Synthesis and complete 1H, 13C and 15N NMR assignment of ...

MAGNETIC RESONANCE IN CHEMISTRY
Magn. Reson. Chem. 2005; 43: 240–245
Published online 17 December 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/mrc.1528
Synthesis and complete 1 H, 13 Cand 15 NNMR
assignment of substituted
isoxazolo[3,4-d]pyridazin-7(6H)-ones
V. Dal Piaz, 1∗∗ A. Graziano, 1 N. Haider 2 and W. Holzer 2∗
1 Dipartimento di Scienze Farmaceutiche, Università di Firenze, Via Ugo Schiff 6, I-50019 Sesto Fiorentino, Italy
2 Department of Pharmaceutical Chemistry, University of Vienna, Althanstraße 14, A-1090 Vienna, Austria
Received 22 July 2004; Accepted 25 October 2004
The synthesis and complete assignment of all hydrogen, carbon and nitrogen NMR signals of several
new isoxazolo[3,4-d]pyridazin-7(6H)-ones is reported. The spectroscopic characterization is extended to
previously described analogues. Copyright © 2004 John Wiley & Sons, Ltd.
KEYWORDS: 1 HNMR; 13 CNMR; 15 NNMR; 13 Cf 1 Hg NOE; INEPT; selective excitation; HMQC; HMBC;
isoxazolo[3,4-d]pyridazinones
INTRODUCTION
The very important role of isoxazole derivatives as reagents
and intermediates in the synthesis of various nitrogen
heterocycles as well as of natural products and complex
molecules has been documented extensively in the
literature. 1,2 Among the different polynuclear isoxazole
types, the isoxazolo[3,4-d]pyridazin-7(6H)-ones (1) proved
to be particularly versatile synthetic precursors of different
classes of 4,5-difunctionalized or heterocycle-annulated
pyridazinones. In fact, the bicyclic system 1 incorporates a
variety of masked functional groups such as amino, nitro,
cyano and alcoholic hydroxy groups as well as carbonyl
functions, which can be exposed easily at the desired stage
of the synthetic pathway by selective cleavage of the N—O
bond under mild conditions. Nitro and amino groups (compounds
2–4; Scheme 1) arise from the isoxazole nitrogen by
oxidative or reductive ring-opening, respectively. Concomitantly,
carbonyl or alcoholic functions are formed from C-3
under these reaction conditions. 3,4
Moreover, alkaline cleavage of the heterocyclic systems
and subsequent rearrangement was found to afford fiveor
seven-membered nitrogen heterocycles bearing amino,
cyano and carbonyl groups (compounds 5–7;Scheme2). 5–7
Pharmaceutically interesting transformation products of
the title compounds include the 5-acyl-4-aminopyridazinones
(2), which, when orally administered, exhibit good
activity and low acute toxicity in animal models of
acute inflammation. 8 On the other hand, the 5-acyl-4-
nitropyridazinones (3), as evaluated in vitro in human
platelet-rich plasma, show potent aggregation inhibitory
Ł Correspondence to: W. Holzer, Department of Pharmaceutical
Chemistry, University of Vienna, Althanstraße 14, A-1090 Vienna,
Austria. E-mail: holzer@merian.pch.univie.ac.at
ŁŁ Correspondence to: V. Dal Piaz, Dipartimento di Scienze
Farmaceutiche, Università di Firenze, Via Ugo Schiff 6, I-50019
Sesto Fiorentino, Italy.
activity, suggesting a possible application as antithrombotic
agents. 8,9
From a synthetic point of view, compounds 3 are very
versatile precursors of different classes of heterocycle-fused
pyridazinones such as pyrazolo-, pyrrolo-, thieno-, pyridoand
pyrimidopyridazinones, which can be obtained from
3 in good yields by treatment with various bifunctional
nucleophiles. 10 Some of these bicyclic derivatives exhibit
potent and selective phosphodiesterase 4 (PDE4) inhibitory
activity, which renders them potentially useful as antiinflammatory
and immunomodulatory agents. 11
In the course of further studies aimed at an extension
of chemical diversity in a compound library featuring the
isoxazolopyridazinone scaffold, the need arose to synthesize
compounds 1a–1c and 1e (Scheme 4). Thus, we present here
the preparation of these novel chemical entities, as well as
their complete 1 H, 13 Cand 15 N NMR assignment. Moreover,
we extended this spectroscopic characterization to several
previously described analogues.
