Diazenedicarboxamides as inhibitors of d-alanine-d-alanine ligase (Ddl)

Article abstract of DOI:10.1016/j.bmcl.2007.01.015

d-Alanine-d-alanine ligase (Ddl) catalyzes the biosynthesis of an essential bacterial peptidoglycan precursor d-alanyl-d-alanine and it represents an important target for development of new antibacterial drugs. A series of semicarbazides, aminocarbonyldiazenecarboxylates, diazenedicarboxamides, and hydrazinedicarboxamides was synthesized and screened for inhibition of DdlB from Escherichia coli. Compounds with good inhibitory activity were identified, enabling us to deduce initial structure-activity relationships. Thirteen diazenedicarboxamides were better inhibitors than d-cycloserine and some of them also possess antibacterial activity, which makes them a promising starting point for further development.

Full text of DOI:10.1016/j.bmcl.2007.01.015

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2047–2054
Diazenedicarboxamides as inhibitors of
D-alanine-D-alanine ligase (Ddl)
Andreja Kovacˇ,a Vita Majce,b Roman Lenarsˇicˇ,b Sergeja Bombek,b Julieanne M. Bostock,c
Ian Chopra,c Slovenko Polancb,* and Stanislav Gobeca,*
aUniversity of Ljubljana, Faculty of Pharmacy, Asˇkercˇeva 7, 1000 Ljubljana, Slovenia
bUniversity of Ljubljana, Faculty of Chemistry and Chemical Technology, Asˇkercˇeva 5, 1000 Ljubljana, Slovenia
cInstitute of Molecular and Cellular Biology and Antimicrobial Research Centre, University of Leeds, Leeds LS 9JT, UK
Received 24 November 2006; revised 27 December 2006; accepted 5 January 2007
Available online 17 January 2007
Abstract—D-Alanine-D-alanine ligase (Ddl) catalyzes the biosynthesis of an essential bacterial peptidoglycan precursor D-alanyl-D-
alanine and it represents an important target for development of new antibacterial drugs. A series of semicarbazides, aminocarbon-
yldiazenecarboxylates, diazenedicarboxamides, and hydrazinedicarboxamides was synthesized and screened for inhibition of DdlB
from Escherichia coli. Compounds with good inhibitory activity were identified, enabling us to deduce initial structure–activity rela-
tionships. Thirteen diazenedicarboxamides were better inhibitors than D-cycloserine and some of them also possess antibacterial
activity, which makes them a promising starting point for further development.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The emergence of bacterial resistance to antibiotic
therapy has become a global health threat.1 To over-
come this problem, new antibacterial agents directed
toward novel targets have to be developed. The best
known and most validated target for antibacterial ther-
apy is the system of enzymes responsible for the con-
struction of peptidoglycan.2,3 The late extracellular
stages of bacterial peptidoglycan biosynthesis are inhib-
ited by b-lactam and glycopeptide antibiotics. In con-
trast, the early intracellular biosynthetic steps have so
far received only marginal attention as potential drug
targets.1
loyl(or L-lysyl)-D-alanine.3 The final intracellular pepti-
doglycan precursor UDP-MurNAc-pentapeptide is
assembled by successive addition of L-Ala, D-Glu, m-
dpm or L-Lys, and D-Ala-D-Ala to UDP-MurNAc by
the action of Mur ligases (MurC, MurD, MurE, and
MurF, respectively). D-Ala-D-Ala ligase (Ddl) is respon-
sible for supplying the MurF substrate, the D-Ala-D-Ala
dipeptide.3 In Escherichia coli, two isoforms of Ddl exist
with 35% amino-acid sequence homology and similar
catalytic efficiencies and substrate recognition features:
DdlA and DdlB.4 In the present work, we focused our
attention on DdlB, which is a more extensively charac-
terized enzyme than DdlA. For example, the crystal
structures of the wild-type E. coli DdlB and Y216F mu-
tant derivate have been previously reported.5,6 In con-
trast, no crystal structures are currently available for
the DdlA isoform. However, both DdlA and DdlB
show similar susceptibility to known inhibitors of D-
Ala-D-Ala ligase validating their potential as novel anti-
bacterial targets.4
Peptidoglycan is an essential macromolecular compo-
nent of the cell wall of both Gram-positive and
Gram-negative bacteria. Its main function is to provide
structural integrity by withstanding the internal osmotic
pressure. The glycan chains are composed of alternating
units of N-acetylglucosamine (GlcNAc) and N-acety-
lmuramic acid (MurNAc). The carboxyl group of Mur-
NAc residues is substituted in most bacteria by a
peptide unit, L-alanyl-c-D-glutamyl-meso-diaminopime-
The dimerization of D-alanine begins with an attack on
the first D-alanine by the c-phosphate of adenosine tri-
phosphate (ATP) to give an acylphosphate. This is
followed by attack by the amino group of the second
D-alanine, which eliminates the phosphate and yields
the product D-alanyl-D-alanine.7,8 In Ddl mutants, this
reaction preferentially uses other amino-acid substrates
Keywords: D-Alanine ligase; Inhibitors; Antibacterials.
