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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Bioorg Med Chem. Author manuscript; available in PMC 2010 May 1.
Published in final edited form as:
PMCID: PMC2696309
NIHMSID: NIHMS105873

Design and Synthesis of a Novel Tyrosine Kinase Inhibitor Template

Abstract

We report the design and synthesis of an insulin receptor kinase family-targeted inhibitor template using the inhibitor conformation observed in an IGF1R/inhibitor co-crystal complex by application of a novel molecular design approach that we have recently published. The synthesis of the template involves a one pot Opatz cyclization reaction that provides a versatile indole ester in good yields. We also developed the required chemistry to elaborate this template with additional substituents and have used this chemistry to prepare some initial compounds that show selective inhibition of anaplastic lymphoma kinase (ALK).

Keywords: Anaplastic lymphoma kinase, ALK, de novo ligand design, kinase template synthesis, insulin receptor kinase family, inhibitor design

Introduction

Often the discovery of new lead structures for drug discovery projects starts from active molecules (hits) that are found from the screening of compound libraries using in vitro assays or cell-based high-throughput screening. However, in some cases, it is possible to design ‘hits’ if sufficient information is available. We have recently published a detailed report on a novel and general method for de novo protein ligand design that uses pharmacophore-based geometry queries, or other similar information, to search virtual hydrocarbon databases for rigid frameworks that match the geometry of the query.1 These new frameworks can then be used as starting points in the design of new structures that contain the constrained pharmacophore interaction features.1 One of the general applications of this design method makes use of a pharmacophore query derived from the bound conformation of an inhibitor, as observed in a protein-inhibitor crystal structure.2 A specific application of this approach to the inactive form of an oncogenic tyrosine kinase has previously been presented.3 The development of new inhibitors of certain tyrosine kinases in the insulin receptor superfamily is of great interest since compounds that demonstrate an appropriate inhibition selectivity profile show promise as potential drugs for the treatment of human malignancies caused by specific gain-of-function oncogenic kinase mutations.48 We report here details of the design of a novel kinase inhibitor template as well as the synthesis of a partially elaborated template and initial activity data.

Using the general published approach,1 we searched a virtual framework library (VFL) using the pharmacophore shown in Figure 1, which was derived from a co-crystal structure of a ligand bound to the non-activated IGF1R kinase domain.2 We utilized a pharmacophore with all of the interaction features converted to hydrophobic for the initial search of the VFL, as previously described.13 This search yielded a large number of polycyclic framework hits that were then subjected to selection based on synthetic and medicinal chemistry expertise. This led to the selection of framework hit 1a (see Figure 2) since it was a close match to four of the five features and also had a synthetically attractive framework. We then targeted the synthesis of compounds containing this core template as a starting point for the design of selective inhibitors of relevant oncogenic tyrosine kinases in the insulin receptor superfamily by the stabilization of the inactive form of their kinase domains.

FIGURE 1
A detailed view of the pharmacophore including measurements in angstroms derived from the bound conformation of a ligand/IGF1R kinase domain co-crystal structure.2,3 This pharmacophore was used to search the virtual framework library. The color coding ...
FIGURE 2
The evolution of scaffold 1c from initial computational hit 1a. Abbreviations: Hyd= hydrogen bond donor; Acc= hydrogen bond acceptor.

Results

As outlined in our retrosynthetic plan (Figure 3) the synthesis of tetracyclic pyridone 1 can start with compounds such as ester 4/5, which can be prepared from commercially available 2-iodoaniline (or 5-chloro-2-iodoaniline, Scheme 1) by iodo-vinyl exchange using phosphine-free, thermal Heck conditions9 in 86/92% yield. Treatment of the resulting ester 4/5 with aldehyde 6 (made from the commercially available ester)10 using modified Opatz conditions11, 12 provided the indole ester 7/8 in 73/68% yield. Deprotection of the t-butyl ester, followed by amide coupling gave indole amide 9/10 in 80/74% overall yield. We initially investigated the direct base-catalyzed cyclization to form the seven-membered lactam13, 14 using 9/10 as a substrate, but the yield of the desired cyclized product was quite low. However, we found that amide reduction followed by base treatment yielded the tetracyclic pyridines 11a/12a and 11b/12b in 34% and 24/21% yields, respectively, over two steps. Acid-promoted cyclization of the amine15 failed to provide the desired tetracyclic compound.

FIGURE 3
The general retrosynthetic approach to scaffold 1.
SCHEME 1
The syntheses of the tetracyclic intermediates 11a/12a and 11b/12b.

Though the cyclization was not regioselective, these compounds were readily separable and we viewed the diversity of these structures as an opportunity for our envisioned kinase inhibitory structure-activity studies on final compounds derived from both of these two templates.

Direct conversion of chlorides 11a/12a and 11b/12b to the corresponding pyridones, using the published acidic conditions (HOAc,16 HCl17) and basic conditions (KOH/DMSO)18 failed to provide the desired products. For this reason, we chose to convert the chloro intermediates to the corresponding ethers, which could serve as a precursor to the pyridone (see Scheme 2). While sodium methoxide (in methanol or toluene)19 under reflux conditions and DMAP yielded only partial chloro to methoxy conversion, we found that barium hydroxide (octahydrate) in the presence of excess DMAP in refluxing methanol provided complete conversion to ethers 13a/14a and 13b in 54/51% and 54% yield (Scheme 2). Though barium hydroxide is used quite often as the base for Suzuki couplings20, 21 and as an ester saponification reagent,22,23 the aforementioned exchange catalyzed by barium hydroxide appears rather new. Ether 13a could readily be converted to pyridone 1,24 in a modest yield of 63% after recrystallization. Structural confirmation of pyridone 1 was achieved via X-ray crystal determination (Figure 5). Using Buchwald conditions, ether 14a was converted to its amino analogue 15. Using a synthetic approach similar to Scheme 1 we synthesized various aryl analogues (Scheme 3).

