Efficient Synthesis of Pyrazinoic Acid Hybrid Conjugates

Abstract

Benzotriazole-activated pyrazinoic acid was utilized as a versatile building block for the efficient and convenient synthesis of novel hybrid conjugates of pyrazinoic acid with secondary amines via amino acid linkers in high yields.

Key words

pyrazinamide - pyrazinoic acid - secondary amines - amino acids - benzotriazole methodology - antituberculosis

Tuberculosis (TB) is a bacterial pathogen caused by Mycobacterium­ tuberculosis, which is known to cause pulmonary infection and to become extremely pervasive within the lungs.[1] [2] [3] TB is considered to be one of the world’s deadliest communicable diseases because of its high virulence and the ability of M. tuberculosis to enter into a dormant­ ­­state, then subsequently undergo reactivation.[3] [4] [5] Pyrazinamide (PZA) is a first-line antituberculosis prodrug that is often used in combinational therapy with drugs such as isoniazide, ethambutol, streptomycin, and rifampicin (Figure [1]).[6–8]

Figure 1 Current antituberculosis drugs

PZA is perceived to inhibit vital ribosomal proteins after being converted into its active constituent, pyrazinoic acid (POA), by the tuberculosis enzyme, pyrazinamidase (PZAase) (Scheme [1]).[9] It may lower the pH of the area surrounding M. tuberculosis to such an extent that the organism is unable to grow. Due to its low lipophilicity, POA cannot be absorbed by the gastrointestinal tract. Fortunately, the drug can be absorbed in the pyrazinamide configuration.

Scheme 1 PZA is converted into POA in the presence of PZAase

One of the drawbacks of using PZA to treat TB is that it inhibits protein synthesis. With prolonged administration of the recommended dose, harmful side effects such as hepatitis, acute hypertension, thrombocytopenia, and gastrointestinal discomfort have been reported.[10] To overcome these issues, several molecular hybridization approaches have been reported for the development of potential antitubercular agents. Most hybridized structures include clinically used drugs such as rifamycin, ethambutol and isoniazid coupled with other hydrophobic structures such as cinnamic acid derivatives.[11] [12] [13] [14] Unfortunately the most promising prodrugs of POA are not stable.[15]

Secondary amines such morpholine, piperidine, N-methylpiperazine, and pyrrolidine are important scaffolds for potential biological molecules. Structural activity relationships (SARs) suggest that these secondary amines play an important role in bioactive molecules.[16] Drug–amino acid conjugates are used to enhance drug delivery and to increase tissue cell penetration. In addition to acting as carriers of these agents, amino acids also amplify their bioavailability while maintaining their bioactive integrity. Recently, we have synthesized and reported several bioconjugates with enhanced biological properties and increased lipophilicity.[17] [18] [19] [20]

In a continuation of our interest in synthesizing amino acid–peptide conjugates with biological significance, we report herein an efficient synthesis of pyrazimide hybrid conjugates, which contain various amino acids and secondary amines, that may decrease the dose of PZA required to fight TB, increase the drug’s lipophilicity and decrease adverse effects.

Table 1 Boc-Protected Amino Acid–Secondary Amine Conjugates 11a–t

Entry	Product 11		Yield (%)	Mp (°C)	[α] d 20 (c 1.0 in MeOH)
1	Boc-Gly-Mor	11a	62	114–116	–
2	Boc-l-Ala-Mor	11b	67	oil	–20.3
3	Boc-dl-Ala-Mor	11b′	74	oil	racemic
4	Boc-l-Val-Mor	11c	75	135–137	–17.5
5	Boc-l-Ile-Mor	11d	81	oil	–23.6
6	Boc-l-Phe-Mor	11e	84	129–131	–15.0
7	Boc-Gly-Pip	11f	90	oil	–
8	Boc-l-Ala-Pip	11g	97	oil	–19.6
9	Boc-l-Val-Pip	11h	78	oil	–20.6
10	Boc-l-Ile-Pip	11i	79	oil	–21.8
11	Boc-l-Phe-Pip	11j	81	122–124	–18.5
12	Boc-Gly-NMP	11k	76	oil	–
13	Boc-l-Ala-NMP	11l	81	oil	–20.0
14	Boc-l-Val-NMP	11m	84	oil	–14.3
15	Boc-l-Ile-NMP	11n	69	oil	–19.6
16	Boc-l-Phe-NMP	11o	83	111–113	–16.5
17	Boc-Gly-Pyr	11p	89	oil	–
18	Boc-l-Ala-Pyr	11q	83	oil	–21.1
19	Boc-l-Val-Pyr	11r	67	oil	–22.3
20	Boc-l-Ile-Pyr	11s	72	oil	–18.6
21	Boc-l-Phe-Pyr	11t	78	126–128	–24.0

POA was activated as its benzotriazolide derivative 1 by following the previously reported procedure[21] and coupled with free amino acids 2 in a mixture of acetonitrile and water (7:3) at 20 °C for 2 h in the presence of 1.5 equivalents of triethylamine to give compounds 3. Attempts to prepare benzotriazole derivatives of POA–amino acid conjugates failed. We were also unsuccessful in coupling compounds 3 with secondary amines using different coupling reagents and ended up with mixtures of compounds as evidenced by TLC (Scheme [2]). We therefore decided to redesign our approach and synthesize bis-conjugates of POA–secondary amines by coupling Boc-protected aminoacylbenzotriazoles[22] with secondary amines. After removing the Boc group with dioxane–HCl, the unprotected conjugates were coupled with benzotriazole-activated POA to produce the desired products in good yields (Scheme [3]). Boc-protected aminoacylbenzotriazoles 6a–d were treated with secondary amines 7–10 in the presence of triethylamine in acetonitrile at 20 °C for 2 h to obtain the conjugates in good yields without loss of chiral integrity (Scheme [3], Table [1]).

