Pyrrolidinedione derivatives as antibacterial agents with a novel mode of action

Article abstract of DOI:10.1016/j.bmcl.2004.12.002

The pseudopeptide pyrrolidinedione natural products moiramide B and andrimid represent a new class of antibiotics that target bacterial fatty acid biosynthesis. Structure-activity relationship (SAR) studies revealed a high degree of variability for the fa

Full text of DOI:10.1016/j.bmcl.2004.12.002

Bioorganic& Medicinal Chemistry Letters 15 (2005) 1189–1192
Pyrrolidinedione derivatives as antibacterial agents
with a novel mode of action
Jens Pohlmann,a Thomas Lampe,a Mitsuyuki Shimada,a Peter G. Nell,a
Josef Pernerstorfer,a Niels Svenstrup,a Nina A. Brunner,b
Guido Schifferb and Christoph Freibergb,*
aDepartment of Medicinal Chemistry, Bayer HealthCare AG, D-42096 Wuppertal, Germany
bDepartment of Anti-Infectives Research, Bayer HealthCare AG, D-42096 Wuppertal, Germany
Received 9 September 2004; revised 2 December 2004; accepted 2 December 2004
Available online 24 December 2004
Abstract—The pseudopeptide pyrrolidinedione natural products moiramide B and andrimid represent a new class of antibiotics that
target bacterial fatty acid biosynthesis. Structure–activity relationship (SAR) studies revealed a high degree of variability for the
fatty acid side chain, allowing optimization of physicochemical parameters, and a restricted SAR for the pyrrolidinedione group,
indicating major relevance of this subunit for efficient target binding.
Ó 2004 Elsevier Ltd. All rights reserved.
Since the introduction of penicillin in the 1940s, antibi-
otics have a history of success in controlling morbidity
and mortality caused by infectious diseases. But, as a
consequence of frequent use, bacterial resistance to
known classes of antibiotics has become a severe global
problem in recent years and presents a continuous clin-
ical challenge.1–3 Resistance can result from modifica-
tion of an antibacterialÕs target or from functional
bypassing of that target, or it can be contingent on
impermeability, efflux or enzymaticinactivation of the
drug.4 There are serious concerns that untreatable
pathogens may develop at an alarming rate in the near
future. Strategies to address this challenge include the
design of improved versions of antibacterial classes al-
ready in clinical use and the use of drug combinations.
The application of these strategies can be quite success-
ful, but a high risk of rapid resistance development re-
mains. Thus, an urgent need for new potent classes of
antibiotics with novel modes of action persists.
identified the target of this compound class to be the
carboxyltransferase subunit of the multimeric bacterial
enzyme acetyl-CoA carboxylase (ACC).8 ACC catalyzes
the first committed step in fatty acid biosynthesis and is
essential for cell growth. Broad structural conservation
among bacteria and a clear distinction from the multi-
functional eukaryotic enzyme make ACC a promising
target for the design of new broad-spectrum antibacteri-
als with a novel mode of action.
The chemical structure of the natural products moir-
amide B and andrimid is quite unique, containing four
characteristic subunits: an unsaturated fatty acid side
chain, the b-amino acid b-(S)-phenylalanine, an L-valine
derived b-ketoamide moiety and the pyrrolidinedione
head group. Since only limited structure–activity rela-
tionship data has been published for this compound
class,9 we explored variations of the two terminal sub-
units, that is, the fatty acid side chain and the pyrrolid-
inedione group (Fig. 1).
The pseudopeptide pyrrolidinedione natural products
moiramide B and andrimid have been described as anti-
biotics with an unknown mode of action.5–7 We recently
O
O
O
NH
Keywords: Pyrrolidine diones; Moiramide B; Andrimid; Acetyl-CoA
carboxylase; Antibiotic.
N
H
N
H
n
O
O
*
Corresponding author. Tel.: +49 202 368461; fax: +49 202 364116;
e-mail: christoph.freiberg@bayerhealthcare.com
Figure 1. Structures of moiramide B (n = 1) and andrimid (n = 2).
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.12.002