RESULTS AND DISCUSSION
Syntheses
The synthesis of the new 4-(2- and 3-thienyl)isoxazolopyridazinones
(1a–1c) was accomplished via a short and convenient
pathway, with the first step based on a 1,3-dipolar cycloaddition
of the nitriloxide 8 (generated in situ from ethyl
chlorooximidoacetate) and the appropriate diketone 9 12 or
10, 13 respectively (Scheme 3). The functionalized isoxazoles
11 and 12 were cyclocondensed with hydrazine hydrate,
affording the isoxazolopyridazinones 1a and 1b, respectively.
Compound 1a,inturn,wasconvertedinto1c by alkylation
under standard conditions. The N-benzoyl compound
1e was obtained from the previously described analogue, 3-
methyl-4-phenylisoxazolo[3,4-d]pyridazin-7(6H)-one 14 (1d),
under similar reaction conditions using benzoyl chloride.
Samples of the known compounds 1f–1l were prepared
using the reported procedures. 3,8,9,14
Copyright © 2004 John Wiley & Sons, Ltd.

Synthesis of substituted isoxazolo[3,4-d]pyridazin-7(6H)-ones 241
Scheme 1. Reductive and oxidative isoxazole ring cleavage in
isoxazolo[3,4-d]pyridazin-7(6H)-ones (1).
Scheme 3. Synthesis of new isoxazolo[3,4-d]pyridazin-7(6H)-
ones 1a–1c and 1e.
Scheme 4. Structure and numbering scheme of isoxazolo[3,4-
d]pyridazin-7(6H)-ones 1a–1l.
Scheme 2. Ring transformations of 1 under alkaline conditions.
1 HNMR, 13 CNMRand 15 N NMR spectral
assignments
The structures and numbering scheme of the newly synthesized
compounds 1a–1c, 1e and the known isoxazolo[3,4-
d]pyridazin-7(6H)-ones 1d and 1f–1l are given in Scheme 4.
The 1 H NMR data are listed in Table 1, 13 C chemical shifts
are presented in Table 2, selected 13 C 1 H spin coupling constants
are given in Table 3 and the 15 N chemical shifts are
summarized in Table 4.
The 1 H NMR spectra of compounds 1a–1l were simple
to analyse because they contain only a few signals. C-
Methyl singlets (C3-CH 3 , C4-CH 3 , υ 2.41–2.90) could be
distinguished from N-methyl lines (N6-CH 3 , υ 3.55–3.69) on
the basis of chemical shift considerations.
In compounds 1f, 1g and 1h two C-methyl groups are
present, which were discriminated via long-range INEPT
experiments with selective excitation. Thus, for instance,
selective excitation of the C3-CH 3 transition in 1g (υ 2.84)
revealed the characteristic downfield signal of C-3 (υ 171.7) as
well as that of C-3a (υ 112.0), whereas in a similar experiment
the C4-CH 3 protons exhibited correlations to C-3a and to C-4
(υ 140.1). Either HMQC or one-dimensional HETCOR spectra
enabled us to identify the carbon atoms directly attached to
the unambiguously assigned proton resonances. However,
the main task in analysis of the 13 C NMR spectra consisted of
assignment of the numerous quaternary carbon atoms. This
could be achieved mainly by a series of long-range INEPT
experiments with selective excitation, optimized for a 13 C 1 H
coupling of 6–8 Hz (outlined in Scheme 5 for 1a) aswell
as on the basis of coupling information from the fully 1 H-
coupled 13 C NMR spectra. With compounds 1a, 1b, 1d and
Copyright © 2004 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2005; 43: 240–245

