Synthesis and antimicrobial activity of the hybrid molecules between amoxicillin and derivatives of benzoic acid

Article abstract of DOI:10.1002/ddr.21739

Due to the increasing problem of bacterial resistance worldwide, the demand for new antibiotics is becoming increasingly urgent. We wished to: (a) prepare hybrid molecules by linking different pharmacophores by chemical bonds; (b) investigate the antib acterial activity of these hybrids using drug-sensitive and drug-resistant pathogens in vitro and vivo. A series of hybrid molecules with a diester structure were designed and synthesized that linked amoxicillin and derivatives of benzoic acid via a methylene bridge. Synthesized compounds were evaluated for activities against Gram-positive bacteria (Staphylococcus aureus American Type Culture Collection [ATCC] 29213, ATCC 11632; methicillin-resistant S. aureus [MRSA] 11; Escherichia coli ATCC 25922) and Gram-negative bacteria (Salmonella LS677, GD836, GD828, GD3625) by microdilution of broth. Synthesized compounds showed good activity against Gram-positive and Gram-negative bacteria in vitro. In particular, amoxicillin-p-nitrobenzoic acid (6d) showed good activity against Salmonella species and had better activity against methicillin-resistant S. aureus (minimum inhibitory concentration [MIC] = 64 μg/ml) than the reference drug, amoxicillin (MIC = 128 μg/ml). Amoxicillin-p-methoxybenzoic acid (6b) had the best antibacterial activity in vivo (ED₅₀ = 13.2496 μg/ml). The hybrid molecules of amoxicillin and derivatives of benzoic acid synthesized based on a diester structure can improve the activity of amoxicillin against Salmonella species and even improve the activity against MRSA.

Full text of DOI:10.1002/ddr.21739

Received: 20 May 2020
DOI: 10.1002/ddr.21739
Revised: 7 August 2020
Accepted: 27 August 2020
R E S E A R C H A R T I C L E
Synthesis and antimicrobial activity of the hybrid molecules
between amoxicillin and derivatives of benzoic acid
Zhonglin Li1
| Hao Lin1 | Junwen Zhou1 | Liangzhu Chen2 | Zhikun Pan2
|
Binghu Fang1,2
1Department of Veterinary Medicine, South
China Agricultural University, Guangzhou,
China
Abstract
Due to the increasing problem of bacterial resistance worldwide, the demand for
new antibiotics is becoming increasingly urgent. We wished to: (a) prepare hybrid
molecules by linking different pharmacophores by chemical bonds; (b) investigate the
antib acterial activity of these hybrids using drug-sensitive and drug-resistant patho-
gens in vitro and vivo. A series of hybrid molecules with a diester structure were
designed and synthesized that linked amoxicillin and derivatives of benzoic acid via a
methylene bridge. Synthesized compounds were evaluated for activities against
Gram-positive bacteria (Staphylococcus aureus American Type Culture Collection
[ATCC] 29213, ATCC 11632; methicillin-resistant S. aureus [MRSA] 11; Escherichia
coli ATCC 25922) and Gram-negative bacteria (Salmonella LS677, GD836, GD828,
GD3625) by microdilution of broth. Synthesized compounds showed good activity
against Gram-positive and Gram-negative bacteria in vitro. In particular, amoxicillin-
p-nitrobenzoic acid (6d) showed good activity against Salmonella species and had bet-
ter activity against methicillin-resistant S. aureus (minimum inhibitory concentration
[MIC] = 64 μg/ml) than the reference drug, amoxicillin (MIC = 128 μg/ml).
Amoxicillin-p-methoxybenzoic acid (6b) had the best antibacterial activity in vivo
(ED50 = 13.2496 μg/ml). The hybrid molecules of amoxicillin and derivatives of
benzoic acid synthesized based on a diester structure can improve the activity of
amoxicillin against Salmonella species and even improve the activity against MRSA.
2Chemical R&D Department, Guangdong
Dahuanong Animal Health Products Co. Ltd.,
Yunfu, China
Correspondence
Binghu Fang, Department of Veterinary
Medicine, South China Agricultural University,
Guangzhou 510642, China.
Email: fangbh@scau.edu.cn
Funding information
National Science Foundation of China, Grant/
Award Number: 31672601; South China
Agricultural University Test Center; Chinese
Academy of Sciences
K E Y W O R D S
amoxicillin, antibacterial activity, benzoic-acid derivative, drug design, hybrid molecule
1
|
INTRODUCTION
health (Bennett, 2010; Goossens
Webb, 2005).
& Herman, 2005; Lim &
The abuse of antibiotics in intensive farming in developing countries
can: (a) result in the residue of antibiotics in animal-derived products;
(b) increase the number of antibiotic-resistant bacteria in the gut of
The growing problem of bacterial resistance has spurred our
interest in discovering new and more efficacious antimicrobial agents
to address the problem of animal-borne bacterial resistance at the
source (Chauhan, Hati, Priyadarshini, & Sen, 2019). However, due to
the long research cycle of traditional methods of new-drug develop-
ment and the expense of research, scholars are beginning to seek new
tools for drug development (Dickson & Gagnon, 2004; Dimasi,
animals (Christy, Sampson, Edson,
& Anthony, 2018; Sørum &
Sunde, 2001). The emergence of multi-drug resistant bacteria, espe-
cially Gram-positive bacteria (e.g., penicillin-resistant Streptococcus
pneumoniae, methicillin-resistant Staphylococcus aureus (MRSA), and
vancomycin-resistant enterococci) poses a major threat to public
Hansen,
& Grabowski, 2003). The concept of pharmacophore
Drug Dev Res. 2020;1–9.
wileyonlinelibrary.com/journal/ddr
© 2020 Wiley Periodicals LLC
1

2
LI ET AL.
FIGURE 1 Benzoic acid and its
derivatives
Benzoic acid (1a)
Anisic acid (1b)
R=H
R=
4-Propoxybenzoic acid (1c) R=
p-Nitrobenzoic acid (1d)
Probenecid (1e)
R=NO2
R=
hybridization has attracted increasing interest from pharmaceutical
chemists. According to the report, a rational drug design method
based on the synthesis of a double ligand or a multi-ligand can pro-
duce a drug with better clinical effects than a single targeted drug.
(Mandal, Moudgil, & Mandal, 2009). Moreover, certain hybrid mole-
cules exhibit better pharmacological advantages and lower toxicity
than simple combination formulations.(Catane, Kaufman, Madajewicz,
Mittelman, & Murphy, 1978; English, Girard, & Haskell, 1984).
