Syntheses and Structure–Activity Relationships of N-Phenethyl-Quinazolin-4-yl-Amines as Potent Inhibitors of Cytochrome bd Oxidase in Mycobacterium tuberculosis

Abstract

The development of cytochrome bd oxidase (cyt-bd) inhibitors are needed for comprehensive termination of energy production in Mycobacterium tuberculosis (Mtb) to treat tuberculosis infections. Herein, we report on the structure-activity-relationships (SAR) of 22 new N-phenethyl-quinazolin-4-yl-amines that target cyt-bd. Our focused set of compounds was synthesized and screened against three mycobacterial strains: Mycobacterium bovis BCG, Mycobacterium tuberculosis H37Rv and the clinical isolate Mycobacterium tuberculosis N0145 with and without the cytochrome bcc:aa₃ inhibitor Q203 in an ATP depletion assay. Two compounds, 12a and 19a, were more active against all three strains than the naturally derived cyt-bd inhibitor aurachin D.

1. Introduction

Tuberculosis (TB) is still a leading cause of death from an infectious agent. According to the World Health Organization’s 2020 Global Tuberculosis Report, over 10 million people became ill with the disease in 2019 [1]. Action must be taken to combat this continuing public health threat. TB is caused by the bacterium Mycobacterium tuberculosis (Mtb) and spread by aerosols of an infected person through a cough or sneeze. Treatment of individuals infected with TB consists of several medications that are taken together for 6–9 months. First-line drugs include isoniazid (INH), rifampin (RIF), ethambutol (EMB) and pyrazinamide (PZA) [1]. Other barriers, such as lengthy treatment regimens and access to care, have made adherence to treatment challenging. Shorter treatment schedules are strongly desired. Despite the advances in drug therapies and modern medications, such as bedaquiline, delamanid and pretomanid, there is a need for comprehensive drug combinations and new treatment regimens to combat drug resistant TB [2]. Consideration of how Mtb survives onslaughts of chemotherapies gives insight into more effective treatment. Investigating the mechanism of action of antibiotics provides critical information that can be used to ultimately cure TB infections [3]. Targets within energy metabolism processes, specifically those involved in the oxidative phosphorylation pathway, have become of increased interest. Mtb can persist in a hypoxic, non-replicating, drug-tolerant state [4]. It has been demonstrated that interrupting oxidative phosphorylation in Mtb is an encouraging strategy [5,6,7]. While compounds have been made to inhibit various targets within the oxidative phosphorylation pathway, limitations have been observed, including the inability to inhibit oxygen respiration and clear Mtb infection due to a redundancy between the terminal oxidases cytochrome bcc:aa₃ (cyt-bcc:aa₃) and cytochrome bd (cyt-bd) [8]. Cytochrome bcc:aa₃ is the main aerobic terminal oxidase in Mtb [9]. The compound Q203 (4, Telacebec^®) inhibits cytochrome bcc by binding to the QcrB subunit, thereby inhibiting the function of the cytochrome bcc:aa₃ super-complex [10]. When cytochrome bcc:aa₃ is inactive, cyt-bd is upregulated [11]. This adaptation provides protection against cytochrome bcc:aa₃ inhibitors such as Q203. We have shown that targeting cyt-bd in combination with Q203 is bactericidal against replicating and non-replicating Mtb [12]. This makes cyt-bd an enticing target for the development of antituberculosis agents to effectively eliminate infection.

Interested in the inhibition of this target, we screened a small set (56) of antibacterial compounds that originated from our decades-old antibacterial program. This led to the identification of cyt-bd compounds like the thieno[3,2-d]pyrimidin-4-amines in which (1) is an example, the in vivo active optimized inhibitor ND-11992 (2) and hit compound 3 (Figure 1) [13].

The N-phenethylquinazolin-4-amine class, exemplified by compound 3 (Figure 1), has an IC₅₀ of 11 µM against Mycobacterium bovis BCG and an IC₅₀ of 27 µM against Mycobacterium tuberculosis H37Rv as determined by our published ATP depletion assay [13]. This assay is purposefully run in the presence and absence of Q203 (4, a selective cyt- bcc:aa₃ inhibitor) to indicate whether a compound inhibits alone or synergizes due to the conditional essentiality of cyt-bd to maintain ATP homeostasis once cyt-bcc:aa₃ is selectively inhibited by Q203. Compound 3 was inactive (IC₅₀ > 25 µM) in the absence of Q203 which is an indication of on-target potency based upon assay design. Similarly, compound 3 was amenable to chemical modifications and structure–activity relationship (SAR) development. Herein, we report our initial design, synthesis, and activity assessment of various quinazoline compounds for activity against cyt-bd of Mycobacterium bovis BCG and Mycobacterium tuberculosis.

2. Materials and Methods

2.1. Chemistry

General. All anhydrous solvents, reagent grade solvents for chromatography and starting materials were purchased from either Aldrich Chemical Co. (Milwaukee, WI, USA) or Fisher Scientific (Suwanee, GA, USA) unless otherwise noted. Water was distilled and purified through a Milli-Q water system (Millipore Corp., Bedford, MA, USA). General methods of purification of compounds involved the use of silica cartridges purchased from Practichem, LLC. (www.practichem.com, last accessed on 23 September 2021) and/or recrystallization. The reactions were monitored by TLC on precoated Merck 60 F254 silica gel plates and visualized using UV light (254 nm). All compounds were analyzed for purity by HPLC and characterized by ¹H and ¹³C NMR using a Bruker Ascend Avance III HD Spectrometer (500 MHz). Chemical shifts are reported in ppm (δ) relative to the residual solvent peak in the corresponding spectra; chloroform δ 7.27 and δ 77.23, methanol δ 3.31 and δ 49.00 and coupling constants (J) are reported in hertz (Hz) (where, s = singlet, bs = broad singlet, d = doublet, dd = double doublet, bd = broad doublet, ddd = double doublet of doublet, t = triplet, tt = triple triplet, q = quartet, m = multiplet) and analyzed using MestreNova NMR data processing. ¹⁹F NMR were run without a standard and are uncorrected. Mass spectra values are reported as m/z. Melting points were measured on a Buchi B-545 melting point instrument and are uncorrected, measured against benzoic acid 118.8–120.2 °C. Compounds 3, 6a–26a appear within US Provisional Patent Application No. 62/783,984. The quinazolines (5, 16–26) and amines (6–15) used in synthesis are all commercially available.

LC-MS method: The liquid chromatography–mass spectrometry method was performed on an Agilent 1290 infinity coupled to Agilent 6538 Ultra High-Definition Quadrupole Time of Flight (UHD-QTOF) instrument. A separation was achieved by using reverse phase Waters Acuity UPLC HSS T3 1.8 µm (2.1 × 100mm) column from Waters (Milford, MA, USA). All solvents were purchased from Fischer Scientific LCMS Optima grade solvents. Water containing 0.1% formic acid was used as mobile phase A and acetonitrile containing 0.1% formic acid was used as mobile phase B. The injection volume was set at 1 µL. Samples were injected in a gradient of 95% mobile phase A and 5% mobile phase B in the initial condition to 5% mobile phase A and 95% mobile phase B in 9 min. The eluent was held at that composition for an additional 3 min and switched back to the initial condition at 12 min.

The MS data acquisition was performed from 50–1000 m/z at 1.0 spectra/sec scan rate. The source gas temperature was set at 350 °C with a flow of 8 l/min. The nebulizer gas was set at 55 psig. The capillary voltage was set at 3500 volts with fragmentor at 100, skimmer at 45 and octopole RF 500 volts. Prior to sample runs, the instrument was calibrated using Agilent low mass calibrant solution.

