MRC letters Received: 3 September 2013
Revised: 30 September 2013
Accepted: 5 October 2013
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/mrc.4025
1H, 13C
NMR studies of new 3-aminophenol isomers linked to pyridinium salts
Santiago Schiaffino-Ortega, Antonio Espinosa, Miguel A. Gallo, L. Carlota López-Cara* and Antonio Entrena* 1
H and 13C NMR spectroscopic data of 20 new non-symmetrical compounds were assigned by a combination of 1D and 2D NMR experiments (DEPT, HSQC, and HMBC). These compounds contain a 4-(N,N-dimethylamino)- or 4-(pyrrolidin-1-yl)pyridinium moiety and a 3-nitro-, 3-amino-, or 3-hydroxyphenyl ring, linked by p-xylene, 4,4′-dimethylbiphenyl, 1,2-bis(p-tolyl)ethane, or 1,4-bis(p-tolyl)butane. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: 1H–13C 1D NMR; 1H–13C 2D NMR (HSQC; HMBC); choline kinase inhibitors; antiproliferative compounds
Introduction Evidence for the requirement of choline kinase-α activity in cancer has been obtained from observations that the expression of this enzyme is really high in many tumor cells.[1] Theoretical studies initially performed on a human choline kinase homology model[2] and further on the X-ray crystal structure of the enzyme indicated that non-symmetrical inhibitors could insert the cationic head into the choline binding site and the adenine moiety into the ATP binding site.[3] In a recent paper, the synthesis and the biological evaluation of a novel family of compounds bearing a 3-aminophenol fragment and a pyridinium salt connected by means of different linkers, which were designed and evaluated as ChoK inhibitors and as antiproliferative agents, have been described.[4] Although the structures of these derivatives have been determined by means of standard spectroscopic techniques (1H and 13C NMR, and MS), a detailed NMR study has been performed in some of them, in order to unequivocally corroborate their structures. This paper reports the 1H and 13C NMR unambiguous signal assignments corresponding to the pyridinium salts, the linkers, and the 3-aminophenol moiety (4a–h and 5a–h) using 1D and 2D NMR techniques. The spectra of nitro derivatives 3e–h, precursors in the synthetic pathway for the preparation of compounds 4a–h, are also included.
Experimental NMR spectra were recorded on 500 MHz 1H and 125 MHz 13C NMR Agilent Direct-Drive (Santa Clara, CA, USA), 400 MHz 1 H NMR Agilent Direct-Drive, or 300 MHz 1H and 75 MHz 13C NMR Agilent Inova-Unity spectrometers at 298 K. Chemical shifts (δ) are quoted in parts per million (ppm) and are referenced to the residual solvent peak: CD3OD, δ = 3.31 ppm (1H), δ = 49.05 ppm (13C); DMSO-d6, δ = 2.50 ppm (1H), δ = 39.5 ppm (13C). Spin multiplicities are given as s (singlet), bs (broad singlet), d (doublet), dd (doublet doublet), ddd (doublet, doublet doublet), t (triplet), dt (doublet triplet), q (quadruplet), and m (multiplet). Coupling
Magn. Reson. Chem. (2013)
constants (J) are given in Hz. The following parameters were used in DEPT experiments: PW (Pulse Width: 135°), 9.0 ms; recycle time, 1 s; 1/2 J (CH) = 4 ms; 65.536 data points acquired and transformed from 1024 scans; spectral width, 15 KHz; and line broadening, 1.3 Hz. HMBC spectra were measured with a pulse sequence gc2hmbc (Standard sequence, Agilent Vnmrj_3.2A software) optimized for 8 Hz (inter-pulse delay for the evolution of longrange couplings: 62.5 ms). The HSQC spectra were measured with a pulse sequence gc2hsqcse (Standard sequence Agilent Vnmrj_3.2A software).
Results and Discussion Scheme 1 represents the previously reported synthetic pathway followed in the preparation of two families of compounds 4a–h and 5a–h.[4] Compounds with general structure 2 were prepared using linkers 1a–d, which were synthetized following the reported route.[4,5] Reaction of compounds 1a–d with a half equivalent of 4-(N,N-dimethylamino)pyridine or 4-pyrrolidinopyridine, in butanone during 3 days at room temperature, yields the corresponding derivatives 2a–h. Compounds 3e–h were obtained by treatment of compounds 2a–h with 3-nitrophenol in the presence K2CO3, with general good yields. Preparation of compounds 5a–h needs an initial protection of the OH group in 3-aminophenol and a further reaction with intermediates 2a–h in the presence of K2CO3, being the OH group liberated during this last reaction. Finally, compounds 4a–h were obtained using two routes depending on the starting intermediate. In this sense, compounds 5a–d were obtained by reaction of 3-aminophenol and intermediates 2a–d in the presence of NaH, while reduction
* Correspondence to: L. C. López-Cara and Antonio Entrena, Departamento de Química Farmacéutica y Orgánica, Facultad de Farmacia, Universidad de Granada, Campus de Cartuja, 18071 Granada, Spain. E-mail: lcarlotalopez@ ugr.es; [email protected] Departamento de Química Farmacéutica y Orgánica, Facultad de Farmacia, Universidad de Granada, Campus de Cartuja, 18071, Granada, Spain
Copyright © 2013 John Wiley & Sons, Ltd.
S. Schiaffino-Ortega et al.
Scheme 1. General synthetic pathway followed in the preparation of final compounds 4a–h and 5a–h. (a) 4-(N,N-dimethylamino)pyridine (DMAP) or 4-pyrrolidinopyridine, butanone, room temperature, 72 h; (b) 3-aminophenol, NaH, dimethylformamide (DMF), reflux, 20 h; (c) 3-nitrophenol, NaH, DMF, reflux, 22 h; (d) Fe/FeSO4/H2O, reflux, 3 h; (e) tert-butyldimethylsilyl chloride, triethylamine, DMAP, dichloromethane, room temperature, 6 h; (f) K2CO3, DMF, 110 °C, 20–24 h.
of the NO2 group in intermediates 3e–h yields the final compounds 5e–h. Structural elucidation of compounds 3, 4, and 5 has been made by routine 1H and 13C NMR techniques. Nevertheless, a definitive assignment of all signals needs the use of several NMR techniques as follows: (i) DEPT experiments to determine the number of protons attached to each carbon atom; (ii) gs-HSQC spectra to determine the 13C resonances of the tertiary, secondary, and primary carbons; and (iii) gs-HMBC sequences to assign the signals of quaternary carbons via two-bond and three-bond interactions. Tables 1–3 show the 1H NMR signals of compounds 3e–h, 4a–h, and 5a–h, respectively, while Tables 4–6 show the corresponding 13C NMR chemical shifts for the same molecules. NMR spectra of all compounds were run in CD3OD solution, except for compounds 4b, 4e, 4h, and 5f, which were registered in DMSO-d6. For this reason, some significant variations are observed in the chemical shifts depending on the solvent. Hydrogen atoms of the 3-substituted phenyl ring (H-2ph, H-5ph, H-4ph, and H-6ph) can be identified in accordance with the estimated values calculated by means of the additive rules for polysubstituted benzenes.[6] 1 H NMR and 13C NMR signals of intermediate nitroderivatives (3e–h) are similar to the signals of the final compounds (4a–h and 5a–h) being the main differences in the terminal benzene ring, which is influenced by the substituent in 3-position (nitro, amino, or hydroxy group). HSQC and HMBC experiments were performed on some compounds of each series, and the results of these experiments have been extrapolated to the other compounds. Tables S1 and S2 in the Supporting information show the HSQC and HMBC correlations, respectively, for compounds 3f, 4c, and 5c. HSQC experiments performed on compounds 3f, 4c, and 5c allowed the unequivocal chemical shifts assignment to the
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carbon atoms of R1 and R2, C-2ph, C-5ph, C-2,6pyr, and C-3,5pyr. These carbon atoms show signals at 41.59, 111.71, 132.67, 144.34, and 110.37 ppm, respectively, in compound 3f; 49.69/ 26.11, 103.34, 130.80, 142.90, and 109.63 ppm, in compound 4c; and 48.46/24.69, 99.55, 129.29, 141.38, and 108.22 ppm in compound 5c. Chemical shifts of the hydrogen atoms of the pyridine ring (H-2,6pyr and H-3,5pyr) coincide with those previously described.3b For the remaining atoms, this information must be coordinated with HMBC experiments simultaneously. The HMBC spectra performed on 3f, 4c, and 5c reveal a correlation between H-2,6pyr and the carbon atom of the –CH2N+– methylene group in the three HMBC spectra (Table S2 and Figs S2–S4 in the Supporting information). This information is the key to identify all other proton and carbon atoms. For the final compounds, when the terminal benzene ring is connected to the linker through the O atom (compounds 4a–h), the 1H and 13C NMR signals of benzene are in ranges of 6.14–7.35 and 99.99–161.31 ppm, respectively, while when is connected through the NH group (compounds 5a–h), the 1 H and 13C NMR signals ranges are 6.02–6.87 and 99.21– 159.16 ppm. Apparently, no big differences in the chemical shifts are observed, but a more in detail study of the chemical shifts clearly shows the differences between both families of isomeric compounds (4a–h and 5a–h). H-2ph appears in a range of 6.22–6.36 ppm in the first family (4a–h), while in the second family (5a–h), the chemical shifts are in a range of 5.99–6.09 ppm. This could be because the addition of the electronic effects in the first family is greater than in the second one. Thus, the H-2ph protons of 3e–h compounds appear less shielded than 4a–h and these ones more than 5a–h because of the mesomeric effects (+M and M) of the 3-position substituent upon these protons. NO2 group exert a M effect (deshielding effect) upon overall phenyl ring, and NH2 and OH groups have a +M effect (shielding effect) being
Copyright © 2013 John Wiley & Sons, Ltd.
