2.1. Assays

2.2. Probe Chemical Characterization

A. Probe Chemical Structure, Physical Parameters and Probe Properties

SID 99437306
CID 46931017
ML205

Molecular Formula: C₁₈H₁₇BrClNO₃

Molecular Weight: 410.69 [g/mol]

Exact Mass: 409.01 [g/mol]

CLogP: 6.74

Topological Polar Surface Area: 66

Solubility (PBS buffer, pH 7.4): 25.6 μg/mL

Stability (% remaining after 48 h): 100%

Purity (LCMS, 215 nm): 100%

Melting Point: 273–276 °C

Physical State: white solid

B. Structure Verification and Purity: ¹H NMR, ¹³C NMR, LCMS and HRMS

Proton and carbon NMR data for ML205 SID 99437306, CID 46931017: Detailed analytical methods and instrumentation are described in section 2.3, entitled “Probe Preparation” under general experimental and analytical details. The numerical experimental proton and carbon data are represented below, along with the associated spectra.

Figure 3Proton spectra for ML205, SID 99437306, CID 46931017

ML205, SID 99437306, CID 46931017 PROTON NMR DATA: ¹H NMR (400 MHz, DMSO-d₆) δ 13.27 (s, 1H), 8.98 (s, 1H), 8.28 (s, 1H), 7.90 (d, J = 8.5 Hz, 2H), 7.60 (d, J = 8.6 Hz, 2H), 1.32 (s, 9H).

Figure 4Carbon spectra for ML205, SID 99437306, CID 46931017

ML205, SID 99437306, CID 46931017 CARBON NMR DATA: ¹³C NMR (100 MHz, DMSO-d₆) δ 167.9, 164.7, 155.1, 141.1, 135.8, 135.7, 131.6, 127.1, 125.7, 122.3, 120.0, 113.2, 34.7, 30.9.

LCMS and HRMS data for ML205, SID 99437306, CID 46931017: Detailed analytical methods and instrumentation are described in section 2.3, entitled “Probe Preparation” under general experimental and analytical details. The numerical experimental LCMS and HRMS data are represented below, along with the associated spectra.

Figure 5LCMS purity data at 215 nm for ML205, SID 99437306, CID 46931017; LCMS retention time: 2.597 min; purity at 215 nm = 100%

User Chromatogram Peak List

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Peak #	Compound Name	RT	Height	Height %	Area	Area %	Area Sum %	Width
1	Cpd 1: C18 H17 Br Cl N O3	2.597	1119.43	100	1266.12	100	100	0.017

Figure 6HRMS data for ML205, SID 99437306, CID 46931017; HRMS m/z calculated for C₁₈H₁₇BrClNO3 [M⁺ - 1] 410.69, found 409.9995

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Compound Label	RT	Mass	Abund	Formula	Tot Mass	Purity Value
Cpd 1: C18 H17 Br Cl N 03	2.592	409.0089	191057	C18 H17 Br Cl N O3	409.008	100

E. Synthetic Route

The probe and analogs were generally synthesized by the method shown (Fig. 8). Commercial acid chlorides were used when available. Otherwise, substituted benzoic acids 1 were converted to the corresponding acid chlorides 2 using oxalyl chloride. The crude acid chlorides were then treated with substituted aminobenzoates 3 in the presence of pyridine to afford amidobenzoates 4. Hydrolysis of the carboxylic esters provided the desired acids 5. Specific experimental details for the probe are detailed in section 2.3 below, entitled, “Probe Preparation.”

Figure 8

General synthetic route for probe and analogue synthesis. Reagents: (a) (COCl)₂, DMF, DCM, 0 °C to rt, 2h; (b) pyridine, THF, 50 °C, 18 h; (c) LiOH, THF/H₂O, rt, 3h.

If a requisite substituted benzoic acid or acid chloride was unavailable, the desired intermediates were prepared by Molander-type coupling of a halogenated benzoate or by Friedel-Crafts alkylation of the appropriate benzoate [13, 14].

F. Submission of Five Related Analogues to the MLSMR

Five analogues have been fully characterized and prepared in sufficient quantity for submission to the MLSMR. The five selected analogs and associated data are shown in Figure 9.

Figure 9

Five analogues selected for submission to support probe ML205, SID 99437306, CID 46931017 and associated data.

2.3. Probe Preparation

General experimental and analytical details: ¹H and ¹³C NMR spectra were recorded on a Bruker AM 400 spectrometer (operating at 400 and 101 MHz respectively) in CDCl₃ with 0.03% TMS as an internal standard or DMSO-d₆. The chemical shifts (δ) reported are given in parts per million (ppm) and the coupling constants (J) are in Hertz (Hz). The spin multiplicities are reported as s = singlet, br. s = broad singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublet and m = multiplet. The LCMS analysis was performed on an Agilent 1200 RRL chromatograph with photodiode array UV detection and an Agilent 6224 TOF mass spectrometer. The chromatographic method utilized the following parameters: a Waters Acquity BEH C-18 2.1 × 50mm, 1.7 μm column; UV detection wavelength = 214 nm; flow rate = 0.4ml/min; gradient = 5 – 100% acetonitrile over 3 minutes with a hold of 0.8 minutes at 100% acetonitrile; the aqueous mobile phase contained 0.15% ammonium hydroxide (v/v). The mass spectrometer utilized the following parameters: an Agilent multimode source which simultaneously acquires ESI+/APCI+; a reference mass solution consisting of purine and hexakis(1H, 1H, 3H-tetrafluoropropoxy) phosphazine; and a make-up solvent of 90:10:0.1 MeOH:Water:Formic Acid which was introduced to the LC flow prior to the source to assist ionization. The melting point was determined on a Stanford Research Systems OptiMelt apparatus.

