New Antibacterial Agents Derived from the DNA Gyrase Inhibitor Cyclothialidine

J . Med. Chem. 2004, 47, 1487-1513

1487

New An tiba cter ia l Agen ts Der ived fr om th e DNA Gyr a se In h ibitor

Cycloth ia lid in e

Peter Angehrn, Stefan Buchmann, Christoph Funk, Erwin Goetschi,* Hans Gmuender,^†Paul Hebeisen,

Dirk Kostrewa,^‡Helmut Link, Thomas Luebbers, Raffaello Masciadri, J oergen Nielsen, Peter Reindl,

Fabienne Ricklin, Anne Schmitt-Hoffmann,^§and Frank-Peter Theil

F. Hoffmann-La Roche Ltd., Pharmaceutical Division, Preclinical Research, CH-4070 Basel, Switzerland

Received September 5, 2003

Cyclothialidine (1, Ro 09-1437) is a potent DNA gyrase inhibitor that was isolated from

Streptomyces filipinensis NR0484 and is a member of a new family of natural products. It acts

by competitively inhibiting the ATPase activity exerted by the B subunit of DNA gyrase but

barely exhibits any growth inhibitory activity against intact bacterial cells, presumably due to

insufficient permeation of the cytoplasmic membrane. To explore the antibacterial potential

of 1, we developed a flexible synthetic route allowing for the systematic modification of its

structure. From a first set of analogues, structure-activity relationships (SAR) were established

for different substitution patterns, and the 14-hydroxylated, bicyclic core (X) of 1 seemed to be

the structural prerequisite for DNA gyrase inhibitory activity. The variation of the lactone

ring size, however, revealed that activity can be found among 11- to 16-membered lactones,

and even seco-analogues were shown to maintain some enzyme inhibitory properties, thereby

reducing the minimal structural requirements to a rather simple, hydroxylated benzyl sulfide

(XI). On the basis of these “minimal structures” a modification program afforded a number of

inhibitors that showed in vitro activity against Gram-positive bacteria. The best activities were

displayed by 14-membered lactones, and representatives of this subclass exhibit excellent and

broad in vitro antibacterial activity against Gram-positive pathogens, including Staphylococcus

aureus, Streptococcus pyogenes, and Enterococcus faecalis, and overcome resistance against

clinically used drugs. By improving the pharmacokinetic properties of the most active

compounds (94, 97), in particular by lowering their lipophilic properties, we were able to identify

congeners of cyclothialidine (1) that showed efficacy in vivo.

In tr od u ction

quinolones⁴and of the coumarin antibiotics⁵aroused a

general interest in inhibitors of this enzyme as potential

antibacterial drugs. DNA gyrase is an A₂B₂holoenzyme

The emergence of bacterial resistance to most of the

antibacterials used clinically represents a major chal-

lenge in today’s antibiotic research, outweighing the

task to develop drugs of higher potency, expanded

spectrum of activity, and improved safety profile. In

particular the spread of multiresistant Gram-positive

bacteria during the last 2 decades is of major concern

in the hospital environment, where the glycopeptides

vancomycin and teicoplanin are considered as the last-

resort drugs against methicillin-resistant Staphylococ-

cus aureus (MRSA).¹First reports on vancomycin-

resistant S. aureus,²however, represent a serious threat

to the therapy of nosocomial infections, which at present

are mainly caused by MRSA and other highly resistant

Gram-positive bacteria. Therefore, the search for new

structural entities and novel targets of attack as a

means to overcome bacterial resistance continues to be

an important research area.

Ch a r t 1

The identification of the bacterial type II topo-

isomerase DNA gyrase³as the biological target of the

that mediates various reactions essential for the bacte-

rial cell, e.g. the introduction of negative superhelical

turns into bacterias circular DNA.^6,7The quinolones, e.g.

ciprofloxacin (2), are DNA gyrase subunit A inhibitors

that directly interfere with the processes between DNA

and the gyrase enzyme.⁴This class of antibacterials has

gained a strong position in the therapy of bacterial

infections. Its use, however, might be hampered in the

future by resistance development and undesired side

* To whom correspondence should be addressed. Phone: +41 61 688

6801. Fax: +41 61 688 8714. E-mail: erwin.goetschi@roche.com.

^†Present address: GeneData AG, PO Box, CH-4016 Basel, Swit-

zerland.

^‡Present address: PSI Paul Scherrer Institut, CH-5232 Villigen,

Switzerland.

^§Present address: BASILEA Pharmaceutica Ltd., PO Box 3255-7

CH-4005 Basel, Switzerland.

Published on Web 02/05/2004

1488 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6

Angehrn et al.

effects.⁷The coumarin antibiotics, such as novobiocin

(3) and coumermycin, competitively inhibit the ATPase

activity conferred by the DNA gyrase B subunit and

thereby abolish the energy-dependent reactions cata-

lyzed by DNA gyrase.⁵Rapid emergence of resistance

as well as poor tolerability has restricted or even

prevented their clinical use.⁸Therefore, the B subunit

of DNA gyrase represents a presently unexploited target

for antibacterial attack.

Nalidixic acid,⁹the progenitor of the quinolones, as

well as the coumarin antibiotics,¹⁰had been discovered

by the traditional search method relying on the growth

inhibition of bacterial cultures. Target-based assays, e.g.

enzyme inhibition assays, have been used less fre-

quently for the identification of a new antibacterial

principle but are playing an increasingly important

role.¹¹They have the advantage of detecting a potential

antibacterial uncoupled from its ability to cross the

bacterial membrane. The possibility to study DNA

gyrase in vitro¹²provided a powerful tool in the search

for new inhibitors of this enzyme, and the discovery of

cyclothialidine (1, Ro 09-1437, Chart 1) represents an

early example of this target-specific drug-finding tech-

nique.^13-15

Cyclothialidine was found by screening microbial

broths for the in vitro inhibition of the supercoiling

activity of DNA gyrase and was eventually isolated in

low yield from the fermentation broth of Streptomyces

filipinensis NR0484.¹³The structure of 1 features a 12-

membered lactone ring that is fused to a highly substi-

tuted benzene ring and partly incorporated in a pen-

tapeptide chain.¹⁴Cyclothialidine was found to be a

potent and selective inhibitor of DNA gyrase and its

mode of action was shown to be inhibition of the ATPase

activity exerted by the B subunit of DNA gyrase.¹⁵The

fact that 1 barely exhibits any growth-inhibitory activity

against intact bacterial cells was tentatively explained

by poor penetration of the bacteria’s cytoplasmic

membrane¹³sa major drawback shared also by other

natural congeners of 1 isolated subsequently.¹⁶Never-

theless, the compound was considered to be a promising

lead structure whose modification might open up a route

to a new class of antibacterials.

We have reported part of our approach in this task

in preliminary form,^17-19and herein, a more compre-

hensive account of this work is given. To explore and

exploit the antibacterial potential of 1, we applied a

strategy addressing in a more or less consecutive

manner the following tasks: (i) development of a flexible

synthetic route allowing the preparation of many ana-

logues of 1, (ii) investigation of the structural require-

ments for DNA gyrase inhibitory activity, (iii) lead

optimization toward in vitro antibacterial activity, and

(iv) optimization for in vivo efficacy.

Ch em istr y

As the limited amount of the natural product avail-

able did not allow for an efficient chemical modification

program, we embarked on a total synthetic approach.

To investigate the role of the substituents both on the

lactone as well as on the benzene ring, preparation of a

number of analogues I retaining the bicyclic core of 1

(Scheme 1) was envisioned. According to the retrosyn-

thetic analysis of 1, a classical lactonization of ω-hy-

droxy acid II (P ) H) is used for the formation of the

macrocycle.¹⁸Disconnection of the peptidic side chains

stepwise or all at oncesleads back to a benzylating

agent, such as benzyl bromide VI or benzaldehyde VIII.

Bromide VI prepared by benzylic bromination of o-

toluate VII is employed to alkylate cysteine derivative

IVa or IVb to afford thioether II or III (P ) protecting

group), respectively. The latter is converted to II by

amidation with carboxylic acid V. Alternatively, thio-

ether III is produced by reacting aldehyde VIII with

thiol IVb in a reductive thiolation.^17b,20,21Benzoic acid

derivative IX serves as common starting material for

Sch em e 1

Antibacterial Agents from Cyclothialidine

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1489

Sch em e 2

Sch em e 3.^aPreparation of Dipeptidic Building Blocks

a

Reagents: (a) DCC, N-methylmorpholine, MeCN; (b) EDC, N-methylmorpholine, MeCN.

VI and VIII. Introduction of a methyl group affords

VII,¹⁸whereas Vielsmeier formylation of IX gives al-

dehyde VIII. Furthermore, this synthetic scheme pro-

vides the option to modify the lactone core of 1 by

variation of building block V. The reductive thiolation

route proved to be particularly valuable for the re-

giospecific preparation of compounds having more com-

plex aromatic substitution patterns.

Our exploratory synthesis of 6a ¹⁸was adapted for the

preparation of the 11-methyl analogue 6b (Scheme 2).

Starting from 6b, the pentapeptide chain of the lead

compound was completed by applying the methodology

used later on for the total synthesis of 1¹⁸to afford

12,14-deoxycyclothialidine (7). The first derivative bear-

ing the original aromatic substitution pattern, 10, was

obtained by fluoride-catalyzed cleavage of the silyl

protecting groups of the intermediate 9, of which the

synthesis from resorcilic acid (8) was previously de-

scribed.¹⁸

The peptidic building blocks 11-18 were prepared,

similarly to 5,¹⁸by reaction of cysteine congeners with

suitably substituted and protected 3-hydroxypropionic

acids, using either DCC or EDC as coupling auxiliary

(Scheme 3).

In Scheme 4 the syntheses of 26a -h and 27-29,

analogues of 10 bearing different substitution patterns,

are outlined. These compounds were prepared in a

manner similar to the procedure applied for the syn-

thesis of 10. Thus, for 11-nor-analogue 26a , 2-methyl-

resorcilic acid (19a )¹⁸was converted to 4-nitrobenzyl

(PNB) ester 20a , and the phenol groups were protected

as silyl ethers. The ester 21a was subjected to bromi-

nation with N-bromosuccinimide (NBS). The resulting

benzyl bromide 22a was coupled with 5 in the presence

of Et₃N, PNB ester 23a was subsequently cleaved by

hydrogenolysis, and the resulting hydroxy acid 24a was

cyclized to lactone 25a using Mitsunobu conditions.²²

Cleavage of the silyl protecting groups afforded 26a .

For the preparation of the monohydroxy derivatives

26b and 26c, 3-hydroxy-2,6-dimethylbenzoic acid²³was

converted to 21b, which was subsequently brominated

with NBS. The crude mixture of the two regioisomeric

monobromides 22b/c was reacted with thiol 5, and

thioethers 23b and 23c were separated by chromatog-

raphy. Corroboration of the structures of the compo-

nents of the mixture 22b/c as well as that of thioethers

23b and 23c was based on NOE experiments. The

isomers displaying a NOE between an aromatic hydro-

gen and the methyl singlet were assigned to structures

22b and 23b, whereas the isomers lacking this NOE

were assigned to structures 22c and 23c, respectively.

The thioethers 23b and 23c were processed further to

26b and 26c, respectively, in analogy to the synthesis

of 26a . Replacing in this scheme thiol 5 by thiols 11-

13 afforded the target compounds 26d -f, respectively.

For the preparation of the regioisomeric monomethoxy

derivatives 26g and 26h , 20d was consecutively mono-

methylated and silylated, and the intermediate 21g was

subjected to NBS bromination. The crude bromination

product (22g/h ), containing approximately 32% and 36%

1490 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6

Angehrn et al.

Sch em e 4^a

a

Reagents and conditions: (a) 4-nitrobenzyl bromide, N,N,N′,N′-tetramethylguanidine, DMF, 20 °C; (b) MeI, K₂CO₃, acetone, 60 °C,

16 h; (c) t-Bu(Me)₂SiCl or thexyl(Me)₂SiCl, Et₃N, DMF; (d) NBS, CCl₄, 90 °C, hν; (e) (i) 5, 11-16, respectively, Et₃N, DCM, 20 °C, (ii) (for

25e,f) pTsOH, MeOH, 60 °C, 1 h; (f) H₂, Pd-C, EtOAc; (g) DEAD, PPh₃, THF, 20 °C; (h) NH₄F, MeOH.

of the regioisomeric bromides 22g and 22h , respectively,

was reacted with 5. The mixture of the two resulting

thioethers 23g and 23h was separated by chromatog-

raphy, and the two methoxy series were individually

processed further to afford the corresponding lactones

26g and 26h , respectively. The regiochemical assign-

ment in this series was based on ¹H-¹³C HMBC

coupling experiments (26g and 26h ) and was eventually

confirmed by an X-ray structure of 25g.²⁴

L-Cys-OMe, 70, or 75 in a reductive thiolation reaction

of 67, using triethylsilane in trifluoroacetic acid/

dichloromethane.^17b,20,21As this new route no longer

required a NBS bromination step, it was possible to

replace the PNB ester by an allyl ester, which later on

could be cleaved more easily with Pd(0) catalysis.

For the preparation of the aldehyde 67, 2-methylre-

sorcilic acid derivative 62²⁶was subjected to Vielsmeyer

formylation, and the 3-methoxy group in the resulting

aldehyde 63 was regiospecifically cleaved due to the

assistance of the neighboring formyl group.²⁷Methyl

ester 64 was converted to allyl ester 67 by consecutive

saponification, alkylative esterification of the resulting

pseudoacid 65 with allyl bromide, and silylation of the

phenol group. For the preparation of the thiol 70, L-Boc-

cystine was coupled with 2 equiv of acetamide oxime,

and the resulting condensation product was then ther-

mally cyclized to the bis-3-methyloxadiazole 69.²⁸The

disulfide function was finally reduced with tributylphos-

phine in 2,2,2-trifluoroethanol. For the preparation of

75, 2-aminoacetonitrile (71) was converted to the ami-

doxime 73, which thereafter was transformed to the

oxadiazole 75 as described for 70.

For the syntheses of the homologous lactones 86a -g

(Scheme 8), amine 76 was reacted in an EDC-mediated

coupling reaction with carboxylic acids 82a -g, which

had been prepared by tritylation of methyl 2-hydroxy-

acetate followed by saponification (82a ²⁹) or by mono-

tritylation of diols 79b-g followed by two sequential

oxidation reactions (82b-g). The trityl protecting group

in the primary amidation products was cleaved off with

pTsOH/MeOH. The resulting allyl esters 83a -g were

converted by Pd(0)-catalyzed ester cleavage to the

corresponding hydroxy acids 84a -g, which were sub-

sequently cyclized to the lactones 85a -g under Mit-

The stereoisomers 27-29 were prepared analogously

to the parent compound 10 but with replacing interme-

diate 5 by thiols 14-16, respectively.

The two isomeric 8-methyl derivatives 32 and 33 were

obtained similarly by reacting the mixture 22g/h with

dipeptides 17 and 18, respectively (Scheme 5). In these

cases, the ring-closing step using Mitsunobu conditions

proceeded with inversion of the configuration at C(8).

The chiral integrity of the purified products was indi-

cated by their NMR spectra lacking the signals of

possible diastereoisomeric products.

Lactones 43 and 47 and the corresponding 7-(Boc-L-

serylamino) derivatives 45 and 48 were prepared start-

ing from hydroxy acid 34 and nitro acid 37, respectively.

In these syntheses, the carboxylic acid function was

protected as a silyl ester during the benzylic bromina-

tion reaction.²⁵

Derivatives 49-52 and 54-61 were prepared by side-

chain modifications of compounds 25g, 26g, 50, 10, and

53,¹⁸as outlined in Scheme 6.

To prepare derivatives of the most active 12-methoxy

series in a more efficient way, we developed a regiospe-

cific synthesis relying on benzaldehyde 67 as starting

point for the elaboration of the sulfanyl side chain

(Scheme 7). Thus, the synthesis of the key intermediates

76, 77, and 78 was achieved by benzylation of thiols

Antibacterial Agents from Cyclothialidine

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1491

Sch em e 5^a

Sch em e 6^a

a

Reagents and conditions: (a) 17 or 18, respectively, Et₃N,

DCM, 20 °C; (b) H₂, Pd-C, EtOAc; (c) DEAD, PPh₃, THF, 20 °C;

(d) NH₄F, MeOH; (e) t-Bu(Me)₂SiCl, Et₃N or imidazole, DMF; (f)

NBS, CCl₄, 90 °C, hν; (g) 5, Et₃N, DCM, 20 °C; (h) TFA; (i) Boc-

L-Ser-OH, EDC, MeCN, 0 °C.

a

Reagents and conditions: (a) NaBH₄, MeOH, THF, 0 °C; (b)

sunobu conditions. Cleaving of the silyl protecting group

afforded the target lactones 86a -g. The corresponding

thioamides 87a -g were obtained by heating 85a -g

with Lawesson’s reagent in toluene and subsequent

cleavage of the silyl protecting group.

The seco-derivatives 90 and 91 were obtained by

reductive thiolation of aldehyde 88 with N-Ac-L-Cys-

OMe. Cleavage of the silyl group afforded the thioether

90, whereas the preceding treatment of 89 with Lawes-

son’s reagent followed by cleavage of the silyl ether gave

91.

For the preparation of 93, 94, 96, and 97, the amine

77 was amidated with 82b or 82d , respectively. The

trityl ethers were cleaved and the resulting products

processed following the standard route used for the

syntheses of 86b,d and 87b,d .

For the synthesis of 103 (Scheme 9), intermediate 77

was amidated with (R)-2-hydroxysuccinic acid-1-methyl

ester.³⁰After cleavage of the allyl ester, hydroxy acid

100 was cyclized under Mitsunobu conditions with

concomitant inversion of the configuration at C(8).

Treatment of the lactone 101 with Lawesson’s reagent

provided thioamide 102. Reduction of the methyl ester

function with NaBH₄in MeOH followed by cleavage of

the silyl protecting group afforded 103.

NH₄F, MeOH; (c) aq NaOH, THF; (d) NH₃, MeOH, 20 °C; (e)

N-hydroxysuccinimide, EDC, MeCN, then allylamine; (f) TFA; (g)

Ac₂O, 60 °C; (h) Boc-L-Pro-OH, Boc-L-3cHyp-OH, BOC-D-3cHyp-

OH, BOC-^L-Ser-OH, or BOC-D-Ser-OH, respectively, EDC, MeCN,

0 °C; (i) (i) (Ph₃)₃COCH₂CH₂COOH, EDC, MeCN, (ii) pTsOH,

MeOH.

respectively. The acetonide group in the resulting

products 106a ,b was hydrolyzed, and the primary

alcohol of diols 107a ,b was protected as the trityl ether

(108a ,b). Cleavage of the allyl ester, lactonization, and

cleavage of the protecting groups afforded the deriva-

tives 110 and 111, respectively.

For the preparation of compounds 114-118, the

amine 78 was converted to the protected thiolactam 112

in analogy to the synthesis of 97. As cleavage of the allyl

ester occurred under concomitant scission of the N-

allyloxycarbonyl protecting group, we used Pd(PPh₃)₄

in the presence of trimethylsilylamine and trimethylsilyl

trifluoroacetate³³in order to prevent allylation of the

unmasked aminomethyl group. The N-protecting group

was subsequently reintroduced with allyl chloroformate

under Schotten-Baumann conditions.

Subjecting 112 to the latter allyl cleavage protocol

gave 113, and subsequent removal of the silyl group

afforded the aminomethyl derivative 114, which was

isolated as the hydrochloride salt.

For the syntheses of the homologous hydroxymethyl

lactones 110 and 111, the amine 77 was amidated with

carboxylic acids 105a , prepared by consecutive hydro-

genation and saponification of 104,³¹and with 105b,⁴⁵

By acylating 113 with acetic anhydride or N-allyloxy-

carbonyl-L-Ala-OH³⁴and subsequently cleaving of the

1492 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6

Angehrn et al.

Sch em e 7^a

a

Reagents and conditions: (a) DMF, POCl₃, DCM, 72 h, 40 °C; (b) BCl₃, DCM, 5 °C, 40 min; (c) 2.5 N KOH; (d) CH₂dCHCH₂Br,

N,N,N′,N′-tetramethylguanidine, DMF; (e) thexyl(Me)₂SiCl, Et₃N, DMF; (f) Et₃SiH, TFA, DCM, 0 °C; (g) ClCOOCH₂CHdCH₂, Et₃N,

MeCN; (h) (NH₂OH)₂‚H₂SO₄, NaOH, MeOH; (i) (L-Boc-Cys-)₂, 1-hydroxypyridin-2(1H)-one, DCC, DMF; (j) toluene (69) or dioxane (74),

100 °C, 7 h; (k) PBu₃, CF₃CH₂OH, H₂O, 0 °C.

Sch em e 8^a

a

Reagents and conditions: (a) Ph₃CCl, pyridine; (b) (COCl)₂, DMSO, DCM, -70 °C, then Et₃N; (c) KMnO₄, aq acetone; (d) (i) EDC,

MeCN, 0 °C, (ii) pTsOH, MeOH, 20 °C; (e) Pd(PPh₃)₄, morpholine, THF, 0 °C; (f) DEAD, PPh₃, THF, 20 °C; (g) NH₄F, MeOH; (h) Lawesson’s

reagent, toluene, 80 °C; (i) thexyl(Me)₂SiCl, Et₃N, DMF; (j) Ac-L-Cys-OMe, Et₃SiH, TFA, DCM, 0 °C; (k) 82b or 82d , respectively, with

conditions (d).

protecting groups, the derivatives 115 and 116, respec-

tively, were obtained. Reductive amination of 113 with

acetone/NaBH₄followed by deprotection of the phenol

group afforded the isopropylamino derivative 117. Ni-

trosation of 113 using NaNO₂in 25% aqueous AcOH

produced a product mixture, from which, after depro-

tection, the hydroxymethyl derivative 118 was isolated.