FIGURE 5
The single crystal X-ray structure of 1, which confirms our structural assignment (see supplementary data).
SCHEME 2
The synthesis of pyridone or methoxy pyridine inhibitors.
SCHEME 3
Synthesis of aryl analogues

Discussion

With this initial set of compounds in hand, we investigated their inhibitory effects on a set of kinases using in vitro enzymatic assays, performed as previously reported.5 As can be seen (TABLE 1), depending on the position of the regioisomer and the substituents, we were able to identify initial compounds with the desirable selectivity for the oncogenic human anaplastic lymphoma kinase (ALK).5,25 For example, compounds such as 24b, 25a and 26a showed selectivity for ALK as compared to the highly homologous (and clinically relevant due to the possibility of iatrogenic diabetes with its inhibition) human insulin receptor kinase (IRK), exhibiting IC50s in the single-digit micromolar range for ALK and greater than the highest compound concentration tested (40 μM) for the IRK. We have previously observed that this degree of selectivity at the hit stage indicates that these ‘hits’ may be converted into more potent and selective compounds. The relative potency of regioisomers such as 25b versus 25a was unexpected and could be due to difference in the kinase domain structure of ALK versus IGF1R, X-ray crystal structure of ALK remain unpublished at this time. The observed potencies are ‘hit-like’ and demonstrates the utility of our framework design method to identify compounds that are suitable starting points for further optimization into more potent selective inhibitors for additional iterations of medicinal chemistry.

TABLE 1
Biochemical evaluation of pyridone 1 and analogs (using the in vitro enzymatic kinase assay previously described in the Supplementary Data).5

Conclusion

We report the details on the specific application of our recently published ‘framework’ design1 of a new kinase inhibitor template and the synthesis of the subject of this design, a novel tetracyclic pyridone tyrosine kinase inhibitor template, which was accomplished in 8 steps.26 This synthesis involves a one pot di-ortho-substituted aldehyde condensation, followed by the cyanide-catalyzed cyclization, which gave the key indole intermediate in >70% yield. In addition, we report the first example of barium hydroxide-catalyzed chloro-to-methoxy pyridine conversion. Finally, we present initial structure-activity data showing that representative examples of compounds based on these templates demonstrate activity and initial selectivity for anaplastic lymphoma kinase (ALK), thus indicating that they are suitable starting points for the development of more active ‘lead-like’ inhibitors through the usual process of ‘hit-to-lead’ chemistry. A future avenue of research will be the exploration of these templates for the discovery of new therapeutic leads against oncogenic tyrosine kinases in the insulin receptor superfamily such as ALK.5, 25

EXPERIMENTAL

General Remarks

All reactions were performed under an atmosphere of nitrogen. Flash chromatography purifications employed silica gel 60 from EMD Chemicals (particle size: 0.040–0.063 mm, 230–400 mesh ASTM). Thin layer chromatography was performed on silica gel with UV-254 indicator (250 micron thickness). All TLC visualizations were conducted with UV light. Nuclear magnetic resonance experiments were conducted using a 400 MHz II instrument (Bruker Avance II). Microwave reactions were conducted using a Biotage Initiator 60 (N = 100–300W, H = 50–150W, VH = 40–90W). HPLC analyses were accomplished using an UPLC/UV/ELSD/SQD (Single Quadrapole Detector) with stationary phase: BEH C18, 1.7 μm, solvents: A: 0.1% formic acid in water, B: 0.1% formic acid in acetonitrile, detector types: PDA (210 to 400 nm) and ELSD.

X-ray crystallographic data

Crystallographic data (excluding structure factors) for the structures in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication nos. CCDC 711726. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, (fax: +44-(0)1223-336033 or e-mail: ku.ca.mac.cdcc@tisoped).

(E)-tert-butyl 3-(2-aminophenyl)acrylate (4)

To a solution of iodoaniline (5.5 g, 25.0 mmol), acrylate (3.8 mL, 26.2 mmol) and sodium bicarbonate (5.3 g, 62.4 mmol) in DMF (8 mL) was added palladium acetate (0.3 g, 1.3 mmol) in one portion. The resulting mixture was heated to 70 °C for 16 h. The mixture was then diluted with EtOAc and filtered through celite. After concentrating under reduced pressure, crude product was purified via column chromatography (EtOAc:Hexanes, 10:90) to provide the ester 4 (4.72 g, 86%) as a yellow solid. mp 76–78 °C. 1H NMR (CDCl3) δ 7.75 (d, J = 15.8 Hz, 1H), 7.38 (dd, J = 7.8, 1.3 Hz, 1H), 7.17 (t, J = 7.5, 1.6 Hz, 1H), 6.78 (t, J = 7.6, 1.5 Hz, 1H), 6.72 (d, J = 8.1 Hz, 1H), 6.32 (d, J = 15.8 Hz, 1H), 3.93 (bs, 2H), 1.56 (s, 9H). 13C NMR (CDCl3) δ 166.7, 145.4, 139.1, 128.1, 120.2, 120.1, 118.9, 116.7, 80.5, 28.3. HRMS (ESI) m/z Calcd for C13H18NO2 (M+1)+ 220.1338, found 220.1332.