Scheme 2 Synthesis of POA hybrid conjugates with secondary heterocyclic amines and amino acid (Route I)

Scheme 3 Synthesis of Boc-protected amino acid–secondary heterocyclic amine conjugates

Boc-protected amino acid–secondary amine conjugates 11a–t were deprotected using a dioxane–HCl mixture at 20 °C for 1 h to give unprotected amino acid–secondary amine conjugates 12a–t. These compounds were then used in the next step without further characterization. The target compounds 5a–t were prepared by coupling the unprotected amino acid–secondary amine conjugates with POA-benzotriazolide 1 in the presence of triethylamine in acetonitrile at 20 °C for 3 h (Scheme [4], Table [2]). All compounds were fully characterized by NMR spectroscopy, HRMS and specific rotation. X-ray diffraction analysis of compound 5a further confirmed the formation of the hybrid conjugate (Figure [2]).

Table 2 POA Hybrid Conjugates 5a–t

Entry	Product 5		Yield (%)	Mp (°C)	[α] d 20 (c 1.0 in MeOH)
1	POA-Gly-Mor	5a	75	180–181	–
2	POA-l-Ala-Mor	5b	69	sticky	–25.4
3	POA-dl-Ala-Mor	5b′	72	oil	racemic
4	POA-l-Val-Mor	5c	76	oil	–19.2
5	POA-l-Ile-Mor	5d	70	oil	–20.3
6	POA-l-Phe-Mor	5e	78	sticky	–25.6
7	POA-Gly-Pip	5f	74	123–125	–
8	POA-l-Ala-Pip	5g	75	oil	–19.9
9	POA-l-Val-Pip	5h	81	oil	–23.6
10	POA-l-Ile-Pip	5i	64	sticky	–22.0
11	POA-l-Phe-Pip	5j	79	oil	–20.1
12	POA-Gly-NMP	5k	69	sticky	–
13	POA-l-Ala-NMP	5l	73	oil	–26.5
14	POA-l-Val-NMP	5m	62	oil	–20.9
15	POA-l-Ile-NMP	5n	64	oil	–23.0
16	POA-l-Phe-NMP	5o	71	oil	–28.1
17	POA-Gly-Pyr	5p	81	148–150	–
18	POA-l-Ala-Pyr	5q	79	oil	–23.0
19	POA-l-Val-Pyr	5r	82	oil	–28.5
20	POA-l-Ile-Pyr	5s	74	oil	–26.6
21	POA-l-Phe-Pyr	5t	73	oil	–25.1

Figure 2 ORTEP diagram of POA-Gly-Mor (5a)

Scheme 4 Synthesis of POA hybrid conjugates with secondary heterocyclic amines and amino acid (Route II)

In conclusion, benzotriazole-activated pyrazinoic acid has been used as a precursor for the efficient synthesis of pyrazinoic acid hybrid conjugates. The hybrid conjugates may be candidates for the development of new antituberculosis agents.

Melting points were determined with a capillary melting-point apparatus equipped with a digital thermometer. Reactions were monitored by using thin-layer chromatography (TLC) on 0.2 mm silica gel F254 plates (Merck). The chemical structures of final products and intermediates were characterized by ¹H and ¹³C NMR spectroscopy with a Bruker NMR spectrometer (500 MHz, 125 MHz). ¹³C NMR spectra were fully decoupled. Chemical shifts are reported in parts per million (ppm) using the deuterated solvent peak or tetramethylsilane as an internal standard. Mass spectrometric analysis was carried out with a high-resolution Biosystems QStar Elite time-of-flight electrospray mass spectrometer or an Agilent 6210 instrument using time-of-flight mass spectrometry (TOF-MS) with electrospray ionization (ESI). Specific rotation measurements were carried out with an Autopol IV polarimeter.

Synthesis of (1H-benzo[d][1,2,3]triazol-1-yl)(pyrazin-2-yl)methanone (POA-Bt, 1)[21]

1H-Benzotriazole (4.0 equiv) was dissolved in anhydrous methylene chloride. Thionyl chloride (1.2 equiv) was added and the mixture was stirred for 30 min. Pyrazinoic acid (1.0 equiv) was added and the reaction mixture was stirred for 2–3 h at r.t. Upon completion of the reaction, 20% aqueous sodium bicarbonate was added and the organic layer was extracted twice with the alkaline solution, washed with brine, and dried over anhydrous magnesium sulfate. After filtration, the solvent was evaporated and the residue crystallized from diethyl ether to yield 1 in good yield.

Synthesis of Secondary Amine–Amino Acid Conjugates 11a–t; General Procedure

A 50 mL round-bottom flask containing a small stir bar was charged with N-(Boc-aminoacyl)benzotriazole 6a–e (1.0 equiv) and secondary amine (morpholine, piperidine, N-methylpiperazine, pyrrolidine) (1.0 equiv) dissolved in acetonitrile (10 mL) along with triethylamine (1.5 equiv). The reaction mixture was stirred at r.t. for 3–4 h and the progress of the reaction was monitored by TLC. After completion of the reaction, the acetonitrile was evaporated under reduced pressure and the residue was extracted with EtOAc. The organic layer was washed with aqueous sodium carbonate and dried over sodium sulfate. After filtration, the EtOAc was evaporated under reduced pressure to obtain the desired amino acid–secondary amine conjugate in good yield.

tert-Butyl (2-Morpholino-2-oxoethyl)carbamate (Boc-Gly-Mor, 11a)

Yield: 62%; colorless microcrystals; mp 114–116 °C.

¹H NMR (500 MHz, CDCl₃): δ = 5.50 (br s, 1 H), 3.95 (d, J=3.5 Hz, 2 H), 3.70–3.62 (m, 6 H), 3.40 (t, J=4.4 Hz, 2 H), 1.45 (s, 9 H).

