Synthesis of Potent Vanin‐1 Inhibitors Labelled With Carbon‐14 and Deuterium

ABSTRACT

Vanin‐1 is an enzyme highly expressed in the kidneys, liver, and small intestines. Inhibition of this enzyme has been postulated as a novel approach in the treatment of several inflammatory diseases. BI 764122, BI 1585773, and BI 1595043 are very potent inhibitors of this enzyme. These three analogs share the same (S)‐N‐(nicotinoyl)pyrrolidin‐3‐yl)‐N‐methyl acetamide moiety. The preparation of these analogs labelled with carbon‐14 was accomplished via carbon‐14 carboxylation with carbon dioxide of corresponding aryl halides, followed by amide bond formation with (S)‐N‐(methyl)‐N‐(pyrrolidin‐3‐yl)acetamide. For metabolic considerations, the radioactive carbon was also incorporated in a cyclopropyl in [ ¹⁴ C]‐BI 764122 synthesis. The deuterium labelling of these three compounds was completed using the common moiety (S)‐N‐(methyl‐d ₃)‐N‐(pyrrolidin‐3‐yl)acetamide‐d ₃, which was prepared in three steps and then coupled to the corresponding aryl acids via amide bond.

Vanin‐1 is an enzyme highly expressed in the kidneys, liver, and small intestines. Inhibition of this enzyme has been postulated as a novel approach in the treatment of several inflammatory diseases. BI 764122, BI 1585773, and BI 1595043 are very potent inhibitors of this enzyme.

1. Introduction

Vanin‐1 or vascular noninflammatory molecule‐1, is a cell surface‐associated, glycosylphosphatidylinositol (GPI)‐anchored protein. It is highly expressed in the kidneys, liver, and small intestines [1, 2]. The only known substrate for vanin‐1 is pantetheine, which is hydrolyzed by this enzyme to pantothenic acid (vitamin B5) and cysteamine (2‐aminoethane‐1‐thiol) (Figure 1) [3]. Both vitamin B5 and cysteamine play major roles in several biological processes. For example, pantothenic acid is necessary for the synthesis of coenzyme A (CoA), an essential cofactor in all living cells, and plays a critical role in many aspects of cellular metabolism, regulation of gene expression, and the biosynthesis of major cellular constituents [4]. The vanin enzymes regulate cellular redox homeostasis, as their reaction products like cysteamine are capable of interfering with glutathione biosynthesis [5, 6, 7].

FIGURE 1.

Enzymatic activity of vanin.

On the other hand, studies in vanin‐1 knockout mice have shown that the knockout animals were more resistant to oxidative stress and showed a lower inflammatory response compared with their wild‐type littermates following challenge with different injury triggers [8]. In addition, vanin‐1 knockout mice are resistant to inflammatory bowel disease [9] and the vanin enzymes have been postulated to play potential roles in other disorders including malaria susceptibility, psoriasis, colitis‐associated colon cancer, and postarterial injury neointimal hyperplasia based on preclinical evidence [10]. Inhibition of vanin‐1 has been pursued as a novel approach for the treatment of IBD, as well as other inflammatory conditions [11, 12, 13, 14, 15, 16, 17].

BI 764122, BI 1585773, and BI 1595043 are very potent inhibitors of vanin enzymatic activity in mice and humans [18, 19]. In this manuscript, we describe their detailed synthesis labelled with carbon‐14 as tools for DMPK studies, and with deuterium for use as an internal standard for bioanalytical studies and metabolism investigations (Figure 2).

FIGURE 2.

Structure of vanin‐1 inhibitors with the common moiety shown in blue.

2. Results and Discussion

In our first synthesis of carbon‐14 labelled BI 764122, a potential metabolic cleavage at the nitrogen, or/and the oxygen prompted us to introduce the radioactive carbon atom in the cyclopropyl ring via a Kulinkovich reaction of a nitrile analog (Scheme 1). The silyl protected 2‐bromoethanol 1 was treated with potassium cyanide‐¹⁴ C in dimethyl sulfoxide to provide the nitrile derivative [ ¹⁴ C]‐2 in quantitative yield. Using Kulinkovich reaction conditions, the nitrile was converted to a cyclopropyl amine [ ¹⁴ C]‐3, followed by Boc protection to [ ¹⁴ C]‐4 in 66% yield over two steps. Removal of the silyl protecting group afforded alcohol [ ¹⁴ C]‐5 in high yield. Mitsunobu's reaction with methyl 6‐chloro‐4‐hydroxynicotinoate (6), followed by removal of the Boc‐protecting group gave [ ¹⁴ C]‐8 in 54% yield over two steps. Palladium‐catalyzed intramolecular C−N coupling gave the Spiro (cyclopropane)oxazepane pyridine derivative [ ¹⁴ C]‐9 in 75% yield [20]. Ester hydrolysis to [ ¹⁴ C]‐10, and amide bond formation with (S)‐N‐methyl‐N‐(pyrrolidin‐3‐yl)acetamide (11) using CDI in dimethyl acetamide gave [ ¹⁴ C]‐BI 764122 in 80% yield after silica gel chromatography with more than 98% radio‐and chemical purities, chiral purity of 99% ee, and a specific activity of 46.64 mCi/mmol (1.72 GBq/mmol).

SCHEME 1.

Synthesis of [ ¹⁴ C]‐BI 764122, cyclopropyl‐¹⁴ C.

In the second carbon‐14 synthesis, the radioactive carbon atom was incorporated at the nicotinic acid‐carboxyl. The commercially available 5‐bromo‐2‐chloropyridin‐3‐ol (12) was first protected using para‐methoxybenzyl chloride to compound 13. Carboxylation at −78°C with [¹⁴C]CO₂ using Turbo‐Grignard in THF gave [ ¹⁴ C]‐14 in good yield. Amide bond formation with (S)‐N‐methyl‐N‐(pyrrolidin‐3‐yl)acetamide (11) afforded [ ¹⁴ C]‐15 in 82% yield. Removal of the protecting group using trifluoroacetic acid, followed by Mitsunobu's reaction as described with amine 5 gave [ ¹⁴ C]‐17 in good yield. Removal of the Boc‐protecting group and palladium‐catalyzed cyclization as mentioned before furnished [ ¹⁴ C]‐BI 764122 in fair yield, and with a specific activity of 55.05 mCi/mmol (2.03 GBq/mmol), and with radioactive and chemical purities higher than 99%, and chiral purity of 99% ee (Scheme 2).

SCHEME 2.

Synthesis of [ ¹⁴ C]‐BI 764122, carbonyl‐¹⁴ C.

To prepare carbon‐14 labelled BI 1585773 [21], the commercially available 2‐amino‐5‐iodopyridine (19) was N‐tert‐butylated using tert‐butyl‐2,2,2‐trichloroacetimidate (20) with copper (II) trifluomethansulfonate in nitromethane in 53% yield [22]. The reported halogen–metal exchange on heterocyclic substrates bearing an acidic proton through a combined use of i‐PrMgCl and n‐BuLi at −20°C was used to introduce the radioactive carbon via ¹⁴CO₂ as postulated in Scheme 3 [23]. Amide bond formation between the produced acid [ ¹⁴ C]‐22 and 11 using a solution of T3P in EtOAc gave [ ¹⁴ C]‐BI 1585773 in 42% yield with a specific activity 55.60 mCi/mmol (2.5 GBq/mmol), and radio and chemical purities higher than 98% and chiral purity of 99% ee.

SCHEME 3.

Synthesis of [ ¹⁴ C]‐BI 1585773.