Magn. Reson. Chem. (2013)
1
Magn. Reson. Chem. (2013)
7.85 (s)
7.74 (t, 2.3)
7.81–7.75 (m)
3f
3g
3h
7.77 (ddd, 8.0, 2.1, 0.9) 7.81–7.75 (m)
7.83 (d, 10.0)
7.82 (d, 8.1)
H-4phd
7.48 (t, 8.1)
7.48 (t, 8.1)
7.52 (t, 8.1)
7.51 (t, 8.2)
H-5ph
1
7.40 (dd, 8.7, 2.3) 7.42 (dd, 8.2, 1.9) 7.36 (ddd, 8.4, 2.5, 1.0) 7.37 (d, 8.2)
H-6phd
5.12 (s)
5.10 (s)
5.25 (s)
5.23 (s)
O–CH2–
7.16 (d, 7.6)
7.15 (d, 8.1) 7.33 (d, 7.6)
7.31 (d, 8.1)
7.56 (d, 8.1)
—
— 7.66 (d, 8.1)
H-3″,5″
H-2″,6″
Benzene″
2.66–2.57 (m) 1.60 (m)
1
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6.22 (m)
6.36 (t, 2.2)
6.36 (t, 2.0)
6.18 6.92 6.39 6.19
—
—
—
— — — —
4bc
4cb
4db
4ec 4fb 4gb 4hc
6.88 7.35 6.97 6.88
(t, 8.0) (m) (m) (t, 8.0)
6.96 (t, 8.0)
6.97 (t, 8.0)
6.93 (m)
6.99 (t, 8.0)
H-5ph
H-6ph
1
7.00f (m) 6.86f (d, 8.0) e 6.33 (m) 6.14e (td, 7.9, 1.9)
6.31e (dt, 9.32, 1.77) 6.30e (dt, 7.8, 1.6) 6.16–6.10e (m)
6.34 (dt, 8.2, 2.1) 6.17e (d, 5.9)
e
H-4ph
4.98 5.15 4.93 4.92
(s) (s) (s) (s)
4.93 (s)
4.94 (s)
5.05 (s)
5.06 (s)
O–CH2–
— 7.45 (d, 7.9) 7.11 (d, 8.1) 7.16 (d, 8.0)
7.12 (d, 7.9)
7.12 (d, 8.0)
— 7.61 (d, 7.9) 7.27 (m) 7.29 (m)
7.31–7.25 (m)
7.27 (d, 8.0)
7.71 (m)
—
— 7.5 (m)
H-3′,5″
H-2″,6′
Benzene″
2.62 (m) 1.61 (m) — — 2.89 (m) 2.58 (m) 1.55 (m)
2.91 (m)
—
—
–(CH2)n′–d
b
H-2′,6′
7.40 7.66 7.27 7.29
(d, 8.2) (d, 7.8) (m) (m)
H-3′,5′
7.20 (d, 7.7)
7.19 (d, 8.2)
7.71 (d, 8.1)
7.55 (d, 8.1)
H-3′,5′
7.45 7.50 7.19 7.22
(d, 8.2) (d, 7.8) (d, 8.1) (d, 8.0)
7.21 (d, 8.0)
7.20 (d, 8.1)
7.50 (m)
7.52 (d, 8.1)
Benzene′
7.31–7.25 (m)
7.25 (d, 8.1)
7.71 (m)
7.40 (d, 8.1)
Multiplicity and coupling constants are given in parentheses. H shifts assignments are in agreement with the HSQC and HMBC spectra. CD3OD was used as solvent in the NMR. c DMSO-d6 was used as solvent in the NMR experiments. d Signals corresponding to the -(CH2)n′- hydrogen atoms of the linker cannot be unequivocally assigned. e These signals are superimposed. f These signals can be interchanged. g These signals correspond to both C-2 and C-5 (N-CH2-) pyrrolidine hydrogen atoms. h These signals correspond to both C-3 and C-4 (N-CH2-CH2-) pyrrolidine hydrogen atoms.
a
(t, 2.1) (m) (t, 2.1) (t, 2.1)
6.38 (t, 2.2)
4a
—
H-2ph
b
3-Aminophenol
H NMR data for compounds 4a–h (δ, ppm; J, Hz)a
Compound –NH2
Table 2.
b
7.28 (d, 7.7)
7.27 (d, 8.2)
7.47 (d, 8.1)
— 2.91–2.86 (m)
7.42 (d, 8.1)
H-2′,6′
Benzene′
—
–(CH2)n′–c
Multiplicity and coupling constants are given in parentheses. H shifts assignments are in agreement with the HSQC and HMBC spectra. CD3OD was used as solvent in the NMR experiments for all compounds. c Signals corresponding to the -(CH2)n′- hydrogen atoms of the linker cannot be unequivocally assigned. d These signals can be interchanged. e These signals correspond to the N–CH3 hydrogen atoms.
a
7.80 (s)
H-2ph
3-Nitrophenol
H NMR data for compounds 3e–h (δ, ppm; J, Hz)a
3e
Compoundb
Table 1.
