Bioorganic & Medicinal Chemistry Letters 24 (2014) 3057–3063

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Synthesis, antimicrobial and molecular docking studies of enantiomerically pure N-alkylated b-amino alcohols from phenylpropanolamines K. Chennakesava Rao a,b,c, Y. Arun b, K. Easwaramoorthi a, C. Balachandran c, T. Prakasam a, T. Eswara Yuvaraj d,⇑, P. T. Perumal b,⇑ a

Malladi Drugs & Pharmaceuticals Limited, Research & Development Centre, Chennai 600 124, TN, India Organic Chemistry Division, CSIR-Central Leather Research Institute, Chennai 600 020, TN, India c Division of Microbiology and Cancer Biology, Entomology Research Institute, Loyola College, Chennai 600 034, TN, India d P.G. & Research Department of Chemistry, The New College (Autonomous), Chennai 600 014, TN, India b

a r t i c l e

i n f o

Article history: Received 4 January 2014 Revised 23 April 2014 Accepted 10 May 2014 Available online 20 May 2014 Keywords: b-Amino alcohols Phenylpropanolamine Reductive amination Antimicrobial activity Disc diffusion method DNA topoisomerase IV

a b s t r a c t Enantiomerically pure N-alkylated b-amino alcohols 1a, 1a0 , 1c, 1c0 , 1d, 1d0 , 1e and 1e0 , with ee 100% have been synthesized from phenylpropanolamines 2. Effect of the neighboring chiral environment on the newly formed chiral center has been studied experimentally and concluded that the newly formed chiral center’s absolute configuration is opposite to the adjacent (a- or b-) chiral environment. The antimicrobial activity of the synthesized b-amino alcohols were screened using in vitro disc diffusion method and variable antimicrobial activities were shown for 1a, 1a0 , 1c, 1c0 , 1d, 1d0 , 1e & 1e0 and amongst them 1d & 1d0 exhibited significant activity against bacteria and fungi. In silico studies revealed all the synthesized b-amino alcohols 1a–e and 1a0 –e0 have shown good binding energies ranging from 7.38 to 6.09 kJ/mol towards the target receptor DNA topoisomerase IV and 1d0 has shown maximum binding energy 7.38 kJ/mol. Ó 2014 Elsevier Ltd. All rights reserved.

Microorganisms can affect the quality of water, air and food which results in sickness of humans, animals and may spread diseases and infections. Antimicrobial agents are chemical substances which kill or prevent the growth of microorganisms such as prokaryotes (bacteria and archaea) and eukaryotes (fungi, algae, protozoa, etc.). Hence a plethora of substances with antimicrobial activity are needed due to the proliferation of toxic microorganisms. Applications of substances with antimicrobial activity have been well known since ancient period. Ancient Greeks and Egyptians have used a few plant extracts as antimicrobials to treat infections.1 Uses of substances with antimicrobial activity like turmeric have a history of more than 2000 years.2 There are many types of natural or synthetic anti-microbial agents are available to treat the diseases caused by microorganisms. b-Amino alcohols have been used as intermediates and chiral auxiliaries in organic synthesis.3 Many b-amino alcohols exhibit a broad spectrum of biological activities4 and some of them are used as active pharmaceutical ingredients (API’s). b-Amino alcohols such as norephedrine (phenylpropanolamine 2) and its isomers;

⇑ Corresponding authors. Tel.: +91 44 2491 3289; fax: +91 44 2491 1589. E-mail address: [email protected] (P.T. Perumal). http://dx.doi.org/10.1016/j.bmcl.2014.05.027 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