For the synthesis of carbon 14 labeled BI 1595043, the chiral iodide analog (30) was first prepared in six steps using a methodology developed for a methyl nicotinoate analog [24]. Thus, the trisubstituted olefin (26) was prepared via a one‐pot borylation of the vinyl bromide (23) to (24) using bis (pinacolato)diborane, followed by a Suzuki cross coupling with the commercially available 2‐amino‐3‐iodo‐5‐nitropyridine (25). Cyclization using concentrated sulfuric acid gave the dihydroazaindole derivative (27) in 63% overall yield in three steps. The nitro group was reduced under 150 psi of hydrogen in EtOAc in good yield. The Sandmeyer reaction of 28 then gave the iodo derivative 29 in 43% yield. The desired enantiomer 30 was separated in 39% recovery using chiral SFC and then used to prepare [ ¹⁴ C]‐BI 1595043 as described before in the synthesis of [ ¹⁴ C]‐BI 585773. The acid [ ¹⁴ C]‐32 was activated with CDI in acetonitrile at 50°C and coupled to (S)‐N‐methyl‐N‐(pyrrolidin‐3‐yl)acetamide (11) in 78% yield after silica gel chromatography purification. The product [ ¹⁴ C]‐BI 1595043 was isolated with a specific activity of 57.62 mCi/mmol (2.13 GBq/mmol) and radio and chemical purities higher than 99%, and chiral purity of 99% ee (Scheme 4).

SCHEME 4.

Synthesis of [ ¹⁴ C]‐BI 1595043.

In the deuterium‐labelled synthesis of these drug candidates, the commercially available (S)‐1‐benzyl‐3‐(Boc‐amino)pyrrolidine (33) was treated with 1.7–2.0 equivalents of lithium aluminum deuterated (ALD) in refluxing THF to give [ ² H ₃ ]‐34 in quantitative yield [25]. The reaction of the secondary amine [ ² H ₃ ]‐34 with deuterated acetyl chloride at low temperature gave [ ² H ₆ ]‐35 in excellent yield. Removal of the benzyl‐protecting group under 400 psi of hydrogen at 50°C in methanol afforded (S)‐N‐(methyl‐d ₃)‐N‐(pyrrolidin‐3‐yl)acetamide‐d ₃ ([ ² H ₆ ]‐11) in quantitative yield. Finally, amide bond formation of [ ² H ₆ ]‐11 with the acids 10, 22, and 32 as described above using CDI or T3P gave [ ² H ₆ ]‐BI 764122, [ ² H ₆ ]‐BI 1585773, and [ ² H ₆ ]‐BI 1595043 in 70%, 63%, and 91% yields respectively after silica gel chromatography purifications (Scheme 5).

SCHEME 5.

Synthesis of deuterium‐labelled vanin‐1 inhibitors.

3. Conclusion

Inhibition of the vanin enzyme has been postulated as a novel target in the treatment of several inflammatory diseases. BI 764122, BI 1585773, and BI 1595043 are potent inhibitors of this enzyme. These three analogs have (S)‐N‐(methyl)‐N‐(pyrrolidin‐3‐yl)acetamide as a common moiety linked via an amide bond to three different aryl carboxylic acids. To access the carbon‐14 labelled versions of these analogs, the corresponding aryl halides were used in a halogen‐[¹⁴C]CO₂ carboxylation to provide the carbon‐14 aryl carboxylic acids, which were then linked to the common moiety using known amidation conditions. Concern about the metabolism of one of these analogs, BI 764122, necessitates the introduction of radioactive carbon in a different site as well. For the stable isotope synthesis, (S)‐N‐(methyl‐d ₃)‐N‐(pyrrolidin‐3‐yl)acetamide‐d ₃ was prepared in three chemical steps in 94% overall yield and then coupled to the known aryl carboxylic acids to afford [ ² H6]‐BI 764122, [ ² H6]‐BI 1585773, and [ ² H6]‐BI 1595043.

4. Materials and Methods

Potassium cyanide‐¹⁴ C with a specific activity 50–60 mCi/mmol was obtained from American Radiolabeled Chemicals Inc. (St. Louis, MO, USA). Carbon‐14 carboxylation of the halides 13, 21, and 30 were performed at ARC (St. Louis, MO, USA). tert‐Butyl(2‐bromoethoxy)dimethylsilane (1), (S)‐1‐Benzyl‐3‐(Boc‐amino)pyrrolidine (33), lithium aluminum deuterated (LAD), and 2‐amino‐3‐iodo‐5‐nitropyridine (25) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Acetyl chloride‐d ₃ (isotopic enrichment 99.4%) was purchased from Cambridge Isotope Laboratories Inc. (Tewksbury, MA, USA). Methyl 6‐chloro‐5‐hydroxypyridine‐3‐carboxylate (6), and 6‐(tert‐butylamino)pyridine‐3 carboxylic acid (22) were purchased from AmBeed (Buffalo Grove, IL, USA). 5‐Bromo‐2‐chloropyridin‐3‐ol (12) was purchased from Combi‐Blocks (San Diego, CA, USA).

4.1. HPLC Method A

Column: Ace3 C18 (2.1 × 75.0 mm, 3 μm). Mobile phase: A: water (0.1% TFA), B: MeCN. Gradient 5% B to 95% B in 8.5 min, flow rate 0.5 mL/min. Column temperature: 25°C. Injection volume: 2 μL. UV detection at 220 nm.

4.2. HPLC Method B

Column Zorbax‐C18 (250 × 4.6 mm, 5 μm). Mobile phase: A: water (0.1% TFA), B: MeCN. Gradient 10% to 100% B in 30 min. Flow rate: 1 mL/min. Column temperature: 25°C. Injection volume: 2 μL. UV detection at 254 nm.

4.3. HPLC Method C

Column: Waters Xbridge BEH C18 (2.1 × 50.0 mm, 2.5 μm). Mobile phase: A: water (0.1% TFA), B: MeCN. Gradient: 5% B to 95% B in 9.5 min. Flow rate: 0.5 mL/min. Column temperature: 30°C. Injection volume: 2 μL. UV detection at 254 nm.

4.4. HPLC Method D

Column: Zorbax Phenyl (2.1 × 50.0 mm, 1.8 μm). Mobile phase: A water (0.1% TFA), B: MeCN. Gradient: 5% B to 98% B in 10 min. Flow rate: 0.5 mL/min. Column temperature: 30°C. Injection volume: 2 μL. UV detection at 254 nm.

4.5. Chiral HPLC A

Column: Chiralcel OD‐3 (4.6 × 150 mm, 3 μm). Mobile phase: A: 5% MeOH/EtOH; B: heptane, isocratic 20% A. Flow rate: 1 mL/min. Injection size: 5 μL. Run time: 15 min. UV detection at 280 nm.

4.6. Chiral HPLC B

Column: Phenomenex/cellulose, LUX‐2 (4.6 × 100 mm, 5 μm). Mobile phase: A: ethanol; B: heptane, isocratic 40/60 A/B. Flow rate: 1 mL/min. Injection size: 5 μL. Run time: 15 min, Detection UV 280 nm.

4.7. Chiral HPLC C

Column: Chromega Chiral CCA (4.6 × 100 mm, 5 μm). Mobile phase A: heptane, B: IPA, gradient: 20% B to 50% B. Flow rate: 1 mL/min. Injection size: 5 μL. Run time: 17 min.

SFC separation method of the desired iodide (30):

Model: Thar/Waters Prep SFC‐80.

Modifier: MeOH. Flow rate 3 mL/min, gradient: 1% B to 3% B in 3 min to 50% B in 5 min, hold at 50% B for 1 min to 1% B in 0.5 min, hold at 1% B for 0.5 min. Run time: 10 min. Column temperature 35°C. Injection volume: 1.5 mL. Column: ES Industries, CCC (30 mm × 250 mm, 5 μm). Temperature: 40°C. UV detection at 220 nm. Total CO₂ flow rate: 80 g/min; co‐solvent flow rate 24 mL/min. Run time: 8 min; post run: 1 min. Mobile phase: A: CO₂ (bone‐dry), B: Isopropanol (HPLC grade). Isocratic: 30% IPA, 70% CO₂. Lower pressure limit: 0 psi; high pressure limit: 6000 psi. Sample concentration 20 mg/mL (DCM/MeOH: 60/40 (v/v)).

