Introduction

Amides of general structures 1 and 2 (Figure 1) have a range of potential applications as ligands for catalysis, in molecular switches and as metal binding agents. When combined with iron(II), ligands of this ilk can promote alkene dihydroxylation and allylic oxidation reactions akin to those mediated by non-heme iron oxidase enzymes (NHIOs)1,2,3,4,5,6,7,8,9; in combination with cobalt(III) or iron(III), they may catalyse conversion of nitriles to primary amide products, as mimics of the metalloenzyme nitrile hydratase10,11,12,13.

Figure 1
figure 1

General structures of bidentate 1 and tridentate 2 amide targets, prepared from picolinic acid 3 and pyridine-2,6-dicarboxylic acid 4.

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These compounds are of interest for potential application in molecular switches and devices that exploit the controlled cis/trans isomerisation of the amide bond14,15,16. They have demonstrated utility in coordination chemistry with transition metals17 and lanthanoids18 and have been applied to radionucleotide extraction19,20,21.

We report herein the synthesis of eleven amides (Figure 2) from picolinic acid 3 and pyridine-2,6-dicarboxylic acid 4 and structural investigations using X-ray crystallography and variable temperature NMR.

Figure 2
figure 2

Structures of target amides 5–8.

For 5a–7a R = Me, 5b–7b R = Et, 5c–7c R = Ph; 8a is derived from the l-valinyl-l-cysteine dipeptide (R1 = iPr, R2 = CH2SBn),</emph> while 8b incorporates the l-cysteinyl-l-valine dipeptide (R1 = CH2SBn, R2 = iPr).

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Results & Discussion

Synthesis

Amides 5a–c, 6a–c, 7a–c, 8a–b were prepared by activating picolinic acid 3 and pyridine-2,6-dicarboxylic acid 4 to the corresponding acid chlorides in situ22, or via diimide-mediated peptide coupling23. Activating picolinic acid 3 with thionyl chloride afforded not only the simple picolinamides 5a–c as expected, but also the 4-chloropicolinamides 6a–c in the same pot. The two products were easily separated by column chromatography, enabling a ‘two for the price of one’ synthesis of new amides.

The mono-amide ligands 5a–c and 6a–c were synthesised from picolinic acid 3 and the corresponding aniline in one pot, via the acid chloride (Figure 3a). Thus acid 3 was treated with thionyl chloride overnight, followed by N-methylaniline, N-ethylaniline or N-diphenylamine and triethylamine in dichloromethane. This route gave the anticipated products 5a–c in low to moderate yields (31–54%) and the 4-chloro derivatives 6a–c, isolated in small but utilisable yields (10–13%). Each pair of compounds was readily separated by column chromatography.

Figure 3
figure 3

Synthesis of target compounds.

(a) Synthesis of mono-amides 5a–c and 6a–c; i. SOCl2, reflux, 16 h; ii. Et3N, N-methylaniline a; N-ethylaniline b or N-diphenylamine c, DCM, rt, 16 h; 5a 35%/6a 13%; 5b 31%/6b 10%; 5c. 54%/6c 10% (yields over two steps for major/minor products). (b) Synthesis of bis-amides 7a–c; i. SOCl2, reflux, 16 h; ii. Et3N, N-methylaniline a, N-ethylaniline b or N-diphenylamine c (2 eq.), DCM, rt, 16 h; 7a 86%, 7b 88%, 7c 90% (over two steps). (c) Synthesis of peptide derivatives 8a–b; iii. EDCI, HOBt, Et3N, l-valinyl-S-benzyl-l-cysteine methyl ester tosylate salt 9 or S-benzyl-l-cysteinyl-l-valine methyl ester 10 (2 eq.), DCM, rt, 22–48 h; 8a 44%, 8b 61%.

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Chlorination of the ring presumably occurs via activation of the pyridine to nucleophilic attack by chloride anion. This could occur during formation of the acid chloride or in the subsequent coupling step. The direct synthesis of 4-chloropicolinyl chloride from picolinic acid using thionyl chloride has been reported previously24,25, although in our own prior work we have converted picolinic acid to picolinoyl chloride with this reagent system, then reacted the acyl chloride with l-proline, without observing ring-chlorinated side products7. Our efforts to characterise the acid chloride intermediate(s) were unsuccessful: we were able to isolate a low-melting orange solid (mp ~ 40–50°C) but this quickly decomposed before it could be further characterised.

