Synthesis and antibacterial activities of novel pleuromutilin derivatives with a substituted pyrimidine moiety

Accepted Manuscript

Synthesis and antibacterial activities of novel pleuromutilin derivatives with a

substituted pyrimidine moiety

Yunpeng Yi, Ximing Xu, Yu Liu, Shuijin Xu, Xin Huang, Jianping Liang, Ruofeng

Shang

PII:

S0223-5234(16)31000-5

10.1016/j.ejmech.2016.11.054

EJMECH 9087

DOI:

Reference:

To appear in: European Journal of Medicinal Chemistry

Received Date: 30 August 2016

Revised Date: 20 November 2016

Accepted Date: 26 November 2016

Please cite this article as: Y. Yi, X. Xu, Y. Liu, S. Xu, X. Huang, J. Liang, R. Shang, Synthesis and

antibacterial activities of novel pleuromutilin derivatives with a substituted pyrimidine moiety, European

Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.11.054.

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ACCEPTED MANUSCRIPT

Synthesis and antibacterial activities of novel pleuromutilin derivatives

with a Substituted Pyrimidine Moiety

Yunpeng Yi¹, Ximing Xu², Yu Liu¹, Shuijin Xu³, Xin Huang¹, Jianping Liang¹, Ruofeng Shang¹*

1 Key Laboratory of New Animal Drug Project of Gansu Province; Key Laboratory of Veterinary Pharmaceutical Development,

Ministry of Agriculture, P. R. China; Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS, 335 Jiangouyan, Lanzhou,

730050, China

2 Institut Pasteur, Unité de Pathogenèse des infections vasculaires, Département de Biologie Cellulaire et Infection, Paris, 75015,

France

3 Yancheng YouHua Pharmaceutical &Chemical Technology Co., Ltd., Yancheng, 224555, China.

Corresponding author: Tel.: +86 931 2115253; fax: +86 931 2115951. E-mail address: shangrf1974@163.com

(Ruofeng Shang).

Author: Tel. +86 931 2115253; fax: +86 931 2115951. E-mail address: yiyp@foxmail.com (Yunpeng Yi).

Address: Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS, No. 335, Qilihe District,

Lanzhou, 730050, P.R. China.

Abstract: The alarming growth of multidrug-resistant bacteria such as methicillin-resistant Staphylococcus

aureus (MRSA) and vancomycin-resistant Enterococci (VRE) has become a major global health hazard. Therefore,

urgent demand for new antibiotics with a unique mechanism of action is very necessary. The present study reports

the design, synthesis, and antibacterial studies of a series of novel pleuromutilin derivatives with substituted

6-amino pyrimidine moieties. Most of the tested compounds exhibited highly potent anti-MRSA or Staphylococcus

aureus (S. aureus) activities. 14-O-[(4,6-Diamino -pyrimidine-2-yl) thioacetyl] mutilin (3) and

14-O-[(2-((3R)-3-Hydroxymethylpiperidine-1-yl)-acetamido-6-aminopyrimidine-2-yl) thioacetyl] mutilin (5h)

were the most active compounds and showed higher antibacterial activities. Compound 3 displayed rapid

bactericidal activity and affected bacterial growth with the same manner as tiamulin fumarate. Docking experiments for

compounds 3 and 5h carried out on the peptidyl transferase center (PTC) of 23S rRNA provided the information

about the binding model. In vivo mouse systemic infection experimental results confirmed the therapeutic efficacy

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of compound 3, with ED₅₀of 4.22 mg/kg body weight against MRSA.

Keywords: Pleuromutilin derivatives, Synthesis, Antibacterial activity, Molecular docking

1. Introduction

The development of novel antibiotics that can be used effectively for a growing number of multidrug-resistant

bacteria has become extremely challenging [1,2]. The emergence of multidrug-resistant bacteria, such as

methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE), has led to many

available drugs reducing or losing curative effect, and remains a global human threat with the potential for

catastrophic consequences in the future [3]. To solve the drastic increase of pathogenic bacteria resistance, there is

pressing need to develop novel antibiotics with a unique mechanism of action against the dreadful pathogens.

Pleuromutilin (Fig. 1), a diterpene natural product from cultures of two species of basidiomycetes, Pleurotus

mutilus and P. passeckerianus, was first discovered and isolated in a crystalline form in 1951[4]. The pleuromutilin

molecule is constituted of a rather rigid 5-6-8 tricyclic carbon skeleton with eight stereogenic centers and a glycolic

acid chain [5-7]. Although, this compound shows modest antibacterial activity [8,9], the chemical modification at

C-14 may improve its biological activity, and thus have led to three drugs: tiamulin, valnemulin and retapamulin

(Fig. 1) [10-14]. While other chemical modifications, including esterification or oxidation of the C-11 hydroxy

group, yield inactive products [15].

Fig. 1. Structural formulas of pleuromutilin, tiamulin, valnemulin and retapamulin.

Pleuromutilin derivatives selectively inhibit bacterial protein synthesis via binding to the 50S subunit of

ribosomes at the acceptor (A) and the donor (P) site [16,17]. Further studies have demonstrated that the

hydrophobic interactions and the hydrogen bonds formed between the nucleotides of domain V and the tricyclic

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core of the tiamulin are the mainly binding modes [18,19]. The cores of pleuromutilin derivatives orient at the

A-site and alter the conformation of U2506 to tightly close the binding pocket. While C-14 extensions point toward

the P-site and alter location of U2585 to preclude tRNA from binding to the P-site [20].

Ling et al. [21] and our previous works [22,23] proposed that heterocyclic rings bearing polar groups at the C-14

side chain of pleuromutilin derivatives raise their antibacterial activity. A series of novel pleuromutilin derivatives

with 6-hydroxy pyrimidine moieties were reported, and their antibacterial studies further confirmed that

pleuromutilin derivatives with primary amines (polar groups) on the terminal C-14 side chains presented improved

activity against resistant Gram-positive bacterial strains [24]. In view of the above findings, we decided to revisit

extension from the pleuromutilin derivatives with pyrimidine moieties. In this article, we describe the design,

synthesis, and antibacterial studies of a series of novel pleuromutilin derivatives with substituted 6-amino

pyrimidine moieties for identification of more efficacious drug candidates.

