Synthesis and Biological Evaluation of Botulinum Neurotoxin A Protease Inhibitors

Abstract

NSC 240898 was previously identified as a botulinum neurotoxin A light chain (BoNT/A LC) endopeptidase inhibitor by screening the National Cancer Institute Open Repository diversity set. Two types of analogs have been synthesized and shown to inhibit BoNT/A LC in a FRET-based enzyme assay, with confirmation in an HPLC-based assay. These two series of compounds have also been evaluated for inhibition of anthrax lethal factor (LF), an unrelated metalloprotease, to examine enzyme specificity of the BoNT/A LC inhibition. The most potent inhibitor against BoNT/A LC in these two series is compound 12 (IC₅₀ = 2.5 µM, FRET assay), which is 4.4-fold more potent than the lead structure, and 11.2-fold more selective for BoNT/A LC versus the anthrax LF metalloproteinase. Structure-activity relationship studies have revealed structural features important to potency and enzyme specificity.

Introduction

Botulinum neurotoxins (BoNTs ^a) secreted by strains of the anaerobic spore-forming bacterial species Clostridium botulinum are the most potent neurotoxins known and are categorized as Category A (highest priority) bioterrorist agents by the Centers for Disease Control and Prevention (CDC).^{1, 2} BoNTs represent a significant bioterrorist threat because they can be used as biological warfare agents in a highly toxic aerosol form or added to food.^2–5 Among the seven BoNT serotypes (A–G), BoNT serotype A (BoNT/A) is the deadliest and the most threatening with a lethal dose of 1.0 ng/kg in humans, and is accompanied by a prolonged half-life.^{2, 6} Structurally, BoNTs are composed of a 100 kDa-heavy chain (HC) and a 50 kDa-light chain (LC) linked by a disulfide bond. The LC is a zinc-dependent endopeptidase that catalyzes the cleavage of a component of the SNARE proteins (soluble N-ethylmaleimide-sensitive factor attachment protein receptor). BoNT serotypes A and E cleave SNAP-25 (synaptosomal-associated protein; 25 kDa); serotypes B, D, F, and G cleave VAMP (vesicle-associated membrane protein, also referred to as synaptobrevin); and serotype C cleaves both SNAP-25 and syntaxin 1.^7–9 BoNT-mediated cleavage of the SNARE proteins interrupts the function of motor nerves via the inhibition of acetylcholine release and produces flaccid paralysis and potential death of the infected patients. ⁶ Even though BoNT toxoid vaccine showed efficacy in the prevention of botulism poisoning, it is only available to consenting military personnel and research workers who are actively handling the toxins. Moreover, the vaccine cannot counter these toxins after they penetrate neurons. Currently, the only therapies for BoNT intoxication include experimental preventative antibodies and long-term supportive care (e.g., mechanical ventilation).^10–12 However, the antibodies can cause severe side effects, such as serum sickness and anaphylaxis, and mechanical ventilation is not practical when large populations are threatened by the toxins. New, small molecule therapeutic BoNT inhibitors, especially those that are active post-exposure (e.g., rescue therapeutics), are vital to the biodefense arsenal.

BoNT/A light chain (BoNT/A LC) has been widely studied as a drug target for the discovery and development of inhibitors of botulinum neurotoxins. Schmidt and Rich et al. ¹³ have developed peptide and peptidomimetic inhibitors of BoNT/A LC around the substrate SNAP-25 cleavage site with submicromolar to low micromolar K_i values. A series of 4-aminoquinolines have been identified, which prevent BoNT/A-induced SNAP-25 cleavage.¹⁴ Boldt et al.¹⁵ have reported that a D-cysteine derivative was a BoNT/A LC inhibitor that also demonstrated cellular activity. Cinnamic acid hydroxamate 1 (Figure 1) was shown to inhibit BoNT/A LC with a K_i value of 0.3 µM and an IC₅₀ value of 0.41 µM.¹⁶ Moe et al.¹⁷ described a series of mercaptoacetamide inhibitors (example 2, Figure 1) active against botulinum neurotoxin A with low micromolar potency. Recently, a quinolinol derivative 3 (Figure 1) has been reported as a BoNT/A LC inhibitor with an IC₅₀ value of 0.5 µM in a tissue-based ex vivo assay¹⁸ and this type of compound inhibits BoNT/A LC in a non-competitive manner.¹⁹ A tetrasubstituted pyrrole inhibitor (compound 4, Figure 1), created by synthesis-based computer-aided molecular design, displayed BoNT/A inhibition with a K_i value of 0.76 ± 0.17 µM and an IC₅₀ value of < 1 µM.²⁰ Burnett et al.²¹ have designed and synthesized a hybrid inhibitor using a three-zone pharmacophore model to link an indole bis-amidine structure²² with a 4-amino-7-chloroquinoline,²³ which exhibits a K_i value of 0.6 µM vs. BoNT/A LC. A series of benzylidene cyclopentenedione-based inhibitors were shown to inactivate BoNT/A LC through covalent modification of the enzyme in a biochemical assay.²⁴

Figure 1.

Small molecule inhibitors of BoNT/A LC.

Indole bis-amidine 5 (NSC 240898, Figure 1) is a potent BoNT/A LC endopeptidase inhibitor identified from screening a collection of the National Cancer Institute’s Open Repository diversity set, using a high throughput fluorescence resonance energy transfer (FRET) assay combined with a molecular modeling approach. The activity of this lead compound has been verified using an HPLC-based confirmatory assay.²² Compound 5 exhibited 75% inhibition of the BoNT/A LC endopeptidase at 20 µM, and no cytotoxicity was detected at concentrations as high as 40 µM. It was also found to be neuron-permeable. These findings show that 5 is a promising lead compound for further optimization. During our ongoing research, Wang et al. ²⁵ reported the synthesis and preliminary structure-activity relationship studies based on compound 5; however, no significant improvement in potency was reported by these authors. Here we report the syntheses of two types of analogs of compound 5 and their inhibitory activities against BoNT/A LC and anthrax LF metalloprotease in FRET-based enzyme assays.

Results and Discussion

Chemistry

Compound 5 is a bis-amidine comprised of an indole core structure with a phenoxyphenyl moiety at the 2-position. To improve the in vitro activity against BoNT/A LC, we designed the two types of analogs shown in Figure 2. Type I inhibitors consist of an indole, benzothiophene or benzimidazole core structure, and bear a 4-(phenoxy)aryl group at the 2-position. The heteroatom on the heterocyclic core was proposed to either interact directly with the active site Zn or to hydrogen-bond with catalytic water molecules.^{26, 27} Type II inhibitors also have an indole or benzothiophene core structure with an aryl group at the 2-position.

Figure 2.

Two types of analogs of BoNT/A LC inhibitor 5.

Type I analogs 10–15 were prepared from dinitrile 9, which was produced via a Cadogan-Sundberg indole synthesis (Scheme 1). 4-Hydroxybenzaldehyde (6) was treated with 4-fluorobenzonitrile to give aldehyde 7, which was then condensed with 4-methyl-3-nitrobenzonitrile to give 8. Stilbene 8 underwent a Cadogan-Sundberg reaction to afford dinitrile 9 in 55% yield. Treatment of dintrile 9 with 4:1 (v/v) TFA/H₂SO₄ gave diamide 10. Interestingly, a mono-amidine product 11 was isolated exclusively when dinitrile 9 was treated with the amidination agent LiN(TMS)₂.

Scheme 1.

Synthesis of Type I analogs 10–15.

The imidate intermediate obtained from the Pinner reaction with dinitrile 9 was treated with N,N-dimethylethylene-diamine to produce the N,N-dimethylethylene-substituted bis-amidine compound 12, which was isolated by HPLC purification. The dinitrile 9 was also treated with diamines in the presence of P₂S₅ to give bis-amidines 13–15.

Dinitriles 23a–c were prepared by using a Suzuki coupling protocol with boronic acids 20a–b and phenoxyarylbromides 19a–c, which were obtained by condensation of compounds 17a–b with arylfluorides 18a–b. The coupling products were further elaborated to Type I analogs 24a–c and 25a–b (Scheme 2). Compounds 24a–b and 25a–b were bis-amidine compounds and 24c was isolated as a mono-imidazoline compound after HPLC purification.

Scheme 2.

Synthesis of Type I analogs 24a–c and 25a–b.

Type I analogs 29 and 32 contain only one imidazoline group on either the indole ring or the phenyl ring. Syntheses of 29 and 32 are shown in Schemes 3 and 4, respectively. Nitrile intermediates 28 and 31 were prepared by Suzuki coupling of corresponding boronic acids 20a and 30 with the 4-phenoxyphenylbromides 27 and 19a, respectively, followed by treatment with ethylenediamine in the presence of P₂S₅ to give imidazolines 29 and 32.