The N-methyl mono-amide 5a has been prepared previously by Habib and Rees, who reported its synthesis, melting point and elemental analysis26 and more recently by Okamoto et al. as part of an investigation into acid-induced conformational changes in aromatic amides14. Habib and Rees prepared 5a via the acid chloride, reacting picolinic acid 3 and thionyl chloride in benzene, then adding N-methylaniline dropwise and heating at reflux; Okamoto activated acid 3 as the mixed anhydride by reaction with ethyl chloroformate and triethylamine, before adding N-methylaniline. The 4-chloro derivative 6a was not isolated in either of these previous syntheses.

Bis-amides 7a–c were prepared in a similar manner, from pyridine-2,6-dicarboxylic acid 4 in one pot (Figure 3b). This gave compounds 7a–c as crystalline solids in excellent yield (86–90%); chlorinated byproducts were not observed from the reactions of dicarboxylic acid 4. Compounds 7a and 7b appear previously in the literature, but details of their synthesis and characterisation are incomplete. Ried and Neidhardt studied “hydrogenolysis” of the N-methyl compound 7a and related quinoline carboxylic acids upon reaction with lithium aluminium hydride27. The N-methyl (7a) and N-ethyl (7b) analogues have been used to generate metal complexes17,18 and in metal extraction experiments19,20,21, while Dobler et al. conducted computational experiments to describe the interaction between ligands of this type and lanthanide cations28. Kapoor and coworkers recently reported synthesis and structural characterisation of related thioamide derivatives29.

In a complementary approach, the peptide derivatives 8a and 8b were prepared from pyridine-2,6-dicarboxylic acid 4 using diimide coupling methodology23. Thus dipeptides l-valinyl-S-benzyl-l-cysteine methyl ester (tosylate salt) 9 and S-benzyl-l-cysteinyl-l-valine methyl ester 10 (prepared from l-cysteine and l-valine via standard methods30) were coupled with 4 to give the peptide derivatives 8a and 8b in moderate yields (Figure 3c).

Crystallographic investigations

The geometry of the amide bond in compounds such as these has received attention previously with a view to potential applications in molecular switches and devices14,15,16. N-Alkylation – specifically N-methylation – has been shown to induce a change from trans-preferential to cis-preferential amides (Figure 4).

Figure 4
figure 4

Cis/trans isomerisation in aromatic amides.

While trans geometry is preferred when R = H, the cis isomer has been shown to predominate when R = Me14,15,16.

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Thus while the amide bond in benzanilide 11 (R = H) is trans, the corresponding bond in N-methylbenzanilide 12 (R = Me) is preferentially cis, both in the crystalline state and in solution16. Likewise crystallographic and NMR characterisation of 5a reported by Okamoto et al. show that the two aromatic groups adopt a cis relationship in that compound too14. To investigate the geometry of the amides prepared in the current study, single crystal X-ray structures were determined for the mono-amides 5b and 5c, 4-chloro mono-amides 6b and 6c and bisamides 7a and 7c (Figures 5 and 6; Supplementary Information).

Figure 5
figure 5

Crystal structures of amides 5b (CCDC-1002446), 5c (1002447), 6b (1002448), 6c (1002449).

Carbon atoms are shown in grey, oxygen in red, nitrogen in blue and hydrogen in white. In 5b and 6b, the cis amide is observed, with the ethyl group syn to the carbonyl oxygen. In 5b, 6b and 6c the pyridine nitrogen is anti to the carbonyl oxygen while in 5c these atoms are synclinal.

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Figure 6
figure 6

Crystal structures of amides 7a (CCDC-1002450) and 7c (1002451).

Carbon atoms are shown in grey, oxygen in red, nitrogen in blue and hydrogen in white. In 7a the methyl group and carbonyl oxygen are cis. The pyridine nitrogen is anti to both carbonyl oxygen atoms in 7c, but syn to one and anti to the other in 7a.

