Discovery of a Teraryl Oxazolidinone Compound (S)-N-((3-(3-Fluoro-4-(4-(pyridin-2-yl)-1H-pyrazol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide Phosphate as a Novel Antimicrobial Agent with Enhanced Safety Profile and Efficacies

Article abstract of DOI:10.1021/acs.jmedchem.5b00152

A series of novel teraryl oxazolidinone compounds was designed, synthesized, and evaluated for their antimicrobial activity and toxicities. The compounds with aromatic N-heterocyclic substituents at the 4-position of pyrazolyl ring showed better antibacterial activity against the tested bacteria than other compounds with different patterns of substitution. Among all potent compounds, 10f exhibited promising safety profile in MTT assays and in hERG K⁺ channel inhibition test. Furthermore, its phosphate was found to be highly soluble in water (47.1 mg/mL), which is beneficial for the subsequent in vivo test. In MRSA systemic infection mice models, 10f phosphate exerted significantly improved survival protection compared with linezolid. The compound also demonstrated high oral bioavailability (F = 99.1%). Moreover, from the results of in vivo toxicology experiments, 10f phosphate would be predicted to have less bone marrow suppression (Chemical Equation).

Full text of DOI:10.1021/acs.jmedchem.5b00152

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Discovery of a teraryl oxazolidinone compound (S)ꢀNꢀ((3ꢀ(3ꢀfluoroꢀ
4ꢀ(4ꢀ(pyridinꢀ2ꢀyl)ꢀ1Hꢀpyrazolꢀ1ꢀyl)phenyl)ꢀ2ꢀoxooxazolidinꢀ5ꢀyl)methyl)
acetamide phosphate as a novel antimicrobial agent with enhanced safety
profile and efficacies
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Tao Yang , Gong Chen , Zitai Sang , Yuanyuan Liu , Xiaoyan Yang , Ying Chang , Haiyue Long
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Wei Ang , Jianying Tang , Zhenling Wang *, Guobo Li , Shengyong Yang , Jingren Zhang ,
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Yuquan Wei , Youfu Luo *
†
State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China
Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan, China.
‡
Sichuan Gooddoctor Pharmaceutical Co., Ltd., Chengdu, Sichuan, China.
§
Centre for Infectious Diseases Research, School of Medicine, Tsinghua University, Beijing, China.
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Abstract
A series of novel teraryl oxazolidinone compounds was designed, synthesized and evaluated for
their antimicrobial activity and toxicities. The compounds with aromatic Nꢀheterocyclic substituents at
the 4ꢀposition of pyrazolyl ring showed better antibacterial activity against the tested bacteria than other
compounds with different pattern of substitution. Among all potent compounds, 10f exhibited promising
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safety profile in MTT assays and in hERG K channel inhibition test. Furthermore, its phosphate was
found to be highly soluble in water (47.1 mg/mL), which is beneficial for the subsequent in vivo test. In
MRSA systemic infection mice models, 10f phosphate exerted significantly improved survival
protection compared with linezolid. The compound also demonstrated high oral bioavailability (F =
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9.1%). Moreover, from the results of in vivo toxicology experiments, 10f phosphate would be predicted
to have less bone marrow suppression.
Keywords
:
o
xazolidinones; antibacterial agents; potency; solubility; toxicity.
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INTRODUCTION:
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The development of bacterial resistance to currently available antibiotics is a growing global health
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threat . Infectious diseases caused by resistant Gramꢀpositive bacteria such as methicillinꢀresistant
Staphylococcus aureus (MRSA), penicillinꢀresistant Streptococcus pneumonia (PRSP), and
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vancomycinꢀresistant Enterococcus faecalis (VRE) are of particular concern . Linezolid (1) (Figure 1),
the first marketed and unique antibacterial agent of oxazolidinone family, has been proven to bind with
the 50S ribosomal subunit to inhibit bacterial translation at the initiation phase of protein synthesis. This
agent demonstrates good activity against all major pathogenic Gramꢀpositive bacteria, including MRSA,
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vancomycinꢀresistant Enterococcus faecium (VREF) and penicillinꢀresistant Streptococcus pneumonia .
The success of linezolid stimulated considerable efforts to search for improved agents in the
oxazolidinone class. Several oxazolidinone drug candidates are currently undergoing clinical
8–9
investigation at different hospitals worldwide . As stated above, linezolid acts by the inhibition of
protein synthesis through binding to 50S subunit of bacterial ribosome, with a similar mechanism
ascribed to the inhibition of mitochondrial protein synthesis in mammals. This mode of action is
fundamentally associated with its biochemical mechanisms responsible for the main adverse effect of
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myelosuppression
. To our knowledge, several novel oxazolidione antibacterial research and
development programs were discontinued because of safety issues. In consequence, improving the
balance between efficacy and toxicities in a campaign on discovery and translational research of novel
oxazolidione drug candidates is crucial.
1
2
Adam and his coꢀworkers have recently performed structural modification of linezolid in Cꢀring
part, attenuating mitochondrial protein synthesis inhibitory side effects, while retaining the desired
antibacterial effect. As such, for the best combination of potency and host tolerability, we focus our
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efforts on the modification of the Cꢀring of linezolid guided by results of docking experiments.
Previously reported modeling studies have demonstrated that oxazolidinone compounds with
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various Cꢀring attachments could fit nicely with the 50S ribosomal subunit . For instance, compounds 3
and 4 (Figure 1) could bind well to the 50S subunit through hydrogen bond or πꢀπ interaction. The
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inducedꢀfit mechanism allows the ribosome pocket to adopt a distinct position to accommodate different
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chemical spaces of oxazolidinone compounds . This in silico hypothesis prompts us to explore the
diverse chemical space of Cꢀring of linezolid.