Scheme 3.

Synthesis of Type I analog 29.

Scheme 4.

Synthesis of Type I analog 32.

Syntheses of Type I analogs 35a–b with a benzimidazole core structure are shown in Scheme 5. Oxidative condensation of 4-cyanophenylenediamine (33) with aldehyde 7 provided dinitrile 34, which was then converted to bis-imidazoline 35a and bis-tetrahydropyrimidine 35b using a P₂S₅-mediated cyclization reaction.

Scheme 5.

Synthesis of Type I analogs 35a–35b.

Syntheses of Type II inhibitors 40a–c, 43a–c and 44a–b with indole core structures are shown in Schemes 6 and 7. Condensation of 4-cyano-2-nitrotoluene (36) with 4-fluorobenzaldehyde (37) gave ortho-nitrostilbene 38, which underwent a Cadogan-Sundberg indole synthesis to provide nitrile 39 as the major product. Subsequently, amide 40a, amidine 40b and imidazoline 45c were prepared using the methods previously described (Scheme 6). Suzuki coupling of boronic acid 20a with aryl bromides 41a–b provided 2-aryl-6-cyanoindoles 42a–b, which were then transformed to Type II inhibitors 43a–c and 44a–b using reaction conditions corresponding to those described in Scheme 6 (Scheme 7).

Scheme 6.

Synthesis of Type II analogs 40a–c.

Scheme 7.

Synthesis of Type II inhibitors 43a–c and 44a–b.

Syntheses of Type II inhibitors 46a–b, 49 and 50 containing a benzothiophene core structure are shown in Schemes 8 and 9. Suzuki coupling of 6-cyanobenzothiophene-2-boronic acid (20b) with 4-bromobenzonitrile (41b) provided dinitrile 45, which was then converted to bis-imidazoline 46a and bis-tetrahydropyrimidine 46b (Scheme 8). Likewise, Suzuki coupling of boronic acid 20b with 2-bromo-5-fluoropyridine (47) provided nitrile 48. Treatment of 48 with H₂SO₄/TFA (4:1) and ethylenediamine gave amide 49 and imidazoline 50, respectively (Scheme 9).

Scheme 8.

Synthesis of Type II inhibitors 46.

Scheme 9.

Synthesis of Type II inhibitors 49 and 50.

Biological evaluation of Type I and Type II inhibitors

The Type I and Type II compounds were evaluated in a fluorescence resonance energy transfer (FRET) assay²⁸ against recombinant BoNT/A LC, and the inhibitory activities are shown in Table 1 and Table 2. FRET data for the most potent compounds were confirmed with a secondary HPLC-based assay (data shown in parentheses in Table 1 and Table 2). To examine enzyme specificity, compounds were also evaluated in a FRET-based anthrax lethal factor (LF) assay using a previously reported procedure.^{28, 29} Anthrax LF, a component of the anthrax tripartite exotoxin, is also a zinc-containing metalloprotease, but it has no significant sequence similarity to BoNT/A LC. Compounds exhibiting specificity for BoNT/A LC were further examined in a larger panel of metalloprotease assays.³⁰

Table 1.

Inhibitory activities of Type I analogs against BoNT/A LC and LF enzymes.

Compd. No.	R1	R2	R3	X	Y	Z	BoNT/A IC50 (µM)a,c	bLF IC50 (µM)b
5	C(=NH)NH2	C(=NH)NH2	H	NH	CH	CH	11 (17)	35
9	CN	CN	H	NH	CH	CH	>100	n.d.d
10	CONH2	CONH2	H	NH	CH	CH	>100	>100
11	C(=NH)NH2	CN	H	NH	CH	CH	>25	>100
12			H	NH	CH	CH	2.5 (4.4)	28
13			H	NH	CH	CH	12.5 (9.4)	43
14			H	NH	CH	CH	21 (43)	>100
15			H	NH	CH	CH	7.3	>100
23a	CN	CN	H	NH	CH	N	>100	>100
23b	CN	CN	Cl	NH	CH	CH	>100	n.d.d
24a			H	NH	CH	N	69 (>100)	55
24b			H	NH	CH	N	67 (>100)	>100
24c		CN	Cl	NH	CH	CH	30	>100
25a			H	S	CH	CH	7.1 (25)	18
25b			H	S	CH	CH	54	>100
29		Cl	Cl	NH	CH	CH	>25 (52)	>100
32	Cl		H	NH	CH	CH	>100	51
35a			H	NH	N	CH	24.5	n.d.d
35b			H	NH	N	CH	28 (41)	n.d.

^aData represent an average value of two experiments in a FRET-based assay with variation less than 10%;

^bdata obtained by a FRET-based assay;

^cdata (in parentheses) represent average value of two experiments in a HPLC-based assay with variation less than 10%;

^dcompound shows strong fluorescence, so an accurate value was not determined.

Table 2.

Inhibitory activities of Type II analogs against BoNT/A LC and LF enzymes.

Compd. No.	R1	R2	X	Y	BoNT/A IC50 (µM)a,c	LF IC50 (µM)b
39	CN	F	NH	CH	>100	>100
40a	CONH2	F	NH	CH	>100	>100
40b	C(=NH)NH2	F	NH	CH	>100	48
40c		F	NH	CH	>100	40
43a	CONH2	OCH3	NH	CH	>100	>100
43b	C(=NH)NH2	OCH3	NH	CH	>100	>100
43c		OCH3	NH	CH	45	61
44a			NH	CH	20	49
44b			NH	CH	>100	>100
46a			S	CH	43 (28)	>100
46b			S	CH	>100	n.d.d
49	CONH2	F	S	N	>100	>100
50			S	N	56	>100

^aData represent an average value of two experiments in a FRET-based assay with variation less than 10%;

^bdata obtained by a FRET-based assay;

^cdata (in parentheses) represent average value of two experiments in a HPLC-based assay with variation less than 10%;

^dcompound shows strong fluorescence, so an accurate value was not determined.

Generally, neutral groups such as nitriles (9, 23a–b, and 39) or amides (10, 40a, 43a, and 49), as substituents on the indole or benzothiophene core structures, diminished the potency against both BoNT/A LC and anthrax LF. Basic groups on the core structures are necessary for botulinum tox in inhibitory activity, and compounds with basic groups on both ends of the scaffold are generally more potent than compounds with one basic group on the scaffold. In the BoNT/A LC assay, for example, bis-amidine 2 exhibited an IC₅₀ value of 11 µM vs. BoNT/A LC, while mono-amidine 11 showed no activity against BoNT/A LC. Mono-imidazoline 24c (BoNT/A LC IC₅₀ = 30 µM) is 2.4-fold less potent than bis-imidazoline 13 (IC₅₀ = 12.5 µM), suggesting that both basic groups may participate in hydrogen bonding or ionic interactions with the target enzyme.²⁵ Type I inhibitors with three-ring scaffolds exhibited more potency than Type II inhibitors with two-ring scaffolds, when bearing the same substituents. For example, Type I inhibitors 13, 14, 25a and 25b with three-ring scaffolds bearing imidazoline or tetrahydropyrimidine moieties were more potent against botulinum neurotoxin than their counterpart Type II inhibitors with two-ring scaffolds, e.g., compounds 44a, 44b, 46a and 46b.

Type I inhibitors with the benzimidazole core structure (35a and 35b) exhibited less potency than Type I inhibitors with indole core structures (13 and 14), which is consistent with an earlier report.²⁵ The more basic benzimidazole core structure may be disruptive to the complementary hydrophobic contact of the indole ring with BoNT/A LC. Additional heteroatoms in the indole ring, such as N and S, may interact with the Zn ion in the BoNT/A LC active site or interact with the catalytic site water molecules. However, replacement of a CH unit in the middle ring of the 2-((phenoxy)phenyl)indole scaffold with nitrogen dramatically decreases the inhibitory activity against BoNT/A LC (for example, 13 vs 24a and 14 vs 24b). The most potent BoNT/A LC inhibitor in the enzyme-based assay is compound 12, which exhibited an IC₅₀ value of 2.5 µM, which is 4.4-fold more potent than the lead compound 4 and 3.6-fold more potent than the cinnamic acid hydroxamate 1 (IC₅₀ = 8.9 µM, FRET-based assay). Compound 1 has been used as a positive control in our assay. The IC₅₀ value reported here for compound 1 is higher than the original reported value (IC₅₀ = 0.41 µM),¹⁶ and similar differences have also been observed by other laboratories.^{17, 22} The difference between these values is probably due to the use of different forms of the SNAP-25 substrate or different concentrations of enzyme. Assay conditions (temperature and incubation time) may also be partly or wholly responsible for these discrepant values.