5. Experimental

5.1. Carbon‐14 Synthesis

5.1.1. Preparation of [¹⁴C]‐BI 764122 With Cyclopropyl‐¹⁴ C

5.1.1.1. 3‐[(tert‐Butyldimethylsilyl)oxy](1‐¹⁴C)propanenitrile, [¹⁴C]‐2

A mixture of tert‐butyl(2‐bromoethoxy)dimethylsilane (1) (1.1 g, 4.60 mmol) and K¹⁴CN (320 mg, 4.76 mmol, 200 mCi) in anhydrous DMSO (2 mL) was stirred at ambient temperature for 48 h. The resulting cloudy mixture was treated with brine (2 mL) and extracted with hexanes (5 mL × 3). The organic layer was passed through a phase separator column and concentrated in vacuo to give 1.1 g (200 mCi) of a colorless oil in quantitative yield. TLC: 10% EtOAc/hexanes, R _f of the bromide 0.57 and R _f of the product 0.32. ¹H NMR (CDCl₃, 500 MHz) δ: 3.83(t, J = 6.50 Hz, 2H), 2.53(t, J = 6.50 Hz, 2H), 0.90(s, 9H), 0.09(s, 6H).

5.1.1.2. 1‐(2‐((tert‐Butyldimethylsilyl)oxy)ethyl)cyclopropane‐1‐Amine‐1‐¹⁴ C, [¹⁴C]‐3

A solution of [ ¹⁴ C]‐2 (0.86 g, 4.60 mmol) in anhydrous ether (20 mL) was added titanium (IV) isopropoxide (1.60 mL, 5.24 mmol) at room temperature under Argon. A solution of ethyl magnesium bromide (3M solution in ether, 3.40 mL, 10.2 mmol) was added slowly. The reaction was exothermic and became dark. Stirring continued for 2 h at room temperature. Boron trifluoride diethyl etherate (1.25 mL, 10.10 mmol) was added in one portion and the resulting mixture was further stirred for 2 h. A solution of 10% NaOH in water (4.5 mL) was added, followed by DCM (30 mL), stirred vigorously, and then extracted with DCM (100 mL × 2), filtered through a phase separator column, and concentrated in vacuo to give 1.2 g (155 mCi) of a slightly brown oil in quantitative yield. The product was used as is in the next step. ¹H NMR (CDCl₃, 400 MHz) δ: 8.42(brs, 2H), 3.78(t, J = 5.41 Hz, 2H), 1.71(t, J = 5.41 Hz, 2H), 0.93(s, 9H), 0.83(s, 2H), 0.66(s, 2H), 0.09(s, 6H).

5.1.1.3. tert‐Butyl N‐(1‐{2‐[(tert‐Butyldimethylsilyl)oxy]ethyl}(1‐¹⁴C)cyclopropyl)carbamate, [¹⁴C]‐4

To a solution of [ ¹⁴ C]‐3 (1.0 g, 4.60 mmol) in THF (16 mL) was added (Boc)₂O (1.90 g, 8.71 mmol), followed by a solution of NaHCO₃ (0.96 g, 11.40 mmol) in water (16 mL). The resulting mixture was stirred for 18 h; then, EtOAc (70 mL) was added and stirred vigorously. The aqueous layer was removed and extracted with EtOAc (50 mL). The combined EtOAc extracts were passed through a phase separator column and concentrated in vacuo to give 2.5 g of an amber oil, which co‐eluted with unlabeled sample and stained brown with a ninhydrin solution. Purification using a gradient EtOAc/DCM (0%–10%) and an 80 g disposable silica gel column gave 963 mg (85 mCi) of a colorless oil in 66% yield over two steps. ¹H NMR (CDCl₃, 400 MHz) δ: 4.91(s, 1H), 3.70(t, J = 6.40 Hz, 2H), 1.67(t, J = 6.40 Hz, 2H), 1.37(s, 9H), 0.84(s, 9H), 0.70(m, 2H), 0.55(m, 2H), −0.10(s, 6H).

5.1.1.4. tert‐Butyl N‐[1‐(2‐Hydroxyethyl)(1‐¹⁴C)cyclopropyl]carbamate [¹⁴C]‐5

A solution of [ ¹⁴ C]‐4 (963 mg, 3.03 mmol) in THF (12 mL) was stirred under Argon in an ice‐bath. A solution of tetrabutylammonium fluoride (1.0 M in THF, 6 mL) was added dropwise and stirring was continued for 14 h. Water (15 mL) was added, and the reaction mixture was extracted with DCM (50 mL × 2). The organic phase was passed through a phase separator column, and the filtrate was concentrated in vacuo to give 800 mg of material which solidified upon standing at room temperature. The crude material was purified on a 40 g disposable silica gel column 0%–10% MeOH/DCM to give 510 mg (71 mCi) of a white solid in 83% yield. ¹H NMR (400 MHz, DMSO‐d ₆): δ 7.00 (br. s, 1H), 4.25 (t, J = 5.60 Hz, 1H), 3.47 (q, J = 6.80 Hz, 2H), 1.59 (t, J = 7.20 Hz, 2H), 1.36 (s, 9H), 0.58–0.55 (m, 2H), 0.51–0.48 (m, 2H).

5.1.1.5. Methyl 5‐(2‐(1‐((tert‐Butoxycarbonyl)amino)[¹⁴C]cyclopropyl)ethoxy)‐6‐Chloronicotinate, [¹⁴C]‐7

Methyl 6‐chloro‐5‐hydroxypyridine‐3‐carboxylate (6) (471 mg, 2.51 mmol), the boc‐protected cyclopropylamine derivative [ ¹⁴ C]‐5 (510 mg, 2.51 mmol) and triphenyl phosphine (987 mg, 3.76 mmol) were charged in a flask. Anhydrous THF (10 mL) was added under N₂, and the resulting colorless solution was stirred at –10°C. Diisopropyl azodicarboxylate (DIAD, 0.741 mL, 3.76 mmol) was added dropwise in a 30‐min period. The resulting yellow solution was stirred for 14 h as the temperature rose to 6°C. Water (24 mL) was added to the reaction flask and extracted with DCM (70 mL × 2). The extracts were passed through a phase separator column and concentrated in vacuo to give 3.07 g of viscous amber oil. This material was purified using an 80 g disposable silica gel column 0%–10% EtOAc in DCM to give 510 mg (31 mCi) of a white solid in 55% yield.

5.1.1.6. Methyl 5‐(2‐(1‐Aminocyclopropyl‐1‐¹⁴C)ethoxy)‐6‐Chloronicotinate, [¹⁴C]‐8

To a solution of [ ¹⁴ C]‐8 (510 mg, 1.37 mmol) in EtOAc (5 mL) was added dropwise a solution of 4 N HCl in dioxane (2 mL). The solution was heated to 64°C and stirred for 6 h, then cooled to room temperature, filtered, and dried under vacuum for 2 h and then under reduced pressure at 35°C for 2 h to give 354 mg (30.76 mCi) of a white solid in 99% yield. ¹H NMR (DMSO‐d ₆, 500 MHz): δ 8.70 (brs, 2H), 8.46 (d, J = 2.00 Hz, 1H), 7.85 (d, J = 2.00 Hz, 1H), 4.39 (t, J = 6.30 Hz, 2H), 3.89 (s, 3H), 2.17 (t, J = 6.30 Hz, 2H), 1.03–1.00 (m, 2H), 0.85–0.83 (m, 2H).

5.1.1.7. Methyl‐2′,3′‐Dihydro‐5′H‐Spiro[cyclopropane‐1,4′‐Pyrido[3,2‐B][1,4]Oxazepine]‐8′‐Carboxylate‐1‐¹⁴ C, [¹⁴C]‐9

The crude [ ¹⁴ C]‐8 (335 mg, 1.08 mmol) and cesium carbonate (777 mg, 2.38 mmol) in anhydrous acetonitrile (5 mL) was degassed three times, and argon was introduced in each cycle. Palladium (II) acetate (4.8 mg, 21.40 μmol) and 1,1′‐bis(diisopropylphosphino)ferrocene (13.55 mg, 32.4 μmol) were added. The mixture was degassed three more times, heated to 83°C, and stirred for 10 h. The mixture was cooled to room temperature and quenched with water (5 mL). The organics were extracted with DCM, filtered through a phase separator and concentrated in vacuo to give 260 mg of a dark solid. Purification on a 25 g disposable silica gel column 0%–40% EtOAc/DCM as eluent gave 100 mg (17.2 mCi) of the unreacted amine, and 153 mg (14 mCi) of product as a white solid in 75% yield based on the reacted amine. ¹H NMR (DMSO‐d ₆, 400 MHz) δ: 8.26 (d, J = 2.0 Hz, 1H), 7.45 (d, J = 2.00 Hz, 1H), 7.03 (s, 1H), 4.19 (t, J = 6.00 Hz, 2H), 3.77 (s, 3H), 1.95(t, J = 6.00 Hz, 2H), 0.85–0.82 (m, 2H), 0.62–0.59 (m, 2H).