Biologica l Resu lts a n d Discu ssion

DNA Gyr a se In h ibition . The DNA gyrase inhibitory

activity of all target compounds was assessed in an in

vitro supercoiling assay in which the introduction of

superhelical turns into a relaxed plasmid was deter-

mined by gel electrophoresis.^12,35For practical reasons,

i.e., to allow more efficiency in the evaluation of new

Antibacterial Agents from Cyclothialidine

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1493

Sch em e 9^a

a

Reagents and conditions: (a) (i) H₂, Pd-C, EtOAc, (ii) KOH, MeOH, 40 °C; (b) EDC, MeCN, 0 °C; (c) 80% AcOH; (d) Ph₃CCl, pyridine;

(e) Pd(PPh₃)₄, morpholine (for 112: TMS-morpholine, TMSOAc), THF, 0 °C; (f) DEAD, PPh₃, THF, 20 °C; (g) Lawesson’s reagent, toluene,

80 °C; (h) pTsOH, MeOH, 60 °C; (i) NH₄F, MeOH; (j) NaBH₄, MeOH; (k) 82d , EDC, CH₃CN, 0 °C; (l) pTsOH, MeOH, 20 °C; (m)

ClCOOCH₂CHCH₂, N-methylmorpholine, DCM; (n) Pd(PPh₃)₄, TMS-DMA, TFA-TMS, DCM; (o) HCl; (p) Ac₂O, pyridine; (q) N-Aloc-L-

Ala-OH, EDC, MeCN, 0 °C; (r) NaBH₄, acetone, aq AcOH, NaOAc, 0 °C; (s) NaNO₂, 25% aq AcOH.

derivatives, a MNEC (maximum noneffective concentra-

tion) value was determined. In general, the MNEC value

of a compound was 3-5 times lower than the IC_50,and

10-20 times lower than the concentration needed for

complete inhibition of the supercoiling reaction.

gyrase inhibitory activity was a clear-cut confirmation

that the phenolic groups of 1 were essential, and

thereafter we chose to remain with the original aromatic

substitution pattern. With the derivative 10, an ana-

logue of 1 featuring truncated peptidic side chains, we

obtained for the first time an active compound, demon-

strating the crucial role of the phenolic hydroxy groups.

The next investigation addressed the specific role of the

different substituents of the bicyclic core. To this end,

the five substituents of 10 were individually discon-

nected by preparing the five tetrasubstituted derivatives

26a -e. It turned out that, with the exception of the 14-

hydroxy group, all the other substituents of 10 could

be omitted without significantly impairing the biological

activity, i.e., only the 14-deoxyderivative 26c was found

to be inactive. A similar bias was also observed for the

two regioisomeric methoxy derivatives 26g and 26h .

Whereas 26g retained the activity of 10, the 14-methoxy

isomer 26h was found to be inactive. On the basis of

According to our strategy, we focused our initial

efforts on revealing the active principle of the lead

structure, thereby depending on a total synthetic ap-

proach. Although the pentapeptide entity was initially

considered also as a synthetic target, our main effort

aimed at simpler derivatives I retaining the bicyclic

lactone core and probing the importance of the various

substituents of 1 (Table 1). Our first synthetic targets,

lactones 6a and 6b, were found to be inactive in the

supercoiling assay, indicating that either the phenolic

hydroxy groups and/or the pentapeptide chain were

important for activity. Starting from 6b, we then

completed the pentapeptide entity of 1. The fact that

12,14-dideoxycyclothialidine (7) was devoid of DNA

1494 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6

Angehrn et al.

Ta ble 1. DNA Gyrase Inhibition (MNEC) and in Vitro Antibacterial Activity (MIC) of Selected Compounds: Variation of R^1-R⁵

MIC^b(µg/mL)

configuration at

MNEC^a

N. meningitidis

M. luteus

compd

R¹

R²

R³

R⁴

R⁵

C(4)

C(7)

(µg/mL)

69480

ATCC8340

1 (cyclothialidine)

0.05

>1

128

nd^c

nd

16

128

nd

32

16

128

nd

32

128

nd

16

128

16

>128

6a

6b

H

Me

H

COOMe

NHBoc

>1

7 (12,14-dideoxy-1)

>100

0.2

0.5

1

10

Me

H

OH

H

OH

OMe

OH

H

OH

H

COOMe

H

COOMe

H

COOMe

NHBoc

H

NHBoc

H

NHBoc

NH-^L-Ser-Boc

NHBoc

NH-^L-Ser-Boc

NHBoc

R

S

26a

26b

26c

26d

26e

26g

26h

26f

43

Me

H

>100

OH

OMe

OH

NH2

OH

1

32

16

0.25

0.2

>1

>128

64

>128

128

20

>1

45

H

0.4

>1

47

H

nd

48

H

>100

>128

128

32

27

28

29

Me

S

R

S

R

S

10

1

4

128

a

b

Supercoiling assay, E. coli gyrase, MNEC ) maximum noneffective concentration. Agar dilution (BB2 medium supplemented with

1% Isovitalex, 7.5% sheep blood, and menadione). Inoculum 10⁴CFU/spot, MIC ) minimum inhibitory concentration. ^cNot determined.

these results, the 14-hydroxylated, bicyclic lactone X

was identified as the partial structure common to all

derivatives found to be active (Figure 1).

than the 4-unsubstituted 26d and by the virtually

abolished activity of 26f.

The replacement of the 14-hydroxy by a 14-amino

group with 47 produced an inactive compound that

could not be improved by the 7-(Boc-L-seryl-amino)

substituent (48). The fact that the 14-hydroxy group

could not be replaced by neither amino nor methoxy

substituents led to the prediction that this group was

acting both as hydrogen-bond donor and acceptor when

binding to the DNA gyrase protein, a behavior not

possible for amino or methoxy substituents. This as-

sumption was eventually confirmed by X-ray structures

of 1 bound to the gyrB protein.³⁶These structures reveal

the crucial role of the 14-hydroxy group, which main-

tains a hydrogen-bond network together with Asp75 and

a tightly bound water molecule, as depicted in Figure

F igu r e 1. Minimal structural requirements for DNA gyrase

inhibitory activity.

However, the other substituents contribute to the

inhibitory activity. Thus, on removing both R⁴and R⁵,

the resulting compound 26f showed only borderline

activity. Removal of the 11-methyl (R¹) and of the 12-

hydroxy group (R²) led in 43 to a loss of activity, which

remarkably was recovered in the 7-(Boc-L-seryl-amino)

derivative 45.

The observation that either of the two substituents

at the macrocycle could be removed without significant

loss of activity had raised the question whether the

entire lactone ring was important for recognition by the

enzyme. The answer was provided by the finding that

27, the enantiomer of 10, was merely inactive, empha-

sizing the importance of the three-dimensional shape

of the lactone. The role of the stereochemistry at

positions 4 and 7 was further probed by preparing the

diastereoisomers 28 and 29. Whereas at C(7) S- and

R-configuration is tolerated, the data obtained indicates

that in particular the R-configuration at C(4) is essential

for optimal activity. This is also supported by the fact

that the 7-unsubstituted 26e exhibits higher activity

F igu r e 2. Binding mode of cyclothialidine at the DNA gyrase

B protein.³⁶

Antibacterial Agents from Cyclothialidine

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1495

Ta ble 2. DNA Gyrase Inhibition (MNEC) and in Vitro Antibacterial Activity (MIC) of Selected Compounds: Variation of R², R⁴-R⁶

MIC^b(µg/mL)

variation

of

MNEC^aN. meningitidis

M. luteus

ATCC8340

S. aureus S. pyogenes

compd R²

R⁴

R⁵

(µg/mL)

69480

887

b15

R²

1

0.05

0.2

2.5

10

0.1

0.25

1

0.4

2.5

0.05

1

0.1

1

128

16

64

nd^c

4

128

16

64

>64

16

8

16

>64

128

64

8

128

8

>64

16

>64

64

>64

16

>64

16

32

64

>64

16

128

nd

>64

32

nd

10

26g

49

50

51

52

86b

54

55

56

57

58

59

60

61

32

33

OH

COOMe

OMe COOMe

OMe CH₂OH

OMe COOH

OMe CONH₂

NHBoc

R⁴

OMe CONHCH₂CHCH₂NHBoc

4

R⁵

OH

COOMe

H

NH₂

NHAc

NH-^L-Pro-Boc

NH-^L-3cHyp-Boc

NH-^D-3cHyp-Boc

NH-^L-Ser-Bo c

NH-^D-Ser-Boc

NHCOCH₂CH₂OH

32

nd

32

nd

64

nd

8

0.2

0.4

4

>64

16

>64

R⁶

16

64

>64

^a,b,cSee Table 1.

2. The information on the exact binding mode of 1 and

of other ligands of the ATP binding site had been used

by others for a biased needle screening aiming at new

DNA gyrase inhibitors.³⁷

Ta ble 3. Variation of the Lactone Ring Size. MNEC and MIC

Values of Selected Compounds

From our experience with a large number of ana-

logues of 1, the binding of the 14-hydroxy group seems

to add a major contribution for their binding to the DNA

gyrase protein. Modification of the substituents on

cyclothialidine’s core structure had revealed X as the

minimal structural requirements for DNA gyrase inhi-

bition. Nevertheless, other substituents add to the

inhibitory potency by providing better binding and

presumably by supplying a stabilizing influence with

regard to the bioactive conformation of the lactone ring.

This is supported by NMR data showing for active

compounds a large and a small coupling constant

between the protons at C(3) and C(4) as a characteristic

feature. This suggests a synclinal/antiperiplanar ar-

rangement of the hydrogens and implies a staggered

(gauche) conformation about CR and Câ of the cysteine

entity. This local conformation is also displayed by 1

when binding to the ATPase site of DNA gyrase.

In our search for compounds of improved antibacterial

activity, we have extensively modified the substituents

R¹, R², and R^4-6. For R¹, methyl was found to be better

than hydrogen, whereas larger alkyl groups led to a

decrease in affinity. Therefore, a number of 11-methyl

derivatives are compiled in Table 2 to describe SAR

found for the other substituents with regard to the

enzyme inhibitory activity. For R², OH ∼ OMe; for R⁴,

COOMe, CONH₂> CH₂OH . COOH. An interesting

feature of R⁵was the striking activity of 3-hydroxypro-

pionylamino substituents, either free as in 61, or as part

of an L-amino acid (57, 59), reminiscent of the 7-(3-

hydroxy-L-prolinoylamino) entity of 1. When embedded

in a D-amino acid residue (58, 60), this motif is less

efficient. In addition, R⁵) H or NHAc allowed good

inhibitory properties (86b, 55).

MICb (µg/mL)

MNEC^a

(µg/mL)

compd

X

n

ring size

S. a. 1

S. a. 2

86a

86b

86c

86d

86e

86f

86g

87a

87b

87c

87d

87e

87f

87g

90

O

S

1

2

3

4

5

6

8

1

2

3

4

5

6

8

-

11

12

13

14

15

16

18

11

12

13

14

15

16

18

-

>1

0.5

2

0.05

2

>1

0.2

0.05

0.4

0.01

0.5

>1

2

>64

64

>64

64

32

4

2

>64

nd^c

>64

2

32

>64

nd

>64

2

4

2

0.5

32

0.25

16

S

O

S

16

nd

>64

16

nd

>64

8

91

-

0.2

^a,cSee Table 1. ^bAgar dilution (Mueller-Hinton agar). Inoculum

10⁴CFU/spot, MIC ) minimum inhibitory concentration. S. a. 1:

S. aureus ATCC25923; S. a. 2: S. aureus Smith.

For R⁶at C(8), a clear preference for a â-methyl group

(32) over a methyl group in the R-position (33) was

observed.

When examining the influence of the lactone ring size

on the biological activity, we were surprised by the

finding that DNA gyrase inhibitory activity was ob-

served over a rather large structural scope, namely from

11- to 16-membered lactones (Table 3). Whereas in the

6-oxo series, activity was found from 12- to 15-mem-

bered lactones (86a -e), the 11- to 16-membered homo-

1496 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6

Angehrn et al.

Ta ble 4. DNA Gyrase Inhibition (MNEC), in Vitro and in Vivo Antibacterial Activity (MIC, ED₅₀) of Selected Compounds.

Comparison with Reference Compounds

MIC (µg/mL)^b

MNEC^a

(µg/mL) 25922 AC739 25923 Smith

E. c.

X. m.

S. a.

S. e.

16/2

E. f.

6

S. p.

b15

ED₅₀

c

compd

R⁴

R⁵

R

X

n

(mg/kg)

10

26g

86b

87b

86d

87d

93

94

96

97

103

110

111

114

115

116

0.2

0.5

>64

64

>64

64

>64

8

16

8

16

4

>64

64

2

128

64

2

64

16

8

0.5

1

64

>64

64

128

32

4

1

4

1

0.25

0.5

2

0.5

1

nd^d

nd

COOMe

NHBoc

O

S

O

S

O

S

O

S

1

3

1

3

1

2

3

H

O

S

H

0.05

0.01

0.05

0.02

0.01

0.004

0.01

0.005

0.002

0.005

0.002

0.001

0.1

4

1

8

1

4

0.5

8

0.5

0.12

2

COOMe

0.25 <0.12

8

nd

3-Me-ODA^e

3-Me-ODA

2

>25

8.5

9.5

12.5

8.5

25

0.25

0.06

2

0.5

0.25 <0.12

1

2

0.12

0.01

0.5

<0.12

0.5

(H)

0.5

8

4

0.25

0.5

0.25

0.5

1

8

0.5

1

0.5

2

NH₂‚HCl

NHAc

NH-^L-Ala‚HCl

NHCH(Me)₂‚HCl

32

>64

4

8

>64

<0.12

1

8

2

25

>12.5

117

118

novobiocin

vancomycin

0.25

0.5

0.25

1

0.12 <0.12

0.5

1

OH

0.25 <0.12

25

3

1.5

0.12

1

0.25

2

4

-

1

a

b

Supercoiling assay, E.coli gyrase, MNEC ) maximum non effective concentration. Agar dilution (Mueller-Hinton medium), inoculum

10⁴CFU/spot, MIC ) minimum inhibitory concentration. Test organisms: E. coli 25922, X. maltophilia IAC 739, S. aureus ATCC 25923,

S. aureus Smith, S. epidermidis 16/2, E. faecalis 6, S. pyogenes b15. ^cIn vivo efficacy, septicaemia in mice (S. aureus Smith), iv

administration. Not determined. ^e3-Methyl-1,2,4-oxadiazol-5-yl.

d

logues 87a -f of the 6-thioxo lactones displayed inhibi-

tory properties. In both series, the 14-membered lactones

(86d , 87d ) were the most active ones, followed by the

12-membered lactones and then by the others. The

improved inhibitory activity was tentatively attributed

to the increased lipophilicity and conformational flex-

ibility of the larger rings, properties even more pro-

nounced in the 6-thioxo series, and obviously optimally

rewarded in the 14-membered lactones.

side chains, such as the hydroxymethyl groups at the

lactone core (103, 110, 111) or amino and hydroxy

functions at the ODA ring (114-118), introduced to

improve the pharmacokinetic properties of the com-

pounds, were in general well-tolerated and did not affect

the excellent enzyme inhibitory activities found in

particular for the 14-membered lactones.

An tiba cter ia l Activity in Vitr o. On the basis of the

minimal structure X, many potent DNA gyrase inhibi-

tors with activities similar to that of the lead compound

have been prepared. However, with regard to the

antibacterial activity, progress was rather modest.

Cyclothialidine itself had been shown to be virtually

inactive, and therefore, first we performed antibacterial

in vitro testing using a set of rather sensitive strains

requiring a test media supplemented with sheep blood

(Tables 1 and 2). The compounds compiled in Table 1

were selected to demonstrate the importance of the

various substituents R¹-R⁵as well as the stereochem-

ical prerequisites of the lactone moiety. Their enzyme

inhibitory activity was matched in a qualitative manner

by the minimum inhibitory concentrations (MICs) de-

termined in particular against Neisseria meningitides

69480 and Micrococcus luteus ATCC8340, confirming

that the antibacterial activity was indeed a consequence

of the inhibition of DNA gyrase. More data is shown in

Table 2. The first synthetic compound found to be active

in the enzyme assay (10) was only slightly better than

1 with regard to antibacterial activity. Methylation of

the 12-hydroxy group, as exemplified by 26g, effected

Considering the high degree of structural variability

tolerated by the enzyme, it was not unexpected that we

eventually identified “seco-compounds”sanalogues lack-

ing the lactone entitysthat also inhibited DNA gyrase.

Compounds 90 and 91 are simple representatives of this

subclass.^17b,38They demonstrate that the lactone entity

is not essential for the binding and that the substructure

common to all congeners of this inhibitor family can now

be reduced further to a hydroxylated benzyl sulfide

entity XI (Figure 1). It can be assumed that in all

compounds found active, this pharmacophore is able to

adopt a similar conformation when binding to DNA

gyrase.

To increase the metabolic stability to enable in vivo

efficacy, the ester function at C(4) was replaced. From

the various five-membered heterocycles examined, the

1,2,4-oxadiazole²⁸(ODA) was found to be the best and

brought a further improvement observed already at the

enzyme level. Thus, the oxadiazoles 93, 94, 96, and 97

were 2-10 times more active in the supercoiling assay

than the corresponding methyl esters (Table 4). Polar

Antibacterial Agents from Cyclothialidine

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1497

Ta ble 5. In Vitro Antibacterial Activities of 97 and Reference Compounds against Multiresistant Gram-Positive Bacteria^a

S. aureus (30)^b

E. faecalis (16)

E. faecium (5)

compound

MIC₅₀

MIC₉₀

range

MIC₅₀

MIC₉₀

range

methicillin

imipenem

ciprofloxacin

erithromycin

tetracyclin

novobiocin

vancomycin

97

32

4

0.5

>16

>64

>16

4->64

0.06->64

0.25->16

0.5->16

4

2

0.12

8

4

0.12

4-8

2-8

1->16

0.25-0.5

1

0.06

2

0.12

0.5-2

0.06-0.12

1-4

0.06-0.25

a

Agar dilution (Mueller-Hinton agar). Inoculum 10⁴CFU/spot; MIC₅₀(MIC₉₀) ) Minimum concentration (in µg/mL) that inhibits at

b

least 50% (90%) of the strains tested. The number of strains is in parentheses.

as an important improvement a modest activity against

S. aureus. Variation of R⁴in the 12-methoxy series

brought about some further progress, e.g. with the allyl

amide 52. Regarding the variation of R⁵, representatives

of the 12-hydroxy series are compiled. These compounds

demonstrate that despite of a good enzyme inhibitory

activity, the effect on bacterial cells remained marginal,

probably due to their highly hydrophilic character. It

can be noted that these R⁵substituents did not perform

better in the 12-methoxy series (data not shown). Some

further progress was marked by the 8-â-methyl deriva-

tive 32.

With this modest level of in vitro antibacterial activity

achieved, we changed the battery of test strains to more

typical pathogens which could be tested in the absence

of blood plasma (Mueller-Hinton media). In Table 3 the

antibacterial performance of the homologues lactones

86a -g and of their corresponding 6-thioxo analogues

87a -g is displayed. Most importantly, the improved

enzyme inhibitory property of the 14-membered lactones

was also matched by a corresponding antibacterial

activity, as can be seen by comparison of 86b with 86d

and of 87b with 87d . The thioacetyl derivative 91,

representing the seco-subclass, exhibited an antibacte-

rial activity corresponding to its activity at the enzyme

level.

series and displays broad-spectrum activity against

Gram-positive bacteria, similar to or better than that

of our reference compounds novobiocin and vancomycin.

Nevertheless, typical Gram-negative bacteria, such as

wild-type strains of Escherichia coli, Klebsiella pneu-

moniae, or Pseudomonas aeruginosa, were not suscep-

tible, although activity was found against selected

strains of Xanthomonas maltophilia, Haemophilus in-

fluenzae, N. meningitides, and Moraxella catarrhalis.

To ensure that the desired target profile can be met by

cyclothialidine congeners, we have tested 97 as a

prototype of this new antibacterial class against a larger

number of Gram-positive isolates (Table 5). The MICs

of 97 against S. aureus, Enterococcus faecalis, and

Enterococcus faecium were considerably lower than

those of vancomycin. Even more important, 97 was

equally inhibitory against antibiotic-susceptible and

multiresistant strains and did not show any cross-

resistance with other structural classes. As a conse-

quence, the MIC₉₀values against multiple-resistant

staphylococci and enterococci were much lower than for

established antibacterial classes, such as â-lactams,

quinolones, macrolides, glycopeptides (vancomycin,

teicoplanin), or rifamycins. Remarkably, clinical isolates

resistant to novobiocin, a drug targeting the same

binding site as 1, were fully susceptible to 97.³⁹

The attachment of hydrophilic side chains to the best

compounds (e.g. 94, 97) in order to lower their lipophilic

character, and concomitantly increase their water solu-

bility, led to a slight decrease of the antibacterial

activity. Thus, the hydroxymethyl substituent of 103 or

111 lowered the in vitro activity to some extent when

compared to that of the parent compounds 94 and 97.

A similar, nondetrimental effect was found for basic and

polar substituents at the methyl group of the oxadiazole

ring, as demonstrated for compounds 114-118. Thus,

tuning of the physicochemical properties of the most

potent compounds appeared to be possible.

The excellent in vitro antibacterial activities of lipo-

philic compounds were significantly reduced by the

presence of plasma proteins (Table 6). For the more

hydrophilic compounds, this effect was less pronounced.

This plasma-shift of the MIC values, due to protein

binding of the compounds⁴⁰and possibly also to a

changed susceptibility of the bacteria, was assumed to

play an important role with regard to the in vivo

efficacy.

Modification work was continued, and key compounds

featuring the major structural changes that led to an

improved antibacterial potency are compiled in Table

4. Starting from 10, the replacement of OH by OMe and

the removal of the NHBoc group afforded 86b, a

derivative displaying a modest activity against S. au-

reus. It was then found that the introduction of a

thioamide function not only improved the DNA gyrase

inhibitory activity but also simultaneously caused a

marked increase of the activity against Gram-positive

strains. Although the MIC values of 87b looked promis-

ing, the methyl ester function due to its metabolical

liability was not suited for in vivo testing. We found an

ODA ring to be an appropriate replacement for the ester

group (93, 94).²⁸The 3-Me-ODA in position 4 indeed not

only retained but improved both the enzyme-inhibitory

and also the antibacterial activity in vitro when com-

pared to the methyl ester group.

