MreB is a cytoskeleton protein present in rod-shaped bacteria that is both essential for bacterial cell division and highly conserved. Because most Gram (-) bacteria require MreB for cell division, chromosome segregation, cell wall morphogenesis, and cell polarity, it is an attractive target for antibacterial drug discovery. As MreB modulation is not associated with the activity of antibiotics in clinical use, acquired resistance to MreB inhibitors is also unlikely. Compounds, such as A22 and CBR-4830, are known to disrupt MreB function by inhibition of ATPase activity. However, the toxicity of these compounds has hindered efforts to assess the in vivo efficacy of these MreB inhibitors. The present study further examines the structure-activity of analogs related to CBR-4830 as it relates to relative antibiotic activity and improved drug properties. These data reveal that certain analogs have enhanced antibiotic activity. In addition, we evaluated several representative analogs (9, 10, 14, 26, and 31) for their abilities to target purified E. coli MreB (EcMreB) and inhibit its ATPase activity. Except for 14, all these analogs were more potent than CBR-4830 as inhibitors of the ATPase activity of EcMreB with corresponding IC50 values ranging from 6.3 ± 2.0 to 29.4 ± 8.8 uM.
Research Article
Novel MreB Inhibitors with Antibacterial Activity Against Gram (-) Bacteria
https://doi.org/10.21203/rs.3.rs-1824695/v1
This work is licensed under a CC BY 4.0 License
You are reading this latest preprint version
MreB inhibitors
Gram (-) Antibiotics
E. coli MreB ATPase Inhibition
Structure Activity Relationships
MreB is the prokaryotic homolog of actin in eukaryotes. The function of MreB within bacteria is associated with its ability to form long filaments, a process similar to actin in eukaryotes [1-5]. These filaments are critical for to the retention of the structure of rod-shaped bacteria, which is dominant among many Gram (-) bacteria [1, 2, 4-6]. MreB is also essential for bacterial cell division [7]. This structural protein is an attractive target for antibacterial drug discovery as it is highly conserved and present in almost all rod-shaped bacteria [8]. Acquired resistance to such novel antibiotics would also be unlikely, as MreB modulation is not associated with the modes of action of antibiotics in clinical use.
Polymerization of MreB requires ATP. Several benzyl thioureas such as A22 and MP265 (1, 2) as well as CBR-4830 and several of its analogs (3-6) have been identified as inhibitors of MreB function (Figure 1) [1, 2, 5, 8-15]. Current theories on the mode of action of these MreB inhibitors suggest that they act by noncompetitively binding to this structural protein, thereby affecting the ATP binding pocket. MreB loss of function is both lethal and pleiotropic [7, 16]. Multiple cellular processes are disrupted including cell shape determination, polar protein localization, and cell division [16-18]. MreB also has a direct role in the segregation of a specific region of the chromosome [18-21].
The MreB inhibitor, A22, is lethal at relatively low doses when administered iv to either rats or mice. Lynch et al reported in 2007 the identification and characterization of CBR-4830 (Figure 1) as a novel antimicrobial agent with activity against efflux-compromised P. aeruginosa [22]. Combined genetic, biochemical and cell morphology studies were performed that demonstrated that the cellular target for CBR-4830 is MreB. A limited structure-activity study was performed with analogs of CBR-4830, 3-6. However, CBR-4830 proved to be the most active analog within this series. CBR-4830 proved not only to be difficult to formulate for iv administration because of poor solubility properties in aqueous vehicles, but it also proved to be lethal at relatively low concentration in mice. Recently, we reported on the enhanced activity of TXH11106 (7) relative to CBR-4830 [23, 24]. In the present study, we initiated studies to develop additional analogs related to CBR-4830 to further improve antibacterial potency and formulation properties relative to CBR-4830.
Our initial studies were focused on examining the impact of changing the fused alicyclic ring of the tetrahydrocarbazole pharmacophore of CBR-4830 to its next higher homologue, a cycloheptane moiety fused to the indole. 2-Bromo-5,6,7,8,9,10-hexahydrocyclohepta[b]indole-6-amine, 8, and the dichloro analogs 9 and 10 were synthesized as outlined in Scheme 1. Commercially available cycloheptanone was condensed with ethyl formate to form 2-(hydroxymethylene)cycloheptan-1-one. Reaction of this intermediate with the appropriate pre-formed diazonium salts provide the hydrazineylidene intermediates, which were converted to 8a and a mixture of 9a and 10a. The mixture of 9a and 10a was converted to their N-Boc derivatives and separated chromatographically, followed by removal of the Boc groups to provide the pure isomers. Reductive amination using ammonium acetate and NaBH3CN provided the 6-amino-5,6,7,8,9,10-hexahydro-cyclohepta[b]indoles 8, 9 and 10.
We also synthesized similarly substituted 1,2,3,4-tetrahydrocyclopenta[b]indol-3-amines, 11–13, which represent analogs that are a one carbon lower homologue of the tetrahydrocarbazole pharmacophore of CBR-4830. The methods used for these syntheses are outlined in Scheme 2. Cyclopentanone was condensed with ethyl formate to provide 2-(hydroxymethylene)cyclopentan-1-one. This intermediate was reacted with the appropriate diazonium salt to give the desired hydrazineylidene intermediates, which were converted under acidic condition to the 1,4-dihydrocyclopenta[b]indole-3(2H)-ones, 11a as well as a mixture of 12a and 13a. The mixture of 12a and 13a was resolved by formation of their N-Boc derivatives (12b and 13b), chromatographic separation, followed by removal of their Boc groups.
The impact of removing two methylene groups and opening the tetrahydrocyclohexyl ring of CBR-4830 on relative antimicrobial activity was also evaluated. The varied 2-(1-aminoethyl)indoles 14–21 were prepared as outlined in Scheme 3.
The Weinreb amide derivatives 14b-21b were formed from the various 2-carboxyindole derivatives and then converted using methyllithium to the methyl ketones 14c-21c. Reductive amination of 14c-21c using ammonium acetate and sodium cyanoborohydride provide 14–21.
The influence of mono- or dimethylation on the amino group as well as methylation of the 1-position of 1-(5-bromo-1-methyl-1H-indol-2-yl)ethan-1-amine was examined. Compounds 22, 23, and 24 were synthesized as outlined in Scheme 4.
Reductive amination was performed using N-methylamine or N,N-dimethylamine, followed by reduction with sodium cyanoborohydride was used to prepare 22 or 23. Methylation of 14c to form the 1-methylindole derivative 24a was accomplished using potassium carbonate in DMF and methyl iodide. Intermediate 24a was then converted 24 using ammonium acetate and sodium cyanoborohydride.
We also examined the impact on activity of having the primary amine of 1-(5-bromo-1-methyl-1H-indol-2-yl)ethan-1-amine attached to a primary carbon as opposed to a secondary carbon. We synthesized (5-bromo-1H-indol-2-yl)methanamine, 25, as outlined in Scheme 5 and evaluated its antibacterial properties.
The preparation of the methylamine derivative 25 was performed starting from the 2-carboxy indole intermediate 14a. This acid was converted to its methyl ester, 25a, using methanol and concentrated HCl. The ester was reduced with lithium borohydride in THF to provide the hydroxymethyl intermediate, 25b. Using Dess-Martin periodinane in dichloromethane, this hydroxymethyl derivative was oxidized to the 2-formylindole derivative, 25c. Reductive amination with ammonium acetate and sodium cyanoborohydride provide the desired 2-aminomethyl indole derivative 25.
The N-(3-aminopropyl) derivatives of 8, 11, and 14 were also synthesized and their relative antibacterial activity compared. The synthetic methods used for their preparation are outlined in Scheme 6.
The ketone intermediates 8a, 11a, and 14c were each treated with N-Boc propylenediamine and then subject to reduction using sodium cyanoborohydride in ethanol. Excellent yields were obtained of each of the N-Boc propylenediamine derivatives, 26a, 27a and 28a. Removal of the N-Boc protecting groups with 4N HCl gave 26–28.
A series of N-acyl derivatives of 28 were also prepared. These included N-acylheteroaryl, N-acyl(3,4-dichlorophenyl), as well as N-acylcyclohexyl derivatives, as illustrated in Scheme 7.
The synthesis of a series carboxamides derived from 28 were prepared by condensation of various carboxylic acids using EDC, HOBt and DIPEA at room temperature. The commercially available carboxylic acids employed for the preparation of 29–33 were 5-chlorofuran-2-crboxylic acid, 5-chlorothiophene-2-carboxylic acid, 4,5-dichlorothiophene-2-carboxylic acid, 3,4-dichlorobenzoic acid and cyclohexane carboxylic acid.
We also prepared the revered amide analog, 34, that is related to 32, as well as the aryloxy analog 35, as illustrated in Scheme 8 and 9. The reversed amide 34 was prepared by first preparing the 4-amino-N-(3,4-dichlorophenyl)butanamide, which was then reacted with 14c and the resulting imine reduce using sodium cyanoborohydride in ethanol to give 34 as outlined in Scheme 8.
The N-[4-(3,4-dichlorophenoxy)butyl] derivative of 14 was prepared from 3-(3,4-dichlorophenoxy)propylamine as illustrated in Scheme 9. Reaction of this amine with 14c, followed by reduction with sodium cyanoborohydride provide 35.
Table 1 provides summarize the intrinsic activity of antibacterial activity against P. aeruginosa, E. coli, K. pneumoniae and A. baumannii for the cyclohepta analogs 8-10, the cyclopenta analogs 11-13, and the ring-opened analogs 14-21, relative to CBR-4830. P. aeruginosa proved relatively resistant in the absence of a bacterial efflux pump inhibitor (EPI). Therefore, the MICs of all of these compounds against P. aeruginosa was also evaluated in the presence of 12.5 µg/ml of an EPI, namely N-(((2S,4R)-4-(aminomethyl)pyrrolidin-2-yl)methyl)-6-(4-fluorophenyl)-1H-indole-2-carboxamide [24]. A similar MIC (1.0 µg/ml) was observed for CBR-4830 against P. aeruginosa PAO1 in the presence of the EPI to that reported against the PAO1 mutant efflux defective strain CB392 ∆(mexAB-oprM) ∆(mexCD-oprJ) [24]. The more potent of the cyclohepta analogs evaluated against P. aeruginosa in the presence of an EPI was 10 with an MIC of 1.0 µg/ml. Among the cyclopenta analogs, the most potent was 13, which had its dichloro substituents at the same relative sites on the fused indole moiety and also exhibited the same MIC value as 10. These two analogs also exhibited the lower MICs against E. coli, with 10 and 13 having MICs of 8 and 16 µg/ml as compared to 8 ug/ml for CBR-4830. Compound 10 had an MIC of 2 µg/ml against K. pneumoniae, which was lower than that observed for 13 and CBR-4830, which had MICs of 8 µg/ml. The MICs of 10 and 13 against A. baumannii were 8 µg/m; which were lower than that observed for CBR-4830 (16 ug/ml). Both 10 and 13 proved to have significantly improved formulation properties and were also less toxic when administered intravenously to mice relative to CBR-4830 (data not shown).
The ring-opened analogs 14-21 also had dramatically improved solubility in aqueous vehicles suitable for intravenous administration and were well tolerated when administered by this route to mice (data not shown). However, their relative intrinsic MICs against P. aeruginosa, E. coli, K. pneumoniae or A. baumanni were disappointing as none of these analogs had MIC values that were less than 32 µg/ml. Only in the presence of an EPI, were notable MICs observed against P. aeruginosa of 8 µg/ml for 14 and 21 and 16 ug/ml for 16, 17 and 20.
The improve physicochemical properties of the ring-opened analogs prompted further studies on their structure-activity relationships. We synthesized and evaluated the N-methyl and N,N-dimethyl amino analogs, 22, and 23, as well as the N1-methyl derivative, 24, of compound 14. We also prepared the methylamino analog, 25, wherein the primary amine was attached to a primary carbon. The antimicrobial activities of these compounds are summarized in Table 2. Only in the case of 25 was notable activity observed against P. aeruginosa in the presence of an EPI. The potency observed was comparable to 14.
A series of N-(3-aminopropane) derivatives were prepared that incorporated the cycloheptyl, cyclopentyl and open-ring analogs of CBR-4830, specifically 26, 27 and 28 (Table 3). Compound 26 had a lower MIC than 8 in terms of its intrinsic MIC against P. aeruginosa (32 versus 128 µg/ml), in the presence of an EPI, the observed MIC was much higher (32 versus 4 µg/ml). The differences between 8 and 26 were not that significant when evaluated in E. coli, K. pneumoniae and A. baumannii. Both 27 and 28 have either similar activity or are less active than their parent primary amines, 11 and 14 against E. coli, K. pneumoniae or A. baumannii. However, both 27 and 28 were notably less active than their parent primary amines when evaluated against P. aeruginosa in the presence of an EPI. Against E. coli, 26 had the same MIC as 8 (16 µg/ml), but it was slightly more active against K. pneumoniae (8 versus 16 µg/ml) and slightly less active against A. baumannii (32 versus 16 µg/ml). Compound 27 did not have remarkable activity against E. coli, K. pneumoniae, and A. baumannii. There were no major differences in the MICs observed for 14 and 28 observed in E. coli, K. pneumoniae or A. baumannii. We extended these studies to include a several N-(3-acylpropanamides) derived from the N-(3-aminopropane), 29-33. The relative antimicrobial activity of these hydrophobic derivatives are summarized in Table 3. None of these analogs exhibited intrinsic activity against P. aeruginosa. In the presence of an EPI, these analogs failed to exhibit potency against P. aeruginosa comparable to 14 or 22 under these assay conditions. There were notable improvements in activity for 29-33 against E. coli, K. pneumoniae and A. baumanni relative to 22. Most notably, 31 had MICs against E. coli, K. pneumoniae and A. baumanni that were 16-, 64-, and 8-fold lower than for 14. The cyclohexyl amide, 33, proved to be less activity than the aryl or heteroaryl amides, 29-32, that were evaluated.
We also prepared 34, the reversed amide analog of 32, as well as the 4-(3,4-dichlorophenoxy)butan-1-amine derivative, 35. Similar activity was observed between 32 and its reversed amide, 34, derivatives. The phenoxy derivative 35 also had similar activity to 34.
