NMR study of 5-substituted pyrazolo[3,4-c]pyridine derivatives

Article abstract of DOI:10.1002/mrc.2226

Substituted pyrazolopyridines are potent inhibitors of phosphodiesterases and cyclin-dependent kinases. In this study, NMR was used to investigate the potential NI-H and N2-H tautomerism of 5-substituted pyrazolo[3,4-c]pyridine derivatives. Six compounds were fully characterized by using ¹H, ¹³C, and ¹⁵N chemical shifts and indirect ¹H-¹³C and ¹H-¹⁵N coupling constants. The ¹H NMR spectra were measured over a broad range of temperatures. All of the compounds were shown to exist predominantly in the NI-H tautomeric form. Complementary quantum-chemical calculations of the chemical shieldings and indirect spin-spin couplings support the structural conclusions drawn. Copyright

Full text of DOI:10.1002/mrc.2226

Research Article
Received: 7 January 2008
Revised: 19 February 2008
Accepted: 19 February 2008
Published online in Wiley Interscience: 28 March 2008
(www.interscience.com) DOI 10.1002/mrc.2226
NMR study of 5-substituted
pyrazolo[3,4-c]pyridine derivatives
a,b
b
b
a
´ ˇ
ˇ
Orestis Tsikouris, Tomas Bartl, Jaromír Tousek, Nikolaos Lougiakis,
Tony Tite,a Panagiotis Marakos,a Nicole Pouli,a Emmanuel Mikrosa and
Radek Marekb∗
Substituted pyrazolopyridines are potent inhibitors of phosphodiesterases and cyclin-dependent kinases. In this study, NMR
was used to investigate the potential N1-H and N2-H tautomerism of 5-substituted pyrazolo[3,4-c]pyridine derivatives. Six
compounds were fully characterized by using 1H, 13C, and 15N chemical shifts and indirect 1H–13C and 1H–15N coupling
constants. The 1H NMR spectra were measured over a broad range of temperatures. All of the compounds were shown to exist
predominantly in the N1-H tautomeric form. Complementary quantum-chemical calculations of the chemical shieldings and
c
indirect spin-spin couplings support the structural conclusions drawn. Copyright ꢀ 2008 John Wiley & Sons, Ltd.
Keywords: NMR; 1H; 13C; 15N; tautomerism; spin-spin coupling constant; purine; pyrazolo[3,4-c]pyridine; quantum-chemical calculation
Introduction
H
7
7a
3a
6
1
N
N
Substituted purines and purine analogs make up an important
group of heterocyclic compounds. The tautomerism of isolated
nucleic acid bases and several purine derivatives have been inves-
tigated extensively.[1] Tautomeric equilibria should be considered
in any study of their binding modes with biological targets.
A number of variously substituted pyrazolopyridines[2,3] have
been shown to be potent inhibitors of phosphodiesterases,[4]
matrix metalloproteinases,[5] glycogen synthase kinase-3,[6] and
cyclin-dependent kinases.[7] Continuing our systematic study of
pyrazolopyridines,[8] we describe here the NMR investigation of a
number of 5-substituted pyrazolo[3,4-c]pyridine derivatives (com-
pounds 1– 6).
N
5
N
N
N
H
2
R
R
3
4
a
b
Scheme 1. N1-H and N2-H tautomeric forms in compounds 1–5.
of tautomerism was investigated systematically by using NMR
spectroscopy, including low-temperature measurements.
Experimental
NMR spectroscopy
H
1: R = Cl
NMR spectra were recorded using a Bruker Avance DRX 500
spectrometer operating at frequencies of 500.13 MHz (1H), and
125.77 MHz (13C), a Bruker Avance 400 spectrometer operating
at 400.13 MHz (1H) and 100.61 MHz (13C), and a Bruker Avance
300 spectrometer operating at frequencies of 300.13 MHz (1H),
75.48 MHz (13C), and 30.41 MHz (15N). The NMR samples were
prepared by dissolving approximately 10 mg of the 5-substituted
pyrazolo[3,4-c]pyridine in 0.55 ml of methanol-d4 or 0.50 ml of
dimethyl sulfoxide (DMSO-d6). The NMR spectra were measured at
thevarioustemperaturesspecifiedinthetextandinthetables. The
N
2: R = OCH3
3: R = H
N
N
4: R = NH2
R
5: R = NHC(O)CH3
H
H
N
N
N
O
6
∗
Correspondence to: Radek Marek, National Center for Biomolecular Research,
Faculty of Science, Masaryk University, Kamenice 5/A4, CZ - 625 00 Brno, Czech
Republic. E-mail: rmarek@chemi.muni.cz
Generally, compounds 1–5 could exist in an N1-H or N2-
H tautomeric form (Scheme 1). The relative populations of
the individual tautomers are influenced by the temperature,
the solvent, and the substitution pattern[9] – which modify the
electron distribution within the molecule. We have thus prepared
several compounds that bear at position 5, substituents with
differing electronic and steric properties. The phenomenon
a
Department of Pharmacy, Division of Pharmaceutical Chemistry, University of
Athens, Panepistimiopolis Zografou, Athens GR-15771, Greece
b
NationalCenterforBiomolecularResearch,MasarykUniversity,Kamenice5/A4,
Brno CZ-62500, Czech Republic
c
Magn. Reson. Chem. 2008, 46, 643–649
Copyright ꢀ 2008 John Wiley & Sons, Ltd.