Lactone ring enlargement, e.g. going from 86b to 86d

or from 87b to 87d , clearly brought about a further

improvement. The outstanding enzyme inhibitory prop-

erties of the 14-membered lactones were also accompa-

nied by a substantial increase in the antibacterial

activity. Combining the most favorable structural ele-

ments, lactone 97 is the most potent compound in this

An tiba cter ia l Effica cy in Vivo. The optimal trans-

lation of the in vitro antibacterial activity into an

adequate in vivo efficacy turned out to be a difficult task.

After compounds exhibiting an MIC value for S. aureus

1498 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6

Angehrn et al.

Ta ble 6. CLOGP, Protein Binding, Effect of Plasma Proteins on the in Vitro Antibacterial Activity, in Vitro Glucuronidation, and

Cytotoxicity of Selected Compounds

in vitro antibacterial activity against S. aureus Smith

MIC

50% plasma^d

(P)

% of total

substrate

cytotoxicity,^f

IC₅₀

(mg/mL)

% bound

BHI agar^c

(B)

ratio

P/B

compound

CLOGP^a

to protein^b

glucuronidated^e

1

86d

97

103

110

114

vancomycin

-2.09

3.00

3.34

1.31

1.80

1.51

<1

>800

>50

24

>50

96

88

94

0.03

0.25

8

25

12

57

91

0.25

1

2

4

2

25^g

a

b

Calculated distribution coefficient, KOW_ClogP. See: Meylan, W. M.; Howard, P. H. J . Pharm. Sci. 1995, 84, 83-92. Binding to

mice plasma protein in percent. ^cMIC (µg/mL) determined in BHI (brain heart infusion) agar. MIC (µg/mL) determined in BHI agar/

d

mouse plasma (1:1). ^eIn vitro glucuronidation estimated under standardized conditions (700 mg of rat liver microsomal protein, 1 mM

UDPGA, and 40 µM substrate. Products formed after 1 h incubation time were analyzed by HPLC and expressed relative to the substrate

added; see Fischer, M. B.; et al. Drug Metab. Rev. 2001, 33, 273-297). ^fAssessed with HeLa cells using the following method: Ohara H.;

g

Terasima T. GANN 1972, 63, 317-327. Data are from the following: Knudsen, J . D. et al., Antimicrob. Agents Chemother. 1997, 41,

1910-1915.

of <8 µg/mL had been identified, their efficacy in a

septicaemia model in mice was tested. Unfortunately,

out of a number of promising derivatives, among them

94 and 97, none was able to cure the infected mice, and

this was rather disappointing. Examination of plasma

and urine samples revealed that our best compounds

displayed unfavorable pharmacokinetics, i.e., they showed

rapidly decreasing plasma concentrations. A major

metabolite, which turned out to be the glucuronide of

the phenolic parent compound, was discovered.⁴¹In

contrast, inactive analogues of rather hydrophilic char-

acter, such as the carboxylic acid 50, displayed a much

longer plasma half-life. It was hypothesized that the

reduction of the lipophilic properties of the best com-

pounds might improve their pharmacokinetic behavior,

and this concept indeed led to vivo efficacy. The hy-

droxymethyl group in the 8â-, 9â-, or 10â position, of

the 12-14 membered lactones 103, 110, and 111,

respectively, did reduce the antibacterial potency by a

factor 8 in comparison to their parent compounds (e.g.

94 and 97). Nevertheless, these compounds were found

to be active in the septicaemia model with an ED₅₀of

8-12 mg/kg (iv administration). A similar performance

was also found for the aminomethyl derivative 114. The

in vivo efficacy of the more hydrophilic derivatives 103,

110, and 114 can be attributed to lower protein binding

and improved pharmacokinetics, both parameters com-

paring favorably with those of 97, but remaining inferior

to those of vancomycin (Table 6, Figure 3). The relatively

high plasma levels achieved with the hydroxymethyl

derivatives 103 and 110 is probably due to a slower

glucuronidation rate, as was demonstrated in an in vitro

experiment in comparison with 114 (Table 6). These

compounds, while exhibiting a moderate in vitro activity

comparable to that of vancomycin, seem to be closer to

this reference compound than 97 and 114 in terms of

hydrophilicity (CLOGP), susceptibility for glucuronida-

tion, and protein binding. The amino derivative 114,

although showing a rapid decline of plasma concentra-

tions due to glucuronidation, still remains efficacious

due to its higher intrinsic antibacterial activity. The

assumption that protein binding (plasma shift of MIC)

and glucuronidation are particularly important for the

more lipophilic derivatives helps to rationalize the in

F igu r e 3. Total plasma concentrations of 97, 103, 110, and

114 in rats (n ) 3-6) after a single intravenous dose of 20

mg/kg (microsuspension in gelatin). Comparison with vanco-

mycin data from Ngeleka et al. (Antimicrob. Agents Chemother.

1989, 33, 1575-1579).

vivo performance of these derivatives.^40,42The examples

given do not yet match the efficacy of vancomycin or

novobiocin, but they indicated means for further opti-

mization by modifying the physicochemical properties.

However, from many more compounds we had to

learn that the window of optimal lipophilicity was rather

small and that the glucuronidation rate was also

depending on structural features of the substrates. This

can be illustrated by compounds 115-118, which showed

only borderline activity in vivo. Whereas the weaker

efficacy of the acylamino derivatives 115 and 116 can

be attributed to their reduced antibacterial potency, the

low efficacy of the rather lipophilic isopropylamino-

methyl derivative 117 and of the rather hydrophilic

hydroxymethyl compound 118 has to be tentatively

explained by unfavorable pharmacokinetics, possibly

due to either high protein binding, large tissue distribu-

tion, and/or rapid elimination, e.g. by glucuronidation.

Antibacterial Agents from Cyclothialidine

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1499

Cytotoxicity in Vitr o. It had been stated that

cyclothialidine did not show any cytotoxic effect against

HeLa cells up to 800 µg/mL, and this was in agreement

with the finding that 1 was not inhibiting mammalian

topoisomerases, e.g. IC₅₀against calf thymus topo-

isomerase II >1000 µg/mL.²¹In view of the more

lipophilic character of our best derivatives, which

undoubtedly allowed them to exert their antibacterial

activity, we wanted to ensure that the increased ability

to penetrate cell membranes did not result in an

unwanted toxic effect against mammalian cells. Thus,

the cytotoxic potential of selected compounds was as-

sessed with HeLa cells (Table 6). The absence of a

significant toxicity confirmed that the affinity of cy-

clothialidine congeners is very specific for the bacterial

type II topoisomerase.

Exp er im en ta l Section

Ch em istr y. Gen er a l. Solvents and chemicals used for

reactions were purchased from commercial suppliers and used

without further purification. Reactions were carried out under

N₂or Ar. If not stated otherwise, aqueous (aq) solutions of

NaHCO₃, Na₂CO₃, and NH₄Cl used in the standard workup

were saturated, aqueous layers were back-extracted with the

organic solvent used, and organic solutions were dried with

Na₂SO₄. Evaporation of solvents and concentration of reaction

mixtures were performed in vacuo at 20-40 °C on a Bu¨chi

rotary evaporator. Thin-layer chromatography (TLC) was

carried out on silica gel glass plates (Kieselgel 60 F254, Merck)

with detection by UV and visualization by spraying with 1%

aq KMnO₄solution or 0.4% aq I₄K₂Pt(II) solution (Meunier,

R.; et al. Bull. Soc. Chim. Biol. 1949, 31, 1144). Normal-phase

silica gel (Silica gel 60, 230-400 mesh, Merck) was used for

preparative chromatography. Melting points (mp) were deter-

mined on a Bu¨chi SMP-20K and are uncorrected. Optical

rotation ([R]_D) was measured on a Perkin-Elmer-241 polarim-

eter, 10 cm, at 20 °C. ¹H NMR spectra were recorded on a

Bruker-AC-250 or on a Bruker-ARX-400. If not stated other-

wise, spectra were recorded at 250 mHz. Chemical shifts are

reported as δ values (ppm) downfield from internal TMS in

the indicated solvent. Coupling constants (J ) are given in Hz;

s ) singlet, d ) doublet, t ) triplet, q ) quartet, m ) multiplet,

br ) broad. Low-resolution EI-mass spectra were recorded with

a Finnigan-MAT SSQ 700 mass spectrometer; ISP-MS and

ISN-MS were recorded on a PE-Sciex API III. High-resolution

mass spectra (HRMS) were determined on a Finnigan-MAT

MAT95. Elemental analyses were carried out in our laboratory

or by Solvias AG (Basel, Switzerland) and results are within

(0.4% of the theoretical values. Target compounds that did

not give satisfactory elemental analyses were analyzed by

HRMS and their purity was assessed by HPLC with two

different experimental systems and was shown to be >95% if

not stated otherwise.

Con clu sion s

The screening technique applied in the discovery of

cyclothialidine provided a lead compound whose out-

standing inhibitory activity against the isolated bacte-

rial enzyme was not translated into an adequate anti-

bacterial activity. A flexible synthetic route has been

developed that allowed us to prepare a variety of

analogues, and the partial structure XI was identified

as the pharmacophoric core of 1. On the basis of this

“minimal structure” many potent inhibitors of DNA

gyrase, belonging to several subclasses, have been

prepared, i.e., bicyclic lactones of different ring sizes as

well as seco-derivatives lacking the lactone ring. Most

importantly, congeners of 1 were found that indeed

displayed considerable in vitro activity against Gram-

positive pathogens. When analyzing the structural

features that brought about the most potent activity

against bacterial cells, it is evident that an increase of

the lipophilic character of the compounds had a positive

impact. At the enzyme level, these structural elements

also contributed to a better affinity via improved

hydrophobic interactions, whereas with regard to the

antibacterial effect, faster permeation of the bacterial

membrane in a presumably passive uptake was benefi-

cial. It became obvious in our investigation that overly

lipophilic compounds showing the most potent in vitro

antibacterial activity did not fulfill the pharmacokinetic

requirements for in vivo efficacy, a general problem for

antibacterials targeting enzymes located in the cyto-

plasm. The optimization for in vivo activity by balancing

physicochemical and antibacterial properties turned out

to be very difficult, and this task was clearly aggravated

by the fact that part of the active compounds was

eliminated by glucuronidation of their essential phenol

group. Nevertheless, by reducing the lipophilic character

of the best compounds through selective introduction

of hydrophilic substituents, derivatives displaying in

vivo efficacy were identified.

Nom en cla tu r e. The nomenclature system was used previ-

ously, and the numbering indicated in formula I (Scheme 1)

was applied for target compounds.^27,28,31

Abbr evia tion s: SiO₂, silica gel; DCM, dichloromethane;

DEAD, diethyl azodicarboxylate; DCC, N,N′-dicyclohexylcar-

bodiimide; EDC, N-(dimethylaminopropyl)-N′-ethylcarbodiim-

ide hydrochloride; NBS, N-bromosuccinimide; TFA, trifluoro-

acetic acid; ^L(D)-3cHyp, cis-3-hydroxy-L(D)-proline; trityl,

triphenylmethyl; thexyl, 1,1,2-trimethylpropyl; R_f, chromato-

graphic retention time.

Meth yl (4R,7S)-7-ter t-bu toxycar bon ylam in o-1,3,4,5,6,7,8,-

10-octa h yd r o-11-m eth yl-6,10-dioxo-9,2,5-ben zoxa th ia a za -

cyclod od ecin e-4-ca r boxyla te (6b) was prepared in five

steps from 2,6-dimethylbenzoic acid using the methodology

described for the synthesis of 6a :¹⁸white powder, mp 150-

1

151 °C; [R]_D) + 31.7° (c ) 1, MeOH); H NMR (DMSO-d₆) δ

1.41 (s, 9H, t-Bu), 2.27 (s, 3H, H-C(11)), 2.85 (dd, 1H, J ) 14,

9, H-C(3)), 3.11 (dd, 1H, J ) 14, 4, H-C(3)), 3.59 (d, 1H, J )

11, H-C(1)), 3.63 (s, 3H, COOMe), 3.96 (d, 1H, J ) 11,

H-C(1)), 4.23 (dd, 1H, J ) 11, 4, H-C(8)), 4.42-4.52 (m, 1H,

H-C(7)), 4.62-4.74 (m, 1H, H-C(4)), 4.86 (dd, 1H, J ) 11, 2,

H-C(8)), 7.15-7.22 (m, 2H, arom H), 7.27 (d, 1H, J ) 8, NH),

7.31 (t, 1H, J ) 7, arom H), 8.28 (d, 1H, J ) 8, NH); MS (ISP)

453.4 (M + H)⁺, 470.2 (M + NH₄)⁺. Anal. (C₂₁H₂₈N₂O₇S) C, H,

N, S.

12,14-Did eoxycycloth ia lid in e (7) was prepared from 6b

in seven steps using the methodology described for the

synthesis of 1:¹⁸white powder; ¹H NMR (400 MHz, D₂O) δ 1.36

(d, 3H, J ) 7.3, Me(Ala)), 2.05-2.16, 2.20-2.31 (2 m, 2H,

H-C(4) (cHyp), 2.33 (s, 3H, Me-C(11)), 2.69 (dd, 1H, J ) 15,

11, H-C(3)), 3.33 (dd, 1H, J ) 15, 4.5, H-C(3)), 3.60 (d, 1H,

J ) 11, H-C(1)), 3.78-3.86 (m, 2H, H-C(5) (cHyp)), 3.86-

3.95 (m, 1H), 3.98-4.07 (m, 2H), 4.11 (q, 1H, J ) 7.3, C(2)

(Ala), 4.40 (dd, 1H, J ) 12, 2, H-C(8)), 4.46 (m, 1H, H-C(2)

(Ser)), 4.70-4.90 (4H, H-C(4), H-C(7), H-C(2)/H-C(3) (cHyp)),

5.64 (dd, J ) 12, 2.5, H-C(8)), 7.26-7.34 (m, 2H, H-C(12),

H-C(14)), 7.42 (t, 1H, J ) 7.6, H-C(13)); HRMS calcd for

(C₂₆H₃₅N₅O₁₀SH⁺) 610.2183, found 610.2181.

Thus, we have demonstrated that the DNA gyrase

inhibitory principle contained in cyclothialidine can be

considered as the basis for a new class of antibacterial

agents. The lack of cytotoxicity against mammalian

cells, shown for optimized derivatives, supports the view

that this class has the potential to provide clinically

useful drugs.

1500 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6

Angehrn et al.

Gen er a l P r ep a r a tion of Com p ou n d s I fr om O-Tr i-

a lk ylsilyl-P r otected P r ecu r sor s. Meth od A-1. Meth yl

(4R,7S)-7-ter t-Bu t oxyca r b on yla m in o-1,3,4,5,6,7,8,10-oc-

ta h yd r o-12,14-d ih yd r oxy-11-m eth yl-6,10-d ioxo-9,2,5-ben -

zoxa th ia a za cyclod od ecin e-4-ca r boxyla te (10). A solution

of methyl (4R,7S)-7-tert-butoxycarbonylamino-12,14-bis[(tert-

butyl)dimethylsilyloxy]-1,3,4,5,6,7,8,10-octahydro-11-methyl-

6,10-dioxo-9,2,5-benzoxa thia a za cyclododecine-4-ca rboxy-

late¹⁸(9, 2.54 g, 3.6 mmol) and NH₄F (0.67 g, 18.0 mmol) in

MeOH (72 mL) was stirred at 20 °C for 0.5 h. The solution

was diluted with EtOAc (150 mL), washed with 5% aq NaCl

(3 × 20 mL), dried, and evaporated. The remaining solid was

crystallized from EtOAc/hexane to give 10 (1.47 g, 84%): white

Meth od B-2. EDC a s Cou p lin g Au xilia r y. N-(3-Tr ityl-

oxyp r op ion yl)cystea m in e (13). To a mixture of cysteamine‚

HCl (0.45 g, 4.0 mmol) and 3-trityloxypropionic acid (82b, 1.33

g, 4.0 mmol) in MeCN (10 mL) were added at 0 °C N-methyl-

morpholine (0.44 mL, 4.0 mmol) and EDC (0.77 g, 4.0 mmol),

and stirring was continued for 3 h at 0 °C. The mixture was

diluted with EtOAc, washed with 1 N aq HCl, 5% aq NaHCO₃,

and brine, dried, and evaporated. The residual oil was chro-

matographed (SiO₂; EtOAc/hexane 1:2) and the purified prod-

uct was crystallized from EtOAc/hexane to give 13 (0.68 g,

1

43%): white solid, mp 151-153 °C; H NMR (CDCl₃) δ 1.29

(t, 1H, J ) 9), 2.48 (t, 2H, J ) 6), 2.56-2.67 (m, 2H), 3.32-

3.47 (m, 4H), 6.64 (br t, 1H), 7.19-7.36 (m, 9H), 7.38-7.49

(m, 6H).

1

solid, mp 122-128 °C (dec); [R]_D) +54.3° (c ) 1, EtOAc); H

NMR (DMSO-d₆) δ 1.42 (s, 9H), 1.89 (s, 3H), 2.84 (dd, 1H, J )

14, 9), 3.02 (dd, 1H, J ) 14, 4), 3.42 (d, 1H, J ) 11), 3.63 (s,

3H), 3.81 (d, 1H, J ) 11), 4.19 (dd, 1H, J ) 11, 4), 4.31-4.41

(m, 1H), 4.46-4.57 (m, 1H), 4.88 (dd, 1H, J ) 11, 2), 6.44 (s,

1H), 7.22 (d, 1H, J ) 8), 8.18 (d, 1H, J ) 8), 9.49 (s, 1H), 9.51

(s, 1H); MS (ISP) 485.5 (M + H)⁺. Anal. (C₂₁H₂₈N₂O₉S) C, H,

N, S.

Using method B-2, compounds 16 and 17 were prepared

similarly.

Meth yl N-(N-ter t-bu toxyca r bon yl-L-ser yl)-^D-cystein a te

(16) was prepared from d-Cys-OMe‚HCl (3.43 g, 20.0 mmol)

and Boc-^L-Ser-OH (4.1 g, 20.0 mmol): white solid (from Et₂O/

hexane) (1.23 g, 20%), mp 104-105 °C; ¹H NMR (CDCl₃)

“identical” with that of 15.

Gen er a l P r oced u r es for th e P r ep a r a tion of Dip ep tid es

11-18. Meth od B-1. DCC a s Cou p lin g Au xilia r y. N-(N-

ter t-Bu toxyca r bon yl-^D-ser yl)cystea m in e (11). To a mix-

ture of cysteamine hydrochloride (5.68 g, 0.05 mol) and Boc-

L-Ser-OH (10.26 g, 0.05 mol) in MeCN (0.15 L) was added at

0 °C N-methylmorpholine (5.5 mL, 0.05 mol). To the stirred

solution was added over 30 min at -10 to -5 °C a solution of

DCC (10.3 g, 0.05 mol) in MeCN (0.15 L), and stirring was

continued for 3 h at 5 °C. The precipitate formed was removed

by filtration and the filtrate was evaporated. The residual oil

was dissolved in EtOAc (0.15 L), and the solution was washed

with 0.5 N aq HCl, 5% aq NaHCO₃, and brine, dried, and

evaporated. The residual oil was chromatographed (SiO₂;

EtOAc/hexane 1:1) and the purified product was crystallized

from EtOAc/hexane to give 11 (3.42 g, 26%): white crystals,

mp 95-96 °C; [R]_D) -18.0° (c ) 0.4, EtOH); ¹H NMR (CDCl₃)

δ 1.43 (t, 1H, J ) 9) superimposed by 1.46 (s, 9H), 2.62-2.73

(m, 2H), 3.14 (br s, 1H), 3.38-3.55 (m, 2H), 3.58-3.73 (m, 1H),

4.07-4.19 (m, 2H), 5.57 (br d, 1H, J ) 8), 7.07 (br s, 1H); MS

(EI) 208 (M - C₄H₈)⁺. Anal. (C₁₀H₂₀N₂O₂S) C, H, N, S.

Meth yl (R)-2-((S)-3-h yd r oxybu tyr yla m in o)-3-m er ca p -

top r op ion a te (17) was prepared from l-Cys-OMe‚HCl (3.43

g, 20.0 mmol) and (S)-3-hydroxy-butyric acid (2.1 g, 20.0

mmol): white crystals (from DCM/hexane) (1.86 g, 42%), mp

84-86 °C; [R]_D) +37.7° (c ) 0.9, EtOAc); ¹H NMR (CDCl₃) δ

1.26 (d, 3H, J ) 6.3), 1.37 (t, 1H, J ) 9), 2.37 (dd, 1H, J ) 15,

8.4), 2.47 (dd, 1H, J ) 15, 3), 2.94-3.14 (m, 2H), 3.48 (br s,

1H), 3.81 (s, 3H), 4.15-4.32 (m, 1H), 4.87-4.96 (m, 1H), 6.74

(d, 1H, J ) 7).

Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s

I fr om Su bstitu ted Ben zoic Acid s VII. (a ) Meth od C.

P r ep a r a tion of 4-Nitr oben zyl Ester s. 4-Nitr oben zyl 3,5-

Dih yd r oxy-2-m eth ylben zoa te (20a ). To a solution of 3,5-

dihydroxy-2-methylbenzoate¹⁸(19a , 33.6 g, 0.20 mol) in DMF

(0.15 L) was added at 10 °C N,N,N′,N′-tetramethylguanidine

(25.2 mL, 0.20 mol). After stirring for 30 min at 20 °C,

4-nitrobenzyl bromide (43.2 g, 0.20 mol) was added, and

stirring was continued for 72 h at 20 °C. The mixture was

concentrated, diluted with EtOAc (0.2 L), and washed with 2

N aq HCl (2 × 0.15 L), aq NaHCO₃(2 × 0.15 L), and brine

(0.15 L). The organic layer was dried and evaporated. The solid

residue was recrystallized from EtOAc/hexane to give 20a (47.1

Using method B-1, compounds 12, 14, 15, and 18 were

prepared similarly.

Meth yl (R)-2-(3-tr ityloxyp r op ion yla m in o)-3-m er ca p -

top r op ion a te (12) was prepared from l-Cys-OMe‚HCl (8.58

g, 0.05 mol) and 3-trityloxypropionic acid (82b, 16.62 g, 0.05

mol): white crystals (from DCM/hexane) (19.32 g, 86%), mp

1

g, 78%): yellow crystals, mp 156-158 °C; H NMR (DMSO-

d₆) δ 2.18 (s, 3H), 5.42 (s, 2H), 6.51/6.72 (2 d, 2 × 1H, J ) 2.5),

7.71/8.27 (2 d, 2 × 2H, J ) 9), 9.37/9.58 (2 s, 2 × 1H).