ephedrine 3 and its isomers; N-methylephedrine 4, etafedrine 5, phenylephrine 6, etc. act as a- and/or b-adrenergic agonists5 (Fig. 1) and well known for their decongestant, bronchodilatory, anorexic, analeptic activities,6 etc. Another category of b-amino alcohols such as betaxalol 7, nadolol 8, pindolol 9, propanolol 10, timolol 11, etc. show anti-hypertensive activity by blocking the a- and/or b-adrenergic receptors6,7 (Fig. 2). Ethambutol 12 and its analogues which are bis-(b-amino alcohol) found to be antibacterial and tuberculostatic agents.8 Various classification of amino alcohols have been examined for their antimicrobial and antifungal activities. Another noteworthy fact is that the synthesis of chiral N-alkylated b-amino alcohols 1 with 100% enantiomeric excess has been documented in the current research work. The phenylpropanolamines 2 used for the synthesis of b-amino alcohols 1 also show decongestant and anorexic activity.9 Hence, the synthesis of enantiomerically pure N-alkylated b-amino alcohols 1 has been accomplished in this work in order to explore their antimicrobial activity against bacteria & fungi. Molecular docking studies have been done against DNA topoisomerase IV for these chiral b-amino alcohols 1. Phenylpropanolamines [(1R,2S)-()-1-phenyl-2-amino-1-propanol 2a and (1S,2R)-(+)-1-phenyl-2-amino-1-propanol 2a0 ] were

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OH

condenses with 2a to give the Schiff’s base 14a and it exists in dynamic equilibrium with its tautomer oxazolidine 15a through inter molecular hydrogen bonding.10–12 During hydrogenation in presence of palladium, hydrogen adds to the imine double bond at low temperatures in syn fashion from the least hindered side to form the N-alkylated b-amino alcohol 1a. In hydrogenation reaction a new chiral center has been generated at the unsymmetrical keto-carbon in the b-amino alcohol 1a. Based on analogy,13,14 the absolute configuration of newly formed chiral center in b-Amino alcohol 1a is assigned as ‘R’ in the case of (1R,2S)-()-1-phenyl-2-amino-1-propanol 2a and ‘S’ in the case of (1S,2R)-(+)-1-phenyl-2-amino-1-propanol 2a0 as shown in Figure 3. The enantiomeric nature of 1a and 1a0 are confirmed by their specific optical rotations which are almost numerically equal but opposite in sign (Table 1). The absolute configuration of 1a was corroborated by the fact that the b-aminoalcohol 1a was prepared through another synthetic route15 (Scheme 3). Condensation reaction performed between (R)-()-phenylacetylcarbinol 16 and (R)-(+)-1-phenylethylamine 17 to form the Schiff’s base 18 followed by asymmetric hydrogenation with palladium catalyst yielded 2-(10 -phenylethylamino)-1-phenylpropanol hydrochloride(b-aminoalcohol 1a). Based on analogy13,14 the newly formed chiral center in 1a is assigned ‘S’ configuration. The specific optical rotations of b-amino alcohol 1a prepared by two different methods (Schemes 1 and 3) were found to be almost numerically equal in value and sign (Table 1). Hence, it is inferred from Tables 1 and 2 that the newly formed chiral center’s (keto-carbon) absolute configuration is opposite to the configuration of the already existing chiral center at a- or bto the new chiral carbon. In the present work ten chiral N-alkylated b-amino alcohols 1a– e & 1a0 –e0 were synthesized by the treatment of 2 with five different unsymmetrical ketones 13 and the analytical results are tabulated in Tables 3 and 4. The b-Amino alcohols 1a, a0 , c, c0 , d, d0 , e & e0 obtained after hydrogenation reaction as free base have different enantiomeric excesses. The % ee of free bases ranges from 94 to 99% ee. On isolation of these b-amino alcohols 1a, a0 , c, c0 , d, d0 , e & e0 as hydrochloride salts, the % ee was found to be 100%. But the b-amino alcohols 1b & b0 obtained are mixture of two diastereomers (1S,2R,20 S & 1S,2R,20 R) and (1R,2S,20 R & 1R,2S,20 S), respectively, as indicated by the chiral purity by HPLC even after their isolation as hydrochloride salts. The formation of the diastereomeric mixture may be due to the lower degree of steric hindrance between the two alkyl groups (methyl & ethyl) of the ketone 13b. The structures of the b-amino alcohols 1 were elucidated16 with the help of analytical techniques such as IR, 1H NMR, 13C NMR,

(1R,2S) d-norephedrine (1S,2R) l-norephedrine (1R,2R) d-norpsuedoephedrine (1S,2S) l-norpsuedoephedrine Norephedrine (Phenylpropanolamine 2 ) NH2

OH

(1R,2S) d-ephedrine (1S,2R) l-ephedrine (1R,2R) d-psuedoephedrine (1S,2S) l-psuedoephedrine

NH

Ephedrine 3 OH

OH

OH N

HO

N

Etafedrine 4

NH

Phenylepherine 6

N-Methylephedrine 5

Figure 1. a- and/or b-adrenergic agonist agents.

