Abstract
Chronic hepatitis B virus (HBV) infection remains a major global health burden. It affects more than 290 million individuals worldwide and is responsible for approximately 900,000 deaths annually. Anti-HBV treatment with a nucleoside analog in combination with pegylated interferon are considered first-line therapy for patients with chronic HBV infection and liver inflammation. However, because cure rates are low, most patients will require lifetime treatment. HBV Capsid Assembly Modulators (CAMs) have emerged as a promising new class of compounds as they can affect levels of HBV covalently closed-circular DNA (cccDNA) associated with viral persistence. SAR studies around the core structure of lead HBV CAM GLP-26 (Fig. 1B) was performed and led to the discovery of non-toxic compound 10a displaying sub-nanomolar anti-HBV activity. Advanced toxicity and cellular pharmacology profiles of compounds 10a were also established and the results are discussed herein.
Keywords: Hepatitis, Antiviral, Virus, HBV Capsid, Drug, Small molecules, CAM, CAE, Ritonavir
Graphical Abstract
1. Introduction
Hepatitis B virus (HBV) remains a global threat and in some parts of the world, it causes severe health crises. Despite vaccines being available for many years now, millions of people worldwide are infected with HBV. Individuals with HBV run the risk of developing more serious life-threatening conditions such as, but not limited to, liver failure and hepatocellular carcinoma. To date, the treatment for individuals with chronic HBV relies on pegylated interferon alpha in combination with one or more nucleoside analogs such as entecavir, tenofovir, disoproxil fumarate or alafenamide, lamivudine, adefovir dipivoxil or telbivudine or just monotherapy with one of these drugs for long durations. These therapies work well in suppressing the replication of HBV, thus preventing virus replication, however these treatments do not cure the patient and lifetime continued therapy are required.1 Therefore, new therapeutic approaches targeting other specific steps of the HBV replication cycle are being evaluated. Among them, Capsid Assembly Modulators (CAMs) disrupting the viral replication by misdirecting HBV capsid assembly, showed promising results both in vitro and in vivo2 and several of them, including GLS4, JNJ-56136379, EDP-514, QL-007, ABI-H3733, ZM-H1505R, ALG-000184, AB-836, VNRX-9945, KL060332, ABI-4334 are currently in Phase I or II clinical development.3 By affecting capsid formation, CAMs can alter entry of HBV capsid and core particles into the cell nucleus, encapsidation of pre-genomic RNA and ultimately lead to reduction of the HBV covalently closed-circular DNA (cccDNA) associated with viral persistence. As part of our HBV CAM discovery program, our group discovered a series of highly potent non-toxic glyoxamoylpyrroloxamide (GLP) derivatives,4,5,6 including lead compound GLP-26 (Fig. 1B). GLP-26 was shown to alter HBV nucleocapsid assembly preventing transport of the capsid to the nucleus and thus inhibiting viral DNA replication. More importantly, in a humanized mouse model, GPL-26 in combination with entecavir, led to sustained antiviral response up to 12 weeks post treatment. 7,8 Despite GLP-26 already high potency, docking of GLP-26 in the reported crystal structure of HBV core protein dimer (PDBID:5T2P) shows the presence of 1) a small hydrophobic pocket near the pyrrole N-Me group formed by F23, W102, Y118, A132 and L140 residues 2) a small space around the propargyl moiety near the W125 and P138 residues susceptible for optimization (Figure 1A). Herein we report the SAR around these two positions (Fig 1B) leading to the discovery of 10a, a compound 200 times more potent than reference GLP-26.
Figure 1:
A. Predicted binding mode of GLP-26 with HBV core dimer with electrostatic potential map of the hydrophobic pocket formed near N-Me group. The residues are shown as lines and GLP-26 is shown as violet sticks form; B. Selected positions for chemical optimization of lead compound GLP-26.
2. Results and Discussion
2.1. Chemistry
Initial N-pyrrole substituted compounds 5a-j were prepared according to the chemistry outlined in Scheme 1. Commercially available pyrrole 1 was alkylated by either, treatment with alkyl bromide and KOH in DMSO when primary carbon chains were introduced (compounds 2a-d, f-j) or treatment with the corresponding boronic acids in presence of Cu(OAc)2,9 in the case of cyclopropyl- N-pyrrole derivative 2e. Formation of amide 3a-j was realized by treating ethyl ester 2a-j with 3,4-difluoroaniline in the presence of trimethyl aluminum. Treatment of compound 3a-j with ethyl oxalyl chloride in the presence of aluminum trichloride led to diketoesters 4a-j, which, after saponification and subsequent CDI mediated coupling with propargyl amine of the crude carboxylic acid, gave compounds 5a-j.
Scheme 1.
Reagents and conditions: a) R-Br, KOH, DMSO, r.t. o/n, 40–95% (cpds 2a-d, f-j); b) R-B(OH)2, 2,2’-bipyridyl, Cu(OAc)2, Na2CO3, DCE, 70°C, o/n, 31% (cpd 2e); c) 3,4-difluoroaniline, AlMe3, DCE, 65°C, o/n, 67–95%; d) ClCOCO2Et, AlCl3, DCM, r.t. o/n, 58–74%; e) 5% NaOH/MeOH:THF r.t. 15 min. then CDI, propargyl amine, DCM r.t. o/n, 30–44%.
GLP-26 analogs 9a-f with substitutions on the propargyl amine group were prepared by following the chemistry shown in Scheme 2. N-Alkylation of pyrrole 1 with methyl iodide was achieved with potassium hydroxide in DMSO to furnish the N-methyl pyrrole 6. Compound 6 was then treated with 3,4-difluoroaniline in the presence of trimethyl aluminum to give amide 7, which was reacted with ethyl oxalyl chloride in presence of AlCl3 to form compound 8. Subsequent saponification of compound 8 followed by HATU coupling with the appropriate amine afforded analogs 9a-f.
Scheme 2.
Reagents and conditions: a) MeI, KOH, DMSO, r.t. o/n; b) 3,4-difluoroaniline, AlMe3, 65°C, r.t.; c) ClCOCO2Et, AlCl3, DCM, r.t. o/n; d) 5% NaOH, MeOH:THF r.t. 15 min. then R1R2 propargyl amine, HATU, DMF, DIPEA, 60–80 °C, o/n.
Compounds combining a N-propargyl pyrrole ring and some of the best substituted propargyl amine moieties identified earlier were prepared according to the chemistry displayed in Scheme 3. Compound 4g was saponified with NaOH and the resulting α-keto acid was coupled the appropriate amine, in presence of HATU and DIPEA, to give access to analogs 10a-b.
Scheme 3.
Reagents and conditions: a) 5% NaOH/MeOH;THF, r.t. 15 min. then R1R1 propargyl amine, HATU, DMF, DIPEA, 60–80°C, o/n.
2.2. Structure activity relationship
The anti-HBV activity in HepAD38 cell and cytotoxicity profile of all the compounds synthesized is summarized in Table 1. The first step of our SAR explored the hydrophobic pocket identified near the N-methyl pyrrole moiety of GLP-26 (Figure 1). Thus, we established that the size of the alkyl group could be extended to ethyl (compound 5a), n-propyl (compound 5b), allyl (compound 5d) or even cyclopropyl (compound 5e) without reducing antiviral activity. On the other hand, addition of larger groups such as a n-butyl (compound 5c) or a benzyl group (compound 5j) lead to a clear decline of potency (EC50 = 87.4 and 2,519 nM, respectively) when compared to reference compound GLP-26 (EC50 = 4.6 nM). Replacement of the methyl group in GLP-26 with a propargyl group (compound 5g) led to a >5 fold increase of activity (EC50 = 0.88 nM). As for the alkyl chains modifications, extension of the propargyl group (compounds 5h-i) was detrimental to the overall potency of the analogs.
Table 1.
Anti-HBV activity in HepAD38 cell and cytotoxicity profile of compounds 5a-j, 9a-f, and 10a-b.
| Compound | Anti-HBV activity (nM) | Cytotoxicity CC50 (μM) | ||||
|---|---|---|---|---|---|---|
|
| ||||||
| EC50 | EC90 | PBM | CEM | Vero | HepG2 | |
| 5a | 7.1 ± 6.2 | 49.4 ± 12.9 | >100 | >100 | >100 | >100 |
| 5b | 9.1 ± 3.7 | 44.0 ± 21.0 | >100 | >100 | >100 | >100 |
| 5c | 87.4 ± 3.7 | 556 ± 147 | >100 | >100 | >100 | >100 |
| 5d | 3.0 ± 1.4 | 128 ± 150 | >100 | >100 | >100 | >100 |
| 5e | 7.5 ± 7.8 | 41.5 ± 12.0 | >100 | >100 | >100 | >100 |
| 5f | 52.2 ± 27.1 | 52.2 ± 27.1 | >100 | >100 | >100 | >100 |
| 5g | 0.88 ± 0.43 | 10.3 ± 6.0 | >100 | >100 | >100 | >100 |
| 5h | 45.2 ± 20.1 | 395 ± 327 | >100 | >100 | >100 | >100 |
| 5i | 826 ± 172 | 3,691 ± 1925 | >100 | >100 | >100 | >100 |
| 5j | 2519 | >10,000 | >100 | >100 | >100 | >100 |
| 9a | 0.71 ± 0.22 | 8.2 ± 0.6 | 64 ± 26 | 25 ± 2 | >100 | 91 ± 12 |
| 9b | 0.15 ± 0.07 | 6.8 ± 3.4 | 61 ± 43 | 10 ± 3 | 64 ± 16 | 84 ± 11 |
| 9c | 0.5 ± 0.3 | 4.6 ± 2.3 | 26 ± 14 | 16 | 42 | 44 ± 35 |
| 9d | 0.8 ± 0.7 | 6.1 ± 2.2 | >100 | >100 | >100 | >100 |
| 9e | 0.3 ± 0.4 | 1.9 ± 1.5 | >100 | >100 | >100 | 98 |
| 9f | 3.5 ± 4.8 | 7.4 ± 9.3 | 23 ± 13 | 15 | 43 | 42 ± 23 |
| 10a | 0.02 ± 0.03 | 1.6 ± 0.8 | >100 | >100 | >100 | >100 |
| 10b | 7.9 ± 1.6 | 60 ± 41 | 53 ± 41 | 76 ± 17 | >100 | 72 ± 39 |
| GLP-26 | 4.6 ± 2.0 | 16.8 ± 7.1 | >100 | >100 | 46 ± 30 | >100 |
The second step of our SAR explored substitutions of the propargyl amine moiety of GLP-26 (Figure 1). Interestingly, substitutions of this position with small alkyl groups led to compounds (Me/Me 9a, Et/Me 9b, Et/Et 9c, cyclopropyl 9d, cyclobutyl 9e) that were 5–30 fold more potent than GLP-26 itself. Compound 9f, with a cyclohexyl group, displayed only a moderate improvement, probably due to the size of the ring.
As a last part of our SAR, we combined our best modifications at both positions and by doing so, were able to identify compound 10a which displayed an antiviral EC50 of 0.02 nM in the HepAD38 cell system. Of note, this compound was devoid of toxicity in a panel of cell systems including PBM, CEM, Vero and HepG2 cells up to 100 μM, denoting a therapeutic index > 5,000,000. To the best of our knowledge, compound 10a is the most potent HBV CAM reported to date in the scientific literature.
