Synthesis of 15 N-labelled 3,5-dimethylpyridine.

15 N-labelled pyridines are liquid- and solid-state nuclear magnetic resonance (NMR) probes for chemical and biological environments because their 15 N chemical shifts are sensitive to hydrogen-bond and protonation states. By variation of the type and number of substituents, different target pyridines can be synthesized exhibiting different pKa values and molecular volumes. Various synthetic routes have been described in the literature, starting from different precursors or modification of other 15 N-labelled pyridines. In this work, we have explored the synthesis of 15 N 15 N-labelled pyridines using a two-step process via the synthesis of alkoxy-3,4-dihydro-2H-pyran as precursor exhibiting already the desired pyridine substitution pattern. As an example, we have synthesized 3,5-dimethylpyridine-15 N (lutidine-15 N) as demonstrated by 15 N-NMR spectroscopy. That synthesis starts from methacrolein, propenyl ether, and 15 N-labelled NH4 Cl as nitrogen source.

INTRODUCTION

15N‐labelled pyridines and related heterocycles are important liquid‐ and solid‐state nuclear magnetic resonance (NMR) probes for chemical and biological environments.1, 2, 3, 4, 5, 6 That feature arises on one hand from the basicity of pyridines and their ability to form hydrogen bonds. On the other hand, 15N chemical shifts are very sensitive to the 15N‐1H distance and can be used to monitor the local H‐bond and protonation state.7, 8, 9 Therefore, 15N‐labelled pyridines have been used to explore the acidity of mesoporous surfaces or of biological environments using high‐resolution solid‐state NMR spectroscopy. Moreover, the mobility of pyridines to jump from one proton donor to another allows one to obtain interesting information about local structures.2

So far, 15N pyridine is the only representative that is commercially available in a 15N‐labelled form. Thus, syntheses of various 15N‐labelled pyridine derivatives with a large range of pKa values have been reported so far, applying several routes as illustrated in Scheme 1. Route I starts from the appropriate pyrylium salt containing already the desired pyridine substituents, using 15NH4Cl as nitrogen source. In Route II, alkoxy‐3,4‐dihydro‐2H‐pyrans exhibiting the desired substituents are firstly synthesized as precursors via a Diels‐Alder addition of vinyl ethers to α,β‐unsaturated carbonyl compounds. The pyrans can then easily be converted into the corresponding 15N‐labelled pyridines using 15NH4Cl. Finally, easily available labelled pyridines can be converted into other derivatives (Route III). Some examples are depicted in Scheme 2.

Scheme 1

Synthetic routes to 15N‐labelled pyridine derivatives

Scheme 2

Overview of the so‐far reported 15N‐labelled pyridine derivatives obtained either by Route II (A), Route I (B), or Route III (C)

Up to date, most 15N‐labelled pyridines have been synthesized following Route I, namely, 2,4.6‐trimethyl‐pyridine 4, also called collidine,10 2,6‐di‐tert‐butyl‐4‐methyl‐pyridine 5,11 4‐dimethylamino‐2,6‐dimethy‐pyridine 6, 12 and 4‐diethylamino‐2,6‐di‐tert‐butyl‐pyridine 7.11 However, Route I is limited to pyridines with aliphatic substitutions at C2 and C6. Route II has been used to synthesize 15N‐labelelled plain pyridine 1 12, 13 and 4‐methyl‐pyridine 2.9 Route III was used for the synthesis of 15N‐labelled 2,4.6‐trimethyl‐3‐nitro‐pyridine 8, 2,4.6‐trimethyl‐3‐bromo‐pyridine9 and 4‐N,N‐dimethylamino‐pyridine 10.12

