Pleuromutilin derivatives having a purine ring. Part 1: New compounds with promising antibacterial activity against resistant Gram-positive pathogens

Article abstract of DOI:10.1016/j.bmcl.2008.05.011

In the course of our research aimed at the discovery of metabolic stable pleuromutilin derivatives with more potent antibacterial activity against Gram-positive pathogens than previous analogues, a series of compounds bearing a purine ring were prepared and evaluated. From SAR studies, we identified two promising compounds 85 and 87, which have excellent in vitro activity against a number of Gram-positive pathogens, including existing drug-resistant strains, and potent in vivo efficacy.

Full text of DOI:10.1016/j.bmcl.2008.05.011

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3556–3561
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pleuromutilin derivatives having a purine ring. Part 1: New compounds
with promising antibacterial activity against resistant Gram-positive pathogens
a
a
b
Yoshimi Hirokawa a, , Hironori Kinoshita a, , Tomoyuki Tanaka , Takanori Nakamura , Koichi Fujimoto ,
*
Shigeki Kashimoto b, Tsuyoshi Kojima b, Shiro Kato a
a Chemistry Research Laboratories, Dainippon Sumitomo Pharma Co., Ltd, Enoki 33-94, Suita 564-0053, Japan
b Pharmacology Research Laboratories, Dainippon Sumitomo Pharma Co., Ltd, 3-1-98 Kasugade Naka, Konohana-ku, Osaka 554-0022, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
In the course of our research aimed at the discovery of metabolic stable pleuromutilin derivatives with
more potent antibacterial activity against Gram-positive pathogens than previous analogues, a series
of compounds bearing a purine ring were prepared and evaluated. From SAR studies, we identified
two promising compounds 85 and 87, which have excellent in vitro activity against a number of
Gram-positive pathogens, including existing drug-resistant strains, and potent in vivo efficacy.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 28 February 2008
Revised 27 April 2008
Accepted 2 May 2008
Available online 4 May 2008
Keywords:
Pleuromutilin
Gram-positive bacteria
MRSA
PRSP
VRE
Purine ring
Metabolic stability
MIC
Sulfide
The increasing use of antibacterial agents for infectious dis-
eases has resulted in the emergence of resistant pathogens, espe-
cially Gram-positive bacteria including methicillin-resistant
Staphylococcus aureus (MRSA), penicillin-resistant Streptococcus
pneumoniae (PRSP), and vancomycin-resistant enterococci
(VRE).1–3 To combat such drug-resistant bacterial strains, there
is an increasing need to discover and develop novel classes of
antibiotics, particularly agents with new mechanisms of action
and consequently no cross-resistance to marketed antibacterial
agents. In our search for promising lead structures that can be
used as new antibiotics, we have focused our attention on the
natural product pleuromutilin4–7 (1), which has good antibacte-
rial activity but insufficient in vivo potency.
karyotic ribosomes, but has no effect on eukaryotic protein synthe-
sis and does not bind to mammalian ribosomes.9 A Sandoz group
prepared a number of semisynthetic pleuromutilin derivatives
and reported initial SAR studies that focused on variations in the
C14 glycolic acid side chain.10–12 As a result, tiamulin (2) and val-
unemulin (3) were successfully developed as therapeutic agent for
veterinary use.13 Further chemical modifications of 1 aimed at pro-
ducing an agent for human use that has sufficient antibacterial effi-
cacy and is less prone to metabolic degradation than 1. These
efforts resulted in the 1980s in the development of azamulin
(4).14 Although 4 showed good in vitro antibacterial activity, its
oral bioavailability was severely limited by atrocious solubility in
water. Thus, 4 entered phase I clinical studies in volunteers but
did not progress further. Recently, researchers at GlaxoSmithKline
identified the novel pleuromutilin analogue retapamulin (5),15
which shows excellent in vitro antibacterial activity and was there-
fore approved in 2007 as a topical antimicrobial agent for treat-
ment of human skin infections. From previous SAR studies on 1,
analogues in which the hydroxyl of the C14 glycolic ester group
in 1 was replaced with a substituent containing the sulfide linkage,
show potent in vitro activity but suffer from being rapidly and
extensively metabolized in vivo because of their strong hydropho-
bic nature. Quite recently, we reported the excellent in vitro and in
The fused 5-6-8 tricyclic diterpenoid 1 was first isolated in 1951
from two basidiomycete species and was characterized as a crys-
talline antibiotic with modest in vitro activity against Gram-posi-
tive bacteria and mycoplasmas.8 The antibiotic
1 selectively
inhibits bacterial protein synthesis through interaction with pro-
* Corresponding author. Tel.: +81 6 6337 5906; fax: +81 6 6337 6010.
