 mL). The organic layer was washed with brine and dried over anhydrous Na₂SO₄. The solvent was evaporated under reduced pressure, and the crude product was then chromatographed on silica gel, with ethyl acetate/hexanes as eluent, to afford the desired TA analogue 46 with hydrogen chloride salt (0.153 g, 87% ) as a yellow solid. ¹H NMR (CDCl₃, 400 MHz) δ 13.73 (br s, 1H), 10.64 (br s, 1H), 9.44 (br s, 1H), 7.31 (d, J = 14.9 Hz, 1H), 7.07 (m, 1H), 6.75 (m, 1H), 6.25 (m, 1H), 5.96 (d, J = 1.8 Hz, 1H), 3.94 (s, 3H), 3.33 (t, J = 6.4 Hz, 2H), 1.94–1.89 (m, 3H), 1.86–1.80 (m, 2H), 1.75–1.45 (m, 6H), 1.32–1.23 (m, 2H); HRMS (ESI) calcd for C₁₈H₂₆N₃O (M + H)⁺ 300.2070; found 300.2058.

Synthesis of tert-Butyl ((1r,4r)-4-(((Z)-(4'-Methoxy-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)amino)cyclohexyl)carbamate (49).

Compound 1 (0.100 g, 0.52 mmol), tert-butyl ((1r,4r)-4-aminocyclohexyl)carbamate (0.225 g, 1.05 mmol), anhydrous Na₂SO₄ (10 g), and anhydrous DCM (50 mL) were placed in a sealed tube with a Teflon-lined cap under an argon atmosphere at room temperature. The reaction mixture was stirred and heated for 72 h at 60 °C. The mixture was filtered by a sintered funnel and washed with DCM (50 mL). The filtrate was concentrated under reduced pressure to give a crude residue, which was further chromatographed on silica gel, with ethyl acetate/hexanes as eluent, to afford the titled compound 49 in excellent yield (0.175 g, 86%) as a yellow solid. ¹H NMR (CDCl_3, 400 MHz) δ 13.70 (br s, 1H), 10.59 (br s, 1H), 9.60 (br s, 1H), 7.36 (d, J = 14.5 Hz, 1H), 7.07 (br s, 1H), 6.74 (br s, 1H), 6.28 (dd, J = 5.3, 2.5 Hz, 1H), 5.93 (br s, 1H), 3.92 (s, 3H), 3.62–3.51 (m, 4H), 3.30 (m, 1H), 2.20 (br s, 1H), 2.07–1.88 (m, 3H), 1.70 (m, 1H), 1.47 (s, 9H); HRMS (ESI) calcd for C₂₁H₃₁N₄O₃ (M + H)⁺ 387.2391; found 387.2377.

Synthesis of Target TA Analogues.

Synthesis and structural characterization of 42–45, 50, 53, 82–91, 93, 94, 102, 103, 115 and 116 were reported in our previous publication¹⁹ that describes the antiplasmodial activity of early-stage TAs. The rest of the new TAs were synthesized and purified by the same procedure as described for 46 from 1, 6–24 (100 mg scale, 1.0 equivalent) and appropriate amines (2.0 equivalents). Note: Majority of the final TA analogues are reported in HCl salt form. Hence, an additional proton, which protonates the nitrogen of ring-B, appears at downfield region in the ¹H NMR spectra.

(1r,4r)-N-((Z)-(4'-Methoxy-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)-4-methylcyclohexan-1-amine Hydrochloride (47).

Yield: 142 mg (84%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.65 (s, 1H), 10.62 (s, 1H), 9.57 (d, J = 15.1 Hz, 1H), 7.42 (d, J = 15.1 Hz, 1H), 7.08 (m, 1H), 6.74 (m, 1H), 6.29 (m, 1H), 5.95 (d, J = 2.3 Hz, 1H), 3.94 (s, 3H), 3.34 (m, 1H), 2.11 (m, 2H), 1.85 (m, 2H), 1.72–1.60 (m, 2H), 1.49–1.41 (m, 1H), 1.09–0.98 (m, 2H), 0.95 (d, J = 6.4 Hz, 3H); HRMS (ESI) calcd for C₁₇H₂₄N₃O (M + H)⁺ 286.1914; found 286.1904.

(1r,4r)-N-((Z)-(4'-Methoxy-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)-4-(trifluoromethyl)cyclohexan-1-amine Hydrochloride (48).

Yield: 176 mg (89%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.98 (br s, 1H), 10.73 (br s, 1H), 9.51 (d, J = 14.8 Hz, 1H), 7.38 (d, J = 14.8 Hz, 1H), 7.10 (m, 1H), 6.77 (m, 1H), 6.30 (m, 1H), 5.97 (d, J = 2.3 Hz, 1H), 3.95 (s, 3H), 3.79 (m, 1H), 2.19–1.97 (m, 5H), 1.89–1.77 (m, 4H); HRMS (ESI) calcd for C₁₇H₂₁F₃N₃O (M + H)⁺ 340.1631; found 340.1621.

(Z)-N-((4'-Methoxy-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)aniline Hydrochloride (51).

Yield: 144 mg (91%) brownish red solid; ¹H NMR (CDCl_3, 400 MHz) δ 14.02 (br s, 1H), 11.26 (d, J = 14.5 Hz, 1H), 10.74 (br s, 1H), 7.82 (d, J = 14.5 Hz, 1H), 7.41 (m, 4H), 7.22 (m, 1H), 7.17 (m, 1H), 6.86 (m, 1H), 6.35 (m, 1H), 6.03 (d, J = 2.2 Hz, 1H), 4.01 (s, 3H); HRMS (ESI) calcd for C₁₆H₁₆N₃O (M + H)⁺ 266.1288; found 266.1278.

(Z)-N-((4'-Methoxy-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)-4-methylaniline Hydrochloride (52).

Yield: 156 mg (94%) brownish red solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.95 (br s, 1H), 11.25 (d, J = 14.6 Hz, 1H), 10.70 (br s, 1H), 7.79 (d, J = 14.6 Hz, 1H), 7.32 (d, J = 8.2 Hz, 2H), 7.21 (d, J = 8.2 Hz, 2H), 7.15 (br s, 1H), 6.83 (br s, 1H), 6.34 (d, J = 2.1 Hz, 1H), 6.01 (d, J = 1.7 Hz, 1H), 4.00 (s, 3H), 2.36 (s, 3H); HRMS (ESI) calcd for C₁₇H₁₈N₃O (M + H)⁺ 280.1444; found 280.1434.

(Z)-N-((4'-Methoxy-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)-4-(trifluoromethoxy)aniline Hydrochloride (54).

Yield: 192 mg (95%) brownish red solid; ¹H NMR (DMSO-d_6, 400 MHz) δ 13.25 (br s, 1H), 12.60 (d, J = 13.7 Hz, 1H), 12.09 (br s, 1H), 8.28 (d, J = 13.7 Hz, 1H), 7.73 (d, J = 8.5 Hz, 2H), 7.46 (d, J = 8.5 Hz, 2H), 7.29 (d, J = 6.2 Hz, 2H), 6.62 (s, 1H), 6.36 (s, 1H), 4.02 (s, 3H); HRMS (ESI) calcd for C₁₇H₁₅F₃N₃O₂ (M + H)⁺ 350.1111; found 350.1099.

(Z)-N-((4'-Methoxy-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)-4-(p-tolyloxy)aniline (55).

Yield: 162 mg (83%) brownish red solid; ¹H NMR (CDCl_3, 400 MHz) δ 7.79 (s, 1H), 7.34 (d, J = 8.8 Hz, 2H), 7.17 (d, J = 8.4 Hz, 2H), 7.12 (dd, J = 2.4, 1.2 Hz, 1H), 7.02 (d, J = 8.8 Hz, 2H), 6.94 (d, J = 8.4 Hz, 2H), 6.80 (dd, J = 3.7, 1.2 Hz, 1H), 6.34 (dd, J = 2.7, 0.97 Hz, 1H), 6.02 (s, 1H), 3.99 (s, 3H), 2.36 (s, 3H); HRMS (ESI) calcd for C₂₃H₂₂N₃O₂ (M + H)⁺ 372.1707; found 372.1695.

(Z)-N-((4'-Methoxy-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)-4-(4-methoxyphenoxy)aniline (56).

