Water-soluble pleuromutilin derivative with excellent in vitro and in vivo antibacterial activity against Gram-positive pathogens

Article abstract of DOI:10.1021/jm8000136

Although earlier pleuromutilin analogues showed potent in vitro antibacterial activity against some Gram-positive pathogens, their in vivo efficacy was low because of insufficient pharmacokinetic properties. We designed novel thioether pleuromutilin derivatives having a purine ring as a polar and water solubilizing group and identified a promising pleuromutilin analogue 6 with good solubility in water (～50 mg/mL). Compound 6 exhibited excellent in vitro and in vivo antibacterial activity against some Gram-positive strains, including drug-resistant pathogens.

Full text of DOI:10.1021/jm8000136

J. Med. Chem. 2008, 51, 1991–1994
1991
Letters
Water-Soluble Pleuromutilin Derivative with
Excellent in Vitro and in Vivo Antibacterial
Activity against Gram-Positive Pathogens
Yoshimi Hirokawa,§,¶ Hironori Kinoshita,*,§
Tomoyuki Tanaka,§ Katsuhisa Nakata,† Noriyuki Kitadai,†
Koichi Fujimoto,‡ Shigeki Kashimoto,‡ Tsuyoshi Kojima,‡
and Shiro Kato§
Chemistry Research Laboratories, Dainippon Sumitomo Pharma
Co., Ltd., Enoki 33-94, Suita, Osaka, 564-0053, Japan, Animal
Health Products, Dainippon Sumitomo Pharma Co., Ltd., 5-51, Ebie
1-chome, Fukushima-ku, Osaka, 553-0001, Japan, and
Pharmacology Research Laboratories, Dainippon Sumitomo
Pharma Co., Ltd., 3-1-98, Kasugade Naka, Konohana-ku,
Osaka, 554-0022, Japan
ReceiVed January 7, 2008
Abstract: Although earlier pleuromutilin analogues showed potent in
vitro antibacterial activity against some Gram-positive pathogens, their
in vivo efficacy was low because of insufficient pharmacokinetic
properties. We designed novel thioether pleuromutilin derivatives having
a purine ring as a polar and water solubilizing group and identified a
promising pleuromutilin analogue 6 with good solubility in water (∼50
mg/mL). Compound 6 exhibited excellent in vitro and in vivo
antibacterial activity against some Gram-positive strains, including drug-
resistant pathogens.
Figure 1. Pleuromutilin derivatives.
to mammalian ribosomes.6,7 During the 1970s and early 1980s,
the Sandoz group prepared a number of semisynthetic pleuro-
mutilin analogues and reported initial structure–activity relation-
ships (SARs) that focused on variations in the C14 glycolic acid
side chain.8–10 As a result, tiamulin (2) was successfully
developed as a therapeutic agent for veterinary use.7 A second
veterinary agent, valunemulin (3), with more potent antibacterial
activity than 2, was launched in 1999.11 Further chemical
modifications of 1 aimed at producing an agent for human use
that has sufficient efficacy and is less prone to metabolic
degradation than 1. These efforts resulted in the 1980s in the
development of azamulin (4), which entered phase I clinical
studies in volunteers.12 Although 4 had good antibacterial
activity, its bioavailability was severely limited by atrocious
solubility in water. Thus, 4 showed a short half-life due to rapid
metabolism and subsequent excretion, and the program for
human therapy was subsequently abandoned.13 Recently, re-
searchers at GlaxoSmithKline have described their efforts in
the development of a pleuromutilin derivative for human use.