Data analysis: The data collected in Agilent LC-MS was analyzed using Agilent Mass Hunter software for HRMS calculation.

General Syntheses of N-Phenethylquinazolin-4-Amine Analogs

General procedure A for base catalyzed S_NAr reaction for the preparation of compounds 6a, 7a, 9a, 12a-26a. In a sealed vial, 4-chloroquinazoline (5, 100 mg, 0.61 mmol, 1 equiv.), 2-[4-(trifluoromethoxy)phenyl]ethylamine (7, 125 mg, 0.61 mmol, 1 equiv.) and potassium carbonate (84 mg, 0.61 mmol, 1 equiv. were combined in DMSO (4 mL). The reaction was heated to 100 °C for 12 h. The reaction mixture was concentrated to dryness. The residue was extracted with CH₂Cl₂ and washed with 5% aqueous acetic acid solution (2x), water and brine. The organic phase was collected, dried over sodium sulfate, filtered, and concentrated in vacuo. Crude material obtained was purified by either silica gel chromatography with a gradient of CH₂Cl₂: ethyl acetate: solvent system (0–80%) or recrystallized from hot isopropanol or acetonitrile to afford the product.

General procedure B for acid catalyzed S_NAr reaction for the preparation of compounds 3, 8a, 10a, 11a. In a sealed vial, desired 4-chloroquinazoline 16–26 (1 equiv.) and 2-[4-(trifluoromethoxy)phenyl]ethylamine (7, 1 equiv.) were dissolved in a 3:1 tetrahydrofuran: 2-propanol solution (4 mL). Next, was added 12 M HCl (~0.4 equiv.). The solution was heated at 70 °C for 24 h. The reaction was concentrated to dryness. The residue was dissolved in CH₂Cl₂ and washed with saturated aqueous NaHCO₃ solution, water, and brine. The organic phase was collected, dried over sodium sulfate, filtered, and concentrated in vacuo. Crude material obtained was purified by either silica gel column chromatography with a gradient of CH₂Cl₂: ethyl acetate: solvent system (0–80%) or recrystallized from hot isopropanol or acetonitrile to afford the product.

N-Phenethylquinazolin-4-amine, 3

Following general procedure B, using 4-chloroquinazoline (5, 100 mg, 0.59 mmol), 2-phenylethylamine (72 mg, 0.59 mmol), and 12 N HCl (11 µL, 0.14 mmol) gave 3 as pale-yellow crystals (43 mg, 28%). mp 171.5–171.9 °C; ¹H (500 MHz, MeOD) δ ppm 8.47 (s, 1H), 8.06 (d, J = 8.3 Hz, 1H), 7.82–7.77 (m, 1H), 7.72 (d, J = 8.3 Hz, 1H), 7.55–7.50 (m, 1H), 7.31–7.26 (m, 4H), 7.23–7.17 (m, 1H), 3.86 (t, J = 7.4 Hz, 2H), 3.04 (t, J = 7.4 Hz, 2H) ¹³C (125 MHz, MeOD) δ ppm 160.0, 154.7, 148.2, 139.3, 132.7, 128.5, 128.1, 126.3, 126.0, 125.9, 121.9, 115.1, 42.5, 34.7. HRMS (EI), M + 1 calculated for C₁₆H₁₅N₃, 250.1339, found 250.1351. These experimental results are consistent with previous reports [14,15,16].

Quinazolin-4-yl{2-[4-(trifluoromethyl)phenyl]ethyl}amine, 6a

Following general procedure A, using 4-chloroquinazoline (5, 100 mg, 0.59 mmol), 2-[4-(trifluoromethyl)phenyl]ethylamine (114 mg, 0.59 mmol) and potassium carbonate (81 mg, 0.59 mmol) gave 6a as gold crystals (79 mg, 38%). mp 160.3–161.0 °C; ¹H (500 MHz, CDCl₃) δ ppm 8.73 (s, 1H), 7.88 (d, J = 7.9 Hz, 1H), 7.77 (ddd, J = 8.4, 7.0, 1.3 Hz), 7.63–7.57 (m, 3H), 7.47 (ddd, J = 8.2, 7.0, 1.2 Hz), 7.40 (d, J = 8.0 Hz, 2H), 5.79 (br.s, 1H), 3.99 (dt, J = 6.9, 5.9 Hz, 2H), 3.14 (t, J = 6.9 Hz, 2H). ¹³C (125 MHz, CDCl₃) δ ppm 159.6, 155.4, 149.5, 143.2 (d, J = 1.5 Hz), 132.7, 129.2, 129.1 (q, J = 32.4 Hz), 128.2, 126.1, 125.6 (q, J = 3.8 Hz), 124.7 (q, J = 271.9 Hz), 120.2, 114.9, 42.1, 35.2. ¹⁹F (470 MHz, CDCl₃) δ ppm −62.4 (s, 3F). HRMS (EI), M + 1 calculated for C₁₇H₁₄F₃N₃, 318.1213, found 318.1229. This compound appears in referenced patent and fungicidal activity was reported [17].

Quinazolin-4-yl{2-[4-(trifluoromethoxy)phenyl]ethyl}amine, 7a

Following general procedure A, using 4-chloroquinazoline (5, 100 mg, 0.59 mmol), 2-[4-(trifluoromethoxy)phenyl]ethylamine (125 mg, 0.59 mmol) and potassium carbonate (84 mg, 0.59 mmol) gave 7a as white crystals (85 mg, 42%). mp 137.4–137.8 °C; ¹H (500 MHz, CDCl₃) δ ppm 8.73 (s, 1H), 7.88 (d, J = 8.2 Hz, 1H), 7.77 (ddd, J = 8.4, 7.0, 1.3 Hz, 1H), 7.60–7.56 (m, 1H), 7.48 (ddd, J = 8.3, 7.0, 1.1 Hz), 7.33–7.26 (m, 2H), 7.20 (d, J = 8.0 Hz, 2H), 5.73 (br.s, 1H), 3.97 (dt, J = 7.0, 5.9 Hz, 2H), 3.08 (t, J = 7.0 Hz, 2H). ¹³C (125 MHz, CDCl₃) δ ppm 159.3, 155.4, 149.5, 148.0 (d, J = 1.8 Hz), 137.7, 132.6, 130.2, 128.8, 126.1, 121.3, 120.6 (q, J = 235.0 Hz), 120.1, 114.9, 42.3, 34.6. ¹⁹F (470 MHz, CDCl₃) δ ppm −59.9 (s, 3F). HRMS (EI), M + 1 calculated for C₁₇H₁₄F₃N₃O, 334.1162, found 334.1155.

[2-(4-Chlorophenyl)ethyl]quinazolin-4-ylamine, 8a

Following general procedure B, using 4-chloroquinazoline (5, 100 mg, 0.59 mmol), 2-(4-chlorophenyl)ethylamine (94 mg, 0.59 mmol) and 12 N HCl (11 µL, 0.14 mmol) gave 8a as yellow crystals (35 mg, 20%). mp 191.0–192.1 °C; ¹H (500 MHz, MeOD) δ ppm 8.47 (s, 1H), 8.05 (dd, J = 8.3, 0.7 Hz, 1H), 7.80 (ddd, J = 8.3, 7.0, 1.3 Hz, 1H), 7.73–7.70 (m, 1H), 7.53 (ddd, J = 8.3, 7.0, 1.3 Hz, 1H), 7.30–7.25 (m, 2H), 3.88–3.83 (m, 2H), 3.03 (t, J = 7.3 Hz, 2H). ¹³C (125 MHz, MeOD) δ ppm 160.0, 154.6, 148.2, 138.1, 132.8, 131.8, 130.2, 128.1, 126.3, 126.1, 121.9, 115.1, 42.2, 34.0. HRMS (EI), M + 1 calculated for C₁₆H₁₄ClN₃, 284.0949, found 284.0950. These experimental results are consistent with previous reports [18,19,20].