Molecular docking of compound 10a with dimeric form of HBV protein showed strong hydrophobic interactions between propargyl group and F23, W102, Y118, A132, L140 residues. Moreover, methyl groups also formed hydrophobic interactions with W125 and P138 residues (Figure 2). In addition, we implemented free energy perturbation (FEP) method to calculate relative binding free energy change (ΔΔG) due to change of N-Me to N-propargyl and additional two methyl groups in GLP-26. FEP has been shown to deliver accurate ligand binding free energies against several protein targets. Compound 10a displayed a negative ΔΔG value of −3.44±0.15 kcal/mol indicative of a better binding affinity than GLP-26.
Figure 2:
Predicted binding mode of compound 10a with HBV core dimer. The residues are shown as lines and compound 10a is shown as green sticks form.
2.3. Cellular Pharmacology
Based on its high potency and favorable toxicity profile, compound 10a was further biologically characterized. Compound 10a displayed only limited conjugation with glutathione (GSH) at 25 μM and did not induce hERG inhibition at 10 μM, two assays predictive of idiosyncratic toxicity risks (Table 2). Furthermore, compound 10a was tested for inhibition and induction of a large panel of cytochrome P450 (CYP) enzymes (Table 3) and except for CYP 2C8, did not show significant effects. Compound 10a also displayed desirable apparent permeability (Papp) in an intestinal epithelial Caco-2 cell monolayer assay (Table 4). However, with half-lives of 2.4 and 9 min respectively, in mouse and human liver microsomes (Table 5), the stability of compound 10a seems suboptimal, and the fast-intrinsic clearance observed in vitro could become a problem during further in vivo evaluation. Combinations of compound 10a with ritonavir, a known inhibitor of certain cytochrome P450 in the liver used as a booster in several combination therapy (e.g., Paxlovid, Technivie), were evaluated in order to circumvent that issue (Table 5). While co-incubation of compound 10a with ritonavir (at ratios up to 1/3) significantly improved compound 10a’s half-life 45-fold in mouse liver microsomes (from 2.4 to 108 min), it had only a two-fold effect on its stability in human liver microsomes (from 9 to 19 min).
Table 2.
Formation of GSH adducts and hERG Inhibition
| Compound | GSH % Reactive metabolites at 25 μM | hERG % Inhibition at 10 μM |
|---|---|---|
| 10a | 0.6 | 18 |
| GLP-26 | 0.8 | 46.6 |
Table 3.
Cell-free CYP inhibition of the GLP analog 10a
| Compound | CYP Inhibition (% at 10 μM) | |||||||
|---|---|---|---|---|---|---|---|---|
|
| ||||||||
| 1A2 | 2B6 | 2C8 | 2C9 | 2C19 | 2D6 | 3A4-M* | 3A4-T* | |
| 10a | 9 | 5 | 95 | 40 | 34 | 8 | 40 | 40 |
| GLP-26 | −9.6 | −1.6 | 54 | 11.7 | 15.9 | 9.5 | −7.2 | 8.3 |
M = midazolam substrate, T = testosterone substrate
Table 4.
Cell permeability of the GLP analog 10a
| Compound | Permeability 10−6 cm/s A-B / B-A | Mean Recovery % | ||
|---|---|---|---|---|
|
| ||||
| A-B | B-A | A-B | B-A | |
| 10a | 33.5 | 10.9 | 100 | 74.6 |
| GLP-26 | 23.5 | 22.8 | 86.3 | 98.7 |
Table 5.
Liver microsome stability of GLP analog 10a
| Compound | Liver microsome stability t1/2 (min) | |
|---|---|---|
| Mouse | Human | |
| 10a | 2.4 | 9 |
| 1 μM 10a + 0.3 μM RTV | <5 | 13 |
| 1 μM 10a + 1 μM RTV | 28.5 | 15 |
| 1 μM 10a + 3 μM RTV | 108 | 19 |
| GLP-26 | 41 | >60 |
3. Conclusion:
Through this work, we were able to identify compound 10a, a sub-nanomolar inhibitor of HBV DNA in vitro. This compound is more than 200 times more potent than reference compound GLP-26 and displays an excellent toxicity profile in a panel of cell systems (therapeutic Index TI > 5 million). Assessment of cardiac risk through hERG inhibition and reactive metabolite formation through GSH adduct generation confirmed the overall safety of compound 10a. Detailed CYP inhibition profile of compound 10a, in a panel of eight relevant CYP enzymes showed no major effects, except for the inhibition of CYP2C8. It is worth noting though, that compound 10a was evaluated at a concentration 500,000 above its EC50, a concentration that would most likely never be reached in vivo. Unfortunately, compound 10a, alone or in combination with booster ritonavir, displays relatively short half lives in human liver microsomes, which could ultimately prevent sufficient in vivo exposure. Efforts to overcome this issue and optimize the overall drug like properties of compound 10a, while maintaining its high potency are ongoing in our laboratory.
4. Experimental section
4.1. Chemistry
Reagents and solvents were purchased from commercial sources and were used as received without further purification unless otherwise noted. Unless otherwise stated, the intermediates and final compounds were obtained from readily available commercial suppliers or synthesized by standard methods known to individuals skilled in the art of chemical synthesis. Intermediates and final compounds were purified by either flash chromatography or preparative TLC. Flash chromatography was performed using Teledyne ISCO combi-flash chromatography instrument model Rf-200. The silica gel cartridges were purchased from Agela Technologies Silica (CS) irregular 40–60 μm 60Å. Preparative TLC plates were purchased from Analtech, GF silica gel plates. 1H, 13C, and 19F NMR spectra were taken on a Bruker AscendTM 400 MHz spectrometer at rt and reported in ppm downfield using residual solvent lines as an internal standard for 1H and 13C NMR. No standard was used for 19F NMR spectra. Signal multiplicities are represented by s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quadruplet), br (broad), bs (broad singlet), m (multiplet). Mass spectra were determined on a Micromass Platform LC spectrometer using electrospray ionization.
4.1.1. General synthetic procedure for compounds 2a-d, f-j:
To a solution of ethyl 3,5-dimethyl-1H-pyrrole-2-carboxylate (1) (1 equiv.) in DMSO (1 M) was added KOH (2 equiv.) and R-Br (3 equiv.) respectively. The mixture was stirred overnight at ambient temperature, after which the reaction mixture was poured into water. The water phase was then extracted with EtOAc. The compound organic phases were dried over anhydrous Na2SO4, filtered, and conc. in vacuo. The crude residue was purified by flash chromatography using to afford the desired compound
4.1.1.1. Ethyl 1-ethyl-3,5-dimethyl-1H-pyrrole-2-carboxylate (2a) - 95 % yield.
1H NMR (400 MHz, CDCl3) δ 5.71 (s, 1H), 4.24 (m, 4H), 2.28 (s, 3H), 2.15 (s, 3H), 1.33 (t, J = 7.2 Hz, 3H), 1.25 (t, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 161.7, 134.4, 129.4, 117.9, 110.9, 59.0, 40.1, 16.2, 14.4, 11.9. HRMS (ESI): m/z [M+1]+ calcd for C11H18NO2: 196.1338, found: 196.1330.
4.1.1.2. Ethyl 3,5-dimethyl-1-propyl-1H-pyrrole-2-carboxylate (2b) - 95% yield.
1H NMR (400 MHz, CDCl3) δ 5.74 (s, 1H), 4.27 (q, J = 7.12 Hz, 2H), 4.14 (t, J = 7.68 Hz, 2H), 2.29 (s, 3H), 2.19 (s, 3H), 1.68 (m, 2H), 1.35 (t, J = 7.12 Hz, 3H), 0.91 (t, J = 7.44 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 161.9, 134.9, 129.6, 118.3, 110.8, 59.2, 46.9, 24.5, 14.5, 12.3, 11.1. HRMS (ESI): m/z [M+1]+ calcd for C12H20NO2: 210.1494, found: 210.1490.
4.1.1.3. Ethyl 1-butyl-3,5-dimethyl-1H-pyrrole-2-carboxylate (2c) – 92% yield.
1H MNR (400 MHz, CDCl3) δ 5.73 (s, 1H), 4.26 (q, J = 7.1 Hz, 2H), 4.12 (t, J = 7.7 Hz, 2H), 2.28 (s, 3H), 2.18 (s, 3H), 1.63 (m, 2H), 1.35 (m, 5H), 0,94 (t, J = 7.3 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 161.8, 134.8, 129.6, 118.2, 110.8, 59.1, 45.3, 33.5, 20.1, 14.5, 13.8, 12.3. HRMS (ESI): m/z [M+1]+ calcd for C13H22NO2: 224.1651, found: 224.1647.
4.1.1.4. Ethyl 1-allyl-3,5-dimethyl-1H-pyrrole-2-carboxylate (2d) - 45% yield.
1H NMR (400 MHz, CDCl3) δ 5.93 (m, 1H), 5.07 (m, 1H), 4.90 (m, 2H), 4.74 (m, 1H), 4.26 (q, J = 7.1 Hz, 2H), 2.30 (s, 3H), 2.17 (s, 3H), 1.34 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 162.0, 135.5, 134.8, 129.7, 59.3, 47.3, 14.5, 12.1. HRMS (ESI): m/z [M+1]+ calcd for C12H17NO2: 208.1338, found: 208.1333.
4.1.1.5. Ethyl 1-(cyclopropylmethyl)-3,5-dimethyl-1H-pyrrole-2-carboxylate (2f) – 94% yield.
1H MNR (400 MHz, CDCl3) δ 5.56 (s, 1H), 4.07 (q, J = 7.1 Hz, 2H), 3.97 (d, J = 6.7 Hz, 2H), 2.09 (s, 3H), 2.00 (s, 3H), 1.15 (t, J = 7.1 Hz, 3H), 0.97 (m, 1H), 0.26 (m, 2H), 0.12 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 162.1, 134.9, 129.7, 118.4, 111.1, 59.2, 48.6, 14.6, 14.5, 12.6, 12.4, 3.5. HRMS (ESI): m/z [M+1]+ calcd for C13H20NO2: 222.1494, found: 222.1487.
4.1.1.6. Ethyl 3,5-dimethyl-1-(prop-2-yn-1-yl)-1H-pyrrole-2-carboxylate (2g) - 51% yield.
1H NMR (400MHz, CDCl3) δ ppm 5.79 (s, 1H), 5.10 (d, J = 2.4 Hz, 2H), 4.29 (q, J = 7.2 Hz, 2H), 2.28 (s, 3H), 2.27 (s, 3H), 2.23 (t, J = 2.4 Hz, 1H), 1.35 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz CDCl3) δ 162.0, 135.5, 118.2, 111.5, 79.3, 71.5, 59.5, 34.5, 14.1, 14.3, 12.1. HRMS (ESI): m/z [M+1]+ calcd for C12H16NO2: 206.1181, found: 206.1183.
