seco-Cyclothialidines: New concise synthesis, inhibitory activity toward bacterial and human DNA topoisomerases, and antibacterial properties

Article abstract of DOI:10.1021/jm0010623

seco-Cyclothialidines are a promising class of bacterial DNA gyrase B subunit inhibitors. A new seco-cyclothialidine derivative containing a dioxazine moiety, BAY 50-7952, was synthesized through a new concise pathway. One key step of the synthesis is the

Full text of DOI:10.1021/jm0010623

J . Med. Chem. 2001, 44, 619-626
619
seco-Cycloth ia lid in es: New Con cise Syn th esis, In h ibitor y Activity tow a r d
Ba cter ia l a n d Hu m a n DNA Top oisom er a ses, a n d An tiba cter ia l P r op er ties§
J oachim Rudolph,*,† Heidi Theis,† Roman Hanke,† Rainer Endermann,‡ Lars J ohannsen,‡ and
Frank-Ulrich Geschke‡
Bayer AG, Central Research, Chemistry for Life Sciences, D-51368 Leverkusen, Germany, and Bayer AG,
Pharma Research Center, D-42096 Wuppertal, Germany
Received August 25, 2000
seco-Cyclothialidines are a promising class of bacterial DNA gyrase B subunit inhibitors. A
new seco-cyclothialidine derivative containing a dioxazine moiety, BAY 50-7952, was synthesized
through a new concise pathway. One key step of the synthesis is the straightforward formation
of the 2-aminothiazole derivative of S-tritylcysteine. In biological tests, BAY 50-7952 and other
known seco-cyclothialidines exhibited high and selective activity toward bacterial DNA gyrase
and toward Gram-positive bacteria. The dioxazine moiety and other similar groups were found
to be important for the ability of the seco-cyclothialidines to penetrate bacterial membranes.
The opposite enantiomer ((S)-form) of BAY 50-7952 was also synthesized, and neither significant
target activity nor in vitro antibacterial activity were found, suggesting a highly selective fit
of the (R)-form. Despite promising in vitro activity, only poor activity was found in the murine
infection model.
Ch a r t 1
In tr od u ction
Due to the rapidly growing number of resistant
bacterial strains, the search for antibacterial agents
with new modes of action will always remain an
important and challenging task.1 In the past, the
bacterial DNA gyrase has drawn much attention as a
selectively addressable target. In particular the fluoro-
quinolones, e.g. ciprofloxacin (1; Chart 1), proved to be
highly successful inhibitors of bacterial DNA gyrase and
were extensively investigated in the pharmaceutical
industry.2
Apart from the quinolones, other naturally occurring
bacterial DNA gyrase inhibitors, such as the coumarins
(e.g. novobiocin (2; Chart 1) and coumermycin), have
been known for a long time. However, no pharmaceuti-
cally useful lead compounds have been derived from
them until now.3,4 In 1994, cyclothialidine (3; Chart 1)
was described as a new potent DNA gyrase-inhibiting
natural product.5 Its structure features a unique 12-
membered lactone ring that is fused to a highly substi-
tuted benzene ring and partly integrated into a pen-
tapeptide chain. Shortly after its discovery, the potential
of this compound was recognized by Hoffmann-La Roche
and has been intensively investigated since that time.6
Different from the quinolones which bind to the A
subunit of the DNA gyrase, the cyclothialidines act as
competitive inhibitors of the B subunit of this enzyme.
Despite their high activity on the target, cyclothiali-
dine and its natural analogues barely exert any bacte-
rial activity against intact bacterial cells, probably due
to insufficient penetration of the cytoplasmic membrane.
In further investigations, it was shown that the lactone
ring is not a mandatory requirement for target activity,
leading to the development of the open-chain analogues.
In fact, these compounds, called seco-cyclothialidines,
were able to penetrate bacterial membranes. The seco-
cyclothialidines were patented by Hoffmann-La Roche,7a
and the compound Ro-61-6653 was later identified as
an interesting lead candidate.7b As shown in Table 1,
Ro-61-6653 exerts excellent activity against Gram-
positive bacteria; however, it is inactive against Gram-
negative strains. Prior to our own investigations, no
results were reported concerning the in vivo antibacte-
rial potential of the seco-cyclothialidines in animal
infection models.
§ Dedicated to Professor K. Barry Sharpless on the occasion of his
60th birthday.
* To whom correspondence should be addressed. Phone: +49 214
30 578 59. Fax: +49 214 30 96 578 59. E-mail: joachim.rudolph.jr@
bayer-ag.de.
† Bayer AG, Central Research.
‡ Bayer AG, Pharma Research Center.
10.1021/jm0010623 CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/10/2001

620 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4
Ta ble 1. In Vitro Antibacterial Activity of Ro-61-6653
Rudolph et al.
pathway that introduces this moiety at a later stage of
the synthetic sequence. Furthermore, since the aromatic
group attached to the thio functionality is highly
substituted and requires a multistep synthesis, we
preferred the introduction of this group at the final stage
of the synthetic pathway.
In the course of our work, we developed a new
improved synthesis of the seco-cyclothialidines which
includes only seven steps (Scheme 2). This synthesis
was applied in the preparation of the seco-cyclothialidine
derivatives BAY 50-7952 and Ro-61-6653.
Starting with the amino acid L-cysteine, the S-trityl
protecting group was attached in the first step (4). To
build the aminothiazole group, different methods were
tried. Originally, we planned to transform the methyl
ester derivative of compound 4 into the corresponding
thiourea, which would serve as the starting material
for a later Hantzsch type reaction to form the corre-
sponding aminothiazole. However, we were unsuccessful
with this approach. Alternatively, a more simple and
direct approach was successfully applied. Compound 4
reacts with ω-thiocyanato-4-nitroacetophenone (5) in
ethanol at 50 °C with good conversion to the aminothia-
zole derivative 6, which could be further used without
purification. It is especially noteworthy that this method
tolerates the presence of a free carboxylic acid group.
