Thiosemicarbazones active against Clostridium difficile

Article abstract of DOI:10.1016/j.bmcl.2008.01.041

A set of closely related furylidene thiosemicarbazones was prepared and screened against various clinically important Gram-positive bacteria. One compound containing an ethylene spacer and a 5-nitrofuryl group was found to have promising activity against Clostridium difficile.

Full text of DOI:10.1016/j.bmcl.2008.01.041

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 1708–1711
Thiosemicarbazones active against Clostridium difficile
Cait Costello, Tarja Karpanen, Peter A. Lambert, Preena Mistry, Katy J. Parker,
Daniel L. Rathbone,* Jiangmeng Ren, Laura Wheeldon and Tony Worthington
School of Life and Health Sciences, Aston University, Birmingham B4 7ET, UK
Received 20 December 2007; revised 11 January 2008; accepted 12 January 2008
Available online 18 January 2008
Abstract—A set of closely related furylidene thiosemicarbazones was prepared and screened against various clinically important
Gram-positive bacteria. One compound containing an ethylene spacer and a 5-nitrofuryl group was found to have promising activ-
ity against Clostridium difficile.
ꢀ 2008 Elsevier Ltd. All rights reserved.
The number of reported cases of Clostridium difficile
associated diarrhoea (CDAD) has increased dramati-
cally over the last decade. In 2005, more than 55,620
cases of CDAD in patients over the age of 65 were re-
ported in England, many of which were associated with
the hypertoxin-producing strain ribotype 027. Not sur-
prisingly, C. difficile is now the leading hospital associ-
ated infection in the UK.1 Furthermore, many strains
of C. difficile are now emerging with increased resistance
and multiple resistance to many common antibiotics
including several fluoroquinolones, erythromycin, clari-
thromycin, fusidic acid and clindamycin.2,3 Such strains
have been associated with outbreaks of CDAD in the
clinical setting. In light of this, there is a clear need to
find novel antimicrobial agents with activity against
both spores and vegetative cells of C. difficile.
We now present the biological activities of a set
of substituted furyl-containing thiosemicarbazones
(Scheme 1) against a panel of Gram-positive bacteria
including Staphylococcus aureus (methicillin-sensitive
and -resistant strains), Staphylococcus epidermidis,
Propionibacterium acnes and C. difficile. This was under-
taken with a view to establishing the influence of the
following structural features on the antimicrobial activ-
ity: the size of the alkyl group at the N-4 position; the
presence of a nitro group at the 5-position on the furan;
the presence of an ethylene spacer group in the furylid-
ene portion.
The thiosemicarbazones were prepared in two steps
using variations on literature procedures.8 The appro-
priate isothiocyanates were added to an excess of hydra-
zine in boiling ethanol to give the corresponding
substituted thiosemicarbazides as crystalline solids that
Arylidene thiosemicarbazones have been explored in
recent years for trypanocidal,4–7 antiamoebic8 and anti-
bacterial9,10 activity. In some of these the 5-nitrofuryl
group has proved to be essential either connected di-
rectly as the thiosemicarbazone derivative or attached
via an ethylene spacer. Biological activity associated
with the 5-nitrofuryl moiety is understood to be a com-
plex sequence of events starting with reduction of the ni-
tro group.11,12 In addition, the corresponding copper
and palladium complexes of these ligands have shown
similar or enhanced activity against the same targets.7,8
S
NH2NH2.H2O /
EtOH/reflux
1
R
NH2
R1NCS
N
H
N
H
2
R
S
O
2
n
O
R
1
N
R
n
O
N
H
N
H
EtOH/reflux
Keywords: Thiosemicarbazone; Clostridium difficile; Antimicrobial;
Nitrofuran.
1 - 12
*
Scheme 1. Preparation of the target thiosemicarbazones (compounds
1–12).
Corresponding author. Tel.: +44 121 204 4002; fax: +44 121 359
0572; e-mail: D.L.Rathbone@aston.ac.uk
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.041