242 V. Dal Piaz et al.
Table 1. The 1 H NMR chemical shifts (υ, ppm) of compounds 1a–1l (in DMSO-d 6 )
Compound H of R 1 HofR 2 HofR 3
1a 2.74 7.73 (H-5 0 ), 7.56 (H-3 0 ), 7.21 (H-4 0 ) a 12.67
1b 2.62 7.92 (H-2 0 ), 7.71 (H-5 0 ), 7.39 (H-4 0 ) b 12.38
1c 2.75 7.76 (H-5 0 ), 7.57 (H-3 0 ), 7.22 (H-4 0 ) c 4.08 (CH 2 ), 1.28 (CH 3 )
1d 2.49 7.60 (o-H), 7.53 (m-H, p-H) 12.66
1e 2.53 7.59 (o-H), 7.54 (m-H, p-H) 8.01 (o-H), 7.73 (p-H), 7.57 (m-H)
1f 2.84 2.41 12.19
1g 2.84 2.43 3.55
1h 2.90 2.50 7.51 (o-H, m-H), 7.39 (p-H)
1i 2.50 7.61 (o-H), 7.54 (m-H, p-H) 3.67
1j 2.83 (CH 2 ), 1.07 (CH 3 ) 7.61 (o-H), 7.55 (m-H, p-H) 4.12 (CH 2 ), 1.29 (CH 3 ) d
1k 2.57 8.76 (H-2,6), 7.64 (H-3,5) 3.69
1l 2.51 7.63 (o-H), 7.54 (m-H, p-H) 4.08 (NCH 2 ), e 1.72 (NCH 2 CH 2 ), 1.33 (CH 2 CH 3 ), 0.89 (CH 3 ) f
a3 J⊲H-3 0 , H-4 0 ⊳ D 3.7Hz, 4 J⊲H-3 0 , H-5 0 ⊳ D 1.2Hz, 3 J⊲H-4 0 , H-5 0 ⊳ D 5.1Hz.
b3 J⊲H-4 0 , H-5 0 ⊳ D 5.0Hz, 4 J⊲H-2 0 , H-4 0 ⊳ D 1.2Hz, 4 J⊲H-2 0 , H-5 0 ⊳ D 2.9Hz.
c3 J⊲H-3 0 , H-4 0 ⊳ D 3.7Hz, 4 J⊲H-3 0 , H-5 0 ⊳ D 1.2Hz, 3 J⊲H-4 0 , H-5 0 ⊳ D 5.1Hz.
d3 J⊲CH 3 , CH 2 ⊳ D 7.1Hz.
e3 J⊲NCH 2 , CH 2 ⊳ D 7.2Hz.
f3 J⊲CH 3 , CH 2 ⊳ D 7.3Hz.
Table 2. The 13 C NMR chemical shifts (υ, ppm) of compounds 1a–1l (in DMSO-d 6 )
Compound C-3 C-3a C-4 C-7 C-7a C of R 1 CofR 2 CofR 3
1a 171.0 110.4 136.8 153.3 151.9 14.3 135.7 (2 0 ), 129.0 (3 0 ),
128.5 (5 0 ), 127.7 (4 0 )
1b 171.1 111.0 138.3 153.5 151.9 13.8 134.7 (3 0 ), 127.6 (4 0 ),
127.0 (5 0 ), 126.7 (2 0 )
1c 171.3 110.3 136.4 151.7 151.8 14.3 135.4 (2 0 ), 129.3 (3 0 ),
128.8 (5 0 ), 127.7 (4 0 )
1d 171.0 110.9 142.5 153.5 152.0 13.7 133.8 (1), 129.6 (4),
128.5 (3,5), 128.4 (2,6)
1e 171.8 111.1 143.3 152.9 152.5 13.6 132.9 (1), 130.2 (4),
128.6 (3,5), 128.5 (2,6)
—
—
44.6 (CH 2 ), 13.3 (CH 3 )
—
170.2 (C O), 134.5 (4),
131.7 (1), 130.6 (2,6), 128.9
(3,5)
1f 171.3 112.0 140.4 153.7 151.6 12.8 18.7 —
1g 171.7 112.0 140.1 152.4 151.3 12.8 18.7 37.5
1h 171.9 111.9 140.9 152.4 152.1 12.9 18.9 140.9 (1), 128.6 (3,5), 127.6
(4), 126.2 (2,6)
1i 171.3 111.0 141.9 152.3 151.7 13.7 133.4 (1), 129.8 (4), 38.0
128.5 (3,5), 128.4 (2,6)
1j 175.2 110.3 142.2 151.8 151.8 20.9 (CH 2 ), 11.1 (CH 3 ) 133.9 (1), 129.8 (4), 44.6 (CH 2 ), 13.3 (CH 3 )
128.5 (3,5), 128.2 (2,6)
1k 171.3 110.5 139.6 152.4 151.7 13.9 150.1 (2,6), 140.8 (4),
122.8 (3,5)
38.1
1l 171.3 110.8 142.0 152.1 151.8 13.7 133.5 (1), 129.8 (4),