(E)-tert-butyl 3-(2-amino-4-chlorophenyl)acrylate (5) was prepared in a similar manner to conditions for ester 2 to give ester 5 (92%) as a yellow solid. mp 63–65 °C. 1H NMR (400 MHz, CDCl3) δ 7.55 (d, 1H, J = 15.8 Hz), 7.20 (d, 1H, J = 8.1 Hz), 6.63 (m, 2H), 6.18 (d, J = 15.8 Hz, 1H), 1.44 (s, 9H), 13C NMR (100 MHz, CDCl3) δ 166.4, 146.2, 137.9, 136.5, 129.2, 120.7, 119.1, 118.6, 116.1, 100.0, 80.7, 28.3. HRMS (ESI) m/z Calcd for C13H16NO2Cl (M+1)+ 253.08695, found 253.08688.

tert-butyl 2-(2-(2,4-dichloro-6-methylpyridin-3-yl)-1H-indol-3-yl)acetate (7)

To a solution of amine 4 (1.4 g, 6.4 mmol) and aldehyde 6 (1.5 g, 7.7 mmol) in EtOH (5 mL) was added HOAc (0.6 mL, 10.2 mmol). After two hrs, the solvent was removed under reduced pressure. The resulting crude imine was treated with potassium cyanide (0.9 g, 14.1 mmol) and additional acetic acid (0.37 mL, 6.4 mmol). The reaction mixture was heated to 70 °C stirred for 22 h. Once deemed complete, the solvent was removed under reduced pressure. Crude product was taken up in EtOAc and washed with sat. aq. NaHCO3 solution. The organic phase was dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude ester was purified via column chromatography (EtOAc:Hexanes, 20:80) to furnish the indole ester 7 (1.84 g, 73%) as a yellow oil. 1H NMR (CDCl3) δ 7.90 (s, 1H), 7.66 (d, J = 7.9 Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.24 (s, 1H), 7.19 (m, 3H), 7.12 (t, J = 7.5 Hz, 1H), 3.43 (d, J = 1.0 Hz, 2H), 2.54 (s, 3H), 1.48 (s, 9H). 13C NMR (CDCl3) δ 170.2, 160.3, 152.4, 147.5, 136.2, 122.8, 127.5, 123.0, 120.1, 119.9, 111.1, 110.1, 80.7, 32.5, 27.9, 24.0. HRMS (ESI) m/z Calcd for C20H21N2O2Cl2 (M+1)+ 391.0980, found 391.0976.

tert-butyl 2-(6-chloro-2-(2,4-dichloro-6-methylpyridin-3-yl)-1H-indol-3-yl)acetate (8) was prepared in a similar manner to the conditions above for 4 to give 8 (68%) as a off-white solid. mp 72–74 °C. 1H NMR (400 MHz, CDCl3) δ 8.21 (s, 1H), 7.79 (d, J = 1.8 Hz, 1H), 7.20 (m, 3H), 3.36 (d, J = 3.3 Hz, 2H), 2.50 (s, 3H), 1.28 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 169.9, 160.6, 152.3, 147.5, 136.5, 129.0, 128.2, 126.3, 123.2, 121.1, 111.1, 110.3, 80.9, 32.4, 27.9, 24.0. HRMS (ESI) m/z Calcd for C20H20N2O2Cl3 (M+1)+ 425.0590, found 425.0571.

2-(2-(2,4-dichloro-6-methylpyridin-3-yl)-1H-indol-3-yl)-N-phenethylacetamide (9)

A solution of indole ester 5 (1.7 g, 4.0 mmol) in dichloromethane (3 mL) was treated with trifluoroacetic acid (3.0 mL, 40.0 mmol). The solution stirred at RT for 1 h. The solvent was then removed under reduced pressure. The crude acid was dissolved in DMF, treated with 4-methylmorpoline (1.3 mL, 12.0 mmol) followed by HBTU (2.3 g, 6.0 mmol) and phenethylamine (0.6 mL, 4.8 mmol) in one portion. After 1 h, the mixture was diluted with EtOAc and washed with sat. aq. NaHCO3 solution, dried with sodium sulfate, filtered and concentrated under reduced pressure. Crude product was purified via column chromatography (EtOAc:Hexanes, 1:1) to yield indole amide 9 (0.50 g, 80%) as an off-white solid. mp 165–167 °C. 1H NMR (CDCl3) δ 8.02 (s, 1H), 7.54 (d, J = 8.0 Hz, 1H), 7.40 (d, J = 8.2 Hz, 1H), 7.29 (t, J = 7.5 Hz 1H), 7.20 - 7.15 (m, 3H), 7.05 - 6.90 (m, 3H), 6.67 (d, J = 6.9 Hz, 2H), 5.62 (s, 1H), 3.47 (q, J = 17.5 Hz, 2H), 3.39-3.22 (m, 2H), 2.55-2.48 (m, 5H). 13C NMR (CDCl3) δ 170.2, 161.3, 152.5, 147.3, 138.3, 135.9, 128.6, 127.5, 126.2, 123.4, 120.6, 119.1, 111.4, 109.4, 40.3, 35.6, 32.6, 24.0. Anal. Calcd for (C24H21Cl2N3O) C, 65.76, H, 4.83, N 9.59. Found: C, 65.22, H, 4.81, N 9.39. HRMS (ESI) m/z Calcd for C24H22N3OCl2 (M+1)+ 438.1140, found 438.1144.

2-(6-chloro-2-(2,4-dichloro-6-methylpyridin-3-yl)-1H-indol-3-yl)-N-phenethylacetamide (10) was prepared in a similar manner to above conditions for 5 to give amide 10 (74%) as a transparent oil. 1H NMR (400 MHz, CDCl3) δ 9.25 (s, 1H), 7.47 - 7.42 (m, 2 H), 7.22 (s, 1H), 7.16 (dd, J = 8.4, 1.4 Hz, 1H), 7.11 - 7.01 (m, 3H), 6.76 (m, 2H), 5.65 (t, J = 5.6 Hz, 1H), 3.53 - 3.27 (m, 4H), 2.78 (s, 5H). 13C NMR (100 MHz, CDCl3) δ 170.2, 161.0, 152.1, 147.2, 138.2, 136.8, 129.4, 128.5, 128.4, 125.8, 123.4, 123.2, 121.3, 120.1, 111.6, 109.1, 40.5, 38.6, 35.4, 32.6, 24.0. HRMS (ESI) m/z Calcd for C24H20N3OCl3 (M+1)+ 472.0750, found 472.0758.