O. Tsikouris et al.
NHBoc
NHAc
NH2
NO2
NHBoc
NHBoc
a
c
b
N
N
N
N
CH3
CH3
CH3
CH3
NH2
NO2
7
8
9
10
d
Ac
Ac
H
N
H
N
N
N
g
e
f
N
N
N
N
N
N
N
N
BocHN
H2N
H2N
AcHN
5
4
12
11
Scheme 2. (a) 1) NaH (60% in hexane), dry THF, rt, 1h, 2) Boc2O, dry THF, rt, 2h; (b) Pd/C 10%, EtOH, 50 psi, 5h; (c) Ac2O, CH2Cl2, rt, 12h; (d) AcOK, Ac2O,
isoamyl nitrite, C6H6, 90 ◦C, 14h; (e) CF3CO2H, CH2Cl2, rt, 16h; (f) NH3 in MeOH, rt, 2h; (g) Ac2O, CH2Cl2, rt, 12h.
package.[16] Basis set 6-31G∗ was used to optimize the geome-
Table 1. 1H- and 15N-NMR chemical shifts (δ in ppm) for compounds
try and basis set 6-311G∗∗ to calculate the chemical shieldings
1–6 in methanol-d4 at 300 K
[gauge-including atomic orbital (GIAO) approach][17] and indi-
Compound
H-3
H-4
H-5
H-7
N-1
N-2
N-6
rect spin–spin coupling constants. Methods were selected based
on our previous results.[18] The polarizable continuum model
(PCM)[19] was employed to simulate solvent effects for calculating
the NMR parameters. The 13C and 15N NMR chemical shifts were
calculated by subtracting the shieldings of the individual atoms
from the shielding of the standard [TMS for 13C (186.40 ppm),[20]
NH3 for 15N (245.07 ppm)[21]]. The 1H–15N coupling constants
were recalculated from the 1H–14N couplings using a factor of
−1.4027 (γN−15/γN−14). The optimized geometries of individual
molecules can be obtained upon request from the author (JT).
1
2b
8.15
8.04
8.20a
7.88
8.11
8.35
7.82
7.08
7.79 8.20a
–
–
8.80 181.3 324.3 294.7
8.62 177.4 320.3 271.3
8.99 181.2 318.6 294.0
8.51 176.8 317.7 268.4
8.76 179.2 318.7 286.6
8.93 187.6 343.3 179.0
3
4
5c
6.85
8.36
7.47
–
–
–
6
a Signal overlap.
b 1H NMR: δCH3 = 3.94 ppm.
c 1H NMR: δCH3 = 2.37 ppm; 15N NMR, δNHCO = 140.2 ppm.
Preparation of compounds
1H and 13C NMR chemical shifts (δ in ppm) were referenced to the
signal of methanol [δ = 3.31 ppm for residual CHD2OD (1H);
δ = 49.20 ppm for CD3OD (13C)] or DMSO [δ = 2.49 ppm
All starting chemicals were purchased from Aldrich Chemical
Co. Melting points were determined on a Bu¨chi apparatus and are
uncorrected.FlashchromatographywasperformedonMercksilica
gel 60 (0.040–0.063 mm). Analytical thin layer chromatography
(TLC) was carried out on precoated (0.25 mm) Merck F-254 silica
gel plates. Elemental analyses were performed on a Perkin-Elmer
PE 240C Elemental Analyzer (Norwalk, CT, USA) and were within
0.4% of the theoretical values.
Compounds 1, 2, and 3 were prepared according to procedures
in the literature.[22,23] The synthesis of 4 and 5 is depicted in
Scheme 2: The compound 7 was first converted to the acetamide
10 by treatment with Boc anhydride, catalytic reduction, and
acetylation.[24]
for residual DMSO-d5 (1H); δ = 39.70 ppm for DMSO-d6
(
13C)].