Compound 5 was easily prepared by treating ω-bromo-
4-nitroacetophenone with sodium thiocyanate (Scheme
3). Alternatively, 5 can be generated in situ and directly
converted to the aminothiazole derivative in the same
pot.9
Our initial goal was the synthesis of known and new
seco-cyclothialidine derivatives to carefully study their
biological profile, especially the activity in animal
models. Since the previously described seco-cyclothia-
lidine synthesis is relatively long and complicated, we
also explored a shorter and more flexible synthetic
pathway. As the starting point for our own investiga-
tions, we focused on varying the carboxylic acid part of
the cysteine core of the seco-cyclothialidines. In Ro-61-
6653, this part is structurally represented by an oxa-
diazole moiety. We speculated that this portion of the
molecule plays an important role for its penetration
properties. On the basis of our speculation, we decided
to synthesize new analogues, including the simplest
case, the corresponding methyl ester, and a derivative
containing a dioxazine ester mimic.8
In the next step of the sequence, the dioxazine moiety
was formed. When we applied the classical way of
introducing this group, reaction of the methyl ester
derivative of 6 with hydroxylamine and subsequent
basic cyclization with dibromoethane, we observed only
decomposition.10 Therefore, we switched to a different
strategy using aminoglycol 7 as building block.11 Com-
pound 7 was attached to the carboxylic acid residue of
6 under standard peptide coupling conditions (EDC,
HOBT). Subsequent cyclization of 8 to 9 was achieved
by using the Burgess reagent. This very mild method
has been widely used for cyclodehydration reactions,
especially for the formation of five-membered rings.12
Resu lts a n d Discu ssion
Ch em istr y. seco-Cyclothialidines are trifunctional-
ized cysteines. According to a known synthesis, Ro-61-
6653 is obtained in a 10-step synthesis starting with
the amino acid cystine (Scheme 1).7a The initial carboxy
functionalization is followed by modification of the side
chain thio group, and the amino group is transformed
into the corresponding aminothiazole in the last part
of the sequence.
In the last part of the synthesis, the S-trityl group
was cleaved (10) and the highly substituted aromatic
portion 1113 was attached via reductive thiolation
reaction, i.e. reaction of the thiol function with the
Since the dioxazine moiety is chemically more labile
than the oxadiazole residue, we explored a synthetic
Sch em e 1

seco-Cyclothialidines
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4 621
Sch em e 2
Sch em e 3
For the synthesis of the methyl ester derivative 22
(BAY 50-7950), S-trityl-L-cysteine (4) was converted to
the methyl ester 20 and subsequently transformed into
the corresponding aminothiazole derivative 21 (Scheme
5). The further course of the synthesis was carried out
analogously as described for the preparation of 13 or
19.
aldehyde group of the aromatic moiety.7,14 Subsequent
silyl deprotection of compound 12 led to the final product
13 (BAY 50-7952). We also prepared the racemate 14
of compound 13. This provided us with the reference
compound needed for chiral HPLC to determine whether
racemization had occurred in the course of the synthesis
of 13. In this determination, we found that the product
13 still contained 90% of the (R)-enantiomer originally
derived from L-cysteine. We also separated the racemate
14 via preparative HPLC to obtain the pure (S)-
enantiomer of 13, BAY 51-4137 (15), and to compare
their antibacterial activities.
Compound Ro-61-6653 (19) was also synthesized via
the improved synthesis, as shown in Scheme 4. Com-
pound 6 was treated with N-hydroxyacetamidine hy-
drochloride15 (16) to form 17. Cyclization of 17 with
Burgess reagent led to compound 18 which was further
converted to the final product Ro-61-6653 (19) in an
analogous manner as in the case of 13.
The sequences developed and described in this report
allow a rapid and flexible access to new cyclothialidine
derivatives. It is especially noteworthy that the synthe-
sis does not require amino and carboxyl protecting
groups, in particular, thanks to a very efficient forma-
tion of the aminothiazole portion.
An tiba cter ia l Activity. (a ) In h ibitor y Effects on
DNA Gyr a se a n d Oth er DNA Top oisom er a ses. The
activity of BAY 50-7952 (13), the methyl ester derivative
BAY 50-7950 (22), and Ro-61-6653 toward the DNA
gyrase target (Micrococcus luteus) (Figure 1) and various
other DNA topoisomerases is shown in Table 2. In the
case of bacterial DNA gyrase, the IC50 values of the (R)-
enantiomers 13, 19, and 22 are all similar and indicate
potent inhibition. For comparison, Novobiocin has an
IC50 of 0.1 µg/mL in the present assay. We were also
interested to find out if the target activity is restricted
Sch em e 4

622 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4
Rudolph et al.
Sch em e 5
The inferior activity of the methyl ester derivative 22
is likely to be caused by poorer penetration of this
compound through the bacterial membrane. As we
speculated earlier, the choice of appropriate ester mim-
ics such as the dioxazine moiety in 13 or the oxadiazole
moiety in 19 has a strong influence on the transport
properties of the seco-cyclothialidines. As expected from
the target tests, no antibacterial activity was observed
for the (S)-enantiomer 15. For compounds 13, 19, and
22, we also carried out MIC determinations in the
presence of blood (Table 3). The antibacterial activity
of all compounds decreased significantly, indicating
strong serum binding.
(c) Th er a p eu tic Effica cy in th e Mu r in e In fection
Mod el. Encouraged by the high target activities and
MIC values, we also studied the in vivo efficacy of
compounds 13, 19, and 22. For this investigation, we
used a murine sepsis model with Staphylococcus aureus
133 as the infecting organism. Compounds were applied
intraperitoneally with 100 mg/kg 30 min after challenge.
As shown in Figure 2, administration of the test
compounds leads in all cases to prolongation of survival
in comparison to an untreated control group. Compound
19 (Ro-61-6653) is more active than compounds 22 and
13. However, in comparison to marketed antibiotics, all
compounds have only minor therapeutic efficacy. As
indicated by the in vitro studies in the presence of blood,
we speculate that a high level of serum binding may
contribute to the low in vivo efficacy. To confirm this
hypothesis, we determined the log P value of compound
13 (BAY 50-7952). We found a value of 3.1 which clearly
demonstrates the lipophilic nature of these compounds.