128.5 (3,5), 128.4 (2,6)
49.0 (NCH 2 ), 29.9
(NCH 2 CH 2 ), 19.3 (CH 2 CH 3 ),
13.5 (CH 3 )
1f, characterized by an NH moiety (R 3 D H), the signal due
to C-7 could be identified independently by heteronuclear
13 Cf 1 Hg NOE difference experiments (irradiation of the NH
resonance enhanced the line of the adjacent C-7 carbon atom).
In cases when no unequivocal assignment of 13 C 1 H coupling
constants was possible (e.g. within the thiophene system
of compounds 1a–1c), two-dimensional (υ,J) long-range
INEPT experiments with selective excitation were applied.
Interestingly, the signal due to C-7a also showed a quartet
splitting (J D 1 Hz, after resolution enhancement) in the
1 H-coupled 13 C NMR spectra. Because this splitting was
observed in compounds carrying no methyl groups attached
toC-4orN-6,itmustoriginatefroma 4 J coupling to the 3-CH 3
protons. This finding was confirmed by the observation that
Copyright © 2004 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2005; 43: 240–245

Synthesis of substituted isoxazolo[3,4-d]pyridazin-7(6H)-ones 243
Table 3. Selected 13 C 1 H spin coupling constants (Hz) of compounds 1a–1l (in DMSO-d6)
Compound
2 J(C3, 3-CH 3)
3 J(C3a, 3-CH 3)
4 J(C7a, 3-CH 3)
1 J(3-CH 3) J of R 2 J of R 3 Other couplings
1a 7.2 2.7 1.1 131.8 a
—
1b 7.2 2.7 1.1 131.7 b
—
1c 7.2 2.8 1.0 131.8 c 1
J⊲NCH 2⊳ D 141.3
2 J⊲NCH 2, CH3⊳ D 4.6
1 J⊲CH 3⊳ D 127.7
2 J⊲CH 3, CH2⊳ D 3.4
3 J(C7, NCH 2) D 2.3
1d 7.1 2.7 1.1 131.7 —
1e 7.2 2.7 1.0 131.9
1f 7.1 2.9 1.1 131.5 1
J D 128.9⊲CH
3⊳ — 3
J(C3a, 4-CH 3) D 2.9
1g 7.1 2.9 1.0 131.6 1
J D 129.1⊲CH
3⊳ 1
J⊲CH
3⊳ D 140.7 3
J(C3a, 4-CH 3) D 2.9
1h 7.1 2.9 1.0 131.6 1
J D 129.3⊲CH
3⊳ 3
J(C3a, 4-CH 3) D 2.9
1i 7.2 2.6 1.1 131.7 1
J⊲CH
3⊳ D 141.0 3
J(C7, NCH 3) D 2.2
1j 7.4 (C3, 3-CH2) 2.1 (C3a, 3-CH2) 1.0 (C7a, 3-CH2) 131.7 (3-CH2), 129.1 (CH3) 1
J⊲NCH 2⊳ D 141.2
2 J⊲NCH 2, CH3⊳ D 4.6
1 J⊲CH 3⊳ D 127.6
2 J⊲CH 3, CH2⊳ D 3.4
3 J(C7, NCH 2) D 2.5
1k 7.2 2.7 131.9 1
J⊲CH
3⊳ D 141.2 3
J(C7, NCH 3) D 2.1
1l 7.2 2.7 131.8 1
J⊲NCH
2⊳ D 140.3
3
J(C7, NCH 2) D 2.3
1.1 1 J⊲CH 3⊳ D 124.7
a Thiophene system:
2 J(C2, H3) D 6.7,
3 J(C2, H4) D 9.8,
3 J(C2, H5) D 6.0;
1 J(C3, H3) D 168.3,
2 J(C3, H4) D 6.1,
3 J(C3, H5) D 9.3;
1 J(C4, H4) D 169.5,
2 J(C4, H3) D 5.0,
2 J(C4, H5) D 4.3;
1 J(C5, H5)
D 188.4, 2 J(C5, H4) D 7.3, 3 J(C5, H3) D 10.4.