Figure 1. Chemical structures of linezolid and other oxazolidinones
8, 13
As indicated by previous studies , biarylꢀ or terarylꢀfeaturing oxazolidinones, like compounds 2,
4, and 5 (Figure 1), are becoming increasingly known for their high potential as novel antimicrobial
candidates. Compound 2, which contains a biaryl substituent at the C ring position of linezolid and
performs an essential function in drug–receptor interaction, has attracted considerable interest. However,
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its teraryl moiety appears to be relative rigid and linear like, and then, this kind of oxazolidinones would
be a small fraction of all teraryl moietyꢀfeaturing oxazolidinone compounds. Elucidating the potential
diversity of the substituents in the linezolid Cꢀring is highly beneficial. The linear conformation of 2
likely enables it to bind well with the pocket and thus exhibit excellent activity. This interesting feature
motivated us to determine whether compounds without a straightꢀline conformation also had good
activity. Thus novel oxazolidinone compounds featuring multiꢀdirectional biaryl C rings were designed
and synthesized with the purpose of discovering oxazolidinone antibacterial agents with enhanced
potency and attenuated toxicology. Herein, an aromaticꢀsubstituted pyrazolyl ring was used as our biaryl
substructure, in which a pyrazolyl ring was retained as the common feature in all target molecules
because of its high efficiency in generating biaryl moieties with multiꢀdirectional projections (Structure
6, Figure 1). The resultant biaryl C ring connects fluorophenyl B ring through CꢀN bond, while various
aromatic rings could be introduced at 3ꢀ, 4ꢀ and 5ꢀ positions of the pyrazolyl ring. The aromatic ring at
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ꢀ position could be achieved through the reaction of aromatic boric acid with 4ꢀhalogenated pyrazolyl
ring, usually by iodination or bromation, under Suzuki−Miyaura reaction conditions. Since the
crossꢀcoupling method would be unsuitable for the synthesis of 3ꢀ and 5ꢀ position resultants. The
pyrazolyl ring can be constructed by simply reacting hydrazine or substituted phenylhydrazine with
2ꢀsubstituted malonaldehyde or the corresponding enaminone to yield the 4ꢀposition and the 3ꢀ or
15
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ꢀposition resultants, respectively
.
Thus, we disclose our studies on the synthesis and antibacterial activity of a novel class of
oxazolidinone compounds that feature a biaryl moiety in C ring position of linezolid. In the primary
screening, 10f was demonstrated as the most potent compound, and we have reported excellent in vitro
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and in vivo antibacterial activity of its hydrochloric acid salt form
. With a tenꢀfold increase, its
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solubility still caused certain limitations in proceeding with in vivo experiments, although considerable
pharmaceutical efforts have been made to address this issue by the addition of various adjuvants such as
(2ꢀhydroxypropyl)ꢀβꢀcyclodextrin, sulfobutyl etherꢀβꢀcyclodextrin, olive oil and tween 80. In general,
two more strategies could be adopted from this point to address the solubility issue: (1) structural
modification by addition of soluble groups to the parent compound, which may cause activity loss and
toxicity increase; (2) preparation of corresponding salts by taking advantage of the compound’s basic
properties. In this paper, we describe our endeavors in medicinal chemistry, acid salt screening and
other relevant translational research of compound 10f.
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CHEMISTRY:
The general synthesis of compounds 9 and 10 are depicted in Scheme 1. A pyrazolyl ring formation
was used to construct an aromatic–pyrazole linked system by condensing intermediate 8 with
3
ꢀ(dimethylamino)ꢀ1ꢀ (aromaticꢀ3ꢀyl)propꢀ2ꢀenꢀ1ꢀone (7) or 2ꢀaromatic malonaldehyde to yield the
final compounds 9a to 9j and 10a to 10f, respectively. Corresponding acetyl aromatic compounds were
condensed with N, NꢀDimethylformamide dimethyl acetal (DMFꢀDMA) to afford the precursor 7 under
microwave conditions. The key intermediate 8 was obtained according to the reported literature with
1
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minor modifications . Compound 10g was synthesized through a long linear route depicted in Scheme
1. Intermediate 3ꢀ(dimethylamino)ꢀ2ꢀ(pyridinꢀ3ꢀyl)acrylonitrile 12 was prepared by condensation of
2
ꢀ(pyridinꢀ3ꢀyl)acetonitrile and DMF−DMA without solvent under microwave irradiation as described
1
9
in published procedures . Compound 12 was reacted with hydrazine in the presence of acetic acid to
give 13 with a high yield. The amine pyrazole compound 13 was subjected to 3, 4ꢀdifluoronitrobenzene
in the presence of K CO , which yielded corresponding regioꢀisomeric mixture 14. The product was
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notably a mixture of 3ꢀamino and 5ꢀamino regioisomers in a ratio of about 3 : 1, which was shown in
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the H NMR spectrum(Figure 2). Separation of the isomeric mixture was not required because the
amino groups would be totally removed in the following reaction step. Deamination of compound 14
was carried out to yield nitro compound 15, in which isoamyl nitrite was used as deamination agent.
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Then, the nitro derivatives 15 were reduced with ion powder and NH Cl complex in ethanol and then
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reacted with benzyl chloroformate to yield compound 16. According to a general inꢀhouse method , the
oxazolidinone ring was constructed in the final step to the target compounds 10g by reaction of 16 with
(
S)ꢀ1ꢀacetamidoꢀ3ꢀchloropropanꢀ2ꢀyl acetate.
Scheme 1. The synthetic route of 5-substituted pyrazolylphenyl oxazolidinones 9a to 9j and
-substituted-pyrazolylphenyloxazolidinones 10a to 10f
4
Reagent and conditions：(a) conc.HCl, EtOH, reflux, 31% to 50%; (b) 8, conc. HCl, EtOH, reflux, 66% to 81%.