Compounds 11a and 11b

A refluxing mixture of amide 9 (0.6 g, 1.3 mmol) and boron trifluoride dimethyl etherate (0.2 mL, 2.2 mmol) in THF (3 mL) was exposed to 1.0 M borane-THF complex (3.9 mL, 3.9 mmol in THF) for 3 h. After this time, the reaction was cooled to 0 °C and quenched with 4.5 N HCl (3 mL). The mixture was stirred for 1 h and then at RT for 1 h. The mixture was then cooled to 0 °C and basified to pH 13 with solid KOH and extracted with CH2Cl2 (10 mL) 3 times. The organic solution was dried (sodium sulfate), filtered and concentrated to afford the amine, which was diluted in DMF (5 mL) and treated triethylamine (0.2 mL, 1.3 mmol). The reaction was heated to 70 °C for 16 h, then quenched via the addition of water (5 mL) at RT. The aqueous layer was extracted with EtOAc (5 mL). The organic layer was dried, filtered and concentrated under reduced pressure. The regioisomers were separated via column chromatography (EtOAc:Hexanes, 9:1 to 4:1) to afford pure 2-chlroro pyridine 11a (0.17 g, 34%) as an off-white solid. mp 165–166°C. 1H NMR (CDCl3) δ 8.95 (s, 1H), 7.41 (d, J = 7.8 Hz, 1H), 7.31 (d, J = 8.1 Hz, 1H), 7.18 - 7.00 (m, 7H), 6.57 (s, 1H), 3.53 (t, J = 7.6 Hz, 2H), 3.46 (t, J = 5.6 Hz, 2H), 2.97 (t, J = 5.6, 2H), 2.86 (t, J = 7.6 Hz, 2H), 2.39 (s, 3H). 13C NMR (CDCl3) δ 161.5, 154.0, 139.8, 139.4, 135.4, 134.7, 127.2, 126.3, 126.0, 122.8, 118.8, 116.7, 115.5, 110.3, 109.2, 53.5, 51.9, 34.7, 27.1, 23.6. HRMS (ESI) m/z Calcd for C24H23N3Cl (M+1)+ 388.1581, found 388.1589. 4-chloro pyridine 11b (0.12 g, 24%) was a yellow solid. Yield: 24%, mp 155–156 °C. 1H NMR (CDCl3) δ 8.92 (s, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.19-7.00 (m, 7H), 6.8 (s, 1H), 3.86 (t, J = 7.7 Hz, 2H), 3.57 (t, J = 7.5 Hz, 2H), 3.09 (t, J = 5.4 Hz, 2H), 3.02 (t, J = 7.7 Hz, 2H), 2.45 (s, 3H). 13C NMR (CDCl3) δ 160.6, 154.8, 147.0, 138.8, 135.4, 128.9, 127.2, 126.7, 123.2, 118.5, 116.1, 113.5, 110.6. 109.5, 54.2, 53.9, 34.7, 26.5, 23.9. HRMS (ESI) m/z Calcd for C20H21N2O2Cl2 (M+1)+ 388.1581, found 388.1563.

Compounds 12a and 12b

These compounds were prepared in a similar manner to the above cyclization from amide 10 to give 2-chloro pyridine 12a (34%) as an off-white solid. mp 152–154 °C. 1H NMR (400 MHz, CDCl3) δ 9.02 (s, 1H), 7.41 (d, J = 1.7 Hz, 1H), 7.36 (d, 1H, J = 8.4 Hz), 7.18 - 7.05 (m, 6H), 6.66 (s, 1H), 3.63 (t, J = 7.4 Hz, 2H), 3.52 (t, J = 5.6 Hz, 2H), 3.00 - 2.92 (m, 4H), 2.46 (s, 3H), 13C NMR (100 MHz, CDCl3) δ 160.5, 155.0, 146.9, 138.7, 135.2, 128.7, 128.5, 128.0, 126.6, 126.3, 120.1, 119.6, 116.0, 113.2, 110.4, 109.5, 54.6, 53.9, 34.4, 26.6, 24.0, HRMS (ESI) m/z Calcd for C24H21Cl2N3 (M+1)+ 422.1191, found 422.1197. 4-chloro pyridine 12b (21%) was a yellow solid. mp 175–177 °C. 1H NMR (400 MHz, CDCl3) δ 8.90 (s, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.35 (d, J = 1.6 Hz, 1H,), 7.17 - 7.13 (m, 5H), 7.08 (dd, J = 8.4, 1.8 Hz, 1H), 6.79 (s, 1H), 3.86 (t, J = 7.6 Hz, 2H), 3.53 (t, J =5.5 Hz, 2H), 3.05 - 2.97 (m, 4H), 2.45 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 161.6, 153.7, 140.2, 139.3, 135.3, 128.9, 128.3, 128.1, 126.5, 120.1, 119.7, 116.7, 115.7, 110.3, 108.7, 53.7, 51.8, 34.7, 26.8, 23.8. HRMS (ESI) m/z Calcd for C24H21Cl2N3 (M+1)+ 422.1191, found 422.1187.

Compound 13a

2-chloro pyridine (0.2 g, 0.4 mmol) and DMAP (0.4 g, 3.6 mmol) was dissolved in methanolic barium hydroxide octahydrate solution (1.2 g, 7.2 mmol Ba(OH)2·8H2O, 8 mL methanol). The resulting solution was heated to reflux for 16 h. The methanol was removed under reduced pressure; the crude product was dissolved in EtOAc and washed with water. The organic solution was dried with sodium sulfate, filtered, concentrated and purified via column chromatography (EtOAc:Hexanes, 1:9) to yield ether 13a (0.07 g, 54%) as a white solid. mp 137–139 °C. 1H NMR (CDCl3) δ 10.1 (s, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.30 - 7.20 (m, 6H), 7.12 (t, J = 7.4 Hz, 1H), 6.48 (s, 1H), 4.18 (s, 3H), 3.64 (t, J = 7.8 Hz, 2H), 3.50 (t, J = 5.4 Hz, 2H), 3.12 (t, J = 5.3 Hz, 2H), 3.03 (t, J = 7.8 Hz, 2H), 2.43 (s, 3H). 13C NMR (CDCl3) δ 160.9, 158.7, 152.1, 139.3, 134.4, 128.6, 128.2, 126.5, 122.1, 118.1, 113.7, 110.3, 105.9, 100.9, 55.2, 53.8, 52.0, 34.7, 27.8, 24.1.Anal. Calcd for (C25H25N3O) C, 78.30, H, 6.57, N 10.96. Found: C, 77.95, H, 6.62, N 10.75.