Yield: 165 mg (81%) brownish red solid; ¹H NMR (CDCl_3, 400 MHz) δ 7.77 (s, 1H), 7.34 (d, J = 8.8 Hz, 2H), 7.13 (dd, J = 2.4, 1.3 Hz, 1H), 7.78 (d, J = 9.0 Hz, 2H), 6.98 (d, J = 8.8 Hz, 2H), 6.91 (d, J = 9.0 Hz, 2H), 6.80 (dd, J = 3.7, 1.1 Hz, 1H), 6.34 (dd, J = 3.7, 1.1 Hz, 1H), 6.01 (s, 1H), 3.99 (s, 3H), 3.83 (s, 3H); HRMS (ESI) calcd for C₂₃H₂₂N₃O₃ (M + H)⁺ 388.1656; found 388.1643.

(Z)-3-Chloro-N-((4'-methoxy-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)aniline Hydrochloride (57).

Yield: 155 mg (88%) brownish red solid; ¹H NMR (DMSO-d_6, 400 MHz) δ 13.21 (br s, 1H), 12.52 (d, J = 13.9 Hz, 1H), 12.10 (br s, 1H), 8.35 (d, J = 13.9 Hz, 1H), 7.78 (s, 1H), 7.58 (d, J = 8.2 Hz, 1H), 7.46 (t, J = 8.2 Hz, 1H), 7.30–7.25 (m, 3H), 6.62 (d, J = 1.9 Hz, 1H), 6.38 (dd, J = 5.2, 2.3 Hz, 1H), 4.03 (s, 3H); HRMS (ESI) calcd for C₁₆H₁₅ClN₃O (M + H)⁺ 300.0898; found 300.0887.

(Z)-N-((4'-Methoxy-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)-3-(trifluoromethoxy)aniline Hydrochloride (58).

Yield: 184 mg (91%) brownish red solid; ¹H NMR (DMSO-d_6, 400 MHz) δ 13.24 (br s, 1H), 12.60 (br s, 1H), 12.12 (br s, 1H), 8.36 (br s, 1H), 7.71–7.19 (m, 6H), 6.63 (br s, 1H), 6.38 (br s, 1H), 4.03 (s, 3H); HRMS (ESI) calcd for C₁₇H₁₅F₃N₃O₂ (M + H)⁺ 350.1111; found 350.1098.

(Z)-N-((4'-Methoxy-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)-2-(trifluoromethoxy)aniline Hydrochloride (59).

Yield: 188 mg (93%) brownish red solid; ¹H NMR (DMSO-d_6, 400 MHz) δ 13.55 (br s, 1H), 12.15 (br s, 1H), 11.75 (d, J = 13.4 Hz, 1H), 8.07 (d, J = 13.4 Hz, 1H), 7.79 (d, J = 7.7 Hz, 1H), 7.53 (d, J = 9.2 Hz, 1H), 7.49 (d, J = 7.7 Hz, 1H), 7.39 (t, J = 7.7 Hz, 1H), 7.31 (s, 1H), 7.27 (s, 1H), 6.65 (s, 1H), 6.39 (br s, 1H), 4.03 (s, 3H); HRMS (ESI) calcd for C₁₇H₁₅F₃N₃O₂ (M + H)⁺ 350.1111; found 350.1099.

(Z)-3,4-Dichloro-N-((4'-methoxy-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)aniline Hydrochloride (60).

Yield: 165 mg (85%) brownish red solid; ¹H NMR (DMSO-d_6, 400 MHz) δ 13.16 (br s, 1H), 12.53 (d, J = 13.9 Hz, 1H), 12.12 (br s, 1H), 8.35 (d, J = 13.9 Hz, 1H), 7.98 (s, 1H), 7.69 (d, J = 8.7 Hz, 1H), 7.60 (d, J = 8.7 Hz, 1H), 7.30 (s, 2H), 6.62 (s, 1H), 6.38 (s, 1H), 4.03 (s, 3H); HRMS (ESI) calcd for C₁₆H₁₄Cl₂N₃O (M + H)⁺ 334.0508; found 334.0495.

(Z)-4-Chloro-3-methoxy-N-((4'-methoxy-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)aniline Hydrochloride (61).

Yield: 175 mg (91%) brownish red solid; ¹H NMR (DMSO-d_6, 400 MHz) δ 13.24 (br s, 1H), 12.51 (d, J = 14.0 Hz, 1H), 12.07 (br s, 1H), 8.40 (d, J = 14.0 Hz, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.44 (d, J = 9.0 Hz, 1H), 7.29 (d, J = 10.0 Hz, 2H), 7.18 (d, J = 8.4 Hz, 1H), 6.62 (s, 1H), 6.36 (s, 1H), 4.02 (s, 3H), 3.94 (s, 3H); HRMS (ESI) calcd for C₁₇H₁₇ClN₃O₂ (M + H)⁺ 330.1004; found 330.0993.

(Z)-4-Fluoro-3-methoxy-N-((4'-methoxy-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)aniline Hydrochloride (62).

Yield: 162 mg (88%) brownish red solid; ¹H NMR (DMSO-d_6, 400 MHz) δ 13.21 (br s, 1H), 12.54 (br s, 1H), 12.05 (br s, 1H), 8.37 (s, 1H), 7.47–7.16 (m, 5H), 6.61 (s, 1H), 6.35 (s, 1H), 4.01 (s, 3H), 3.93 (s, 3H); HRMS (ESI) calcd for C₁₇H₁₇FN₃O₂ (M + H)⁺ 314.1299; found 314.1286.

(Z)-N-((4'-Methoxy-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)-2,3-dihydrobenzo[b][1,4]dioxin-6-amine Hydrochloride (63).

Yield: 132 mg (93%) brownish red solid; ¹H NMR (DMSO-d_6, 400 MHz) δ 13.10 (s, 1H), 12.45 (d, J = 14.3 Hz, 1H), 11.98 (s, 1H), 8.21 (d, J = 14.3 Hz, 1H), 7.22 (br s, 3H), 7.11 (dd, J = 6.5, 2.3 Hz, 1H), 6.93 (d, J = 8.6 Hz, 1H), 6.57 (d, J = 1.0 Hz, 1H), 6.34 (d, J = 1.0 Hz, 1H), 4.28 (dd, J = 5.1, 3.1 Hz, 4H), 4.00 (s, 3H); HRMS (ESI) calcd for C₁₈H₁₈N₃O₃ (M + H)⁺ 324.1343; found 324.1328.

(Z)-N-((4'-(4-(Trifluoromethoxy)phenoxy)-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)hexan-1-amine (64).

Yield: 106 mg (85%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 7.55 (s, 1H), 7.31 (d, J = 9.0 Hz, 2H), 7.24 (d, J = 9.0 Hz, 2H), 7.06 (br s, 1H), 6.65 (br s, 1H), 6.27 (d, J = 2.5 Hz, 1H), 5.82 (s, 1H), 3.57 (t, J = 7.2 Hz, 2H), 1.82 (m, 2H), 1.47–1.42 (m, 2H), 1.37–1.35 (m, 4H), 0.92 (t, J = 6.9 Hz, 3H); HRMS (ESI) calcd for C₂₂H₂₅F₃N₃O₂ (M + H)⁺ 420.1893; found 420.1879.

(Z)-N-((4'-(4-(Trifluoromethoxy)phenoxy)-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)undecan-1-amine Hydrochloride (65).

Yield: 126 mg (81%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.98 (br s, 1H), 10.56 (br s, 1H), 10.05 (br s, 1H), 7.56 (d, J = 12.2 Hz, 1H), 7.31 (d, J = 8.9 Hz, 2H), 7.24 (d, J = 8.9 Hz, 2H), 7.07 (br s, 1H), 6.66 (br s, 1H), 6.27 (m, 1H), 5.81 (s, 1H), 3.57 (t, J = 6.7 Hz, 2H), 1.83 (m, 2H), 1.47–1.42 (m, 2H), 1.33–1.28 (m, 14H), 0.89 (t, J = 6.4 Hz, 3H); HRMS (ESI) calcd for C₂₇H₃₅F₃N₃O₂ (M + H)⁺ 490.2676; found 490.2663.

(Z)-N-((4'-(4-(Trifluoromethoxy)phenoxy)-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)cyclohexanamine Hydrochloride (66).

Yield: 117 mg (87%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.95 (br s, 1H), 10.56 (br s, 1H), 10.10 (br s, 1H), 7.62 (d, J = 15.3 Hz, 1H), 7.31 (d, J = 8.8 Hz, 2H), 7.24 (d, J = 8.8 Hz, 2H), 7.08 (m, 1H), 6.65 (m, 1H), 6.27 (m, 1H), 5.82 (d, J = 2.4 Hz, 1H), 3.49 (m, 1H), 2.14 (m, 2H), 1.96–1.92 (m, 2H), 1.74–1.64 (m, 2H), 1.43–1.29 (m, 4H); HRMS (ESI) calcd for C₂₂H₂₃F₃N₃O₂ (M + H)⁺ 418.1737; found 418.1724.