We evaluated six representative analogs (8, 9, 10, 14, 26, and 31) for their abilities to target purified E. coli MreB (EcMreB) and inhibit its ATPase activity. All six compounds inhibited the ATPase activity of EcMreB (Figure 1), with corresponding IC50 values ranging from 6.3 ± 2.0 µM for analog 31 to 120 ± 17.0 µM for analog 14 (Table 4). Based on IC50 values, the EcMreB-inhibiting potency of the six analogs tested followed the hierarchy: 31 > 9 ≈26 ≈ 10 > 8 > 14.
|
Table 4. IC50 values for inhibition of the ATPase activity of EcMreB by select analogs. |
|
|
Compound |
IC50 (μM) |
|
8 |
44.1 ± 7.8 |
|
9 |
24.5 ± 4.7 |
|
10 |
29.4 ± 8.8 |
|
14 |
120 ± 17.0 |
|
26 |
25.1 ± 7.0 |
|
31 |
6.3 ± 2.0 |
|
EcMreB = E. coli MreB; IC50 = the compound concentration that inhibits the ATPase activity of EcMreB by 50%. IC50 values were derived from fits of the velocity vs. [compound] plots shown in Figure 1, with the indicated uncertainties reflecting the standard deviation of the fitted curves from the experimental data points. |
|
The data in Table 1 indicate that cyclohepta and cyclopenta analogs related to CBR-4830 retain significant antibacterial activity. CBR-4830 proved difficult to formulate (requiring 40% propylene glycol to achieve a concentration of 2 mg/ml) and did produce severe toxic effects when administered to mice b.i.d. at a dose of 400 ug/mouse (data not shown). Several of the promising cyclohepa and cyclopenta analogs could be readily formulated using 10 mM citrate in water and were well tolerated when administered at more than twice this dose. The actual significance of these differences, however, needs to be examined in the context of comparative pharmacokinetic studies which have not been performed. The ring-opened analogs of CBR-4830 were significantly less active and only exhibited modest activity when evaluated against P. aeruginosa in the presence of a bacterial efflux pump inhibitor, such as N-(((2S,4R)-4-(aminomethyl)pyrrolidin-2-yl)methyl)-6-(4-fluorophenyl)-1H-indole-2-carboxamide. The ring-open series of compounds, like the cyclohepta and cyclopenta analogs, had favorable formulation properties and were well tolerated in mice. Efforts to improve antibacterial activity among the ring-opened analog 8, by either mono- or demethylation of the amino substituent as in 22 and 23, or methylation of the indole nitrogen as in the case of 24, failed to enhance activity (Table 2). Changing the 1-aminoethyl of 8 to a aminomethyl group as in 25 did restore activity against P. aeruginosa in the presence of the EPI, N-(((2S,4R)-4-(aminomethyl)pyrrolidin-2-yl)methyl)-6-(4-fluorophenyl)-1H-indole-2-carboxamide. However, no significant activity was observed against E. coli, K. pneumoniae or A. baumannii (Table 2).
The impact of attaching a 3-aminopropyl linkage to the amine of cycloheptyl, cyclopentyl or ring-opened analogs of CBR-4830 on antibacterial activity was also evaluated (Table 3). In the case of the cycloheptyl analog 26, there was an enhancement in intrinsic activity against P. aeruginosa and a modest increase in activity against K. pneumoniae relative to 8. In the case of the cyclopenta analog 27, there was a notable loss in activity observed against P. aeruginosa when evaluated in the presence of an EPI and no increase in activity against E. coli, K. pneumoniae or A. baumanni relative to 11. Modification of the ring-opened analog by attaching this linkage as in 28 primarily resulted in a loss of the activity that was previously observed with 14 in the presence of an EPI against P. aeruginosa.
We examined the effect of modifying the terminal amine of 28 by forming various hydrophobic amide derivatives as a possible means of enhancing the antibacterial properties of these ring-opened analogs. While these analogs 29–33 did not exhibit significant intrinsic activity against P. aeruginosa, their activity in the presence of an EPI was improved relative to 28. More significantly, their intrinsic antibacterial activity was increased against E. coli and K. pneumoniae, and to a less extent against A. baumannii. Similar improvements in activity were observed with the reversed amide derivative 34 and the oxyphenyl analog 35.
We also sought to validate MreB as the antibacterial target of our analogs. To this end, we selected six representative analogs and evaluated the compounds for their abilities to inhibit the catalytic (ATPase) activity of purified E. coli MreB (EcMreB). Significantly, all six compounds tested exhibited concentration-dependent inhibition of EcMreB ATPase activity (Fig. 1), establishing their antibacterial actions as MreB inhibitors. In addition, the relative EcMreB-inhibiting potencies of the compounds (as reflected by the magnitudes of the IC50 values shown in Table 4) were generally correlated with their corresponding antibacterial activities, further validating MreB as the antibacterial target of our compounds. Among the analogs tested, 31 was the most potent inhibitor of EcMreB activity. Significantly, this analog also exhibited the best antibacterial activity against K. pneumoniae of all compounds evaluated here and was among the best agents versus E. coli and A. baumannii as well. Analog 31 may thus represent an important reference compound for the development of next-generation MreB inhibitors.
The structure activity studies performed with these varied analogs of CBR-4830 indicate that in many instances significant antibacterial activity can be retained. In addition, it is possible to reduce the toxicity associated with CBR-4830 and improve its formulation properties. The extreme toxicity of A22 and CBR-4830 has hindered efforts to demonstrate the in vivo efficacy of these MreB inhibitors. In view of their unique mechanism of action, such agents would be anticipated to be useful against multidrug-resistant bacteria. The data on the structure-activity of these CBR-4830 derivatives suggest that the development of next-generation MreB inhibitors could overcome this limitation and provide a means for demonstrating the potential clinical efficacy and utility of MreB inhibitors as antibiotics. Evaluation of comparative pharmacokinetic parameters and efficacy in vivo remain essential for assessing the potential of this novel class of antibiotics.
Chemistry: General Methods
All reactions, unless otherwise stated, were done under nitrogen atmosphere. Reaction monitoring and follow-up were done using aluminum backed Silica G TLC plates with UV254 (Sorbent Technologies), visualizing with ultraviolet light. Flash column chromatography was done on a Combi Flash Rf Teledyne ISCO using hexane, ethyl acetate, dichloromethane, and methanol. The 1H (400 MHz) and 13C (100 MHz) NMR spectra were done in CDCl3, Methanol-d4, and DMSO-d6 and recorded on a Bruker Avance III (400 MHz) Multinuclear NMR Spectrometer. Data is expressed in parts per million relative to the residual nondeuterated solvent signals, spin multiplicities are given as s (singlet), d (doublet), dd (doublet of doublets), t (triplet), dt (doublet of triplets), q (quartet), m (multiplet), and bs (broad singlet), and coupling constants (J) are reported in Hertz. Analytical HPLC was performed on a Shimadzu LC-20AT Prominence liquid chromatograph using a 150 x 4.6 mm Princeton SPHER-100 RP C18 1000A 5 um column using 0% water for 2 minutes and a 0-100% water/methanol gradient over a 5-minute period and 5 minutes at 100% methanol at a 2.0 ml/minute flow rate monitoring uv absorbance at 254 and 296 nm. Using this method of analysis, the purity of all compounds used in bioassays was determined to be ≥ 95%. Mass spectrometry (MS) was performed by electrospray (ESI) ionization using a Shimadzu 2020 LC-MS quadrupole mass spectrometer using a 0-100% water/methanol gradient over a 3-minute period. HRMS experiments were conducted using the Waters ACQUITY UPLC – Synapt G2 HRMS (Milford, MA) to determine the accurate mass values of final compounds. UPLC separation was performed on an ACQUITY UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) with 0.1% formic acid in water and 0.1% formic acid in methanol as mobile phase A and B, respectively. The column temperature was maintained at 40°C. The gradient was set from 5% to 95% B over 3 minutes at a flow rate of 0.25 mL/min. Extensive fragmentation under the conditions used for these HRMS analyses did not allow for the detection of parent ions. HRMS data obtained under these conditions are provided for several of the compounds evaluated in Supplemental Materials.
2-Bromo-5,6,7,8,9,10-hexahydrocyclohepta[b]indol-6-amine (8)
2-Bromo-7,8,9,10-tetrahydrocyclohepta[b]indol-6(5H)-one (100 mg, 0.36 mmol), ammonium acetate (277 mg, 3.60 mmol) and sodium cyanoborohydride (113 mg, 1.80 mmol) were dissolved in ethanol (10 mL). The mixture was stirred for overnight at 60 oC. The reaction mixture was acidified with 6N HCl, and it was washed with ethyl acetate. Then, the aqueous layer was basified with NaOH, and it was extracted with ethyl acetate. The organic layer was washed with brine, and it was dried over Na2SO4. The organic layer was concentrated under reduced pressure, and the residue was purified on an ISCO chromatograph (0-10% methanol/ dichloromethane + 0.1% NH4OH) to give the product as a white solid (34 mg, 34%); 1H NMR (300 MHz) (CD3OD) δ 10.76 (bs, 1H), 7.51 (d, J = 2 Hz, 1H), 7.24 (d, J = 8 Hz, 1H), 7.03 (dd, J = 8 Hz, J = 2 Hz, 1H), 4.01-3.97 (m, 1H), 2.86-2.81 (m, 1H), 2.58-2.56 (m, 1H), 1.99-1.84 (m, 2H), 1.71-1.50 (m, 4H); LC/MS RT = 2.57 (M-H-: 277/279).
(E)-2-(Hydroxymethylene)cycloheptan-1-one
Cycloheptanone (2.95 mL, 25 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL), and it was cooled to 0 oC. A solution of 1.0 M LiHMDS in tetrahydrofuran (30 mL, 30 mmol) was slowly added, and it was stirred for 5 minutes at 0 oC. Ethyl formate (2.42 mL, 30 mmol) was slowly added, and it was stirred for 2 hours at 0 oC. The reaction mixture was diluted with ethyl acetate, and it was washed with 6N HCl and brine. The organic layer was dried over Na2SO4. The organic layer was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-20% ethyl acetate/hexane) to give product as a colorless oil (1.30 g, 37%); 1H NMR (300 MHz) (CDCl3) δ 7.59 (d, J = 9 Hz, 1H), 2.51-2.47 (m, 2H), 2.23-2.19 (m, 2H), 1.73-1.54 (m, 6H).
(E)-2-(2-(4-Bromophenyl)hydrazineylidene)cycloheptan-1-one
To a solution of 4-bromoaniline (1.60 g, 9.30 mmol) in concentrated hydrochloric acid (2 mL), a solution of sodium nitrite (642 mg, 9.30 mmol) in water (4 mL) was added slowly at 0 oC. The mixture was stirred for 30 minutes at 0 oC. In a separate round bottom flask, (E)-2-(hydroxymethylene)cycloheptan-1-one (1.30 g, 9.30 mmol) was dissolve in methanol (12 mL). To the mixture, a solution of sodium acetate (1.91 g, 23.3 mmol) in water (5 mL) was added slowly at 0 oC. The mixture was stirred for 20 minutes at 0 oC. Then, the freshly prepared diazonium salt solution was slowly added. The mixture was stirred for additional 30 minutes at 0 oC, and the formed yellow suspension was filtered to give the product as a yellow solid (2.17 g, 79%); 1H NMR (300 MHz) (DMSO-d6) δ 13.27 (s, 1H), 7.43 (d, J = 9 Hz, 2H), 7.23 (d, J = 9 Hz, 2H), 2.62-2.56 (m, 4H), 1.68 (m, 6H).
2-Bromo-7,8,9,10-tetrahydrocyclohepta[b]indol-6(5H)-one (8a)
(E)-2-(2-(4-Bromophenyl)hydrazineylidene)cycloheptan-1-one (2.17 g, 7.35 mmol) was dissolved in a mixture of concentrated hydrochloric acid (2 mL) and acetic acid (8 mL), and it was stirred for 30 minutes at 130 oC. The resulting dark brown suspension was diluted with ethyl acetate, and it was washed with 10% NaOH and brine. The organic layer was dried over Na2SO4, and it was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-20% ethyl acetate/hexane) to give product as a yellow solid (472 mg, 23%); 1H NMR (300 MHz) (CDCl3) δ 8.90 (bs, 1H), 7.79 (s, 1H), 7.41 (dd, J = 9 Hz, J = 2 Hz, 1H), 7.26-7.23 (m, 1H), 3.11-3.07 (m, 2H), 2.86-2.82 (m, 2H), 2.11-1.99 (m, 4H).
1,2-Dichloro-5,6,7,8,9,10-hexahydrocyclohepta[b]indol-6-amine (9).
1,2-Dichloro-7,8,9,10-tetrahydrocyclohepta[b]indol-6(5H)-one (99 mg, 0.37 mmol), ammonium acetate (285 mg, 3.70 mmol) and sodium cyanoborohydride (116 mg, 1.85 mmol) were dissolved in ethanol (10 mL). The reaction mixture was stirred for overnight at 60 oC. After removal of solvent, the reaction mixture was diluted with ethyl acetate. The organic layer was washed with 10% sodium hydroxide and brine, and it was dried over sodium sulfate. The organic layer was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-10% methanol/dichloromethane + 0.1% NH4OH) to give product as a white solid. The solid was then treated with 4N hydrochloric acid in dioxane, and it was stirred for 30 minutes at room temperature. The white suspension was concentrated under reduced pressure, and the resulting residue was suspended in ethyl acetate. The suspension was filtered to give hydrochloric acid salt of product as a white solid (42 mg, 37%); 1H NMR (300 MHz) (DMSO-d6) δ 11.65 (bs, 1H), 8.42 (bs, 3H), 7.37 (d, J = 8 Hz, 1H), 7.25 (d, J = 9 Hz, 1H), 4.58 (m, 1H), 3.65-3.60 (m, 2H), 3.12-2.98 (m, 2H), 1.97-1.89 (m, 4H); LC/MS RT = 2.90 (M-H-: 267/269).
tert-Butyl 1,2-dichloro-6-oxo-7,8,9,10-tetrahydrocyclohepta[b]indole-5(6H)-carboxylate (9a) and tert-Butyl 2,3-dichloro-6-oxo-7,8,9,10-tetrahydrocyclohepta[b]indole-5(6H)-carboxylate (10a)
To a solution of 3,4-dichloroaniline (1.53 g, 9.42 mmol) in concentrated hydrochloric acid (10 mL), a solution of sodium nitrite (650 mg, 9.42 mmol) in water (20 mL) was added slowly at 0 oC. The mixture was stirred for 30 minutes at 0 oC. In a separate round bottom flask, (E)-2-(hydroxymethylene)cycloheptan-1-one (1.32 g, 9.42 mmol) was dissolve in methanol (12 mL). To the mixture, a solution of sodium acetate (1.93 g, 23.55 mmol) in water (5 mL) was added slowly at 0 oC. The mixture was stirred for 20 minutes at 0 oC. Then, the freshly prepared diazonium salt solution was slowly added. The mixture was stirred for additional 30 minutes at 0 oC. The reaction mixture was diluted with ethyl acetate, and the organic layer was washed with saturated sodium bicarbonate followed brine. The organic layer was concentrated under reduced pressure and the resulted dark brown oil was carried next step without further purification.