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C. Costello et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1708–1711
1709
precipitated out of the reaction mixture upon cooling.
These were sufficiently clean to be used in the next step
without purification. The thiosemicarbazides were sus-
pended in ethanol and heated at reflux for 2 h with
1.3 mequiv of the appropriate furyl aldehyde to give
the target compounds 1–12 (Scheme 1 and Table 1).
Again, these compounds precipitated cleanly out of
the reaction mixture. By this simple procedure the
nitrofuranyl compounds (1–4, 6, 8, 10, 12) were
obtained in 59–93% yield and the furanyl compounds
(5, 7, 9, 11) in 27–57% yield. The compounds were char-
acterised by melting point, infra-red spectroscopy, mass
spectrometry (+ve electrospray accurate mass) and pro-
ton NMR spectroscopy and had properties consistent
with the proposed structures.
ing for 24 h at 37 ꢁC against an initial panel of organ-
isms in using 2.5 · 107 CFU/mL. Anaerobic micro-
organisms were incubated in an anaerobic cabinet.
Controls consisted of incubations of the specified organ-
isms as above but omitting the test compounds. Com-
pound 8 was tested further against an expanded panel
of organisms. The resulting minimum inhibitory concen-
trations are presented in Tables 2 and 3. Vancomycin
susceptibility was investigated for a subset of organisms
and the results are presented in Table 2. A suspension of
each organism (at a concentration of McFarland 0.5)
was used to inoculate the entire surface of appropriate
agar plates. Vancomycin E-test strips (AB BIODISK,
Solna, Sweden) were placed in the middle of the agar
plates and incubated under the appropriate conditions:
Staphylococci, Nutrient agar (Oxoid, UK) plates,
37 ꢁC, aerobic, 24 h; C. difficile, Wilkins Chlagren agar
WCA (Oxoid, UK) plates, 37 ꢁC, anaerobic, 48 h;
P. acnes, nutrient agar plates, 37 ꢁC, anaerobic, 48 h.
The test compounds were solubilised in DMSO and
diluted with nutrient broth (Oxoid, UK) prior to screen-
Compounds 1–12 were screened against an initial panel
of Gram-positive organisms in nutrient broth that in-
cluded representative strains of C. difficile, S. epidermi-
dis, S. aureus (including MRSA strains) and P. acnes
(Table 2). Compound 8 was significantly more active
against each of the different types of organism. This
can be seen in Figure 1 where the reciprocal of the aver-
aged MIC values for the different types of organism for
each of the compounds has been presented as a bar
chart. Excluding P. acnes, the structure–activity profile
was quite sharp. Apart from compound 8 only com-
pounds 1, 2 and 6 had noticeable antimicrobial activity.
Each of these four compounds has the 5-nitrofuran moi-
ety. For P. acnes, again, compound 8 was the most
active but this time moderate antimicrobial activity
was spread across the test compounds with the excep-
tion of 3, 7 and 9.
Table 1. Structures of the compounds synthesised and screened in this
study
Compound
R1
n
R2
cLogPa
Reference
1
2
Me
Et
0
0
0
0
1
1
1
1
1
1
1
1
NO2
NO2
NO2
NO2
H
2.1
2.4
2.8
2.9
1.7
2.5
2.1
2.8
2.5
3.2
2.5
3.3
6,7,13
6,7,13
7,8
3
i-Pr
t-Bu
Me
Me
Et
4
5
6
NO2
H
6,7,13
6,7,13
14
7
8
Et
NO2
H
9
i-Pr
i-Pr
t-Bu
t-Bu
10
11
12
NO2
H
NO2
15
a CAChe WorkSystem Pro Version 6.1.12.33 (Fujitsu Ltd).
Table 2. Minimum inhibitory concentration (lg mLÀ1) of compounds 1–12 against an initial panel of organisms
Organisms Compound
1
2
3
4
5
6
7
8
9
10
11
12
Vancomycin
C. difficile NCTC11204 >256
64–128 >256
64–128 64–128 64–128 >256
>256 128–256 >256 128–256 >256
128–256 >256
64–128 >256 4–8
>256
>256 >256 >256
0.5
C. difficile O27
C. difficile Z162
64–128 32–64 64–128 8–16 64–128 64–128 64–128 64–128 0.75
32–64 >256 0.125– >256
0.25
>256 >256 128–256
C. difficile Z229
S. epidermidis
NCTC 11047
64–128 8–16 128–256 16–32 128–256 16–32 64–128 4–8
16–32 32–64 64–128 128–256 >256 32–64 >256 4–8
128–256 64–128 64–128 64–128
>256
64–128 >256 >256
2
S. epidermidis 9
S .epidermidis 4
128–256 64–128 >256
32–64 32–64 >256
S. aureus ATCC6538 64–128 32–64 64–128 64–128 >256
>256
>256
>256
64–128 >256 1–2
>256
>256
>256
>256 >256 >256
64–128 >256 >256
>256 >256 >256
128–256 32–64 >256 4–8
32–64 >256 4–8
1.5
S. aureus H1
S. aureus K1
MRSA A1
64–128 16–32 128–256 >256
128–256 64–128 >256
64–128 64–128 >256
>256
64–128 >256
16–32 >256 16–32 >256
256
>256 64–128
>256 >256 4–8
64–128 >256 2–4
64–128 >256 4–8
64–128 >256 4–8
>256
>256
>256
>256
>256 >256 32–64
>256 >256 >256
>256 >256 >256
>256 >256 >256
>256
>256
>256
>256
>256
>256
MRSA C3
MRSA E1
>256 64–128 >256
64–128 64–128 >256
P. acnes NCTC737
P. acnes B5
P. acnes C3
P. acnes C5
16–32 16–32 64–128 64–128 32–64 16–32 16–32 4–8
32–64 32–64 64–128 16–32 0.5
>256 8–16 >256 32–64 64–128 32–64
32–64 2–4 1–2
64–128 32–64 >256
16–32 2–4 32–64
64–128 32–64 64–128 16–32 1–2
32–64 32–64 1–2
16–32 16–32 1–2
32–64 4–8
8–16
64–128 32–64 32–64 64–128 64–128 1–2
16–32