1

78-80 °C; H NMR (CDCl₃) δ 1.29 (t, 1H, J ) 9), 2.52 (t, 2H,

(b ) Met h od D. Tr ia lk ylsilyla t ion of P h en olic OH

Gr ou p s. 4-Nitr oben zyl 3,5-Bis[(ter t-bu tyl)d im eth ylsilyl-

oxy]-2-m eth ylben zoa te (21a ). To a stirred mixture of 20a

(3.03 g, 10.0 mmol) and (t-Bu)Me₂SiCl (4.52 g, 30.0 mol) in

DMF (10 mL) was added at 0 °C Et₃N (4.2 mL, 30.0 mmol), a

precipitate being formed immediately. The reaction mixture

was stirred at 0 °C for 6 h and then diluted with EtOAc (100

mL), extracted with 1 N aq HCl (50 mL) and H₂O (4 × 50 mL),

dried, and evaporated. The residual oil was crystallized from

hexane to give 13 (3.20 g, 60%): white crystals, mp 72-73

J ) 5.7), 2.97 (dd, 2H, J ) 9, 4), 3.42 (t, 2H, J ) 5.7), 3.71 (s,

3H), 4.68-4.95 (m, 1H), 7.14 (d, 1H, 7.4), 7.19-7.34 (m, 9H),

7.42-7.48 (m, 6H).

Meth yl N-(N-ter t-bu toxyca r bon yl-^D-ser yl)-D-cystein a te

(14) was prepared from d-Cys-OMe‚HCl (1.72 g, 10.0 mmol)

and Boc-^D-Ser-OH (2.05 g, 10.0 mmol): white crystals (from

Et₂O/hexane) (1.82 g, 56%), mp 73-75 °C; [R]_D) -4.3° (c ) 1,

EtOAc); ¹H NMR (CDCl₃) “identical” with that of methyl N-(N-

tert-butoxycarbonyl-^L-seryl)-L-cysteinate¹⁸(5).

Meth yl N-(N-ter t-bu toxyca r bon yl-^D-ser yl)-L-cystein a te

(15) was prepared from ^L-Cys-OMe‚HCl (18.9 g, 0.11 mol) and

Boc-^D-Ser-OH (22.6 g, 0.11 mol): white solid (from Et₂O/

hexane) (12.2 g, 34%), mp 104-106 °C; ¹H NMR (CDCl₃) δ 1.47

(s, 9H) superimposed by 1.49 (t, 1H, J ) 9), 2.90-3.05 (br s,

1H), 3.02 (dd, 2H, J ) 8, 4), 3.77 (dd, 1H, J ) 12, 4), 3.79 (s,

3H), 4.11 (dd, 1H), 4.19-4.29 (m, 1H), 4.84 (dt, 1H, J ) 8, 4),

5.60 (br d, 1H, J ) 8), 7.43 (br d, J ) 8).

Meth yl (R)-2-((R)-3-h yd r oxybu tyr yla m in o)-3-m er ca p -

top r op ion a te (18) was prepared from l-Cys-OMe‚HCl (8.2 g,

48.0 mmol) and (R)-3-hydroxybutyric acid (5.0 g, 48.0 mmol):

white crystals (from EtOAc/hexane) (3.41 g, 32%), mp 64-66

°C; [R]_D) -1.3° (c ) 0.8, EtOAc); ¹H NMR (CDCl₃) δ 1.26 (d,

3H, J ) 6.2), 1.42 (t, 1H, J ) 9), 2.37 (dd, 1H, J ) 15, 8.4),

2.48 (dd, 1H, J ) 15, 3.3), 3.02 (dd, 2H, J ) 9, 4.3), 3.44 (d,

1H, J ) 3.6), 3.80 (s, 3H), 4.14-4.30 (m, 1H), 4.87-4.95 (m,

1H), 6.84 (d, 1H, J ) 8).

1

°C; H NMR (CDCl₃) δ 0.20 (s, 6H), 0.22 (s, 6H), 0.98 (s, 9H),

1.02 (s, 9H), 2.33 (s, 3H), 5.41 (s, 2H), 6.50/7.00 (2 d, 2 × 1H,

J ) 2.5), 7.59/8.25 (2 d, 2 × 2H, J ) 8.5).

(c) Meth od E. Ben zylic Br om in a tion of 2-Meth ylben -

zoa tes. 4-Nitr oben zyl 2-(Br om om eth yl)-3,5-bis[(ter t-bu -

tyl)d im eth ylsilyloxy]-6-m eth ylben zoa te (22a ). To a solu-

tion of 21a (10.64 g, 20 mmol) in CCl₄(100 mL) was added

NBS (3.56 g, 20 mmol). The stirred suspension was heated at

reflux and irradiated with light (250 W) until all the insoluble

material (succinimide) was floating at the surface of the

solution (40 min). The mixture was cooled to 0 °C. Insoluble

material was removed by filtration, the solution was evapo-

rated, and the residual material was crystallized from hexane

to give 22a (11.54 g, 94%): white crystals, mp 101-103 °C;

¹H NMR (CDCl₃) δ 0.22 (s, 6H), 0.30 (s, 6H), 0.98 (s, 9H), 1.06

(s, 9H), 4.96 (s, 2H), 5.46 (s, 2H), 6.51/7.06 (2 d, 2 × 1H, J )

2.5), 7.62/8.25 (2 d, 2 × 2H, J ) 8.5).

Antibacterial Agents from Cyclothialidine

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1501

(d ) Meth od F . Alk yla tive Th iola tion of 2-Br om om eth -

ylben zoa tes. Meth yl N-[N-ter t-Bu toxyca r bon yl-^L-ser yl]-

S-[2,4-bis[(ter t-bu tyl)d im eth ylsilyloxy]-6-(4-n itr oben zyl-

oxyca r bon yl)-ben zyl]-^L-cystein a te (23a ). A stirred solution

of 22a (6.1 g, 10.0 mmol) and 5¹⁸(3.22 g, 10 mmol) in DCM

(80 mL) was cooled to 0 °C and treated with Et₃N (1.4 mL, 10

mmol). Stirring was continued for 1 h at 0 °C and for 1 h at

20 °C. The solution was washed with 5% aq NaHCO₃and

brine, dried, and evaporated. The residual oil (9.68 g) was

chromatographed (SiO₂; EtOAc/hexane 1:2) to give 23a (5.66

g, 66%), colorless foam: ¹H NMR (CDCl₃) δ 0.19/0.26 (2 s, 2 ×

6H), 0.97/1.03 (2 s, 2 × 9H), 1.43 (s, 9H); 2.86-3.10 (m, 3H),

3.65-3.75 (m, 1H) superimposed by 3.70 (s, 3H), 4.00-4.18

(m, 2H), 4.38 (m, 1H), 4.81 (m, 1H), 5.49 (s, 2H), 5.59 (d, 1H),

6.52/7.04 (2 d, 2 × 1H, J ) 2.5), 7.40 (br d, 1H, J ) 8), 7.63/

8.25, (2 d, 2 × 2H, J ) 9).

4-Nitr oben zyl 3-[Dim eth yl(th exyl)silyloxy]-2,6-d im e-

th ylben zoa te (21b). Using method D (21a ), 20b (0.91 g, 3.0

mmol) was silylated with (thexyl)Me₂SiCl (0.65 mL, 3.3 mmol)

to give 21b (0.99 g, 74%): white crystals (from hexane), mp

77-78 °C; ¹H NMR CDCl₃) δ 0.23 (s, 6H), 0.94 (d, 6H, J )

6.8), 0.96 (s, 6H), 1.76 (m, 1H), 2.11/2.19 (2 s, 2 × 3H), 5.44 (s,

1H), 6.73/6.88 (2 d, 2 × 1H, J ) 8), 7.59/8.23 (2 d, 2 × 2H, J

) 9).

4-Nitr oben zyl 2/6-(Br om om eth yl)-3-[d im eth yl(th exyl)-

silyloxy]-6/2-m eth ylben zoate (22b/c). Using method E (22a),

21b (0.89 g, 2.0 mmol) was brominated with NBS (0.36, 2.0

mmol) to give a light-yellow oil (1.02 g) consisting (NMR

analysis) of 21b (0.32 g), 22b (0.26 g, 25%), 22c (0.36 g, 34%),

and 4-nitrobenzyl 2,6-bis(bromomethyl)-3-[dimethyl(thexyl)-

silyloxy)]benzoate (0.08 g). This mixture was directly used in

the next step. 22b: ¹H NMR (CDCl₃) (partial) δ 0.26 (s, 6H),

2.14 (s, 3H), 4.49 (s, 2H), 5.49 (s, 2H), 6.79/7.12 (2 d, 2 × 1H,

J ) 8.8), 7.66/8.24 (2 d, 2 × 2H, J ) 9). 22c: ¹H NMR (CDCl₃)

(partial) δ 0.32 (s, 6H), 2.22 (s, 3H), 4.56 (s, 2H), 5.49 (s, 2H),

6.80/7.04 (2 d, 2 × 1H, J ) 8.8), 7.67/8.24 (2 d, 2 × 2H, J ) 9).

Dibromo side product: ¹H NMR (CDCl₃) (partial) δ 0.33 (s,

6H), 4.50/4.59 (2 s, 2 × 2H), 5.54 (s, 2H). (A NOE was observed

between the aromatic methyl group and one of the phenyl

hydrogens for the compound that was assigned structure 22b,

whereas for 22c no NOE was found.)

(e) Meth od G. Hyd r ogen a tive Clea va ge of 4-Nitr oben -

zyl Ester s. Meth yl N-(N-ter t-Bu toxyca r bon yl-^L-ser yl)-S-

[2,4-bis[(ter t-bu tyl)d im eth ylsilyloxy]-6-ca r boxylben zyl]-

L-cystein a te (24a ). A mixture of 23a (5.66 g, 6.64 mmol) and

5% Pd-C (1.13 g) in EtOAc (70 mL) was stirred under H₂at

20 °C for 18 h. The reaction mixture was filtered and the

solution was evaporated. The residual oil was stirred with

MeOH (60 mL) for 1 h at 20 °C. The mixture was evaporated

and the residual oil was dissolved in EtOAc (100 mL). Insoluble

material was removed by filtration, and the solution was

washed with 1 N aq HCl (30 mL) and brine (2 × 20 mL), dried,

and evaporated to give 24a (4.30 g, 90%): white foam; ¹H NMR

(300 MHz, DMSO-d₆) δ 0.20 (s, 6H), 0.24/0.25 (2 s, 2 × 3H),

0.95/0.99 (2 s, 2 × 9H), 1.38 (s, 9H), 2.68-2.87 (m, 2H), 3.40-

3.60 (m, 2H) superimposed by 3.58 (s, 3H), 4.04/4,13 (2 d, 2 ×

1H, J ) 12) superimposed by 3.95-4.07 (m, 1H), 4.28-4.46

(m, 1H), 4.72-4.80 (m, 1H), 6.44 (d, 1H, J ) 2.7), 6.58 (d, 1H,

J ) 8), 6.89 (d, 1H, J ) 2.7), 8.15 (d, 1H, J ) 8), 13.05 (br s,

1H); MS (ISN) 715.5 (M - H)^-.

Met h yl N-(N-ter t-Bu t oxyca r b on yl-^L-ser yl)-S-[6- a n d

4-(d im eth yl(th exyl)silyloxy)-2-((4-n itr oben zyloxyca r bo-

n yl)-3-m et h ylben zyl)-^L-cyst ein a t e (23b a n d 23c). Using

method F (23a ), the crude bromination mixture 22b/c [1.0 g,

containing 22b (0.25 g, 0.48 mmol) and 22c (0.35 g, 0.67

mmol)] was reacted with 5 (0.64 g, 2.0 mmol) to give, after

chromatographic separation (SiO₂; EtOAc/hexane 1:2), 23b

(0.26 g, 71%) and 23c (0.39 g, 76%). 23b: colorless foam; TLC

1

R_f) 0.31 (EtOAc/hexane 1:1); H NMR (400 MHz, CDCl₃) δ

0.26/0.28 (2 s, 2 × 3H), 0.94 (d, 6H, J ) 6.8), 0.98 (s, 6H), 1.45

(s, 9H), 1.75 (m, 1H), 2.22 (s, 3H), 2.92 (d, 2H, J ) 5), 3.01 (br

t, 1H), 3.65 (m, 1H), 3.70 (s, 3H), 3.81/3.89 (2 d, 2 × 1H, J )

13), 4.06 (m, 1H), 4.22 (m, 1H), 4.70 (m, 1H), 5.49 (s, 1H), 5.60

(br d, 1H), 6.79/6.98 (2 d, 2 × 1H, J ) 8.4), 7.03 (br d, 1H),

7.67/8.25 (2 d, 2 × 2H, J ) 8.8) (NOE between s at 2.22 and

d at 6.98). 23c: colorless foam; TLC: R_f) 0.23 (EtOAc/hexane

1:1); ¹H NMR (400 MHz, CDCl₃) δ 0.25 (s, 6H), 0.94 (d, 6H, J

) 6.8), 0.97 (s, 6H), 1.45 (s, 9H), 1.75 (m, 1H), 2.13 (s, 3H),

2.77 (dd, 1H, J ) 14, 7), 2.90 (dd, 1H, J ) 14, 5), 2.95 (m, 1H),

3.71 (m, 1H) superimposed by 3.70 (d, 1H, J ) 9.6) and 3.72

(s, 3H), 3.76 (d, 1H, J ) 9.6), 4.11/4.27/4.45 (3 m, 3 × 1H),

5.47 (s, 1H), 5.59 (br d, 1H), 6.77/7.01 (2 d, 2 × 1H, J ) 8.4),

7.18 (br d, 1H), 7.64/8.25 (2 d, 2 × 2H, J ) 8.8) (No NOE

between s at 2.13 and d at 6.77 or 7.01).

(f) Meth od H-1. Cycliza tion of ω-Hyd r oxy Ca r boxylic

Acid s. Meth yl (4R,7S)-7-ter t-Bu toxyca r bon yla m in o-12,-

14-b is[(ter t-b u t yl)d im et h ylsilyloxy]-1,3,4,5,6,7,8,10-oc-

ta h yd r o-6,10-d ioxo-9,2,5-ben zoxa th ia a za cyclod od ecin e-

4-ca r boxyla te (25a ). To a solution of 24a (4.3 g, 6.0 mmol)

in toluene (150 mL) were added at 0 °C TPP (2.04 g, 7.8 mmol)

and 95% DEAD (1.28 mL, 7.8 mmol). The reaction mixture

was stirred for 15 min at 0 °C and for 12 h at 20 °C and then

evaporated. The residual oil was dissolved in DCM and

submitted to chromatographic purification (SiO₂; EtOAc/

hexane 1:1) to give 25a (3.36 g, 80%): amorphous solid; ¹H

NMR (300 MHz, CDCl₃) δ 0.20 (s, 6Η), 0.24/0.27 (2 s, 2 × 3H),

0.98/1.04 (2 s, 2 × 9H), 1.49 (s, 9H), 2.95-3.08 (m, 1H), 3.20

(dd, 1H, J ) 14, 5), 3.76 (s, 3H), 3.86/4.13 (2 d, 2 × 1H, J )

10.5), 4.48 (dd, 1H, J ) 11.4, 2); 4.54-4.64 (m, 1H), 4.81-

4.93 (m, 1H), 5.03 (dd, 1H, J ) 11.4, 3), 6.46/6.90 (2 d, 2 ×

1H, J ) 2.4), 7.21 (d, 1H, J ) 8); MS (ISP) 699.5 (M + H)⁺.

Meth yl (4R,7S)-7-ter t-bu toxycar bon ylam in o-1,3,4,5,6,7,8,-

10-oct a h yd r o-12,14-d ih yd r oxy-6,10-d ioxo-9,2,5-b en zox-

a th ia a za cyclod od ecin e-4-ca r boxyla te (26a ) was prepared

from 25a (1.40 g, 2.0 mmol) using method A-1 (10): white solid

(from EtOAc/hexane) (0.80 g, 85%), mp 146-150 °C; ¹H NMR

(300 MHz, DMSO-d₆) δ 1.42 (s, 9H), 2.88 (dd, 1H, J ) 14, 11),

3.04 (dd, 1H, J ) 14, 4), 3.64 (s, 3H), 3.84/4.22 (2 d, 2 × 1H,

J ) 10), 4.29 (dd, 1H, J ) 10, 2), 4.30-4.44 (m, 1H), 4.57-

4.72 (m, 1H), 4.75 (dd, 1H, J ) 10, 3), 6.50/6.71 (2 d, 2 × 1H,

J ) 2.4), 7.39 (d, 1H, J ) 8), 8.25 (d, 1H, J ) 8), 9.57/9.77 (2

s, 2 × 1H); MS (ISP) 471.3 (M + H)⁺. Anal. (C₂₀H₂₆N₂O₉S‚

0.3H₂O) C, H, N, S.

Meth yl (4R,7S)-7-ter t-bu toxycar bon ylam in o-1,3,4,5,6,7,8,-

10-octa h yd r o-14-h yd r oxy-11-m eth yl-6,10-d ioxo-9,2,5-ben -

zoxa th ia a za cyclod od ecin e-4-ca r boxyla te (26b) was pre-

pared from 23b (0.25 g, 0.33 mmol) by consecutively applying

methods G (24a ), H-1 (25a ), and A-1 (10): white crystals (from

1

EtOAc/hexane) (46 mg); H NMR (DMSO-d₆) δ 1.41 (s, 9H),

2.12 (s, 3H), 2.89 (dd, 1H, J ) 14, 9), 3.08 (dd, 1H, J ) 14, 4),

3.52 (d, 1H, J ) 11), 3.63 (s, 3H), 3.93 (d, 1H, J ) 11), 4.17

(dd, 1H, J ) 11, 3), 4.31-4.43 (m, 1H), 4.50-4.64 (m, 1H),

4.93 (dd, 1H, J ) 11, 2), 6.80/6.97 (2 d, 2 × 1H, J ) 8), 7.28 (d,

1H, J ) 8), 8.09 (d, 1H, J ) 8), 9.69 (s, 1H); MS (ISP) 469.1

(M + H)⁺. Anal. (C₂₁H₂₈N₂O₈S) C, H, N, S.

Meth yl (4R,7S)-7-ter t-bu toxycar bon ylam in o-1,3,4,5,6,7,8,-

10-octa h yd r o-12-h yd r oxy-11-m eth yl-6,10-d ioxo-9,2,5-ben -

zoxa th ia a za cyclod od ecin e-4-ca r boxyla te (26c) was pre-

pared from 23c (0.38 g, 0.50 mmol), using the reaction

sequence of the preparation of 26b from 23b: white solid (79

mg); ¹H NMR (DMSO-d₆) δ 1.41 (s, 9H), 2.03 (s, 3H), 2.80 (dd,

1H, J ) 14, 9), 3.07 (dd, 1H, J ) 14, 4), 3.48 (d, 1H, J ) 11),

3.63 (s, 3H), 3.81 (d, 1H, J ) 11), 4.22 (dd, 1H, J ) 11, 3),

4.28-4.40 (m, 1H), 4.47-4.60 (m, 1H), 4.83 (dd, 1H, J ) 11,

2), 6.82/6.98 (2 d, 2 × 1H, J ) 8), 7.23 (d, 1H, J ) 8), 8.25 (d,

1H, J ) 8), 9.70 (s, 1H); MS (ISP) 469.4 (M + H)⁺. Anal.

(C₂₁H₂₈N₂O₈S) C, H, N, S.

Syn th esis of Meth yl (4R,7S)-7-ter t-bu toxycar bon ylam i-

n o-1,3,4,5,6,7,8,10-octa h yd r o-14-h yd r oxy-11-m eth yl-6,10-

d ioxo-9,2,5-b e n zoxa t h ia a za cyclod od e cin e -4-ca r b oxy-

late (26b). 4-Nitr oben zyl 3-h ydr oxy-2,6-dim eth ylben zoate

(20b) was prepared from 3-hydroxy-2,6-dimethylbenzoic acid²⁶

(19b, 0.67 g, 4.0 mol), using method C (20a ): pale-yellow

crystals (from EtOAc/hexane) (0.92 g, 76%), mp 124-126 °C;

¹H NMR (CDCl₃) δ 2.15 (s, 3 H), 2.19 (s, 3H), 4.80 (s, 1H),

5.45 (s, 2H), 6.74/6.92 (2 d, 2 × 1H, J ) 8), 7.62 (d, 2H, J ) 9),

7.62/8.25 (2 d, 2 × 2H, J ) 9).

1502 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6

Angehrn et al.

ter t-Bu tyl (S)-1,3,4,5,6,7,8,10-octah ydr o-12,14-dih ydr oxy-

11-m eth yl-6,10-dioxo-9,2,5-ben zoxath iaazacyclododecin e-

7-ca r ba m a te (26d ) was prepared from 11 (1.23 g, 5.0 mmol)

and crude 4-nitrobenzyl 2-(bromomethyl)-3,5-bis[(tert-butyl)-

dimethylsilyloxy]-6-methylbenzoate (22d )¹⁸(3.37 g containing

ca. 2.1 g, 3.35 mmol, of 22d ) by consecutively applying methods

F (23a ), G (24a ), H-1 (25a ), and A-1 (10): white solid (from

(15.8 g) consisting (NMR analysis) of 21g (ca. 2.5 g), 22g (5.06

g, 32%), 22h (5.69 g, 36%), and 4-nitrobenzyl 2,6-bis(bromo-

methyl)-3-[(tert-butyl)dimethylsilyloxy]-5-methoxybenzoate (2.5

g). 22g: ¹H NMR (CDCl₃) δ 0.24 (s, 6H), 1.01 (s, 9H), 2.05 (s,

3H), 3.84 (s, 3H), 4.54 (s, 2H), 5.48 (s, 2H), 6.41 (s, 1H), 7.67/

8.24 (2 d, 2 × 2H, J ) 8). 22h : ¹H NMR (CDCl₃) δ 0.30 (s,

6H), 1.05 (s, 9H), 2.04 (s, 3H), 3.78 (s, 3H), 4.53 (s, 2H), 5.48

(s, 2H), 6.39 (s, 1H), 7.67/8.24 (2 d, 2 × 2H, J ) 8). 2,6-Bis-

(bromomethyl) side product: ¹H NMR (CDCl₃) δ 0.32 (s, 6H),

1.04 (s, 9H), 3.87 (s, 3H), 4.51 (s, 2H), 5.54 (s, 2H), 6.44 (s,

1H), 7.71/8.24 (2 d, 2 × 2H, J ) 8).