Almost all of the active Type I inhibitors are more selective for BoNT/A LC, as indicated by poorer anthrax LF inhibitory activity. Compounds 12 and 15 were the most selective BoNT/A LC inhibitors, demonstrating over ten-fold selectivity versus the LF. The most potent Type II inhibitor, compound 44a, also exhibited more than five-fold selectivity for BoNT/A LC versus anthrax LF. Moreover, no inhibitory activities were observed for compounds 12 and 44a against metalloproteases BoNT/B, human MMP-1 and MMP-9 (data not shown), further demonstrating the high specificity of these compounds for BoNT/A LC.³⁰

Molecular Modeling

Several X-ray structures of BoNT/A LC complexed with small peptides or small molecules are now available, and they have revealed an unexpected conformational flexibility in the enzyme active site.^{26, 27, 31, 32} To better understand the binding mode and rationalize the improved potency of the 2-(phenoxyphenyl)indole series of compounds (Type I derivatives), we performed a molecular modeling study with compound 12 and BoNT/A LC. Realizing that all of the four basic moieties of compound 12 may be ionized at physiological pH, and that combinations of ionized species can exist in the aqueous physiological solution, four ionized states (mono-, di-, tri- and tetra-ionized states) of compound 12 were first generated by the Schrödinger Epik program. These states were then docked into the BoNT/A LC active site along with the free base form. The +4 positively charged state showed the highest docking score. To better account for the protein flexibility, the best conformations of each ionized state of the inhibitor were energy minimized in the bound state, with no restriction of the protein structure within 8Å from the bound ligand. Further relative free binding energy calculations suggested that the +4 positively charged state is predominantly populated in the binding complexes in aqueous solution. The lowest energy binding mode of the +4 positively charged state is shown in Fig. 3. The two protonated amidine moieties form hydrogen bonds or ionic interactions with Asp370 and Pro239. One of the amidine groups is hydrogen-bonded with a structural water molecule, and one of the protonated N,N-dimethylamino moieties is buried in a hydrophilic site surrounded by Thr214, Glu351, Asn362 and Arg363 The other N,N-dimethylamino moiety points toward the solvent accessible region, which may produce a favorable solvation binding energy. Moreover, the indole NH is hydrogen-bonded with the main chain carbonyl oxygen atom of Glu257, which further enhances the ligand binding affinity. Compared to the proposed binding mode of NSC-240898 reported earlier, ^{20, 21} the overall more favorable binding mode of compound 12 in the BoNT/A LC active site may provide a molecular basis for the improvement of potency.

Figure 3.

Proposed binding mode for Type I inhibitor 12 (shown in a green stick model). Oxygen atoms are red, nitrogen atoms are blue, hydrogen atoms are white and Zn is cyan. BoNT/A LC is displayed as a ribbon structure.

Interestingly, compound 12 apparently does not directly coordinate with the Zn ion, which is consistent with the finding that the inhibitory activity of compound 12 is independent of the Zn concentration (data not shown). Replacing the indole core of compounds 13 and 14 with the similarly hydrophobic benzothiophene core, e.g., compounds 25a and 25b, improves the inhibitory activity for BoNT/A LC. However, replacement of the indole core with the more basic and hydrophilic benzimidazole core, e.g., compounds 35a and 35b, gives lower affinity compounds for BoNT/A LC. Additionally, replacement of the central phenyl ring with a basic pyridyl ring also leads to decreased inhibitory activity for the BoNT/A LC, e.g., for compounds 24a and 24b. The proposed binding mode also suggests that small substituents on the scaffold may be tolerated. Compounds 24c and 29, which contain substituents on the phenoxy ring, both maintain inhibitory activity for BoNT/A LC.

Conclusion

To optimize the potency of the lead compound 5, we have synthesized two series of compounds with three-ring or two-ring scaffolds using various synthetic methods. The biological activities of the two series of compounds have been evaluated in FRET-based BoNT/A LC and anthrax LF assays. Structure-activity relationship studies have demonstrated that basic groups on both ends of the scaffolds are necessary for potency, and that additional hydrogen-bonding or ionic interactions may be incorporated by adding polar or basic substitutions to the amidine moieties. Hydrophobic cores, e.g., indole or benzothiophene cores, are required for interaction with the enzyme active site, whereas polar atom replacement in the phenyl linker may alter the electronic environment to ultimately decrease potency. The SAR that we have identified will help to further optimize existing leads and allow the design of new small molecule BoNT/A LC inhibitors. The optimization effort has led to the identification of a potent BoNT/A LC inhibitor 12, which displays an IC₅₀ value of 2.5 µM in the BoNT/A LC assay, is 4.4-fold more potent than the original lead compound 5 and three-fold more potent than the cinnamic acid hydroxamate 1 (IC₅₀ = 8.9 µM, FRET assay). Moreover, compound 12 also showed cellular activity in a chick neuronal assay.³⁰ Compound 12 is highly specific, is ten-fold more selective over the metalloprotease anthrax LF, and does not inhibit BoNT/B LC, human metalloprotease MMP-1 or MMP-9.²⁶ In summary, we have discovered a potent, highly specific lead candidate which may be suitable for further development as a therapeutic agent for BoNT/A LC, which is a bioterrorist threat of increasing importance.

Experimental Section

General Procedures

All commercially obtained solvents and reagents were used as received. Melting points were determined in open capillary tubes with an EZ-Melt (Stanford Research Systems) apparatus and are uncorrected. ¹H NMR spectra were determined on a Bruker 300 MHz instrument. Chemical shifts are given in δ values referenced to the internal standard tetramethylsilane. LC-MS analyses were performed by CreaGen Biosciences, Inc. (Woburn, MA) using a Shimadzu LC-10 AD VP HPLC, with Waters micromass quattro ultima triple-quad MS. High resolution mass spectra were recorded on an Agilent LC/MSD TOF high accuracy instrument at Scripps Research Institute. Elemental analyses were performed by Columbia Analytical Services. All of the compounds tested in vitro showed >95% purity by LC-MS except compounds 23b (93%) and 24c (90%).

4-(4-Formylphenoxy)benzonitrile (7)

4-Hydroxybenzaldehyde (6, 5.04 g, 41.3 mmol), 4-fluorobenzonitrile (5.0 g, 41.3 mmol) and K₂CO₃ (5.8 g, 42 mmol) were mixed in DMF (35 mL) and heated to 150–160 °C using a heating mantle. After 3 h the reaction mixture was cooled, and 2N NaOH solution (150 mL) was added followed by ice water (200 mL). The product precipitated, and was collected by filtration and dried to give a brown solid (8.0 g, 87% yield). R_f = 0.30 (3:1 hexane/EtOAc), mp = 94–95 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 9.91 (s, 1H), 7.92 (d, J = 8.4 Hz, 4 H), 7.85 (d, J = 8.5 Hz, 4H), 7.22 (d, J = 3.7 Hz, 4H), 7.19 (d, J = 3.6 Hz, 4H).

(E)-1-(4-cyano-2-nitrophenyl)-2-(4-(4-cyanophenoxy)phenyl)ethene (8)

4-Methyl-3-nitrobenzonitrile (5.08 g, 31.4 mmol) and 4-(4-formylphenoxy)benzonitrile (7.0 g, 31.4 mmol) were heated together to 150 °C until the compounds melted. Sulfolane (8 mL) and piperidine (1.5 mL) were added and the resulting solution was stirred at 150 °C for 4 h, and cooled to room temperature to yield a yellow solid (7.74 g, 67% yield after collection and drying. R_f = 0.15 (4:1 hexane/EtOAc), mp = 193–194 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 8.55 (s, 1H), 8.20 (s, 2H), 7.87 (d, J = 8.7 Hz, 2H), 7.75 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 16.0 Hz, 1H), 7.44 (d, J = 16.0 Hz, 1H), 7.21 (d, J = 9.8 Hz, 2H), 7.18 (d, J = 9.0 Hz, 2H).

2-(4-(4-Cyanophenoxy)phenyl)indole-6-carbonitrile (9)

Compound 8 (5.0 g, 13.6 mmol) was suspended in triethyl phosphite (30 mL), and heated to reflux using a heating mantle for 1 h. The solution was cooled to room temperature and excess triethyl phosphite was removed by distillation under vacuum. The residue was purified by flash chromatography using 25% ethyl acetate in hexane to yield a light yellow solid 9 (2.5 g, 55% yield): R_f = 0.21 (3:1 hexane/EtOAc), mp = 254–256 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 12.17 (s, 1H), 8.02 (d, J = 8.6 Hz, 2H), 7.88 (d, J = 8.5 Hz, 2H), 7.84 (s, 1H), 7.71 (d, J = 8.2 Hz, 1H), 7.35 (dd, J = 0.7, 8.3 Hz, 1H), 7.30 (d, J = 8.6 Hz, 2H), 7.20 (d, J = 8.5 Hz, 2H), 7.08 (s, 1H).