¹³C NMR (125 MHz, CDCl₃): δ = 167.5, 156.0, 80.0, 66.9, 66.5, 45.0, 42.4, 42.3, 28.5.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₁H₂₀N₂O₄: 244.1424; found: 244.1425.

tert-Butyl (S)-(1-Morpholino-1-oxopropan-2-yl)carbamate (Boc-l-Ala-Mor, 11b)

Yield: 67%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 5.51 (br s, 1 H), 4.59 (br s, 1 H), 3.67–3.47 (m, 8 H), 1.43 (s, 9 H), 1.29 (d, J=5.8 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 171.5, 155.3, 79.8, 67.0, 66.8, 46.1, 42.6, 28.6, 19.5.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₂H₂₂N₂O₄: 258.1584; found: 258.1580.

tert-Butyl (1-Morpholino-1-oxopropan-2-yl)carbamate (Boc-dl-Ala-Mor, 11b′)

Yield: 74%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 5.57 (d, J=5.8 Hz, 1 H), 4.54 (d, J=5.8 Hz, 1 H), 3.62–3.40 (m, 8 H), 1.37 (s, 9 H), 1.24 (d, J=7 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 171.4, 155.1, 79.6, 66.8, 66.6, 46.0, 42.4, 28.4, 19.2.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₂H₂₂N₂O₄: 258.1584; found: 258.1582.

tert-Butyl (S)-(3-Methyl-1-morpholino-1-oxobutan-2-yl)carbamate (Boc-l-Val-Mor, 11c)

Yield: 75%; colorless microcrystals; mp 135–137 °C.

¹H NMR (500 MHz, CDCl₃): δ = 5.37 (d, J=7.9 Hz, 1 H), 4.34 (t, J=7.9 Hz, 1 H), 3.64–3.48 (m, 8 H), 1.89–1.83 (m, 1 H), 1.36 (s, 9 H), 0.88 (d, J=6.6 Hz, 3 H), 0.83 (d, J=6.6 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 170.8, 155.9, 79.4, 66.9, 66.7, 54.6, 46.3, 42.4, 31.4, 28.3, 19.6, 17.3.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₄H₂₆N₂O₄: 286.1896; found: 286.1894.

tert-Butyl ((2S,3S)-3-Methyl-1-morpholino-1-oxopentan-2-yl)carbamate (Boc-l-Ile-Mor, 11d)

Yield: 81%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 5.32 (d, J=7.9 Hz, 1 H), 4.39 (t, J=7.9 Hz, 1 H), 3.68–3.48 (m, 8 H), 2.30–2.23 (m, 1 H), 1.65–1.64 (m, 1 H), 1.50–1.45 (m, 1 H), 1.38 (s, 9 H), 0.88–0.81 (m, 6 H).

¹³C NMR (125 MHz, CDCl₃): δ = 171.1, 155.9, 79.6, 66.9, 66.8, 54.0, 46.5, 42.5, 38.1, 28.4, 24.1, 15.9, 11.4.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₅H₂₈N₂O₄: 300.2049; found: 300.2054.

tert-Butyl (S)-(1-Morpholino-1-oxo-3-phenylpropan-2-yl)carbamate (Boc-l-Phe-Mor, 11e)

Yield: 84%; colorless microcrystals; mp 129–131 °C.

¹H NMR (500 MHz, CDCl₃): δ = 7.29–7.19 (m, 5 H), 5.44 (d, J=7.5 Hz, 1 H), 4.82–4.78 (m, 1 H), 3.62–3.28 (m, 6 H), 3.05–2.89 (m, 4 H), 1.43 (s, 9 H).

¹³C NMR (125 MHz, CDCl₃): δ = 170.5, 155.2, 136.5, 129.7, 128.8, 127.3, 80.0, 66.7, 66.3, 51.0, 46.2, 42.4, 40.7, 28.5.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₈H₂₆N₂O₄: 334.1898; found: 334.1909.

tert-Butyl (2-Oxo-2-(piperidin-1-yl)ethyl)carbamate (Boc-Gly-Pip, 11f)

Yield: 90%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 5.56 (br s, 1 H), 3.94 (d, J=3.5 Hz, 2 H), 3.56 (t, J=5 Hz, 2 H), 3.31 (t, J=5 Hz, 2 H), 1.65–1.63 (m, 6 H), 1.45 (s, 9 H).

¹³C NMR (125 MHz, CDCl₃): δ = 167.9, 164.3, 79.6, 45.9, 44.2, 41.9, 28.4, 25.9, 24.8, 24.6.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₂H₂₂N₂O₃: 242.1633; found: 242.1641.

tert-Butyl (S)-(1-Oxo-1-(piperidin-1-yl)propan-2-yl)carbamate (Boc-l-Ala-Pip, 11g)

Yield: 97%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 5.61 (d, J=8.4 Hz,1 H), 4.64–4.58 (m, 1 H), 3.62–3.38 (m, 4 H), 1.68–1.53 (m, 6 H), 1.44 (s, 9 H), 1.29 (d, J=6.7 Hz, 1 H).