5.39 5.39 5.31 5.33
(s) (s) (s) (s)
5.29 (s)
5.28 (s)
5.45 (s)
5.38 (s)
–CH2–N
+
5.31 (s)
5.31 (s)
5.42 (s)
5.39 (s)
+
H-2,6pyr
8.40 8.23 8.19 8.37
(d, (d, (d, (d,
7.8) 5.8) 7.8) 7.8)
8.16 (d, 7.5)
8.14 (d, 7.6)
8.44 (d, 7.1)
7.04 7.00 6.97 7.03
(d, 7.8) (m) (m) (d, 7.7)
6.80 (d, 7.5)
6.82 (d, 7.6)
6.93 (m)
6.88 (d, 7.6)
H-3,5pyr
6.97 (d, 7.0)
6.97 (d, 7.9)
7.02 (d, 7.6)
Pyridine
8.20 (d, 6.9)
8.20 (d, 7.8)
8.26 (d, 7.6)
H-3,5pyr 7.00 (d, 7.7)
Pyridine
8.23 (d, 7.7)
H-2,6pyr
8.22 (d, 7.7)
–CH2–N
3.57 2.14 3.51 2.01 3.52 2.10 3.50 2.09 3.17 3.22 3.21 3.17
(m)g (m)h (m)g (m)h (m)g (m)h (m)g (m)h (s) (s) (s) (s)
R1, R2
3.22 (s)
3.22 (s)
3.25 (s)
3.25 (s)
R1, R2e
NMR studies of new 3-aminophenol isomers
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6.09 (t, 2.2)
6.02 (s)
6.08 (s)
6.05 (s)
5.99 (t, 2.1) 6.07 (s)
6.09 (t, 2.1)
—
—
—
—
8.87 (bs)
—
—
5cb
5db
5eb
5fc
5gb
5hb
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6.80 (t, 8.3) 6.86 (t, 8.0) 6.83 (t, 9.0) 6.79 (t, 7.9) 6.83 (t, 8.3) 6.85 (t, 7.9)
6.81 (t, 8.3) 6.87 (t, 8.0)
H-5ph
6.13 (ddd, 8.0, 2.2, 0.8)
6.04 (dd, 7.9, 1.8) 6.11 (d, 2.2)
6.14 (dd, 8.0, 1.8) 6.10 (d, 7.2)
6.15 (ddd, 8.1, 2.2, 0.9) 6.07 (d, 10.0)
6.08 (d, 10.0)
H-4phe
3-Aminophenol
1
6.06 (ddd, 7.8, 2.3, 0.9)
5.95 (dd, 8.0, 2.2) 6.04 (d, 2.3)
6.06 (dd, 8.0, 1.8) 6.02 (d, 7.2)
6.01 (d, 10.0)
6.06 (ddd, 8.1, 2.2, 0.9)
6.02 (d, 10.0)
H-6phe
—
6.16 (t, 6.0) —
—
—
4.18 (s)
4.17 (s)
4.24 (s)
4.27 (s)
4.20 (s)
4.14 (s)
4.30 (s)
—
—
4.24 (s)
NH–CH2–
—
–NH– CH2–
7.03 (d, 8.1)
7.01 (d, 8.2)
7.60 (d, 8.3)
—
7.22 (d, 7.9)
6.98 (d, 8.1)
7.48–7.38 (m)
—
H-2″,6′ —
H-3′,5″
7.26 (d, 8.1)
7.24 (d, 8.2)
7.42 (d, 8.3)
—
7.05 (d, 7.9)
7.15 (d, 8.1)
7.53 (d, 8.4)
Benzene″
2.57 (m) 1.57 (m)
2.83 (m)
b
—
2.67–2.52 (m) 1.67–1.47 (m) —
2.80 (m)
—
—
–(CH2)n′–d
Multiplicity and coupling constants are given in parentheses. H shifts assignments are in agreement with the HSQC and HMBC spectra. CD3OD was used as solvent in the NMR. c DMSO-d6 was used as solvent in the NMR experiments. d Signals corresponding to the -(CH2)n′- hydrogen atoms of the linker cannot be unequivocally assigned. e These signals can be interchanged. f These signals correspond to both C-2 and C-5 (N-CH2-) pyrrolidine hydrogen atoms. g These signals correspond to both C-3 and C-4 (N-CH2-CH2-) pyrrolidine hydrogen atoms.
a
5a
5bb
H-2ph
6.03 (s)
–OH
H NMR data for compounds 5a–h (δ, ppm; J, Hz)a
—
1
b
Compound
Table 3.
7.19 (d, 8.1) 7.19 (d, 8.0) 7.32 (d, 8.4) 7.47 (d, 8.2) 7.19 (d, 8.1) 7.21 (d, 8.2)
7.31 (d, 8.2) 7.63 (d, 8.3)
H-2′,6′
7.17 (d, 8.2)
7.14 (d, 8.1)
7.69 (d, 8.2)
7.40 (d, 8.4)
7.25 (d, 8.0)
7.11 (d, 8.1)
7.48–7.38 (m)
7.39 (d, 8.2)
H-3′,5′
Benzene′
5.27 (s)
5.25 (s)
5.44 (s)
5.32 (s)
5.27 (s)
5.20 (s)
5.35 (s)
5.28 (s)
–CH2–N
+
8.04 (d, 7.8) 8.14 (d, 7.6) 8.19 (d, 7.9) 8.45 (d, 7.8) 8.11 (d, 7.9) 8.14 (d, 7.9)
8.14 (d, 7.6) 8.18 (d, 7.8)
H-2,6pyr
6.71 (d, 7.8) 6.80 (d, 7.6) 6.95 (d, 7.9) 7.06 (d, 7.8) 6.89 (d, 7.9) 6.90 (d, 7.9)
6.76 (d, 7.6) 6.81 (d, 7.8)
H-3,5pyr
Pyridine
(m)g (m)g (m)f (m)g
3.18 (s)
3.17 (s)
3.18 (s)
3.43(m)g 2.03 (m)g 3.57–3.46 (m)f 2.14–1.98 (m)g 3.21 (s)
3.55–3.40 2.12–2.02 3.56–3.46 2.13–2.03
R1, R2
S. Schiaffino-Ortega et al.
Copyright © 2013 John Wiley & Sons, Ltd.
Magn. Reson. Chem. (2013)
13
Magn. Reson. Chem. (2013)
111.75
109.02 111.71 109.37
C-2ph
151.79
149.18 151.84 149.36
C-3ph
124.11
121.43 124.07 121.79
C-4ph d
132.63
130.04 132.67 130.30
C-5ph
117.87
115.43 117.99 115.52
C-6phd
72.85
69.54 72.46 70.42
–O–CH2–
136.26
— 138.65 134.12
— 142.45 141.66 145.10
C-4″
C-1″
Copyright © 2013 John Wiley & Sons, Ltd.
13
100.26
100.27 99.99 100.16
103.28
103.28 100.33 103.34
C-2ph
149.87
147.73 148.83 149.74
150.12
150.17 149.96 150.08
C-3ph
106.96
107.11 109.96 106.87
109.61
109.78 107.08 109.63
129.45
129.50 131.86 129.31
130.81
130.83 129.50 130.80
C-5ph
102.16
102.11 105.06 102.05
105.72
105.61 102.18 105.78
C-6phe
68.57
68.14 70.88 68.41
70.70
70.03 68.27 70.68
O–CH2–
133.00
135.07 135.11 133.05
133.51
135.59 134.95 133.66
C-1′
142.83
138.14 142.73 141.98
145.19
140.44 140.18 144.29
C-4′
127.89
128.06 129.93 127.79
129.35
129.37 128.52 129.30
b
C-2′,6′
Benzene′
Assignments of C chemical shifts are in agreement with the HSQC and HMBC spectra. CD3OD was used as solvent in the NMR. c DMSO-d6 was used as solvent in the NMR experiments. d Signals corresponding to the -(CH2)n′- carbon atoms of the linker cannot be unequivocally assigned. e These signals are interchangeable. f These signals correspond to both C-2 and C-5 (N-CH2-) pyrrolidine carbon atoms. g These signals correspond to both C-3 and C-4 (N-CH2-CH2-) pyrrolidine carbon atoms.
a
159.34
159.15 161.10 159.34
4ec 4fb 4gb
4hc
161.31
4db
C-1ph
C-4phe
3-Aminophenol
C NMR data for compounds 4a–h (δ, ppm)a
161.04 159.26 161.30
13
4ab 4bc 4cb
Compound
Table 5.
b
C-3′,5′
128.88
128.00 128.77 128.84
130.45
— — 38.36 38.36 36.30 36.27 31.99 31.91 — — 36.28 36.28 34.52 34.48 30.45 30.40
C-1″
134.72
— 137.60 134.81
— 140.98 140.56 141.52
136.30
143.28
— 137.14 136.60
C-4″
128.18
— 128.08 128.13
129.48
— 126.68 129.66
127.56
— 129.10 127.39
128.64
— 128.06 128.59
59.19
59.12 61.50 59.04
61.66
61.54 59.14 61.64
+
141.90
141.96 143.15 141.78
142.90
143.01 141.99 142.90
107.91
107.98 109.17 107.79
109.64
109.70 108.60 109.63
C-3,5pyr
155.82
155.87 157.92 155.70
155.12
155.20 153.05 155.15
C-4pyr
R1, R2
41.59
38.93 41.59 39.27
R1, R2
39.46
30.65 40.49 39.35
49.68f 26.10g
49.72f 26.12g 48.31f 24.63g 49.69f 26.11g
159.22
156.61 159.26 156.79
C-4pyr
110.32
107.72 110.37 107.96
C-3,5pyr
Pyridine
Pyridine
144.26
141.69 144.34 141.89
C-2,6pyr
C-2,6pyr
62.87
59.99 62.67 60.43
+
–CH2–N
–CH2–N
130.65
128.16 130.15 128.28
C-3′,5′
C-3″,5″
131.68
128.16 131.17 129.48
C-2′,6′
C-2″,6″
Benzene″
146.46
134.67
C-4′ 137.62 144.10 143.07
C-1′
Benzene′
134.67 136.52 132.51
— 138.63 142.18
— — 37.20 37.20 37.56 30.54 33.25 33.22
–(CH2)n′–c
–(CH2)n′–d
130.94
— 130.59 128.72
C-3″,5″
129.33 127.22 130.67
130.14
— 129.47 127.73
C-2″,6″
Benzene″
Assignments of C chemical shifts are in agreement with the HSQC and HMBC spectra. CD3OD was used as solvent in the NMR experiments for all compounds. c Signals corresponding to the -(CH2)n′- carbon atoms of the linker cannot be unequivocally assigned. d These signals can be interchanged.