E-mail address: hironori-kinoshita@ds-pharma.co.jp (H. Kinoshita).
Present address: Faculty of Pharmacy, Osaka Ohtani University, 3-11-1 Nishikiori-
kita, Tondabayashi, Osaka 584-8540, Japan.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.011

Y. Hirokawa et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3556–3561
3557
vivo antibacterial activity of the structurally novel pleuromutilin
analogue 6 having a purine ring as a polar and water solubilizing
group.16 The excellent in vivo efficacy of 6 showing good solubility
in water may reflect good metabolic stability. In this communica-
tion we describe the synthesis and in vitro and in vivo antibacterial
activities of these pleuromutilin derivatives having 4-piperidin-
ethio moiety (see Fig. 1).
The 3-(6-substituted purin-9-yl)propionic ethyl esters 46–53 were
prepared by the reverse method described for synthesis of 28–37,
that is, reaction of 7f with nitrogen-containing heteroalicycles
bearing N-Boc substituent, followed by alkylation of the resultants
38–45 with ethyl acrylate gave the desired esters 46–53.
The pleuromutilin derivatives 55–89 shown in Tables 1–3 were
prepared as illustrated in Scheme 2. Acid hydrolysis of the resul-
tant tert-butyl esters 8a, 10a, 11a, 12b, 13c, 14c, 16c, 17d, and
19–26 using trifluoroacetic acid (TFA) afforded the corresponding
(purin-7- or -9-yl)carboxylic acids. The 3-(purin-9-yl)propionic
acids having N-Boc substituent in nitrogen-containing heteroalicy-
cles were obtained by alkaline hydrolysis of the corresponding
ethyl esters 28–37 and 46–53. Condensation of the (purin-7- or
-9-yl)carboxylic acids with 5410,11 in the presence of benzotria-
zole-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate
as a coupling agent, and in the case of compounds having N-Boc
substituent, successive acid hydrolysis gave 55–89 as a free base
or a hydrochloride in moderate to good yields. The free base com-
pounds 68, 69, 76, and 83 having a basic nitrogen were treated
with HCl in AcOEt to prepare the corresponding hydrochlorides.
The chemical structures of all pleuromutilin derivatives obtained
were confirmed by 1H NMR and mass spectra and the purity was
demonstrated by HPLC analysis. The pleuromutilin derivatives ob-
tained as hydrochlorides showed good solubility in water
(ꢀ50 mg/mL).
The purine-carboxylic esters 8a–11a, 12b, 13c–16c, 17d, 18e,
19–37, and 46–53 were prepared as shown in Scheme 1. Reaction
of purine (7a) and 2-aminopurine (7c) with tert-butyl bromoace-
tate, tert-butyl 3-bromopropionate or tert-butyl acrylate, and
tert-butyl propiolate gave a mixture of the corresponding 9- and
7-substituted purine esters 8a, 10a, 14c and 9a, 11a, 15c, respec-
tively. After separation of the mixture by silica gel column chroma-
tography, the less polar 9-substituted purine esters 8a (51%), 10a
(19%), and 14c (57%) and the more polar 7-substituted purine es-
ters 9a (32%), 11a (4%), and 15c (23%) were obtained.17 Treatment
of 6-amino-, 2-amino-, and 2,6-diaminopurine (7b–d) with tert-
butyl 3-bromopropionate or tert-butyl acrylate and tert-butyl 4-
bromobutyrate regioselectively furnished the 9-substituted purine
esters 12b, 13c, 16c, and 17d. The 3-(2-amino-6-substituted purin-
9-yl)propionic esters 19–26 were prepared by treatment of 18e,
which was obtained by reaction of 2-amino-6-chloropurine (7e)
with tert-butyl acrylate, with methylamine, dimethylamine, and
nitrogen-containing heteroalicycles, such as pyrrolidine, morpho-
line, piperazine, and piperidine rings. On the other hand, the 3-
(2-amino-6-substituted purin-9-yl)propionic ethyl esters 28–37
having N-Boc substituent in the nitrogen-containing heteroalicy-
cles were obtained by reaction of the corresponding ethyl ester
27, which was prepared from 7e and ethyl acrylate, with
nitrogen-containing heteroalicycles bearing N-Boc substituent.
