M. Markowicz-Piasecka, et al.
BioorganicChemistryxxx(xxxx)xxxx
(C10H14FN5O2S *0.10 EtOH): C, 42.38; H, 5.13; N, 23.76; Found: C,
+H)+: Calcd 304.78, Found 304.20. Anal. Calcd for (C10H14ClN5O2S
*0.10 MeOH): C, 39.52; H, 4.73; N, 22.81; Found: C, 39.92; H, 4.67; N,
22.49.
42.59; H, 4.93; N, 23.82.
2.2.9. N1,N1-Dimethyl-N4-(4-fluorobenzenesulfonamide)-biguanidine (9)
Compound 9 was prepared from 4-fluorobenzenesulfonyl chloride
to yield off-white solid, 0.35 g, (63%). 1H NMR ((CD3)2SO): δ ppm 8.01
(bs, 2H), 7.85–7.80 (m, 2H), 7.36–7.31 (m, 2H), 6.94 (bs, 1H), 6.72 (bs,
2.2.3. N1,N1-Dimethyl-N4-(4-chlorobenzenesulfonamide)-biguanidine (3)
Compound 3 was prepared from 4-chlorobenzenesulfonyl chloride
to yield off-white solid, 0.34 g, (58%). 1H NMR ((CD3)2SO): δ ppm 8.00
(bs, 2H), 7.75 (d, J = 8.7 Hz, 2H), 7.58 (d, J = 8.7 Hz, 2H), 6.99 (bs,
1H), 6.69 (bs, 1H), 2.91 (s, 6H); 13C NMR ((CD3)2SO): δ ppm 159.71,
158.36, 143.11, 136.04, 128.88 (2C), 127.69 (2C), 36.55 (2C). MS
(ESI+) for C10H15ClN5O2S (M+H)+: Calcd 304.78, Found 304.16.
Anal. Calcd for (C10H14ClN5O2S*0.1 (CH3)2CO): C, 39.96; H, 4.69; N,
22.62; Found: C, 40.13; H, 4.21; N, 23.07.
1H), 2.91 (s, 6H); 13C NMR ((CD3)2SO):
δ ppm 163.43 (d,
J = 249.9 Hz), 159.72, 158.35, 140.70 (d, J = 3.3 Hz), 128.53 (d,
J = 9.4 Hz, 2C), 115.82 (d, J = 22.2 Hz, 2C), 36.51 (2C). MS (ESI+) for
C
10H15FN5O2S (M+H)+: Calcd 288.32, Found 288.10. Anal. Calcd for
(C10H14FN5O2S *0.10 EtOH): C, 42.38; H, 5.13; N, 23.76; Found: C,
42.64; H, 5.11; N, 23.42.
2.2.4. N1,N1-Dimethyl-N4-(2-bromobenzenesulfonamide)-biguanidine (4)
Compound 4 was prepared from 2-bromobenzenesulfonyl chloride
to yield off-white solid, 0.50 g, (74%). 1H NMR ((CD3)2SO): δ ppm 8.10
(bs, 2H), 8.06 (dd, J = 7.7; 1.6 Hz, 1H), 7.77 (dd, J = 8.0; 0.9 Hz, 1H),
7.52 (dt, J = 7.7; 0.8 Hz, 1H), 7.45 (dt, J = 7.8; 1.6 Hz, 1H), 6.97 (bs,
1H), 6.68 (bs, 1H), 2.92 (s, 6H); 13C NMR ((CD3)2SO): δ ppm 159.42,
158.28, 140.64, 134.97, 132.84, 129.44, 127.63, 119.26, 36.51 (2C).
MS (ESI+) for C10H15BrN5O2S (M+H)+: Calcd 349.23, Found 348.00;
350,00. Anal. Calcd for (C10H14BrN5O2S *0.65 MeOH): C, 34.66; H,
4.53; N, 18.98; Found: C, 35.11; H, 4.33; N, 18.72.
2.3. Biological material for stability studies
Rat and mouse liver and brain S9 fraction were prepared by col-
lecting fresh tissues from the animals in compliance with the European
Commission Directives 2010/63/EU and 86/609 and approved by the
Institutional Animal Care and Use Committee of University of Eastern
Finland. The liver and brain homogenates were prepared by homo-
genizing freshly-collected rat or mouse liver or brain with 50 mM Tris-
buffered saline (TBS) (pH 7.4) (1:4 w/v). The homogenates were cen-
trifuged at 9 000 rpm for 20 min at 4 °C and the supernatant was col-
lected. Protein concentrations of both fractions were determined by
Bio-Rad Protein Assay, based on Bradford dye-binding method
(EnVision, Perkin Elmer, Waltham, MA, USA). All biological material
was stored at −80 °C until used.
2.2.5. N1,N1-Dimethyl-N4-(3-bromobenzenesulfonamide)-biguanidine (5)
Compound 5 was prepared from 3-bromobenzenesulfonyl chloride
to yield off-white solid, 0.59 g, (88%). 1H NMR ((CD3)2SO): δ ppm 7.97
(bs, 2H), 7.90 (s, 1H), 7.79–7.74 (m, 2H), 7.48 (t, J = 7.9 Hz, 1H), 6.91
(bs, 2H), 2.92 (s, 6H); 13C NMR ((CD3)2SO): δ ppm 159.64, 158.35,
2.4. High-performance liquid chromatography (HPLC) analyses
146.32, 134.11, 131.15, 128.16, 124.79, 121.65, 36.55 (2C). MS (ESI+
)
for C10H15BrN5O2S (M+H)+: Calcd 349.23, Found 348.00; 350,00.
