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1H, 13C, and 15N NMR conformational characterization of a series of 2-acetylthiazolethiosemicarbazone compounds
Journal of Molecular Structure 1157 (2018) 8e13
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1
H, 13C, and 15N NMR conformational characterization of a series of 2-acetylthiazolethiosemicarbazone compounds
William R. Carroll*, Dylan M. Gardner, Elizabeth R. Melton, Shana T. Murphy, Arielle K. Buckner, Madison S. Fulmer, William G. Qualls, Edward C. Lisic Department of Chemistry, Tennessee Technological University, Cookeville, TN, USA, 38505
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 July 2017 Received in revised form 14 November 2017 Accepted 15 November 2017 Available online 12 December 2017
A series of new 2-acetylthiazolethiosemicarbazone molecules belonging to a family of pharmaceutically relevant molecules was synthesized and characterized. These molecules showed atropisomeric properties and occupied two distinct conformations at room temperature. Each of these configurations was characterized by NMR and identified as both internally hydrogen bonded and non-hydrogen bonded forms. The knowledge of these two forms and their identities may be of use to those seeking to employ this family of molecules in a pharmaceutical context. Published by Elsevier B.V.
Keywords: Thiosemicarbazones Nuclear magnetic resonance Conformational analysis
1. Introduction Thiosemicarbazone compounds have been the subject of intense research for the last twenty years due to their biological and medicinal properties. These compounds have been used even earlier as drugs against leukemia and to help cure leprosy, tuberculosis, and smallpox [1e5]. Additionally, these compounds also act as high affinity multi-dentate chelating agents ligands for a wide selection of transition metals where they have been characterized in a variety of context [1,6e9]. A specific subset of thiosemicarbazones known as a-(N)heterocyclic thiosemicarbazones have been the focus of much of the recent interest due to the ability of some of them to destroy the function of the ribonucleotide reductase enzyme, which is important in cell replication [2,10e15]. Interestingly, the Cu(II) complexes of these a-(N)-heterocyclic thiosemicarbazone ligands are extremely effective in the inhibition of the topoisomerase IIa enzyme, this is one of the areas of the most intense research [16e23]. The topoisomerase IIa enzyme is of particular importance in the area of anti-cancer research since it is absolutely essential for human DNA replication and it is a target for chemotherapy drugs such as etoposide [24].
* Corresponding author. Tennessee Technological University, Department of Chemistry, P.O. Box 5055, Cookeville, TN 38505. E-mail address: [email protected] (W.R. Carroll). https://doi.org/10.1016/j.molstruc.2017.11.061 0022-2860/Published by Elsevier B.V.
A new series compounds have moiety and show the existence of solution.
of five a-(N)-heterocyclic thiosemicarbazone been synthesized that contain a thiazole ring evidence, as obtained by NMR spectroscopy, of two conformations of these compounds in
2. Experimental 2.1. Syntheses 2.1.1. ATZ-MTSC (3a) (E)eN-methyl-2-[1-(thiazol-2-yl)-ethylidene] hydrazinecarbothioamide To a stirred solution of 2-acetylthiazole (1, 7.77 104 mmol, 0.0989 g) and 4-methyl-3-thiosemicarbazide (2a, 7.77 103 mmol, 0.0816 g) in 50 mL of isopropanol was added one drop of sulfuric acid. The reaction mixture was heated to 60 C for 16 h. A yellow crystalline precipitate formed and was collected via gravity filtration to yield ATZ-MTSC (3a, 5.82 104 mmol, 0.1247 g, 74.9% yield). Ratio of major to minor was 5.4:1.0. 3a major 1H NMR (500 MHz, DMSO-d6) d 10.61 (s, 1H), 8.32e8.27 (m, 1H), 7.88 (d, J ¼ 3.2 Hz, 1H), 7.80 (dd, J ¼ 3.2, 0.9 Hz, 1H), 3.05 (d, J ¼ 4.5 Hz, 3H), 2.42 (s, 3H). 13C NMR (126 MHz, DMSO) d 178.52, 166.98, 144.03, 143.24, 122.42, 31.32, 13.68. 3a minor 1H NMR (500 MHz, DMSOd6) d 13.29 (s, 1H), 8.82 (q, J ¼ 3.3 Hz, 1H), 8.26 (dd, J ¼ 3.3, 1.1 Hz, 1H), 8.09 (dd, J ¼ 3.3, 1.3 Hz, 1H), 3.02 (d, J ¼ 4.6 Hz, 3H), 2.44 (s, 3H). 13 C NMR (126 MHz, DMSO) d 178.03, 161.81, 143.95, 131.84, 122.59,
W.R. Carroll et al. / Journal of Molecular Structure 1157 (2018) 8e13
31.07, 21.88. Theoretical MS m/z (relative intensity) for C7H9N4S2 [3a-Hþ]: 213.0 (100%). Actual MS m/z (relative intensity): 212.9 (100%). 2.1.2. ATZ-ETSC (3b) (E)eN-ethyl-2-[1-(thiazol-2-yl)-ethylidene] hydrazinecarbothioamide To a stirred solution of 2-acetylthiazole (1, 4.66 104 mmol, 0.5927 g) and 4-ethyl-3-thiosemicarbazide (2b, 0.462 104 mmol, 0.5502 g) in 50 mL of isopropanol was added one drop of sulfuric acid. The reaction mixture was heated to 60 C for 16 h. A yellowwhite crystalline precipitate formed and was collected via gravity filtration to yield ATZ-ETSC (3b, 4.40 104 mmol, 1.004 g, 95.2% yield). Ratio of major to minor was 5.0:1.0. 3b major 1H NMR (500 MHz, DMSO-d6) d 13.29 (s, 1H), 8.82 (q, J ¼ 3.3 Hz, 1H), 8.26 (dd, J ¼ 3.3, 1.1 Hz, 1H), 8.09 (dd, J ¼ 3.3, 1.3 Hz, 1H), 3.02 (d, J ¼ 4.6 Hz, 3H), 2.44 (s, 3H). 13C NMR (126 MHz, DMSO) d 177.50, 166.97, 144.04, 143.28, 122.39, 38.65, 14.28, 13.68. 3b minor 1H NMR (500 MHz, DMSO-d6) d 13.25 (s, 1H), 8.83 (t, J ¼ 6.2 Hz, 1H), 8.25 (dd, J ¼ 3.4, 1.2 Hz, 1H), 8.12e8.03 (m, 1H), 3.59e3.55 (m, 2H), 2.44 (s, 3H), 1.16e1.09 (m, 3H). 