(Z)-N-((4'-(4-(Trifluoromethoxy)phenoxy)-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)cycloheptanamine Hydrochloride (67).

Yield: 124 mg (89%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.98 (br s, 1H), 10.57 (br s, 1H), 10.14 (m, 1H), 7.60 (d, J = 15.2 Hz, 1H), 7.31 (d, J = 8.8 Hz, 2H), 7.25–7.22 (m, 2H), 7.08 (m, 1H), 6.64 (m, 1H), 6.27 (m, 1H), 5.81 (d, J = 2.2 Hz, 1H), 3.68 (m, 1H), 2.19–2.11 (m, 2H), 1.99–1.92 (m, 2H), 1.90–1.83 (m, 2H), 1.68–1.63 (m, 4H), 1.59–1.51 (m, 2H); HRMS (ESI) calcd for C₂₃H₂₅F₃N₃O₂ (M + H)⁺ 432.1893; found 432.1882.

(Z)-N-((4'-(4-(Trifluoromethoxy)phenoxy)-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)cyclooctanamine Hydrochloride (68).

Yield: 132 mg (92%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 14.00 (s, 1H), 10.58 (br s, 1H), 10.08 (m, 1H), 7.60 (d, J = 15.2 Hz, 1H), 7.31 (d, J = 8.8 Hz, 2H), 7.23 (m, 2H), 7.08 (d, J = 1.2 Hz, 1H), 6.64 (m, 1H), 6.27 (m, 1H), 5.81 (d, J = 2.2 Hz, 1H), 3.72 (m, 1H), 2.09–2.01 (m, 4H), 1.92–1.84 (m, 2H), 1.70–1.54 (m, 8H); HRMS (ESI) calcd for C₂₄H₂₇F₃N₃O₂ (M + H)⁺ 446.2050; found 446.2037.

(Z)-1-Cycloheptyl-N-((4'-(4-(trifluoromethoxy)phenoxy)-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)methanamine Hydrochloride (69).

Yield: 123 mg (86%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 14.03 (s, 1H), 10.56 (s, 1H), 9.98 (m, 1H), 7.52 (d, J = 15.0 Hz, 1H), 7.32 (d, J = 8.8 Hz, 2H), 7.26–7.23 (m, 2H), 7.08 (m, 1H), 6.66 (m, 1H), 6.27 (m, 1H), 5.82 (d, J = 2.3 Hz, 1H), 3.42 (t, J = 6.4 Hz, 2H), 1.99 (m, 1H), 1.90–1.84 (m, 2H), 1.76–1.70 (m, 2H), 1.67–1.61 (m, 2H), 1.58–1.50 (m, 4H), 1.37–1.30 (m, 2H); HRMS (ESI) calcd for C₂₄H₂₇F₃N₃O₂ (M + H)⁺ 446.2050; found 446.2038.

(3s,5s,7s)-N-((Z)-(4'-(4-(Trifluoromethoxy)phenoxy)-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)adamantan-1-amine Hydrochloride (70).

Yield: 123 mg (82%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.95 (br s, 1H), 10.60 (br s, 1H), 10.22 (d, J = 15.7 Hz, 1H), 7.65 (d, J = 15.7 Hz, 1H), 7.32 (d, J = 8.8 Hz, 2H), 7.27–7.23 (m, 2H), 7.08 (m, 1H), 6.63 (m, 1H), 6.26 (m, 1H), 5.82 (d, J = 2.2 Hz, 1H), 2.27 (br s, 3H), 2.07 (d, J = 2.5 Hz, 6H), 1.80–1.72 (m, 6H); HRMS (ESI) calcd for C₂₆H₂₇F₃N₃O₂ (M + H)⁺ 470.2050; found 470.2039.

(Z)-4-methyl-N-((4'-(4-(Trifluoromethoxy)phenoxy)-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)cyclohexan-1-amine Hydrochloride (71) (mixture of two isomers).

Yield: 126 mg (91%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 14.19 (br s, 0.6H), 13.92 (br s, 0.4H), 10.73 (br s, 0.6H), 10.54 (br s, 0.4H), 10.13 (m, 0.6H), 9.90 (m, 0.4H), 7.64–7.59 (m, 1H), 7.32 (d, J = 9.0 Hz, 2H), 7.26–7.21 (m, 2H), 7.08 (m, 1H), 6.64 (m, 1H), 6.27 (m, 1H), 5.81 (m, 1H), 3.74 (m, 0.6H), 3.43 (m, 0.4H), 2.17 (m, 1H), 2.04 (m, 1H), 1.85–1.81 (m, 3H), 1.77–1.65 (m, 2H), 1.51–1.44 (m, 0.7H), 1.11–1.00 (m, 3H), 0.98 (m, 1H); HRMS (ESI) calcd for C₂₃H₂₅F₃N₃O₂ (M + H)⁺ 432.1893; found 432.1882.

(1r,4r)-N-((Z)-(4'-(4-(Trifluoromethoxy)phenoxy)-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)-4-(trifluoromethyl)cyclohexan-1-amine Hydrochloride (72).

Yield: 135 mg (87%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 14.27 (s, 1H), 10.66 (br s, 1H), 10.03 (d, J = 10.6 Hz, 1H), 7.59 (d, J = 14.9 Hz, 1H), 7.33 (d, J = 8.7 Hz, 2H), 7.26–7.24 (m, 2H), 7.10 (m, 1H), 6.67 (m, 1H), 6.28 (m, 1H), 5.81 (d, J = 2.1 Hz, 1H), 3.88 (m, 1H), 2.23–2.14 (m, 3H), 2.12–2.03 (m, 2H), 1.93–1.82 (m, 4H); HRMS (ESI) calcd for C₂₃H₂₂F₆N₃O₂ (M + H)⁺ 486.1611; found 486.1597.

(Z)-N-((4'-(3,5-Difluorophenoxy)-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)cycloheptanamine Hydrochloride (73).

Yield: 128 mg (88%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 14.09 (br s, 1H), 10.56 (br s, 1H), 10.29 (br s, 1H), 7.58 (d, J = 13.2 Hz, 1H), 7.09 (br s, 1H), 6.79–6.68 (m, 4H), 6.29 (m, 1H), 5.96 (s, 1H), 3.67 (m, 1H), 2.18–2.12 (m, 2H), 1.99–1.92 (m, 2H), 1.90–1.84 (m, 2H), 1.68–1.63 (m, 4H), 1.57–1.51 (m, 2H); HRMS (ESI) calcd for C₂₂H₂₄F₂N₃O (M + H)⁺ 384.1882; found 384.1869.

(3s,5s,7s)-N-((Z)-(4'-(3,5-Difluorophenoxy)-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)adamantan-1-amine Hydrochloride (74).

Yield: 133 mg (84%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 14.06 (br s, 1H), 10.59 (br s, 1H), 10.36 (d, J = 15.5 Hz, 1H), 7.62 (d, J = 15.5 Hz, 1H), 7.09 (br s, 1H), 6.79–6.71 (m, 3H), 6.67 (br s, 1H), 6.28 (m, 1H), 5.96 (d, J = 1.8 Hz, 1H), 2.27 (br s, 3H). 2.06 (br s, 6H), 1.80–1.72 (m, 6H); HRMS (ESI) calcd for C₂₅H₂₆F₂N₃O (M + H)⁺ 422.2038; found 422.2025.

(1r,4r)-N-((Z)-(4'-(3,5-Difluorophenoxy)-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)-4-methylcyclohexan-1-amine Hydrochloride (75).

Yield: 121 mg (83%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 14.04 (br s, 1H), 10.52 (br s, 1H), 10.28 (br s, 1H), 7.59 (d, J = 12.9 Hz, 1H), 7.09 (br s, 1H), 6.78–6.68 (m, 4H), 6.29 (m, 1H), 5.96 (s, 1H), 3.46 (m, 1H), 2.16–2.12 (m, 2H), 1.89–1.86 (m, 2H), 1.77–1.66 (m, 2H), 1.48 (m, 1H), 1.10–1.00 (m, 2H), 0.95 (t, J = 6.4 Hz, 3H); HRMS (ESI) calcd for C₂₂H₂₄F₂N₃O (M + H)⁺ 384.1882; found 384.1869.

(Z)-N-((4'-(4-Chlorophenoxy)-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)cycloheptanamine Hydrochloride (76).