¹³C NMR (125 MHz, CDCl₃): δ = 171.0, 155.3, 79.5, 46.6, 46.4, 43.4, 28.6, 28.6, 28.5, 26.6, 25.7, 24.7, 19.7

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₃H₂₄N₂O₃: 256.1787; found: 256.1792.

tert-Butyl (S)-(3-Methyl-1-oxo-1-(piperidin-1-yl)butan-2-yl)carbamate (Boc-l-Val-Pip, 11h)

Yield: 78%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 5.37 (d, J=7.5 Hz, 1 H), 4.42 (t, J=7.5 Hz, 1 H), 3.54–3.41 (m, 4 H), 1.89–1.84 (m, 1 H), 1.60–1.45 (m, 6 H), 1.37 (s, 9 H), 0.90 (d, J=6.7 Hz, 3 H), 0.81 (d, J=6.7 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 170.8, 155.9, 79.4, 66.9, 66.7, 54.6, 46.3, 42.4, 31.4. 28.3, 19.6, 17.3.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₅H₂₈N₂O₃: 284.2105; found: 284.2100.

tert-Butyl ((2S,3S)-3-Methyl-1-oxo-1-(piperidin-1-yl)pentan-2-yl)carbamate (Boc-l-Ile-Pip, 11i)

Yield: 79%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 5.33 (d, J=7.9 Hz, 1 H), 4.50 (t, J=7.9 Hz, 1 H), 3.59–3.49 (m, 4 H), 3.20–3.17 (m, 1 H), 1.70–1.53 (m, 8 H), 1.43 (s, 9 H), 0.93 (d, J=5.8 Hz, 3 H), 0.88 (t, J=7.5 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 170.7, 156.1, 79.5, 54.4, 47.1, 43.3, 38.5, 28.6, 26.8, 25.8, 24.7, 24.0, 16.2, 11.7.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₆H₃₀N₂O₃: 298.2260; found: 298.2258.

tert-Butyl (S)-(1-Oxo-3-phenyl-1-(piperidin-1-yl)propan-2-yl)carbamate (Boc-l-Phe-Pip, 11j)

Yield: 81%; colorless microcrystals; mp 122–124 °C.

¹H NMR (500 MHz, CDCl₃): δ = 7.30–7.20 (m, 5 H), 5.48 (d, J=8.5 Hz, 1 H), 4.89–4.85 (m, 1 H), 3.52–3.48 (m, 2 H), 3.28–3.23 (m, 1 H), 3.06–2.97 (m, 3 H), 1.51–1.42 (m, 15 H).

¹³C NMR (125 MHz, CDCl₃): δ = 169.9, 155.2, 136.8, 129.8, 128.6, 127.0, 79.7, 51.1, 46.7, 43.2, 40.5, 28.5, 26.1, 25.5, 24.5.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₉H₂₈N₂O₃: 332.2100; found: 332.2104.

tert-Butyl (2-(4-Methylpiperazin-1-yl)-2-oxoethyl)carbamate (Boc-Gly-NMP, 11k)

Yield: 76%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 5.54 (br s, 1 H), 3.95 (d, J=4 Hz, 2 H), 3.64 (t, J=4.7 Hz, 2 H), 3.40 (t, J=4.7 Hz, 2 H), 2.41–2.38 (m, 4 H), 1.45 (s, 9 H).

¹³C NMR (125 MHz, CDCl₃): δ = 169.7, 164.4, 79.4, 54.7, 54.5, 45.9, 44.2, 42.2, 41.9, 28.4.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₂H₂₃N₃O₃: 257.1739; found: 257.1740.

tert-Butyl (S)-(1-(4-Methylpiperazin-1-yl)-1-oxopropan-2-yl)carbamate (Boc-l-Ala-NMP, 11l)

Yield: 81%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 5.53 (d, J=7.6 Hz,1 H), 4.55–4.50 (m, 1 H), 3.63–3.40 (m, 4 H), 2.35–2.30 (m, 4 H), 2.23 (s, 3 H), 1.36 (s, 9 H), 1.22 (d, J=6.9 Hz, 1 H).

¹³C NMR (125 MHz, CDCl₃): δ = 171.2, 155.2, 79.6, 55.1, 54.7, 46.2, 46.0, 45.4, 42.0, 28.5, 19.5.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₃H₂₅N₃O₃: 271.1896; found: 271.1899.

tert-Butyl (S)-(3-Methyl-1-(4-methylpiperazin-1-yl)-1-oxobutan-2-yl)carbamate (Boc-l-Val-NMP, 11m)

Yield: 84%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 5.34 (d, J=7.2 Hz, 1 H), 4.30 (d, J=7.2 Hz, 1 H), 3.53–3.39 (m, 4 H), 2.27–2.22 (m, 4 H), 2.15 (s, 3 H), 1.82–1.76 (m, 1 H), 1.29 (s, 9 H), 0.81 (d, J=6.7 Hz, 3 H), 0.74 (d, J=6.7 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 170.4, 155.8, 79.3, 55.2, 54.7, 45.9, 45.6, 41.9, 31.5, 28.3, 19.7, 17.1.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₅H₂₉N₃O₃: 299.2211; found: 299.2208.

tert-Butyl ((2S,3S)-3-Methyl-1-(4-methylpiperazin-1-yl)-1-oxopentan-2-yl)carbamate (Boc-l-Ile-NMP, 11n)

Yield: 69%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 5.26 (d, J=8.2 Hz, 1 H), 4.41 (t, J=8.2 Hz, 1 H), 3.66–3.46 (m, 4 H), 2.35–2.32 (m, 4 H), 2.24 (s, 3 H), 2.15–2.10 (m, 1 H), 1.65–1.60 (m, 1 H), 1.49–1.45 (m, 1 H), 1.29 (s, 9 H), 0.91–0.80 (m, 6 H).

¹³C NMR (125 MHz, CDCl₃): δ = 170.9, 156.0, 79.6, 55.4, 54.9, 54.3, 46.1, 45.9, 42.1, 38.4, 28.5, 24.1, 16.1, 11.6.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₆H₃₁N₃O₃: 313.2365; found: 313.2366.

tert-Butyl (S)-(1-(4-Methylpiperazin-1-yl)-1-oxo-3-phenylpropan-2-yl)carbamate (Boc-l-Phe-NMP, 11o)

Yield: 83%; white microcrystals; mp 111–113 °C.