The 15N NMR chemical shifts were referenced to 1 M urea in
DMSO-d6 (δ = 77.0 ppm)[10] and are reported relative to liquid
ammonia.[11,12] No susceptibility correction was applied. The
temperature was calibrated using the methanol sample.
1
A 5-mm QNP [13C/19F/31P{ H}] or a 5-mm multinuclear inverse
BBI [1H{BB}] probe with a self-shielded z-gradient coil was used to
measure the 1H, 13C, and heteronuclear shift correlation spectra.
1H NMR spectra: pulse 90◦, relaxation delay 10 s, number of
scans 4–16, resolution <0.005 ppm per point. 13C NMR spectra:
pulse 45◦, relaxation 3 s, number of scans 512–8192, resolution
<0.01 ppm per point. The 1H–13C GSQMBC[13] NMR experiment
tert-Butyl-N-(1-acetylpyrazolo[3,4-c]pyridin-5-yl) carbamate
(11)
was adjusted for a long-range coupling of 7.5 Hz. 1H–15
N
GSQMBC and 1H–15N gs-HMBC[14,15] NMR experiments were
adjusted for couplings ranging from 9 to 11 Hz (corresponding to
55.6–45.5 ms). Computer processing was performed with Bruker
TopSpinsoftware.Indirectspin–spincouplingconstants(reported
in hertz) are determined with an accuracy of 0.5 Hz for 1JH–C and
Potassium acetate (425 mg, 4.33 mmol) and acetic anhydride
(1.25 ml, 13.22 mmol) were added to a solution of 10 (850 mg,
3.21 mmol)[24] in dry benzene (20 ml) under argon. The reaction
mixture was heated to boiling, isoamyl nitrite (1 ml, 7.44 mmol)
wasadded, andtheresultingmixturewasrefluxedat80 ◦Cfor14 h.
The insoluble material was then filtered off, the solvent vacuum-
evaporated, and the residue purified by column chromatography
(silica gel), using a mixture of cyclohexane : ethyl acetate (8 : 2,
v/v) as eluent, to give 11 (770 mg, 87% yield), mp 188 ◦C
(EtOAc). 1H-NMR (400 MHz, CDCl3) chemical shifts in ppm: 1.62
0.3 Hz for nJH–C and nJH–N
.
Quantum-chemical calculations
Calculations were performed at the DFT level, using the
B3LYP functional as implemented in the Gaussian 03 program
c
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Copyright ꢀ 2008 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2008, 46, 643–649

NMR study of pyrazolo[3,4-c]pyridines
Table 2. 13C-NMR chemical shifts (δ in ppm) and 1JH–C coupling constants (Hz) for compounds 1–6 in methanol-d4 at 300 K
Compound
C-3 (1JH3–C3
)
C-3a
C-4 (1JH4–C4
)
C-5
C-7 (1JH7–C7
)
C-7a
1
134.39 (195.3)
133.97 (192.2)
134.85 (192.2)
132.60a (191.2)
134.82 (192.2)
134.65 (197.4)
131.42
132.62
128.51
132.23
130.40
136.75
115.17 (172.7)
96.51 (166.9)
116.59 (167.0)
96.56 (164.1)
105.27 (171.8)
101.17 (173.0)
141.30
160.15
138.82
153.78
144.54a
154.17
135.34 (187.8)
132.52 (184.5)
135.77 (184.5)
132.40a (182.2)
133.99 (185.2)
126.53 (192.7)
137.86
135.98
138.47
134.76
136.70
133.53
2b
3
4
5c
6
a Value obtained from the 2D 1H–13C chemical shift correlation spectrum.
b δCH3 = 55.48 ppm.
c δCH3 = 23.96 ppm, δNHCO = 171.86 ppm.
H-4
H-7
H-3
ppm
180
200
220
240
260
280
300
H-3/N-1
(J = 6.6 Hz)
H-3/N-2
(J = 12.6 Hz)
H-4/N-6
H-7/N-6
320
340
8.9
8.8
8.7
8.6
8.5
8.4
8.3
8.2
8.1
8.0
7.9
7.8
ppm
Figure 1. 1H–15N GSQMBC spectrum of 1 in methanol-d4 at 300 K.
(s, 9H, (CH3)3), 2.78 (s, 3H, COCH3), 8.11 (s, 1H, H-3), 8.36 (s,
1H, H-4), 9.51 (s, 1H, H-7), 9.78 (brs, 1H, D2O exchang., NH).