4.1.1.7. Ethyl 1-(but-2-yn-1-yl)-3,5-dimethyl-1H-pyrrole-2-carboxylate (2h) – 41% yield.
1H NMR (400 MHz, CDCl3) δ 5.76 (s, 1H), 5.03 (br d, J = 2.4 Hz, 2H), 4.28 (q, J = 7.1 Hz, 2H), 2.27 (s, 3H), 2.26 (s, 3H), 1.74 (t, J = 2.3 Hz, 3H), 1.35 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 161.9, 135.4, 129.9, 118.0, 111.1, 78.9, 74.6, 59.3, 56.8, 34.7, 14.4, 12.0, 3.5. HRMS (ESI): m/z [M+1]+ calcd for C13H18NO2: 220.1338, found 220.1335.
4.1.1.8. Ethyl 3,5-dimethyl-1-(pent-2-yn-1-yl)-1H-pyrrole-2-carboxylate (2i) - 50% yield.
1H NMR (400 MHz, CDCl3) δ 5.76 (s, 1H), 5.06 (t, J = 1.2 Hz, 2H), 4.28 (q, J = 7.2 Hz, 2H), 2.27 (s, 3H), 2.26 (s, 3H), 2.12 (qt, J = 7.6, 2.4 Hz, 2H), 1.35 (t, J = 7.2 Hz, 3H), 1.07 (t, J = 7.6 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 161.9, 135.5, 129.9, 118.0, 111.1, 84.8, 74.8, 59.3, 34.8, 14.4, 13.7, 12.3. HRMS (ESI): m/z [M+1]+ calcd for C14H20NO2: 234.1494, found 234.1494.
4.1.1.9. Ethyl 1-benzyl-3,5-dimethyl-1H-pyrrole-2-carboxylate (2j) - 80% yield.
1H NMR (400 MHz, CDCl3) δ 7.21 (m, 3H), 6.90 (m, 2H), 5.57 (s, 2H), 5.54 (s, 1H), 4.20 (q, J = 7.12 Hz, 2H), 2.34 (s, 3H), 2.10 (s, 3H), 1.25 (t, J = 7.12 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 161.4, 138.2, 133.6, 128.7, 128.3, 126.8, 125.8, 118.8, 111.4, 100.9, 59.8, 49.3, 14.0, 12.3. HRMS (ESI): m/z [M+1]+ calcd for C16H20NO2: 258.1494, found 258.1489.
4.1.2. Ethyl 1-cyclopropyl-3,5-dimethyl-1H-pyrrole-2-carboxylate (2e).
A round bottom flask was charged with ethyl 3,5-dimethyl-1H-pyrrole-2-carboxylate (1) (1.66 g, 9.93 mmol), cyclopropyl boronic acid (1.81 g, 21.1 mmol), Cu(OAc)2-H2O (2.24 g, 11.2 mmol), 2,2’-dipyridyl (1.70 g, 10.8 mmol), Na2CO3 (2.36 g, 22.3 mmol), and 25 mL of DCE. The mixture was stirred at 70°C overnight after which the reaction was quenched with sat. aqueous NH4Cl. The aqueous phase was extracted with DCM (3 × 15 mL). The combined organic phases were washed with brine, dried over anhydrous Na2SO4, filtered and conc. in vacuo. The crude residue purified by flash chromatography using an eluent of 10% EtOAc in hexanes to afford 2e (0.64g, 3.09 mmol, 31% yield). 1H NMR (400 MHz, CDCl3) δ 5,68 (s, 1H), 4.29 (q, J = 7.1 Hz, 2H), 3.19 (m, 1H), 2.27 (s, 3H), 2.24 (s, 3H), 1.35 (t, J = 7.1 Hz, 3H), 1.06 (m, 2H), 0.17 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 161.5, 137.3, 128.9, 120.7, 110.3, 59.4, 28.1, 14.5, 13.8, 13.6, 9.5. HRMS (ESI): m/z [M+1]+ calcd for C12H18NO2: 208.1338, found: 208.1331.
4.1.3. General synthetic procedure for compounds 3a-j:
To a solution of pyrrole (2) (1 equiv.) and 3,4-difluoroaniline (2 equiv.) in dichloroethane (0.1 M) was cooled to 0°C and was treated with trimethyl aluminum (2.0 M in toluene, 2 equiv.) The solution was allowed to slowly come to ambient temperature over 2 hr and then heated to reflux under nitrogen for 24 hr after which time the solution was poured into water and extracted three times with dichloromethane. The combined organic phase was dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography to give the desired compound (3).
4.1.3.1. N-(3,4-Difluorophenyl)-1-ethyl-3,5-dimethyl-1H-pyrrole-2-carboxamide (3a) - 77% yield.
1H NMR (400 MHz, CDCl3) δ 7.68 (m, 1H), 7.29 (bs, 1H), 7.09 (m, 2H), 5.77 (s, 1H), 4.24 (q, J = 7.2 Hz, 2H), 2.36 (s, 3H), 2.23 (s, 3H), 1.31 (t, J = 7.2 Hz, 3H). 19F NMR (377 MHz, CDCl3) δ −135.8 (d, J = 21.9 Hz, 1F), −143.5 (d, J = 21.8 Hz, 1F). 13C NMR (101 MHz, CDCl3) δ 160.4, 151.4 (d, J = 13.3 Hz), 149.0 (d, J = 13.2 Hz), 147.9 (d, J = 12.8 Hz), 145.5 (J = 13.0 Hz), 134.8 (m), 134.0, 122.2, 121.8, 117.2 (d, J = 18.4 Hz), 115.2 (m), 110.6, 109.5 (d, J = 21.9 Hz), 40.1, 16.5, 14.0, 12.0. HRMS (ESI): m/z [M+1]+ calcd for C15H17F2N2O: 279.1309, found 279.1304.
4.1.3.2. N-(3,4-Difluorophenyl)-3,5-dimethyl-1-propyl-1H-pyrrole-2-carboxamide (3b) - 95% yield.
1H NMR (400 MHz, CDCl3) δ 7.67 (m, 2H), 7.30 (s, 2H), 7.08 (m, 2H), 5.76 (s, 1H), 4.13 (t, J = 7.6 Hz, 2H), 2.34 (s, 3H), 2.21 (s, 3H), 1.70 (m, 2H), 0.90 (t, J = 7.44 Hz, 3H). 19F NMR (377 MHz, CDCl3) δ −135.8 (d, J = 21.8 Hz, 1F), −143.5 (d, J = 21.8 Hz, 1F). 13C NMR (101 MHz, CDCl3) δ 160.5, 151.4 (d, J = 13.2 Hz), 149.0 (d, J = 13.2 Hz), 147.9 (d, J = 12.9 Hz), 145.5 (d, J = 13.0 Hz), 134.9 (m), 134.4, 122.2, 122.0, 117.2 (d, J = 17.3 Hz), 115.2 (m), 110.4, 109.5 (d, J = 21.6 Hz), 46.6, 24.7, 14.0, 12.2, 11.2. HRMS (ESI): m/z [M+1]+ calcd for C16H19F2N2O: 293.1465, found 293.1462.
4.1.3.3. 1-Butyl-N-(3,4-difluorophenyl)-3,5-dimethyl-1H-pyrrole-2-carboxamide (3c) - 92% yield.
1H NMR (400 MHz, CDCl3) δ 7.68 (m, 1H), 7.26 (bs, 1H), 7.10 (m, 2H), 5.77 (s, 1H), 4.18 (t, J = 7.68 Hz, 2H), 2.35 (s, 3H), 2.22 (s, 3H), 1.65 (m, 2H), 1.33 (m, 2H), 0.93 (t, J = 7.36 Hz, 3H). 19F NMR (377 MHz, CDCl3) δ −135.8 (d, J = 21.8 Hz, 1F), −143.5 (d, J = 21.8 Hz, 1F). 13C NMR (101 MHz, CDCl3) δ 160.4, 151.4 (d, J = 13.2 Hz), 149.0 (d, J = 13.2 Hz), 147.9 (d, J = 12.9 Hz), 145.5 (d, J = 13.0 Hz), 134.9 (m), 134.4, 122.2, 122.0, 117.2 (d, J = 17.3 Hz), 115.2 (m), 110.4, 109.5 (d, J = 21.6 Hz), 45.0, 33.6, 20.1, 14.1, 13.9, 12.2. HRMS (ESI): m/z [M+1]+ calcd for C17H21F2N2O: 307.1622, found 307.1619.
4.1.3.4. 1-Allyl-N-(3,4-difluorophenyl)-3,5-dimethyl-1H-pyrrole-2-carboxamide (3d) - 89% yield.
1H NMR (400 MHz, CDCl3) δ 7.68 (m, 1H), 7.32 (bs, 1H), 7.08 (m, 2H), 5.97 (m, 1H), 5.11 (dd, J = 10.36, 1.24 Hz, 1H), 4.85 (m, 2H), 4.79 (dd, J = 17.12, 1.28 Hz, 1H), 2.36 (s, 3H), 2.19 (s, 3H). 19F NMR (377 MHz, CDCl3) δ −135.8 (d, J = 21.8 Hz, 1F), −143.5 (d, J = 21.8 Hz, 1F). 13C NMR (101 MHz, CDCl3) δ 160.4, 151.4 (d, J = 13.3 Hz), 148.9 (d, J = 13.2 Hz), 147.9 (d, J = 13.0 Hz), 145.5 (d, J = 12.8 Hz), 134.8 (m), 122.6, 122.3, 117.2 (d, J = 1.05 Hz), 117.0 (d, J = 1.11 Hz), 115.3, 113.1 (m), 110.7, 109.6, 109.4, 47.2,13.9, 12.0. HRMS (ESI): m/z [M+1]+ calcd for C16H17F2N2O: 291.1309, found 291.1306.
4.1.3.5. 1-Cyclopropyl-N-(3,4-difluorophenyl)-3,5-dimethyl-1H-pyrrole-2-carboxamide (3e) - 85% yield.
1H NMR (400 MHz, CDCl3) δ 7.70 (m, 1H), 7.41 (bs, 1H), 7.07 (m, 2H), 5.67 (s, 1H), 3.21 (m, 1H), 2.27 (s, 3H), 2.23 (s, 3H), 1.04 (m, 2H), 0.75 (m, 2H). 19F NMR (377 MHz, CDCl3) δ −135.8 (d, J = 21.8 Hz, 1F), −143.4 (d, J = 21.8 Hz, 1F), 13C NMR (101 MHz, CDCl3) δ 160.3, 151.4 (d, J = 13.3 Hz), 149.0 (d, J = 13.3 Hz), 147.9 (d, J = 13.0 Hz), 145.5 (d, J = 12.7 Hz), 143.3 (m), 136.3, 124.6, 122.6, 117.2 (d, J = 18.6 Hz), 115.0 (m), 109.4, 109.2, 27.5, 13.2, 12.9, 8.9. HRMS (ESI): m/z [M+1]+ calcd for C16H17F2N2O: 291.1309, found 291.1301.
4.1.3.6. 1-(Cyclopropylmethyl)-N-(3,4-difluorophenyl)-3,5-dimethyl-1H-pyrrole-2-carboxamide (3f) - 94% yield.