F igu r e 1. Gel photographs of reactions with M. luteus DNA
gyrase: rel, relaxed DNA; sc, supercoiled DNA; lane 1, 100
ng relaxed DNA only; lanes 2-12, relaxed DNA and DNA
gyrase; lanes 3-12, 1:2 dilutions of test compound starting
from 1.25 mg/L in lane 3, except for BAY 51-4137, where test
compound concentrations are indicated above the gel picture.
only to the (R)-enantiomers, as already known for the
cyclothialidines.6b,c Therefore, as a representative of the
(S)-enantiomers, BAY 51-4137 (15), the enantiomer of
BAY 50-7952, was tested and found to be only weakly
active, as expected.
The active compounds inhibit DNA gyrase from
Gram-positive M. luteus and from Gram-negative E. coli
equally well. In contrast, the three active compounds
inhibit human type II DNA topoisomerase only at very
high concentrations and are essentially inactive against
eukaryotic type I DNA topoisomerases. In addition, only
very weak activity is observed against E. coli type I DNA
topoisomerases.
(b) Min im a l In h ibitor y Con cen tr a tion s for Va r i-
ou s Ba cter ia . As shown in Table 3, the dioxazine
compound BAY 50-7952 (13) and Ro-61-6653 (19) both
exhibit excellent activity against Gram-positive strains.
No activity was observed against Gram-negative strains.
Su m m a r y of th e Biologica l Resu lts a n d
Con clu sion
The seco-cyclothialidines derivatives described in this
report show strong and highly selective target activity
toward the bacterial DNA gyrase B subunit and exert
high inhibitory activity against Gram-positive bacteria.
No substantial activity was observed against bacterial
topoisomerase I or human topoisomerases I and II. We
also confirmed that only the (R)-enantiomers exhibit
activity, whereas the corresponding (S)-enantiomers are
only weakly active in the target and completely inactive
in the whole cell assay. Among the three compounds

seco-Cyclothialidines
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4 623
Ta ble 2. Inhibitory Effects of Selected seco-Cyclothialidine Derivatives Against Bacterial DNA Gyrase
Ta ble 3. In Vitro Antibacterial Activities of Selected
seco-Cyclothialidine Derivativesa
MIC (µg/mL)
strain
13
15
19
22
S. epidermidis 193
S. ha¨molyticus 146
S. saprophyticus 2916 e0.12 (16)
S. aureus 133
S. aureus 48 N
e0.12 (8)
e0.12 (16)
>64 e0.12 (8)
0.5 (>64)
1 (>64)
1 (>64)
1 (>64)
1 (>64)
1 (>64)
2 (>64)
1 (>64)
1 (>64)
1 (>64)
1 (>64)
1 (>64)
2 (>64)
2 (>64)
2 (>64)
2 (>64)
1 (>64)
>64
>64
>64
>64
>64
>64
0.25 (16)
0.25 (16)
0.25 (16)
0.25 (16)
0.25 (16)
0.25 (16)
0.25 (16)
0.25 (16)
0.25 (16)
0.25 (16)
e0.12 (16)
0.25 (16)
0.25 (32)
0.25 (16)
0.25 (32)
0.5 (16)
0.5 (64)
0.5 (64)
0.5 (64)
0.5 (64)
>64 (>64) >64
>64 (>64) >64
>64 (>64) >64
>64 (>64) >64
>64 (>64) >64
S. aureus 9TV
S. aureus 44 (25508)
S. aureus 25701
S. aureus 25470
S. pyogenes Wacker
S. pyogenes 4851
S. pyogenes 4333
S. pyogenes 7029
E. faecium L4001
E. faecium 27266
E. faecalis 27251
E. faecalis 27253
P. vulgaris 1017
P. mirabilis 1235
K. pneumoniae 8085
K. pneumoniae 63
E. coli Neumann
>64 e0.12 (16)
>64
>64
>64
0.25 (16)
0.25 (32)
0.25 (16)
64 e0.12 (32)
>64
0.5 (16)
0.5 (32)
0.5 (32)
0.5 (32)
0.25 (64)
>64
>64
>64
>64
F igu r e 2. Therapeutic efficacy of selected seco-cyclothialidine
derivatives. Murine sepsis model with S. aureus 133 ip therapy
with 100 mg/kg, 30 min post-infection.
>64 (>64) >64 (>64)
>64 (>64) >64 (>64)
>64 (>64) >64 (>64)
>64 (>64) >64 (>64)
>64 (>64) >64 (>64)
Exp er im en ta l Section
Ch em istr y. Gen er a l. Melting points were obtained on a
Gallenkamp melting point apparatus and are uncorrected.
Optical rotations were measured on a Perkin-Elmer 241
polarimeter. 1H NMR spectra were recorded on Bruker DPX400
spectrometers in CDCl3 or DMSO-d6 using the solvent signals
as internal references. The chemical shifts are reported in δ
(ppm) relative to TMS and the coupling constants (J ) are given
in hertz (Hz). Electrospray-high-resolution mass spectrometry
(ESI-HRMS) was performed on an Finnigan-MAT 900 double
focusing mass spectrometer. The ESI source was operated in
the positive ion mode, spray needle voltage 3.5 kV, nitrogen
sheath gas pressure 500 kPa, and the heated metal capillary
held at 250 °C.
All reactions were monitored by analytical TLC on aluminum-
backed TLC plates coated with silica gel (Merck Si 60 F254),
and visualization of spots was accomplished by UV detection
at 254 nm. Column chromatography was carried out using
Amicon silica gel (30-70 µm). Preparative HPLC racemate
separations were performed on a Gilson-Abimed M 305 system
(flow: 30 mL/min) with a stationary silica gel phase based on
poly(N-methacryloyl-L-isoleucine-3-pentylamide) (10 µm) and
a
MIC values in the presence of 10% horse blood are given in
parentheses.
investigated, the nature of the residue at the carboxylic
acid moiety of the cysteine core unit does not have a
strong influence on the target activity. However, a
significant activity difference can be observed in the
whole cell bacterial test. The derivatives bearing a
dioxazine or oxadiazole moiety are strikingly more
active than the corresponding methyl ester derivative,
suggesting a pivotal role of this portion for the transport
properties of the seco-cyclothialidines. Despite the high
antibacterial activity of the compounds investigated,
only weak activity was found in the murine infection
model. Correspondingly, we found high levels of serum
binding which is likely to be caused by the lipophilic
nature of the compounds.