(Bronfenbrenner & Favour, 1945). In recent years, studies have shown
that probenecid can increase the serum concentration and prolong
the duration of action of BLAs such as penicillin by inhibiting the
secretion of these weak organic acids in the proximal and distal
tubules of the kidney. Probenecid is a derivative of benzoic acid used
to treat chronic gout, and acts by competitively inhibiting renal-
tubular reabsorption of uric acid and increasing its excretion (Brown,
Zemcov,
&
Clarke, 1993; Overbosch, Gulpen, Hermans,
&
Currently, β-lactam antibiotics (BLAs) are the most important
anti-infective drugs used in the clinical treatment of animals. The
semi-synthetic penicillin amoxicillin was developed in the 1960s. The
spectrum and effect of amoxicillin against most bacteria in vitro are
basically identical to those of ampicillin. However, the bactericidal
effect of amoxicillin against various bacteria is faster and stronger
than that of ampicillin. Amoxicillin has been used widely in veterinary
clinics because of its stability, rapid absorption in the body, strong
bactericidal power, few side-effects, diverse preparations, convenient
combination with other drugs, and low price.
Mattie, 1988; Tanizaki et al., 2015).
To investigate the antibacterial activity and pharmacokinetic char-
acteristics of the heterozygous molecules of amoxicillin and deriva-
tives of benzoic acid (Figure 1), we linked amoxicillin and benzoic-acid
derivatives via a methylene bridge. The synthetic route is shown in
Scheme 1. We also investigated the antibacterial activity of hybrids
using drug-sensitive and drug-resistant pathogens by standard
methods in vitro. Results indicated that these molecules had excellent
properties against Gram-negative bacteria such as Salmonella species.
Also, compound 6d had good activity against MRSA.
Studies have shown that BLAs inhibit the formation of bacterial
cell walls. The target molecules for the action of BLAs on bacterial cell
walls are penicillin-binding proteins because of the stereochemical
similarity between β-lactam and D-alanine-dialanine substrates
(Yocum, Waxman, Rasmussen, & Strominger, 1979). In this way, a
BLA can form a lethal covalent penicillin–enzyme complex with a tran-
speptidase, which is used to block normal transpeptidation in bacteria
(Wilke, Lovering, & Strynadka, 2005). Pharmacodynamic studies have
shown that BLAs are time-dependent antibacterial drugs, and that the
key to enhancing their efficacy is to maximize the time when the
plasma concentration is greater than the minimum inhibitory concen-
2
|
MATERIALS AND METHODS
| Chemicals and instruments
2.1
The starting materials, reagents, and solvents used in the present
study were purchased from J & K Chemicals (Beijing, China) or Sigma–
Aldrich (Saint Louis, MO). Infrared (IR) spectroscopy was carried out
on an Affinity-1 spectrometer (Shimadzu, Kyoto, Japan) using
potassium-bromide disks. Proton nuclear magnetic resonance (1H
NMR) spectroscopy was undertaken on a spectrometer (AVANCE
AV600; Bruker Billerica, MA) in dimethylsulfoxide (DMSO). Mass
spectroscopy (MS) was carried out with an Orbitrap™ Elite system
(Thermo Fisher Scientific, Waltham, MA) using the electrospray ioni-
zation (ESI) method.
tration (MIC) (Hohlfelder, Kubiak, Degrado, Reardon,
Szumita, 2016).
&
Benzoic acid is widely found in nature in the form of free acids,
esters, or derivatives thereof. It has been well documented that vari-
ous derivatives of benzoic acid have great potential for antibacterial,
antiviral, and anti-inflammatory effects (Inglis et al., 2007; Sapra,
Kumar, Kakkar, & Narasimhan, 2014; Selvam, Breitenbach, Borysko, &
Drach, 2009). In the 1940s, it was reported that benzoic acid
(Figure 1) increased the blood level of penicillin by 4–8-fold, and that
the time benzoic acid remained in the blood was extended
The preparation of target compounds is outlined in Scheme 1.
Identification of the structures of the synthesized compounds was
supported by data from IR, 1H NMR, and 13C NMR spectroscopy. The
results of HR-MS (ESI) were in agreement with the proposed
structures.

LI ET AL.
3
SCHEME 1 Synthesis of hybrid molecules between amoxicillin and derivatives of benzoic acid. Reagents and conditions:
(a) Chloroiodomethane, triethylamine, DMF, room temperature, stirring for 5 hr. (b) NaI, acetone aqueous solution, room temperature, stirring in
the dark for 18 hr. (c) Methyl acetoacetate, DMF, K2CO3, stirring for 2 hr at room temperature followed by another 4 hr at 0 ꢀC. (d) DMF, 0–5 ꢀC,
stirring for 4 hr. (e) Acetone aqueous solution, 10% HCl, stirring for 0.5 hr
2.2
|
General procedure for the synthesis of
HR-MS (ESI) Calcd for C9H9ClO3 (M + H+): 201.5408; Found:
200.5411; purity:98.64% (determined by HPLC).
benzoic chloromethyl ester (2a–e)
Chloromethyl 4-ethoxybenzoate (2c) White solid; yield: 46.63%; Rf:
0.65; IR (KBr, cm−1): 2,968.45, 2,877.79, 1,735.93 (C=O), 1,606.70,
1,510.26, 1,438.90, 1,332.81, 1,257.59, 1,168.86, 1,080.14, 1,010.70;
1H NMR (600 MHz, Chloroform-d) δ 8.03 (d, 2H), 1H NMR (600 MHz,
Chloroform-d) δ 8.03 (d, J = 8.2 Hz, 2H), 6.94 (d, J = 8.2 Hz, 2H), 5.95
(s, 2H, CH2Cl), 3.99 (s, 2H), 1.85 (q, J = 7.1 Hz, 2H), 1.06 (t, J = 7.4 Hz,
3H); 13C NMR (151 MHz, Chloroform-d) δ 164.24, 163.81, 132.22,
120.64, 114.34, 69.79, 69.20, 22.41, 10.42.
To a solution of 1a (7.32 g, 60 mmol) in dimethylformamide
(50 ml) was added triethylamine (8 ml, 58 mmol) and
chloroiodomethane (28 ml, 0.38 mol). The mixture was stirred for
5 hr at room temperature. After dilution with ethyl acetate
(100 ml), the mixture was washed with water (3 × 100 ml) and
then by brine (100 ml). The mixture was dried and evaporated in
vacuo to leave the desired compound: a yellowish oil. The residue
underwent chromatography using petroleum ether/ethyl acetate
(4:1, v:v).