13C NMR (126 MHz, DMSO) d 176.98, 161.81, 143.95, 131.91, 122.59, 38.62, 21.88, 14.30. Theoretical MS m/z (relative intensity) for C8H11N4S2 [3b-Hþ]: 227.1 (100%). Actual MS m/z (relative intensity): 227.0 (100%). 2.1.3. ATZ-BzTSC (3c) (E)eN-benzyl-2-[1-(thiazol-2-yl)-ethylidene] hydrazinecarbothioamide To a stirred solution of 2-acetylthiazole (1, 2.68 mmol, 0.3400 g) and 4-benzyl-3-thiosemicarbazide (2c, 2.70 mmol, 0.4901 g) in 50 mL of isopropanol was added one drop of sulfuric acid. The reaction mixture was heated to 60 C for 16 h. A yellow crystalline precipitate formed and was collected via gravity filtration to yield ATZ-BzTSC (3c, 2.13 mmol, 0.62 g, 79.7% yield). Ratio of major to minor was 5.3:1.0. 3c major 1H NMR (500 MHz, DMSO-d6) d 10.76 (s, 1H), 8.79 (t, J ¼ 6.2 Hz, 1H), 7.88 (d, J ¼ 3.2 Hz, 1H), 7.79 (d, J ¼ 3.2 Hz, 1H), 7.41e7.20 (m, 5H), 4.87 (d, J ¼ 6.1 Hz, 2H), 2.45 (s, 3H). 13C NMR (126 MHz, DMSO) d 178.39, 166.89, 144.50, 143.30, 139.00, 128.24, 127.20, 126.83, 122.51, 46.93, 13.79. 3c minor 1H NMR (500 MHz, DMSO-d6) d 13.38 (s, 1H), 9.37 (t, J ¼ 6.4 Hz, 1H), 8.27 (d, J ¼ 3.2 Hz, 1H), 8.10 (d, J ¼ 3.0 Hz, 1H), 7.42e7.20 (m, 5H), 4.83 (d, J ¼ 6.2 Hz, 2H), 2.45 (s, 3H). 13C NMR (126 MHz, DMSO) d 177.89, 161.80, 143.97, 138.91, 132.38, 128.19, 127.38, 126.85, 122.73, 46.86, 21.90. Theoretical MS m/z (relative intensity) for C13H13N4S2 [3c-Hþ]: 289.1 (100%). Actual MS m/z (relative intensity): 289.1 (100%). 2.1.4. ATZ-PTSC (3d) (E)eN-phenyl-2-[1-(thiazol-2-yl)-ethylidene] hydrazinecarbothioamide To a stirred solution of 2-acetylthiazole (1, 4.08 mmol, 0.5186 g) and 4-phenyl-3-thiosemicarbazide (2d, 4.08 mmol, 0.6820 g) in 50 mL of isopropanol was added one drop of sulfuric acid. The reaction mixture was heated to 60 C for 16 h. A yellow crystalline precipitate formed and was collected via gravity filtration to yield ATZ-PTSC (3d, 3.44 mmol, 0.9508 g, 84.3% yield). Ratio of major to minor was 4.8:1.0. 3d major 1H NMR (500 MHz, DMSO-d6) d 11.02
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(s, 1H), 9.93 (s, 1H), 7.91 (d, J ¼ 3.1 Hz, 1H), 7.82 (d, J ¼ 3.1 Hz, 1H), 7.68e7.57 (m, 2H), 7.38 (t, J ¼ 7.9 Hz, 2H), 7.30e7.16 (m, 1H), 2.50 (s, 3H). 13C NMR (126 MHz, DMSO) d 176.83, 166.78, 145.10, 143.35, 138.90, 128.24, 125.41, 125.06, 122.75, 13.97. 3d minor 1H NMR (500 MHz, DMSO-d6) d 13.57 (s, 1H), 10.45 (s, 1H), 8.29 (d, J ¼ 3.3 Hz, 1H), 8.13 (d, J ¼ 3.2 Hz, 1H), 7.64e7.62 (m, 2H), 7.34e7.29 (m, 2H), 7.12 (t, J ¼ 7.5 Hz, 1H), 2.51 (s, 3H). 13C NMR (126 MHz, DMSO) d 176.34, 161.79, 144.02, 138.72, 132.81, 128.17, 125.51, 125.26, 122.96, 21.82. Theoretical MS m/z (relative intensity) for C12H11N4S2 [3d-Hþ]: 275.1 (100%). Actual MS m/z (relative intensity): 275.1 (100%). 2.1.5. ATZ-tBTSC (3e) (E)eN-tert-butyl-2-[1-(thiazol-2-yl)ethylidene]hydrazinecarbothioamide To a stirred solution of 2-acetylthiazole (1, 4.34 mmol, 0.5514 g) and 4-tertbutyl-3-thiosemicarbazide (2e, 4.29 mmol, 0.6320 g) in 50 mL of isopropanol was added one drop of sulfuric acid. The reaction mixture was heated to 60 C for 16 h. A yellow crystalline precipitate formed and was collected via gravity filtration to yield ATZ-tBTSC (3e, 3.90 mmol, 0.9995 g, 90.9% yield). Ratio of major to minor was 8.0:1.0. 3e major 1H NMR (500 MHz, DMSO-d6) d 10.62 (s, 1H), 7.88 (d, J ¼ 3.2 Hz, 1H), 7.77 (d, J ¼ 3.2 Hz, 1H), 7.71 (s, 1H), 2.42 (s, 3H), 1.54 (s, 9H). 13C NMR (126 MHz, DMSO) d 176.05, 167.16, 143.48, 142.76, 122.27, 52.78, 28.30, 13.55. 3e minor 1H NMR (500 MHz, DMSO-d6) d 13.22 (s, 1H), 8.26 (d, J ¼ 3.2 Hz, 1H), 8.10 (d, J ¼ 3.2 Hz, 1H), 7.88 (s, 1H), 2.43 (s, 3H), 1.53 (s, 9H). 13C NMR (126 MHz, DMSO) d 175.53, 161.78, 143.99, 131.66, 122.79, 52.96, 28.38, 21.98. Theoretical MS m/z (relative intensity) for C10H15N4S2 [3e-Hþ]: 255.1 (100%). Actual MS m/z (relative intensity): 255.1 (100%). 3. Results and discussion This study examines a series of thiosemicarbazones with potential anti-microbial and anti-cancer activity. Each of these thiosemicarbazones includes a conserved thiazole and central thiosemicarbazone core. To the thioamide nitrogen of each molecule is attached a moiety that varies from methyl (3a), ethyl (3b), benzyl (3c), phenyl (3d), and tert-butyl (3e) as described in Fig. 1. Acetylthiazole thiosemicarbazones (ATZ-TSC's) were synthesized in the typical manner for other thiosemicarbazone derivatives [25,26]. The reaction used to form the thiosemicarbazone was an acid catalyzed imine formation that produced the desired thiosemicarbazones in high yield and purity. Chemicals were purchased from Sigma-Aldrich and Fischer chemical companies and used without further preparation unless otherwise noted. These molecules were characterized by 1H, 13C DEPTQ-135, 1He1H COSY, 1 He13C HSQC, 1He15N HSQC, 1He13C HMBC, and 1He15N HMBC NMR techniques as well as mass spec. NMR spectroscopy was carried out at the Center for Structural Chemistry, Tennessee Technological University (USA). The spectra reported here were measured with a Bruker Avance III HD 500 spectrometer at 500.13 MHz (1H), 50.69 MHz (15N) and 125.03 MHz (13C) at 25 C equipped with a PRODIGY cryoprobe. For these measurements, the
Fig. 1. Synthesis of the series of 2-acetylthiazolethiosemicarbazones 3a-3e.
