Design, synthesis, and structure–activity relationship studies of novel pleuromutilin derivatives having a piperazine ring

Received Date : 07-Feb-2016

Revised Date : 18-May-2016

Accepted Date : 31-May-2016

Article type : Research Article

Design, synthesis and structure-activity relationship

studies of novel pleuromutilin derivatives having a

piperazine ring

Jian Luo, Qiu-E Yang, Yuan-Yuan Yang, You-Zhi Tang*, Ya-Hong Liu*

Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development and Safety

Evaluation, College of Veterinary Medicine, South China Agricultural University, Guangzhou

5

10642, China

Correspondence should be addressed:

College of Veterinary Medicine, South China Agricultural University, No. 483 Wushan

Road, Tianhe District, Guangzhou 510642, China

You-Zhi Tang,

Fax: +86 20 85280665; Email: youzhitang@scau.edu.cn

or Ya-Hong Liu,

Fax: +86 20 85284896; Email: gale@scau.edu.cn

This article has been accepted for publication and undergone full peer review but has not

been through the copyediting, typesetting, pagination and proofreading process which may

lead to differences between this version and the Version of Record. Please cite this article as

an 'Accepted Article', doi: 10.1111/cbdd.12799

Abstract

A series of novel pleuromutilin derivatives possessing piperazine moieties were synthesized

under mild conditions. The in vitro antibacterial activities of these derivatives against

Staphylococcus aureus and Escherichia coli were tested by the agar dilution method.

Structure-activity relationship (SAR) studies resulted in compounds 11b, 13b and 14a with

the most potent in vitro antibacterial activity among the series (MIC = 0.0625-0.125 μg/ml).

The binding of compounds 11b, 13b and 14a to the Escherichia coli ribosome was

investigated by molecular modeling, and it was found that there is a reasonable correlation

between the binding free energy and the antibacterial activity.

Introduction

The rate of new antibiotics reach the market is declining, and at the same time, the rate of

multi-drug resistant strains is increasing(1). Therefore, the discovery and development of new

antibiotics with a novel mechanism of action has become more and more important to the

medical community(2). Natural products or semisynthetic derivatives of natural products

usually play a vital role for developing new antibacterial agents (3, 4).

Pleuromutilin (1, Fig. 1), a 5-6-8 tricycle diterpene natural product with a modest

antibacterial activity, was first isolated from two basidiomycete species in 1951(5, 6).

Pleuromutilin selectively inhibits bacterial protein synthesis by binding to the ribosomes, but

does not bind to mammalian ribosomes(7). Further studies have shown that this class of

antibiotics interact with the 23S rRNA of the 50S bacterial ribosome subunit and displays

lack of cross-resistance with antibiotics currently in market(2, 8, 9). This new mechanism of

action of pleuromutilin derivatives have let them lead compounds in the discovery and

development of novel antibacterial agents for the treatment of the problem of bacterial

resistance(1, 2, 10). Throughout the 1970s, chemists at Sandoz reported initial structure-

activity relationship (SAR) studies on a series of semisynthetic pleuromutilin analogous (11-

3). Further studies have shown that modification of the C14 position of pleuromutilin could

1

led to compounds with improved antimicrobial activity(14). Additionally, the de novo

syntheses of analogues of pleuromutilin, which allow the core of the pleuromutilin to be

varied, have been reported recently (15, 16).

More than 3000 semisynthetic pleuromutilin analogous with C14 glycol ester side chains

have been synthesized and studied(10). Luckily, three of them were marketed by

pharmaceutical companies. Tiamulin (2, Fig. 1) and valnemulin (3, Fig. 1) were approved as

therapeutic agents for pig and poultry in 1979 and 1999, respectively(17). Although

retapamulin (4, Fig. 1) was approved by the Food and Drug Administration (FDA) in 2007

for human use(18), it was limited to the topical treatment of skin infections. The sulfide

linkage in these three pleuromutilin analogues yield potent in vitro antibacterial activities, as

well as limited oral bioavailability in vivo of these derivatives for their strong hydrophobic

nature (7).

In order to overcome this problem, along with previous SAR studies (1, 7, 19), we have

focused our efforts on the development of novel pleuromutilin antibiotics with amine

substituents incorporated into the C14 side chain (20). Then, tiamulin is used as a lead

compound. The aim was first of all to make an isometric exchange of the sulfur in the linker

with nitrogen in order to decrease the hydrophobicity of the linker. Considering piperazine

ring plays a vital role for increasing antibacterial activities of quinolones (21, 22). We

envisaged that the development of pleuromutilin derivatives with piperazine ring as linker

and various benzene ring as substituents incorporated into the C14 side chain may increase

hydrogen bonding and ∏-∏ stacking interactions and thereby increase their antibacterial

properties.

For the present sutdy, we report the design, synthesis, and SAR studies of novel pleuromutilin

derivatives with one piperazine ring as linker with similar substituents incorporated into the

C14 side chain. To explore the antibacterial mechanisms of these derivatives, several

compounds were chosen for molecular docking studies.

Experimental

Materials

Pleuromutilin (>90% pure) was obtained from Great Enjoyhood Biochemical Co Ltd

(Daying, China). Piperazine derivatives were purchased from J&K Scientific Ltd. The other

reagents were all of analytical grade and purchased from Guangzhou Chemical Reagent

Factory (Guangzhou, China). Melting points were determined on a Shenguang X-4 apparatus

1

13

(

China) and are uncorrected. H-NMR and C-NMR spectra were measured on Bruker AV-

4

00 or Bruker AV-600 spectrometer in CDCl . Chemical shift values (δ) were given in p.p.m.

3

and tetramethylsilane was used as internal standard. Fourier transform infrared (FTIR) were

obtained with a Nicolet 6700 Research FTIR spectrometer using KBr pellets. Mass spectra

were recorded with Agilent 6430 and LTQ-Orbitrap mass spectrometer (Thermo Fisher)

using the electro spray ionization (ESI) method.

Synthesis

A general synthesis strategy based on the usual 22-O-tosylpheuromutilin and piperazine

derivatives were used (Scheme 1).

2

2-O-tosylpleuromutilin (1a)

A solution of pleuromutilin 1 (10.0g, 26.4 mmol) in pyridine (30.0 mL) was stirred at 0 ℃

in a three-necked round bottom flask, and p-toluenesulfonyl chloride (5.5 g, 29.0 mmol) was

added. The solution was stirred at 0 ℃ for 3h. CHCl (50 mL) and Ice-cold water (50 mL)

3

were added to the solution. The organic phase was washed with a 2 M aqueous solution of

H SO , a saturated aqueous solution of NaHCO and water, respectively. Then the organic

2

4

3

phase was dried with anhydrous Na SO for 2 h and evaporated in vacuum. The residue was

2

4

precipitated from isopropanol to give a white solid (10.3 g, 73.0%) (20, 23).

The synthetic route of pleuromutilin derivatives having a piperazine ring

A solution of compound 1a (1.3 g, 2.4 mmol) in acetonitrile (10 mL) was refluxed for 0.5h.

NaI and Na CO were added and the solution was refluxed for 2 h and then gave the iodide

2

3

2

2-deoxypleuromutilin. A solution of piperazine derivatives in acetonitrile were added in

above mixture. The pleuromutilin derivatives having a piperazine ring were obtained by the

nucleophilic attack of piperazine derivatives on iodide 22-deoxypleuromutilin. After

completion of the reaction, water (50 mL) was added to the solution, which was then

extracted with CHCl (50 mL). The organic phase was washed with water for twice, dried

3

(

Na SO ) and concentrated under reduced pressure to give the crude product. The crude

2 4

product was purified by silica gel column chromatography using petroleum ether/ethyl

acetate (2:1) as eluent to give a pure product.