Probe compound Ml205, SID 99437306, CID 46931017 was prepared by coupling 2-amino-5-bromo-4-chlorobenzoate with 4-tert-butylbenzoyl chloride, followed by hydrolysis (Figure 10).

Figure 10

Assembly of probe compound ML205, SID 99437306, CID 46931017. Reagents: a) pyridine, THF, 50 °C, 18 h, 68%; b) LiOH, THF/H₂O, rt, 3 h, 93%.

Methyl 2-(4-tert-butylbenzamido)-5-bromo-4-chlorobenzoate. Methyl 2-amino-5-bromo-4-chlorobenzoate (Ace Synthesis, CAS # 765211-09-4) and 4-tert-butylbenzoyl chloride (Sigma-Aldrich, CAS # 1710-98-1) were obtained commercially. In a 40 mL vial was combined sequentially methyl 2-amino-5-bromo-4-chlorobenzoate (0.21 g, 0.81 mmol, 1 eq.), 4-tert-butylbenzoyl chloride (0.22 mL, 1.21 mmol, 1.5 eq.) and pyridine (0.098 mL, 1.21 mmol, 1.5 eq.) in THF (2 mL). The resulting colorless solution was stirred under nitrogen at 50 °C for 18 hours. The reaction was removed from heat, cooled to rt, diluted with EtOAc (5 mL) and quenched with saturated aqueous NaHCO₃ (5 mL). After extracting with EtOAc (2 × 5 mL), the crude material was adsorbed onto silica. Purification by silica gel chromatography (0–25% EtOAc:Hex ramp over 20 min) afforded the desired product methyl 2-(4-tert-butylbenzamido)-5-bromo-4-chlorobenzoate as a white solid (0.26 g, 0.55 mmol, 68% yield). ¹H NMR (400 MHz, CDCl₃) δ11.95 (s, 1H), 9.22 (s, 1H), 8.31 (s, 1H), 7.96 (d, J = 8.6 Hz, 2H), 7.55 (d, J = 8.6 Hz, 2H), 3.99 (s, 3H), 1.36 (s, 9H).

Probe

SID 99437306

CID 46931017

ML205

PROBE: 2-(4-tert-butylbenzamido)-5-bromo-4-chlorobenzoic acid. (ML205, SID 99437306, CID 46931017): To a mixture of methyl 2-(4-tert-butylbenzamido)-5-bromo-4-chlorobenzoate (0.26 g, 0.55 mmol, 1 eq.) and LiOH (0.092 g, 3.84 mmol, 7 eq.) in water (2 mL) was added THF (2 mL) to give a colorless solution. After stirring the solution for 3 h at rt, the reaction was acidified with 1.0 M aq. HCl to pH 2 – 3 and then extracted with CH₂Cl₂ (2 × 5 mL). The separated organic extracts were combined, dried (MgSO₄), filtered and concentrated to produce the desired 2-(4-tert-butylbenzamido)-5-bromo-4-chlorobenzoic acid as a white solid (0.23 g, 0.57 mmol, 93 % yield). ¹H NMR (400 MHz, DMSO-d₆) δ 13.27 (s, 1H), 8.98 (s, 1H), 8.28 (s, 1H), 7.90 (d, J = 8.5 Hz, 2H), 7.60 (d, J = 8.6 Hz, 2H), 1.32 (s, 9H). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.9, 164.7, 155.1, 141.1, 135.8, 135.7, 131.6, 127.1, 125.7, 122.3, 120.0, 113.2, 34.7, 30.9. LCMS retention time: 2.592 min, purity at 215 nm = 100%. HRMS m/z calculated for C₁₈H₁₇BrClNO3 [M⁺ - 1] 410.69, found 409.99. White solid, mp 273 – 276 °C.

Analogue

SID 99222690

CID 45436131

ANALOGUE 1: 4-chloro-2-(4-isopropylbenzamido)benzoic acid (SID 99222690/CID 45436131): Isolated 42 mg, yield 97% of 4-chloro-2-(4-isopropylbenzamido)benzoic acid as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 12.24 (s, 1H), 8.84 (d, J = 2.1 Hz, 1H), 8.06 (d, J = 8.6 Hz, 1H), 7.88 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 8.3 Hz, 2H), 7.27 (dd, J₁ = 8.6 Hz, J₂ = 2.2 Hz, 1H), 2.99 (m, J = 6.9 Hz, 1H), 1.24 (d, J = 7.0 Hz, 6H). ¹³C NMR (125 MHz, DMSO-d₆) δ169.37, 164.89, 153.33, 142.28, 138.73, 133.00, 131.66, 127.24, 127.04, 122.72, 119.06, 115.03, 33.43, 23.55. LCMS retention time: 2.327 min, purity at 215 nm = 98.2%. HRMS m/z calculated for C₁₇H₁₆ClNO₃ [M⁺ + 1] 317.0818, found 318.0891. White solid, mp 217 – 220 °C.

Analogue

SID 99380766

CID 46916165

ANALOGUE 2: 5-bromo-4-chloro-2-(4-methoxybenzamido)benzoic acid (SID 99380766/CID 46916165): Isolated 132 mg, yield 98 % of 5-bromo-4-chloro-2-(4-methoxybenzamido)benzoic acid as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ12.10 (s, 1H), 8.98 (s, 1H), 8.26 (s, 1H), 7.90 (d, J = 8.8 Hz, 2H), 7.13 (d, J = 8.8 Hz, 2H), 3.86 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ 168.22, 164.38, 162.63, 141.22, 138.48, 135.48, 129.08, 125.82, 120.81, 117.02, 114.35, 113.77, 55.56. LCMS retention time: 2.345 min; purity at 215 nm = 100%. HRMS m/z calculated for C₁₈H₁₈ClNO₃ [M⁺ + 1] 382.9565, found 383.9633. White solid, mp 258 – 261 °C.

