Pleuromutilin derivatives having a purine ring. Part 3: Synthesis and antibacterial activity of novel compounds possessing a piperazine ring spacer

Article abstract of DOI:10.1016/j.bmcl.2008.10.127

SAR studies on the water-soluble thioether pleuromutilin analogue 6, which has excellent in vitro and in vivo antibacterial activities, led to discovery of the novel pleuromutilin derivatives having a piperazine ring spacer. These derivatives displayed po

Full text of DOI:10.1016/j.bmcl.2008.10.127

Bioorganic & Medicinal Chemistry Letters 19 (2009) 175–179
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pleuromutilin derivatives having a purine ring. Part 3: Synthesis and
antibacterial activity of novel compounds possessing a piperazine ring spacer
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:
SAR studies on the water-soluble thioether pleuromutilin analogue 6, which has excellent in vitro and
in vivo antibacterial activities, led to discovery of the novel pleuromutilin derivatives having a piperazine
ring spacer. These derivatives displayed potent and well-balanced in vitro antibacterial activity against
various drug-susceptible and -resistant Gram-positive bacteria. In particular, the promising pleuromuti-
lin analogues 37 and 40 were found to exhibit strong in vivo efficacy against Staphylococcus aureus Smith.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 26 August 2008
Revised 23 October 2008
Accepted 28 October 2008
Available online 5 November 2008
Keywords:
Pleuromutilin
Gram-positive bacteria
MRSA
PRSP
VRE
Purine ring
Spacer
MIC
Piperazine ring
The antibiotic pleuromutilin1–4 (1), having an unusual fused
5-6-8 tricyclic diterpenoid structure, was first isolated in 1951
from two basidiomycete species and was characterized as a crys-
talline antibiotic with modest in vitro antibacterial activity against
Gram-positive pathogens and mycoplasmas and weak in vivo effi-
cacy.5 Pleuromutilin (1) selectively inhibits bacterial protein
synthesis through interaction with prokaryotic ribosomes, but
has no effect on eukaryotic protein synthesis and does not bind
to mammalian ribosomes.6 To find a novel class of antibiotics with
a new mechanism of action, a Sandoz group reported initial struc-
ture–activity relationship (SAR) studies on a number of semisyn-
thetic pleuromutilin derivatives.7–9 Further studies led to the
development of tiamulin (2) and valnemulin (3) as therapeutic
agents for veterinary use.10 Chemical modifications of 1, with the
aim of producing an agent for use in human, resulted in the
1980s in the development of azamulin (4).11 Although 4 showed
good in vitro antibacterial activity, its oral bioavailability was
severely limited by atrocious solubility in water.12 Recently,
researchers at GlaxoSmithKline identified the new pleuromutilin
analogue retapamulin (5)13 with excellent in vitro antibacterial
activity, and 5 was approved in 2007 as a topical antimicrobial
agent for treatment of human skin infections. From previous SAR
studies on 1, analogues in which the hydroxyl of the C14 glycolic
ester group is replaced with a substituent containing the sulfide
linkage show potent in vitro antibacterial activity, but suffer from
being rapidly and extensively metabolized in vivo because of their
strong hydrophobic nature.14 To overcome this problem, we de-
signed structurally novel pleuromutilin derivatives having a purine
ring as a polar and water solubilizing group and identified the no-
vel pleuromutilin analogue 6 with potent in vitro and in vivo anti-
bacterial activities, good solubility in water, and appreciable
metabolic stability. Further structural modification of the 4-piperi-
dinethio moiety as a central spacer of the first generation 6 led to
discovery of the promising pleuromutilin analogue 7 having a
piperazine ring spacer (see Fig. 1) refer to part 215 in this series.