2. Results and Discussion

2.1. Chemistry

The synthetic approaches for the preparation of target pleuromutilin derivatives and their intermediates are

illustrated in Scheme 1. The pleuromutilin 1 was converted into the known 22-O-tosylpleuromutilin 2, a key

intermediate for synthesizing almost all pleuromutilin derivatives. Compound 14-O-[(4,6-diamino -pyrimidine-2-yl)

thioacetyl] mutilin (3) was prepared by nucleophilic substitution of 2 with 4,6-diamino-2-mercapto pyrimidine

under basic conditions in 95% yield. The key intermediate, 14-O-[(2-chloroacetamide-6-aminopyrimidine-2-yl)

thioacetyl] mutilin 4, was prepared in 72% yield by commercially available chloracetyl chloride and 3 with an aim

to construct acetamide linker between the tertiary amine and 6-aminopyrimidine. The target compounds 5a-i were

directly obtained in 43-74% yield from 4 and various secondary amines in the presence of triethylamine. The

1

structures of the synthesized derivatives were characterized by IR, H NMR, ¹³C NMR and HRMS spectra

(Supplementary data). For further confirming the structure and conformation, a crystal of compound 3 was obtained

as a colorless, block-like from a solution of ethanol and acetone by slow evaporation method at room temperature

(Fig. 2) [25].

2.2. Antibacterial activity

The synthesized pleuromutilin derivatives 3, 5a-i and tiamulin fumarate used as reference drug were screened for

their in vitro antibacterial activity against methicillin-resistant Staphylococcus aureus ATCC 29213

(MRSA-29213), methicillin-resistant Staphylococcus aureus ATCC 33591 (MRSA-33591), Staphylococcus aureus

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CMCC (B) 26003 (S. aureus), Bacillus subtilis CMCC (B) 63501 (B. subtilis), and Escherichia coli CMCC (B)

44102 (E. coli). The minimum inhibitory concentrations (MICs) of all screened compounds were listed in Table 1.

All the tested compounds exhibited excellent antibacterial efficiency against MRSA-29213, MRSA-33591 and S.

aureus, with MIC values ranging from 0.0125 to 1, from 0.0625 to 1 and from 0.0625 to 0.25 ꢀg/mL, respectively.

However, these compounds showed comparatively higher MIC values for B. subtilis and E. coli, with MIC values

ranging from 1 to 4 and from 2 to 8 ꢀg/mL respectively, than that for MRSA or S. aureus. It can be observed that

compound 3 and 5h showed the highest antibacterial activities for all the five strains than the other synthesized

compounds and tiamulin fumarate.

Scheme 1. General synthetic scheme for the pleuromutilin derivatives

Fig. 2. ORTEP diagram for compound 3 with ellipsoids set at 75% probability

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Table 1. in vitro antibacterial activity (MIC) of the synthesized pleuromutilin derivatives.

MICs (ꢀg/mL)

Compounds

MRSA-29213

MRSA-33591 S. aureus

B. subtilis E. coli

3

0.125

1

0.0625

0.5

0.0625

0.25

2

4

2

4

2

1

2

8

4

8

4

8

4

8

4

5a

5b

0.5

0.25

0.5

0.125

0.25

5c

0.5

5d

0.25

0.125

0.25

0.125

1

0.5

5e

1

0.125

0.0625

0.125

0.0625

5f

0.25

0.125

0.5

5g

5h

5i

Tiamulin

0.25

0.125

The antibacterial activities against the above-mentioned five bacterial strains were evaluated by Oxford cup

assays. The zones of inhibition for two concentrations (320 and 160 ꢀg/mL) are reported as diameters of growth

inhibition (Supplementary data). The results correspond with MICs obtained by agar dilution method as a whole.

Compound 3 and 5h showed the best growth inhibition against the pathogens particularly MRSA-29213,

MRSA-33591 and S. aureu, the others showed moderate growth of strains except E. coli.

Almost all the synthesized compounds, as well as reference drug, presented slightly higher activity against S.

aureus than MRSA in vitro. However, this anti-bacterial difference for the two strains behind the result were not

clear. A possible explanation for that may be the membrane permeability alterations of MRSA, which may prohibit

influx of certain antimicrobials [26].

Time kill studies were performed to probe the effect of compound 3 and tiamulin fumarate at two different

concentrations (1 × MIC and 6 × MIC) against the S. aureu (Fig. 3, A) and MRSA-29213 (Fig. 3, B). Time-kill

kinetics revealed that the two compounds displayed a concentration-dependent effect, and killed bacteria rapidly at

6 × MICs, achieving a > 6 log reduction in 4 h. However, they were limited to being bacteriostatic (3-4 log₁₀

CFU/mL) at 1 × MICs. Compared to tiamulin fumarate at 6 × MICs, compound 3 displayed more rapid bactericidal

kinetics against S. aureu with the same MIC (0.0625 μg/mL), but the similar kinetics against MRSA-29213 with

only a 2-fold improvement in MIC (0.125 ꢀg/mL). These studies suggested that compound 3 affected bacterial

growth with the same manner as tiamulin fumarate.

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Fig. 3. Time-kill kinetics of compound 3 against S. aureus (A) and MRSA-29213 (B). Mean values of the

CFU/mL were obtained from the triplicate measurements.

2.3. Molecular docking study

In order to survey the binding model of the synthesized compounds 3, 5a-i within the binding pocket of 50S

ribosomal subunit and to understand their structure-activity relationship, we performed molecular docking using

Homdock software [27]. The crystal structure (PDB ID: 1XBP) [28] was used for modeling. Tiamulin within 1XBP

was used to compare experimental and predicted binding modes and validate our docking protocol. Upon flexible

docking into the 50S ribosomal subunit, the 10 compounds present a similar binding mode consistent with that of

tiamulin, with a RMSD range of 0.87 to 1.28 Å within the binding site (Fig. 4 and Table 2). The best pose was

selected from the10 poses simulated by the software. The results revealed hydrogen bonding played the most

important role in the binding of the compounds to 1XBP, with the binding free energies (ꢁGb) in the range of −9.22

to −13.28 kcal/mol (Table 3).

Out of above ten compounds, 3 and 5h exhibited high binding affinity (-12.44 and -13.28 kcal/mol, ꢁGb), which

were in agreement with they antibacterial activity. For the two docking models (Fig. 5), hydrogen bonds formed

between hydroxyl groups (eight-membered ring), C=O (ester) and N (pyrimidine) of compounds 3 and 5h and the

residue of G-2484 and G-2044 were the key interactions. In addition, one hydrogen bond was formed between the

NH₂(pyrimidine ring) of compound 3 with C-2046, and two hydrogen bonds were formed between the hydroxyl

group (piperidine ring) of the compound 5h with A-2418. However, no π−π or cation-π interactions were found

between the pyrimidine rings of the two compounds, as well as the other compounds, with the residues.