Scheme 2. The synthetic route of 4-substituted pyrazolylphenyloxazolidinones 10g
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Reagent and conditions：(a) DMFꢀDMA, 100 °C, 30 min, 72%; (b) Hydrazine hydrate, acetic acid, EtOH, reflux, 24 h,
46%; (c) 3,4ꢀdifluoronitrobenzene, K CO , DMSO, 4 h, 69%; (d) Isopropyl nitrite, DMF, 75 °C, 82%; (e) Fe, NH Cl,
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EtOH, H O, 60 °C; (f) CbzꢀCl, K CO , DCM, overnight, 74%; (g) tꢀBuOLi, MeOH,
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(S)ꢀ1ꢀacetamidoꢀ3ꢀchloropropanꢀ2ꢀyl acetate, THF, 0 °C ꢀ r.t., 17 h, 56%.
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Figure 2. Component ratio determination of regioisomers of 14 by H NMR in CDCl
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Compounds 22 were obtained through a linear synthetic route starting from 7, as depicted in
Scheme 3. Starting material 7 was condensed with hydrazine hydrate in refluxed ethanol to yield
intermediate 18 which was the precursor of the next nucleophilic S Ar reaction. Referring to the
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synthetic route depicted in Scheme 2, compound 18 and 3, 4ꢀdifluoronitrobenzene were reacted in the
presence of K CO to afford the nitro derivatives 19, which were reduced with ion powder and NH Cl
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system in ethanol and successively mixed with benzyl chloroformate to yield compound 21. In addition,
target compound 22 was obtained through reaction of (S)ꢀ1ꢀacetamidoꢀ3ꢀchloropropanꢀ2ꢀyl acetate with
compound 21 in moderate to good yields.
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Scheme 3. The synthetic route of 4-substituted-pyrazolylphenyloxazolidinones 22a to 22c
Reagent and conditions: (a) Hydrazine hydrate, EtOH, reflux, 83% to 88%; (b) 3,4ꢀdifluoronitrobenzene, DMSO,
K CO , r.t., overnight, 64% to 83%; (c) Fe, NH Cl, EtOH, H O, 60 °C, 4 h; (d) CbzꢀCl, K CO , DCM, overnight, 66%
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to 78% (steps of c and d); (e) tꢀBuOLi, (S)ꢀ1ꢀacetamidoꢀ3ꢀchloropropanꢀ2ꢀyl acetate, MeOH, THF, 0 °C to r.t., 17 h,
34% to 42%.
After obtaining the most potent compound 10f, further attempts of structural refinement were
carried out to seek more potent compounds. First, several malonaldehyde compounds were obtained
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using amide formation reaction or esterification , in which 5ꢀcarboxylpyridinꢀ2ꢀyl malonaldehyde
was used as starting material (Scheme 4). Then, various substituted malonaldehyde intermediates were
reacted with key intermediates 8 for the preparation of final product 24 using the same procedures for
compounds 9 and 10. Second, a series of Nꢀcontaining aromatic rings was introduced to replace the
pyridyl ring, which afforded the corresponding final product 28. As illustrated in Scheme 5, the building
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blocks for the substituted pyrazole were purchased or prepared. For the synthesis of compound 26,
oxidation of primary alcohol 2ꢀ(quinolinꢀ2ꢀyl)propaneꢀ1, 3ꢀdiol (25), synthesized through reported
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procedures , was accomplished using Parikh–Doering condition . Intermediate 27, the chemical
equivalent of its malonaldehyde form, was obtained in a single step by Vilsmeier reaction with good
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yield . Four malonaldehydes including two purchased materials or their equivalent sources were
reacted with key intermediate 8 to yield the final products 28a-28d.
Scheme 4. The synthetic route of 4-substituted pyrazolylphenyl oxazolidinones 24a to 24d
Reagent and conditions: (a) HOBt, EDCI, NH Cl (for synthesis of 24a) or methylamine hydrochloride (for synthesis of
4
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4b) or morpholine (for synthesis of 24c), 4ꢀmethylmorpholine, DMF, 60 °C, overnight, 47% to 65%; (b) MeI, K CO ,
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DMF, 60 °C, 52%; (c) conc. HCl, 8, EtOH, reflux, 4 h, 68% to 75%.
Scheme 5. The synthetic route of 4-substituted pyrazolylphenyl oxazolidinones 28a to 28d
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Reagent and conditions: (a) 2ꢀmethylquinoline, formalin, 130 °C, 7 h, 45%; (b) PySO , DMSO, Et N, methylene
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chloride, 0 °C to r.t., 2 h, 40%; (c) 8, conc.HCl, EtOH, reflux 4h, 57% to 64%; (d) DMF, POCl , 2ꢀmethylpyrazine, 0°C,
3
1
h, 60°C, overnight, 49%.
Scheme 6. The synthetic route of 4-substituted pyrazolylphenyl oxazolidinones 32a-b
Reagent and conditions: (a) 4ꢀfluoronitrobenzene (for synthesis of 29a) or 3,4,5ꢀtrifluoronitrobenzene (for synthesis of
29b), K CO , DMSO, 72% to 87%; (b) Fe, NH Cl, EtOH, H O, 60 °C, 4h; (c) CbzꢀCl, K CO , DCM, overnight, 71%
2
3
4
2
2
3
to 78%; (d) tꢀBuOLi, (S)ꢀ1ꢀacetamidoꢀ3ꢀchloropropanꢀ2ꢀyl acetate, MeOH, THF, 0 °C to r.t., 17 h, 39% to 47%.