1190
J. Pohlmann et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1189–1192
O
O
O
O
N
O
a, b
b
a
N
R1
OH
O
OBn
NH
HO
BocHN
BocHN
BocHN
BocHN
O
1
2
O
3a-g O
O
O
O
O
O
O
4a
4f
5a
5f
O
N
R1 = OBn
CH3
c
O
N
O
N
R1
a
cyclopropyl
CH2CH2OCH3
OCH2CH3
N(CH3)Cbz
N(CH3)2
BocHN
N
Cbz
N
H
O
O
O
O
4a-g
Scheme 2. Reagents and conditions: (a) H2, Pd/C (10%), EtOH; (b) 20-
bromoacetophenone, Et3N, CH3CN.
Scheme 1. Reagents and conditions: (a) acetyl chloride, 60 °C; (b)
H2NR1, CDI, CH2Cl2, 0– 45 °C; (c) (1) N-Boc-L-valine, CDI, THF; (2)
3a–g, LiHMDS, THF, ꢀ65 °C.
The introduction of the b-phenylalanine and fatty acid
subunits is shown in Scheme 3. Acidic treatment of the
Boc-protected b-ketoamide pyrrolidinediones yielded
primary amines 6a–f, ready for amide formation under
standard HATU (O-(7-azabenzotriazol-1-yl)-N,N,N0,
N0-tetramethyluronium hexafluorophosphate) peptide
coupling conditions. The final products 11a–i and
12a–f were obtained from 6a–f either by a stepwise
procedure consisting of coupling with N-Boc-b-(S)-
phenylalanine, acidic deprotection and final introduc-
tion of the fatty acid 9, or alternatively, by coupling
with a preformed b-amino acid–fatty acid building
block 10.
Our chemical synthesis was based on previously pub-
lished procedures.9–12 As shown in Scheme 1, treatment
of (S)-(ꢀ)-methylsuccinic acid 1 with acetyl chloride
yielded anhydride 2. The formation of diverse N-substi-
tuted pyrrolidinediones 3a–g was achieved by reaction
with the appropriate primary amines, hydroxylamines
or hydrazines in the presence of N,N0-carbonyldiimid-
azole (CDI). The next step involved the diastereoselec-
tive formation of the b-ketoamide moiety. For this
conversion, N-Boc-L-valine was first treated with CDI
to form the imidazole derivative. Then, the activated
amino acid was mixed with the pyrrolidinedione in tetra-
hydrofurane and the resulting concentrated solution was
slowly added to a lithium hexamethyldisilazide solution
at low temperature. This protocol allowed an in situ
quench of the labile pyrrolidinedione enolate. Although
the b-ketoamide moiety of the products 4a–g can equili-
brate between the trans-, cis- and enolic-forms, 1H
NMR examination revealed the trans-isomers to be
dominant in the equilibrium mixtures. The other iso-
mers were hardly detectable in most cases.
Compounds 13–15 with variations of the pyrrolidinedi-
one 4-methyl group and the piperidine dione derivatives
16a–c were prepared according to the prescribed proce-
dure, starting from the appropriate dicarboxylic acids or
anhydrides.
All compounds were tested for target activity against the
carboxyltransferase AccAD subunits derived from
Staphylococcus aureus and Escherichia coli, representing
Gram-positive and Gram-negative pathogens, respec-
tively. Antibacterial activity was measured versus S.
aureus and Streptococcus pneumoniae as Gram-positive
bacteria, and versus two E. coli strains, one of them
being an efflux pump deletion strain.14
Removal of the N-benzyloxy group in 4a was achieved
by hydrogenation and subsequent treatment of the N-
hydroxy derivative with 20-bromoacetophenone and tri-
ethylamine.13 The Cbz-group of hydrazine derivative 4f
was removed by standard hydrogenation (Scheme 2).
O
N
O
N
O
N
O
N
a
a
b
R1
R1
O
O
R1
R1
BocHN
R1 = H
H2N
BocHN
N
H
H2N
N
H
O
O
O
O
O
O
O
O
4b-e,g
6a-f
7a-f
8a-f
5a,f
CH3
O
cyclopropyl
CH2CH2OCH3
c
c
O
O
OH
R
n
O
OCH2CH3
NH(CH3)
N(CH3)2
N
H
OH
R
n
O
O
9
R1
10
N
N
H
R
N
n
H
O
O
11a-i Table 1
12a-f Table 2
Scheme 3. Reagents and conditions: (a) HCl, 1,4-dioxane; (b) N-Boc-b-(S)-phenylalanine, HATU, i-Pr2EtN, CH2Cl2/DMF; (c) HATU, i-Pr2EtN,
CH2Cl2/DMF.

