Solubility-driven optimization of (pyridin-3-yl) benzoxazinyl- oxazolidinones leading to a promising antibacterial agent

Article abstract of DOI:10.1021/jm4000598

The solubility-driven structural modification of (pyridin-3-yl) benzoxazinyl-oxazolidinones is described, which resulted in the development of a new series of benzoxazinyl-oxazolidinone analogues with high antibacterial activity against Gram-positive pathogens, including that against linezolid-resistant strains and low hERG inhibition. With regard to structure-activity relationship (SAR) trends among the various substituents on the pyridyl ring, relatively small and nonbasic substituents were preferable to sterically demanding or basic substituents. Oxazolidinone ring substitution on the pyridyl ring generated analogues with antibacterial activity superior to imidazolidinone ring. Solubility was enhanced by the incorporation of polar groups, especially when compounds were converted to their prodrugs. Among the prodrugs, compound 85 exhibited excellent solubility and a good pharmacokinetic profile. In a MRSA systemic infection model, compound 85 displayed an ED ₅₀ = 5.00 mg/kg, a potency that is 2-fold better than that of linezolid.

Full text of DOI:10.1021/jm4000598

Article
pubs.acs.org/jmc
Solubility-Driven Optimization of (Pyridin-3-yl) Benzoxazinyl-
oxazolidinones Leading to a Promising Antibacterial Agent
Bin Guo,† Houxing Fan,† Qisheng Xin,† Wenjing Chu,† Hui Wang,‡ Yanqin Huang,§ Xiaoyan Chen,†
and Yushe Yang*,†
†State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203,
China
‡Department of Microbiology, China Pharmaceutical University, Nanjing, Jiangsu Province 210009, China
§MicuRx Pharmaceuticals, Inc., Shanghai 201203, China
S
* Supporting Information
ABSTRACT: The solubility-driven structural modification of
(pyridin-3-yl) benzoxazinyl-oxazolidinones is described, which
resulted in the development of a new series of benzoxazinyl-
oxazolidinone analogues with high antibacterial activity against
Gram-positive pathogens, including that against linezolid-
resistant strains and low hERG inhibition. With regard to
structure−activity relationship (SAR) trends among the
various substituents on the pyridyl ring, relatively small and nonbasic substituents were preferable to sterically demanding or
basic substituents. Oxazolidinone ring substitution on the pyridyl ring generated analogues with antibacterial activity superior to
imidazolidinone ring. Solubility was enhanced by the incorporation of polar groups, especially when compounds were converted
to their prodrugs. Among the prodrugs, compound 85 exhibited excellent solubility and a good pharmacokinetic profile. In a
MRSA systemic infection model, compound 85 displayed an ED50 = 5.00 mg/kg, a potency that is 2-fold better than that of
linezolid.
soluble compounds face other challenges, for example, high
concentrations of poorly soluble drugs in organisms may result in
crystallization and acute toxicity, as in the case of uric acid and
gout. Currently, there are only two oxazolidinone compounds
undergoing clinical evaluation: tedizolid phosphate (2), which is
the phosphate prodrug of bioactive parent tedizolid (3),15 and
radezolid (4).16
INTRODUCTION
■
The emergence and worldwide spread of bacterial pathogens
resistant to existing antimicrobials has become a serious problem
in hospitals and in the community.1−3 In particular, multidrug-
resistant Gram-positive bacteria including methicillin-resistant
Staphylococcus aureus (MRSA) and Staphylococcus epidermidis
(MRSE), vancomycin-resistant Enterococci (VRE), and pen-
icillin-resistant Streptococcus pneumoniae (PRSP) are the major
concern. Oxazolidinones, a new class of synthetic antibacterial
agents, have shown good activity against a broad range of Gram-
positive bacteria such as MRSA, MRSE, and VRE. Linezolid4 (1,
Figure 1) was the first and only member of this class to achieve
FDA approval and has resulted in important and new treatment
options for infections caused by Gram-positive pathogens.