13C-NMR (50 MHz, CDCl3) chemical shifts in ppm: 22.61(CH3CO),
28.49 [(CH3)3CO], 81.35 [(CH3)3CO], 101.98 (C-4), 132.35 (C-3a),
133.94 (C-7a), 136.28 (C-7), 139.16 (C-3), 147.75 (C-5), 153.06
(OCONH), 170.29 (COCH3). Partial elemental analysis calculated
for C13H16N4O3: C, 56.51; H, 5.84; N, 20.28. Found: C, 56.39; H, 6.05;
N, 20.16.
for 16 h. The pH was then adjusted to 8 with a saturated solution
of NaHCO3, the organic solvent was vacuum-evaporated, and the
residuewasextractedwithethylacetate. Theorganicextractswere
dehydrated with Na2SO4 and vacuum concentrated to dryness,
and the residue was purified by column chromatography (silica
gel), using a mixture of cyclohexane : ethyl acetate (6 : 4, v/v) as
eluent, to give 12 (242 mg, 80% yield), mp 193–194 ◦C (EtOAc).
1H-NMR (400 MHz, DMSO-d6) chemical shifts in ppm: 2.64 (s, 3H,
COCH3), 6.00 (brs, 2H, D2O exchang., NH2), 6.73 (s, 1H, H-4), 8.31 (s,
1H, H-3), 8.99 (s, 1H, H-7). 13C-NMR (50 MHz, DMSO-d6) chemical
shifts in ppm: 22.14 (CH3), 95.04 (C-4), 128.79 (C-7a), 134.42 (C-3a),
135.03 (C-7), 139.32 (C-3), 156.45 (C-5), 169.51 (COCH3). Partial
elemental analysis calculated for C8H8N4O: C, 54.54; H, 4.58; N,
31.80. Found: C, 54.78; H, 4.66; N, 31.59.
1-Acetylpyrazolo[3,4-c]pyridin-5-yl-amine (12)
Trifluoracetic acid (3.5 ml, 47.12 mmol) was added to a solution
of 11 (470 mg, 1.7 mmol) in dry dichloromethane (20 ml) at 0 ◦C,
and the resulting solution was stirred at laboratory temperature
c
Magn. Reson. Chem. 2008, 46, 643–649
Copyright ꢀ 2008 John Wiley & Sons, Ltd.
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O. Tsikouris et al.
H-3
H-7
ppm
130
H-7/C-7
H-7/C-3a
H-3/C-3a
135
140
145
150
155
160
H-7/C-7a
H-3/C-7a
H-3/C-3
H-7/C-3a
H-7/C-7a
H-3/C-3a
H-3/-7a
H-7/C-5
8.9 8.8 8.7 8.6 8.5 8.4 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6
ppm
Figure 2. Portion of 1H–13C GSQMBC spectrum of 2 in methanol-d4 at 300 K with additional details of correlation signals H3–C3a, H3–C7a and H7–C3a,
H7–C7a.
Table 3. Selected nJH–C coupling constants (Hz) for compounds 1–6
in methanol-d4 at 300 K
Table 4. SelectednJH–N couplingconstants(Hz)forcompounds 1–6,
13, and 14 in methanol-d4 at 300 K
3JH3–N1
3JH4–N6
2JH7–N6
2
3
2
3
3
2
Compound JH3–C3a JH3–C7a JH4–C3a JH4–C7a JH7–C3a JH7–C7a
Compound
2JH3–N2
1
2
3
4
5
6
11.4
11.4
12.4
11.3
11.2
11.8
4.0
3.4
5.4
3.8
4.3
3.8
2.0
2.0
4.6
3.0
5.9
6.0
6.5
6.1
5.7
7.0
4.2
4.8
4.1
4.3
4.1
4.8
7.1
6.6
8.5
6.4
7.4
2.4
1
12.6
12.4
12.7
12.5
11.9
12.5
14.8
4.4
6.6
6.5
7.3
7.2
8.2
7.0
8.0
2.3
2.0
3.1
10.4
9.8
9.8
10.4
10.5
2.7
–
2
a
3
–
4
2.5
a
a
–
–
5
–
a
6
3.0
13c
14c
–
b
a Not determined.
b
–
–
a Not determined.
b Not published.
1H-Pyrazolo[3,4-c]pyridine (3)
c Data from Ref. [8].