[2-(4-(Pentafluoro-(λ)⁶-sulfaneyl]quinazolin-4-ylamine, 9a

Following general procedure A, using 4-chloroquinazoline (5, 70 mg, 0.42 mmol) and 2-(4-(pentafluoro-(λ)⁶-sulfaneyl)phenyl)ethan-1-amine (105 mg, 0.42 mmol) and potassium carbonate (59 mg, 0.42 mmol) gave 9a as white crystals (97 mg, 61%). mp 169.2–170.7 °C; ¹H (500 MHz, CDCl₃) δ ppm 8.73 (s, 1H), 7.89 (d, J = 8.3 Hz, 1H), 7.80–7.70 (m, 3H), 7.61 (d, J = 8.2 Hz, 1H), 7.51–7.46 (m, 1H), 7.37 (d, J = 8.3 Hz, 2H), 5.80 (br.s, 1H), 3.99 (q, J = 6.6 Hz, 2H), 3.14 (t, J = 6.9 Hz, 2H). ¹³C (125 MHz, CDCl₃) δ ppm 159.3, 155.3, 152.5 (quintet, J = 17.6 Hz), 149.5, 143.2, 132.7, 129.1, 128.8, 126.3 (quintet, J = 4.5 Hz), 120.2, 114.9, 42.1, 34.9. ¹⁹F (470 MHz, CDCl₃) δ ppm 84.8 (pent, J = 150.0 Hz, 1F), 63.1 (d, J = 150.0 Hz, 4F). HRMS (EI), M + 1 calculated for C₁₆H₁₄F₅N₃S, 376.0901, found 376.0908.

[2-(4-Methylpheny)ethyl]quinazolin-4-ylamine, 10a

Following general procedure B, using 4-chloroquinazoline (5, 100 mg, 0.59 mmol), 2-(4-methylphenyl)ethan-1-amine (82 mg, 0.59 mmol) and 12 N HCl (11 µL, 0.14 mmol) gave 10a as white crystals (25 mg, 15%). mp 183.6–184.6 °C; ¹H (500 MHz, CDCl₃) δ ppm 8.71 (s, 1H), 7.86 (d, J = 8.2 Hz, 1H), 7.74 (ddd, J = 8.3, 7.0, 1.3 Hz, 1H), 7.56 (d, J = 7.8 Hz, 1H), 7.44 (ddd, J = 8.2, 7.0, 1.1 Hz, 1H), 5.79 (br.s, 1H), 3.95 (dt, J = 6.7, 5.8 Hz, 2H), 3.02 (t, J = 6.8 Hz, 2H), 2.37 (s, 3H). ¹³C (125 MHz, CDCl₃) δ ppm 159.3, 155.4, 149.3, 136.3, 135.7, 132.6, 129.5, 128.7, 128.6, 126.0, 120.3, 115.0, 42.3, 34.8, 21.1. HRMS (EI), M + 1 calculated for C₁₇H₁₇N₃, 264.1495, found 264.1496.

[2-(4-Methoxyphenyl)ethyl]quinazolin-4-ylamine, 11a

Following general procedure B, using 4-chloroquinazoline (5, 100 mg, 0.59 mmol), 2-(4-methoxyphenyl)ethylamine (91 mg, 0.59 mmol) and 12 N HCl (11 µL, 0.14 mmol) gave 11a as off-white crystals (74 mg, 43%). mp 172.3–172.9 °C; ¹H (500 MHz, CDCl₃) δ ppm 8.71 (s, 1H), 7.86 (d, J = 8.3 Hz, 1H), 7.74 (ddd, J = 8.3, 7.0, 1.3 Hz, 1H), 7.56 (d, J = 8.3 Hz, 1H), 7.45 (ddd, J = 8.2, 7.0, 1.2 Hz, 1H), 7.20 (dt, J = 8.6, 2.9 Hz, 2H), 6.90 (dt, J = 8.6, 2.9 Hz, 2H), 5.78 (s, 1H), 3.93 (dt, J = 6.8, 5.7 Hz, 2H), 3.83 (s, 3H), 3.01 (t, J = 6.8 Hz, 2H). ¹³C (125 MHz, CDCl₃) δ ppm 159.3, 158.4, 155.4, 149.4, 132.6, 130.8, 129.8, 128.6, 126.0, 120.3, 115.0, 114.2, 55.3, 42.4, 34.3. HRMS (EI), M + 1 calculated for C₁₇H₁₇N₃O, 280.1444, found 280.1458.

{2-[4-(tert-Butyl)phenyl]ethyl}quinazolin-4-ylamine, 12a

Following general procedure A, using 4-chloroquinazoline (5, 100 mg, 0.59 mmol), 2-[4-(tert-butyl)phenyl]ethylamine (110 mg, 0.59 mmol) and potassium carbonate (81 mg, 0.59 mmol) gave 12a as yellow crystals (58 mg, 29%). mp 149.5–151.3 °C; ¹H (500 MHz, CDCl₃) δ ppm 8.71 (s, 1H), 7.86 (dd, J = 8.4, 0.6 Hz, 1H), 7.74 (ddd, J = 8.4, 7.0, 1.3 Hz, 1H), 7.57 (dd, J = 8.3, 0.8 Hz, 1H), 7.45 (ddd, J = 8.2, 7.0, 1.2 Hz, 1H), 7.39 (dt, J = 8.3, 2.3 Hz, 2H), 7.22 (dt, J = 8.3, 2.2 Hz, 2H), 5.81 (br.s, 1H), 3.96 (dt, J = 6.8, 5.7 Hz, 2H), 3.04 (t, J = 6.8 Hz, 2H), 1.35 (s, 3H). ¹³C (125 MHz, CDCl₃) δ ppm 159.3, 155.5, 149.6, 149.5, 135.8, 132.5, 128.7, 128.5, 125.9, 125.7, 120.3, 115.0, 42.3, 34.7, 34.5, 31.4. HRMS (EI), M + 1 calculated for C₂₀H₂₃N₃, 306.1965, found 306.1967. These experimental results are consistent with a previous report [21].

Quinazolin-4-yl{2-[3-(trifluoromethoxy)phenyl]ethyl}amine, 13a

Following general procedure A, using 4-chloroquinazoline (5, 100 mg, 0.59 mmol), 3-(trifluoromethoxy)phenylamine (125 mg, 0.59 mmol) and potassium carbonate (84 mg, 0.59 mmol) gave 13a as white crystals (136 mg, 67%). mp 144.0–144.9 °C; ¹H (500 MHz, CDCl₃) δ ppm 8.72 (s, 1H), 7.87 (d, J = 7.8 Hz), 7.76 (ddd, J = 8.4, 7.0, 1.3 Hz, 1H), 7.60 (ddd, J = 8.2, 7.0, 1.2 Hz, 1H), 7.39–7.35 (m, 1H), 7.22–7.19 (m, 1H), 7.16–7.12 (m, 1H), 5.83 (br.s, 1H), 3.97 (dt, J = 6.9, 5.9 Hz, 2H), 3.09 (t, J = 6.9 Hz, 2H). ¹³C (125 MHz, CDCl₃) δ ppm 159.3, 155.4, 149.5 (q, J = 1.8 Hz), 141.3, 132.6, 130.1, 128.7, 127.3, 126.1, 121.4, 120.5 (q, J = 257.2 Hz), 120.2, 119.1, 114.9, 42.1, 35.1. ¹⁹F (470 MHz, CDCl₃) δ ppm -57.7 (s, 3F). HRMS (EI), M + 1 calculated for C₁₇H₁₄F₃N₃O, 306.1965, found 306.1967.