Unfortunately, a short time after its launch, linezolid-resistant
Staphylococcus aureus and Enterococcus faecium began to
emerge.5−7 The success of linezolid in treating bacterial
infections and the emergence of linezolid-resistant strains have
prompted significant interest in the development of new
oxazolidinone drugs with a broad antimicrobial spectrum and
activity especially against linezolid-resistant strains. Numerous
reviews have been published by different groups on oxazolidi-
none antibacterial research.8−12 Significant efforts have been
made which have led to the clinical advancement of more than a
dozen oxazolidinones. However, most of these oxazolidinones
showed poor solubility which resulted in difficulties in
developing an intravenous formulation.13,14 In addition, poorly
In our previous efforts in the development of oxazolidinones,17
we first reported a highly potent benzoxazinyl-oxazolidinone
series as typified by the candidate compound 5. Compared to
linezolid, compound 5 showed a 3- to 4-fold increase in efficacy
in vivo and an excellent pharmacokinetic (PK) profile. However,
the poor solubility of compound 5 limited the development of its
intravenous formulation and led to difficulty in purification. This
prompted us to undertake SAR studies to maintain the
antimicrobial activity, including that against linezolid-resistant
strains, and good PK profile of compound 5, while significantly
increasing its aqueous solubility. In our tricyclic [6,6,5] fused
benzoxazinyl-oxazolidinones, the pyridyl ring was preferable for
the C ring. Therefore, we focused on modifications around the
different substituent groups and their position on the pyridyl ring
and the 3-position side chain of the tricyclic core structure. Our
strategy to improve solubility included: (1) the incorporation of
polar groups; (2) disruption of molecular planarity; (3) the
Received: January 12, 2013
Published: February 21, 2013
© 2013 American Chemical Society
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Figure 1. Structures of oxazolidinone compounds.
α
Scheme 1. General Synthesis of Key Intermediate 13
α
Reagent and conditions: (a) cis-2-butene-1,4-diol, NaH, THF, 0 °C to room temp, 70%; (b) (i) Zn powder, NH4Cl, THF, room temp, (ii) CbzCl,
NaHCO3, THF:H2O = 2:1, 0 °C to room temp, 74% for two steps; (c) L-(+)DET, TBHP, Ti(OiPr)4, 4 Å molecular sieves, CH2Cl2, −40 °C, 72%;
(d) TBDMSCl, DMAP, imidazole, DMF, 0 °C, 78%; (e) nBuLi, THF, −78 °C to room temp, 71%; (f) bis(pinacolato)diboron,
n
Pd(dppf)Cl2·CH2Cl2, KOAc, DMSO, 80 °C, 32.4%; (g) Bu4NF, THF, 0 °C to room temp, 85%.
prodrug strategy. Herein, we report the discovery of a promising
antibacterial agent (85), the prodrug of compound 47, which has
greater in vitro and in vivo activity than linezolid, significantly
improved solubility, a good PK profile, and reduced hERG
channel inhibition.
reaction between compound 11 and bis(pinacolato)diboron,
catalyzed with Pd(dppf)Cl2·CH2Cl2, yielded compound 12 and
the partly deprotected product 13. Compound 12 was converted
to 13 by further deprotection.
The synthesis of the biaryl benzoxazinyl-oxazolidinones is
outlined in Scheme 2. They were assembled by the Suzuki
coupling reaction of key intermediate boric acid ester 13 with a
series of bromide-substituted heteroaromatic fragments. Com-
pounds 21 and 23 were generated from compounds 20 and 22
after reduction with sodium borohydride or PPh3. Compounds
25 and 54 were obtained from 24 and 53 via cleavage of the N-
Boc groups after Suzuki coupling. Compound 31 was
synthesized from 30 with ethoxyamine hydrochloride using
K2CO3 as the base at 40 °C.
The preparation of chiral fragments for compounds 47 and 48
is illustrated in Scheme 3. The route chosen for the chiral pyridyl
fragments was based on previous literature.19 The reaction of 2,5-
dibromopyridine with Weinreb amide 61 generated chloroace-
tylpyridine (compound 62). After the Noyori asymmetry
CHEMISTRY
■
The general synthesis of key intermediate 13 is depicted in
Scheme 1. The preparation involved a nucleophilic aromatic
substitution reaction between cis-2-butene-1,4-diol and 4-
bromo-2-fluoro-1-nitrobenzene (6) to give compound 7.