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1710
C. Costello et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1708–1711
Table 3. Minimum inhibitory concentration (lg mLÀ1) of compound 8
against an expanded panel of organisms
Organism
MIC
C. difficile Z16
C. difficile Z21
C. difficile Z 66
C. difficile Z150
C. difficile Z158
C. difficile Z159
C. difficile Z160
C. difficile Z161
C. difficile Z163
C. difficile Z164
C. difficile Z166
C. difficile Z167
C. difficile Z168
C. difficile Z169
C. difficile Z170
C. difficile Z171
C. difficile Z172
C. difficile Z173
C. difficile Z175
C. difficile Z176
C. difficile Z177
C. difficile Z179
C. difficile Z251
C. difficile Z387
C. difficile Z1568
C. difficile Z1577
C. difficile Z1578
C. difficile Z1579
C. difficile Z1580
C. difficile Z1591
8–16
16-32
4–8
4–8
2–4
8–16
8–16
0.5–1
2–4
1–2
2–4
2–4
2–4
Figure 1. Bar chart of averaged 1/MIC values (mL lgÀ1
compounds 1–12 for the various classes of organisms tested.
) for
2–4
2–4
2–4
4–8
8–16
0.5–1
2–4
Thiosemicarbazones of 5-nitro-2-furaldehyde also con-
taining N-4 dialkyl/cycloalkyl substituents are known
to have antifungal properties and activity against
mainly Gram-positive bacteria.9 For example, the N-4
diethyl analogue of compound 2 has good activity
against Escherichia coli, Micrococcus pyrogenes, Strep-
tococcus pyogenes, Baccillus subtilis and moderate
activity against Trichophyton mentagrophytes and Can-
dida albicans. Although these are different organisms
from those presented in this article it appears that
the addition of a second alkyl substituent at the N-4
position of the thiosemicarbazones without the ethyl-
ene spacer (n = 0) improves activity against Gram-posi-
tive bacteria.
2–4
2–4
1–2
16–32
1–2
0.5–1
1–2
2–4
1–2
1–2
S. epidermidis NCTC 9865
S. epidermidis RP62A
S. epidermidis 3
4–8
2–4
2–4
2–4
4–8
1–2
S. epidermidis 7A
S. epidermidis 8A
S. epidermidis 10
Compound 8 was tested against an expanded panel of
organisms (Table 3). It had good activity against the
organisms mentioned above. For C. difficile in particular
the reference strain NCTC 11204 was inhibited at 4–
8 lg/mL and a set of 33 clinical strains exhibited mini-
mum inhibitory concentrations in the range 0.125–0.25
to 16–32 lg/mL. In addition, no activity was observed
against the Gram-negative microorganism Acinetobacter
spp. or C. albicans yeast strains. The mechanism of ac-
tion of the lead compound 8 is unknown at this time.
On the basis that the LogP values for the inactive com-
pounds 3, 4, 6, 9, 10 and 11 (Table 1) are very similar to
that of the active compound 8, it may be expected that
the cellular penetrations of these compounds are similar.
Thus the observed SAR may indeed reflect interactions
at particular targets rather than differences in cellular
penetration. In addition previous authors6 have investi-
gated the acute in vivo toxicity of compounds 2 and 8.
These indicated nontoxic effects on the basis of clinical
biochemistry, haematology and the histological studies
of organs.
S. epidermidis 11
S. aureus T1
S. aureus V1
4–8
2–4
S. aureus H1
64–128
4–8
4–8
S. aureus MSSA2
S. aureus MSSA3
MRSA A3
MRSA B1
MRSA B3
MRSA D1
MRSA C1
4–8
4–8
4–8
4–8
2–4
EMRSA 15
4–8
P. acnes B3
P. acnes C1
P. acnes D1
P. acnes D3
8–16
8–16
4–8
4–8
C. albicans 1013781
C. albicans 1012155
C. albicans 1009116
C. albicans 1011377
C. albicans 1015391
128–256
128–256
128–256
128–256
128-256
In summary, amongst a set of closely related thiosemi-
carbazones, compound 8 was found to have promising
antimicrobial activity against clinically important
Gram-positive skin organisms. Its key structural fea-
tures included an ethyl group at N-4, an ethylene spacer
in the furylidene portion and a nitro group at the 5-po-
sition of the furan ring.
Acinetobacter spp. 1
Acinetobacter spp. 2
Acinetobacter spp. 3
Acinetobacter spp. 4
Acinetobacter spp. 5
128–256
128–256
128–256
128–256
128–256