Compound 13b

This compound was prepared in a similar manner to the above methoxy displacement to give ether 13b (51%) as an off-white solid. mp 80–82 °C. 1H NMR (CDCl3) δ 9.75 (s, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H), 7.26 - 7.23 (m, 4H), 7.19 (t, J = 8.0 Hz, 2H), 7.10 (t, J = 7.0 Hz, 1H), 6.42 (s, 1H) 4.06 (s, 3H), 3.88 (t, J = 7.8 Hz, 2H), 3.53 (t, J = 5.3 Hz, 2H), 3.12 - 3.08 (m, 4H), 2.48 (s, 3H). 13C NMR (CDCl3) δ 140.6, 134.7, 129.0, 128.4, 128.1, 126.1, 122.1, 118.8, 118.4, 113.4, 110.3, 98.5, 56.0, 54.5, 50.9, 34.9, 29.7, 27.8, 24.8. HRMS (ESI) m/z Calcd for C25H26N3O (M+1)+ 384.2076, found 384.2069.

Compound 14a

This compound was prepared in a similar manner to the above methoxy displacement to give ether 14a (54%) as a clear oil. 1H NMR (400 MHz, CDCl3) δ 9.98 (s, 1H), 7.28 (d, J = 1.8 Hz, 1H) 7.27 (d, J = 8.7 Hz, 1H), 7.18 - 7.07 (m, 7H), 6.96 (dd, J = 8.4, 1.8 Hz, 1H), 6.38 (s, 1H), 4.05 (s, 3H), 3.54 (t, J = 7.7 Hz, 2H), 3.37 (t, J = 5.2 Hz, 2H), 2.95 - 2.86 (m, 4H), 2.32 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 159.1, 156.8, 150.6, 137.4, 132.9, 127.0, 126.8, 125.9, 125.1, 124.7, 117.7, 117.1, 111.9, 108.4, 98.8, 53.4, 52.0, 50.2, 32.8, 25.8, 22.4. HRMS (ESI) m/z Calcd for C25H25N3OCl (M+1)+ 418.1686, found 418.1680.

3-Methyl-6-N-phenethyl-5,6,7,12-tetrahydroindolo[2,1-d]pyrido[4,3-b]azepin-1(2H)-one (1)

To a solution of O-methyl pyridine (0.05 g, 140μmol) in dioxane (0.2 mL) was added 4M HCl (aq.). The mixture was heated to 65 °C for 5 h. Crude product was purified via recrystallization (EtOAc:Hexanes) to afford pyridone 1 (32 mg, 63%) as a tan solid. Yield: 63%, mp 244 °C. 1H NMR (CDCl3) δ 12.4 (s, 1H), 9.94 (s, 1H), 7.49 (d, J = 7.5 Hz, 1H), 7.41 (d, J = 7.5 Hz, 1H), 7.36 - 7.24 (m, 6H), 7.17 (t, J = 7.0 Hz, 1H), 7.09 (t, J = 7. 0 Hz, 1H), 5.85 (s, 1H), 3.65 (t, J = 7.6 Hz, 2H), 3.51 (t, J = 4.8 Hz, 2H), 3.12 (t, J = 4.9 Hz, 2H), 3.08 (t, J = 7.6 Hz, 2 H), 2.30 (s, 3H). 13C NMR (CDCl3) δ 165.7, 158.1, 140.9, 138.7, 133.7, 132.1, 128.8, 128.0, 126.7, 121.1, 118.5, 117.5, 111.2, 110.6, 100.4, 99.8, 55.4, 51.3, 34.8, 29.7, 26.9, 19.1.

Compound 15

A mixture of 14a (0.08 g, 0.2 mmol), dicyclohexyl(2′,4′,6′-triisopropylbiphenyl-2-yl)phosphine (X-Phos®, 6 mg, 0.01 mmol), and Pd2(dba)3 (16 mg, 0.02 mmol) was treated with a solution of N-methyl piperazine (0.03 mL, 0.2 mmol) in THF (1 mL). This solution was treated with LiHMDS (0.6 mL, 1M in THF) in one portion. The reaction was heated to 70 °C in a sealed tube overnight. The reaction mixture was dissolved in EtOAc, washed with sat. aq. NaHCO3 solution, dried over magnesium sulfate, filtered and concentrated under reduced pressure. Crude product was purified via reverse phase HPLC. Ether 15 (11 mg, 10%) was a tan oil. 1H NMR (400 MHz, CDCl3) δ 10.4 (s, 1H), 8.25 (s, 1H), 7.30 - 7.25 (m, 6H), 6.96 (d, J = 1.9 Hz, 1H), 6.75 (dd, J = 8.6, 2.0 Hz, 1H), 4.05 (s, 3H), 3.57 (t, J = 7.8 Hz, 4H), 3.44 (t, J = 5.2 Hz, 4H), 3.12 (t, J = 4.8 Hz, 4H), 3.00 - 2.92 (m, 5H), 2.32 (s, 3H), 2.24 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 163.6, 160.2, 158.1, 150.8, 147.5, 139.4, 135.6, 128.8, 127.0, 126.1, 121.8, 117.9, 112.9, 110.9, 105.4, 100.5, 97.5, 54.8, 54.3, 53.3, 51.5, 50.0, 45.7, 33.7, 27.2, 23.8. HRMS (ESI) m/z Calcd for C30H35N5O (M+1)+ 482.2920, found 482.2912.