Yield: 130 mg (89%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.95 (br s, 1H), 10.57 (br s, 1H), 10.10 (br s, 1H), 7.60 (d, J = 15.0 Hz, 1H), 7.42 (d, J = 8.9 Hz, 2H), 7.15 (d, J = 8.9 Hz, 2H), 7.07 (br s, 1H), 6.62 (br s, 1H), 6.26 (m, 1H), 5.78 (d, J = 2.3 Hz, 1H), 3.67 (m, 1H), 2.19–2.11 (m, 2H), 1.98–1.83 (m, 4H), 1.67–1.64 (m, 4H), 1.57–1.51 (m, 2H); HRMS (ESI) calcd for C₂₂H₂₅ClN₃O (M + H)⁺ 382.1681; found 382.1670.

(3s,5s,7s)-N-((Z)-(4'-(4-Chlorophenoxy)-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)adamantan-1-amine Hydrochloride (77).

Yield: 134 mg (84%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.91 (br s, 1H), 10.60 (br s, 1H), 10.18 (d, J = 15.8 Hz, 1H), 7.65 (d, J = 15.8 Hz, 1H), 7.42 (d, J = 8.6 Hz, 2H), 7.17 (d, J = 8.6 Hz, 2H), 7.07 (br s, 1H), 6.61 (br s, 1H), 6.26 (m, 1H), 5.78 (d, J = 2.1 Hz, 1H), 2.26 (br s, 3H), 2.06 (s, 6H), 1.80–1.72 (m, 6H); HRMS (ESI) calcd for C₂₅H₂₇ClN₃O (M + H)⁺ 420.1837; found 420.1825.

(1r,4r)-N-((Z)-(4'-(4-Chlorophenoxy)-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)-4-methylcyclohexan-1-amine Hydrochloride (78).

Yield: 121 mg (83%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.90 (s, 1H), 10.54 (br s, 1H), 10.08 (m, 1H), 7.62 (d, J = 15.3 Hz, 1H), 7.42 (d, J = 8.8 Hz, 2H), 7.15 (d, J = 8.8 Hz, 2H), 7.07 (br s, 1H), 6.63 (br s, 1H), 6.27 (m, 1H), 5.78 (d, J = 2.0 Hz, 1H), 3.42 (m, 1H), 2.17–2.12 (m, 2H), 1.89–1.86 (m, 2H), 1.76–1.66 (m, 2H), 1.51 (m, 1H), 1.11–1.00 (m, 2H), 0.95 (d, J = 6.5 Hz, 3H); HRMS (ESI) calcd for C₂₂H₂₅ClN₃O (M + H)⁺ 382.1681; found 382.1669.

(Z)-N-((4'-(4-(Trifluoromethoxy)phenyl)-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)cycloheptanamine Hydrochloride (79).

Yield: 128 mg (91%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 14.40 (br s, 1H), 10.76 (br s, 1H), 10.68 (br s, 1H), 7.46 (d, J = 8.4 Hz, 2H), 7.38–7.35 (m, 3H), 7.11 (br s, 1H), 6.81 (br s, 1H), 6.65 (s, 1H), 6.32 (m, 1H), 3.66 (m, 1H), 2.16–2.11 (m, 2H), 2.01–1.92 (m, 2H), 1.89–1.84 (m, 2H), 1.66–1.64 (m, 4H), 1.54–1.52 (m, 2H); HRMS (ESI) calcd for C₂₃H₂₅F₃N₃O (M + H)⁺ 416.1944; found 416.1931.

(3s,5s,7s)-N-((Z)-(4'-(4-(Trifluoromethoxy)phenyl)-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)adamantan-1-amine Hydrochloride (80).

Yield: 133 mg (87%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 14.38 (br s, 1H), 10.81 (d, J = 15.1 Hz, 1H), 10.71 (br s, 1H), 7.47–7.45 (m, 3H), 7.38 (d, J = 8.1 Hz, 2H), 7.11 (br s, 1H), 6.81 (br s, 1H), 6.65 (d, J = 1.7 Hz, 1H), 6.32 (m, 1H), 2.27 (br s, 3H), 2.05 (m, 6H), 1.80–1.71 (m, 6H); HRMS (ESI) calcd for C₂₆H₂₇F₃N₃O (M + H)⁺ 454.2101; found 454.2086.

(1r,4r)-4-Methyl-N-((Z)-(4'-(4-(trifluoromethoxy)phenyl)-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)cyclohexan-1-amine Hydrochloride (81).

Yield: 120 mg (85%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 14.34 (br s, 1H), 10.74 (br s, 1H), 10.64 (m, 1H), 7.46 (d, J = 8.4 Hz, 2H), 7.41–7.35 (m, 3H), 7.11 (br s, 1H), 6.81 (br s, 1H), 6.65 (s, 1H), 6.32 (m, 1H), 3.42 (m, 1H), 2.13–2.10 (m, 2H), 1.88–1.85 (m, 2H), 1.80–1.70 (m, 2H), 1.48 (m, 1H), 1.09–0.98 (m, 2H), 0.94 (d, J = 6.4 Hz, 3H); HRMS (ESI) calcd for C₂₃H₂₅F₃N₃O (M + H)⁺ 416.1944; found 416.1931.

(Z)-1-Cycloheptyl-N-((3',4'-dimethyl-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)methanamine Hydrochloride (92).

Yield: 158 mg (89%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.73 (br s, 1H), 10.80 (br s, 1H), 10.12 (m, 1H), 7.25 (d, J = 15.1 Hz, 1H), 7.09 (m, 1H), 6.79 (m, 1H), 6.31 (m, 1H), 3.38 (t, J = 6.5 Hz, 2H), 2.19 (s, 3H), 2.18 (s, 3H), 1.95 (m, 1H), 1.86–1.80 (m, 2H), 1.73–1.66 (m, 2H), 1.64–1.58 (m, 2H), 1.55–1.47 (m, 4H), 1.32–1.23 (m, 2H); HRMS (ESI) calcd for C₁₉H₂₈N₃ (M + H)⁺ 298.2278; found 298.2266.

(1r,4r)-N-((Z)-(3',4'-Diethyl-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)-4-methylcyclohexan-1-amine Hydrochloride (95).

Yield: 148 mg (92%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.59 (br s, 1H), 10.87 (d, J = 15.0 Hz, 1H), 10.33 (br s, 1H), 7.39 (br s, 1H), 7.09–7.08 (m, 1H), 6.79 (m, 1H), 6.30 (m, 1H), 3.39 (m, 1H), 2.68 (q, J = 7.5 Hz, 2H), 2.59 (q, J = 7.6 Hz, 2H), 2.11–2.08 (m, 2H), 1.85–1.82 (m, 2H), 1.72–1.65 (m, 2H), 1.46–1.41 (m, 1H), 1.22–1.17 (m, 6H), 1.07–0.96 (m, 2H), 0.92 (d, J = 6.5 Hz, 3H); HRMS (ESI) calcd for C₂₀H₃₀N₃ (M + H)⁺ 312.2434; found 312.2423.

(Z)-N-((3',4'-Dimethyl-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)-4-methylaniline Hydrochloride (96).

Yield: 155 mg (93%) brownish red solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.86 (br s, 1H), 11.69 (d, J = 14.3 Hz, 1H), 10.89 (m, 1H), 7.74 (d, J = 14.3 Hz, 1H), 7.31 (d, J = 8.1 Hz, 2H), 7.17 (br s, 1H), 7.13 (d, J = 8.1 Hz, 2H), 6.85 (br s, 1H), 6.35 (m, 1H), 2.31 (s, 3H), 2.26 (s, 3H), 2.16 (s, 3H); HRMS (ESI) calcd for C₁₈H₂₀N₃ (M + H)⁺ 278.1652; found 278.1643.

(Z)-4-Chloro-N-((3',4'-dimethyl-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)aniline Hydrochloride (97).

Yield: 166 mg (94%) brownish red solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.82 (br s, 1H), 11.64 (d, J = 14.3 Hz, 1H), 10.86 (br s, 1H), 7.71 (d, J = 14.3 Hz, 1H), 7.33 (d, J = 8.8 Hz, 2H), 7.23 (d, J = 8.8 Hz, 2H), 7.19 (br s, 1H), 6.89 (br s, 1H), 6.37 (m, 1H), 2.25 (s, 3H), 2.13 (s, 3H); HRMS (ESI) calcd for C₁₇H₁₇ClN₃ (M + H)⁺ 298.1106; found 298.1097.

(Z)-N-((3',4'-Dimethyl-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)-4-(trifluoromethoxy)aniline Hydrochloride (98).