5.1.1.8. 2′,3′‐Dihydro‐5′H‐Spiro[cyclopropane‐1,4′‐Pyrido[3,2‐B][1,4]oxazepine]‐8′‐Carboxylic‐1‐¹⁴ C Acid, [¹⁴C]‐10

[ ¹⁴ C]‐9 (93 mg, 0.39 mmol) was treated with methanol (3 mL) and water (1 mL). Lithium hydroxide (38 mg, 1.66 mmol) was added, and the mixture was heated to 62°C and stirred for 2 h. The reaction solution was cooled to room temperature and concentrated to remove most of the methanol. The remaining was treated slowly with aqueous 1 M HCl (1.60 mL) to give a thick white mixture. The mixture was stirred for 15 min, filtered, rinsed with water and heptane, and dried under vacuum for 14 h. The solid was further dried under reduced pressure at 33°C for 2 h to give 87 mg (8.5 mCi) of a white solid in 99% yield. ¹H NMR (DMSO‐d ₆, 400 MHz) δ: 12.59 (br.s, 1H), 8.25 (d, J = 2.00 Hz, 1H), 7.45 (d, J = 1.60 Hz, 1H), 6.90 (s, 1H), 4.18 (t, J = 6.00 Hz, 2H), 1.94 (t, J = 6.00 Hz, 2H), 0.84–0.81 (m, 2H), 0.61–0.58 (m, 2H).

5.1.1.9. (S)‐N‐(1‐(2′,3′‐Dihydro‐5′H‐Spiro[cyclopropane‐1,4′‐Pyrido[3,2‐B][1,4]Oxazepine]‐8′‐Carbonyl‐1‐¹⁴ C)pyrrolidin‐3‐Yl)‐N‐Methylacetamide, [¹⁴C]‐BI 764122

A solution of [ ¹⁴ C]‐10 (87 mg, 0.39 mmol) in N,N‐dimethyl acetamide (DMAC, 4 mL) was added under nitrogen 1,1′‐carbonyldiimidazole (CDI) (82 mg, 0.51 mmol) in one portion. The reaction was stirred at room temperature for 1.5 h, then (S)‐N‐methyl‐N‐(pyrrolidin‐3‐yl)acetamide hydrochloride (11) (84 mg, 0.47 mmol) was added in one portion under Argon. The resulting solution was heated to 32°C and stirred for 2 h. The reaction was cooled to room temperature, treated with water (4 mL), extracted with DCM (10 mL × 3), filtered through a phase separator column, and concentrated in vacuo. Purification on a 40 g disposable silica gel column 0%–10% MeOH/DCM gave 109 mg (6.77 mCi) of product in 80% yield with a specific activity of 46.64 mCi/mmol. HPLC method A: t _R = 3.85 min (99.79%). Radioactive purity: t _R = 3.97 min (99.25%). Chiral HPLC method A: t _R = 9.35 min (99.93%). ¹H NMR with rotamers (DMSO‐d ₆, 500 MHz) δ: 7.92(s, 1H), 7.27(s, 1H), 6.45(s, NH), 4.49–5.03(brs, 1H), 4.17(t, J = 7.10 Hz, 2H), 3.52–3.67(m, 2H), 3.42–3.60(brs, 2H), 2.87(s, 3H), 2.06(s, 3H), 1.99(brs, 2H), 1.92(brs, 2H), 0.80(s, 2H), 0.58(s, 2H).

5.1.2. Synthesis of [¹⁴C]‐BI 764122, With Carbonyl‐¹⁴ C

5.1.2.1. 5‐Bromo‐2‐Chloro‐3‐((4‐Methoxybenzyl)oxy)pyridine, 13

A solution of 5‐bromo‐2‐chloropyridin‐3‐ol (12) (3.0 g, 14.40 mmol) in DMF (25 mL) was added potassium carbonate (3.98 g, 28.80 mmol), followed by a dropwise addition of 4‐methoxybenzyl chloride (2.145 mL, 15.80 mmol). The mixture was stirred for 14 h, then treated with water (30 mL), and stirred for 1.5 h. The resulting solid was collected by vacuum filtration and dried under suction to give 4.6 g of a white solid in 93% yield. ¹H NMR (DMSO‐d ₆, 400 MHz) δ: 8.52 (s, 1H), 7.92 (s, 1H), 6.96 (d, J = 7.25 Hz, 2H), 6.88 (d, J = 7.25 Hz, 2H), 5.14 (s, 2H), 3.85 (s, 3H).

5.1.2.2. 6‐Chloro‐5‐((4‐Methoxybenzyl)oxy)nicotinic Acid‐3‐¹⁴ C, [¹⁴C]‐14

A solution of 13 (3.0 g, 9.13 mmol) in THF (30 mL) was cooled to −20°C. A solution of iPrMgCl/LiCl (1.3 M in THF, 12.64 mL) was then added dropwise and stirred for 2 h then further cooled to −80°C before [¹⁴C]CO₂ (500 mCi) was transferred to the reaction flask. The reaction was then allowed to warm to room temperature slowly with stirring. The mixture was cooled back down to −20°C, then Et₂O (45 mL) was added, followed by 6 N aqueous HCl (20 mL). The mixture was warmed to room temperature, and the aqueous layer was removed and extracted with EtOAc (50 mL × 2), dried over MgSO₄, filtered, and concentrated to give an off white solid. The solid was diluted with IPA (20 mL) and warmed to 85°C with stirring. An additional amount of IPA (5 mL) was added to dissolve all the solids. The mixture was held at this temperature for 10 min, then allowed to cool to room temperature, and stirred for 14 h. The solid was collected by vacuum filtration and dried under suction for 30 min to give 2.42 g (450 mCi) of a white solid in 90% yield. HPLC method B: t _R = 15.49 min (100%). Radiochemical purity t _R = 15.55 min (96%).

5.1.2.3. (S)‐N‐(1‐(6‐Chloro‐5‐((4‐Methoxybenzyl)oxy)nicotinoyl‐3‐¹⁴ C)pyrrolidin‐3‐Yl)‐N‐Methylacetamide, [¹⁴C]‐15

CDI (393.5 mg, 2.43 mmol) was added to a solution [ ¹⁴ C]‐14 (552 mg, 1.87 mmol) in DMAC (5 mL) at room temperature. The mixture was stirred for 1 h, and then 11 (400 mg, 2.24 mmol) was added in one portion. The mixture was stirred at room temperature for 14 h, then treated with water (20 mL), and extracted with EtOAc (20 mL × 3). The combined organic extracts were washed sequentially with water (30 mL × 2), brine (20 mL), filtered, and concentrated to give 1.26 g (165 mCi) of a light brown oil in 82% yield. Radiochemical purity t _R = 6.18 min (95.82%).

5.1.2.4. (S)‐N‐(1‐(6‐Chloro‐5‐Hydroxynicotinoyl‐3‐¹⁴ C)pyrrolidin‐3‐Yl)‐N‐Methylacetamide, [¹⁴C]‐16

TFA (10 mL) was added to a solution of [ ¹⁴ C]‐15 (1.26 g, 3.01 mmol) in DCM (10 mL) at room temperature. After stirring for 30 min, the mixture was concentrated. The residue was purified by CombiFlash Companion (80 g, 0%–10% MeOH/CH₂Cl₂) to give 882 mg (162 mCi) of [ ¹⁴ C]‐16 in quantitative yield as a colorless oil. Radiochemical purity t _R = 4.12 min (99.07%).