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2
3
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Compounds 32a and 32b were synthesized to determine whether or not the fluorine atom affects
antibacterial activity. As depicted in Scheme 6, a linear synthetic route was adopted to yield the final
compound 32. Nitro derivatives 29 were obtained through a nucleophilic S Ar reaction, which used
N
2
ꢀ(1Hꢀpyrazolꢀ4ꢀyl)pyridine and corresponding fluoronitrobenzenes as starting materials. Target
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
compounds 32 were synthesized in moderate yields through the route described in Scheme 2.
Scheme 7. The synthetic route of 4-substituted pyrazolylphenyl oxazolidinones 33 and 34
Reagent and conditions: (a) mCPBA, DCM, K CO , r.t., 18 h, 71%; (b) TMSCN, dimethylcarbamyl chloride, DCM,
2
3
r.t., 48 h, 46%.
Meanwhile, to improve the aqueous solubility, the cyano group was intended to be introduced into
pyridyl ring (Scheme 7). The N atom of pyridine ring of 10f was oxidized by mCPBA to give compound
2
6
27
3
3 . Then, compound 33 was subjected to TMSCN condition to afford compound 34 .
Scheme 8. The synthetic route of 4-substituted-pyrazolylphenyl oxazolidinones 40a to 40c
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7
8
9
10
11
12
13
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15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Reagent and conditions: (a) NaNO , HCl, CuCl, or HBr, CuBr or HBF , H O, 5 h, 25% to 31%; (b)
2
4
2
3
,4ꢀdifluoronitrobenzene, K CO , DMSO, 4 h, 82% to 89%; (c) Fe, NH Cl, EtOH, H O, 60 °C; (d) CbzꢀCl, K CO ,
2 3 4 2 2 3
DCM, overnight, 62% to 71%; (e) tꢀBuOLi, (S)ꢀ1ꢀacetamidoꢀ3ꢀchloropropanꢀ2ꢀyl acetate, MeOH, THF, 0 °C to r.t., 17
h, 22% to 36%.
Based on 10f, a series of compounds was synthesized to determine whether one halogen atom
substituted at the pyrazolyl ring could affect antibacterial activity. As depicted in Scheme 8, target
compounds 40 were also synthesized through a relatively long linear synthetic route. Intermediate 35
2
0
were prepared in high yield as previously described . Sandmeyer condition was used to introduce
2
8
29
halogen atom to the pyrazoyl ring and produce chlorinated 36b or brominated 36c , whereas
3
0
fluoroaromatic 36a was obtained through Balz–Schiemann reaction . Halogenated compounds 36 were
then reacted with 3,4ꢀdifluoronitrobenzene in the presence of K CO to yield the corresponding nitro
2
3
derivatives 37. As shown in Scheme 2, three sequential transformations were performed using the nitro
derivatives as starting materials to yield the final compounds 40.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
RESULTS AND DISCUSSION
Table 1. In Vitro Antibacterial Activity of Substituted Pyrazolylphenyl Oxazolidinones
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
MIC (ꢁg/mL)
Compd
9a
Ar
b
c
d
e
f
g
h
i
S.a
S.a
S.a
S.a
S.a
S.a
E.c
P.a
8
16
8
8
16
16
>32
>32
>32
>32
9b
16
16
32
32
32
16
9
c
16
16
8
16
8
32
16
16
32
16
16
32
8
32
16
32
>32
>32
>32
>32
>32
>32
9d
9e
9f
16
16
16
32
32
16
32
32
>32
>32
9g
8
4
4
4
8
4
8
4
8
4
8
4
>32
>32
>32
>32
>32
>32
9h
9
i
32
16
32
32
32
32
9
j
16
8
16
8
16
16
>32
>32
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3
4
5
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7
8
9
10a
4
4
4
4
8
8
4
8
4
4
8
8
4
8
8
8
8
8
>32
>32
>32
>32
>32
>32
10b
10c
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
0d
8
8
1
4
8
1
8
4
1
4
8
1
8
4
1
8
8
2
>32
>32
>32
>32
>32
>32
1
0e
1
0f
1
0g
4
4
2
4
4
2
4
8
2
4
4
2
4
8
2
4
8
2
>32
>32
>32
>32
>32
>32
22a
22b
2
2c
4
2
4
1
4
2
4
2
8
2
8
2
>32
>32
>32
>32
LZN
ꢀ
a
Abbreviations: MIC is defined as the minimum concentration of a compound that inhibits growth by 99%. Identical
values were obtained for each compound in three replicates by visual investigation as stated in the experimental section;
b
c
d
e
f
g
MSSA ATCC25923; MSSA ATCC29213; MSSA ATCC6538; MSSA CMCC26003; MRSA ATCC33591;
h
i
MRSAATCC43300; Escherichia coli ATCC25922; Pseudomonas aeruginosa ATCC27853.
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Regions subjected to separate SAR investigation
As designed, aromatic rings were introduced to the 3ꢀ, 4ꢀ and 5ꢀposition of the pyrazolyl ring to
generate compounds 22a to 22c, 10a to 10g and 9a to 9j, respectively, which are responsible for the
generation of three types of C ring moieties (with different space projections). All oxazolidinone
analogues prepared were tested for their in vitro activity against various Gramꢀpositive and
Gramꢀnegative bacteria. MIC values were determined using the standard agar dilution method. The
results of these studies are summarized in Table 1 and Table 2.
Compounds with aromatic ring substituents at the 5ꢀposition of the pyrazolyl ring generally
showed weak activity against all test bacteria, whereas strong activity was observed when aromatic
rings were connected to the 3ꢀ or 4ꢀposition of the ring. Thus, we focused on 10 and 22, where the
aromatic substituents of C ring moieties were similar to those in compounds 9. Compounds bearing
pyridyl groups (10a, 10f, and 10g) displayed stronger activity than those bearing phenyl or substituted
phenyl groups (10b, 10c and 10d). These results implied that the N atom attached at different positions
of the pyridyl ring may be the hydrogen bond acceptor responsible for the activity. Then, the 3ꢀ position
of the pyrazolyl ring was introduced with heterocyclic substituents such as 2ꢀpyridyl, 3ꢀpyridyl and
4
ꢀpyridyl to yield compounds 22. This set of oxazolidinones showed moderate to good antibacterial
activity. Therefore, we speculated that the N atom of the pyridyl group may contribute to the biological
activity possibly because of its participation in hydrogen bonding.