¹H NMR (500 MHz, CDCl₃): δ = 7.29–7.18 (m, 5 H), 5.45 (d, J=8.2 Hz, 1 H), 4.85–4.80 (m, 1 H), 3.60–3.52 (m, 2 H), 3.34–3.28 (m, 1 H), 2.99–2.92 (m, 3 H), 2.33–2.17 (m, 7 H), 1.42 (m, 9 H).

¹³C NMR (125 MHz, CDCl₃): δ = 170.2, 155.2, 136.6, 129.8, 128.7, 127.2, 79.9, 54.7, 54.5, 51.1, 46.0, 45.6, 42.0, 40.7, 28.6.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₉H₂₉N₃O₃: 347.2211; found: 347.2220.

tert-Butyl (2-Oxo-2-(pyrrolidin-1-yl)ethyl)carbamate (Boc-Gly-Pyr, 11p)

Yield: 89%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 5.38 (br s, 1 H), 4.07 (d, J=4.3 Hz, 2 H), 3.56–3.38 (m, 4 H), 2.03–1.84 (m, 4 H).

¹³C NMR (125 MHz, CDCl₃): δ = 168.3, 163.1, 79.6, 46.9, 46.3, 43.1, 28.5, 26.3, 24.5.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₁H₂₀N₂O₃: 228.1475; found: 228.1477.

tert-Butyl (S)-(1-Oxo-1-(pyrrolidin-1-yl)propan-2-yl)carbamate (Boc-l-Ala-Pyr, 11q)

Yield: 83%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 5.48 (br s, 1 H), 4.47–4.42 (m, 1 H), 3.63–3.40 (m, 4 H), 1.99–1.86 (m, 4 H), 1.43 (s, 9 H), 1.31 (d, J=6.7 Hz, 1 H).

¹³C NMR (125 MHz, CDCl₃): δ = 171.4, 155.3, 79.6, 48.0, 46.5, 46.1, 28.6, 26.2, 24.3, 18.9.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₂H₂₂N₂O₃: 242.1630; found: 242.1637.

tert-Butyl (S)-(3-Methyl-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl)carbamate (Boc-l-Val-Pyr, 11r)

Yield: 67%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 5.26 (d, J=7.8 Hz, 1 H), 4.15 (t, J=7.8 Hz, 1 H), 3.60–3.30 (m, 4 H), 1.87–1.75 (m, 5 H), 1.37 (s, 9 H), 0.86 (d, J=6.7 Hz, 3 H), 0.83 (d, J=6.7 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 170.7, 155.8, 79.2, 57.0, 46.6, 45.7, 31.3, 28.3, 26.0, 24.1, 19.5, 17.5.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₄H₂₆N₂O₃: 270.1945; found: 270.1946.

tert-Butyl ((2S,3S)-3-Methyl-1-oxo-1-(pyrrolidin-1-yl)pentan-2-yl)carbamate (Boc-l-Ile-Pyr, 11s)

Yield: 72%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 5.23 (d, J=8.5 Hz, 1 H), 4.27 (t, J=8.5 Hz, 1 H), 3.74–3.69 (m, 1 H), 3.56–3.20 (m, 4 H), 1.67–1.86 (m, 4 H), 1.73–1.55 (m, 2 H), 1.43 (s, 9 H), 0.93–0.86 (m, 6 H).

¹³C NMR (125 MHz, CDCl₃): δ = 171.2, 156.0, 79.6, 56.6, 48.1, 46.9, 46.0, 38.2, 28.6, 26.2, 24.4, 15.8, 11.5.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₅H₂₈N₂O₃: 284.2101; found: 284.2111.

tert-Butyl (S)-(1-Oxo-3-phenyl-1-(pyrrolidin-1-yl)propan-2-yl)carbamate (Boc-l-Phe-Pyr, 11t)

Yield: 78%; colorless microcrystals; mp 126–128 °C.

¹H NMR (500 MHz, CDCl₃): δ = 7.29–7.22 (m, 5 H), 5.43 (d, J=7.8 Hz, 1 H), 4.63–4.58 (m, 1 H), 3.48–3.33 (m, 3 H), 3.28–3.23 (m, 1 H), 3.01–2.94 (m, 2 H), 2.63–2.57 (m, 1 H), 1.82–1.56 (m, 4 H), 1.44 (m, 9 H).

¹³C NMR (125 MHz, CDCl₃): δ = 170.1, 155.3, 136.8, 129.7, 128.5, 127.0, 79.8, 53.8, 46.4, 45.9, 40.5, 28.6, 26.0, 24.2.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₈H₂₆N₂O₃: 318.1944; found: 318.1943.

Synthesis of Bisconjugates 5a–t; General Procedure

The secondary amine–amino acid conjugate was stirred in 4 M HCl–dioxane solution for 1 h. The dioxane was evaporated under reduced pressure and the residue was treated with diethyl ether. The resulting solid was treated without further purification with the benzotriazole derivative of pyrazinoic acid in the presence of triethylamine (1.5 equiv) in acetonitrile (10 mL). The reaction mixture was stirred at 20 °C for 4–6 h, monitoring by TLC. Upon completion of reaction, the acetonitrile was evaporated and the residue was extracted with EtOAc. The organic layer was washed with aqueous sodium carbonate and dried over anhydrous sodium sulfate. After filtration, the EtOAc was evaporated under reduced pressure to obtain the desired conjugates in good yields.

N-(2-Morpholino-2-oxoethyl)pyrazine-2-carboxamide (POA-Gly-Mor, 5a)

Yield: 75%; colorless microcrystals; mp 180–181 °C.

¹H NMR (500 MHz, CDCl₃): δ = 9.32 (s, 1 H), 8.70 (br s, 1 H), 8.65 (br s, 1 H), 8.53 (br s, 1 H), 4.23 (d, J=4.3 Hz, 2 H), 3.68–3.63 (m, 6 H), 3.46–3.44 (m, 2 H).