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C. Costello et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1708–1711
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10. Bilgin, A. A.; Erol, D. D.; Yulug, N.; Istanbullu, I. Fabad
Farm. Bilimler Derg 1989, 14, 79.
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14. 4-(i-Propyl)-1-[3-(5-nitrofuryl)propenilidene]thiosemi car-
bazide (10): Dark red powder. 86% Yield. Mp 219.4–
222.0 ꢁC (corrected). 1H NMR (d6-DMSO; d DMSO =
2.50 ppm) 1.18 (6H, d, J = 6.9 Hz, Me2), 4.47 (1H, m,
CHMe2), 7.04 (3H, overlapping m, Furan-H3 and
N@CHACH@CH), 7.75 (1H, d, J = 3.8 Hz, Furan-H4),
7.86 (1H, dd, J = 6.95, 1.90 Hz, N@CHACH), 8.15 (1H, d,
J = 8.9 Hz, Me2CHNH), 11.62 (s, 1H, NHAN@C) ppm.
IR (KBr) 3358, 3320, 3123, 2971, 1531, 1464, 1347, 1231,
1190 cm À1. High-resolution MS (+electrospray): (M+H)+
calcd 283.0865, found 283.0845.
15. 4-(tert-Butyl)-1-[3-(5-nitrofuryl)propenilidene]thiosemi
carbazide (12): Orange powder. 93% Yield. Mp dec
209.5 ꢁC (corrected). 1H NMR (d6-DMSO;
d
DMSO = 2.50 ppm), 1.51 (9H, s, Me3), 7.02 (2H, over-
lapping m, N@CHACH@CH), 7.08 (d, J = 3.8 Hz, 1H,
Furan-H3), 7.57 (1H, s, NHCMe3), 7.76 (1H, d,
J = 4.4 Hz, Furan-H4), 7.87 (dd, J = 5.06 3.16 Hz, 1H,
N@CHACH), 11.56 (s, 1H, NHAN@C) ppm. IR (KBr)
3312, 3142, 2973, 1529, 1509, 1466, 1417, 1390, 1355, 1260,
1241 cmÀ1. High-resolution MS (+electrospray): (M+H)+
calcd 297.1021, found 297.1013.