b Thiophene system:
1 J(C2, H2) D 188.2,
3 J(C2, H4) D 8.3,
3 J(C2, H5) D 4.5;
2 J(C3, H2) D 3.8,
2 J(C3, H4) D 4.8,
3 J(C3, H5) D 10.2;
1 J(C4, H4) D 169.8,
2 J(C4, H5) D 5.6,
3 J(C4, H2) D 8.6;
1 J(C5,
H5) D 187.9, 2 J(C5, H4) D 8.5, 3 J(C5, H2) D 5.5.
c Thiophene system:
2 J(C2, H3) D 6.7,
3 J(C2, H4) D 9.9,
3 J(C2, H5) D 6.1;
1 J(C3, H3) D 168.5,
2 J(C3, H4) D 6.1,
3 J(C3, H5) D 9.4;
1 J(C4, H4) D 169.7,
2 J(C4, H3) D 5.2,
2 J(C4, H5) D 4.4;
1 J(C5, H5)
D 188.3, 2 J(C5, H4) D 7.3, 3 J(C5, H3) D 10.4.
Copyright © 2004 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2005; 43: 240–245

244 V. Dal Piaz et al.
Table 4. The 15 N NMR chemical shifts (υ, ppm) of compounds
1a–1l (in DMSO-d 6 )
Compound N-1 N-5 N-6
1a 1.2 71.5 190.5
1b 0.9 70.3 190.8
1c 0.9 66.4 182.5
1d 0.7 69.0 190.2
1e 1.4 71.2 166.0
1f 0.1 74.6 192.0
1g 0.2 65.6 195.2
1h 1.4 66.4 180.7
1i 0.8 60.5 193.5
1j 0.5 63.0 181.0
1k a 0.7 57.7 192.4
1l 0.4 62.5 183.0
a Pyridine-N:
61.2 ppm.
Scheme 5. Key assignments of the quaternary carbon atoms in
1a based on long-range 13 C/ 1 H correlations (full arrows) and a
heteronuclear NOE (dashed arrow).
upon selective excitation of the 3-CH 3 transition in the longrange
INEPT spectra the C-7 signal was enhanced, albeit to
a small extent (small coupling).
Assignment of the 15 N NMR signals due to N-5 and
N-6 was based on INEPT (refocused and decoupled) or gs-
HMBC experiments. Moreover, INEPT spectra optimized to
a 1 J⊲ 15 N, 1 H⊳ coupling constant of 90–100 Hz revealed the
signal due to N-6 in compounds 1a, 1b, 1d and 1f (R 3 D H),
thus confirming a pyridazinone and not a hydroxypyridazine
structure. Finally, the signal due to N-1 had to be determined
in one-pulse 15 N experiments because this nitrogen atom
lacks couplings suitable for polarization transfer.
EXPERIMENTAL
All melting points were determined on a Büchi apparatus and
are uncorrected. The 1 H NMR spectra of intermediates were
recorded with a Varian Gemini 200 instrument (200 MHz);
chemical shifts are reported in ppm using the solvent signal
as an internal standard. Extracts were dried over Na 2 SO 4
and the solvents were removed under reduced pressure.