[2-(3-Ethoxyphenyl)ethyl]quinazolin-4-ylamine, 14a

Following general procedure A, using 4-chloroquinazoline (5, 151 mg, 0.92 mmol), 3-ethoxyphenylethylamine (155 mg, 0.92 mmol) and potassium carbonate (127 mg, 0.92 mmol) gave 14a as off-white crystals (211 mg, 78%). mp 148.5–148.8 °C; ¹H (500 MHz, CDCl₃) δ ppm 8.71 (s, 1H), 7.86 (d, J = 8.3 Hz, 1H), 7.74 (ddd, J = 8.3, 7.0, 1.2 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.44 (ddd, J = 8.0, 7.0, 1.1 Hz, 1H), 6.86 (d, J = 7.5 Hz, 1H), 6.83–6.79 (m, 2H), 5.79 (br.s, 1H), 4.02 (q, J = 7.0 Hz, 2H), 3.96 (dt, J = 6.7, 5.8 Hz, 2H), 1.42 (t, J = 7.0 Hz, 3H). ¹³C (125 MHz, CDCl₃) δ ppm 159.4, 159.3, 155.5, 149.5, 140.4, 132.5, 129.8, 128.7, 126.0, 121.0, 120.3, 115.1, 115.0, 112.6, 63.4, 42.1, 35.3, 14.8. HRMS (EI), M + 1 calculated for C₁₈H₁₉N₃O, 294.1601, found 294.1616.

Quinazolin-4-yl{2-[2-(trifluromethyl)phenyl]ethyl}amine, 15a

Following general procedure A outlined, using 4-chloroquinazoline (5, 100 mg, 0.59 mmol), 2-(2-trifluoromethylphenyl)ethylamine (117 mg, 0.59 mmol) and potassium carbonate (81 mg, 0.59 mmol) gave 15a as a yellow flaky solid (68 mg, 34%). mp 134.4–137.4 °C; ¹H (500 MHz, CDCl₃) δ ppm 8.66 (s, 1H), 8.12–8.05 (m, 1H), 7.95 (d, J = 8.3 Hz, 1H), 7.80–7.74 (m, 1H), 7.66 (d, J = 7.8 Hz, 1H), 7.54–7.49 (m, 1H), 7.49–7.46 (m, 1H), 7.37–7.32 (m, 1H), 4.04 (dt, J = 6.9, 6.7 Hz, 2H), 3.30 (t, J = 7.2 Hz, 2H). ¹³C (125 MHz, CDCl₃) δ ppm 159.9, 153.5, 145.7, 137.3, 133.6, 132.0, 131.7, 128.9 (q, J = 29.7 Hz), 126.9, 126.1 (q, J = 5.7 Hz), 126.8, 125.7, 124.5 (q, J = 254.0 Hz), 121.9, 114.4, 42.7, 31.8. ¹⁹F (470 MHz, CDCl₃) δ ppm -59.2 (s, 3F). HRMS (EI), M + 1 calculated for C₁₇H₁₄F₃N₃, 318.1213, found 318.1220. This compound appears in referenced patent and fungicidal activity was reported [17].

(6-Fluoroquinazolin-4-yl){2-[4-(trifluormethoxy)phenyl]ethyl}amine, 16a

Following general procedure A, using 4-chloro-6-fluoroquinazoline (16, 100 mg, 0.55 mmol), 2-[4-(trifluromethoxy)phenyl]ethylamine (112 mg, 0.55 mmol), and potassium carbonate (76 mg, 0.55 mmol) gave 16a as a white solid (99 mg, 51%). mp 171.6–172.6 °C; ¹H (500 MHz, CDCl₃) δ ppm 8.70 (s, 1H), 7.89 (dd, J = 9.1, 5.3 Hz, 1H), 7.52 (ddd, 9.1, 8.2, 2.7 Hz, 1H), 7.32–7.29 (m, 2H), 7.23–7.19 (m, 3H), 5.57 (br.s, 1H), 3.96 (dt, J = 7.0, 5.8 Hz, 2H), 3.08 (t, J = 7.0 Hz, 2H). ¹³C (125 MHz, CDCl₃) δ ppm 160.0 (d, J = 248.9 Hz), 159.0, 148.1 (q, J = 1.9 Hz), 146.5, 137.5, 131.4 (d, J = 8.4 Hz), 130.1, 122.2 (d, J = 24.4 Hz), 121.5, 121.3, 120.5 (q, J = 255.5 Hz), 115.2 (d, J = 7.9 Hz), 104.7 (d, J = 22.7 Hz), 42.4, 34.6. ¹⁹F (470 MHz, CDCl₃) δ ppm -57.9 (s, 3F), -112.3 (ddd, J = 5.4, 8.5, 8.5 Hz, 1F). HRMS (EI), M + 1 calculated for C₁₇H₁₃F₄N₃O, 352.1068, found 352.1078. This compound was shown to inhibit autophagy in referenced patent [22].

(6-Methylquinazolin-4-yl){2-[4-(trifluormethoxy)phenyl]ethyl}amine, 17a

Following general procedure A, using 4-chloro-6-methylquinazoline (17, 100 mg, 0.56 mmol), 2-[4-(trifluoromethoxy)phenyl]ethylamine (115 mg, 0.56 mmol), and potassium carbonate (77 mg, 0.56 mmol) gave 17a as an off-white solid (134 mg, 69%). mp 134.3–135.0 °C; ¹H (500 MHz, CDCl₃) δ ppm 8.67 (s, 1H), 7.77 (d, J = 8.5 Hz, 1H), 7.58 (dd, J = 8.5, 1.6 Hz, 1H), 7.36 (s, 1H), 7.31–7.27 (m, 2H), 5.76 (br.s, 1H), 3.94 (dt, J = 6.9, 5.9 Hz, 2H), 3.08 (t, J = 7.0 Hz, 2H), 2.50 (s, 3H). ¹³C (125 MHz, CDCl₃) δ ppm 158.9, 154.6, 148.0 (q, J = 1.8 Hz), 147.8, 137.8, 136.1, 134.5, 130.2, 128.5, 121.2, 120.5 (q, J = 256.9 Hz), 119.4, 114.8, 42.3, 34.7, 21.7. ¹⁹F (470 MHz, CDCl₃) δ ppm -57.9 (s, 3F). HRMS (EI), M + 1 calculated for C₁₈H₁₆F₃N₃O, 348.1318, found 348.1336.