5.1.2.5. tert‐Butyl (S)‐(1‐(2‐((2‐Chloro‐5‐(3‐(N‐Methylacetamido)pyrrolidine‐1‐Carbonyl‐¹⁴ C)pyridin‐3‐Yl)oxy)ethyl)cyclopropyl)carbamate, [¹⁴C]‐17

DIAD (0.88 mL, 4.41 mmol) was added dropwise to a mixture of [ ¹⁴ C]‐16 (0.88 g, 2.94 mmol), Ph₃P (1.16 g, 4.41 mmol), and 5 (592 mg, 2.94 mmol) in THF at 0°C with stirring. The ice bath was removed, and the mixture was allowed to warm to room temperature, and stirring was continued overnight. The mixture was concentrated and purified by CombiFlash Companion (80 g, 0%–100% EtOAc/DCM). The desired fractions were combined and concentrated to give 1.12 g (79%) of [ ¹⁴ C]‐17 as an off‐white foam. The material was continued to the next step. Radiochemical purity t _R = 6.50 min (70.58%).

5.1.2.6. (S)‐N‐(1‐(5‐(2‐(1‐Aminocyclopropyl)ethoxy)‐6‐Chloronicotinoyl‐3‐Carbonyl‐¹⁴ C)pyrrolidin‐3‐Yl)‐N‐Methylacetamide, [¹⁴C]‐18

A 4 N HCl solution in dioxane (2.32 mL) was added to a solution of [ ¹⁴ C]‐17 (1.12 g, 2.32 mmol) in EtOAc (10 mL) at room temperature. A white precipitate was formed instantly. The mixture was stirred for 14 h, then concentrated, treated with 1 N aqueous NaOH (20 mL), and extracted with DCM (20 mL × 3). The combined extracts were filtered through a phase separator and concentrated. The residue was purified by CombiFlash Companion (40 g, 0%–15% MeOH/CH₂Cl₂/0.1% Et₃N) to give 630 mg (88.3 mCi) of a colorless oil in 71% yield. Radiochemical purity t _R = 4.39 min (94.09%).

5.1.2.7. (S)‐N‐(1‐(2′,3′‐Dihydro‐5′H‐Spiro[cyclopropane‐1,4′‐Pyrido[3,2‐B][1,4]Oxazepine]‐8′‐Carbonyl‐¹⁴ C)pyrrolidin‐3‐Yl)‐N‐Methylacetamide, [¹⁴C]‐BI 764122

A mixture of Pd (OAc)₂ (12 mg, 0.05 mmol), 1,1′‐Bis (diisopropylphosphino)ferrocene (44.6 mg, 0.11 mmol), and Cs₂CO₃ (208.4 mg, 0.64 mmol) were weighed into two separate 24 mL reaction vials. The above material [ ¹⁴ C]‐18 (203 mg, 0.53 mmol) in MeCN (6.5 mL) was added to each vial and degassed. The vials were capped and heated to 85°C for 14 h. The mixtures were cooled to room temperature. Each reaction was worked up separately by diluting with water (20 mL) and extracting with DCM (20 mL × 3). The combined extracts from each vial were filtered through a phase separator and concentrated to give 552 mg of dark oil in 99.7% crude yield. The crude product was purified by CombiFlash Companion (40 g, 0%–10% MeOH/DCM). The desired fractions were combined and concentrated to a viscous oil. The oil was diluted with IPA (1.5 mL) and warmed to 85°C and stirred at this temperature for 15 min. The mixture was removed from the oil bath, then BI 764122 seeds ca. 0.5 mg were added, and the mixture was allowed to cool to room temperature and stirred for 46 h. The solid was collected by vacuum filtration and washed with cold IPA (3 mL) and heptane (5 mL) to give 407 mg (65.05 mCi) of an off‐white solid in 73% recovery, with a specific activity of 55.1 mCi/mmol; radiopurity: 99.81%. UV purity at 220 nm: 98.97%. The mother liquor was transferred to a tarred vial and concentrated under a stream of argon to give 92 mg (14.72 mCi) as a brown oil. HPLC method C: t _R  = 4.15 min (99.03%) and for rad t _R  = 4.38 min (99.5%). Chiral HPLC method A: t _R = 8.47 min (99.99%). ¹H NMR with rotamers (DMSO‐d ₆, 500 MHz) δ: 7.92(s, 1H), 7.27(s, 1H), 6.45(s, NH), 4.49–5.03(m, 1H), 4.17(t, J = 7.10 Hz, 2H), 3.52–3.67(m, 2H), 3.42–3.60(m, 2H), 2.87(s, 3H), 2.06(s, 3H), 1.99(brs, 2H), 1.92(brs, 2H), 0.80(s, 2H), 0.58(s, 2H).

5.1.3. Synthesis of [¹⁴C]‐BI 1585773

5.1.3.1. N‐(tert‐Butyl)‐5‐Iodopyridin‐2‐Amine (21)

2‐Amino‐5‐iodopyridine (19) (4.40 g, 20.00 mmol) and tert‐butyl‐2,2,2‐trichloroacetimidate (20) (18.0 g, 82.40 mmol) were dissolved in nitromethane (50 mL), and Cu (OTf)₂ (720 mg, 1.99 mmol) was added under nitrogen at room temperature to give a green mixture. The mixture was stirred for 36 h and then diluted with EtOAc (300 mL) and transferred to a separatory funnel and treated with a saturated solution of NaHCO₃ (300 mL). The aqueous phase was extracted again with EtOAc (300 mL), and the combined extracts were dried over MgSO₄, filtered and concentrated in vacuo to give a brown solid. DCM (20 mL) was added to precipitate the trichloroacetamide byproduct. The mixture was filtered, and the crude product was purified using a 120 g disposable silica gel column and up to 10% EtOAc/DCM to give 2.94 g of a yellow oil in 53% yield. ¹H NMR (CDCl₃, 500 MHz) δ: 8.20(d, J = 2.00 Hz, 1H), 7.53(dd, J = 2.0, 8.5 Hz, 1H), 6.26(d, J = 8.50 Hz, 1H), 1.40(s, 9H). ¹³C NMR (CDCl₃, 125.75 MHz) δ: 157.06, 153.60, 144.23, 111.05, 67.97, 50.95, 29.25.

5.1.3.2. 6‐(tert‐Butylamino)pyridine‐3‐¹⁴C‐Carboxylic Acid, [¹⁴C]‐22

A solution of the iodide 21 (280 mg, 1.01 mmol) in anhydrous THF (5 mL) was stirred at −25°C under nitrogen. A solution of 2 M isopropyl magnesium chloride in THF (0.7 mL, 1.40 mmol) was added dropwise at −25°C. The resulting solution was stirred at this temperature for 2 h, then a solution of 2.5 M n‐BuLi in hexanes (0.81 mL, 2.03 mmol) was added dropwise. The resulting solution was stirred for 1 h at −20°C, and then cooled to −80°C before [¹⁴C]CO₂ (300 mCi) was transferred slowly to the reaction flask. The mixture was stirred overnight as the temperature rose to 5°C. The reaction mixture was cooled down to −20°C again and water (5 mL) was added. The ice bath was removed, and the mixture was warmed to room temperature and extracted with EtOAc. The EtOAc extracts were dried over MgSO₄, filtered, and concentrated in vacuo to 156 mg of a residue in 79% yield, which was used as is in the next step. ¹H NMR (CDCl₃, 400 MHz) δ: 8.70 (d, J = 2.00 Hz, 1H), 8.13 (dd, J = 2.00, 8.80 Hz, 1H), 6.60 (d, J = 8.50 Hz, 1H), 1.49 (s, 9H).