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The structures of the N-methyl (7a) and N-ethyl (5b, 6b) compounds reveal cis amide geometry in all cases: the aromatic groups (N-phenyl and pyridyl) are cis to each other and the methyl or ethyl substituent is cis to the carbonyl group. There is also a general preference for the pyridine nitrogen to sit anti to the carbonyl oxygen(s). Among the mono-amides, these groups are anticlinal in 5b (the O–C–C–N dihedral angle is 123.9°), 6b (126.5°) and 6c (137.6°), but synclinal in 5c (56.7°) (Figure 5). Of the bis-amide structures, the pyridine nitrogen is anticlinal to both carbonyls in the tetraphenyl compound 7c: there are two inequivalent molecules of 7c in the crystal structure, which exhibit dihedral angles around the bond in question of 141.6° and 131.9°/139.1° and 149.8° respectively. However in the dimethyl compound 7a, the pyridine nitrogen is anti to one of the amide carbonyls (137.2°) but syn to the other (−57.2°), which – in combination with the two cis amide bonds – positions the two phenyl groups in close proximity and an edge-to-face arrangement (Figure 6).

Variable temperature NMR experiments

In light of the recent work by Okamoto et al. using 1H NMR to follow cis/trans isomerisation in related aromatic amides14, we were interested to note evidence for slow conformational change in the 1H NMR spectra of compounds 7a–c. The room temperature 1H NMR spectra of 7a–c are generally poorly resolved with considerable line broadening (in contrast to the spectra of corresponding mono-amides 5a–c in which equivalent line broadening is not observed – see Supplementary Information). Variable temperature 1H NMR data for the ethyl substituted ligand 7b (Figure 7) show that signals resolve as the temperature is increased, confirming that the observed line broadening arises due to slow conversion between amide conformational isomers at room temperature. For example the signal at ~ 3.7 ppm, due to the methylene protons of the ethyl group, is a broad apparent singlet at 300 K but a clearly resolved quartet at 350 K (see inset in Figure 7).

Figure 7
figure 7

Variable temperature 1H NMR spectra of bis-amide ligand 7b (400 MHz, d8-toluene), confirming slow conformational change at room temperature.

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Conclusion

Amides derived from picolinic acid 3 and pyridine-2,6-dicarboxylic acid 4 have potential applications in catalysis, coordination chemistry and molecular switches. These compounds are readily prepared via the acid chloride or applying peptide coupling reagents. X-Ray crystal structures reveal that the generally preferred geometry of these amides positions the aromatic groups cis to each other and the pyridine nitrogen anti to the carbonyl oxygen. Variable temperature NMR experiments indicate slow cis/trans isomerisation in solution for the bis-amide series.

Methods

Amide synthesis

General procedure 1

Thionyl chloride (8.0 mL, 109 2 mmol) was added to picolinic acid 3 (1.00 g, 8.20 mmol) and the resulting suspension was refluxed for 16 h. The orange coloured solution was reduced in vacuo to give the acid chloride as a bright orange oil. The oil was dissolved in dry DCM (40 mL) and cooled to 0°C. A solution of N-alkylaniline (16.20 mmol) and triethylamine (2.20 mL, 16.20 mmol) in dry DCM (20 mL) was added via cannula. The resulting purple coloured solution was stirred at 0°C for 20 min and at room temperature for 16 h after which time the solution had become dark brown. The solution was washed with half-saturated aqueous ammonium chloride solution (2 × 12 mL), water (2 × 6 mL) and dried (Na2SO4), then concentrated in vacuo.

General procedure 2

Thionyl chloride (4.0 mL, 60 mmol) was added to 2,6-pyridinedicarboxylic acid 4 (0.50 g, 3.0 mmol) and the resulting suspension was refluxed under an argon atmosphere for 16 h to give a clear yellow solution. Excess thionyl chloride was removed in vacuo and the acid chloride was dissolved in dry CH2Cl2 (10 mL) and cooled to 0°C. A solution of N-alkylaniline (12.0 mmol) and triethylamine (0.84 mL, 6.0 mmol) in dry DCM (2.5 mL) was added via cannula. The resulting mixture was stirred at room temperature for 16 h during which time a white precipitate formed. The suspension was washed with half-saturated aqueous ammonium chloride solution (2 × 6 mL) and water (2 × 3 mL), then dried (Na2SO4) and concentrated in vacuo.