1

EtOAc/hexane) (110 mg), mp 215 °C (dec); H NMR (DMSO-

d₆) δ 1.39 (s, 9H), 1.89 (s, 3H), 2.42-2.59 (m, lH), 2.65-2.75

(m, lH), 2.98-3.15 (m, lH), 3.50-3.70 (m, 1H) superimposed

by 3.58/3.66 (2 d, 2 × 1H, J ) 11), 4.26-4.55 (m, 3H), 4.26-

4.55 (m, 3H), 6.44 (s, lH), 7.03 (d, 1H, J ) 7), 8.13-8.24 (m,

lH), 9.45/9.49 (2 s, 2 x lH); MS (ISP) 427 (M + H)⁺. Anal.

(C₁₉H₂₆N₂O₇S) C, H, N, S.

Meth yl N-[N-ter t-Bu toxyca r bon yl-^L-ser yl]-S-[6/4-[(ter t-

b u t yl)d im et h ylsilyloxy]-4/6-m et h oxy-3-m et h yl-2-(4-n i-

tr oben zyloxycar bon yl)ben zyl]-^L-cystein ate (23g an d 23h ).

Using method F (23a ), the crude mixture 22g/h (15.0 g,

containing ca. 4.8 g, 9.2 mmol, of 22g, and 5.4 g, 10.3 mmol,

of 22h ) was reacted with 5 (9.63 g, 30 mmol), and the product

mixture was separated by chromatography (SiO₂; EtOAc/

hexane 2:3) to give 23g (5.15 g, 27%) and 23h (4.90 g, 26%).

23g: white foam; TLC R_f) 0.27 (EtOAc/hexane 1:1); ¹H NMR

(CDCl₃) δ 0.25/0.26 (2 s, 2 × 3H), 1.02 (s, 9H), 1.45 (s, 9H),

2.05 (s, 3H), 2.91 (d, 2H, J ) 5.4), 3.00 (br t, 1H), 3.62-3.72

(m, 1H), 3.70 (s, 3H), 3.74 (d, 1H, J ) 12), 3.77 (s, 3H), 3.81

(d, 1H, J ) 12), 4.02-4.12 (m, 1H), 4.16-4.28 (m, 1H), 4.66-

4.76 (m, 1H), 5.49 (s, 2H), 5.59 (d, 1H, J ) 8), 6.40 (s, 1H),

7.06 (d, 1H, J ) 8), 7.66/8.28 (2 d, 2 × 2H, J ) 8.8). 23h : white

Meth yl (R)-1,3,4,5,6,7,8,10-Octa h yd r o-12,14-d ih yd r oxy-

11-m eth yl-6,10-dioxo-9,2,5-ben zoxath iaazacyclododecin e-

4-ca r boxyla te (26e). Using method F (23a ), 12 (1.35 g, 3.0

mmol) and crude 22d ¹⁸(2.0 g containing ca. 1.25 g, 2.0 mmol,

of 22d ) were coupled and the product was purified by chro-

matography (SiO₂, EtOAc/hexane 1:2) to give a white foam

(0.72 g) [TLC R_f) 0.87 (EtOAc/hexane 1:1)]. To cleave off the

trityl group, a solution of this material and pTsOH‚H₂O (20

mg) in MeOH (10 mL) was heated to 60 °C for 1 h. The solution

was evaporated and the residue was chromatographed (SiO₂;

EtOAc/hexane 1:1) to give 23e (0.38 g): white foam; TLC R_f

) 0.10 (EtOAc/hexane 1:1); ¹H NMR (CDCl₃) δ 0.21/0.22/0.24/

0.25 (4 s, 4 × 3H), 1.00/1.01 (2 s, 2 × 9H), 2.03 (s, 3H), 2.32-

2.50 (m, 2H), 2.84 (dd, 1H, J ) 14, 4), 3.01 (dd, 1H, J ) 14, 5),

3.69 (s, 3H), 3.72 (d, 1H, J ) 12), 3.77-3.88 (m, 3H), 4.70-

4.80 (m, 1H), 5.47 (s, 2H), 6.36 (s, 1H), 6.60 (d, 1H, J ) 8),

7.66/8.26 (2 d, 2 × 2H, J ) 9). Subjecting 23e in a consecutive

manner to methods G (24a ), H-1 (25a ), and A-1 (10) afforded

26e: white solid (48 mg), mp 184-188 °C (dec); ¹H NMR (400

mHz, DMSO-d₆) δ 1.86 (s, 3H), 2.40-2.47 (m, 1H), 2.58 (dd,

1H, J ) 15, 11), 2.84-2.93 (m, 1H), 3.06 (dd, 1H, J ) 15, 4),

3.65 (s, 3H), 3.66-3.69 (m, 2H), 4.47-4.53 (m, 1H), 4.59-4.69

(m, 2H), 6.42 (s, 1H), 8.55 (d, 1H, J ) 9), 9.45 (s, 1H), 9.49 (s,

1H). Anal. (C₁₆H₁₉NO₇S) C, H, N, S.

1

foam; TLC R_f) 0.20 (EtOAc/hexane 1:1); H NMR (CDCl₃) δ

0.23 (s, 6H), 1.01 (s, 9H), 1.45 (s, 9H), 2.03 (s, 3H), 2.80 (dd,

1H, J ) 14, 6), 2.96 (dd, 1H, J ) 14, 4), 3.08 (br t, 1H), 3.62-

3.76 (m, 1H), 3.72 (s, 3H), 3.74 (d, 1H, J ) 12), 3.80 (s, 3H),

3.81 (d, 1H, J ) 12), 4.06-4.17 (m, 1H), 4.22-4.32 (m, 1H),

4.75-4.87 (m, 1H), 5.47 (s, 2H), 5.59 (d, 1H, J ) 8), 6.42 (s,

1H), 7.17 (d, 1H, J ) 8), 7.65/8.25 (2 d, 2 × 2H, J ) 9).

Meth yl (R)-14-[(ter t-Bu tyl)dim eth ylsilyloxy]-1,3,4,5,6,7,8,-

10-oct a h yd r o-12-m et h oxy-11 -m et h yl-6,10-d ioxo-9,2,5-

ben zoxa th ia a za cyclod od ecin e-4-ca r boxyla te (25g). Using

method G (24a ), 23g (5.15 g, 6.72 mmol) was hydrogenated.

The crude hydroxy acid (24g, 3.57 g, 84%, pale-yellow foam)

was cyclized using method H-1 (25a ) to give 25g (1.83 g,

53%): white crystals (from EtOAc/hexane), mp 156-158 °C;

¹H NMR (DMSO-d₆) δ 0.25/0.26 (2 s, 2 × 3H), 1.02 (s, 9H),

1.41 (s, 9H), 1.96 (s, 3H), 2.81 (dd, 1H, J ) 14, 10), 3.06 (dd,

1H, J ) 14, 4), 3.51 (d, 1H, J ) 11), 3.62 (s, 3H), 3.77 (s, 3H),

3.85 (d, 1H, J ) 11), 4.17 (dd, 1H, J ) 11, 4), 4.33-4.41/4.49-

4.63 (2 m, 2 × 1H), 4.99 (dd, 1H, J ) 11, 2), 6.43 (s, 1H), 7.14

(d, 1H, J ) 8), 8.23 (d, 1H, J ) 8). The structure of 25g was

confirmed by X-ray analysis.²⁴

1,3,4,5,6,7,8,10-Octa h yd r o-12,14-d ih yd r oxy-11-m eth yl-

6,10-d ioxo-9,2,5-ben zoxa th ia a za cyclod od ecin e (26f). Us-

ing the procedure of the preparation of 26e, but replacing the

thiol 12 by 13, the lactone 26f was prepared (110 mg): white

solid (from EtOAc/hexane), mp 215-220 °C; ¹H NMR (DMSO-

d₆) δ 1.87 (s, 3H), 2.53-2.66 (m, 4H), 3.26-3.34 (m, 2H), 3.63

(s, 2H), 4.49-4.59 (m, 2H), 6.41 (s, 1H), 7.65 (t, 1H, J ) 6),

9.41 (s, 1H), 9.47 (s, 1H). Anal. (C₁₄H₁₇NO₅S‚0.25H₂O) C, H,

N, S.

4-Nitr oben zyl 3-Hyd r oxy-5-m eth oxy-2,6-d im eth ylben -

zoa te (20g). A mixture of 4-nitrobenzyl 3,5-dihydroxy-2,6-

dimethylbenzoate¹⁸(20d , 95.1 g, 0.30 mol), methyl iodide (22.5

mL, 0.36 mol), and K₂CO₃(49.8 g, 0.36 mol) in acetone (0.6 L)

was heated at reflux for 16 h. Insoluble material was removed

by filtration, and the filtrate was diluted with EtOAc (0.5 L),

washed with 1 N aq HCl and brine, dried, and evaporated.

Repeated crystallizations of the crude product (from EtOAc/

DCM, DCM, and DCM/hexane) afforded 20g (32.5 g, 32.7%):

Met h yl (R)-1,3,4,5,6,7,8,10-oct a h yd r o-14-h yd r oxy-12-

m et h oxy-11-m et h yl-6,10-d ioxo-9,2,5-b en zoxa t h ia a za cy-

clod od ecin e-4-ca r boxyla te (26g) was prepared from 25g by

applying method A-1 (10): white crystals (from EtOAc/

1

hexane), mp 138-142 °C (dec); H NMR (400 MHz, CDCl₃) δ

1.41 (s, 9H), 1.94 (s, 3H), 2.90 (dd, 1H, J ) 14, 10), 3.04 (dd,

1H, J ) 14, 4), 3.40 (d, 1H, J ) 11), 3.63 (s, 3H), 3.71 (s, 3H),

3.81 (d, 1H, J ) 11), 4.24 (dd, 1H, J ) 11, 4), 4.30-4.38/4.48-

4.58 (2 m, 2 × 1H), 4,87 (dd, 1H, J ) 11, 3), 6.53 (s, 1H), 7.27

(d, 1H, J ) 8), 8.15 (d, 1H, J ) 8), 9.73 (s, 1H); MS (ISP) 499.3

(M + H)⁺. Anal. (C₂₂H₃₀N₂O₉S‚0.05 H₂O) C, H, N, S. The

structure assignment of 26g is in agreement with a ¹H-¹³C

HMBC NMR experiment which showed a correlation between

C(15) and the phenolic hydrogen.

1

white crystals, mp 157-158 °C; H NMR (CDCl₃) δ 2.03/2.06

(2 × 3H), 3.77 (s, 3H), 4.71 (s, 1H), 5.44 (s, 2H), 6.42 (s, 2H),

7.61/8.24 (2 d, 2 × 2H, J ) 8.8). In addition, 4-nitrobenzyl 3,5-

dimethoxy-2,6-dimethylbenzoate (35.4 g, 34%; white solid, mp

165-167 °C) and starting material (20d , 12.0 g, 12.6%) were

isolated.

4-Nitr oben zyl 3-[(ter t-Bu tyl)d im eth ylsilyloxy]-5-m eth -

oxy-2,6-d im eth ylben zoa te (21g). Working similarly as de-

scribed in method D (21a ), 20g (28.8 g, 87 mmol) was reacted

with (t-Bu)Me₂SiCl (15.7 g, 104 mmol) to give 21g (36.8 g,

95%): white crystals (from hexane), mp 81-82 °C; ¹NMR

(CDCl₃) δ 0.21 (s, 6H), 1.01 (s, 9H), 2.02/2.04 (2 s, 2 × 3H),

3.76 (s, 3H), 5.43 (s, 2H), 6.38 (s, 1H), 7.61/8.24 (2 d, 2 × 2H,

J ) 8.8).

4-Nitr oben zyl 3-[(ter t-Bu tyl)d im eth ylsilyloxy]-5-m eth -

oxy-2/6-(br om om eth yl)-6/2-m eth ylben zoa te (22g/22h ). Us-

ing method E (22a ), 21g (13.36 g, 30 mmol) was brominated

with NBS (5.34 g, 30 mmol) in CCl₄(150 mL) to give an oil

Meth yl (R)-1,3,4,5,6,7,8,10-Octa h yd r o-12-h yd r oxy-14-

m et h oxy-11-m et h yl-6,10-d ioxo-9,2,5-b en zoxa t h ia a za cy-

clod od ecin e-4-ca r boxyla te (26h ). By applying the reaction

sequence used to prepare 26g from 23g, 23h was converted

to 26h : white solid (from EtOAc/hexane), mp 125-130 °C

(dec); ¹H NMR (400 mHz, DMSO-d₆) δ 1.41 (s, 9H), 1.99 (s,

3H), 2.90 (dd, 1H, J ) 14, 9), 3.04 (dd, 1H, J ) 14, 4), 3.40 (d,

1H, J ) 11), 3.63 (s, 3H), 3.71 (s, 3H), 3.81 (d, 1H, J ) 11),

4.22 (dd, 1H, J ) 11, 4), 4.32-4.40/4.50-4.58 (2 m, 2 × 1H),

4.87 (dd, 1H, J ) 11, 3), 6.53 (s, 1H), 7.27 (d, 1H, J ) 8), 8.14

(d, 1H, J ) 8), 9.72 (s, 1H); MS (ISP) 499.3 (M + H)⁺. Anal.

(C₂₂H₃₀N₂O₉S) C, H, N, S. The structure assignment of 26h is

Antibacterial Agents from Cyclothialidine

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1503

in agreement with a ¹H-¹³C HMBC NMR experiment which

showed a correlation between C(11) and the phenolic hydrogen.

Meth yl (4S,7R)-7-ter t-bu toxycar bon ylam in o-1,3,4,5,6,7,8,-

10-octah ydr o-12,14-dih ydr oxy-11-m eth yl-6,10-dioxo-9,2,5-

ben zoxath iaazacyclododecin e-4-car boxylate (27) was pre-

pared from 14 (1.61 g, 5.0 mmol) and crude 22d ¹⁸(4.0 g

containing ca. 2.50 g, 4.0 mmol, of 22d ) using sequentially

methods F (23a ), G (24a ), H-1 (25a ), and A-1 (10): white solid

(from EtOAc/hexane) (85 mg), mp 125-130 °C; [R]_D) -53.8°

g, 0.263 mol), and imidazole (34 g, 0.5 mol) in DMF (100 mL)

was heated to 70 °C for 6 h. The cold mixture was poured onto

ice and then extracted with hexane (2 × 200 mL). The organic

layer was washed with H₂O, 5% aq NaHCO₃, H₂O, and brine,

dried, and evaporated, and the remaining oil was crystallized

from hexane to give 35 (41.9 g, 88%): white crystals, mp 89-

90 °C; ¹NMR (CDCl₃) δ 0.21 (s, 6H), 0.37 (s, 6H), 1.01 (s, 9H),

1.02 (s, 9H), 2.46 (s, 3H), 6.93 (dd, 1H, J ) 8, 1), 7.09 (t, 1H,

J ) 8), 7.51 (dd, 1H, J ) 8, 1).

(ter t-Bu tyl)d im eth ylsilyl 3-[(ter t-Bu tyl)d im eth ylsilyl-

oxy]-2-br om om eth ylben zoa te (36). Using method E (22a ),

35 (39.8 g, 104 mmol) was brominated with NBS (18.6 g, 104

mmol) for 3 h to give 36 (40.2 g, 84%): white crystals (from

hexane), mp 73-74 °C; ¹NMR (CDCl₃) δ 0.30/0.40 (2 s, 2 ×

6H), 1.02/1.07 (2 s, 2 × 9H), 5.08 (s, 2H), 6.99 (dd, 1H, J ) 8,

1), 7.22 (t, 1H, J ) 8), 7.56 (dd, 1H, J ) 8, 1).

1

(c ) 0.8, EtOAc); H NMR “identical” to that of 10; MS (ISP)

485 (M + H)⁺. Anal. (C₂₁H₂₈N₂O₉S) C, H, N, S.

Compounds 28 and 29 were prepared similarly.

Meth yl(4R,7R)-7-ter t-bu toxycar bon ylam in o-1,3,4,5,6,7,8,-

10-octah ydr o-12,14-dih ydr oxy-11-m eth yl-6,10-dioxo-9,2,5-

ben zoxath iaazacyclododecin e-4-car boxylate (28) was pre-

pared from 15 (1.61 g, 5.0 mmol) and crude 22d ¹⁸(3.37 g,

containing ca. 2.1 g, 3.35 mmol, of 22d ): white solid (from

EtOAc/hexane) (150 mg), mp 198-200 °C; [R]_D) -62.5° (c )

Met h yl N-(N-ter t-Bu t oxyca r b on yl-^L-ser yl)-S-[6-[(ter t-

bu tyl)d im eth ylsilyloxy]-2-ca r boxylben zyl]-^L-cystein a te

(40). Using method F (23a ), 36 (2.5 g, 5.44 mmol) was reacted

with 5 (1.77 g, 5.5 mmol). The reaction solution was washed

with NaOAc/AcOH buffer (pH 4) and with brine, dried, and

evaporated. The residue was chromatographed (SiO₂; EtOAc,

then EtOAc-EtOH 5:1) to give 40 (1.0 g, 31%): white crystals

1

0.7, EtOAc); H NMR (DMSO-d₆) δ 1.40 (s, 9H), 1.88 (s, 3H),

2.58 (dd, 1H, J ) 14, 11), 3.04 (dd, 1H, J ) 14, 4), 3.61 (d, 1H,

J ) 11), 3.66 (s, 3H), 3.77 (d, 1H, J ) 11), 4.30-4.49 (m, 2H),

4.53-4.73 (m, 2H), 6.44 (s, 1H), 7.05 (d, 1H, J ) 8), 8.82 (d,

1H, J ) 8), 9.46 (s, 1H), 9.49 (s, 1H); MS (ISP) 485 (M + H)⁺.

Anal. (C₂₁H₂₈N₂O₉S) C, H, N, S.

1

(from DCM/hexane), mp 130-135 °C (dec); H NMR (DMSO-

Meth yl (4S,7S)-7-ter t-bu toxycar bon ylam in o-1,3,4,5,6,7,8,-

10-octah ydr o-12,14-dih ydr ox y-11-m eth yl-6,10-dioxo-9,2,5-

ben zoxath iaazacyclododecin e-4-car boxylate (29) was pre-

pared from 16 (0.97 g, 3.0 mmol) and crude 22d ¹⁸(2.0 g

containing ca. 1.25 g, 2.0 mmol, of 22d ): white solid (from

EtOAc/hexane) (96 mg), mp 195-198 °C; [R]_D) +60.5° (c )

d₆) δ 0.24/0.26 (2 s, 2 × 3H), 1.01 (s, 9H), 1.40 (s, 9H), 2.74-

2.95 (m, 2H), 3.50-3.68 (m, 2H) superimposed by 3.60 (s, 3H),

4.03-4.17 (m, 2H), 4.40 (d, 1H, J ) 11), 4.48-4,62 (m, 1H),

6.80 (d, 1H, J ) 8), 7.09 (t, 1H, J ) 8), 7.17 (d, 1H, J ) 8),

7.27 (d, 1H, J ) 8), 8.56 (d, 1H, J ) 8).

Meth yl(4R,7S)-7-ter t-Bu toxycar bon ylam in o-1,3,4,5,6,7,8,-

10-oct a h yd r o-14-h yd r oxy-6,10-d ioxo-9,2,5-b en zoxa t h ia -

a za cyclod od ecin e-4-ca r boxyla te (43). Using methods H-1

(25a ) and A-1 (10), 40 (0.73 g, 1.25 mmol) was cyclized and

the silyl group of the purified product 42 was subsequently

cleaved off to give 43 (63 mg, 11%): white foam; ¹H NMR

(DMSO-d₆) δ 1.42 (s, 9H), 2.92 (dd, 1H, J ) 14, 10), 3.08 (dd,

1H, J ) 14, 4), 3.64 (s, 3H), 3.93 (d, 1H, J ) 10), 4.23 (dd, 1H,

J ) 10, 2), 4.32-4.44 (m, 2H), 4.57-4.83 (m, 2H), 7.04 (d, 1H,

J ) 8), 7.18 (t, 1H, J ) 8), 7.27 (d, 1H, J ) 8), 7.45 (d, 2H, J

) 8), 8.31 (d, 2H, J ) 8), 9.98 (s, 1H); HRMS calcd for

(C₂₀H₂₆N₂O₈SNa⁺) 477.1308, found 477.1306.

1

0.7, EtOAc); H NMR “identical” to that obtained for 28; MS

(ISP) 485 (M + H)⁺. Anal. (C₂₁H₂₈N₂O₉S) C, H, N, S.

4-Nitr oben zyl 3-[Dim eth yl(th exyl)silyloxy]-2-[(R)-[2-

((S)-3-h yd r oxybu tyr yla m in o)-2-m eth oxyca r bon yleth yl]-

su lfa n ylm eth yl]-5-m eth oxy-6-m eth ylben zoa te (30). Using

method F (23a ), the crude mixture 22g/h (1.57 g, containing

ca. 0.50 g, 0.96 mmol, of 22g) was reacted with 17 (0.66 g, 3.0

mmol). The less polar product was isolated by chromatography

(SiO₂; EtOAc/hexane 1:2) to give 30 (0.47 g, 24%): white foam;

1

TLC R_f) 0.20 (EtOAc/hexane 1:1); H NMR (CDCl₃) δ 0.24/

0.27 (2 s, 2 × 3H), 1.02 (s, 9H), 1.19 (d, 3H, J ) 6.3), 2.05 (s,

3H), 2.14-2.28 (m, 2H), 2.79 (dd, 1H, J ) 14, 4.4), 3.04 (dd,

1H, J ) 14, 5.2), 3.69 (s, 3H), 3.70 (d, 1H, J ) 12.8), 3.78 (s,

3H), 3.88 (d, 1H, J ) 12.8), 4.00-4.14 (m, 1H), 4.68-4.77 (m,

1H), 5.43/5.50 (2 d, 2 × 1H, J ) 14), 6.39 (s, 1H), 6.53 (d, 1H,

J ) 8), 7.66/8.28 (2 d, 2 × 2H, J ) 8.8).