*
Corresponding authors. Tel.: +386 1 47 69 585; fax: +386 1 42 58
031; e-mail addresses: slovenko.polanc@fkkt.uni-lj.si; gobecs@ffa.
uni-lj.si
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.01.015

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A. Kovac et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2047–2054
2048
with a D-configuration: VanA and VanB use D-lactate
and VanC uses D-serine.9 This modification of the D-
Ala-D-Ala terminus leads to reduction in the binding
affinity of vancomycin and confers vancomycin
resistance.9
Unsymmetrical diazenedicarboxamides are usually pre-
pared from alkyl aminocarbonyldiazenecarboxylates.
The latter are easily available by the oxidation of the cor-
responding 1,4-disubstituted semicarbazides (Scheme 1).
Thus, the addition of alkyl hydrazinecarboxylate 2 to the
isocyanate 1 results in the formation of 1,4-disubstituted
semicarbazide 3.21 Oxidation of 3 to give 4 can be per-
formed with either N-bromosuccinimide/pyridine22 or
ceric(IV) ammonium nitrate (CAN).23 The most conve-
nient route to unsymmetrical diazenedicarboxamides 6
(R1 5 R2) is a substitution of the alkoxy group in the
diazene 4 employing a primary amine 5 as a nucleo-
phile.24 On the other hand, the synthesis of symmetrical
diazenedicarboxamides of type 6 (R1 = R2) involves
treatment of dialkyl diazenedicarboxylates 7 with two
equivalents of the appropriate primary amine 5. A reduc-
tion of any diazenedicarboxamide with various thiols
leads to the formation of product 8.25
As Ddl is essential for bacteria development and has no
human counterpart, it is an important target for devel-
opment of new antibacterial drugs.10 The most impor-
tant inhibitor of Ddl is no doubt a structural analog
of D-alanine, the antitubercular agent D-cycloser-
ine.4,11,12 A series of phosphinates, phosphonates, and
phosphonamidates have been developed as transition-
state analog inhibitors or as analogs of D-alanyl phos-
phate.13–16 It was shown that transition-state mimetics
can be phosphorylated by Ddl and inhibit the reaction
by tight binding to the enzyme after this phosphoryla-
tion. Although their antibacterial activities are low, they
enabled the crystallographic determination of complexes
of E. coli DdlB with ADP/phosphorylated phosphinate
(pdb code 2DLN)5 and ADP/phosphorylated phospho-
nate (pdb code 1IOV).6 Using the de novo structure-
based molecular design software SPROUT, a cyclopro-
pane derivative was developed as an inhibitor of DdlB.17
Recently, an allosteric inhibitor of D-alanine-D-alanine
ligase from Staphylococcus aureus was discovered by
high-throughput screening, and it was co-crystallized
with the enzyme.10 In addition, the crystal structure of
the apo form of Ddl from Thermus caldophilus has just
been resolved, providing insight into the substrate-in-
duced conformational changes, which could be impor-
tant for inhibitor design.18
Target compounds 3, 4, 6, and 8 were tested for inhibi-
tory activity on DdlB from E. coli.35 The results are pre-
sented as residual activities (RA) of the enzyme in the
presence of 500 lM of each compound (Tables 1, 2,
and 4), and for the more active compounds, as IC50 val-
ues (Table 3). In a series of semicarbazides (3, Table 1),
aminocarbonyldiazenecarboxylates (4, Table 3), and
hydrazinedicarboxamides (8, Table 4), only some
aminocarbonyldiazenecarboxylates displayed moderate
inhibition at 500 lM, while the remainder were inactive.