General procedure 3

Pyridine-2,6-dicarboxylic acid 4 (0.10–0.30 g, 1 eq.), dipeptide amine (as the free amine or tosylate salt, 2 eq.), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 2 eq.) and 1-hydroxybenzotriazole (HOBt, 2 eq.) were dissolved in DCM (10–30 mL) and triethylamine (2 eq. for free amine, 4 eq. for tosylate salt) was added. The reaction mixture was stirred at room temperature for 22–48 h while monitored by TLC. Additional DCM or chloroform (10–20 mL) was added and the solution washed with equivalent volumes of water, 1 m hydrochloric acid, saturated sodium bicarbonate (aqueous) and brine, dried (MgSO4) then concentrated in vacuo.

N-Methyl-N-phenylpicolinamide 5a and 4-Chloro-N-methyl-N-phenylpicolinamide 6a

Picolinic acid 3 (1.0 g, 8.2 mmol) and N-methylaniline (1.76 mL, 16.2 mmol) were coupled using thionyl chloride (Procedure 1). TLC of the crude mixture showed the presence of two products, which were separated by flash column chromatography (petroleum benzine/ethyl acetate, 1:1) to afford 5a (0.60 g, 35%) as a white crystalline solid and 6a (0.27 g, 13%) as a thick, clear, colourless oil.

Data for N-methyl-N-phenylpicolinamide 5a in agreement with literature14; see Supplementary Information for details.

Data for 4-chloro-N-methyl-N-phenylpicolinamide 6a: Rf 0.40 (petroleum benzine/ethyl acetate, 1:1); νmax (CHCl3, cm−1) 3060 (w), 2997 (m), 1662 (s), 1581 (s), 1353(s), 1303(s); δH (400 MHz, (CD3)2CO) 3.43 (3H, s, NCH3), 6.97–7.19 (6H, m, NC6H5, 1 × pyr-CH), 7.45 (1Η, s, 1 × pyr-CH), 8.12 (1H, bs, 1 × pyr-CH); δC (100 MHz, (CD3)2CO) 38.0, 124.6, 124.9, 127.5, 127.9, 129.9, 144.7, 145.3, 150.5, 157.7, 167.9; m/z (ES+) 247 (65%, [MH]+ for 35Cl), 249 (20%, [MH]+ for 37Cl); HRMS (ES+) C13H11ClN2NaO+ ([M+Na]+ for 35Cl) requires 269.04540, found 269.04591.

N-Ethyl-N-phenylpicolinamide 5b and 4-Chloro-N-ethyl-N-phenylpicolinamide 6b

Picolinic acid 3 (1.0 g, 8.2 mmol) and N-ethylaniline (2.0 mL, 16.2 mmol) were coupled using thionyl chloride (Procedure 1). The crude product was purified by flash column chromatography (petroleum benzine/ethyl acetate, 1:1) to afford 5b (0.57 g, 31%) and 6b (0.21 g, 10%) as white solid products.

Data for N-ethyl-N-phenylpicolinamide 5b: Rf 0.30 (petroleum benzine/ethyl acetate, 1:1); mp: 87–92°C; νmax (CHCl3, cm−1) 3089, 3064 (w), 2950 (s) 1600 (s), 1492 (s), 1377 (s), 1272 (s); δH (400 MHz, (CD3)2CO) 1.17 (3Η, t, J = 7.0 Hz, NCH2CH3), 3.95 (2H, q, J = 7.0 Hz, NCH2CH3), 7.12−7.23 (6H, m, NC6H5, 1 × pyr-CH), 7.48 (1H, d, J = 7.5 Hz, 1 × pyr-CH), 7.70 (1H, bs, 1 × pyr-CH), 8.26 (1H, bs, 1 × pyr-CH); δC (100 MHz, (CD3)2CO) 12.3, 44.3, 123.2, 126.3, 128.0, 128.6, 136.1, 142.9, 148.0, 155.3, 167.8; m/z (ES+) 227 (26%, [MH]+), 249 (17%, [MNa]+), 475 (100%, [2MNa]+); HRMS (ES+) C14H14N2ONa+ ([M + Na]+) requires 249.09984, found 249.09967.