HR-MS (ESI) Calcd for C11H13ClO3 (M + H+): 229.6723; Found:
229.6726; purity:97.51% (determined by HPLC)550.
Chloromethyl 4-nitrobenzoate (2d) was prepared according to step
1. White solid; yield: 36.74%; Rf: 0.77; IR (KBr, cm−1): 3,005.10 (ben-
zene ring), 1,737.86, 1,604.77, 1,525.69, 1,433.11, 1,338.60,
1,253.73, 1,082.07, 1,010.70; 1H NMR (600 MHz, Chloroform-d) δ
8.35–8.27 (dd, 4H), 6.00 (s, 2H, CH2Cl); 13C NMR (151 MHz,
Chloroform-d) δ 162.86, 151.09, 133.93, 131.21, 123.74, 69.52.
HR-MS (ESI) Calcd for C8H6ClNO4 (M + H+): 216.5890; Found:
216.5887; purity:98.32% (determined by HPLC).
Chloromethyl benzoate (2a) White solid; yield: 45.42%; Rf: 0.75; IR
(KBr, cm−1): 2,987.74, 1,741.72 (C=O), 1,597.06, 1,444.68, 1,342.46,
1,255.66, 1,174.65, 1,078.21; 1H NMR (600 MHz, Chloroform-d) δ
8.10–8.06 (d, 2H), 7.61 (t, J = 7.4, 1.4 Hz, 1H), 7.46 (t, J = 7.8 Hz, 2H),
5.96 (s, 2H, CH2Cl); 13C NMR (151 MHz, Chloroform-d) δ 164.54,
133.94, 130.06, 128.62, 69.30.
HR-MS (ESI) Calcd for C8H7ClO2 (M + H+): 171.5273; Found:
171.5276; purity:97.85% (determined by HPLC).
Chloromethyl 4-(N,N-dipropylsulfamoyl)benzoate (2e) was prepared
according to step 1. White solid; yield: 32.52%; Rf: 0.0.72; IR (KBr,
cm−1): 2,972.31, 2,935.66, 2,873.94, 1,747.51 (C=O), 1,597.06,
1,450.47, 1,394.53, 1,342.46, 1,251.80, 1,157.29, 1,078.21; 1H NMR
(600 MHz, Chloroform-d) δ 8.19 (d, J = 8.6 Hz, 2H), 7.90 (d,
J = 8.6 Hz, 2H), 5.97 (s, 2H, CH2Cl), 3.13–3.07 (m, 4H), 1.58–1.51 (m,
4H), 0.86 (t, J = 7.4 Hz, 6H). (t, J = 7.4 Hz, 6H); 13C NMR (151 MHz,
Chloroform-d) δ 163.34, 145.29, 131.82, 130.67, 127.15, 69.44,
Chloromethyl 4-methoxybenzoate (2b) White solid; yield: 45.42%;
Rf: 0.71; IR (KBr, cm−1): 2,970.38, 2,845.00, 1,734.01 (C=O),
1,606.70, 1,510.26, 1,448.54, 1,334.74, 1,259.52, 1,172.72, 1,082.07,
1,024.20; 1H NMR (600 MHz, Chloroform-d) δ 8.04–8.00 (d, 2H),
6.92–6.90 (d, 2H), 5.93 (s, 2H, CH2Cl), 3.83 (s, 3H); 13C NMR
(151 MHz, Chloroform-d) δ 164.19, 132.24, 120.92, 113.89, 69.22,
55.51.

4
LI ET AL.
49.91, 21.91, 11.12.
condensed with the carboxylate ion of compound 2 (0.114 mol) at
HR-MS (ESI) Calcd for C14H20ClNO4S (M + H+): 334.8270; Found:
5
ꢀC to obtain compound 5a–e. The crude product obtained under-
334.8276.
went chromatography using petroleum ether/ethyl acetate (1:1, v:v).
The condensation product was dissolved in an aqueous acetone
solution. The pH of the solution was adjusted to 2.5 with 10% HCl,
deprotected and, then, the acetone in the solution was removed by
vacuum spinning. Next, NaCl was added to the solution until it was
saturated, followed by extraction of an acetone/ethyl acetate solu-
tion (1:2, v:v). The organic layer was removed and dried under
reduced pressure. Finally, the crude product was purified by column
chromatography using silica gel to afford compound 6a–e. The crude
product obtained underwent chromatography using ethyl acetate/
methanol (5:1, v:v).
2.3
|
Synthesis of (Z)-6-(2-(4-hydroxyphenyl)-
2-([4-methoxy-4-oxobut-2-en-2-yl]amino)acetamido)-
3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-
2-carboxylic acid (3)
To a suspension of potassium carbonate (7.6 g, 55 mM) in 10 ml of
dimethylformamide was added 10.8 ml (100 mM) of methyl
acetoacetate and 20.97 g (50 mM) of amoxicillin trihydrate. The reac-
tion mixture was stirred for 2 hr at room temperature followed by
another 4 hr at 0 ꢀC. The solvent was decanted and the residue
washed with 25 ml of diethyl ether. The residue was dissolved in
15 ml of acetone and the insoluble material filtered through celite.
The filtrate was diluted with 15 ml of isopropanol and evaporated in
vacuo to remove acetone. Recrystallization was induced by scratching,
and the resulting suspension was maintained for 12 hr at 0 ꢀC. The
residue underwent chromatography using ethyl acetate/methanol
(3:2, v:v). And the precipitated solid was washed with cold isopropanol
and diethyl ether to give a fluffy white solid.
