N-substituted 3-acetyltetramic acid derivatives as antibacterial agents

Article abstract of DOI:10.1021/jm701356q

In order to expand the structure-activity relationship of tetramic acid molecules with structural similarity to the antibiotic reutericyclin, 22 compounds were synthesized and tested against a panel of clinically relevant bacteria. Key structural changes

Full text of DOI:10.1021/jm701356q

J. Med. Chem. 2008, 51, 1487–1491
1487
N-Substituted 3-Acetyltetramic Acid Derivatives as Antibacterial Agents
Raghunandan Yendapally, Julian G. Hurdle, Elizabeth I. Carson, Robin B. Lee, and Richard E. Lee*
Department of Pharmaceutical Sciences, College of Pharmacy, UniVersity of Tennessee Health Science Center,
847 Monroe AVenue Room 327, Memphis, Tennessee 38163
ReceiVed October 30, 2007
In order to expand the structure–activity relationship of tetramic acid molecules with structural similarity to the antibiotic
reutericyclin, 22 compounds were synthesized and tested against a panel of clinically relevant bacteria. Key structural
changes on the tetramic acid core affected antibacterial activity. Various compounds in the N-alkyl 3-acetyltetramic
acid series exhibited good activity against Gram-positive bacterial pathogens including Bacillus anthracis, Propioni-
bacterium acnes, Enterococcus faecalis, and both Methicillin-sensitive and -resistant Staphylococcus aureus.
Introduction
salts were used as starting materials for the synthesis of
compounds 9a-e and D-amino acid ester salts were used as
starting materials for the synthesis of compounds 9f,g. N-
Substituted secondary amines 8a-g were synthesized by
reductive amination reactions of the amino acid ester salts with
various alkyl and aryl aldehydes in THF in the presence of
MgSO4 and Et3N for 5 h, and the resulting imines were reduced
using NaBH4 in methanol for 30 min.17 Subsequently, secondary
amines 8a-g were converted into their respective cyanoamides
by reacting them with the cyanoacetic acid in DCM in the
presence of DIC or DCC and HOBt at rt for 6 h to afford
cyanoamide intermediates. The cyanoamides were then cyclized
into tetramic acids by using Amberlyst A-26 hydroxide resin
in methanol at rt for 2 h.16 The use of a solid-phase resin during
the cyclization step eliminates the need for column purification
by acting both as a reaction catalyst and as scavenger resin for
the product. The unreacted amides and other impurities were
simply removed by subsequent washings with methanol. The
final products were obtained by elution of the resin with
methanol/ TFA at rt for 20 min.
To explore the importance of the substituents at the 1-position
on reutericyclin, several stable N-substituted 3-acetyl tetramic acids
were synthesized (Scheme 2). Secondary amines 8a-o that were
obtained by reductive amination were subsequently reacted with
diketene in DCM in the presence of catalytic amounts of Et3N at
reflux temperature to yield their respective ꢀ-keto amides.18 These
amides were subsequently cyclized into 3-acetyltetramic acids19
10a-o using Amberlyst A-26 hydroxide resin.16 The structural
determination of 3-acyltetramic acids is complex because they exist
in different tautomeric forms. These tautomeric forms can exist in
both internal and external pairs (Figure 3).20 Internal tautomeriza-
tion involves the rapid transfer of a hydroxyl proton, which is
difficult to detect in an NMR time scale. However, NMR does
detect the ratios of external tautomers. Steyn et al. have analyzed
the X-ray crystallography data and reported that tetramic acids
primarily exist as exoenol tautomer (11d) (Figure 3).21 Thus, for
the sake of consistency in the structures through out this paper,
3-acetyltetramic acids are represented as exoenol tautomer (11d).