In contrast, very potent inhibitors were obtained in the
series of diazenedicarboxamides (6, compounds in Table
3 are ordered according to their synthesis). With the sys-
tematic variation of substituents R1 and R2, we were
able to deduce some initial structure–activity relation-
ships (SARs). In the set of compounds where the R1
substituent is 2-chloroethyl, all compounds were good
inhibitors of DdlB, with IC50 values between 119 and
236 lM (compounds 6aA, 6aB, and 6aD). Similar inhib-
itory activity was observed also for cyclohexyl derivative
6bD. Promising activities were obtained if the R1 substi-
tuent is phenyl and R2 is 2-, 3- or 4-picolyl (compounds
6cB, 6cC, and 6cD; IC50 values 111, 36, and 106 lM,
As a part of our efforts toward the discovery of new
small molecule inhibitors of the early steps of pepti-
doglycan biosynthesis,17,19,20 we screened our in-house
bank of compounds against DdlB from E. coli. We
found that some diazenedicarboxamides inhibited
the enzyme, prompting us to synthesize a small fo-
cused library of structurally related compounds and
to evaluate their enzyme inhibitory and antibacterial
activities.
O
O
O
O
O
H2NNHCOR' (2)
oxidation
R1N
R1NHCNHNHCOR'
R1NHCN
C
O
NCOR'
3
4
1
H2NR2 (5)
O
O
O
O
RSH
R1NHCN NCNHR2
R1NHCNHNHCNHR2
6
8
O
O
R'OCN NCOR'
7
Scheme 1. Synthesis of semicarbazides (3), aminocarbonyldiazenecarboxylates (4), diazenedicarboxamides (6), and hydrazinedicarboxamides (8).

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A. Kovac et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2047–2054
2049
Table 1. DdlB inhibitory activity of semicarbazides 3
Table 2. DdlB inhibitory activity of aminocarbonyldiazenecarboxy-
lates 4
O
H
N
H
N
R2
O
R1
N
H
O
H
N
N
R2
1
R
N
O
O
O
Compound
R1
R2
% inhibition at
500 lMa
Compound
R1
R2
% inhibition at
500 lMa
3a26
Me
Me
5
4a26
Me
Et
7
Cl
Cl
3b27
3c28
3d29
1
4c31
1
Et
Et
1
1
4d32
Et
Et
40
33
4e33
3e30
Et
Et
0
0
4f33
Et
37
7
3f
4g
t-Bu
Cl
a Results represent means of two independent experiments. Standard
deviations were within 10% of the means.
a Results represent means of two independent experiments. Standard
deviations were within 10% of the means.
To investigate the possible binding mode, a representa-
tive inhibitor, compound 6cB, was docked into the DdlB
active site (pdb code 1IOV), using AutoDock 3.0 with
the Lamarckian genetic algorithm.36 AutoDock calcu-
lated that the inhibitor could have a novel binding mode
as it occupies the binding sites of the phosphorylated
phosphinate inhibitor and partly also of ADP (Fig. 1).
As the binding site that accommodates the phosphory-
lated phosphinate is too small to bind our diazenedi-
carboxylates, the predicted binding pose is not
surprising. In addition, the carbonyl groups of the inhib-
itor could interact with Mg2+ ions present in the active
site of the enzyme. The predicted final docked energy
was ꢀ13.37 kcal/mol.
respectively). Compound 6cE with phenyl and the more
flexible N,N-dimethylamino-2-ethyl residue was com-
pletely inactive. The best inhibitors were those where
the R1 is a substituted phenyl. Introduction of an m-
chloro substituent to R1 of compound 6cC resulted in
a 2-fold increase in DdlB inhibition (compound 6eC,
IC50 = 15 lM). If the halogen atom is introduced either
to the meta or para position of 6cD, the activity also im-
proves (IC50 values of compounds 6eD and 6fD were 33
and 73 lM, respectively). Compounds 6hA and 6iA
where R1 is 4-alkylphenyl and R2 is 2-chloroethyl show
very good DdlB inhibition (IC50 values 49 and 25 lM,
respectively). If we compare the activities of these two
compounds with inhibitors 6aB and 6aD, we can con-
clude that in a series of 2-chloroethyl derivatives, inhibi-
tion is optimized if the second substituent of the
diazenedicarboxamide core is 4-alkylphenyl rather than
picolyl. The combination of 4-t-butylphenyl (as R1) and
4-picolyl (as R2) substituents yielded an inactive com-
pound, 6jD. When both substituents R1 and R2 were
3-picolyl, the IC50 value of the symmetrical derivative
6kC remained above 120 lM. When we compare the
inhibitory activities of our diazenedicarboxylates with
the positive control, we saw that six of our compounds
are one order of magnitude more potent as inhibitors
than D-cycloserine. In our assay, the IC50 value for D-cy-
closerine was 314 lM.