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2
3
After achieving the most potent compound 10f, we conducted extensive structure–activity
relationship (SAR) studies on pyrazolyl functionality and Bꢀring. We selected 10f as the parent structure
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
to remain unchanged.
3
1, 32
Fluorination is beneficial in many cases
. Thus, we assessed the activity of fluorinated
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
compounds (32a and 32b). As illustrated in Table 2, in Bꢀring subtypes, 10f was more potent than 32a
and 32b. Thus, the monofluorinated derivative was superior to the fluorideꢀfree or difluorinated ones.
Halogen atoms, including fluorine, chlorine and bromine atoms, were introduced at 3ꢀ position of
pyrazole ring of compound 10f through Sandmeyer reactions. However, the halogenated analogs 40a to
4
0c had twoꢀfold to fourꢀfold decrease in antibacterial activity than 10f (Table 2). We speculated that
the added hydrophobicity and steric bulk of halogens on the pyrazole ring affects the antibacterial
activity.
Further development and potential applications of 10f are hindered by its low aqueous solubility.
Disruption of molecular planarity or addition of waterꢀsoluble group was considered to improve the
3
3
aqueous solubility of 10f . Compound 34, in which a cyano group was attached at the orthoꢀpostion of
the pyridyl group, was synthesized and evaluated for its antibacterial activity. Its synthetic intermediate
3
3 was also evaluated for comparison (Table 2). However, compounds 33 and 34 exhibited
approximately twoꢀfold to fourꢀfold lower antibacterial activity than 10f.
Conversely, a carboxyl group was introduced at the 5ꢀposition of the pyridyl ring for another
optimization endeavor. However, the resultant compound 10e showed lower antimicrobial activity than
10f. The extremely poor solubility of the compound in both organic and aqueous solvents may be the
key underlying reason. Thus, a series of analogues was designed and synthesized by transforming
carboxyl group through amidation and esterification. Alterations to these moieties or groups can directly
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
improve the solubility of the compound to some extent and they supply potential linking methods for
further derivation, such as preparation of activityꢀbased profiling probes for identification of biological
targets. This particular modification was beneficial, as shown in Table 2. The esterified compound (24d)
showed stronger activity than linezolid, with MIC values ranging from 0.5 ꢁg/mL to 1 ꢁg/mL. In
addition, amidated compounds bearing a relatively small substituent (compounds 24a and 24b) at this
site displayed good to moderate in vitro antibacterial activity compared with that of linezolid. However,
morpholine ring substitution at the same site resulted in reduced activity (compound 24c).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
On the basis of the aboveꢀmentioned hydrogen bonding for structural optimization, several other
heteroaromatic moieties were selected to mimic the interactions of pyridyl rings in accordance with
bioisosterism theory. For this purpose, the core structure was remained unchanged, and the 4ꢀposition of
the pyrazolyl ring was modified. In consequence, a series of Arꢀsubstituted pyrazolyl oxazolidinone
derivatives at the 4ꢀposition were synthesized and evaluated for their in vitro antibacterial activity, in
which the Ar substituents include pyrazinyl, quinolyl and quinoxalinyl groups. Among the
aforementioned analogues, as shown in Table 2, 28b and 28c displayed excellent antibacterial activity
against all tested Gramꢀpositive bacteria, while 28d exhibited a reduced activity with MIC values of
only 2 to 4 ꢁg/mL. Compound 28a with a quinolyl group to replace the pyridyl ring appeared
unexpectedly ineffective, which is far beyond to our prediction and is under further investigation. By
contrast, compound 28c, which features a quinoxalinyl group that contains an extra nitrogen atom at the
4
ꢀposition of the quinolyl group, exhibited excellent activity. The extra nitrogen atom, which was the
unique distinction between two oxazolidinones, was considered to be important for the action. Far from
being exhaustive, this initial exploration suggests that the hydrogen bonding responsible for activity is
more pronounced when the sixꢀmember aromatic ring contains more hydrogen acceptor N atoms, which
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allow greater degrees of freedom and probabilities to approach the site. Consequently, this region can
tolerate a relatively wide range of chemical space.
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. In Vitro Antibacterial Activity of Substituted Pyrazolylphenyl Oxazolidinones
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
MIC (ꢁg/mL)
Compd
Ar
X
R1
R2
b
c
d
e
f
g
h
i
S.a
S.a S.a
S.a
S.a
S.a
E.c
P.a
2
4a
H
H
H
H
F
F
F
F
H
H
H
H
1
1
2
4
1
1
2
4
1
1
1
1
>32 >32
>32 >32
>32 >32
>32 >32
2
4b
2
8
2
8
1
2
8
1
2
4
1
2
4c
2
4d
0.5
2
8a
H
H
F
F
H
H
>32 >32 >32 >32 >32 >32 >32 >32
2
8b
1
1
1
1
1
1
1
1
0.5
0.5
1
1
>32 >32
>32 >32
2
8c
H
H
H
H
F
F
H
H
H
F
2
8d
4
8
8
2
8
4
2
8
4
4
8
8
4
8
8
4
4
4
>32 >32
>32 >32
>32 >32
3
2a
H
F
3
2b
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
3
3
4
H
H
F
F
H
H
4
4
2
4
2
4
4
8
2
4
4
8
>32 >32
>32 >32
>32 >32
>32 >32
>32 >32
4
0a
F
F
F
F
F
H
H
H
H
4
4
8
2
2
4
4
4
8
4
4
8
4
8
8
4
8
8
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
0b
Cl
Br
4
0c
1
0f
H
ꢀ
1
2
1
1
1
2
1
2
1
2
2
2
>32 >32
>32 >32
LZN
ꢀ
ꢀ
a
Abbreviations: MIC is defined as the minimum concentration of a compound that inhibits growth by 99%. Identical
values were obtained for each compound in three replicates by visual investigation as stated in the experimental section;
b
c
d
e
f
g
MSSA ATCC25923; MSSA ATCC29213; MSSA ATCC6538; MSSA CMCC26003; MRSA ATCC33591;
h
i
MRSAATCC43300; Escherichia coli ATCC25922; Pseudomonas aeruginosa ATCC27853.