Reduction of the nitro group and Cbz protection of the resulting
aniline gave intermediate 8. Then, Sharpless asymmetric
epoxidation of 8 afforded compound 9. The hydroxyl group of
9 was then protected using tert-butyldimethylsilyl chloride
(TBDMSCl) to give compound 10. The core tricyclic ring
scaffold 11 was constructed through a tandem cyclization of 10
18
n
with BuLi as the base at −78 °C. The Miyaura coupling
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α
Scheme 2. Synthesis of the Biaryl Benzoxazinyl-oxazolidinones
α
Reagent and conditions: (a) ArBr, Pd(PPh3)4, Cs2CO3, dioxane/H2O, 80 °C; (b) NaBH4, CH2Cl2/MeOH, room temp; (c) PPh3, THF/H2O, 45
°C to room temp; (d) CF3COOH, CH2Cl2/MeOH, room temp; (e) ethoxyamine hydrochloride, K2CO3, CH2Cl2/MeOH, 40 °C.
α
Scheme 3. Asymmetry Synthesis of Chiral Fragments (R)- and (S)-67
α
Reagent and conditions: (a) nBuLi, toluene, −78 °C, 73%; (b)HCOOH, Et3N, dichloro(p-cymene)ruthenium(II)dimer, (1S,2S)-(+)-N-(4-
toluenesulphonyl)-1,2- ethane diamine, MTBE, room temp, 89%; (c) HCOOH, Et3N, dichloro(p-cymene) ruthenium(II)dimer, (1R,2R)-(+)-N-(4-
toluenesulphonyl)-1,2-ethane diamine, MTBE, room temp, 74%; (d) NaN3, DMF, 90 °C, 83% for (S)-64 and 82% for (R)-64; (e) PPh3, THF/H2O,
45 °C to room temp, 81% for (S)-65 and 78% for (R)-65; (f) CDI, DMAP, DMF, room temp, 87% for (S)-66 and 81% for (R)-66; (g) MeI, NaH,
THF, 0 °C to room temp, 91% for (S)-67 and 92% for (R)-67.
reduction of 62, chiral chlorohydrins (R)-63 and (S)-63 were
obtained, respectively, and the ee value was raised to 99.5%
through recrystallization in ethyl acetate/hexane. The chlorine of
compound 63 was substituted by azide to give 64. Then
reduction of compound 64 with PPh3 yielded the desired
compound 65. Oxazolidinone ring compound 66 was prepared
by an intramolecular condensation reaction using the N,N′-
carbonyldiimidazole (CDI) as the agent in DMF. Finally, a
methyl group was introduced into compound 66 to obtain the
target fragments (S)-67 and (R)-67.
The preparation of C-3-substituted amine compound 70 from
33 is illustrated in Scheme 4. First, the hydroxyl group in 33 was
converted into the corresponding mesylate group with mesyl
chloride. Then the resulting compound 68 was reacted with
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α
Scheme 4. Synthesis of Compound 70
α
Reagent and conditions: (a) MsCl, Et3N, CH2Cl2, 0 °C to room temp, 77%; (b) NaN3, DMF, 90 °C, 90%; (c) PPh3, THF/H2O, 45 °C to room
temp, 54%.
α
Scheme 5. Synthesis of Prodrugs of Compound 38
α
Reagent and conditions: (a) Pd2(dba)3, xantphos, Cs2CO3, toluene, 100 °C, 88%; (b) KOAc, DMF, 80 °C, 92%; (c) K2CO3, MeOH, room temp,
70%; (d) (i) (BnO)2PN(iPr)2, 4,5-dicyanoiazole, CH2Cl2, room temp, (ii) 3-chloroperbenzoic acid, CH2Cl2, 0 °C, 92%, for two steps; (e)
intermediate 13, Pd(PPh3)4, Cs2CO3, dioxane/H2O, 80 °C, 34%; (f) 10% Pd/C, H2, room temp, 86%; (g) sodium 2-ethylhexanoate or arginine,
acetone/H2O, room temp, 62% for 78 or 65% for 79.