2

2-(4-(2-Methoxy-phenyl)-piperazin-1-yl)-22-deoxypleuromutilin (11a)

Prepared according to the above general preparation of pleutromutilin derivatives in scheme

1

. Light pink powder; yield: 64.0%; m.p.: 55.0-57.4℃; H-NMR spectrum (400 MHz; d -

1

CDCl TMS): δ (ppm)6.93 (4H, m), 6.54 (1H, dd, J 10.8, 17.2, H19), 5.81 (1H, d, J 8.8,

3;

H14), 5.35(1H, d, J 10.8, H20), 5.21 (1H, d, J 17.2, H20), 3.86 (3H, s), 3.36 (1H, dd, J 6.8,

0.8, H11), 3.17 (6H, m, H22), 2.75 (4H, m), 2.22 (5H, m, H2, H4, H10, H13),1.78 (1H, dd,

J 2.0, 14.4, H6), 1.66 (5H, m, H1, H7, H8,),1.46(3H, s, H15), 1.34 (3H, m, H8, H13, 11-OH),

1

13

1

.13 (1H, m, H8),1.17 (3H, s, H18), 0.88 (3H, d, J 6.8 H17), 0.75 (3H, d, J 6.8, H16); C

NMR (100 MHz, d -CDCl ):δ (ppm) 217.08 (C3),169.14 (C21), 152.29, 141.26, 139.14

1

3

(C19), 122.99, 121.04, 118.31, 117.29 (C20), 111.28, 74.61 (C11), 68.27 (C14), 60.16, 58.22

(C4), 55.39, 53.42, 50.35 (C22), 45.48 (C9), 45.02 (C13), 43.98 (C12), 41.81 (C5), 36.78

(C6), 36.07 (C10), 34.49 (C2), 30.47 (C8), 26.86 (C7), 26.36 (C18), 24.89 (C1), 16.76 (C16),

-

1

2

4.93 (C15), 11.50 (C17); IR (KBr, cm ) 3504, 2926, 1728, 1500, 1450, 1286, 1240, 1211,

+

034, 918, 753; HR-MS (ESI): Calcd for C H N O (M-H ): 553.3636; Found: 553.3632.

3

48

2

5

2-(4-(3-Methoxy-phenyl)-piperazin-1-yl)-22-deoxypleuromutilin (11b)

Prepared according to the above general preparation of pleutromutilin derivatives in scheme

1

. Light pink powder; yield: 78.0%; m.p.: 55.0-57.4℃; H-NMR spectrum (400 MHz; d -

1

CDCl TMS): δ (ppm) 7.16 (1H, m), 6.49 (4H, m, H19), 5.81 (1H, d, J 8.8, H14), 5.35 (1H,

3;

d, J 10.8, H20), 5.20 (1H, d, J 17.2, H20), 3.78 (3H, s),3.23 (6H, m, H11, H22), 2.71 (4H, d,

J 22.4), 2.21 (6H, m, H2, H4, H10, H13, 11-OH), 1.64 (6H, m, H1, H7, H8, H6), 1.45 (3H, s,

H15),1.34 (3H, m, H8, H13)1.17 (3H, s, H18),0.88 (3H, d, J 6.8 H17),0.75 (3H, d, J 6.8,

13

H16); C NMR (100 MHz, d -CDCl ):δ (ppm) 217.04 (C3), 168.98 (C21), 160.59, 152.55,

1

3

1

5

3

39.11 (C19), 129.79,117.29 (C20), 108.93, 104.50, 102.61, 74.61 (C11), 68.38 (C14),59.94,

8.20 (C4), 55.18, 52.95,48.85 (C22), 45.48 (C9), 45.05 (C13), 43.97 (C12), 41.80 (C5),

6.76 (C6), 36.07 (C10), 34.48 (C2), 30.46 (C8), 26.85 (C7), 26.38 (C18), 24.87 (C1), 16.76

-1

(

C16), 14.92 (C15), 11.50 (C17); IR (KBr, cm ) 3504, 2938, 1727, 1598, 1495, 1450, 1381,

+

1

5

2

294, 1256, 1204, 1176, 976, 825, 765, 688; HR-MS (ESI): Calcd for C H N O (M-H ):

3

48

2

5

53.3636; Found: 553.3630.

2-(4-(4-Methoxy-phenyl)-piperazin-1-yl)-22-deoxypleuromutilin (11c)

Prepared according to the above general preparation of pleutromutilin derivatives in scheme

1

. Light pink powder; yield: 58.0%; m.p.: 55.0-57.4℃; H-NMR spectrum (400 MHz; d -

1

CDCl TMS): δ (ppm) 6.90 (2H), 6.84 (2H, d, J 8.8), 6.53 (1H, dd, J 10.8, 17.2, H19), 5.81

3;

(

1H, d, J 8.4, H14), 5.35 (1H, dd, J 1.6, 11.2, H20),5.21 (1H, dd, J 1.6, 17.6, H20), 3.77（

H, s）3.36 (1H, dd, J 6.4, 10.8, H11), 3.24 (1H, d, J 17.2, H22),3.11 (5H, m, H22), 2.73

4H, d, J 23.2),2.21 (5H, m, H2, H4, H10, H13), 1.78 (1H, dd, J 2.8, 14.4, H6), 1.59 (5H, m,

3

(

H1, H7, H8,),1.46 (3H, s, H15), 1.30 (2H, m, H8, H13), 1.17 (3H, s, H18), 0.88 (3H, d, J 7.2

13

H17), 0.75 (3H, d, J 6.8, H16); C NMR (100 MHz, d -CDCl ):δ (ppm) 217.05 (C3),

1

3

1

71.11,169.04 (C21), 151.17,139.13 (C19), 118.34, 117.29 (C20), 114.48, 74.61 (C11), 68.35

C14), 59.98, 58.21 (C4), 55.57, 53.13, 50.48 (C22), 45.48 (C9), 45.05 (C13), 43.98 (C12),

1.81 (C5), 36.77(C6), 36.07 (C10), 34.48 (C2), 30.47 (C8), 26.86 (C7), 26.37 (C18), 24.87

(

4

-1

(

C1), 16.76 (C16), 14.92 (C15), 11.50 (C17); IR (KBr, cm ) 3444, 2936, 1734, 1511, 1454,

383, 1285, 1244, 1201, 1153, 1116, 1019, 918, 823; HR-MS (ESI): Calcd for C H N O

5

1

3

48

2

+

(

M-H ): 553.3636; Found: 553.3633.