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W.R. Carroll et al. / Journal of Molecular Structure 1157 (2018) 8e13
substances were dissolved in the appropriate deuterated solvent, and the chemical shifts were referenced to the solvent residual peak. Coupling constants (J) are given in Hertz. 1H NMR experiments were acquired using Bruker's standard PROTON (zg30) NMR pulse sequence with the following parameters: Relaxation delay, 1s; 90 pulse, 12.0 ms; spectral width, 10,000 Hz; number of data points, 32 K; and digital resolution, 0.153 Hz/point. All mass spectrometry data was taken with a Varian 300/310/320-MS LC/MS Quadrupole Mass Spectrometer in negative mode using APCI. The corona current was set to 5.00 mA, while the shield potential was set to 600.00 V. The housing, drying gas, and vaporizer gas temperatures were set to 50 C, 150 C, and 350 C respectively. The drying, nebulizing, and vaporizer gas pressures were each set to 12.0 psi, 55.0 psi, and 17.0 psi. For the mass spectrometry data, each sample was dissolved in minimal amounts of dimethyl sulfoxide and then diluted to 10 ppm in methanol. In each case this thiosemicarbazone was produced and confirmed by Mass Spec; however, a second set of NMR signals with similar couplings was observed with slightly displaced chemical shifts. The relative abundance of this second set of signals varied between the different derivatives. The presence of these minor and major sets of peaks may be a result of a conformational equilibrium that may rotate to present a possible internal hydrogen bond resulting in two conformations of these molecules of varying relative abundance. The conformational equilibrium of the thiosemicarbazone family of molecules has been explored in previous studies via theoretical computational studies, variable temperature NMR, and using 1H NMR in variable solvents [28,29]. In this study we examine the 15N NMR chemical shifts, 1H NMR in variable solvents, and NOESY spectrum. To elucidate the configuration of the major and minor sets of peaks and determine if a hydrogen bond was present, the chemical shifts of each signal was assigned for the major and minor conformers separately and 2D NMR spectra of heteronuclear correlations between 1H and 13C, as well as 1H and 15N were recorded. This allowed the observation of any changes in chemical environment between the nitrogens in the major and minor set of signals. For the purposes of discussion, a letter label has been applied to each hydrogen and a number label has been applied to each carbon and nitrogen shown in Fig. 2. Numbering on the variable parts of the molecule was carried out sequentially progressing away from the thiosemicarbazone. Signals for the thiazole ring hydrogens were found as two doublets between 7.7 and 8.3 ppm in both the major and minor forms (Table 1). The signals from the minor form were shifted upfield on average 0.31 ppm for H(a) and 0.38 ppm for H(b). This is consistent with the nitrogen on the thiazole ring forming a
hydrogen bond with the neighboring thiosemicarbazone hydrazinic NeH H(d). Carbon signals for the thiazole ring carbons C(1) and C(2) appeared near 122 ppm and 143 ppm respectively (Table 2). The minor signal was shifted an average of 0.26 ppm for C(1) and 0.65 ppm for C(2) downfield. With C(2) being more closely localized next to N(1) the larger downfield shift may be expected. The thiosemicarbazone hydrazinic NeH hydrogen H(d) appeared between 10.5 and 13.6 ppm in each molecule. The minor form of H(d) was shifted downfield an average of 2.63 ppm also supporting its participation in a hydrogen bond in the minor form. Parts of these molecules that should not experience substantial changes in their chemical environment with the formation of an internal hydrogen bond such as the methyl hydrogens H(c) appeared between 2.4 ppm and 2.5 ppm. The minor signals were shifted an average of 0.01 ppm downfield reinforcing the local nature of these chemical shift changes. The only molecule previously reported in the literature from this series is the ethyl derivative 3b the crystal structure of which shows an internal hydrogen bond between the hydrazinic nitrogen N(2) and the thioamide NeH H(e) [27]. In the molecules reported here H(e) appears between 8.2 ppm and 10.5 ppm. The signal for the minor form of H(e) shifted on average 0.54 ppm downfield supporting the presence of this hydrogen bond. Thus far the minor and major forms of the series of molecules reported here can be visualized as an internally hydrogen bonded and non-hydrogen bonded configurations as shown in Fig. 3. In the thiosemicarbazone family of molecules this conformational equilibrium has been attributed to the imine E/Z conformational equilibrium [28,29]. The nature of the rotation of the thiazole ring is not apparent in the form on the right in Fig. 3 however the relative basicity of the nitrogen would suggest that the thiazole ring nitrogen would preferentially participate in a hydrogen bond over the ring sulfur in the form on the left in Fig. 3. To ascertain the exact form of the major and minor configurations of this series of thiosemicarbazones the nitrogen chemical shifts were examined. This was achieved via 1He15N HSQC and 1 He15N HMBC NMR with nitrogens N(1)eN(4) being clearly visible in all but one spectrum. The use of nitrogen chemical shift changes with internal hydrogen bonding has precedent in the investigation of hydrogen bonding within imidazoles [30,31]. This existing 15N NMR investigation into imidazoles provides an expectation for the changes in chemical shift an internal hydrogen bond to the thiazole ring would cause as well. In imidazoles it was observed that when the sp2 hybridized nitrogen of the imidazole hydrogen bonds it was shifted upfield. This was attributed to a decrease in the pyridine-like character and increase in the pyrrole-like character of the nitrogen. In this
Fig. 2. Enumeration of Hydrogens a-i, carbons 1e11, and nitrogens 1e4 in the 2-acetylthiazolethiosemicarbazones.
W.R. Carroll et al. / Journal of Molecular Structure 1157 (2018) 8e13
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Table 1 1 H NMR data for compounds 3a-e (d in ppm; J in Hz)a. Compound
A
B
3a (major) 3a (minor) 3b (major) 3b (minor) 3c (major) 3c (minor) 3d (major) 3d (minor) 3e (major) 3e (minor)
7.80 (dd, 3.2, 0.9 Hz) 8.09 (dd, 3.3, 1.3) 7.79 (dd, 3.3, 1.5) 8.12e8.03 (m) 7.79 (d, 3.2) 8.10 (d, 3.0) 7.82 (d, 3.1) 8.13 (d, 3.2) 7.77 (d, 3.2) 8.10 (d, 3.2)
7.88 8.26 7.88 8.25 7.88 8.27 7.91 8.29 7.88 8.26
C (d, 3.2) (dd, 3.3, 1.1) (d, 3.2) (dd, 3.4, 1.2) (d, 3.2) (d, 3.2) (d, 3.1) (d, 3.3) (d, 3.2) (d, 32)
2.42 2.44 2.42 2.44 2.45 2.45 2.50 2.51 2.42 2.43
D (s) (s) (s) (s) (s) (s) (s) (s) (s) (s)
10.61 13.29 10.57 13.25 10.76 13.38 11.02 13.57 10.62 13.22
(s) (s) (s) (s) (s) (s) (s) (s) (s) (s)
E
F
G
H
8.32e8.27 (m) 8.82 (q, 3.3) 8.28 (t, 5.9) 8.83 (t, 6.2) 8.79 (t, 6.2) 9.37 (t, 6.4) 9.93 (s) 10.45 (s) 7.71 (s) 7.88 (s)
3.05 (d, 4.5) 3.02 (d, 4.6) 3.61 (dt, 12.9, 7.0) 3.59e3.55 (m) 4.87 (d, 6.1) 4.83 (d, 6.2) 7.68e7.57 (m) 7.64e7.62 (m) 1.54 (s) 1.53 (s0
1.16 (t, 7.0) 1.16e1.09 (m) 7.41e7.20 (m) 7.42e7.20 (m) 7.38 (t, 7.9) 7.34e7.29 (m)
7.30e7.16 (m) 7.12 (t, 7.5)
DMSO-d6 was used as solvent in the NMR experiments. a Multiplicity and coupling constants are given in parentheses. 1H shift assignments are in agreement with HSQC and HMBC spectra.