As we wanted to obtain 3,5‐dimethyl‐pyridine‐15N (3) as molecular sensor for comparison with pyridine‐15N (1) and collidine‐15N (4), we explored the most suitable route to synthesize 3. We could not use Route I as pyrylium salts without substituents in 2‐ and 6‐ position are rare and not very stable.14 In addition, it is not possible to obtain 3 from 1 via Route III. Therefore, we checked in more detail Route II. That route had been used to synthesize pyridine‐15N (1). The required precursor 3,4‐dihydro‐2‐methoxy‐2H‐pyran is commercially available and can be synthesized in solution at high pressures up to 15 000 bar15 or under milder conditions using either dry‐state adsorption conditions16 or an ytterbium catalyst.17 The original synthesis of Longley and Emerson18 did not use a solvent or additives but only the neat reactants, heating them up to about 200 °C in a normal laboratory autoclave. The pressure achieved was not reported, but they probably did not exceed about 15 bar.18 Therefore, that method seemed to us preferable as only small quantities of the pyran are needed. We found that this method was suitable and succeeded to synthesize in a similar way also 4‐methyl‐pyridine‐15N (2).9Therefore, we want to describe here in more detail how to prepare pyridines for which commercial precursors are not available, using the example of 15N‐labelled 3,5‐dimethylpyridine (3).

RESULTS AND DISCUSSION

In the first stage of this work, we checked out alternative routes starting from unlabelled 3,5‐dimethyl pyridine, but these efforts were not successful.

As precursor of the Diels‐Alder reaction, we used methacrolein 11 and ethyl 1‐propenyl ether 12 leading to 2‐ethoxy‐3,4‐dihydro‐3,5‐dimethyl‐2H‐pyran 13 (Scheme 3). Methacrolein was stabilized with a small amount of hydroquinone to avoid polymerization. Nuclear magnetic resonance spectroscopy revealed a cis/trans mixture of compound 13 in the ratio of 2:3 (Figure 1); the chemical shifts are listed and compared to literature values in Table 1. The chemical shifts and coupling constants of the two 2H‐pyran ring isomers fit very well to previous reports for 2‐benzoyloxy‐3,4‐dihydro‐3,5‐dimethyl‐2H‐pyran from Yamamoto et al19 and 2‐methoxy‐3,4‐dihydro‐3,5‐dimethyl‐2H‐pyran from Descotes et al.20 Although the diastereomers could potentially be separated by chromatography, a separation was not required because both diastereomers are an in situ source of 1,5‐pentane‐dial that is generated in the initial part of the second step of the synthesis. A side product of the reaction was the Diels‐Alder reaction of methacrolein with itself forming 3,4‐dihydro‐2H‐pyran‐2‐carbaldehyde, which was however reduced by using an excess of the dienophile and was separated by distillation.

Scheme 3

Synthetic route to 15N‐labelled lutidine used in this work

Figure 1

1H NMR spectrum of 2‐ethoxy‐3,4‐dihydro‐3,5‐dimethyl‐2H‐pyran 13 consisting of a 2:3 cis/trans mixture measured in DMSO‐d . For clarity, only one enantiomer is shown for each diastereomer (2R,3R for trans and 2S,3R for cis). The dominating trans form shows a larger 3 J H2H3 scalar coupling. Signals between 1.5 and 2.2 ppm were only tentatively assigned

Table 1

Chemical shifts of the mixture of 2‐ethoxy‐3,4‐dihydro‐3,5‐dimethyl‐2H‐pyran diastereomers and comparison with values of cis and trans 2‐benzoyloxy‐3,4‐dihydro‐3,5‐dimethyl‐2H‐pyran from Yamamoto et al19 and 2‐methoxy‐3,4‐dihydro‐3,5‐dimethyl‐2H‐pyran from Descotes et al20

Atom	Observed (DMSO‐d 6) trans	Observed (DMSO‐d 6) cis	Yamamoto (CDCl3) trans	Yamamoto (CDCl3) cis	Descotes (CCl4) trans	Descotes (CDCl4) cis
H2	4.52, d, J = 4.6 Hz	4.73, d, J = 2.3 Hz	4.60, d, J = 4.0 Hz	4.79, d, J = 2.1 Hz	4.35, d, J = 3.4 Hz	4.52, d, J = 1.7 Hz
H3	1.5‐2.2 ov a	1.5‐2.2 ov a	1.98 dddt	1.90 m
H41	1.5‐2.2 ov a	1.5‐2.2 ov a	2.24 dd	1.76 m
H42	1.5‐2.2 ov a	1.5‐2.2 ov a	1.53 dd	0.96 m
H6	6.02, d, J = 1.4 Hz	5.99, d, J = 1.4 Hz	6.03, d, J = 1.2 Hz	6.02, d, J = 1.2 Hz	5.91	5.91
H7	0.90, d, J = 6.9 Hz	0.92, d, J = 6.5 Hz	0.96, d, J = 7.0 Hz	1.00, d, J = 6.4 Hz	0.91‐1.51	0.96‐1.52
H8	1.50 ov	1.50 ov	1.54, d, J = 1.2 Hz	1.55, d, J = 1.2 Hz	0.91‐1.51	0.96‐1.52
H1’/H1”	3.50‐3.70 ov	3.50‐3.70 ov	4.82 d, 4.57 d	4.78 d, 4.55 d	3.32	3.33
H2’	1.13 ov	1.13 ov	‐	‐
C2	100.1	97.9	99.8	97.8
C3	29.9 b	30.5	30.3	31.0
C4	29.8 b	29.4	30.2	29.7
C5	107.0	108.1	108.2	109.7
C6	134.1	134.0	134.0	133.8
C7	16.2	15.8	16.5	16.2
C8	18.0	17.9	18.4	18.3
C1’	62.9	62.9	69.4	69.1
C2’	15.0	14.9