Merck F 254 commercial plates were used for analytical thinlayer
chromatography to follow the course of the reactions.
Silica gel 60 (Merck 70–230 mesh) was used for column
chromatography.
Compounds
Preparation of ethyl 5-methyl-4-[(2-thienyl)carbonyl]-
isoxazole-3-carboxylate (11)
To a cooled and stirred solution of sodium ethoxide (0.78 g,
11.5 mmol) in absolute EtOH (35 ml) a solution of 1-(2-
thienyl)butane-1,3-dione (1.94 g, 11.5 mmol) in the same
solvent (45 ml) was added dropwise. Then, a solution of ethyl
chloro(hydroximino)acetate (1.75 g, 11.5 mmol) in absolute
EtOH (15 ml) was added over a period of 45 min. After
solvent evaporation, the residue was washed with cold 0.5 N
NaOH and water. The solid obtained (2.08 g, 68%) was
purified by crystallization from EtOH: m.p. 46–48 °C. 1 H
NMR (CDCl 3 ): υ 1.15 (t, 3H, J D 7.2Hz,CH 2 CH 3 ), 2.55 (s, 3H,
CH 3 ), 4.20 (q, 2H, J D 7.2Hz,CH 2 CH 3 ), 7.20–7.70 (m, 3H,
thienyl-H); Anal. calcd. for C 12 H 11 NO 4 S (265.3): C, 54.33; H,
4.18; N, 5.28. Found: 54.11; H, 4.39; N, 5.45.
Preparation of ethyl 5-methyl-4-[(3-thienyl)carbonyl]-
isoxazole-3-carboxylate (12)
Compound 12 was synthesized in 63% yield following
the above procedure, starting from the appropriate 1-(3-
thienyl)butane-1,3-dione, and the oil purified by column
chromatography (cyclohexane–ethyl acetate, 3 : 1). Anal.
calcd. for C 12 H 11 NO 4 S (265.3): C, 54.33; H, 4.18; N, 5.28.
Found: C, 54.61; H, 4.02; N, 5.19.
Preparation of 3-methyl-4-(2-thienyl)isoxazolo[3,4-
d]pyridazin-7(6H)-one (1a)
To a cooled and stirred solution of the ester 11 (0.25 g,
0.94 mmol) dissolved in EtOH (1.7 ml), hydrazine hydrate
(0.1 ml, 2.05 mmol) was added. After 30 min of stirring,
the precipitate was collected by filtration to afford 1a
(0.136 g, 62%), m.p. 219–220 °C (from EtOH). Anal. calcd.
for C 10 H 7 N 3 O 2 S (233.3): C, 51.48; H, 3.03; N, 18.01. Found: C,
51.72; H, 3.25; N, 18.29.
Preparation of 3-methyl-4-(3-thienyl)isoxazolo[3,4-
d]pyridazin-7(6H)-one (1b)
Compound 1b was synthesized in 48% yield following the
above procedure, starting from the ester 12 and using the
same molar ratio: m.p. 237–239 °C (from EtOH). Anal. calcd.
for C 10 H 7 N 3 O 2 S (233.3): C, 51.48; H, 3.03; N, 18.01. Found: C,
51.64; H, 2.92; N, 17.89.
Preparation of 6-ethyl-3-methyl-4-(2-thienyl)isoxazolo-
[3,4-d]pyridazin-7(6H)-one (1c)
A mixture of 1a (0.14 g, 0.6 mmol), anhydrous K 2 CO 3
(0.17 g, 1.2 mmol) and bromoethane (0.14 g, 1.34 mmol) in
anhydrous DMF (1.7 ml) was stirred at 90 °C for1h.After
cooling and dilution with cold water (15 ml), the precipitate
1c (0.13 g, 83%) was collected by filtration and recrystallized
from water (with gentle heating): m.p. 86–87 °C. Anal. calcd.
for C 12 H 11 N 3 O 2 S (259.1): C, 55.63; H, 4.28; N, 16.22. Found:
C, 55.43; H, 4.07; N, 16.41.