13
162.03
3h
a
159.14 161.95 159.59
C-1ph
3-Nitrophenol
C NMR data for compounds 3e–h (δ, ppm)a
3e 3f 3g
Compoundb
Table 4.
NMR studies of new 3-aminophenol isomers
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C-1ph
13
99.79
99.21 99.99 99.84
100.92
99.55
99.52
158.02
158.04 158.83 158.03
159.16
157.77
157.75
157.77
C-3ph
105.26
103.85 104.66 105.30
106.40
105.00
104.95
104.81
129.59
129.34 130.10 129.59
130.66
129.29
129.27
129.32
C-5ph
103.98
103.36 104.08 103.96
105.11
103.67
103.75
103.72
C-6phe
53.70
45.99 46.79 47.29
47.95
48.27
48.42
46.62
NH–CH2–
141.00
— 141.01 139.91
142.12
139.62
137.70
— 141.15 138.01
138.84
137.73
140.11
—
— 141.78
C-4″
C-1″
b
128.32
— 127.32 128.49
129.44
128.22
127.46
—
C-2″,6″
Benzene″
Assignments of C chemical shifts are in agreement with the HSQC and HMBC spectra. CD3OD was used as solvent in the NMR. c DMSO-d6 was used as solvent in the NMR experiments. d Signals corresponding to the -(CH2)n′- carbon atoms of the linker cannot be unequivocally assigned. e These signals are interchangeable. f These signals correspond to both C-2 and C-5 (N-CH2-) pyrrolidine carbon atoms. g These signals correspond to both C-3 and C-4 (N-CH2-CH2-) pyrrolidine carbon atoms.
a
150.48
5hb
151.64
5db
149.78 150.64 150.46
150.19
5cb
5eb 5fc 5gb
150.07
99.44
C-2ph
C-4phe
3-Aminophenol
C NMR data for compounds 5a–h (δ, ppm)a
149.86
13
5bb
5a
b
Compound
Table 6.
127.23
— 127.85 127.19
128.33
126.89
126.56
—
C-3″,5″
132.23
133.95 135.39 132.37
133.47
132.17
133.54
— 37.07 36.87 36.25 36.22 32.00 31.87 — — 37.34 37.13 35.14 35.10 30.89 30.74
133.16
C-1′ —
–(CH2)n′–d
144.05
141.32 138.35 143.15
145.21
142.88
138.35
141.78
C-4′
128.32
127.92 129.30 128.34
129.33
127.97
128.41
128.05
C-2′,6′
Benzene′
129.31
127.62 128.39 129.50
130.45
129.23
127.34
127.78
C-3′,5′
60.48
59.11 59.77 60.44
61.66
60.21
60.10
60.15
+
–CH2–N
141.80
141.93 142.76 141.76
142.88
141.38
141.56
141.48
107.93
107.97 108.76 107.93
109.63
108.22
108.28
108.25
C-2,6pyr C-3,5pyr
Pyridine
156.73
155.85 156.64 156.70
155.12
153.62
153.74
153.63
C-4pyr
39.25
39.75 40.50 39.29
48.29f 24.69g 48.29f 24.69g 48.46f 24.69g 49.68f 26.10g
R1, R2
S. Schiaffino-Ortega et al.
Copyright © 2013 John Wiley & Sons, Ltd.
Magn. Reson. Chem. (2013)
NMR studies of new 3-aminophenol isomers higher in 4a–h than in 5a–h compounds. H-4ph and H-6ph suffers the same effect for the same reason. The same behavior is observed in 13C NMR signals of C-2ph, C-4ph, and C-6ph, which appear more shielded in 5a–h than in 4a–h derivatives and their direct precursors (3e–h). 1 H NMR signals of H-4ph and H-6ph in compounds 4a–h are superimposed and appear as multiplets, and for this reason, these signals cannot be completely assigned, except in 4f, where the signals are well resolved. In compounds 5a–h, the 1H NMR signals of H-4ph and H-6ph are well resolved, but they cannot be unequivocal assigned through the HMBC and HMQC techniques. Consequently, these signals can be considered interchangeable. C-4ph and C-6ph are interchangeable in both families as well. Finally, the methylene group connected to the ether bond in 4a–h compounds appears less shielded as in 1H NMR as in 13C NMR signals, than when this methylene is connected to amino group in the 5a–h compounds. In the rest of the molecule, no significant differences have been found. The 1H and 13C NMR chemical shifts of the linker and the cationic head are very similar in isomeric compounds of families 4 and 5. In conclusion, two families of structural isomers bearing 3-aminophenol moiety (4a–h and 5a–h) and their direct precursors (3e–h) have been unequivocally identified by 1D and 2D NMR techniques. The only differences between both isomers are the chemical shift of H-2ph, H-4ph, and H-6ph and of C-2ph, C-4ph, and C-6ph respectively in the 3-aminophenol moiety, which appears more deshielding for 4a–h compounds than for 5a–h. Thus, only one difference between two isomers is enough to unequivocally identify them.
References [1] a) M. Bañez-Coronel, A. Ramírez de Molina, A. Rodríguez-González, J. Sarmentero, M. A. Ramos, M. A. García-Cabezas, L. García-Oroz, J. C. Lacal, Curr. Cancer Drug Targets 2008, 8, 709–719; b) E. Hernando, J. Sarmentero-Estrada, T. Koppie, C. Belda-Iniesta, V. Ramírez de Molina, P. Cejas, C. Ozu, C. Le, J. J. Sánchez, M. González-Barón, J. Koutcher, C. Cordón-Cardó, B. H. Bochner, J. C. Lacal, A. Ramírez de Molina, Oncogene 2009, 28, 2425–2435; c) W. Cun See Too, M. Teng Wong, L. Ling Few, M. Konrad, PLoS ONE, 2010, 5(9), e12999; d) D. Gallego-Ortega, T. Gómez del Pulgar, F. Valdés-Mora, A. Cebrián, J. C. Lacal, Adv. Enzyme Regul. 2011, 51, 183–194. [2] a) E. Malito, N. Sekulic, W. C. Too, M. Konrad, A. Lavie, J. Mol. Biol. 2006, 364, 136–151; b) L. Milanese, A. Espinosa, J. M. Campos, M. A. Gallo, A. Entrena, ChemMedChem 2006, 1, 1216–1228. [3] a) B. Rubio-Ruiz, A. Conejo-García, P. Ríos-Marco, M. P. CarrascoJimenez, J. Segovia, C. Marco, M. A. Gallo, A. Espinosa, A. Entrena, Eur. J. Med. Chem. 2012, 50, 154–162; b) B. Rubio-Ruiz, A. ConejoGarcía, M. A. Gallo, A. Espinosa, A. Entrena, Magn. Reson. Chem. 2012, 50, 466–469. [4] S. Schiaffino-Ortega, L. C. López-Cara, P. Ríos-Marco, M. P. CarrascoJimenez, C. Marco, M. A. Gallo, A. Espinosa, A. Entrena. Biorg. Med. Chem. 2013. doi:10.1016/j.bmc.2013.09.003. [5] a) H. A. Staab, M. Haenel, Chem. Ber. 1973, 106, 2190–2202; b) D. J. Cram, H. Steinberg, J. Am. Chem. Soc. 1951, 73, 5691–5704. [6] M. Hesse, H. Meier, B. Zeeh, Spektroskopische Methoden in der Organischen Chemie, 5th edn, Georg Thieme Verlag, Stuttgart- Nueva, York, 1995.
Supporting Information
Acknowledgements This work was supported by a project from the ‘Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía’ (P07-CTS-03210)
Magn. Reson. Chem. (2013)
and the ‘Ministerio de Ciencia e Innovación’ (SAF2009-11955). The award of grant from the ‘Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía’ to S. S. O. is gratefully acknowledged.
Additional supporting information may be found in the online version of this article at the publisher’s web site.