2-(4-(4-Carbamoylphenoxy)phenyl)-1H-indole-6-carboxamide (10)

Compound 9 (130 mg, 0.39 mmol) was dissolved in CH₂Cl₂/hexanes (16 ml, 1:1) and conc H₂SO₄ (8 mL) was added at 0 °C. The resulting solution was stirred for 24 h and poured onto ice (20 g). EtOAc (10 mL) was added, and the precipitates were collected and washed with H₂O to obtain a light yellow solid 5 (119 mg, 83% yield). R_f = 0.11 (1:9 MeOH/CHCl₃), mp = 170–171 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 11.78 (s, 1H), 7.97-7.92 (m, 7H), 7.53 (m, 2H), 7.32 (s, 1H), 7.20 (d, J = 8.5 Hz, 2H), 7.19 (s, 1H), 7.10 (d, 8.5 Hz, 2H), 6.93 (s, 1H). Anal. Calcd for C₂₂H₁₇N₃O₃·0.8H₂O: C, 68.49; H, 4.86; N, 10.89. Found: C, 68.52; H; 4.68; N, 10.84.

2-(4-(4-Cyanophenoxy)phenyl)-1H-indole-6-carboximidamide hydrochloride salt (11)

Compound 9 (200 mg, 0.60 mmol) was dissolved in THF (10 mL) at room temperature, and LiN(TMS)₂ (1.0 g, 6.0 mmol) was added. The resulting solution was stirred overnight, after which time TLC showed the disappearance of the starting material. The reaction solution was poured over ice, and the precipitated solid was washed with cold water. The crude product was dissolved in a minimum amount of MeOH and treated with 2M HCl in diethyl ether, and the diamidine HCl salt was precipitated, filtered and dried to give compound 11 (176 mg, 83%). R_f = 0.11 (80:18:2 CHCl₃/CH₃OH/CH₃NH₂), mp = 198–199 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 7.98 (d, J = 8.8 Hz, 2H), 7.84 (d, J = 8.5 Hz, 2H), 8.31 (s, 1H), 7.69 (d, J = 8.3 Hz, 1H), 7.32 (dd, J = 1.4, 8.2 Hz, 1H), 7.18 (d, J = 8.8 Hz, 2H), 7.09 (d, J = 8.8 Hz, 2H), 7.03 (s, 1H). HRMS (ESI-TOF) m/z calcd for C₂₂H₁₇N₄O 353.1402 (M+H)⁺, found 353.1401.

N'-(2-(Dimethylamino)ethyl)-2-(4-(4-(N'-2-(dimethylamino)ethyl)carbamimidoyl)phenoxy)phenyl)-1H-indole-6-carboximidamide (12)

Compound 9 (200 mg, 0.6 mmol) was gently stirred in anhydrous ethanol (50 ML) at 0 °C and HCl gas was purged into the solution until saturation occurred. The resulting solution was stirred at room temperature for 2 days (reaction bottle was capped tightly) and the solvent and HCl were evaporated to dryness. To the residue dissolved in anhydrous ethanol (20 mL) was added N,N-dimethylethylenediamine (526 mg, 6.0 mmol) and the resulting solution was stirred at room temperature for 24 h. The reaction mixture was evaporated at 35 °C and dried at room temperature under high vacuum for 24 h to give a crude product, which was purified by reverse phase HPLC on a C-18 column using a 21-minute method with a gradient of 10–60% acetonitrile-H₂O to give the title compound (143 mg, yield 25%). Mp = 92–93 °C; ¹H NMR (DMSO-d₆) δ 12.29 (s, 1H), 10.25 (br, 2H), 9.81 (br, 1H), 9.72-9.66 (m, 3H), 9.28 (s, 1H), 9.14 (s, 1H), 8.06 (d, J = 8.7 Hz, 2H), 7.89 (s, 1H), 7.86 (d, J = 1.8 Hz, 2H), 7.75 (d, J = 8.4 Hz, 1H), 7.43 (dd, J = 9. 1.2 Hz, 4H), 7.08 (s, 1H), 3.83 (br, 4H), 3.45 (d, J = 6.3 Hz, 4H), 2.91 (s, 6H), 2.89 (s, 6H). LC-MS (+ESI): m/z 512.5 (M+H)⁺; HPLC T_R = 10.01 min. Anal. Calcd for C₃₀H₃₇N₇O·4TFA·H₂O: C, 46.30; H, 4.40; N, 9.95. Found: C, 46.06; H, 4.40; N, 9.68.

6-(4,5-Dihydro-1H-imidazol-2-yl)-2-(4-(4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy)phenyl)-1H-indole (13)

Compound 9 (153 mg, 0.46 mmol) and P₂S₅ (55.6 mg, 0.25 mmol) were dissolved in ethylenediamine (3 mL) in a sealed tube, and the tube was heated to 120 °C in an oil bath for 2 h. The tube was cooled to room temperature, and the green solution was poured into water (90 mL). After 15 min the solids were collected and rinsed with cold water to give 13 as an off-white solid (195 mg, 93% yield). R_f = 0.09 (80:18:2 CHCl₃/CH₃OH/CH₃NH₂), mp = 290–291 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 11.75, 7.94 (d, J = 7.1 Hz, 2H), 7.87 (s, 1H), 7.86 (d, J =7.1 Hz, 1H), 7.85 (d, J = 8.8 Hz, 2H), 7.53 (s, 2H), 7.17 (d, J = 8.8 Hz, 2H), 7.10 (d, J = 6.9 Hz, 1H), 7.09 (d, J = 8.8 Hz, 2H), 6.90 (s, 1H), 3.63 (s, 4H), 3.60 (s, 4H). HRMS (ESI-TOF) m/z calcd for C₂₆H₂₄N₅O 422.1981 (M+H)⁺, found 422.1979.

6-(1,4,5,6-Tetrahydropyrimidin-2-yl)-2-(4-(4-(1,4,5,6-tetrahydropyrimidin-2-yl)phenoxy)phenyl)-1H-indole (14)

Compound 9 (400 mg, 1.20 mmol) and P₂S₅ (133 mg, 0.59 mmol) were dissolved in 1,3-diaminopropane (15 mL) in a sealed tube. The tube was heated to 120 °C in an oil bath for 2 h. After cooling to room temperature, the green solution was poured into water (100 mL) and after 15 min the solids were collected and rinsed with cold water to give 14 as a white solid (526 mg, 98 % yield). Mp = 183–184 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 8.03 (d, J = 8.7 Hz, 2H), 7.85 (s, 1H), 7.81 (d, J = 8.7 Hz, 2H), 7.67 (d, J = 8.4 Hz, 1H), 7.36 (dd, J = 1.2, 9.0 Hz, 1H), 7.18 (d, J = 8.7 Hz, 2H), 7.1 (d, J = 8.7 Hz, 2H), 6.99 (s, 1H), 3.49 (t, J = 5.4 Hz, 4H), 3.39 (t, J = 5.7Hz, 4H), 1.95 (t, J = 4.2Hz, 2H), 1.78 (t, J = 5.1 Hz, 2H). HRMS (ESI-TOF) m/z calcd for C₂₈H₂₈N₅O 450.2294 (M+H)⁺, found 450.2281.

2-(4-(4-(6-(5-Hydroxy-1,4,5,6-tetrahydropyrimidin-2-yl)-1H-indol-2-yl)phenoxy)phenyl)-1,4,5,6-tetrahydropyrimidin-5-ol (15)

Compound 9 (200 mg, 0.6 mmol), phosphorus pentasulfide (66 mg, 0.3 mmol) and 2-hydroxy-1,3-diaminopropane (2 g) were stirred at 120 °C under nitrogen in a sealed tube for 2 h. The reaction mixture was cooled to room temperature and poured into excess water, stirred for 15 min and the resulting grey precipitate was collected by filtration and air-dried to give 15 (241 mg, 84%). Mp = 223–224 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 12.64 (br, 1H), 8.06 (d, J = 8.1 Hz, 2H), 7.89 (s, 1H), 7.84 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 8.1 Hz, 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.16 (dd, J = 8.7, 21 Hz, 4H), 7.00 ( s, 1H), 4.20 (s, 1H), 3.99 (s, 1H), 3.57-3.35 (m, 7H), 3.23 (dd, J = 4.5, 12 Hz, 3H). Anal. Calcd for C₂₈H₂₇N₅O₃·2CF₃COOH·1.5H₂O: C, 52.18; H, 4.38; N, 9.51. Found: C, 52.10; H; 4.19; N, 9.57.