Table 2 13 C NMR data for compounds 3a-e (d in ppm; J in Hz)a. Compound
1
2
3
4
5
6
7
8
9
10
11
3a (major) 3a (minor) 3b (major) 3b (minor) 3c (major) 3c (minor) 3d (major) 3d (minor) 3e (major) 3e (minor)
122.42 122.59 122.39 122.59 122.51 122.73 122.75 122.96 122.27 122.79
143.24 143.95 143.28 143.95 143.3 143.97 143.35 144.02 143.48 143.99
166.98 161.81 166.97 161.81 166.89 161.8 166.78 161.79 167.16 161.78
144.03 131.84 144.04 131.91 144.5 132.38 145.1 132.81 142.76 131.66
13.68 21.88 13.68 21.88 13.79 21.9 13.97 21.82 13.55 21.98
178.52 178.03 177.5 176.98 178.39 177.89 176.83 176.34 176.05 175.53
31.32 31.07 38.65 38.62 46.93 46.86 138.9 138.72 52.78 52.96
14.28 14.3 139 138.91 125.06 125.26 28.3 28.38
127.2 127.38 128.24 128.17
128.24 128.19 125.41 125.51
126.83 126.85
DMSO-d6 was used as solvent in the NMR experiments. a Multiplicity and coupling constants are given in parentheses.13C shift assignments are in agreement with HSQC and HMBC spectra.
Fig. 3. Hydrogen bonded and non-hydrogen bonded forms of compounds 3a-3e.
system, the thiazole ring is conjugated through the attached thiosemicarbazone providing a possible route for similar changes. In this system, the thiazole nitrogen N(1) appears between 304 ppm and 312 ppm (Table 3). In the minor form the resonance shifts an average of 4.55 ppm upfield. This upfield shift is consistent with the hydrogen bond reducing the sp2 character of N(1) and increasing its sp3 character. The protonation of the thiazole ring and resulting resonance stabilized tautomer that would be one of the expected contributing resonance forms of the molecule in the hydrogen-bonded conformation. This tautomer would also effect the carbons connecting the thiazole to the thiourea as they are included in a C]C double bond instead of a C]N double bond. This can be observed in carbons C(3) and C(4) which occur between 161 ppm and 167 ppm for the signal from C(3) and between 131 ppm and 145 ppm for the signal from C(4). In each case the minor conformation was shifted an average of 5.16 ppm for the signal from C(3) and 11.97 ppm for the signal from C(4) upfield. Additional evidence of this hydrogen bonded form is apparent in the NOESY spectrum of compound 3a. In the NOESY spectrum of
compound 3a the major compound shows clear cross peaks between the hydrogens H(d) and H(c) in Fig. 2 as expected from the major form depicted on the right in Fig. 3. The minor form of hydrogen H(d) showed a cross peak with hydrogen H(b) on the
Table 3 15N NMR data for compounds 3a-d (d in ppm)a. Compound
1
2
3
4
3a (major) 3a (minor) 3b (major) 3b (minor) 3c (major) 3c (minor) 3d (major) 3d (minor) 3e (major) 3e (minor)
311.85 307.56 310.95 307.7 311.61 306.46 311.82 306.32 308.83 304.14
311.97 316.76 311.64 316.79 311.37
166.18 170.29 166.12 170.31 166.76 170.62 169.64 173.53 167.63 172.14
106.92 106.68 122.36 122.02 119.39 119.48 130.67 129.96 137.53 135.85
311.69 316.67 308.79 317.55
DMSO-d6 was used as solvent in the NMR experiments. N shift assignments are derived from HSQC and HMBC spectra.
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thiazole ring indicating that the minor form shows a clear organization placing the hydrogen bonding H(d) in close proximity to the thiazole ring. This signal and the other NH peaks showed exchange peaks with residual water in DMSO. EXSY cross peaks indicating the slow equilibration of these two conformers at room temperature were observed between the major and minor conformers for each resolved peak. With the internally hydrogen bonded and non-hydrogen bonded forms identified the relative abundance of each conformer may help establish their relative stability in solution. In each case the ratio between the major and minor conformers was 4.8:1.0 to 5.4:1.0 which is characteristic of an energy difference of 0.93e1.00 kcal/mol at 25 C. These measurements were repeated in chloroform and showed a difference of 11.6:1.0 to 0.5:1.0 which indicates a difference of energy between 1.5 and 0.4 kcal/mol at 25 C. Notably these molecules were much less soluble in chloroform and so these measurements may be skewed. Such measurements are established in a variety of context to determine the strength of non-covalent interactions under carefully controlled conditions [32]. While it may be tempting to attribute this difference in energy exclusively to the strength of the hydrogen bond it is worth noting that differences in energy between two conformers may often be dependent on many other factors such as the relative solubility of each form [33]. The complex nature of the forces that contribute to the equilibrium populations of these conformations of these molecules makes it difficult to isolate the cause of any change in population to a single phenomenon however comparison of the steric and electronic effects of the different substituents may provide some insight. The ratios of major to minor forms in DMSO and chloroform were determined and included in the supplementary material. In DMSO no trend was observed between these ratios and their substituents steric or electronic properties. In chloroform these values show a correlation with more sterically bulky groups favoring the hydrogen bonded form. Electronic effects showed no correlation in either solvent's data. 4. Conclusions With the interpretation of the information provided here we can not only establish the identity and purity of a series of thiazole derivatized thiosemicarbazones but we can identify the configurations they exhibit in solution as both internally hydrogen bonded and non-hydrogen bonded. The knowledge of these two forms and their identities may be of use to those seeking to employ these a-(N)-heterocyclic thiosemicarbazones molecules in a pharmaceutical context. The exact biological activity of each conformation may be challenging to isolate as they interconvert readily but the population of each configuration is certainly effected by the solvent system. Acknowledgements We would like to thank the National Science Foundation for funding the purchase of the FT-NMR used in this research, (National Science Foundation (NSF) Major Research Instrument (MRI 1531870) and the URECA! Grant Program at TTU for awarding funds to Ms. A. Buckner to buy necessary supplies and funds to present portions of this research at the April 2017 ACS national meeting in San Francisco, CA. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.molstruc.2017.11.061.