The residue was dissolved in formic acid (10 mL), and it was stirred for 2 hours at 100 oC. The resulting dark brown suspension was diluted with ethyl acetate, and it was washed with 10% NaOH and brine. The organic layer was dried over sodium sulfate, and it was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-20% ethyl acetate/hexane) to give a mixture of two regioisomers, 1,2-dichloro-7,8,9,10-tetrahydrocyclohepta[b]indol-6(5H)-one and 2,3-dichloro-7,8,9,10-tetrahydrocyclohepta[b]indol-6(5H)-one as a beige solid (212 mg, 8%).
To a solution of a mixture of the two regioisomers (212 mg, 0.79 mmol) in tetrahydrofuran (10 mL), Boc anhydride (345 mg, 1.58 mmol) and DMAP (89 mg, 0.79 mmol) were added. The reaction mixture was stirred for 3 hours. The reaction mixture was diluted with ethyl acetate, and it was washed with saturated ammonium chloride, followed by brine. The organic layer was dried over sodium sulfate, and it was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-10% ethyl acetate/hexane) to give tert-butyl 1,2-dichloro-6-oxo-7,8,9,10-tetrahydrocyclohepta[b]indole-5(6H)-carboxylate (152 mg, 49%). 1H NMR (300 MHz) (CDCl3) δ 7.93 (d, J = 9 Hz, 1H), 7.41 (d, J = 9 Hz, 1H), 3.42-3.38 (m, 2H), 2.87-2.83 (m, 2H), 1.97-1.95 (m, 4H), 1.55 (s, 9H). along with tert-butyl 2,3-dichloro-6-oxo-7,8,9,10-tetrahydrocyclohepta[b]indole-5(6H)-carboxylate (59 mg, 20%). 1H NMR (300 MHz) (CDCl3) δ 8.20 (s, 1H), 7.62 (s, 1H), 2.91 (m, 2H), 2.85 (m, 2H), 2.02-1.98 (m, 4H), 1.57 (s, 9H).
1,2-Dichloro-7,8,9,10-tetrahydrocyclohepta[b]indol-6(5H)-one (9a)
To a solution of tert-butyl 1,2-dichloro-6-oxo-7,8,9,10-tetrahydrocyclohepta[b]indole-5(6H)-carboxylate (152 mg, 0.41 mmol) in dichloromethane (5 mL), trifluoroacetic acid (1 mL) was added. The reaction mixture was stirred for an hour at room temperature. After removal of solvent, the mixture was diluted with ethyl acetate, and it was washed with 10% sodium hydroxide and brine. The organic layer was dried over sodium sulfate, and it was concentrated under reduced pressure to give product as a white solid (99 mg, 90%); 1H NMR (300 MHz) (CDCl3) δ 9.08 (bs, 1H), 7.35 (d, J = 9 Hz, 1H), 7.20 (d, J = 9 Hz, 1H), 3.58-3.54 (m, 2H), 2.87-2.83 (m, 2H), 2.11-2.04 (m, 2H), 1.99-1.91 (m, 2H).
2,3-Dichloro-7,8,9,10-tetrahydrocyclohepta[b]indol-6(5H)-one (10a)
To a solution of tert-butyl 2,3-dichloro-6-oxo-7,8,9,10-tetrahydrocyclohepta[b]indole-5(6H)-carboxylate (943 mg, 2.64 mmol) in dichloromethane (25 mL), trifluoroacetic acid (5 mL) was added. The reaction mixture was stirred for 2 hours at room temperature. After removal of solvent, the mixture was diluted with ethyl acetate, and it was washed with 10% sodium hydroxide and brine. The organic layer was dried over sodium sulfate, and it was concentrated under reduced pressure to give product as a white solid (675 mg, 95%); 1H NMR (300 MHz) (CDCl3) δ 8.90 (bs, 1H), 7.74 (s, 1H), 7.48 (s, 1H), 3.10-3.06 (m, 2H), 2.87-2.83 (m, 2H), 2.11-2.05 (m, 2H), 2.01-1.99 (m, 2H).
2,3-Dichloro-5,6,7,8,9,10-hexahydrocyclohepta[b]indol-6-amine (10).
2,3-Dichloro-7,8,9,10-tetrahydrocyclohepta[b]indol-6(5H)-one (100 mg, 0.37 mmol), ammonium acetate (285 mg, 3.70 mmol) and sodium cyanoborohydride (116 mg, 1.85 mmol) were dissolved in ethanol (10 mL). The reaction mixture was stirred for overnight at 60 oC. After removal of solvent, the reaction mixture was diluted with ethyl acetate. The organic layer was washed with 10% sodium hydroxide and brine, and it was dried over sodium sulfate. The organic layer was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-10% methanol/dichloromethane + 0.1% NH4OH) to give product as a white solid. The solid was then treated with 4N hydrochloric acid in dioxane, and it was stirred for 30 minutes at room temperature. The white suspension was concentrated under reduced pressure, and the resulting residue was suspended in ethyl acetate. The suspension was filtered to give hydrochloric acid salt of product as a white solid (76 mg, 67%); 1H NMR (300 MHz) (DMSO-d6) δ 11.36 (bs, 1H), 8.44 (bs, 3H), 7.78 (s, 1H), 7.62 (s, 1H), 4.56 (m, 1H), 3.55-3.26 (m, 2H), 2.89-2.71 (m, 4H), 1.93-1.76 (m, 2H); LC/MS RT = 2.91 (M-H-: 267/269).
7-Bromo-1,2,3,4-tetrahydrocyclopenta[b]indol-3-amine (11)
7-Bromo-1,4-dihydrocyclopenta[b]indol-3(2H)-one (100 mg, 0.40 mmol), ammonium acetate (308 mg, 4.00 mmol) and sodium cyanoborohydride (126 mg, 2.00 mmol) were dissolved in ethanol (10 mL). The mixture was stirred for overnight at 60 oC. An additional ammonium acetate (308 mg, 4.00 mmol) and sodium cyanoborohydride (126 mg, 2.00 mmol) along with catalytic amount of acetic acid were added. The mixture was stirred for 5 hours at 85 oC. The reaction mixture was acidified with 6N HCl, and it was washed with ethyl acetate. Then, the aqueous layer was basified with NaOH, and it was extracted with ethyl acetate. The organic layer was washed with brine, and it was dried over Na2SO4. The organic layer was concentrated under reduced pressure, and the residue was purified on an ISCO chromatograph (0-10% methanol/dichloromethane + 0.1% NH4OH) to give the product as a white solid (17 mg, 17%); 1H NMR (300 MHz) (CD3OD) δ 7.48 (d, J = 1 Hz, 1H), 7.22 (d, J = 9 Hz, 1H), 7.12 (dd, J = 9 H, J = 2 Hz, 1H), 4.43-4.41 (m, 1H), 2.94-2.83 (m, 2H), 2.78-2.62 (m, 1H), 2.21-2.12 (m, 1H); LC/MS RT = 2.57 (M-H-: 249/251).
5 (E)-2-(Hydroxymethylene)cyclopentan-1-one
Cyclopentanone (2.21 mL, 25 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL), and it was cooled to 0 oC. A solution of 1.0 M LiHMDS in tetrahydrofuran (30 mL, 30 mmol) was slowly added, and it was stirred for 5 minutes at 0 oC. Ethyl formate (2.42 mL, 30 mmol) was slowly added, and it was stirred for 2 hours at 0 oC. The reaction mixture was diluted with ethyl acetate, and it was washed with 6N HCl and brine. The organic layer was dried over Na2SO4, and it was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-30% ethyl acetate/hexane) to give product as a white solid (772 mg, 28%); 1H NMR (300 MHz) (CDCl3) δ 7.21 (s, 1H), 2.56-2.51 (m, 2H), 2.43-2.37 (m, 2H), 2.01-1.94 (m, 2H).
(E)-2-(2-(4-Bromophenyl)hydrazineylidene)cyclopentan-1-one
To a solution of 4-bromoaniline (1.07 g, 6.23 mmol) in concentrated hydrochloric acid (2 mL), a solution of sodium nitrite (430 mg, 6.23 mmol) in water (4 mL) was added slowly at 0 oC. The mixture was stirred for 30 minutes at 0 oC. In a separate round bottom flask, 5 (E)-2-(hydroxymethylene)cyclopentan-1-one (700 mg, 6.23 mmol) was dissolve in methanol (12 mL). To the mixture, a solution of sodium acetate (1.28 g, 15.58 mmol) in water (5 mL) was added slowly at 0 oC. The mixture was stirred for 20 minutes at the temperature. Then, the freshly prepared diazonium salt solution was slowly added. The mixture was stirred for additional 30 minutes at the temperature, and the formed yellow suspension was filtered to give the product as a yellow solid (1.47 g, 89%); 1H NMR (300 MHz) (DMSO-d6) δ 10.03 (s, 1H), 7.42 (d, J = 9 Hz, 2H), 7.19 (d, J = 9 Hz, 2H), 2.65-2.60 (m, 2H), 2.34-2.29 (m, 2H), 1.99-1.95 (m, 2H).
7-Bromo-1,4-dihydrocyclopenta[b]indol-3(2H)-one (11a)
(E)-2-(2-(4-Bromophenyl)hydrazineylidene)cyclopentan-1-one (1.47 g, 5.50 mmol) was dissolved in a mixture of concentrated hydrochloric acid (2 mL) and acetic acid (8 mL), and it was stirred for 30 minutes at 130 oC. The resulting dark brown suspension was diluted with ethyl acetate, and it was washed with 10% NaOH and brine. The organic layer was dried over Na2SO4, and it was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-20% ethyl acetate/hexane) to give product as a beige solid (116 mg, 8%); 1H NMR (300 MHz) (CDCl3) δ 8.80 (bs, 1H), 7.87 (d, J = 2 Hz, 1H), 7.48 (dd, J = 9 H, J = 2 Hz, 1H), 7.35 (d, J = 9 Hz, 1H), 3.10-3.07 (m, 2H), 3.03-3.00 (m, 2H).
6,7-Dichloro-1,2,3,4-tetrahydrocyclopenta[b]indol-3-amine. (12).
6,7-Dichloro-1,4-dihydrocyclopenta[b]indol-3(2H)-one (148 mg, 0.62 mmol), ammonium acetate (956 mg, 12.4 mmol) and sodium cyanoborohydride (195 mg, 3.1 mmol) were dissolved in ethanol (20 mL). The reaction mixture was stirred for overnight at 60 oC. After removal of solvent, the reaction mixture was diluted with ethyl acetate. The organic layer was washed with 10% sodium hydroxide and brine, and it was dried over sodium sulfate. The organic layer was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-10% methanol/dichloromethane + 0.1% NH4OH) to give product as a white solid (93 mg, 62%); 1H NMR (300 MHz) (MeOD) δ 7.46 (s, 1H), 7.45 (s, 1H), 4.43-4.39 (m, 1H), 2.93-2.79 (m, 2H), 2.71-2.61 (m, 1H), 2.21-2.12 (m, 1H); LC/MS RT = 2.74 (M-H-: 239/241).
6,7-Dichloro-1,4-dihydrocyclopenta[b]indol-3(2H)-one (12a)
To a solution of tert-butyl 6,7-dichloro-3-oxo-2,3-dihydrocyclopenta[b]indole-4(1H)-carboxylate (571 mg, 1.68 mmol) in dichloromethane (25 mL), trifluoroacetic acid (5 mL) was added. The reaction mixture was stirred for an hour at room temperature. After removal of solvent, the mixture was diluted with ethyl acetate, and it was washed with 10% sodium hydroxide and brine. The organic layer was dried over sodium sulfate, and it was concentrated under reduced pressure to give product as a white solid (148 mg, 37%); 1H NMR (300 MHz) (DMSO-d6) δ 11.98 (bs, 1H), 8.06 (s, 1H), 7.64 (s, 1H), 3.02-2.99 (m, 2H), 2.90-2.87 (m, 2H).
7,8-Dichloro-1,2,3,4-tetrahydrocyclopenta[b]indol-3-amine (13)
7,8-Dichloro-1,4-dihydrocyclopenta[b]indol-3(2H)-one (100 mg, 0.42 mmol), ammonium acetate (647 mg, 8.40 mmol) and sodium cyanoborohydride (132 mg, 2.10 mmol) were dissolved in ethanol (10 mL). The reaction mixture was stirred for overnight at 60 oC. After removal of solvent, the reaction mixture was diluted with ethyl acetate. The organic layer was washed with 10% sodium hydroxide and brine, and it was dried over sodium sulfate. The organic layer was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-10% methanol/dichloromethane + 0.1% NH4OH) to give product as a white solid (41 mg, 41%); 1H NMR (300 MHz) (MeOD) δ 7.22 (d, J = 9 Hz, 1H), 7.09 (d, J = 9 Hz, 1H), 4.47-4.44 (m, 1H), 3.16-3.07 (m, 1H), 2.95-2.83 (m, 2H), 2.24-2.16 (m, 1H); LC/MS RT = 2.74 (M-H-: 239/241).
7,8-Dichloro-1,4-dihydrocyclopenta[b]indol-3(2H)-one (13a)
To a solution of tert-butyl 7,8-dichloro-3-oxo-2,3-dihydrocyclopenta[b]indole-4(1H)-carboxylate (121 mg, 0.36 mmol) in dichloromethane (5 mL), trifluoroacetic acid (1 mL) was added. The reaction mixture was stirred for an hour at room temperature. After removal of solvent, the mixture was diluted with ethyl acetate, and it was washed with 10% sodium hydroxide and brine. The organic layer was dried over sodium sulfate, and it was concentrated under reduced pressure to give product as a white solid (27 mg, 31%); 1H NMR (300 MHz) (CDCl3) δ 9.61 (bs, 1H), 7.41 (d, J = 9 Hz, 1H), 7.36 (d, J = 9 Hz, 1H), 3.35-3.32 (m, 2H), 3.09-3.03 (m, 2H).
tert-Butyl 7,8-dichloro-3-oxo-2,3-dihydrocyclopenta[b]indole-4(1H)-carboxylate (12b) and
tert-Butyl 6,7-dichloro-3-oxo-2,3-dihydrocyclopenta[b]indole-4(1H)-carboxylate (13b)
To a suspension of 3,4-dichloroaniline (8.10 g, 50.00 mmol) in water (30 mL), concentrated hydrochloric acid (12.5 mL) was slowly added at 0 oC. Then, a solution of sodium nitrite (3.45 g, 50.00 mmol) in water (35 mL) was slowly added at 0 oC. The mixture was stirred for 30 minutes at 0 oC. This freshly prepared diazonium salt solution was slowly added to a solution of 2-oxocyclopentane-1-carboxylic acid (6.40 g, 50.00 mmol) in concentrated hydrochloric acid (4.58 mL). The mixture was stirred for additional 30 minutes at 0 oC to give a yellow suspension. The suspension was filtered to give intermediate, (E)-2-(2-(3,4-dichlorophenyl)hydrazineylidene)-cyclopentan-1-one, as an orange solid.