Analogue

SID 99460893

CID 46947875

ANALOGUE 3: 2-(4-tert-butylbenzamido)-4,5-dichlorobenzoic acid. (SID 99437306/CID 46947875 was prepared according to the following scheme:

Reagents: a) NaOH, NaOCl, MeOH, 80 °C, 2 h, 60%; b) 4-tert-butylbenzoyl chloride, TMS-diazomethane, CH₂Cl₂/MeOH, rt 30 min, 87%; c) CH₃CN, 150 °C, 30 min, MW, 32%; d) LiOH, THF/H₂O, rt, 3 h, 96%.

2-Amino-4,5-dichlorobenzoic acid. The starting material 4,5-dichlorophthalimide (CAS# 15997-89-4) was purchased from Sigma-Aldrich. In a vial was added the 4,5-dichlorophthalimide (1.93 g, 8.92 mmol, 1 eq.) and MeOH (10 mL). The 10.0 M NaOH (3.6 mL, 35.6 mmol, 4 eq.) and 10 % w/w NaOCl solution (6.08 mL, 9.81 mmol, 1.1 eq) were added and the reaction stirred for 2 hours at 80 °C. The reaction was then cooled to rt and was poured into 220 mL of 1.0 M HCl. After extracting with CHCl₃ (3 × 200 mL) the crude material was adsorbed onto silica. Purification by silica gel chromatography (0–100% EtOAc:Hex ramp over 20 min) afforded the desired product 2-amino-4,5-dichlorobenzoic acid as a white solid (1.09 g, 5.30 mmol, 60% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 7.77 (s, 1H), 7.10 (s, 1H).

Methyl 2-amino-4,5-dichlorebenzoate. To a vial was added 2-amino-4,5-dichlorobenzoic acid (0.66 g, 3.21 mmol, 1 eq.) and DCM/MeOH (3 mL/2 mL). A solution of 2.0 M TMS-diazomethane in hexanes (2.89 mL, 5.78 mmol, 1.8 eq.) was added dropwise and the reaction stirred for 30 minutes at rt. The crude material was then adsorbed onto silica. Purification by silica gel chromatography (0–20% EtOAc:Hex ramp over 20 min) afforded the desired product methyl 2-amino-4,5-dichlorobenzoate as a white solid (0.61 g, 2.80 mmol, 87% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.92 (s, 1H), 7.79 (s, 1H), 5.77 (brs, 2H), 3.87 (s, 3H).

Methyl 2-(4-tert-butylbenzamido)-4,5-dichlorobenzoate. To a microwave vial was added the methyl 2-amino-4,5-dichlorobenzoate (0.11 g, 0.48 mmol, 1 eq.), 4-tert-butylbenzoyl chloride (CAS# 1710-98-1) (0.096 mL, 0.527 mmol, 1.1 eq.) and MeCN (2.0 mL). The vial was sealed and the reaction stirred in a microwave at 150 °C for 30 minutes. The reaction was then cooled to rt and was diluted with EtOAc (10 mL) and washed with saturated NaHCO₃ (10 mL). The crude material was then adsorbed onto silica. Purification by silica gel chromatography (0–15% EtOAc:Hex ramp over 10 min) afforded the desired product methyl 2-(4-tert-butylbenzamido)-4,5-dichlorobenzoate as a white solid (0.059 g, 0.16 mmol, 32% yield). ¹H NMR (400 MHz, CDCl₃) δ 11.94 (brs, 1H), 9.22 ( s, 1H), 8.15 (s, 1H), 7.96 (d, J = 8.6 Hz, 2H), 7.55 (d, J = 8.6 Hz, 2H), 3.99 (s, 3H), 1.36 (s, 9H).

Analogue

SID 99460893

CID 46947875

ANALOGUE 3: 2-(4-tert-butylbenzamido)-4,5-dichlorobenzoic acid. (SID 99437306/CID 46947875) To a mixture of methyl 2-(4-tert-butylbenzamido)-4,5-dichlorobenzoate (0.019 g, 0.050 mmol, 1 eq.) and LiOH (0.010 g, 0.35 mmol, 7 eq.) in water (1 mL) was added THF (1 mL) to give a colorless solution. After stirring the solution for 3 h at rt, the reaction was acidified with 1.0 M aq. HCl to pH 2 – 3 and then extracted with CH₂Cl₂ (2 × 5 mL). The separated organic extracts were combined, dried (MgSO₄), filtered and concentrated to produce the desired 2-(4-tert-butylbenzamido)-4,5-dichlorobenzoic acid as a white solid (0.017 g, 0.048 mmol, 96% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 12.17 (brs, 1H), 9.00 (s, 1H), 8.16 (s, 1H), 7.88 (d, J = 8.6 Hz, 2H), 7.63 (d, J = 8.6 Hz, 2H), 1.33 (s. 9H). ¹³C NMR (125 MHz, DMSO-d₆) δ 168.2, 164.9, 155.7, 140.6, 136.4, 132.4, 131.0, 127.0, 126.0, 124.5, 121.1, 117.2, 34.8, 30.9. LCMS retention time: 2.514 min; Purity at 215 nm = 97.9%. HRMS m/z calculated for C₁₈H₁₇Cl₂NO₃ [M⁺ - 1] 365.06, found 364.05. White solid, mp 242 – 245 °C.