J. Pohlmann et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1189–1192
Table 1. IC50 and MIC values of fatty acid side chain derivatives
1191
O
O
O
NH
N
H
N
H
R
n
O
O
Compound
R
n
IC50 (nM)
MIC (lg/mL)
E. coli
S. aureus
E. coli HN818
E. coli Neumann
S. aureus 133
S. pneumoniae G9A
11a
11b
11c
11d
11e
11f
11g
11h
11i
CH3
CH3
2
3
1
1
1
1
1
1
0
6
13
2
96
305
317
151
75
4
32
>64
64
2
32
>64
>64
>64
32
8
8
32
8
CO2H
4-Imidazolyl
Ph
>64
>64
8
>64
>64
32
6
9
3-CH3O–Ph
3,4-Di-Cl–Ph
4-Ph–Ph
6-F-2-pyridyl
4
43
2
32
8
4
16
32
7
288
540
50
1
2
37
1
>64
1
>64
8
2
16
16
8
Variation of the fatty acid side chain revealed that quite
diverse substituents were tolerated in this position
(Table 1). Natural product moiramide B 11a displayed
IC50 values of 6 nM versus E. coli AccAD and 96 nM
versus the corresponding S. aureus enzyme. Synthetic
compounds with relatively small, polar residues like car-
boxylicacid 11c or imidazole 11d were similar potent on
target level as derivatives with significantly larger, highly
lipophilicresidues like biphenyl compound 11h. In gen-
eral, IC50 values were below 10 nM in most cases for the
E. coli enzyme, whereas higher concentrations of at least
50 nM were usually needed to inhibit S. aureus ACC.
3-Methoxycinnamic acid compound 11f and 5-fluoro-
nicotinic acid derivative 11i were the most potent com-
pounds with IC50 values of 4 and 1 nM versus the E.
coli enzyme, and 43 and 50 nM versus the S. aureus en-
zyme, respectively. Despite these rather small deviations
in in vitro target potency, major differences in antibac-
terial activity were observed. Carboxylic acid derivative
11c, for example, did not show any antibacterial activity
even at high concentrations, despite being potent on tar-
get level, especially against the E. coli enzyme
(IC50 = 2 nM). Biphenyl derivative 11h, on the other
hand, exhibited good antibacterial activity against
Gram-positive bacteria although it was the least potent
compound on target level. Apparently, the physico-
chemical properties of test compounds significantly
affected antibacterial activity. This was probably due
to hampered penetration of polar compounds into the
bacterial cell. All compounds showed increased antibac-
terial activity against the E. coli HN818 efflux pump
deletion strain compared to the E. coli Neumann wild
type strain, indicating that pyrrolidinediones were sub-
strates for this efflux mechanism.
In contrast to the broad structural variations tolerated
at the fatty acid side chain, a much more concise struc-
ture–activity relationship was revealed for the pyrrol-
idinedione group (Table 2). N-Methyl derivative 12a
showed a 2- to 4-fold loss of potency regarding IC50 val-
ues and MICs compared to moiramide B 11a. Further
decrease in potency was observed with larger carbon
linked substituents at the pyrrolidinedione nitrogen
atom. It is interesting to note, that oxygen or nitrogen
Table 2. IC50 and MIC values of pyrrolidinedione group derivatives
R2 R3
O
O
O
R1
N
N
H
N
H
O
O
Compound R1
R2
R3
IC50 (nM)
MIC (lg/mL)
E. coli S. aureus E. coli HN818 E. coli Neumann S. aureus 133 S. pneumoniae G9A
11a
12a
12b
12c
12d
12e
12f
13a
14a
15a
H
CH3
CH3
CH3
CH3
H
6
25
96
211
4
16
32
>64
>64
>64
>64
>64
>64
>64
>64
>64
8
16
32
>64
>64
64
H
Cyclopropyl
H
60
2560
1303
123
>64
>64
64
>64
64
CH2CH2OCH3 CH3
H
150
100
14
OCH2CH3
NHCH3
N(CH3)2
CH3
CH3
CH3
CH3
H
H
8
32
16
H
105
238
8
4
H
33
4
4
16
H
>200
>200
54
>1000
>1000
900
>64
>64
>64
>64
>64
>64
>64
>64
>64
CH3
H
CH2CH3
CH3
H
CH3
a Mixture of two diastereomers regarding pyrrolidinedione C3/4 stereochemistry.