OH O

OH

H N

O

O

OH HO

O

HO

Pindolol 10

HN

Betaxolol 7

H N

O H N

N N S

Nadolol 8

N

OH H N O

Timolol 11 OH O

H N HO

Propanolol 9

H N

OH N H Ethambutol 12

Figure 2. a- and/or b- adrenergic receptors.

treated with various unsymmetrical ketones 13 in presence of an acid to form the corresponding Schiff’s base 14 followed by asymmetric hydrogenation at 0–10 °C with palladium on carbon yielded the corresponding N-alkylated b-amino alcohols 1 (Scheme 1). A possible reaction mechanism for the formation of b-aminoalcohol 1a is mentioned in Scheme 2. Protonated acetophenone 13a OH (R)

(S)

NH2

R1

O

(R)

R2 2a

(S)

R1

O

2a'

13

N

R R2

1. Pd/C, H2 , 0-10 °C, 8-12 h

(R)

(R)

(S)

OH

110 °C, 16-24 h

R2 14a'-e'

.HCl

1a-e 100% ee: 1a ,1c, 1d & 1e

R1

N

H N (R) R2 R1

2. HCl

OH PTSA/Toluene (S)

R2

1

14a-e

13

NH2

(S)

110 °C, 16-24 h

OH (R)

OH

OH PTSA/Toluene

1. Pd/C, H2 , 0-10 °C, 8-12 h 2. HCl

(S)

(R)

H N (S) R2

R1 1a'-e'

.HCl

100% ee: 1a ', 1c', 1d' & 1e' 1a & a'; R 1 = methyl, R 2 = phenyl 1 2 1c & c'; R = methyl, R = isobutyl 1d & d'; R 1 = ethyl, R 2 = phenyl 1e & e'; R 1 = methyl, R 2 = p-methoxyphenyl

Scheme 1. Synthesis of N-alkylated b-aminoalcohols 1 from phenylpropanolamines.

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OH

OH (R)

(S)

OH

NH 2

H+

(R)

(S)

H N

OH 2a

OH2

H+ -H2 O

13a

OH (R)

(S)

H N

(R)

(S)

N

14a

(R) (R)

O

H

O

(S)

H H N (R)

Pd/C

(R)

H (S)

HH

N

0 to 10 °C syn addition

1a

15a Scheme 2. Plausible mechanism for the formation of chiral b-amino alcohol 1a.

OH (R)

(S)

H N

OH (R)

(S)

.HCl 1a (1R,2S,1'R)-(-)-2-(1'-Phenylethylamino)-1-phenyl-1-propanol.HCl

(R)

H N

(S)

.HCl 1a' (1S,2R,1'S)-(+)-2-(1'-Phenylethylamino)-1-phenyl-1-propanol.HCl

Figure 3. Structures of b-amino alcohols 1a and 1a0 .

Mass spectral data and elemental analysis as illustrated for compound 1a. In the IR spectrum, the broad bands at 3362 cm1, 2968 cm1 and 2831 cm1 correspond to –OH, aromatic C–H and aliphatic C–H stretchings, respectively, the weak band at 2500 cm1 corresponds to –NH2+ stretching, the medium bands appearing at 1589 cm1 and 1454 cm1 correspond to benzenoid bands of phenyl rings, the short band at 1213 cm1 accounts for the C–O stretching, the medium bands at 766 cm1 and 704 cm1 represent the C–H out of plane bending of mono-substituted benzene ring of b-amino alcohol 1a. In 1H NMR spectrum, the broad signals at d: 9.73 & 9.37 ppm confirmed the presence of two – NH2+ protons, the signals between d: 7.80–7.17 ppm shows ten aromatic protons, the doublet signal at d: 6.20 ppm shows the presence of one –CH proton in-between hydroxy and phenyl groups, the broad signal at d: 5.25 ppm confirms the presence of one –OH proton, the multiplet signal at d: 4.53 ppm accounts for

Table 1 Specific optical rotation of 1a & 1a0

* **

Compound

Absolute configuration of b-aminoalcohol 1

[a]25 D (c = 1% methanol)

1a* 1a0 * 1a**

(1R,2S,10 R)(1S,2R,10 S)(1R,2S,10 R)-

39.2° +39.5° 40.0°

Prepared as per Scheme 1. Prepared as per Scheme 3.