(6-Methoxyquinazolin-4-yl){2-[4-(trifluormethoxy)phenyl]ethyl}amine, 18a

Following general procedure A, using 4-chloro-6-methoxyquinazoline (18, 100 mg, 0.51 mmol), 2-[4-(trifluoromethoxy)phenyl]ethylamine (105 mg, 0.51 mmol), and potassium carbonate (71 mg, 0.51 mmol) gave 18a as a white solid (93 mg, 50%). mp 144.7–145.2 °C; ¹H (500 MHz, CDCl₃) δ ppm 8.64 (s, 1H), 7.81 (d, J = 9.1 Hz, 1H), 7.41 (dd, J = 9.1, 2.5 Hz, 1 Hz), 7.32–7.26 (m, 2H), 7.20 (d, J = 8.1 Hz, 2H), 6.82 (d, J = 2.5 Hz, 1H), 5.65 (br.s, 1H), 3.95 (dt, J = 6.8, 6.2 Hz, 2H), 3.87 (s, 3H), 3.08 (t, J = 7.0 Hz, 2H). ¹³C (125 MHz, CDCl₃) δ ppm 158.6, 157.6, 153.4, 148.0 (q, J = 1.8 Hz), 144.9, 137.9, 130.3, 130.2, 123.6, 121.2, 120.5 (q, J = 258.4 Hz), 115.3, 99.8, 55.6, 42.3, 34.7. ¹⁹F (470 MHz, CDCl₃) δ ppm -57.9 (s, 3F). HRMS (EI), M + 1 calculated for C₁₈H₁₆F₃N₃O₂, 364.1267, found 364.1276.

(7-Fluoroquinazolin-4-yl){2-[4-(trifluormethoxy)phenyl]ethyl}amine, 19a

Following general procedure A, using 4-chloro-7-fluoroquinazoline (19, 100 mg, 0.55 mmol), 2-[4-(trifluoromethoxy)phenyl]ethylamine (112 mg, 0.55 mmol), and potassium carbonate (76 mg, 0.55 mol) gave 19a as off-white crystals (107 mg, 56%). mp 128.9–129.7 °C; ¹H (500 MHz, CDCl₃) δ ppm 8.69 (s, 1H), 7.60 (dd, J = 9.1, 5.6 Hz, 1H), 7.49 (dd, J = 9.8, 2.5 Hz, 1H), 7.32–7.26 (m, 2H), 7.24–7.17 (m, 3H), 5.74 (br.s, 1H), 3.96 (dt, J = 6.9, 5.9 Hz, 2H), 3.07 (t, J = 7.0 Hz, 2H). ¹³C (125 MHz, CDCl₃) δ ppm 165.0 (d, J = 253.3 Hz), 159.1, 156.4, 151.6 (d, J = 13.1 Hz), 148.1 (q, J = 2.0 Hz), 137.6, 130.1, 122.8 (d, J = 10.4 Hz), 121.3, 120.5 (q, J = 256.9 Hz), 115.8 (d, J = 24.9 Hz), 112.9 (d, J = 20.4 Hz), 111.8, 42.3, 34.6. ¹⁹F (470 MHz, CDCl₃) δ ppm -57.9 (s, 3F), -105.3 (ddd, J = 5.3, 9.2, 9.2 Hz, 1F). HRMS (EI), M + 1 calculated for C₁₇H₁₃F₄N₃O, 352.1068, found 352.1072.

(7-Chloroquinazolin-4-yl){2-[4-(trifluormethoxy)phenyl]ethyl}amine, 20a

Following general procedure A, using 4,7-dichloroquinazoline (20, 100 mg, 0.51 mmol), 2-[4-(trifluoromethoxy)phenyl]ethylamine (105 mg, 0.51 mmol), and potassium carbonate (78 mg, 0.56 mol) gave 20a as off-white flaky crystals (72 mg, 50%). mp 153.0–153.4 °C; ¹H (500 MHz, MeOD) δ ppm 8.47 (s, 1H), 8.03 (d, J = 8.6 Hz, 1H), 7.69 (d, J = 2.1 Hz, 1H), 7.49 (dd, J = 8.6, 2.1 Hz, 1H), 7.36 (dt, J = 8.6, 2.7 Hz, 2H), 7.21–7.16 (m, 2H), 3.86 (t, J = 7.3 Hz, 2H), 3.06 (t, J = 7.3 Hz, 2H). ¹³C (125 MHz, MeOD) δ ppm 159.8, 155.8, 149.3, 147.7 (q, J = 1.7 Hz), 138.6, 130.1, 126.4, 125.4, 123.9, 120.7, 120.5 (q, J = 255.1 Hz), 113.6, 42.2, 33.9. ¹⁹F (470 MHz, MeOD) δ ppm -59.6 (s, 3F). HRMS (EI), M + 1 calculated for C₁₇H₁₃ClF₃N₃O, 368.0772, found 368.0767.

(7-Bromoquinazolin-4-yl){2-[4-(trifluormethoxy)phenyl]ethyl}amine, 21a

Following general procedure A, using 4-chloro-7-bromoquinazoline (21, 75 mg, 0.30 mmol), 2-[4-(trifluoromethoxy)phenyl]ethylamine (62 mg, 0.30 mmol), and potassium carbonate (41 mg, 0.30 mmol) gave 21a as off-white crystals (52 mg, 42%). mp 151.8–152.8 °C; ¹H (500 MHz, CDCl₃) δ ppm 8.68 (s, 1H), 8.06 (s, 1H), 7.56 (s, 2H), 7.32–7.26 (m, 2H), 7.20 (d, J = 8.0 Hz, 2H), 6.18 (br.s, 1H), 3.97 (dt, J = 6.9, 6.1 Hz, 2H), 3.09 (t, J = 7.0 Hz, 2H). ¹³C (125 MHz, CDCl₃) δ ppm 159.4, 155.7, 149.4, 148.1 (q, J = 1.6 Hz), 137.4, 130.4, 130.1, 129.7, 127.4, 122.2, 121.3, 120.5 (q, J = 257.0 Hz), 113.4, 42.5, 34.5. ¹⁹F (470 MHz, CDCl₃) δ ppm -57.9 (s, 3F). HRMS (EI), M + 1 calculated for C₁₇H₁₃BrF₃N₃O, 412.0267, found 412.0246.

{2-[4-(Trifluoromethoxy)phenyl]ethyl}[7-(trifluoromethyl)quinazolin-4-yl]amine, 22a

Following general procedure A, using 7-(trifluoromethyl)-4-chloroquinazoline (22, 100 mg, 0.45 mmol), 2-[4-(trifluoromethoxy)phenyl]ethylamine (93 mg, 0.45 mmol), and potassium carbonate (69 mg, 0.50 mol) gave 22a as off-white crystals (82 mg, 55%). mp 149.7–152.3 °C; ¹H (500 MHz, MeOD) δ ppm 8.44 (d, J = 8.4 Hz, 1H), 8.41–8.37 (m, 1H), 8.01 (s, 1H), 7.86 (dd, J = 8.4, 1.5 Hz, 1H), 7.42 (dt, J = 8.6, 2.8 Hz, 2H), 7.31–7.25 (m, 2H), 3.22 (t, J = 7.7 Hz, 2H), 3.02 (t, J = 7.7 Hz, 2H). ¹³C (125 MHz, MeOD) δ ppm 160.4, 148.3 (q, J = 1.8 Hz), 147.3, 135.8, 135.8 (q, J = 32.9 Hz), 130.2, 127.7, 125.0, 123.4 (q, J = 272.4 Hz), 123.1 (q, J = 3.6 Hz), 122.8 (m), 121.2, 120.5 (q, J = 255.5 Hz), 119.5, 40.3, 32.4. ¹⁹F (470 MHz, MeOD) δ ppm -59.6 (s, 3F), -64.8 (s, 3F). HRMS (EI), M + 1 calculated for C₁₈H₁₃F₆N₃O, 402.1036, found 402.1025.