The ability to effectively treat bacterial infectious diseases
through antimicrobial chemotherapy is being severely undermined
by the widespread emergence of antibiotic resistance among
bacterial pathogens. These drug-resistant bacteria are a major cause
of morbidity and mortality in both hospital and community
settings.1 Thus, there is an urgent need to develop new chemo-
therapeutic agents to treat increasingly common, clinically relevant,
drug-resistant strains such as Staphylococcus aureus, Streptococcus
pyogenes, Streptococcus pneumoniae, Mycobacterium tuberculosis,
and Enterococcus faecalis.2 One approach to develop novel
therapeutic agents is by employing diversity-oriented synthesis of
natural product-like libraries.3 Naturally occurring tetramic acid
derivatives are of great interest because of their broad spectrum of
antibacterial activity.4 However, there is currently no drug in the
market that contains this type of scaffold.4 Tetramic acid is a key
constituent of various natural products such as reutericyclin 1,5
tenuazonic acid 2,6 streptolydigin 3,7 “4 (PF1052)”,8 and eryth-
roskyrine 59 (Figure 1), all of which exhibit antibacterial activity.
In most cases, the tetramic acid core is present as a 3-acyl
derivative. Although quite a number of studies have focused on
synthesizing and isolating naturally occurring tetramic acids, there
are only a few reports6,10–12 regarding the structure–activity
relationships (SAR) of these compounds with emphasis on their
antibacterial properties. Reutericyclin was isolated from Lactoba-
cillus reuteri,13 and the chemical synthesis of this molecule has
been demonstrated using racemic14 and enantiomerically specific
routes.15 However, it has an N-substituted unstable R,ꢀ-unsaturated
acyl side chain attached to the teramic acid core. In the present
study, the SAR of N-substituted tetramic acids, similar in structure
to reutericyclin, was explored by introducing a variety of chemically
stable N-alkyl, N-aryl or N-alkenyl side chains and evaluating their
antibacterial properties. A series of N-substituted 3-cyanotetramic
acids (6) and N-substituted 3-acetyltetramic acids (7) (Figure 2)
were successfully synthesized using a solid-supported reagent. The
antibacterial susceptibility of these compounds and the structure-
–activity relationships developed are discussed herein.
Chemistry
Results and Discussion
To investigate the importance of acyl substituents at the
3-position on the tetramic acid core, N-substituted 3-cyanotet-
ramic acid derivatives 9a-g (Scheme 1) were synthesized using
the method described by Kulkarni et al.16 L-Amino acid ester
To explore the structure–activity relationship of tetramic acids,
22 compounds were synthesized and evaluated for their
antibacterial properties. The compounds were tested against a
wide panel of clinically relevant bacteria consisting of M.
tuberculosis, Escherichia coli, S. aureus, E. faecalis, Bacillus
anthracis, Bacillus subtilis, Pseudomonas aeruginosa, S. pyo-
* To whom correspondence should be addressed. (Richard E. Lee) Tel:
(901) 448 6018. Fax: (901) 448 6828. E-mail: relee@utmem.edu.
10.1021/jm701356q CCC: $40.75
2008 American Chemical Society
Published on Web 02/19/2008

1488 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Brief Articles
Figure 1. Structures of 1, reutericyclin; 2, tenuazonic acid; 3, streptolydigin; 4, PF1052; and 5, erythroskyrine.
are believed to act.23,24 S. pyogenes and S. pneumoniae were
the least susceptible of the classical Gram-positive species and
displayed a trend very similar to M. tuberculosis. Both strep-
tococci and M. tuberculosis possess complex cell wall envelopes
that might have enabled these to be more refractory to
penetration by tetramic acids that the other tested Gram-positive
species.25,26 We also observed that stereochemistry did not affect
the antibacterial properties of tetramic acid compounds. In the
N-substituted 3-cyanotetramic acids series, the compounds that
were synthesized from D-amino acids ester salts (9f and 9g)
and their corresponding l-amino acid ester salt analogues (9e
and 9c) did not exhibit any antibacterial properties. Similarly,
in the 3-acetyltetramic acids, the compounds synthesized from
D-amino acids ester salts (10f,g,o) and their corresponding
L-amino acid ester salt analogues (10e,c,b) had similar activities.