4-(4-Bromophenoxy)benzonitrile (19a)

4-Bromophenol (17a, 4.0 g, 23.1 mmol) and 4-fluorobenzonitrile (18a, 2.8 g, 23.1 mmol) were heated together to 150 °C until compound melted. Piperidine (1.0 mL) was added and the resulting solution was stirred at 150 °C for 4 h and cooled to room temperature. The precipitate was collected by filtration and washed with water to yield a red solid (5.2 g, 82% yield). R_f = 0.54 (1:9 EtOAc/Hexane), mp = 78–79 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 7.86 (d, J = 8.7 Hz, 2H), 7.65 (d, J = 8.7 Hz, 2H), 7.16-7.11 (m, 4H).

2-Bromo-5-(4-cyanophenoxy)-pyridine (19b)

Compound 19b was prepared in the same fashion as was 19a, and was obtained as white solid. Mp = 118–119 °C; ¹H NMR (300 MHz, CDCl₃) δ 8.22 (d,1H, J = 3 Hz), 7.67 (ddd, J = 2.4, 4.5, 14.1 Hz, 2H), 7.53 (d, J = 8.7 Hz, 1H), 7.28 (dd, J = 3, 8.7 Hz, 1H), 7.06 (ddd, J = 2.7, 4.8, 11.7 Hz, 2H).

1-Bromo-4-(2-chloro-4-cyanophenoxy)benzene (19c)

Compound 19c prepared in the same fashion as was 19a, and was obtained as a white solid. Mp = 161–162 °C; ¹H NMR (300 MHz, CDCl₃) δ 7.95 (d, J = 2.4 Hz, 1H), 7.66 (dd, J = 2.1, 7.5 Hz, 1H), 7.49 (ddd, J = 3.3, 5.4, 12.3 Hz, 2H), 6.95 (d, J = 8.7 Hz, 1H), 6.89 (ddd, J = 3.3, 5.7, 12.3 Hz, 2H).

General synthesis of 23a–c

A mixture of corresponding boronic acid 20 (1.0 g, 1 equiv), compound 19 (1 equiv), tetrakistriphenylphosphinepalladium (0.1 equiv), and sodium carbonate (2 equiv) in 10% ethanol in toluene (50 mL) was heated at reflux under argon for 4 h. The reaction mixture was cooled to room temperature and evaporated to dryness. The residue was dissolved in ethyl acetate, washed thrice with water, followed by brine, dried over magnesium sulfate and evaporated to give the crude product. Recrystallization from toluene gave compounds 22a, 22b and 23c, respectively. Compound 22a or 22b (0.5 g, 1 equiv) was stirred in a mixture of trifluoroacetic acid (10 mL) and 4N HCl in dioxane (2 mL) at room temperature for 1 h. The solvent was evaporated. The residue was dissolved in ethyl acetate, washed with potassium carbonate solution, water and brine, dried over magnesium sulfate and the solvent was evaporated. The crude product was recrystallized to give 23a or 23b.

2-(5-(4-Cyanophenoxy)pyridin-2-yl)-1H-indole-6-carbonitrile (23a)

Compound 23a was obtained as a white solid. R_f = 0.20 (3:1 hexane:EtOAc), mp = 239–240 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 12.25 (s, 1H), 8.59 (d, J = 2.7 Hz, 1H), 8.19 (d, J = 8.7 Hz, 1H), 7.91 (d, J = 1.8 Hz, 1H), 7.90 (d, J = 2.4 Hz, 2H), 7.77 (dd, J = 3.0, 11.4 Hz, 2H), 7.35 (dd, J = 1.2, 8.1, 1H), 7.28 (dd, J = 4.5, 6.3 Hz, 2H), 7.26 (s, 1H). HRMS (ESI-TOF) m/z calcd for C₂₁H₁₃N₄O 337.1089 (M+H)⁺, found 337.1091.

2-(4-(2-Chloro-4-cyanophenoxy)phenyl)-1H-indole-6-carbonitrile (23b)

Compound 23b was obtained as a white solid. Mp = 123–125 °C, ¹H NMR (300 MHz, DMSO-d₆) δ 12.14 (br, 1H), 8.13 (d, J = 2.1 Hz, 1H), 7.97 (d, J = 8.7 Hz, 2H), 7.89 (dd, J = 2.1, 6 Hz, 1H), 7.82 (s, 1H), 7.70 (d, J = 8.1 Hz, 1H), 7.34 (dd, J = 1.5, 6 Hz, 1H), 7.21-7.16 (m, 3H), 7.04 (s, 1H).

2-(4-(4-Cyanophenoxy)phenyl)benzo[b]thiophene-6-carbonitrile (23c)

Compound 23c was obtained as a white solid. Mp = 219–220 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 8.61 (d, J = 0.6 Hz, 1H), 8.01 (d, J = 7.5 Hz, 2H), 7.92 (t, J = 4.2 Hz, 4H), 7.76 (dd, J = 1.2, 9.0 Hz, 1H), 7.28 (d, J = 8.7 Hz, 2H), 7.22 (d, J = 9.0 Hz, 2H).

6-(4,5-Dihydroimidazol-2-yl)-2-(5-(4-(4,5-dihydroimidazol-2-yl)phenoxy)pyridine-2-yl)indole (24a)

The synthetic procedure used was the same as described for compound 13. Compound 24a was obtained as a light brown solid. R_f = 0.01 (80:18:2 CHCl₃/CH₃OH/CH₃NH₂), mp ≥300 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 11.91 (s, 1H), 8.48 (d, J = 2.4 Hz, 1H), 8.08 (d, J = 8.7 Hz, 1H), 7.93 (s, 1H), 7.88 (d, J = 9.0 Hz, 2H), 7.62 (dd, J = 3.0, 9.0 Hz, 1H), 7.54 (d, J = 11.7 Hz, 1H), 7.50 (dd, J = 1.2, 8.4 Hz, 1H), 7.16 (d, J = 8.7 Hz, 1H), 7.14 (s, 2H), 3.61 (br, 10H). HRMS (ESI-TOF) m/z calcd for C₂₅H₂₃N₆O 423.1933 (M+H)⁺, found 423.1920.

6-(3,4,5,6-Tetrahydropyrimidin-2-yl)-2-(4-(4-(3,2,5,6-tetrahydropyrimidin-2-yl)phenoxy)phenyl)indole (24b)

The synthetic procedure used was the same as described for compound 14. Compound 24b was obtained as a light yellow powder. Mp = 183–184 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 8.03 (d, 2H), 7.85 (s, 1H), 7.81 (d, 2H), 7.67 (d, 1H), 7.36 (dd, 1H), 7.18 (d, 2H), 7.10 (d, 2H), 6.99 (s, 1H), 3.49(t, 4H), 3.39 (t, 4H), 1.95 (t, 2H), 1.78 (t, 2H). HRMS (ESI-TOF) m/z calcd for C₂₇H₂₇N₆O 451.2246 (M+H)⁺, found 451.2227.

2-(4-(2-Chloro-4-cyanophenoxy)phenyl)-6-(4,5-dihydro-1H-imidazol-2-yl)indole (24c)

The synthetic procedure used was the same as described for compound 13. Compound 24c was purified by a ten-minute method by reverse phase HPLC using a C-18 column and isolated as a tan powder. Mp = 167–168 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 12.3 (s, 1H), 8.15-8.11 (m, 1H), 8.05 (d, J = 5.1 Hz, 2H), 8.01 (s, 1H), 7.91 (dd, J = 1.8, 9 Hz, 1H), 7.72 (d, J = 8.4 Hz, 1H),7.58 (d, J = 8.1 Hz, 1H), 7.51 (s, 1H), 7.19-7.17 (m, 3H), 7.04 (s, 1H), 3.95 (s, 4H).

2-(4-(4-(6-(4,5-Dihydro-1H-imidazol-2-yl)benzo[b]thiophen-2-yl)phenoxy)phenyl)-4,5-dihydro-1H-imidazole (2TFA salt) (25a)

The synthetic procedure used was the same as described for compound 13. Compound 25a was treated with TFA to give the di-TFA salt. Mp = 147–148 °C, ¹H NMR (300 MHz, DMSO-d₆) δ 10.59 (d, J = 3.9 Hz, 2 H), 10.51 (d, J = 3.9 Hz, 2 H), 8.64 (s, 1H), 8.12-7.89 (m, 7H), 7.32 (dd, J = 4.5, 8.7 Hz, 4H), 4.04 (d, J = 14.4 Hz, 8H). HRMS (ESI-TOF) m/z calcd for C₂₆H₂₃N₄OS 439.1593 (M+H)⁺, found 439.1596.