α
Scheme 6. Synthesis of Prodrugs of Compounds 33 and 47
α
Reagent and conditions: (a) (i) (BnO)2PN(iPr)2, 4,5-dicyanoiazole, CH2Cl2, room temp, (ii) 3-chloroperbenzoic acid, CH2Cl2, 0 °C, 75% for 80 or
72% for 81; (b) 10% Pd/C, H2, room temp, 65% for 82 or 80% for 83; (c) sodium 2-ethylhexanoate or arginine, acetone/H2O, room temp.
sodium azide in DMF to afford compound 69. Finally, reduction
of 69 with PPh3 provided compound 70.
Because of the good in vitro antibacterial activity of
compounds 33, 38, and 47, their various prodrugs were
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synthesized in order to improve water solubility for intravenous
administration.
Table 1. In Vitro Antibacterial Activity of Substituted Pyridyl
Benzoxazinyl-oxazolidinones
The phosphate prodrugs of 38 were synthesized from 60 as
illustrated in Scheme 5. First, the Buchwald−Hartwig coupling
reaction of 60 with 71 resulted in 72, and then nucleophilic
substitution of 72 by potassium acetate afforded compound 73.
The ester 73 was hydrolyzed with K2CO3 as the base in methanol
to give compound 74. Then the hydroxyl group in 74 was
converted to a dibenzyl phosphate ester 75. The Suzuki coupling
reaction of 75 with key intermediate 13 gave 76, and then benzyl
group was cleaved in 10% Pd/C under H2 to give mono-
phosphate ester 77. Finally, compound 77 was reacted with
sodium 2-ethylhexanoate or arginine to obtain phosphate salts 78
or 79.
As shown in Scheme 6, to synthesize the phosphate prodrug of
33, the hydroxyl group in 33 was first converted to a dibenzyl
phosphate ester 80, and then benzyl group was cleaved in 10%
Pd/C under H2 to give monophosphate ester 82. Finally, 82 was
reacted with sodium 2-ethylhexanoate to obtain disodium
phosphate salt 84. In addition, various amino acid-conjugated
ester salt compounds of 33 were synthesized. Unfortunately,
these compounds decomposed either under reaction conditions
or during purification by column chromatography (data not
shown). Using the same synthetic procedures as those in Scheme
6, the phosphate prodrugs of 47 were prepared
RESULTS AND DISCUSSION
■
In Vitro Antibacterial Activity. The target compounds
were evaluated for their antibacterial activity against a panel of
susceptible and resistant Gram-positive organisms, including
linezolid-resistant strains. The results of these studies are
summarized in Tables 1−4.
First, a series of analogues with hydroxyl or amino groups were
designed and synthesized. The introduction of these polar
groups have two purposes: (1) they can, to an extent, directly
improve the water solubility of compounds, (2) they supply
potent sites for further modification, such as prodrug design or
formation of salts. As shown in Table 1, for alcohols, compounds
bearing a relatively small substituent (compounds 14, 15, and
17) displayed good in vitro antibacterial activity and were similar
to that of linezolid. Substituents that were more sterically
demanding yielded reduced activity (16 and 18). Dialcohols
were more potent than the corresponding monoalcohols (19 vs
15). Oxidation of alcohols to ketones lowered the antibacterial
activity, and if ketones were converted further into oximes, the
activity was lost (30 and 31). However, it is worth noting that α-
hydroxy ketone compound 20 exhibited activity several times
more potent than that of linezolid and was the most potent of this
series, with MIC values of 0.25, 0.125−0.5, 0.125−0.5, 0.25−0.5,
0.125−0.5, and 1 μg/mL against MSSA, MRSA, MSSE, MRSE,
PRSP, and Enterococcus faecalis, respectively. Most of analogues
containing substituents with basic groups, no matter the basic
groups were simple alkyl (23 and 25), or nonaromatic rings (26−
28, 51−52, and 54−55), had activity that was lower than that of
linezolid. The best compound within the basic series was
compound 25 with MIC values of only 1−4 μg/mL against all the
tested strains, which was equipotent to linezolid.