¹³C NMR (125 MHz, CDCl₃): δ = 166.4, 163.3, 147.5, 144.3, 143.1, 66.8, 66.5, 45.1, 42.5. 41.2.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₁H₁₄N₄O₃: 250.1068; found: 250.1074.

(S)-N-(1-Morpholino-1-oxopropan-2-yl)pyrazine-2-carboxamide (POA-l-Ala-Mor, 5b)

Yield: 68%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 9.34 (s, 1 H), 8.74–8.70 (m, 2 H), 8.50 (br s, 1 H), 5.13–5.08 (m, 1 H), 3.69–3.53 (m, 8 H), 1.45 (d, J=6.9 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 170.8, 162.6, 147.5, 144.3, 143.0, 66.9, 66.7, 46.2, 45.4, 42.8, 19.0.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₂H₁₆N₄O₃: 264.1221; found: 264.1225.

(S)-N-(1-Morpholino-1-oxopropan-2-yl)pyrazine-2-carboxamide (POA-dl-Ala-Mor, 5b′)

Yield: 72%; gum.

¹H NMR (500 MHz, CDCl₃): δ = 9.33 (s, 1 H), 8.74–8.68 (m, 2 H), 8.50 (br s, 1 H), 5.16–5.07 (m, 1 H), 3.69–3.55 (m, 8 H), 1.44 (d, J=6.9 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 170.8, 162.4, 147.5, 145.0, 143.1, 66.9, 66.7, 46.2, 45.5, 42.7, 19.0.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₂H₁₆N₄O₃: 264.1221; found: 264.1222.

(S)-N-(3-Methyl-1-morpholino-1-oxobutan-2-yl)pyrazine-2-carboxamide (POA-l-Val-Mor, 5c)

Yield: 76%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 9.37 (s, 1 H), 8.76 (br s, 1 H), 8.58 (br s, 1 H), 8.50 (d, J=8.7 Hz, 1 H), 5.00–4.97 (m, 1 H), 3.77–3.60 (m, 8 H),2.19–2.13 (m, 1 H), 1.05 (d, J=6.6 Hz, 3 H), 1.00 (d, J=6.6 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 169.8, 163.0, 147.3¸ 144.3, 142.7, 66.7, 66.6, 53.2, 46.3, 42.4, 31.7, 19.7, 17.5.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₄H₂₀N₄O₃: 292.1535; found: 292.1534.

N-((2S,3S)-3-Methyl-1-morpholino-1-oxopentan-2-yl)pyrazine-2-carboxamide (POA-l-Ile-Mor, 5d)

Yield: 70%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 9.37 (s, 1 H), 8.76 (br s, 1 H), 8.57 (br s, 1 H), 8.46 (d, J=7.9 Hz, 1 H), 5.02 (t, J=7.9 H, 1 H), 3.76–3.52 (m, 8 H), 1.94–1.93 (m, 1 H), 1.64–1.59 (m, 1 H), 1.23–1.16 (m, 1 H), 1.02–0.91 (m, 6 H).

¹³C NMR (125 MHz, CDCl₃): δ = 170.3, 163.0, 147.5, 144.5, 142.9, 67.0, 66.9, 52.8, 46.7, 42.7, 38.4, 24.4, 16.1, 11.4.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₅H₂₂N₄O₃: 306.1694; found: 306.1692.

(S)-N-(1-Morpholino-1-oxo-3-phenylpropan-2-yl)pyrazine-2-carboxamide (POA-l-Phe-Mor, 5e)

Yield: 78%; gum.

¹H NMR (500 MHz, CDCl₃): δ = 9.36 (s, 1 H), 8.75 (br s, 1 H), 8.59–856 (m, 2 H), 7.33–7.26 (m, 5 H), 5.35–5.30 (m, 1 H), 3.61–3.32 (m, 6 H), 3.20–3.09 (m, 2 H), 3.00–2.89 (m, 2 H).

¹³C NMR (125 MHz, CDCl₃): δ = 169.7, 162.6, 147.6, 144.5, 143.0, 136.2, 129.8, 128.9, 127.6, 66.7, 66.2, 49.8, 46.3, 42.5, 40.4.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₈H₂₀N₄O₃: 340.1536; found: 340.1535.

N-(2-Oxo-2-(piperidin-1-yl)ethyl)pyrazine-2-carboxamide (POA-Gly-Pip, 5f)

Yield: 74%; colorless microcrystals; mp 123–125 °C.

¹H NMR (500 MHz, CDCl₃): δ = 9.38 (s, 1 H), 8.77–8.74 (m, 2 H), 8.58 (br s, 1 H), 4.27 (d, J=4.3 Hz, 2 H), 3.63 (t, J=5.2 Hz, 2 H), 3.41 (t, J=5.2 Hz, 2 H), 1.69–1.59 (m, 6 H).

¹³C NMR (125 MHz, CDCl₃): δ = 165.8, 163.3, 147.5, 144.4, 143.1, 45.8, 43.4, 41.4, 41.4, 26.4, 25.6, 24.6.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₂H₁₆N₄O₂: 248.1273; found: 248.1275.

(S)-N-(1-Oxo-1-(piperidin-1-yl)propan-2-yl)pyrazine-2-carboxamide (POA-l-Ala-Pip, 5g)

Yield: 75%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 9.28 (s, 1 H), 8.74 (d, J=7.2 Hz, 1 H), 8.66 (br s, 1 H), 8.50 (br s, 1 H), 5.04–4.98 (m, 1 H), 3.61–3.38 (m, 4 H), 1.61–1.45 (m, 6 H), 1.37 (d, J=6.9 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 169.9, 162.1, 147.2, 144.1, 142.8, 46.5, 45.3, 43.3, 26.4, 25.5, 24.4, 19.0.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₃H₁₈N₄O₂: 262.1429; found: 262.1431.