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Fig. 4. Superimposition of tiamulin (red) and the best conformations of compounds 3 (green), 5a (blue), 5b

(yellow), 5c (magenta), 5d (cyan), 5e (orange), 5f (wheat), 5g (white), 5h (brown) and 5i (pink) docked to the

binding pocket of ribosome (1XBP).

Fig. 5. Docking modes of compounds 3 (A) and 5h (B) into 1XBP. Important residues are drawn in stick with

orange. Hydrogen bonds are showed as dashed red lines.

2.4. In vivo efficacy in mouse model

After being infected with MRSA-29213 (1 × 10⁹CFU in 0.1 mL saline), the mice were intravenously treated

with different doses of 3 dissolved in 3.0% vehicle and tiamulin fumarate used as the reference drug. Figure 6

showed the time related death and survival of the infected mice when treated with different doses of 3 and tiamulin

fumarate dissolved in vehicle at a volume of 0.1 mL given in a single intravenous injection in the tail vein. The

results demonstrated dose dependent effects on the survival against MRSA-29213 bearing mice, with ED₅₀of 4.22

and 5.94 mg/kg body weight, respectively. Thus, compound 3 showed the higher antibacterial activity than that of

tiamulin against MRSA-29213 in the mouse systemic model and might act as a potent antibacterial drug.

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Table 2. Bind freeenergy, number of noncovalent molecular interaction and RMSD.

Noncovalent molecular interaction

ꢁG_b

(kcal/mol) interaction

H-bonding

RMSD

(Å)

Compound

Atom of Compound

Residue

Distance(Å)

OH (8-membered ring)

C=O (ester)

G-2484

G-2044

G-2484

G-2044

A-2482

A-2045

G-2044

A-2045

C-2565

A-2045

A-2482

G-2484

G-2044

A-2418

G-2044

U-2564

G-2484

G-2044

2.0

2.1

2.3

2.4

2.1

2.3

2.2

1.9

2.1

2.3

1.7

2.4

2.2

2.3

1.9

2.4

2.7

2.3

2.4

2.2

2.3

1.8

2.0

1.7

2.6

1.9

2.2

2.4

2.1

2.3

2.4

1.8

2.4

2.5

2.3

2.4

2.0

1.8

2.1

2.2

2.3

H-bonding

3

-12.44

C=O (ester)

0.87

0.97

N (pyrimidine ring)

NH₂(pyrimidine ring)

OH (8-membered ring)

C=O (ester)

5a

-12.01

C=O (ester)

N (pyrimidine ring)

NH₂(pyrimidine ring)

C=O (ester)

5b

5c

5d

-10.30

-9.22

-9.42

1.05

1.02

1.28

N (pyrimidine ring)

NH₂(pyrimidine ring)

C=O (ester)

N (pyrimidine ring)

NH₂(pyrimidine ring)

C=O (ester)

5e

-10.77

0.99

N (pyrimidine ring)

NH₂(pyrimidine ring)

NH₂( pyrimidine ring )

N (pyrolidine )

5f

-9.78

-9.81

1.08

1.18

NH₂(pyrimidine ring)

OH (8-membered ring)

C=O (amido bond)

N (pyrimidine ring)

NH₂(pyrimidine ring)

OH (piperidine)

5g

5h

-13.28

1.19

OH (piperidine)

C=O (5-membered ring )

NH₂(pyrimidine ring)

OH (piperidine)

5i

-11.39

-10.20

1.25

0.87

OH (8-membered ring)

C=O (ester)

Tiamulin

C=O (ester)

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Fig. 6. Efficacy of compound 3 (A) and tiamulin fumarate (B)in mouse systemic infection model.

3. Conclusion

In summary, we have synthesized a series of novel pleuromutilin derivatives possessing 6-amino pyrimidine

moieties. These derivatives were initially evaluated for their in vitro antibacterial activities against four

Gram-positive strains (MRSA-29213, MRSA-33591, S. aureus and B. subtilis) and a Gram-negative strain (E. coli).

All the screened compounds showed excellent in vitro antibacterial activity against MRSA-29213, MRSA-33591

and S. aureu . Compounds 3 and 5h were the most active antibacterial agents against Gram-positive bacteria,

especially the MRSA-33591 and S. aureus in vitro. In time-to-kill assays, 3 showed rapid bactericidal activity

against S. aureu and MRSA at the higher concentration, and its effect on bacterial growth was suggested in the

same manner as that of tiamulin fumarate. Docking studies for compounds 3 and 5h revealed the binding free

energies (ꢁG_b) of −12.44 and −13.28 kcal/mol, with an RMSD of 0.92 and 1.18 Å, respectively. It is important to

note that compound 3 showed higher efficacy than that of tiamulin fumarate against MRSA-29213 when chosen for

the further evaluation in vivo activity using systemic infection mode in mice.

4. Materials and methods

4.1. Chemistry

The reagents and solvents were obtained commerically and used without further purification. Infrared (IR)

spectra were obtained on a Thermo Nicolet NEXUS-670 spectrometer using KBr thin films, and the absorptions are

reported in cm^-1. ¹H NMR spectra were recorded using Bruker 400 MHz spectrometers in CDCl₃or DMSO-d6. The

chemical shifts (δ) were reported in parts per million (ppm) relative to tetramethylsilane. ¹³C NMR spectra were

recorded on 100 MHz spectrometers. Purity of the compounds and completion of reactions were monitored by thin

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layer chromatography (TLC) on silica gel plates (GF254; Qingdao Haiyang Chemical Co., Ltd, Shandong, China),

followed by visualization after spraying with a 0.05% KMnO4 aqueous solution or under UV illumination directly.

High-resolution mass spectra (HRMS) were obtained with a Bruker Daltonics APEX II 47e mass spectrometer

equipped with an electrospray ion source.