Analogue

SID 99380773

CID 46916168

ANALOGUE 4: 4-(tert-butyl)-N-(5-chloro-2-(1H-tetrazol-5-yl)phenyl)benzamide (SID 99380773/CID 46916168): Isolated 67 mg, 61% yield of 4-(tert-butyl)-N-(5-chloro-2-(1H-tetrazol-5-yl)phenyl)benzamide as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 11.73 (s, 1H), 8.78 (d, J = 2.2 Hz, 1H), 8.04 (d, J = 8.5 Hz, 1H), 8.02 – 7.96 (m, 2H), 7.67 – 7.60 (m, 2H), 7.45 (dd, J = 8.5, 2.2 Hz, 1H), 1.34 (s, 9H). ¹³C NMR (101 MHz, DMSO-d₆) δ 165.12, 155.47, 138.43, 136.25, 131.18, 129.98, 127.21, 125.77, 123.77, 120.63, 111.64, 97.71, 34.78, 30.83. LCMS retention time: 2.514 min; LCMS purity at 215 nm = 100%. HRMS m/z calculated for C₁₈H₁₈ClN₅O [M⁺+1]: 356.1273, found 356.1264. White solid, mp 231–234 °C.

Reagents: (a) NaN₃, Et₃N•HCl, PhMe, MWl, 110 °C

The requisite 5-chloro-2-(1H-tetrazol-5-yl)aniline precursor to analogue 4 (CID 46916168; SID 99380773) was prepared according to a previously reported procedure [15].

Analogue

SID 103023816

CID 49766619

ANALOGUE 5: 2-(4-tert-butylbenzamido)-4-chloro-5-fluorobenzoic acid (SID 103023816/CID 49766619): This analogue was prepared according to the scheme shown below:

Reagents and conditions: a) acetic anhydride, acetic acid, reflux, 2 h; b) SOCl₂, CH₂Cl₂, 45 °C for 2 h, 51% for two steps; c) 3-chloro-4-fluoroaniline, KHCO₃, CH₂Cl₂, −10 °C - rt, 20 h; d) hydroxylamine-HCl, EtOH/H₂O, reflux, 2 h, 45% for two steps; e) H₂SO₄, 80°C, 3 h; f) NaOH, H₂O₂, 80 °C, 3 h, 54% for two steps; g) TMS-diazomethane, CH₂Cl₂/MeOH, rt, 30 min, 82%; h) 4-tert-butylbenzoyl chloride, CH₃CN, 150 °C, 50 min, MW, 56%; i) LiOH, THF/H₂O, rt, 3 h, 94%.

2,2-diacetoxyacetyl chloride. The requisite starting materials glyoxylic acid monohydrate was purchased from Sigma-Aldrich (CAS # 563-96-2). To a vial was added glyoxylic acid monohydrate (2.05 g, 22.3 mmol, 1 eq.), acetic ahydride (21.0 mL, 223 mmol, 10 eq.) and acetic acid (5 mL). The colorless mixture was heated under reflux for 2 hr. The reaction was then cooled to rt, and the solvents were removed by rotary evaporation. The remaining volatiles were azeotroped with toluene and the crude material was solubilized in CH₂Cl₂. Thionyl chloride (5.68 mL, 78.1 mmol, 3.5 eq.) was added to the mixture and the reaction stirred at 45 °C for 2 hr. The reaction was cooled to rt, the volatiles were removed by rotary evaporation and the remaining material was isolated as the desired product, 2,2-diacetoxyacetyl chloride, as a brown oil (2.21 g, 11.4 mmol, 51% yield). ¹H NMR (400 MHz, CDCl₃) δ 6.89 (s, 1H), 2.19 (s, 6H).

N-(3-chloro-4-fluorophenyl)-2-(hydroxyimino)acetamide. The requisite starting material 3-chloro-4-fluoroaniline was purchased from Sigma-Aldrich (CAS # 367-21-5). To a vial was added the 3-chloro-4-fluoroaniline (2.71 g, 18.6 mmol, 1 eq.), potassium hydrogen-carbonate (9.31 g, 93.0 mmol, 5 eq.) in CH₂Cl₂ (35 mL), and the reaction was cooled to −10 °C. The 2,2-diacetoxyacetyl chloride (4.69 g, 24.2 mmol, 1.3 eq.) in CH₂Cl₂ (15 mL) was added dropwise and the reaction was allowed to warm to rt and stirred for 20 h. The reaction was filtered and rinsed with CH₂Cl₂ and the filtrate was concentrated. The crude material was then dissolved in EtOH:H₂O (50 mL:20 mL) and hydroxylamine-HCl (6.46 g, 93.0 mmol, 5 eq.) was added. The reaction stirred at reflux for 2 hr, cooled to rt, and the EtOH was removed by rotary evaporation to form a precipitate. The solid was filtered and rinsed with water (50 mL) and dried on the filter to produce N-(3-chloro-4-fluorophenyl)-2-(hydroxyimino)acetamide as an off-white solid (1.83 g, 8.43 mmol, 45% yield). ¹H NMR (400 MHz, CDCl₃) δ12.25 (s, 1H), 10.38 (s, 1H), 7.99 (dd, J₁ = 6.8 Hz, J₂ = 2.6 Hz, 1H), 7.65 – 7.60 (m, 2H), 7.40 (t, J = 9.1 Hz, 1H).