Like 6, the originally identified 7 displayed excellent in vitro anti-
bacterial activity against a number of Gram-positive pathogens,
including methicillin-resistant Staphylococcus aureus (MRSA), pen-
icillin-resistant Streptococcus pneumoniae (PRSP), and vancomycin-
resistant enterococci (VRE). Furthermore, its in vivo efficacy against
S. aureus Smith systemic infection model in mice was essentially
equivalent to that of the marketed antibiotic vancomycin (VCM)
(Table 1). The microbiological profile observed with the prototype
* 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.2008.10.127

176
Y. Hirokawa et al. / Bioorg. Med. Chem. Lett. 19 (2009) 175–179
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
O
N
R1 = Et
NH2
N
S
4
Azamulin ( ); R =
Valnemulin (3)
;
R =
Me
N
S
N
H
H
Me
Me
O
NH2
R1 = CH=CH2
N
N
N
Me
• 2HCl
N
N
7
8
;
R =
N
S
Retapamulin (5)
;
R =
O
NH2
R1 = CH=CH2
N
N
R1 = CH=CH2
N
R = OTs, R1 = CH=CH2
R3
;
R4
N1
N
N
R
1 = CH=CH2
6
;
R =
S
N
HCl
25-31
H
;
N2
R =
NH2
N
• 2HCl
R1 = CH=CH2
N
R5
R6
(CH2)n
Figure 1. Structure of pleuromutilin derivatives.
7 is of clear interest, showing potent antibacterial activity against
relevant bacterial strains in particular Staphylococci and Strepto-
cocci, with the exception of a borderline activity against Haemoph-
ilus influenzae. In this letter, we describe the synthesis and in vitro
and in vivo antibacterial activities of novel pleuromutilin deriva-
tives having a piperazine ring spacer.
broth microdilution assay method,17 the novel pleuromutilin
derivatives 32–57 were tested against well characterized drug-sus-
ceptible and -resistant Gram-positive and -negative bacteria
including methicillin-susceptible S. aureus (MSSA), MRSA, penicil-
lin-susceptible S. pneumoniae (PSSP), PRSP, VRE, Streptococcus
pyogenes, Moraxella catarrhalis, and H. influenzae, all of which are
common serious respiratory tract pathogen. In addition, com-
pounds minimum inhibitory concentration (MIC) values were
compared with those of the previously reported pleuromutilin ana-
logue 715 and VCM. The pleuromutilin derivatives 32–57, except
53 and the reference compounds, displayed good to excellent
in vivo efficacy against S. aureus Smith (MSSA) systemic infection
model in mice. The lead compound 7 exhibited potent in vitro anti-
bacterial activity comparable to that of VCM and had an interesting
profile with good balanced in vitro activity against Gram-positive
and -negative pathogens, except for a borderline activity against
H. influenzae. Additionally, 7 revealed almost the same in vivo effi-
cacy as VCM. In general, all the newly prepared pleuromutilin
The new purine-propionic acids 14–17 with ( )-3-[N-(tert-butox-
ycarbonyl)-N-ethyl]amino, (R)- and (S)-3-[N-(tert-butoxycarbonyl)-
N-methyl]aminomethyl, and ( )-3-dimethylaminopyrrolidines and
4-(tert-butoxycarbonyl)aminomethylpiperidine at the 6-position
of the purine ring, except the previously described 3-(6-substituted
purin-9-yl)propionic acids,16 were prepared by the method de-
scribed in a previous issue of this letter16 (Scheme 1). In brief, reac-
tion of 6-chloropurine 9 with the corresponding pyrrolidine and
piperidine derivatives, followed by alkylation of the resultants
10–13 with ethyl 3-bromopropionate or ethyl acrylate gave the de-
sired esters. Alkaline hydrolysis of the ethyl esters produced 14–17.
The piperazine and hexahydro-1,4-diazepine derivatives 25–31
(see Fig. 1) were prepared as illustrated in Scheme 2. Reaction of
the commercially available piperazines 18–22, 18R, and 18S, the
N-Boc-protected hexahydro-1,4-diazepine 23, and the N-Boc-pro-
tected piperazines 24, 24R, and 24S with the mutilin 14-tosyloxyac-
etate 87,8 (see Fig. 1) followed by successive treatment with HCl
afforded the desired 25–31 as di-hydrochlorides.
derivatives having
a piperazine ring spacer retained potent
in vitro antibacterial activity with only slight differences. Interest-
ingly, PSSP, PRSP, S. pyogenes, and M. catarrhalis strains were highly
susceptible, while H. influenzae strain exhibited somewhat higher
MIC value. Lead optimization, aimed at improving the potency of
7, proceeded stepwise.