Compounds that exhibited DdlB inhibitory activities
were further tested for their antimicrobial activities
(Table 3).37 The minimal inhibitory concentrations
(MICs) of each compound were determined against
E. coli 1411,38 and SM1411,39 an acrAB deficient deriv-
ative of 1411 that exhibits increased susceptibility to a
range of antimicrobial agents,40,41 and S. aureus 8325-4.42
2-Chloroethyl derivatives 6aA, 6aB, 6hA, and 6iA,
and the symmetrical 3-picolyl derivative 6kC did not
prevent the growth of any of the bacteria strains under
investigation. Compound 6aD prevented the growth of
E. coli 1411 and E. coli SM1411 at 128 lg/mL, but not
of S. aureus 8325-4. All of the remaining DdlB inhibitors

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A. Kovac et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2047–2054
2050
Table 3. DdlB inhibitory activity of diazenedicarboxamides 6
O
H
N
N
R2
R1
N
N
H
O
6
R1
R2
IC50 (lM)
MICa (lg/mL)
E. coli 1411 AcrABꢀ
a
Compound
6aA34
E. coli 1411
S. aureus 8325-4
133
>256
>256
>256
Cl
Cl
Cl
N
6aB
119
>256
>256
>256
>256
128
64
N
N
6aD
6bD
6cB32
6cC
6cD
6cE
6eC
6eD
6fD
236
121
111
36
128
256
64
128
256
64
Cl
N
N
64
64
128
128
ND
256
64
N
106
>500
15
64
32
N
ND
64
ND
64
N
Cl
N
N
33
64
64
Cl
F
73
64
64
128
6hA
49
>256
>256
>256
Cl

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A. Kovac et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2047–2054
2051
Table 3 (continued)
R1
R2
IC50 (lM)
MICa (lg/mL)
a
Compound
E. coli 1411
E. coli 1411 AcrABꢀ
S. aureus 8325-4
6iA
6jD
25
>256
>256
>256
Cl
N
>500
ND
ND
ND
N
N
6kC
123
314
>256
16
>256
16
>256
32
D-cycloserineb
a Results represent means of two independent experiments. Standard deviations were within 10% of the means. ND, not done.
b Positive control.
Table 4. DdlB inhibitory activity of hydrazinedicarboxamides 8
O
H
N
H
N
R2
R1
N
H
N
H
O
8
Compound
R1
R2
% inhibition at 500 lMa
N
8cC
0
N
8cE
8eC
2
3
N
Cl
a Results represent means of two independent experiments. Standard deviations were within 10% of the means.
prevented the growth of all three bacteria strains, with
MICs between 32 and 256 lg/mL. The greater activity
seen against E. coli may be due to increased uptake of
the compound into these cells or to differences in the
susceptibility of the Ddl enzymes in E. coli and S. aure-
us. It is interesting to note that the best MICs were ob-
tained for the compounds where both substituents of the
diazenedicarboxamide core are aromatic. We can also
see that there is no strict correlation between DdlB
inhibitory activities and in vitro antimicrobial activities.
For this reason we have initiated further studies to
investigate the modes of antimicrobial action of our
inhibitors.
To conclude, we report the synthesis and activity of a
series of new diazenedicarboxamides as inhibitors of
DdlB from E. coli. Thirteen compounds were better
inhibitors than D-cycloserine, and five of them had
IC50 values below 50 lM and MIC values as low as
64 lg/mL against E. coli and S. aureus. As Ddl is essen-
tial and universal in prokaryotes, we predict antimicro-
bial activity against a wide range of bacteria. These
inhibitors are structurally distinct from both ADP and
D-alanine, so we expect that they inhibit Ddl by a novel
binding mode. As our diazenedicarboxamides also have
in vitro antimicrobial activities, they constitute a prom-
ising starting point for further investigations.