α
Abbreviations: MIC, minimum inhibitory concentration; MSSA,
methicillin-sensitive Staphylococcus aureus, 5 strains; MRSA, methi-
cillin-resistant Staphylococcus aureus, 6 strains; MSSE, methicillin-
sensitive Staphylococcus epidermidis, 5 strains; MRSE, methicillin-
resistant Staphylococcus epidermidis, 5 strains; PRSP, penicillin-resistant
Streptococcus pneumoniae, 4 strains; E.f., Enterococcus faecalis, 6 strains.
resulted in analogues 38−41. As shown in Table 2, analogues
substituted at the 5-position of the oxazolidinone ring with
hydroxymethyl (38−40) showed similar activity to the
unsubstituted parent 32. Moreover, the same potency of
compound 39 and 40 showed that the absolute configuration
of this chiral center hardly had an impact on the antibacterial
activity. The replacement of the hydroxymethyl of 38 with
aminomethyl (41) resulted in reduced activity compared with
For further modification, nonaromatic ring substituents were
introduced into the 3-pyridyl core structure, as shown in Table 2.
Molecule 32 showed excellent antibacterial activity, which was 2-
to 4-fold greater than that of linezolid. To improve water
solubility, further polar groups were introduced into the 5-
position of the oxazolidinone ring of compound 32 which
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Table 2. In Vitro Antibacterial Activity of Substituted Pyridyl
Benzoxazinyl-oxazolidinones
Table 3. In Vitro Antibacterial Activity of Substituted Pyridyl
Benzoxazinyl-oxazolidinones
α
Abbreviations: MIC, minimum inhibitory concentration; MSSA,
methicillin-sensitive Staphylococcus aureus, 5 strains; MRSA, methi-
cillin-resistant Staphylococcus aureus, 6 strains; MSSE, methicillin-
sensitive Staphylococcus epidermidis, 5 strains; MRSE, methicillin-
resistant Staphylococcus epidermidis, 5 strains; PRSP, penicillin-resistant
Streptococcus pneumoniae, 4 strains; E.f., Enterococcus faecalis, 6 strains;
LZ, linezolid.
α
Abbreviations: MIC, minimum inhibitory concentration; MSSA,
methicillin-sensitive Staphylococcus aureus, 5 strains; MRSA, methi-
cillin-resistant Staphylococcus aureus, 6 strains; MSSE, methicillin-
sensitive Staphylococcus epidermidis, 5 strains; MRSE, methicillin-
resistant Staphylococcus epidermidis, 5 strains; PRSP, penicillin-resistant
Streptococcus pneumoniae, 4 strains. E.f., Enterococcus faecalis, 6 strains.
displayed excellent activity against all the tested strains, while 37
almost lost its antibacterial activity. It is worth noting that
replacement of the hydroxymethyl at the 3-position of the [6,6,5]
tricyclic core structure with aminomethyl resulted in loss of
antibacterial activity (33 vs 70).
32. On the basis of the bioisosterism approach, some
imidazolidinone ring substituted analogues (42−44) of com-
pound 32 were synthesized and their in vitro antibacterial activity
was evaluated. Unfortunately, most compounds only displayed
moderate activity and were less potent than linezolid. In addition,
the link position between the oxazolidinone ring and the pyridyl
ring was changed from 3-substituted to 5-substituted (45−48).
The antibacterial activity results demonstrated that, for the 5-
substituted oxazolidinone series, compounds bearing a smaller
N-substituent such as methyl exhibited more potent activity (45
vs 46). The obtained lead compound 46 was further fine-tuned
by structure−activity relationship (SAR) studies on the absolute
configuration of the oxazolidinone substituent. This led to the
discovery of compound 47 with greater activity than compound
48, which indicates that the S-configuration is preferable.
Disruption of molecular planarity can improve water
solubility,20 therefore, a methyl group was introduced at the
para-position of the meta-pyridyl ring (Table 3). Unfortunately,
although the resulting compounds 58 and 59 retained the
excellent antibacterial activity of the unsubstituted parent
compounds 33 and 32 against Staphylococcus aureus and
Staphylococcus epidermidis, their activity against Enterococcus
faecalis was significantly reduced. Analogues substituted on the
pyridyl ring with −CN and −CF3 electron-withdrawing moieties
(33 and 37) exhibited quite different activity. Compound 33
Following the clinical use of linezolid, linezolid-resistant
strains of Staphylococcus aureus and Enterococcus faecium began to
emerge. Therefore, one of the hallmarks in the next generation of
oxazolidinones is the demonstration of significant improvements
in activity against linezolid-resistant strains. The antibacterial
activity of some highly active pyridyl benzoxazinyl-oxazolidi-
nones was further evaluated against linezolid-resistant strains. As
shown in Table 4, all compounds, especially compounds 33, 38,
and 47, were more potent than linezolid against common
linezolid-resistant pathogens. Compound 47 exhibited a 4- to 8-
fold increase in activity compared with linezolid against all the
linezolid-resistant strains tested. It is worth noting that the
antibacterial activity of compound 47 against linezolid-resistant
strains was greater than that of tedizolid, which is the only
oxazolidinone at clinical phase III, especially against linezolid-
resistant Staphylococcus aureus.