Copyright © 2013 John Wiley & Sons, Ltd.
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Revised: 30 September 2013
Accepted: 5 October 2013
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/mrc.4025
1H, 13C
NMR studies of new 3-aminophenol isomers linked to pyridinium salts
Santiago Schiaffino-Ortega, Antonio Espinosa, Miguel A. Gallo, L. Carlota López-Cara* and Antonio Entrena* 1
H and 13C NMR spectroscopic data of 20 new non-symmetrical compounds were assigned by a combination of 1D and 2D NMR experiments (DEPT, HSQC, and HMBC). These compounds contain a 4-(N,N-dimethylamino)- or 4-(pyrrolidin-1-yl)pyridinium moiety and a 3-nitro-, 3-amino-, or 3-hydroxyphenyl ring, linked by p-xylene, 4,4′-dimethylbiphenyl, 1,2-bis(p-tolyl)ethane, or 1,4-bis(p-tolyl)butane. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: 1H–13C 1D NMR; 1H–13C 2D NMR (HSQC; HMBC); choline kinase inhibitors; antiproliferative compounds
Introduction Evidence for the requirement of choline kinase-α activity in cancer has been obtained from observations that the expression of this enzyme is really high in many tumor cells.[1] Theoretical studies initially performed on a human choline kinase homology model[2] and further on the X-ray crystal structure of the enzyme indicated that non-symmetrical inhibitors could insert the cationic head into the choline binding site and the adenine moiety into the ATP binding site.[3] In a recent paper, the synthesis and the biological evaluation of a novel family of compounds bearing a 3-aminophenol fragment and a pyridinium salt connected by means of different linkers, which were designed and evaluated as ChoK inhibitors and as antiproliferative agents, have been described.[4] Although the structures of these derivatives have been determined by means of standard spectroscopic techniques (1H and 13C NMR, and MS), a detailed NMR study has been performed in some of them, in order to unequivocally corroborate their structures. This paper reports the 1H and 13C NMR unambiguous signal assignments corresponding to the pyridinium salts, the linkers, and the 3-aminophenol moiety (4a–h and 5a–h) using 1D and 2D NMR techniques. The spectra of nitro derivatives 3e–h, precursors in the synthetic pathway for the preparation of compounds 4a–h, are also included.
Experimental NMR spectra were recorded on 500 MHz 1H and 125 MHz 13C NMR Agilent Direct-Drive (Santa Clara, CA, USA), 400 MHz 1 H NMR Agilent Direct-Drive, or 300 MHz 1H and 75 MHz 13C NMR Agilent Inova-Unity spectrometers at 298 K. Chemical shifts (δ) are quoted in parts per million (ppm) and are referenced to the residual solvent peak: CD3OD, δ = 3.31 ppm (1H), δ = 49.05 ppm (13C); DMSO-d6, δ = 2.50 ppm (1H), δ = 39.5 ppm (13C). Spin multiplicities are given as s (singlet), bs (broad singlet), d (doublet), dd (doublet doublet), ddd (doublet, doublet doublet), t (triplet), dt (doublet triplet), q (quadruplet), and m (multiplet). Coupling
Magn. Reson. Chem. (2013)
constants (J) are given in Hz. The following parameters were used in DEPT experiments: PW (Pulse Width: 135°), 9.0 ms; recycle time, 1 s; 1/2 J (CH) = 4 ms; 65.536 data points acquired and transformed from 1024 scans; spectral width, 15 KHz; and line broadening, 1.3 Hz. HMBC spectra were measured with a pulse sequence gc2hmbc (Standard sequence, Agilent Vnmrj_3.2A software) optimized for 8 Hz (inter-pulse delay for the evolution of longrange couplings: 62.5 ms). The HSQC spectra were measured with a pulse sequence gc2hsqcse (Standard sequence Agilent Vnmrj_3.2A software).
Results and Discussion Scheme 1 represents the previously reported synthetic pathway followed in the preparation of two families of compounds 4a–h and 5a–h.[4] Compounds with general structure 2 were prepared using linkers 1a–d, which were synthetized following the reported route.[4,5] Reaction of compounds 1a–d with a half equivalent of 4-(N,N-dimethylamino)pyridine or 4-pyrrolidinopyridine, in butanone during 3 days at room temperature, yields the corresponding derivatives 2a–h. Compounds 3e–h were obtained by treatment of compounds 2a–h with 3-nitrophenol in the presence K2CO3, with general good yields. Preparation of compounds 5a–h needs an initial protection of the OH group in 3-aminophenol and a further reaction with intermediates 2a–h in the presence of K2CO3, being the OH group liberated during this last reaction. Finally, compounds 4a–h were obtained using two routes depending on the starting intermediate. In this sense, compounds 5a–d were obtained by reaction of 3-aminophenol and intermediates 2a–d in the presence of NaH, while reduction
* Correspondence to: L. C. López-Cara and Antonio Entrena, Departamento de Química Farmacéutica y Orgánica, Facultad de Farmacia, Universidad de Granada, Campus de Cartuja, 18071 Granada, Spain. E-mail: lcarlotalopez@ ugr.es; [email protected] Departamento de Química Farmacéutica y Orgánica, Facultad de Farmacia, Universidad de Granada, Campus de Cartuja, 18071, Granada, Spain
Copyright © 2013 John Wiley & Sons, Ltd.
S. Schiaffino-Ortega et al.
Scheme 1. General synthetic pathway followed in the preparation of final compounds 4a–h and 5a–h. (a) 4-(N,N-dimethylamino)pyridine (DMAP) or 4-pyrrolidinopyridine, butanone, room temperature, 72 h; (b) 3-aminophenol, NaH, dimethylformamide (DMF), reflux, 20 h; (c) 3-nitrophenol, NaH, DMF, reflux, 22 h; (d) Fe/FeSO4/H2O, reflux, 3 h; (e) tert-butyldimethylsilyl chloride, triethylamine, DMAP, dichloromethane, room temperature, 6 h; (f) K2CO3, DMF, 110 °C, 20–24 h.
of the NO2 group in intermediates 3e–h yields the final compounds 5e–h. Structural elucidation of compounds 3, 4, and 5 has been made by routine 1H and 13C NMR techniques. Nevertheless, a definitive assignment of all signals needs the use of several NMR techniques as follows: (i) DEPT experiments to determine the number of protons attached to each carbon atom; (ii) gs-HSQC spectra to determine the 13C resonances of the tertiary, secondary, and primary carbons; and (iii) gs-HMBC sequences to assign the signals of quaternary carbons via two-bond and three-bond interactions. Tables 1–3 show the 1H NMR signals of compounds 3e–h, 4a–h, and 5a–h, respectively, while Tables 4–6 show the corresponding 13C NMR chemical shifts for the same molecules. NMR spectra of all compounds were run in CD3OD solution, except for compounds 4b, 4e, 4h, and 5f, which were registered in DMSO-d6. For this reason, some significant variations are observed in the chemical shifts depending on the solvent. Hydrogen atoms of the 3-substituted phenyl ring (H-2ph, H-5ph, H-4ph, and H-6ph) can be identified in accordance with the estimated values calculated by means of the additive rules for polysubstituted benzenes.[6] 1 H NMR and 13C NMR signals of intermediate nitroderivatives (3e–h) are similar to the signals of the final compounds (4a–h and 5a–h) being the main differences in the terminal benzene ring, which is influenced by the substituent in 3-position (nitro, amino, or hydroxy group). HSQC and HMBC experiments were performed on some compounds of each series, and the results of these experiments have been extrapolated to the other compounds. Tables S1 and S2 in the Supporting information show the HSQC and HMBC correlations, respectively, for compounds 3f, 4c, and 5c. HSQC experiments performed on compounds 3f, 4c, and 5c allowed the unequivocal chemical shifts assignment to the
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carbon atoms of R1 and R2, C-2ph, C-5ph, C-2,6pyr, and C-3,5pyr. These carbon atoms show signals at 41.59, 111.71, 132.67, 144.34, and 110.37 ppm, respectively, in compound 3f; 49.69/ 26.11, 103.34, 130.80, 142.90, and 109.63 ppm, in compound 4c; and 48.46/24.69, 99.55, 129.29, 141.38, and 108.22 ppm in compound 5c. Chemical shifts of the hydrogen atoms of the pyridine ring (H-2,6pyr and H-3,5pyr) coincide with those previously described.3b For the remaining atoms, this information must be coordinated with HMBC experiments simultaneously. The HMBC spectra performed on 3f, 4c, and 5c reveal a correlation between H-2,6pyr and the carbon atom of the –CH2N+– methylene group in the three HMBC spectra (Table S2 and Figs S2–S4 in the Supporting information). This information is the key to identify all other proton and carbon atoms. For the final compounds, when the terminal benzene ring is connected to the linker through the O atom (compounds 4a–h), the 1H and 13C NMR signals of benzene are in ranges of 6.14–7.35 and 99.99–161.31 ppm, respectively, while when is connected through the NH group (compounds 5a–h), the 1 H and 13C NMR signals ranges are 6.02–6.87 and 99.21– 159.16 ppm. Apparently, no big differences in the chemical shifts are observed, but a more in detail study of the chemical shifts clearly shows the differences between both families of isomeric compounds (4a–h and 5a–h). H-2ph appears in a range of 6.22–6.36 ppm in the first family (4a–h), while in the second family (5a–h), the chemical shifts are in a range of 5.99–6.09 ppm. This could be because the addition of the electronic effects in the first family is greater than in the second one. Thus, the H-2ph protons of 3e–h compounds appear less shielded than 4a–h and these ones more than 5a–h because of the mesomeric effects (+M and M) of the 3-position substituent upon these protons. NO2 group exert a M effect (deshielding effect) upon overall phenyl ring, and NH2 and OH groups have a +M effect (shielding effect) being
Copyright © 2013 John Wiley & Sons, Ltd.