(E)-tert-butyl 3-(2-amino-4-bromophenyl)acrylate (17) was prepared in a similar manner to conditions for 2 to give ester 17 (92%) as yellow solid. mp 63–65 °C. 1H NMR (400 MHz, CDCl3) δ 7.62 (d, J = 15.8 Hz, 1H), 7.48 (d, J = 2.3 Hz, 1H), 7.23 (dd, J = 8.5, 2.2 Hz, 1H), 6.59 (d, J = 8.5 Hz, 1H), 6.29 (d, J = 15.7 Hz, 1H), 3.96 (s, 2H), 1.55 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 166.2, 155.3, 144.3, 137.5, 133.4, 130.3, 121.9, 121.5, 118.2, 110.6, 80.8, 28.2. HRMS (ESI) m/z Calcd for C13H17NO2Br (M+1)+ 298.0443, found 298.0442.

tert-butyl 2-(5-bromo-2-(2-chloro-4-iodopyridin-3-yl)-1H-indol-3-yl)acetate (18) was prepared in a similar manner to the conditions above for 4 to give indole ester 18 (66%) as a yellow solid. mp 181–184 °C. 1H NMR (400 MHz, CDCl3) δ 8.05 (s, 1H), 8.00 (d, J = 5.2 Hz, 1H), 7.83 (s, 1H), 7.79 (d, J = 5.2 Hz, 1H), 7.28 (dd, J = 8.6, 1.8 Hz, 1H), 7.24 – 7.18 (m, 2H), 3.36 (d, J = 4.6 Hz, 2H), 1.31 (s, 9H).13C NMR (101 MHz, CDCl3) δ 169.6, 151.7, 149.9, 134.6, 134.0, 133.2, 133.2, 129.4, 126.1, 123.1, 114.8, 113.5, 112.8, 108.8, 81.2, 32.5, 28.1. HRMS (ESI) m/z Calcd for C19H18N2O2ClBrI (M+1)+ 546.9285, found 546.9272.

2-(5-bromo-2-(2-chloro-4-iodopyridin-3-yl)-1H-indol-3-yl)-N-((S)-1-phenylethyl)acetamide (19) was prepared in a similar manner to above conditions for 5 to give amide 19 (68%) as a tan oil. 1H NMR (400 MHz, CDCl3) δ 8.50 (d, J = 9.9 Hz, 1H), 8.03 (dd, J = 5.2, 2.2 Hz, 1H), 7.75 (dd, J = 6.2, 5.4 Hz, 1H), 7.68 (dd, J = 8.8, 1.8 Hz, 1H), 7.33 (dt, J = 8.6, 1.9 Hz, 1H), 7.24 (dd, J = 10.0, 1.2 Hz, 1H), 7.20 –7.10 (m, 4H), 7.01 (m, 2H), 5.84 (d, J = 8.0 Hz, 1H), 4.99 (qn, J = 7.2 Hz, 1H), 3.46 (dd, J = 17.5, 5.5 Hz, 1H), 3.36 (dd, J = 17.5, 4.5 Hz, 1H), 1.25 (dd, J = 6.9, 5.0 Hz, 3H). 13C NMR (CDCl3) δ 168.9, 151.4, 151.4, 150.4, 142.9, 142.8, 135.0, 133.3, 132.7, 127.3, 126.1, 122.3, 114.7, 114.0, 113.2, 108.1, 108.0, 49.0, 33.0, 21.8. HRMS (ESI) m/z Calcd for C23H19N3OClBrI (M+1)+ 593.9445, found 593.9445.

2-(5-bromo-2-(2-chloro-4-iodopyridin-3-yl)-1H-indol-3-yl)-N-(2-(4-hydroxyphenyl)ethyl) acetamide (20) was prepared in a similar manner to above conditions for 5 to give amide 20 (39%) as a brown oil. 1H NMR (400 MHz, Acetone) δ 10.64 (s, 1H), 8.20 (bs, 1H) 8.15 (t, J = 4.9 Hz, 1H), 8.04 (t, J = 4.9 Hz, 1H), 7.98 (d, J = 2.4 Hz, 1H), 7.47 (dd, J = 8.6, 4.4 Hz, 1H), 7.36 (m, 1H), 6.83 (m, 2H), 6.63 (m, 3H), 3.46 (t, J = 5.3 Hz, 2H), 3.31 (m, 2H), 2.58 (m, 2H). 13C NMR (101 MHz, Acetone) δ 169.8, 156.7, 151.9, 150.9, 136.3, 136.0, 134.4, 134.3, 130.8, 130.6, 130.5, 126.1, 123.4, 116.1, 115.6, 114.3, 113.2, 109.3, 41.8, 35.7, 33.4. HRMS (ESI) m/z Calcd for C22H19N3O2ClBrI (M+1)+ 609.9394, found 609.9421.

2-(5-bromo-2-(2-chloro-4-iodopyridin-3-yl)-1H-indol-3-yl)-N-(2,2-diphenylethyl)acetamide (21) was prepared in a similar manner to above conditions for 5 to give amide 21 (94%) as a clear oil. 1H NMR (400 MHz, CDCl3) δ 8.34 (s, 1H), 7.95 (d, J = 5.2 Hz, 1H), 7.70 (d, J = 5.2 Hz, 1H), 7.65 (d, J = 1.7 Hz, 1H), 7.37 (dd, J = 8.6, 1.8 Hz, 1H), 7.25 (d, J = 8.6 Hz, 1H), 7.05 – 6.97 (m, 6H), 6.90 – 6.84 (m, 4H), 5.53 (t, J = 5.4 Hz, 1H), 3.82 (t, J = 7.6 Hz, 1H), 3.73 (t, J = 6.7 Hz, 2H), 3.33 (d, J = 5.2 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 169.7, 151.4, 150.3, 141.5, 141.4, 135.1, 134.7, 133.0, 132.6, 128.8, 128.6, 128.0, 127.9, 126.8, 126.7, 122.1, 114.7, 114.1, 113.1, 107.8, 50.4, 43.6, 32.7. HRMS (ESI) m/z Calcd for C29H23N3OClBrI (M+1)+ 669.9758, found 669.9736.