2-(4-(4-(6-(1,4,5,6-Tetrahydropyrimidin-2-yl)benzo[b]thiophen-2-yl)phenoxy)phenyl)-1,4,5,6-tetrahydropyrimidine (25b)

The synthetic procedure used was the same as used for compound 14. Compound 25b was obtained as a white solid. Mp = 239–240 °C; ¹HNMR (300 MHz, DMSO-d₆) δ 8.31 (s, 1H), 7.85-7.78 (m, 7H), 7.14 (d, J = 8.4 Hz, 2H), 7.08 (d, J = 8.7 Hz, 2H), 3.42-3.34 (m, 8H), 1.78-1.68 (m, 4H). HRMS (ESI-TOF) m/z calcd for C₂₈H₂₇N₄OS 467.1906 (M+H)⁺, found 467.1895.

1-(4-Bromophenoxy)-2,4-dichlorobenzene (27)

The synthetic procedure used was the same as that used for compound 19c. Compound 27 was obtained as a pale brown oil. R_f = 0.76 (5% EtOAc/hexane); ¹H NMR (300 MHz, CDCl₃) δ 7.45 (d, 1H), 7.41 (dd, 2H), 7.19 (dd, 1H), 6.91 (d, 1H), 6.81 (dd, 2H).

tert-Butyl 6-cyano-2-(4-(2,4-dichlorophenoxy)phenyl)-1H-indole-1-carboxylate (28)

A mixture of N-tert-butoxycarbonyl-6-cyano-indole-2-boronic acid 20a (500 mg, 1.8 mmol), compound 27 (668 mg, 2.1 mmol), tetrakistriphenylphosphinepalladium (40 mg, 0.035 mmol), sodium carbonate (371 mg, 3.5 mmol) in 10 % ethanol in toluene (18 mL) was heated at reflux under argon for 1.5 h. The reaction mixture was cooled to room temperature, washed thrice with water, followed by brine, dried over magnesium sulfate and the solvent was evaporated. The crude product was purified by flash chromatography using 8% EtOAc in hexane to obtain a light yellow solid (411 mg, 49% yield). Mp = 122–124 °C; ¹H NMR (300 MHz, CDCl₃) δ 8.56 (br, 1H), 7.59 (d, J = 7.8 Hz, 1H), 7.49 (s, 2H), 7.38 (d, J = 8.7 Hz, 2H), 7.26 (dd, J = 2.4, 8.7 Hz, 1H), 7.03 (d, J = 8.7 Hz, 1H), 6.98 (d, J = 9Hz, 2H), 6.58 (s, 1H), 1.37 (s, 9H).

2-(4-(2,4-Dichlorophenoxy)phenyl)-6-(4,5-dihydro-1H-imidazol-2-yl)-1H-indole (29)

Compound 28 (15 mg, 0.31 mmol), phosphorus pentasulfide (17 mg, 0.078 mmol) and ethylenediamine (5 mL) were stirred at 120 °C under nitrogen in a sealed tube for 2 h. The reaction mixture was cooled to room temperature and poured into excess water, stirred for 15 min and the white precipitate was collected by filtration and dried in air to obtain an off-white powder (84.5 mg, 64% yield). R_f = 0.17 (80:18:2 CHCl₃/CH₃OH/CH₃NH₂), mp = 219–220 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 11.71 (s, 1H),7.91 (d, J = 9 Hz, 2H), 7.86 (s, 1H), 7.81 (d, J = 2.7 Hz, 1H), 7.52 (s, 2H), 7.47 (dd, J = 2.4, 8.7 Hz, 1H), 7.20 (d, J = 8.7 Hz, 1H), 7.10 (d, J = 8.7 Hz, 2H), 6.89 (s, 1H), 3.62 (s, 4H). LC-MS (+ESI): m/z 422.3 (M+H)⁺. Anal. Calcd for C₂₃H₁₇Cl₂N₃O·0.2H₂O: C, 64.86; H, 4.12; N, 9.87. Found: C, 64.72; H; 4.11; N, 9.74.

tert-Butyl 6-chloro-2-(4-(4-cyanophenoxy)phenyl)-1H-indole-1-carboxylate (31)

Compound 30 (1.0 g, 3.4 mmol), compound 19a (1.11 g, 4.1 mmol), tetrakistriphenylphosphinepalladium (79 mg, 0.068 mmol) and sodium carbonate (742 mg, 7 mmol) in 10% ethanol in toluene (36 mL) were heated at reflux under argon for 1.5 h. The reaction mixture was cooled to room temperature, washed thrice with water, followed by brine, dried over magnesium sulfate and the solvent was evaporated. The crude product was purified by flash chromatography using 1:1 dichloromethane:hexane to obtain a light yellow solid (604 mg, 40% yield). Mp = 148–149 °C; ¹H NMR (300 MHz, CDCl₃) δ 8.3 (s, 1H), 7.64 (d, J = 9.0 Hz, 2H), 7.46 (dd, J = 3.0, 9.0 Hz, 3H), 7.24 (dd, J = 3.0, 9.0 Hz, 1H), 7.09 (t, J = 9.0 Hz, 4H), 6.53 (s, 1H), 1.39 (s, 9H).

6-Chloro-2-(4-(4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy)phenyl)-1H-indole (32)

Compound 31 (15 mg, 3.4 mmol), phosphorus pentasulfide (19 mg, 0.084 mmol) and ethylenediamine (5 mL) were stirred at 120 °C under nitrogen in a sealed tube for 2 h. The reaction mixture was cooled to room temperature and poured into excess water, stirred for 15 min and the resulting precipitate was collected by filtration and dried in air to give a white solid (84.9 mg, 65% yield). R_f = 0.17 (80:18:2 CHCl₃/CH₃OH/CH₃NH₂), mp = 271–272 °C; ¹H NMR (300 MHz, DMSO-d₆), δ 11.69 (s, 1H), 7.89 (d, J = 8.7 Hz, 2H), 7.86 (d, J = 8.7 Hz, 2H), 7.54 (d, J = 9.0 Hz, 1H), 7.39 (d, J = 1.5 Hz, 1H), 7.17 (d, J = 9.0 Hz, 2H), 7.09 (d, J = 9.0 Hz, 2H), 7.01 (dd, J = 1.8, 8.4 Hz, 1H), 6.85 (d, J = 1.5 Hz, 1H), 3.6 (s, 4H). HRMS (ESI-TOF) m/z calcd for C₂₃H₁₉ClN₃O 388.1217 (M+H)⁺, found 388.1208.

2-(4-(4-Cyanophenoxy)phenyl)-1H-benzo[d]imidazole-6-carbonitrile (34)

A mixture of 3,4-diaminobenzonitrile (33, 500 mg, 3.8 mmol), 4-(4-cyanophenoxy)benzaldehyde (7, 848 mg, 3.8 mmol) and 40% aqueous sodium bisulfite (2.2 mL, 8.3 mol) in ethanol (20 mL) was stirred at 80 °C for 19 h. A white precipitate appeared. The supernatant dark brown solution was decanted and evaporated to dryness. The crude product was washed twice with hot toluene to give a white solid (1.31g, 100%). Mp = 222–223 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 8.36 (d, J = 8.7 Hz, 2H), 8.21 (s, 1H), 7.97 (d, J = 8.4 Hz, 2H), 7.83 (d, J = 8.4 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.39 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H). LC-MS (+ESI): m/z 336.3 (M⁺).

6-(4,5-Dihydro-1H-imidazol-2-yl)-2-(4-(4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy)phenyl)-1H-benzo[d]imidazole (35a)

The synthetic procedure used was the same as used for compound 13. Compound 35a was obtained as a grey powder. Mp = 257–260 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 8.26 (d, J = 9.0 Hz, 2H), 8.15 (s, 1H), 7.91 (d, J = 9.0 Hz, 2H), 7.77 (dd, J = 1.5, 8.4 Hz, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.24 (d, J = 9 Hz, 2H), 7.18 (d, J = 8.7 Hz, 2H), 3.82 (s, 4H), 3.67(s, 4H). LCMS (+ESI): m/z 423.43 (M+H)⁺.

6-(1,4,5,6-Tetrahydropyrimidin-2-yl)-2-(4-(4-(1,4,5,6-tetrahydropyrimidin-2-yl)phenoxy)phenyl)-1H-benzo[d]imidazole (35b)

The synthetic procedure used was the same as used for compound 14. Compound 35b was obtained as a white solid. Mp = 247–250 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 8.27 (d, J = 8.7 Hz, 2H), 7.89 (s, 1H), 7.78 (d, J = 8.7 Hz, 2H), 7.53 (d, J = 9 Hz, 1H), 7.38 ( d, J =8.1 Hz, 1H), 7.11 (d, J = 8.7 Hz, 2H), 7.06 (d, J = 8.7 Hz, 2H), 3.45 (t, J = 5.5 Hz, 4H), 3.36 (t, J = 5.4 Hz, 4H), 1.89 (t, J = 5.4 Hz, 2H), 1.72 (t, J = 5.4 Hz, 2H). HRMS (ESI-TOF) m/z calcd for C₂₇H₂₇N₆O 451.2246 (M+H)⁺, found 451.2247.