Magn. Reson. Chem. (2013)
1
Magn. Reson. Chem. (2013)
7.85 (s)
7.74 (t, 2.3)
7.81–7.75 (m)
3f
3g
3h
7.77 (ddd, 8.0, 2.1, 0.9) 7.81–7.75 (m)
7.83 (d, 10.0)
7.82 (d, 8.1)
H-4phd
7.48 (t, 8.1)
7.48 (t, 8.1)
7.52 (t, 8.1)
7.51 (t, 8.2)
H-5ph
1
7.40 (dd, 8.7, 2.3) 7.42 (dd, 8.2, 1.9) 7.36 (ddd, 8.4, 2.5, 1.0) 7.37 (d, 8.2)
H-6phd
5.12 (s)
5.10 (s)
5.25 (s)
5.23 (s)
O–CH2–
7.16 (d, 7.6)
7.15 (d, 8.1) 7.33 (d, 7.6)
7.31 (d, 8.1)
7.56 (d, 8.1)
—
— 7.66 (d, 8.1)
H-3″,5″
H-2″,6″
Benzene″
2.66–2.57 (m) 1.60 (m)
1
Copyright © 2013 John Wiley & Sons, Ltd.
6.22 (m)
6.36 (t, 2.2)
6.36 (t, 2.0)
6.18 6.92 6.39 6.19
—
—
—
— — — —
4bc
4cb
4db
4ec 4fb 4gb 4hc
6.88 7.35 6.97 6.88
(t, 8.0) (m) (m) (t, 8.0)
6.96 (t, 8.0)
6.97 (t, 8.0)
6.93 (m)
6.99 (t, 8.0)
H-5ph
H-6ph
1
7.00f (m) 6.86f (d, 8.0) e 6.33 (m) 6.14e (td, 7.9, 1.9)
6.31e (dt, 9.32, 1.77) 6.30e (dt, 7.8, 1.6) 6.16–6.10e (m)
6.34 (dt, 8.2, 2.1) 6.17e (d, 5.9)
e
H-4ph
4.98 5.15 4.93 4.92
(s) (s) (s) (s)
4.93 (s)
4.94 (s)
5.05 (s)
5.06 (s)
O–CH2–
— 7.45 (d, 7.9) 7.11 (d, 8.1) 7.16 (d, 8.0)
7.12 (d, 7.9)
7.12 (d, 8.0)
— 7.61 (d, 7.9) 7.27 (m) 7.29 (m)
7.31–7.25 (m)
7.27 (d, 8.0)
7.71 (m)
—
— 7.5 (m)
H-3′,5″
H-2″,6′
Benzene″
2.62 (m) 1.61 (m) — — 2.89 (m) 2.58 (m) 1.55 (m)
2.91 (m)
—
—
–(CH2)n′–d
b
H-2′,6′
7.40 7.66 7.27 7.29
(d, 8.2) (d, 7.8) (m) (m)
H-3′,5′
7.20 (d, 7.7)
7.19 (d, 8.2)
7.71 (d, 8.1)
7.55 (d, 8.1)
H-3′,5′
7.45 7.50 7.19 7.22
(d, 8.2) (d, 7.8) (d, 8.1) (d, 8.0)
7.21 (d, 8.0)
7.20 (d, 8.1)
7.50 (m)
7.52 (d, 8.1)
Benzene′
7.31–7.25 (m)
7.25 (d, 8.1)
7.71 (m)
7.40 (d, 8.1)
Multiplicity and coupling constants are given in parentheses. H shifts assignments are in agreement with the HSQC and HMBC spectra. CD3OD was used as solvent in the NMR. c DMSO-d6 was used as solvent in the NMR experiments. d Signals corresponding to the -(CH2)n′- hydrogen atoms of the linker cannot be unequivocally assigned. e These signals are superimposed. f These signals can be interchanged. g These signals correspond to both C-2 and C-5 (N-CH2-) pyrrolidine hydrogen atoms. h These signals correspond to both C-3 and C-4 (N-CH2-CH2-) pyrrolidine hydrogen atoms.
a
(t, 2.1) (m) (t, 2.1) (t, 2.1)
6.38 (t, 2.2)
4a
—
H-2ph
b
3-Aminophenol
H NMR data for compounds 4a–h (δ, ppm; J, Hz)a
Compound –NH2
Table 2.
b
7.28 (d, 7.7)
7.27 (d, 8.2)
7.47 (d, 8.1)
— 2.91–2.86 (m)
7.42 (d, 8.1)
H-2′,6′
Benzene′
—
–(CH2)n′–c
Multiplicity and coupling constants are given in parentheses. H shifts assignments are in agreement with the HSQC and HMBC spectra. CD3OD was used as solvent in the NMR experiments for all compounds. c Signals corresponding to the -(CH2)n′- hydrogen atoms of the linker cannot be unequivocally assigned. d These signals can be interchanged. e These signals correspond to the N–CH3 hydrogen atoms.
a
7.80 (s)
H-2ph
3-Nitrophenol
H NMR data for compounds 3e–h (δ, ppm; J, Hz)a
3e
Compoundb
Table 1.