The intermediate was then dissolved in acetonitrile (50 mL), and 1.8M sulfuric acid was added. The reaction mixture was stirred for overnight at 75 oC. The resulting dark brown suspension was diluted with ethyl acetate, and it was washed with 10% NaOH and brine. The organic layer was dried over sodium sulfate, and it was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-20% ethyl acetate/hexane) to give a mixture of two regioisomers, 7,8-dichloro-1,4-dihydrocyclopenta[b]indol-3(2H)-one and 6,7-dichloro-1,4-dihydrocyclopenta[b]indol-3(2H)-one as a dark brown solid (493 mg, 10%).
To a solution of a mixture of the two regioisomers (493 mg, 2.05 mmol) in tetrahydrofuran (25 mL), Boc anhydride (895 mg, 4.10 mmol) and DAMP (230 mg, 2.05 mmol) were added. The reaction mixture was stirred for 3 hours. The reaction mixture was diluted with ethyl acetate, and it was washed with saturated ammonium chloride, followed by brine. The organic layer was dried over sodium sulfate, and it was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-10% ethyl acetate/hexane) to give tert-butyl 7,8-dichloro-3-oxo-2,3-dihydrocyclopenta[b]indole-4(1H)-carboxylate (121 mg, 17%). 1H NMR (300 MHz) (CDCl3) δ 8.15 (d, J = 9 Hz, 1H), 7.48 (d, J = 9 Hz, 1H), 3.26-3.22 (m, 2H), 3.02-2.99 (m, 2H), 1.68 (s, 9H). along with tert-butyl 6,7-dichloro-3-oxo-2,3-dihydrocyclopenta[b]indole-4(1H)-carboxylate (160 mg, 23%). 1H NMR (300 MHz) (CDCl3) δ 8.47 (s, 1H), 7.73 (s, 1H), 3.01-3.00 (m, 4H), 1.69 (s, 9H).
1-(5-Bromo-1H-indol-2-yl)ethan-1-amine (14)
1-(5-Bromo-1H-indol-2-yl)ethan-1-one (95 mg, 0.40 mmol), ammonium acetate (308 mg, 4.00 mmol) and sodium cyanoborohydride (126 mg, 2.00 mmol) were dissolved in ethanol (5 mL). The mixture was stirred for overnight at 60 oC. The reaction mixture was diluted with ethyl acetate. The organic layer was washed with 10% NaOH and brine, and it was dried over Na2SO4. The organic layer was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-10% methanol/dichloromethane + 0.1% NH4OH) to give the product as a yellow oil (32 mg, 33%);1H NMR (300 MHz) (DMSO-d6) δ 11.07 (bs, 1H), 7.57 (d, J = 2 Hz, 1H), 7.24 (d, J = 9 Hz, 1H), 7.08 (dd, J = 8 Hz, J = 2 Hz, 1H), 6.18 (s, 1H), 4.11-4.04 (m, 1H), 1.34 (d, J = 7 Hz, 3H); LC/MS RT = 2.50 (M-H-: 237/239).
5-Bromo-N-methoxy-N-methyl-1H-indole-2-carboxamide (14b)
5-Bromo-1H-indole-2-carboxylic acid (1.0 g, 4.17 mmol), N,O-dimethylhydroxylamine hydrochloride (611 mg, 6.26 mmol), HOBt (563 mg, 4.17 mmol), EDC hydrochloride (1.68 g, 8.76 mmol) and triethylamine (2.32 mL, 16.68 mmol) were dissolved in anhydrous DMF (40 mL). The mixture was stirred for overnight at room temperature. The reaction mixture was diluted with ethyl acetate, and the organic layer was washed with water, saturated NH4Cl, 10% NaOH and brine. The organic layer was dried over Na2SO4, and it was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-100% ethyl acetate/hexane) to give product as a white solid (800 mg, 68%); 1H NMR (300 MHz) (CDCl3) δ 9.33 (bs, 1H), 7.83 (s, 1H), 7.38 (dd, J = 9 Hz, J = 1 Hz, 1H), 7.31 (d, J = 9 Hz, 1H), 7.15 (s, 1H), 3.85 (s, 3H), 3.43 (s, 3H).
1-(5-Bromo-1H-indol-2-yl)ethan-1-one (14c)
5-Bromo-N-methoxy-N-methyl-1H-indole-2-carboxamide (600 mg, 2.12 mmol) was dissolved in anhydrous tetrahydrofuran (50 mL). The mixture was cooled to -78 oC, then, a solution of 1.6 M methyllithium in diethyl ether (4.00 mL, 6.36 mmol) was added. The mixture was stirred for 2 hours at -78 oC. An additional solution of 1.6 M methyllithium in diethyl ether (4.00 mL, 6.36 mmol) was added. The mixture was stirred for an hour at -78 oC. The reaction was stopped by addition of water (10 mL). After removal of the solvent, the reaction mixture was diluted with ethyl acetate. The organic layer was washed with saturated NH4Cl and brine, and it was dried over Na2SO4. The organic layer was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-20% ethyl acetate/hexane) to give product as a white solid (344 mg, 68%); 1H NMR (300 MHz) (CDCl3) δ 9.19 (bs, 1H), 7.85 (s, 1H), 7.42 (dd, J = 9 Hz, J = 2 Hz, 1H), 7.31 (d, J = 8 Hz, 1H), 7.12 (s, 1H), 2.60 (s, 3H).
1-(1H-indol-2-yl)ethan-1-amine (15).
1-(1H-Indol-2-yl)ethan-1-one (100 mg, 0.63 mmol), ammonium acetate (486 mg, 6.30 mmol) and sodium cyanoborohydride (198 mg, 3.15 mmol) were dissolved in ethanol (10 mL). The mixture was stirred for overnight at 60 oC. The reaction mixture was diluted with ethyl acetate. The organic layer was washed with 10% NaOH and brine, and it was dried over Na2SO4. The organic layer was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (100% dichloromethane followed by 10% methanol/dichloromethane + 0.1% NH4OH) to give the product as a colorless oil (85 mg, 84%);1H NMR (300 MHz) (CD3OD) δ 7.44 (d, J = 8 Hz, 1H), 7.29 (d, J = 8 Hz, 1H), 7.03 (t, J = 8 Hz, 1H), 6.94 (t, J = 7 Hz, 1H), 6.29 (s, 1H), 4.24-4.17 (m, 1H), 1.51 (d, J = 7 Hz, 3H); LC/MS RT = 2.19 (M-H-: 159).
N-Methoxy-N-methyl-1H-indole-2-carboxamide (15b)
1H-Indole-2-carboxylic acid (1.0 g, 6.21 mmol), N,O-dimethylhydroxylamine hydrochloride (909 mg, 9.32 mmol), HOBt (839 mg, 6.21 mmol), EDC hydrochloride (2.5 g, 13.04 mmol) and triethylamine (3.46 mL, 24.84 mmol) were dissolved in anhydrous DMF (40 mL). The mixture was stirred for overnight at room temperature. The reaction mixture was diluted with ethyl acetate, and the organic layer was washed with water, saturated NH4Cl, 10% NaOH and brine. The organic layer was dried over Na2SO4, and it was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-30% ethyl acetate/hexane) to give product as a white solid (450 mg, 35%); 1H NMR (300 MHz) (CDCl3) δ 9.30 (bs, 1H), 7.70 (d, J = 8 Hz, 1H), 7.44 (d, J = 8 Hz, 1H), 7.31 (t, J = 8 Hz, 1H), 7.25 (s, 1H), 7.14 (t, J = 8 Hz, 1H), 3.85 (s, 3H), 3.44 (s, 3H).
1-(1H-Indol-2-yl)ethan-1-one (15c)
N-Methoxy-N-methyl-1H-indole-2-carboxamide (450 mg, 2.20 mmol) was dissolved in anhydrous tetrahydrofuran (50 mL). The mixture was cooled to -78oC, then, a solution of 1.6 M methyllithium in diethyl ether (4.13 mL, 6.60 mmol) was added. The mixture was stirred for 2 hours at -78oC. An additional solution of 1.6 M methyllithium in diethyl ether (4.13 mL, 6.60 mmol) was added. The mixture was stirred for an hour at -78oC. The reaction was stopped by addition of water (10 mL). After removal of the solvent, the reaction mixture was diluted with ethyl acetate. The organic layer was washed with saturated NH4Cl and brine, and it was dried over Na2SO4. The organic layer was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-20% ethyl acetate/hexane) to give product as a white solid (290 mg, 83%); 1H NMR (300 MHz) (CDCl3) δ 9.37 (bs, 1H), 7.72 (d, J = 8 Hz, 1H), 7.45 (d, J = 8 Hz, 1H), 7.38-7.33 (m, 1H), 7.22-7.14 (m, 2H), 2.62 (s, 3H).
1-(5-Fluoro-1H-indol-2-yl)ethan-1-amine (16)
1-(5-Fluoro-1H-indol-2-yl)ethan-1-one (100 mg, 0.56 mmol), ammonium acetate (432 mg, 5.60 mmol) and sodium cyanoborohydride (176 mg, 2.80 mmol) were dissolved in ethanol (10 mL). The mixture was stirred for overnight at 60 oC. The reaction mixture was acidified with 6N HCl, and it was washed with ethyl acetate. Then, the aqueous layer was basified with NaOH, and it was extracted with ethyl acetate. The organic layer was washed with brine, and it was dried over Na2SO4. The organic layer was concentrated under reduced pressure, and the residue was purified on an ISCO chromatograph (0-10% methanol/dichloromethane + 0.1% NH4OH) to give the product as a white solid (61 mg, 60%);1H NMR (300 MHz) (CD3OD) δ 7.25-7.21 (m, 1H), 7.10 (d, J = 8 Hz, 1H), 6.79 (t, J = 8 Hz, 1H), 6.28 (s, 1H), 4.22-4.17 (m, 1H), 1.49 (d, J = 6 Hz, 3H); LC/MS RT = 2.33 (M-H-: 177).
5-Fluoro-N-methoxy-N-methyl-1H-indole-2-carboxamide (16b)
5-Fluoro-1H-indole-2-carboxylic acid (1.0 g, 5.58 mmol), N, O-dimethylhydroxylamine hydrochloride (816 mg, 8.37 mmol), HOBt (754 mg, 5.58 mmol), EDC hydrochloride (2.25 g, 11.7 mmol) and triethylamine (3.11 mL, 22.3 mmol) were dissolved in anhydrous DMF (50 mL). The mixture was stirred for overnight at room temperature. The reaction mixture was diluted with ethyl acetate, and the organic layer was washed with water, saturated NH4Cl, 10% NaOH and brine. The organic layer was dried over Na2SO4, and it was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-50% ethyl acetate/hexane) to give product as a white solid (770 mg, 62%); 1H NMR (300 MHz) (CDCl3) δ 9.94 (bs, 1H), 7.41-7.36 (m, 1H), 7.32 (dd, J = 9 Hz, J = 2 Hz, 1H), 7.19 (s, 1H), 7.06 (td, J = 9 Hz, J = 3 Hz, 1H), 3.85 (s, 3H), 3.46 (s, 3H).
1-(5-Fluoro-1H-indol-2-yl)ethan-1-one (16c)
5-Fluoro-N-methoxy-N-methyl-1H-indole-2-carboxamide (300 mg, 1.35 mmol) was dissolved in anhydrous tetrahydrofuran (25 mL). The mixture was cooled to -78 oC, then, a solution of 1.6 M methyllithium in diethyl ether (2.53 mL, 4.05 mmol) was added. The mixture was stirred for 2 hours at -78 oC. The reaction was stopped by addition of water (10 mL). After removal of the solvent, the reaction mixture was diluted with ethyl acetate. The organic layer was washed with saturated NH4Cl and brine, and it was dried over Na2SO4. The organic layer was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-20% ethyl acetate/hexane) to give product as a white solid (220 mg, 92%); 1H NMR (300 MHz) (CDCl3) δ 9.18 (bs, 1H), 7.39-7.32 (m, 2H), 7.16-7.08 (m, 2H), 2.60 (s, 3H).
1-(5-(Trifluoromethyl)-1H-indol-2-yl)ethan-1-amine (17).
1-(5-Trifluoromethyl-1H-indol-2-yl)ethan-1-one (100 mg, 0.44 mmol), ammonium acetate (339 mg, 4.40 mmol) and sodium cyanoborohydride (138 mg, 2.20 mmol) were dissolved in ethanol (10 mL). The mixture was stirred for overnight at 60 oC. The reaction mixture was acidified with 6N HCl, and it was washed with ethyl acetate. Then, the aqueous layer was basified with NaOH, and it was extracted with ethyl acetate. The organic layer was washed with brine, and it was dried over Na2SO4. The organic layer was concentrated under reduced pressure, and the residue was purified on an ISCO chromatograph (0-10% methanol/dichloromethane + 0.1% NH4OH) to give the product as a white solid (42 mg, 42%);1H NMR (300 MHz) (DMSO-d6) δ 11.34 (bs, 1H), 7.79 (s, 1H), 7.46 (d, J = 9 Hz, 1H), 7.27 (d, J = 8 Hz, 1H), 6.36 (s, 1H), 4.15-4.09 (m, 1H), 1.37 (d, J = 6 Hz, 3H); LC/MS RT = 2.50 (M-H-: 227).
5-(Trifluoromethyl)-1H-indole-2-carboxylic acid (17a)
2-Iodo-4-(trifluoromethyl)aniline (1.0 g, 3.48 mmol), pyruvic acid (0.74 mL, 10.44 mmol), DABCO (1.17 g, 10.44 mmol) and Pd(OAc)2 (79 mg, 0.35 mmol) were dissolved in anhydrous DMF (10 mL). The mixture was purged with nitrogen, and it was stirred for 4 hours at 110 oC. The reaction mixture was diluted with ethyl acetate, and the organic layer was washed with 1N HCl and brine. The organic layer was dried over Na2SO4, and it was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-50% ethyl acetate/hexane) to give product as a beige solid (484 mg, 54%); 1H NMR (300 MHz) (CD3OD) δ 11.66 (bs, 1H), 7.98 (s, 1H), 7.57 (d, J = 9 Hz, 1H), 7.47 (d, J = 9 Hz, 1H), 7.26 (s, 1H).