Anal. Calcd for (C10H14BrN5O2S *0.1 Et2O): C, 35.12; H, 4.25; N, 19.69;
Found: C, 35.26; H, 4.08; N, 19.36.
Concentrations of metformin derivatives were determined by HPLC.
The apparatus was formed of an Agilent 1100 binary pump (Agilent
Technologies Inc., Wilmington, DE, USA), a 1100 micro vacuum de-
gasser, a HP 1050 Autosampler, a HP 1050 variable wavelength de-
tector, operated at 235 nm. The chromatographic separations were
achieved on an Agilent Zorbax SB-C18 analytical column
(4.6 mm × 150 mm, 5 μm) (Agilent Technologies Inc., Wilmington, DE,
USA) by using isocratic elution of water containing 0.1% formic acid
(pH ca. 3.0) and acetonitrile containing 0.1% formic acid with a ratio of
75:25 (v/v) for compounds 1, 4, 7–9 and with a ratio of 70:30 (v/v) for
compounds 2, 3, 5 and 6. The retention times of the compounds were
ca. 2.8–3.9 min at the flow rate of 1.0 ml/min at room temperature. The
lower limit of quantification for the compounds was 0.5 μM. These
HPLC methods were also accurate, precise and selective over the range
1–100 μM.
2.2.6. N1,N1-Dimethyl-N4-(4-bromobenzenesulfonamide)-biguanidine (6)
Compound 6 was prepared from 4-bromobenzenesulfonyl chloride
to yield off-white solid, 0.46 g, (68%). 1H NMR ((CD3)2SO): δ ppm 7.98
(bs, 2H), 7.74–7.68 (m, 4H), 6.99 (bs, 1H), 6.69 (bs, 1H), 2.91 (s, 6H);
13C NMR ((CD3)2SO): δ ppm 159.70, 158.37, 143.52, 131.82 (2C),
127.84 (2C), 124.90, 36.55 (2C). MS (ESI+) for C10H15BrN5O2S (M
+H)+
: Calcd 349.23, Found 348;14; 350.14. Anal. Calcd for
(C10H14BrN5O2S*0.1 (CH3)2CO): C, 34.94; H, 4.10; N, 19.78; Found: C,
35.02; H, 3.74; N, 19.59.
2.2.7. N1,N1-Dimethyl-N4-(2-fluorobenzenesulfonamide)-biguanidine (7)
Compound 7 was prepared from 2-fluorobenzenesulfonyl chloride
to yield off-white solid, 0.40 g, (72%). 1H NMR ((CD3)2SO): δ ppm 8.03
(bs, 2H), 7.82 (dt, J = 7.6; 1.5 Hz, 1H), 7.62–7.58 (m, 1H), 7.36–7.29
(m, 2H), 7.00 (bs, 1H), 6.70 (bs, 1H), 2.92 (s, 6H); 13C NMR ((CD3)2SO):
2.5. Stabilities of metformin sulfonamide derivatives
Enzymatic and chemical stabilities of metformin sulfonamide deri-
vatives in rat liver and brain S9 fractions, in human plasma or in TBS
buffer (pH 7.4) were determined at 37 °C. Stability in 0.1 M NaOH was
determined at RT. The incubation mixtures were prepared by mixing
liver or brain S9 fraction (final protein concentration 1.0 mg/mL) with
TBS buffer (pH 7.4) and 10 mmol/L stock solution of studied com-
pounds in DMSO (the final concentration of compounds were
100 µmol/L and the DMSO concentration 2%). The mixtures were in-
cubated for six hours and the samples (100 µL) were withdrawn at
appropriate intervals. The proteins in the samples were precipitated
with ice-cold acetonitrile (100 µL) and the samples were centrifuged for
5 min at 12 000 rpm at room temperature. The supernatants were
collected and analyzed by the HPLC method described above. In che-
mical stability study with TBS buffer, the S9 fractions were replaced
with same volume of buffer and in study with 0.1 M NaOH, the reaction
mixture contained only 0.1 M NaOH and 10 mmol/L studied com-
pound.
δ
ppm 159.16, 158.97 (d, J = 142.1 Hz), 157.16, 133.94 (d,
J = 8.3 Hz), 131.64 (d, J = 15.1 Hz), 128.69, 124.24 (d, J = 3.6 Hz),
117.02 (d, J = 21.6 Hz), 36.52 (2C). MS (ESI+) for C10H15FN5O2S (M
+H)+: Calcd 288.32, Found 288.10. Anal. Calcd for (C10H14FN5O2S
*0.15 EtOH *0.05 MeOH): C, 42.63; H, 5.27; N, 23.34; Found: C, 42.82;
H, 5.07; N, 23.64.
2.2.8. N1,N1-Dimethyl-N4-(3-fluorobenzenesulfonamide)-biguanidine (8)
Compound 8 was prepared from 3-fluorobenzenesulfonyl chloride
to yield off-white solid, 0.19 g, (34%). 1H NMR ((CD3)2SO): δ ppm 7.98
(bs, 2H), 7.60 (t, J = 7.7 Hz, 1H), 7.58–7.54 (m, 2H), 7.43–7.38 (m,
1H), 6.97 (bs, 1H), 6.76 (bs, 1H), 2.92 (s, 6H); 13C NMR ((CD3)2SO): δ
ppm 157.98 (d,
J = 244.52 Hz), 159.67, 158.36, 146.46 (d,
J = 6.3 Hz), 131.14 (d, J = 7.6 Hz), 121.92 (d, J = 2.7 Hz), 118.38 (d,
J = 21.3 Hz), 112.84 (d, J = 23.8 Hz), 36.53 (2C). MS (ESI+) for
C
10H15FN5O2S (M+H)+: Calcd 288.32, Found 288.10. Anal. Calcd for
3