N-(2-(1H-indol-3-yl)ethyl)-2-(5-bromo-2-(2-chloro-4-iodopyridin-3-yl)-1H-indol-3-yl)acetamide (22) was prepared in a similar manner to above conditions for 5 to give amide 22 (58%) as a tan solid. mp: 240–242 °C. 1H NMR (400 MHz, DMSO) δ 11.45 (s, 1H), 10.77 (s, 1H), 8.16 (d, J = 5.2 Hz, 1H), 8.08 (d, J = 5.1 Hz, 1H) 7.94 (d, J = 1.9 Hz, 1H), 7.84 (t, J = 5.7 Hz, 1H), 7.49 (d, J = 7.9 Hz, 1H), 7.35 (d, J = 8.6 Hz, 1H), 7.32 (d, J = 8.1 Hz, 1H), 7.30 (dd, J = 8.6, 2.0 Hz, 1H), 7.06 – 7.03 (m, 2H), 6.95 (dt, J = 7.5, 1.0 Hz, 1H), 3.30 (m, 5H), 2.72 (t, J = 7.5 Hz, 2H). 13C NMR (101 MHz, DMSO) δ 169.1, 150.3, 149.9, 136.2, 134.9, 134.4, 133.3, 133.2, 129.5, 127.1, 124.2, 122.5, 122.4, 120.9, 118.2, 116.5, 113.3, 111.7, 111.4, 108.1, 31.9, 25.3, HRMS (ESI) m/z Calcd for C25H20N4OClBrI (M+1)+ 632.9554, found 632.9537.

Compounds 23a/b

These compounds were prepared in a similar manner to the above cyclization from amide 10 except the cyclization step was heated to 170 °C for 1 h using microwave heating (absorbance: normal) to provide 23a (11%) as a tan solid. mp: 142–146 °C. 1H NMR (400 MHz, CDCl3) δ 9.00 (s, 1H), 7.83 (d, J = 5.7 Hz, 1H), 7.51 (s, 1H), 7.29 - 7.22 (m, 7H), 6.68 (d, J = 5.7 Hz, 1H), 4.96 (q, J = 6.8 Hz, 1H), 3.49 (t, J = 5.5 Hz, 2H), 2.93 (dt, J = 16.7, 5.7 Hz, 1H), 2.72 (dt, J = 16.7, 5.4 Hz, 1H), 1.67 (d, J = 6.9 Hz, 3H), 13C NMR (101 MHz, CDCl3) δ 159.8, 147.0, 144.2, 139.6, 132.5, 128.4, 127.8, 127.4, 126.6, 125.9, 125.2, 120.4, 114.7, 113.7, 111.5, 111.1, 110.0, 58.2, 47.6, 26.4, 17.4. HRMS (ESI) m/z Calcd for C23H20N3BrCl (M+1)+ 452.0529, found 452.0509. Compound 23b was a tan oil (7%). 1H NMR (400 MHz, CDCl3) δ 8.38 (s, 1H), 7.61 (d, J = 4.9 Hz, 1H), 7.47 (d, J = 1.6 Hz, 1H), 7.23 (m, 8H), 5.83 (q, J = 6.9 Hz, 1H), 3.40 (ddd, J = 14.5, 6.5, 4.4 Hz, 1H), 3.18 (ddd, J = 14.3, 8.2, 3.6 Hz, 1H), 2.78 (ddd, J = 16.7, 6.2, 3.7 Hz, 1H), 2.46 (ddd, J = 16.7, 8.2, 4.3 Hz, 1H), 1.61 (d, J = 6.9 Hz, 3H), 13C NMR (101 MHz, CDCl3) δ 160.9, 143.0, 140.7, 132.3, 129.0, 128.9, 127.3, 126.9, 126.6, 126.1, 125.0, 120.7, 114.7, 114.4, 111.6, 111.0, 100.9, 54.5, 45.1, 25.9, 15.5, HRMS (ESI) m/z Calcd for C23H20N3BrI (M+1)+ 543.9885, found 543.9873.