N-Methoxy-N-methyl-5-(trifluoromethyl)-1H-indole-2-carboxamide (17b)
5-(Trifluoromethyl)-1H-indole-2-carboxylic acid (484 mg, 2.11 mmol), N, O-dimethyl-hydroxylamine hydrochloride (309 mg, 3.17 mmol), HOBt (285 mg, 2.11 mmol), EDC hydrochloride (849 mg, 4.43 mmol) and triethylamine (1.18 mL, 8.44 mmol) were dissolved in anhydrous DMF (50 mL). The mixture was stirred for overnight at room temperature. The reaction mixture was diluted with ethyl acetate, and the organic layer was washed with water, saturated NH4Cl, 10% NaOH and brine. The organic layer was dried over Na2SO4, and it was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-50% ethyl acetate/hexane) to give product as a white solid (402 mg, 70%); 1H NMR (300 MHz) (CDCl3) δ 9.97 (bs, 1H), 8.01 (s, 1H), 7.53 (m, 2H), 7.32 (s, 1H), 3.86 (s, 3H), 3.45 (s, 3H).
1-(5-(Trifluoromethyl)-1H-indol-2-yl)ethan-1-one (17c)
N-Methoxy-N-methyl-5-(trifluoromethyl)-1H-indole-2-carboxamide (300 mg, 1.10 mmol) was dissolved in anhydrous tetrahydrofuran (20 mL). The mixture was cooled to -78 oC, then, a solution of 1.6 M methyllithium in diethyl ether (2.06 mL, 3.30 mmol) was added. The mixture was stirred for 2 hours at -78 oC. The reaction was stopped by addition of water (10 mL). After removal of the solvent, the reaction mixture was diluted with ethyl acetate. The organic layer was washed with saturated NH4Cl and brine, and it was dried over Na2SO4. The organic layer was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-20% ethyl acetate/hexane) to give product as a white solid (197 mg, 80%); 1H NMR (300 MHz) (CDCl3) δ 9.40 (bs, 1H), 8.03 (s, 1H), 7.59-7.51 (m, 2H), 7.28 (s, 1H), 2.64 (s, 3H).
1-(5-Methoxy-1H-indol-2-yl)ethan-1-amine (18).
1-(5-Methoxy-1H-indol-2-yl)ethan-1-one (100 mg, 0.42 mmol), ammonium acetate (324 mg, 4.20 mmol) and sodium cyanoborohydride (132 mg, 2.10 mmol) were dissolved in ethanol (10 mL). The mixture was stirred for overnight at 60 oC. The reaction mixture was acidified with 6N HCl, and it was washed with ethyl acetate. Then, the aqueous layer was basified with NaOH, and it was extracted with ethyl acetate. The organic layer was washed with brine, and it was dried over Na2SO4. The organic layer was concentrated under reduced pressure, and the residue was purified on an ISCO chromatograph (0-10% methanol/dichloromethane + 0.1% NH4OH) to give the product as a white solid (58 mg, 58%);1H NMR (300 MHz) (DMSO-d6) δ 10.66 (bs, 1H), 7.15 (d, J = 9 Hz, 1H), 6.90 (d, J = 2 Hz, 1H), 6.61 (dd, J = 9 Hz, J = 2 Hz, 1H), 6.09 (s, 1H), 4.08-4.02 (m, 1H), 3.69 (s, 3H), 1.33 (d, J = 6 Hz, 3H); LC/MS RT = 2.17 (M-H-: 189).
N,5-Dimethoxy-N-methyl-1H-indole-2-carboxamide (18b)
5-Methoxy-1H-indole-2-carboxylic acid (1.0 g, 5.23 mmol), N, O-dimethylhydroxylamine hydrochloride (766 mg, 7.85 mmol), HOBt (707 mg, 5.23 mmol), EDC hydrochloride (2.10 g, 10.98 mmol) and triethylamine (2.92 mL, 20.92 mmol) were dissolved in anhydrous DMF (50 mL). The mixture was stirred for overnight at room temperature. The reaction mixture was diluted with ethyl acetate, and the organic layer was washed with water, saturated NH4Cl, 10% NaOH and brine. The organic layer was dried over Na2SO4, and it was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-50% ethyl acetate/hexane) to give product as a white solid (743 mg, 60%); 1H NMR (300 MHz) (CDCl3) δ 9.78 (bs, 1H), 7.35 (d, J = 9 Hz, 1H), 7.17 (s, 1H), 7.1 (d, J = 2 Hz, 1H), 6.98 (dd, J = 9 Hz, J = 2 Hz, 1H), 3.85 (s, 3H), 3.84 (s, 3H), 3.46 (s, 3H).
1-(5-Methoxy-1H-indol-2-yl)ethan-1-one (18c)
N,5-Dimethoxy-N-methyl-1H-indole-2-carboxamide (300 mg, 1.28 mmol) was dissolved in anhydrous tetrahydrofuran (20 mL). The mixture was cooled to -78oC, then, a solution of 1.6 M methyllithium in diethyl ether (2.40 mL, 3.84 mmol) was added. The mixture was stirred for 2 hours at -78oC. The reaction was stopped by addition of water (10 mL). After removal of the solvent, the reaction mixture was diluted with ethyl acetate. The organic layer was washed with saturated NH4Cl and brine, and it was dried over Na2SO4. The organic layer was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-20% ethyl acetate/hexane) to give product as a white solid (180 mg, 74%); 1H NMR (300 MHz) (CDCl3) δ 8.91 (bs, 1H), 7.31 (d, J = 9 Hz, 1H), 7.12-7.08 (m, 2H), 7.03 (dd, J = 9 Hz, J = 3 Hz, 1H), 3.85 (s, 3H), 2.58 (s, 3H).
1-(5-(Trifluoromethoxy)-1H-indol-2-yl)ethan-1-amine (19)
1-(5-(Trifluoromethoxy)-1H-indol-2-yl)ethan-1-one (100 mg, 0.41 mmol), ammonium acetate (316 mg, 4.10 mmol) and sodium cyanoborohydride (129 mg, 2.05 mmol) were dissolved in ethanol (10 mL). The mixture was stirred for overnight at 60 oC. The reaction mixture was acidified with 6N HCl, and it was washed with ethyl acetate. Then, the aqueous layer was basified with NaOH, and it was extracted with ethyl acetate. The organic layer was washed with brine, and it was dried over Na2SO4. The organic layer was concentrated under reduced pressure, and the residue was purified on an ISCO chromatograph (0-10% methanol/dichloromethane + 0.1% NH4OH) to give the product as a white solid (48 mg, 48%);1H NMR (300 MHz) (DMSO-d6) δ 11.15 (bs, 1H), 7.38 (s, 1H), 7.35 (d, J = 9 Hz, 1H), 6.94 (d, J = 8 Hz, 1H), 6.27 (s. 1H), 4.14-4.07 (m, 1H), 1.36 (d, J = 7 Hz, 3H); LC/MS RT = 2.55 (M-H-: 243).
5-(Trifluoromethoxy)-1H-indole-2-carboxylic acid (19a)
2-Iodo-4-(trifluoromethoxy)aniline (1.0 g, 3.30 mmol), pyruvic acid (0.70 mL, 9.90 mmol), DABCO (1.11 g, 9.90 mmol) and Pd(OAc)2 (74 mg, 0.33 mmol) were dissolved in anhydrous DMF (10 mL). The mixture was purged with nitrogen, and it was stirred for 4 hours at 110 oC. The reaction mixture was diluted with ethyl acetate, and the organic layer was washed with 1N HCl and brine. The organic layer was dried over Na2SO4, and it was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-50% ethyl acetate/hexane) to give product as a beige solid (592 mg, 73%); 1H NMR (300 MHz) (DMSO-d6) δ 12.02 (bs, 1H), 7.64 (s, 1H), 7.49 (d, J = 9 Hz, 1H), 7.20 (d, J = 6 Hz, 1H), 7.13 (s, 1H).
N-Methoxy-N-methyl-5-(trifluoromethoxy)-1H-indole-2-carboxamide (19b)
5-(Trifluoromethoxy)-1H-indole-2-carboxylic acid (592 mg, 2.41 mmol), N, O-dimethyl-hydroxylamine hydrochloride (353 mg, 3.62 mmol), HOBt (326 mg, 2.41 mmol), EDC hydrochloride (970 mg, 5.06 mmol) and triethylamine (1.18 mL, 8.44 mmol) were dissolved in anhydrous DMF (50 mL). The mixture was stirred for overnight at room temperature. The reaction mixture was diluted with ethyl acetate, and the organic layer was washed with water, saturated NH4Cl, 10% NaOH and brine. The organic layer was dried over Na2SO4, and it was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-70% ethyl acetate/hexane) to give product as a white solid (378 mg, 54%); 1H NMR (300 MHz) (CDCl3) δ 9.33 (bs, 1H), 7.55 (s, 1H), 7.42 (d, J = 9 Hz, 1H), 7.24 (s, 1H), 7.18 (d, J = 9 Hz, 1H), 3.85 (s, 3H), 3.44 (s, 3H).
1-(5-(Trifluoromethoxy)-1H-indol-2-yl)ethan-1-one (19c)
N-Methoxy-N-methyl-5-(trifluoromethoxy)-1H-indole-2-carboxamide (378 mg, 1.31 mmol) was dissolved in anhydrous tetrahydrofuran (20 mL). The mixture was cooled to -78 oC, then, a solution of 1.6 M methyllithium in diethyl ether (2.46 mL, 3.93 mmol) was added. The mixture was stirred for 2 hours at -78 oC. The reaction was stopped by addition of water (10 mL). After removal of the solvent, the reaction mixture was diluted with ethyl acetate. The organic layer was washed with saturated NH4Cl and brine, and it was dried over Na2SO4. The organic layer was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-20% ethyl acetate/hexane) to give product as a white solid (259 mg, 81%); 1H NMR (300 MHz) (CDCl3) δ 9.35 (bs, 1H), 7.57 (s, 1H), 7.44 (d, J = 9 Hz, 1H), 7.26-7.20 (m, 2H), 2.62 (s, 3H).
1-(4,6-Dichloro-1H-indol-2-yl)ethan-1-amine (20).
1-(4,6-Dichloro-1H-indol-2-yl)ethan-1-one (100 mg, 0.44 mmol), ammonium acetate (339 mg, 4.40 mmol) and sodium cyanoborohydride (138 mg, 2.20 mmol) were dissolved in ethanol (10 mL). The mixture was stirred for overnight at 60 oC. The reaction mixture was acidified with 6N HCl, and it was washed with ethyl acetate. Then, the aqueous layer was basified with NaOH, and it was extracted with ethyl acetate. The organic layer was washed with brine, and it was dried over Na2SO4. The organic layer was concentrated under reduced pressure, and the residue was purified on an ISCO chromatograph (0-10% methanol/dichloromethane + 0.1% NH4OH) to give the product as a white solid (55 mg, 54%);1H NMR (300 MHz) (DMSO-d6) δ 7.32 (s, 1H), 7.06 (s, 1H), 6.27 (s, 1H), 4.12-4.01 (m, 1H), 1.35 (d, J = 8 Hz, 3H); LC/MS RT = 2.61 (M-H-: 227/229).
4,6-Dichloro-N-methoxy-N-methyl-1H-indole-2-carboxamide (20b)
4,6-Dichloro-1H-indole-2-carboxylic acid (500 mg, 2.17 mmol), N, O-dimethylhydroxylamine hydrochloride (423 mg, 4.34 mmol), HOBt (332 mg, 2.17 mmol), EDC hydrochloride (874 mg, 4.56 mmol) and triethylamine (1.32 mL, 8.68 mmol) were dissolved in anhydrous DMF (40 mL). The mixture was stirred for overnight at room temperature. The reaction mixture was diluted with ethyl acetate, and the organic layer was washed with water, saturated NH4Cl, 10% NaOH and brine. The organic layer was dried over Na2SO4, and it was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-50% ethyl acetate/hexane) to give product as a white solid (505 mg, 85%); 1H NMR (300 MHz) (DMSO-d6) δ 12.10 (bs, 1H), 7.45 (d, J = 1 Hz, 1H), 7.24 (d, J = 1 Hz, 1H), 7.09 (s, 1H), 3.80 (s, 3H), 3.33 (s, 3H).
1-(4,6-Dichloro-1H-indol-2-yl)ethan-1-one (20c)
4,6-Dichloro-N-methoxy-N-methyl-1H-indole-2-carboxamide (500 mg, 1.83 mmol) was dissolved in anhydrous tetrahydrofuran (25 mL). The mixture was cooled to -78 oC, and, then, a solution of 1.6 M methyllithium in diethyl ether (3.43 mL, 5.49 mmol) was added. The mixture was stirred for 2 hours at -78 oC. The reaction was stopped by addition of water (10 mL). After removal of the solvent, the reaction mixture was diluted with ethyl acetate. The organic layer was washed with saturated NH4Cl and brine, and it was dried over Na2SO4. The organic layer was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-20% ethyl acetate/hexane) to give product as a white solid (278 mg, 67%); 1H NMR (300 MHz) (DMSO-d6) δ 12.20 (bs, 1H), 7.41 (s, 1H), 7.39 (s, 1H), 7.27 (s, 1H), 2.57 (s, 3H).
1-(5,6-Difluoro-1H-indol-2-yl)ethan-1-amine (21).
1-(5,6-Difluoro-1H-indol-2-yl)ethan-1-one (150 mg, 0.77 mmol), ammonium acetate (594 mg, 7.70 mmol) and sodium cyanoborohydride (242 mg, 3.85 mmol) were dissolved in ethanol (10 mL). The mixture was stirred for overnight at 60 oC. The reaction mixture was acidified with 6N HCl, and it was washed with ethyl acetate. Then, the aqueous layer was basified with NaOH, and it was extracted with ethyl acetate. The organic layer was washed with brine, and it was dried over Na2SO4. The organic layer was concentrated under reduced pressure, and the residue was purified on an ISCO chromatograph (0-10% methanol/dichloromethane + 0.1% NH4OH) to give the product as a white solid (69 mg, 46%);1H NMR (300 MHz) (DMSO-d6) δ 11.49 (bs, 1H), 8.49 (bs, 2H), 7.57-7.50 (m, 1H), 7.44-7.38 (m, 1H), 6.50 (s, 1H), 4.58-4.51 (m, 1H), 1.58 (d, J = 6 Hz, 3H); LC/MS RT = 2.47 (M-H-: 195).
