K. Chennakesava Rao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3057–3063
3063
13. Cervinka, O.; Hilbert, O.; Struzka, V.; Svatos, A.; Vodnansky, M.; Jakal, V. L-
Ephedrine 1986, Czech CS233442.
dilutions that were added to each medium in 96 well plates. An inoculums of
100 from each well was inoculated. The antifungal agents ketoconazole for
fungi and Streptomycin for bacteria were included in the assays as positive
controls. For fungi, the plates were incubated for 48–72 h at 28 °C and for
bacteria the plates were incubated for 24 h at 37 °C. The MIC for fungi was
defined as the lowest extract concentration, showing no visible fungal growth
after incubation time. 5 lL of tested broth was placed on the sterile MHA plates
for bacteria and incubated at respective temperature. The MIC for bacteria was
determined as the lowest concentration of the compound inhibiting the visual
growth of the test cultures on the agar plate.
(c) Minimum bacterial concentration (MBC) study: Freshly prepared tubes
containing serial twofold dilutions of synthesized compounds in 5 mL of MHB
15. Prakasam, T.; Srinivasan, P.S.; Bhanumathi, A.; Ramana, D.V.; Hiteshkumar, B.N.
IN 249376 Oct 21, 2011.
(range, 1000
l
g/mL, 500
l
g/mL, 250
lg/mL, 125 lg/mL, 62.5 lg/mL, 31.25 lg/
mL, 15.62 g/mL and 7.81
l
lg/mL) were inoculated beneath the surface with
5 ꢁ 105 to 1 ꢁ 106 cells in 0.1 mL of MHB, mixed by flushing’s and incubated
without shaking or agitation. After 20 h of incubation, all broths were
examined for visual turbidity or growth of small colonies on the bottom of
tubes and again vortexed. The tubes were re-incubated for a further 4 h and
vortexed again and all tubes without visual turbidity. The MBC was considered
the lowest concentration of synthesized compounds which prevented growth
and reduced the inoculum by P99.9% within 24 h, irrespective of counts of
survivors at higher antibiotic concentrations and the lowest concentration of
the compound inhibiting the visual growth of the test cultures on the agar
plate. For fungi, the plates were incubated for 48–72 h at 28 °C and for bacteria
the plates were incubated for 24 h at 37 °C.
16. See the Supplementary data.
24. General procedure for the preparation of compounds 1a–e and 1a0–e0:
Phenylpropanolamine 2 (53 mmol), unsymmetrical ketone 13 (55 mmol) and
p-toluenesulfonic acid (5 mmol) were refluxed with toluene (50 mL) for about
16 h. The formed water in the reactions is removed using Dean-Stark apparatus
and the progress of the reaction was monitored by TLC (EtOAc/hexane, 3:7).
After completion of the reaction, mass was cooled and washed with 5% sodium
bicarbonate solution followed by water. Toluene was distilled off completely
under reduced pressure to yield the Schiff’s base 14 as thick syrup. This Schiff’s
base 14 was used immediately for the reductive amination due to its poor
stability. The Schiff’s base 14 was taken in a Paar hydrogenator flask along with
isopropyl alcohol (50 mL) and 10% palladium on carbon catalyst (0.5 g) and
cooled to about 5 °C. The Paar hydrogenator was shaken at 40–50 psi pressure
23. (a) Disc diffusion method: Antimicrobial activities were carried out using disc
diffusion method. Petri plates were prepared with 20 mL of sterile Mueller
Hinton Agar(MHA) (Hi-media, Mumbai). The test cultures were swabbed on
the top of the solidified media and allowed to dry for 10 min and a specific
amount of synthesized b-aminoalcohol 1 at 1 mg/disc was added to each disc
separately. The loaded discs were placed on the surface of the medium and left
for 30 min at room temperature for compound diffusion. Negative control was
prepared using respective solvents. Streptomycin was used as positive control
against bacteria. Ketoconazole was used as positive control of fungi. The plates
were incubated for at 37 °C for 24 h and for bacteria and at 28 °C for 48 h for
fungi. Zones of inhibition were recorded in mm and experiment was repeated
twice. Bacterial inoculums were prepared by growing cells in Mueller Hinton
broth (MHB) (Himedia) for 24 h at 37 °C. The filamentous fungi were grown on
sabouraud dextrose agar (SDA) slants at 28 °C for 10 days and the spores were
collected using sterile doubled distilled water and homogenized. Yeast was
grown on sabouraud dextrose broth (SDB) at 28 °C for 48 h.
of hydrogen gas and
a temperature between 0 °C and 10 °C till the
consumption of hydrogen ceases. Progress of the reaction was monitored by
using TLC (EtOAc/hexane, 3:7). After completion of the reaction, reaction mass
was filtered and the filtrate was treated with dry HCl gas to get white solid
which was filtered and dried under vacuum at 80 °C afford pure b-
aminoalcohols 1a–e and 1a0–e0 as hydrochloride salts. (1R,2S,10R)-(ꢀ)-2-(10-
Phenylethylamino)-1-phenyl-1-propanol HCl 1a: white solid; mp 198–200 °C;
yield 85%; purity by HPLC 99.6%; ee = 100%; specific optical rotation ꢀ39.2°
(c = 1.0, CH3OH @ 25 °C); IR (KBr) (cmꢀ1): 3362, 2968, 2831, 2500, 1589, 1454,
1213, 766, 704; 1H NMR (300 MHz, DMSO-d6) dH: 0.94 (d, 3H, J = 3.9 Hz),1.70
(d, 3H, d, J = 3.9 Hz), 2.86 (q, 1H), 4.53 (m, 1H), 5.25 (br s, 1H), 6.20 (d, 1H,
J = 2.4 Hz), 7.17–7.80 (m, 10H), 9.37 & 9.73 (br s, 2H); 13C NMR (75 MHz,
DMSO-d6) dC: 8.47, 20.58, 55.19, 56.88, 70.45, 125.63–141.18; ESI-MS m/
z = 256 [M+H]+. Anal. Calcd for C17H22ClNO: C, 69.97; H, 7.60; N, 4.80. Found: C,
70.03; H, 7.57; N, 4.77.
(b) Minimum inhibition concentration (MIC) study: The minimum amount of a
drug required to inhibit the growth of bacteria in vitro method is called as
minimum inhibitory concentration (MIC). The MIC studies of the prepared
chiral-aminoalcohols 1 were performed according to the standard reference
methods for bacteria9 for filamentous fungi (CLSI, 2008) and yeasts (NCCLS/
CLSI, 2002) and significant values were observed against bacteria and fungi
(Table 6). The required concentrations (1000
125 g/mL, 62.5 g/mL, 31.25 g/mL, 15.62
compound were dissolved in DMSO (2%) and diluted to give serial two-fold
l
g/mL, 500
l
g/mL, 250
lg/mL,
l
l
l
l
g/mL and 7.81
lg/mL) of the