Data for 4-chloro-N-ethyl-N-phenylpicolinamide 6b: Rf 0.40 (petroleum benzine/ethyl acetate, 1:1); mp: 80–83°C; νmax (CHCl3, cm−1) 3001 (w), 1650 (s), 1593 (s), 1554(m), 1492(m), 1311(m); δH (400 MHz, (CD3)2CO) 1.18 (3Η, t, J = 7.0 Hz, NCH2CH3), 3.93 (2H, q, J = 7.0 Hz, NCH2CH3), 7.17–7.29 (6H, m, NC6H5, 1 × pyr-CH), 7.57 (1H, s, 1 × pyr-CH), 8.22 (1H, bs, 1 × pyr-CH); δC (100 MHz, (CD3)2CO) 13.1, 45.3, 124.4, 127.6, 129.1, 129.7, 143.4, 144.5, 150.4, 157.8, 167.4; m/z (ES+) 261 (100%, [MH]+ for 35Cl), 263 (30%, [MH]+ for 37Cl); HRMS (ES+) C14H14ClN2O+ ([MH]+ for 35Cl) requires 261.07912, found 261.07938.

N,N-Diphenylpicolinamide 5c and 4-Chloro-N,N-diphenylpicolinamide 6c

Picolinic acid 3 (1.0 g, 8.2 mmol) and N-phenylaniline (2.74 g, 16.2 mmol) were coupled using thionyl chloride (Procedure 1). The crude product was purified by flash column chromatography (petroleum benzine/ethyl acetate, 1:1) to afford 5c (1.21 g, 54%) and 6c (0.25 g, 10%) as white solid products.

Data for N,N-diphenylpicolinamide 5c: Rf 0.30 (petroleum benzine/ethyl acetate, 1:1); mp: 129–132°C; νmax (KBr, cm−1) 3058 (w), 1670 (s), 1587 (m), 1488 (m); δH (400 MHz, (CD3)2CO) 7.19–7.32 (10Η, m, N(C6H5)2), 7.63 (1H, d, J = 1.0 Hz, 1 × pyr-CH), 7.77 (1H, t, J = 7.5 Hz, 1 × pyr-CH), 8.28–8.30 (2H, m, pyr-CH); δC (100 MHz, (CD3)2CO) 124.5, 124.7, 126.9, 127.8, 129.4, 136.9, 143.8, 148.9, 154.9, 169.2; m/z (ES+) 275 (100%, [MH]+), 297 (55%, [MNa]+); HRMS (ES+) C18H14N2ONa+ ([M + Na]+) requires 297.09984, found 297.09958.

Data for 4-chloro-N,N-diphenylpicolinamide 6c: Rf 0.40 (petroleum benzine/ethyl acetate, 1:1); mp: 122–124°C; νmax (CHCl3, cm−1) 3031 (m), 3024 (m), 1666 (s), 1643(s), 1593 (s), 1569(s) 1492 (s), 1404(s), 1350(s); δH (400 MHz, (CD3)2CO) 7.19−7.36 (11Η, m, N(C6H5)2, 1 × pyr-CH), 7.73 (1H, d, J = 2.0 Hz, 1 × pyr-CH), 8.27 (1H, d, J = 5.0 Hz, 1 × pyr-CH); δC (100 MHz, (CD3)2CO) 123.9, 124.1, 126.5, 127.6, 128.8, 143.3, 143.7, 149.6, 156.6, 166.9; m/z (ES+) 309 (90%, [MH]+ for 35Cl), 311 (35%, [MH]+ for 37Cl); HRMS (ES+) C18H13ClN2ONa+ ([M + Na]+ for 35Cl) requires 331.06111, found 331.06075.