(S)-N-(3-Methyl-1-oxo-1-(piperidin-1-yl)butan-2-yl)pyrazine-2-carboxamide (POA-l-Val-Pip, 5h)

Yield: 81%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 9.31 (s, 1 H), 8.68 (br s, 1 H), 8.52–8.49 (m, 2 H), 5.00–4.97 (m, 1 H), 3.59–3.51 (m, 4 H), 2.12–2.05 (m, 1 H), 1.61–150 (m, 6 H), 0.98 (d, J=6.6 Hz, 3 H), 0.91 (d, J=6.6 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 169.5, 163.0, 147.3, 144.5, 142.9, 53.5, 47.1, 43.4, 32.1, 26.7, 25.7, 24.6, 20.0, 17.5.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₅H₂₂N₄O₂: 290.1742; found: 290.1749.

N-((2S,3S)-3-Methyl-1-oxo-1-(piperidin-1-yl)pentan-2-yl)pyrazine-2-carboxamide (POA-l-Ile-Pip, 5i)

Yield: 64%; gum.

¹H NMR (500 MHz, CDCl₃): δ = 9.32 (s, 1 H), 8.73 (br s, 1 H), 8.52 (br s, 1 H), 8.43 (d, J=7.9 Hz, 1 H), 5.04 (t, J=7.9 H, 1 H), 3.54–3.49 (m, 4 H),1.87–1.85 (m, 1 H), 1.15–1.08 (m, 8 H), 0.92 (d, J=7.3 Hz, 1 H), 0.79 (d, J=7.3 Hz, 1 H).

¹³C NMR (125 MHz, CDCl₃): δ = 170.6, 163.3, 147.5, 144.7, 142.6, 53.2, 47.3, 43.6, 38.4, 26.5, 25.6, 24.3, 24.0, 16.0, 11.4.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₆H₂₄N₄O₂: 304.1896; found: 304.1899.

(S)-N-(1-Oxo-3-phenyl-1-(piperidin-1-yl)propan-2-yl)pyrazine-2-carboxamide (POA-l-Phe-Pip, 5j)

Yield: 79%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 9.35 (s, 1 H), 8.73 (br s, 1 H), 8.62–8.56 (m, 2 H), 7.34–7.24 (m, 5 H), 5.40–5.35 (m, 1 H), 3.57–3.47 (m, 4 H), 2.94–2.78 (m, 2 H), 1.54–1.43 (m, 6 H).

¹³C NMR (125 MHz, CDCl₃): δ = 169.1, 162.4, 147.4, 144.4, 142.9, 138.0, 129.5, 128.6, 127.2, 52.5, 49.9, 46.3, 43.2, 26.2, 25.6, 24.4.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₉H₂₂N₄O₂: 338.1743; found: 338.1746.

N-(2-(4-Methylpiperazin-1-yl)-2-oxoethyl)pyrazine-2-carboxamide (POA-Gly-NMP, 5k)

Yield: 69%; gum.

¹H NMR (500 MHz, CDCl₃): δ = 9.34 (s, 1 H), 8.71 (br s, 1 H), 8.68 (br s, 1 H), 8.54 (br s, 1 H), 4.26 (d, J=4.3 Hz, 2 H), 3.75–3.60 (m, 4 H), 2.42–2.35 (m, 4 H), 2.31 (s, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 169.4, 163.3, 147.8, 144.5, 143.4, 55.3, 54.8, 46.4, 46.1, 45.9, 40.8.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₂H₁₇N₅O₂: 263.1385; found: 263.1386.

(S)-N-(1-(4-Methylpiperazin-1-yl)-1-oxopropan-2-yl)pyrazine-2-carboxamide (POA-l-Ala-NMP, 5l)

Yield: 73%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 9.32 (s, 1 H), 8.72–8.70 (m, 2 H), 8.53 (br s, 1 H), 5.07–5.04 (m, 1 H), 3.72–3.51 (m, 4 H), 2.44–2.34 (m, 4 H), 2.27 (s, 3 H), 1.42 (d, J=6.7 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 170.3, 162.3, 147.4, 144.3, 142.9, 55.1, 54.6, 46.0, 45.4, 45.3, 42.2, 19.1.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₃H₁₉N₅O₂: 277.1536; found: 277.1541.

(S)-N-(3-Methyl-1-(4-methylpiperazin-1-yl)-1-oxobutan-2-yl)pyrazine-2-carboxamide (POA-l-Val-NMP, 5m)

Yield: 62%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 9.37 (s, 1 H), 8.75 (br s, 1 H), 8.57 (br s, 1 H), 8.52 (d, J=8.9 Hz, 1 H), 5.03–5.00 (m, 1 H), 3.76–3.61 (m, 4 H), 2.46–2.38 (m, 4 H), 2.31 (s, 3 H), 2.17–2.13 (m, 1 H), 1.04 (d, J=6.7 Hz, 3 H), 0.98 (d, J=6.7 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 169.8, 163.3, 147.5, 144.4, 143.0, 55.4, 54.9, 53.6, 46.2, 46.0, 42.3, 32.1, 20.1, 17.7.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₅H₂₃N₅O₂: 305.1852; found: 305.1860.

N-((2S,3S)-3-Methyl-1-(4-methylpiperazin-1-yl)-1-oxopentan-2-yl)pyrazine-2-carboxamide (POA-l-Ile-NMP, 5n)

Yield: 64%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 9.32 (s, 1 H), 8.70 (br s, 1 H), 8.52 (br s, 1 H), 8.45 (d, J=7.8 Hz, 1 H), 5.01 (t, J=7.8 H, 1 H), 3.72–3.60 (m, 4 H), 2.42–2.36 (m, 4 H), 2.27 (s, 3 H), 1.89–1.88 (m, 1 H), 1.57–1.53 (m, 1 H), 1.18–1.13 (m, 1 H), 0.98–0.86 (m, 6 H).