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Hasinoff, The anticancer thiosemicarbazones Dp44mT and triapine lack inhibitory effects as catalytic inhibitors or poisons of DNA topoisomerase IIa, Biochem. Pharmacol. 84 (2012) 52e58. [19] A.Y. Lukmantara, D.S. Kalinowski, N. Kumar, D.R. Richardson, Structureeactivity studies of 4-phenyl-substituted 20 -benzoylpyridine thiosemicarbazones with potent and selective anti-tumour activity, Organ. Biomol. Chem. 11 (2013) 6414e6425. [20] R. Pingaew, S. Prachayasittikul, S. Ruchirawat, Synthesis, cytotoxic and antimalarial activities of benzoyl thiosemicarbazone analogs of isoquinoline and related compounds, Molecules 15 (2010) 988e996. [21] I.C. Mendes, J.P. Moreira, N.L. Speziali, A.S. Mangrich, J.A. Takahashi, H. Beraldo, N (4)-tolyl-2-benzoylpyridine thiosemicarbazones and their copper (II) complexes with significant antifungal activity: crystal structure of N (4)-para-tolyl-2-benzoylpyridine thiosemicarbazone, J. Braz. Chem. Soc. 17 (2006) 1571e1577. [22] J.T. Wilson, X. Jiang, B.C. 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1
H, 13C, and 15N NMR conformational characterization of a series of 2-acetylthiazolethiosemicarbazone compounds
William R. Carroll*, Dylan M. Gardner, Elizabeth R. Melton, Shana T. Murphy, Arielle K. Buckner, Madison S. Fulmer, William G. Qualls, Edward C. Lisic Department of Chemistry, Tennessee Technological University, Cookeville, TN, USA, 38505
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 July 2017 Received in revised form 14 November 2017 Accepted 15 November 2017 Available online 12 December 2017
A series of new 2-acetylthiazolethiosemicarbazone molecules belonging to a family of pharmaceutically relevant molecules was synthesized and characterized. These molecules showed atropisomeric properties and occupied two distinct conformations at room temperature. Each of these configurations was characterized by NMR and identified as both internally hydrogen bonded and non-hydrogen bonded forms. The knowledge of these two forms and their identities may be of use to those seeking to employ this family of molecules in a pharmaceutical context. Published by Elsevier B.V.
Keywords: Thiosemicarbazones Nuclear magnetic resonance Conformational analysis
1. Introduction Thiosemicarbazone compounds have been the subject of intense research for the last twenty years due to their biological and medicinal properties. These compounds have been used even earlier as drugs against leukemia and to help cure leprosy, tuberculosis, and smallpox [1e5]. Additionally, these compounds also act as high affinity multi-dentate chelating agents ligands for a wide selection of transition metals where they have been characterized in a variety of context [1,6e9]. A specific subset of thiosemicarbazones known as a-(N)heterocyclic thiosemicarbazones have been the focus of much of the recent interest due to the ability of some of them to destroy the function of the ribonucleotide reductase enzyme, which is important in cell replication [2,10e15]. Interestingly, the Cu(II) complexes of these a-(N)-heterocyclic thiosemicarbazone ligands are extremely effective in the inhibition of the topoisomerase IIa enzyme, this is one of the areas of the most intense research [16e23]. The topoisomerase IIa enzyme is of particular importance in the area of anti-cancer research since it is absolutely essential for human DNA replication and it is a target for chemotherapy drugs such as etoposide [24].
* Corresponding author. Tennessee Technological University, Department of Chemistry, P.O. Box 5055, Cookeville, TN 38505. E-mail address: [email protected] (W.R. Carroll). https://doi.org/10.1016/j.molstruc.2017.11.061 0022-2860/Published by Elsevier B.V.
A new series compounds have moiety and show the existence of solution.
of five a-(N)-heterocyclic thiosemicarbazone been synthesized that contain a thiazole ring evidence, as obtained by NMR spectroscopy, of two conformations of these compounds in
2. Experimental 2.1. Syntheses 2.1.1. ATZ-MTSC (3a) (E)eN-methyl-2-[1-(thiazol-2-yl)-ethylidene] hydrazinecarbothioamide To a stirred solution of 2-acetylthiazole (1, 7.77 104 mmol, 0.0989 g) and 4-methyl-3-thiosemicarbazide (2a, 7.77 103 mmol, 0.0816 g) in 50 mL of isopropanol was added one drop of sulfuric acid. The reaction mixture was heated to 60 C for 16 h. A yellow crystalline precipitate formed and was collected via gravity filtration to yield ATZ-MTSC (3a, 5.82 104 mmol, 0.1247 g, 74.9% yield). Ratio of major to minor was 5.4:1.0. 3a major 1H NMR (500 MHz, DMSO-d6) d 10.61 (s, 1H), 8.32e8.27 (m, 1H), 7.88 (d, J ¼ 3.2 Hz, 1H), 7.80 (dd, J ¼ 3.2, 0.9 Hz, 1H), 3.05 (d, J ¼ 4.5 Hz, 3H), 2.42 (s, 3H). 13C NMR (126 MHz, DMSO) d 178.52, 166.98, 144.03, 143.24, 122.42, 31.32, 13.68. 3a minor 1H NMR (500 MHz, DMSOd6) d 13.29 (s, 1H), 8.82 (q, J ¼ 3.3 Hz, 1H), 8.26 (dd, J ¼ 3.3, 1.1 Hz, 1H), 8.09 (dd, J ¼ 3.3, 1.3 Hz, 1H), 3.02 (d, J ¼ 4.6 Hz, 3H), 2.44 (s, 3H). 13 C NMR (126 MHz, DMSO) d 178.03, 161.81, 143.95, 131.84, 122.59,
W.R. Carroll et al. / Journal of Molecular Structure 1157 (2018) 8e13