(E)-1-(4-Fluorophenyl)-2-(4-cyano-2-nitrophenyl)ethene (38)

4-Methyl-3-nitrobenzonitrile (36, 4.0 g, 24.7 mmol) and 4-fluorobenzaldehyde (37, 3.06 g, 24.7 mmol) were heated together to 150 °C until melt occurred. Piperidine (1.23 mL) was added and the resulting solution was stirred at 150 °C for 4 h. After cooling to room temperature, cold MeOH was added to the residue, and filtration yielded a red solid (6.2 g, 96% yield). R_f = 0.43 (3:1 hexane/EtOAc), mp = 137–138 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 8.26+8.19 (2d, J = 1.6, 1.7 Hz, 1H) 7.90 (d, J = 8.2 Hz, 1H), 7.84+7.75 (2dd, J = 1.6, 17.2, Hz, 1H), 7.57-7.39 (m, 3H), 7.22+7.7.15 (2d, J = 5.9, 6.4 Hz, 1H), 7.09+6.90 (2d, J = 8.6, 8.9 Hz, 1H).

2-(4-Fluorophenyl)-1H-indole-6-carbonitrile (39)

Compound 38 (6.0 g, 22.4 mmol) was suspended in triethyl phosphite (70 mL) and heated to 150–160 °C for 1.5 h. The reaction mixture was cooled to room temperature and excess triethyl phosphite was removed by distillation under vacuum. The residue was recrystallized from methanol to give an off-white solid (39, 2.4 g, 45% yield): R_f = 0.44 (3:1 hexane/EtOAc), mp = 110–111 °C; (300 MHz, DMSO-d₆) δ 12.15 (s, 1H), 8.00-7.95 (m, 2H), 7.83 (s, 1H), 7.71 (d, J = 8.3 Hz, 1H), 7.40-7.33 (m, 3H), 7.06 (s, 1H).

2-(4-Fluorophenyl)-1H-indole-6-carboxamide (40a)

The synthetic procedure used was the same as used for compound 10. Compound 40a was obtained as a grey solid. R_f = 0.51 (80:18:2 CHCl₃/CH₃OH/CH₃NH₂), mp = 236–237 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 11.79 (s, 1H), 7.95 (t, J = 6.0 Hz, 4H), 7.55 (s, 2H), 7.34 (t, J = 8.8 Hz, 2H), 7.19 (s, 1H), 6.94 (s, 1H). HRMS (ESI-TOF) m/z calcd for C₁₅H₁₂FN₂O 255.0934 (M+H)⁺, found 255.0927.

2-(4-Fluorophenyl)-1H-indole-6-carboximidamide (40b)

The synthetic procedure used was similar as used for compound 12. Compound 40b was obtained as a green powder. Mp = 262–263 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 9.2-8.6 (br, 4H), 7.99 (dd, 2H), 7.88 (s, 1H), 7.69 (d, 1H), 7.40 (d, 1H), 7.33 (t, 2H), 7.02 (s, 1H). HRMS (ESI-TOF) m/z calcd for C₁₅H₁₃FN₃ 254.1094 (M+H)⁺, found 254.1091.

6-(4,5-Dihydro-1H-imidazol-2-yl)-2-(4-fluorophenyl)-1H-indole (40c)

The synthetic procedure used was the same as used for compound 13. Compound 40c was obtained as a light brown solid. R_f = 0.01 (80:18:2 CHCl₃/CH₃OH/CH₃NH₂), mp = 227–228 °C; ¹H NMR (300 MHz, DMSO-d₆) δ δ11.71 (s, 1H), 7.94 (d, J = 5.4 Hz, 1H), 7.91 (d, J = 5.4 Hz, 1H), 7.86 (s, 1H), 7.52 (s, 2H), 7.32 (t, J = 8.7 Hz, 2H), 6.91 (s, 1H), 6.7 (br, 1H), 3.61 (s, 4H). HRMS (ESI-TOF) m/z calcd for C₁₇H₁₅FN₃ 280.1250 (M+H)⁺, found 280.1241.

tert-Butyl 6-cyano-2-(4-methoxyphenyl)-1H-indole-1-carboxylate (42a)

The synthetic procedure used was the same as used for compound 23a. Compound 42a was obtained as a white solid. Mp = 206–207 °C; ¹H NMR (300 MHz, CDCl₃) 8.59 (s, br, 1H), 7.69 (d, J = 0.6 Hz, 1H), 7.64-7.60 (m, 3H), 7.34 (dd, J = 1.2, 1.5 Hz, 1H), 7.00 (dd, J = 0.9, 2.1 Hz, 2H), 6.76 (dd, J = 0.9, 2.1 Hz, 1H), 3.87 (s, 3H). LC-MS (+ESI): m/z 249.2 (M+H)⁺.

tert-Butyl 6-cyano-2-(4-cyanophenyl)-1H-indole-1-carboxylate (42b)

The synthetic procedure used was the same as used for compound 23a. Compound 42b was obtained as a white solid. Mp = 279–280 °C; ¹H NMR (300 MHz, CDCl₃) δ 8.57 (s, 1H), 7.74 (dd, J = 2.1, 6.0 Hz, 2H), 7.65 (dd, J = 0.6, 8.1 Hz, 1H), 7.55 (dd, J = 2.1, 6.0 Hz, 2H), 7.51 (d, J = 1.5 Hz, 1H), 6.68 (s, 1H), 1.37 (s, 9H). LC-MS (+ESI): m/z 344.1 (M+H)⁺.

2-(4-Methoxyphenyl)-1H-indole-6-carboxamide (43a)

The synthetic procedure used was the same as used for compound 10. Compound 43a was obtained as a tan powder. Mp = 258–259 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 11.65 (s, 1H), 7.93 (s, H), 7.84 (d, J = 9.0 Hz, 3H), 7.52-7.13 (m, 4H), 7.05 (d, J = 9.0 Hz, 1H), 6.82 (s, 1H), 3.82 (s, 3H). HRMS (ESI-TOF) m/z calcd for C₁₆H₁₅N₂O₂ 267.1134 (M+H)⁺, found 267.1124.

2-(4-Methoxyphenyl)-1H-indole-6-carboximidamide (43b)

The synthetic procedure used was the same as used for compound 12. Compound 43b was obtained as a pale yellow powder. R_f = 0.07 (80:18:2 CHCl₃/CH₃OH/CH₃NH₂), mp = 290–291 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 12.19 (br, 1H), 9.08 (br, 3H), 7.9 (d, J = 9 Hz, 3H), 7.68 (d, J = 8 Hz, 1H), 7.43 (dd, J = 1.5, 9.0 Hz, 1H), 7.08 (d, J = 6 Hz, 2H), 6.95 (s, 1H), 3.83 (s, 3H). HRMS (ESI-TOF) m/z calcd for C₁₆H₁₆N₃O 266.1293 (M+H)⁺, found 266.1293.

2-(4-Methoxyphenyl)-6-(4,5-dihydro-1H-imidazol-2-yl)-1H-indole (43c)

The synthetic procedure used was the same as used for compound 13. Compound 38c was obtained as a light yellow powder. R_f = 0.41 (80:18:2 CHCl₃/CH₃OH/CH₃NH₂), mp = 241–242 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 11.63 (br, 1H), 7.85 (d, J = 5.1 Hz, 2H), 7.81 (s, 1H), 7.49 (s, 2H), 7.04 (d, J = 8.7 Hz, 2H), 6.8 (s, 1H), 3.81 (s, 3H), 3.6 1(s, 4H). HRMS (ESI-TOF) m/z calcd for C₁₈H₁₈N₃O 292.1450 (M+H)⁺, found 292.1449.

6-(4,5-Dihydro-1H-imidazol-2-yl)-2-(4-(4,5-dihydro-1H-imidazol-2-yl)phenyl)-1H-indole (44a)

The synthetic procedure used was the same as used for compound 13. Compound 44a was obtained as a light yellow powder. R_f = 0.34 (80:18:2 CHCl₃/CH₃OH/CH₃NH₂), mp = 289–291°C; ¹H NMR (300 MHz, DMSO-d₆) δ 11.88 (s, 1H), 7.97–7.86 (m, 4H), 7.6–7.52 (m, 2H), 7.05 (s, 1H), 3.65 (d, J = 9.0 Hz, 8H). LC-MS (+ESI): m/z 330.4 (M+H)⁺. Anal. Calcd for C₂₀H₁₉N₅·0.7H₂O: C, 70.24; H, 6.01; N, 20.48. Found: C, 70.47; H; 5.73; N, 20.04.