Initial screening for antibacterial activity18 led to identification
of the 3-(purin-9-yl)propionamide 55, which showed potent in vi-
tro activity against methicillin-susceptible S. aureus Smith (MSSA),
S. aureus KMP9 (MRSA), penicillin-susceptible S. pneumoniae I
(PSSP), and Enterococcus faecium KU1778 (VRE). Although 55 dis-
played similar activity against both susceptible (MSSA, PSSP) and
resistant (MRSA, VRE) strains regardless of their susceptibility to
other classes of antibiotics, its in vivo efficacy was characterized
by a higher ED50 value (>3.13 mg/kg) against S. aureus Smith sys-
temic infection model in mice. We therefore set out to investigate
the influence of changes in the position and substituent, such as
the amino group of the purine ring or the ethylene chain on the
in vitro and in vivo antibacterial activities, while keeping the muti-
lin framework with its 4-piperidinylthio moiety as a spacer intact
(Table 1). The 3-(purin-7-yl)propionamide 56 as a regioisomer of
lead compound 55 showed slightly decreased in vitro activity.
Shortening of the ethylene chain in 55 (giving 57) caused a signif-
icant decrease in activity against all strains. Introduction of an ami-
no group into the 6-position at the purine ring as in 58 led to
poorer MIC values. On the other hand, the regioisomer 59 of 58,
that is, 3-(2-aminopurin-9-yl)propionamide was essentially equi-
potent to 55. Quite surprisingly, 59 exhibited dramatic improve-
ment of in vivo efficacy (ED50 = <3.13 mg/kg) compared with 55,
56, and 58. Extension of the ethylene chain (giving 60) and inser-
tion of double bond (giving 61) in the ethylene chain of 59 had
no favorable influence on the in vitro or in vivo activity.
Me
R1
OH
O
Me
R-CH2CO
14
Me
Me
O
Pleuromutilin (1); R = OH, R1 = CH=CH2
Tiamulin (2); R = -SCH2CH2NEt2, R1 = CH=CH2
Me
Me
O
Valunemulin (3);
R = -S
Me
N
H
Me
NH2
R1 = CH=CH2
N N
Influence of a change in the substituent at the 6-position in the
2-aminopurine ring of 59 was next examined (Table 2). Introduc-
tion of an amino group as in 62 substantially retained the in vitro
activity against all strains compared with that of 59, but the in vivo
efficacy was not improved. Substitution by a methylamino or a
dimethylamino group, or by a pyrrolidine or a morpholine ring
(giving 63–66, respectively) provided no favorable effect on the
in vitro or in vivo activity. On the other hand, introduction of a
piperazine ring (yielding 6) improves in vivo efficacy.
In addition to MSSA, MRSA, PSSP, and VRE shown in Tables 1
and 2, MIC values of the pleuromutilin analogues 59, 6, and 67–
89 against S. pneumoniae KT2524 (PRSP), Streptococcus pyogenes,
Moraxella catarrhalis, and Haemophilus influenzae, all of which are
common serious respiratory tract pathogen and their in vivo effi-
cacy in mice are illustrated in Table 3, which also includes the
R1 = Et
Azamulin (4);
R = -S
NH2
N
H
Me
N
Retapamulin (5);
R =
O
-S
R1 = CH=CH2
N
N
R1 = CH=CH2
N
6;
R =
-S
-S
N
HCl
H2N
N
N
NH
R1 = CH=CH2
54; R =
NH
Figure 1. Structure of pleuromutilin derivatives.