31.07, 21.88. Theoretical MS m/z (relative intensity) for C7H9N4S2 [3a-Hþ]: 213.0 (100%). Actual MS m/z (relative intensity): 212.9 (100%). 2.1.2. ATZ-ETSC (3b) (E)eN-ethyl-2-[1-(thiazol-2-yl)-ethylidene] hydrazinecarbothioamide To a stirred solution of 2-acetylthiazole (1, 4.66 104 mmol, 0.5927 g) and 4-ethyl-3-thiosemicarbazide (2b, 0.462 104 mmol, 0.5502 g) in 50 mL of isopropanol was added one drop of sulfuric acid. The reaction mixture was heated to 60 C for 16 h. A yellowwhite crystalline precipitate formed and was collected via gravity filtration to yield ATZ-ETSC (3b, 4.40 104 mmol, 1.004 g, 95.2% yield). Ratio of major to minor was 5.0:1.0. 3b major 1H NMR (500 MHz, DMSO-d6) d 13.29 (s, 1H), 8.82 (q, J ¼ 3.3 Hz, 1H), 8.26 (dd, J ¼ 3.3, 1.1 Hz, 1H), 8.09 (dd, J ¼ 3.3, 1.3 Hz, 1H), 3.02 (d, J ¼ 4.6 Hz, 3H), 2.44 (s, 3H). 13C NMR (126 MHz, DMSO) d 177.50, 166.97, 144.04, 143.28, 122.39, 38.65, 14.28, 13.68. 3b minor 1H NMR (500 MHz, DMSO-d6) d 13.25 (s, 1H), 8.83 (t, J ¼ 6.2 Hz, 1H), 8.25 (dd, J ¼ 3.4, 1.2 Hz, 1H), 8.12e8.03 (m, 1H), 3.59e3.55 (m, 2H), 2.44 (s, 3H), 1.16e1.09 (m, 3H). 13C NMR (126 MHz, DMSO) d 176.98, 161.81, 143.95, 131.91, 122.59, 38.62, 21.88, 14.30. Theoretical MS m/z (relative intensity) for C8H11N4S2 [3b-Hþ]: 227.1 (100%). Actual MS m/z (relative intensity): 227.0 (100%). 2.1.3. ATZ-BzTSC (3c) (E)eN-benzyl-2-[1-(thiazol-2-yl)-ethylidene] hydrazinecarbothioamide To a stirred solution of 2-acetylthiazole (1, 2.68 mmol, 0.3400 g) and 4-benzyl-3-thiosemicarbazide (2c, 2.70 mmol, 0.4901 g) in 50 mL of isopropanol was added one drop of sulfuric acid. The reaction mixture was heated to 60 C for 16 h. A yellow crystalline precipitate formed and was collected via gravity filtration to yield ATZ-BzTSC (3c, 2.13 mmol, 0.62 g, 79.7% yield). Ratio of major to minor was 5.3:1.0. 3c major 1H NMR (500 MHz, DMSO-d6) d 10.76 (s, 1H), 8.79 (t, J ¼ 6.2 Hz, 1H), 7.88 (d, J ¼ 3.2 Hz, 1H), 7.79 (d, J ¼ 3.2 Hz, 1H), 7.41e7.20 (m, 5H), 4.87 (d, J ¼ 6.1 Hz, 2H), 2.45 (s, 3H). 13C NMR (126 MHz, DMSO) d 178.39, 166.89, 144.50, 143.30, 139.00, 128.24, 127.20, 126.83, 122.51, 46.93, 13.79. 3c minor 1H NMR (500 MHz, DMSO-d6) d 13.38 (s, 1H), 9.37 (t, J ¼ 6.4 Hz, 1H), 8.27 (d, J ¼ 3.2 Hz, 1H), 8.10 (d, J ¼ 3.0 Hz, 1H), 7.42e7.20 (m, 5H), 4.83 (d, J ¼ 6.2 Hz, 2H), 2.45 (s, 3H). 13C NMR (126 MHz, DMSO) d 177.89, 161.80, 143.97, 138.91, 132.38, 128.19, 127.38, 126.85, 122.73, 46.86, 21.90. Theoretical MS m/z (relative intensity) for C13H13N4S2 [3c-Hþ]: 289.1 (100%). Actual MS m/z (relative intensity): 289.1 (100%). 2.1.4. ATZ-PTSC (3d) (E)eN-phenyl-2-[1-(thiazol-2-yl)-ethylidene] hydrazinecarbothioamide To a stirred solution of 2-acetylthiazole (1, 4.08 mmol, 0.5186 g) and 4-phenyl-3-thiosemicarbazide (2d, 4.08 mmol, 0.6820 g) in 50 mL of isopropanol was added one drop of sulfuric acid. The reaction mixture was heated to 60 C for 16 h. A yellow crystalline precipitate formed and was collected via gravity filtration to yield ATZ-PTSC (3d, 3.44 mmol, 0.9508 g, 84.3% yield). Ratio of major to minor was 4.8:1.0. 3d major 1H NMR (500 MHz, DMSO-d6) d 11.02
9
(s, 1H), 9.93 (s, 1H), 7.91 (d, J ¼ 3.1 Hz, 1H), 7.82 (d, J ¼ 3.1 Hz, 1H), 7.68e7.57 (m, 2H), 7.38 (t, J ¼ 7.9 Hz, 2H), 7.30e7.16 (m, 1H), 2.50 (s, 3H). 13C NMR (126 MHz, DMSO) d 176.83, 166.78, 145.10, 143.35, 138.90, 128.24, 125.41, 125.06, 122.75, 13.97. 3d minor 1H NMR (500 MHz, DMSO-d6) d 13.57 (s, 1H), 10.45 (s, 1H), 8.29 (d, J ¼ 3.3 Hz, 1H), 8.13 (d, J ¼ 3.2 Hz, 1H), 7.64e7.62 (m, 2H), 7.34e7.29 (m, 2H), 7.12 (t, J ¼ 7.5 Hz, 1H), 2.51 (s, 3H). 13C NMR (126 MHz, DMSO) d 176.34, 161.79, 144.02, 138.72, 132.81, 128.17, 125.51, 125.26, 122.96, 21.82. Theoretical MS m/z (relative intensity) for C12H11N4S2 [3d-Hþ]: 275.1 (100%). Actual MS m/z (relative intensity): 275.1 (100%). 2.1.5. ATZ-tBTSC (3e) (E)eN-tert-butyl-2-[1-(thiazol-2-yl)ethylidene]hydrazinecarbothioamide To a stirred solution of 2-acetylthiazole (1, 4.34 mmol, 0.5514 g) and 4-tertbutyl-3-thiosemicarbazide (2e, 4.29 mmol, 0.6320 g) in 50 mL of isopropanol was added one drop of sulfuric acid. The reaction mixture was heated to 60 C for 16 h. A yellow crystalline precipitate formed and was collected via gravity filtration to yield ATZ-tBTSC (3e, 3.90 mmol, 0.9995 g, 90.9% yield). Ratio of major to minor was 8.0:1.0. 3e major 1H NMR (500 MHz, DMSO-d6) d 10.62 (s, 1H), 7.88 (d, J ¼ 3.2 Hz, 1H), 7.77 (d, J ¼ 3.2 Hz, 1H), 7.71 (s, 1H), 2.42 (s, 3H), 1.54 (s, 9H). 13C NMR (126 MHz, DMSO) d 176.05, 167.16, 143.48, 142.76, 122.27, 52.78, 28.30, 13.55. 3e minor 1H NMR (500 MHz, DMSO-d6) d 13.22 (s, 1H), 8.26 (d, J ¼ 3.2 Hz, 1H), 8.10 (d, J ¼ 3.2 Hz, 1H), 7.88 (s, 1H), 2.43 (s, 3H), 1.53 (s, 9H). 13C NMR (126 MHz, DMSO) d 175.53, 161.78, 143.99, 131.66, 122.79, 52.96, 28.38, 21.98. Theoretical MS m/z (relative intensity) for C10H15N4S2 [3e-Hþ]: 255.1 (100%). Actual MS m/z (relative intensity): 255.1 (100%). 3. Results and discussion This study examines a series of thiosemicarbazones with potential anti-microbial and anti-cancer activity. Each of these thiosemicarbazones includes a conserved thiazole and central thiosemicarbazone core. To the thioamide nitrogen of each molecule is attached a moiety that varies from methyl (3a), ethyl (3b), benzyl (3c), phenyl (3d), and tert-butyl (3e) as described in Fig. 1. Acetylthiazole thiosemicarbazones (ATZ-TSC's) were synthesized in the typical manner for other thiosemicarbazone derivatives [25,26]. The reaction used to form the thiosemicarbazone was an acid catalyzed imine formation that produced the desired thiosemicarbazones in high yield and purity. Chemicals were purchased from Sigma-Aldrich and Fischer chemical companies and used without further preparation unless otherwise noted. These molecules were characterized by 1H, 13C DEPTQ-135, 1He1H COSY, 1 He13C HSQC, 1He15N HSQC, 1He13C HMBC, and 1He15N HMBC NMR techniques as well as mass spec. NMR spectroscopy was carried out at the Center for Structural Chemistry, Tennessee Technological University (USA). The spectra reported here were measured with a Bruker Avance III HD 500 spectrometer at 500.13 MHz (1H), 50.69 MHz (15N) and 125.03 MHz (13C) at 25 C equipped with a PRODIGY cryoprobe. For these measurements, the
Fig. 1. Synthesis of the series of 2-acetylthiazolethiosemicarbazones 3a-3e.