2.4.1
|
(Benzoyloxy)methyl(Z)-
6-(2-(4-hydroxyphenyl)-2-([4-methoxy-4-oxobut-2-en-
2-yl]amino)acetamido)-3,3-dimethyl-7-oxo-4-thia-
1-azabicyclo[3.2.0]heptane-2-carboxylate (5a)
Light-yellow solid; yield: 72.68%; Rf: 0.69; IR (KBr, cm−1):
3,356.14 (OH), 3,064.89, 2,974.23, 1,778.37 (C=O), 1,666.50,
1,600.92, 1,508.33, 1,448.54, 1,269.16, 1,168.86; 1H NMR
(600 MHz, Chloroform-d) δ 9.07 (d, J = 7.0 Hz, 1H, CONH), 8.07
(d, J = 7.7 Hz, 2H), 7.63 (t, J = 7.5 Hz, 1H), 7.48 (t, J = 7.7 Hz, 2H),
7.17 (d, J = 8.3 Hz, 2H), 6.91 (d, J = 9.0 Hz, 1H, NH), 6.72 (d,
J = 8.4 Hz, 2H), 6.07 (dd, J = 39.7, 5.7 Hz, 2H, OCH2O), 5.64 (dd,
J = 8.9, 4.1 Hz, 1H), 5.57–5.54 (m, 1H), 5.03 (d, J = 6.9 Hz, 1H),
4.67 (s, 1H), 4.48 (s, 1H), 3.67–3.64 (m, 3H), 1.93 (s, 3H), 1.56 (s,
3H), 1.46 (s, 3H); 13C NMR (151 MHz, Chloroform-d) δ 172.89
(C=O), 170.95 (C=O), 170.70 (C=O), 166.43 (C=O), 164.90 (C=O),
160.11, 156.70, 134.00, 130.04, 128.64, 128.36, 116.37, 85.94,
80.03 (OCH2O), 70.11, 68.01, 61.20, 60.41, 58.81, 50.47, 26.72,
21.03, 19.78.
(Z)-6-(2-(4-hydroxyphenyl)-2-([4-methoxy-4-oxobut-2-en-2-yl]
amino)acetamido)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]hep-
tane-2-carboxylic acid (3): White solid; yield: 86.98%; Rf: 0.76; IR
(KBr, cm−1): 3,302.13 (OH), 2,964.59, 1,772.58 (C=O), 1,664.57,
1,597.06, 1,508.33, 1,446.61, 1,392.61, 1,269.16, 1,172.72, 1,134.14;
1H NMR (600 MHz, DMSO-d6) δ 10.70 (s, 1H, COOH), 9.25 (d,
J = 8.3 Hz, 1H,CONH), 8.86 (d, J = 8.5 Hz, 1H, NH), 7.13 (d,
J = 8.1 Hz, 2H), 6.75 (d, J = 8.2 Hz, 2H), 5.48–5.44 (m, 1H), 5.42–5.37
(m, 1H), 5.30–5.26 (m, 1H), 4.45 (s, 1H), 3.88 (s, 1H), 3.52 (s, 3H), 1.76
(s, 3H), 1.56 (s, 3H), 1.42 (s, 3H); 13C NMR (151 MHz, DMSO-d6)
δ:173.43, 170.67, 169.93, 169.83, 160.65, 158.30, 128.91, 127.79,
116.00, 83.41, 74.63, 66.94, 64.87, 58.23, 57.57, 50.01, 31.63,
27.96, 19.66.
HR-MS (ESI) Calcd for C29H31N3O9S (M + H+): 598.6390; Found:
598.6393; purity: 98.56% (determined by HPLC).
HR-MS (ESI) Calcd for C21H25N3O7S (M + H+): 464.5050; Found:
464.5057; purity:97.41% (determined by HPLC).
2.4.2
|
([4-Methoxybenzoyl]oxy)methyl (Z)-
6-(2-(4-hydroxyphenyl)-2-([4-methoxy-4-oxobut-2-en-
2-yl]amino)acetamido)-3,3-dimethyl-7-oxo-4-thia-
1-azabicyclo[3.2.0]heptane-2-carboxylate (5b)
2.4
|
General procedure for the synthesis of the
hybrid molecules between amoxicillin and the
derivatives of benzoic acid (5a–e, 6a–e)
Light-yellow solid; yield: 68.96%; Rf: 0.54; IR (KBr, cm−1): 3,363.86
(OH), 2,972.31, 2,368.59, 1,782.23 (C=O), 1,604.77, 1,512.19,
1,446.61, 1,265.30, 1,170.79, 1,091.71, 1,022.27; 1H NMR (600 MHz,
Chloroform-d) δ 9.09 (d, J = 7.0 Hz, 1H, CONH), 8.02 (d, J = 8.5 Hz,
2H), 7.19 (d, J = 8.1 Hz, 2H), 6.95 (d, J = 8.7 Hz, 2H), 6.87 (d,
J = 9.2 Hz, 1H, NH), 6.74 (d, J = 8.1 Hz, 2H), 6.09–6.00 (m, 2H,
OCH2O), 5.64 (dd, J = 9.1, 4.2 Hz, 1H), 5.55 (d, J = 4.0 Hz, 1H), 5.03
(d, J = 6.9 Hz, 1H), 4.67 (s, 1H), 4.48 (d, J = 1.3 Hz, 1H), 3.89 (d,
Anhydrous sodium iodide (3.1 g, 32 mmol) was added to EtOAc
(15 ml) and stirred for 16 hr at room temperature. Then, the temper-
ature of the system was lowered to 0–5 ꢀC, the pH was adjusted to
3.0–7.3 with sodium hydrogen carbonate under stirring, and the
solution decolorized by dropwise addition of a 0.1 mol/L sodium
thiosulfate solution. During stirring, 15 ml of water was added
dropwise to the solution to precipitate white crystals. and the resi-
due was washed with an aqueous acetone solution (30 ml; 1:1, v:v)
to give compounds 4a–e. Then, compound 4a–e (0.1 mol) was
J = 1.3 Hz, 3H), 3.65 (s, 3H), 1.94 (s, 3H), 1.55 (s, 3H), 1.46 (s, 3H); 13
C
NMR (151 MHz, Chloroform-d) 172.84 (C=O), 170.92 (C=O),
δ

LI ET AL.
5
170.62 (C=O), 166.48 (C=O), 164.54 (C=O), 164.24, 160.05, 156.61,
132.23, 128.39, 128.19, 120.64, 116.35, 113.95, 85.98, 79.87
(OCH2O), 70.12, 68.01, 64.87, 61.21, 55.52, 50.45, 31.58,
26.71, 19.78.
2.4.5
|
((4-(N,N-Dipropylsulfamoyl)benzoyl)oxy)
methyl (Z)-6-(2-(4-hydroxyphenyl)-2-([4-methoxy-
4-oxobut-2-en-2-yl]amino)acetamido)-3,3-dimethyl-
7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-
2-carboxylate (5e)
HR-MS (ESI) Calcd for
Found: 628.6646; purity:98.69% (determined by HPLC).