6-(3,4,5,6-Tetrahydropyrimidin-2-yl)-2-(4-(3,4,5,6-tetrahydropyrimidin-2-yl)phenyl)-1H-indole (44b)

The synthetic procedure used was the same as used for compound 14. Compound 44b was obtained as a light yellow powder. R_f = 0.04 (80:18:2 CHCl₃/CH₃OH/CH₃NH₂), mp >300 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 8.03 (d, J = 8.4 Hz, 2H), 7.88 (d, J = 8.4 Hz, 2H), 7.86 (s, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.37 (dd, J = 1.5, 8.4 Hz, 1H), 7.12 (s, 1H), 3.48 (t, J = 5.7 Hz, 4H), 3.41 (t, J = 5.4 Hz, 4H), 1.96–1.93 (m, 2H), 1.79-1.76 (m, 2H). LC-MS (+ESI): m/z 358.3 (M+H)⁺.

2-(4-Cyanophenyl)benzo[b]thiophene-6-carbonitrile (45)

A mixture of 6-cyanobenzo[b]thiophen-2-ylboronic acid (20b, 1 g, 4.9 mol), 4-bromobenzonitrile (41b, 1.08 g, 5.9 mol), (2-biphenyl)-di-tert-butylphosphine (146 mg, 0.49 mmol), Pd(OAc)₂ (55 mg, 0.25 mmol) and K₂CO₃ (1.35 g, 9.8 mmol) in DMF (30 mL) was heated in a sealed tube under argon atmosphere at 100 °C for 4h, cooled to room temperature and poured into an excess of water. The precipitate obtained was collected by filtration, washed with water and dried at 50 °C under vacuum. The product was further purified by trituration with hot MeOH to yield a light tan powder (1.28 g, 90% yield). R_f = 0.49 (80:18:2 CHCl₃/CH₃OH/CH₃NH₂), mp = 262–263 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 8.67 (s, 1H), 8.22 (s, 1H), 8.07–7.96 (m, 5H), 7.79 (dd, J = 1.5, 8.4 Hz, 1H).

2-(4-(6-(4,5-Dihydro-1H-imidazol-2-yl)benzo[b]thiophen-2-yl)phenyl)-4,5-dihydro-1H-imidazole (46a)

The synthetic procedure used was the same as used for compound 13. Compound 46a was obtained as a light yellow powder. R_f = 0.41 (80:18:2 CHCl₃/CH₃OH/CH₃NH₂), mp > 300 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 8.39 (s, 1H), 8.01 (s, 1H), 7.94–7.85 (m, 6H), 7.0 (br, 1H), 3.64 (d, J = 4.2 Hz, 8H). HRMS (ESI-TOF) m/z calcd for C₂₀H₁₉N₄S 347.1330 (M+H)⁺, found 347.1330.

2-(4-(6-(1,4,5,6-Tetrahydropyrimidin-2-yl)benzo[b]thiophen-2-yl)phenyl)-1,4,5,6-tetrahydropyrimidine (2TFA salt) ( 46b)

The synthetic procedure used was the same as used for compound 14. Compound 46b was obtained as a light brown powder. R_f = 0.08 (80:18:2 CHCl₃/CH₃OH/CH₃NH₂), mp = 227–228 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 10.09 (s, 4H), 8.47 (s, 1H), 8.25 (s, 1H), 8.11 (d, J = 8.4 Hz, 3H), 7.86 (d, J = 8.4 Hz, 2H), 7.72 (dd, J = 1.8, 8.4 Hz, 1H), 3.54 (br, 8H), 2.01 (br, 4H). HRMS (ESI-TOF) m/z calcd for C₂₂H₂₃N₄S 375.1643 (M+H)⁺, found 375.1642.

2-(5-Fluoro-2-pyridyl)-6-benzo[b]thiphenecarbonitrile (48)

The synthetic procedure used was the same as used for compound 23c. Compound 48 was obtained as a light yellow powder. Mp = 228–229 °C; ¹H NMR (300 MHz, CDCl₃) δ 8.52 ((d, J = 3Hz, 1H), 8.18 (s, 1H), 7.79–7.87 (m, 3H), 7.58 (dd, J = 1.5, 6 Hz, 1H), 7.51 (dd, J = 8.1, 16.8 Hz, 1H). LC-MS (+ESI): m/z 255.3 (M+H)⁺.

2-(5-Fluoro-2-pyridyl)-6-benzo[b]thiophenecarboxamide (49)

The synthetic procedure used was the same as used for compound 10. Compound 49 was obtained as a light yellow powder. Mp = 250–252 °C; ¹H NMR (300 MHz, DMSO-d⁶) δ 8.64 (d, 1H, J = 2.7 Hz), 8.52 (s, 1H), 8.26-8.19 (m, 2H), 8.08 (br, 1H), 7.95-7.86 (m, 2H), 7.45 (br, 1H). HRMS (ESI-TOF) m/z calcd for C₁₄H₁₀FN₂OS 273.0498 (M+H)⁺, found 273.0495.

N¹–(6-(6-(4,5-Dihydro-1H-imidazol-2-yl)benzo[b]thiophen-2-yl)pyridine-3-yl)ethane-1,2-diamine (50)

Compound 48 (220 mg, 0.87m mol), phosphorus pentasulfide (48 mg, 0.22 mmol) and ethylenediamine (5 mL) were stirred at 120 °C under nitrogen in a sealed tube for 2 h. The reaction mixture was cooled to room temperature and poured into excess water, stirred for 15 min and the precipitate was collected by filtration and dried in air. Purification by a ten-minute method using reverse phase HPLC on a C-18 column gave 50 as a light yellow solid (87 mg, 30%). Mp = 211–212 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 10.59 (s, 2H), 8.56 (s, 1H), 8.07-7.94 (m, 6H), 7.86 (d, J = 8.7 Hz, 1H), 7.13 (dd, J = 2.7, 9.0 Hz, 1H), 4.04 (s, 4H), 3.42 (t, J = 6.3 Hz, 2H), 3.04 (t, J = 5.4 Hz, 2H). HRMS (ESI-TOF) m/z calcd for C₁₈H₂₀N₅S 338.1439 (M+H)⁺, found 338.1435.

BoNT/A LC FRET Assay

The BoNT/A LC FRET-based assay was originally developed by Schmidt.²³ Test compound, 20 µM SNAP-25 (aa 187–203) peptide substrate of sequence SNRTRIDEAN[DnpK]RA[daciaC]RML (Peptides International, Louisville, KY), and 10 ng of BoNT/A LC (List Biological Laboratories, Campbell, CA) were incubated at 37 °C for 40 min. in the presence of buffer (50 mM HEPES–0.05% Tween, pH 7.4) in a volume of 100 µL. The reactions were stopped with acetic acid (0.5% [final]) prior to measuring the fluorescence of the cleaved substrate at 485 nm following excitation at 398 nm in a Molecular Devices (Sunnyvale, CA) plate reader. IC₅₀ values were obtained by dose-response measurements.

Anthrax lethal factor FRET Assay

The anthrax lethal factor FRET-based assay was previously reported.²⁸ The reaction mixture contained 20 µM peptide substrate (KKVYPYPME) with a fluorogenic coumarin group at the N-terminus and a 2,4-dinitrophenyl (DNP) quenching group at the C terminus (Peptides International, Louisville, KY), LF (50 ng) (List Biological Laboratories), 20 mM HEPES, pH 8.2, 0.05% Tween-20 and the test compound. The assay mixture was incubated at 30 °C for 15 min. The reactions were stopped with acetic acid (0.5% [final]) prior to measuring the fluorescence of the cleaved substrate at 395 nm following excitation at 324 nm in the Molecular Devices plate reader. IC₅₀ values were obtained by dose-response measurements.

Molecular modeling

The molecular modeling study was performed with the Schrödinger computational software package (Schrödinger, Inc., New York). X-ray coordinates of BoNT/A LC (PDB code: 2G7N) were used for this modeling study. The ionic states of compound 12 were generated by Epik version 1.6, the docking study was performed with Glide 5.0, and energies were minimized by Macromodel. For each state of compound 12, 10 solutions were generated and subsequently ranked according to the glide score. The best poses of ionic charged states were further thoroughly energy minimized (RMSD = 0.05 Å) and the relative free binding energies were calculated according to the following equation: ΔE = E_complex − (E_enzyme + E_ligand).

ABRREVIATIONS

BoNTs

Botulinum neurotoxins

BoNT/A

botulinum neurotoxin serotype A

BoNT/B

botulinum neurotoxin serotype B

BoNT/A HC

BoNT/A heavy chain

BoNT/A LC

BoNT/A light chain

LF

lethal factor

FRET

fluorescence resonance energy transfer

HPLC

high performance liquid chromatography

MMPs

matrix metalloproteases