5.1.3.3. (S)‐N‐(1‐(6‐(tert‐Butylamino)nicotinoyl‐Carbonyl‐¹⁴ C)pyrrolidin‐3‐Yl)‐N‐Methylacetamide, [¹⁴C]‐BI 1585773

A mixture of [ ¹⁴ C]‐22 (156 mg, 0.79 mmol) and 11 (190 mg, 0.98 mmol) in EtOAc (15 mL) was stirred at room temperature under nitrogen. Triethylamine (0.93 mL, 6.66 mmol) was added, and the mixture was further stirred for 30 min. A solution of T3P (50% solution in EtOAc, 1.06 mL, 1.82 mmol) was then added dropwise. The mixture was stirred for 14 h. A saturated solution of NaHCO₃ (50 mL) was added to the thick mixture to give a solution. EtOAc (50 mL) was added, and the mixture was stirred vigorously. After letting the bi‐phasic solution settle, the upper organic layer was removed, and the aqueous layer was extracted one more time with EtOAc (50 mL). The combined organic extracts were filtered through a phase separator column and concentrated in vacuo to give 250 mg of a residue. Purification on a 40 g disposable silica gel column 0%–10% MeOH/DCM gave 107 mg of an off‐white solid in 42% yield with a specific activity of 55.59 mCi/mmol. HPLC method C: Radiochemical purity t _R  = 3.28 min (98.40%), chemical purity, t _R  = 3.20 (99.30%). Chiral HPLC method B: t _R  = 9.48 min (99.9% ee). ¹H NMR (DMSO‐d ₆, 500 MHz) d: 7.51(s, 1H), 6.65(d, J = 4.50 Hz, 1H), 6.48(d, J = 9.0 Hz, 1H), 5.03(brs, 0.5H), 4.73(brs, 0.5H), 3.42–3.75(m, 4H), 2.86(s, 3H), 1.85–2.06(m, 5H), 1.39(s, 9H).

5.1.4. Synthesis of [¹⁴C]‐BI 1595043

5.1.4.1. (E)‐3‐(2‐(4‐Fluorophenyl)prop‐1‐En‐1‐Yl)‐5‐Nitropyridin‐2‐Amine (26)

In a two‐necked round bottomed flask was charged with vinyl bromide 23 (1.08 g 5.00 mmol), KOAc (0.98 g, 10.00 mmol), B₂Pin₂ (1.40 g, 5.50 mmol) and PdCl₂(dppf).DCM (122 mg, 0.15 mmol) under nitrogen, followed by IPA (5 mL). The flask was evacuated and refilled with nitrogen three times, then heated to 90°C and stirred for 1 h. The reaction was cooled to 40°C, and 2‐amino‐3‐iodo‐5‐nitropyridine (25) (1.33 g, 5.00 mmol) was added, followed by IPA (2 mL) and K₂CO₃ (2.76 g, 20.00 mmol) dissolved in water (6 mL). The mixture was heated to 85°C and stirred for 17 h. The dark reaction mixture was cooled to room temperature, diluted with EtOAc (20 mL), and filtered through a short pad of Celite. The dark solution was washed with water (100 mL), and the organic phase was dried over MgSO₄, filtered and concentrated in vacuo to give 1.37 g of a dark solid in 100% crude yield. A portion of this material (103 mg) was dissolved in DCM and applied on top of a 25 g disposable silica gel column and eluted with up to 30% EtOAc/DCM to give 36 mg a bright yellow solid. ¹H NMR (DMSO‐d ₆, 500 MHz) δ: 8.83(s, 1H), 8.01(s, 1H), 7.76(t, J = 10.00 Hz, 2H), 7.23(t, J = 10.00 Hz, 2H), 6.57(s, 1H), 5.76(s, 2H), 2.17(s, 3H). ¹³C NMR (DMSO‐d ₆, 125.75 MHz) δ: 161.82, 161.34, 145.67, 139.93, 135.51, 131.95, 129.66, 129.03, 128.73, 128.67, 120.49, 115.52, 115.35, 17.84. ¹⁹F NMR (DMSO‐d ₆, 470.60 MHz) δ: −114.81.

5.1.4.2. 2‐(4‐Fluorophenyl)‐2‐Methyl‐5‐Nitro‐2,3‐Dihydro‐1H‐Pyrrolo[2,3‐B]Pyridine (27)

The crude 26 (663 mg, 2.43 mmol) was placed in a vial. Concentrated sulfuric acid (3 mL, 98%, 55.20 mmol) was added to give a dark mixture. After stirring for 1 h, the black viscous oil was poured in ice‐water (50 g) and treated with 5 M aqueous NaOH until pH = 8–9. The mixture was extracted with EtOAc (200 mL × 3), and the combined extracts were dried over MgSO₄, filtered, and concentrated in vacuo to give 860 mg of a sandy solid. The solid was adsorbed onto silica gel (5 g) with EtOAc (20 mL) in vacuo, transferred to a cartridge and purified on a silica gel column (40 g) 0%–30% EtOAc/DCM to give 420 mg of a yellow‐orange solid in 63% yield. ¹H NMR (DMSO‐d ₆, 500 MHz) δ: 9.01(s, 1H), 8.78(d, J = 2.00 Hz, 1H), 7.95(s, 1H), 7.48(dt, J = 5.50, 8.00 Hz, 2H), 7.20(t, J = 8.90 Hz, 2H), 3.30(d, J = 15.00 Hz, 1H), 3.17(d, J = 15.00 Hz, 1H), 1.62(s, 3H). ¹³C NMR (DMSO‐d ₆, 125.75 MHz) δ: 165.96, 162.49, 147.29, 143.94, 136.17, 134.2, 127.50, 127.43, 126.40, 115.66, 115.49, 65.26, 53.8, 30.11.

5.1.4.3. 2‐(4‐Fluorophenyl)‐2‐Methyl‐2,3‐Dihydro‐1H‐Pyrrolo[2,3‐B]Pyridin‐5‐Amine (28)

The nitro compound 27 (390 mg, 1.43 mmol) was dissolved in ethyl acetate (10 mL) (heating was used to dissolve the material). The material was transferred to a reaction flask containing 100 mg of 5% Pt/C. The mixture was stirred under 150 psi of hydrogen for 8 h at 25°C. The colorless mixture, which turned yellow upon exposure to the air, was filtered through a short pad of Celite and rinsed with methanol and concentrated in vacuo to give 283 mg of orange‐brown solid in 81% yield. The solid was kept under nitrogen at −25°C but still turned dark. ¹H NMR (MeOD, 500 MHz) δ: 7.51(m, 2H), 7.36(m, 1H), 7.05(m, 2H), 6.97(s, 1H), 3.18(d, J = 15.00 Hz, 1H), 3.06(d, J = 15.00 Hz, 1H), 1.61(s, 3H). ¹³C NMR (MeOD, 125.75 MHz) δ: 162.55, 156.84, 144.46, 135.35, 131.07, 128.33, 126.79, 123.70, 121.2, 114.46, 114.29, 63.99, 54.6, 28.40. ¹⁹F NMR (MeOD, 470.60 MHz) δ: −119.26.

5.1.4.4. 2‐(4‐Fluorophenyl)‐5‐Iodo‐2‐Methyl‐2,3‐Dihydro‐1H‐Pyrrolo[2,3‐B]Pyridine (29)

To a solution of 28 (277 mg, 1.14 mmol) in acetonitrile (5 mL), was added p‐toluenesulfonic acid monohydrate (660 mg, 3.42 mmol). The mixture was cooled to 10°C. A solution of potassium iodide (480 mg, 2.89 mmol) and sodium nitrite (158 mg, 2.29 mmol) in water (1 mL) was added slowly. After stirring for 10 min at 10°C, the ice bath was removed, and the dark mixture was stirred at room temperature for 1 h. Water (20 mL) was added, followed by a saturated aqueous solution of NaHCO₃ (6 mL) until pH = 9–10, followed by a saturated aqueous solution of Na₂S₂O₃ (10 mL). The honey‐colored mixture was extracted with EtOAc (100 mL × 3), and the combined extracts were dried over MgSO₄, filtered and concentrated in vacuo to give 310 mg of a dark solid. The crude product was purified using a 40 g disposable silica gel column 0%–30% EtOAc/DCM to give 172 mg of a residue in 43% yield. LCMS: t _R = 1.74 min (100%), m/z = 355 (100%). Chiral HPLC method C: desired enantiomer, t _R  = 5.85 (49.63%). Undesired enantiomer t _R  = 6.27 min (50.36%). ¹H NMR (DMSO‐d ₆, 500 MHz) δ: 8.23(s, 1H), 7.91(s, 1H), 7.48(m, 3H), 7.16(t, J = 7.50 Hz, 2H), 3.22(d, J = 15.00 Hz, 1H), 3.03(d, J = 15.00 Hz, 1H), 1.53(s, 3H). ¹³C NMR (DMSO‐d ₆, 125.75 MHz) δ: 162.39, 160.37, 151.60, 144.85, 139.00, 128.28, 127.63, 124.49, 115.39, 114.72, 77.18, 64.36, 53.72, 30.07.