(7-Methoxyquinazolin-4-yl){2-[4-(trifluormethoxy)phenyl]ethyl}amine, 23a

Following general procedure A, using 7-(methoxy)-4-chloroquinazoline (23, 100 mg, 0.54 mmol), 2-[4-(trifluoromethoxy)phenyl]ethylamine (110 mg, 0.54 mmol), and potassium carbonate (82 mg, 0.59 mol) gave 23a as colorless crystals (68 mg, 51%). mp 158.0–159.0 °C; ¹H (500 MHz, MeOD) δ ppm 8.40 (s, 1H), 7.94 (d, J = 9.1 Hz, 1H), 7.36 (dt, J = 8.7, 2.8 Hz, 2H), 7.21–7.16 (m, 2H), 7.11 (dd, J = 9.1, 2.6 Hz, 1H), 7.08 (d, J = 2.5 Hz, 1H), 3.93 (s, 3H), 3.83 (t, J = 7.4 Hz, 2H), 3.05 (t, J = 7.4 Hz, 2H). ¹³C (125 MHz, MeOD) δ ppm 163.5, 159.6, 155.0, 150.6, 147.7 (q, J = 1.7 Hz), 138.7, 130.1, 123.5, 120.7, 120.6 (q, J = 255.1 Hz), 117.3, 109.1, 105.5, 54.7, 42.1, 34.1. ¹⁹F (470 MHz, MeOD) δ ppm -59.5 (3F). HRMS (EI), M + 1 calculated for C₁₈H₁₆F₃N₃O₂, 364.1267, found 364.1259.

(2-Methylquinazolin-4-yl){2-[4-(trifluormethoxy)phenyl]ethyl}amine, 24a

Following general procedure A, using 4-chloro-2-methylquinazoline (24, 100 mg, 0.56 mmol), 2-[4-(trifluoromethoxy)phenyl]ethylamine (115 mg, 0.56 mmol), and potassium carbonate (77 mg, 0.56 mmol) gave 24a as a pale yellow solid (153 mg, 79%). mp 124.4–127.2 °C; ¹H (500 MHz, CDCl₃) δ ppm 7.81 (d, J = 8.3 Hz, 1H), 7.74–7.68 (m, 1H), 7.60 (d, J = 8.1 Hz, H), 7.42–7.37 (m, 2H), 7.20 (d, J = 8.0 Hz, 2H), 5.91 (br.s, 1H), 3.96 (dt, J = 6.9, 6.0 Hz, 2H), 3.07 (t, J = 7.0 Hz, 2H), 2.69 (s, 3H). ¹³C (125 MHz, CDCl₃) δ ppm 164.3, 159.3, 149.4, 148.0 (q, J = 1.7 Hz), 137.8, 132.7, 130.2, 127.5, 125.3, 121.2, 120.5 (q, J = 256.9 Hz), 120.3, 112.8, 42.2, 34.7, 26.5. ¹⁹F (470 MHz, CDCl₃) δ ppm -57.9 (s, 3F). HRMS (EI), M + 1 calculated for C₁₈H₁₆F₃N₃O, 348.1318, found 348.1335.

(2-Cyclopropylquinazolin-4-yl){2-[4-(trifluoromethoxy)phenyl]ethyl}amine, 25a

Following general procedure A, using 4-chloro-2-cyclopropylquinazolne (25, 100 mg, 0.49 mmol), 2-[4-(trifluoromethoxy)phenyl]ethylamine (100 mg, 0.49 mmol) and potassium carbonate (67 mg, 0.48 mmol) gave 25a as a tan solid (119 mg, 65%). mp 205.1–205.5 °C; ¹H (500 MHz, MeOD) δ ppm 8.24 (d, J = 8.5 Hz, 1H), 8.02–7.97 (m, 1H), 7.74–7.68 (m, 2H), 7.36 (dt, J = 8.6, 2.7 Hz, 2H), 7.20 (d, J = 7.8 Hz, 2H), 3.99 (t, J = 7.1 Hz, 2H), 3.08 (t, J = 7.1 Hz, 2H), 2.23–2.16 (m, 1H), 1.44–1.32 (m, 4H). ¹³C (125 MHz, MeOD) δ ppm 166.5, 160.8, 147.9, 138.3, 137.9, 135.7, 130.2, 127.7, 123.3, 120.9, 120.5 (q, J = 255.2 Hz), 118.0, 111.9, 42.9, 33.8, 14.5, 11.2. ¹⁹F (470 MHz, MeOD) δ ppm -59.6 (s, 3F). HRMS (EI), M + 1 calculated for C₂₀H₁₈F₃N₃O, 374.1475, found 374.1493.

(7-Fluoro-2-methylquinazolin-4-yl){2-[4-(trifluormethoxy)phenyl]ethyl}amine, 26a

Following general procedure A, using 4-chloro-7-fluoro-2-methylquinazoline (26, 75 mg, 0.37 mmol), 2-[4-(trifluoromethoxy)phenyl]ethylamine (77 mg, 0.37 mmol), and potassium carbonate (52 mg, 0.37 mmol) gave 26a as a white solid (58 mg, 43%). mp 121.7–124.7 °C; ¹H (500 MHz, CDCl₃) δ ppm 14.78 (s, 1H), 10.62 (s, 1H), 9.10 (dd, J = 9.1, 5.2 Hz, 1H), 7.84 (dd, J = 8.6, 2.4 Hz, 1H), 7.30–7.22 (m, 1H), 7.19–7.13 (m, 2H), 7.06–7.02 (m, 2H), 4.14 (q, J = 6.9 Hz, 2H), 3.22 (t, J = 7.4 Hz, 2H), 2.77 (s, 3H). ¹³C (125 MHz, CDCl₃) δ ppm 166.1 (d, J = 259.4 Hz), 162.0, 160.0, 149.3 (q, J = 2.0 Hz), 140.7, 140.5 (d, J = 12.9 Hz), 129.8, 128.7 (d, J = 10.3 Hz), 127.4, 121.4, 120.4 (q, J = 257.1 Hz), 119.1, 116.9 (d, J = 23.9 Hz), 108.6, 104.7 (d, J = 25.6 Hz), 43.1, 34.6, 22.4. ¹⁹F (470 MHz, CDCl₃) δ ppm -57.8 (s, 3F), -98.3 (ddd, J = 4.8, 8.1, 8.1 Hz, 1F). HRMS (EI), M + 1 calculated for C₁₈H₁₅F₄N₃O, 366.1224, found 366.1199.