¹³C NMR (125 MHz, CDCl₃): δ = 169.9, 162.9, 147.4, 144.4, 142.9, 55.3, 54.7, 52.9, 46.0, 42.2, 38.4, 24.3, 16.1, 11.4.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₆H₂₅N₅O₂: 319.2005; found: 319.2000.

(S)-N-(1-(4-Methylpiperazin-1-yl)-1-oxo-3-phenylpropan-2-yl)pyrazine-2-carboxamide (POA-l-Phe-NMP, 5o)

Yield: 71%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 9.34 (s, 1 H), 8.73 (br s, 1 H), 8.58–854 (m, 2 H), 7.30–7.19 (m, 5 H), 5.30–5.24 (m, 1 H), 3.72–3.61 (m, 4 H), 3.20–3.09 (m, 2 H), 2.47–2.38 (m, 4 H), 2.24 (s, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 170.1, 162.8, 147.5, 144.7, 143.2, 136.5, 129.3, 128.7, 127.2, 55.3, 54.7, 53.5, 46.5, 46.2, 42.6, 40.8.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₉H₂₃N₅O₂: 353.1853; found: 353.1850.

N-(2-Oxo-2-(pyrrolidin-1-yl)ethyl)pyrazine-2-carboxamide (POA-Gly-Pyr, 5p)

Yield: 81%; colorless microcrystals; mp 148–150 °C.

¹H NMR (500 MHz, CDCl₃): δ = 9.31 (s, 1 H), 8.68–8.64 (m, 2 H), 8.52 (br s, 1 H), 4.15 (d, J=4.3 Hz, 2 H), 3.50–3.40 (m, 4 H), 2.00–1.78 (m, 4 H).

¹³C NMR (125 MHz, CDCl₃): δ = 166.1, 163.3, 147.4, 144.3, 143.0, 68.1, 46.2, 45.7, 42.1, 26.1, 24.3.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₁H₁₄N₄O₂: 234.1117; found: 234.1121.

(S)-N-(1-Oxo-1-(pyrrolidin-1-yl)propan-2-yl)pyrazine-2-carboxamide (POA-l-Ala-Pyr, 5q)

Yield: 79%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 9.29 (s, 1 H), 8.67 (br s, 1 H), 8.59 (br s, 1 H), 8.49 (br s, 1 H), 4.89–4.85 (m, 1 H), 3.64–3.40 (m, 4 H), 1.97–1.82 (m, 4 H), 1.41 (d, J=6.4 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 170.4, 162.4, 147.3, 144.3, 142.9, 47.0, 46.6, 46.2, 26.2, 24.2, 18.4.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₂H₁₆N₄O₂: 248.1271; found: 248.1279.

(S)-N-(3-Methyl-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl)pyrazine-2-carboxamide (POA-l-Val-Pyr, 5r)

Yield: 82%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 9.32 (s, 1 H), 8.69 (br s, 1 H), 8.52 (br s, 1 H), 8.38 (d, J=8.2 Hz, 1 H), 4.74 (t, J=8.2 Hz, 1 H), 3.77–3.39 (m, 4 H), 2.18–2.11 (m, 1 H), 1.98–1.82 (m, 4 H), 1.00 (d, J=6.6 Hz, 3 H), 0.97 (d, J=6.6 Hz, 3 H).

¹³C NMR (125 MHz, CDCl₃): δ = 169.9, 163.0, 147.4, 144.5, 142.9, 56.1, 47.0, 46.1, 31.8, 26.2, 24.4, 19.8, 18.1.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₄H₂₀N₄O₂: 276.1586; found: 276.1582.

N-((2S,3S)-3-Methyl-1-oxo-1-(pyrrolidin-1-yl)pentan-2-yl)pyrazine-2-carboxamide (POA-l-Ile-Pyr, 5s)

Yield: 74%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 9.34 (s, 1 H), 8.68 (br s, 1 H), 8.57 (br s, 1 H), 8.49 (d, J=7.8 Hz, 1 H), 5.04 (t, J=7.8 H, 1 H), 3.66–3.42 (m, 4 H), 2.01–1.88 (m, 5 H), 1.62–1.58 (m, 1 H), 1.17–1.12 (m, 1 H), 1.01–0.89 (m, 6 H).

¹³C NMR (125 MHz, CDCl₃): δ = 170.3, 163.1, 147.6, 144.2, 142.5, 52.9, 45.7, 42.6, 38.1, 25.0, 24.8, 24.5, 16.2, 11.7.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₅H₂₂N₄O₂: 290.1743; found: 290.1746.

(S)-N-(1-Oxo-3-phenyl-1-(pyrrolidin-1-yl)propan-2-yl)pyrazine-2-carboxamide (POA-l-Phe-Pyr, 5t)

Yield: 73%; oil.

¹H NMR (500 MHz, CDCl₃): δ = 9.33 (s, 1 H), 8.73 (br s, 1 H), 8.59–855 (m, 2 H), 7.35–7.23 (m, 5 H), 5.14–5.09 (m, 1 H), 3.75–3.34 (m, 4 H), 3.03–2.93 (m, 2 H), 2.03–1.72 (m, 4 H).

¹³C NMR (125 MHz, CDCl₃): δ = 169.0, 162.3, 147.3, 144.2, 142.8, 136.2, 129.2, 128.3, 126.6, 52.5, 45.9, 45.8, 39.7, 24.0, 23.8.

HRMS (ESI): m/z[M + H]⁺ calcd for C₁₈H₂₀N₄O₂: 324.1588; found: 324.1585.

Acknowledgment

We thank the Augusta University and Pamplin Student Research & Travel Fund for financial support.

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