C30H33N3O10S
(M + H+): 628.6650;
Light-yellow solid; yield: 76.76%; Rf: 0.59; IR (KBr, cm−1): 3,365.78
(OH), 2,968.45, 2,374.37, 1,782.23 (C=O), 1,664.57, 1,602.85,
1,510.26, 1,450.47, 1,336.67, 1,269.16, 1,163.08, 1,087.85; 1H NMR
(600 MHz, Chloroform-d) δ 9.07 (d, J = 7.0 Hz, 1H, CONH), 8.18 (d,
J = 8.1 Hz, 2H), 7.91 (d, J = 8.2 Hz, 2H), 7.17 (d, J = 8.1 Hz, 2H), 6.99
(d, J = 8.8 Hz, 1H, NH), 6.75–6.67 (m, 2H), 6.07 (dd, J = 24.9, 5.7 Hz,
2H,OCH2O), 5.63 (dd, J = 8.8, 4.1 Hz, 1H), 5.55 (d, J = 4.0 Hz, 1H),
5.03 (d, J = 7.0 Hz, 1H), 4.66 (s, 1H), 4.50 (d, J = 13.6 Hz, 1H), 3.64 (s,
3H), 3.12 (t, J = 7.7 Hz, 4H), 1.93 (s, 3H), 1.61–1.52 (m, 7H), 1.46 (s,
3H), 0.88 (t, J = 7.3 Hz, 6H); 13C NMR (151 MHz, DMSO-d6) δ172.82
(C=O), 171.03 (C=O), 170.83 (C=O), 166.35 (C=O), 163.65 (C=O),
160.17, 156.84, 145.35, 131.62, 130.68, 128.34, 127.94, 127.22,
116.36, 85.88, 80.23 (OCH2O), 70.08, 68.02, 64.80, 61.17, 58.90,
50.50, 49.95, 31.60, 26.72, 21.94, 19.80, 11.14.
2.4.3
|
([4-Ethoxybenzoyl]oxy)methyl (Z)-
6-(2-(4-hydroxyphenyl)-2-([4-methoxy-4-oxobut-2-en-
2-yl]amino)acetamido)-3,3-dimethyl-7-oxo-4-thia-
1-azabicyclo[3.2.0]heptane-2-carboxylate (5c)
Light-yellow solid; yield: 73.56%; Rf: 0.0.76; IR (KBr, cm−1):
3,356.14 (OH), 2,968.45, 1,780.30 (C=O), 1,600.92, 1,512.19,
1,444.68, 1,265.30, 1,166.93, 1,085.92; 1H NMR (600 MHz,
Chloroform-d) δ 9.08 (d, J = 6.9 Hz, 1H, CONH), 8.00 (d, J = 7.6 Hz,
2H), 7.18 (d, J = 8.0 Hz, 2H), 6.93 (d, J = 8.7 Hz, 2H), 6.88 (d,
J = 9.0 Hz, 1H, NH), 6.74 (d, J = 7.5 Hz, 2H), 6.04 (dd, J = 39.5,
5.6 Hz, 2H, OCH2O), 5.64 (dd, J = 8.8, 4.0 Hz, 1H), 5.55 (d,
J = 4.1 Hz, 1H), 5.03 (d, J = 7.0 Hz, 1H), 4.67 (s, 1H), 4.47 (s, 1H),
4.00 (t, J = 6.5 Hz, 2H), 3.65 (s, 3H), 1.94 (s, 3H), 1.85 (q,
J = 7.0 Hz, 2H), 1.55 (s, 3H), 1.46 (s, 3H), 1.06 (t, J = 7.4 Hz, 3H);
13C NMR (151 MHz, Chloroform-d) δ 172.85 (C=O), 170.94 (C=O),
170.66 (C=O), 166.49 (C=O), 164.59 (C=O), 163.87, 160.08,
156.66, 132.21, 128.38, 128.13, 120.32, 116.35, 114.39, 85.95,
79.85 (OCH2O), 70.12, 69.81, 68.00, 64.88, 61.21, 60.41, 58.80,
50.46, 31.57, 26.71, 22.41, 19.78, 10.42.
HR-MS (ESI) Calcd for
Found: 761.8734; purity: 97.31% (determined by HPLC).
C
35H44N4O11S2 (M + H+): 761.8740;
2.4.6
|
(Benzoyloxy)methyl 6-(2-amino-
2-(4-hydroxyphenyl)acetamido)-3,3-dimethyl-7-oxo-
4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate (6a)
HR-MS (ESI) Calcd for
C
32H37N3O10
S
(M + H+): 656.7790;
Light-yellow solid; yield: 87.48%; solubility: 574.06 mg/ml; Rf: 0.73;
IR (KBr, cm−1): 3,409.5 (NH2), 3,201.3 (OH), 2,975.6, 2,613.1, 1,770.3
(C=O), 1,689.3, 1,606.4, 1,515.8, 1,459.8, 1,268.9, 1,089.6; 1H NMR
(600 MHz, DMSO-d6) δ 9.83 (s, 1H, OH), 9.28 (d, J = 7.5 Hz, 1H,
CONH), 8.69 (s, 2H, NH2), 7.96 (d, J = 7.7 Hz, 2H), 7.71 (t, J = 7.4 Hz,
1H), 7.56 (t, J = 7.6 Hz, 3H), 7.28 (d, J = 8.1 Hz, 2H), 6.79 (d,
J = 8.2 Hz, 2H), 6.03 (dd, J = 47.9, 6.0 Hz, 2H, OCH2O), 5.58 (dd,
J = 7.0, 3.9 Hz, 1H), 5.45 (d, J = 4.1 Hz, 1H), 4.95 (s, 1H), 4.42 (s, 1H),
1.47 (s, 3H), 1.31 (s, 3H); 13C NMR (151 MHz, DMSO-d6) δ 173.33
(C=O), 168.51 (C=O), 166.81 (C=O), 164.78 (C=O), 158.65, 134.74,
129.93, 129.57, 128.60, 124.18, 115.77, 80.83 (OCH2O), 70.10,
67.39, 64.38, 58.67, 54.98, 29.99, 26.66.
Found: 656.7194; purity: 98.75% (determined by HPLC).