5,6-Difluoro-N-methoxy-N-methyl-1H-indole-2-carboxamide (21b)
5,6-Difluoro-1H-indole-2-carboxylic acid (500 mg, 2.54 mmol), N, O-dimethylhydroxylamine hydrochloride (496 mg, 5.08 mmol), HOBt (389 mg, 2.54 mmol), EDC hydrochloride (1.02 g, 5.33 mmol) and triethylamine (1.54 mL, 10.2 mmol) were dissolved in anhydrous DMF (20 mL). The mixture was stirred for overnight at room temperature. The reaction mixture was diluted with ethyl acetate, and the organic layer was washed with water, saturated NH4Cl, 10% NaOH and brine. The organic layer was dried over Na2SO4, and it was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-50% ethyl acetate/hexane) to give product as a white solid (367 mg, 60%); 1H NMR (300 MHz) (CDCl3) δ 9.33 (bs, 1H), 7.45-7.38 (m, 1H), 7.23-7.17 (m, 2H), 3.85 (s, 3H), 3.42 (s, 3H).
1-(5,6-Difluoro-1H-indol-2-yl)ethan-1-one (21c)
5,6-Difluoro-N-methoxy-N-methyl-1H-indole-2-carboxamide (350 mg, 1.46 mmol) was dissolved in anhydrous tetrahydrofuran (20 mL). The mixture was cooled to -78 oC, then, a solution of 1.6 M methyllithium in diethyl ether (2.73 mL, 4.38 mmol) was added. The mixture was stirred for 2 hours at -78 oC. The reaction was stopped by addition of water (10 mL). After removal of the solvent, the reaction mixture was diluted with ethyl acetate. The organic layer was washed with saturated NH4Cl and brine, and it was dried over Na2SO4. The organic layer was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-20% ethyl acetate/hexane) to give product as a white solid (207 mg, 73%); 1H NMR (300 MHz) (CDCl3) δ 9.24 (bs, 1H), 7.47-7.41 (m, 1H), 7.26-7.14 (m, 2H), 2.58 (s, 3H).
1-(5-Bromo-1H-indol-2-yl)-N-methylethan-1-amine (22).
To a mixture of 1-(5-bromo-1H-indol-2-yl)ethan-1-one (50 mg, 0.21 mmol) in ethanol (5 mL), a solution of 2.0 M methylamine in tetrahydrofuran (1.05 mL, 2.10 mmol) was added. The mixture was treated with catalytic amount of acetic acid, and it was stirred for an hour at 60 oC. Then, the mixture was treated with sodium cyanoborohydride (66 mg, 1.05 mmol), and it was stirred for overnight at 60 oC. The reaction mixture was acidified with 6N HCl, and it was washed with ethyl acetate. Then, the aqueous layer was basified with NaOH, and it was extracted with ethyl acetate. The organic layer was washed with brine, and it was dried over Na2SO4. The organic layer was concentrated under reduced pressure, and the residue was purified on an ISCO chromatograph (0-10% methanol/dichloromethane + 0.1% NH4OH) to give the product as a colorless oil (53 mg, 100%); 1H NMR (300 MHz) (CDCl3) δ 9.79 (bs, 1H), 7.67 (s, 1H), 7.27-7.26 (m, 2H), 6.41 (s, 1H), 4.35-4.32 (m, 1H), 2.43 (s, 3H), 1.71 (d, J = 7 Hz, 3H); LC/MS RT = 2.60 (M-H-: 251/253).
1-(5-Bromo-1H-indol-2-yl)-N,N-dimethylethan-1-amine (23).
To a mixture of 1-(5-bromo-1H-indol-2-yl)ethan-1-one (75 mg, 0.32 mmol) in ethanol (10 mL), a solution of 2.0 M N,N-dimethylamine in tetrahydrofuran (1.60 mL, 3.20 mmol) was added. The mixture was treated with catalytic amount of acetic acid followed by sodium cyanoborohydride (101 mg, 1.69 mmol), and it was stirred for overnight at 60 oC. The reaction mixture was acidified with 6N HCl, and it was washed with ethyl acetate. Then, the aqueous layer was basified with NaOH, and it was extracted with ethyl acetate. The organic layer was washed with brine, and it was dried over Na2SO4. The organic layer was concentrated under reduced pressure, and the residue was purified on an ISCO chromatograph (0-10% methanol/dichloromethane + 0.1% NH4OH) to give the product as a colorless oil (12 mg, 14%); 1H NMR (300 MHz) (CDCl3) δ 8.81 (bs, 1H), 7.66 (s, 1H), 7.26-7.20 (m, 2H), 6.24 (s, 1H), 3.82-3.80 (m, 1H), 2.25 (s, 6H), 1.40 (d, J = 7 Hz, 3H); LC/MS RT = 2.62 (M-H-: 265/267).
1-(5-Bromo-1-methyl-1H-indol-2-yl)ethan-1-amine (24).
1-(5-Bromo-1-methyl-1H-indol-2-yl)ethan-1-one (91 mg, 0.36 mmol), ammonium acetate (277 mg, 3.60 mmol) and sodium cyanoborohydride (113 mg, 1.80 mmol) were dissolved in ethanol (10 mL). Then, catalytic amount of acetic acid was added. The mixture was stirred for overnight at 60 oC. The reaction mixture was diluted with ethyl acetate. The organic layer was washed with 10% NaOH and brine, and it was dried over Na2SO4. The organic layer was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-10% methanol/dichloromethane + 0.1% NH4OH) to give the product as a white solid (57 mg, 63%);1H NMR (300 MHz) (CD3OD) δ 7.62 (d, J = 2 Hz, 1H), 7.28 (d, J = 9 Hz, 1H), 7.21 (dd, J = 9 Hz, J = 2 Hz, 1H), 6.44 (s, 1H), 4.44-4.38 (m, 1H), 3.75(s, 3H), 1.56 (d, J = 7 Hz, 3H); LC/MS RT = 2.49 (M-NH2+:236/238).
1-(5-Bromo-1-methyl-1H-indol-2-yl)ethan-1-one (24a)
1-(5-Bromo-1H-indol-2-yl)ethan-1-one (100 mg, 0.42 mmol), K2CO3 (116 mg, 0.84 mmol) and methyl iodide (52 µL, 0.84 mmol) were dissolved in anhydrous DMF (5 mL). The mixture was stirred for 3 hours at 60 oC. After the mixture was cooled to room temperature, it was diluted with ethyl acetate. The organic layer was washed with water and brine, and it was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-50% ethyl acetate/hexane) to give product as a white solid (91 mg, 86%); 1H NMR (300 MHz) (CDCl3) δ 7.82 (d, J = 2 Hz, 1H), 7.45 (dd, J = 9 Hz, J = 2 Hz, 1H), 7.27 (d, J = 9 Hz, 1H), 7.20 (s, 1H), 4.05 (s, 3H), 2.61 (s, 3H).
(5-Bromo-1H-indol-2-yl)methanamine (25).
5-Bromo-1H-indole-2-carbaldehyde (55 mg, 0.25 mmol) was dissolved in ethanol (25 mL), and it was treated with ammonium acetate (193 mg, 2.50 mmol). The mixture was stirred for an hour at room temperature. Then, sodium cyanoborohydride (79 mg, 1.25 mmol) was added, and it was stirred for 3 hours at room temperature. The reaction mixture was diluted with ethyl acetate, and it was washed with saturated NaHCO3 and brine. The organic layer was dried over Na2SO4, and it was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-10% methanol/dichloromethane + 0.1% NH4OH) to give (5-bromo-1H-indol-2-yl)methanamine as a white solid (26 mg, 46%). 1H NMR (300 MHz) (DMSO-d6) δ 11.25 (bs, 1H), 7.72 (d, J = 2 Hz, 1H), 7.36 (d, J = 8 Hz, 1H), 7.19 (dd, J = 9 Hz, J = 2 Hz, 1H), 6.46 (s, 1H), 4.11 (s, 2H); LC/MS RT = 2.52 (M-H-: 223/225). along with bis((5-bromo-1H-indol-2-yl)methyl)amine as a white solid (26 mg, 49%). 1H NMR (300 MHz) (CDCl3) δ 7.65 (s, 2H), 7.26-7.21 (m, 4H), 6.31 (s, 2H), 3.97 (s, 4H); LC/MS RT = 2.97 (M-H-: 430/432/434).
Methyl 5-bromo-1H-indole-2-carboxylate (25a)
5-Bromo-1H-indole-2-carboxylic acid (1.5 g, 6.25 mmol) was dissolved in methanol (100 mL), and it was treated with catalytic amount of concentrated sulfuric acid. The mixture was refluxed overnight. After removal of solvent, it was diluted with ethyl acetate. The organic layer was washed with 10% NaOH and brine, and it was dried over Na2SO4. The organic layer was concentrated under reduced pressure, and the residue was purified on an ISCO chromatograph (0-50% ethyl acetate/hexane) to give product as a white solid (1.19 g, 75%); 1H NMR (300 MHz) (CDCl3) δ 8.97 (bs, 1H), 7.83 (s, 1H), 7.40 (dd, J = 9 Hz, J = 2 Hz, 1H), 7.30 (d, J = 9 Hz, 1H), 7.14 (s, 1H), 3.95 (s, 3H).
(5-Bromo-1H-indol-2-yl)methanol (25b)
Methyl 5-bromo-1H-indole-2-carboxylate (200 mg, 0.79 mmol) and lithium borohydride (86 mg, 3.95 mmol) were dissolved in anhydrous tetrahydrofuran (10 mL) at 0 oC, and it was stirred for overnight at room temperature. The reaction mixture was diluted with ethyl acetate, and it was washed with saturated NH4Cl and brine. The organic layer was dried over Na2SO4, and it was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-50% ethyl acetate/hexane) to give product as a white solid (149 mg, 83 %); 1H NMR (300 MHz) (CDCl3) δ 8.37 (bs, 1H), 7.69 (s, 1H), 7.28-7.21 (m, 2H), 6.34 (d, J = 1 Hz, 1H), 4.84 (s, 2H).
5-Bromo-1H-indole-2-carbaldehyde (25c)
(5-Bromo-1H-indol-2-yl)methanol (100 mg, 0.44 mmol) and Dess-Martin periodinane (280 mg, 0.66 mmol) were dissolved in dichloromethane (10 mL), and it was stirred for 15 minutes at room temperature. The reaction mixture was diluted with ethyl acetate, and it was washed with 10% sodium thiosulfate, saturated NaHCO3 and brine. The organic layer was dried over Na2SO4, and it was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-10% ethyl acetate/hexane) to give product as a white solid (55 mg, 56%); 1H NMR (300 MHz) (CDCl3) δ 9.85 (s, 1H), 9.05 (bs, 1H), 7.90 (s, 1H), 7.47 (dd, J = 9 Hz, J = 2 Hz, 1H), 7.34 (d, J = 9 Hz, 1H), 7.21 (s, 1H).
N1-(2-bromo-5,6,7,8,9,10-hexahydrocyclohepta[b]indol-6-yl)propane-1,3-diamine (26).
To a solution of tert-butyl (3-((2-bromo-5,6,7,8,9,10-hexahydrocyclohepta[b]indol-6-yl)amino)propyl)carbamate (614 mg, 1.41 mmol) in methanol (3 mL), 4N HCl in dioxane (6 mL, 24 mmol) was added. The reaction mixture was stirred for 1.0 hour at room temperature. After removal of solvent, the reaction mixture was diluted with ethyl acetate. The organic layer was washed with 10% sodium hydroxide and brine, and it was dried over sodium sulfate. The organic layer was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-10% methanol/dichloromethane + 0.1% NH4OH) to give the product as yellow oil (202 mg, 43%); 1H NMR (300 MHz) (CDCl3) δ 10.02 (bs, 1H), 7.58 (s, 1H), 7.15-7.14 (m, 2H), 3.87-3.83 (m, 1H), 2.96-2.75 (m, 6H), 2.63-2.51 (m, 1H), 2.12-2.02 (m, 2H), 1.94-1.88 (m, 1H), 1.74-1.62 (m, 4H); LC/MS RT = 2.52 (M+H+: 336/338).
tert-Butyl (3-((2-bromo-5,6,7,8,9,10-hexahydrocyclohepta[b]indol-6-yl)amino)propyl)carbamate (26a)
2-Bromo-7,8,9,10-tetrahydrocyclohepta[b]indol-6(5H)-one (500 mg, 1.80 mmol), N-Boc propylenediamine (941 mg, 5.40 mmol) and sodium cyanoborohydride (566 mg, 9.00 mmol) were dissolved in ethanol (20 mL). Catalytic amount of acetic acid was added. The reaction mixture was stirred for overnight at 60 oC. After removal of solvent, the reaction mixture was diluted with ethyl acetate. The organic layer was washed with 10% sodium hydroxide and brine, and it was dried over sodium sulfate. The organic layer was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-100% ethyl acetate/hexane) to give the product as yellow oil (614 mg, 78%); 1H NMR (300 MHz) (CDCl3) δ 9.51 (bs, 1H), 7.61 (s, 1H), 7.36-7.25 (m, 2H), 5.14-5.12 (m, 1H), 4.62-4.61 (m, 1H), 3.25-2.80 (m, 6H), 2.45-2.43 (m, 1H), 2.12-1.82 (m, 7H), 1.39 (s, 9H); LC/MS RT = 3.13 (M+H+: 436/438).
N1-(7-bromo-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)propane-1,3-diamine (27).
To a solution of tert-butyl (3-((7-bromo-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)amino)-propyl)carbamate (817 mg, 2.00 mmol) in methanol (3 mL), 4N HCl in dioxane (6 mL, 24 mmol) was added. The reaction mixture was stirred for an hour at room temperature. After removal of solvent, the reaction mixture was diluted with ethyl acetate. The organic layer was washed with 10% sodium hydroxide and brine, and it was dried over sodium sulfate. The organic layer was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-10% methanol/dichloromethane + 0.1% NH4OH) to give the product as a white solid (616 mg, 100%); 1H NMR (300 MHz) (CDCl3) δ 9.25 (bs, 1H), 7.58 (s, 1H), 7.26-7.18 (m, 2H), 4.37-4.33 (m, 1H), 2.88-2.65 (m, 8H), 2.12-2.04 (m, 1H), 2.04-1.97 (m, 2H); LC/MS RT = 2.50 (M+H+: 308/310).
tert-Butyl (3-((7-bromo-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)amino)propyl)carbamate (27a)
7-Bromo-1,4-dihydrocyclopenta[b]indol-3(2H)-one (500 mg, 2.00 mmol), N-Boc propylenediamine (1.05 g, 6.00 mmol) and sodium cyanoborohydride (628 mg, 10.00 mmol) were dissolved in ethanol (20 mL). A catalytic amount of acetic acid was added. The reaction mixture was stirred for overnight at 60 oC. After removal of solvent, the reaction mixture was diluted with ethyl acetate. The organic layer was washed with 10% sodium hydroxide and brine, and it was dried over sodium sulfate. The organic layer was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-100% ethyl acetate/hexane) to give the product as a foamy yellow solid (817 mg, 100%); 1H NMR (300 MHz) (CDCl3) δ 9.16 (bs, 1H), 7.63 (s, 1H), 7.33-7.32 (m, 2H), 5.07-5.03 (m, 1H), 4.83 (m, 1H), 3.26-3.24 (m, 2H), 3.04-2.85 (m, 5H), 2.60-2.54 (m, 1H), 1.98-1.96 (m, 2H), 1.39 (s, 9H); LC/MS RT = 3.08 (M+H+: 408/410).