Yield: 193 mg (95%) brownish red solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.93 (br s, 1H), 11.66 (d, J = 14.1 Hz, 1H), 10.90 (br s, 1H), 7.68 (d, J = 14.1 Hz, 1H), 7.44 (d, J = 9.0 Hz, 2H), 7.20–7.18 (m, 3H), 6.92 (br s, 1H), 6.38 (m, 1H), 2.25 (s, 3H), 2.16 (s, 3H); HRMS (ESI) calcd for C₁₈H₁₇F₃N₃O (M + H)⁺ 348.1318; found 348.1306.

(Z)-N-((3',4'-Dimethyl-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)-4-(4-methoxyphenoxy)aniline Hydrochloride (99).

Yield: 199 mg (89%) brownish red solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.86 (br s, 1H), 11.80 (d, J = 14.6 Hz, 1H), 10.86 (br s, 1H), 7.67 (d, J = 14.6 Hz, 1H), 7.38 (d, J = 8.9 Hz, 2H), 7.16 (br s, 1H), 6.98–6.87 (m, 7H), 6.36 (m, 1H), 3.81 (s, 3H), 2.24 (s, 3H), 2.16 (s, 3H); HRMS (ESI) calcd for C₂₄H₂₄N₃O₂ (M + H)⁺ 386.1863; found 386.1850.

(Z)-N-((3',4'-Dimethyl-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)-4-(p-tolyloxy)aniline Hydrochloride (100).

Yield: 187 mg (87%) brownish red solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.92 (br s, 1H), 11.85 (d, J = 14.6 Hz, 1H), 10.88 (br s, 1H), 7.67 (d, J = 14.6 Hz, 1H), 7.40 (d, J = 8.9 Hz, 2H), 7.18 (s, 1H), 7.15 (d, J = 8.4 Hz, 2H), 6.99 (d, J = 8.9 Hz, 2H), 6.91 (d, J = 8.4 Hz, 2H), 6.88 (br s, 1H), 6.36 (m, 1H), 2.34 (s, 3H), 2.26 (s, 3H), 2.19 (s, 3H); HRMS (ESI) calcd for C₂₄H₂₄N₃O (M + H)⁺ 370.1914; found 370.1902.

(Z)-N-((3-(1H-Pyrrol-2-yl)-4,5,6,7-tetrahydro-1H-isoindol-1-ylidene)methyl)cyclohexanamine Hydrochloride (101).

Yield: 141 mg (91%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.72 (br s, 1H), 10.75 (br s, 1H), 9.99 (br s, 1H), 7.29 (d, J = 14.6 Hz, 1H), 7.10 (br s, 1H), 6.71 (br s, 1H), 6.29 (m, 1H), 3.34 (m, 1H), 2.65–2.63 (m, 4H), 2.80–2.04 (m, 2H), 1.92–1.79 (m, 6H), 1.70–1.60 (m, 3H), 1.39–1.24 (m, 3H); HRMS (ESI) calcd for C₁₉H₂₆N₃ (M + H)⁺ 296.2121; found 296.2111.

(Z)-1-(3-(1H-Pyrrol-2-yl)-4,5,6,7-tetrahydro-1H-isoindol-1-ylidene)-N-(cycloheptylmethyl)methanamine Hydrochloride (104).

Yield: 141 mg (84%) brownish red solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.79 (br s, 1H), 10.75 (br s, 1H), 9.89 (br s, 1H), 7.17 (d, J = 14.5 Hz, 1H), 7.10 (m, 1H), 6.72 (m, 1H), 6.30 (m, 1H), 3.35 (t, J = 6.3 Hz, 2H), 2.65–2.62 (m, 4H), 1.92 (m, 1H), 1.85–1.79 (m, 6H), 1.72–1.65 (m, 2H), 1.59–1.57 (m, 2H), 1.53–1.46 (m, 4H), 1.37–1.22 (m, 2H); HRMS (ESI) calcd for C₂₁H₃₀N₃ (M + H)⁺ 324.2434; found 324.2423.

(Z)-N-((3-(1H-Pyrrol-2-yl)-4,5,6,7-tetrahydro-1H-isoindol-1-ylidene)methyl)-4-methylaniline Hydrochloride (105).

Yield: 147 mg (93%) brownish red solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.87 (br s, 1H), 11.47 (d, J = 14.6 Hz, 1H), 10.81 (br s, 1H), 7.69 (d, J = 14.6 Hz, 1H), 7.27 (d, J = 8.5 Hz, 2H), 7.16 (br s, 1H), 7.07 (d, J = 8.5 Hz, 2H), 6.77 (br s, 1H), 6.34 (d, J = 3.0 Hz, 1H), 2.74 (t, J = 5.6 Hz, 2H), 2.59 (t, J = 5.6 Hz, 2H), 2.28 (s, 3H), 2.14–1.82 (m, 4H); HRMS (ESI) calcd for C₂₀H₂₂N₃ (M + H)⁺ 304.1808; found 304.1797.

(Z)-N-((3-(1H-Pyrrol-2-yl)-4,5,6,7-tetrahydro-1H-isoindol-1-ylidene)methyl)-4-chloroaniline Hydrochloride (106).

Yield: 151 mg (90%) brownish red solid; ¹H NMR (DMSO-d_6, 400 MHz) δ 13.42 (br s, 1H), 12.53 (d, J = 13.9 Hz, 1H), 11.19 (br s, 1H), 8.45 (d, J = 13.9 Hz, 1H), 7.70 (d, J = 8.7 Hz, 2H), 7.55 (d, J = 8.7 Hz, 2H), 7.27 (br s, 1H), 7.11 (br s, 1H), 6.37 (br s, 1H), 2.84 (t, J = 6.2 Hz, 2H), 2.69 (t, J = 6.2 Hz, 2H), 1.81–1.79 (m, 4H); HRMS (ESI) calcd for C₁₉H₁₉ClN₃ (M + H)⁺ 324.1262; found 324.1251.

(Z)-N-((3-(1H-Pyrrol-2-yl)-4,5,6,7-tetrahydro-1H-isoindol-1-ylidene)methyl)-4-(trifluoromethoxy)aniline Hydrochloride (107).

Yield: 174 mg (91%) brownish red solid; ¹H NMR (CDCl_3, 400 MHz) δ 14.01 (br s, 1H), 11.59 (d, J = 14.3 Hz, 1H), 10.84 (br s, 1H), 7.60 (d, J = 14.3 Hz, 1H), 7.42 (d, J = 9.1 Hz, 2H), 7.20 (s, 1H), 7.19 (d, J = 9.1 Hz, 2H), 6.86 (br s, 1H), 6.36 (m, 1H), 2.74 (t, J = 5.4 Hz, 2H), 2.63 (t, J = 5.4 Hz, 2H), 1.84–1.83 (m, 4H); HRMS (ESI) calcd for C₂₀H₁₉F₃N₃O (M + H)⁺ 374.1475; found 374.1465.

(Z)-N-((3-(1H-Pyrrol-2-yl)-4,5,6,7-tetrahydro-1H-isoindol-1-ylidene)methyl)-4-(4-methoxyphenoxy)aniline Hydrochloride (108).

Yield: 182 mg (87%) brownish red solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.97 (br s, 1H), 11.66 (d, J = 14.6 Hz, 1H), 10.81 (br s, 1H), 7.59 (d, J = 14.6 Hz, 1H), 7.36 (d, J = 9.1 Hz, 2H), 7.17 (br s, 1H), 6.98–6.94 (m, 4H), 6.81 (d, J = 9.1 Hz, 2H), 6.82 (br s, 1H), 6.35 (m, 1H), 3.81 (s, 3H), 2.73 (t, J = 5.2 Hz, 2H), 2.66 (t, J = 5.2 Hz, 2H), 1.87–1.56 (m, 4H); HRMS (ESI) calcd for C₂₆H₂₆N₃O₂ (M + H)⁺ 412.2020; found 412.2007.

(Z)-N-((3-(1H-Pyrrol-2-yl)-4,5,6,7-tetrahydro-1H-isoindol-1-ylidene)methyl)-4-(p-tolyloxy)aniline Hydrochloride (109).

Yield: 173 mg (86%) brownish red solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.93 (br s, 1H), 11.64 (d, J = 14.6 Hz, 1H), 10.80 (br s, 1H), 7.60 (d, J = 14.6 Hz, 1H), 7.36 (d, J = 8.8 Hz, 2H), 7.16 (br s, 1H), 7.14 (d, J = 8.4 Hz, 2H), 6.96 (d, J = 8.9 Hz, 2H), 6.89 (d, J = 8.4 Hz, 2H), 6.81 (br s, 1H), 6.35 (m, 1H), 2.73 (t, J = 5.9 Hz, 2H), 2.64 (t, J = 5.4 Hz, 2H), 2.34 (s, 3H), 1.86–1.82 (m, 4H); HRMS (ESI) calcd for C₂₆H₂₆N₃O (M + H)⁺ 396.2070; found 396.2059.