1H NMR (400 MHz, CDCl3) δ 7.68 (m, 1H), 7.30 (bs, 1H), 7.10 (m, 2H), 5.79 (s, 1H), 4.15 (d, J = 6.72 Hz, 2H), 2.35 (s, 3H), 2.25 (s, 3H), 2.26 (m, 1H), 0.47 (m, 2H), 0.31 (m, 2H). 19F NMR (377 MHz, CDCl3) δ −135.8 (d, J = 21.8 Hz, 1F), −143.4 (d, J = 21.8 Hz, 1F). 13C NMR (101 MHz, CDCl3) δ 160.8, 151.4 (d, J = 13.3 Hz), 149.0 (d, J = 13.3 Hz), 148.0 (d, J = 13.0 Hz), 145.5 (d, J = 13.0 Hz), 134.8 (m), 134.2, 122.3, 117.3, 117.1, 115.1 (m), 110.6, 109.6, 109.4, 48.5,13.9, 12.5, 3.7. HRMS (ESI): m/z [M+1]+ calcd for C17H19F2N2O: 305.1465, found 305.1456.
4.1.3.7. N-(3,4-Difluorophenyl)-3,5-dimethyl-1-(prop-2-yn-1-yl)-1H-pyrrole-2-carboxamide (3g) - 78% yield.
1H NMR (400 MHz, CDCl3) δ 7.47 (m, 1H), 7.37 (s, 1H), 7.09 (m, 2H), 5.81 (s, 1H), 5.07 (d, J = 2.4 Hz, 2H), 2.34 (s, 3H), 2.30 (s, 3H), 2.27 (t, J = 2.4 Hz, 1H). 19F NMR (377 MHz, CDCl3) δ −135.79 (d, J = 24 Hz, 1F), −143.23 (d, J = 24 Hz, 1F). 13C NMR (101 MHz, CDCl3) δ 160.31, 151.46, 151.33, 149.00, 148.87, 148.06, 147.93, 145.63, 145.50, 134.95, 134.73, 134.70, 134.64, 134.61, 123.37, 121.91, 117.25, 117.08, 115.32, 111.21, 109.74, 109.52, 79.24, 71.94, 34.24, 13.85, 11.99. HRMS (ESI): m/z [M+1]+ calcd for C16H15F2N2O: 289.1152, found 289.1148.
4.1.3.8. 1-(But-2-yn-1-yl)-N-(3,4-difluorophenyl)-3,5-dimethyl-1H-pyrrole-2-carboxamide (3h) - 60% yield.
1H NMR (400 MHz, CDCl3) δ 7.71 (m, 1H), 7.38 (bs, 1H), 7.10 (m, 2H), 5.80 (s, 1H), 4.99 (bm, 2H), 2.34 (s, 3H), 2.30 (s, 3H),1.78 (t, J = 2.28 Hz, 3H). 19F NMR (377 MHz, CDCl3) δ −135.8 (d, J = 21.8 Hz, 1F), −143.4 (d, J = 21.8 Hz, 1F). 13C NMR (101 MHz, CDCl3) δ 160.3, 151.4 (d, J = 13.2 Hz), 149.0 (d, J = 13.2 Hz), 147.9 (d, J = 12.9 Hz), 145.5 (d, J = 13.2 Hz), 123.3, 122.0, 117.3, 117.1, 115.0 (m), 110.9, 109.6, 109.4, 79.9, 74.5, 34.7, 13.8, 12.1, 3.7. HRMS (ESI): m/z [M+1]+ calcd for C17H17F2N2O: 303.1309, found 303.1306
4.1.3.9. N-(3,4-Difluorophenyl)-3,5-dimethyl-1-(pent-2-yn-1-yl)-1H-pyrrole-2-carboxamide (3i) −87% yield.
1H NMR (400 MHz, CDCl3) δ 7.71 (m, 1H), 7.40 (bs, 1H), 7.10 (m, 2H), 5.80 (s, 1H), 5.01 (t, J = 2.2 Hz, 2H), 2.34 (s, 3H), 2.30 (s, 3H), 2.16 (m, 2H), 1.08 (t, J = 7.48 Hz, 3H). 19F NMR (377 MHz, CDCl3) δ −135.8 (d, J = 21.8 Hz, 1F), −143.4 (d, J = 21.8 Hz, 1F). 13C NMR (101 MHz, CDCl3) δ 160.3, 151.4 (d, J = 13.2 Hz), 149.0 (d, J = 13.2 Hz), 147.9 (d, J = 12.8 Hz), 145.5 (d, J = 13.1 Hz), 134.8 (m), 123.3,122.1, 117.2, 117.6, 115.1 (m), 110.9, 109.6, 109.4, 85.8, 74.7, 34.7, 13.7, 12.4, 12.0. HRMS (ESI): m/z [M+1]+ calcd for C18H19F2N2O: 317.1465, found 317.1464.
4.1.3.10. 1-Benzyl-N-(3,4-difluorophenyl)-3,5-dimethyl-1H-pyrrole-2-carboxamide (3j) - 64% yield.
1H NMR (400 MHz, CDCl3) δ 7.60 (m, 1H), 7.24 (m, 4H), 7.05 (q, J = 8.88 Hz, 1H), 6.98 (m, 3H), 5.86 (s, 1H), 5.51 (s, 2H), 2.39 (s, 3H), 2.14 (s, 3H). 19F NMR (377 MHz, CDCl3) δ −135.8 (d, J = 21.8 Hz, 1F), −143.4 (d, J = 21.8 Hz, 1F). 13C NMR (101 MHz, CDCl3) δ 160.4, 151.4 (d, J = 13.2 Hz), 148.9 (d, J = 13.3 Hz), 147.9 (d, J = 13.0 Hz), 145.5 (d, J = 12.7 Hz), 138.6, 135.1, 134.8 (d, J = 2.97 Hz), 134.7 (d, J = 2.95 Hz), 138.6, 135.1, 134.8 (d, J = 2.97 Hz), 134.7 (d, J = 2.95 Hz), 48.1, 14.0, 12.3. HRMS (ESI): m/z [M+1]+ calcd for C20H19F2N2O: 341.1465, found 341.1462.
4.1.4. General synthetic procedure for compounds 4a-j:
A solution of compound (3a-j) (1 equiv.) and aluminum trichloride (3 equiv.) in DCM (0.1 M) was cooled to 0°C and ClCOCO2Et (3 equiv.) was added in a dropwise fashion over 30 min. The solution was allowed to slowly come to ambient temperature and was stirred under nitrogen for 24 hr, after which time the solution was poured over cold aqueous NH4Cl. The aqueous phase was extracted three time with DCM. The combined organic phase was dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography to give the desired compound (4a-j).
4.1.4.1. Ethyl 2-(5-((3,4-Difluorophenyl)carbamoyl)-1-ethyl-2,4-dimethyl-1H-pyrrol-3-yl)-2-oxoacetate (4a) - 62% yield.
1H NMR (400 MHz, CDCl3) δ 8.05 (bs, 1H), 7.72 (m, 1H), 7.21 (m, 1H), 7.12 (q, J = 8.84 Hz, 1H), 4.35 (q, J = 7.16 Hz, 2H), 4.14 (q, J = 7.16 Hz, 2H), 2.42 (s, 3H), 2.30 (s, 3H), 1.37 (t, J = 7.16 Hz, 3H), 1.33 (t, J = 7.16 Hz, 3H). 19F NMR (377 MHz, CDCl3) δ −135.5 (d, J = 21.8 Hz, 1F), −142.2 (d, J = 21.8 Hz, 1F). 13C NMR (101 MHz, CDCl3) δ 183.3, 166.0, 160.2, 151.3 (d, J = 13.3 Hz), 148.9 (d, 13.3 Hz), 148.3 (d, J = 12.8 Hz), 145.9 (d, J = 12.9 Hz), 141.5, 134.2 (m), 126.0, 122.9, 117.3 (d, J = 18.4 Hz), 116.4, 115.6 (m), 109.7 (d, J = 21.9 Hz), 62.2, 40.2, 16.0, 13.9, 11.5, 11.4. HRMS (ESI): m/z [M+1]+ calcd for C19H21F2N2O4: 379.1469, found 379.1468.
4.1.4.2. Ethyl 2-(5-((3,4-difluorophenyl)carbamoyl)-2,4-dimethyl-1-propyl-1H-pyrrol-3-yl)-2-oxoacetate (4b) - 58% yield.
1H NMR (400 MHz, CDCl3) δ 7.81 (bs, 1H), 7.72 (m, 1H), 7.20 (m, 1H), 7.14 (q, J = 8.52 Hz, 1H), 4.36 (q, J = 7.16 Hz, 2H), 4.08 (q, J = 7.16 Hz, 2H), 2.43 (s, 3H), 2.33 (s, 3H), 1.71 (m, 2H), 1.38 (t, J = 7.16 Hz, 3H), 0.92 (t, J = 7.16 Hz, 3H). 19F NMR (377 MHz, CDCl3) δ −135.4 (d, J = 21.8 Hz, 1F), −142.1 (d, J = 21.8 Hz, 1F). 13C NMR (101 MHz, CDCl3) δ 183.3, 165.9, 160.1, 151.4 (d, J = 13.3 Hz), 148.9 (d, J = 13.3 Hz), 148.4 (d, J = 12.7 Hz), 145.0 (d, J = 13.0 Hz), 141.8, 134.1 (m), 126.0, 122.9, 117.4 (d, J = 18.6 Hz), 116.4, 115.5 (m), 109.7 (d, J = 21.8 Hz), 62.2, 46.4, 24.1, 14.0, 11.7, 11.1. HRMS (ESI): m/z [M+1]+ calcd for C20H23F2N2O4: 393.1626, found 393.1622.
4.1.4.3. Ethyl 2-(1-butyl-5-((3,4-difluorophenyl)carbamoyl)-2,4-dimethyl-1H-pyrrol-3-yl)-2-oxoacetate (4c) - 63% yield.
1H NMR (400 MHz, CDCl3) δ 7.97 (bs, 1H), 7.72 (m, 1H), 7.23 (m, 1H), 7.14 (m, 1H), 4.41 (q, J = 7.16 Hz, 2H), 4.35 (q, J = 7.16 Hz, 2H), 2.42 (s, 3H), 2.30 (s, 3H), 1.66 (m, 2H), 1.37 (m, 5H), 0.92 (t, J = 7.16 Hz, 3H). 19F NMR (377 MHz, CDCl3) δ −135.5 (d, J = 21.7 Hz, 1F), −142.2 (d, J = 21.6 Hz, 1F). 13C NMR (101 MHz, CDCl3) δ 183.3, 165.9, 160.1, 151.4 (d, J = 13.3 Hz), 148.9 (d, J = 13.3 Hz), 148.4 (d, J = 12.7 Hz), 145.0 (d, J = 13.0 Hz), 141.8, 134.1 (m), 126.0, 122.9, 117.4 (d, J = 18.6 Hz), 116.4, 115.5 (m), 109.7 (d, J = 21.8 Hz), 62.1, 44.8, 32.9, 19.9, 13.9, 11.6, 11.5. HRMS (ESI): m/z [M+1]+ calcd for C21H25F2N2O4: 407.1782, found 407.1782.