Their work led to identification of the novel retapamulin (5)14
(Figure 1), which has excellent in vitro activity and was
approved in 2007 as a topical antimicrobial agent for treatment
of skin infections. Although semisynthetic pleuromutilin ana-
logues generally exhibit potent antibacterial activity, they suffer
from being rapidly and extensively metabolized in vivo because
of their strong hydrophobic nature.3
Antibacterial resistance by hospital-acquired Gram-positive
bacterial pathogens, such as methicillin-resistant Staphylococcus
aureus (MRSA), penicillin-resistant Streptococcus pneumoniae
(PRSP), and vancomycin-resistant enterococci (VRE), has
become a serious medical problem.1,2 Although antibacterial
agents including linezolid, quinupristin/dalfopristin, and teico-
planin are now available for the treatment of infections caused
by resistant bacteria, these agents produce undesirable side
effects and their efficacy is restricted because of development
of resistant mutants. It is therefore necessary to identify and
develop new antibacterial agents with novel mechanisms of
action. For one approach in this search for novel antibiotics
agents, we reassessed an earlier antibiotic pleuromutilin (1)
analogue, which was not successfully developed for human
therapy at that time.3
The antibiotic 1, having an unusual tricyclic diterpenoid
structure, was first isolated in 1951 from two basidiomycetes
species and was characterized as a crystalline antibiotic with
modest in vitro activity against Gram-positive pathogens and
mycoplasmas and weak in vivo activity.4,5 Further studies have
shown that this antibiotic selectively inhibits bacterial protein
synthesis through interaction with prokaryotic ribosomes but
has no effect on eukaryotic protein synthesis and did not bind
In the course of our program aimed at the discovery of a
pleuromutilin derivative for human use with potent antibacterial
activity, good solubility in water, and metabolic stability over
previous analogues, we designed the structurally novel thioether
pleuromutilin analogues having a a purine ring as the polar and
water solubilizing group. On the basis of our results of SARs,15
we identified a promising compound 6 with good solubility in
water. Herein, we describe the excellent in vitro and in vivo
antibacterial activity of 6.
* To whom correspondence should be addressed. Phone: +81-6-6337-
5906. Fax: +81-6-6337-6010. E-mail: hironori-kinoshita@ds-pharma.co.jp.
§
Chemistry Research Laboratories.
Current address: Faculty of Pharmacy, Osaka Ohtani University, 3-11-1
¶
Nishikiori-kita, Tondabayashi, Osaka 584-8540, Japan.
†
The purine-propionic acid moiety 10 of 6 was prepared as
shown in Scheme 1. Regioselective reaction of 7-amino-6-
Animal Health Products.
Pharmacology Research Laboratories.
‡
10.1021/jm8000136 CCC: $40.75
2008 American Chemical Society
Published on Web 03/11/2008

1992 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Letters
Scheme 1a
tococci, and Enterococci strains are presented in Table 1. In
addition to these strains, their antibacterial activity against
Staphylococcus epidermidis, Streptococcus pyogenes, Entero-
coccus faecalis, Moraxella (Branhamella) catarrhalis, and
Haemophilus influenzae was also evaluated (Table 1).
The pleuromutilin analogues 4 and 6 exhibited remarkable
antibacterial activity against all Gram-positive pathogens includ-
ing MRSA, PRSP, and VRE, except for E. faecalis. In fact, 4
and 6 were not effective against the E. faecalis strain. Although
4 and 6 revealed almost the same profile of in vitro activity
against susceptible and resistant Gram-positive microorganisms
(MSSAa vs MRSA, PSSP vs PRSP, and VSE vs VRE), 6 showed
a particularly potent antibacterial activity against S. pyogenes
(MIC ) 0.032 µg/mL). Compound 6 was also found to be more
active against MRSA than all the marketed antibacterial agents
tested here except for VCM. More importantly, 6 exhibited
excellent antibacterial activity against susceptible and resistant
S. pneumoniae with an MIC of 0.063 µg/mL and appeared as
a possible alternative to the weak EM, MINO, and LVFX.
Furthermore, 6 was found to have excellent antibacterial activity
against S. epidermidis and relatively good potency against Gram-
negative organisms such as M. catarrhalis and H. influenzae,
both of which are common serious respiratory tract pathogens.