5.39 5.39 5.31 5.33
(s) (s) (s) (s)
5.29 (s)
5.28 (s)
5.45 (s)
5.38 (s)
–CH2–N
+
5.31 (s)
5.31 (s)
5.42 (s)
5.39 (s)
+
H-2,6pyr
8.40 8.23 8.19 8.37
(d, (d, (d, (d,
7.8) 5.8) 7.8) 7.8)
8.16 (d, 7.5)
8.14 (d, 7.6)
8.44 (d, 7.1)
7.04 7.00 6.97 7.03
(d, 7.8) (m) (m) (d, 7.7)
6.80 (d, 7.5)
6.82 (d, 7.6)
6.93 (m)
6.88 (d, 7.6)
H-3,5pyr
6.97 (d, 7.0)
6.97 (d, 7.9)
7.02 (d, 7.6)
Pyridine
8.20 (d, 6.9)
8.20 (d, 7.8)
8.26 (d, 7.6)
H-3,5pyr 7.00 (d, 7.7)
Pyridine
8.23 (d, 7.7)
H-2,6pyr
8.22 (d, 7.7)
–CH2–N
3.57 2.14 3.51 2.01 3.52 2.10 3.50 2.09 3.17 3.22 3.21 3.17
(m)g (m)h (m)g (m)h (m)g (m)h (m)g (m)h (s) (s) (s) (s)
R1, R2
3.22 (s)
3.22 (s)
3.25 (s)
3.25 (s)
R1, R2e
NMR studies of new 3-aminophenol isomers
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6.09 (t, 2.2)
6.02 (s)
6.08 (s)
6.05 (s)
5.99 (t, 2.1) 6.07 (s)
6.09 (t, 2.1)
—
—
—
—
8.87 (bs)
—
—
5cb
5db
5eb
5fc
5gb
5hb
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6.80 (t, 8.3) 6.86 (t, 8.0) 6.83 (t, 9.0) 6.79 (t, 7.9) 6.83 (t, 8.3) 6.85 (t, 7.9)
6.81 (t, 8.3) 6.87 (t, 8.0)
H-5ph
6.13 (ddd, 8.0, 2.2, 0.8)
6.04 (dd, 7.9, 1.8) 6.11 (d, 2.2)
6.14 (dd, 8.0, 1.8) 6.10 (d, 7.2)
6.15 (ddd, 8.1, 2.2, 0.9) 6.07 (d, 10.0)
6.08 (d, 10.0)
H-4phe
3-Aminophenol
1
6.06 (ddd, 7.8, 2.3, 0.9)
5.95 (dd, 8.0, 2.2) 6.04 (d, 2.3)
6.06 (dd, 8.0, 1.8) 6.02 (d, 7.2)
6.01 (d, 10.0)
6.06 (ddd, 8.1, 2.2, 0.9)
6.02 (d, 10.0)
H-6phe
—
6.16 (t, 6.0) —
—
—
4.18 (s)
4.17 (s)
4.24 (s)
4.27 (s)
4.20 (s)
4.14 (s)
4.30 (s)
—
—
4.24 (s)
NH–CH2–
—
–NH– CH2–
7.03 (d, 8.1)
7.01 (d, 8.2)
7.60 (d, 8.3)
—
7.22 (d, 7.9)
6.98 (d, 8.1)
7.48–7.38 (m)
—
H-2″,6′ —
H-3′,5″
7.26 (d, 8.1)
7.24 (d, 8.2)
7.42 (d, 8.3)
—
7.05 (d, 7.9)
7.15 (d, 8.1)
7.53 (d, 8.4)
Benzene″
2.57 (m) 1.57 (m)
2.83 (m)
b
—
2.67–2.52 (m) 1.67–1.47 (m) —
2.80 (m)
—
—
–(CH2)n′–d
Multiplicity and coupling constants are given in parentheses. H shifts assignments are in agreement with the HSQC and HMBC spectra. CD3OD was used as solvent in the NMR. c DMSO-d6 was used as solvent in the NMR experiments. d Signals corresponding to the -(CH2)n′- hydrogen atoms of the linker cannot be unequivocally assigned. e These signals can be interchanged. f These signals correspond to both C-2 and C-5 (N-CH2-) pyrrolidine hydrogen atoms. g These signals correspond to both C-3 and C-4 (N-CH2-CH2-) pyrrolidine hydrogen atoms.
a
5a
5bb
H-2ph
6.03 (s)
–OH
H NMR data for compounds 5a–h (δ, ppm; J, Hz)a
—
1
b
Compound
Table 3.
7.19 (d, 8.1) 7.19 (d, 8.0) 7.32 (d, 8.4) 7.47 (d, 8.2) 7.19 (d, 8.1) 7.21 (d, 8.2)
7.31 (d, 8.2) 7.63 (d, 8.3)
H-2′,6′
7.17 (d, 8.2)
7.14 (d, 8.1)
7.69 (d, 8.2)
7.40 (d, 8.4)
7.25 (d, 8.0)
7.11 (d, 8.1)
7.48–7.38 (m)
7.39 (d, 8.2)
H-3′,5′
Benzene′
5.27 (s)
5.25 (s)
5.44 (s)
5.32 (s)
5.27 (s)
5.20 (s)
5.35 (s)
5.28 (s)
–CH2–N
+
8.04 (d, 7.8) 8.14 (d, 7.6) 8.19 (d, 7.9) 8.45 (d, 7.8) 8.11 (d, 7.9) 8.14 (d, 7.9)
8.14 (d, 7.6) 8.18 (d, 7.8)
H-2,6pyr
6.71 (d, 7.8) 6.80 (d, 7.6) 6.95 (d, 7.9) 7.06 (d, 7.8) 6.89 (d, 7.9) 6.90 (d, 7.9)
6.76 (d, 7.6) 6.81 (d, 7.8)
H-3,5pyr
Pyridine
(m)g (m)g (m)f (m)g
3.18 (s)
3.17 (s)
3.18 (s)
3.43(m)g 2.03 (m)g 3.57–3.46 (m)f 2.14–1.98 (m)g 3.21 (s)
3.55–3.40 2.12–2.02 3.56–3.46 2.13–2.03
R1, R2
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Magn. Reson. Chem. (2013)
13
Magn. Reson. Chem. (2013)
111.75
109.02 111.71 109.37
C-2ph
151.79
149.18 151.84 149.36
C-3ph
124.11
121.43 124.07 121.79
C-4ph d
132.63
130.04 132.67 130.30
C-5ph
117.87
115.43 117.99 115.52
C-6phd
72.85
69.54 72.46 70.42
–O–CH2–
136.26
— 138.65 134.12
— 142.45 141.66 145.10
C-4″
C-1″
Copyright © 2013 John Wiley & Sons, Ltd.
13
100.26
100.27 99.99 100.16
103.28
103.28 100.33 103.34
C-2ph
149.87
147.73 148.83 149.74
150.12
150.17 149.96 150.08
C-3ph
106.96
107.11 109.96 106.87
109.61
109.78 107.08 109.63
129.45
129.50 131.86 129.31
130.81
130.83 129.50 130.80
C-5ph
102.16
102.11 105.06 102.05
105.72
105.61 102.18 105.78
C-6phe
68.57
68.14 70.88 68.41
70.70
70.03 68.27 70.68
O–CH2–
133.00
135.07 135.11 133.05
133.51
135.59 134.95 133.66
C-1′
142.83
138.14 142.73 141.98
145.19
140.44 140.18 144.29
C-4′
127.89
128.06 129.93 127.79
129.35
129.37 128.52 129.30
b
C-2′,6′
Benzene′
Assignments of C chemical shifts are in agreement with the HSQC and HMBC spectra. CD3OD was used as solvent in the NMR. c DMSO-d6 was used as solvent in the NMR experiments. d Signals corresponding to the -(CH2)n′- carbon atoms of the linker cannot be unequivocally assigned. e These signals are interchangeable. f These signals correspond to both C-2 and C-5 (N-CH2-) pyrrolidine carbon atoms. g These signals correspond to both C-3 and C-4 (N-CH2-CH2-) pyrrolidine carbon atoms.
a
159.34
159.15 161.10 159.34
4ec 4fb 4gb
4hc
161.31
4db
C-1ph
C-4phe
3-Aminophenol
C NMR data for compounds 4a–h (δ, ppm)a
161.04 159.26 161.30
13
4ab 4bc 4cb
Compound
Table 5.
b
C-3′,5′
128.88
128.00 128.77 128.84
130.45
— — 38.36 38.36 36.30 36.27 31.99 31.91 — — 36.28 36.28 34.52 34.48 30.45 30.40
C-1″
134.72
— 137.60 134.81
— 140.98 140.56 141.52
136.30
143.28
— 137.14 136.60
C-4″
128.18
— 128.08 128.13
129.48
— 126.68 129.66
127.56
— 129.10 127.39
128.64
— 128.06 128.59
59.19
59.12 61.50 59.04
61.66
61.54 59.14 61.64
+
141.90
141.96 143.15 141.78
142.90
143.01 141.99 142.90
107.91
107.98 109.17 107.79
109.64
109.70 108.60 109.63
C-3,5pyr
155.82
155.87 157.92 155.70
155.12
155.20 153.05 155.15
C-4pyr
R1, R2
41.59
38.93 41.59 39.27
R1, R2
39.46
30.65 40.49 39.35
49.68f 26.10g
49.72f 26.12g 48.31f 24.63g 49.69f 26.11g
159.22
156.61 159.26 156.79
C-4pyr
110.32
107.72 110.37 107.96
C-3,5pyr
Pyridine
Pyridine
144.26
141.69 144.34 141.89
C-2,6pyr
C-2,6pyr
62.87
59.99 62.67 60.43
+
–CH2–N
–CH2–N
130.65
128.16 130.15 128.28
C-3′,5′
C-3″,5″
131.68
128.16 131.17 129.48
C-2′,6′
C-2″,6″
Benzene″
146.46
134.67
C-4′ 137.62 144.10 143.07
C-1′
Benzene′
134.67 136.52 132.51
— 138.63 142.18
— — 37.20 37.20 37.56 30.54 33.25 33.22
–(CH2)n′–c
–(CH2)n′–d
130.94
— 130.59 128.72
C-3″,5″
129.33 127.22 130.67
130.14
— 129.47 127.73
C-2″,6″
Benzene″
Assignments of C chemical shifts are in agreement with the HSQC and HMBC spectra. CD3OD was used as solvent in the NMR experiments for all compounds. c Signals corresponding to the -(CH2)n′- carbon atoms of the linker cannot be unequivocally assigned. d These signals can be interchanged.