The above reaction was performed again on a 5.0 g scale of 28 to give about 3.0 g of product 29 in 38% yield. Separation of 2.9 g of the racemic gave 1.14 g (39.3%) recovery of (R)‐2‐(4‐fluorophenyl)‐5‐iodo‐2‐methyl‐2,3‐dihydro‐1H‐pyrrolo[2,3‐b]pyridine (30). HPLC method A: 97.2%, and chiral HPLC method C (99.5% ee). The other enantiomer (S)‐2‐(4‐fluorophenyl)‐5‐iodo‐2‐methyl‐2,3‐dihydro‐1H‐pyrrolo[2,3‐b]pyridine (31) recovered in 46.8% (1.36 g), HPLC 99.9%, and chiral HPLC (99.5% ee).

5.1.4.5. (R)‐2‐(4‐Fluorophenyl)‐2‐Methyl‐2,3‐Dihydro‐1H‐Pyrrolo[2,3‐B]Pyridine‐5‐Carboxylic‐¹⁴ C Acid, [¹⁴C]‐32

A solution of the iodide 30 (354 mg, 1.00 mmol) in anhydrous THF (5 mL) was stirred at −25°C under nitrogen. A 2 M solution of isopropyl magnesium chloride in THF (0.80 mL, 1.60 mmol) was added dropwise at −25°C. The resulting solution was stirred at this temperature for 2 h, then a 2 M solution of n‐BuLi in hexanes (0.80 mL, 1.60 mmol) was added dropwise. The resulting solution was stirred for 1 h at −20°C, then cooled to −80°C. [¹⁴C]CO₂ (200 mCi) was transferred to the reaction flask. The resulting mixture was stirred for 1 h at −80°C then warmed to room temperature. Then cooled back to −20°C and quenched with water (5 mL) and warmed to room temperature. EtOAc (15 mL) was added, and the mixture was stirred vigorously for a few minutes then left to settle. The organic layer was removed and the aqueous was extracted with EtOAc (10 mL × 2). The combined EtOAc extracts were dried over MgSO₄, filtered and concentrated in vacuo to give 191 mg of a yellow film, which was purified on a 25 g disposable silica gel column 0%–30% MeOH/DCM to give 125 mg of pure material. Substantial amounts of the product were observed in the aqueous layer, which was filtered and concentrated in vacuo to 0.4 g of a white solid. This solid was triturated with ethanol and filtered one more time. The filtrate was concentrated then dissolved in methanol and DCM and applied on a 25 g disposable silica gel column and eluted with up to 30% MeOH/DCM. The pure product was combined with that from the first purification and concentrated to give 233 mg (47 mCi) in 85% yield. HPLC method B: Radiochemical purity: t _R  = 9.47 min (98.43%).

5.1.4.6. N‐((S)‐1‐((R)‐2‐(4‐Fluorophenyl)‐2‐Methyl‐2,3‐Dihydro‐1H‐Pyrrolo[2,3‐B]Pyridine‐5‐Carbonyl‐¹⁴ C)pyrrolidin‐3‐Yl)‐N‐Methylacetamide, [¹⁴C]‐BI 1595043

To a mixture of [ ¹⁴ C]‐32 (150 mg, 0.55 mmol) and CDI (97.21 mg, 0.60 mmol) in a screw cap vial was added anhydrous MeCN (5 mL). The mixture was heated to 50°C and stirred for 2 h under nitrogen atmosphere. The pyrrolidine 11 (107 mg, 0.60 mmol) was added to the reaction flask at 50°C and stirred for another 3 h at 50°C. After cooling to room temperature, water (6 mL) was added, and the mixture was extracted with EtOAc (20 mL × 2). The combined organic extracts were filtered through a phase separator column and concentrated in vacuo to give 340 mg of a yellow residue. Flash chromatography purification using 40 g disposable silica gel column and up to 10% MeOH/DCM gave 170 mg of a white sugar like product in 78% yield, with a specific activity of 57.62 mCi/mmol, and total radioactivity of 21.18 mCi. HPLC method D: Chemical purity: t _R = 5.77 min (99.23%), Radiopurity: t _R = 5.98 min (99.80%). Chiral HPLC method C: t _R = 7.53 min (99.73% ee). ¹H NMR (DMSO‐d ₆, 500 MHz) δ: 8.04(s, 1H), 7.75(s, 1H, NH), 7.50(dd, J = 2.50, 8.50 Hz, 2H), 7.43(s, 1H), 7.17(t, J = 8.50 Hz, 2H), 5.02(brs, 0.5H), 4.48(brs, 0.5H), 3.37–3.72(m, 5H), 3.06(d, J = 15.00 Hz, 1H), 2.07(s, 3H), 1.95–2.06(m, 5H), 1.55(s, 3H).

5.1.5. Synthesis of [²H₆]‐BI 764122, [²H₆]‐BI 158577 and [²H₆]‐BI 1595043

5.1.5.1. (S)‐1‐Benzyl‐N‐(Methyl‐d ₃)pyrrolidin‐3‐Amine, [²H₃]‐34

LiAlD₄ (760 mg, 18.00 mmol) was added portion wise to a 3‐neck round bottom flask containing THF (30 mL) and the mixture was heated at 65°C. A solution of (S)‐(‐)‐1‐benzyl‐3‐(Boc‐amino)pyrrolidine (33) (3.0 g, 10.85 mmol) in THF (30 mL) was then added dropwise to the above mixture maintaining the internal temperature of the reaction mixture at around 70°C. Heating was continued at 65°C for 2.5 h, then the reaction mixture was cooled to room temperature and quenched with water (1 mL), 15% aqueous NaOH (1 mL), and finally with water (3 mL). The mixture was treated with THF (30 mL) and stirred for 30 min, then filtered through a short pad of Celite and rinsed with THF (50 mL). The filtrate was concentrated in vacuo to give 2.1 g of a colorless oil in quantitative yield. m/z = 194.6 (100%). ¹H NMR (CDCl₃, 400 MHz) δ: 7.21–7.32 (m, 5H), 3.60 (s, 2H), 3.20 (m, 1H), 2.70 (dd, J = 8.0, 4.0 Hz, 1H), 2.63 (m, 1H), 2.51 (m, 1H), 2.36 (dd, J = 4.0, 8.0 Hz, 1H), 2.11 (m, 1H), 1.57 (m, 2H). ¹³C NMR (CDCl₃, 100.6 MHz) δ: 139.08, 128.82, 128.22, 126.90, 60.54, 60.49, 59.07, 53.07, 34.07 (m), 31.87.

5.1.5.2. (S)‐N‐(1‐Benzylpyrrolidin‐3‐Yl)‐N‐(Methyl‐d ₃)acetamide‐d ₃ Hydrochloride Salt, [²H₆]‐35

To a solution of [ ² H ₃ ]‐34 (2.0 g, 10.35 mmol) in THF (40 mL) at 5°C was added CD₃COCl (1.3 mL, 17.60 mmol) dropwise and the mixture was stirred at 0°C–5°C for 2 h to obtain a white precipitate. The mixture was concentrated at 35°C under reduced pressure to give 2.46 g of [ ² H ₆ ]‐35 as a white solid in quantitative yield. m/z 239.5 (100%). ¹H NMR (MeOD, 500 MHz) δ: 7.48–7.57(m, 5H), 4.52(m, 1H), 4.41(m, 1H), 4.28(d, J = 8.0, 4.0 Hz, 1H), 3.96(m, 1H), 3.84(m, 1H), 3.72(m, 1H), 3.60(m, 1H), 3.47(m, 2H), 3.24(m, 1H), 2.58(m, 1H), 2.19–2.31(m, 1.5 H), 1.97–2.06(m, 1.43 H). ¹³C NMR (CDCl₃, 100.6 MHz) δ: (172.05, 171.52), (130.76, 130.45), 130.05, 129.99, 129.83, 129.41, (129.33, 129.28), (59.18, 58.82), 58.37, (53.50, 53.35), (53.05, 52.95), 52.58, 50.79, 38.93(m), 31.32(m), 28.22, 27.02, (22.69, 21.64).