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J. Pohlmann et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1189–1192
Table 3. IC50 and MIC values of piperidinedione derivatives
R2
O
R1
O
O
O
NH
N
H
N
H
O
Compound
R1
R2
IC50 (nM)
MIC (lg/mL)
E. coli
S. aureus
E. coli HN818
E. coli Neumann
S. aureus 133
S. pneumoniae G9A
16a
16b
16c
H
H
>300
>300
295
>1000
>1000
>1000
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
CH3
H
H
CH3
linked substituents were better tolerated. Compounds
12d–f were similar potent against the S. aureus enzyme
as unsubstituted moiramide B. The 4-(S)-methyl group
of the pyrrolidinedione moiety seems to be essential
for activity. Removal of that methyl group (13) or
replacement by an ethyl group (14) caused total loss of
activity. Only dimethyl derivative 15 still showed IC50
values at the concentration range tested. However, the
potency was 10-fold reduced compared to parent com-
pound moiramide B. Six-membered piperidinedione
derivatives were evaluated, as well (Table 3). Neither
nonmethylated derivative 16a, nor 4-methyl compound
16b showed any activity. Only 5-methyl derivative 16c
was slightly active in the E. coli enzyme assay.
subunits that have not been addressed in this study, will
show, if a significant improvement of potency compared
to the natural products is feasible. A detailed under-
standing of the structure–activity relationship for all
subunits will allow a complete evaluation of the poten-
tial of pyrrolidinediones as a new structural class with
a novel mode of action for antibacterial therapy.
References and notes
1. Gold, H. S.; Moellering, R. C. New Engl. J. Med. 1996,
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4. Livermore, D. M. Clin. Infect. Dis. 2003, 36(Suppl. 1),
S11.
In summary, the structure–activity relationship for the
fatty acid side chain and the pyrrolidinedione moiety re-
flect that these two groups play entirely different roles in
binding to the target.
5. Needham, J.; Kelly, M. T.; Ishige, M.; Andersen, R. J.
J. Org. Chem. 1994, 59, 2058.
6. Fredenhagen, A.; Tamura, S. Y.; Kenny, P. T. M.;
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Microbios. 1994, 78, 7.
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A high degree of variability was found for the fatty acid
side chain, which apparently is not involved in pivotal
binding interactions with the bacterial acetyl-CoA
carboxylase. Nevertheless, despite limited influence on
enzyme inhibition, a significant impact on antibacterial
activity was observed for the side chain variations. Cell
penetration issues appear to be responsible for this ef-
fect. Consequently, variation of the fatty acid side chain
should allow optimization of the physicochemical pro-
file during the drug discovery process. On the other
hand, only limited variations were tolerated in the pyr-
rolidinedione head group. Most derivatives showed a
significant decrease or even total loss of target- and anti-
bacterial activity. Only few selected substituents at the
pyrrolidinedione nitrogen atom were not detrimental
for inhibitory potency. These results indicate major
relevance of the 4-(S)-methyl pyrrolidinedione group
for efficient target binding. Synthetic variation of the
b-amino acid and the b-ketoamide moiety, the two
11. Dixon, D. J.; Davies, S. G. Chem. Commun. 1996, 1797.
12. Davies, S. G.; Dixon, D. J. J. Chem. Soc., Perkin Trans. 1
1998, 2635.
´
13. Consonni, P.; Favara, D.; Omodei-Sale, A.; Bartolini, G.;
Ricci, A. J. Chem. Soc., Perkin Trans. 2 1983, 967.
14. For carboxyltransferase assay conditions, see: Blanchard,
C. Z.; Waldrop, G. L. J. Biol. Chem. 1998, 273, 19140,
MIC values were determined according to NCCLS
guidelines.