OH (R)

O

H 2N

(R)

PTSA Toluene, 110 °C

16

17

the presence of –CH proton in-between amino and phenyl groups, another quintet signal at d: 2.86 ppm indicates the presence of –CH proton in-between amino and methyl groups and the doublet signals at d: 1.70 & 0.94 shows the presence of six protons of two methyl groups of b-amino alcohol 1a. In 13C NMR spectrum, the signals present in the range of d: 141.18–125.63 ppm shows the presence of aromatic carbons, the signal at d: 70.45, 56.88 & 55.19 ppm confirms the presence of three –CH carbons adjacent to hydroxy, amino and phenyl groups, respectively, and the signals at d: 20.58 & 8.47 ppm indicates the presence of two methyl groups of b-amino alcohol 1a. A diagnostic peak observed at m/z: 256 in the mass spectrum of b-amino alcohol 1a corresponds to the protonated molecular ion [M+H]+. The structure deduced from X-ray crystallographic study of the single crystal of the b-amino alcohol 1c0 is unambiguously confirms the structure that derived from spectroscopic data (Fig. 4). Crystal data, data collection and refinement parameters of b-amino alcohol 1c0 are given in Table 5. The synthesized chiral b-amino alcohols 1a–e & 1a0 –e0 were screened for the antimicrobial activity at 1 mg/disc using in vitro disc diffusion method,17 minimum inhibitory concentration18 (MIC) study at 1 lg/mL and minimum bacterial concentration19 (MBC) study at 1 lg/mL against eleven bacteria and two fungi and the results are tabulated in Tables 5–7. The results revealed that chiral b-amino alcohols 1a,c–e & 1a0 ,c0 –e0 have shown good antimicrobial activity and significant MIC values against standard streptomycin and ketoconazole, These b-amino alcohols 1 also have shown good MBC values against eleven bacteria and two fungi. But the b-amino alcohols 1b & 1b0 showed very poor activity against bacteria and fungi, due to the presence of mixture of diastereomers as discussed above. The reason for the potential antimicrobial activity of b-amino alcohols 1d & 1d0 may be the ‘p-methoxy’ substitution which enhances the in vitro activity against gram-positive results in the potential antimicrobial potency20 when compare with other b-amino alcohols. The following bacteria and fungi were used for the experiment. Bacteria: Bacillus subtilis MTCC 441, Micrococcus luteus MTCC 106, Enterobacter aerogenes MTCC 111, Staphylococcus aureus MTCC 96, Salmonella typhimurium MTCC 1251, Klebsiella pneumoniae MTCC 109, Proteus

OH (R)

(E) N (R)

1. Pd/C, H 2, 0 to 10 °C, 6 h 2. HCl, ether

18 Scheme 3. Synthesis of b-aminoalcohol 1a from R-()-phenylacetylcarbinol 16.

OH (R)

(S)

H N

(R)

.HCl 1a

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Table 2 Comparison of absolute configurations of 14a, 14a0 & 18 and 1a & 1a0 Compound

Absolute configuration of Schiff’s bases 14 & 18 at carbon C1

*

14a 14a0 * 18**

C2

R S R

S R —

Compound

C1

0

*

— — R

1a 1a0 * 1a**

Absolute configuration of b-amino alcohol 1 at carbon C1

C2

C10

R S R

S R S

R S R

Bold letters indicates compounds and newly formed chiral centres. Prepared as per Scheme 1. Prepared as per Scheme 3.