2-Amino-4-chloro-5-fluorobenzoic acid. To a vial was added the N-(3-chloro-4-fluorophenyl)-2-(hydroxyimino)acetamide and concentrated sulfuric acid (30 mL). The reaction then stirred for 3 hr at 80°C, cooled to rt and poured onto ice. The product was extracted with CHCl₃ (3 × 200 mL) and the organic layers were combined and concentrated by rotary evaporation. The crude material was solubilized in aqueous 2.5 M NaOH (20 mL), 30 % w/w hydrogen peroxide (2.50 mL, 24.3 mmol, 3 eq.) was added and the reaction stirred at 80°C for 3 h, cooled to rt and was acidified carefully with concentrated HCl to pH 3. The resulting precipitate was filtered and rinsed with water to produce 2-amino-4-chloro-5-fluorobenzoic acid as a reddish-brown solid (0.86 g, 4.60 mmol, 54% yield). ¹H NMR (400 MHz, DMSO-d₆) δ7.55 (d, J = 10.3 Hz, 1H), 6.93 (d, J = 6.5 Hz, 2H).

Methyl 2-amino-4-chloro-5-fluorobenzoate. To a vial was added 2-amino-4-chloro-5-fluorobenzoic acid (0.046 g, 0.24 mmol, 1 eq.) and CH₂Cl₂/MeOH (3 mL/2 mL). A solution of 2.0 M TMS-diazomethane in hexanes (0.22 mL, 0.44 mmol, 1.8 eq.) was added dropwise, and the reaction stirred for 30 minutes at rt. The crude material was then adsorbed onto silica. Purification by silica gel chromatography (0–20% EtOAc:Hex ramp over 20 min) afforded the desired product methyl 2-amino-4-chloro-5-fluorobenzoate as a white solid (0.041 g, 0.20 mmol, 82 % yield). ¹H NMR (400 MHz, CDCl₃) δ7.62 (d, J = 9.8 Hz, 1H), 6.71 (d, J = 6.1 Hz, 1H), 3.87 (s, 3H).

Methyl 2-(4-tertbutylbenzamido)-4-chloro-5-fluorobenzoate. To a microwave vial was added the methyl 2-amino-4-chloro-5-fluorobenzoate (0.076 g, 0.37 mmol, 1 eq.), 4-tert-butylbenzoyl chloride (CAS# 1710-98-1) (0.082 mL, 0.45 mmol, 1.2 eq.) and MeCN (2.0 mL). The vial was sealed and the reaction stirred in a microwave at 150 °C for 50 minutes. The reaction was then cooled to rt and was diluted with EtOAc (10 mL) and washed with saturated aqueous NaHCO₃ (10 mL). The crude material was then adsorbed onto silica. Purification by silica gel chromatography (0–15% EtOAc:Hex ramp over 10 min) afforded the desired product methyl 2-(4-tertbutylbenzamido)-4-chloro-5-fluorobenzoate as a white solid (0.076 g, 0.21 mmol, 56% yield). ¹H NMR (400 MHz, CDCl₃) δ 11.89 (s, 1H), 9.17 (d, J = 7.1 Hz, 1H), 7.96 (d, J = 8.7 Hz, 2H), 7.84 (d, J = 9.5 Hz, 1H), 7.55 (d, J = 8.6 Hz, 2H), 3.98 (s, 3H), 1.36 (s, 9H).

Analogue

SID 103023816

CID 49766619

ANALOGUE 5: 2-(4-tert-butylbenzamido)-4-chloro-5-fluorobenzoic acid (SID 103023816/CID 49766619): To a mixture of methyl 2-(4-tertbutylbenzamido)-4-chloro-5-fluorobenzoate (0.076 g, 0.21 mmol, 1 eq.), and LiOH (0.035 g, 1.5 mmol, 7 eq.) in water (2 mL) was added THF (2 mL) to give a colorless solution. After stirring the solution for 3 h at rt, the reaction was acidified with 1.0 M aq. HCl to pH 2 – 3 and then extracted with CH₂Cl₂ (2 × 5 mL). The separated organic extracts were combined, dried (MgSO₄), filtered and concentrated to produce the desired 2-(4-tertbutylbenzamido)-4-chloro-5-fluorobenzoic acid as a white solid (0.069 g, 0.196 mmol, 94% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 12.06 (s, 1H), 8.92 (d, J = 7.08 Hz, 1H), 7.96 (d, J = 9.8 Hz, 1H), 7.88 (d, J = 8.6 Hz, 2H), 7.62 (d, J = 8.6 Hz, 2H), 1.33 (s, 9H). ¹³C NMR (125 MHz, DMSO-d₆) δ 168.3, 164.7, 155.5, 152.0 (d, J = 244.1 Hz), 138.1 (d, J = 2.8 Hz), 131.1, 127.0, 125.9, 125.0 (d, J = 18.1 Hz), 121.4, 118.5 (d, J = 23.0 Hz), 117.3 (d, J = 5.6 Hz), 34.8, 30.9. LCMS retention time = 3.641 min; purity at 215 nm = 98.3%. HRMS m/z calculated for C₁₈H₁₇ClFNO₃ [M⁺ - 1] 349.09, found 350.09. White solid, mp 250 – 252 °C.