In particular, its antibacterial activity against S. epidermidis was
comparable to that of EM and MINO but significantly superior
to that of VCM or CTRX. VCM, the traditionally last resort
for serious infections caused by Gram-positive pathogens, is
not active against VCM-resistant Gram-positive organisms
including E. faecium. On the other hand, 6 exhibited superior
antibacterial activity against VCM-resistant E. faecium strain
(MIC ) 0.063 µg/mL) compared to CTRX, EM, or LVFX. In
general, 6 displayed potent antibacterial activity against MRSA,
PRSP, and VRE pathogens, while marketed antibacterial agents
were less active than 6 or inactive against these pathogens.
As shown in Table 2, 6 and five reference agents, i.e.,
ampicillin (ABPC), clarithromycin (CAM), MINO, VCM, and
LVFX, were further evaluated for their in vitro activity against
nine anaerobe and two mycoplasma strains. While 6 showed
excellent activity against all anaerobe and mycoplasma strains,
the reference agents appeared to have moderate to weak activity
against some anaerobe and/or mycoplasma strains.
a Reagent and conditions: (i) ethyl acrylate or ethyl 3-bromopropionate,
K2CO3, DMF, 80 °C, 64%; (ii) tert-butyl 1-piperazinecarboxylate, N,N-
diisopropylethylamine, 2-propanol, reflux, 20 h, 92%; (iii) 2 M NaOH/
MeOH, reflux, 2 h, then citric acid/H2O, 94%.
Scheme 2a
a Reagent and conditions: (i) di-tert-butyl dicarbonate, CHCl3, room temp,
2 h; (ii) diisopropyl azodicarboxylate, Ph3P, THF, -20 °C, 0.5 h then
thioacetic acid, -10 °C to room temp, 0.5 h, 98%.
Scheme 3a
a Reagent and conditions: (i) 13, t-BuOK, MeOH, reflux, 2 h, then 14,
0 °C to room temp, overnight, 98%; (ii) 30% HCl/EtOH, room temp, 2 h,
98%; (iii) 10, benzotriazole-1-yloxytris(pyrrolidino)phosphonium hexafluo-
rophosphate, Et3N, DMF, room temp, 2 h, 72%; (iv) 4 M HCl/AcOEt, room
temp, 2 h, 97%.
chloropurine (7) with ethyl acrylate or ethyl 3-bromopropionate
gave the 9-purine propionic ester 8 in moderate yield. Treatment
of 8 with 1-(tert-butoxycarbonyl)piperazine in refluxing 2-pro-
panol followed by alkaline hydrolysis of the resulting ester 9
produced 10 in good yield.
Next, 6, 4, CTRX, EM, MINO, VCM, and LVFX were
examined for their in vivo efficacy (ED50) against lethal S.
aureus (MSSA and MRSA) and S. pneumoniae (PSSP and
PRSP) systemic infection model in mice (Table 3). Compounds
4 and 6 and VCM showed moderate to potent protective effects
against MRSA and PRSP strains compared with other marketed
antibacterial agents. However, as in vitro evaluation, the in vivo
antibacterial activity of 6 was more potent than that of 4.
Interestingly, 6 and VCM exhibited superior ED50 values (about
1-2 mg/kg) when given intravenously regardless of the
frequency of dosing (once or twice), indicating the potential of
6 as an antibacterial agent with efficacy equal to that of VCM.
The other reference agents showed relatively poor in vivo
antibacterial efficacy against drug-resistant strains.
The efficacy of compounds 4 and 6, given orally twice a day,
against S. aureus Smith (MSSA) was next evaluated. While 4
had an ED50 of 13.3 mg/kg, po, the ED50 of 6 was more than
50 mg/kg, po (data not shown).
Finally, the efficacy of 6 and the reference agents EM and
VCM was examined in a pulmonary infection model in which
The intermediate piperidine thioester 13 was prepared by
Mitsunobu reaction of 1-(tert-butoxycarbonyl)-4-hydroxypip-
eridine (12), which was obtained in excellent yield from reaction
of 4-hydroxypiperidine (11) and di-tert-butyl dicarbonate, with
thioacetic acid (Scheme 2).