Meth yl (4R,7S)-7-Am in o-14-[(ter t-bu tyl)d im eth ylsilyl-

oxy]-1,3,4,5,6,7,8,10-octa h yd r o-6,10-d ioxo-9,2,5-ben zoxa -

th ia a za cyclod od ecin e-4-ca r boxyla te (44). A solution of 42

(0.4 g, 0.7 mmol) in TFA (4 mL) was stirred at 0 °C for 0.5 h.

The solvent was evaporated. The residue was dissolved in

DCM and the solution was washed with aq NaHCO₃, dried,

and evaporated. The residual material was chromatographed

(EtOAc) to give 44 (165 mg, 51%): white foam; ¹H NMR

(DMSO-d₆) δ 0.25/0.26 (2 s, 2 × 3H), 1.03 (s, 9H), 2.31 (br s,

2H), 2.91 (dd, 1H, J ) 14, 11), 3.06 (dd, 1H, J ) 14, 4), 3.60-

3.72 (m, 1H), 3.66 (s, 3H), 3.91 (d, 1H, J ) 10), 4.19 (dd, 1H,

J ) 11, 3), 4.68 (d, 1H, J ) 10), 4.69-4.82 (m, 1H), 4.89 (dd,

1H, J ) 11, 2), 7.08 (dd, 1H, J ) 8, 1), 7.28 (t, 1H, J ) 8), 7.40

(dd, 1H, J ) 8, 1), 8.62 (d, 1H, J ) 8).

Met h yl (4R,7S)-7-[[N-(ter t-Bu t oxyca r b on yl)-^L-ser yl]-

a m in o]-14-h yd r oxy-1,3,4,5,6,7,8,10-octa h yd r o-6,10-d ioxo-

9,2,5-ben zoxa th ia a za cyclod od ecin e-4-ca r boxyla te (45). A

mixture of 44 (147 mg, 0.31 mmol), Boc-^L-Ser-OH (64 mg, 0.31

mmol), and EDC (60 mg, 0.31 mmol) in MeCN (3 mL) was

stirred at 0 °C for 2 h. The solution was diluted with EtOAc,

washed with 5% aq NaHCO₃, 0.1 N aq HCl, and brine, dried,

and evaporated. The residue was stirred with NH₄F (46 mg,

1.25 mmol) in MeOH (3 mL) for 0.5 h at 20 °C. The solution

was diluted with EtOAc, washed with 5% aq NaCl, dried, and

evaporated. The residue was chromatographed (SiO₂; EtOAc)

to give 45 (118 mg, 70%): white crystals (from DCM/Et₂O),

mp 120-130 °C (dec); ¹H NMR (DMSO-d₆) δ 1.36 (s, 9H), 2.69

(dd, 1H, J ) 14, 11), 3.12 (dd, 1H, J ) 14, 4), 3.44-3.72 (m,

2H), 3.63 (s, 3H), 3.95 (d, 1H, J ) 10), 4.16-4.28 (m, 1H), 4.34

(dd, 1H, J ) 11, 2), 4.44 (d, 1H, J ) 10), 4.54-4.70 (m, 2H),

4.85 (dd, 1H, J ) 11, 3), 5.53 (t, 1H, J ) 5), 6.84 (d, 1H, J )

7), 7.08 (d, 1H, J ) 7), 7.20 (t, 1H, J ) 7), 7.36 (d, 1H, J ) 7),

Meth yl (4R,8R)-1,3,4,5,6,7,8,10-Octa h yd r o-14-h yd r oxy-

12-m eth oxy-8,11-d im eth yl-6,10-d ioxo-9,2,5-ben zoxa th ia -

a za cyclod od ecin e-4-ca r boxyla te (32). Using consecutively

methods G (24a ), H-1 (25a ), and A-1 (10), 30 (0.46 g, 0.7 mmol)

was converted to 32 (0.07 g): white solid (from EtOAc/hexane),

mp 217-222 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 1.32 (d, 3H,

J ) 6.4), 1.91 (s, 3H), 2.48 (dd, 1H, J ) 14, 2), 2.62 (dd, 1H, J

) 14, 10.5), 2.69 (dd, 1H, J ) 14, 12), 3.10 (dd, 1H, J ) 14,

4.6), 3.34 (d, 1H, J ) 11), 3.65 (s, 3H), 3.72 (s, 3H), 3.99 (d,

1H, J ) 11), 4.65-4.73 (m, 1H), 5.65-5.75 (m, 1H), 6.50 (s,

1H), 8.45 (d, 1H, J ) 8.8), 9.68 (s, 1H); MS (ISP) 398 (M +

H)⁺. Anal. (C₁₈H₂₃NO₇S‚0.2H₂O) C, H, N.

Meth yl (4R,8S)-1,3,4,5,6,7,8,10-octa h yd r o-14-h yd r oxy-

12-m eth oxy-8,11-d im eth yl-6,10-d ioxo-9,2,5-ben zoxa th ia -

a za cyclod od ecin e-4-ca r boxyla te (33) was obtained from

the crude mixture 22g/h (3.15 g, containing ca. 1.0 g, 1.92

mmol, of 22g) and 18 (1.33 g, 6.0 mmol) in analogy to the

preparation of 32: white solid (from EtOAc/hexane) (0.11 g),

1

mp 123-125 °C; H NMR (DMSO-d₆) δ 1.41 (d, 3H, J ) 7),

1.91 (s, 3H), 2.45 (dd, 1H, J ) 14, 9), 2.75 (dd, 1H, J ) 14, 4)

superimposed by 2.77 (dd, 1H, J ) 14, 12), 3.14 (dd, 1H, J )

14, 4.5), 3.31 (d, 1H, J ) 10), 3.62 (s, 3H), 3.71 (s, 3H), 3.92 (d,

1H, J ) 10), 4.28/5.46 (2 m, 2 × 1H), 6.46 (s, 1H), 8.56 (d, 1H,

J ) 7), 9.68 (s, 1H); MS (ISP) 398 (M + H)⁺. Anal. (C₁₈H₂₃

NO₇S) C, H, N, S.

-

(ter t-Bu tyl)d im eth ylsilyl 3-[(ter t-Bu tyl)d im eth ylsilyl-

oxy]-2-m eth ylben zoa te (35). A mixture of 3-hydroxy-2-

methylbenzoic acid (34) (19.0 g, 0.125 mol), (t-Bu)Me₂SiCl (39.5

1504 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6

Angehrn et al.

8.55/8.60 (2 d, 2 × 1H, J ) 8), 10.02 (s, 1H); HRMS calcd for

7.59 (d, 1H, J ) 8), 9.68 (s, 1H); MS (ISP) 471 (M + H)⁺. Anal.

(C₂₁H₃₀N₂O₈S‚0.4H₂O) C, H, N.

(C₂₃H₃₁N₃O₁₀SNa⁺) 564.1628, found 564.1632.

(ter t-Bu tyl)dim eth ylsilyl 3-Nitr o-2-m eth ylben zoate (38).

To 3-nitro-2-methylbenzoic acid (37, 1.81 g, 10.0 mmol) and

(t-Bu)Me₂SiCl (1.66 g, 11 mmol) in DMF (8 mL) was added

Et₃N (1.53 mL), and the mixture was stirred at 20 °C for 24

h. Hexane (80 mL) was added and the mixture was washed

with 5% aq NaHCO₃and with brine. The organic layer was

dried and evaporated, and the remaining oil was crystallized

from hexane to give 38 (1.62 g, 55%): white crystals, mp 60-

(4R,7S)-7-(ter t-Bu toxyca r bon yla m in o)-1,3,4,5,6,7,8,10-

octa h yd r o-14-h yd r oxy-12-m eth oxy-11-m eth yl-6,10-d ioxo-

9,2,5-ben zoxath iaazacyclododecin e-4-car boxylic Acid (50).

A solution of 26g (0.25 g, 0.5 mmol) in 80% aq THF (6.5 mL)

was adjusted to pH 12 by the addition of 1 N NaOH (ca. 0.4

mL), and 0.1 N NaOH (6 mL) was then added at 20 °C at such

a rate that the pH did not exceed 12 (autotitration; 20 min).

The pH was lowered to 9 by the addition of 1 N aq HCl, and

the solution was washed with EtOAc. The aqueous phase was

set to pH 2.5 and subsequently extracted with EtOAc. The

organic layer was washed with H₂O, dried, and evaporated to

give 50 (0.21 g, 88%): white crystals (from acetone/hexane),

mp 172-175 °C (dec); ¹H NMR (DMSO-d₆) δ 1.41 (s, 9H), 1.94

(s, 3H), 2.91 (dd, 1H, J ) 14, 8), 3.05 (dd, 1H, J ) 14, 4), 3.32

(d, 1H, J ) 11), 3.72 (s, 3H), 3.92 (d, 1H, J ) 11), 4.18 (dd,

1H, J ) 11, 2.5), 4.26-4.36/4,40-4.52 (2 m, 2 × 1H), 4.92 (dd,

1H, J ) 11, 2), 6.51 (s, 1H), 7.38 (d, 1H, J ) 7), 7.89 (d, 1H, J

) 7), 9.72 (s, 1H), 13.10 (br s, 1H); MS (ISN) 483.4 (M - H)^-.

Anal. (C₂₁H₂₈N₂O₉S) C, H, N, S.

ter t-Bu tyl (4R,7S)-4-Ca r ba m oyl-1,3,4,5,6,7,8,10-octa h y-

d r o-14-h yd r oxy-12-m et h oxy-11-m et h yl-6,10-d ioxo-9,2,5-

ben zoxa th ia a za cyclod od ecin e-7-ca r ba m a te (51). A solu-

tion of 26g (0.2 g, 0.4 mmol) in 5.5 N NH₃-MeOH (12 mL)

was kept at 20 °C for 16 h. The solvent was evaporated and

the residue crystallized from MeOH/Et₂O to give 51 (0.13 g,

70%): white solid; ¹H NMR (DMSO-d₆) δ 1.41 (s, 9H), 1.93 (s,

3H), 2.76 (dd, 1H, J ) 14, 9), 3.02 (dd, 1H, J ) 14, 4), 3.36 (d,

1H, J ) 11), 3.72 (s, 3H), 3.93 (d, 1H, J ) 11), 4.17 (dd, 1H, J

) 12, 3), 4.19-4.30/4.36-4.48 (2 m, 2 × 1H), 4.96 (dd, 1H, J

) 12, 2), 6.50 (s, 1H), 7.27/7.40 (2 s, 2 × 1H), 7.53 (d, 1H, J )

7), 7.88 (d, 1H, J ) 8), 9.71 (s, 1H); MS (ISP) 484.4 (M + H)⁺.

Anal. (C₂₁H₂₉N₃O₈S) C, H, N, S.

1

62 °C; H NMR (CDCl₃) δ 0.40 (s, 6H), 1.01 (s, 9H), 2.65 (s,

3H), 7.38 (t, 1H, J ) 8), 7.83/8.04 (2 d, 2 × 1H, J ) 8).

(ter t-Bu tyl)d im eth ylsilyl 2-(Br om om eth yl)-3-n itr oben -

zoa te (39). Using method E (22a ), 38 (1.48 g, 5.0 mmol) was

brominated with NBS (0.89 g, 5.0 mmol) to give 39 (1.39 g,

74%): white crystals (from hexane), mp 98-100 °C; ¹H NMR

(CDCl₃) δ 0.43 (s, 6H), 1.02 (s, 9H), 5.20 (s, 2H), 7.53 (t, 1H, J

) 8), 7.94/8.11 (2 d, 2 × 1H, J ) 8).

Met h yl N-(N-ter t-Bu t oxyca r b on yl-^L-ser yl)-S-[6-n it r o-

2-ca r boxylben zyl]-^L-cystein a te (41). Using method F (23a ),

39 (1.12 g, 3.0 mmol) was reacted with 5 (0.97 g, 3.0 mmol).

The solvents were evaporated, and the residue was stirred with

NH₄F (0.3 g, 8.1 mmol) in MeOH (40 mL) for 0.5 h at 20 °C.

The solvent was evaporated and the residue was taken up in

Et₂O (50 mL). The solution was extracted with aq NaHCO₃

(45 mL), and the basic extracts were acidified (pH 2) by the

addition of 3 N aq HCl. The precipitated product was extracted

with EtOAc, and the organic layer was washed with brine,

dried, and evaporated to give 41 (0.69 g, 46%): white foam;

¹H NMR (DMSO-d₆) δ 1.38 (s, 9H), 2.72-2.92 (m, 2H), 3.40-

3.60 (m, 2H) superimposed by 3.59 (s, 3H), 3.94-4.06 (m, 1H),

4.23/4.33 (2 d, 2 × 1H, J ) 12), 4.38-4.46 (m, 1H), 4.80 (br s,

1H), 6.62 (d, 1H, J ) 8), 7.61 (t, 1H, J ) 8), 8.04 (d, 2H, J )

8), 8.19 (d, 1H, J ) 8), 13.75 (br s, 1H).

(4R,7S)-7-ter t-Bu t oxyca r b on yla m in o-14-h yd r oxy-12-

m eth oxy-11-m eth yl-6,10-d ioxo-1,3,4,5,6,7,8,10-octa h yd r o-

9,2,5-ben zoxa th ia a za cyclododecin e-4-ca r boxylic Acid Al-

lyla m id e (52). A mixture of 50 (97 mg, 0.2 mmol), N-hydroxy-

succinimide (35 mg, 0.3 mmol), and EDC (58 mg, 0.3 mmol)

in MeCN (6 mL) was stirred at 0 °C for 3 h. A solution of

allylamine (0.03 mL, 0.4 mmol) in MeCN (1 mL) was added,

and stirring was continued for 1.5 h at 20 °C. The mixture

was diluted with EtOAc, washed with 0.1 N aq HCl, 5% aq

NaHCO₃, and brine, dried, and evaporated. The residue was

chromatographed (SiO₂; EtOAc-hexane 1:2), and the silyl

protecting groups in the purified product were cleaved off using

method A-1 (10) to give 52 (30 mg, 29%): white solid (from

EtOAc/hexane); ¹H NMR (DMSO-d₆) δ 1.41 (s, 9H), 1.93 (s,

3H), 2.72 (dd, 1H, J ) 14, 10), 3.03 (dd, 1H, J ) 14, 4), 3.42

(d, 1H, J ) 11), 3.62-3.74 (m, 2H) superimposed by 3.72 (s,

3H), 3.92 (d, 1H, J ) 11), 4.18 (dd, 1H, J ) 11, 3), 4.23-4.34/

4.44-4.58 (2 m, 2 × 1H), 4.96 (dd, 1H, J ) 11, 2), 5.01 (d, 1H,

J ) 10), 5.08 (d, 1H, J ) 18), 5.68-5.86 (m, 1H), 6.51 (s, 1H),

7.48 (d, 1H, J ) 7), 7.96 (d, 1H, J ) 8), 8.06 (t, 1H, J ) 5),

9.71 (s, 1H); MS (ISP) 524.5 (M + H)⁺. Anal. (C₂₄H₃₃N₃O₈S)

C, H, N, S.

Meth yl (4R,7S)-14-Am in o-7-ter t-bu toxyca r bon yla m in o-

1,3,4,5,6,7,8,10-octah ydr o-6,10-dioxo-9,2,5-ben zoxath iaaza-

cyclod od ecin e-4-ca r boxyla te (47). Using method H-1 (25a ),

41 (0.6 g, 1.2 mmol) was cyclized and the purified product (46,

0.25 g, 43%) was hydrogenated in EtOAc (12 mL) for 18 h at

20 °C in the presence of 5% Pd-C (0.06 g). The catalyst was

filtered off and the solution was evaporated to give 47 (0.17 g,

31% from 41): white foam; ¹H NMR (DMSO-d₆) δ 1.43 (s, 9H),

2.97 (dd, 1H, J ) 14, 10), 3.13 (dd, 1H, J ) 14, 4), 3.63 (d, 1H,

J ) 11), 3.64 (s, 3H), 4.20-4.50 (m, 3H), 4.60-4.80 (m, 2H),

5.30 (s, 2H), 6.86 (d, 1H, J ) 8), 6.95-7.10 (m, 2H), 7.48 (d,

1H, J ) 8), 8.14 (d, 1H, J ) 8); HRMS calcd for (C₂₀H₂₇N₃O₇-

SNa⁺) 476.1468, found 476.1470.

Meth yl (4R,7S)-14-Am in o-7-[[N-(ter t-bu toxyca r bon yl)-

L-ser yl]a m in o]-1,3,4,5,6,7,8,10-octa h yd r o-6,10-d ioxo-9,2,5-

ben zoxath iaazacyclododecin e-4-car boxylate (48). In anal-

ogy to the preparation of 45, 47 (91 mg, 0.2 mmol) was

1

converted to 48 (68 mg, 63%): white foam; H NMR (DMSO-

d₆) δ 1.36 (s, 9H), 2.72 (dd, 1H, J ) 14, 11), 3.19 (dd, 1H, J )

14, 4), 3.45-3.75 (m, 4H) superimposed by 3.63 (s, 3H), 4.15-

4.27 (m, 1H), 4.33-4.45 (m, 2H), 4.50-4.70 (m, 2H), 4.76 (dd,

1H, J ) 11, 2), 5.30 (s, 2H), 5.43 (t, 1H, J ) 5), 6.80 (d, 1H, J

) 8), 6.87 (t, 1H, J ) 6), 7.06-7.13 (m, 2H), 8.44 (d, 1H, J )

8), 8.57 (d, 1H, J ) 8); HRMS calcd for (C₂₃H₃₂N₄O₉SNa⁺)

563.1788, found 563.1792.

ter t-Bu tyl (4R,7S)-1,3,4,5,6,7,8,10-Octah ydr o-14-h ydr oxy-

4-(h ydr oxym eth yl)-12-m eth oxy-11-m eth yl-6,10-dioxo-9,2,5-

ben zoxa th ia a za cyclod od ecin e-7-ca r ba m a te (49). To a

stirred solution of 25g (123 mg, 0.20 mmol) in MeOH-THF

(1:1, 1.4 mL) was added at 0 °C over 5 min NaBH₄(78 mg, 2.0

mmol), and stirring was continued for 40 min. The mixture

was diluted with EtOAc and washed with 1 N aq HCl and

brine. The organic layer was dried and evaporated. The residue

was treated with NH₄F/MeOH (method A-1) to give 49 (56 mg,

60%): white crystals (from EtOAc/hexane), mp 135-140 °C

(dec); ¹H NMR (DMSO-d₆) δ 1.41 (s, 9H), 1.92 (s, 3H), 2.51

(dd, 1H, J ) 14, 11), 2.83 (dd, 1H, J ) 14, 4), 3.26-3.42 (m,

2H), 3.45 (d, 1H, J ) 11), 3.72 (s, 3H), 3.85 (d, 1H, J ) 11),

4.15 (dd, 1H, J ) 11, 2.5), 4.22-4.34 (m, 1H), 4.79 (t, 1H, J )

5), 4.95 (dd, 1H, J ) 11, 2), 6.51 (s, 1H), 7.13 (d, 1H, J ) 8),

Meth yl (4R,7S)-7-Am in o-1,3,4,5,6,7,8,10-octa h yd r o-12,-

14-dih ydr oxy-11-m eth yl-6,10-dioxo-9,2,5-ben zoxath iaaza-

cyclod od ecin e-4-ca r boxyla te (54). A solution of 10 (14 mg,

0.03 mmol) in TFA (0.3 mL) was stirred at 0 °C for 0.5 h. The

solution was diluted with H₂O (3 mL), and the pH was set to

4 by the addition of 2 N NaOH. The solution was subjected to

reverse phase column chromatography (MCI-Gel CHP20P,

Mitsubishi Ltd.) using 0.1% aq AcOH/0-15% MeCN as eluent.

The product fraction was lyophilized to give 54 (3 mg, 27%):

1

white powder; H NMR (400 MHz, DMSO-d₆) δ 1.87 (s, 3H)

superimposed by 1.88 (br s, 2H), 2.85 (dd, 1H, J ) 15, 10),

3.06 (dd, 1H, J ) 15, 4), 3.39 (d, 1H, J ) 11), 3.66 (s, 3H), 3.82

(d, 1H, J ) 11), 4.09 (dd, 1H, J ) 11, 3), 4.58-4.66 (m, 1H),

5.11 (dd, 1H, J ) 11, 2), 6.46 (s, 1H), 8.46 (d, 1H, J ) 7), 9.55

(br s, 2H); HRMS calcd for (C₁₆H₂₀N₂O₇SH⁺) 385.1069, found

385.1065.

Meth yl (4R,7S)-7-Aceta m id o-1,3,4,5,6,7,8,10-octa h yd r o-

12,14-d ih yd r oxy-11-m eth yl-6,10-d ioxo-9,2,5-ben zoxa th ia -

Antibacterial Agents from Cyclothialidine

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1505

a za cyclod od ecin e-4-ca r boxyla te (55). A solution of 53¹⁸

(0.73 g, 1.2 mmol) in Ac₂O (20 mL) and pyridine (0.1 mL) was

heated to 60 °C for 1 h. The solvents were evaporated, and

the remaining oil was subjected to method A-1 (10) to give 55

(0.39 g, 76%): white crystals (from MeOH/Et₂O), mp 140-145

°C (dec); ¹H NMR (DMSO-d₆) δ 1.90 (s, 3H), 1.94 (s, 3H), 2.88

(dd, 1H, J ) 14, 11), 3.08 (dd, 1H, J ) 14, 4) 3.50 (d, 1H, J )

11), 3.63 (s, 3H), 3.76 (d, 1H, J ) 11), 4.08 (dd, 1H, J ) 12, 4),

4.37-4.49/4.61-4.70 (2 m, 2 × 1H), 5.07 (dd, 1H, J ) 12, 2),

6.45 (s, 1H), 8.25 (d, 1H, J ) 8), 8.29 (d, 1H, J ) 8), 9.49/9.51

(2 s, 2 × 1H); MS (ISP) 427 (M + H)⁺. Anal. (C₁₈H₂₂N₂O₈S) C,

H, N, S.