4.1.4.4. Ethyl 2-(1-allyl-5-((3,4-difluorophenyl)carbamoyl)-2,4-dimethyl-1H-pyrrol-3-yl)-2-oxoacetate (4d) - 54% yield.
1H NMR (400 MHz, CDCl3) δ 7.81 (bs, 1H), 7.71 (m, 1H), 7.18 (m, 1H), 7.12 (q, J = 8.44 Hz, 1H), 5.93 (m, 1H), 5.20 (d, J = 10.4 Hz, 1H), 4.91 (d, J = 17.2 Hz, 1H), 4.79 (m, 2H), 4.36 (q, J = 7.16 Hz, 2H), 2.40 (s, 3H), 2.35 (s, 3H), 1.38 (t, J 7.16 Hz, 3H). 19F NMR (377 MHz, CDCl3) δ −135.4 (d, J = 21.8 Hz, 1F), −142.0 (d, J = 21.8 Hz, 1F). 13C NMR (101 MHz, CDCl3) δ 183.4, 165.8, 159.9, 151.5 (d, J = 13.4 Hz), 148.9 (d, J = 13.2 Hz), 148.4 (d, J = 12.8 Hz), 145.9 (d, J = 13.0 Hz), 142.3, 134.1 (m), 132.9, 126.0, 123.2, 117.3 (d, J = 18.4 Hz), 117.0, 116.6, 115.5 (m), 109.8 (d, J = 21.8 Hz), 62.2, 47.1, 14.0, 11.7, 11.5. HRMS (ESI): m/z [M+1]+ calcd for C20H21F2N2O4: 391.1469, found 391.1464.
4.1.4.5. Ethyl 2-(1-cyclopropyl-5-((3,4-difluorophenyl)carbamoyl)-2,4-dimethyl-1H-pyrrol-3-yl)-2-oxoacetate (4e) - 53% yield.
1H NMR (400 MHz, CDCl3) δ 8.87 (bs, 1H), 7.75 (m, 1H), 7.34 (m, 1H), 7.13 (q, J = 9.04 Hz, 1H), 4.34 (q, J = 7.04 Hz, 2H), 3.30 (m, 1H), 2.46, (s, 3H), 2.14 (s, 3H), 1.36 (t, J = 7.12 Hz, 3H), 1.11 (m, 2H), 0.90 (m, 2H). 19F NMR (377 MHz, CDCl3) d −135.7 (d, J =21.8 Hz, 1F), −141.9 (d, J = 21.8 Hz, 1F). 13C NMR (101 MHz, CDCl3) δ 183.4, 160.9, 158.3, 151.3 (d, J = 13.3 Hz), 148.8 (d, J = 13.3 Hz), 146.0 (d, J = 12.7 Hz), 144.6, 143.3 (m), 128.0, 122.6, 117.3 (d, J = 18.2 Hz), 115.9 (m), 115.0, 109.8 (d, J = 21.8 Hz), 63.4, 62.5, 27.8, 13.8, 12.8, 10.7, 8.4. HRMS (ESI): m/z [M+1]+ calcd for C20H21F2N2O4: 391.1469, found 391.1469.
4.1.4.6. Ethyl 2-(1-(cyclopropylmethyl)-5-((3,4-difluorophenyl)carbamoyl)-2,4-dimethyl-1H-pyrrol-3-yl)-2-oxoacetate (4f) - 63% yield.
1H NMR (400 MHz, CDCl3) δ 7.73 (bs, 1H), 7.71 (m, 1H), 7.15 (m, 2H), 4.38 (q, J = 7.16 Hz, 2H), 4.10 (d, J = 6.88 Hz, 2H), 2.48 (s, 3H), 2.35 (s, 3H), 1.39 (t, J = 7.16 Hz, 3H), 1.06 (m, 1H), 0.52 (m, 2H), 0.33 (m, 2H). 19F NMR (377 MHz, CDCl3) δ −135.2 (d, J = 21.5 Hz, 1F), −141.8 (d, J = 21.7 Hz, 1F). 13C NMR (101 MHz, CDCl3) δ 183.3, 165.9, 160.4, 151.4 (d, J = 13.3 Hz), 149.0 (d, J = 13.4 Hz), 148.5 (d, J = 12.9 Hz), 146.0 (d, J = 13.0 Hz), 141.9, 134.0 (m), 125.9, 123.0, 117.4 (d, J = 18.3 Hz), 116.5, 115.5 (m), 109.8 (d, J = 21.8 Hz), 62.2, 46.4, 14.0, 11.9, 11.6, 3.9. HRMS (ESI): m/z [M+1]+ calcd for C21H23F2N2O4: 405.1626, found 405.1616.
4.1.4.7. Ethyl 2-(5-((3,4-difluorophenyl)carbamoyl)-2,4-dimethyl-1-(prop-2-yn-1-yl)-1H-pyrrol-3-yl)-2-oxoacetate (4g) - 74% yield.
1H NMR (400 MHz, CDCl3) δ 7.95 (bs, 1H), 7.72 (m, 1H), 7.22 (m, 1H), 7.13 (q, J = 8.72 Hz, 1H), 4.97 (s, 2H), 4.36 (q, J = 7.16 Hz, 2H), 2.50 (s, 3H), 3.37 (t, J = 2.4 Hz, 1H), 2.33 (s, 3H), 1.38 (t, J = 7.24 Hz, 3H). 19F NMR (377 MHz, CDCl3) δ −135.4 (d, J = 21.8 Hz, 1F), −141.8 (d, J = 21.8 Hz, 1F). 13C NMR (101 MHz, CDCl3 δ 183.3, 165.6, 159.7, 151.3 (d, J = 13.4 Hz), 148.9 (d, J = 13.3 Hz), 148.4 (d, J = 12.8 Hz), 146.0 (d, J = 13.2 Hz), 142.3, 134.0 (m), 125.4, 123.7, 117.4, 117.2, 117.1, 115.7 (m), 109.9 (d, J = 21.9 Hz), 62.3, 34.4, 13.9, 11.6. HRMS (ESI): m/z [M+1]+ calcd for C20H19F2N2O4: 389.1313, found 389.1311.
4.1.4.8. Ethyl 2-(1-(but-2-yn-1-yl)-5-((3,4-difluorophenyl)carbamoyl)-2,4-dimethyl-1H-pyrrol-3-yl)-2-oxoacetate (4h) - 63% yield.
1H NMR (400 MHz, CDCl3) δ 7.97 (bs, 1H), 7.71 (m, 1H), 7.20 (m, 1H), 7.15 (q, J = 8.88 Hz, 1H), 4.88 (m, 2H), 4.38 (q, J = 7.16 Hz, 2H), 2.82 (s, 3H), 2.34 (s, 3H), 1.79 (t, J = 2.4 Hz, 3H), 1.40 (t, J = 7.16 Hz, 3H). 19F NMR (377 MHz, CDCl3) δ −135.3 (d, J = 21.7 Hz, 1F), −141.6 (d, J = 20.7 Hz, 1F). 13C NMR (101 MHz, CDCl3) δ 160.3, 151.4 (d, J = 13.2 Hz), 149.0 (d, J = 13.2 Hz), 147.9 (d, J = 12.9 Hz), 145.5 (d, J = 13.2 Hz), 123.3, 122.0, 117.3, 117.1, 115.0 (m), 110.9, 109.9, 81.9, 72.8, 63.8, 62.4, 35.0, 13.9, 11.7, 3.5. HRMS (ESI): m/z [M+1]+ calcd for C21H21F2N2O4: 403.1469, found 403.1471.
4.1.4.9. Ethyl 2-(5-((3,4-difluorophenyl)carbamoyl)-2,4-dimethyl-1-(pent-2-yn-1-yl)-1H-pyrrol-3-yl)-2-oxoacetate (4i) - 71% yield.
1H NMR (400 MHz, CDCl3) δ 8.08 (bs, 1H), 7.73 (m, 1H), 7.23 (m, 1H), 7.13 (q, J = 8.88 Hz, 1H), 4.89 (m, 2H), 4.36 (q, J = 7.16 Hz, 2H), 2.50 (s, 3H), 2.31 (s, 3H), 2.15 (m, 2H), 1.38 (t, J = 7.16 Hz, 3H), 1.07 (t, J = 7.52 Hz, 3H). 19F NMR (377 MHz, CDCl3) δ −135.5 (d, J = 21.8 Hz, 1F), −142.1 (d, J = 21.8 Hz, 1F). 13C NMR (101 MHz, CDCl3) δ 183.4, 165.8, 159.9, 151.3 (d, J = 13.2 Hz), 148.9 (d, J = 13.2 Hz), 148.4 (d, J = 12.7 Hz), 145.2 (d, J = 13.0 Hz), 142.2, 134.2 (m), 125.7, 123.6, 117.3 (d, J = 18.3 Hz), 116.8, 115.6 (m), 109.8 (d, J = 21.8 Hz),87.5, 72.9, 62.2, 35.0, 13.9, 12.3, 11.4. HRMS (ESI): m/z [M+1]+ calcd for C22H23F2N2O4: 417.1626, found 417.1627.
4.1.4.10. Ethyl 2-(1-benzyl-5-((3,4-difluorophenyl)carbamoyl)-2,4-dimethyl-1H-pyrrol-3-yl)-2-oxoacetate (4j) - 68% yield.
1H NMR (400 MHz, CDCl3) δ 7.58 (m, 1H), 7.26 (m, 4H), 7.09 (q, J = 8.88 Hz, 1H), 6.99 (m, 3H), 5.44 (s, 2H), 4.39 (q, J = 7.16 Hz, 2H), 2.43 (bs, 6H), 1.40 (t, J = 7.16 Hz, 3H). 19F NMR (377 MHz, CDCl3) δ −135.2 (d, J = 21.8 Hz, 1F), −141.7 (d, J = 21.8 Hz, 1F). 13C NMR (101 MHz, CDCl3) δ 183.4, 165.9, 160.0, 151.3 (d, J = 13.3 Hz), 148.8 (d, J = 13.3 Hz), 148.4 (d, J = 12.7 Hz), 145.9 (d, J = 13.0 Hz), 142.5, 136.4, 134.0 (m), 129.0, 127.8, 126.6, 126.3, 123.4, 117.2 (d, J = 18.4 Hz), 116.7, 115.7 (m), 109.8 (d, J = 21.9 Hz), 62.3, 48.2, 13.9, 11.9, 11.6. HRMS (ESI): m/z [M+1]+ calcd for C24H23F2N2O4: 441.1626, found 441.1627.
4.1.5. General synthetic procedure for compounds 5a-j:
A solution of (4a-j) (1 equiv.) in MeOH (0.1 M) and THF (0.5 M) was cooled to 0°C and 5% aqueous NaOH (0.1 M) was added slowly. The solution was stirred at ambient temperature for 20 min., then MeOH and THF were removed under vacuum. The resulting aqueous residue was partitioned between H2O and EtOAc. The organic phase was discarded. The aqueous solution was acidified to pH 4–5 by slowly adding 1N HCl. The mixture was then extracted 3 times with EtOAc. The combined organic phases were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was then taken up in DMF (0.1 M), and CDI (3 equiv.) was added. After stirring the solution for 30 min. under nitrogen at ambient temperature, the desired amine (5 equiv.) was added, and the reaction was stirred overnight. The reaction mixture was poured over water (50 mL) and extracted 3 times with EtOAc. The combined organic phase was dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography to give the desired compound (5a-j)
4.1.5.1. N-(3,4-Difluorophenyl)-1-ethyl-3,5-dimethyl-4-(2-oxo-2-(prop-2-yn-1-ylamino)acetyl)-1H-pyrrole-2-carboxamide (5a) - 35% yield.