The selectivity indices for the compounds were calculated
as the ratio of the cytotoxic IC50 against Vero monkey kidney
cell line and the MIC against the wild type drug-sensitive strain
S. aureus 8325. Compounds demonstrating good antimicrobial
action (10b,i-n,o) also exhibited a favorable selectivity index
(g10), with the exception of 10i (SI ) 8.8). These findings
indicate that further studies are warranted to investigate their
usefulness as antibacterials agents in the treatment and preven-
tion of infections caused by Gram-positive pathogens.27
Figure 2. General structures of 6, N-substituted 3-cyanotetramic acids,
and 7, N-substituted 3-acetyltetramic acids.
genes, S. pneumoniae, and Propionibacterium acnes. Results
arising from the antibacterial screens reveal a clear SAR as it
relates to the ability of different chemical structures to inhibit
bacterial growth. None of the tetramic acids synthesized were
active against E. coli and P. aeruginosa (MIC > 200 µg/mL),
indicating that these compounds are primarily effective against
Gram-positive bacteria. This finding is not surprising and may
in part reflect the inability of these compounds to penetrate the
complex outer membrane barrier of Gram-negative bacteria.22
Compounds in the N-substituted 3-cyano series (9a-g) were
inactive against all strains except for 9b, which interestingly
was the most active compound in the entire series of 22
compounds when tested against M. tuberculosis (MIC of 3.12
µg/mL).
The N-substituted 3-acetyltetramic acids (10a-o), in contrast
to their cyano counterparts, were active against many clinically
relevant Gram-positive bacterial species (Table 1), suggesting
the requirement of an acidic proton for anti-Gram-positive
activity. In this class of compounds, N-substitution appears to
play a critical role in engendering antibacterial activity. Tetramic
acids bearing n-decyl side chains were generally the most active
species (10b,i,n,o). Nevertheless, compounds possessing other
lipophilic substituents such as the bicyclic ring system (10l)
and the branched unsaturated systems (10m) also demonstrated
good antibacterial activities. Even replacement of n-decyl with
a biphenyl ring system (10k) did not affect antibacterial activity;
however, reduced activity was associated with compound 10a,
which carries a single phenyl ring and the compounds with aryl
ring systems (10d and 10e). Shortening of chain lengths from
n-decyl (10b) to n-butyl (10j) also led to decreased activity in
all strains except E. faecalis and P. acnes. However, this
substitution did seem to lower the toxicity of the compound
(IC50 254 µg/mL). It appears that R1 substitutions at the
5-position do not play a major role in determining antibacterial
activity since the isobutyl (10b), isopropyl (10i), and benzyl
(10n) side chain tetramic acids exhibited similar trends in their
activities. The long n-decyl chain most likely facilitates
compound entry into the cell membrane where the compounds
Conclusions
In summary, we have developed tetramic acid molecules which
exhibit good antibacterial properties. Compounds in this series were
easily synthesized enabling facile development of this class. The
synthesis of tetramic acids bearing a cyano group at the third
position has previously been reported.16 However, we have shown
for the first time that incorporation of the 3-cyano group hinders
the antibacterial activity of tetramic acid compounds. This study
found that the 3-acetyl functionality is important for the antibacterial
activity of tetramic acids and defines the 3-acetyltetramic acid
derivatives as an interesting class of compounds with good activity
against Gram-positive pathogens.
The antimicrobial mechanism of action for tetramic acids for
the most part is not well characterized. A prior study with
reutericyclin has shown that its antibacterial action results from
the molecule acting as a proton ionophore to cause dissipation of
the transmembrane pH of bacteria.23,24 The 3-acetyl derivatives
described in this study are closely related to reutericyclin but
possess chemically stable N-alkyl, N-aryl, or N-alkenyl side chains.