2.2. Biological Assessments

Mycobacterium bovis BCG and Mycobacterium tuberculosis strains were routinely cultured in Middlebrook 7H9 medium (Becton Dickson and Company Limited, Franklin Lakes, NJ, USA) supplemented with 0.05% tween 80, 0.5% glycerol, bovine serum albumin fraction V, D-glucose, and NaCl. M. tuberculosis clinical isolate N0145 was a gift from Sebastien Gagneux (Swiss Tropical and Public Health Institute at the University of Basel). Prior to use, bacteria cells were pelleted and resuspended in medium without glycerol and adjusted to a cell density of circa 10⁷ CFU/mL. The test compounds were tested in the presence or absence of Q203. Prior to the assay, Q203 or DMSO (solvent control) was added to the bacteria cultures. Q203 was used in excess (at 100 nM) to completely inhibit the function of the cytochrome bcc:aa₃, thereby revealing the activity of cyt-bd in the assay. The Q203 ATP IC₅₀ for M. bovis BCG, M. tuberculosis H37Rv, and M. tuberculosis N0145 are 2.6, 1.0, and 2.5 nM, respectively (Supplementary Materials Figure S61). The test compounds were tested in eight concentrations, starting at 25 µM, in two-fold dilutions (0.2–25.0 µM) in the presence or absence of 100 nM Q203. 1 µL of test compounds of varying concentrations was added to each well of 96-well white plates, and 100 µL of bacterial culture was subsequently added. The assay plates were incubated at 37 °C for 15 h, after which the BacTiter-Glo™ (Promega, Madison, WI, USA) reagent was added. Following a 12-min incubation, the luminescence of each plate was measured using a BioTek Cytation 3 Cell Imaging Multiple mode reader. IC₅₀ values were determined using GraphPad Prism 9.

3. Results and Discussion

Our initial efforts were to probe the effect of alteration and modification of the phenethylaniline moiety around a fixed quinazoline core. This was done by syntheses of 10 compounds and screening against M. bovis BCG and Mtb strains (H37Rv and N0145) to assess activity against mycobacterial cyt-bd (

Table 1. In vitro activity of phenethyl-quinazolin-4-yl-amines (3, 6a–15a) and control compounds bedaquiline (BDQ), ND-11992 (2), and aurachin D against three mycobacterial strains (M. bovis BCG, M. tuberculosis H37Rv, and M. tuberculosis N0145).

Compound	Clog p	Mol. Wt.	Replicating ATP IC50 (µM)
			BCG		H37Rv		N0145
			–Q203	+Q203	–Q203	+Q203	–Q203	+Q203
3	3.98	249.31	>25	11.2 ± 1.574	>25	27.2 ± 4.613	>25	6.9 ± 0.752
6a	4.86	317.31	>25	5.1 ± 0.501	>25	16.9 ± 3.758	>25	2.6 ± 0.581
7a	5.01	333.31	>25	2.1 ± 0.205	>25	11.0 ± 2.081	>25	1.1 ± 0.719
8a	4.69	283.76	>25	12.5 ± 1.952	>25	29.7 ± 5.336	>25	8.2 ± 0.808
9a	5.21	375.36	>25	3.9 ± 0.490	>25	14.5 ± 2.691	>25	2.3 ± 0.853
10a	4.48	263.34	>25	6.7 ± 1.116	>25	20.7 ± 3.624	>25	3.8 ± 0.813
11a	3.9	279.34	>25	13.7 ± 2.107	>25	>25	>25	7.1 ± 0.653
12a	5.81	305.42	>25	0.8 ± 0.144	>25	5.8 ± 0.616	>25	0.1 ± 0.017
13a	5.01	333.31	>25	7.1 ± 0.849	>25	22.5 ± 3.912	>25	3.8 ± 0.733
14a	4.43	293.36	>25	5.8 ± 0.867	>25	19.3 ± 5.703	>25	3.2 ± 0.204
15a	4.86	317.31	>25	3.9 ± 0.311	>25	17.0 ± 2.945	>25	3.2 ± 0.795
1	5.85	313.44	>25	5.8 ± 1.06	>25	18.9 ± 9.03	>25	8.5 ± 2.38
2	6.69	381.36	>25	0.7 ± 0.103	>25	6.3 ± 0.349	>25	0.3 ± 0.049
BDQ	7.25	555.52	0.17 ± 0.019	0.11 ± 0.026	0.06 ± 0.019	0.04 ± 0.024	0.05 ± 0.003	0.03 ± 0.001
Aurachin D	6.79	363.55	>25	2.9 ± 0.317	5.5 ± 1.107	7.3 ± 1.973	>25	1.5 ± 0.065

IC₅₀ values were determined by ATP depletion in the presence and absence of Q203 (see Section 2.2 for details). IC₅₀ values were determined using GraphPad Prism 9. The values reflected in the table represent the average and standard deviation, which were calculated from the IC₅₀ values of replicates from two experimental repeats. cLogP was calculated by Perkin Elmer ChemDraw Professional 16.0. Bedaquiline (BDQ), ND-11992 (2) and aurachin D were used as positive control compounds.

). A representative compound from this set (7a) served as the foundation toward the design of a second set of 11 compounds that focused on modification of the quinazoline core and were subsequently screened for cyt-bd activity (

Table 2. Screening of the second set of N-phenethylquinazolin-4-amines 16a–26a against cyt-bd of M. bovis BCG and M. tuberculosis H37Rv by ATP readout along with known inhibitors ND-11992 (2), aurachin D and bedaquiline (BDQ) as positive controls.

Compound	Clog p	Mol. Wt.	Replicating ATP IC50 (µM)
			BCG		H37Rv		N0145
			–Q203	+Q203	–Q203	+Q203	–Q203	+Q203
16a	5.19	351.3	>25	13.1 ± 0.651	>25	28.8 ± 5.054	>25	7.1 ± 0.207
17a	5.51	347.33	>25	7.5 ± 0.580	>25	22.3 ± 5.796	>25	3.4 ± 0.559
18a	5.41	363.33	>25	26.9 ± 1.231	>25	>25	>25	17.1 ± 0.220
19a	5.19	351.3	>25	0.8 ± 0.077	>25	7.6 ± 1.079	>25	0.2 ± 0.057
20a	5.76	367.75	>25	>25	>25	>25	>25	>25
21a	5.91	412.21	>25	6.7 ± 0.591	>25	19.5 ± 2.356	>25	4.9 ± 0.226
22a	5.95	401.31	>25	>25	>25	>25	>25	>25
23a	5.41	363.33	>25	>25	>25	>25	>25	>25
24a	5.51	347.33	12.2 ± 0.737	3.1 ± 0.369	7.5 ± 1.025	11.7 ± 1.252	8.2 ± 0.654	2.9 ± 0.140
25a	5.95	373.37	8.5 ± 1.529	4.0 ± 0.604	7.6 ± 1.026	11.7 ± 1.036	7.3 ± 1.052	2.1 ± 0.347
26a	5.69	365.32	27.6 ± 9.513	6.5 ± 0.751	23.6 ± 7.807	16.9 ± 3.822	32.7 ± 2.700	4.6 ± 0.219
2	6.69	381.36	>25	0.7 ± 0.103	>25	6.3 ± 0.349	>25	0.3 ± 0.049
BDQ	7.25	555.52	0.17 ± 0.019	0.11 ± 0.026	0.06 ± 0.019	0.04 ± 0.024	0.05 ± 0.003	0.03 ± 0.001
Aurachin D	6.79	363.55	>25	2.9 ± 0.317	5.5 ± 1.107	7.3 ± 1.973	>25	1.5 ± 0.065

IC₅₀ values were determined by ATP depletion in the presence and absence of Q203 (see 2.2 for details). IC₅₀ values were determined using GraphPad Prism 9. The values given in the table represent the average and standard deviation, which were calculated from the IC₅₀ values of replicates from two experimental repeats. cLogP was calculated by Perkin Elmer ChemDraw Professional 16.0. Bedaquiline (BDQ), ND-11992 (2) and aurachin D were used as positive control compounds.

).

3.1. Chemical Syntheses of the First Set of N-Phenethylquinazolin-4-Amines (6a–15a) and the Second Set of N-Phenethylquinazolin-4-Amines (16a–26a)

The first set of compounds (6a–15a) were prepared by reaction of 4-chlorquinazoline (5) with ten commercially available amines (6–15) using S_NAr conditions (basic or acidic, Scheme 1). These compounds were evaluated for their activity against cyt-bd in M. bovis BCG using an ATP readout (Table 1).