To explore the possible interaction modes of teraryl oxazolidinone with D. radiodurans 50S
ribosomal subunit (PDB code: 3DLL), a molecular flexible docking study was conducted, and the
results are shown in Figure 4. As depicted, the Cꢀ5 side chains of both compounds (28b and 10f) went
deep into the pocket, which was inconsistent with binding mode of linezolid (C) for the hydrogen bond
formed between the hydrogen atom of the amide group and residue C2505. A π stack interaction may
occur between residue U2585 and pyridyl ring or pyrazinyl ring, while a hydrogen bond, as depicted in
Figure 4ꢀC, formed between residue U2585 and oxygen atom of morpholine ring of Linezolid. The
nitrogen atom of the pyridyl ring of 10f provided hydrogen bond interaction with residue C2067 (Figure
4B), as well as the orthoꢀposition nitrogen atom of the pyrazinyl ring of 28b. However, the pyrazinyl
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ring of 28b rotated to offer extra hydrogen bond interaction between the metaꢀposition nitrogen atom
and residue A2439 (Figure 4A). This hydrogen bond may be the best explanation for its improved in
vitro activities. In addition, another hydrogen bond formed between residue C2066 and Nꢀ2 atom of
pyrazolyl ring of both compounds.
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. In silico docking models of the compound–target complex. Compound 28b (A, colored by cyan), 10f (B,
colored by cyan) and Linezolid (C, colored by yellow) interacting with residues in the binding site of 50S ribosomal
subunit.
Table 3. Mean CC of Selected Compounds and Linezolid in HEK 293 Cells, L02 Cells, THP-1
5
0
Cells and K562 Cells after Incubation for 48 h
a
CC (ꢁM; ꢁg/mL )
5
0
Compd
L02
HEK 293
97.3 ± 4.5;
38.5±1.8
THPꢀ1
> 160;
> 63.3
> 160;
> 70.1
> 160;
> 63.4
K562
> 160;
> 63.3
> 160;
> 70.1
> 160;
> 63.4
7
2.9 ± 3.2;
10f
2
8.8 ±1.2
> 160;
24.6 ± 3.6;
10.1 ±1.6
69.2 ± 4.1;
27.4 ± 1.6
24a
>
70.1
1.1 ± 4.3;
28.2 ± 1.7
7
28b
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Journal of Medicinal Chemistry
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9
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
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5
2.4 ± 3.8;
3.4 ± 1.7
49.5 ± 8.6;
0.4 ± 2.9
> 160;
> 71.4
> 160;
> 160;
> 71.4
> 160;
> 54
2
8c
2
> 71.4
> 160;
> 54
1
72.4 ± 5.5;
24.4 ± 1.9
LZN
5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
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8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
For better reading, ꢁg/mL values for cytotoxicity are also concluded.
At an early stage, the safety profile identification of compounds could be conducted by cytotoxicity
analysis performed with several mammalian cells, like K562 and HEK 293 cells, which could partially
3
4
reflect in vitro myelosuppression ability . Thus, with several potent compounds available, we evaluated
their cytotoxicity on HEK 293 and K562 cell lines, as well as with L02 and THPꢀ1 cell lines. All CC50
values were calculated after 48 h incubation. As shown in Table 4, for HEK 293 and K562 cell lines, all
tested compounds displayed low cytotoxicity, except for compound 24a, which inhibited HEK 293 cell
lines at a relative high degree with a CC value of 24.6 ꢁM. Then, THPꢀ1 and L02 cell lines were not
5
0
significantly inhibited, and all tested compounds expressed a different degree of cytotoxicity against
L02 cell line. In addition, linezolid presented low cytotoxicity to the tested cell lines. Compounds 10f
and 28b showed satisfactory cytotoxicity against all tested cell lines and were selected for further
investigation.
Table 4. Characterization of Various Salts of Selected Oxazolidinone Compounds
Characterization
Salts
aqueous solubility
a
c
d
size (nm)
appearance
ꢀ
pH
ꢀ
b
(
mg/mL)
1
0f
ꢀ
0.3
.
1
0f HBr
898
0.6
paleꢀyellow solid
2.19
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Journal of Medicinal Chemistry
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8
9
1
0f H SO
2248
264
971
0.7
668
ꢀ
1.8
4.3
1.1
47.1
2.0
0.05
0.1
0.06
ꢀ
paleꢀyellow solid
1.77
2.04
1.89
2.75
2.10
ꢀ
2
4
.
10f HCl
paleꢀyellow solid
white solid
yellow solid
white solid
ꢀ
.
1
0f TsOH
.
1
0f H PO
3
4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
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36
37
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39
40
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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
.
10f Oxalic acid
2
8b
.
2
8b H PO
684
687
ꢀ
paleꢀyellow solid
paleꢀyellow solid
viscous solid
ꢀ
2.01
3.1
ꢀ
3
4
.
28b HCl
.