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2052
Figure 1. Superimposition of the computer model of compound 6eC (in magenta) on the X-ray structure of phosphorylated phosphinate inhibitor (in
red), ADP (in yellow) and Mg2+ (in cyan) bound to DdlB. The highest ranked position of the inhibitor, as calculated by AutoDock 3.0, is presented.
Gadebusch, H.; Weissberger, B.; Valiant, M. E.; Mellin, T.
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Acknowledgments
This work was supported by the European Union FP6
Integrated Project EUR-INTAFAR (Project No.
LSHM-CT-2004-512138) under the thematic priority
of Life Sciences, Genomics and Biotechnology for
Health, the British Society of Antimicrobial Chemother-
apy and the British Council. The support from the Min-
istry of Higher Education, Science and Technology of
the Republic of Slovenia and the Slovenian Research
Agency (P1-0208, P1-0230, J1-6693) is also acknowl-
edged. The authors thank Dr. Chris Berrie for critical
reading of the manuscript.
17. Besong, G. E.; Bostock, J. M.; Stubbings, W.; Chopra, I.;
Roper, D. I.; Lloyd, A. J.; Fishwick, C. W. G.; Johnson,
A. P. Angew. Chem. Int. Ed. 2005, 44, 2.
18. Lee, J. H.; Na, Y.; Song, H.-E.; Kim, D.; Park, B.-H.;
Rho, S.-H.; Im, Y. J.; Kim, M.-K.; Kang, G. B.; Lee,
D.-S.; Eom, S. H. Proteins 2006, 64, 1078.
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19. Strancar, K.; Blanot, D.; Gobec, S. Bioorg. Med. Chem.
Lett. 2006, 16, 343.
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20. Humljan, J.; Kotnik, M.; Boniface, A.; Solmajer, T.;
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istry 1997, 36, 2531.
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8. Shi, Y.; Walsh, C. T. Biochemistry 1995, 34, 2768.
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1996, 4, 401.
10. Liu, S.; Chang, J. S.; Herberg, J. T.; Horng, M.-M.;
Tomich, P. K.; Lin, A. H.; Marotti, K. R. Proc. Natl.
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11. Neuhaus, F. C.; Lynch, J. L. Biochemistry 1964, 3, 471.
12. Strominger, J. L.; Ito, E.; Threnn, R. H. J. Am. Chem.
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13. Parsons, W. H.; Patchett, A. A.; Bull, H. G.; Schoen, W.
R.; Taub, D.; Davidson, J.; Combs, P. L.; Springer, J. P.;
21. Procedure for the preparation of aminocarbonylhydrazine-
carboxylates: synthesis of ethyl (4-fluorophenyl)aminocar-
bonylhydrazinecarboxylate (3f).
7
9
H
H
O
2
12
8
N
N
1
3
11
N
O
13
O
H
6
4
10
F
5
3f
solution of 4-fluorophenyl isocyanate (0.675 mL,
A
6 mmol) in acetonitrile (4 mL) was added drop-wise to a
stirred solution of ethyl hydrazinecarboxylate (0.625 g,
6 mmol) in acetonitrile (4 mL) at 0 ꢁC. The suspension was
stirred for 10 min at 0 ꢁC and then for 10 min at rt. Solid
material was filtered off and washed with diethyl ether
(10 mL), to provide 3f (1.369 g, 95% yield): mp
190–191.5 ꢁC (methanol); IR 3260, 3060, 2980, 1730,
1680, 1630, 1560, 1240, 1030, 850 cmꢀ1
;
1H NMR
(DMSO-d6) d 1.19 (t, 3H, J = 7.1 Hz, H-13), 4.06 (q, 2H,
J = 7.1 Hz, H-12) 7.08 (m, 2H, H-3, H-5), 7.48 (m, 2H, H-
2, H-6), 8.02 (s, 1H, H-7), 8.75 (s, 1H) and 8.90 (s, 1H):

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A. Kovac et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2047–2054
2053
H-9 and H-10; 13C NMR (DMSO-d6) d 14.5 (C-13), 60.5
(C-12), 115.0 (d, J = 22.4 Hz, C-3, C-5), 120.3 (d,
J = 5.2 Hz, C-2, C-6), 136.0 (d, J = 2.3 Hz, C-1), 155.7
and 156.9: C-8 and C-11, 157.3 (d, J = 238.0 Hz, C-4); MS
(EI) m/z 241 (M+, 13), 137 (32), 110 (36), 104 (100), 83
(25). Anal. Calcd for C10H12FN3O3 (241.22): C, 49.79; H,
5.01; N, 17.42. Found: C, 49.72; H, 4.99; N 17.69.