N1-(1-(5-Bromo-1H-indol-2-yl)ethyl)propane-1,3-diamine (28).
To a solution of tert-butyl (3-((1-(5-bromo-1H-indol-2-yl)ethyl)amino)propyl)carbamate (817 mg, 2.06 mmol) in methanol (3 mL), 4N HCl in dioxane (6 mL, 24 mmol) was added. The reaction mixture was stirred for an hour at room temperature. After removal of solvent, the reaction mixture was diluted with ethyl acetate. The organic layer was washed with 10% sodium hydroxide and brine, and it was dried over sodium sulfate. The organic layer was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-10% methanol/dichloromethane + 0.1% NH4OH) to give the product as colorless oil (332 mg, 54%); 1H NMR (300 MHz) (CDCl3) δ 9.22 (bs, 1H), 7.65 (s, 1H), 7.26-7.20 (m, 3H), 6.24 (s, 1H), 4.05-4.01 (m, 1H), 2.83-2.54 (m, 4H), 1.67-1.60 (m, 2H), 1.45 (d, J = 6 Hz, 3H); LC/MS RT = 2.49 (M+H+: 296/298).
tert-Butyl (3-((1-(5-bromo-1H-indol-2-yl)ethyl)amino)propyl)carbamate (28a)
1-(5-Bromo-1H-indol-2-yl)ethan-1-one (500 mg, 2.10 mmol), N-Boc propylenediamine (1.10 g, 6.30 mmol) and sodium cyanoborohydride (314 mg, 10.50 mmol) were dissolved in ethanol (20 mL). Catalytic amount of acetic acid was added. The reaction mixture was stirred for overnight at 60 oC. After removal of solvent, the reaction mixture was diluted with ethyl acetate. The organic layer was washed with 10% sodium hydroxide and brine, and it was dried over sodium sulfate. The organic layer was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-100% ethyl acetate/hexane) to give the product as colorless oil (817 mg, 99%); 1H NMR (300 MHz) (CDCl3) δ 9.50 (bs, 1H), 7.68 (s, 1H), 7.37-7.26 (m, 3H), 6.43 (s, 1H), 5.04 (m, 1H), 4.35-4.33 (m, 1H), 3.30-3.28 (m, 2H), 2.80-2.76 (m, 2H), 1.85-1.80 (m, 2H), 1.75 (d, J = 7 Hz, 3H), 1.46 (s, 9H).
N-(3-((1-(5-bromo-1H-indol-2-yl)ethyl)amino)propyl)-4,5-dichlorothiophene-2-carboxamide (29).
5-Chlorofuran-2-carboxylic acid (120 mg, 0.82 mmol), EDC hydrochloric acid (157 mg, 0.82 mmol), HOBt (55 mg, 0.41 mmol) and DIPEA (0.23 mL, 1.26 mmol) were dissolved in DMF (5 mL). After 5 minutes of stirring, N1-(1-(5-bromo-1H-indol-2-yl)ethyl)propane-1,3-diamine (120 mg, 0.41 mmol) was added, and it was stirred overnight at room temperature. The reaction mixture was diluted with ethyl acetate, and it was washed with 1N hydrochloric acid, 10% sodium hydroxide and brine. The organic layer was dried over sodium sulfate. The organic layer was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-10% methanol/dichloromethane + 0.1% NH4OH) to give the product as a white solid (54 mg, 31%); 1H NMR (300 MHz) (CDCl3) δ 9.02 (bs, 1H), 7.63 (s, 1H), 7.18 (m, 2H), 7.11 (d, J = 3 Hz, 1H), 7.05 (bs, 1H), 6.32 (d, J = 3 Hz, 1H), 6.24 (s, 1H), 4.03-3.98 (m, 1H), 3.76-3.69 (m, 1H), 3.51-3.35 (m, 1H), 2.73-2.68 (m, 1H), 2.60-2.54 (m, 1H), 1.72-1.69 (m, 2H), 1.49 (d, J = 6 Hz, 3H); LC/MS RT = 2.95 (M+H+: 424/426/428).
N-(3-((1-(5-bromo-1H-indol-2-yl)ethyl)amino)propyl)-5-chlorothiophene-2-carboxamide (30).
5-Chlorothiophene-2-carboxylic acid (249 mg, 1.53 mmol), EDC hydrochloric acid (196 mg, 1.02 mmol), HOBt (69 mg, 0.51 mmol) and DIPEA (0.27 mL, 1.53 mmol) were dissolved in DMF (2 mL). After 5 minutes of stirring, N1-(1-(5-bromo-1H-indol-2-yl)ethyl)propane-1,3-diamine (150 mg, 0.51 mmol) was added, and it was stirred overnight at room temperature. The reaction mixture was diluted with ethyl acetate, and it was washed with 1N hydrochloric acid, 10% sodium hydroxide and brine. The organic layer was dried over sodium sulfate. The organic layer was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-10% methanol/dichloromethane + 0.1% NH4OH) to give the product as a white solid (29 mg, 13%); 1H NMR (300 MHz) (CDCl3) δ 9.07 (bs, 1H), 7.64 (s, 1H), 7.26-7.06 (m, 2H), 6.79-6.78 (m, 2H), 6.26 (s, 1H), 4.05-4.00 (m, 1H), 3.66-3.60 (m, 1H), 3.51-3.39 (m, 1H), 2.75-2.71 (m, 1H), 2.62-2.54 (m, 1H) 1.73-1.70 (m, 2H), 1.51 (d, J = 7 Hz, 3H); LC/MS RT = 3.05 (M+H+: 440/442/444).
N-(3-((1-(5-bromo-1H-indol-2-yl)ethyl)amino)propyl)-4,5-dichlorothiophene-2-carboxamide (31).
4,5-Dichlorothiophene-2-carboxylic acid (162 mg, 0.82 mmol), EDC hydrochloric acid (157 mg, 0.82 mmol), HOBt (55 mg, 0.41 mmol) and DIPEA (0.23 mL, 1.26 mmol) were dissolved in DMF (5 mL). After 5 minutes of stirring, N1-(1-(5-bromo-1H-indol-2-yl)ethyl)propane-1,3-diamine (120 mg, 0.41 mmol) was added, and it was stirred overnight at room temperature. The reaction mixture was diluted with ethyl acetate, and it was washed with 1N hydrochloric acid, 10% sodium hydroxide and brine. The organic layer was dried over sodium sulfate. The organic layer was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-10% methanol/dichloromethane + 0.1% NH4OH) to give the product as a white solid (63 mg, 32%); 1H NMR (300 MHz) (CDCl3) δ 8.88 (bs, 1H), 7.64 (s, 1H), 7.26-7.12 (m, 3H), 7.06 (bs, 1H), 6.26 (s, 1H), 4.04-3.98 (m, 1H), 3.65-3.58 (m, 1H), 3.49-3.40 (m, 1H), 2.75-2.69 (m, 1H), 2.63-2.57 (m, 1H) 1.74-1.68 (m, 2H), 1.49 (d, J = 7 Hz, 3H); LC/MS RT = 3.05 (M+H+: 474/476/478).
N-(3-((1-(5-bromo-1H-indol-2-yl)ethyl)amino)propyl)-3,4-dichlorobenzamide (32).
3,4-Dichlorobenzoic acid (32 mg, 0.17 mmol), EDC hydrochloric acid (65 mg, 0.34 mmol), HOBt (23 mg, 0.17 mmol) and DIPEA (0.09 mL, 0.51 mmol) were disolved in dichloromethane (5 mL). After 5 minutes of stirring, N1-(1-(5-bromo-1H-indol-2-yl)ethyl)propane-1,3-diamine (50 mg, 0.17 mmol) was added, and it was stirred overnight at room temperature. The reaction mixture was diluted with ethyl acetate, and it was washed with 1N hydrochloric acid, 10% sodium hydroxide and brine. The organic layer was dried over sodium sulfate. The organic layer was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-10% methanol/dichloromethane + 0.1% NH4OH) to give the product as colorless oil (14 mg, 18%); 1H NMR (300 MHz) (CDCl3) δ 9.97 (bs, 1H), 7.88 (d, J = 2 Hz, 1H), 7.64 (s, 1H), 7.53 (dd, J = 8 Hz, J = 2Hz, 2H), 7.42 (d, J = 8 Hz, 1H), 7.25-7.18 (m, 2H), 4.35-4.28 (m, 1H), 3.63-3.48 (m, 2H), 2.81-2.69 (m, 2H), 1.94-1.90 (m, 2H), 1.72 (d, J = 7 Hz, 3H); LC/MS RT = 3.12 (M+H+: 468/470/472).
N-(3-((1-(5-bromo-1H-indol-2-yl)ethyl)amino)propyl)-cyclohexanecarboxamide (33)
Cyclohexane carboxylic acid (22 mg, 0.17 mmol), EDC hydrochloric acid (65 mg, 0.34 mmol), HOBt (23 mg, 0.17 mmol) and DIPEA (0.09 mL, 0.51 mmol) were dissolved in dichloromethane (5 mL). After 5 minutes of stirring, N1-(1-(5-bromo-1H-indol-2-yl)ethyl)propane-1,3-diamine (50 mg, 0.17 mmol) was added, and it was stirred overnight at room temperature. The reaction mixture was diluted with ethyl acetate, and it was washed with 1N hydrochloric acid, 10% sodium hydroxide and brine. The organic layer was dried over sodium sulfate. The organic layer was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-10% methanol/dichloromethane + 0.1% NH4OH) to give the product as colorless oil (55 mg, 80%); 1H NMR (300 MHz) (CDCl3) δ 9.28 (bs, 1H), 7.64 (d, J = 2 Hz, 1H), 7.26 (d, J = 9 Hz, 1H), 7.19 (dd, J = 8 Hz, J = 2 Hz, 1H), 6.23 (d, J = 1 Hz, 1H), 5.68 (bs, 1H), 4.00-3.96 (m, 1H), 3.54-3.47 (m, 1H), 3.27-3.20 (m, 1H), 2.62-2.54 (m, 1H), 2.49-2.41 (m, 1H), 2.04-2.00 (m, 1H), 1.79-1.57 (m, 6H), 1.47 (d, J = 7 Hz, 3H), 1.38-1.14 (m, 6H); LC/MS RT = 2.88 (M+H+: 406/408).
4-((1-(5-Bromo-1H-indol-2-yl)ethyl)amino)-N-(3,4-dichlorophenyl)butanamide (34).
To a solution of 1-(5-bromo-1H-indol-2-yl)ethan-1-one (243 mg, 1.02 mmol) in ethanol (10 mL), 4-amino-N-(3,4-dichlorophenyl)butanamide (253 mg, 1.02 mmol) and sodium cyanoborohydride (320 mg, 5.10 mmol) were added. The reaction mixture was stirred for 60 oC for 24 hours. The mixture was diluted with ethyl acetate, and the organic layer was washed with 10% sodium hydroxide, saturated ammonium chloride, and brine. The organic layer was then dried over sodium sulfate, and it was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (10% methanol/dichloromethane + 1% NH4OH) to give product as colorless oil (73 mg, 15%); 1H NMR (300 MHz, DMSO-d6) d 11.30 (bs, 1H), 10.25 (bs, 1H), 8.00-7.99 (m, 1H), 7.72 (s, 1H), 7.60-7.55 (m, 1H), 7.49-7.44 (m, 1H), 7.38-7.36 (m, 1H), 7.23-7.21 (m, 1H), 6.48 (s, 1H), 4.33 (m, 1H), 2.76-2.60 (m, 2H), 2.42 (m, 2H), 1.84 (m, 2H), 1.57 (m, 3H).
tert-Butyl (4-((3,4-dichlorophenyl)amino)-4-oxobutyl)carbamate
To a solution of 4-((tert-butoxy carbonyl)amino)butanoic acid (1.0 g, 4.9 mmol) in dichloromethane (50 mL), EDC hydrochloric acid (1.98 g, 10.3 mmol), HOBt (665 mg, 4.9 mmol) and DIPEA (1.80 mL, 10.33 mmol) were added. After the mixture stirred for 5 minutes, 3,4-dichloroaniline (797 mg, 4.92 mmol) was added. The mixture was then stirred for overnight at room temperature. The mixture was then diluted with dichloromethane, and the organic layer was washed with 1N hydrochloric acid, 10% sodium hydroxide, saturated ammonium chloride and brine. The organic layer was then dried over sodium sulfate, and it was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-30% ethyl acetate/hexane) to give product as a white solid (757 mg, 44%); 1H NMR (300 MHz, CDCl3) d 9.47 (bs, 1H), 7.92 (s, 1H), 7.48 (d, J = 9 Hz, 1H), 7.37 (d, J = 9 Hz, 1H), 4.87 (m, 1H), 3.27-3.24 (m, 2H), 2.41-2.37 (m, 2H), 1.89-1.87 (m, 2H), 1.49 (s, 9H).
4-Amino-N-(3,4-dichlorophenyl)butanamide
To a solution of tert-butyl (4-((3,4-dichlorophenyl)amino)-4-oxobutyl)carbamate (500 mg, 1.44 mmol) in methanol (0.36 mL), 4N hydrochloric acid (3.60 mL, 14.40 mmol) was added. The reaction mixture was stirred for 2 hours at room temperature. The mixture was diluted with ethyl acetate, and the organic layer was washed with 10% sodium hydroxide saturated ammonium chloride and brine. The organic layer was then dried over sodium sulfate, and it was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-100% ethyl acetate/hexane followed by 10% methanol/dichloromethane + 1% NH4OH) to give product as colorless oil (253 mg, 71%); 1H NMR (300 MHz, CDCl3) d 9.85 (bs, 1H),7.78 (d, J = 2Hz, 1H), 7.38-7.32 (m, 2H), 2.90-2.86 (m, 2H), 2.54-2.49 (m, 2H), 1.80-1.84 (m, 2H), 1.64 (bs, 2H).
N-(1-(5-Bromo-1H-indol-2-yl)ethyl)-4-(3,4-dichlorophenoxy)butan-1-amine (35).