Compound 6 was prepared as shown in Scheme 3. Reaction
of mutilin 14-tosyloxyacetate8,9 14 with the 4-piperidinethiol,
which was generated by treatment of the thioester 13 with
t-BuOK/MeOH, followed by deprotection of Boc group in the
resultant 15 afforded 16 in excellent yield. Condensation of 16
with 10 in the presence of benzotriazole-1-yloxytris(pyrrolidi-
no)phosphonium hexafluorophosphate as a coupling agent and
successive acid hydrolysis of the resultant 17 gave the desired
6 as a hydrochloride with good solubility in water (∼50 mg/
mL).
The in vitro antibacterial activity of 6, the earlier pleuromutilin
analogue 4, and other marketed antibacterial agents, i.e., a
cephalosporin ceftriaxone (CTRX), a macrolide erythromycin
(EM), a tetracycline minocycline (MINO), a glycopeptide
vancomycin (VCM), and a quinolone levofloxacin (LVFX), was
determined by broth microdilution according to standard pro-
cedures.16 Their minimal inhibitory concentrations (MICs)
against drug-susceptible and/or -resistant Staphylococci, Strep-
a Abbreviations: MSSA, methicillin-susceptible S. aureus; PSSP, penicil-
lin-susceptible S. pneumoniae; VSE, vancomycin-susceptible enterococci.

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 1993
Table 1. In Vitro Antibacterial Activity of 6, 4, and Reference Agents (CTRX, EM, MINO, VCM, and LVFX)
MIC (µg/mL)
organism
6
4
CTRX
EM
0.063
0.125
>128
32
0.063
0.016
MINO
VCM
LVFX
0.125
0.125
>128
>128
0.25
S. aureus FDA 209P (MSSA)
S. aureus Smith (MSSA)
S. aureus KT0116 (MRSA)a
S. aureus KMP9 (MRSA)a
S. epidermidis ATCC12228
S. pneumoniae ATCC49619 (PSSP)
S. pneumoniae KT2524 (PRSP)b
S. pyogenes ATCC12344
E. faecalis ATCC29212
0.063
0.25
0.5
0.25
0.5
1
2
2
0.063
0.063
16
0.5
1
1
0.5
1
0.25
0.5
0.5
4
>128
0.5
2
>128
4
0.063
0.063
0.063
0.032
0.25
0.5
0.5
0.25
128
0.5
0.25
0.032
0.5
2
0.125
0.032
8
0.063
1
0.063
0.063
0.25
0.5
0.063
0.5
8
0.5
0.5
4
64
0.063
0.032
0.5
0.016
4
>128
0.008
2
1
64
E. faecium ATCC19434 (VSE)
E. faecium KU1778 (VRE)c
M. catarrhalis K1209
0.063
0.063
0.032
0.125
4
0.5
>128
>128
>128
0.25
0.008
0.032
2
64
H. influenzae TH13
>128
a A clinical isolate of MRSA. b A clinical isolate of PRSP. c A clinical isolate of VRE.
Table 2. In Vitro Antibacterial Activity of 6 and Reference Agents (ABPC, CAM, MINO, VCM, and LVFX) against Anaerobe and Mycoplasma
Strainsa
MIC (µg/mL)
organism
6
ABPC
CAM
MINO
VCM
LVFX
anaerobes
Staphylococcus saccharolyticus GAI 5520
Streptococcus intermedius GAI 7416
Atopobium parVulum GAI 5542
Peptostreptococcus asaccharolyticus GAI 5534
Peptostreptococcus anaerobius GAI 5506
Peptostreptococcus magnus GAI 5528
Clostridium perfringens GAI 5526
Propionibacterium acnes GAI 5568
Propionibacterium granulosum GAI 7414
mycoplasma
0.06
0.25
0.25
0.032
0.032
0.063
0.063
0.008
0.016
0.008
0.125
0.125
0.25
0.125
0.25
0.063
0.125
0.063
0.063
0.125
0.25
0.25
2
1
2
0.125
0.5
0.25
2
0.125
0.125
0.25
0.25
0.25
<0.004
<0.004
>128
<0.004
2
1
16
0.125
0.032
0.125
0.063
0.125
0.25
0.25
0.25
0.5
0.5
0.5
<0.004
<0.004
Mycoplasma pneumoniae FH Liu
Mycoplasma pneumoniae Mac
0.008
0.008
NT
NT
0.016
0.016
0.25
0.25
>128
>128
0.125
0.063
a NT: Not tested.