624 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4
Rudolph et al.
ethyl acetate as eluent (UV detection: 280 nm). Analytical
HPLC racemate separations were performed on a J asco system
(flow: 1 mL/min) with a Kromasil CHI-TBB stationary phase
(5 µm) and n-heptane/THF 1:1 (v/v) as eluent (UV detection:
280 nm).
S-Tr ityl-L-cystein e (4). L-Cysteine hydrochloride (10.0 g,
63.4 mmol) and trityl chloride (27.0 g, 96.9 mmol) were stirred
in 40 mL DMF for 2 days at room temperature. A 10% sodium
acetate solution (350 mL) was then added, and the precipitate
was filtered and washed with distilled water. Afterward, the
residue was stirred in acetone at 50 °C for 30 min and filtered
after cooling. The residue was washed with little acetone and
diethyl ether. After drying in vacuo, 20.5 g (89%) of 4 was
obtained as a white powder: mp 195 °C dec; 1H NMR (DMSO-
d6) δ 2.49, (dd, J ) 9 Hz, 12 Hz, 1H, CH2), 2.58 (dd, J ) 4.4
Hz, 12 Hz, 1H, CH2), 2.91 (m, 1H, CH), 7.22-7.36 (m, 15H,
arom H).
(dd, J ) 6 Hz, 12 Hz, 1H, CH2), 2.83 (dd, J ) 6 Hz, 12 Hz, 1H,
CH2), 4.05 (m, 2H, dioxazine-CH2), 4.31 (m, 1H, CH), 4.32 (m,
2H, dioxazine-CH2), 5.42 (d, J ) 8 Hz, 1H, NH), 6.93 (s, 1H,
thiazole-H), 7.18-7.29, 7.39-7.44 (m, 15H, trityl-H), 7.89 (d,
J ) 9 Hz, 2H, arom H), 8.21 (d, J ) 9 Hz, 2H, arom H); 13C
NMR (CDCl3) δ 34.1, 50.9, 63.7, 67.0, 105.8, 124.0, 126.5, 126.9,
128.0, 129.6, 140.6, 144.4, 146.8, 148.9, 154.9, 167.0; MS (ESI,
+) m/z 243 (100%), 609 (100%, [M + H]+).
(2R)-2-(5,6-Dih yd r o-1,4,2-d ioxa zin -3-yl)-2-{[4-(4-n itr o-
p h en yl)-1,3-th ia zol-2-yl]a m in o}eth a n eth iol (10). In an
inert atmosphere, compound 9 (3.74 g, 4.93 mmol) was
dissolved in 35 mL CH2Cl2. Afterward, triethylsilane (2.6 mL,
16.2 mmol) and trifluoroacetic acid (37.4 mL) were added
subsequently. After 2 h, the solvent was removed in vacuo,
and the crude product was absorbed onto silica gel. The
product was purified via column chromatography using an
EtOAc/cyclohexane 1:2 w 2:3 gradient (1% triethylamine was
added to each solvent). The product was obtained as a bright
yellow powder: yield 1.37 g (76%); mp 189 °C; 1H NMR
(DMSO-d6) δ 3.03 (m, 1H, CH2), 3.12 (m, 1H, CH2), 4.12 (m,
2H, dioxazine-CH2), 4.41 (m, 2H, dioxazine-CH2), 4.77 (m, 1H,
CH), 6.96 (d, J ) 8 Hz, 1H, NH), 7.00 (s, 1H, thiazole-H), 7.98
ω-Th iocya n o-4-n itr oa cetop h en on e (5). ω-Bromo-4-ni-
troacetophenone (30.0 g, 123.2 mmol) and sodium thiocyanate
(11.0 g, 135.8 mmol) were dissolved in 300 mL acetonitrile and
stirred for 3 h at 45 °C. After removal of the solvent, 50 mL
distilled water was added, and the mixture was extracted three
times with ethyl acetate. The organic layers were dried with
Na2SO4, and the solvent was removed in vacuo. The product
was obtained as an ocre, finely crystalline powder in high
(d, J ) 9 Hz, 2H, arom H), 8.22 (d, J ) 9 Hz, 2H, arom H); 13
C
NMR (DMSO-d6) δ 25.6, 57.9, 63.3, 64.5, 107.0, 123.9, 126.3,
140.6, 146.0, 147.4, 154.7, 167.2; MS (ESI, +) m/z 367 [M +
H]+.
1
purity: yield 27.0 g (99%); mp 118.5 °C; H NMR (DMSO-d6)
δ 3.35 (s, 2H, CH2), 8.25 (d, J ) 13 Hz, 2H, arom H), 8.39 (d,
Meth yl 2-Br om o-6-{[((2R)-2-(5,6-dih ydr o-1,4,2-dioxazin -
3-yl)-2-{[4-(4-n itr op h en yl)-1,3-th ia zol-2-yl]a m in o}eth yl)-
su lfa n yl]m et h yl}-5-{[d im et h yl(1,1,2-t r im et h ylp r op yl)-
silyl]oxy}-3-m eth oxyben zoa te (12). In an inert atmosphere,
trifluoroacetic acid (1.5 mL) was cooled to 0 °C and 2-bromo-
5-dimethylthexylsilyloxy-6-formyl-3-methoxybenzoic acid meth-
J ) 13 Hz, 2H, arom H).
N-[4-(4-Nitr oph en yl)th iazol-2-yl]-S-tr ityl-L-cystein e (6).