Linezolid HCl
Linezolid
ꢀ
2.7
ꢀ
a
The particle size distribution of prepared solution was determined by a Malvern NanoꢀZS 90 laser particle size
b
c
analyzer after equilibration for 2 min. In mg/mL, determined in water, at 30 °C. Visual observation of products after
d
filtration at room temp (within 12 h). pH determination was carried out for the saturation solution of corresponding
.
salts with pH electrode, whereas pH of 10f H PO was detected at concentration of 4.71 mg/mL.
3
4
Considering the basicity of the pyridyl ring in compound 10f, it was arbitrarily prepared in our
previous work as its hydrochloric acid salt with which in vivo experiments at low dosage, like
pharmacodynamics research, could be accomplished. Its hydrochloric acid salt could form
nanoassemblies in aqueous solution by selfꢀassembly method in pure water, with an average particle
1717
size of 264.6 nm , and observed solubility was elevated from 0.3 mg/mL to 4 mg/mL. This improved
solubility still did not meet the demand of drug administration in the toxicology evaluation and other in
vivo experiments even aided by a variety of pharmaceutical adjuvant. In light of the results, to further
improve the aqueous solubility, a panel of common acids were screened for the salts preparation of
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Journal of Medicinal Chemistry
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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compounds 10f and 28b, including hydrochloric acid, pꢀtoluene sulfonic acid, sulfuric acid, oxalic acid,
hydrobromic acid and phosphoric acid. All these salts were examined for aqueous solubility, as well as
the particle size and pH value of their aqueous solution. As illustrated in Table 4, most of the salts
appeared to be aggregated and featured poor solubility in water, except for 10f phosphate, with
solubility of 47.1 mg/mL. The solubility of 10f phosphate appeared to be significantly enhanced from
that of its base form and was higher than that of linezolid. The observed particle size is extremely tiny
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(0.7 nm) which indicates the real solution in water in form of single molecule and meets the requirement
of intravenous injection development. However, efforts toward enhancing the solubility of 28b through
this method failed even though phosphate appeared as the most soluble salt, with an aqueous solubility
of 0.1 mg/mL.
+
Table 5. hERG K Channel Inhibition of Selected Compounds
+
hERG K Channel Inhibition
Compd
a
IC (ꢁM)
5
0
1
0f
> 40
> 40
> 40
0.13
28b
Linezolid
Cisapride
a
Measured in hERGꢀexpressing CHO cells using Qpatch 16X assay.
In most cases, noncardiovascular drugs with high liability to block the human etherꢀàꢀgoꢀgo related
+
gene (hERG) K channel and delayed cardiac repolarization can induce QT interval prolongation, which
3
5
is frequently associated with potentially lethal arrhythmias . In addition, marketed drugs owning to this
deficiency have been called for withdrawal. Thus, QT prolongation risk was defined as one of the major
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safety concerns in the course of drug development. The in vitro hERG channel activity in mammalian
2
3
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
6
cell lines can be tested to predict QT prolongation risk . Consequently, to estimate the risk, potent
compounds 10f and 28b were selected to evaluate their affinity for the hERG channel using Qpatch
system, in which linezolid was tested for comparison. As shown in Table 5, both 10f and 28b displayed
low hERG inhibition compared with the control drug, cisapride.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. The survival rates of 10f phosphate and 28b phosphate in Mice Systemic Infection Models
Compounds 10f phosphate and 28b phosphate were tested in comparison with linezolid in a
staphylococcal systemic infection model using MRSA strain ATCC33591 in mice. The dissolution of
2
8b phosphate was aided by sulfobutyl etherꢀβꢀcyclodextrin. However, difficulties in administration still
existed, which may result in lower bioavailability and high standard deviations in subjects’ groups. As
shown in Figure 5, the survival rate of compound 28b phosphate was poorer than that of linezolid in the
MRSA infection model at all dosage cohorts following intravenous administration, which may be
caused by the lower exposure of 28b resulted from the poor aqueous solubility of its phosphate. By
contrast, when 10f phosphate was intravenously administered, all dosage cohorts that treated
MRSAꢀinfected mice presented high survival rates over those treated with linezolid at the corresponding
dosage. Consequently, 10f phosphate displayed better in vivo activity against MRSA strain ATCC33591
than linezolid and 28b phosphate.
Table 6. Antimicrobial Activity of 10f phosphate against 120 Clinically Isolated Strains
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1
2
2
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2
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2
2
2
2
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6
MIC (ꢁg/mL)
Strains
Compd
a
MIC50
MIC90 MICMode
MICRange
0.5ꢀ1
1
.
10f H PO
1
1
1
1
1
1
3
4
MSSA (14)
MRSA (23)
Linezolid
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
.
1
0f H PO
1
1
1
0.5ꢀ1
0.5ꢀ1
0.5ꢀ1
0.5ꢀ1
0.5ꢀ1
0.5ꢀ1
0.25ꢀ0.5
0.25ꢀ1
0.5ꢀ1
0.5ꢀ1
1ꢀ2
3
4
Linezolid
1
1
1
.
1
0f H PO
1
1
1
3
4
PSSP (13)
Linezolid
1
1
1
.
1
0f H PO
0.5
0.5
0.25
0.25
1
1
0.5
0.5
0.25
0.25
1
3
4
PRSP/PISP (14)
Linezolid
1
.
Streptococcus
pyogenes (15)
Streptococcus
agalactiae (17)
10f H PO
0.5
1
3
4
Linezolid
.
10f H PO
1
3
4
Linezolid
1
1
1
.
1
0f H PO
1
1
1
3
4
VSE (12)
VRE (12)
Linezolid
1
1
1
1ꢀ2
.
1
0f H PO
1
1
1
1ꢀ2
3
4
Linezolid
1
1
1
1ꢀ2
a
MICmode is the value that is repeated most often in the data set of MIC values against a series of bacterial isolates.