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W.R. Carroll et al. / Journal of Molecular Structure 1157 (2018) 8e13
substances were dissolved in the appropriate deuterated solvent, and the chemical shifts were referenced to the solvent residual peak. Coupling constants (J) are given in Hertz. 1H NMR experiments were acquired using Bruker's standard PROTON (zg30) NMR pulse sequence with the following parameters: Relaxation delay, 1s; 90 pulse, 12.0 ms; spectral width, 10,000 Hz; number of data points, 32 K; and digital resolution, 0.153 Hz/point. All mass spectrometry data was taken with a Varian 300/310/320-MS LC/MS Quadrupole Mass Spectrometer in negative mode using APCI. The corona current was set to 5.00 mA, while the shield potential was set to 600.00 V. The housing, drying gas, and vaporizer gas temperatures were set to 50 C, 150 C, and 350 C respectively. The drying, nebulizing, and vaporizer gas pressures were each set to 12.0 psi, 55.0 psi, and 17.0 psi. For the mass spectrometry data, each sample was dissolved in minimal amounts of dimethyl sulfoxide and then diluted to 10 ppm in methanol. In each case this thiosemicarbazone was produced and confirmed by Mass Spec; however, a second set of NMR signals with similar couplings was observed with slightly displaced chemical shifts. The relative abundance of this second set of signals varied between the different derivatives. The presence of these minor and major sets of peaks may be a result of a conformational equilibrium that may rotate to present a possible internal hydrogen bond resulting in two conformations of these molecules of varying relative abundance. The conformational equilibrium of the thiosemicarbazone family of molecules has been explored in previous studies via theoretical computational studies, variable temperature NMR, and using 1H NMR in variable solvents [28,29]. In this study we examine the 15N NMR chemical shifts, 1H NMR in variable solvents, and NOESY spectrum. To elucidate the configuration of the major and minor sets of peaks and determine if a hydrogen bond was present, the chemical shifts of each signal was assigned for the major and minor conformers separately and 2D NMR spectra of heteronuclear correlations between 1H and 13C, as well as 1H and 15N were recorded. This allowed the observation of any changes in chemical environment between the nitrogens in the major and minor set of signals. For the purposes of discussion, a letter label has been applied to each hydrogen and a number label has been applied to each carbon and nitrogen shown in Fig. 2. Numbering on the variable parts of the molecule was carried out sequentially progressing away from the thiosemicarbazone. Signals for the thiazole ring hydrogens were found as two doublets between 7.7 and 8.3 ppm in both the major and minor forms (Table 1). The signals from the minor form were shifted upfield on average 0.31 ppm for H(a) and 0.38 ppm for H(b). This is consistent with the nitrogen on the thiazole ring forming a
hydrogen bond with the neighboring thiosemicarbazone hydrazinic NeH H(d). Carbon signals for the thiazole ring carbons C(1) and C(2) appeared near 122 ppm and 143 ppm respectively (Table 2). The minor signal was shifted an average of 0.26 ppm for C(1) and 0.65 ppm for C(2) downfield. With C(2) being more closely localized next to N(1) the larger downfield shift may be expected. The thiosemicarbazone hydrazinic NeH hydrogen H(d) appeared between 10.5 and 13.6 ppm in each molecule. The minor form of H(d) was shifted downfield an average of 2.63 ppm also supporting its participation in a hydrogen bond in the minor form. Parts of these molecules that should not experience substantial changes in their chemical environment with the formation of an internal hydrogen bond such as the methyl hydrogens H(c) appeared between 2.4 ppm and 2.5 ppm. The minor signals were shifted an average of 0.01 ppm downfield reinforcing the local nature of these chemical shift changes. The only molecule previously reported in the literature from this series is the ethyl derivative 3b the crystal structure of which shows an internal hydrogen bond between the hydrazinic nitrogen N(2) and the thioamide NeH H(e) [27]. In the molecules reported here H(e) appears between 8.2 ppm and 10.5 ppm. The signal for the minor form of H(e) shifted on average 0.54 ppm downfield supporting the presence of this hydrogen bond. Thus far the minor and major forms of the series of molecules reported here can be visualized as an internally hydrogen bonded and non-hydrogen bonded configurations as shown in Fig. 3. In the thiosemicarbazone family of molecules this conformational equilibrium has been attributed to the imine E/Z conformational equilibrium [28,29]. The nature of the rotation of the thiazole ring is not apparent in the form on the right in Fig. 3 however the relative basicity of the nitrogen would suggest that the thiazole ring nitrogen would preferentially participate in a hydrogen bond over the ring sulfur in the form on the left in Fig. 3. To ascertain the exact form of the major and minor configurations of this series of thiosemicarbazones the nitrogen chemical shifts were examined. This was achieved via 1He15N HSQC and 1 He15N HMBC NMR with nitrogens N(1)eN(4) being clearly visible in all but one spectrum. The use of nitrogen chemical shift changes with internal hydrogen bonding has precedent in the investigation of hydrogen bonding within imidazoles [30,31]. This existing 15N NMR investigation into imidazoles provides an expectation for the changes in chemical shift an internal hydrogen bond to the thiazole ring would cause as well. In imidazoles it was observed that when the sp2 hybridized nitrogen of the imidazole hydrogen bonds it was shifted upfield. This was attributed to a decrease in the pyridine-like character and increase in the pyrrole-like character of the nitrogen. In this
Fig. 2. Enumeration of Hydrogens a-i, carbons 1e11, and nitrogens 1e4 in the 2-acetylthiazolethiosemicarbazones.
W.R. Carroll et al. / Journal of Molecular Structure 1157 (2018) 8e13
11
Table 1 1 H NMR data for compounds 3a-e (d in ppm; J in Hz)a. Compound
A
B
3a (major) 3a (minor) 3b (major) 3b (minor) 3c (major) 3c (minor) 3d (major) 3d (minor) 3e (major) 3e (minor)
7.80 (dd, 3.2, 0.9 Hz) 8.09 (dd, 3.3, 1.3) 7.79 (dd, 3.3, 1.5) 8.12e8.03 (m) 7.79 (d, 3.2) 8.10 (d, 3.0) 7.82 (d, 3.1) 8.13 (d, 3.2) 7.77 (d, 3.2) 8.10 (d, 3.2)
7.88 8.26 7.88 8.25 7.88 8.27 7.91 8.29 7.88 8.26
C (d, 3.2) (dd, 3.3, 1.1) (d, 3.2) (dd, 3.4, 1.2) (d, 3.2) (d, 3.2) (d, 3.1) (d, 3.3) (d, 3.2) (d, 32)
2.42 2.44 2.42 2.44 2.45 2.45 2.50 2.51 2.42 2.43
D (s) (s) (s) (s) (s) (s) (s) (s) (s) (s)
10.61 13.29 10.57 13.25 10.76 13.38 11.02 13.57 10.62 13.22
(s) (s) (s) (s) (s) (s) (s) (s) (s) (s)
E
F
G
H
8.32e8.27 (m) 8.82 (q, 3.3) 8.28 (t, 5.9) 8.83 (t, 6.2) 8.79 (t, 6.2) 9.37 (t, 6.4) 9.93 (s) 10.45 (s) 7.71 (s) 7.88 (s)
3.05 (d, 4.5) 3.02 (d, 4.6) 3.61 (dt, 12.9, 7.0) 3.59e3.55 (m) 4.87 (d, 6.1) 4.83 (d, 6.2) 7.68e7.57 (m) 7.64e7.62 (m) 1.54 (s) 1.53 (s0
1.16 (t, 7.0) 1.16e1.09 (m) 7.41e7.20 (m) 7.42e7.20 (m) 7.38 (t, 7.9) 7.34e7.29 (m)
7.30e7.16 (m) 7.12 (t, 7.5)
DMSO-d6 was used as solvent in the NMR experiments. a Multiplicity and coupling constants are given in parentheses. 1H shift assignments are in agreement with HSQC and HMBC spectra.