N2, N6-Dimethyl-N2, N6-diphenylpyridine-2,6-dicarboxamide 7a

2,6-Pyridinedicarboxylic acid 4 (0.50 g, 3.0 mmol) and N-methylaniline (1.28 mL, 12.0 mmol) were coupled using thionyl chloride (Procedure 2). The crude orange oil was triturated with hexane to yield the title compound as a white crystalline solid (0.89 g, 86%); Rf 0.20 (petroleum benzine/ethyl acetate, 1:1); mp: 148–155°C; νmax (KBr, cm−1) 3053 (w), 2969 (m), 2934 (w), 1651 (s), 1596 (m), 1585 (m); δH (400 MHz, (CD3)2CO) 3.31 (6H, s, 2 × NCH3), 7.04 (4H, br app. s, 4 of 2 × NC6H5), 7.16−7.30 (7H, m, 6 of 2 × NC6H5, 1 × pyr-CH), 7.66 (2H, br app s, 2 × pyr-CH); δC (100 MHz, (CD3)2CO) 37.2, 123.5, 126.3, 126.8, 128.9, 136.7, 144.5, 153.6, 167.3; m/z (ES+) 346 (73%, [MH]+), 368 (100%, [MNa]+); HRMS (ES+) C21H20N3O2+ ([MH]+) requires 346.15501, found 346.15501.

N2, N6-Diethyl-N2, N6-diphenylpyridine-2,6-dicarboxamide 7b

2,6-Pyridinedicarboxylic acid 4 (0.50 g, 3.0 mmol) and N-ethylaniline (1.5 mL, 12.0 mmol) were coupled using thionyl chloride (Procedure 2). The crude product was purified by flash column chromatography (petroleum benzine/ethyl acetate, 1:1)to yield 7b (0.99 g, 88%) as a white solid; Rf 0.20 (hexane/ether, 1:1); mp: 105–115°C; νmax (KBr, cm−1) 3055 (w), 2970 (m), 2931 (w), 1650 (s), 1596 (m), 1585 (m); δH (400 MHz, (CD3)2CO) 1.11 (6H, br app s, 2 × CH2CH3), 3.84 (4H, br app s, 2 × CH2CH3), 6.90–7.15 (4H, br app s, 4 of 2 × NC6H5), 7.16–7.35 (7H, m, 7 of 2 × NC6H5, 1 × pyr-CH), 7.58 (2H, br app s, 2 × pyr-CH); δC (100 MHz, (CD3)2CO) 13.3, 45.2, 124.2, 127.4, 129.0, 129. 7, 137.3, 143.6, 154.6, 167.7; m/z (ES+) 374 (50%, [MH]+), 396 (100%, [MNa]+); HRMS (ES+) C23H24N3O2+ ([MH]+) requires 374.18631, found 374.18631.

N2, N2, N6, N6-Tetraphenylpyridine-2,6-dicarboxamide 7c

2,6-Pyridinedicarboxylic acid 4 (0.50 g, 3.0 mmol) and N-phenylaniline (2.05 g, 12.0 mmol) were coupled using thionyl chloride (Procedure 2). The crude product was purified by flash column chromatography (CH2Cl2/diethyl ether, 10:1) to give 7c (1.26 g, 90%); Rf 0.25 (DCM/ether, 10:1); mp: 215–219°C; νmax (CHCl3, cm−1) 2999 (w), 1658 (s), 1639 (s), 1589 (s), 1485 (m), 1335 (s); δH (400 MHz, (CD3)2CO) 7.09 (8H, d, J = 7.5 Hz, 8 of 4 × NC6H5), 7.23–7.27 (4H, m, 4 of 4 × NC6H5), 7.32–7.36 (8H, m, 6 of 2 × NC6H5, 2 × pyr-CH), 7.58 (2H, d, J = 7.5 Hz, 2 of 2 × NC6H5), 7.77–7.81 (1H, m, 1 × pyr-CH); δC (100 MHz, (CD3)2CO) 125.4, 127.1, 128.5, 129.7, 137.9, 144.7, 153.8, 167.9; m/z (ES+) 470 (100%, [MH]+), 492 (43%, [MNa]+); HRMS (ES+) C31H24N3O2+ ([MH]+) requires 470.18631, found 470.18615.