Meth yl (4R,7S)-7-[(R)-2-(ter t-bu toxyca r bon yla m in o)-3-

h yd r oxyp r op ion a m id o]-1,3,4,5,6,7,8,10-octa h yd r o-12,14-

d ih yd r oxy-11-m et h yl-6,10-d ioxo-9,2,5-b en zoxa t h ia a za -

cyclod od ecin e-4-ca r boxyla te (60): white solid (from Et₂O/

hexane, 13 mg, 23%); ¹H NMR (DMSO-d₆) two rotamers (5:1),

data of major rotamer, δ 1.34 (s, 9H), 1.89 (s, 3H), 2.71 (dd,

1H, J ) 14, 12), 3.11 (dd, 1H, J ) 14, 4), 3.48 (d, 1H, J ) 11),

3.50-3.65 (m, 2H) superimposed by 3.61 (s, 3H), 3.74 (d, 1H,

J ) 11), 4.00-4.16 (m, 2H), 4.42-4.58 (m, 1H), 4.58-4.72 (m,

1H), 5.02 (t, 1H, J ) 6), 5.08-5.19 (m, 1H), 6.45 (s, 1H), 6.97

(d, 1H, J ) 8), 8.08 (d, 1H, J ) 9), 8.24 (d, 1H, J ) 9), 9.51/

9.53 (2 s, 2 × 1H); MS (ISP) 572 (M + H)⁺; HRMS calcd for

(C₂₄H₃₃N₃O₁₁SNa⁺) 594.1733, found 594.1733.

Meth yl (4R,7S)-7-[(2S,3R)-1-(ter t-Bu toxyca r bon yl)-3-

h yd r oxy-2-p yr r olid in eca r b oxa m id o]-1,3,4,5,6,7,8,10-oc-

ta h yd r o-12,14-d ih yd r oxy-11-m eth yl-6,10-d ioxo-9,2,5-ben -

zoxath iaazacyclododecin e-4-car boxylate (57). The acylation

of the amine 53 with Boc-^L-3cHyp-OH (EDC/MECN/0 °C) was

previously described.¹⁸The silyl protecting groups in the

purified coupling product (83 mg, 0.1 mmol) were cleaved off

using method A-1 (10) to give 57 (37 mg, 56% from 53): white

Met h yl (4R,7S)-1,3,4,5,6,7,8,10-oct a h yd r o-12,14-d ih y-

dr oxy-7-(3-h ydr oxypr opion am id o)-11-m eth yl-6,10-dioxo-

9,2,5-ben zoxa t h ia a za cyclod od ecin e-4-ca r boxyla t e (61):

1

white solid (from MeOH/Et₂O/hexane, 20 mg, 43%); H NMR

(DMSO-d₆) δ 1.90 (s, 3H), 2.28-48 (m, 2H), 2.81 (dd, 1H, J )

14, 11), 3.08 (dd, 1H, J ) 14, 4), 3.49 (d, 1H, J ) 11), 3.56-

3.70 (m, 2H) superimposed by 3.62 (s, 3H), 3.78 (d, 1H, J )

11), 4.11 (dd, 1H, J ) 12, 3), 4.38-4.50/4.60-4.70 (2 m, 2 ×

1H), 4.74 (t, 1H, J ) 5), 5.11 (dd, 1H, J ) 12, 2), 6.45 (s, 1H),

8.25 (d, 1H, J ) 8), 8.29 (d, 1H, J ) 8), 9.51/9.53 (2 s, 2 × 1H);

HRMS calcd for (C₁₉H₂₄N₂O₉SNa⁺) 457.1281, found 457.1281.

1

solid (from MeOH/EtOAc/hexane), mp 180-183 °C (dec); H

NMR (DMSO-d₆) two rotamers (ca. 5:4), data of major rotamer,

δ 1.30 (s, 9H), 1.74-2.00 (m, 2H) superimposed by 1.89 (s, 3H),

2.58 (dd, 1H, J ) 14, 12), 3.04 (dd, 1H, J ) 14, 4), 3.06-3.23

(m, 1H), 3.40-3.58 (m, 1H) superimposed by 3.48 (d, 1H, J )

11), 3.60 (s, 3H), 3.87 (d, 1H, J ) 11), 3.97 (dd, 1H, J ) 11, 3),

4.32-4.70 (m, 4H), 5.52 (dd, 1H, J ) 11, 2), 6.04 (d, 1H, J )

3), 6.46 (s, 1H), 8.38 (d, 1H, J ) 8), 8.49 (d, 1H, J ) 7), 9.52 (s,

2H); HRMS calcd for (C₂₆H₃₅N₃O₁₁SNa⁺) 620.1890, found

620.1890.

P r ep a r a tion of Bu ild in g Block s 76-78. Meth yl 2-F or m -

yl-3,5-d im eth oxy-6-m eth ylben zoa te (63). To a solution of

DMF (0.34 L, 4.41 mol) in DCM (1 L) was slowly added POCl₃

(0.404 L, 4.41 mol). The mixture was stirred for 1.5 h at 20

26

°C. A solution of methyl 3,5-dimethoxy-2-methylbenzoate

(62, 618 g, 2.95 mol) in DCM (0.2 L) was added over 10 min.

After being heated for 72 h at reflux temperature, the reaction

mixture was cooled and poured into ice-water (3 L). The

mixture was extracted with DCM (3.6 L), and the organic layer

was washed with aq Na₂CO₃(2 L) and H₂O (2 × 2 L), dried,

and evaporated. The solid residue was recrystallized from

EtOAc/hexane to give 63 (646 g, 92%): white solid, mp 164-

Starting from 53 (0.1 mmol), but replacing in the above

procedure Boc-^L-3cHyp-OH by either Boc-L-Pro-OH, Boc-D-

3cHyp-OH,¹⁸Boc-L-Ser-OH, Boc-D-Ser-OH, or Ph₃COCH₂CH₂-

COOH (82b), respectively, the derivatives 56, 58, 59, 60, and,

after cleavage of the trityl protecting group as described in

the synthesis of 23e, 61 were prepared, respectively.

Meth yl (4R,7S)-7-[(S)-1-(ter t-bu toxyca r bon yl)-2-p yr r o-

lid in eca r b oxa m id o]-1,3,4,5,6,7,8,10-oct a h yd r o-12,14-d i-

h yd r oxy-11-m et h yl-6,10-d ioxo-9,2,5-b en zoxa t h ia a za cy-

clod od ecin e-4-ca r boxyla te (56): white solid (from MeOH/

EtOAc/hexane, 11 mg, 19%), mp 208-210 °C; ¹H NMR (DMSO-

d₆) two rotamers (3:2), data of major rotamer, δ 1.72-2.22 (m,

4H) superimposed by 1.91 (s, 3H), 2.89 (dd, 1H, J ) 14, 9),

3.09 (dd, 1H, J ) 14, 4), 3.24-3.41 (m, 2H), 3.48 (d, 1H, J )

11), 3.62 (s, 3H), 3.76 (d, 1H, J ) 11), 4.14 (dd, 1H, J ) 11, 4),

4.22-4.38 (m, 2H), 4.64-4.75 (m, 1H), 4.93 (dd, 1H, J ) 11,

2.5), 6.45 (s, 1H), 8.28 (d, 1H, J ) 7), 8.40 (d, 1H, J ) 8), 9.52/

9.55 (2 s, 2 × 1H); HRMS calcd for (C₂₆H₃₅N₃O₁₀SNa⁺)

604.1941, found 604.1944.

1

165 °C; H NMR (CDCl₃) δ 2.04 (s, 3H), 3.93 (s, 3H), 3.95 (s,

3H), 6.43 (s, 1H), 10.28 (s, 1H).

Meth yl 2-F or m yl-3-h yd r oxy-5-m eth oxy-6-m eth ylben -

zoa te (64). A 1 M BCl₃-DCM solution (0.8 L) was added over

40 min at 5-10 °C to a solution of 63 (95.3 g, 0.40 mol) in

DCM (0.25 L). The mixture was warmed to 20 °C over 0.5 h,

and stirring was continued for 4 h. The clear solution was

poured into ice-water (1.5 L), and the layers were separated.

The organic layer was washed with H₂O, dried, and evapo-

rated, and the solid residue was recrystallized from EtOAc/

hexane to give 64 (77.3 g, 86%): white crystals, mp 118-119

1

°C; H NMR (DMSO-d₆) δ 1.91 (s, 3H), 3.80 (s, 3H), 3.86 (s,

3H), 6.58 (s, 1H), 10.08 (s, 1H), 11.07 (s, 1H).

(R S )-3,4-D ih y d r o x y -6-m e t h o x y -7-m e t h y l-1,3-d ih y -

d r oisoben zofu r a n -1-on e (65). A stirred mixture of 64 (44.8

g, 0.20 mol) and 2.5 N aq KOH (0.2 L) was warmed to 75 °C

over 0.5 h and subsequently cooled to 5 °C. Upon addition of

10 N aq HCl, a precipitate formed which was isolated by

filtration, washed with H₂O, and dried. Trituration with

EtOAc/hexane afforded pseudoacid 65 (40.1 g, 98%): white

Met h yl (4R,7S)-7-[(2R,3S)-1-(ter t-b u t oxyca r b on yl)-3-

h yd r oxy-2-p yr r olid in eca r boxa m i d o]-1,3,4,5,6,7,8,10-oc-

ta h yd r o-12,14-d ih yd r oxy-11-m eth yl-6,10-d ioxo-9,2,5-ben -

zoxa th ia a za cyclod od ecin e-4-ca r boxyla te (58): white solid

(from MeOH/EtOAc/hexane, 32 mg, 54%), mp 193-195 °C

1

(dec); H NMR (DMSO-d₆) two rotamers (5:4), data of major

rotamer, δ 1.30 (s, 9H), 1.74-1.96 (m, 2H) superimposed by

1.85 (s, 3H), 2.63 (dd, 1H, J ) 14, 12), 3.30-3.52 (m, 4H), 3.61

(d, 1H, J ) 11), 3.63 (s, 3H), 4.00-4.17 (m, 1H), 4.17 (d, 1H,

J ) 5), 4.39-4.53 (m, 1H), 4.75-4.87 (m, 1H), 5.19 (dd, 1H, J

) 12, 2), 5.59-5.72 (m, 1H), 6.45 (s, 1H), 7.56 (d, 1H, J ) 8),

8.38 (d, 1H, J ) 8), 9.53/9.55 (2 s, 2 × 1H); HRMS calcd for

(C₂₆H₃₅N₃O₁₁SNa⁺) 620.1890, found 620.1893.

Meth yl (4R,7S)-7-[(S)-2-(ter t-bu toxyca r bon yla m in o)-3-

h yd r oxyp r op ion a m id o]-1,3,4,5,6,7,8,10-octa h yd r o-12,14-

d ih yd r oxy-11-m et h yl-6,10-d ioxo-9,2,5-b en zoxa t h ia a za -

cyclod od ecin e-4-ca r boxyla te (59): white solid (from MeOH/

Et₂O/hexane, 22 mg, 38%); ¹H NMR (DMSO-d₆) δ 1.36 (s, 9H),

1.90 (s, 3H), 2.72 (dd, 1H, J ) 14, 12), 3.08 (dd, 1H, J ) 14, 4),

3.46-3.72 (m, 2H) superimposed by 3.49 (d, 1H, J ) 11) and

3.61 (s, 3H), 3.80 (d, 1H, J ) 11), 4.10 (dd, 1H, J ) 12, 3),

4.14-4.28 (m, 1H), 4.39-4.57 (m, 1H), 4.59-4.69 (m, 1H),

5.21-5.31 (m, 2H), 6.46 (s, 1H), 6.83 (d, 1H, J ) 9), 8.31 (d,

1H, J ) 7), 8.37 (d, 1H, J ) 9), 9.51/9.53 (2 s, 2 × 1H); HRMS

calcd for (C₂₄H₃₃N₃O₁₁SNa⁺) 594.1733, found 594.1733.

1

solid, mp >255 °C (dec); H NMR (DMSO-d₆) δ 2.30 (s, 3H),

3.79 (s, 3H), 6.45 (d, 1 H, J ) 8.5), 6.71 (s, 1H), 7.64 (d, 1 H,

J ) 8.5), 10.08 (s, 1H).

Allyl 2-For m yl-3-h ydr oxy-5-m eth oxy-6-m eth ylben zoate

(66). To a solution of 65 (42.0 g, 0.2 mol) in DMF (1 L) was

added N,N,N′,N′-tetramethylguanidine (25.2 mL, 0.2 mol).

After stirring for 0.5 h, allyl bromide (33.8 mL, 0.4 Mol) was

added, and stirring was continued for 3 h. The mixture was

evaporated and the residual oil was dissolved in EtOAc (0.7

L). The solution was washed with brine, dried, and evaporated.

The solid residue was crystallized twice from tert-butyl methyl

1

ether to give 66 (43.3 g, 86%): white solid, mp 74-75 °C; H

NMR (DMSO-d₆) δ 1.92 (s, 3H), 3.86 (s, 3H), 4.77 (d, 2H, J )

6), 5.26 (d, 1H, J ) 10), 5.37 (d, 1H, J ) 17), 5.90-6.10 (m,

1H), 6.59 (s, 1H), 10.09 (s, 1H), 11.07 (s, 1H).

Allyl 2-F or m yl-5-m eth oxy-6-m eth yl-3-(d im eth yl(th ex-

yl)silyloxy)ben zoa te (67). Using method D (21a ), 66 (5.0 g,

0.02 mol) was silylated with (thexyl)Me₂SiCl (4.33 mL, 0.022

1506 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6

Angehrn et al.

mol) to give 67 (7.1 g, 90%): white solid (from hexane); mp

81-82 °C; ¹H NMR (CDCl₃) δ 0.32 (s, 6H), 0.94 (d, 6H, J ) 7),

0.98 (s, 6H), 1.70-1.83 (m, 1H), 2.06 (s, 3H), 3.85 (s, 3H), 4.88

(d, 2H, J ) 6), 5.29 (d, 1H, J ) 10), 5.40 (d, 1H, J ) 16), 6.00-

6.16 (m, 1H), 6.31 (s, 1H), 10.24 (s, 1H).

°C tributylphosphine (15.4 mL, 62 mmol), and stirring was

continued for 2 h at 0 °C. The solvent was evaporated and the

residue was chromatographed (SiO₂; EtOAc/hexane 1:1) to give

75 (23.1 g, 79%): white crystals (from EtOAc/hexane), mp 55-

58 °C; ¹H NMR (CDCl₃) δ 0.95 (t, 1H, J ) 8), 1.46 (s, 9H),

2.97-3.23 (m, 1H), 4.50/4.61 (2 d, 2 × 2H, J ) 6), 5.23-5.38

(m, 1H) superimposed by 5.22/5.32 (2 d, 2 × 1H, J ) 10), 5.45

(br s, 1H), 5.59 (d, 1H, J ) 8), 5.84-6.00 (m, 1H).

Bis[(R)-2-ter t-bu toxyca r bon yla m in o-2-(3-m eth yl-1,2,4-

oxa d ia zol-5-yl)-eth yl] Disu lfid e (69). To a solution of Boc-

L-cystine (248.0 g, 0.56 mol), acetamidoxime (68, 89.0 g, 1.20

mol), and 1-hydroxypyridin-2(1H)-one (2.9 g, 0.026 mol) in THF

(1 L) was added at 0 °C over 30 min a solution of DCC (250 g,

1.21 mol) in THF (0.8 L). The mixture was stirred for 16 h

while the temperature was allowed to warm to 20 °C. The

mixture was cooled to 0 °C and the precipitate was removed

by filtration. The filtrate was concentrated in vacuo to a

volume of about 0.5 L and then diluted with EtOAc (0.8 L).

Upon the addition of H₂O (1 L), a precipitate formed which

was isolated by filtration to give Boc-^L-cystine bis(acetami-

doxime) ester (246.6 g, 79%) as white crystals, mp 134-136

°C. This material was taken up in toluene (0.8 L) and the

mixture was heated at reflux for 3 h, the water formed being

removed by a Dean-Stark trap. Addition of hexane (3 L) to

the cooled mixture led to crystallization of the product 69

(215.0 g, 74%) as white crystals, mp 130-131 °C.

(R)-2-Mer ca p to-1-(3-m eth yl-1,2,4-oxa d ia zol-5-yl)-eth yl-

ca r ba m ic Acid ter t-Bu tyl Ester (70). To a stirred solution

of 69 (30 g, 58 mmol) in trifluoroethanol (50 mL) and H₂O (6

mL) was added over 30 min at 0-5 °C tributylphosphine (18.1

mL, 14.84 g, 73.3 mmol), and the mixture was stirred for 3 h

at 0 °C. The solvent was evaporated and the residue was

chromatographed (SiO₂; EtOAc/hexane 1:3) to give 70 (24.1 g,

80%): colorless oil; ¹H NMR (CDCl₃) δ 1.47(s, 9H), 2.41(s, 3H),

2.96-3.24 (m, 2H), 5.24-5.34 (m, 1H), 5.45-5.55 (br d, 1H, J

) 8).

N-Allyloxyca r bon yla m in oa ceton itr ile (72). To a stirred

suspension of aminoacetonitrile hydrochloride (71, 110.0 g,

1.19 mol) in MeCN (1.2 L) was slowly added Et₃N (167.3 mL,

1.2 mol) at 20 °C. Allyl chloroformate (127.6 mL, 1.2 mol) was

then added at 20 °C with cooling, followed by another portion

of Et₃N (167.3 mL, 1.2 mol), and stirring was continued for 2

h. Solid material was filtered off, and the solvents were

evaporated. The residue was dissolved in EtOAc, and the

solution was washed with H₂O, aq NaHSO₄, aq NaHCO₃, and

brine, dried, and evaporated, and the residual oil was distilled

to give 72 (132.6 g, 64%) as a colorless oil, bp 100 °C/0.07 mbar.

N-(Hyd r oxyca r ba m im id oylm eth yl)ca r ba m ic Acid Al-

lyl Ester (73). To a solution of 72 (126.0 g, 0.9 mol) in MeOH

(0.9 L) was added over 15 min with cooling in an ice bath a

solution of hydroxylamine sulfate (73.9 g, 0.9 mol) and NaOH

(36.0 g, 0.9 mol) in H₂O (0.18 L), and the mixture was stirred

for 15 h at 20 °C. The pH of the suspension was lowered from

8.7 to 7.0 by addition of 37% aq HCl. Solid material was filtered

off and the solution was evaporated. Crystallization of the

residue from EtOAc afforded 73 (128.8 g, 83%) as white

crystals, mp 92-93 °C.

(R)-2-Mer capto-1-(3-allyloxycar bon ylam in om eth yl-1,2,4-

oxa d ia zol-5-yl)eth ylca r ba m ic Acid ter t-Bu tyl Ester (75).

To a solution of Boc-^L-cystine (194.1 g, 0.44 mol), 73 (167.9 g,

0.97 mol), and 1-hydroxypyridin-2(1H)-one (2.5 g, 0.023 mol)

in DMF (2 L) was added at 0-20 °C over 15 min a solution of

DCC (200 g, 0.97 mol) in DMF (0.3 L), and the mixture was

stirred for 3 h at 20 °C. The precipitate that formed was

removed by filtration. The filtrate was concentrated to a

volume of 0.5 L, diluted with EtOAc (4 L), and then washed

with 5% aq NaCl, a precipitate being formed. The aqueous

phase was separated, and the precipitate was isolated by

filtration, triturated with MeOH (4 l), and dried. The white

solid obtained (285 g, mp 147.5-149 °C) was heated in dioxane

(1 L) at reflux for 7 h. The solvent was evaporated and the

residue was crystallized twice, from t-BuOMe and from EtOAc/

t-BuOMe, to give bis-[(R)-2-tert-butoxycarbonylamino-2-(3-

allyloxycarbonylaminomethyl-1,2,4-oxadiazol-5-yl)ethyl] di-

sulfide (74, 178.2 g, 68%) as white crystals, mp 118-128 °C.

To a stirred solution of this material (30 g, 0.042 mol) in

trifluoroethanol (90 mL) and H₂O (6.4 mL) was added at 0-5

Gen er a l P r oced u r es for th e Red u ctive Th iola tion of

Ald eh yd e 67. Meth od I-1. Allyl (R)-2-[2-Am in o-2-(m eth -

oxyca r bon yl)eth ylsu lfa n ylm eth yl]-3-[d im eth yl(th exyl)-

silyloxy]-5-m eth oxy-6-m eth ylben zoa te (76). To a stirred

solution of 67 (3.14 g, 8.0 mmol) in TFA (16 mL), cooled to 0

°C, was added ^L-Cys-OMe‚HCl (2.34 g, 13.6 mmol). After 2 min,

Et₃SiH (2.54 mL, 16 mmol) was added, and stirring was

continued at 0 °C for 0.5 h. The mixture was evaporated and

the residual oil was taken up in EtOAc. The solution was

successively washed with H₂O, aq Na₂CO₃, and brine, dried,

and evaporated. Chromatography of the crude product (SiO₂;

EtOAc/hexane 1:1) afforded 76 (2.7 g, 66%): colorless foam;

1

TLC R_f) 0.47 (EtOAc); H NMR (CDCl₃) δ 0.29 (s, 6H), 0.94

(d, 6H, J ) 7), 1.00 (s, 6H), 1.63 (br s, 2H), 1.77 (m, 1H), 2.09

(s, 3H), 2.65 (dd, 1H, J ) 13.5, 8), 2.90 (dd, 1H, J ) 13.5, 4.5),

3.50 (dd, 1H, J ) 8, 4.5), 3.70 (s, 3H), 3.77 (s, 3H), 3.79 (s,

2H), 4.83 (d, 2H, J ) 6), 5.30 (d, 1H, J ) 10), 5.43 (d, 1H, J )

17), 5.97-6.15 (m, 1H), 6.38 (s, 1H).