The preparation of this compound has been reported
previously.[22] Selected NMR data are listed below. 1H-NMR
(500 MHz, methanol-d4) chemical shifts in ppm at 304 K: 7.80
(d, 1H, H-4), 8.20 (d, 1H, H-5), 8.21 (s, 1H, H-3), 8.99 (s, 1H, H-7); at
215 K: 7.89 (d, 1H, H-4), 8.23 (d, 1H, H-5), 8.29 (s, 1H, H-3), 9.04 (s, 1H,
H-7); at 171 K: 7.93 (d, 1H, H-4), 8.25 (d, 1H, H-5), 8.34 (s, 1H, H-3),
9.08 (s, 1H, H-7). 13C-NMR (125 MHz, methanol-d4) chemical shifts
in ppm at 171 K: 117.10 (C-4), 128.41 (C-7a), 134.84 (C-3), 135.77
(C-7), 138.06 (C-3a), 138.53 (C-5).
in vacuo, and the residue was purified by column chromatography
(silica gel), using a mixture of cyclohexane : ethyl acetate (2 : 8, v/v)
as eluent, to give pure 4 (80 mg, 88% yield), mp 236–237 ◦C (dec)
(EtOAc). 1H-NMR (400 MHz, DMSO-d6) chemical shifts in ppm: 5.34
(brs, 2H, D2O exchange, NH2), 6.62 (s, 1H, H-4), 7.84 (s, 1H, H-3), 8.50
(s, 1H, H-7), 12.98 (brs, 1H, D2O exchange, NH). 13C-NMR (50 MHz,
DMSO-d6) chemical shifts in ppm: 92.63 (C-4), 130.05 (C-3a), 131.15
(C-3), 131.96 (C-7), 132.66 (C-7a), 152.99 (C-5). 1H-NMR (500 MHz,
methanol-d4) chemical shifts in ppm at 171 K: 6.90 (brs, 1H, H-4),
8.02 (brs, 1H, H-3), 8.56 (brs, 1H, H-7). Partial elemental analysis
calculated for C6H6N4: C, 53.72; H, 4.51; N, 41.77. Found: C, 53.52;
H, 4.37; N, 41.63.
Pyrazolo[3,4-c]pyridin-5-yl-amine (4)
Compound 12 (120 mg, 0.68 mmol) was added to a saturated
solutionofammoniainmethanol(15 ml),andtheresultingmixture
was stirred at room temperature for 2 h. The solvent was removed
c
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Copyright ꢀ 2008 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2008, 46, 643–649

NMR study of pyrazolo[3,4-c]pyridines
N-(Pyrazolo[3,4-c]pyridin-5-yl)acetamide (5)
purified by column chromatography (silica gel), using a mixture
of dichloromethane : methanol (100 : 3, ◦v/v) as eluent, to give
compound6(35 mg, 55%yield). mp>250 C(dec)(EtOH). 1H-NMR
(400 MHz, DMSO-d6) chemical shifts in ppm: 7.28 (1H, s, H-4), 8.30
(1H, s, H-3), 8.96 (1H, s, H-7). 13C-NMR (50 MHz, DMSO-d6) chemical
shifts in ppm: 99.00 (C-4), 127.58 (C-7), 132.41 (C-7a), 133.34 (C-
3), 134.06 (C-3a), 153.59 (C-5). 1H-NMR (500 MHz, methanol-d4)
chemical shifts in ppm at 303 K: 7.47 (d, 1H, H-4), 8.34 (d, 1H, H-3),
8.92 (t, 1H, H-7); at 171 K: 7.52 (bs, 1H, H-4), 8.38 (bs, 1H, H-3), 9.18
(bs, 1H, H-7). Partial elemental analysis calculated for C6H5N3O: C,
53.33; H, 3.73; N, 31.10. Found: C, 53.02; H, 3.67; N, 30.84.
Acetic anhydride (0.04 ml, 0.37 mmol) was added to a solution
of 4 (50 mg, 0.37 mmol) in dry dichloromethane (5 ml), and
the resulting solution was stirred at laboratory temperature for
12 h. The solvent was vacuum-evaporated, and the residue was
purified by column chromatography (silica gel), using a mixture
of cyclohexane : ethyl acetate (1 : 1, v/v) as eluent, to give 5
(40 mg, 60% yield). mp >250 ◦C (dec) (EtOAc). 1H-NMR (400 MHz,
DMSO-d6) chemical shifts in ppm: 2.09 (s, 3H, COCH3), 8.16 (s,
1H, H-3), 8.38 (s, 1H, H-4), 8.79 (s, 1H, H-7), 10.38 (brs, 1H, D2O
exchange, NHCO), 13.46 (brs, 1H, D2O exchange, NH). 13C-NMR
(50 MHz, DMSO-d6) chemical shifts in ppm: 23.82 (CH3), 101.95
(C-4), 128.39 (C-3a), 132.53 (C-7), 133.18 (C-3), 134.60 (C-7a), 143.65
(C-5), 168.58 (COCH3). 1H-NMR (500 MHz, methanol-d4) chemical
shifts in ppm at 304 K: 2.19 (s, 3H, CH3), 8.11 (s, 1H, H-3), 8.36 (s, 1H,
H-4), 8.76 (s, 1H, H-5); at 204 K: 2.20 (s, 3H, CH3), 8.19 (s, 1H, H-3),
8.50 (s, 1H, H-4), 8.81 (s, 1H, H-5); at 171 K: 2.21 (s, 3H, CH3), 8.22
(s, 1H, H-3), 8.55 (s, 1H, H-4), 8.84 (s, 1H, H-5). 13C-NMR (125 MHz,
methanol-d4) chemical shifts at 171 K: 23.87 (CH3), 104.61 (C-4),
130.40 (C-7a), 134.17 (C-7), 134.67 (C-3), 136.24 (C-3a), 144.84 (C-5),
172.13 (COCH3). Partial elemental analysis calculated for C8H8N4O:
C, 54.54; H, 4.58; N, 31.80. Found: C, 54.61; H, 4.49; N, 31.95.