14-O-[(4,6-Diamino -pyrimidine-2-yl) thioacetyl] mutilin (3)

To a solution of 4,6-dihydroxy-2-mercaptopyrimidine (1.44 g, 10 mmol) in 25 mL methanol, 10 M NaOH (1.1 mL,

11 mmol) was added and stirred for 30 min. A solution of 22-O-tosylpleuromutilin (5.85, 11 mmol) in 30 mL of

DCM was added dropwise to the reaction mixture. The mixture was stirred for 36 h at room temperature. Then the

reaction mixture was evaporated under reduced pressure to dryness, and the crude product was extracted with a

solution of ethyl acetate (60mL) and water (20mL) and treated with saturated NaHCO₃. The target compound 3 was

then precipitated and purified by flash silica column chromatography (ethyl acetate: ethanol 20:1 v/v) to yield 4.77

1

g (95%). IR (KBr): 3475, 3377, 2933, 1728, 1619, 1582, 1547, 1465, 1309, 1153, 1117, 1019 cm^-1. H NMR (400

MHz, DMSO) δ 6.20-6.11 (m, 1H), 6.06 (d, J = 15.9 Hz, 3H), 5.52 (d, J = 7.8 Hz, 1H), 5.18 (d, J = 33.9 Hz, 1H),

5.04 (dd, J = 24.4, 14.5 Hz, 2H), 4.50 (d, J = 5.8 Hz, 1H), 4.03 (dd, J = 14.1, 7.1 Hz, 1H), 3.80 (d, J = 9.7 Hz, 1H),

3.42 (s, 1H), 2.39 (s, 1H), 2.17 (dd, J = 25.8, 15.3 Hz, 1H), 2.13-1.93 (m, 4H), 1.79-1.53 (m, 2H), 1.51-1.12 (m,

9H), 1.11-0.89 (m, 4H), 0.82 (d, J = 6.5 Hz, 3H), 0.62 (d, J = 6.4 Hz, 3H). ¹³C NMR (101 MHz, DMSO) δ 217.6,

168.3, 167.3, 163.8, 141.2, 115.8, 79.6, 73.1, 70.0, 60.2, 57.7, 45.4, 44.5, 44.0, 42.0, 36.8, 34.5, 33.3, 30.6, 29.0,

27.1, 24.9, 21.2, 16.6, 15.0, 12.0. HRMS (ES) calcd [M + H]+ for [C₂₆H₃₈N₄O₄S 503.2687, found 503.2689.

General procedure for the synthesis of compounds 5a-i

To a solution of compound 3 (1.51 g, 3 mmol) and 4-methylmorpholine (0.61 g, 6 mmol) in 20 mL dry DCM,

chloroacetyl chloride (0.51 g, 4.5 mmol) in 10 mL dry DCM was slowly dropped at 0 ^oC. The reaction mixture was

stirred for 1 h. After the reaction, the solution was washed with ammonium chloride one time, followed by water

three times. Then the organic layer was dried with Na₂SO₄overnight and rotary evaporated to dryness. The crude

residue obtained was purified by column chromatography (petroleum ether: ethyl acetate = 1:6) to afford the

desired compound 4 in 72 % yield as a white solid. IR (KBr): 3388, 2927, 1719, 1625, 1553, 1509, 1459, 1406,

1288, 1153, 1117 cm^-1. ¹H NMR (400 MHz, CDCl₃) δ 8.57 (s, 1H), 6.94 (s, 1H), 6.43 (dd, J = 17.4, 11.0, 1H), 5.66

(d, J = 8.4, 1H), 5.18 (dd, J = 60.3, 14.3, 2H), 5.06 (s, 1H), 4.07 (s, 2H), 3.77 (d, J = 16.3, 1H), 3.70-3.60 (m, 2H),

3.29 (dd, J = 9.7, 6.6, 1H), 2.29-2.20 (m, 1H), 2.19-2.01 (m, 2H), 1.95 (dd, J = 15.9, 8.6, 1H), 1.69 (d, J = 14.2, 1H),

1.52 (ddd, J = 44.3, 27.8, 15.1, 4H), 1.39-1.25 (m, 4H), 1.18 (t, J = 7.0, 3H), 1.13-0.96 (m, 4H), 0.79 (d, J = 6.9,

3H), 0.67 (d, J = 6.9, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 216.12, 168.19, 167.12, 164.23, 163.17, 154.68, 138.27,

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116.04, 87.13, 73.57, 68.69, 57.14, 44.44, 43.51, 42.93, 41.70, 40.87, 35.75, 34.98, 33.47, 29.40, 25.89, 25.37, 23.81,

17.41, 15.84, 13.90, 10.44. HRMS (ES) calcd [M + H]+ for [C₂₈H₃₉ClN₄O₅S 579.2402, found 579.2407.

Secondary amines (4.5 mmol) was added to the solution of compound 4 (1.73 g, 3 mmol) and triethylamine (0.61

g, 6 mmol) in methanol (60 mL) and stirred at 45 ^oC for 2 h. Then the solvent was evaporated in vacuum from the

reaction mixture. The residue was added DCM (60 mL) and quenched with saturated aqueous NH₄Cl (30 mL). The

organic layer was separated, washed with water (20 mL for three times), dried with anhydrous Na₂SO₄and rotary

evaporated to dryness. The crude residue was purified by silica gel column chromatography (petroleum ether and

ethyl acetate) to afford the desired compounds 5a-i.

14-O-[(2-(bis(Methyl)-amino)-acetamido-6-aminopyrimidine-2-yl) thioacetyl] mutilin (5a)

Compound 5a was prepared according to the general procedure with a reaction time of 2 hours. The crude

product was purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 1:4) to give 43%

yield (0.76 g) of 5a as a white solid. IR (KBr): 3345, 2935, 1720, 1625, 1593, 1550, 1498, 1457, 1287 cm^-1; ¹H

NMR (400 MHz, CDCl₃) δ 9.36 (s, 1H), 7.09 (d, J = 15.1 Hz, 1H), 6.51 (dd, J = 17.4, 11.0 Hz, 1H), 5.76 (t, J =

16.9 Hz, 1H), 5.36-5.14 (m, 2H), 5.04-4.82 (m, 2H), 3.93-3.70 (m, 2H), 3.35 (s, 1H), 3.06 (s, 1H), 2.37-2.30 (m,

6H), 2.25-2.13 (m, 2H), 2.02 (dt, J = 13.1, 12.4 Hz, 3H), 1.79-1.72 (m, 1H), 1.69-1.48 (m, 4H), 1.48-1.42 (m, 4H),

1.38-1.24 (m, 3H), 1.17-1.08 (m, 4H), 0.86 (d, J = 7.0 Hz, 3H), 0.74 (dd, J = 15.9, 5.4 Hz, 3H); ¹³C NMR (101

MHz, CDCl₃) δ 216.03, 169.57, 167.92, 167.15, 163.04, 155.27, 138.26, 116.05, 86.86, 73.60, 68.57, 62.64, 57.16,

44.96, 44.45, 43.51, 42.95, 40.87, 35.79, 34.99, 33.46, 32.86, 29.42, 25.91, 25.37, 23.82, 15.82, 13.89, 10.42;

HRMS (ES) calcd [M + H]⁺for [C₃₀H₄₅N₅O₅S 588.3214, found 588.3219.