Compound 4 (15.0 g, 41.3 mmol) was dissolved in 400 mL
ethanol, and compound 5 (10.1 g, 45.4 mmol) was subsequently
added. After stirring for 6 h at 55 °C, the solvent was removed
in vacuo. The crude material (yellow powder) was used without
further purification. NMR data of a purified sample: 1H NMR
(DMSO-d6) δ 2.59 (m, 1H, CH2), 2.68 (m, 1H, CH2), 3.99 (m,
1H, CH), 6.98 (s, 1H, thiazole-H), 7.14-7.34 (m, 15H, trityl-
H), 8.0 (d, J ) 9 Hz, 2H, arom H), 8.22 (d, J ) 9 Hz, 2H, arom
H).
yl ester (11;13
0.385 g, 0.955 mmol) was added. A suspension
of compound 10 (0.35 g, 0.955 mmol) and triethylsilane (0.14
g, 1.2 mmol) in 2 mL CH2Cl2 was added. The mixture was
stirred for 18 h at 0 °C. After removal of the solvent, ethyl
acetate was added to the mixture, and it was washed succes-
sively with water, saturated NaHCO3 solution, and brine. The
organic layer was dried with Na2SO4 and purified via column
chromatography using an EtOAc/cyclohexane 1:2 w 2:1 gradi-
ent. The product 12 was obtained in a yield of 0.35 g (47%) as
a dark-yellow powder: mp 63 °C (phase change); 1H NMR
(CDCl3) δ 0.25 (s, 6H, SiCH3), 0.91 (d, J ) 6.9 Hz, 6H, TDS-
CH3), 0.97 (s, 6H, TDS-CH3), 1.70 (m, 1H, TDS-H), 3.07 (d,
J ) 6 Hz, 2H, CH2), 3.68 (d, J ) 13 Hz, 1H, CH2), 3.76 (s, 3H,
OCH3), 3.82 (d, J ) 13 Hz, 1H, CH2), 3.96 (s, 3H, OCH3), 4.08
(m, 2H, dioxazine-CH2), 4.34 (m, 2H, dioxazine-CH2), 4.60 (m,
1H, CH), 5.80 (br, 1H, NH), 6.33 (s, 1H, arom H), 6.92 (s, 1H,
thiazole-H), 7.94 (d, J ) 9 Hz, 2H, arom H), 8.22 (d, J ) 9 Hz,
2H, arom H); 13C NMR (CDCl3) δ 0.0, 20.5, 22.1, 27.3, 35.8,
36.3, 54.7, 57.5, 58.3, 65.7, 66.8, 103.0, 106.2, 107.9, 121.9,
126.0, 128.4, 139.5, 142.7, 148.8, 150.7, 156.1, 157.0, 157.5,
169.2, 170.0; MS (ESI, +) m/z 782 [M + H]+.
Meth yl 2-Br om o-6-{[((2R)-2-(5,6-dih ydr o-1,4,2-dioxazin -
3-yl)-2-{[4-(4-n itr op h en yl)-1,3-th ia zol-2-yl]a m in o}eth yl)-
su lfan yl]m eth yl}-5-h ydr oxy-3-m eth oxyben zoate (BAY 50-
7952, 13). Compound 12 (0.6 g, 0.767 mmol) was dissolved in
20 mL methanol. Ammonium fluoride (280 mg, 7.67 mmol, 10
equiv) was then added. After addition of 100 mL ethyl acetate,
the reaction mixture was washed with 60 mL H2O. The
aqueous layer was extracted once more with ethyl acetate (50
mL). The combined organic layers were washed with 60 mL
brine and dried with Na2SO4. After removal of the solvent in
vacuo, the residue was dissolved in ethyl acetate and precipi-
tated by the addition of hexane. The precipitate was filtered
and washed with hexane. Compound 13 was obtained as bright
yellow powder: yield 0.41 g (82.8%); mp 93 °C (phase change);
1H NMR (CDCl3) δ 2.99 (dd, J ) 14 Hz, 6.3 Hz, 1H, CH2), 3.08
(dd, J ) 14 Hz, 6.3 Hz, 1H, CH2), 3.74 (s, 3H, OCH3), 3.76 (d,
J ) 14 Hz, 1H, CH2), 3.83 (d, J ) 14 Hz, 1H, CH2), 3.95 (s,
3H, OCH3), 4.10 (m, 2H, dioxazine-CH2), 4.39 (m, 2H, diox-
azine-CH2), 4.75 (m (br), 1H, CH), 5.96 (br, 1H, NH), 6.44 (s,
1H, arom H), 6.93 (s, 1H, thiazole-H), 7.90 (d, J ) 9 Hz, 2H,
arom H), 8.21 (d, J ) 9 Hz, 2H, arom H); 13C NMR (CDCl3) δ
N1-(2-Hydr oxyeth oxy)-N2-[4-(4-n itr oph en yl)-1,3-th iazol-
2-yl]-S-tr ityl-L-cystein a m id e (8). The crude product 6 (10.0
g, ∼17.6 mmol) was dissolved in 50 mL DMF and cooled to
-30 °C. HOBT (4.56 g, 30 mmol) and EDC (5.70 g, 30 mmol)
were then added. After 15 min of stirring, a solution of
aminoglycol (1.93 g, 25 mmol) in 50 mL DMF was added. The
reaction mixture was allowed to warm to room temperature
and stirred for 14 h. Ethyl acetate (250 mL) was added, and
the solution was washed with water (200 mL). The aqueous
layer was extracted again with ethyl acetate (150 mL). The
combined organic layers were successively extracted with 5%
citric acid solution, 10% NaHCO3 solution and twice with
brine. The organic layer was dried with Na2SO4, and the
solvent was removed in vacuo. The crude material (yellow
powder) was used without further purification. Analytical data
of a purified sample: mp 100 °C (phase change); 1H NMR
(CDCl3) δ 2.82 (dd, J ) 5.5 Hz, 13.6 Hz, 1H, CH2), 2.96 (dd, J
) 5.5 Hz, 13.6 Hz, 1H, CH2), 3.63 (m, 2H, CH2), 3.88 (m, 2H,
CH2), 3.94 (dd, J ) 5.5 Hz, 6 Hz, 1H, CH), 4.88 (d, J ) 6 Hz,
1H, NH), 6.95 (s, 1H, thiazole-H), 7.21-7.34, 7.45-7.50 (m,
15H, trityl-H), 7.87 (d, J ) 9 Hz, 2H, arom H), 8.23 (d, J ) 9
Hz, 2H, arom H), 9.11 (s, 1H, NH); 13C NMR (CDCl3) δ 32.8,
55.7, 59.2, 67.6, 78.5, 106.5, 124.1, 126.3, 127.1, 128.2, 129.5,
140.0, 144.1, 146.9, 148.5, 167.0, 170.5; MS (ESI, +) m/z 627
[M + H]+.