*

**

Table 3 (1R,2S,10 R)-b-Amino alcohols (1a–e) from 2a from Scheme 1 Compound

R1

R2

[a]25 D (c = 1%, methanol)

Mp (°C)

HPLC (area%)

ee (%)

Yield (%)

1a 1b 1c 1d 1e 1a**

Methyl Methyl Methyl Ethyl Methyl Methyl

Phenyl Ethyl iso-Butyl Phenyl 4-Methoxyphenyl Phenyl

39.2° 28.2° 31.8° 22.5° 38.9° 40.0°

198–200 197–199 225–227 193–195 172–176 198–200

99.6 81.0 97.4 99.0 99.6 99.4

100 # 100 100 100 100

85 74 90 74 80 76

# mixture of diastereomer. Prepared from Scheme 3.

**

Table 4 (1S,2R,10 S)-b-Amino alcohols (1a0 –e0 ) from 2a0 from Scheme 1 Compound

R1

R2

[a]25 D (c = 1% methanol)

Mp (°C)

HPLC (area%)

ee (%)

Yield (%)

1a0 1b0 1c0 1d0 1e0

Methyl Methyl Methyl Ethyl Methyl

Phenyl Ethyl iso-Butyl Phenyl 4-Methoxyphenyl

+39.5° +32.8° +33.6° +23.1° +37.5°

200–202 204–206 228–230 197–200 175–177

99.9 88.0 99.6 99.0 99.9

100 # 100 100 100

78 78 68 80 65

# mixture of diastereomer.

vulgaris MTCC 1771, Staphylococcus aureus MTCC 96 (MRSA-methicillin resistant), Salmonella paratyphi-B, Pseudomonas aeruginosa MTCC 741 and Shigella flexneri MTCC 1457. Fungi: Candida albicans MTCC 227 and Malassesia pachydermatis. The reference cultures were obtained from Institute of Microbial Technology (IMTECH), Chandigarh 160036, India. All the synthesized b-amino alcohols 1 were subjected to molecular docking studies using the AutoDock Tools (ADT)21 version 1.5.6 and AutoDock version 4.2.5.1 docking program to investigate the potential binding mode of inhibitors. DNA topoisomerase IV receptor is required for maintenance of proper DNA topology during transcription and replication in bacteria.22 The DNA topoisomerase IV structure was obtained from the Protein Data Bank (PDB ID: 4EMV). The co-crystallized ligand in the DNA topoisomerase IV structure was removed. The polar hydrogen atoms were then added, lower occupancy residue structures were deleted and any incomplete side chains were replaced using ADT. Further ADT was used to remove crystal water, Gasteiger charges were added to each atom and the non-polar hydrogen atoms were merged to the protein structure. The distance between donor and acceptor atoms that form a hydrogen bond was defined as 1.9 Å with a tolerance of 0.5 Å, and the acceptor–hydrogen–donor angle was not less than 120°. The structures were then saved in PDBQT file format for further studies in ADT. Ligand 2D structures were drawn using ChemDraw Ultra 7.0 (ChemOffice 2002). Chem3D Ultra 7.0 was used to convert 2D structure into 3D and the energy minimized using semi-empirical AM1 method. Minimize energy to minimum RMS gradient of 0.100 was set in each iteration. All structures were saved as .pdb file format for input to ADT. All the ligand structures were then saved in PDBQT file format, to carry out docking in ADT.

A grid box with dimension of 40  40  40 Å3 with 0.375 Å spacing and centered on 14.860, 29.555, 6.941 was created around the binding site of ligand on DNA topoisomerase IV using ADT. The center of the box was set at ligand center and grid energy calculations were carried out. For the AutoDock docking calculation, default parameters were used and 10 docked conformations were generated for each compound. The energy calculations were done using genetic algorithms. All dockings were taken into 2.5 million energy evaluations were performed for each of the test molecules. In order to verify the reproducibility of the docking calculations, the bound ligand was extracted from the complex and submitted for one-ligand run calculation. This reproduced top scoring 10 conformations falling within root-mean-square deviation (rmsd) value of 0.58–1.53 Å from bound X-ray conformation for DNA topoisomerase IV, suggesting that, this method is valid enough to be used for docking studies of other compounds. Docking of different ligands to protein was performed using AutoDock, following the same protocol used in as that of validation study. Docked ligand conformations were analysed in terms of energy, hydrogen bonding, and hydrophobic interaction between ligand and receptor protein DNA topoisomerase IV. Detailed analyses of the ligand–receptor interactions were carried out, and final coordinates of the ligand and receptor were saved. For display of the receptor with the ligand binding site, PyMol software was used. From the docking scores, the free energy of binding (FEB) of all compounds were calculated (Table 8). The docking of synthesized b-amino alcohols 1 with receptor DNA topoisomerase IV exhibited well established bonds with one or more amino acids in the receptor active pocket. The active pocket consisted of 13 amino acid residues as ASN51, ALA52,