3.1. Summary of Screening Results

The Pittsburgh Molecular Libraries Screening Center (PMLSC, MLSCN phase) screened 220,223 compounds, available as part of the NIH Molecular Libraries Roadmap Initiative, at a single concentration (10 μM) for small molecule inhibitors of TbHK1 (AID 1430). The TbHK1 coupled assay was optimized and validated for HTS by screening the LOPAC set. Compounds were assayed in duplicate at a single concentration (10 μM) and reproducibility between the duplicate screens is represented in Figure 12A (R² = 0.96). The HTS assay performed robustly (average Z-factors of 0.80 ± 0.1) and identified 239 compounds as primary actives (> 50% inhibition at 10 μM), for an overall hit rate of 0.1% (Fig 12B). The 239 active compounds were cherry-picked from DPI (of which 212 were available), and the initial inhibitory activity was confirmed in the primary TbHK1 assay (AID 2560) to afford 29 confirmed compounds. The compounds were then tested against the reporter enzyme, G6PDH, to verify that they did not interfere with the assay format (AID 2516). This effort afforded 16 hit compounds that were then assessed via a 20 point IC₅₀ value determination (AID 1632) [7]. These assays are bundled in PubChem under summary AID 2600.

Figure 12

A: plot of percent inhibition for duplicate screen of the 1280 LOPAC screening deck. LOPAC compounds (blue), minimum control, which should equal ~100% inhibition of signal readout (red), IC₅₀ control compounds (yellow), and maximum control compounds, (more...)

Of the 16 hits obtained from the HTS effort, three scaffolds were suitable from the perspective of lacking reactive functionality, being synthetically accessible, and demonstrating a reasonable activity profile (Figure 13).

Figure 13

HTS scaffolds and associated data prior to validation from resynthesized solid materials.

The two sulfonylbenzoates were resynthesized and upon retest, both compounds failed to confirm. Representatives from the isobenzothiazolinones and the one benzamidobenzoic acid were validated from resynthesized powder material.

3.2. Dose Response Curves for Probe ML205, SID 99437306, CID 46931017

The dose response curves for probe ML205, SID 99437306, CID 46931017 are depicted in Figure 14. The probe demonstrated submicromolar activity versus rTbHK1 (IC₅₀ = 0.98 μM, Fig. 14B). Probe ML205 (SID 99437306) did not show activity in the IMR90 growth inhibition assay or the G6PDH counterscreen (> 25 μM for both, Figure 14C and 14D, respectively).

Figure 14

A. probe structure; B. dose response curve for rTbHK1 inhibition by probe ML205, SID 99437306; C. dose response curve for IMR90 growth inhibition by probe – no EC₅₀ determined over the concentration range evaluated (EC₅₀ > 25 μM); (more...)

3.3. Scaffold/Moiety Chemical Liabilities

The benzamidobenzoic acid scaffold and its derivatives have been easily handled in terms of stability to reaction conditions, exposure to acid or base, heating, and general manipulation. Most are isolated as stable solid materials. We have not observed decomposition nor have we experienced any chemical liability with these compounds. The structure does not contain moieties that are known generally to be reactive. The stability data that was collected has shown that after 48 hrs, 100% of the starting material remains (see section 2.2D, Figure 7).

Figure 7

Stability data depicted as a graph showing the loss of ML205 with time over a 48 hr period. The probe appears to be stable under the experimental conditions, as the percent of compound remaining was 100%.

3.4. SAR Tables and Discussion

Incipient SAR was observed from the initial HTS set of isobenzothiazolinones (see Figure 13), so a chemistry effort was launched around this primary scaffold (SID 17387000/CID 164981). Chemistry was developed to support the synthesis of compounds, and a total of 86 compounds were submitted for testing related to SID 17387000. The only clear trend observed with this chemotype was that the presence of the sulfur was necessary for any TbHK1 in vitro activity, thus implicating its participation in possible covalent interactions with nucleophilic residues, as we have had experience with this particular chemotype in the context of other projects and shown this to be true. While compounds were identified in the series with improved in vitro TbHK1 activity (SID 85285422, TbHK1 IC₅₀ = 1.03 ± 0.17 μM) and lacking G6PDH activity (IC₅₀ > 25 μM), clear SAR trends were difficult to draw out after a rigorous structural modification effort.

These results prompted the team to pursue the singleton benzamidobenzoic acid as a secondary chemotype. Over the course of the pursuing analogs of SID 24798131/CID 1254149, the parent hit has been tested 9 times in triplicate, demonstrating consistent in vitro TbHK1 activity on average of IC₅₀ = 9.12 μM (n = 27) and a lack of activity in the G6PDH counterscreen, IC₅₀ > 25 μM (n = 27). The chemotype was aggressively modified in the shaded regions of the diagram as part of our SAR strategy. A total of 102 compounds have been tested to date that relate to this scaffold. Of these, 83 were synthesized and 19 were purchased. All compounds, regardless of source, were purified and analyzed by LCMS and NMR for structural integrity and purity prior to assessment in assays.

A. Carboxylic acid isosteres

In most cases, replacement of the carboxylic acid with traditional isosteres resulted in loss of TbHK1 potency. Primary amides, sulfonamides, esters, or the use of an oxadiazolone were incorporated to mimic the hydrogen bonding and acidic nature of the carboxylic acid; however, all of these were found to be inferior to the parent. Installation of a nitro group, nitrile, a hydroxyl group or hydrogen at that position also resulted in no observable potency on TbHK1. Comparable activity to the carboxylic acid was achieved on substitution with a tetrazole, resulting in a TbHK1 IC₅₀ = 10.4 μM (SID 99344412/CID 16124577). All compounds in this set did not interfere in the G6PDH counterscreen and possessed EC₅₀s in the IMR90 cytotoxicity assay of > 25 μM (Table 2).

Table 2

SAR summary for compounds with carboxylic acid isosteric replacements.