(Z)-N-((5-Ethyl-3',4'-dimethyl-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)cycloheptanamine Hydrochloride (110).

Yield: 148 mg (92%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.59 (br s, 1H), 10.80 (br s, 1H), 10.03 (br s, 1H), 7.28 (t, J = 14.9 Hz, 1H), 6.71 (t, J = 3.3 Hz, 1H), 6.05 (br s, 1H), 3.64 (m, 1H), 2.81 (q, J = 7.6 Hz, 2H), 2.18 (s, 3H), 2.17 (s, 3H), 2.14–2.08 (m, 2H), 1.98–1.83 (m, 4H), 1.65–1.64 (m, 4H), 1.56–1.48 (m, 2H), 1.37 (t, J = 7.6 Hz, 3H); HRMS (ESI) calcd for C₂₀H₃₀N₃ (M + H)⁺ 312.2434; found 312.2424.

(Z)-N-((5-Isobutyl-3',4'-dimethyl-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)cycloheptanamine Hydrochloride (111).

Yield: 144 mg (94%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.61 (br s, 1H), 10.80 (br s, 1H), 10.03 (m, 1H), 7.27 (d, J = 14.8 Hz, 1H), 6.72 (t, J = 3.0 Hz, 1H), 6.01 (br s, 1H), 3.64 (m, 1H), 2.62 (d, J = 7.6 Hz, 2H), 2.18 (s, 3H), 2.17 (s, 3H), 2.14–2.06 (m, 3H), 1.98–1.83 (m, 4H), 1.66–1.60 (m, 4H), 1.56–1.50 (m, 2H), 0.97 (d, J = 6.7 Hz, 6H); HRMS (ESI) calcd for C₂₂H₃₄N₃ (M + H)⁺ 340.2747; found 340.2737.

(Z)-N-((5-Ethyl-3',4'-dimethyl-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)cyclooctanamine Hydrochloride (112).

Yield: 159 mg (95%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.62 (br s, 1H), 10.82 (br s, 1H), 10.01 (br s, 1H), 7.28 (d, J = 14.9 Hz, 1H), 6.71 (t, J = 3.0 Hz, 1H), 6.05 (br s, 1H), 3.66 (m, 1H), 2.78 (q, J = 7.6 Hz, 2H), 2.19 (s, 3H), 2.17 (s, 3H), 2.05–1.99 (m, 4H), 1.93–1.84 (m, 2H), 1.71–1.52 (m, 8H), 1.37 (t, J = 7.6 Hz, 3H); HRMS (ESI) calcd for C₂₁H₃₂N₃ (M + H)⁺ 326.2591; found 326.2579.

(Z)-N-((4-Ethyl-3',4'-dimethyl-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)cycloheptanamine Hydrochloride (113).

Yield: 140 mg (87%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.61 (br s, 1H), 10.56 (br s, 1H), 10.21 (br s, 1H), 7.33 (d, J = 15.0 Hz, 1H), 6.91 (br s, 1H), 6.64 (br s, 1H), 3.63 (m, 1H), 2.55 (q, J = 7.6 Hz, 2H), 2.19 (s, 3H), 2.18 (s, 3H), 2.16–2.10 (m, 2H), 1.98–1.82 (m, 4H), 1.64–1.63 (m, 4H), 1.55–1.49 (m, 2H), 1.23 (t, J = 7.6 Hz, 3H); HRMS (ESI) calcd for C₂₀H₃₀N₃ (M + H)⁺ 312.2434; found 312.2423.

(Z)-N-((3,3',4',5-Tetramethyl-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)cycloheptanamine Hydrochloride (114).

Yield: 144 mg (90%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 12.86 (br s, 1H), 11.11 (br s, 1H), 9.50 (m, 1H), 7.39 (d, J = 15.0 Hz, 1H), 5.83 (br s, 1H), 3.64 (m, 1H), 2.33 (s, 3H), 2.22 (s, 3H), 2.20 (s, 3H), 2.14–2.06 (m, 5H), 2.01–1.94 (m, 2H), 1.89–1.84 (m, 2H), 1.64–1.63 (m, 4H), 1.55–1.49 (m, 2H); HRMS (ESI) calcd for C₂₀H₃₀N₃ (M + H)⁺ 312.2434; found 312.2422.

(Z)-N-((5-Ethyl-4'-methoxy-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)cycloheptanamine Hydrochloride (117).

Yield: 147 mg (92%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.55 (br s, 1H), 10.62 (br s, 1H), 9.33 (br s, 1H), 7.33 (d, J = 14.7 Hz, 1H), 6.65 (t, J = 3.0 Hz, 1H), 6.01 (br s, 1H), 5.89 (d, J = 1.8 Hz, 1H), 3.92 (s, 3H), 3.58 (m, 1H), 2.76 (q, J = 7.6 Hz, 2H), 2.11–2.05 (m, 2H), 1.92–1.80 (m, 4H), 1.66–1.63 (m, 4H), 1.55–1.48 (m, 2H), 1.37 (t, J = 7.6 Hz, 3H); HRMS (ESI) calcd for C₁₉H₂₈N₃O (M + H)⁺ 314.2227; found 314.2214.

(Z)-N-((4'-Methoxy-5-(4-methoxyphenyl)-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)cycloheptanamine Hydrochloride (118).

Yield: 120 mg (83%) yellow solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.67 (br s, 1H), 11.07 (br s, 1H), 9.22 (m, 1H), 7.74 (d, J = 8.2 Hz, 2H), 7.27 (d, J = 15.1 Hz, 1H), 6.81 (d, J = 8.2 Hz, 2H), 6.68 (br s, 1H), 6.44 (br s, 1H), 5.86 (br s, 1H), 3.85 (br s, 3H), 3.76 (br s, 3H), 3.55 (m, 1H), 2.01–1.97 (m, 2H), 1.86–1.75 (m, 4H), 1.50–1.45 (m, 6H); HRMS (ESI) calcd for C₂₄H₃₀N₃O₂ (M + H)⁺ 392.2333; found 392.2319.

(Z)-N-((4'-Methoxy-5-(4-methoxybenzyl)-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)cyclopentanamine Hydrochloride (119).

Yield: 124 mg (93%) yellow solid; ¹H NMR (CDCl_3, 600 MHz) δ 13.58 (br s, 1H), 10.62 (br s, 1H), 9.37 (m, 1H), 7.33 (d, J = 15.0 Hz, 1H), 7.27 (d, J = 8.5 Hz, 2H), 6.86 (d, J = 8.5 Hz, 2H), 6.63 (t, J = 3.2 Hz, 1H), 5.96 (t, J = 2.8 Hz, 1H), 5.87 (t, J = 2.2 Hz, 1H), 3.98 (s, 2H), 3.93 (br s, 4H), 3.79 (s, 3H), 2.07–2.03 (m, 2H), 1.97–1.87 (m, 4H), 1.71–1.67 (m, 2H); HRMS (ESI) calcd for C₂₃H₂₈N₃O₂ (M + H)⁺ 378.2176; found 378.2164.

(Z)-N-((4'-Methoxy-5-(4-methoxybenzyl)-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)cyclohexanamine Hydrochloride (120).

Yield: 127 mg (92%) yellow solid; ¹H NMR (CDCl_3, 600 MHz) δ 13.58 (br s, 1H), 10.64 (br s, 1H), 9.36 (m, 1H), 7.35 (d, J = 15.1 Hz, 1H), 7.28 (d, J = 8.6 Hz, 2H), 6.85 (d, J = 8.6 Hz, 2H), 6.63 (t, J = 3.2 Hz, 1H), 5.96 (t, J = 3.2 Hz, 1H), 5.87 (t, J = 2.2 Hz, 1H), 3.98 (s, 2H), 3.91 (s, 3H), 3.79 (s, 3H), 3.38 (m, 1H), 2.07–2.04 (m, 2H), 1.91–1.89 (m, 2H), 1.66–1.59 (m, 4H), 1.39–1.28 (m, 2H); HRMS (ESI) calcd for C₂₄H₃₀N₃O₂ (M + H)⁺ 392.2333; found 392.2320.

(Z)-N-((4'-Methoxy-5-(4-methoxybenzyl)-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)cycloheptanamine Hydrochloride (121).