It is therefore plausible that the 3-acetyl group of compounds, in
contrast with the 3-cyano derivatives, enables the translocation of
protons across the membranes of susceptible bacteria, thereby

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1489
Scheme 1a
a Reagents: (a) (i) aldehydes, Et3N, MgSO4, THF, rt, 5 h; (ii) NaBH4, MeOH, rt, 30 min; (b) cyanoacetic acid, HOBt, DIC or DCC, CH2Cl2, rt, 6 h; (c)
(i) Amberlyst A-26 hydroxide resin, MeOH, rt, 2 h; (ii) MeOH, TFA, rt, 20 min. *Indicates racemizations might have occurred during synthesis.
Scheme 2a
a Reagents: (a) diketene or 50% diketene in CH2Cl2, CH2Cl2, Et3N, 6 h, reflux; (b) (i) Amberlyst A-26 hydroxide resin, MeOH, rt, 2 h; (ii) MeOH, TFA,
rt, 20min. *Indicates racemizations might have occurred during synthesis.
can be modulated by introducing different groups at the N-
substituted position. Additional studies are underway to characterize
the mode of action of these compounds and investigate their
potential for medical use as topical agents for the control of
superficial infections caused by Gram-positive pathogens.
Experimental Section
General Procedure for Preparation of Secondary Amines
(8a-o). To a stirred solution of amino acid HCl salt (1 equiv) in
THF were added MgSO4 (1.7 equiv), aldehyde (2 equiv), and Et3N
(1 equiv). The reaction was then allowed to stir at rt under argon
for 5 h. The reaction mixture was then filtered and the eluent
evaporated to give the crude imine intermediate. The imine was
directly redisolved in methanol, and sodium borohydride (2 equiv)
was slowly added to the reaction mixture. The reaction was stirred
at rt for 30 min before being quenched with excess 1 N NaOH and
extracted with ethyl acetate. The ethyl acetate extracts were
combined, washed with brine, dried over Na2SO4, and concentrated
under vacuum. The residue was purified by flash column chroma-
tography using a petroleum ether to ethyl acetate gradient elution
to afford pure products.
The preparation of (2S,3S)-methyl 2-(benzylamino)-3-methyl-
pentanoate (8a) is given as a representative example.
(2S,3S)-Methyl 2-(Benzylamino)-3-methylpentanoate (8a). Syn-
thesized according to the above general procedure using L-isoleucine
methyl ester hydrochloride (1 g, 5.5 mmol), THF (20 mL), MgSO4
(1.12 g, 9.35 mmol), benzaldehyde (1.12 mL, 11.0 mmol), Et3N
(767 µL, 5.5 mmol), sodium borohydride (416 mg, 11.0 mmol),
Figure 3. Structures of internal and external pairs of tetramic acids.
altering pH dependent intracellular reactions. The chemical structure
of side chains introduced at the N-substituted position also appears
to influence the antibacterial action of 3-acetyl tetramic acids.
Activity was improved by introducing more lipophilic substituents,
a property that may reflect better partitioning of bacterial cell
membranes by these molecules and is consistent with the proposed
antibacterial mode of action of this type of tetramic acids as proton
ionophores.
Previously, it has been suggested that the challenge in the
development of tetramic acids as antibiotic agents lies in the
potential toxicity of these compounds.4 Results arising from this
study, however, showed that the cytopathic effects of this class

1490 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Brief Articles
Table 1. Structures and Activities of N-Substituted 3-Acetyltetramic Acid Derivativesa
a Key: TB, M. tuberculosis H37Rv; BA, B. anthracis Sterne 34F2; BS, B. subtilis ATCC 23857; EF, E. faecalis ATCC 33186; MRSA, methicillin-
resistant S. aureus ATCC 33591; MSSA, methicillin sensitive S. aureus 8325 ATCC 35556; P. acnes ATCC 6919; SP, S. pyogenes ATCC 700294; SPn,
S. pneumoniae DAW30; cytotoxicity IC50, concentration which reduces viability of Vero kidney cells by 50%; selectivity index (SI), cytotoxicity IC50
divided by MIC against the methicillin sensitive strain S. aureus 8325; NA, not assessed.