2.4.4
|
([4-Nitrobenzoyl]oxy)methyl (Z)-
6-(2-(4-hydroxyphenyl)-2-([4-methoxy-4-oxobut-2-en-
2-yl]amino)acetamido)-3,3-dimethyl-7-oxo-4-thia-
1-azabicyclo[3.2.0]heptane-2-carboxylate (5d)
Light-yellow solid; yield: 71.24%; Rf: 0.47; IR (KBr, cm−1): 3,369.64
(OH), 2,974.23, 2,374.37, 1,780.30 (C=O), 1,670.35, 1,600.92,
1,521.84, 1,444.68, 1,269.16, 1,166.93, 1,087.85; 1H NMR
(600 MHz, Chloroform-d) δ 9.07 (d, J = 6.8 Hz, 1H, CONH), 8.29
(dd, J = 51.0, 8.0 Hz, 5H), 7.18 (d, J = 7.6 Hz, 2H), 6.91 (d,
J = 8.8 Hz, 1H, NH), 6.73 (d, J = 7.6 Hz, 2H), 6.12–6.07 (m, 2H,
OCH2O), 5.65 (dd, J = 8.8, 4.1 Hz, 1H), 5.55 (d, J = 4.0 Hz, 1H),
5.03 (d, J = 7.0 Hz, 1H), 4.68 (s, 1H), 4.50 (s, 1H), 3.65 (s, 3H), 1.94
(s, 3H), 1.57 (s, 3H), 1.47 (s, 3H); 13C NMR (151 MHz, Chloroform-
d) δ 172.85 (C=O), 170.85 (C=O), 170.72 (C=O), 166.30 (C=O),
163.12 (C=O), 160.09, 156.68, 151.14, 133.70, 131.19, 128.35,
128.04, 123.80, 116.36, 116.00, 86.01, 80.32 (OCH2O), 70.08,
68.06, 64.79, 61.20, 60.41, 58.88, 52.36, 50.48, 49.84, 31.61,
30.13, 19.78.
HR-MS (ESI) Calcd for C24H25N3O7S (M + H+): 499.5380; Found:
499.5367; purity: 97.11% (determined by HPLC).
2.4.7
|
([4-Methoxybenzoyl]oxy)methyl
6-(2-amino-2-(4-hydroxyphenyl)acetamido)-
3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-
2-carboxylate (6b)
Light-yellow solid; yield: 86.34%; solubility: 142.19 mg/ml; Rf: 0.72;
IR (KBr, cm−1): 3,459.7 (NH2), 3,214.8 (OH), 2,973.7, 2,591.9, 1,772.3
(C=O), 1,722.1, 1,666.2, 1,606.4, 1,515.8, 1,457.9, 1,263.1, 1,168.7,
1,095.4; 1H NMR (600 MHz, DMSO-d6) δ9.77 (s, 1H, OH), 9.27 (d,
HR-MS (ESI) Calcd for
Found: 643.6357; purity: 98.86% (determined by HPLC).
C29H30N4O11S
(M + H+): 643.6360;

6
LI ET AL.
J = 7.6 Hz, 1H, CONH), 8.64 (s, 2H, NH2), 7.92 (d, J = 8.1 Hz, 2H),
7.27 (d, J = 8.0 Hz, 2H), 7.08 (d, J = 8.1 Hz, 2H), 6.78 (d, J = 7.8 Hz,
2H), 6.00 (dd, J = 51.1, 6.0 Hz, 2H,OCH2O), 5.59 (dd, J = 7.8, 4.1 Hz,
1H), 5.45 (d, J = 4.0 Hz, 1H), 4.94 (s, 1H), 4.41 (s, 1H), 3.84 (s, 3H),
1.47 (s, 3H), 1.30 (s, 3H); 13C NMR (151 MHz, DMSO-d6) δ 173.35
(C=O), 168.41 (C=O), 166.83 (C=O), 164.48 (C=O), 164.36, 158.65,
132.20, 129.64, 124.05, 120.63, 115.80, 114.86, 80.60 (OCH2O),
70.12, 67.33, 64.39, 58.60, 56.12, 55.02, 29.87, 26.66.
2.4.10
|
((4-(N,N-Dipropylsulfamoyl)benzoyl)oxy)
methyl 6-(2-amino-2-(4-hydroxyphenyl)acetamido)-
3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-
2-carboxylate (6e)
Light-yellow solid; yield: 87.25%; solubility: 442.48 mg/ml; Rf: 0.75;
IR (KBr, cm−1): 3,405.7 (NH2), 3,209.1 (OH), 2,967.9, 299.6, 1,762.6
(C=O), 1,687.4, 1,606.4, 1,515.8, 1,463.7, 1,334.5, 1,270.9, 1,159; 1H
NMR (600 MHz, DMSO-d6) δ 9.79 (s, H, OH) 9.24 (s, 1H, CONH),
8.30 (s, 2H, NH2), 8.14 (d, J = 8.1 Hz, 2H), 7.97 (d, J = 8.1 Hz, 2H),
7.27 (d, J = 8.2 Hz, 2H), 6.78 (d, J = 8.2 Hz, 2H, OCH2O), 6.08 (dd,
J = 39.4, 6.5 Hz, 2H, OCH2O), 5.58 (d, J = 4.0 Hz, 1H), 5.46 (d,
J = 4.1 Hz, 1H), 4.91 (s, 1H), 4.45 (s, 1H), 3.05 (dd, J = 8.5, 6.5 Hz, 4H),
1.50–1.41 (m, 7H), 1.32 (s, 2H), 0.79 (t, J = 7.3 Hz, 6H); 13C NMR
(151 MHz, DMSO-d6) δ 173.41 (C=O), 169.05 (C=O), 166.80 (C=O),
163.80 (C=O), 158.50, 144.85, 131.99, 130.98, 129.47, 127.86,
124.98, 115.71, 81.13 (OCH2O), 70.05, 67.47, 64.40, 58.70, 55.25,
50.00, 30.14, 26.68, 21.99, 11.39.
HR-MS (ESI) Calcd for C25H27N3O8S (M + H+): 530.5640; Found:
530.5637; purity: 98.34% (determined by HPLC).