hERG K+ Channel Inhibition. In most cases, compounds
with high affinity for the hERG ion channel can induce QT
interval prolongation frequently associated with potentially lethal
arrhythmias.21 Consequently, the in vitro hERG channel activity
in mammalian cell lines can be tested to predict QT prolongation
risk. Compound 5, the highly potent oxazolidinone we
previously reported, also had an unsuitable inhibition activity
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As shown in Table 7, different phosphate salts had an
important influence on the pharmacokinetic properties of
prodrugs. The pharmacokinetic profiles of 84 were not as good
as parent compound 33, which indicated that prodrug 84 may be
partly metabolized before it is converted into 33. Compared to
38, its prodrugs 78 and 79 did not show notable improvements in
pharmacokinetic properties.
For compound 47, its prodrugs 85 and 86 were rapidly
converted into 47 after intravenous injection or oral admin-
istration. After oral administration, the Tmax was shorter and was
0.44 and 0.38 h for 85 and 86, respectively. The AUC of 85 was
significantly improved by 57% compared with 47 following oral
administration, while the AUC of 85 was about 1.34-fold greater
than that of 47 after intravenous injection. This may explain why
the oral bioavailability of 85 only improved from 44.8% to 52.5%.
In addition, the T1/2 of compound 85 was longer following
intravenous or oral administration.
In Vivo Efficacy of Compound 85. Compound 85 was
tested in comparison to linezolid in a staphylococcal systemic
infection model using both MRSA and MSSA strains in mice. As
shown in Table 8, the efficacy of compound 85 was not as good as
that of linezolid in the MSSA infection model following
intravenous administration. This may be because compound
85 had a relatively shorter MRT (mean retention time) of 1.25 h
after intravenous administration. Nonetheless, when adminis-
tered orally, lower dosages of 85 were required to cure both the
MSSA and MRSA infections compared to linezolid. In the MSSA
and MRSA infection models, the ED50 values for 85 were 6.65
and 5.00 mg/kg for oral administration, compared to 8.28 and
9.87 mg/kg for linezolid, respectively.
Table 4. In Vitro Activity of Selected Pyridyl Benzoxazinyl-
oxazolidinones against Linezolid-Resistant Bacteria
α
MIC (μg/mL)
compd
19
S.a.
LRSA
LRSE
LREFL
LREFA
0.5
0.25
0.5
0.25
1
16
8
8
4
2
4
8
4
2
2
8
4−8
8
4−8
4−8
4−8
2−4
8
20
32
16
4
4−8
4
33
35
16
8
8
38
0.5
0.5
0.25
1
4
4
47
4
2
2
tedizolid
linezolid
8−16
>16
2−4
16
2−4
16
α
Abbreviations: MIC, minimum inhibitory concentration; S.a.,
Staphylococcus aureus ATCC 29213; LRSA, linezolid-resistant Staph-
ylococcus aureus, 3 strains; LRSE, linezolid-resistant Staphylococcus
epidermidis, 1 strain; LREFL, linezolid-resistant Enterococcus faecalis, 3
strains; LREFA, linezolid-resistant Enterococcus faecium, 3 strains.
for the hERG channel, with IC50 = 2.78 μM. Therefore, to
estimate the risk, potent compounds were selected to evaluate
their affinity for the hERG channel. As shown in Table 5, all
tested compounds displayed significantly less hERG inhibition
than compound 5.