With high efficacy in vivo, we subjected 10f phosphate to an antimicrobial spectrum evaluation, in
which 120 Gramꢀpositive isolate strains were used. As concluded in Table 6, 10f phosphate displayed
excellent antimicrobial activity against all test isolates, which implied that 10f phosphate could be a
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Journal of Medicinal Chemistry
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wideꢀspectrum antiꢀGramꢀpositive bacteria agent.
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 7. Pharmacokinetic Parameters of Compounds 10f and 10f phosphate
Dose
mg/kg/day)
10 mg
Cmax
(ꢁg/ml)
5.8
AUC0ꢀ∞
(ꢁg.h/mL)
157.1
clearance
F
Compd
route
tmax (h) t (h)
1/2
(
(mL/h/kg) (%)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
po
iv
11
13
0.9
0.08
76.7
0.05
1
0f
10 mg
33.1
4.2
0.12
2.4
204.8
po
iv
10 mg
14.7
8.7
81.7
0.15
99.1
0.12
.
1
0f H PO
3
4
10 mg
12.3
0.12
82.3
The pharmacokinetic properties of 10f phosphate and its parent compound 10f were measured in
SD rats following oral administration and intravenous injection, in which experiments
2ꢀhydroxypropyl)ꢀβꢀcyclodextrin was employed as vehicle. As illustrated in Table 7, although 10f
(
featured relatively poor solubility, its pharmacokinetic profiles were claimed acceptable with moderate
halfꢀlife and oral bioavailability. However, its phosphate form upgraded some of PK properties,
including better halfꢀlife of 14.7 h and excellent comparable oral bioavailability to linezolid of which
3
7
the oral bioavailability was reported as 100% . Gaining insights from these results, research works on
tissue distribution and metabolism of 10f phosphate are underway in our laboratory and will be reported
in due course.
Figure 6. Histomorphological assay of main organs of BALB/c mice one week after 10f phosphate treatment
Acute toxicity information on 10f phosphate was determined by 7ꢀday studies. Mice received 10f
phosphate through oral administration at doses of 448 and 896 mg/kg/day. Body weights of mice were
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Journal of Medicinal Chemistry
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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monitored daily. In both dosage cohorts, no evident signs of adverse results were observed in body
weight, behavior or feeding. Furthermore, H&E staining to sections of the heart, liver, kidney, spleen
and lung of mice in the 896 mg/kg group were observed under a microscope (Figure 6) and revealed that
no histopathological changes were found in major organs after oral administration of 10f phosphate.
Among various adverse events of oxazolidinones, hematologic toxicity, which includes anemia,
thrombocytopenia, and hematocytopenia, merits particular consideration. Reversible myelosuppression
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
8–39
has been suggested as a potential explanation for this observation
.
To ascertain myelosuppressive potential of compound 10f phosphate in vivo, a 4ꢀweek
repeatedꢀdose study in mice was conducted. Compound 10f phosphate was administered orally twice
daily (30 rats/group) at high dosage of 448 mg/kg/day, respectively, for 4 weeks. The linezolid
comparator was dosed using a similar method at 300 mg/kg/day. As depicted in Figure 7, some mice in
both 10f phosphate and linezolid groups were deceased by day 12, but 10f phosphate presented a
relatively high survival rate of 93.3% during the test period. Notably, no apparent signs of
myelosuppression including cachexia and body weight loss appeared in mice treated with both
compounds. Meanwhile, blood samples were collected for assessment of hematologic toxicity and the
results are concluded in Table 8.
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10
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43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 7. The survival rates of 10f phosphate and linezolid on normal mice
a
Table 8. Hematology Parameters of Compound 10f phosphate in Mice
9
12
9
WBC (10 /L)
RBC (10 /L)
PLT (10 /L)
Groups
Normal
Day 14
Day 28
4.06
Day 14
Day 28
9.46
Day 14
Day 28
1844
4.85
3.17
2.16
9.78
6.37
7.99
1728
1761
1747
10f phosphate (448 mg/kg)
3.29
8.36
1771
Linezolid (300 mg/kg)
2.88
9.03
1506
a
WBC, white blood cells; RBC, red blood cells; PLT, platelets.
As shown in Table 8, results have revealed a pronounced drop in the white blood cell (WBC) count
in the 300 mg/kg/day cohort for linezolid. In contrast, 10f phosphate induced a slight decrease in WBC
count. Also, red blood cell (RBC) count was also determined. Both compounds resulted in a decrease by
day 14. However, by day 28, slight rise in RBC count occurred in both cohorts, which indicates the
minor myelosuppression would be reversed at the end of administration. Reductions in platelet count,
which is one of the most pertinent indicators, were observed in linezolid cohorts, but not at 10f
phosphate groups, thus indicating lower thrombocytopenia potential for our compound (Table 8). The
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6
aforementioned data indicated a minor attenuated hematological toxicity for compound 10f phosphate in
comparison with linezolid including the propensity to cause thrombocytopenia, which is denoted as a
warning in the prescribing information for linezolid.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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8
9
0
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0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 8. Granulocyte count after administration of 10f phosphate and linezolid at days 7, 14 and 28, respectively. *, P
<
0.05, denote significant difference compared with normal by tꢀtest.
To our knowledge, myelosuppression was related to low granulocyte count. To assess bone marrow
depression of 10f phosphate, bone marrow cytological examination was accomplished and granulocytes
were counted. As depicted in Figure 8, linezolid induced a downtrend of granulocytes and reached nadir
at day 28, which was consistent with the result of hematology evaluation, that is, a decrease in WBC
count. By contrast, no obvious decline was observed during the treatment of 10f phosphate, which
implied that a small risk factor for the leukopenia is associated with 10f phosphate use.
CONCLUSION
An effort of critical balancing potencyꢀ and toxicologyꢀdriven structural modification was made to
find a candidate that shows improved potency and reduced myelosuppression along with good PK
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