3558
Y. Hirokawa et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3556–3561
R2
a; R2 = R3 = H
R2
N
t
R2
XCO2 Bu
b; R2 = NH2, R3 = H
c; R2 = H, R3 = NH2
d; R2 = R3 = NH2
e; R2 = Cl, R3 = NH2
f; R2 = Cl, R3 = H
N
i)
7
N
N
N
N
N
N
+
N
R3
N
N
H
R3
R3
9
N
7a-f
t
XCO2 Bu
9a; X = CH2, 32%
11a; X = CH2CH2, 4%
15c; X = CH=CH, 23%
ii)
iii)
8a; X = CH2, 51%
10a; X = CH2CH2, 19%
12b; X = CH2CH2, 31%
13c; X = CH2CH2, 78%
14c; X = CH=CH, 57%
16c; X = (CH2)3, 85%
17d; X = CH2CH2, 68%
18e; X = CH2CH2, 94%
or iv)
R2
Cl
N
N
N
R2
N
N
N
N
N
N
N
ii)
H2N
H
38-45
CO2Et
27
iv)
N
N
19-26
H2N
t
CO2 Bu
Me
NH
iii)
19; R2 = -NHMe
20; R2 = -NMe2
21; R2 =
R2
24; R2 =
25; R2 =
26; R2 =
N
N
N
R2
N
N
N
N
N
NMe2
22; R2 =
N
N
N
O
N
N
H2N
N
N
CO2Et
23; R2 =
28-37
Me
NMe2
CO2Et
46-53
H
N
NHBoc
32, 42, 50; R2 =
34, 44, 52; R2 =
35, 45, 53; R2 =
36; R2 =
Boc
28, 38, 46; R2 =
29, 39, 47; R2 =
N
N
N
N
N Boc
H
N
NBoc
Me
42R, 50R; R2 =
42S, 50S; R2 =
Boc
N
NHBoc
NHBoc
N
N
H
N
Boc
Me
30, 40, 48; R2 =
31, 41, 49; R2 =
N
NHBoc
Boc
N
N
N
Boc
37; R2 =
Boc
N
N
33, 43, 51; R2 =
N
N
Me
Me
Scheme 1. Reagents and conditions: (i) BrCH2CO2tBu, Br(CH2)3CO2tBu, CH2@CHCO2tBu (Br(CH2)2CO2tBu) or CH„CCO2tBu, K2CO3 (Na2CO3), DMF (aq THF), 50–80 °C (reflux),
i
4–24 h; (ii) R2H, K2CO3 (80 °C) or DBU (rt), DMF, 15 h; (iii) CH2@CHCO2Et, K2CO3, DMF, 80 °C, 24 h, 65%; (iv) R2H, Pr2NEt, 2-PrOH, 20 h, >90%.
Table 1
In vitro and in vivo antibacterial activities of 55–61
CH2
Me
OH
O
S
Me
O
N
X
N
X1
X2
N
Me
Me
O
X4
X3
O
Compound
X1
X2
X3
X4
X
MICa (lg/mL)
MSSAb
MSSAa
MRSAc
PSSPd
VREe
ED50 (mg/kg, iv)
f
55
56
57
58
59
60
61
CH
N
CH
C-NH2
CH
N
CH
N
N
N
CH
N
CH
CH
C-NH2
C-NH2
C-NH2
N
CH
N
N
N
(CH2)2
(CH2)2
CH2
(CH2)2
(CH2)2
(CH2)3
CH@CH
0.05
0.1
0.2
0.2
0.05
0.1
0.05
0.1
0.2
0.39
0.05
0.2
0.1
0.1
0.05
0.1
0.2
0.39
0.05
0.1
>3.13
>3.13
NTg
>3.13
<3.13
>3.13
>3.13
1.56
0.78
0.05
0.78
0.39
CH
CH
N
N
N
N
0.1
0.2
0.2
a
Minimum inhibitory concentration (MIC): lowest concentration of compound that inhibits visible growth of the organism.
b
c
d
e
f
MSSA, methicillin-susceptible S. aureus Smith.
MRSA, S. aureus KMP9.
PSSP, penicillin- susceptible S. pneumoniae I.
VRE, E. faecium KU1778.
The efficacy criterion, ED50, was calculated as the dose at which mice survival rate was 50%. Mice were inoculated with each organism intraperitoneally. Medication was
given intravenously once, 1 h after inoculation.
g
NT, not tested.
activity of the earlier pleuromutilin analogue 4 and the marketed
antibacterial agent vancomycin (VCM) for comparison. Compound
59, which showed potent in vitro activity displayed strong in vivo
efficacy with an ED50 value of 2.89 mg/kg. This value was much
lower than that of 4 but approximately threefold higher than that
of VCM. All compounds with the nitrogen-containing heteroalicy-

Y. Hirokawa et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3556–3561
3559
Table 2
In vitro and in vivo antibacterial activities of 6, 59, and 62–66
CH2
Me
OH
O
S
N
Me
O
R2
N
N
N
Me
Me
O
N
O
H2N
Compound
R2
MICa (lg/mL)
MSSAb
f
MSSAb
MRSAc
PSSPd
VREe
ED50 (mg/kg, iv)
59
62
63
64
H
0.05
0.05
0.1
0.05
0.1
0.2
0.05
0.05
0.05
0.1
<3.13
>3.13
>3.13
>3.13
NH2
NHMe
NMe2
0.025
0.025
0.05
0.1
0.1
0.1
65
66
0.39
0.2
0.39
0.2
0.2
0.39
0.2
>3.13
NTg
N
N
N
0.1
O
6
0.25
0.5
0.063
0.25
<3.13
NH
a–gSee Table 1.