Pyridine-2,6-dicarboxylic acid bis(l-valinyl-S-benzyl-l-cysteine methyl ester)carboxamide 8a

2,6-Pyridinedicarboxylic acid 4 (0.10 g, 0.58 mmol) and l-valinyl-S-benzyl-l-cysteine methyl ester tosylate salt 9 (0.60 g, 1.2 mmol) were coupled using EDCI/HOBt (Procedure 3) to give 8a as a yellow oil (0.20 g, 44%) after purification by column chromatography (cyclohexane/ethyl acetate, 1:4); Rf 0.65 (cyclohexane/ethyl acetate, 1:1); = + 1.5 (c = 2.0, CHCl3); νmax (thin film) 3290 (s), 1745 (s), 1659 (s), 1530 (s); δH (300 MHz, CDCl3) 1.06 (12H, 2d, J = 6.5 Hz, 2 × CH(CH3)2), 2.25–2.37 (2H, m, 2 × CH(CH3)2), 2.86–2.88 (4H, m, 2 × CH2SCH2Ph), 3.66 (4H, s, 2 × SCH2Ph), 3.74 (6H, s, 2 × OCH3), 4.55 (2H, dd, J = 9.0, 7.0 Hz, 2 × CHNH), 4.80 (2H, dt, J = 7.5, 5.5 Hz, 2 × CHCH2S), 6.90 (2H, d, J = 7.5 Hz, 2 × NH), 7.17–7.70 (10H, m, 2 × C6H5), 7.88 (1H, m, 1 × pyr-CH), 8.34 (2H, d, J = 7.5 Hz, 2 × pyr-CH), 8.71 (2H, d, J = 9.0 Hz, 2 × NH); δC (75.4 MHz, CDCl3) 19.4, 28.4, 34.2, 36.6, 52.7, 58.9, 59.9, 127.3, 128.3, 128.7, 128.9, 137.6, 139.1, 148.6, 148.6, 163.7, 170.9; m/z (ES+) 780 (100%, [MH]+); HRMS (ES+) C39H50N5O8S2 ([MH]+) requires 780.3101, found 780.3112.

Pyridine-2,6-dicarboxylic acid bis(S-benzyl-l-cysteinyl-l-valine methyl ester)carboxamide 8b

2,6-Pyridinedicarboxylic acid 4 (0.25 g, 1.5 mmol) and S-benzyl-l-cysteinyl-l-valine methyl ester 10 (1.20 g, 3.0 mmol) were coupled using EDCI/HOBt (Procedure 3) to give 8b as a yellow oil (1.13 g, 61%), after purification by column chromatography (cyclohexane/ethyl acetate, 1:1); Rf 0.55 (cyclohexane/ethyl acetate 1:1); = -7.6 (c = 2.0, CHCl3); νmax (thin film) 3420 (s, br), 3290 (s, br), 1740 (s), 1649 (s), 1538 (w); δH (300 MHz, CDCl3) 0.89 (12H, 2d, J = 4.5 Hz, 2 × CH(CH3)2), 2.11–2.22 (2H, m, 2 × CH(CH3)2), 2.91 (2H, dd, J = 14.0, 7.5 Hz, 2 of (2 × CH2S)), 3.01 (2H, dd, J = 14.0, 6.5 Hz, 2 of (2 × CH2S)), 3.74 (6H, s, 2 × OCH3), 3.84 (4H, s, 2 × SCH2Ph), 4.51 (2H, dd, J = 8.5, 5.0 Hz, 2 × CHNH), 4.69–4.77 (2H, m, 2 × CHCH2S), 6.94 (2H, d, J = 8.5 Hz, 2 × NH), 7.17–7.70 (10H, m, 2 × C6H5), 8.02–8.10 (2H, m, 2 × pyr-CH), 8.36 (1H, d, J = 7.5 Hz, 1 × pyr-CH), 8.83 (2H, d, J = 8.0 Hz, 2 × NH); δC (75.4 MHz, CDCl3) 17.8, 31.1, 33.4, 36.6, 52.2, 52.7, 57.6, 125.3, 127.2, 128.6, 129.1, 138.0, 138.6, 163.5, 163.6, 170.2, 171.9; m/z (ES+) 780 (50%, [MH]+); HRMS (ES+) C39H50N5O8S2 ([MH]+) requires 780.3101, found 780.3098.