The pleuromutilin analogues 32–57 shown in Table 1 were pre-
pared as follows. Condensation of the 3-(6-substituted purin-9-
yl)propionic acids including 14–17 with the known pleuromutilin
analogues16 having a piperazine ring and 25–31 in the presence
of benzotriazole-1-yloxytris(pyrrolidino)phosphonium hexafluoro-
phosphate as a coupling agent and Et3N, and successive acid treat-
ment gave the desired compounds 32–57 as di-hydrochlorides in
moderate to good yields. The chemical structures of all pleuromu-
tilin analogues obtained were confirmed by 1H NMR and mass
spectra and the purity was demonstrated by HPLC analysis. The
pleuromutilin derivatives 32–57 formed as di-hydrochlorides
showed good solubility in water (ꢀ50 mg/mL). Using standard
Influence of the 6-substituent on the purine ring was first
examined. The 6-(3-methylamino, 3-ethylamino, and 3-dimethyla-
minopyrrolin-1-yl)purine analogues 32–34 had essentially the
same in vitro antibacterial activity as 7, which bears a 3-aminopyr-
rolidine ring, although their in vivo efficacy was lower than that of
7. Replacement of the amino group of 7 with an aminomethyl or a
methylaminomethyl substituent (yielding 35 or 36, respectively)
resulted in a slight decrease in in vitro and in vivo antibacterial
activities. These results indicate that the in vivo efficacy is highly
sensitive to small structural changes at the 3-position of the pyrr-
olidino ring. Compounds in vivo efficacy following substitution at
the 3-pyrrolidine ring decreased generally in this order NH2

Y. Hirokawa et al. / Bioorg. Med. Chem. Lett. 19 (2009) 175–179
177
Table 1
In vitro and in vivo antibacterial activities of 7 and 32À57.
CH2
O
Me
N1
OH
N
N
O
Q
N2
Me
N
O
R2
Me
Me
N
• 2HCl
O
R2
MICa
(lg/mL)
MSSAb ED50 (mg/kg, iv)
j
Compound
Q
MSSAb
MRSAc
PSSPd
PRSPe
S. p.f
VREg
M. c.h
H. i.i
NH2
N1
N2
7
0.25
1
0.063
0.063
0.032
0.5
0.25
4
0.80
N
NHMe
NHEt
NMe2
N1
N1
N1
N2
N2
N2
32
33
34
0.25
0.25
0.25
1
0.063
0.125
0.125
0.125
0.125
0.063
0.032
0.063
0.063
0.25
0.25
0.125
0.25
4
4
4
1.30
2.21
2.94
N
N
N
N
0.5
0.25
0.125
0.125
N1
N2
NH2
35
0.25
2
0.063
0.125
0.032
1
0.5
4
1.21
N1
N1
N1
N1
N1
N1
N2
N2
N2
N2
N2
N2
NHMe
36
37
38
39
40
41
42
43
44
45
0.25
0.25
0.25
0.125
0.25
0.25
0.5
2
0.125
0.125
0.125
0.063
0.125
0.125
0.063
0.032
0.032
0.125
0.125
0.063
0.125
0.063
0.125
0.125
0.063
0.032
0.063
0.125
0.032
0.032
0.063
0.032
0.032
0.032
0.032
0.032
0.032
0.063
1
0.5
4
1.10
0.83
1.18
1.65
0.97
1.51
2.51
1.39
1.47
2.21
N
N
2
0.5
0.125
0.125
0.063
0.125
0.125
0.25
0.5
2
NHMe
NHMe
2
0.5
N
N
0.25
1
0.063
0.25
0.25
0.25
0.125
0.125
0.25
2
NH
2
N
N
NH2
NH2
1
2
NH2
1
4
N1
N2
N