1H NMR (DMSO-d6) δ 10.5 (s, 1H), 9.14 (t, J = 5.64 Hz, 1H), 7.84 (m, 1H), 7.42 (m, 2H), 4.09 (q, J = 7.16 Hz, 2H), 4.00 (dd, J = 5.64, 2.48 Hz, 2H), 3.18 (t, J = 2.48 Hz, 1H), 2.42 (s, 3H), 2.22 (s, 3H), 1.22 (t, J = 7.16 Hz, 3H). 19F NMR (377 MHz, DMSO-d6) δ 187.8, 167.6, 160.7, 150.6 (d, J = 13.2 Hz), 148.2 (d, J = 13.4 Hz), 147.2 (d, J = 12.8 Hz), 144.8 (d, J = 15.7 Hz), 140.4, 136.4 (m), 126.5, 122.8, 118.0 (d, J = 17.7 Hz), 116.8, 114.8 (m), 109.1 (d, J = 21.6 Hz), 80.8, 73.9, 28.1, 16.4, 11.9, 11.5. HRMS (ESI): m/z [M+1]+ calcd for C20H20F2N3O3: 388.1473, found 388.1470.
4.1.5.2. N-(3,4-Difluorophenyl)-3,5-dimethyl-4-(2-oxo-2-(prop-2-yn-1-ylamino)acetyl)-1-propyl-1H-pyrrole-2-carboxamide (5b) - 36% yield.
1H NMR (400 MHz, CDCl3) δ 7.71 (m, 1H), 7.46 (bs, 1H), 7.13 (m, 2H), 7.03 (m, 1H), 4.17 (dd, J = 5.56, 2.52 Hz, 2H), 4.11 (m, 2H), 2.42 (s, 3H), 2.37 (s, 3H), 2.30 (t, J = 2.52 Hz, 1H), 1.71 (m, 2H), 0.93 (t, J = 4.40 Hz, 3H). 19F NMR (377 MHz, CDCl3) δ −135.2 (d, J = 21.8 Hz, 1F), −142.1 (d, J = 21.8 Hz, 1F). 13C NMR (101 MHz, CDCl3) δ 186.2, 163.6, 160.1, 151.5 (d, J = 13.4 Hz), 149.0 (d, J = 13.1 Hz), 148.4 (d, J = 12.6 Hz), 145.9 (d, J = 12.9 Hz), 141.6, 134.1 (m), 126.4, 123.4, 117.9, 117.4 (d, J = 17.4 Hz), 115.3 (m), 109.7 (d, J = 21.8 Hz), 78.4, 72.3, 46.5,29.3, 24.2, 12.6, 12.0, 11.2. HRMS (ESI): m/z [M+1]+ calcd for C21H22F2N3O3: 402.1629, found 402.1627.
4.1.5.3. 1-Butyl-N-(3,4-difluorophenyl)-3,5-dimethyl-4-(2-oxo-2-(prop-2-yn-1-ylamino)acetyl)-1H-pyrrole-2-carboxamide (5c). 36% yield.
1H NMR (400 MHz, DMSO-d6) δ 10.5 (s, 1H), 9,14 (t, J = 5.68 Hz, 1H), 7.85 (m, 1H), 7.42 (m, 2H), 4.09 (t, J = 7.24 Hz, 2H), 4.00 (dd, J = 5.68, 2.48 Hz, 2H), 3.18 (t, J = 2.48 Hz, 1H), 2.42 (s, 3H), 2.22 (s, 3H), 1.56 (m, 2H), 1.23 (m, 2H), 0.85 (t, J = 7.28 Hz, 3H). 19F NMR (377 MHz, DMSO-d6) δ −137.2 (d, J = 23.2 Hz, 1F), −144.2 (d, J = 23.2 Hz, 1F). 13C NMR (101 MHz, DMSO-d6) δ 187.9, 167.6, 160.8, 150.6 (d, J = 13.1 Hz), 148.2 (d, J = 13.5 Hz), 147.2 (d, J = 12.7 Hz), 144.7 (d, J = 12.9 Hz), 140.7, 136.3 (m), 126.7, 122.8, 118.0 (d, J = 17.8 Hz), 116.7, 116.5 (m), 109.1 (d, J = 21.5 Hz), 80.6, 73.9, 32.8, 28.1, 19.7, 14.0, 11.9, 11.7. HRMS (ESI): m/z [M+1]+ calcd for C22H24F2N3O3: 416.1786, found 416.1789.
4.1.5.4. 1-Allyl-N-(3,4-difluorophenyl)-3,5-dimethyl-4-(2-oxo-2-(prop-2-yn-1-ylamino)acetyl)-1H-pyrrole-2-carboxamide (5d) - 30% yield.
1H NMR (400 MHz, DMSO-d6) δ 10.5 (s, 1H), 9.19 (t, J = 5.68 Hz, 1H), 7.86 (m, 1H), 7.42 (m, 2H), 5.92 (m, 1H), 5.15 (d, J = 10.3, 1H), 4.85 (d, J =17.2 Hz, 1H), 4.79 (d, J = 4.52 Hz, 2H), 4.02 (dd, J = 5.56, 2.4 Hz, 2H), 3.18 (t, J = 2.4 Hz, 1H), 2.39 (s, 3H), 2.27 (s, 3H). 19F NMR (377 MHz, DMSO-d6) δ −137.1 (d, J = 23.2 Hz, 1F), −144.2 (d, J = 23.1 Hz, 1F). 13C NMR (101 MHz, DMSO-d6) δ 187.9, 167.5, 160.6, 150.6 (d, J = 13.3 Hz), 148.2 (d, J = 13.2 Hz), 147.2 (d, J = 12.6 Hz), 144.8 (d, J = 12.6 Hz), 141.1, 136.3 (m), 134.2, 126.8, 123.0, 117.9 (d, J = 17.8 Hz), 116.9, 116.4 (m),109.1 (d, J = 21.8 Hz), 80.5, 73.8, 46.6, 28.1, 12.0, 11.6. HRMS (ESI): m/z [M+1]+ calcd for C21H20F2N3O3: 400.1473, found 400.1473.
4.1.5.5. 1-Cyclopropyl-N-(3,4-difluorophenyl)-3,5-dimethyl-4-(2-oxo-2-(prop-2-yn-1-ylamino)acetyl)-1H-pyrrole-2-carboxamide (5e) - 37% yield.
1H NMR (400 MHz, DMSO-d6) δ 10.5 (s, 1H), 9.15 (t, J = 5.16 Hz, 1H), 7.87 (m, 1H), 7.43 (m, 2H), 4.00 (m, 2H), 3.18 (m, 1H), 2.50 (bs, 4H), 2.16 (s, 3H), 1.03 (m, 2H), 0.74 (m, 2H). 19F NMR (377 MHz, DMSO-d6) d −137.1 (d, J = 23.1 Hz, 1F), −144.3 (d, J = 23.1 Hz, 1F). 13C NMR (101 MHz, DMSO-d6) δ 187.8, 167.6, 160.5, 157.4, 150.7 (d, J = 13.2 Hz), 148.2 (d, J = 13.2 Hz), 147.2 (d, J = 144.8 (d, J = 12.6 Hz), 142.6, 136.4 (m), 121.6, 118.0 (d, J = 17.8 Hz), 116.4, 115.9 (m), 108.8 (d, J = 21.7 Hz), 82.8, 80.7, 73.8, 73.0, 29.3, 28.0,12.9, 11.3, 8.4. HRMS (ESI): m/z [M+1]+ calcd for C21H20F2N3O3: 400.1473, found 400.1472.
4.1.5.6. 1-(Cyclopropylmethyl)-N-(3,4-difluorophenyl)-3,5-dimethyl-4-(2-oxo-2-(prop-2-yn-1-ylamino)acetyl)-1H-pyrrole-2-carboxamide (5f) - 42% yield.
1H NMR (400 MHz, CDCl3) δ 7.71 (m, 1H), 7.53 (bs, 1H), 7.14 (m, 1H), 7.13 (m, 2H), 7.07 (m, 1H), 4.14 (dd, J = 5.56, 2.52 Hz, 2H), 4.11 (d, J = 6.88 Hz, 2H), 2.45 (s, 3H), 2.36 (s, 3H), 2.30 (t, J = 2.52 Hz, 1H), 0.50 (m, 2H), 0.32 (m, 2H). 19F NMR (377 MHz, CDCl3) δ −135.2 (d, J = 21.8 Hz, 1F), −142.0 (d, J = 21.7 Hz, 1F). 13C NMR (101 MHz, CDCl3) δ 186.2, 163.7,160.5, 151.5 (d, J = 13.3 Hz), 149.0 (d, J = 13.2 Hz), 148.4 (d, J = 13.0 Hz), 141.6, 134.1 (m), 125.5, 123.4, 118.0, 117.4 (d, J = 18.5 Hz), 115.4 (m), 109.7 (d, 21.9 Hz),78.4, 72.3, 48.4, 29.3, 12.5, 12.3, 12.0, 3.9. HRMS (ESI): m/z [M+1]+ calcd for C22H22F2N3O3: 414.1629, found 414.1632.
4.1.5.7. N-(3,4-Difluorophenyl)-3,5-dimethyl-4-(2-oxo-2-(prop-2-yn-1-ylamino)acetyl)-1-(prop-2-yn-1-yl)-1H-pyrrole-2-carboxamide (5g) - 35% yield.
1H NMR (DMSO-d6) δ 10.53 (s, 1H), 9.21 (t, J = 5.6 Hz, 1H), 7.88 (m, 1H), 7.44 (m, 2H), 5.03 (d, J = 2 Hz, 2H), 4.02 (dd, J = 2.4, 5.6 Hz, 2H), 3.42 (t, J = 2.4 Hz, 1H), 3.2 (t, J = 2.4 Hz, 1H), 2.47 (s, 3H), 2.26 (s, 3H). 19F NMR (DMSO-d6) δ −137.15 (d, J = 28 Hz, 1F), −144.14 (d, J = 24 Hz, 1F). 13C NMR (DMSO-d6) δ 187.93, 167.31, 160.30, 150.68, 150.55, 148.26, 148.13, 147.33, 147.19, 144.91, 144.79, 141.23, 136.28, 136.20, 126.17, 123.69, 118.05, 117.87, 117.26, 116.51, 109.29, 109.08, 80.50, 78.99, 76.07, 73.86, 34.06, 28.12, 11.97, 11.69. HRMS (ESI): m/z [M+1]+ calcd for C21H18F2N3O3: 398.1316, found 398.1316.
4.1.5.8. 1-(But-2-yn-1-yl)-N-(3,4-difluorophenyl)-3,5-dimethyl-4-(2-oxo-2-(prop-2-yn-1-ylamino)acetyl)-1H-pyrrole-2-carboxamide (5h) - 44% yield.