Compounds 24a/b through 26a/b

These compounds were prepared in a similar manner to the above cyclization from amide 10. Compound 24a was a tan solid (22%). mp: 217–219 °C. 1H NMR (400 MHz, Acetone) δ 10.29 (s, 1H), 8.07 (s, 1H), 8.02 (s, 1H), 7.95 (d, J = 5.6 Hz, 1H), 7.66 (d, J = 1.9 Hz, 1H), 7.46 (d, J = 8.6 Hz, 1H), 7.28 (dd, J = 8.6, 1.9 Hz, 1H), 7.07 (d, J = 5.7 Hz, 1H), 7.05 (d, J = 8.4 Hz, 2H), 6.65 (d, J = 8.5 Hz, 2H), 3.65 (m, 4H), 3.12 (t, J = 5.8 Hz, 2H), 2.91 (t, J = 7.7 Hz, 2H). 13C NMR (101 MHz, Acetone) δ 217.5, 214.9, 213.8, 212.7, 212.3, 206.1, 206.1, 161.3, 156.8, 149.1, 146.3, 135.3, 130.7, 130.7, 130.4, 130.2, 126.2, 121.8, 116.4, 116.1, 116.0, 113.8, 112.5, 111.3, 55.6, 55.2, 33.9, 26.8. HRMS (ESI) m/z Calcd for C23H20N3OBrCl (M+1)+ 468.0478, found 468.0469. Compound 24b was a brown solid (10%). mp: 208–212 °C. 1H NMR (400 MHz, DMSO) δ 10.80 (s, 1H), 9.17 (s, 1H), 7.76 (d, J = 4.9 Hz, 1H), 7.73 (d, J = 1.7 Hz, 1H), 7.47 (d, J = 4.9 Hz, 1H), 7.40 (d, J = 8.6 Hz, 1H), 7.30 (dd, J = 8.6, 1.9 Hz, 1H), 7.04 (d, J = 8.4 Hz, 2H), 6.64 (d, J = 8.4 Hz, 2H), 3.72 (t, J = 7.7 Hz, 2H), 3.58 (t, J = 5.4 Hz, 2H), 3.08 (t, J = 5.6 Hz, 2H), 2.85 (t, J = 7.7 Hz, 2H). 13C NMR (101 MHz, DMSO) δ 161.2, 155.5, 144.2, 134.2, 131.5, 129.8, 129.5, 129.4, 127.1, 124.8, 120.8, 117.1, 115.0, 114.1, 113.3, 111.1, 104.9, 52.5, 33.1, 25.5. HRMS (ESI) m/z Calcd for C23H20N3OBrI (M+1)+ 559.9835, found 559.9857. Compound 25a was an off-white solid (24%). mp: 112–114 °C. 1H NMR (400 MHz, Acetone) δ 10.13 (s, 1H), 7.99 (d, J = 5.6 Hz, 1H), 7.33 (d, J = 1.9 Hz, 1H), 7.28 (dd, J = 8.6, 0.4 Hz, 1H), 7.13 (dd, J = 8.6, 1.9 Hz, 1H), 7.00 (dd, J = 7.6, 1.6 Hz, 5H), 6.89 (t, J = 1.8 Hz, 1H), 6.87 (dd, J = 5.8, 1.4 Hz, 3H), 6.84 (t, J = 1.5 Hz, 1H), 6.83 (t, J = 3.0 Hz, 1H), 4.18 (d, J = 8.6, 6.9 Hz, 1H), 4.09 (d, J = 3.9 Hz, 2H), 3.48 (t, J = 5.8 Hz, 2H), 2.57 (d, J = 5.8 Hz, 2H). 13C NMR (101 MHz, Acetone) δ 161.1, 149.2, 146.3, 143.5, 135.3, 130.4, 129.9, 128.9, 128.9, 127.1, 125.9, 121.9, 117.4, 116.6, 113.6, 112.1, 112.1, 58.8, 57.2, 50.6, 26.4, HRMS (ESI) m/z Calcd for C29H24N3ClBr (M+1)+ 528.0842, found 528.0848. Compound 25b was a tan oil (3%). 1H NMR (400 MHz, CDCl3) δ 8.23 (s, 1H), 7.60 (d, J = 4.9 Hz, 1H), 7.35 (d, J = 1.7 Hz, 1H), 7.29 (d, J = 5.0 Hz, 1H), 7.23 (dd, J = 8.6, 1.8 Hz, 1H), 7.19 (s, 2H), 7.14 (d, J = 8.5 Hz, 1H), 6.96 (m, 10H), 4.25 (m, 3H), 3.37 (t, J = 5.7 Hz, 2H), 2.40 (t, J = 5.7 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 161.3, 144.1, 142.9, 133.3, 129.8, 129.7, 128.2, 128.1, 128.1, 126.2, 125.9, 121.9, 117.8, 115.9, 112.3, 111.8, 102.3, 57.4, 54.6, 49.9, 25.8. HRMS (ESI) m/z Calcd for C29H24N3BrI (M+1)+ 620.0198, found 620.0211. Compound 26a was a light brown solid (30%). mp: 248–252 °C. 1H NMR (400 MHz, DMSO) δ 10.99 (s, 1H), 10.81 (s, 1H), 7.98 (d, J = 5.6 Hz, 1H), 7.69 (s, 1H), 7.56 (d, J = 7.4 Hz, 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.32 (d, J = 7.8 Hz, 1H), 7.25 (d, J = 6.7 Hz, 1H), 7.17 (s, 1H), 7.06 (m, 2H), 6.95 (t, J = 7.1 Hz, 1H), 3.64 (bs, 4H), 3.10 (bs, 2H), 3.06 (bs, 2H). 13C NMR (101 MHz, DMSO) δ 160.1, 147.9, 145.4, 136.1, 134.2, 129.2, 128.8, 127.1, 124.8, 122.9, 120.9, 120.6, 118.3, 114.8, 114.5, 113.3, 111.3, 111.3, 111.0, 110.39, 54.2, 53.1, 25.5, 23.1. HRMS (ESI) m/z Calcd for C25H21N4ClBr (M+1)+ 491.0638, found 491.0646. Compound 26b was a tan solid (5%). mp: 208–212 °C. 1H NMR (400 MHz, DMSO) δ 10.76 (s, 2H), 7.75 (t, J = 4.3 Hz, 1H), 7.69 (bs, 1H), 7.63 (m, 1H), 7.44 (t, J = 4.7, 1H), 7.36 (dd, J = 8.3, 5.3 Hz, 1H), 7.31 (dd, J = 7.9, 4.8 Hz, 1H), 7.25 (m, 1H), 7.11 (bs, 1H), 7.05 (m, 1H), 6.96 (m, 1H), 3.80 (m, 2H), 3.60 (m, 2H), 3.08 (m, 4H). 13C NMR (101 MHz, DMSO) δ 161.3, 144.2, 136.2, 134.3, 131.5, 129.4, 127.3, 127.0, 124.8, 122.5, 120.8, 118.5, 118.2, 116.9, 114.1, 113.3, 112.1, 111.3, 111.2, 104.9, 52.3, 51.3, 25.6, 23.6. HRMS (ESI) m/z Calcd for C25H21N4BrI (M+1)+ 582.9994, found 583.0003.

FIGURE 4
A right-left stereoview of a low energy conformation of 1 (the methyl group on the pyridine is omitted for simplicity) with an overlay of the pharmacophore from Figure 1 generated in MOE. The color coding is as follows Green= Aromatic (Aro); Purple= Hydrogen-bond ...

Acknowledgments

We would like to thank Antonio DePasquale and Arnold L. Rheingold of the Department of Chemistry at the University of California, San Diego for the X-ray structure determination of 8a. This work was supported in part by NCI grant CA69129 (S.W.M.) and by the American Lebanese Syrian Associated Charities (ALSAC), St. Jude Children’s Research Hospital.

Footnotes

Supplementary data: All additional experimental details including methods for the kinase inhibition determination, NMR spectra and HPLC purity data are included in the Supplementary Data.

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