Table 5. hERG K+ Channel Inhibition of Selected Compounds
compd
hERG K+ channel inhibition
20
32
33
38
47
5
18.4% @ 30 μM
13.3% @ 30 μM
IC50 = 18.35 μM
IC50 > 40 μM
CONCLUSION
■
Starting with benzoxazinyl-oxazolidinones represented by
compound 5, an extensive plan of solubility-driven structural
modifications was undertaken with the goal of maintaining the
high antimicrobial activity including that against linezolid-
resistant strains and good PK profile of compound 5 while
significantly increasing aqueous solubility. The SAR study
demonstrated that relatively small and nonbasic substituents
were preferable to sterically demanding or basic substituents. On
the basis of the lead compound 32, a further SAR study was
performed, and this resulted in some excellent compounds
possessing potent antibacterial activity such as compounds 38,
39, 40, 46, and 47. Analogues with a methyl group at the para-
position of the meta-pyridyl ring retained excellent antibacterial
activity against Staphylococcus aureus and Staphylococcus
epidermidis; however, their activity against Enterococcus faecalis
was significantly reduced. Compared to compound 5, selected
compounds exhibited reduced hERG ion channel inhibition.
Several prodrugs of the highly active compounds 33, 38, and 47
exhibited promising results, especially compound 85. This
compound exhibited high in vivo activity for treating MSSA
and MRSA infected mice. Moreover, it had high aqueous
solubility and an excellent pharmacokinetic profile that displayed
rapid conversion to the active antibacterial compound, a long
half-life, and good oral bioavailability. These favorable drug
properties led to the promotion of 85 as a promising drug
candidate. Further preclinical evaluation of compound 85 is
currently underway.
IC50 > 40 μM
IC50 = 2.78 μM
Water Solubility of Prodrugs. As compounds 33, 38, and
47 exhibited excellent in vitro antibacterial activities, including
that against linezolid-resistant strains, their prodrugs were
synthesized in order to dramatically improve their solubility for
intravenous administration. Because of the instability of the
amino acid-conjugated ester prodrugs, these prodrugs were not
included (data not shown). The phosphate salts were
synthesized and evaluated for water solubility. The solubilities
of 78−79 and 84−86 are shown in Table 6. All compounds
showed high water solubility. Therefore, all compounds were
subjected to pharmacokinetic studies.
Table 6. Solubility of Prodrugs in Water
compd
solubility (mg/mL)
78
79
84
85
86
534
210
15
227
196
Pharmacokinetic Properties. The pharmacokinetic prop-
erties of prodrugs 78−79, 84−86 and their parent compounds
38, 33, and 47 were measured in rats following oral
administration and intravenous injection. The study results are
summarized in Table 7.
EXPERIMENTAL SECTION
Minimum Inhibitory Concentration Testing. The minimum
inhibitory concentrations of the novel compounds against Gram-
■
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e
Table 7. Comparison of the Pharmacokinetic Properties of Parent Compounds and Their Prodrugs in Rats
compd
route
dose (mg/kg)
50
Cmax (ng/mL)
Tmax (h)
T1/2 (h)
AUC (ng·h/mL)
CLz (L/h/kg)
F (%)
a
33
84
38
po
po
po
iv
3015
15.2
332
4.50
1.31
0.38
4.69
3.17
2.25
1.08
1.61
1.00
1.45
0.26
1.93
2.51
4.61
4.88
3.88
9.43
33419
81.1
NT
b
10
NT
10
1491
2805
1227
3999
914
27.5
5.6
2.00
2.03
2.78
3.22
2.42
3.37
c
78
79
47
85
86
po
iv
15
650
416
809
524
170
0.25
0.25
1.00
0.44
0.38
19.7
32.6
44.8
52.5
34.8
c
10
c
po
iv
15
c
10
1803
2167
1556
1665
3165
523
po
iv
15
5
d
po
iv
10
d
10
d
po
10
d
iv
10
1445
a
b
NT: not tested. A dose of 10 mg/kg of 84 is equivalent to 7.2 mg/kg of 33. All values were measured based on the amount of the active
c
antibacterial agent 33 detected. A dose of 15 and 10 mg/kg of 78 is equivalent to 11.5 and 7.7 mg/kg of 38, respectively. A dose of 15 and 10 mg/kg
of 79 is equivalent to 7.4 and 4.9 mg/kg of 38, respectively. All values were measured based on the amount of the active antibacterial agent 38
detected. A dose of 10 mg/kg of 85 is equivalent to 7.6 mg/kg of 47. A dose of 10 mg/kg of 86 is equivalent to 4.8 mg/kg of 47. All values were
d
e
measured based on the amount of the active antibacterial agent 47 detected. Abbreviations: Cmax, the peak plasma concentration of a drug after
administration; Tmax, time to reach Cmax; T1/2, elimination half-life; AUC, area under the concentration−time curve; Clz, clearance; F, bioavailability.