14-O-[(2-(bis (Ethyl)-amino) acetamido-6-aminopyrimidine-2-yl) thioacetyl] mutilin (5b)

Compound 5b was prepared according to the general procedure with a reaction time of 2 hours. The crude

product was purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 1:4) to give 52%

yield (0.96 g) of 5b as a white solid. IR (KBr): 3348, 2934, 1725, 1625, 1591, 1550, 1500, 1459, 1400, 1287, 1199,

1

1153, 1117 cm^-1; H NMR (400 MHz, CDCl₃) δ 9.53 (d, J = 20.9 Hz, 1H), 7.12-7.01 (m, 1H), 6.50 (dt, J = 20.5,

10.3 Hz, 1H), 5.74 (d, J = 8.5 Hz, 1H), 5.24 (ddd, J = 18.5, 14.2, 3.7 Hz, 2H), 4.99 (d, J = 46.1 Hz, 2H), 3.95-3.64

(m, 2H), 3.35 (dd, J = 9.2, 6.8 Hz, 1H), 3.13 (s, 1H), 2.92 (d, J = 29.8 Hz, 1H), 2.62 (q, J = 7.0 Hz, 3H), 2.37-2.27

(m, 1H), 2.27-2.06 (m, 3H), 2.01 (dd, J = 14.9, 7.5 Hz, 1H), 1.73 (dd, J = 17.5, 10.6 Hz, 1H), 1.70-1.47 (m, 4H),

1.44 (s, 4H), 1.39 -1.20 (m, 3H), 1.14 (t, J = 6.7 Hz, 4H), 1.07 (t, J = 7.1 Hz, 5H), 0.86 (d, J = 7.0 Hz, 3H), 0.75 (d,

J = 6.9 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 216.03, 171.03, 167.87, 167.17, 163.04, 155.25, 138.25, 116.06,

86.80, 73.60, 68.55, 57.16, 47.77, 44.45, 43.54, 42.96, 40.87, 35.78, 34.99, 33.47, 32.90, 29.43, 25.90, 25.37, 23.82,

ACCEPTED MANUSCRIPT

15.83, 13.90, 11.04, 10.43; HRMS (ES) calcd [M + H]⁺for C₃₂H₄₉N₅O₅S 616.3527, found 616.3521.

14-O-[(2-(Pyrrolidine-1-yl)-acetamido-6-aminopyrimidine-2-yl) thioacetyl] mutilin (5c)

Compound 5c was prepared according to the general procedure with a reaction time of 2 hours. The crude product

was purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 1:4) to give 49% yield (0.90

g) of 5c as a white solid. IR (KBr): 3357, 2934, 1720, 1624, 1592, 1550, 1500, 1465, 1200, 1018, 939 cm⁻¹; 1H

NMR (400 MHz, CDCl₃) δ 9.25 (s, 1H), 7.04 (d, J = 15.2 Hz, 1H), 6.52-6.34 (m, 1H), 5.67 (d, J = 8.5 Hz, 1H),

5.29-5.07 (m, 2H), 4.90 (d, J = 25.6 Hz, 2H), 3.83-3.64 (m, 2H), 3.33-3.25 (m, 1H), 3.19 (s, 1H), 2.59 (s, 4H),

2.28-2.20 (m, 1H), 2.19-2.07 (m, 2H), 2.07-1.90 (m, 3H), 1.78 (s, 4H), 1.69 (dd, J = 14.4, 2.4 Hz, 1H), 1.63-1.41

(m, 4H), 1.40-1.33 (m, 4H), 1.23 (dt, J = 14.5, 9.4 Hz, 3H), 1.10-1.01 (m, 4H), 0.80 (d, J = 7.0 Hz, 3H), 0.66 (t, J =

13.1 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 216.02, 169.96, 167.86, 167.13, 163.03, 155.34, 138.27, 116.04,

86.88, 73.60, 68.53, 58.96, 57.15, 53.60, 44.45, 43.50, 42.95, 40.87, 35.79, 34.99, 33.46, 32.89, 29.43, 25.90, 25.37,

23.82, 23.08, 15.81, 13.90, 10.43; HRMS (ES) calcd [M + H]⁺for C₃₂H₄₇N₅O₅S 614.3370, found 614.3371.

14-O-[(2-(Piperidine-1-yl)-acetamido-6-aminopyrimidine-2-yl) thioacetyl] mutilin (5d)

Compound 5d was prepared according to the general procedure with a reaction time of 2 hours. The crude

product was purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 1:4) to give 47%

yield (0.87 g) of 5d as a white solid. IR (KBr): 3369, 2933, 1734, 1605, 1581, 1549, 1511, 1458, 1401, 1299, 1251,

1

1160, 1116, 1069, 1024, 846 cm^-1; H NMR (400 MHz, CDCl₃) δ 9.31 (d, J = 15.1 Hz, 1H), 7.05-6.96 (m, 1H),

6.54-6.37 (m, 1H), 5.66 (t, J = 8.4 Hz, 1H), 5.28-5.08 (m, 2H), 4.96-4.75 (m, 2H), 3.78-3.64 (m, 2H), 3.34-3.24 (m,

1H), 2.97 (s, 1H), 2.42 (s, 4H), 2.24 (dd, J = 13.2, 6.3 Hz, 1H), 2.19-2.06 (m, 2H), 1.95 (dt, J = 13.8, 12.6 Hz, 3H),

1.68 (d, J = 14.1 Hz, 1H), 1.61-1.54 (m, 6H), 1.48-1.40 (m, 3H), 1.38 (d, J = 5.2 Hz, 4H), 1.29-1.18 (m, 3H),

1.11-1.03 (m, 4H), 0.80 (d, J = 7.0 Hz, 3H), 0.67 (dd, J = 10.8, 4.5 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 216.00,

169.81, 167.88, 167.13, 163.04, 155.29, 138.26, 116.03, 86.85, 73.60, 68.53, 62.03, 57.16, 53.96, 44.45, 43.55,

42.95, 40.87, 35.77, 34.99, 33.46, 32.92, 29.42, 25.90, 25.37, 24.94, 23.81, 22.56, 15.82, 13.90, 10.42; HRMS (ES)

calcd [M + H]⁺for C₃₃H₄₉N₅O₅S 628.3527, found 628.3524.