2.4.8
|
([4-Ethoxybenzoyl]oxy)methyl 6-(2-amino-
2-(4-hydroxyphenyl)acetamido)-3,3-dimethyl-7-oxo-
4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate (6c)
Light-yellow solid; yield: 88.47%; solubility: 220.38 mg/ml; Rf: 0.77;
IR (KBr, cm−1): 3,398.1 (NH2), 3,205.1 (OH), 2,967.9, 2,599.6, 1772.3
(C=O), 1,693.2, 1,604.5, 1,513.8, 1,467.6, 1,261.2, 1,168.7, 1,087.7;
1H NMR (600 MHz, DMSO-d6) δ 9.78 (s, 1H, OH), 9.25 (d, J = 7.5 Hz,
1H, CONH), 8.56 (s, 2H, NH2), 7.90 (d, J = 8.3 Hz, 2H), 7.27 (d,
J = 8.1 Hz, 2H), 7.06 (d, J = 8.3 Hz, 2H), 6.78 (d, J = 8.0 Hz, 2H), 5.99
(dd, J = 50.3, 6.1 Hz, 2H, OCH2O), 5.59 (d, J = 4.9 Hz, 1H), 5.45 (d,
J = 3.9 Hz, 1H), 4.92 (s, 1H), 4.41 (d, J = 1.5 Hz, 2H), 4.02 (t,
J = 6.9 Hz, 2H), 1.47 (s, 3H), 1.30 (s, 3H), 0.97 (t, J = 7.4 Hz, 3H); 13C
NMR (151 MHz, DMSO-d6) δ 173.39 (C=O), 168.62 (C=O), 166.84
(C=O), 164.36 (C=O), 163.83, 158.60, 132.20, 129.58, 124.37,
120.44, 115.76, 115.24, 80.58 (OCH2O), 70.02 (d, J = 29.2 Hz), 67.37,
64.38, 60.20, 58.61, 55.09, 29.92, 26.66, 22.31, 10.73.
HR-MS (ESI) Calcd for
Found: 663.7737; purity: 97.24% (determined by HPLC).
C
30H38N4O9S2 (M + H+): 663.7730;
2.5
|
Pharmacology
The MIC of hybrid molecules was determined by dilution in a liquid
medium distributed in a 96-well plate according to the standard pro-
tocol set by the Clinical and Laboratory Standards Institute. The
results of antibacterial testing of hybrid molecules (6a–e) against a
panel of selected bacteria (S. aureus American Type Culture Collection
[ATCC] 29,213, ATCC 11632; MRSA 11; E. coli ATCC 25922; Salmo-
nella LS677, GD836, GD828, GD3625) are reported in Table 1. These
plates were incubated for 24 hr at 37 ꢀC. The lowest concentration
which inhibited the growth of the test organism was considered to be
the MIC (μg/ml). Amoxicillin was used as the control drug.
HR-MS (ESI) Calcd for C27H31N3O8S (M + H+): 558.6180; Found:
558.6174; purity: 96.83% (determined by HPLC).
2.4.9
|
([4-Nitrobenzoyl]oxy)methyl 6-(2-amino-
2-(4-hydroxyphenyl)acetamido)-3,3-dimethyl-7-oxo-
4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate (6d)
2.6
|
Systemic infections in mice
Light-yellow solid; yield: 89.67%; solubility: 484.84 mg/m; Rf: 0.72; IR
(KBr, cm−1): 3,428.8 (NH2), 3,224.4 (OH), 2,975.6, 2,611.1, 1,766.5
(C=O), 1,691.3, 1,610.3, 1,525.4, 1,353.8, 1,270.9, 1,089.6; 1H NMR
(600 MHz, DMSO-d6) δ 9.78 (s, 1H, OH), 9.26 (d, J = 7.6 Hz, 1H,
CONH), 8.59 (s, 2H, NH2), 8.37 (d, J = 8.1 Hz, 2H), 8.20 (d, J = 8.0 Hz,
2H), 7.27 (d, J = 7.9 Hz, 2H), 6.78 (d, J = 7.9 Hz, 2H), 6.07 (dd,
J = 38.6, 6.2 Hz, 2H, OCH2O), 5.59 (t, J = 5.6 Hz, 1H), 5.46 (d,
Female ICR mice weighing about 18–22 g (Changsha, China) were
infected intraperitoneally with 0.5 ml of bacterial suspension. The
strains and the challenge dose per mouse were as follows:
S. enteritidis GD836, 1.35 × 109 cfu. Salmonella enteritidis GD836
were injected as suspensions in normal saline. 6a–e (hydrochloride)
and amoxicillin (Sodium salt) were orally administered in 0.2 ml of
aqueous solution at 0.5 and 4 hr after pathogen inoculation. The num-
ber of mice that survived in each experimental group was monitored
up to 48 hr after pathogen inoculation, and the 50% effective dose
(ED50) of the drug-treated animals were determined by the probit
method. Each experimental group consisted of 10 animals and four
different doses of the drug was evaluated per compound, and physio-
logical saline was used as the vehicle control. (Bliss, 1934).
J = 4.1 Hz, 1H), 4.94 (s, 1H), 4.45 (s, 1H), 1.48 (s, 3H), 1.31 (s, 3H); 13
C
NMR (151 MHz, DMSO-d6) δ 173.29 (C=O), 168.52 (C=O), 166.76
(C=O), 163.45 (C=O), 158.62, 151.21, 134.00, 131.47, 129.62,
124.61, 124.21, 115.77, 81.24 (OCH2O), 70.06, 67.40, 64.41, 58.70,
55.04, 30.06, 26.70.
HR-MS (ESI) Calcd for C24H24N4O9S (M + H+): 545.5350; Found:
545.5356; purity: 98.34% (determined by HPLC).

LI ET AL.
7
TABLE 1 In vitro antimicrobial activities of compound 6a–e based on MIC values
MIC (μg/ml)
Gram-Positive
Gram-Negative
S. aureus
S. aureus
S. aureus
E. coli
S. enteritidis
Compound
ATCC 29213
ATCC 11632
MRSA 11
ATCC 25922
LS677
GD836
GD828
GD3625
6a
4
4
4
4
4
2
8
>128
128
128
64
16
8
2
2
4
2
2
2
4
4
8
2
4
8
4
4
8
2
4
8
8
4
2
2
4
2
6b
8
6c
16
8
16
8
6d
6e
8
128
>128
8
Amoxicillin
4
4
3
|
RESULTS AND DISCUSSION
| Chemistry
TABLE 2 Efficacy in systemic infections in mice
ED50 (mg/kg)
3.1
Compound
S. enteritidis GD836
14.6575
Our research focuses on the design, synthesis, and bioactivity evalua-
tion of a series of hybrid molecules between amoxicillin and deriva-
tives of benzoic acid. The excellent antibacterial activity of
sultamicillin proves that hybrid molecules connected by methylene
bridges have great development potential (Friedel, Campoli-
Richards, & Goa, 1989). In addition, a series of studies have shown
that benzoic acid derivatives play a positive role in enhancing the
antibacterial properties of compounds.(Begini et al., 2020; Filho,
Kronenberger, Ferreira, Trossini,& Marcio, 2020; Sharma, Parvinder,
Kakkar, & Khatkar, 2020), and the latest research shows that benzoic
acid derivatives with para-substituents have has potential as anti-
biofilm agent (Campbell et al., 2020; Zarafu, 2020). These studies led
us to design and synthesise a series of hybrid molecules which were
linked via a methylene bridge in order to investigate the effect of the
presence of benzoic acid derivatives on the biological activity.