α
mg/kg for compounds 85 and linezolid was used as a control. The ED50
was calculated 24 h after treatment by the method of Reed Muench23
and using the Hill equation (Graphpad Prism 5.0; Graphpad Software,
Inc., San Diego, CA, USA).
Table 8. In Vivo Efficacy (ED50) of 85 in the Mouse Systemic
Infection Models
ED50 MRSA (mg/kg)
(ATCC 33591)
ED50 MSSA (mg/kg) (ATCC 19636)
85 Linezolid
85
Linezolid
ASSOCIATED CONTENT
* Supporting Information
■
po
iv
po
8.28
iv
po
po
S
Experimental details and pharmacokinetic studies. This material
is available free of charge via the Internet at http://pubs.acs.org.
6.65
14.15
7.07
5.00
9.87
α
ED50 is the dose at which 50% of infections had been successfully
treated after oral administration.
AUTHOR INFORMATION
Corresponding Author
*Phone: 86-21-5080 6786. Fax: 86-21-5080 6786. E-mail:
ysyang@mail.shcnc.ac.cn.
■
positive bacteria were tested using linezolid as a positive control.
Minimum inhibitory concentration (MIC) values were determined
using an agar dilution method according to the methods of National
Committee for Clinical Laboratory Standards (NCCLS).22 Compounds
were dissolved in 50% water in DMSO to prepare a stock solution that
had a concentration of 320 μg/mL. Serial 2-fold dilutions were prepared
from the stock solution with sterile water and then 10-fold diluted with
Mueller−Hinton (MH) agar medium to provide concentration ranges
of 16−0.03125 μg/mL. The tested organisms were grown in MH broth
medium at 35 °C for 8 h and were adjusted to the turbidity of the 0.5
McFarland standard. The bacterial suspensions were inoculated onto
the drug-supplemented MH agar plates with a multipoint inoculator and
incubated at 35 °C for 16 h.
S. aureus Systemic Infection Model. Compound 85 and linezolid
were studied in a mouse systemic infection model. Institute of Cancer
Research mice (female, Sippr/BK Lab Animal Ltd.) weighing 20−22 g
were used in the study, with six mice in each group. A lethal systemic S.
aureus infection was given to the mice by the injection of 0.5 mL of an
inoculum of S. aureus 107−108 CFU/mL via intraperitoneal injection.
Compounds were administered orally or via intravenous injection 1 h
after infection at doses of 2.5, 5, 10, and 20 mg/kg for compounds 85,
and linezolid was used as a control. The ED50 was calculated 48 h after
treatment by the method of Reed Muench23 and using the Hill equation
(Graphpad Prism 5.0; Graphpad Software, Inc., San Diego, CA, USA).
MRSA Infection Model.24 Female Institute of Cancer Research
mice (B&K Universal Group Limited), weighing between 23 and 25 g,
were rendered neutropenic by treatment with 150 mg/kg of
cyclophosphamide intraperitoneally 4 days prior to infection, and a
further 100 mg/kg was given 2 days prior to infection. Neutropenic
animals were inoculated intraperitoneally with ∼0.5 mL of an inoculum
containing 106 CFU/mL of bacteria. Mice were administered with the
test compounds orally 1 h after infection at doses of 5, 10, 20, and 40
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank the National Natural Science Foundation of China
(no. 81172922) for financial support.
■
ABBREVIATIONS USED
■
MRSA, methicillin-resistant Staphylococcus aureus; MRSE,
methicillin-resistant Staphylococcus epidermidis; VRE, vancomy-
cin-resistant Enterococci; FDA, Food and Drug Administration;
TBDMSCl, tert-butyldimethylsilyl chloride; CbzCl, benzyl
chloroformate; SAR, structure−activity relationship; hERG,
human ether-a-go-go related gene; QT, the length of time
between the start of the Q wave and end of the T wave on an
electrocardiogram
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