2

2-(4-(2-Methyl-phenyl)-piperazin-1-yl)-22-deoxypleuromutilin (12a)

Prepared according to the above general preparation of pleutromutilin derivatives in scheme

1

. Light pink powder; yield: 64.0%;m.p.: 55.0-57.4℃; H-NMR spectrum (400 MHz; d -

1

CDCl TMS): δ (ppm) 7.16 (2H), 7.00 (2H), 6.54 (1H, dd, J 11.2, 17.6, H19), 5.82 (1H, d, J

3;

8

3

.4, H14), 5.35 (1H, d, J 11.2, H20), 5.21 (1H, d, 17.6, H20), 3.37 (1H, dd, J 6.4, 10.4, H11),

.26 (1H, d, J 17.2, H22), 3.13 (1H, d, J 16.8, H22), 2.98 (4H), 2.73 (4H, d, J 27.6), 2.382

(1H, t, J 6.8, H2), 2.29 (3H, s), 2.15 (4H, m, H2, H4, H10, H13), 1.45 (9H, m, H1, H6, H7,

H8, H13, 11-OH),1.47 (3H, s, H15),1.17 (3H, s, H18),0.88 (3H, d, J 6.8, H17), 0.76 (3H, d, J

1

3

6

1

6

4

.4, H16); C NMR (100 MHz, d -CDCl ):δ (ppm) 217.08 (C3), 169.14 (C21),

1 3

51.42,139.15 (C19), 132.64, 131.03, 126.59, 123.22, 119.12, 117.29 (C20), 74.62 (C11),

8.26 (C14),60.03,58.23 (C4), 53.46,51.48 (C22), 45.49 (C9), 45.08 (C13),43.97 (C12),

1.81 (C5), 36.79 (C6),36.09 (C10), 34.49 (C2), 30.48 (C8),26.86 (C7), 26.39 (C18), 24.88

-1

(

C1), 17.83,16.76 (C16), 14.94 (C15), 11.51 (C17); IR (KBr, cm ) 3498, 2950, 1727, 1491,

1

7

452, 1421, 1402, 1381, 1281, 1240, 1208, 1172, 1149, 1123, 1037, 1021, 916, 819, 763,

+

24; HR-MS (ESI): Calcd for C H N O (M-H ): 537.3687; Found: 537.3684.

3

48

2

4

2

2-(4-(3-Methyl-phenyl)-piperazin-1-yl)-22-deoxypleuromutilin (12b)

Prepared according to the above general preparation of pleutromutilin derivatives in scheme

1

. Light pink powder; yield: 72.0%; m.p.: 55.0-57.4℃; H-NMR spectrum (400 MHz; d -

1

CDCl TMS): δ (ppm) 7.15 (1H, t, J 7.6), 6.71 (3H, m), 6.53 (1H, dd, J 10.8, 17.2, H19),

3;

5

.81 (1H, d, J 8.4, H14),5.35 (1H, dd, J 1.2, 10.8, H20), 5.21 (1H, dd, J 1.2, 17.6, H20), 3.36

1H, dd, J 6.4, 10.4, H11),3.24 (5H, m, H22), 3.10 (1H, d, J 17.2, H22), 2.71 (4H, d, J

2.4),2.36 (1H, t, J 7.2, H2), 2.31 (3H, s),2.15 (4H, m, H2, H4, H10, H13), 1.53 (9H, m, H1,

H6, H7, H8, H13, 11-OH),1.46 (3H, s, H15), 1.17 (3H, s, H18), 0.88 (3H, d, J 7.2, H17),0.75

(

2

13

(

3H, d, J 6.8, H16); C NMR (100 MHz, d -CDCl ):δ (ppm) 217.05 (C3), 169.01 (C21),

1 3

1

6

4

51.25,139.12 (C19), 138.82, 128.97, 120.72,117.30 (C20), 117.06, 113.28, 74.61 (C11),

8.38 (C14),59.99,58.21 (C4), 53.07,49.02 (C22), 45.48 (C9), 45.06 (C13),43.98 (C12),

1.81 (C5), 36.77 (C6),36.08 (C10), 34.48 (C2), 30.47 (C8),26.86 (C7), 26.38 (C18), 24.87

-1

(

C1), 21.76,16.76 (C16), 14.92 (C15), 11.50 (C17); IR (KBr, cm ) 3556, 2934, 1735, 1602,

1

494, 1453, 1363, 1294, 1190, 1152, 1116, 1016, 939, 844, 773, 693; HR-MS (ESI): Calcd

+

for C H N O (M-H ): 537.3687; Found: 537.3682.

3

48

2

4

2

2-(4-(4-Methyl-phenyl)-piperazin-1-yl)-22-deoxypleuromutilin (12c)

Prepared according to the above general preparation of pleutromutilin derivatives in scheme

1

. Light pink powder; yield: 58.0%; m.p.: 55.0-57.4℃; H-NMR spectrum (400 MHz; d -

1

CDCl TMS): δ (ppm) 7.07 (2H, d, J 8.0), 6.84 (2H, d, J 7.0), 6.53 (1H, dd, J 10.8, 17.2,

3;

H19),5.81 (1H, d, J 8.8, H14), 5.35 (1H, dd, J 1.6, 11.2, H20), 5.20 (1H, dd, J 1.2, 17.6,

H20), 3.36 (1H, dd, J 6.4, 10.0, H11), 3.23 (5H, m, H22), 3.10 (1H, d, J 17.2, H22), 2.72 (4H,

d, J 22.8), 2.36 (1H, t, J 6.8, H2), 2.27 (3H, s), 2.14 (4H, m, H2, H4, H10, H13), 1.78 (1H,

dd, J 2.8, 14.4, H6), 1.59 (4H, m, H1, H7),1.46 (3H, s, H15),1.33 (3H, m, H8, H13, 11-OH),

13

1

.17 (3H, s, H18),1.12 (1H, m, H8),0.88 (3H, d, J 6.8 H17), 0.75 (3H, d, J 6.8, H16); C

NMR (100 MHz, d -CDCl ):δ (ppm) 217.05 (C3), 169.01 (C21), 149.11, 139.12 (C19),

1

3

1

29.66, 117.30 (C20), 116.54, 74.61 (C11), 68.36 (C14), 59.96, 58.21(C4), 53.05, 49.53

(

C22), 45.48 (C9), 45.05 (C13), 43.98 (C12), 41.81 (C5), 36.77 (C6), 36.08 (C10), 34.48

C2), 31.58, 30.47 (C8), 26.86 (C7), 26.38 (C18), 24.87 (C1), 20.42, 16.76 (C16), 14.92

-1

C15), 11.50 (C17); IR (KBr, cm )3563, 2933, 1734, 1615, 1515, 1453, 1412, 1383, 1303,

+

1

5

236, 1196, 1152, 1115, 1019, 915, 811; HR-MS (ESI): Calcd for C H N O (M-H ):

3

48

2

4

37.3687; Found: 537.3680.

2

2-(4-(2-Hydroxy-phenyl)-piperazin-1-yl)-22-deoxypleuromutilin (13a)

Prepared according to the above general preparation of pleutromutilin derivatives in scheme

1

. Light pink powder; yield: 92.0%; m.p.: 55.0-57.4℃; H-NMR spectrum (400 MHz; d -

1

CDCl TMS): δ (ppm) 7.17 (1H, dd, J 1.6, 8.0), 7.08 (1H, m), 6.94 (1H, dd, J 1.2, 8.0), 6.86

3;

(1H, m), 6.53 (1H, dd, J 10.8, 17.2, H19), 5.82 (1H, d, J 8.4, H14), 5.36 (1H, dd, J 1.2, 10.8,

H20), 5.21 (1H, dd, J 1.2, 17.2, H20),3.37 (1H, dd, J 6.8, 10.0, H11), 3.28 (1H, d, J 16.8,

H22), 3.15 (1H, d, J 17.2, H22), 2.95 (4H, m), 2.77 (4H, d, J 22.0), 2.37 (1H, t, J 6.8, H2),