14-O-[(2-(Morpholine-4-yl)-acetamido-6-aminoprimidine-2-yl) thioacetyl] mutilin (5e)

Compound 5e was prepared according to the general procedure with a reaction time of 2 hours. The crude

product was purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 1:3) to give 51%

yield (0.96 g) of 5e as a white solid. IR (KBr): 3422, 2919, 1719, 1637, 1547, 1500, 1466, 1287, 1118, 1047

cm^-1.¹H NMR (400 MHz, CDCl3) δ 9.26 (s, 1H), 6.99 (s, 1H), 6.44 (dd, J = 17.4, 11.0 Hz, 1H), 5.67 (d, J = 8.4 Hz,

1H), 5.18 (dd, J = 58.3, 14.2 Hz, 2H), 4.92 (s, 2H), 3.71 (dd, J = 26.5, 17.1 Hz, 2H), 3.29 (s, 1H), 3.03 (s, 1H),

ACCEPTED MANUSCRIPT

2.81-2.65 (m, 2H), 2.31 (dd, J = 20.0, 10.0 Hz, 2H), 2.27-2.21 (m, 1H), 2.18-2.00 (m, 3H), 2.00-1.92 (m, 2H), 1.87

(dd, J = 26.1, 14.8 Hz, 3H), 1.69 (d, J = 11.9 Hz, 1H), 1.67-1.61 (m, 2H), 1.60-1.40 (m, 4H), 1.40-1.30 (m, 4H),

1.20 (dd, J = 18.7, 11.5 Hz, 3H), 1.04 (d, J = 17.7 Hz, 4H), 0.80 (d, J = 6.8 Hz, 3H), 0.67 (d, J = 6.8 Hz, 3H); ¹³C

NMR (101MHz, CDCl₃) δ216.00, 168.67, 167.99, 167.07, 163.05, 155.11, 138.28, 116.04, 86.88, 73.59, 68.57,

65.77, 61.70, 57.14, 52.76, 44.45, 43.54, 42.95, 40.87, 35.76, 34.99, 33.46, 32.91, 29.41, 25.90, 25.39, 23.81, 15.84,

13.91, 10.42; HRMS (ES) calcd [M + H]⁺for C₃₂H₄₇N₅O₆S 630.3319, found 630.3315.

14-O-[(2-((3S)-3-Hydroxypyrrolidine-1-yl)-acetamido-6-aminopyrimidine-2-yl) thioacetyl] mutilin (5f)

Compound 5f was prepared according to the general procedure with a reaction time of 2 hours. The crude

product was purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 1:2) to give 65%

yield (1.23 g) of 5f as a white solid. IR (KBr): 3448, 2930, 2930, 1724, 1629, 1593, 1549, 1500, 1466, 1287, 1150,

1118 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 9.34 (s, 1H), 7.08 (d, J = 15.2 Hz, 1H), 6.49 (dt, J = 20.0, 10.0 Hz, 1H),

5.74 (d, J = 8.5 Hz, 1H), 5.24 (ddd, J = 32.5, 14.2, 4.1 Hz, 2H), 4.99 (d, J = 29.7 Hz, 2H), 3.82 (dt, J = 30.8, 10.8

Hz, 2H), 3.72 (s, 1H), 3.35 (t, J = 7.3 Hz, 1H), 3.07 (s, 1H), 2.84 (d, J = 11.4 Hz, 2H), 2.33 (dd, J = 13.7, 6.9 Hz,

1H), 2.21 (dd, J = 13.9, 10.4 Hz, 3H), 2.10 (s, 1H), 2.07-1.97 (m, 2H), 1.73 (dd, J = 22.9, 11.8 Hz, 3H), 1.63 (s, 1H),

1.58-1.49 (m, 4H), 1.46-1.40 (m, 4H), 1.36-1.24 (m, 3H), 1.18-1.07 (m, 4H), 0.87 (d, J = 7.0 Hz, 3H), 0.75 (d, J =

6.9 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 216.20, 169.56, 167.92, 167.25, 163.09, 155.14, 138.22, 116.06, 86.94,

73.60, 68.63, 63.96, 61.69, 57.16, 53.79, 44.45, 43.52, 42.95, 40.88, 35.76, 35.73, 34.96, 33.48, 32.91, 32.87, 29.42,

25.88, 25.39, 25.07, 23.80, 23.43, 15.87, 15.81, 13.92, 10.42; HRMS (ES) calcd [M + H]⁺for C₃₂H₄₉N₅O₆S

630.3320, found 630.3329.

14-O-[(2-(4-Hydroxypiperidine-1-yl)-acetamido-6-aminopyrimidine-2-yl) thioacetyl] mutilin (5g)

Compound 5g was prepared according to the general procedure with a reaction time of 2 hours. The crude

product was purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 1:2) to give 60%

1

yield (1.16 g) of 5g as a white solid. IR (KBr): 3446, 3367, 2928, 2860, 1719, 1685, 1295, 1117, 1012 cm^-1; H

NMR (400 MHz, CDCl₃) δ 9.22 (s, 1H), 7.05 (s, 1H), 6.51 (dd, J = 17.4, 11.0 Hz, 1H), 5.74 (d, J = 8.4 Hz, 1H),

5.25 (dd, J = 57.9, 14.2 Hz, 2H), 4.95 (s, 2H), 3.88-3.69 (m, 6H), 3.41-3.29 (m, 1H), 3.11 (s, 2H), 2.71-2.47 (m,

4H), 2.42-2.27 (m, 1H), 2.27-2.12 (m, 2H), 2.11-1.97 (m, 2H), 1.84-1.68 (m, 2H), 1.67-1.47 (m, 4H), 1.47-1.39 (m,

4H), 1.39-1.19 (m, 3H), 1.18-1.06 (m, 4H), 0.86 (d, J = 7.0 Hz, 3H), 0.74 (d, J = 6.9 Hz, 3H); ¹³C NMR (101 MHz,

CDCl₃) δ 216.09, 169.39, 167.93, 167.15, 163.06, 155.19, 138.27, 116.05, 86.88, 73.60, 68.57, 61.19, 59.39, 57.16,

50.38, 44.45, 43.53, 42.95, 40.88, 35.77, 34.98, 33.21, 29.42, 25.39, 23.81, 15.83, 13.93, 13.19, 10.43; HRMS (ES)

calcd [M + H]⁺for C₃₃H₄₉N₅O₆S 644.3476, found 644.3473.