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Method validation using crystallised and docked ligand

Binding mode of all the synthesized compounds

Binding mode of compound 1d

Interaction of compound 1d with active site amino acids

Binding mode of compound 1b

Interaction of compound 1b with active site amino acids

Figure 4. Molecular docking images with receptor DNA topoisomerase IV.

Table 5 Antimicrobial activity of synthesized b-amino alcohols 1a–e & 1a0 –e0 using disc diffusion method (Zone of inhibition in mm) (1 mg/disc) Compound

1a 1b 1c 1d 1e 1a0 1b0 1c0 1d0 1e0 C

Bacteria

Fungi

B. subtilis

M. luteus

E. aerogenes

S. aereus

S. typhimurium

K. pneumoniae

P. vulgaris

S. aureus MRSA

S. paratyphiB

S. flexneri

P. aeruginosa

Candida albicans

Malassesia pachydermatis

14 NA 15 20 14 16 NA 12 22 18 30

14 NA 16 21 15 15 10 13 23 19 26

13 NA 14 21 14 14 NA 12 20 18 22

16 10 17 22 15 15 NA 13 24 17 14

11 10 14 20 13 12 10 10 19 14 18

13 NA 16 23 18 15 NA 13 24 17 20

12 NA 15 21 17 13 10 14 20 18 30

11 NA 16 20 15 14 NA 15 22 18 30

14 10 13 19 16 13 NA 10 20 17 30

16 NA 15 21 18 12 NA 10 20 17 30

16 NA 17 18 15 10 10 11 16 14 30

14 NA 17 22 17 15 NA 13 20 16 28

15 NA 16 24 18 14 10 12 22 19 26

NA-no activity, C-streptomycin (standard antibacterial agent) C-ketoconazole (standard antifungal agent).

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Table 6 Minimum inhibitory concentration (MIC) of synthesized b-aminoalcohols 1a,c–e & 1a0 , c0 –e0 (1 lg/mL) Compound

1a 1c 1d 1e 1a0 1c0 1d0 1e0 C

Bacteria

Fungi

B. subtilis

M. luteus

E. aerogenes

S. aereus

S. typhimurium

K. pneumoniae

P. vulgaris

S. aureus MRSA

S. paratyphiB

S. Flexneri

P. aeruginosa

Candida albicans

Malassesia pachydermatis

125 125 31.25 125 62.5 250 31.25 62.5 6.25

125 62.5 31.25 125 125 250 31.25 62.5 6.25

125 125 31.25 125 125 250 31.25 62.5 25

62.5 62.5 31.25 125 125 250 15.62 62.5 6.25

250 125 31.25 250 250 500 62.5 125 30

250 62.5 15.62 62.5 125 250 31.25 62.5 6.25

250 125 31.25 62.5 250 125 31.25 62.5 25

250 62.5 31.25 125 125 125 31.25 62.5 6.25

125 125 62.5 125 250 250 62.5 125 62.5

250 125 15.62 31.25 250 250 31.25 62.5 6.25

62.5 62.5 62.5 125 250 250 62.5 125 25

125 62.5 31.25 62.5 125 250 31.25 62.5 25

125 62.5 31.25 62.5 125 250 31.25 62.5 25

C-streptomycin (standard antibacterial agent) C-ketoconazole (standard antifungal agent).