2

.16 (5H, m, H2, H4, H10, H13), 1.78 (1H, dd, J 2.8, 14.4, H6),1.60 (4H, m, H1, H7), 1.46

(3H, s, H15), 1.32 (3H, m, H8, H13, 11-OH), 1.18 (3H, s, H18), 1.13 (1H, m, H8), 0.89 (3H,

13

d, J 7.2, H17), 0.75 (3H, d, J 6.8, H16); C NMR (100 MHz, d -CDCl ):δ (ppm) 217.03

1

3

(

C3), 171.13，169.02 (C21), 151.48,139.12 (C19), 126.57, 121.53, 120.09, 117.31 (C20),

14.08,74.62 (C11), 68.41 (C14), 60.38,59.77, 58.21 (C4), 53.48, 52.34 (C22),45.48 (C9),

5.06 (C13), 43.98 (C12), 41.81 (C5), 36.75 (C6),36.09 (C10), 34.47 (C2), 30.46 (C8),26.85

1

4

-1

(

C7), 26.40 (C18), 24.87 (C1),16.74 (C16), 14.92 (C15), 11.50 (C17); IR (KBr, cm ) 3419,

2

937, 1734, 1589, 1439, 1455, 1375, 1283, 1250, 1195, 1152, 1116, 1019, 927, 756; HR-MS

+

(

ESI): Calcd for C H N O (M-H ): 539.3479; Found: 539.3477.

3

2

46

2

5

2

2-(4-(3-Hydroxy-phenyl)-piperazin-1-yl)-22-deoxypleuromutilin (13b)

Prepared according to the above general preparation of pleutromutilin derivatives in scheme

1

. Light pink powder; yield:76.0%; m.p.: 55.0-57.4℃; H-NMR spectrum (400 MHz; d -

1

CDCl TMS): δ (ppm) 7.09 (1H, t, J 8.0),6.47 (3H, m), 6.32 (1H, dd, J 2.0, 8.0, H19), 5.81

3;

(1H, d, J 8.4, H14), 5.45 (1H, br),5.34 (1H, dd, J 1.2, 10.8, H20), 5.20 (1H, dd, J 1.2, 17.2,

H20), 3.36 (1H, dd, J 6.4, 10.8, H11), 3.23(5H, m, H22), 3.11(1H, d, J 17.2, H22), 2.71 (4H,

d, J 22.0), 2.36 (1H, t, J 6.8, H2),2.16 (4H, m, H2, H4, H10, H13),1.78 (1H, dd, J 2.8, 14.4,

H6), 1.60 (4H, m, H1, H7), 1.45 (3H, s, H15),1.33 (3H, m, H8, H13, 11-OH), 1.17 (3H, s,

13

H18), 1.12 (1H, m, H8), 0.88 (3H, d, J 6.8, H17), 0.74 (3H, d, J 6.8, H16); C NMR (100

MHz, d -CDCl ):δ (ppm) 217.22 (C3), 169.00 (C21), 156.70, 152.63,139.05 (C19), 129.98,

1

3

117.36 (C20), 108.62,106.81, 103.22, 74.64 (C11), 68.46 (C14), 59.89, 58.22 (C4),

5

3

2.90(C22), 48.74,45.48 (C9), 45.05 (C13), 43.97 (C12), 41.81 (C5), 36.76 (C6),36.06 (C10),

4.49 (C2), 30.47 (C8),26.85 (C7), 26.37 (C18), 24.86 (C1),16.77 (C16), 14.93 (C15), 11.52

-1

(

C17); IR (KBr, cm ) 3375, 2935, 2862, 1731, 1599, 1453, 1193, 976, 866, 756, 689; HR-

+

MS (ESI): Calcd for C H N O (M-H ): 539.3479; Found: 539.3477.

3

2

46

2

5

2

2-(4-(4-Hydroxy-phenyl)-piperazin-1-yl)-22-deoxypleuromutilin (13c)

Prepared according to the above general preparation of pleutromutilin derivatives in scheme

1

. Light pink powder; yield:64.0%; m.p.: 55.0-57.4℃; H-NMR spectrum (400 MHz; d -

1

CDCl TMS): δ (ppm) 6.862-6.841 (2H, d, J 8.4), 6.76 (2H, d, J 8.8), 6.55 (1H, dd, J 11.2,

3;

1

7.6, H19), 5.81 (1H, d, J 8.4, H14), 5.34 (1H, dd, J 1.2, 10.8, H20), 5.22 (1H, dd, J 1.2,

7.6, H20), 5.15 (1H, br), 3.36 (1H, dd, J 6.4, 10.8, H11), 3.24 (1H, d, J 16.8, H22), 3.11

(

5H, m, H22), 2.75 (4H, d, J 22.0), 2.36 (1H, t, J 6.8, H2), 2.17 (4H, m, H2, H4, H10, H13),

.78 (1H, dd, J 2.8, 14.4, H6), 1.61 (4H, m, H1, H7), 1.45 (3H, s, H15), 1.32 (3H, m, H8,

H13, 11-OH), 1.17 (3H, s, H18), 1.12 (1H, m, H8), 0.88 (3H, d, J 7.2, H17), 0.74 (3H, d, J

1

13

7

.2, H16); C NMR (100 MHz, d -CDCl ):δ (ppm) 217.18 (C3), 171.22, 69.05 (C21),

1 3

1

5

50.10,139.08 (C19), 118.68, 117.32 (C20), 115.91,74.63 (C11), 68.40 (C14), 60.43,59.92,

8.220\ (C4), 53.08, 50.63 (C22),45.48 (C9), 45.04 (C13), 43.97 (C12), 41.81 (C5), 36.76

(C6),36.06 (C10), 34.49 (C2), 30.46 (C8),26.85 (C7), 26.36 (C18), 24.86 (C1),16.76 (C16),

-

1

5

4.93 (C15), 11.51 (C17); IR (KBr, cm ) 3382, 2931, 1732, 1589, 1493, 1454, 1375, 1283,

+

250, 1152, 1116, 1019, 927, 812, 754; HR-MS (ESI): Calcd for C H N O (M-H ):

3

2

46

2

5

39.3479; Found: 539.3483.

2

2-(4-(2-Nitro-phenyl)-piperazin-1-yl)-22-deoxypleuromutilin (14a)

Prepared according to the above general preparation of pleutromutilin derivatives in scheme

1

. Light pink powder; yield: 52.0%; m.p.: 55.0-57.4℃; H-NMR spectrum (400 MHz; d -

1

CDCl TMS): δ (ppm) 7.75 (1H, d, J 8.0), 7.47 (1H, t, J 7.6), 7.15 (1H, d, J 8.4), 7.05 (1H, t,

3;

J 7.6), 6.52 (1H, dd, J 10.8, 17.2, H19), 5.81 (1H, d, J 8.4, H14), 5.34 (1H, d, J 11.2, H20),

.21 (1H, dd, J 17.6, H20),3.36 (1H, dd, J 6.8, 10.8, H11), 3.24 (1H, d, J 16.8, H22), 3.13

5H, m, H22),2.71 (4H, d, J 22.8), 2.36 (1H, t, J 6.8, H2), 2.15 (4H, m, H2, H4, H10, H13),

.78 (1H, d, J 14.4, H6), 1.60 (4H, m, H1, H7), 1.46 (3H, s, H15), 1.31 (3H, m, H8, H13, 11-

OH), 1.17(3H, s, H18), 1.13 (1H, m, H8), 0.88 (3H, d, J 6.8, H17), 0.74 (3H, d, J 6.8, H16);

5

(

1

13

C NMR (100 MHz, d -CDCl ):δ (ppm) 217.01 (C3), 169.00 (C21), 145.92, 143.65,139.10

1

3

(C19), 133.47, 125.80, 121.99, 121.10, 117.31 (C20), 74.61 (C11), 68.41 (C14), 59.87,58.20

(C4), 52.92,51.49 (C22),45.49 (C9), 45.05 (C13), 43.97 (C12), 41.80 (C5), 36.75 (C6),36.07

(C10), 34.47 (C2), 30.46 (C8),26.85 (C7), 26.38 (C18), 24.87 (C1),16.76 (C16), 14.91 (C15),

-1

1

1.50 (C17); IR (KBr, cm )3500, 2931, 1727, 1601, 1527, 1452, 1382, 1358, 1286, 1209,

+

1

151, 1124, 1036, 1019, 916; HR-MS (ESI): Calcd for C H N O (M-H ): 568.3381;

3

2

45

3

6

Found: 568.3380.