N
N
N
NH2
NH2
NH2
Me
0.25
0.125
0.25
0.25
0.25
0.5
0.125
0.125
0.25
2
N1
N2
Me
2
N1
Me
N2
8
N1
Me
N2
N2
NH2
46
0.25
0.5
0.032
0.032
0.016
0.125
0.125
4
1.10
N1
N
(continued on next page)

178
Y. Hirokawa et al. / Bioorg. Med. Chem. Lett. 19 (2009) 175–179
Table 1 (continued)
R2
MICa
(l
g/mL)
MSSAb ED50 (mg/kg, iv)
j
Compound
Q
MSSAb
MRSAc
PSSPd
PRSPe
S. p.f
VREg
M. c.h
H. i.i
Me
NH2
47
48
0.125
0.25
0.032
0.032
0.032
0.016
0.063
0.125
0.125
2
1.56
1.20
N1
Me
N2
N2
N
N
NHMe
0.125
1
0.063
0.25
0.125
2
N1
Me
49
50
51
0.25
0.25
0.25
0.5
0.063
0.063
0.063
0.063
0.063
0.063
0.032
0.032
0.032
0.25
0.125
0.125
0.125
2
4
4
1.10
1.66
2.95
NHMe
N1
Me
N2
N2
N2
N
N
0.5
0.125
0.125
NH2
N1
NH2
Et
0.25
N1
N
N
NH2
Me
Me
N1
Me
N1
52
53
0.125
0.125
0.125
0.125
0.032
0.016
0.032
0.016
0.016
0.016
0.063
0.063
0.032
0.063
4
4
2.32
N2
NH2
NH2
N2
>3.13
N
N
Me
Me
N1
N2
54
0.25
0.25
0.063
0.063
0.032
0.125
0.125
4
3.13
Me
Me
NH2
NH2
55
56
0.25
0.25
0.5
0.5
0.063
0.125
0.063
0.063
0.032
0.032
0.125
0.125
0.125
0.125
2
4
1.42
1.28
N1
N1
N2
N
N
Me
N2
Me
NH2
57
0.25
1
0.5
0.5
0.032
0.25
0.063
0.5
0.016
0.5
0.063
>128
0.063
64
2
1.42
0.88
N1
N2
N
VCM
>128
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.
S. pneumoniae ATCC49619.
S. pneumoniae KT2524.
S. pyogenes ATCC12344.
VRE, E. faecium KU1778.
M. catarrhalis K1209.
g
h
i
H. influenzae TH13.
j
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.
R2
R2
Cl
N
(7) > CH2NHMe (36) > CH2NH2 (35) > NHMe (32) >> NHEt (33) >>
NMe2 (34). As for 36, both compounds 37 and 38 with the optically
active 3-methylaminomethyl substituent showed approximately
similar in vitro antibacterial activity to 36 having the racemic pyr-
rolidine ring. However, the in vivo efficacy of 37 was slightly better
than that of the racemic compound 36 or 38. The ED50 value
(0.83 mg/kg) of 37 was essentially equivalent to that of 7 or
VCM. On the other hand, replacement of the pyrrolidine ring in 7
with a piperazine (giving 39), a 4-aminopiperidine (giving 40), or
a 4-aminomethylpiperidine (giving 41) ring uniformly retained
in vitro antibacterial activity against all strains, but the in vivo effi-
cacy of 39 and 41 was not favorable. Compound 40 exhibited po-
tent in vivo efficacy with ED50 value of 0.97 mg/kg, which is
slightly less than that of 7.
N
N
N
N
ii)
iii)
i)
N
N
N
N
N
H
N
H
N
N
CO2H
10-13
14-17
9
Et
Boc
10, 14; R2
=
N
12, 16; R2
13, 17; R2
=
=
N
N
CH2NHBoc
NMe2
NBoc
Me
11R, 15R; R2
11S, 15S; R2
=
=
N
N
NBoc
Me
Scheme 1. Reagents and conditions: (i) R2H, iPr2NEt, iPrOH, reflux, 15 h; (ii) BrCH2CH2-
CO2Et (CH2@CHCO2Et), K2CO3, DMF, 80 °C, 24 h; (iii) 2 N NaOH/MeOH, reflux, 3 h.