The second set of compounds (16a–26a) was prepared by reacting eight functionalized 4-chloroquinazolines (16–26) with 4-(trifluoromethoxy)benzylamine (7) using standard S_NAr conditions (Scheme 2). As with the first set, the activity was determined against cyt-bd in M. bovis BCG and M. tuberculosis H37Rv by ATP readout (Table 2).

3.2. Structure–Activity Relationship Studies

The structure–activity-relationship (SAR) studies revealed that the cyt-bd of M. bovis BCG was more sensitive to inhibition than M. tuberculosis H37Rv (Table 1). This mirrors the trend observed previously and is indicative that there is a greater expression of cyt-bd in the lab-adapted M. tuberculosis H37Rv strain relative to the M. bovis BCG or “fresh” Mtb clinical isolate N0145 [13]. This is further supported by the activity of aurachin D [7], a known synergistic cyt-bd inhibitor of Mtb, which had greater activity against M. bovis BCG in our ATP assay relative to M. tuberculosis H37Rv (2.9 µM vs. 5.5 µM, respectively; Table 1). In addition, based on our assay, compounds 6a–15a are all specific inhibitors of cyt-bd as they show no activity against wild-type (WT) M. bovis BCG or M. tuberculosis H37Rv (Mtb-H37Rv), whereas the positive control bedaquiline (BDQ), a selective ATP synthase inhibitor, remains very potent against both strains (Table 1). Interestingly, aurachin D showed the expected synergy with Q203 against mycobacterial strains but was unexpectedly active against wild-type Mtb-H37Rv (Table 1).

SAR trends revealed that the pendant aryl group can accommodate a variety of steric and electronic changes and retain good activity (Table 1). Evaluation of the compounds with electron withdrawing substituents 6a (p-CF₃), 7a, (p-OCF₃), 8a (p-Cl), 9a (p-SF₅), indicated that 7a is the most active compound with IC₅₀ values of 2.1 µM against M. bovis BCG and 11 µM against Mtb H37Rv. We explored electron withdrawing groups at the meta- and ortho-positions of the pendant aryl group by comparing compound sets 7a (para-OCF₃) and 13a (meta-OCF₃) and 6a (para-CF₃) and 15a (ortho-CF₃). We found that the meta-OCF₃ compound (13a) position was less active compared to the para-OCF₃ (17a). Whereas incorporation of substituents in the ortho-position gave slightly improved potency against M. bovis but equal activity (within error) in Mtb-H37Rv as demonstrated by comparing 6a and 15a. When evaluating the compounds with electron donating substituents, 10a (p-CH₃), 11a (p-OCH₃), and 12a (p-tert-butyl), 12a was found to be significantly more active with IC₅₀ values of 0.8 µM against M. bovis BCG and 5.8 µM against Mtb H37Rv. That is an improvement over thieno[3,2-d]pyrimidine (1) and activity on par with the optimized quinazoline ND-11992 (2). This is particularly interesting as it follows the Topliss aromatic substitution decision tree pattern [23] where one would first evaluate the phenyl (3), then para-chloro (8a) and if equally potent (as it is in our assays) then prepare the tert-butyl (12a) to derive the most potent compound. While the Topliss approach can often lead to compounds with improved activity it also directs the syntheses of compounds which often have metabolic liabilities (CH₃, OCH₃, t-butyl) or toxic pharmacophores (NH₂, NO₂, I). For this reason, we chose 7a which bears a para-trifluoromethoxy group (which is prevalent in various anti-TB drugs such as delamanid, pretomanid, and telacebec) as the basis for our second set of compounds (Table 2) because it possesses good activity (IC₅₀′s of 2.1 and 11 μM against BCG and Mtb-H37Rv, respectively).

The SAR trends for alteration of the quinazoline core suggest an interplay between both steric and electronic effects (Table 2). For instance, the 6-position was probed with three substituents-fluoro (16a), methyl (17a) and methoxy (18a)—which resulted in a wide spectrum of activity. The 6-methyl analog (17a) was the most active against M. bovis BCG (IC₅₀ of 7.5 µM) but weakly active against Mtb H37Rv (IC₅₀ of 22 µM). Whereas the fluoro (16a) and methoxy (18a) analogs had diminished activity against M. bovis BCG (IC₅₀ of 13 µM and 27 µM, respectively) and both were >25 µM against Mtb H37Rv. The 7-position was explored with five different substituents-fluoro (19a), chloro (20a), bromo (21a), trifluoromethoxy (22a) and methoxy (23a)—resulting in only two active compounds (19a and 21a). The 7-fluoro analog (19a) displayed good activity against both mycobacterial strains (IC₅₀ of 0.8 µM and 7.6 µM; respectively) and the 7-bromo (21a) showed much better activity against M. bovis BCG than Mtb H37Rv (IC₅₀ of 7 µM and 20 µM; respectively). The most interesting SAR was observed with substitution of the quinazoline 2-position with methyl (24a) and cyclopropyl (25a) groups. Both compounds displayed good activity range (3–12 µM) with or without cyt-bcc:aa₃ inhibition, suggesting possible dual modes of action. Lastly, one compound (26a) bore both 7-fluoro and 2-methyl quinazoline substituents which biased activity back towards cyt-bd (IC₅₀ of 7 and 17 µM with Q203, and 28 µM and 24 µM without Q203; respectively)

Our previous work had shown that the laboratory adapted Mtb H37Rv strain over expresses cyt-bd resulting in higher IC₅₀ values than those observed using a clinical Mtb isolate [24]. These higher IC₅₀ values render compound ranking more challenging. To gain greater insight on the activity of these compounds, they were re-screened against the clinical Mtb isolate N0145 strain. As observed previously with the thieno[3,2-d]pyrimidin-4-amine class of cyt-bd oxidase inhibitors [13], these compounds displayed greatly improved activity against the Mtb clinical isolate likely due to lower expression of cyt-bd within clinical isolates as compared to lab-adapted strains like H37Rv [12]. Two compounds, 7a and 12a, from the first set were more active than or comparable in activity to aurachin D (0.1 and 1.1 µM vs. 1.5 µM, respectively). While six additional compounds (6a, 9a, 10a, 13a–15a) had IC₅₀ values below 5 µM against N0145-Mtb. In the second set of compounds, only 19a had activity superior to aurachin D (0.2 µM vs. 1.5 µM, respectively) and four additional compounds (17a, 21a, 24a–26a) had activity below 5 µM. The two most potent compounds, 12a and 19a, had slightly improved activity relative to ND-11992 which was active in the murine infection model of tuberculosis. However, preliminary metabolic stability assessment of these compounds against human and rat microsomes indicate much more rapid metabolism than that of ND-11992 (data not shown).

4. Conclusions

The described N-phenethylquinazolin-4-amine class of compounds are mycobacterial cyt-bd inhibitors. Through focused SAR, the activity of the hit compound 3 (IC₅₀ = 6.9 µM against Mtb N0145) was improved with 14 of the 22 analogs (64%) and two, 12a and 19a, had sub-micromolar activity (0.1 and 0.2 µM against Mtb N0145, respectively). Future work includes identification of more efficacious compounds through additional SAR around the quinazoline core. These results will be reported in due course.

5. Patents

US Provisional Application No. 62/783,984.