Table 2 13 C NMR data for compounds 3a-e (d in ppm; J in Hz)a. Compound
1
2
3
4
5
6
7
8
9
10
11
3a (major) 3a (minor) 3b (major) 3b (minor) 3c (major) 3c (minor) 3d (major) 3d (minor) 3e (major) 3e (minor)
122.42 122.59 122.39 122.59 122.51 122.73 122.75 122.96 122.27 122.79
143.24 143.95 143.28 143.95 143.3 143.97 143.35 144.02 143.48 143.99
166.98 161.81 166.97 161.81 166.89 161.8 166.78 161.79 167.16 161.78
144.03 131.84 144.04 131.91 144.5 132.38 145.1 132.81 142.76 131.66
13.68 21.88 13.68 21.88 13.79 21.9 13.97 21.82 13.55 21.98
178.52 178.03 177.5 176.98 178.39 177.89 176.83 176.34 176.05 175.53
31.32 31.07 38.65 38.62 46.93 46.86 138.9 138.72 52.78 52.96
14.28 14.3 139 138.91 125.06 125.26 28.3 28.38
127.2 127.38 128.24 128.17
128.24 128.19 125.41 125.51
126.83 126.85
DMSO-d6 was used as solvent in the NMR experiments. a Multiplicity and coupling constants are given in parentheses.13C shift assignments are in agreement with HSQC and HMBC spectra.
Fig. 3. Hydrogen bonded and non-hydrogen bonded forms of compounds 3a-3e.
system, the thiazole ring is conjugated through the attached thiosemicarbazone providing a possible route for similar changes. In this system, the thiazole nitrogen N(1) appears between 304 ppm and 312 ppm (Table 3). In the minor form the resonance shifts an average of 4.55 ppm upfield. This upfield shift is consistent with the hydrogen bond reducing the sp2 character of N(1) and increasing its sp3 character. The protonation of the thiazole ring and resulting resonance stabilized tautomer that would be one of the expected contributing resonance forms of the molecule in the hydrogen-bonded conformation. This tautomer would also effect the carbons connecting the thiazole to the thiourea as they are included in a C]C double bond instead of a C]N double bond. This can be observed in carbons C(3) and C(4) which occur between 161 ppm and 167 ppm for the signal from C(3) and between 131 ppm and 145 ppm for the signal from C(4). In each case the minor conformation was shifted an average of 5.16 ppm for the signal from C(3) and 11.97 ppm for the signal from C(4) upfield. Additional evidence of this hydrogen bonded form is apparent in the NOESY spectrum of compound 3a. In the NOESY spectrum of
compound 3a the major compound shows clear cross peaks between the hydrogens H(d) and H(c) in Fig. 2 as expected from the major form depicted on the right in Fig. 3. The minor form of hydrogen H(d) showed a cross peak with hydrogen H(b) on the
Table 3 15N NMR data for compounds 3a-d (d in ppm)a. Compound
1
2
3
4
3a (major) 3a (minor) 3b (major) 3b (minor) 3c (major) 3c (minor) 3d (major) 3d (minor) 3e (major) 3e (minor)
311.85 307.56 310.95 307.7 311.61 306.46 311.82 306.32 308.83 304.14
311.97 316.76 311.64 316.79 311.37
166.18 170.29 166.12 170.31 166.76 170.62 169.64 173.53 167.63 172.14
106.92 106.68 122.36 122.02 119.39 119.48 130.67 129.96 137.53 135.85
311.69 316.67 308.79 317.55
DMSO-d6 was used as solvent in the NMR experiments. N shift assignments are derived from HSQC and HMBC spectra.
15
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W.R. Carroll et al. / Journal of Molecular Structure 1157 (2018) 8e13
thiazole ring indicating that the minor form shows a clear organization placing the hydrogen bonding H(d) in close proximity to the thiazole ring. This signal and the other NH peaks showed exchange peaks with residual water in DMSO. EXSY cross peaks indicating the slow equilibration of these two conformers at room temperature were observed between the major and minor conformers for each resolved peak. With the internally hydrogen bonded and non-hydrogen bonded forms identified the relative abundance of each conformer may help establish their relative stability in solution. In each case the ratio between the major and minor conformers was 4.8:1.0 to 5.4:1.0 which is characteristic of an energy difference of 0.93e1.00 kcal/mol at 25 C. These measurements were repeated in chloroform and showed a difference of 11.6:1.0 to 0.5:1.0 which indicates a difference of energy between 1.5 and 0.4 kcal/mol at 25 C. Notably these molecules were much less soluble in chloroform and so these measurements may be skewed. Such measurements are established in a variety of context to determine the strength of non-covalent interactions under carefully controlled conditions [32]. While it may be tempting to attribute this difference in energy exclusively to the strength of the hydrogen bond it is worth noting that differences in energy between two conformers may often be dependent on many other factors such as the relative solubility of each form [33]. The complex nature of the forces that contribute to the equilibrium populations of these conformations of these molecules makes it difficult to isolate the cause of any change in population to a single phenomenon however comparison of the steric and electronic effects of the different substituents may provide some insight. The ratios of major to minor forms in DMSO and chloroform were determined and included in the supplementary material. In DMSO no trend was observed between these ratios and their substituents steric or electronic properties. In chloroform these values show a correlation with more sterically bulky groups favoring the hydrogen bonded form. Electronic effects showed no correlation in either solvent's data. 4. Conclusions With the interpretation of the information provided here we can not only establish the identity and purity of a series of thiazole derivatized thiosemicarbazones but we can identify the configurations they exhibit in solution as both internally hydrogen bonded and non-hydrogen bonded. The knowledge of these two forms and their identities may be of use to those seeking to employ these a-(N)-heterocyclic thiosemicarbazones molecules in a pharmaceutical context. The exact biological activity of each conformation may be challenging to isolate as they interconvert readily but the population of each configuration is certainly effected by the solvent system. Acknowledgements We would like to thank the National Science Foundation for funding the purchase of the FT-NMR used in this research, (National Science Foundation (NSF) Major Research Instrument (MRI 1531870) and the URECA! Grant Program at TTU for awarding funds to Ms. A. Buckner to buy necessary supplies and funds to present portions of this research at the April 2017 ACS national meeting in San Francisco, CA. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.molstruc.2017.11.061.
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