Y. Hirokawa et al. / Bioorg. Med. Chem. Lett. 19 (2009) 175–179
179
R3
H N1
R5
R4
R4
N2
R3
O
tBu
ii)
i)
18-22
iii)
i)
23, 24
N2
R6
H
N1
O
25-29
H
30, 31
(CH2)n
R5
18, 25; R3 = Me, R4 = R5 = R6 = H
23, 30; R3 = R4 = R5 = H, n = 1
18R, 25R; R3 = Me, R4 = R5 = R6 = H
18S, 25S; R3 = Me, R4 = R5 = R6 = H
19, 26; R3 = Et, R4 = R5 = R6 = H
20, 27; R3 = R4 = Me, R5 = R6 = H
21, 28; R3 = R5 = Me, R4 = R6 = H
22, 29; R3 = R6 = Me, R4 = R5 = H
24, 31; R3 = Me, R4 = R5 = H, n = 0
24R, 31R; R3 = Me, R4 = R5 = H, n = 0
24S, 31S; R3 = Me, R4 = R5 = H, n = 0
Scheme 2. Reagents and conditions: (i) 8, K2CO3, NaI, MeCN, reflux, 16 h; (ii) 4 M HCl/AcOEt, rt, quant; (iii) 30% HCl/EtOH, rt, 2 h, quant.
Compound 42, bearing the hexahydro-1,4-diazepine ring as a
central spacer, showed in vitro antibacterial activity practically
comparable to that of 7 against all strains, but its in vivo efficacy
was not improved. Next, we studied the influence of a substituent
in the piperazine ring on the antibacterial activity. Introduction of
a methyl group into the piperazine-carbon atom (N1) on the side of
the purine ring of 7 (yielding the racemic compound 43) caused a
slight increase in in vitro antibacterial activity. Compound 43
appeared to have good in vitro profile against Staphylococci and
Streptococci. However, the in vivo efficacy was less than that of 7.
Comparison of the antibacterial activity of 44, having an (S)-meth-
ylpiperazine spacer, to that of 45, having an (R)-methylpiperazine
spacer, indicated that the former compound was more potent.
However, 44 showed no improvement in in vivo efficacy when
compared to 7. As for 44 bearing the racemic 3-aminopyrrolidine
ring, compounds 46 and 47 with the optically active 3-aminopyr-
rolidine ring were prepared to probe the steric effect of the amino
group in 44. Neither of these analogues differed significantly from
the parent analogue 44 in vitro antibacterial activity, but the
in vivo efficacy of 46 with (S)-3-aminopyrrolidine was more potent
than that of 44, and the ED50 value was 1.10 mg/kg. The in vivo effi-
cacy of 47 having the 6-[(R)-3-aminopyrrolidin-1-yl]purine moiety
was substantially weaker than that of 44. On the other hand, the
racemic and the (R)-(3-methylaminomethylpyrrolidin-1-yl)purine
derivatives 48 and 49 showed potent in vivo efficacy, and the
ED50 value of 49 was 1.10 mg/kg. Compound 50 bearing the
4-aminopiperidine ring showed an in vitro antibacterial activity
practically comparable to that of 7 against all strains, but its
in vivo efficacy was not improved.
methyl group in the piperazine ring spacer did not significantly
influence the antibacterial activity.
In summary, further studies aimed at the development of pleu-
romutilin derivatives for use in human led to identification of a no-
vel class of pleuromutilin analogues having a piperazine ring
spacer. As a result of SAR, compounds 37 and 40 showing not only
excellent in vitro antibacterial activity against MRSA, PRSP, VRE, S.
pyogenes, and M. catarrhalis, but also potent in vivo efficacy were
identified. The excellent in vivo efficacy of both compounds, which
have good solubility in water, may reflect good pharmacokinetics
and ADME properties. Therefore, this new class of pleuromutilin
appears to have a very promising profile for the treatment of infec-
tions caused by respiratory pathogens.
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Wayne, PA, 2000.
Introduction of an ethyl group or two methyl groups into the
piperazine ring spacer of 7 (yielding racemic compound 51 or
52–54, respectively) significantly decreased the in vivo efficacy de-
spite high in vitro antibacterial activity.
Finally, introduction of a methyl group into the piperazine-
carbon atom on the side (N2) of the mutilin ring of 7 gave the
racemic compound 55, whose in vitro and in vivo antibacterial
activities was comparable to that of the corresponding counter-
part 43. Next, the stereochemistry at the methyl group on the
pyrrolidine ring of 55 was examined. Compound 56 with (S)-
methylpiperazine and the (R)-methylpiperazine analogue 57
showed no improvement in the in vitro and in vivo antibacterial
activities compared with the racemic compound 55. For methyl-
piperazine analogues, the position and stereochemistry of the