22. Typical preparation of aminocarbonyldiazenecarboxylates:
synthesis of t-butyl (2-chloroethyl)aminocarbonyldiazene-
carboxylate (4g).
(M++H, 30), 123 (22), 107 (33). Anal. Calcd for
C14H12ClN5O2 (317.73): C, 52.92; H, 3.81; N, 22.04.
Found: C, 53.14; H, 3.88; N, 21.72.
25. Reduction of diazenedicarboxamides: synthesis of N-(3-
chlorophenyl)-N0-(3-picolyl)hydrazine-1,2-dicarboxamide
(8eC).
7
9
H
H
O
2
18
13
14
8
3
Cl
N
N
1
11
N
N
N
3
7
4
O
H
H
15
H
O
CH3
6
17
6
10
12
1
CH3
16
5
N
N
4
5
8eC
Cl
N
O
2
CH3
A suspension of the diazene 6eC (794 mg, 2.5 mmol) in
acetone (10 mL) was treated with 1-thioglycerol
(0.505 mL, 5.2 mmol). The reaction mixture was stirred
at rt for 15 min. The solid material was filtered off and
washed with acetone to provide the product 8eC (770 mg;
96% yield): mp 206–208 ꢁC (methanol); IR 3299, 3226,
O
4g
NBS (979 mg; 5.5 mmol) was slowly added at rt to a
stirred mixture of t-butyl (2-chloroethyl)aminocarbonyl-
hydrazinecarboxylate (1.19 g, 5 mmol) and pyridine
(0.81 mL, 10 mmol) in CH2Cl2 (7 mL). After continuous
stirring at rt for 30 min, HCl (1:1, 15 mL) was added and
two phases were separated. A CH2Cl2 solution was treated
successively with 5% aqueous solution of Na2S2O3 (7 mL),
saturated solution of NaHCO3 (2· 7 mL), and water
(15 mL), then dried over anhydrous Na2SO4 and evapo-
rated to dryness to give 4g (1.14 g, 97% yield): mp
73–74 ꢁC (dichloromethane/diethyl ether); IR 3266, 3059,
2991, 1763, 1734, 1564, 1537, 1435, 1372, 1255, 1194, 1144,
2925, 1667, 1594, 1542, 1427, 1324, 1259 cmꢀ1; H NMR
1
(DMSO-d6) d 4.27 (d, 2H, J = 6.1 Hz, H-13), 6.99 (ddd,
1H, J1 = 7.9 Hz, J2 = 2.1 Hz, J3 = 2.0 Hz, H-6), 7.18 (t,
1H, J = 6.1 Hz, H-12), 7.26 (dd, 1H, J1 = 8.3 Hz,
J2 = 7.9 Hz, H-5), 7.32 (ddd, 1H, J1 = 7.8 Hz,
J2 = 4.8 Hz, J3 = 0.8 Hz, H-16), 7.40 (m, 1H, H-4), 7.68
(ddd, 1H, J1 = 7.8 Hz, J2 = 2.3 Hz, J3 = 1.8 Hz, H-15),
7.74 (dd, 1H, J1 = 2.1 Hz, J2 = 2.0 Hz, H-2), 7.90 (s, 1H)
and 8.08 (s, 1H): H-9 and H-10, 8.43 (dd, 1H, J1 = 4.8 Hz,
J2 = 1.8 Hz, H-17), 8.50 (m, 1H, H-18), 8.93 (s, 1H, H-7);
13C NMR (DMSO-d6) d 40.4 (C-13), 117.0 and 118.0: C-2
and C-6, 121.4 (C-4), 123.2 (C-16), 130.1 (C-5), 132.9 (C-
15), 134.8 and 135.9: C-3 and C-14, 141.3 (C-1), 147.8 and
148.6: C-17 and C-18, 155.8 and 158.7: C-8 and C-11; MS
(FAB) 320 (M++H, 24), 107 (30), 69 (84). Anal. Calcd for
C14H14ClN5O2.14MeOH: C, 52.22; H, 4.61; N, 21.37.