Table 7 Minimum bacterial concentration (MBC) of synthesized b-aminoalcohols 1a,c–e & 1a0 , c0 –e0 (1 lg/mL) Compound

1a 1c 1d 1e 1a0 1c0 1d0 1e0

Bacteria

Fungi

B. subtilis

M. luteus

E. aerogenes

S. aereus

S. typhimurium

K. pneumoniae

P. vulgaris

S. aureus MRSA

S. paratyphiB

S. Flexneri

P. aeruginosa

Candida albicans

Malassesia pachydermatis

125 250 31.25 250 62.5 250 62.5 125

250 62.5 62.5 250 125 500 31.25 125

250 125 62.5 125 250 250 31.25 125

62.5 125 31.25 250 250 250 31.25 125

500 250 31.25 500 250 500 62.5 250

500 62.5 15.62 125 250 250 62.5 125

500 250 31.25 62.5 500 125 62.5 125

500 125 31.25 250 125 250 31.25 125

250 250 125 125 500 500 62.5 250

500 250 31.25 31.25 500 500 62.5 125

125 125 62.5 250 500 500 125 250

125 62.5 31.25 62.5 125 250 31.25 62.5

125 62.5 31.25 62.5 125 250 31.25 62.5

NA-no activity, C-streptomycin (standard antibacterial agent) C-ketoconazole (standard antifungal agent).

GLU55, VAL76, ASP78, ARG81, GLY82, MET83, PRO84, ILE98, VAL122, THR172 and VAL174. In silico studies revealed all the synthesized molecules showed good binding energy towards the target receptor DNA topoisomerase IV, ranging from 7.83 to 6.09 kJ/mol. Among all the compounds docked, compound 1d, which fits exactly in the active site and forms four hydrogen bonds with three amino acids namely ASP78, GLY82 and THR172, exhibits very high binding energy value of 7.83 kJ/mol. But compound 1b, which does not exactly fit in the active site, forms only one hydrogen bond with ASP78 amino acid, exhibits low binding energy value of 6.09 kJ/mol (Fig. 4). In summary, have reported the synthesis of chiral b-amino alcohols 1 with ee 100% by reductive amination from phenylpropanolamines 2. These chiral b-amino alcohols 1 were evaluated for their antimicrobial activities, MIC & MBC studies against eleven bacteria

Table 8 Binding energy of synthesized b-amino alcohols 1 with receptor DNA topoisomerase IV

a

Compound

Binding energya (kcal/mol) DNA topoisomerase IV

1a 1a0 1b 1b0 1c 1c0 1d 1d0 1e 1e0

7.52 6.76 6.09 7.16 7.25 7.34 7.83 7.38 7.36 7.57

Calculated by AutoDock.

and two fungi and found the synthesized chiral b-amino alcohols 1 have shown potential antimicrobial activity.23,24 Amongst all the compounds screened, 1d and 1d0 showed very good activity against tested bacteria and fungi. Molecular docking studies revealed that compound 1d exhibits good binding with the active site of the docked receptor DNA topoisomerase IV. Acknowledgment The authors thank the management of Malladi Drugs & Pharmaceuticals Limited for providing support to carry out this research work. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014. 05.027. References and notes 1. Wainwright, M. Mycologist 1989, 3, 21. 2. Chaturvedi, T. P. Indian J. Dent. Res. 2009, 20, 107. 3. (a) Roos, G. Compendium of Chiral Auxiliary Applications; Academic Press: New York, 2002; (b) Ager, D. J.; Prakash, I.; Schaad, D. R. Chem. Rev. 1996, 96, 835; (c) Myers, A. G.; Yang, B. H.; Chen, H.; McKinstry, L.; Kopecky, D. J.; Gleason, J. L. J. Am. Chem. Soc. 1997, 119, 6496. 4. (a) Angelina, M. A.; Thiago, N.; Bianca, S. F.; Pedro, P. C.; Vania, L. S.; Claudio, G. D.; Mireille, L. H. Bioorg. Med. Chem. Lett. 2013, 23, 2883; (b) Fabien, A.; Bernard, T.; Eric, H.; Daniel, C.; Alain, C.; Sandrine, B. J. Chem. Soc., Dalton Trans. 2010, 39, 8982; (c) Imzil, L.; Elamrani, K.; Charaf, A.; Bouzoubaa, M.; Leclerc, G. J. Pharm. Belg. 1993, 48, 335. 5. (a) Declerck, I.; Himpens, B.; Droogmans, G.; Casteels, R. Eur. J. Physiol. 1990, 417, 117; (b) Weinberger, M. M. Pediatr. Clin. North Am. 1975, 22, 121. 6. Maryadele, J. O’Neil The Merck Index: An Encyclopedia of Chemicals, Drugs and Biological, 14th ed.; Merck Research Laboratories, 2006.