1
and methanol (30 mL) to give 8a (880 mg, 68%). H NMR (500
MHz, CDCl3): δ 0.88–0.943 (6H, m), 1.16–1.3 (1H, m), 1.55–1.64
(1H, m), 1.68–1.88 (2H, m), 3.13 (1H, d, J ) 6.10 Hz), 3.62 (1H,
d, J ) 12.93 Hz), 3.74 (3H, s), 3.84 (1H, d, J ) 12.93 Hz),
7.24–7.29 (1H, m), 7.31–7.39 (4H, m). ESI-MS: 258.0 (M + 23).
General Procedure for Preparation of N-Substituted 3-Cy-
anotetramic Acid Derivatives (9a-g). To a solution of substituted
amino acids (1 equiv) in CH2Cl2 were added cyanoacetic acid (1.12 equiv),
HOBt (1.12 equiv), and either DIC or DCC (1.4 equiv), and the mixture
was stirred for 6 h at rt. The reaction mixture was subsequently filtered,
diluted with CH2Cl2, and washed with water, saturated NaHCO3, and brine.
The organic fraction was dried over Na2SO4 and concentrated. This was
purified by flash column chromatography using a petroleum ether to ethyl
acetate gradient elution to afford amides. To the solution of amide (1 equiv)
in methanol (10 mL) was added Amberlyst A-26 resin (4.2 meq/gm, 3
equiv), and the reaction was stirred at rt under argon for 2 h. Resin
containing the product was filtered and washed with methanol (3 × 10
mL). The resin was then stirred for 30 min with methanol (10 mL) and

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1491
TFA (400 µL), filtered, and washed with methanol (3 × 10 mL).
Concentration of the eluent afforded the desired products.
The preparation of (S)-1-benzyl-5-sec-butyl-4-hydroxy-2-oxo-
2,5-dihydro-1H-pyrrole-3-carbonitrile (9a) is given as a representa-
tive example.
References
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(S)-1-Benzyl-5-sec-butyl-4-hydroxy-2-oxo-2,5-dihydro-1H-
pyrrole-3-carbonitrile (9a). Compound 9a was synthesized ac-
cording to the above general procedure using (2S,3S)-methyl
2-(benzylamino)-3-methylpentanoate 8a (880 mg, 3.74 mmol),
CH2Cl2 (20 mL), cyanoacetic acid (358 mg, 4.19 mmol), HOBt
(566 mg, 4.19 mmol), and DCC (1.08 g, 5.23 mmol) to give amide
(650 mg, 57%). To the solution of amide (180 mg, 0.596 mmol) in
methanol (10 mL) was added Amberlyst A-26 resin (425 mg, 1.78
mmol), and the reaction was carried out as described in the above
general procedure to give 9a (140 mg, 87%). 1H NMR (500 MHz,
CD3OD): δ 0.78 (3H, d, J ) 6.86 Hz), 0.88 (3H, d, J ) 7.96 Hz),
1.42–1.6 (2H, m), 1.9–2.08 (1H, m), 3.94 (1H, d, J ) 3.02 Hz),
4.18 (1H, d, J ) 15.37 Hz), 5.01 (1H, d, J ) 15.37 Hz), 7.27 (2H,
d, J ) 6.86 Hz), 7.72–7.33 (1H, m), 7.34–7.39 (2H, m). ESI-MS:
(3) Shang, S.; Tan Derek, S. Advancing chemistry and biology through
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antibiotic tetramic acid reutericyclin. Synlett 2000, (8), 1131–1132.
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268.9 (M - 1). IR νmax(cm-1): 2225.86, 1642.35, 1570.15 cm-1
.
[R]26.2D -62.2 (c ) 1, MeOH). HPLC1: tR 5.87 min, purity >99%.
HPLC2: tR 5.11 min, purity 98%.