To a solution of 1-(5-bromo-1H-indol-2-yl)ethan-1-one (190 mg, 0.80 mmol) in ethanol (10 mL), 4-(3,4-dichlorophenoxy)butan-1-amine (187 mg, 0.80 mmol) and sodium cyanoborohydride (251 mg, 4.00 mmol) were added. The reaction mixture was stirred for 60 oC for 24 hours. The mixture was diluted with ethyl acetate, and the organic layer was washed with 10% sodium hydroxide, saturated ammonium chloride and brine. The organic layer was then dried over sodium sulfate, and it was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (10% methanol/dichloromethane + 1% NH4OH) to give product as colorless oil (47 mg, 13%); 1H NMR (300 MHz, CDCl3) d 9.11 (bs, 1H), 7.71 (s, 1H), 7.31-7.28 (m, 3H), 6.91 (s, 1H), 6.62 (d, J = 9 Hz, 1H), 6.44 (s, 1H), 4.11-4.39 (m, 1H), 3.87 (m, 2H), 2.78 (m, 2H), 1.81 (m, 4H), 1.72 (d, J = 7 Hz, 3H).
2-(4-(3,4-Dichlorophenoxy)butyl)isoindoline-1,3-dione
To a solution of 2-(4-bromobutyl)isoindoline-1,3-dione (500 mg, 1.77 mmol) in DMF (5 mL), 3,4-dichlorophenol (289 mg, 1.77 mmol) and potassium carbonate (245 mg, 1.77 mmol) were added. The reaction mixture was stirred for 5 hours at room temperature. The mixture was diluted with ethyl acetate, the organic layer was washed with water and brine. The organic layer was then dried over sodium sulfate, and it was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-30% ethyl acetate/hexane) to give product as colorless oil (545 mg, 84%); 1H NMR (300 MHz, CDCl3) d 7.89-7.86 (m, 2H), 7.76-7.73 (m, 2H), 7.29 (d, J = 9 Hz, 1H), 6.98 (d, J = 3 Hz, 1H), 6.75 (dd, J = 9 Hz, J = 3 Hz, 1H), 4.00-3.96 (m, 2H), 3.81-3.77 (m, 2H), 1.89-1.87 (m, 4H).
4-(3,4-Dichlorophenoxy)butan-1-amine
To a solution of 2-(4-(3,4-dichlorophenoxy)butyl)isoindoline-1,3-dione (545 mg, 1.50 mmol) in methanol (15 mL), hydrazine monohydrate (0.15 mL, 3.00 mmol) was added at room temperature. The reaction mixture was stirred for 2 hours at 60 oC. The white suspension was formed, and the suspension was filtered. The suspension was concentrated under reduced pressure, and the residue was diluted with ethyl acetate. The organic layer was washed with 10% sodium hydroxide and brine. The organic layer was then dried over sodium sulfate, and it was concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0-100% ethyl acetate/hexane followed by 10% methanol/dichloromethane + 1% NH4OH) to give product as colorless oil (187 mg, 53%); 1H NMR (300 MHz, CDCl3) d 7.31 (d, J = 9 Hz, 1H), 6.99 (d, J = 3 Hz, 1H), 6.75 (dd, J = 9 Hz, J = 3 Hz, 1H), 3.97-3.92 (m, 2H), 2.80-2.75 (m, 2H), 1.85-1.80 (m, 2H), 1.66-1.58 (m, 2H), 1.23 (bs, 2H).
Intrinsic MIC Assays
MIC assays were conducted in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines for broth microdilution. A 96-well plate containing cation-adjusted Mueller-Hinton (CAMH) broth with 2-fold serial dilution of compounds was inoculated with log-phase bacteria at 5x105 CFU/mL. The final volume in each well was 100 µL. Each compound was tested in duplicate. The microtiter plates were incubated in an aerobic environment for 18 hours at 37 °C. Then the bacterial growth was tested by reading the plate with a VersaMax plate reader (Molecular Devices, Inc.) at 600 nm. The MIC was defined as the lowest compound concentration that inhibited 90% of bacterial growth.
The intrinsic MIC of the experimental EPIs was tested with the method described above. The 2-fold serial dilution begins with 100 µg/mL of tested compound in the first column of the 96-well plates. The following Gram-negative bacterial strains were included in these assays:
Escherichia coli ATCC 25922
Klebsiella pneumoniae ATCC 13883 and ATCC 10031
Pseudomonas aeruginosa PAO1
Acinetobacter baumannii ATCC 19606
MIC Assays in the Presence of a Bacterial Efflux Inhibitor
The EPI assay for the purposes of these studies represents a MIC assay in which the MIC of the antibiotic against the bacteria is tested in the presence of an experimental efflux pump inhibitor (EPI), N-(((2S,4R)-4-(aminomethyl)pyrrolidin-2-yl)methyl)-6-(4-fluorophenyl)-1H-indole-2-carboxamide. The highest concentration of the EPI present in the assay typically is ½ of the intrinsic MIC of the compound. If the intrinsic MIC of the EPI is greater than 100 µg/mL, the EPI assay was tested with 50 µg/mL. Using serial dilutions of the EPI, its enhancement of antibiotic activity was then evaluated. The relative EPI activity was decided by comparing the MIC of the antibiotic in the presence of the EPI compound with the intrinsic MIC of the antibiotic alone. For comparative purposes, we used this EPI at concentration of 12.5 µg/mL against varying concentration of our test compounds.
ATPase Assays with E. coli MreB (EcMreB)
The cloning and expression of E. coli MreB as well as all ATPase assays were conducted as described previously (23).
Notes
The authors declare the following competing financial interest(s): Dr. LaVoie and Dr. Pilch are co-founders of TAXIS Pharmaceuticals and Dr. Parhi is a shareholder and therefore have a financial interest in the company.
Acknowledgement
This research was supported in part by Research Agreement between Rutgers University and TAXIS Pharmaceuticals, Inc.
- Figge, RM, Divakaruni, AV, Gober, JW. MreB, the cell shape-determining bacterial actin homologue, co-ordinates cell wall morphogenesis in Caulobacter crescentus. Mol. Microbiol. 2004;51(5): 1321-1332.
- Strahl, H, Bürmann, F, Hamoen. LW. The actin homologue MreB organizes the bacterial cell membrane. Nat. Commun. 2013;5(3442): 1-11.
- Wang, H, Xie, L, Luo, H, Xie, J. Bacterial cytoskeleton and implications for new antibiotic targets. J. Drug Target. 2016;24(5): 392-398.
- Busiek, K, Margolin, W. Bacterial Actin and Tubulin Homologs in Cell Growth and Division. Curr. Biol. 2015;25(6); R243-R254.
- Foss, MH, Eun, Y-J, Weibel, DB. Chemical-biological studies of subcellular organization in bacteria. Biochemistry 2011;50:7719-7734.
- White, CL, Gober, JW. MreB: pilot or passenger of cell wall synthesis? Trends Microbiol. 2012; 20(2), 74-79.
- Fenton, AK, Gerdes, K. Direct Interaction of FtsZ and MreB is required for septum synthesis and cell division in Escherichia coli. EMBO J. 2013;32(13): 1953-1965.
- Vollmer, W. The prokaryotic cytoskeleton: a putative target for inhibitors and antibiotics? Appl. Microbiol. Biotechnol. 2006;73: 37-47.
- Noguchi, N, Yanagimoto, K, Nakaminami, H, Wakabayashi, M, Iwai, N, Wachi, M, Sasatsu, M. Anti-infectious effect of S-benzylisothiourea compound A22, which inhibits the actin-like protein, MreB, in Shigella flexneri. Biol. Pharm. Bull. 2008;31(7): 1327-1332.
- Barker, CA, Allison, SE, Zlitni, S, Nguyen, ND, Das, R, Melacini, G, Capretta, AA, Brown, ED. Degradation of MAC13243 and studies of the interaction of resulting thiourea compounds with the lipoprotein targeting chaperone LolA. Bioorg. Med. Chem. Lett. 2013; 23: 2426-2431.
- Bonez, PC, Ramos, AP, Nascimento, K, Copetti, PM, Souza, ME, Rossi, GG, Agertt, VA, Sagrillo, MR, Santos, RCV, Campos, MMA. Antibacterial, cyto and genotoxic activities of A22 compound ((S-3, 4-dichlorobenzyl) isothiourea hydrochloride). Microb. Pathog. 2016;99: 14-18.
- Iwai, N, Fujii, T, Nagura, H, Wachi, M, Kitazume, T. Structure-activity relationship study of the bacterial actin-like protein MreB inhibitors: Effects of substitution of benzyl group in S-benzylisothiourea. Biosci. Biotechnol. Biochem. 2007;71(1): 246-248.
- Iwai, N, Ebata, T, Nagura, H, Kitazume, T, Nagai, K, Wachi, M. Structure-activity relationship of S-benzylisothiourea derivatives to induce spherical cells in Escherichia coli. Biosci. Biotechnol. Biochem., 2004; 68(11): 2265-2269.
- Nicholson, A, Perry, JD, James, AL, Stanforth, SP, Carnell, S, Wilkinson, K, Kahn, CMA, De Soyza, A, Gould, FK. In vitro activity of S-(3,4-dichlorobenzyl)isothiourea hydrochloride and novel structurally related compounds against multidrug-resistant bacteria, including Pseudomonas aeruginosa and Burkholderia cepacia complex. Int. J. Antimicrob. Agents 2012;39: 27-32.
- Bean, GJ, Flickinger, ST, Westler, WM, McCully, ME, Sept, D, Weibel, DB, Amann, KJ. A22 disrupts the bacterial actin cytoskeleton by directly binding and inducing a low-affinity state in MreB. Biochemistry, 2009;48(22): 4852-4857.
- Takacs, CN, Poggio, S, Charbon, G, Pucheault, M, Vollmer, W, Wagner, CJ. MreB drives de novo rod morphogenesis in Caulobacter crescentus via remodeling of the cell wall. J. Bacteriol. 2010: 1671-1684.
- Gerdes, K, Møller-Jensen, J, Ebersbach, G, Kruse, T, Nordström, K. Bacterial mitotic machineries. Cell 2004;116(3): 359-366.
- Kruse, T, Gerdes, K. Bacterial DNA segregation by the actin-like MreB protein. Trends Cell Biol. 2005;15(7): 343-345.
- Kruse, T, Blagoev, B, Lobner-Olesen, A, Wachi, M, Sasaki, K, Iwai, N, Mann, M, Gerdes, K. Actin homolog MreB and RNA polymerase interact and are both required for chromosome segregation in Escherichia coli. Genes Dev. 2006;20: 113-124.
- Kruse, T, Moller-Jensen, J, Lobner-Olesen, A, Gerdes, K. Dysfunctional MreB inhibits chromosome segregation in Escherichia coli. EMBO J. 2003;22(19): 5283-5292.
- Gitai, Z, Dye, NA, Reisenauer, A, Wachi, M, Shapiro, L. MreB actin-mediated segregation of a specific region of a bacterial chromosome. Cell 2005;120: 329-341.
- Robertson, GT, Doyle, TB, Du, Q, Duncan, L, Mdluli, KE, Lynch, AS. A novel indole compound that inhibits Pseudomonas aeruginosa growth by targeting MreB is a substrate for MexAB-OprM. J. Bacteriol. 2007;189(19): 6870-6881.
- Bryan, EJ, Sagong HY, Parhi AK, Grier MC, Roberge JC, LaVoie EJ, Pilch, DS. TXH11106: A third-generation MreB inhibitor with enhanced activity against a broad range of Gram-negative bacterial pathogens. Antibiotics 2022;11: 693-709.
- LaVoie, E, Parhi, A, Sagong, HY, inventors; Therapeutic compounds and methods to treat infection.US 0155507 A1, 2020 May 21.
Tables 1 to 3 are available in the Supplementary Files section
Scheme 1 to 9 are available in the Supplementary Files section.
- MreBInhibitorsSupplementaryMaterial.docx
- scheme1.png
Scheme 1. Reagents and conditions: a) LiHMDA, ethyl formate, 0 °C (37%); b) NaNO2, conc. HCl, 0 °C; c) MeOH, NaOAc, 0 °C (79%); d) conc. HCl, AcOH, 130 °C (23% for 8a), (9a and 10a (8%); e) Boc anhydride, DMAP, THF, rt, (49% 9b and 20% 10b); f) TFA in DCM (1:5), rt, (90% for 9a and 95% for 10a); g) NaBH3CN, NH4OAc, EtOH, 60 °C, 34% for 8, 37% for 9, and 67% for 10.
- scheme2.png
Scheme 2. Reagents and conditions: a) LiHMDA, ethyl formate, 0 °C (37%); b) NaNO2, conc. HCl, 0 °C; c) MeOH, NaOAc, 0 °C (28%); d) conc. HCl, AcOH, 130 °C (8% for 11a), (12a and 13a (10%); e) Boc anhydride, DMAP, THF, rt, (17% 12b and 23% 13b); f) TFA in DCM (1:5), rt, (31% for 12a and 37% for 13a); g) NaBH3CN, NH4OAc, EtOH, 60 °C, 17% for 11, 41% for 12, and 62% for 13.
- scheme3.png
Scheme 3. Reagents and conditions: a) CH3ONHCH3·HCl, EDC·HCl; b) CH3Li, THF/EtO; c) NH4OAc, NaBH3CN, EtOH.
- scheme4.png
Scheme 4. Reagents and conditions: a) i (CH3NH, THF, AcOH cat., 60 °C, ii NaBH3CN, 60 °C, (100% for 22); i (CH3)2N, THF, AcOH cat., 60 °C, ii NaBH3CN, 60 °C (14% for 23), c) MeI, K2CO3, DMF (86% for 24a); d) NaBH3CN, NH4OAc, cat. AcOH, EtOH, 60 °C (63% for 24).
- scheme5.png
Scheme 5. Reagents and conditions: a) MeOH, conc. HCl, reflux, (75%); b) LiBH4, THF, rt, (83%); c) DMP, DCM, rt, (56%); d) NH4OAc, EtOH, NaBH3CN, rt, (46%).
- scheme6.png
Scheme 6. Reagents and conditions: a) N-Boc Propylenediamine, NaBH3CN, cat. AcOH, EtOH (78% for 26a, 100% for 27a and 99% for 28a); b) 4N HCl in dioxane/MeOH (43% for 26, 100% for 27 and 54% for 28).
- scheme7.png
Scheme 7. Reagents and conditions: a) EDC·HCl, HOBt, DIPEA, rt, (31% for 29, 13% for 30, 22% for 31, 18% for 32 and 80% for 33).
- scheme8.png
Scheme 8. Reagents and conditions: a) MeOH, 4N HCl in dioxane (71%); b) NaBH3CN, EtOH, 60 °C, (15%).
- scheme9.png
Scheme 9. Reagents and conditions: a) Hydrazine, MeOH, 60 °C (53%); b) NaBH3CN, EtOH, 60 °C, (13%).
- GraphicalAbstract.png
Graphical Abstract
- Tables.docx