N-[(1R)-1-(5,6-Dih yd r o-1,4,2-d ioxa zin -3-yl)-2-(tr itylsu l-
fa n yl)eth yl]-N-[4-(4-n itr op h en yl)-1,3-th ia zol-2-yl]a m in e
(9). The crude product 8 (5.0 g, ∼8 mmol) was dissolved in
200 mL THF, and Burgess reagent [(methoxycarbonylsulfa-
moyl)triethylammonium N-betaine] (2.1 g, 8.8 mmol) was
added. After refluxing for 3 h, the solvent was removed in
vacuo. The crude product was absorbed onto silica gel and
purified via column chromatography using an EtOAc/cyclo-
hexane 2:1 mixture as the eluent. The product was obtained
as a dark yellow powder: yield 2.66 g (total yield from three
steps: 44%); mp 77 °C (phase change); 1H NMR (CDCl3) δ 2.73

seco-Cyclothialidines
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4 625
29.2, 33.2, 52.8, 55.9, 56.3, 63.8, 65.0, 99.7, 102.4, 106.0, 113.6,
124.0, 126.5, 137.6, 140.4, 146.8, 148.8, 155.5, 155.9, 156.2,
156.3, 166.7, 167.1, 168.1, 178.0; MS (ESI, +) m/z 638 [M +
H]+. Anal. (C24H22BrN5O7S2) C, H, N.
167.4, 167.9; MS (ESI, +) m/z 640 [M + H]+. Anal. (C24H23
-
S-Tr ityl-L-cystein e Meth yl Ester Hyd r och lor id e (20).
S-Trityl-L-cysteine (50 g, 0.138 mol) was added to 1000 mL
methanol to form a suspension. After cooling to 0-5 °C, thionyl
chloride (75 mL, 1.03 mmol) was added, and the reaction
mixture was allowed to warm to room temperature. The
mixture was then refluxed for 5 h, cooled, and evaporated in
vacuo. The residue was dried for 2 d at 30 °C in high vacuum.
The product 20 was obtained as a white, finely crystalline
solid: yield 55.52 g (97.2%); mp 78 °C (phase change); 1H NMR
(DMSO-d6) δ 2.60 (m, 2H, CH2), 3.71 (s, 3H, CO2CH3), 3.85
(dd, J ) 6 Hz, 6 Hz, 1H, CH), 7.28-7.41 (m, 15H, trityl-H),
8.50 (br, 3H, NH3+).
N-[4-(4-Nitrophenyl)thiazol-2-yl]-S-trityl-L-cysteine Meth-
yl Ester (21). Compound 20 (2.67 g, 6.46 mmol) was dissolved
in 50 mL ethanol, and triethylamine (0.65 g, 6.46 mmol, 0.9
mL) was added. Afterward, compound 5 (1.44 g, 6.46 mmol)
was added, and the mixture was stirred at 50 °C for 16 h. The
solvent was removed in vacuo, and the residue was dissolved
in ethyl acetate and subsequently washed with water. The
aqueous layer was extracted three times with ethyl acetate,
and the combined organic layers were dried with Na2SO4. The
product 21 was obtained as a golden yellow solid (3.6 g, 96%,
purity 90-95%). It was used without further purification: mp
131-133 °C; 1H NMR (400 MHz, DMSO-d6) δ 2.60 (m, 1H,
CH2), 2.71 (m, 1H, CH2), 3.60 (s, 3H, OCH3), 4.32 (m, 1H, CH),
7.20-7.33 (m, 15H, trityl-H), 7.52 (s, 1H, thiazole-H), 7.99 (d,
J ) 8.9 Hz, 2H, ArH), 8.26 (d, J ) 8.9 Hz, 2H, ArH), 8.36 (d,
J ) 7.8 Hz, 1H, NH); 13C NMR (100.4 MHz, CDCl3) δ 33.5,
52.8, 56.3, 67.0, 105.8, 124.0, 126.4, 126.9, 128.0, 129.5, 140.6,
144.3, 146.8, 148.9, 166.6, 171.3.
BrN4O8S2) C, H, N.
Meth yl 2-Br om o-6-{[((2S)-2-(5,6-dih ydr o-1,4,2-dioxazin -
3-yl)-2-{[4-(4-n itr op h en yl)-1,3-th ia zol-2-yl]a m in o}eth yl)-
su lfan yl]m eth yl}-5-h ydr oxy-3-m eth oxyben zoate (BAY 51-
4137, 15, en a n tiom er of 13). Starting from DL-cysteine, the
corresponding racemate 14 of 13 was synthesized in the same
manner and in similar yields as 13. The racemic mixture was
separated via chiral HPLC (conditions given in the general
experimental part). Under preparative HPLC conditions, the
(R)-enantiomer 13 and the (S)-enantiomer 15 have retention
times of 18.0 and 23.5 min, respectively. Under analytical
conditions, the (R)-enantiomer 13 and the (S)-enantiomer 15
have retention times of 7.67 and 8.42 min, respectively.
Compound 15 was obtained in an optical purity of 99.5%
according to HPLC analysis.