Table 3
In vitro and in vivo antibacterial activities of 6, 59, and 67–89
CH2
Me
OH
O
S
N
Me
O
R2
N
N
N
Me
Me
O
HCl
N
R3
O
Compound
R2
H
R3
MICa (lg/mL)
MSSAb
MSSAb
0.05
MRSAc
0.05
PSSPd
PRSPe
S. p.f
VREg
0.05
M. c.h
H. i.i
ED50 (mg/kg, iv)
j
59
6
NH2
NH2
0.125
0.063
0.063
0.063
0.25
2
2.89
0.25
0.5
0.063
0.032
0.063
0.032
0.016
0.063
0.125
0.032
0.125
0.25
1
1
2
1.86 (1.51)k
N
NH
NH
N
67
68
H
0.063
0.125
0.063
0.125
0.032
0.125
0.032
0.063
2.94
8.83
N
N
NH2
Me
Me
NH
69
70
71
72
73
74
75
76
NH2
NH2
H
0.25
0.25
0.125
0.25
0.125
0.5
0.25
1
0.063
0.063
0.016
0.063
0.016
0.125
0.063
0.063
0.063
0.125
0.032
0.063
0.032
0.25
0.032
0.063
0.016
0.032
0.016
0.063
0.032
0.032
0.125
0.25
0.063
0.125
0.063
0.125
0.032
0.25
2
2
4
2
1
2
2
2
2.21
1.86
1.50
2.21
2.21
1.41
2.21
1.72
N
N
NH2
0.25
1
0.063
0.25
N
N
NH2
NH2
NH2
H
NH2
0.125
2
0.032
0.5
N
N
NH2
H
NHMe
0.125
0.25
0.25
0.25
0.063
0.063
0.063
0.125
0.125
0.063
N
N
NHMe
NMe2
NH2
(continued on next page)

3560
Y. Hirokawa et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3556–3561
Table 3 (continued)
Compound
R2
R3
MICa (lg/mL)
MSSAb
j
MSSAb
0.5
MRSAc
1
PSSPd
0.125
PRSPe
S. p.f
VREg
M. c.h
H. i.i
ED50 (mg/kg, iv)
NH2
77
78
79
80
81
82
83
84
85
86
87
88
89
NH2
H
0.125
0.063
0.125
0.25
1
3.05
1.47
1.30
1.01
1.72
1.61
3.00
2.84
0.59
1.01
0.76
3.04
3.97
N
N
N
N
N
N
NH2
0.063
0.125
0.063
0.25
0.063
0.25
0.5
0.125
0.5
0.25
1
0.016
0.032
0.016
0.063
0.016
0.063
0.063
0.016
0.125
0.063
0.063
0.063
0.032
0.032
0.016
0.063
0.032
0.063
0.125
0.032
0.25
0.016
0.016
0.008
0.032
0.016
0.063
0.032
0.008
0.032
0.016
0.063
0.125
0.032
0.125
0.063
0.25
0.032
0.125
0.5
0.032
0.125
0.063
0.125
0.063
0.125
0.125
0.063
0.25
1
2
1
1
2
2
4
1
4
2
1
2
NH2
H
NH2
H
NHMe
NHMe
NMe2
NH2
H
0.25
0.25
2
NH2
NH2
H
N
N
NH2
NH2
0.125
0.5
0.5
4
0.125
0.5
N
N
NHMe
NH2
H
NHMe
0.25
0.5
0.5
1
0.063
0.125
0.125
0.125
0.25
0.5
0.125
0.25
N
N
NH2
NH2
NH2
0.5
1
0.25
N
NHMe
4
0.5
1
2
0.5
0.5
0.5
0.25
0.5
0.25
0.032
64
0.5
(6.88)k
0.88
VCM
0.5
0.25
>128
>128
a–c, g,
d
j
See Table 1a–c, e, f.
S. pneumoniae ATCC49619.
S. pneumoniae KT2524.
S. pyogenes ATCC12344.
M. catarrhalis K1209.
H. influenzae TH13.
e
f
h
i
k
Mice were inoculated with each organism and medication was given intravenously twice, 1 and 4 h after inoculation.