5.1.5.3. (S)‐N‐(methyl‐d ₃)‐N‐(Pyrrolidin‐3‐Yl)acetamide‐d ₃ Hydrochloride Salt, [²H₆]‐11

A solution of [ ² H ₆ ]‐35 (2.0 g, 7.30 mmol) in methanol (30 mL) was transferred to a hydrogenation reactor and 10% Pd/C (1.0 g) was added at room temperature. The mixture was evacuated and backfilled with nitrogen 3 times, followed once by hydrogen and stirred at 50°C for 12 h under 400 psi of H₂. The mixture was then filtered through a short pad of Celite and concentrated in vacuo to give 1.79 g of crude [ ² H ₆ ]‐11 as a yellowish oil in quantitative yield. m/z 185.9. ¹H NMR (MeOD, 400 MHz) δ: 4.63(m, 1H), 3.64(m, 1H), 3.43(m, 2H), 3.24(m, 1H), 2.38(m, 1H), 2.20(m, 1H). ¹³C NMR (CDCl₃, 100.6 MHz) δ: 172.92, 56.24, 45.46, 44.41, 34.30(m), 27.31, 20.30(m).

5.1.5.4. (S)‐N‐(1‐(2′,3′‐Dihydro‐5′H‐Spiro[cyclopropane‐1,4′‐Pyrido[3,2‐B][1,4]Oxazepine]‐8′‐Carbonyl)pyrrolidin‐3‐Yl)‐N‐(Methyl‐d ₃)acetamide‐d ₃, [²H₆]‐BI 764122

A stirred mixture of 10 (1.80 g, 7.54 mmol) in DMAC (11 mL) was added CDI (1.73 g, 10.02 mmol) in portions under N₂, and the stirring was continued for 1 h at room temperature. A solution of [ ² H ₆ ]‐11 (1.32 g, 6.78 mmol) in DMAC (4 mL) was added in portions and the mixture was stirred at 35°C for 3 h. Water (3.6 mL) was then added and stirring continued for 14 h. The mixture was extracted with EtOAc (30 mL × 3), and the combined organics were concentrated. The crude product was purified by chromatography on SiO₂ (100% DCM to 10% MeOH/DCM) to obtain 1.66 g of [ ² H ₆ ]‐BI 764122 in 70% yield as a white solid. HPLC method A: t _R = 3.92 min (98.14%). Chiral HPLC method A, t _R  = 9.43 min (99.91%). HRMS m/z [M + H]⁺: [C₁₈H₁₉ ²H₆N₄O₃]⁺: 351.2297 (95.60%); [C₁₈H₂₀ ²H₅N₄O₃]⁺: 350.2234 (4.40%). ¹HNMR (DMSO‐d ₆, 600 MHz) δ: 7.95(s, 1H), 7.29(s, 1H), 6.48(s, NH), 4.44–5.10(br, 1H), 4.18(t, J = 7.10 Hz, 2H), 3.32–3.65(br, 4H), 1.80–2.25(br, 4H), 0.81(s, 2H), 0.58(s, 2H). ¹³C NMR (DMSO‐d ₆, 151 MHz) δ: (170.44, 168.77), 155.38, 142.02, 140.90, 126.45, 122.88, 69.98, (54.69, 50.27, 49.26, 47.35, 44.44), 37.68, 34.67, (28.47, 26.44, 25.89, 21.51), 13.49.

5.1.5.5. (S)‐N‐(1‐(6‐(tert‐Butylamino)nicotinoyl)pyrrolidin‐3‐Yl)‐N‐(Methyl‐d ₃)acetamide‐d ₃, [²H₆]‐BI 1585773

To a mixture of 22 (1.0 g 5.00 mmol) and [ ² H ₆ ]‐11 (1.07 g 5.50 mmol) in EtOAc (10 mL) was added triethylamine (3.48 mL, 25.00 mmol) and stirred at room temperature under N₂ for 20 min. A solution of T3P (50% in EtOAc, 6 mL, 10.00 mmol) was then added dropwise and the mixture was stirred for 14 h. The reaction mixture was then diluted with EtOAc (30 mL), quenched with water (10 mL) and stirred to obtain a bi‐phasic mixture. The layers were then separated, and the aqueous layer was further extracted with EtOAc (10 mL × 2). The combined organics were washed once with water (20 mL), followed by brine (10 mL), dried over Na₂SO₄, filtered and concentrated. The mixture was purified by silica gel chromatography (100% EtOAc to 10% MeOH/EtOAc) to obtain 1.02 g (63% yield) as a white foam. HPLC method B: t _R = 3.19 min (99.46%). Chiral HPLC method B: t _R  = 9.48 min (98.2% ee). HRMS: [M + H]⁺: [C₁₇H₂₁ ²H₆N₄O₂]⁺ (93.9%); [C₁₇H₂₂ ²H₅N₄O₂]⁺ (5.5%); [C₁₇H₂₃ ²H₄N₄O₂]⁺ (0.7%). ¹H NMR (DMSO‐d ₆, 400 MHz) δ: 8.22(m, 1H), 7.51(m, 1H), 6.66(d, J = 4.00 Hz, 1H), 6.48(d, J = 8.80, 1H), 4.96(m, 1H), 3.37–3.69(m, 4H), 1.88–2.04(m, 2H), 1.39(s, 9H). ¹³C NMR (DMSO‐d ₆, 100.6 MHz) δ: 170.89, 17,025, 159–94, 148.00, 147.89, 136.17, 136.11, 119.17, 109.36, 59.33(m), 58.37, 39.38, 26.42(m).

5.1.5.6. N‐((S)‐1‐((R)‐2‐(4‐Fluorophenyl)‐2‐Methyl‐2,3‐Dihydro‐1H‐Pyrrolo[2,3‐B]Pyridine‐5‐Carbonyl)pyrrolidin‐3‐Yl)‐N‐(Methyl‐d ₃)acetamide‐d ₃, [²H₆]‐BI 1595043

A mixture of 32 (1.96 g, 6.36 mmol) and CDI (1.2 g, 7.40 mmol) in a two‐necked round bottom flask (100 mL) was added anhydrous MeCN (15 mL). The mixture was heated to 50°C and stirred for 1 h under nitrogen atmosphere. To a solution of [ ² H ₆ ]‐11 (1.60 g, 8.66 mmol) in MeCN (5 mL) was added to the reaction flask at 50°C. The mixture was stirred for another 2 h at 50°C. After cooling to room temperature, water (30 mL) was added, and the mixture was extracted with EtOAc (100 mL × 2). The combined organic extracts were dried over MgSO₄, filtered and concentrated in vacuo to give 2.82 g of a white solid. Flash chromatography purification using 120 g disposable silica gel column and up to 10% MeOH/DCM gave 2.33 g of a white sugar like product in 91% yield. HPLC method B: t _R = 4.59 min (98.87%). Chiral HPLC method B: t _R 9.85 min (98.32%). HRMS: [M + H]⁺: [C₂₂H₂₀ ²H₆N₄O₂F]⁺ (96.7%); [C₂₂H₂₁ ²H₅N₄O₂F]⁺ (3.3%). ¹H NMR (DMSO‐d ₆, 500 MHz) δ: 8,04(d, J = 10.00 Hz, 1H), 7.75(d, J = 2.00 Hz, 1H), 7.50(dd, J = 2.00, 10.00 Hz, 2H), 7.43(d, J = 2.00 Hz, 1H), 7.17(t, J = 2.00, 10.00 Hz, 2H), 4.45(m, 1H), 3.17–3.68(m, 6H), 3.05(d, J = 15.00 Hz, 1H), 1.85–2.04(m, 2H), 1.35(s, 3H). ¹³C NMR (DMSO‐d ₆, 100.6 MHz) δ: 170.90, 170.26, 167.89, 164.17, 162.31, 160.38, 147.30, 144.96, 144.94, 131.18, 131.11, 127.59, 127.53, 121.35, 120.24, 115.40, 115.23, 64.16, 52.74(m), 43.57, 30.22, 26.60(m). ¹⁹F NMR (DMSO‐d ₆, 470.6 MHz) δ: −116.86.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

Data are available on request from the authors.