B. Benzoic Acid Substitution

The 4-chlorophenyl substituent was targeted as a means of modulating the pKa of the benzoic acid and assessing the effect of different groups on the ring. Exchange of the 4-chloro group with 4-bromine was beneficial, resulting in a slight improvement in TbHK1 potency as compared to the parent (entry 2, SID 97302149/CID 46245547, IC₅₀ = 6.9 μM); however other halides (entries 3–4), electron withdrawing or donating substituents (entries 5–6), larger substituents such as phenyl (entry 8), simple methyl substitution at the 4-position (entry 9), including removal of the 4-chloro group altogether (entry 10), resulted in erosion or complete loss of TbHK1 potency (Table 3). Moving the 4-chloro group to the 5-position was also not tolerated (entry 7). The effect of introducing other substituents on this ring while retaining the 4-chloro group was also surveyed. The introduction of a bromine at the 5-position notably improved potency (entry 12, IC₅₀ = 4.6 μM) as compared to the parent (entry 1) while the installation of a 5-methyl group (entry 13) resulted in a marginal loss in potency. Interestingly, the 5 position appears to tolerate some substitution in concert with the 4-chloro group and may represent an opportunity to further refine the potency profile of this scaffold. Compounds with substitutions at either the 3- and 6- positions with the 4-chloro group retained are being prepared, as the 4-substituent has been determined to be critical to maintaining reasonable TbHK1 potency, possibly as a function of both size and electronics, and at present, some advantage has been made by functionalizing alternative positions (C3, C5 or C6). All completed compounds in this set did not interfere in the G6PDH counterscreen and possessed EC₅₀s in the IMR90 cytotoxicity assay of > 25 μM.

Table 3

SAR summary for substitution on the benzoic acid moiety.

C. Amide Linker Modifications

The amide linker portion of the scaffold was particularly sensitive to modification. Any attempt to methylate the nitrogen (entry 2, Table 4), migrate the amide and associated appendage to the 3-position of the benzoic acid moiety (entry 5), truncate or elongate the linker region (entries 3, 6–7), or replace the amide with a sulfonamide resulted in complete loss of TbHK1 potency.

Table 4

SAR summary for substitution of the amide linker region.

Other analogs in this set that were pursued included compounds that featured a fused amide moiety (Figure 15). Fusion of the amide either to the methoxyphenyl appendage or the benzoic acid region was not tolerated.

Figure 15

Fused amide analogs and associated data.

This data suggests, in concert with the data collected for carboxylic acid isosteric replacements in Table 2 and 3-substitution on the benzoic acid moiety (see Table 3, entry 11), that interference with hydrogen bonding (possibly between the acid functionality and the amide N-H) is detrimental.

D. Amidoaryl Modifications

The parental 4-methoxyphenyl moiety (parent: SID 24798131/CID 1254149) was probed extensively through structural variation to determine if any potency could be gained on TbHK1 as compared to the hit compound (Table 5). Migration of the 4-methoxy group to either the 3- or 2-positions on the ring resulted in progressively worse potencies, respectively. Comparable potency to that of the parent was obtained with a benzodioxole group (entry 7, TbHK1 IC₅₀ = 9.4 μM); however, the similarly substituted 3,4-dimethoxyphenyl derivative (entry 6) demonstrated nearly a 2-fold loss in potency.

Table 5

SAR summary for modification of the amidoaryl component.

A series of analogues were also prepared that replaced the 4-methoxyphenyl group with various heterocycles or non-aromatic, cyclic entities that positioned polar functionality in the region where the 4-methoxy substituent of the parent resides (entries 9–17). In all cases, complete loss of TbHK1 activity was observed. Analogously, replacement of the complete methoxyphenyl moiety with linear aliphatic units was not tolerated (entries 19–23). Simple alterations (4-methoxy group → 4-Cl, 4-CF₃, or 4-H) were also inferior.

Guided by the fact that polar functionality occupying similar space to the 4-methoxy substituent was undesirable, the team pursued analogues bearing lipophilic groups in that region of space. The 4-ethylphenyl derivative was the first of these that produced a 3-fold boost in potency (SID 25734182/CID 97302135, entry 34, TbHK1 IC₅₀ = 3.0 μM). This region of space was further characterized with several analogues bearing branched or cyclic alkyl groups at the 3- or 4-positions (entries 32–42).

Migration of branched groups to the 3-position of the ring, again, resulted in loss of observable TbHK1 activity (> 25 μM); however, a few analogues were identified with improved TbHK1 activity when the 4-substituent was modified, specifically SID 99222689/CID 883362 (R = 4-t-butylphenyl, IC₅₀ = 2.1 μM) and SID 99344415/CID 46891921 (R = 4-t-pentylphenyl, IC₅₀ = 2.4 μM). Clearly, the data suggests that small, branched, lipophilic groups off of C4 are advantageous. That binding region also appears to concurrently accommodate some small lipophilic group off of C3, as evidenced by a few ring-fused analogs that occupy C3 and C4 simultaneously (entries 43–45). For these compounds, there was some modest compromise in TbHK1 activity, but these changes were still well-tolerated (IC₅₀ range = 4.8 – 5.4 μM). A handful of analogs further exploring this region of space are currently in development to see if any enhancement of potency can be achieved. All compounds surveyed to date did not interfere in the G6PDH counterscreen and possessed EC₅₀s in the IMR90 cytotoxicity assay of > 25 μM.