Found: C, 52.15; H, 4.61; N, 21.50.
1
1057, 957, 825 cmꢀ1; H NMR (CDCl3) d 1.63 (s, 9H, H-
7), 3.75 (m, 2H, H-2), 3.83 (m, 2H, H-1), 6.75 (broad, 1H,
H-3); 13C NMR (CDCl3) d 27.7 (C-7), 42.6 and 42.7: C-1
and C-2, 87.1 (C-6), 159.0 and 160.0: C-4 and C-5; MS
(FAB) m/z 236 (M++H, 5), 57 (100). Anal. Calcd for
C8H14ClN3O3 (235.67): C, 40.77; H, 5.99; N, 17.83.
Found: C, 40.78; H, 6.09; N 17.64.
ˇ
ˇ
23. Kosmrlj, J.; Kocevar, M.; Polanc, S. J. Chem. Soc., Perkin
Trans. 1 1998, 3917.
ˇ
26. Bombek, S.; Pozˇgan, F.; Kocevar, M.; Polanc, S. J. Org.
24. Procedure for the preparation of diazenedicarboxamides:
synthesis of N-(3-chlorophenyl)-N0-(3-picolyl)diazene-1,2-
dicarboxamide (6eC).
Chem. 2004, 69, 2224.
27. Bombek, S.; Pozˇgan, F.; Kocevar, M.; Polanc, S. New
J. Chem. 2005, 29, 948.
28. Zinner, G.; Deucker, W. Arch. Pharm. 1961, 249, 370.
29. Strickler, J. C.; Pirkle, W. H. J. Org. Chem. 1966, 31, 3444.
30. Bollbuck, G.; Stroh, H. H.; Barnikow, G. J. Prakt. Chem.
1971, 313, 773.
ˇ
7
H
N
O
2
11
16
8
3
12
Cl
N
1
9
N
N
N
31. Knight, G. T.; Loadman, M. J. R.; Saville, B.; Wildgoose,
J. J. Chem. Soc., Chem. Commun. 1974, 193.
4
13
O
H
15
6
10
5
14
ˇ
ˇ ˇ
ˇ
32. Pieters, L.; Kosmrlj, J.; Lenarsic, R.; Kocevar, M.; Polanc,
6eC
S. ARKIVOC 2001, 42 (Part V).
ˇ ˇ
Ethyl (3-chlorophenyl)aminocarbonyldiazenecarboxylate
(1.534 g, 6 mmol) was slowly added to the stirred solution
of 3-picolylamine (0.61 mL, 6 mmol) in acetonitrile (2 mL)
at 0 ꢁC. The reaction mixture was stirred at 0 ꢁC for
15 min. The solid material was filtered off and washed with
acetonitrile to give the diazene 6eC (1.412 g, 74%): mp
129–130 ꢁC (ethyl acetate); IR 3338, 2939, 1741, 1713,
1546, 1504, 1428, 1189 cmꢀ1; 1H NMR (DMSO-d6) d 4.54
(d, 2H, J = 6.0 Hz, H-11), 7.27 (ddd, 1H, J1 = 8.0 Hz,
J2 = 2.1 Hz, J3 = 2.0 Hz, H-6), 7.41 (ddd, 1H, J1 = 7.8 Hz,
J2 = 4.8 Hz, J3 = 0.9 Hz, H-14), 7.45 (dd, 1H, J1 = 8.2 Hz,
J2 = 8.0 Hz, H-5), 7.66 (ddd, 1H, J1 = 8.2 Hz, J2 = 2.1 Hz,
J3 = 2.0 Hz, H-4), 7.78 (ddd, 1H, J1 = 7.8 Hz, J2 = 2.4 Hz,
J3 = 1.7 Hz, H-13), 7.86 (dd, 1H, J1 = 2.1 Hz, J2 = 2.0 Hz,
H-2), 8.52 (dd, 1H, J1 = 4.8 Hz, J2 = 1.7 Hz, H-15), 8.60
(dd, 1H, J1 = 2.4 Hz, J2 = 0.9 Hz, H-16), 9.63 (t, 1H,
J = 6.0 Hz, H-10), 11.55 (s, 1H, H-7); 13C NMR (DMSO-
d6) d 41.2 (C-11), 118.1 and 119.0: C-2 and C-6, 123.6 (C-
4), 124.7 (C-14), 130.9 (C-5), 133.4 and 133.6: C-3 and
C-13, 135.4 (C-12), 138.9 (C-1), 148.6 and 148.9: C-15 and
C-16, 158.2 and 161.7: C-8 and C-9; MS (FAB) 318
ˇ
33. Lenarsic, R.; Kocevar, M.; Polanc, S. J. Org. Chem. 1999,
64, 2558.