Results and Discussion
Low-temperature NMR spectroscopy
Low-temperature NMR spectroscopy was used to study the
tautomeric equilibria of compounds 1–6. It has been shown
previously that separate signals for the individual tautomers of
substitutedpurines[25] andpurineanalogs[8] canbedetectedatlow
temperatures. In order to examine this phenomenon, compounds
3–6 were studied at temperatures of 304–171 K. No significant
changes in the 1H NMR spectra were observed on decreasing the
temperature. Thus, since only one set of 1H and 13C NMR signals
was obtained for samples investigated at low temperature, these
compounds probably exist in one predominant tautomeric form
1,6-Dihydro-1H-pyrazolo[3,4-c]pyridin-5-one (6)
Sodium iodide (200 mg, 1.335 mmol) and trimethylsilylchloride
(500 µl, 3.93 mmol) were added to a solution of 2 (70 mg,
0.469 mmol) in dry acetonitrile (20 ml) under an atmosphere
of argon, and the resulting solution was refluxed for 17 h. The
solvent was then evaporated to dryness, and the residue was
1
or undergo a rapid chemical exchange process. The H and 13C
chemical shifts of compounds 3–6 at various temperatures can be
found in the section Experimental.
Table 5. Selected experimental (in methanol-d4) and calculated (with PCMMeOH) JH–C coupling constants (Hz) for compounds 1, 2, and 6 (a: N1-H
tautomer, b: N2-H tautomer)
Compound
1JH3–C3
1JH4–C4
1JH7–C7
2JH3–C3a
3JH3–C7a
2JH4–C3a
3JH4–C7a
3JH7–C3a
2JH7–C7a
1 (exp)
195.3
186.9
193.3
172.7
164.0
164.3
187.8
180.6
178.1
11.4
11.7
8.3
4.0
3.1
6.1
2.0
1.3
1.2
5.9
5.3
4.6
4.2
4.0
4.1
7.1
6.8
7.0
1a (calcd)
1b (calcd)
2 (expt)
192.2
184.6
191.2
166.9
158.4
158.4
184.5
176.3
174.5
11.4
11.6
8.3
3.4
3.2
6.2
2.0
1.4
1.3
6.0
5.6
4.9
4.8
4.2
4.2
6.6
6.4
6.7
2a (calcd)
2b (calcd)
a
6 (expt)
6 (calcd)
197.4
186.6
173.0
157.5
192.7
179.4
11.8
11.9
3.8
3.1
–
7.0
7.6
4.8
5.7
2.4
0.8
1.2
a Not determined.
Table 6. Experimental (in methanol-d4) and calculated (with PCMMeOH
constants (experimental data are absolute values) for compounds 1, 2, and 6 (a, N1-H tautomer; b, N2-H tautomer)
)
15N-NMR chemical shifts (δ in ppm) and selected indirect 1H–15N coupling
Compound
N-1
N-2
ꢀδ (N2-N1)
N-6
2JH3–N2
3JH3–N1
3JH4–N6
2JH7–N6
1 (expt)
181.3
186.7
304.9
324.3
347.6
236.4
143.0
160.9
−68.5
294.7
315.9
317.2
12.6
−13.0
−4.9
6.6
−8.0
0.3
2.0
−0.6
−0.7
10.4
−11.4
−12.1
1a (calcd)
1b (calcd)
2 (expt)
177.4
182.0
300.6
320.3
345.6
234.5
142.9
163.6
−66.1
271.3
283.2
291.2
12.4
−13.0
−5.2
6.5
−8.0
0.4
3.1
−0.6
−0.6
9.8
−10.8
−11.4
2a (calcd)
2b (calcd)
6 (expt)
6 (calcd)
187.6
176.0
343.3
359.9
155.7
179.0
183.5
12.5
7.0
3.0
2.7
183.9
−13.2
−8.1
−2.2
−1.8
c
Magn. Reson. Chem. 2008, 46, 643–649
Copyright ꢀ 2008 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/mrc