1H NMR (400 MHz, DMSO-d6) δ 10.5 (s, 1H), 9.13 (t, J = 5.68 Hz, 1H), 7.80 (m, 1H), 7.37 (m, 2H), 4.89 (d, J = 2.32 Hz, 2H), 3.94 (dd, J = 5.68, 2.48 Hz, 2H), 3.13 (t, J = 2.48 Hz, 1H), 2.40 (s, 3H), 2.18 (s, 3H), 1.68 (t, 2.28 Hz, 3H). 19F NMR (377 MHz, DMSO-d6) δ −137.1 (d, J = 23.1 Hz, 1F), −144.2 (d, J = 23.1 Hz, 1F). 13C NMR (101 MHz, DMSO-d6) δ 160.3, 151.4 (d, J = 13.2 Hz), 149.0 (d, J = 13.2 Hz), 147.9 (d, J = 12.9 Hz), 145.5 (d, J = 13.2 Hz), 123.3, 122.0, 117.3, 117.1, 115.0 (m), 110.9, 109.9, 81.3, 80.5, 74.4, 55.4, 34.4, 28.1, 12.0, 11.7, 3.4. HRMS (ESI): m/z [M+1]+ calcd for C22H20F2N3O3: 412.1473, found 412.1475.
4.1.5.9. N-(3,4-Difluorophenyl)-3,5-dimethyl-4-(2-oxo-2-(prop-2-yn-1-ylamino)acetyl)-1-(pent-2-yn-1-yl)-1H-pyrrole-2-carboxamide (5i) - 39% yield.
1H NMR (400 MHz, DMSO-d6) δ 10.5 (s, 1H), 9.19 (t, J = 5.68 Hz, 1H), 7.87 (m, 1H), 7.43 (m, 2H), 4.96 (bs, 2H), 4.01 (dd, J = 5.64, 2.44 Hz, 2H), 3.19 (t, J = 2.48 Hz, 1H), 2.46 (s, 3H), 2.34 (s, 3H), 2.12 (m, 2H), 0.96 (t, J = 7.48 Hz, 3H). 19F NMR (377 MHz, DMSO-d6) δ −137.2 (d, J = 23.1 Hz, 1F), −144.2 (d, J = 23.1 Hz, 1F). 13C NMR (101 MHz, CDCl3) δ 183.4, 165.8, 159.9, 151.3 (d, J = 13.2 Hz), 148.9 (d, J = 13.2 Hz), 148.4 (d, J = 12.7 Hz), 145.2 (d, J = 13.0 Hz), 142.2, 134.2 (m), 125.7, 123.6, 117.3 (d, J = 18.3 Hz), 116.8, 115.6 (m), 109.8 (d, J = 21.8 Hz), 86.8, 80.5, 74.6, 73.9, 34.4, 28.1, 13.9, 12.0. HRMS (ESI): m/z [M+1]+ calcd for C23H22F2N3O3: 426.1629, found 426.1629.
4.1.5.10. 1-Benzyl-N-(3,4-difluorophenyl)-3,5-dimethyl-4-(2-oxo-2-(prop-2-yn-1-ylamino)acetyl)-1H-pyrrole-2-carboxamide (5j) - 32% yield.
1H NMR (400 MHz, CDCl3) δ 7.60 (m, 1H), 7.41 (bs, 1H), 7.26 (m, 2H), 7.90 (m, 3H), 7.01 (d, J = 6.84, 2H), 5.45 (s, 2H), 4.16 (dd, J = 5.56, 2.52 Hz, 2H), 2.40 (s, 3H), 2.36 (s, 3H), 2.29 (t, J = 2.52 Hz, 1H). 19F NMR (377 MHz, CDCl3) δ −135.3 (d, J = 21.6 Hz, 1F), −142.0 (d, J = 21.8 Hz, 1F). 13C NMR (101 MHz, CDCl3) δ 186.2, 163.4, 160.0, 151.4 (d, J = 13.3 Hz), 148.9 (d, J = 133.3 Hz), 148.4 (d, J = 13.0 Hz), 145.9 (d, J = 12.8 Hz),142.2, 136.6, 133.9 (m), 126.9, 127.7, 126.3, 125.9, 123.8, 118.3, 117.4 (d, J = 18.4 Hz), 115.4 (m), 109.7 (d, J = 21.9 Hz), 78.4, 72.3, 48.1, 29.3, 12.6, 12.3. HRMS (ESI): m/z [M+1]+ calcd for C25H22F2N3O3: 450.1629, found 450.1626.
4.1.6. General synthetic procedure for compounds 9a-f:
A solution of 86 (1 equiv.) in MeOH (0.1 M) and THF (0.5 M) was cooled to 0°C and 5% aqueous NaOH (0.1 M) was added slowly. The solution was stirred at ambient temperature for 20 min, then MeOH and THF were removed under vacuum. The resulting aqueous layer was washed with EtOAc. The organic phase was discarded. The aqueous solution was acidified to pH 4–5 by the slow addition of 1N HCl. The mixture was then extracted 3 times with EtOAc and the combined organic phase was dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was then taken up in DMF and HATU (3 equiv.), DIPEA (6 equiv.) and the desired substituted amine (4 equiv.) were added. The resulting mixture was stirred at 60–80°C overnight and was quenched with cold H2O. The water layer was then extracted twice with EtOAc. The organic layers were finally combined, washed with water, brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by flash chromatography (hexane /ethyl acetate 10:1 to 0:100) to afford product 3a-f.
4.1.6.1. N-(3,4-Difluorophenyl)-1,3,5-trimethyl-4-(2-((2-methylbut-3-yn-2-yl)amino)-2-oxoacetyl)-1H-pyrrole-2-carboxamide (9a) - 83% yield.
1H NMR (400 MHz, CDCl3) δ 8.13 (s, 1H), 7.74–7.69 (m, 1H), 7.26–7.22 (m, 1H), 7.11 (dd, J = 18.4 Hz, 8.8 Hz, 1H), 6.99 (s, 1H), 3.64 (s, 3H), 2.38 (s, 1H), 2.35 (s, 3H), 2.29 (s, 3H), 1.69 (s, 6H).13C NMR (101 MHz, CDCl3) δ 187.2, 163.8, 160.3, 151.3, 148.8, 148.2, 145.8, 142.0, 134.3, 126.1, 123.8, 117.3, 115.6, 109.7, 85.9, 70.0, 47.7, 32.3, 28.6, 12.3, 12.1. 19F NMR (377 MHz, CDCl3) δ −135.6 to −135.7 (m, 1 F), −142.3 to −142.4 (m, 1F). HRMS (ESI) m/z calcd for C21H22F2N3O3 [M+H] +: 402.1629; found 402.1629.
4.4.6.2. N-(3,4-Difluorophenyl)-1,3,5-trimethyl-4-(2-(3-methylpent-1-yn-3-ylamino)-2-oxoacetyl)-1H-pyrrole-2-carboxamide (9b) - 86% yield.
1H NMR (400 MHz, CDCl3) δ 8.00 s, 1H), 7.74–7.68 (m, 1H), 7.24–7.19 (m, 1H), 7.11 (dd, J = 18.5 Hz, 8.8 Hz, 1H), 6.92 (s, 1H), 3.65 (s, 3H), 2.40 (s, 1H), 2.36 (s, 3H), 2.31 (s, 3H), 2.12–2.06 (m, 1H), 1.89–1.84 (m, 1H),1.67 (s, 3H), 1.05 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 187.2, 163.5, 160.2, 151.3, 148.8, 148.2, 145.8, 142.0, 134.3, 126.0, 123.7, 117.4, 115.6, 109.7, 84.8, 71.2, 52.2, 33.1, 32.3, 25.9, 12.2, 8.7. 19F NMR (377 MHz, CDCl3) δ −135.5 to −135.6 (m, 1 F), −142.3 to −142.4 (m, 1F). HRMS (ESI) m/z calcd for C22H24F2N3O3 [M+H] +: 416.1786; found 416.1786.
4.1.6.3. N-(3, 4-Difluorophenyl)-4-(2-(3-ethylpent-1-yn-3-ylamino)-2-oxoacetyl)-1,3,5-trimethyl-1H-pyrrole-2-carboxamide (9c) - 76% yield.
1H NMR (400 MHz, CDCl3) δ 7.77 (s, 1H), 7.73–7.68 (m, 1H), 7.20–7.08 (m, 2H), 6.84 (s, 1H), 3.66 (s, 3H), 2.42 (s, 1H), 2.37 (s, 3H), 2.34 (s, 3H), 2.20–2.11 (m, 2H), 1.94–1.85 (m, 2H), 1.03 (t, J = 7.4 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 187.1, 163.0, 160.2, 151.4, 148.9, 148.3, 145.8, 142.0, 134.3, 125.9, 123.7, 117.5, 115.4, 109.6, 83.9, 72.4, 57.2, 32.3, 30.3, 12.5, 12.2, 8.6. 19F NMR (377 MHz, CDCl3) δ −135.4 to −135.5 (m, 1 F), −142.3 to −142.4 (m, 1F). HRMS (ESI) m/z calcd for C23H26F2N3O3 [M+H] +: 430.1942; found 430.1944.
4.1.6.4. N-(3,4-Difluorophenyl)-4-(2-(1-ethynylcyclopropylamino)-2-oxoacetyl)-1,3,5-trimethyl-1H-pyrrole-2-carboxamide (9d) - 73% yield.
1H NMR (400 MHz, DMSO-d6) δ 10.43 (s, 1H), 9.30 (s, 1H), 7.90–7.85 (m, 1H), 7.45–7.38 (m, 2H), 3.59 (s, 3H), 3.08 (s, 1H), 2.40 (s, 3H), 2.23 (s, 3H). 1.18 (dd, J = 8.04 Hz, 5.56 Hz, 2H), 1.04 (dd, J = 6.96 Hz, 4.48 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 188.0, 168.0, 160.5, 150.6, 148.2, 147.2, 144.8, 141.3, 136.4, 127.3, 122.8, 117.9, 116.5, 109.1, 85.6, 69.8, 32.3, 22.1, 16.9, 12.0. 19F NMR (377 MHz, DMSO-d6) δ - 137.1 to −137.2 (m, 1 F), −144.2 to −144.3 (m, 1F). HRMS (ESI) m/z calcd for C21H20F2N3O3 [M+H] +: 400.1473; found 400.1476.
4.1.6.5. N-(3,4-Difluorophenyl)-4-(2-(1-ethynylcyclobutylamino)-2-oxoacetyl)-1, 3, 5-trimethyl-1H-pyrrole-2-carboxamide (9e) – 62% yield.
1H NMR (400 MHz, DMSO-d6) δ 10.42 (s, 1H), 9.21 (s, 1H), 7.90–7.85 (m, 1H), 7.47–7.38 (m, 2H), 3.60 (s, 3H), 3.33 (s, 1H), 7.90–7.85 (m, 1H), 2.45 (s, 3H), 2.42–2.38 (m, 3H), 2.27 (s, 3H), 2.02–1.91 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 180.0, 166.8, 160.5, 150.6, 148.2, 147.2, 144.8, 141.3, 136.4, 127.2, 122.9, 117.9, 116.4, 109.1, 86.9, 72.9, 48.6, 35.3, 32.3, 16.3, 12.0. 19F NMR (377 MHz, DMSO-d6) δ −137.2 to −137.3 (m, 1 F), −144.2 to −144.4 (m, 1F). HRMS (ESI) m/z calcd for C22H22F2N3O3 [M+H] +: 414.1629; found 414.1632.