2

2-(4-(3-Nitro-phenyl)-piperazin-1-yl)-22-deoxypleuromutilin (14b)

Prepared according to the above general preparation of pleutromutilin derivatives in scheme

1

. Light pink powder; yield: 62.0%; m.p.: 55.0-57.4℃; H-NMR spectrum (400 MHz; d -

1

CDCl TMS): δ (ppm) 7.68 (2H, m), 7.37 (1H, t, J 8.4), 7.17 (1H, dd, J 2.0, 8.0), 6.52 (1H,

3;

dd, J 11.2, 17.2, H19), 5.82 (1H, d, J 8.4, H14), 5.35 (1H, dd, J 1.6, 11.2, H20),5.21 (1H, dd,

J 1.6, 17.2, H20), 3.25 (7H, m, H11, H22), 2.75 (4H, d, J19.2), 2.36 (1H, t, J 6.8, H2), 2.15

(4H, m, H2, H4, H10, H13), 1.79 (1H, dd, J 2.8, 14.4, H6), 1.59 (4H, m, H1, H7),1.46 (3H,

s, H15), 1.32 (2H, m, H8, H13), 1.18 (3H, s, H18),1.13 (1H, m, H8), 0.89 (3H, d, J 6.8, H17),

13

0

.75 (3H, d, J 6.8, H16); C NMR (100 MHz, d -CDCl ):δ (ppm) 217.00 (C3), 168.88

1 3

(

C21), 151.66, 149.30,139.09 (C19), 129.72, 121.06,117.31 (C20), 113.75, 109.76, 74.61

C11), 68.53 (C14),59.78,58.19 (C4), 52.51,48.16 (C22), 45.47 (C9), 45.07 (C13),43.98

C12), 41.80 (C5), 36.73 (C6),36.08 (C10), 34.47 (C2), 30.45 (C8),26.85 (C7), 26.34 (C18),

(

-

1

2

1

4.87 (C1),16.77 (C16), 14.91 (C15), 11.50 (C17); IR (KBr, cm ) 3554, 2926, 1732, 1616,

525, 1455, 1343, 1293, 1241, 1201, 1175, 1114, 1016, 934, 866, 847, 759, 736, 667; HR-

+

MS (ESI): Calcd for C H N O (M-H ): 568.3381; Found: 568.3378.

3

2

45

3

6

2

2-(4-(4-Nitro-phenyl)-piperazin-1-yl)-22-deoxypleuromutilin (14c)

Prepared according to the above general preparation of pleutromutilin derivatives in scheme

1

. Light pink powder; yield: 72.0%; m.p.: 55.0-57.4℃; H-NMR spectrum (400 MHz; d -

1

CDCl TMS): δ (ppm) 8.13 (2H, d, J 9.6), 6.82 (2H, d, J 9.2), 6.51 (1H, dd, J 11.2, 17.6,

3;

H19),5.82 (1H, d, J 8.4, H14),5.35 (1H, dd, J 1.2, 10.8, H20), 5.21 (1H, dd, J 1.2, 17.2, H20),

.11 (11H, m, H11, H22), 2.35 (1H, t, J 6.8, H2),2.15 (4H, m, H2, H4, H10, H13), 1.78 (1H,

dd, J 2.8, 14.4, H6), 1.58 (5H, m, H1, H7, 11-OH), 1.45 (3H, s, H15), 134 (2H, m, H8, H13),

3

13

1

.18 (3H, s, H18),1.13 (1H, m, H8), 0.89 (3H, d, J 7.2, H17), 0.74 (3H, d, J 6.8, H16); C

NMR (100 MHz, d -CDCl ):δ (ppm) 216.92 (C3), 168.88 (C21), 151.66, 149.30, 139.07

1

3

(C19), 125.96, 121.06, 117.32 (C20), 112.85, 109.76, 74.60 (C11), 68.53 (C14), 59.16,58.16

(

C4), 52.19,46.82 (C22), 45.46 (C9), 45.05 (C13), 43.98 (C12), 41.78 (C5), 36.68 (C6),36.09

C10), 34.45 (C2), 30.42 (C8),26.85 (C7), 26.41 (C18), 24.86 (C1),16.77 (C16), 14.88 (C15),

(

-1

1

1.49 (C17); IR (KBr, cm ) 3453, 2932, 1131, 1597, 1503, 1453, 1383, 1323, 1241, 1203,

+

1

5

2

153, 1116, 1013, 920, 829, 755, 692, 666; HR-MS (ESI): Calcd for C H N O (M-H ):

32 45 3 6

68.3381; Found: 568.3382.

2-(4-(2-Chloro-phenyl)-piperazin-1-yl)-22-deoxypleuromutilin (15a)

Prepared according to the above general preparation of pleutromutilin derivatives in scheme

1

. Light pink powder; yield: 36.0%; m.p.:213.0-213.4℃; H-NMR spectrum (600 MHz; d -

1

CDCl TMS): δ (ppm) 7.35 (1H, dd, J 1.2, 8.0), 7.22 (1H, dt, J 1.2, 7.8), 7.057 (1H, dd, J

3;

1

5

.8, 13.8), 6.97 (1H, dt, J 1.2, 7.8), 6.54 (1H, dd, J 11.4, 17.4, H19), 5.82 (1H, d, J 8.4, H14),

.35 (1H, dd, J 1.8, 10.8, H20), 5.21 (1H, dd, J 1.2, 17.4, H20), 3.37 (1H, dd, J 5.4, 10.8,

H11), 3.26 (1H, d, J 16.8, H22), 3.13 (5H, m, H22), 2.79(2H, d, J 4.2), 2.72 (2H, d, J 4.2),

.37 (1H, m, H2), 2.18 (4H, m, H2, H4, H10, H13), 1.78 (1H, d, J 3.0, 14.4, H6), 1.60 (4H,

m, H1, H7), 1.47 (3H, s, H15), 1.36 (2H, m, H8, H13), 1.18 (3H, s, H18), 1.13 (1H, m, H8),

2

1

3

0

2

.89 (3H, d, J 6.6, H17), 0.76 (3H, d, J 7.2, H16); C NMR (150 MHz, d -CDCl ):δ (ppm)

1 3

17.00 (C3), 169.08 (C21), 149.22, 139.72 (C19), 130.61, 128.83, 127.59, 123.72, 120.46,

17.23 (C20), 74.61 (C11), 68.33 (C14), 60.00,58.21 (C4), 53.21,50.95 (C22), 45.48 (C9),

5.08 (C13), 43.98 (C12), 41.81 (C5), 36.77 (C6),36.09 (C10), 34.47 (C2), 30.47 (C8),26.86

1

4

-

1

(

C7), 26.43 (C18), 24.87 (C1),16.74 (C16), 14.93 (C15), 11.49 (C17); IR (KBr, cm ) 3500,

953, 1727, 1480, 1292, 1211, 1151, 1020, 916, 766. HR-MS (ESI): Calcd for C H ClN O

4

2

3

2

45

2

+

(

M-H ) : 557.3141; Found: 557.3145.