34. Kraebel, C. M.; Davis, S. M. J. Chem. Eng. Data 1969, 14,
133.
35. The D-Ala-adding activity of DdlB ligase was monitored by
the detection of orthophosphate generated during the
reaction based on the colorimetric malachite green method
described by Walsh, A. et al. J. Bacteriol. 1999, 181, 5395.
Assays were performed at 37 ꢁC in a mixture (final volume:
50 ll) containing 38.5 mM Hepes, pH 8.0, 3.25 mM MgCl2,
6.5 mM (NH4)2SO4, 700 lM D-Ala, 500 lM ATP, purified
DdlB (diluted in 20 mM Hepes, pH 7.2, and 1 mM
dithiothreitol), and 500 lM test compound (IC50 values
were determined for a range of inhibitor concentrations).
All compounds were soluble in the assay mixture containing
5% DMSO. After 30 min of incubation, 100 lL Biomolꢂ
reagent was added. After 5 min, absorbance was read at
650 nm. Residual activity was calculated with respect to a
similar assay without inhibitor. To exclude possible
non-specific (promiscuous) inhibitors, representative

ˇ
A. Kovac et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2047–2054
2054
compounds 3c, 4c, 6aD, 6bD, 6cC, 6cD, 6eD, 6fD, 6iA, and
16 h at 37 ꢁC in a Spectramax 384 plus microwell plate
reader (Molecular Devices, Abingdon, UK), running
SOFTmax PRO 3.1.1 software. Optical density readings
(600 nm) were taken at 10 min intervals. Plates were
shaken for 30 s before each reading. The MIC was taken
as the lowest concentration of antimicrobial agent that
prevented growth.
6jD were tested also in the presence of Tween (0.003%),
Triton X-114 (0.005%), and SDS (420 lM), as described by
McGovern, S. L. et al. J. Med. Chem. 2003, 46, 4265, and
Ryan, A. J. et al. J. Med. Chem. 2003, 46, 3448. No
significant differences were found when compared to
measurements without detergents.
36. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.;
Hart, W. E.; Belew, R. K.; Olson, A. J. J. Comp. Chem.
1998, 19, 1639.
38. Miller, K.; O’Neill, A. J.; Chopra, I. J. Antimicrob.
Chemother. 2002, 49, 925.
39. O’Neill, A. J.; Bostock, J. M.; Morais Moita, A.; Chopra,
I. J. Antimicrob. Chemother. 2002, 50, 839.
40. Sulavik, M. C.; Houseweart, C.; Cramer, C.; Jiwani, N.;
Murgolo, N.; Greene, J.; DiDomenico, B.; Shaw, K. J.;
Miller, G. H.; Hare, R.; Shimer, G. Antimicrob. Agents
Chemother. 2001, 45, 1126.
41. Xiong, L. Q.; Kloss, P.; Douthwaite, S.; Andersen, N. M.;
Swaney, S.; Shinabarger, D. L.; Mankin, A. S. J.
Bacteriol. 2000, 182, 5325.
37. Minimal inhibitory concentrations (MICs) of compounds
were determined by broth microdilution in IsoSensitest
broth (Oxoid, Basingstoke, UK) using an inoculum of 104
cells per mL for E. coli or 106 cells per mL for S. aureus.
The antimicrobial agents were prepared in a 2-fold
dilution series in 50% dimethyl sulfoxide (Sigma–Aldrich,
Dorset, UK). Microwell plates with 96 wells (Nunc, Fisher
Scientific, Loughborough, UK) containing the antimicro-
bial agent and bacterial suspension were incubated for
42. Novick, R. Virology 1967, 33, 155.