6a
6b
13.2496
6c
16.6281
6d
14.3089
6e
16.9435
Amoxicillin
17.5997
The MIC values of the hybrid molecules of the five newly synthe-
sized amoxicillin–benzoic acid analogs indicated that certain hybrid
molecules had comparable or better activity against Salmonella species
than amoxicillin. Compound 6d exhibited the most potent inhibitory
activity against Salmonella enteritidis GD828 (MIC = 2 μg/ml), and Sal-
monella enteritidis GD836 (MIC = 2 μg/ml), which were fourfold more
potent than that of amoxicillin, respectively. Conversely, the in vitro
activity of most of the test compounds against E. coli species (Gram-
positive bacteria) was comparable with (or even weaker) than that of
amoxicillin. However, compound 6d was superior to that of amoxicillin
against MRSA 11 (MIC = 64 μg/ml).
The synthesis of new compounds is shown in Scheme 1. Com-
pound 2a–e was converted to the corresponding iodomethyl deriva-
tive by the Finkelstein reaction to give compound 4a–e. Then,
compound 4a–e was condensed with the carboxylate ion of com-
pound 2 at 0–5 ꢀC to give compound 5a–e. The condensation product
was dissolved in an aqueous acetone solution. The pH of this solution
was adjusted to 2.5 with 10% HCl, the protecting group was removed,
and then the acetone in the solution was removed by vacuum spin-
ning. Then, excess NaCl was added to the remaining solution to pre-
cipitate the product. Finally, the crude product was purified by
column chromatography using silica gel to afford compound 6a–e.
3.3
|
In vivo antibacterial activity
The protective effect of 6a–e against systemic infections with Salmo-
nella enteritidis GD836 in mice were compared with Amoxicillin
(Table 2).
Compound 6b showed the greatest efficacy of all the compounds
tested against systemic infections caused by Salmonella enteritidis
GD836. Although 6a–e has good in vitro activity against Salmonella
enteritidis GD836, their in vivo antibacterial activity is not signifi-
cantly superior to amoxicillin. This unexpected loss of in vivo activity
of these derivatives may partly be due to the poor bioavailability of
3.2
|
In vitro antibacterial activity
The antimicrobial activity of hybrids (6a–e) in vitro was evaluated.
Their MIC values against Gram-positive and Gram-negative bacteria
were determined by comparison with that of amoxicillin (Table 1).

8
LI ET AL.
Chauhan, D., Hati, S., Priyadarshini, R., & Sen, S. (2019). Transcriptome
analysis predicts mode of action of benzimidazole molecules 1 against
Staphylococcus aureus uams-1. Drug Development Research, 80,
490–503. https://doi.org/10.1002/ddr.21523
these hybrid molecules when administered orally, because they may
have poor stability in the digestive tract.
Christy, M. L., Sampson, M., Edson, M., & Anthony, O. (2018). Antibiotic
use in agriculture and its consequential resistance in environmental
sources: Potential public health implications. Molecules, 23(4), 795.
https://doi.org/10.3390/molecules23040795
4
|
CONCLUSIONS
Five hybrid molecules similar to amoxicillin–benzoic acid were synthe-
sized and evaluated. Amoxicillin–benzoic acid-like hybrid molecules
based on a diester structure could improve the activity of amoxicillin
against Salmonella species. Compound 6d had good activity against
MARSA 11. Our findings provided a new approach to researching and
developing a novel antibiotic.
Dickson, M., & Gagnon, J. P. (2004). Key factors in the rising cost of new
drug discovery and development. Nature Reviews Drug Discovery, 3(5),
417–429. https://doi.org/10.1038/nrd1382
Dimasi, J. A., Hansen, R. W., & Grabowski, H. G. (2003). The price of inno-
vation: New estimates of drug development costs. Journal of Health
Economics, 22, 151–185. https://doi.org/10.1016/S0167-6296(02)
00126-1
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sultamicillin in mice, rats, and dogs. Antimicrobial Agents and Chemo-
therapy, 25(5), 599–602. https://doi.org/10.1128/aac.25.5.599
Filho, R. P., Kronenberger, T., Ferreira, G. M., Trossini, G. H. G., & Marcio
Vinícius Bertacine Dias. (2020). Design, synthesis and biological activ-
ity of novel substituted 3-benzoic acid derivatives as mtdhfr inhibitors.
ACKNOWLEDGMENTS
The present study had financial support from the National Science
Foundation of China (31672601), NMR data were provided by South
China Agricultural University Test Center. Data from IR spectroscopy
were provided by Guangzhou Chemistry Institute of Chinese Acad-
emy of Sciences.
Bioorganic
& Medicinal Chemistry, 28, 115600. https://doi.org/10.
1016/j.bmc.2020.115600
Friedel, H. A., Campoli-Richards, D. M., & Goa, K. L. (1989). Sultamicillin.
Drugs, 37, 491–522. https://doi.org/10.2165/00003495-198937040-
00005
CONFLICT OF INTEREST
Goossens, & Herman. (2005). European status of resistance in nosocomial
infections. Chemotherapy, 51(4), 177–181. https://doi.org/10.1159/
000086919
The authors declare no conflicts of interest.
DATA AVAILABILITY STATEMENT
Hohlfelder, B., Kubiak, D. W., Degrado, J. R., Reardon, D. P.,
&
The data used to support the findings of this study are available from
the corresponding author upon request.
Szumita, P. M. (2016). Implementation of a prolonged infusion guide-
line for time-dependent antimicrobial agents at a tertiary academic
medical center. American Journal of Therapeutics, 23(6), e1768–e1773.
https://doi.org/10.1097/MJT.0000000000000377
ORCID
Inglis, J., Criado, G., Andrews, M., Feldmann, M., Williams, R., & Selley, M.
(2007). The anti-allergic drug, n-(30,40-dimethoxycinnamonyl) anthra-
nilic acid, exhibits potent anti-inflammatory and analgesic properties in
arthritis. Rheumatology, 46(9), 1428–1432. https://doi.org/10.1093/
rheumatology/kem160
Zhonglin Li
https://orcid.org/0000-0001-5498-2503
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