E. Tandem Modifications

Those structural features found to improve TbHk1 activity were combined in anticipation of a synergistic effect (Table 6). For instance, exchange of the chlorine of the parent (entry 1) for a bromine was determined to be acceptable, as was the optimization of the amidoaryl moiety with branched alkyl groups off of the 4-position. In the course of our investigation, the bromine and 4-ethylphenyl or 4-tert-butylphenyl combination were studied (entries 2 and 3), affording an improved TbHK1 activity of IC₅₀ = 5.5 μM and 2.4 μM, respectively. As it has been shown, the best amidoaryl 4-substituent is a t-butyl group. This alteration was coupled with other features such as the tetrazole (entry 4), which was tolerated in place of the carboxylic acid. Additionally, the substitution pattern of the benzoic acid moiety of R₁ = Cl, R₂ = Br (entry 7) was discovered to be the most beneficial optimization, resulting in a submicromolar inhibition of TbHK1 and culminating in the declaration of a probe compound for this project.

Table 6

Survey of combined effects of optimized functionality.

3.5. Cellular Activity

Compounds were evaluated in a human IMR90 growth inhibition assay (AID 449725) to assess mammalian cytotoxicity. All compounds in the series possessed EC₅₀s in the IMR90 cytotoxicity assay of > 25 μM. Data from the whole parasite BSF assay, while not required for probe criteria, was collected for many of the compounds in this series. Most compounds were assessed at a single concentration of 10 μM for percent growth inhibition of parasites and all registered in the range of 0–55%. The SAR profile relating to the percent BSF inhibition is not quite clear at this point in time; however, the compounds do show toxicity towards parasites to varying degrees. Select examples are shown in Figure 16 and in the preceding SAR tables.

Figure 16

Select examples of cellular IMR90 and percent BSF inhibition data.

3.6. Profiling Assays

The probe compound ML205, SID 99437306, CID 46931017 and the purified prior art compound quercetin (SID 99460891/CID 5280343) were profiled at 5 μM in a 50-member kinase panel to assess differences in selectivity between the two compounds [16].

For most of the kinases assayed, both the probe and quercetin showed relatively low (0–10%) inhibition. In the case of PIM1, quercetin showed significant inhibition (~66%) while the probe compound demonstrated inhibition at ~ 20%. Profiling data for all kinases was plotted as percent activity remaining vs. kinases profiled, represented below:

Human glucokinase was not available as part of the panel, but the Assay Provider assessed compounds for this target at 10 μM (AID 492951). The probe compound did not significantly inhibit hGlk (IC₅₀ = 48.3 μM) while quercetin inhibited the same target with an IC₅₀ of 2.2 μM.

Probe

SID 99437306

CID 46931017

ML# 205

hGlk IC₅₀ = 48.3 ± 6.9 μM

Quercetin

SID 99460891

CID 5280343

hGlk IC₅₀ = 2.2 ± 0.5 μM

4.1. Comparison to existing art and how the new probe is an improvement

The HTS effort that identified a viable hit scaffold and the optimization of the parent hit SID 24798131/CID 1254149 with rTbHK1 IC₅₀ = 9.1 μM to a probe that inhibits rTbHK1 in the submicromolar range is a significant achievement given the limited leads and therapeutics available. This project has delivered a probe that meets all of the criteria set forth in the CPDP and significantly improves upon prior art in the (1) potency on the in vitro rTbHK1 target, (2) structural improvements to avoid reactive functionality, and (3) better selectivity against hGlk (a related human kinase) and PIM1 (important in cell cycle regulation and apoposis). Given that quercetin has been shown to inhibit a variety of targets, thus limiting its use with respect to definitive hexokinase pathway investigation, the increased value of the probe defined here is in its ability to avoid these off-target liabilities [8].

4.2. Mechanism of Action Studies

In an attempt to better understand the nature of the inhibition associated with these compounds on TbHK1, an additional study was completed by the assay provider, Dr. James Morris (Fig. 17). Preliminary studies suggest that SID 99437306/CID 46931017 is a mixed inhibitor of TbHK1 with respect to ATP with a K_i of 0.695 μM. This is notable because screens for inhibitors of kinases have tended to yield competitive inhibitors that are frequently ATP analogues – SID 99437306/CID 46931017 may represent a novel, non-ATP binding site scaffold for probe improvement against TbHK1.

Figure 17

Michaelis-Menten and Lineweaver-Burk plots showing mixed inhibition of TbHK1 with respect to ATP using ML205, SID 99437306, CID 46931017.

4.3. Planned Future Studies

A probe advancement proposal was submitted to the NIH as of October 13, 2010, and has been subsequently approved. While some regions of the scaffold represented by probe ML205 were not amenable to SAR modification without adversely affecting potency, optimization was possible in four sectors that were investigated. There remain a few diversification points which were not included in this effort which may lead to improved profile and opportunity to refine physiochemical parameters. The following specific SAR plans are being implemented to address potency and cellular permeability.

Figure 18Planned SAR optimization for extended probe characterization

The areas highlighted above will be investigated to meet the goals of the project. Unaddressed areas of the structure are not being modified as these were extensively studied in the probe project leading to the development of ML205; however, these regions may be altered in the event that structural variation elsewhere gains sufficient activity. In the probe enhancement phase, the probe will be compared to quercetin and assessed in the following ways:

Compounds will be provided by the KU SCC in sufficient quantities (500 mg – 1 gram) and purity for the proposed studies and will likely be further refined structurally to tune physiochemical and pharmacokinetic parameters necessary for adequate analysis in rodent models. Specifically, the probe will be assessed by the KU SCC and structurally optimized to:

1. possess suitable aqueous solubility, stability, and cellular permeability
2. possess reasonable microsomal stability, plasma protein binding and plasma stability
3. be assessed for in vivo pharmacokinetics