2

2-(4-(3-Chloro-phenyl)-piperazin-1-yl)-22-deoxypleuromutilin (15b)

Prepared according to the above general preparation of pleutromutilin derivatives in scheme

1

. Light pink powder; yield: 60.0%; m.p.:65.1-65.8℃; H-NMR spectrum (600 MHz; d -

1

CDCl TMS): δ (ppm) 7.15 (1H, t, J 7.8), 6.86 (1H, t, J 1.8), 6.80(1H, dd, J 1.8, 7.8), 6.77

3;

(1H, dd, J 2.4, 8.4), 6.52 (1H, dd, J 10.8, 17.4, H19), 5.81 (1H, d, J 8.4, H14), 5.35 (1H, dd,

J 1.8, 11.4, H20), 5.21 (1H, dd, J 1.8, 17.4, H20), 3.37 (1H, dd, J 6.6, 10.8, H11), 3.25 (5H,

m, H22), 3.11 (1H, d, J 16.8, H22), 2.74 (2H, m), 2.67(2H, m), 2.37 (1H, m, H2), 2.23 (2H,

m, H10, H13), 2.09 (2H, m, H2, 11-OH), 2.04(1H, s, H4), 1.78 (1H, dd, J 3.0, 14.4, H6 ),

1

.61 (4H, m, H1, H7), 1.46 (3H, s, H15), 1.37 (1H, m, H8), 1.31 (1H, d, J 16.2, H13), 1.18

13

(

3H, s, H18), 1.13 (1H, m, H8), 0.89 (3H, d, J 6.6, H17), 0.75 (3H, d, J 7.2, H16); C NMR

(

150 MHz, d -CDCl ):δ (ppm) 216.99 (C3), 168.92 (C21), 152.21, 139.15 (C19), 134.95,

1

3

1

5

30.02, 119.31, 117.23 (C20), 115.82, 113.89, 74.60 (C11), 68.46(C14), 59.86,58.12 (C4),

2.74,48.45 (C22), 45.47 (C9), 45.09 (C13), 43.98 (C12), 41.80 (C5), 36.74 (C6),36.08

(C10), 34.46 (C2), 30.45 (C8),26.85 (C7), 26.44 (C18), 24.87 (C1),16.74 (C16), 14.91 (C15),

-1

1

1.49 (C17); IR (KBr, cm ) 3457, 2936, 1734, 1594, 1454, 1384, 1200, 1152, 1016, 939.

+

HR-MS (ESI): Calcd for C H ClN O (M-H ): 557.3141; Found: 557.3140.

32

45

2

4

2

2-(4-(4-Chloro-phenyl)-piperazin-1-yl)-22-deoxypleuromutilin (15c)

Prepared according to the above general preparation of pleutromutilin derivatives in scheme

1

. Light pink powder; yield: 72.0%; m.p.: 55.0-57.4℃; H-NMR spectrum (400 MHz; d -

1

CDCl TMS): δ (ppm) 7.19 (2H, d, J 8.8), 6.82 (2H, d, J 8.8), 6.52 (1H, dd, J 10.8, 17.2,

3;

H19),5.81 (1H, d, J 8.4, H14), 5.34 (1H, dd, J 1.2, 11.2, H20),5.21 (1H, dd, J 1.2, 17.6, H20),

3

2

.36 (1H, dd, J 6.4, 10.4, H11), 3.22 (5H, m, H22), 3.10 (1H, d, J 16.8, H22), 2.70 (4H, m),

.36 (1H, t, J 7.2, H2), 2.15 (4H, m, H2, H4, H10, H13), 1.800-1.756 (1H, dd, J 2.8, 14.4,

H6), 1.59 (5H, m, H1, H7, 11-OH), 1.45(3H, s, H15), 1.34 (2H, m, H8, H13), 1.17 (3H, s,

13

H18), 1.12 (1H, m, H8), 0.88 (3H, d, J 6.8, H17), 0.74 (3H, d, J 6.8, H16); C NMR (100

MHz, d -CDCl ):δ (ppm) 217.04 (C3), 169.01 (C21), 149.85, 139.12 (C19),128.95, 124.59,

1

3

117.29 (C20), 74.60 (C11), 68.39 (C14), 61.30, 59.93,58.20 (C4), 52.85,48.98 (C22), 45.47

(

C9), 45.06 (C13), 43.97 (C12), 41.80 (C5), 36.75 (C6),36.07 (C10), 34.47 (C2), 30.46

C8),26.85 (C7), 26.39 (C18), 24.86 (C1),16.76 (C16), 14.92 (C15), 11.50 (C17); IR (KBr,

(

-1

cm ) 3557, 2936, 1732, 1596, 1496, 1451, 1384, 1292, 1234, 1202, 1153, 1115, 1018, 916,

+

8

18, 754; HR-MS (ESI): Calcd for C H ClN O (M-H ): 557.3141; Found: 557.3142.

3

2

45

2

4

2

2-(4-phenyl-piperazin-1-yl)-22-deoxypleuromutilin (16)

Prepared according to the above general preparation of pleutromutilin derivatives in scheme

1

. Light pink powder; yield: 50.1%; m.p.:76.0-76.6℃; H-NMR spectrum (600 MHz; d -

1

CDCl TMS): δ (ppm) 7.19 (2H), 6.85 (2H), 6.79 (1H), 6.46 (1H, dd, J 11.4, 17.4, H19),

3;

5

6

.74 (1H, d, J 8.4, H14), 5.27 (1H, d, J 11.4, H20), 5.13 (1H, dd, 17.4, H20), 3.29 (1H, dd, J

.6, 9.6, H11), 3.17 (5H，H22), 3.04 (1H, d, J 17.4, H22), 2.66 (4H, m), 2.29 (1H, t, J 6.6,

H2), 2.10 (4H, m, H2, H4, H10, H13), 1.71 (1H, dd, J 3.0, 14.4, H6), 1.51 (5H, m, H1, H7,

H8),1.28 (3H, m, H13, 11-OH), 1.39 (3H, s, H15), 1.10 (3H, s, H18), 0.81 (3H, d, J 17.2,

13

H17), 0.68 (3H, d, J 7.2, H16); C NMR (150 MHz, d -CDCl ):δ (ppm) 217.01 (C3), 168.94

1

3

(

C21), 151.16, 139.15 (C19), 129.13, 119.87, 117.26 (C20), 116.20, 74.62 (C11), 68.44

C14), 59.92, 58.21 (C4), 52.99, 48.97 (C22), 45.48 (C9), 45.08 (C13), 43.99 (C12), 41.82

C5), 36.76 (C6), 36.09 (C10), 34.47 (C2), 31.58 (C8), 30.470, 26.89 (C7), 26.42 (C18),

(

-

1

2

1

5

2

4.88 (C1), 16.76 (C16), 14.93 (C15), 11.49 (C17); IR (KBr, cm ) 3452, 2935, 1732, 1599,

+

453, 1202, 1153, 1017, 917, 759, 692; HR-MS (ESI): Calcd for C H N O (M-H ):

3

2

46

2

4

23.3530; Found: 523.3524.