GeneralProcedureforPreparationofN-Substituted3-Acetyltet-
ramic Acids (10a-o). To a solution of substituted amino acid (1 equiv)
in CH2Cl2 were added diketene (1 equiv) and Et3N (five drops), and the
mixture was then heated under reflux for 6 h. The reaction mixture was
then cooled, diluted with CH2Cl2, and washed with dilute hydrochloric
acid followed by water. The CH2Cl2 fraction was dried over Na2SO4 and
concentrated. This was then purified by flash column chromatography
using a petroleum ether to ethyl acetate gradient elution to afford the desired
intermediate products that were then used directly in the next step. To the
solution of amide (1 equiv) in methanol (10 mL) was added Amberlyst
A-26 resin (4.2 mequiv/g, 3 equiv), and the reaction was stirred at rt under
argon for 2 h. The product containing resin was filtered and washed with
methanol (3 × 10 mL). The resin was then stirred for 30 min with
methanol (10 mL) and TFA (400 µL), filtered, and washed with methanol
(3 × 10 mL). Concentration of the eluent afforded the desired products.
The preparation of (S,Z)-1-benzyl-5-sec-butyl-3-(1-hydroxyeth-
ylidene)pyrrolidine-2,4-dione (10a) is given as a representative
example.
(S,Z)-1-Benzyl-5-sec-butyl-3-(1-hydroxyethylidene)pyrrolidine-
2,4-dione (10a). Compound 10a was synthesized according to the
above general procedure using (2S,3S)-methyl 2-(benzylamino)-3-
methylpentanoate 8a (500 mg, 2.12 mmol), CH2Cl2 (30 mL),
diketene (165 µL, 2.12 mmol), and Et3N (five drops) to give amide
(420 mg, 62%). To the solution of amide (420 mg, 1.31 mmol) in
methanol (10 mL) was added Amberlyst A-26 resin (936 mg, 3.93
mmol), and the reaction was carried out as described in the above
general procedure to give 10a (310 mg, 82%). 1H NMR (500 MHz,
CDCl3): δ 0.78–0.94 (6H, m), 1.5–1.66 (2H, m), 1.9–2.2 (1H, m),
2.46 (2.2H, s, Me 3-acetyl major tautomer), 2.58 (0.8H, s, Me
3-acetyl minor tautomer), 3.59 and 3.76 (1H, 2ds, J ) 3.29 Hz),
3.94–4.4 (1H, m), 5.24–5.38 (1H, m), 7.22–7.28 (2H, m), 7.31–7.42
1
(3H, m). H NMR (500 MHz, CD3OD): δ 0.76 (3H, d, J ) 6.86
Hz), 0.87 (3H, t, J ) 7.41 Hz), 1.46–1.66 (2H, m), 1.9–2.4 (1H,
m), 2.47 (3H, s), 3.72–3.8 (1H, bs), 4.21 (0.81 H, d, J ) 15.1 Hz)
and 4.35 (0.19 H, d, J ) 15.31 Hz), 5.01 (0.81H, d, J ) 15.37
Hz), 5.12 (0.19 H, d, J ) 15.1 Hz), 7.29–7.41 (5H, m). ESI-MS:
286 (M - 1). IR νmax (cm-1): 1709.26, 1615.06 cm-1. [R]27.3
D
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Biochem. 1995, 64, 29–63.
-87.0 (c ) 1%, MeOH). HPLC1: tR 7.17 min, purity >99%.
HPLC2: tR 6.10 min, purity 97%.
Acknowledgment. This work is supported by Grant No.
AI057836 from the National Institutes of Health. We thank Dr.
Sucheta Kudrimoti help with the preparation of this manuscript.
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Supporting Information Available: Experimental procedure for
the synthesis and spectral data of compounds 8b-o, 9b-g, and
10b-o and experimental procedures for MIC determination and
cytotoxicity assay. This material is available free of charge via the
Internet at http://pubs.acs.org.
JM701356Q