K. Chennakesava Rao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3057–3063 7. William, H. F. Circulation 2003, 107, 117; (b) Nash, D. T. Clin. Cardiol. 1990, 13, 764. 8. Yendapally, R.; Lee, R. E. Bioorg. Med. Chem. Lett. 2008, 18, 1607. 9. Nicolas, A. F. J. Pharmacol. Exp. Ther. 2005, 1, 313. 10. Pastushenko, E. V.; Safiulova, G. I. Chem. Heterocycl. Compd. 1994, 30, 741. 11. Fulop, B. G.; Mattinen, J.; Pihlaja, K. Tetrahedron 1989, 45, 4317. 12. (a) Sreekumar, R.; Pillai, C. N. Tetrahedron: Asymmetry 1993, 4, 2095; (b) Edward, L. E.; Frank, S. C.; James, M. S. J. Am. Chem. Soc. 1950, 72, 2718. 13. Cervinka, O.; Hilbert, O.; Struzka, V.; Svatos, A.; Vodnansky, M.; Jakal, V. LEphedrine 1986, Czech CS233442. 14. Newmann, P. In Optical Resolution, Procedures for Chemical Compound, Vol. 1: Amines and Related Compounds; A Publication of the Optical Resolution Information Center, Manhattan College: Riverdale, New York, 1978. 15. Prakasam, T.; Srinivasan, P.S.; Bhanumathi, A.; Ramana, D.V.; Hiteshkumar, B.N. IN 249376 Oct 21, 2011. 16. See the Supplementary data. 17. Balachandran, C.; Duraipandiyan, V.; Al-Dhabi, N. A.; Balakrishna, K.; Kalia, N. P.; Rajput, V. S.; Khan, I. A.; Ignacimuthu, S. Indian J. Microbiol. 2012, 52, 676. 18. Duraipandiyan, V.; Ignacimuthu, S. J. Ethnopharmacol. 2009, 123, 494. 19. Yilmaz, M. T. Turk. J. Med. Sci. 2012, 42, 1423. 20. (a) Peterson, R. L. Clin. Infect. Dis. 2001, 33, S180; (b) Takei, M.; Fukuda, H.; Kishii, R.; Kadowaki, Y.; Atobe, Y.; Hosaka, M. Antimicrob. Agents Chemother. 2002, 46, 3337. 21. http://autodock.scripps.edu/resources/references. 22. Manchester, J. I.; Dussault, D. D.; Rose, J. A.; Boriack-Sjodin, P. A.; UriaNickelsen, M.; Ioannidis, G.; Bist, S.; Fleming, P.; Hull, K. G. Bioorg. Med. Chem. Lett. 2012, 22, 5150. 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. (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 lg/mL, 500 lg/mL, 250 lg/mL, 125 lg/mL, 62.5 lg/mL, 31.25 lg/mL, 15.62 lg/mL and 7.81 lg/mL) of the compound were dissolved in DMSO (2%) and diluted to give serial two-fold

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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 (range, 1000 lg/mL, 500 lg/mL, 250 lg/mL, 125 lg/mL, 62.5 lg/mL, 31.25 lg/ mL, 15.62 lg/mL and 7.81 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. 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 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 baminoalcohols 1a–e and 1a0 –e0 as hydrochloride salts. (1R,2S,10 R)-()-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) (cm1): 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.

Synthesis, antimicrobial and molecular docking studies of enantiomerically pure N-alkylated β-amino alcohols from phenylpropanolamines.

Enantiomerically pure N-alkylated β-amino alcohols 1a, 1a', 1c, 1c', 1d, 1d', 1e and 1e', with ee 100% have been synthesized from phenylpropanolamines...
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