2-(4-Methyl-piperazin-1-yl)-22-deoxypleuromutilin (17)

Prepared according to the above general preparation of pleutromutilin derivatives in scheme

1

. Light pink powder; yield: 32.0%; m.p.: 55.0-57.4℃; H-NMR spectrum (400 MHz; d -

1

CDCl TMS): δ (ppm) 6.52 (1H, dd, J 11.2, 17.6, H19), 5.79 (1H, d, J 8.4, H14),5.33 (1H,

3;

dd, J 1.2, 10.8, H20), 5.20 (1H, dd, J 1.2, 17.6, H20), 3.35 (1H, dd, J 6.8, 8.8, H11),3.17 (1H,

d, J 17.2, H22),3.04 (1H, d, J 17.2, H22), 2.60 (8H, m) 2.36 (1H, d, J 5.4, H4), 2.33 (3H, s),

2

.14 (4H, m, H2, H10, H13),1.61 (6H, m, H1, H6, H7, H8,),1.44 (3H, s, H15),1.30 (1H, m,

H8),1.29(1H, d, J 12, H13),1.16 (3H, s, H18), 0.88 (3H, d, J 7.2 H17), 0.73 (3H, d, J 6.8,

13

H16); C NMR (100 MHz, d -CDCl ):δ (ppm) 217.06 (C3), 169.08 (C21), 139.13 (C19),

1

3

117.26 (C20), 74.60 (C11), 68.29 (C14), 59.93, 58.20 (C4), 54.71, 52.79 (C22), 45.81, 45.49

(C9), 45.02 (C13), 43.97 (C12), 41.79 (C5), 36.75 (C6), 36.05 (C10), 34.48 (C2), 30.46 (C8),

-

1

2

3

6.84 (C7), 26.35 (C18), 24.86 (C1), 16.73 (C16), 14.91 (C15), 11.50 (C17); IR (KBr, cm )

503, 2930, 1727, 1480, 1452, 1292, 1209, 1151, 1123, 1038, 916, 765; HR-MS (ESI): Calcd

+

for C H N O (M-H ): 461.3374; Found: 461.3373.

2

7

44

2

4

Minimal inhibitory concentration (MIC) testing

MIC values of these novel pleuromutilin derivatives, and pleuromutilin were determined by

agar dilution in accordance with the “Clinical and Laboratory Standards Institute”(CLSI,

2

008).The following reference strains were used for quality control: Staphylococcus aureus

ATCC 29213.

Preparation of stock and working solutions of pleuromutilin derivatives

Stock solutions of these compounds were prepared in N,N-Dimethylformamide (DMF) at the

concentrations of 5120 µg/ml. The working solutions (256 µg/ml) were obtained by diluting

stock solutions in sterile Mueller Hinton broth.

Preparation solution of bacteria

After all three strains were all recovered, single colony selected was incubated on Mueller

Hinton agar respectively, followed by incubation for 18-24 h at 37℃. Then, inocula were

prepared by transferring several colonies of bacteria to saline. The suspensions were mixed

for 15 s and then corrected to 0.5 McF arland standard using saline. Further dilutions in saline

5

were made to get the required working suspensions (10 CFU/ml).

The determination of MIC

The test was performed in 96-well plate. All dates were tested in duplicate in each plate. 100

µl of Mueller Hinton broth was added into all the wells of the 96-well plate. 100 µl of the

working solutions (256 µg/ml) of compounds were added into the wells in rows A to H in

column 1 and well mixed. Then 100 µl of the mixture in column 1 was inhaled to add into the

wells in column 2, well mixed. The similar operation was repeated until the 10th row of wells

was filled. 100 µl of excess medium was discarded from the wells in column 10. 100 µl of

the bacteria solution was added to the wells in rows A to H in columns 1 to 10, well mixed.

Two columns served as drug-free controls (no cultures were added in one column and drugs

replaced by blank solvent in the other column). Tiamulin was used as positive controls

against bacteria. The final concentration of DMF in the first well column was 1.25%.

Initially, preliminary analyses were conducted with 1.25% (v/v) DMF/MHB and this did not

affect neither the growth of the tested bacteria nor the determination of MIC. The

concentration of drugs in each row of well were 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 µg/ml

respectively. Each 96-well plate was covered and incubated for 18-24 h at 37℃. The MIC

value was defined as the lowest concentration of the sample which inhibits the visible growth

of test bacteria.

Molecular Modeling

For all these compounds bearing a same pleuromutilin scaffold, similarity-based molecular

docking was performed to analyze the different activities of compounds. It has been revealed

that tiamulin and valnemulin bind to domain V of 23S rRNA in the peptidyl transferase

center (PTC) of Escherichia coli ribosomes (24). Thus, the peptidyl transferase center (PTC)

ribosome model was constructed that consists of all residues within 30 Åfrom the PTC

binding site. Because of the lack of crystal structures of Escherichia coli in complex with

pleuromutilin analogues, the PTC model was constructed based on the crystal structure of

Deinococcus radiodurans in complex with tiamulin (PDB ID: 1XBP) (9, 25). AutoDockTools

were used for docking study. Briefly, the receptor used for docking was extracted from the

crystal structure of 1XBP in which the tiamulin was removed. The binding site of tiamulin in

1

XBP was set as docking position. The compounds were prepared with Avogadro 1.1.1 (25),

with a 5000 steps Steepest Descent as well as 1000 steps Conjugate Gradients geometry

optimization using MMFF94 force field.

Results and Discussion

Chemistry

All those pleuromutilin derivatives were prepared as shown in Scheme 1 from 22-O-

tosylpheuromutilin (1a) and piperazine derivatives following the method reported by

Hirokawa et. al. (7). In brief, KI was added into the reaction solution reaction to transfer the

2

2-O-tosylpheuromutilin into iodide compound, which is much easier to substitute than the

tosyl group. Actually, in addition to the Scheme 1, all those compounds could be synthesized

directly from 1a with much longer time reactions. However, this method was given up for

lower yield.

All those compounds were purified by silica column chromatography. Melt points of those

compounds were measured using the Shenguang X-4 apparatus (China) apparatus and are

uncorrected, which were used to ensure the purity of those compounds. The structures of

1

13

those compounds were characterized by FT-IR, H-NMR, C-NMR and HR-MS.

Antibacterial activity

The antibacterial actives of those pleuromutilin derivatives were evaluated against

Staphylococcus aureus ATCC 29213, Staphylococcus aureus which was isolated from

GuangDong Province in 2013 and Escherichia coli ATCC25922 using agar dilution method.

The Minimum inhibitory concentrations (MIC) of those 17 new pleuromutilin derivatives,

11a-c, 12a-c, 13a-c, 14a-c, 15a-c 16 and 17, as well as tiamulin used as positive control, were

determined in duplicated at pH 7.40. The results of these studies are listed in Table 1.