Yield: 135 mg (95%) yellow solid; ¹H NMR (CDCl_3, 600 MHz) δ 13.59 (br s, 1H), 10.66 (br s, 1H), 9.38 (m, 1H), 7.33 (d, J = 15.3 Hz, 1H), 7.27 (d, J = 8.5 Hz, 2H), 6.86 (d, J = 8.5 Hz, 2H), 6.62 (t, J = 2.9 Hz, 1H), 5.96 (t, J = 2.8 Hz, 1H), 5.87 (d, J = 2.1 Hz, 1H), 3.98 (s, 2H), 3.91 (s, 3H), 3.78 (s, 3H), 3.57 (m, 1H), 2.10–2.06 (m, 2H), 1.90–1.80 (m, 4H), 1.66–1.63 (m, 4H), 1.53–1.49 (m, 2H); HRMS (ESI) calcd for C₂₅H₃₂N₃O₂ (M + H)⁺ 406.2489; found 406.2475.

(Z)-N-((4'-Methoxy-5-(4-methoxybenzyl)-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)cyclooctanamine Hydrochloride (122).

Yield: 132 mg (90%) yellow solid; ¹H NMR (CDCl_3, 600 MHz) δ 13.62 (br s, 1H), 10.68 (br s, 1H), 9.35 (m, 1H), 7.33 (d, J = 15.0 Hz, 1H), 7.27 (d, J = 8.5 Hz, 2H), 6.86 (d, J = 8.5 Hz, 2H), 6.62 (t, J = 2.9 Hz, 1H), 5.96 (t, J = 2.8 Hz, 1H), 5.87 (d, J = 2.2 Hz, 1H), 3.98 (s, 2H), 3.91 (s, 3H), 3.79 (s, 3H), 3.60 (m, 1H), 2.04–1.93 (m, 4H), 1.88–1.83 (m, 2H), 1.67–1.51 (m, 8H); HRMS (ESI) calcd for C₂₆H₃₄N₃O₂ (M + H)⁺ 420.2646; found 420.2631.

(3s,5s,7s)-N-((Z)-(4'-Methoxy-5-(4-methoxybenzyl)-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)adamantan-1-amine Hydrochloride (123).

Yield: 137 mg (89%) yellow solid; ¹H NMR (CDCl_3, 600 MHz) δ 13.57 (br s, 1H), 10.68 (br s, 1H), 9.49 (d, J = 14.9 Hz, 1H), 7.41 (d, J = 15.5 Hz, 1H), 7.28 (d, J = 8.5 Hz, 2H), 6.86 (d, J = 8.5 Hz, 2H), 6.61 (t, J = 2.9 Hz, 1H), 5.96 (t, J = 2.9 Hz, 1H), 5.87 (d, J = 2.5 Hz, 1H), 3.98 (s, 2H), 3.91 (s, 3H), 3.78 (s, 3H), 2.22 (br s, 3H), 2.00 (d, J = 2.4 Hz, 6H), 1.76–1.70 (m, 6H); HRMS (ESI) calcd for C₂₈H₃₄N₃O₂ (M + H)⁺ 444.2646; found 444.2632.

(Z)-4-Chloro-N-((4'-methoxy-5-(4-methoxybenzyl)-1H,5'H-[2,2'-bipyrrol]-5'-ylidene)methyl)aniline Hydrochloride (124).

Yield: 138 mg (94%) brownish red solid; ¹H NMR (CDCl_3, 400 MHz) δ 13.85 (br s, 1H), 11.11 (d, J = 14.3 Hz, 1H), 10.65 (br s, 1H), 7.64 (d, J = 14.3 Hz, 1H), 7.37–7.28 (m, 6H), 6.89 (d, J = 8.5 Hz, 2H), 6.77 (d, J = 2.8 Hz, 1H), 6.05 (d, J = 2.8 Hz, 1H), 5.94 (br s, 1H), 4.03 (s, 2H), 3.78 (s, 3H), 3.80 (s, 3H); HRMS (ESI) calcd for C₂₄H₂₃ClN₃O₂ (M + H)⁺ 420.1473; found 420.1462.

BIOLOGICAL EXEPRIMENTS

Infection of J774A.1 Macrophages.

Macrophages (from ATCC) were grown in tissue culture flasks in DMEM plus 10% heat inactivated bovine fetal bovine serum until confluence. L. mexicana (strain MNYC/B2/62/M379, ATTC) expressing red-shifted firefly luciferase from an integrated ribosomal (28S) RNA locus⁴⁸ (see section below on mouse infections for details of strain construction) were inoculated into RPMI plus 10% heat inactivated fetal bovine serum (FBS) containing 5 μg/mL hemin and 0.1 mM xanthine at a density of 2 × 10⁵ cells/mL and grown at 26 °C for 8 days to stationary phase, ~5–6 × 10⁷ cells/mL. They were then examined microscopically for the presence of metacyclic promastigotes with long flagella and short cell bodies. The parasites were pelleted and resuspended in a volume of PBS that is sufficient to cover the macrophages. Macrophages were infected at a 10:1 ratio of parasites to macrophages by removing medium from the macrophages and adding the parasites in PBS. They were incubated for 10 min at 37 °C in a 5% CO₂ atmosphere. Minimal Essential Media (GIBCO) plus 10% heat inactivated FBS was added at half the volume of the parasite suspension in PBS, and the flask was incubated at 37 °C in 5% CO² atmosphere overnight (~16 h). The medium was removed from the infected macrophages and the monolayer was washed 3× with PBS at 37 °C to remove parasites that has not been internalized. Macrophages were then resuspended in a small volume of warm PBS using a cell scraper, counted, and adjusted to 5 × 10⁴ cells/mL with MEM 10% HI-FBS. A 200 μL aliquot of the cell suspension (1 × 10⁴ cells) was pipetted into each well of a 96-well plate and incubated for 3 h at 37 °C to reattach macrophages to the plates.

Dose-Response Curves for Test Compounds.

For measurement of sensitivity of intracellular amastigotes to each compound, 2 μL of compound at the appropriate concentration in DMSO was added to the well, and the opaque plate was incubated at 37 °C in 5% CO₂ for 96 h. Medium was removed from each well, and a 50 μL aliquot of 1:100 firefly luciferase substrate in assay buffer (Promega Firefly Luciferase Assay System) was added and the luminescence signal was read immediately in a Wallac Victor² 1420 Multilabel Counter, using the instrument luminescence program and 1 sec reads. Typically, compounds were applied in serial 3-fold dilutions to cover a range of 10 μM to 1 nM. Data were log transformed and EC₅₀ values were determined using GraphPad Prism 8 (GraphPad Software) and the sigmoidal dose-response with variable slope fitting.

The SYBR Green assay was used for the measurement of sensitivity macrophages. Uninfected macrophages were diluted to 5 × 10⁴ cells/mL in DMEM 10% HI-FBS. A 200 μL aliquot of the cell suspension (1 × 10⁴ cells) was pipetted into each well of a 96-well black plate (Greiner Bio-One, 655086) and incubated for 3 h at 37 °C to reattach macrophages to the plates. Then, compounds were added. After 96 h the medium was removed, the stock SYBR Green II (10,000×) was diluted to 1:1000× in 1% Triton in PBS, and 100 μL was added. Samples were incubated in the dark at room temperature for 1 h with moderate shaking. The plate was then read on the plate reader using Ex 485 nM, Em 535 using a 0.1 s exposure.

Animals and Ethical Statements (Oregon Health & Science University).

All animal work was carried out under the approved Institutional Animal Care and Use Committee protocol IBC-03-07 at the Oregon Health & Sciences University. The facility is accredited by AAALAC International, has a Public Health Service Animal Welfare Assurance, and complies with the Animal Welfare Act Regulations.

In Vivo Antileishmanial Efficacy in a Murine Model.

L. mexicana and L. donovani promastigotes expressing red-shifted firefly luciferase were prepared as follows. The 1647 bp red-shifted firefly luciferase gene PpyRE9H⁵⁹ was cloned into the Leishmania expression vector pRP-H⁶⁰ containing a neomycin resistance gene. Following linearization by SwaI restriction digestion, this construct, which contains an rRNA promoter and 28S rRNA sequence flanking the luciferase and neomycin resistance genes, was transfected into the parasites and selected with G418 to achieve integration into the 28S rRNA locus. For in vivo studies, the L. mexicana parasites were inoculated into RPMI plus 10% FBS plus 0.5 μg/mL hemin and 0.1 mM xanthine as above at a density of 2 × 10⁵ cells/mL. Parasites were grown at 26 °C for 8 days to stationary phase ~5–6 × 10⁷ cells/mL, and parasites were examined microscopically for the presence of metacyclics. Parasites were pelleted, washed twice with sterile PBS, and resuspended to a density of 5 × 10⁷ cells/mL in sterile PBS. Female BALB/c mice weighing about 20 g were injected in one hind footpad with 20 μL of resuspended parasites (1 × 10⁶ parasites). Footpad widths were measured with calipers weekly. At 20 days pi, mice were treated with the relevant compound by resuspending at the relevant dose in PEG-400 and delivering 100 μL by oral gavage. Compounds were delivered daily for 10 days. Lesion sizes were determined by subtracting the footpad width at day 19 (just before treatment) from the width at the subsequent time of measurement.