ACCEPTED MANUSCRIPT

14-O-[(2-((3R)-3-Hydroxymethylpiperidine-1-yl)-acetamido-6-aminopyrimidine-2-yl) thioacetyl] mutilin

(5h)

Compound 5h was prepared according to the general procedure with a reaction time of 2 hours. The crude product

was purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 1:2) to give 67% yield (1.32

g) of 5h as a white solid. IR (KBr): 3448, 2930, 1720, 1625, 1593, 1550, 1500, 1466, 1400, 1286, 1201, 1153, 1117

cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 9.33 (d, J = 5.2 Hz, 1H), 6.98 (t, J = 8.4 Hz, 1H), 6.42 (dd, J = 17.3, 11.0 Hz,

1H), 5.66 (dd, J = 8.3, 2.3 Hz, 1H), 5.33-5.03 (m, 2H), 4.96 (d, J = 18.1 Hz, 2H), 3.84-3.63 (m, 2H), 3.58-3.48 (m,

2H), 3.33-3.23 (m, 1H), 3.01 (qd, J = 16.8, 4.6 Hz, 2H), 2.67 (dd, J = 30.2, 10.1 Hz, 2H), 2.23 (dd, J = 17.0, 9.9 Hz,

3H), 2.19-2.06 (m, 3H), 2.05 – 1.99 (m, 1H), 1.99-1.82 (m, 3H), 1.68 (d, J = 13.7 Hz, 2H), 1.61 (d, J = 7.4 Hz, 1H),

1.49 (ddd, J = 49.0, 15.8, 7.2 Hz, 4H), 1.40-1.34 (m, 4H), 1.31-1.18 (m, 3H), 1.04 (d, J = 17.5 Hz, 4H), 0.80 (d, J =

7.0 Hz, 3H), 0.71-0.60 (m, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 216.10, 169.69, 167.87, 167.14, 163.04, 155.27,

138.24, 116.06, 86.88, 73.60, 68.53, 61.67, 59.39, 57.16, 53.36, 44.45, 43.54, 42.94, 40.88, 38.15, 35.76, 34.98,

33.47, 32.93, 31.29, 30.71, 29.42, 25.89, 25.38, 23.81, 15.84, 13.94, 13.19, 10.43; HRMS (ES) calcd [M + H]⁺for

C₃₄H₅₁N₅O₆S 658.3628, found 658.3626.

14-O-[(2-(4-Hydroxyethylpiperidine-1-yl)-acetamido-6-aminopyrimidine-2-yl) thioacetyl] mutilin (5i)

Compound 5i was prepared according to the general procedure with a reaction time of 2 hours. The crude

product was purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 1:2) to give 74%

yield (1.49 g) of 5i as a white solid. IR (KBr): 3448, 2932, 1720, 1625, 1593, 1550, 1466, 1400, 1287, 1201, 1154,

1117, 1018 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ9.32 (s, 1H), 6.99 (s, 1H), 6.43 (dd, J = 17.4, 11.0 Hz, 1H), 5.65 (d,

J = 8.4 Hz, 1H), 5.28-5.07 (m, 2H), 4.95 (s, 2H), 4.39 (dd, J = 6.8, 4.8 Hz, 1H), 4.11-3.99 (m, 1H), 3.80 – 3.61 (m,

2H), 3.28 (s, 1H), 3.24 (d, J = 4.2 Hz, 1H), 2.94 (dd, J = 14.7, 8.1 Hz, 1H), 2.75 (dt, J = 17.7, 7.5 Hz, 2H), 2.50 (dd,

J = 14.8, 8.6 Hz, 1H), 2.30-2.09 (m, 4H), 2.02 (s, 1H), 1.98 (s, 1H), 1.97-1.86 (m, 1H), 1.79 (dt, J = 13.7, 5.8 Hz,

1H), 1.68 (d, J = 13.9 Hz, 1H), 1.63-1.42 (m, 4H), 1.42-1.35 (m, 4H), 1.30 (dd, J = 18.2, 15.6 Hz, 2H), 1.23-1.12

(m, 4H), 1.09-1.01 (m, 4H), 0.80 (d, J = 7.0 Hz, 3H), 0.66 (d, J = 6.9 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ

216.07, 169.52, 167.83, 167.21, 163.09, 155.19, 138.28, 116.04, 86.91, 73.59, 70.41, 68.63, 62.20, 59.39, 58.55,

57.14, 51.95, 44.45, 42.95, 40.88, 35.76, 34.98, 34.21, 33.47, 29.41, 25.90, 25.39, 23.81, 20.03, 15.79, 13.91, 13.19,

10.42; HRMS (ES) calcd [M + H]⁺for C₃₄H₅₃N₅O₆S 672.3789, found 672.3783.

4.2. Biological evaluation

MIC Testing

As described previously [22-24], The MIC values were tested using the agar dilution method according to the

ACCEPTED MANUSCRIPT

National Committee for Clinical Laboratory Standards (NCCLS, 4^thed). Briefly, compounds were dissolved in 25%

DMSO to a solution with concentration of 1280 µg/mL. Tiamulin fumarate used as a reference drug was directly

dissolved in 10 mL distilled water. All the solutions were then diluted two-fold with distilled water to provide 11

dilutions (final concentration is 0.625 µg/mL). A 2 mL volume of the 2-fold serial dilution of each test

compound/drug was incorporated into 18 mL hot MuellereHinton agar medium, which resulted in the final

concentration of each dilutions decreasing ten fold. Inoculums, including four Gram (+),MRSA-29213,

MRSA-33591, S. aureus and B. subtilis, and one Gram (-), E. coli were prepared from blood slants and adjusted to

approximately 10⁵-10⁶CFU/mL with sterile saline (0.90% NaCl). A 10 µL amount of bacterial suspension was

spotted onto Muellere-Hinton agar plates containing serial dilutions of the compounds/drug. The plates were

incubated at 36.5 ^oC for 24-48 h. The same procedure was repeated in triplicate.

Oxford Cup Assays

Inoculums were prepared in 0.9% saline using McFarland standard and spread uniformly on nutrient agar plates. The

320 and 160 µg/mL of all the compounds prepared in the same manner as the MIC test were added individually into the

Oxford cups which were placed at equal distances above the agar surfaces. The zone of inhibition for each concentration

was measured after a 24 -36 h cubation at 37 °C. The same procedure was repeated in triplicate.

Bactericidal Time-Kill Kinetics

S. aureus and MRSA-29213 were cultured in MuellereHinton broth at 37 °C for 6 h with shaking and then

diluted to approximately 1.5 ×10⁶and 1.4 ×10⁷CFU/mL, respectively. Test compound 3 and tiamulin fumarate

having the final concentrations of 1 × MIC and 6 × MIC was inoculated with the aliquots of bacteria resuspended in

fresh media. After specified time intervals (0, 1, 2, 4, 6, 12 and 24 h), 20 ꢀL aliquots were serially diluted 10-fold

in 0.9% saline, plated on sterile MuellereHinton agar plates, and incubated at 37 °C for 24 h. The viable colonies

were counted and represented as log₁₀(CFU/mL). The same procedure was repeated in triplicate.