Individual assignment could not be achieved because of overlapping signals (ov: overlap).

Assignment might be swapped.

In the second step, the dihydropyran mixture was converted to 3,5‐dimethylpyridine according to Scheme 4. 15N‐labelled 3,5‐dimethylpyridine was isolated as an aqueous azeotrope by steam distillation of the basified reaction mixture, after volatile substances were initially removed by distillation of the acidic reaction mixture. Methylene chloride was used to extract the product from the azeotrope with yields of ~ 55% relative to the amount of the 15N isotope used.

Scheme 4

Proposed mechanism of the formation of 3,5‐dimethylpyridine in analogy to Whaley and Ott13

NMR characterization of 3,5‐dimethylpyridine

The 1H NMR spectrum of 15N‐labelled 3,5‐dimethylpyridine was identical to the unlabelled compound, except that the protons adjacent to the 15N nucleus show a splitting of 10.6 Hz due to the 2 J HN scalar coupling. The coupling is in agreement with previous reports measured in the same solvent.21 The observed 1H and 13C resonances agree with previously reported values measured in the same solvent,22 except that we assigned the 13C signal at 137.0 ppm to C3/C5 and 132.4 ppm to C4 (swapped in Pazderski et al). Our 13C assignment agrees also with the data measured in DMSO‐d and D2O despite small deviations due to the different solvents.23, 24 Although the 13C spectrum of labelled and unlabelled 3,5‐dimethylpyridine looked virtually identical, a closer interpretation revealed a small splitting of two signals due to small n J CN scalar couplings. Interestingly, no splitting was observed for the two carbons directly adjacent to the nitrogen nucleus, but the signals of C3, C5, and C4 showed a splitting. Values of 3.1 Hz for 2 J CN and of 3.5 Hz for 3 J CN were observed, which are in a similar range as those observed in 15N‐labelled pyridine ( J = –2.53 Hz and 3 J CN = –3.85 Hz 25). 1 J CN was too small to be detectable in a splitting in agreement with a 1 J CN of 0.67 Hz observed for 15N‐labelled pyridine.26 The observed 15N resonance of –69.7 ppm referenced to CH3NO2 agrees well with previously reported chemical shifts measured at natural abundance.21, 22 A comparison with 15N chemical shifts of other methyl‐substituted pyridine derivatives is given in Supplementary Table S1.

In addition to NMR spectroscopy, mass spectrometry confirmed the chemical identity of compound 3, whose mass spectrum differed from the unlabelled 3,5‐dimethylpyridine,27 only for the 15N‐containing fragments.

CONCLUSION

15N‐labelled 3,5‐dimethylpyridine could be conveniently synthesized in two steps starting from methacrolein, 1‐ethoxypropene, and 15NH4Cl.

EXPERIMENTAL

Unlabelled reagents were purchased from Sigma‐Aldrich. 15N‐labelled NH4Cl was purchased from Chemotrade Chemiehandelsgesellschaft (Leipzig, Germany).

NMR spectroscopy and mass spectrometry

Unless stated otherwise, NMR spectra were recorded either on a Bruker AMX 500 or a Bruker AMSY 270 with CDCl3 as solvent at 298K. 1H and 13C chemical shifts were referenced to TMS. The solvent signals of signals were set for DMSO‐d to 2.49 ppm (1H) and 39.51 ppm (13C) and for CDCl3 to 7.24 ppm (1H) and 77.2 ppm (13C). 15N resonances were indirectly referenced to CH3NO2, using a saturated solution of 15NH4Cl in H2O (~5.64 M) with a chemical shift of –352.89 ppm.28 Mass spectra were recorded on a Varian MAT 711.