O. Tsikouris et al.
Table 7. Experimental (in methanol-d4) and calculated (with PCMMeOH
)
13C-NMR chemical shifts (δ in ppm) for compounds 1, 2, and 6 (a, N1-H
tautomer; b, N2-H tautomer) and theoretical populations of two tautomers (%) calculated using the Boltzmann equation
Compound
C-3
C-3a
C-4
C-5
C-7
C-7a
ꢀδ (C7a-C3)
% at 300 K
1 (expt)
134.39
141.73
132.17
131.42
138.71
133.99
115.17
124.31
124.18
141.30
155.54
155.82
135.34
140.28
152.39
137.86
144.43
153.04
3.5
2.7
–
1a (calcd)
1b (calcd)
98.9
1.1
20.9
;2 (expt)
133.97
140.61
129.91
132.62
140.02
135.18
96.51
104.99
102.14
160.15
168.33
168.17
132.52
138.05
150.88
135.98
141.90
151.64
2.0
1.3
–
2a (calcd)
2b (calcd)
99.5
0.5
21.7
;6 (expt)
6 (calcd)
134.65
139.78
136.75
147.95
101.17
109.88
154.17
165.55
126.53
127.25
133.53
133.81
−1.1
−6.0
–
a
–
a Not calculated.
Ambient-temperature NMR spectroscopy
OCH3
N
OCH3
COCH3
Since we did not observe any signal broadening or splitting at low
temperatures, a full set of NMR data was recorded for compounds
N
N
N
N
COCH3
N
1
1–6 at 300 K. The H and 15N NMR chemical shifts for 1–6 are
summarized in Table 1, the 13C NMR chemical shifts and single-
bond coupling constants (1JH,C) in Table 2, and the heteronuclear
long-range coupling constants in Tables 3 and 4.
13
14
The NMR signals of three aromatic protons were assigned by
using 2D shift correlation experiments. The interaction of H-3
with nitrogens N-1 and N-2 was observed in the 1H–15N GSQMBC
spectrum (Fig. 1). This undoubtedly distinguishes H-3 from the
other two hydrogens of the ring system. H-4 and H-7 are both
coupled with N-6 but H-7 is expected to be more deshielded
because of its position in the molecule. The assignment was
confirmed unequivocally by using a NOESY experiment (mixing
time 700 ms) for compound 2. A NOE (nuclear Overhauser effect)
cross-peakwasobtainedbetweentheprotonsofthemethylgroup
and H-4.
15N NMR chemical shifts were obtained by using long-range
1H–15N chemical shift correlation experiments at the natural
abundance of the 15N isotope. The assignment of N-6 is
straightforward because of its coupling with H-7 and H-4. The
1H–15N coupling constants were obtained from the antiphase
(GSQMBC) and also the in-phase (gs-HMBC) splitting; the two
values thus obtained for each coupling constant were then
averaged to obtain the results presented in Table 4.
Several NMR parameters were analyzed and compared to
determine the predominant tautomer in solution. Firstly, values
n
of JH3–N were compared with those reported recently for
The signals of the six carbon atoms that form the heterocyclic
skeleton were observed in the 13C NMR spectrum. The carbons of
the aromatic CH groups (C-3, C-4, and C-7) were easily assigned by
observing their correlation peaks with the corresponding protons.
The one-bond coupling constants measured for all compounds
are summarized in Table 2.
the N-1 and N-2 substituted analogs 13 and 14.[8] Coupling
constants JH3–N2 = 11.9–12.7 Hz and JH3–N1 = 6.5–8.2 Hz were
obtained for compounds 1–6. These values agree well with those
obtained for regioisomer 13 (JH3–N2 = 14.8; JH3–N1 = 8.0 Hz). In
contrast, JH3–N2 = 4.4 Hz and JH3–N1 = 2.3 Hz were measured
for regioisomer 14. Based on these tendencies observed for
the indirect spin–spin coupling constants, we conclude that our
compounds exist predominantly or exclusively in the N1-H form.
Thevaluesofthe1H–15Ncouplingconstantsforthetwotautomers
a (N1-H) and b (N2-H) calculated by using the DFT method (see
Table 6)correspondnicelywiththeexperimentaldataandsupport
our conclusion.