Table 3. In Vivo Antibacterial Activity of 6, 4, and Reference Agents (CTRX, EM, MINO, VCM, and LVFX)
ED50 (mg/kg/dose)a
organism
6
4
CTRX
EM
2.55
>100
1.48
>100
MINO
VCM
LVFX
S. aureusSmith (MSSA)
S. aureusKT0116 (MRSA)
S. pneumoniae ATCC49619 (PSSP)
S. pneumoniae KT2524 (PRSP)
1.86 (1.51)
2.15 (0.91)
1.54 (1.34)
1.55 (1.88)
(6.88)
(5.20)
(12.9)
(17.7)
3.72
>100
0.22
0.515
50
0.968
50
0.88
1.01
1.06
2.37
0.78
>100
24.7
27.5
>100
a The efficacy criterion, ED50, was calculated as the dose at which mice survival rate was 50%. Mice were inoculated intraperitoneally. Medication was
given intravenously once, 1 h after infection. Values in parentheses represent ED50 given as intravenous twice, 1 and 4 h after infection.
consecutive days provided significant protection (83-67%
survival rate) against lethal pulmonary challenge with S.
pneumoniae ATCC6303. Although the treatment dose was
reduced to 6.25 mg/kg, 6 and VCM provided potent protective
effect with 50% survival in both cases. In general, the in vivo
efficacy of 6 was comparable to that of VCM and somewhat
higher than that of EM. Clearly, in vitro antibacterial activity
is not the only parameter that influences in vivo efficacy in this
study, as evidenced by identical MIC values of 6 and EM against
S. pneumoniae ATCC6303. Pharmacokinetics and ADME
properties also have considerable impact on efficacy.
In summary, 6 with good solubility in water not only
conferred in vitro antibacterial activity against a number of
Gram-positive and several Gram-negative pathogens, including
MRSA, PRSP, VRE, anaerobes, and mycoplasmas, but also
showed potent in vivo efficacy in mice. Furthermore, 6 was
very effective at preventing mortality induced by penicillin-
susceptible S. pneumoniae ATCC6303 and exhibited ap-
proximately the same antibacterial activity as VCM in this
animal model. S. aureus, S. pneumoniae, H. influenzae, M.
catarrhalis, S. pyogenes, and mycoplasma are known to cause
Figure 2. Dose–response study showing prevention by 6, EM, and
VCM of mortality in mice exposed to a lethal challenge of S.
pneumoniae ATCC6303 by intratracheal instillation.
mice were inoculated with penicillin-susceptible S. pneumoniae
ATCC6303. As shown in Figure 2, twice daily intraperitoneal
treatment with 25 mg/kg of 6 (MIC ) 0.032 µg/mL), VCM
(MIC ) 0.5 µg/mL), or EM (MIC ) 0.032 µg/mL) for 3

1994 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Letters
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main causative pathogen of community-acquired pneumoniae.
Because 6 showed potent in vitro and in vivo antibacterial
activity against almost all of the main causative pathogens of
community-acquired pneumoniae tested, it is suggested that 6
has potential as an antibacterial agent for use in humans. In
future publications, we will give extensive details on the in vitro
SARs and in vivo antibacterial activity of this compound. In
addition, a more comprehensive study directed at optimization
for an even more potent in vivo efficacy will be reported in
due course.
(9) Egger, H.; Reinshagen, H. New pleuromutilin derivatives with
enhanced antimicrobial activity: I. J. Antibiot. 1976, 29, 915–922.
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