Synthesis of 2‐ethoxy‐3,4‐dihydro‐3,5‐dimethyl‐2H‐pyran 13

14 g (0.2 mol) of methacrolein 11, 26 g (0.3 mol) of 1‐ethoxypropene 12, and 0.1 g of hydroquinone (0.25% of mixture) were heated in a 200‐ml autoclave (high‐pressure laboratory autoclave model II from Carl Roth, Germany) at 190 °C for 16 hours. During that time, the pressure first rose to 15 bar and then fell to 8 bar. After cooling, the reaction mixture was distilled under reduced pressure of 48 mbar, yielding at 97°C 18.8 g of a fruity‐smelling colorless oil. The product 13 was further purified by column chromatography (Al2O3, hexane/ethyl acetate 10:1, column dimensions 40 × 6 cm). Yield: 11.7 g (74.8 mmol; 37%). nD 20 = 1.4420. TLC (Al2O3 hexane/ethyl acetate 10:1): Rf = 0.727. 1H‐NMR (DMSO‐d ): 6.02 (s, 0.59H, H6trans), 5.99 (s, 0.41H, H6cis), 4.73 (d, 0.41H, H2cis, J = 2.3 Hz), 4.52 (d, 0.59H, H2trans, J = 4.6 Hz), 3.5‐3.7 (m, 2H, CH CH3), 1.5‐2.6 (m, 3H, H3/H4), 1.5 (s, 3H, 5‐CH3), 1.12 (q, 3H, CH2 CH ), 0.91 (2×d, 3H, 3‐CH3). 13C‐NMR (DMSO‐d ): 134.1 (C6), 108.1 (C5cis), 107.0 (C5trans), 100.0 (C2trans), 97.9 (C2cis), 62.9 (CH CH3), 29.4‐30.4 (C3 and C4), 17.9 (5‐CH3), 16.2 (3‐CH3 trans), 15.7 (3‐CH3 cis), 15.0 CH2 CH ). MS (EI): 156 (18, M+), 111 (19), 86 (100, retro‐Diels‐Alder), 58 (90).

Synthesis of 3,5‐dimethylpyridine 3

In a three‐necked flask equipped with a reflux condenser, an addition funnel, and a magnetic stirrer, 150 ml of deionized water were poured, followed by 4.4 ml of concentrated H2SO4, 15 g (39.7 mmol) of methylene blue, and 2 g (36.7 mmol) of 15NH4Cl. The solution was brought to reflux, and a solution of 5.78 g (37 mmol) of 2‐ethoxy‐dihydro‐3,5‐dimethyl‐2H‐pyran 13 in 5 ml of ethanol was added dropwise over a period of 1 hour and refluxed for 17 hours. After cooling, 150 ml of deionized water was added and the mixture was distilled until the odor of glutaraldehyde disappeared in the distillate (ca. 200 ml). After cooling of the remaining reaction mixture, 250 ml of 1.3 M NaOH were added gradually and distilled until ~200 ml of distillate was collected. 0.1 g of Na2CO3 was added, and CH2Cl2 was used to extract the organic base. The combined organic layers were dried with Na2SO4, and the solvent was removed with a rotary evaporator. Yield: 2.18 g (20.2 mmol; 55%). 1H‐NMR (CDCl3): 8.21 (d, 2H, H2/H6, 2 J NH = 10.6 Hz), 7.26 (s, 1H, H4), 2.25 (s, 6H, CH3), 13C‐NMR (CDCl3): 147.3 (C2/C6), 137.0 (d, 3 J NC = 3.5 Hz, C4), 132.4 (d, 2 J NC = 3.1 Hz, C3/C5), 18.1 (CH3). 15N‐NMR (CDCl3): –69.7 ppm referenced to CH3NO2. MS (EI): 108 (100, C7H9 15N+), 93 (21), 79 (35), 77 (11).

Table S1 Comparison of 15N chemical shifts of pyridine derivatives measured in CDCl3 at 298 K and referenced to external CH3NO2

Click here for additional data file.