A 2D 1H–13C GSQMBC experiment[13] was used to assign
the signals of the quaternary carbons. A portion of the 1H–13
C
GSQMBC spectrum with additional details of the H3–C3a, H3–C7a
and H7–C3a, H7–C7a cross-peaks is shown in Fig. 2. The 1H–13
C
coupling constants were extracted from the antiphase patterns
of the cross-peaks[26] obtained in the GSQMBC spectra and are
summarized in Table 3. In order to distinguish between C-3a
2
3JH3–C7a
,
2JH4–C3a
,
Further, the differences between the chemical shifts of N-1
and N-2 were employed to confirm unequivocally the structure
drawnforthepredominanttautomer(Table 6). Theexperimentally
measured ꢀδ (N2-N1) values for compounds 1 and 2 are 143 ppm
and 142 ppm, respectively. The values calculated for 1a (160 ppm)
and2a(163 ppm)usingDFTagreewellwiththeexperimentaldata,
whereas the values calculated for the N-2 tautomers (−68 ppm for
1b, −66 ppm for 2b) differ significantly from the experimental
results. Finally, for all of the compounds the two nitrogen
resonances were assigned to the N-1 and N-2 atoms on the
basis of the chemical shifts. A hydrogen-bearing nitrogen atom is
more shielded than its nonprotonated counterpart.[1] Therefore,
the more shielded resonance was assigned to N-1.
and C-7a, the coupling constants JH3–C3a
,
3JH4–C7a, JH7–C3a, and JH7–C7a were analyzed. According to the
DFT calculations, the values of the coupling constants JH3–C3a
and 2JH7–C7a are larger for all compounds than those of 3JH3–C7a
3
2
2
3
and JH7–C3a (see Table 5). The only exception is compound 6
which has a different electron distribution caused by the C
O
group at position C-5. For this compound, the couplings of both
protons (H-3 and H-7) are larger with C-3a than with C-7a. These
observations allow us to assign C-3a and C-7a unequivocally.
The values of other coupling constants are also in agreement
with the theoretical data (Table 5). Finally, C-5 is assigned to the
remaining resonance of the quaternary carbon found in the range
of 141–160 ppm.
c
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Copyright ꢀ 2008 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2008, 46, 643–649

NMR study of pyrazolo[3,4-c]pyridines
The structural conclusions formulated above from the 15N NMR
data are also supported by the values of the 13C chemical shifts.
Protonation of a nitrogen atom increases the shielding of a
neighboring carbon atom by about 10 ppm.[1] For compounds
1–6, the tautomeric migration of a proton should affect mainly
the C-7a and C-3 resonances. This effect is clearly evident from
the DFT calculations (see Table 7). The experimentally measured
differences in the chemical shifts ꢀδ (C7a-C3) are 3.5 ppm for 1
and 2.0 ppm for 2. In accordance with the experimental results,
the calculated ꢀδ values for the N-1 tautomers are 2.7 ppm (1a)
and 1.3 ppm (2a). In contrast, the values ꢀδ (C7a-C3) of 20.9 ppm
(1b) and 21.7 ppm (2b) were calculated for the N-2 tautomers.
Finally, the calculated energies support our conclusion that the
N-1 form predominates (ꢀE(b − a) is 2.7 kcal mol−1 for 1 and
3.2 kcal mol−1 for 2).[9] According to the Boltzmann distribution
(Table 7), at a temperature of 300 K, the difference in energy
corresponds to an a : b ratio of about 99 : 1, with the population of
the minor tautomer near or even below the limit of detection
for conventional NMR spectroscopy. These results represent
additional support for the conclusions made from the NMR data.
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Conclusions
Using low-temperature NMR spectroscopy, it was found that in
methanol all compounds either exist in one highly dominating
tautomeric form or undergo a rapid chemical exchange process,
evenattemperaturesaslowas171 K. ThepredominanceoftheN1-
H form at laboratory temperature was determined by comparing
the experimental 2JNH, 3JHN, ꢀδN(N2-N1), and ꢀδC(C7a-C3) values
for our compounds with those published for the N-acyl analogs
13 and 14 and the values calculated using DFT. Theoretically
calculated difference in energy favors the N1-H tautomer (a in
Scheme 1), with about 99% of the population at 300 K. All of the
compounds 1–6 were fully characterized by 1H, 13C, and 15N
chemical shifts and by 1H–13C and 1H–15N coupling constants.
G. A. Voth,
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M. A. Al-Laham, C. Y. Peng, A. Nanayakkara, M. Challacombe,
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Acknowledgements
This work was supported by the Ministry of Education of the Czech
Republic (grants MSM0021622413 and LC06030 to TB, JT, and RM)
and by the Ministry of Industry, Energy, and Technology of Greece
(Research Grant, Program ENTER). We express our gratitude to the
staff members of the supercomputing centers in Brno and Prague
for CPU time.
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c
Magn. Reson. Chem. 2008, 46, 643–649
Copyright ꢀ 2008 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/mrc