4.3. Molecular modeling

Docking studies were performed using Homdock software in the Chil²package [27]. The starting conformations

of compounds were obtained from alignment to tiamulin in the X-ray crystal stucture (PDB ID: 1XBP) [28] which

was used as a control to evaluate the accuracy of the docking performance. We performed the molecular

superposition using the graph-based molecular alignment (GMA) method. Then the optimization of interaction

between the receptor and compounds to remove partial overlap using Glamdock, and the binding affinity was

evaluated by Chil²Score. All the compounds were built with Avogadro software [29], including a 5000 steps

Steepest Descent and 1000 steps Conjugate Gradients optimization using MMFF94 force field.

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4.4. Mouse systemic infection model

The in vivo efficacy of compound 3 was determined in 6-8 weeks old Balb/c mice (Laboratory Animal Center of

Lanzhou University, Lanzhou, China). All mice were maintained at the central animal facility at the Lanzhou

Institute of Husbandry and Pharmaceutical Sciences of CAAS, with a constant 12 h light-dark cycle and free access

to food and water. After acclimating on site for at least one week, mice were rendered neutropenic upon treatment

with 150 mg/kg cyclophosphamide intraperitoneally for four days and with 100 mg/kg for one day prior to

inoculation, respectively. The neutropenic mice (10 per group) were anesthetized with isoflurane and then received a

0.5 mL MRSA-29213 inoculum of 10⁹CFU/mL via intraperitoneal (ip) injection. About 1 h after infection, the

mice were then intravenously (iv) administered compound

3 dissolved in 0.5 mL vehicle (ethyl

Oleate:1,2-Propanediol:Tween-80 sterile:water = 4:10:31:55) at doses of 2, 3, 4.46, 6.64, and 10 mg/kg body

weight. Tiamulin fumarate was used as a reference drug in the same manner at the same doses as 3. Mice were

euthanized by cervical dislocation under anesthesia with 5% isoflurane and were placed in 70% ethanol for 10

minutes at pre-determined experimental endpoints, including consistent or rapid body weight loss exceeding 20%

of the body weight, severe mucous excretion from eyes, severe respiratory distress, ulceration/infection of the

injection site or obvious distress for any reason. The healthy survival of mice at 72 h after infection was used as the

endpoint. All animals were observed twice daily for symptoms and mortality for the animal procedures. The

survival of the mice at 7 d after infection was used as the end-point, and the ED₅₀was calculated by the Bliss

method [30].

4.5. Ethics Statement

All animal experiments were performed in accordance with the Ethical Principles in Animal Research and were

approved by the Committee for Ethics in Laboratory Animal Center of Lanzhou University (number:

SCXK2013-0002).

Acknowledgments

The authors are grateful for financial support from National Key Technology Support Program (No.

2015BAD11B02) and Agricultural Science and Technology Innovation Program (ASTIP, No.

CAAS-ASTIP-2014-LIHPS-04).

Appendix A. Supplementary data

Supplementary data related to this article can be found.

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Table 1. in vitro antibacterial activity (MIC) of the synthesized pleuromutilin derivatives.

MICs (µg/mL)

Compounds

MRSA-29213 MRSA-33591 S. aureus

B. subtilis E. coli

3

5a

0.125

1

0.0625

0.5

0.0625

0.25

2

4

2

4

2

1

2

8

4

8

4

8

4

8

4

5b

0.5

0.25

0.5

0.125

0.25

5c

0.5

5d

0.25

0.125

0.25

0.125

1

0.5

5e

1

0.125

0.0625

0.125

0.0625

5f

0.25

0.125

0.5

5g

5h

5i

Tiamulin

0.25

0.125

ACCEPTED MANUSCRIPT

Table 3. Bind freeenergy, number of noncovalent molecular interaction and RMSD.

Noncovalent molecular interaction

Atom of Compound

∆G_b

RMSD

(Å)

Compound

(kcal/mol)

interaction

H-bonding

Residue

Distance(Å)

OH (8-membered ring)

C=O (ester)

G-2484

G-2044

G-2484

G-2044

A-2482

A-2045

G-2044

A-2045

C-2565

A-2045

A-2482

G-2484

G-2044

A-2418

G-2044

U-2564

2.0

2.1

2.3

2.4

2.1

2.3

2.2

1.9

2.1

2.3

1.7

2.4

2.2

2.3

1.9

2.4

2.7

2.3

2.4

2.2

2.3

1.8

2.0

1.7

2.6

1.9

2.2

2.4

2.1

2.3

2.4

1.8

2.4

2.5

2.3

2.4

2.0

1.8

2.1

2.2

2.3

H-bonding

3

-12.44

-12.01

C=O (ester)

0.87

0.97

N (pyrimidine ring)

NH₂(pyrimidine ring)

OH (8-membered ring)

C=O (ester)

5a

C=O (ester)

N (pyrimidine ring)

NH₂(pyrimidine ring)

C=O (ester)

5b

5c

5d

-10.30

-9.22

-9.42

1.05

1.02

1.28

N (pyrimidine ring)

NH₂(pyrimidine ring)

C=O (ester)

N (pyrimidine ring)

NH₂(pyrimidine ring)

C=O (ester)

5e

-10.77

0.99

N (pyrimidine ring)

NH₂(pyrimidine ring)

NH₂( pyrimidine ring )

N (pyrolidine )

5f

-9.78

-9.81

1.08

1.18

NH₂(pyrimidine ring)

OH (8-membered ring)

C=O (amido bond)

N (pyrimidine ring)

NH₂(pyrimidine ring)

OH (piperidine)

5g

5h

-13.28

1.19

OH (piperidine)

C=O (5-membered ring )

NH₂(pyrimidine ring)

OH (piperidine)

5i

-11.39

-10.20

1.25

0.87

OH (8-membered ring)

C=O (ester)

G-2484

G-2044

Tiamulin

C=O (ester)

ACCEPTED MANUSCRIPT

Article Doi

Synthesis and antibacterial activities of novel pleuromutilin derivatives with a substituted pyrimidine moiety

DOI: 10.1016/j.ejmech.2016.11.054

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Article abstract of DOI:10.1016/j.ejmech.2016.11.054

Full text of DOI:10.1016/j.ejmech.2016.11.054

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