R2
N
R2
N
N
N
N
N
ii)
XCO2H
iii)
i)
XCO2 Bu(Et)
N
N
55-89
R3
R3
t
8a, 10a, 11a, 12b,
13c, 14c, 16c, 17d,
19-26, 28-37, 46-53
Scheme 2. Reagents and conditions: (i) TFA, CH2Cl2, rt, 2 h or 2 N NaOH/MeOH, reflux, 2 h; (ii) 54, benzotriazole-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate,
Et3N, DMF, rt, 2 h; (iii) 30% HCl/EtOH, rt, 2 h or 4 M HCl/AcOEt.
cles at the 6-position in the purine ring were found to possess good
to excellent in vitro activity against all strains compared with VCM.
Furthermore, their in vivo efficacy was clearly superior to that of 4.
As the 2-amino-6-piperazinylpurine analogue 6 shown in Table
2 displayed more potent in vivo efficacy than 59, we decided to
prepare its related piperazine derivatives. Removal of the 2-amino
group from 6 (giving 67) exhibited a higher in vitro activity against
all strains, but the in vivo efficacy was less than that of 6. Introduc-
tion of a methyl group into the piperazine ring of 6 (yielding 68 and
69) did not improve the in vitro or in vivo activity. Replacement of
the piperazine ring in 6 and 67 with a 4-aminopiperidine ring (giv-
ing 70 and 71, respectively) generally had no favorable influence
on the in vitro activity. However, 70 showed an in vivo efficacy
comparable to that of 6 and the in vivo efficacy of 71 was almost
twofold more potent than that of 67. The 3-aminopiperidine deriv-
atives 72 and 73 had an in vitro activity comparable to that of 70

Y. Hirokawa et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3556–3561
3561
and 71, respectively, but reduced in vivo efficacy. Introduction of a
methyl group into the amino group on the 4-aminopiperidine ring
of 70 and 71 (yielding 74, 76, and 75, respectively) caused a slight
decrease or increase in in vitro activity, while the in vivo efficacy of
74 was more potent than that of 70. The in vivo efficacy of 75 and
76 did not improve. Furthermore, replacement of the piperazine
ring in 6 with racemic 3-aminopyrrolidine (giving 77) or 3-ami-
nomethylpyrrolidine (giving 84) ring led to a decrease in both
the in vitro and in vivo antibacterial activities. On the other hand,
the ( )-6-(3-aminopyrrolidin-1-yl)purine 78 and the ( )-6-(3-ami-
nomethylpyrrolidine)purine 85 displayed excellent in vivo efficacy
compared with 67 although their in vitro activity was comparable
to that of 67. In particular, 85 conferred the highest in vivo activity
with an efficacy more potent than that of VCM (ED50 = 0.59 mg/kg).
Next, the stereochemistry at the 3-position of the pyrrolidine ring
of 78 was examined. Both compounds 79 and 80 with R and S con-
figurations, respectively, as well as the racemic compound 78
showed excellent in vitro and in vivo activities. Introduction of a
methyl group at the amino group of 77, 78, 84, and 85 (giving
81, 82, 86, and 87, respectively) provided favorable influence on
the in vivo efficacy, thus retaining (as in 82 and 87) or slightly
increasing the activity (as in 81 and 86). Compound 87 showed
excellent in vitro and in vivo antibacterial activities and was essen-
tially equipotent to 85. By contrast, the in vivo efficacy of the 3-
dimethylaminopyrrolidine analogue 83 did not improve compared
to that of 77. Replacement of the piperidine ring of 6 by 3-amino-
and 3-methylaminoazetizine rings (giving 88 and 89, respectively)
resulted in a significant decrease in in vivo efficacy.
when compared to azamulin (4) and VCM. The excellent in vivo
efficacy of these compounds, which also have good solubility in
water, reflects good pharmacokinetics and ADME properties (data
not shown). It is therefore believed that compounds 85 and 87
have potential as novel antibacterial agents for use in human. A
more comprehensive study directed at optimization of these com-
pounds will be reported in due course.
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As part of our research to develop novel pleuromutilin deriva-
tives for human use, the polar and water solubilizing purine ring
was introduced into the pleuromutilin C14 side chain. From SAR
studies, we found that compounds 85 and 87 show not only excel-
lent in vitro antibacterial activity against MRSA, PRSP, VRE, S. pyog-
enes, M. catarrhalis, and H. influenzae but also potent in vivo efficacy