The activities of compounds 16 and 17 were evaluated (bioactivities: 16>17) at first. This

observation suggests that the replacement of the methyl on the nitrogen atom of the

piperazine ring with benzene ring increase the electron densities of C14 side chain of

pleuromutilin derivatives, which resulted in compound 16 possessing higher antibacterial

activities compared with compound 17. A series of novel pleuromutilin derivatives containing

thiadiazole moieties with substituted phenyl have been synthesized by Shang et. al.(26). The

substitution position in the benzene ring attached on the thiadiazole appeared to have

influence on the antibacterial activity of these pleuromutilin derivatives. For the potent

activity exhibited by the benzene ring in the pleuromutilin derivatives, as well as the results

from Shang et. al.(26), further research on benzene ring was carried on.

Using the piperazine derivatives 16 and 17 as lead compounds, our research focused on the

substitution pattern on the benzene ring attached on the piperazine ring of those pleuromutilin

derivatives. Both electron withdrawing and donating groups on the ortho-, meta-, and para-

position of the benzene ring were introduced to explore the SAR (Table 1). It can be observed

that most compounds exhibited relatively moderate antibacterial activities against both

Staphylococcus aureus ATCC 29213 and Staphylococcus aureus(Guangdong isolated). It

could also be obtained that all these 17 compounds displayed pretty weak activities against E.

coli.

The ortho- substituted benzene derivatives with CH O- and CH - (compounds 11a and

3

1

2a) exhibited much less potency compared to compounds 16 and 17. The ortho-OH

substituted compound 13a showed the same antibacterial activities as 16 and tiamulin

against ATCC 29213 and Staphylococcus aureus (Guangdong isolated).The ortho-NO₂

substituted derivative 14a was the most active compound in this series. However,

replacement of the nitro group in compound 14a with chlorine group at the same position,

which resulted the compound 15a dramatically reduced the antibacterial activity.

The meta- substituted benzene derivatives with CH O-, CH - and NO - substituents showed

3

2

enhanced antibacterial activities (11b, 13b and 14b compared with 16). Compounds 11b and

3b displayed superior antibacterial activities to that of compound 16 and tiamulin against

1

ATCC 29213 and Staphylococcus aureus (Guangdong isolated). Compound 14b displayed

higher antibacterial activity than tiamulin against ATCC 29213 as well as the same activity as

tiamulin against Staphylococcus aureus (Guangdong isolated). The meta- substituted benzene

derivatives with CH - and Cl- (compounds 12b and 15b) showed less potency compared

3

to tiamulin.

Generally, all the para- substituted benzene derivatives bearing methoxyl, methyl, hydroxyl,

nitro and chlorine substituents (11c, 12c, 13c, 14c and 15c) showed moderate antibacterial

activities. Compound 14c, bearing nitro group in the para-position showed higher activity

compared to other para-position substituted derivatives. The para-position appeared to have

less influence on the antibacterial activity of these pleuromutilin derivatives bearing a phenyl

piperazine.

Molecular Docking Study

Three tested pleuromutilin derivatives with excellent antibacterial activities, 11b, 13b and

1

4a, were selected for molecular docking investigations. Compound 11b was found to form a

strong hydrogen bond with ribosome in the docking studies. As shown in Fingure 2, the

oxygen atom of 11b (on C3) formed one H-bond with the hydroxyl group of G2484 (O/OH

distance: 1.9Å). The binding free energy of 11b with ribosome was calculated to be -12.99

kcal/mol. Compound 13b, with the same antibacterial activity as 11b, demonstrated a similar

binding mode to that of 11b. From Figure 3, two strong hydrogen bond were observed

through the interaction of 13b with G2044 (O/NH, 1.7Å) and G2484 (H/OH, 2.6Å). Although

the binding free energy of 13b with ribosome (-12.12 kcal/mol) was higher than that of 11b,

the antibacterial activity of 13b was the same as that of 11b. One possible reason was that the

replacement of the methoxyl substituent in 11b with a hydroxy substituent on the benzene

ring gave 13b that yield better solubility.

The molecular docking results of 14a are shown in Fig. 4. The binding free energy of 14a

with ribosome was calculated to be -6.94 kcal/mol. Only one hydrogen bond was observed

through the interaction of 14a with G2044 (O/NH, 1.7Å). However, there are 9 residues

around 14a with distance less than 3Åin this docking mode. The interaction between those

residues and 14a might be the main reason that why 14a possessed the best antibacterial

activities than all the other derivatives in this paper.

Conclusions

A series of 17 novel pleuromutilin derivatives with piperazine as a linker including various

phenyl substituents into the C14 side chain has been reported. The obtained MIC values

revealed that all the synthesized derivatives possessed excellent antibacterial activity. SAR

studies have shown that a benzene substituent bearing meta-hydroxy, meta-methoxy and

ortho-nitro groups lead to increased antibacterial activity in vitro. Among the derivatives

prepared, compounds 11b, 13b and 14a exhibited the most potent activity. Docking studies

were performed on these three compounds. The results showed that the binding free energies

were -12.99, -12.12 and -6.94 kcal/mol, respectively. The discovery of compounds 11b, 13b

and 14a and the SAR, and docking studies reported here will provide insight into further

optimization of pleuromutilin derivatives.

Acknowledgements

This work was supported by the Pearl River S&T Noval Program of Guangzhou (No.

2

014J2200042) and the Natural Science Foundation of Guangdong Province (No.

S2012030006590).

Figure Captions

Scheme 1 Reagent and conditions: (i) p-toluenesulfonyl chloride, pyridine, 0℃, 3h, 63.5%;

(

ii) NaI, K CO , acetonitrile, reflux; (iii) piperazine derivatives, acetonitrile, reflux.

2 3

Table 1 MIC (μg/ml) for Staphylococcus aureus (S. aureus) ATCC 29213, Staphylococcus

aureus(S. aureus) (Guangdong isolated) andEscherichia coli (E. coli) ATCC25922.

Figure 1 Structure of pleuromutilin (1), tiamulin (2), valnemulin (3) and retapamulin (4).

Figure 2 Result of the docking of 11b (green) into the PTC model binding site. Residue

numbers shown are according to E. coli 23s RNA numbering.

Figure 3 Result of the docking of 13b (yellow) into the PTC model binding site. Residue

numbers shown are according to E. coli 23s RNA numbering.

Figure 4 Result of the docking of 14a (purple) into the PTC model binding site. Residue

numbers shown are according to E. coli 23s RNA numbering.

Figure 1

R

N

1

6

HO

O

TsO

O

3

O

3

N

O

6

O

15

3

2

1

7

2

1

O

(i)

(ii)

(iii)

22

8

2

1

4

O

1

4

O

5

4

1

4

9

1

8

2

0

17

OH

1

9

OH

1

1a

Scheme 1

S. aureus ATCC

S. aureus

Compounds

R

E. coli ATCC25922

2

9213

(Guangdong isolated)

11a

64

>128

O

11b

0.125

>128

O

1

1c

0.5

64

0.5

64

>128

O

1

2a

2b

1

0.5

>128

1

2c

3a

2

>128

0.25

OH

1

3b

0.125

>128

OH

NO₂

Cl

1

3c

4a

2

>128

HO

0.0625

NO₂

1

4b

0.125

0.25

>128

1

4c

5a

0.25

1

0.5

1

>128

O N

2

Cl

1

5b

5c

0.5

>128

1

>128

Cl

1

6

7

0.25

1

8

>128

128

H C

3

Tiamulin

0.25

Table 1

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Article Doi

Design, synthesis, and structure–activity relationship studies of novel pleuromutilin derivatives having a piperazine ring

DOI: 10.1111/cbdd.12799

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Article abstract of DOI:10.1111/cbdd.12799

Full text of DOI:10.1111/cbdd.12799

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