N1-[(1Z)-N-Hydr oxyeth an im idoyl]-N2-[4-(4-n itr oph en yl)-
1,3-th ia zol-2-yl]-S-tr ityl-L-cystein a m id e (17). The crude
product 6 (13.4 g, 23.0 mmol) was dissolved in 120 mL DMF,
and the solution was cooled to -30 °C. HOBT (5.95 g, 39.1
mmol) and EDC (7.43 g, 39.1 mmol) were then added. After
15 min, a solution of N-hydroxyacetamidine hydrochloride15
(3.6 g, 32.6 mmol) and triethylamine (3.3 g, 32.6 mmol, 4.6
mL) in 120 mL DMF was slowly added. The reaction mixture
was allowed to warm to room temperature and stirred for 14
h. Water (200 mL) was added subsequently, and the mixture
was extracted twice with 200 mL ethyl acetate. The combined
organic layers were successively washed 5% citric acid, 10%
NaHCO3 solution and twice with brine. The organic layer was
dried with Na2SO4, and the solvent was removed in vacuo.
Crude yield: 13.3 g. The crude material was used without
further purification. Analytical data of a purified sample: mp
The further conversion of 21 to 22 was carried out in an
analogous manner and in similar yields as described for the
preparation of 13 from 9.
1
100 °C (phase change); H NMR (CDCl3) δ 1.88 (s, 3H, CH3),
2.71 (dd, J ) 5.5 Hz, 13.5 Hz, 1H, CH2), 2.84 (dd, J ) 5.5 Hz,
13.5 Hz, 1H, CH2), 4.50 (m, 1H, CH), 7.00 (s, 1H, thiazole-H),
7.16-7.42 (m, trityl-H), 7.92 (d, J ) 9 Hz, 2H, arom H), 8.19
(d, J ) 9 Hz, 2H, arom H); 13C NMR (CDCl3) δ 16.8, 33.7, 56.0,
67.3, 106.1, 124.0, 126.5, 127.0, 128.1, 129.5, 140.3, 144.2,
146.9, 148.6, 156.8, 167.0, 168.4.
BAY 50-7950 (22). Bright yellow powder: mp 67 °C (phase
change); H NMR (CDCl3) δ 3.04 (dd, J ) 14 Hz, 6 Hz, 1H,
1
CH2), 3.26 (dd, J ) 14 Hz, 4.7 Hz, 1H, CH2), 3.72 (d, J ) 14
Hz, 1H, CH2), 3.77 (s, 3H, OCH3), 3.80 (d, J ) 14 Hz, 1H, CH2),
3.81 (s, 3H, OCH3), 3.97 (s, 3H, OCH3), 4.86 (br, 1H, CH), 5.97
(br, 1H, NH), 6.42 (s, 1H, arom H), 6.94 (s, 1H, thiazole-H),
7.90 (d, J ) 9 Hz, 2H, arom H), 8.23 (d, J ) 9 Hz, 2H, arom
H); 13C NMR (CDCl3) δ 29.7, 32.8, 52.9, 53.0, 56.4, 57.4, 99.8,
102.3, 106.1, 113.2, 124.0, 126.5, 137.6, 140.3, 140.5, 146.8,
148.8, 155.8, 156.3, 167.1, 168.0, 171.5; MS (ESI, +) m/z 614
[M + H]+. Anal. (C23H22BrN3O8S2) C, H, N.
N-[(1R)-1-(3-Meth yl-1,2,4-oxa d ia zol-5-yl)-2-(tr itylsu lfa -
n yl)eth yl]-N-[4-(4-n itr oph en yl)-1,3-th iazol-2-yl]am in e (18).
Compound 17 (4 g, 6.41 mmol) was dissolved in THF (160 mL)
and treated with Burgess reagent [(methoxycarbonylsulfa-
moyl)triethylammonium N-betaine] (1.68 g, 7.05 mmol). After
refluxing for 3 h, the mixture was cooled to room temperature,
and the solvent was removed in vacuo. The crude product was
absorbed onto silica gel and purified via column chromatog-
raphy using EtOAc/cyclohexane 1:3. The product 18 was
obtained as a bright yellow powder: yield 0.97 g (25% over
three steps starting from 4); mp 90 °C (phase change); 1H NMR
(CDCl3) δ 2.38 (s, 3H, oxadiazole-CH3), 2.98 (m, 2H, CH2), 4.95
(m, 1H, CH), 5.36 (br, 1H, NH), 6.93 (s, 1H, thiazole-H), 7.18-
7.41, 7.40-7.44 (m, 15H, trityl-H), 7.82 (d, J ) 9 Hz, 2H, arom
H), 8.20 (d, J ) 9 Hz, 2H, arom H); 13C NMR (CDCl3) δ 11.6,
34.9, 51.1, 67.6, 106.3, 124.0, 126.4, 127.1, 128.0, 129.4, 140.3,
144.0, 146.8, 148.8, 166.1, 167.2, 177.9; MS (ESI, +) m/z 606
[M + H]+.
Biology. 1. DNA Top oisom er a se Tests. M. luteus DNA
gyrase and calf thymus DNA topoisomerase I were purchased
from Life Technologies (Karlsruhe, Germany), E. coli DNA
gyrase from Lucent Ltd. (Leicester, U.K.), and human DNA
topoisomerases I and II from TopoGEN (Columbus, OH). The
E. coli DNA topoisomerase I gene was cloned in the pET28a
vector (Novagen), and the resulting N-terminally His-tagged
enzyme was overproduced in E. coli BL21(DE3)pLys and
purified on Ni-NTA support (Qiagen). All reactions were
performed in buffers as suggested by the respective manufac-
turers. Supercoiled pBR322 plasmid DNA (Life Technologies)
was used as substrate for the enzymatic reaction. For DNA
gyrase tests, the plasmid was first relaxed using calf thymus
DNA topoisomerase I and then purified using standard
procedures.16
Test compounds were diluted to the desired concentrations
in 2 µL DMSO. Topoisomerases (1 unit) and 100 ng substrate
DNA were added in the respective reaction buffers to a final
volume of 20 µl. DNA gyrase and E. coli DNA topoisomerase
I reactions were incubated at 37 °C for 60 min, the eukaryotic
enzyme reactions were incubated for 30 min. Reactions were
stopped by addition of 3 µL stop solution (0.3% SDS, 0.3%
bromophenyl blue, 40% sucrose). DNA topoisomers were
separated by electrophoresis on 1% agarose gels at 45 V for
15 h. Gels were then stained in 1 µg/mL ethidium bromide for
10 min followed by 20 min destaining in water. The gels were
photographed under UV light and visually inspected and
scored for IC50 values.