Preparation of 6-benzoyl-3-methyl-4-phenylisoxazolo[3,4-
d]pyridazin-7(6H)-one (1e)
To a stirred suspension of 1d 14 (0.545 g, 2.4 mmol) in
anhydrous toluene (20 ml), pyridine (0.19 g, 2.4 mmol) and
Copyright © 2004 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2005; 43: 240–245

Synthesis of substituted isoxazolo[3,4-d]pyridazin-7(6H)-ones 245
benzoyl chloride (0.337 g, 2.4 mmol) were added in this
order. The mixture was refluxed for 30 h. After cooling,
the precipitate was removed by filtration and the filtrate
was evaporated to dryness. The residue (0.651 g, 82%) was
recrystallized from EtOH: m.p. 161–162 °C. Anal. calcd. for
C 19 H 13 N 3 O 3 (331.3): C, 68.88; H, 3.95; N, 12.68. Found: C,
69.11; H, 4.07; N, 12.93.
NMR measurements
All 1 HNMRand 13 C NMR spectra of the title compounds
were recorded on a Varian UnityPlus NMR spectrometer
(300 MHz for 1 H, 75 MHz for 13 C) at 28 °C fromDMSOd
6 solutions (¾0.3–0.5 M) using a 5 mm direct detection
broadband probe and deuterium lock. The centre of the
solvent signal was used as an internal standard, which
was related to tetramethylsilane with υ 2.49 ppm ( 1 H)
and υ 39.5 ppm ( 13 C). The recording conditions were the
following: 1 H NMR: pulse width 6 µs (35°), acquisition
time 5 s, digital resolution 0.2 Hz per data point, spectral
width 15 ppm, 16 transients, relaxation delay 5 s; broadband
decoupled 13 C NMR spectra: pulse width 7 µs (45°),
acquisition time 2 s, digital resolution 0.5 Hz per data point,
spectral width 250 ppm, 2048 transients, relaxation delay
2 s, exponential multiplication with 1.0 Hz line broadening
factor before fourier transformation; gated decoupled 13 C
NMR spectra: as above but acquisition time 2.5 s, digital
resolution 0.4 Hz per data point, 8192 transients, relaxation
delay 2.5 s, resolution enhancement by Gaussian weighting
(lb D 0.15, gf D 0.7) before fourier transformation. Full
and unambiguous assignments wereachievedbycombined
application of various standard NMR techniques. Thus,
fully 1 H-coupled 13 C NMR spectra (gated decoupling),
heteronuclear 13 Cf 1 Hg NOE difference spectroscopy, 15–17
one-dimensional HETCOR, 18 long-range INEPT spectra with
selective excitation 19 and a two-dimensional variant of
the latter 20 and (occasionally) HMQC spectra 21 were used
for this purpose. 15 N NMR spectra were obtained on a
Bruker Avance 500 instrument (50.69 MHz) equipped with a
5 mm broadband observe probe at 300 K using one-pulse
[pulse width 7 µs (50°), relaxation delay 10 s] or INEPT
techniques 22 (optimized for a 15 N 1 H coupling of 90 Hz or
8 Hz, respectively, relaxation delay 5 s). Referencing was
done against external, neat nitromethane. The acquisition
time was 1 s, digital resolution 1 Hz per data point, spectral
width 400 ppm, 4000–8000 transients. In compound 1k,
N-5 was unequivocally distinguished from the pyridine-
Nviaan 15 N 1 Hgs-HMBCexperiment 23 [Bruker standard
program ‘inv4gplplrndqf’, 4096 ð 256 data matrix, 10 ppm
for 1 H, 400 ppm for 15 N, 32 transients accumulated per
t 1 increment; 65 ms delay for the evolution of the 15 N 1 H
long-range coupling (optimized for J D 8 Hz), zero-filling to
1000 data points in the F1 dimension, sine multiplication in
both dimensions].
Acknowledgement
We are grateful to Dr Csaba Szantay Jr (Gedeon Richter Ltd,
Budapest, Hungary) for providing a pulse sequence 17 for the 13 Cf 1 Hg
NOE difference experiments.
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