13
162.03
3h
a
159.14 161.95 159.59
C-1ph
3-Nitrophenol
C NMR data for compounds 3e–h (δ, ppm)a
3e 3f 3g
Compoundb
Table 4.
NMR studies of new 3-aminophenol isomers
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C-1ph
13
99.79
99.21 99.99 99.84
100.92
99.55
99.52
158.02
158.04 158.83 158.03
159.16
157.77
157.75
157.77
C-3ph
105.26
103.85 104.66 105.30
106.40
105.00
104.95
104.81
129.59
129.34 130.10 129.59
130.66
129.29
129.27
129.32
C-5ph
103.98
103.36 104.08 103.96
105.11
103.67
103.75
103.72
C-6phe
53.70
45.99 46.79 47.29
47.95
48.27
48.42
46.62
NH–CH2–
141.00
— 141.01 139.91
142.12
139.62
137.70
— 141.15 138.01
138.84
137.73
140.11
—
— 141.78
C-4″
C-1″
b
128.32
— 127.32 128.49
129.44
128.22
127.46
—
C-2″,6″
Benzene″
Assignments of C chemical shifts are in agreement with the HSQC and HMBC spectra. CD3OD was used as solvent in the NMR. c DMSO-d6 was used as solvent in the NMR experiments. d Signals corresponding to the -(CH2)n′- carbon atoms of the linker cannot be unequivocally assigned. e These signals are interchangeable. f These signals correspond to both C-2 and C-5 (N-CH2-) pyrrolidine carbon atoms. g These signals correspond to both C-3 and C-4 (N-CH2-CH2-) pyrrolidine carbon atoms.
a
150.48
5hb
151.64
5db
149.78 150.64 150.46
150.19
5cb
5eb 5fc 5gb
150.07
99.44
C-2ph
C-4phe
3-Aminophenol
C NMR data for compounds 5a–h (δ, ppm)a
149.86
13
5bb
5a
b
Compound
Table 6.
127.23
— 127.85 127.19
128.33
126.89
126.56
—
C-3″,5″
132.23
133.95 135.39 132.37
133.47
132.17
133.54
— 37.07 36.87 36.25 36.22 32.00 31.87 — — 37.34 37.13 35.14 35.10 30.89 30.74
133.16
C-1′ —
–(CH2)n′–d
144.05
141.32 138.35 143.15
145.21
142.88
138.35
141.78
C-4′
128.32
127.92 129.30 128.34
129.33
127.97
128.41
128.05
C-2′,6′
Benzene′
129.31
127.62 128.39 129.50
130.45
129.23
127.34
127.78
C-3′,5′
60.48
59.11 59.77 60.44
61.66
60.21
60.10
60.15
+
–CH2–N
141.80
141.93 142.76 141.76
142.88
141.38
141.56
141.48
107.93
107.97 108.76 107.93
109.63
108.22
108.28
108.25
C-2,6pyr C-3,5pyr
Pyridine
156.73
155.85 156.64 156.70
155.12
153.62
153.74
153.63
C-4pyr
39.25
39.75 40.50 39.29
48.29f 24.69g 48.29f 24.69g 48.46f 24.69g 49.68f 26.10g
R1, R2
S. Schiaffino-Ortega et al.
Copyright © 2013 John Wiley & Sons, Ltd.
Magn. Reson. Chem. (2013)
NMR studies of new 3-aminophenol isomers higher in 4a–h than in 5a–h compounds. H-4ph and H-6ph suffers the same effect for the same reason. The same behavior is observed in 13C NMR signals of C-2ph, C-4ph, and C-6ph, which appear more shielded in 5a–h than in 4a–h derivatives and their direct precursors (3e–h). 1 H NMR signals of H-4ph and H-6ph in compounds 4a–h are superimposed and appear as multiplets, and for this reason, these signals cannot be completely assigned, except in 4f, where the signals are well resolved. In compounds 5a–h, the 1H NMR signals of H-4ph and H-6ph are well resolved, but they cannot be unequivocal assigned through the HMBC and HMQC techniques. Consequently, these signals can be considered interchangeable. C-4ph and C-6ph are interchangeable in both families as well. Finally, the methylene group connected to the ether bond in 4a–h compounds appears less shielded as in 1H NMR as in 13C NMR signals, than when this methylene is connected to amino group in the 5a–h compounds. In the rest of the molecule, no significant differences have been found. The 1H and 13C NMR chemical shifts of the linker and the cationic head are very similar in isomeric compounds of families 4 and 5. In conclusion, two families of structural isomers bearing 3-aminophenol moiety (4a–h and 5a–h) and their direct precursors (3e–h) have been unequivocally identified by 1D and 2D NMR techniques. The only differences between both isomers are the chemical shift of H-2ph, H-4ph, and H-6ph and of C-2ph, C-4ph, and C-6ph respectively in the 3-aminophenol moiety, which appears more deshielding for 4a–h compounds than for 5a–h. Thus, only one difference between two isomers is enough to unequivocally identify them.
References [1] a) M. Bañez-Coronel, A. Ramírez de Molina, A. Rodríguez-González, J. Sarmentero, M. A. Ramos, M. A. García-Cabezas, L. García-Oroz, J. C. Lacal, Curr. Cancer Drug Targets 2008, 8, 709–719; b) E. Hernando, J. Sarmentero-Estrada, T. Koppie, C. Belda-Iniesta, V. Ramírez de Molina, P. Cejas, C. Ozu, C. Le, J. J. Sánchez, M. González-Barón, J. Koutcher, C. Cordón-Cardó, B. H. Bochner, J. C. Lacal, A. Ramírez de Molina, Oncogene 2009, 28, 2425–2435; c) W. Cun See Too, M. Teng Wong, L. Ling Few, M. Konrad, PLoS ONE, 2010, 5(9), e12999; d) D. Gallego-Ortega, T. Gómez del Pulgar, F. Valdés-Mora, A. Cebrián, J. C. Lacal, Adv. Enzyme Regul. 2011, 51, 183–194. [2] a) E. Malito, N. Sekulic, W. C. Too, M. Konrad, A. Lavie, J. Mol. Biol. 2006, 364, 136–151; b) L. Milanese, A. Espinosa, J. M. Campos, M. A. Gallo, A. Entrena, ChemMedChem 2006, 1, 1216–1228. [3] a) B. Rubio-Ruiz, A. Conejo-García, P. Ríos-Marco, M. P. CarrascoJimenez, J. Segovia, C. Marco, M. A. Gallo, A. Espinosa, A. Entrena, Eur. J. Med. Chem. 2012, 50, 154–162; b) B. Rubio-Ruiz, A. ConejoGarcía, M. A. Gallo, A. Espinosa, A. Entrena, Magn. Reson. Chem. 2012, 50, 466–469. [4] S. Schiaffino-Ortega, L. C. López-Cara, P. Ríos-Marco, M. P. CarrascoJimenez, C. Marco, M. A. Gallo, A. Espinosa, A. Entrena. Biorg. Med. Chem. 2013. doi:10.1016/j.bmc.2013.09.003. [5] a) H. A. Staab, M. Haenel, Chem. Ber. 1973, 106, 2190–2202; b) D. J. Cram, H. Steinberg, J. Am. Chem. Soc. 1951, 73, 5691–5704. [6] M. Hesse, H. Meier, B. Zeeh, Spektroskopische Methoden in der Organischen Chemie, 5th edn, Georg Thieme Verlag, Stuttgart- Nueva, York, 1995.
Supporting Information
Acknowledgements This work was supported by a project from the ‘Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía’ (P07-CTS-03210)
Magn. Reson. Chem. (2013)
and the ‘Ministerio de Ciencia e Innovación’ (SAF2009-11955). The award of grant from the ‘Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía’ to S. S. O. is gratefully acknowledged.
Additional supporting information may be found in the online version of this article at the publisher’s web site.
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