The further conversion of 18 to 19 was carried out in an
analogous manner and in similar yields as described for the
preparation of 13 from 9.
Meth yl 2-Br om o-5-h yd r oxy-3-m eth oxy-6-{[((2R)-2-(3-
m eth yl-1,2,4-oxa d ia zol-5-yl)-2-{[4-(4-n itr op h en yl)-1,3-th i-
a zol-2-yl]a m in o}et h yl)su lfa n yl]m et h yl}b en zoa t e (BAY
50-7954, 19). Bright yellow powder: mp 115 °C; 1H NMR
(CDCl3) δ 2.42 (s, 3H, oxadiazole-CH3), 3.12 (dd, J ) 14.4 Hz,
6.9 Hz, 1H, CH2), 3.25 (dd, J ) 14.4 Hz, 5.2 Hz, 1H, CH2),
3.74 (d, J ) 4.1 Hz, 1H, CH2), 3.80 (s, 3H, OCH3), 3.83 (d, J )
4.1 Hz, 1H, CH2), 3.99 (s, 3H, OCH3), 5.59 (br, 1H, CH), 6.08
(br, 1H, NH), 6.47 (s, 1H, arom H), 6.97 (s, 1H, thiazole-H),
7.89 (d, J ) 9 Hz, 2H, arom H), 8.23 (d, J ) 9 Hz, 2H, arom
H); 13C NMR (CDCl3) δ 11.6, 29.4, 33.9, 52.0, 53.0, 56.4, 102.2,
106.5, 113.4, 124.1, 126.5, 137.6, 140.2, 146.9, 148.8, 155.7,

626 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4
Rudolph et al.
lidines revealed by X-ray crystallography. EMBO J . 1996, 15,
1412-1420. (d) Yamaji, K.; Masubuchi, M.; Kawahara, F.;
Nakamura, Y.; Nishio, A.; Matsukuma, S.; Fujimoro, M.; Na-
kada, N.; Watanabe, J .; Kamiyama, T. Cyclothialidine analogues,
novel DNA gyrase inhibitors. J . Antibiot. 1997, 50, 402-411.
(6) (a) Preparation of benzoxathiaazabicyclododecines as novel DNA
gyrase inhibitors. Hoffmann-La Roche, WO 9218490 A1, Apr 9,
1992. (b) Goetschi, E.; Angehrn, P.; Gmuender, H.; Hebeisen,
P.; Link, H.; Masciadri, R.; Nielsen, J .; Reindl, P.; Ricklin, F.
The DNA gyrase inhibitor cyclothialidine: progenitor of a new
class of antibacterial agents. Med. Chem.: Today Tomorrow,
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Blackwell: Oxford, U.K., 1997; pp 263-270. (c) Goetschi, E.;
Angehrn, P.; Gmuender, H.; Hebeisen, P.; Link, H.; Masciadri,
R.; Nielsen, J . Cyclothialidine and its congeners: a new class of
DNA gyrase inhibitors. Pharmacol. Ther. 1993, 60, 367-380.
(d) Goetschi, E.; J enny, C. J .; Reindl, P.; Ricklin, F. Total
synthesis of cyclothialidine. Helv. Chim. Acta 1996, 79, 2219-
2234.
(7) (a) Preparation of mono- and bicyclic DNA gyrase inhibitors.
Hoffmann-La Roche, EP 675122 A2, Oct 4, 1995. (b) Hebeisen,
P.; Angehrn P.; Gmuender, H.; Goetschi, E.; Link, H.; Luebbers,
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(8) See, for example: (a) Stetter, J .; Fuehrer, W.; Brandes, W.,
Hydroximic acid esters and their use as fungicides. Bayer AG,
EP56161 A2, J uly 21, 1982. (b) Heinemann, U.; Gayer, H.;
Gerdes, P.; Kru¨ger, B.-W.; Markert, R.; Seitz, T.; Gallenkamp,
B.; Stelzer, U.; Tiemann, R.; Mauler-Machnik, A.; Dutzmann,
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2. MIC Deter m in a tion (m in im a l in h ibitor y con cen tr a -
tion ). The MICs were determined by the agar dilution method
using IsoSensitest agar or Mueller Hinton agar supplemented
with 10% horseblood and IsoVitaleX. Overnight cultures were
grown in BHI broth (staphylococci, enterobacteria) or BHI
broth supplemented with 10% bovine serum (streptococci,
enterococci). After dilution (1:100), agar plates containing the
test compounds (concentration range: 0.125-64 µg/mL) were
inoculated with the test bacteria by a Denley inoculator. Plates
were incubated aerobically at 37 °C and read after ap-
proximately 20 h. The MIC was considered the first concentra-
tion with no visible growth.
3. System ic In fection w ith S. a u r eu s 133. Bacteria from
an overnight culture were grown to the logarithmic growth
phase in BHI broth. The culture was precipitated and washed
twice with phosphate-buffered saline. Bacteria were suspended
in phosphate-buffered saline containing 10% mucin to a cell
number of 4 × 106 cells/mL. Female CFW1 mice (20 g) were
infected with 0.25 mL of the bacterial suspension. The test
compounds were dosed ip at 30 min post-infection. The
survival in each treatment group was monitored for 6 days.
Ack n ow led gm en t. We thank W. Kreiss and B.
Gallenkamp for the complementing target tests and
supplying generous amounts of aminoglycol, respec-
tively. We are grateful to R. Grosser for the chiral HPLC
separations and to G. Thielking for the log P determina-
tions. Additionally, we thank D. Backhaus and A. Plant
for valuable discussions. Finally, the authors appreciate
the helpful comments and suggestions made by the
reviewers.
(10) As the main cleavage product 4-(4-nitrophenyl)-1,3-thiazol-2-
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(11) (a) U. Stelzer, personal communication. (b) Aminoglycol was
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sulfides from carbonyl compounds with thiols and triethylsilane.
Synthesis 1992, 465-466.
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