4.1.6.6. N-(3,4-Difluorophenyl)-4-(2-(1-ethynylcyclohexylamino)-2-oxoacetyl)-1, 3, 5-trimethyl-1H-pyrrole-2-carboxamide (9f) - 78% yield.
1H NMR (400 MHz, CDCl3) δ 8.24 (s, 1H), 7.75–7.69 (m, 1H), 7.28–7.24 (m, 1H), 7.11 (dd, J = 18.6 Hz, 8.8 Hz, 1H), 6.91 (s, 1H), 3.62 (s, 3H), 2.43 (s, 1H), 2.34 (s, 3H), 2.28 (s, 3H), 2.20–2.17 (m, 2H), 1.83–1.78 (m, 2H), 1.72–1.59 (m, 5H), 1.33–1.25 (m, 1H). 13C NMR (101 MHz, CDCl3) δ 187.3, 163.9, 160.3, 151.3, 148.8, 148.2, 145.8, 142.0, 134.4, 126.0, 123.9, 117.2, 115.7, 109.7, 84.3, 72.2, 51.9, 36.6, 32.3, 25.1, 22.3, 12.2. 19F NMR (377 MHz, DMSO-d6) δ −135.7 to −135.8 (m, 1 F), −142.4 to −142.5 (m, 1F). HRMS (ESI) m/z calcd for C24H26F2N3O3 [M+H] +: 442.1942; found 442.1946.
4.1.7. General synthetic procedure for compounds 10a-b:
A solution of 4g (1 equiv.) in MeOH (0.1 M) and THF (0.5 M) was cooled to 0°C and 5% aqueous NaOH (0.1 M) was added slowly. The solution was stirred at ambient temperature for 20 min, then MeOH and THF were removed under vacuum. The resulting aqueous layer was washed with EtOAc. The organic phase was discarded. The aqueous solution was acidified to pH 4–5 by slowly adding 1N HCl. The mixture was then extracted 3 times with EtOAc and the combined organic phase was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was then taken up in DMF and HATU (3 equiv.), DIPEA (6 equiv.) and the desired substituted amine (4 equiv.) were added. The resulting mixture was stirred at 60–80°C overnight and was quenched with cold H2O. The water layer was then extracted twice with EtOAc. The organic layers were finally combined, washed with water, brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by flash chromatography to afford product 10a-b.
4.1.7.1. N-(3,4-Difluorophenyl)-3,5-dimethyl-4-(2-((2-methylbut-3-yn-2-yl)amino)-2-oxoacetyl)-1-(prop-2-yn-1-yl)-1H-pyrrole-2-carboxamide (10a) - 32% yield.
1H NMR (DMSO-d6) δ 10.53 (s, 1H), 8.83 (s, 1H), 7.89 (dd, J = 2.4, 7.6 Hz, 1H), 7.49–7.43 (m, 2H), 5.05 (d, J = 2.0 Hz, 2H), 3.41 (t, J = 2.4 Hz, 1H), 3.22 (s, 4H), 2.31 (s, 3H), 1.59 (s, 6H). 19F NMR (DMSO-d6) δ −137.13 (d, J = 28 Hz, 1F), −144.14 (d, J = 24 Hz, 1F). 13C NMR (DMSO-d6) δ 187.9, 167.0, 160.3, 150.7, 150.6, 148.3, 148.1, 147.3, 147.2, 144.9, 144.8, 141.2, 136.3, 136.3, 136.3, 136.2, 126.1, 123.8, 118.0, 117.8, 117.3, 116.5, 109.3, 109.0, 87.3, 79.0, 76.0, 72.0, 46.8, 34.0, 29.2, 12.1, 11.7. HRMS (ESI) m/z calcd for C23H22F2N3O3 [M+H] +: 426.1629; found 426.1627.
4.1.7.2. N-(3,4-difluorophenyl)-4-(2-((3-ethylpent-1-yn-3-yl)amino)-2-oxoacetyl)-3,5-dimethyl-1-(prop-2-yn-1-yl)-1H-pyrrole-2-carboxamide (10b) - 36% yield.
1H NMR (CDCl3) δ 7.73–7.69 (m, 1H), 7.65 (s, 1H), 7.17–7.09 (m, 2H), 6.88 (s, 1H), 5.01 (d, J = 2.4 Hz, 2H), .45 (s, 3H), 2.43 (s, 1H), 2.37 (s, 3H), 2.31 (t, J = 2.4 Hz, 1H), 2.21–2.13 (m, 2H), 1.96–1.87 (m, 2H), 1.04 (t, J = 7.2 Hz, 6H). 19F NMR (CDCl3) δ −135.31 (d, J = 24 Hz, 1F), −147.04 (d, J = 20 Hz, 1F). 13C NMR (CDCl3) δ 187.2, 162.4, 159.9, 151.5, 151.3, 149.0, 148.9, 148.46, 146.0, 145.9, 141.9, 134.1, 134.0, 124.7, 124.3, 118.4, 117.4, 117.2, 115.5, 115.5, 115.4,109.9,109.7, 83.9, 73.2, 72.4, 57.2, 34.3, 30.3, 12.6, 12.0, 8.6. HRMS (ESI) m/z calcd for C25H26F2N3O3 [M+H] +: 454.1942; found 454.1941.
4.2. Anti-HBV activity in HepAd38
The HBV 7-day assay was performed in HepAD38 wild type (HepAD38) cells as previously described.10 Briefly, HepAD38 cells were seeded onto 96-well plates and incubated for two days at 37°C in a humidified 5% CO2 atmosphere. On day two, medium was removed and cells were washed with 1X phosphate buffer saline (PBS). Forty mM stock solutions of the compounds were prepared for the assay. The desired aliquot of the solution was diluted in medium without tetracycline and added in duplicate at various concentrations to the wells. On day seven, total DNA was extracted using DNeasy 96 Tissue kit (Qiagen), and HBV DNA was amplified by RT-PCR.11 Antiviral activity was measured by determining the average threshold cycle for the HBV amplification of the compounds (alone or in combination), which was subtracted from the average cycle of the untreated-tetracycline control (ΔCT). Drugs were first tested individually for effective concentration, which inhibited 50% and 90% of HBV DNA replication (EC50 and EC90) using CalcuSyn software program (Biosoft, Cambridge, UK).
4.3. Cytotoxicity assays.
Cytotoxicity was determined by using the CellTiter 96 non-radioactive cell proliferation colorimetric assay (Promega) in human peripheral blood mononuclear (PBM) cells and in human T lymphoblast (CEM), African green monkey kidney (Vero), and human hepatocellular carcinoma (HepG2) cells. Toxicity levels were measured as the concentration of test compound that inhibited cell growth by 50% (CC50).
4.4. Formation of Glutathione (GSH) adducts
The test compound (25 μM) was incubated with human liver microsomes (1 mg/mL), in the presence of 1 mM reduced glutathione (GSH) and 0 or 2 mM NADPH in 100 mM potassium phosphate, pH 7.4 buffer with 3 mM MgCl2. After 60 min incubation at 37°C, the samples were processed and analyzed by LC/MS/MS analysis. Abundance of Reactive metabolite as percent was calculated based on the peak area of GSH adduct in the presence of NADPH and the peak area of the parent compound in the absence of NADPH.
4.5. hERG Inhibition
The hERG assays were run using an automated patch clamp system at either Charles River (GLP-26) or Metrion (compound 10a) in CHO cells at room temperature using a concentration of up to 10 μM test article (n ≥ 3 per compound).
4.6. Cell permeability
Permeability of the test compound at 2 μM was determined bidirectionally across Caco-2 cell monolayer at pH 7.4. The assay was conducted in duplicate. Final DMSO concentration was adjusted to less than 1%. The plate was incubated for 2 hr in CO2 incubator at 37°C, with 5% CO2 at saturated humidity without shaking. The processed samples were analyzed by LC/MS/MS analysis for concentrations of in starting solution, donor solution, and receiver solution. After the transport assay, lucifer yellow rejection assay was applied to determine the Caco-2 cell monolayer integrity.
4.7. Liver microsome stability
The stability of test compound (10a) in mouse and human liver microsomes, with or without ritonavir, was evaluated according to the following procedure. Briefly, 1 μM of 10a with or without ritonavir (0.3, 1 and 3 μM) were exposed to 1 mg/mL mouse or human liver microsome (XenoTech) in a reaction mixture (final 500 μL) containing 100 mM potassium phosphate buffer (pH 7.4) and 5 mM MgCl2. NADPH (final 1 mM) was added to the mixture to initiate the reaction at 37°C. At selected times of 0, 5, 15, 30, 45, 60, and 90 min, 50 μl aliquots were taken and the reaction stopped by mixing with 200 μl of ice-cold methanol. The samples were centrifuged and the supernatant were subjected to LC-MS/MS analysis after a 1:1 dilution with H2O. Propranolol was used as a positive control.
4.8. CYP inhibition
Appropriate known substrates of CYP450s were incubated in human liver microsomes (0.05–0.2 mg/mL) in a buffer containing 1 mM NADPH in 100 mM potassium phosphate, pH 7.4 with 3 mM MgCl2 at 37°C, in the presence or absence of the test compound (10 μM). The assay was conducted in duplicate. At the end of the incubations, the reactions were terminated with the addition of the quenching solution containing the internal standards. The extracted samples were subjected to LC/MS/MS analysis for the formation of each of the signature metabolites of the CYP substrates.
4.9. Molecular modeling
GLP-26 and compound 10a were docked in the reported crystal structure of HBV core protein dimer bound to class-II CAM, SBA (PDBID:5T2P) using the Glide SP module of Schrödinger.
Alchemical FEP calculations were performed using the academic Desmond Ligand FEP module with the OPLS-2005 forcefield with the TIP4P water model, the details of which are described elsewhere.12,13 Briefly, the perturbation was carried out over twelve λ windows. FEP production runs were conducted over 50 ns per window for the protein-ligand complex and 5 ns per window for the ligand alone. The replica exchange method offered enhanced sampling over the course of the production runs,14 and error was calculated using the bootstrap methodology.15
Supplementary Material
Highlights:
Despite numerous drugs to treat chronic hep B infections, there is no cure
Highly potent CAMs could be used to develop novel curative approaches
SAR around known HBV CAM GLP-26 has been performed
Novel HBV CAM displaying sub-nanomolar activity were identified.
Acknowledgments and Funding
This work was supported by funding from NIH (RO1-AI-132833), in part by 1-RO1-AI-148740, and Emory Center for AIDS Research (5P30-AI-50409).
Footnotes
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Conflict of interest
Drs. Schinazi, Amblard, Bassit, and Emory University, are entitled to equity and royalties related to anti-HBV products licensed to Aligos Therapeutics, Inc., being further evaluated in the research described in this paper. Emory University has reviewed and approved the terms of this arrangement per its conflict-of-interest policies.
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