For in vivo imaging system (IVIS), mice infected with L. mexicana expressing red-shifted firefly luciferase were injected intraperitoneally with D-luciferin (PerkinElmer Health Science, Inc.) reconstituted at a concentration of 30 mg/mL in Dulbecco’s PBS without Mg²⁺ or Ca²⁺ and filtered using a 0.2 μm filter unit. The luciferin solution was warmed to 37 °C and injected at a dose of 300 mg/mL. Mice were anesthetized with 5% isoflurane and maintained in a sedated state with 2–5–3.5% isoflurane administered by nose cone. Images were taken 10 min after injection with luciferin using an IVIS Spectrum instrument (Revvity Inc., Small Animal Research Imaging Core, Oregon Health & Science University) with 1 min scans. Data settings included the following: F-stop 1, no emission filter, 22 cm field of view, CCD exposure time automatically determined by instrument for optimal signal, data binning 8. For data analysis, Living Image software version 4.7.3.20616 (Revvity, Inc.) was employed. Areas of interest surrounding the infected and uninfected hind footpads were quantified, and the luminescence units of the uninfected footpad were subtracted from those of the infected footpad to determine relative luminescence units for each mouse.

In Vitro Cytotoxicity Assessments.

To identify toxicity to mammalian cells, a dimethylthiazol-diphenyltetrazolium bromide (MTT) assay using human hepatocarcinoma cells (HepG2) was employed. HepG2 cells were cultured in complete minimal essential medium (MEM; Gibco-Invitrogen, No. 11090-099) prepared by supplementing MEM with 0.19% sodium bicarbonate (Gibco-BRL No. 25080-094), 10% heat-inactivated fetal bovine serum (FBS; Gibco-Invitrogen No. 16000-036), 2 mM L-glutamine (Gibco-Invitrogen, No. 25030-081), 0.1 mM MEM nonessential amino acids (Gibco-Invitrogen, No. 11140-050), 0.009 mg/mL insulin (Sigma No. I1882), and 1.76 mg/mL bovine serum albumin (Sigma No. A1470). Cell viability was assessed using trypan blue. 96-well plates were seeded with 2.5 × 10⁴ cells in 170 μL of culture medium per well and incubated at 37 °C overnight in a humidified 5% CO₂ atmosphere. Test compound plates were prepared with a Biomek 4000 automated laboratory station and 96-well plates containing 11 duplicate 1.6-fold serial dilutions of each test compound suspended in dimethyl sulfoxide (DMSO). A 30 μL sample of the diluted test compound was then added to 170 μL of medium per well. Cells were exposed to test compound concentrations ranging from 10 to 0.15 μg/mL for 48 h. After incubation, 30 μL of a 1.5 mg/mL solution of MTT diluted in complete MEM media was added to each well; all plates were subsequently incubated in the dark for 1 h at room temperature. Next, the media and test compounds in each well were removed and plates were then left to dry in a hood for 15 min. 60 μL of acidified isopropyl alcohol was added to dissolve the formazan dye crystals created by the reduction of MTT. Plates were placed on a three-dimensional (3-D) rotator for 15–30 min. Absorbance was determined with the Perkin Elmer EnSight plate reader. All experiments were run in triplicates, and the IC₅₀ values were determined using GraphPad Prism software using the nonlinear regression equation (sigmoidal dose–response, variable slope).

In Vitro Metabolic Stability.

The half-lives of lead TAs in mouse and human liver microsomes were determined, as described previously.^{50, 51} All samples were tested in human and mouse liver microsomal preparations. Sample stocks at 10 or 20 μM (depending on solubility) in DMSO were diluted to a final concentration of 1 μM with a mixture containing 0.5 mg/mL prewarmed pooled human or mouse liver microsomes (BD Gentest), 1.3 mM NADP (Sigma), 3.3 mM MgCl₂ (Sigma), and 100 mM potassium phosphate buffer (pH 7.4) using a TECAN Genesis robotic liquid handler. The reaction was started with the addition of 1 U/mL glucose-6-phosphate dehydrogenase (G6PD). The mixture was incubated on a shaking platform at 37 °C, and aliquots were taken and quenched with the addition of an equal volume of cold acetonitrile at 0, 10, 20, 30, and 60 minutes. Samples were centrifuged at 3700 rpm for 10 min at 20 °C to remove debris. Sample quantification was carried out by LC/MS, and the metabolic half-life was calculated by log plots of the total ion chromatography area remaining.

Animals and Ethical Statements (Walter Reed Army Institute of Research).

Male ICR-CD1 mice (Charles River Laboratories. Inc. Raleigh, NC) were used in this study. All animals were assigned a study number with an individual ear tag. Cards attached to each animal cage were also used to identify the study groups. All animals were quarantined for stabilization for 7 days prior to infection. Animals were housed in a designated room with food and water supplied ad libitum and in 12:12 light/dark cycle, in a room with temperature ranging from 64–79 °F, and 34–68% relative humidity.

Research was conducted under the approved Institutional Animal Care and Use Committee. The facility is accredited by AAALAC International, has a Public Health Service Animal Welfare Assurance, and complies with the Animal Welfare Act Regulations and other federal statutes relating to research animals.

In Vivo Pharmacokinetic (PK) Studies.

PK studies were conducted using the previously established methods.^{53, 54} Briefly, male ICR-CD1 mice weighing 23 to 28 g (Charles River Laboratories. Inc. Raleigh, NC) were used for the PK evaluations. Compound 110 was formulated in 50% PEG-400:50% sterile water and given orally using intragastric (IG) administration. A single 40 mg/kg dose of experimental compound 110 was administered to three mice in each experimental endpoint. At each time point, a terminal whole blood sample was collected by cardiac puncture using heparinized syringes (Heparin, Hospira, Lake Forest, IL). Following the separation of appropriate aliquots, plasma was obtained from the whole blood via centrifugation at 16,200 rpm for 10 minutes at 4 °C. Liver samples were also collected and weighed at each experimental time point. All tissue samples were immediately preserved on dry ice and later stored at −80 °C until analytical work was performed. Drug concentrations were determined for each sample taken from animals dosed with test compounds. PK parameters such as time to maximum plasma concentration (T_max), maximum plasma concentration (C_max), the elimination half-life (t_1/2), area under the curve (AUC), etc. were obtained using noncompartmental analysis via the Phoenix-WinNonlin software package (version 6.4; Pharsight Crop., Mountain View, CA).

AMES Assay.

Mutagenicity evaluation was assessed using the Ames assay^{55, 56} (EBPI, Ontario, Canada) against S. typhimurium TA100 and TA98, with and without S9 activation. Tester strain S. typhimurium (TA100 and TA98) cultures were inoculated from the lyophilized pellets and grown in nutrient broth provided by EBPI. Each inoculated flask (125- or 150-mL volume) was placed overnight in the incubator at 37 °C. The positive controls for the assay were 5 μg/mL 2-aminoanthracene for incubations with an S9 mix and a mixture of 1 μg/mL 4-nitroquinoline N-oxide and 2 μg/mL 2-nitrofluoroene for incubations without the S9 mix. The tester strains (2.5 mL) were mixed with exposure medium and, where appropriate, 2.0 mL of S9 to give a final volume of 18 mL. An aliquot (0.2 mL) of the mixture was then added to each well of a 96-well plate. The test compound (10 μL/well) was then added to each well. The 96-well plates were covered and sealed in zip-lock bags and incubated for 96 h at 37 °C. Plates were scored visually, and the positive wells against background mutations were recorded.

ABBREVIATIONS

EC₅₀

half maximal effective concentration

IC₅₀

half-maximal inhibitory concentration

HLM

human liver microsomes

MBC

4-methoxy-2,2'-bipyrrole-5-carboxaldehyde

MEM

minimal essential medium

MLM

mouse liver microsomes

PK

pharmacokinetics

PG

prodigiosin or prodiginine

SAR

structure-activity relationship

TA

tambjamine

TAs

tambjamines

TI

therapeutic index