Meth od I-2. Allyl (R)-2-[2-Am in o-2-(3-m eth yl-1,2,4-oxa -

d ia zol-5-yl)et h ylsu lfa n ylm et h yl]-3-[d im et h yl(t h exyl)si-

lyloxy]-5-m eth oxy-6-m eth ylben zoa te (77). To a solution of

67 (13.8 g, 35.0 mmol) in TFA (38 mL), cooled to 0 °C, was

added over 15 min a solution of 70 (13.7 g, 53 mmol) and

HSiEt₃(8.4 mL, 53 mmol) in DCM (35 mL). The solution was

kept at 0 °C for 18 h. The mixture was subjected to a workup

as described for 76. Chromatography of the crude product

(SiO₂; EtOAc/hexane 1:3) afforded 77 (14.1 g, 69%): pale-

yellow oil; TLC R_f) 0.34 (EtOAc/hexane 2:1); ¹H NMR (CDCl₃)

δ 0.29 (s, 6H), 0.93 (d, 6H, J ) 7); 0.99 (s, 6H), 1.66-1.88 (m,

1H), 1.80 (br s, 2H), 2.09 (s, 3H), 2.38 (s, 3H), 2.80 (dd, 1H, J

) 13, 8), 2.96 (dd, 1H, J ) 13, 5), 3.77/3.83 (2 d, 2 × 1H, J )

12), 3.79 (s, 3H), 4.10 (dd, 1H, J ) 7, 4.5), 4.82 (dd, 2H, J )

6), 5.32 (d, 1H, J ) 10), 5.44 (d, 1H, J ) 16), 5.95-6.15 (m,

1H), 6.39 (s, 1H). (Addition of TAE shift reagent gave no

indication of the presence of the enantiomer). Allyl 5-methoxy-

2,6-dimethyl-3-(dimethyl(thexyl)silyloxy)benzoate (1.7 g) was

isolated as the major side product: colorless oil; TLC R_f) 0.67

1

(EtOAc/hexane 2:1); H NMR (CDCl₃) δ 0.24 (s, 6H), 0.94 (d,

6H, J ) 7), 0.97 (s, 6H), 1.68-1.82 (m, 1H), 2.07/2.08/3.75 (3

s, 3 × 3H), 4.81 (d, 2H, J ) 6), 5.29 (d, 1H, J ) 10), 5.41 (d,

1H, J ) 17), 5.86-6.14 (m, 1H), 6.36 (s, 1H).

Allyl (R)-2-[2-Am in o-2-(3-allyloxycar bon ylam in om eth yl-

1,2,4-oxa d ia zol-5-yl)-et h ylsu lfa n ylm et h yl]-3-[d im et h yl-

(th exyl)silyloxy]-5-m eth oxy-6-m eth ylben zoa te (78). Using

method I-2 (77), but replacing thiol 70 by 75, 67 (5.22 g, 13.3

mmol) was converted to 78 (5.4 g, 64%): pale-yellow oil; TLC

R_f) 0.39 (EtOAc/hexane 2:1); ¹H NMR (CDCl₃) δ 0.29 (s, 6H),

0.93 (d, 6H, J ) 7), 0.99 (s, 6H), 1.67-1.88 (m, 1H), 1.79 (br s,

2H), 2.08 (s, 3H), 2.81 (dd, 1H, J ) 14, 8), 3.03 (dd, 1H, J )

14, 5), 3.73 (d, 1H, J ) 12), 3.77 (s, 3H), 3.81 (d, 1H, J ) 12),

4.07-4.17 (m, 1H), 4.50/4.58/4.80 (3 d, 3 × 2H, J ) 6), 5.22/

5.26 (2 d, 2 × 1H, J ) 10), 5.30-5.45 (m, 1H), 5.37/5.44 (2 d,

2 × 1H, J ) 18), 5.83-6.14 (m, 2H), 6.39 (s, 1H).

Gen er a l P r oced u r e for th e Syn th esis of ω-Tr ityloxy-

a lk a n oic Acid s. 5-Tr ityloxyp en ta n oic Acid (82d ). (a )

5-Tr ityloxyp en ta n ol (80d ). To a solution of 1,5-pentanediol

(79d , 150 mL, 1.50 mol) in pyridine (0.75 L) was added at 0

°C Ph₃CCl (334.2 g, 1.5 mol). The mixture was stirred for 0.5

h at 0 °C followed by 16 h at 20 °C. Solvents were evaporated,

and the residue was partitioned between EtOAc (1.5 L) and

H₂O (0.6 L). The organic layer was washed successively with

1 N aq HCl, aq NaHCO₃, and brine, dried, and evaporated.

The crude product was purified by chromatography. t-BuOMe/

hexane (1:3) eluted the bis-tritylated side product (TLC: R_f)

0.8 (EtOAc/hexane 1:1)) and EtOAc/hexane (1:1) eluted the

product, which was crystallized from hexane to give 80d (242.2

Antibacterial Agents from Cyclothialidine

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1507

g, 47%): white crystals, mp 68-69 °C; TLC: R_f) 0.4 (EtOAc/

hexane 1:1); ¹H NMR (CDCl₃) δ 1.23 (br t, 1H), 1.38-1.72 (m,

6H), 3.07 (t, 2H, J ) 6.5), 3.56-3.66 (m, 2H), 7.17-7.34 (m,

9H), 7.40-7.49 (m, 6H).

0.30 (2 s, 2 × 3H), 0.95 (d, 6H, J ) 7), 0.99 (s, 6H), 1.50-1.82

(m, 6H), 2.09 (s, 3H), 2.10-2.28 (m, 2H), 2.77 (dd, 1H, J ) 14,

4), 3.08 (dd, 1H, J ) 14, 5), 3.61 (t, 2H, J ) 6), 3.69 (d, 1H, J

) 13), 3.70 (s, 3H), 3.77 (s, 3H), 3.88 (d, 1H, J ) 13), 4.68-

4.78 (m, 1H), 4.85 (d, 2H, J ) 6), 5.33 (d, 1H, J ) 10), 5.46 (d,

1H, J ) 18), 5.98-6.15 (m, 1H), 6.37 (s, 1H), 6.44 (d, 1H, J )

8).

(b) Meth od K. Clea va ge of Allyl Ester s. (R)-3-[Dim eth -

yl(t h exyl)silyloxy]-2-[2-(5-h yd r oxyp r op a n oyla m in o)-2-

(m et h oxyca r b on yl)et h ylsu lfa n ylm et h yl]-5-m et h oxy-6-

m eth ylben zoic Acid (84d ). To a solution of 83d (79.0 g, 0.129

mol) in THF (1 L) were added morpholine (56.3 mL, 0.645 mol)

and Pd(PPh₃)₄(0.75 g, 0.65 mmol). The mixture was stirred

at 0 °C for 2 h, and thereafter most of the solvent was

evaporated. EtOAc (0.5 L) was added and the solution was

washed with 2 N aq HCl and brine, dried, and evaporated to

give 84d (73.9 g, “100%”): colorless oil; ¹H NMR (CHCl₃) δ

0.28/0.29 (2 s, 2 × 3H), 0.95 (d, 6H, J ) 7), 1.00 (s, 6H), 1.55-

1.88 (m, 6H), 2.17 (s, 3H), 2.25-2.50 (m, 2H), 2.91 (dd, 1H, J

) 13, 4), 3.05 (dd, 1H, J ) 13, 5), 3.62-3.82 (m, 2H), 3.70 (d,

1H, J ) 11), 3.73 (s, 3H), 3.77 (s, 3H), 3.88 (d, 1H, J ) 11),

4.66-4.76 (m, 1H), 6.36 (s, 1H), 6.68 (d, 1H, J ) 8).

(b) 5-Tr ityloxyp en ta n a l (81d ). A solution of DMSO (75

mL, 1.05 mol) in DCM (0.12 L) was added over 25 min at -65

to -70 °C to a solution of oxalyl chloride (41.5 mL, 0.48 mol)

in DCM (0.75 L). Stirring was continued for 10 min, and then

a solution of 80d (103.8 g, 0.30 mol) in DCM (0.45 L) was added

over 25 min at -65 to -70 °C. After stirring for 10 min, Et₃N

(168 mL, 1.20 mol) was added over 5 min at -70 °C. Stirring

was continued for 20 min, and the mixture was then warmed

to 10 °C over 1 h and washed with H₂O (1.6 L), and the

aqueous layer was extracted with DCM (0.8 L). The organic

layer was dried and evaporated, and the residue was crystal-

lized from hexane (0.25 L) to give 5-trityloxypentanal (81d ,

77.5 g, 75%): white crystals, mp 41 °C; ¹H NMR (CDCl₃) δ

1.60-1.80 (m, 2H), 2.39 (dt, 2H, J ) 2, 7), 3.08 (t, 2H, J ) 6),

7.17-7.34 (m, 9H), 7.40-7.49 (m, 6H), 9.73 (t, 1H, J ) 2).

(c) 5-Tr ityloxyp en ta n oic Acid (82d ). To a mixture of 81d

(86.1 g, 0.25 mol) in acetone (0.63 L) and H₂O (0.25 L) was

added portionwise, over 25 min, KMnO₄(39.5 g, 0.25 mol), the

temperature being kept below 28 °C. Stirring was continued

at 20 °C for 2 h. The pH of the mixture was set to 5 by the

addition of 3 N aq HCl, and 40% aq NaHSO₃(ca. 1.5 L) was

added over 45 min until the mixture became colorless, the pH

being kept at 5. The mixture was acidified to pH 2 and

extracted with EtOAc (1.5 L). The organic layer was washed

with H₂O, dried, and evaporated. The residue was crystallized

from EtOAc/hexane to give 82d (70.4 g, 78%): white crystals,

(c) Meth od H-2. Cycliza tion of ω-Hyd r oxyca r boxylic

Acid s. Meth yl (R)-1,3,4,5,6,7,8,9,10,12-Deca h yd r o-16-[d i-

m eth yl(th exyl)silyloxy]-14-m eth oxy-13-m eth yl-6,12-dioxo-

11,2,5-b en zoxa t h ia a za cyclot et r a d ecin e-4-ca r b oxyla t e

(85d ). To a stirred solution of 84d (73.9 g, ca. 0.129 mol) and

PPh₃(43.2 g, 0.168 mol) in THF (2.5 L) was added at 0 °C,

over 10 min, DEAD (26.4 mL, 0.168 mol). Stirring was

continued for 1 h at 0 °C and for 15 h at 20 °C. The solution

was evaporated, and the residue was stirred with DCM (0.2

L)/hexane (0.3 L) for 1 h at 0 °C. The crystals (diethyl

hydrazodicarboxlate, 19.2 g) were filtered off, the filtrate was

evaporated, and the residue was chromatographed (SiO₂;

1

mp 146-148 °C; H NMR (CDCl₃) δ 1.59-1.82 (m, 4H), 2.34

(t, 2H, J ) 7), 3.08 (t, 2H, J ) 6), 7.18-7.34 (m, 9H), 7.39-

7.48 (m, 6H), 10.54 (br s, 1H).

The trityloxy acid 82a was prepared as described previ-

ously.²⁹The trityloxy acids 82b,c and 82e-g were prepared

using the procedure described for the synthesis of 82d from

79d .

1

EtOAc/hexane 1:1) to give 85d (55.9 g, 78%): foam; H NMR

(CDCl₃) δ 0.28/0.29 (2 s, 2 × 3H), 0.95 (d, 6H, J ) 7), 0.99 (s,

6H), 1.71-1.98 (m, 4H), 2.07 (s, 3H), 2.24-2.55 (m, 2H), 2.87-

3.04 (m, 2H), 3.67 (d, 1H, J ) 11), 3.73 (s, 3H), 3.77 (s, 3H),

3.93 (d, 1H, J ) 11), 4.45-4.58 (m, 2H), 4.60-4.70 (m, 1H),

6.36 (s, 1H), 6.46 (d, 1H, J ) 8).

3-Tr ityloxyp r op ion ic a cid (82b) (starting from 1,3-pro-

panediol): white crystals (EtOAc/hexane), mp 163-165 °C (lit.⁴³

1

mp 164-166); H NMR (CDCl₃) δ 2.64 (t, 2H, J ) 6), 3.24 (t,

2H, J ) 6), 7.17-7.32 (m, 9H), 7.36-7.45 (m, 6H).

Meth od A-2. Liber a tion of O-Tr ia lk ylsilyl-P r otected

P h en ols. Met h yl (R)-1,3,4,5,6,7,8,9,10,12-Deca h yd r o-16-

h ydr oxy-14-m eth oxy-13-m eth yl-6,12-dioxo-11,2,5-ben zoxa-

th ia a za cyclotetr a d ecin e-4-ca r boxyla te (86d ). To a solu-

tion of 85d (55.4 g, 0.1 mol) in MeOH (0.6 L) was added NH₄F

(3,7 g, 0.1 mol) and the mixture was stirred at 20 °C for 40

min. The mixture was concentrated to a volume of ca. 0.2 L,

diluted with EtOAc (0.6 L), and washed with 5% aq NaCl (3

× 0.1 L). The organic layer was dried and evaporated, and

the solid residue was recrystallized from MeOH/Et₂O to give

4-Tr ityloxybu tyr ic a cid (82c) (starting from 1,4-butane-

diol): white crystals (EtOAc/hexane), mp 138-140 °C; ¹H NMR

(CDCl₃) δ 1.88-2.02 (m, 2H), 2.49 (t, 2H, J ) 7), 3.13 (t, 2H,

J ) 6), 7.18-7.33 (m, 9H), 7.38-7.47 (m, 6H), 11.20 (br s, 1H).

6-Tr ityloxyh exa n oic a cid (82e) (starting from 1,6-hex-

anediol): white crystals (from EtOAc/hexane), mp 117-119 °C;

¹H NMR (300 MHz, CDCl₃) δ 1.38-1.52 (m, 2H), 1.57-1.73

(m, 4H), 2.34 (t, 2H, J ) 7), 3.06 (t, 2H, J ) 6), 7.16-7.32 (m,

9H), 7.40-7.48 (m, 6H), 10.60 (br s, 1H).

7-Tr ityloxyh ep ta n oic a cid (82f) (starting from 1,7-hep-

tanediol): colorless oil; ¹H NMR (CDCl₃) δ 1.20-1.45 (m, 4H),

1.52-1.68 (m, 4H), 2.32 (t, 2H, J ) 7), 3.04 (t, 2H, J ) 6),

7.18-7.34 (m, 9H), 7.38-7.48 (m, 6H).

1

86d (33.82 g, 82%): white crystals, mp 165-67 °C; H NMR

(DMSO-d₆) δ 1.60-2.03 (m, 5H) superimposed by 1.90 (s, 3H),

2.34-2.48 (m, 1H), 2.68 (dd, 1H, J ) 13, 10), 3.04 (dd, 1H, J

) 13, 4), 3.64 (s, 3H), 3.65 (d, 1H, J ) 11), 3.69 (s, 3H), 3.75

(d, 1H, J ) 11), 4.00-4.12 (m, 1H), 4.33-4.58 (m, 2H), 6.51

(s, 1H), 8.33 (d, 1H, J ) 8), 9.73 (s, 1H); MS (ISP) 412.2 (M +

H)⁺. Anal. (C₁₉H₂₅NO₇S) C, H, N, S.

9-Tr it yloxyn on a n oic a cid (82g) (starting from 1,9-

nonanediol): colorless oil; ¹H NMR (CDCl₃) δ 1.15-1.45 (m,

8H), 1.52-1.68 (m, 4H), 2.35 (t, 2H, J ) 8), 3.04 (t, 2H, J )

7), 7.16-7.33 (m, 9H), 7.38-7.48 (m, 6H).

Using the procedures of the reaction sequence 76 f 83d f

84d f 85d f 86d , but replacing 82d by 82a -c and 82e-g,

respectively, the following lactones were prepared.

Gen er a l P r oced u r e for th e Syn th esis of La cton es 86a -

g. (a ) Meth od J . Acyla tion of Am in es 76-78. Allyl (R)-3-

[Dim eth yl(th exyl)silyloxy]-2-[2-(5-h ydr oxypen tan oylam i-

n o)-2-(m eth oxycar bon yl)eth ylsu lfan ylm eth yl]-5-m eth oxy-

6-m eth ylben zoa te (83d ). To a suspension of 76 (76.8 g, 0.15

mol) and 82d (64.9 g, 0.18 mol) in MeCN (0.6 L), cooled to 0

°C, was added EDC (34.5 g, 0.18 mol). The mixture was stirred

at 0 °C for 8 h. The solvent was evaporated and the remaining

oil was dissolved in EtOAc (0.8 L). The solution was washed

successively with 1 N aq HCl and brine, dried, and evaporated.

The solution of the residual oil (146.6 g) and pTsOH‚H₂O (5.7

g, 0.03 mmol) in MeOH (0.3 L) was stirred at 20 °C for 3 h.

The precipitated crystals were filtered off (Ph₃COMe), and the

filtrate was evaporated. The residue was chromatographed

(SiO₂; EtOAc/hexane 1:1) to afford 83d (79.2 g, 86%): foam;

Met h yl (4R)-3,4,5,6,7,9-h exa h yd r o-1H -13-h yd r oxy-11-

m et h oxy-10-m et h yl-6,9-d ioxo-8,2,5-b en zoxa t h ia a za cy-

clou n d ecin e-4-ca r boxyla te (86a ) was prepared starting

from 76 and 82a : white crystals (from t-BuOMe/hexane), mp

179-182 °C; ¹H NMR (DMSO-d₆) δ 1.99 (s, 3H), 2.99 (dd, 1H,

J ) 14, 6), 3.02 (dd, 1H, J ) 14, 6), 3.44 (d, 1H, J ) 11), 3.63

(s, 3H), 3.74 (s, 3H), 4.11 (d, 1H, J ) 11), 4.63-4.76 (m, 1H),

4.76/4.86 (2 d, 2 × 1H, J ) 13), 6.55 (s, 1H), 7.83 (d, 1H, J )

8), 9.90 (s, 1H); MS (ISN) 368.3 (M - H)^-. Anal. (C₁₆H₁₉NO₇S)

C, H, N, S.

Met h yl (R)-1,3,4,5,6,7,8,10-oct a h yd r o-14-h yd r oxy-12-

m eth oxy-11-m eth yl-6,10-d ioxo-9 ,2,5-ben zoxa th ia a za cy-

clod od ecin e-4-ca r boxyla te (86b) was prepared starting

from 76 and 82b: white crystals (from MeOH/Et₂O/hexane),

1

TLC R_f) 0.13 (EtOAc/hexane 1:1); H NMR (CDCl₃) δ 0.27/

1508 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6

Angehrn et al.

mp 212-214 °C; ¹H NMR (DMSO-d₆) δ 1.90 (s, 3H), 2.38-

2.50 (m, 1H), 2.59 (dd, 1H, J ) 14, 11), 2.83-3.00 (m, 1H),

3.07 (dd, 1H, J ) 14, 4), 2.63-3.77 (m, 2H) superimposed by

3.65/3.71 (2 s, 2 × 3H), 4.47-4.73 (m, 3H), 6.49 (s, 1H), 8.56

(d, 1H, J ) 8), 9.69 (s, 1H); MS (ISP) 384 (M + H) ⁺. Anal.

(C₁₇H₂₁NO₇S) C, H, N, S.

5.04 (d, 1H, J ) 14), 5.28-5.39 (m, 1H), 5.40 (d, 1H, J ) 14),

6.54 (s, 1H), 9.67 (d, 1H, J ) 8), 9.94 (s, 1H); MS (ISN) 384.3

(M-H)^-. Anal. (C₁₆H₁₉NO₆S₂) C, H, N, S.

Met h yl (R)-1,3,4,5,6,7,8,10-oct a h yd r o-14-h yd r oxy-12-

m eth oxy-11-m eth yl-10-oxo-6-th ioxo-9,2,5-ben zoxath iaaza-

cyclod od ecin e-4-ca r boxyla te (87b): white crystals (from

1

Meth yl (R)-3,4,5,6,7,8,9,11-octa h yd r o-1H-15-h yd r oxy-

13-m eth oxy-12-m eth yl-6,11-d ioxo-10,2,5-ben zoxa th ia a za -

cyclotr id ecin e-4-ca r boxyla te (86c) was prepared starting

from 76 and 82c: white crystals (from MeOH/Et₂O), mp 126-

EtOAc/hexane), mp 162-164 °C; H NMR (DMSO-d₆) δ 1.90

(s, 3H), 2.79-2.93 (m, 1H) superimposed by 2.85 (dd, 1H, J )

14, 11), 3.21 (dd, 1H, J ) 14, 4), 3.34-3.50 (m, 1H), 3.58 (d,

1H, J ) 11), 3.67/3.71 (2 s, 2 × 3H), 3.75 (d, 1H, J ) 11), 4.57-

4.68/4.80-4.94/5.23-5.34 (3 m, 3 × 1H), 6.49 (s, 1H), 9.72 (s,

1H), 10.58 (d, 1H, J ) 8); MS (ISP) 399.9 (M + H)⁺. Anal.

(C₁₇H₂₁NO₆S₂) C, H, N, S.

1

129 °C; H NMR (DMSO-d₆) δ 1.91 (s, 3H) superimposed by

1.86-2.12 (m, 2H), 2.16-2.44 (m, 2H), 2.53 (dd, 1H, J ) 14,

12), 2.90 (dd, 1H, J ) 14, 4), 3.62 (s, 3H), 3.73 (s, 3H), 3.74 (d,

1H, J ) 12), 3.88 (d, J ) 12), 4.14-4.38 (m, 2H), 4.38-4.49

(m, 1H), 6.52 (s, 1H), 8.32 (d, 1H, J ) 8.5), 9.70 (s, 1H); MS

(ISN) 396.2 (M - H)^-. Anal. (C₁₈H₂₃NO₇S) C, H, N, S.

Met h yl (R)-3,4,5,6,7,8,9,11-oct a h yd r o-1H -15-h yd r oxy-

13-m eth oxy-12-m eth yl-11-oxo-6-th ioxo-10,2,5-ben zoxath ia-

azacyclotr idecin e-4-car boxylate (87c): white crystals (from

1

Met h yl (R)-3,4,5,6,7,8,9,10,11,13-d eca h yd r o-1H -17-h y-

d r oxy-15-m eth oxy-14-m eth yl-6,13-d ioxo-12,2,5-ben zoxa -

th ia a za cyclop en ta d ecin e-4-ca r boxyla te (86e) was pre-

pared starting from 76 and 82e: white crystals (from MeOH/

EtOAc/hexane), mp 93-96 °C; H NMR (DMSO-d₆) δ 1.92 (s,

3H), 2.02-2.36 (m, 2H), 2.56-2.72 (m, 2H), 2.84-2.98 (m, 1H),

3.05 (dd, 1H, J ) 14, 4), 3.64 (s, 3H), 3.66 (d, 1H, J ) 12), 3.73

(s, 3H), 3.83 (d, 1H, J ) 12), 4.14-4.36 (m, 2H), 4.90-5.02

(m, 1H), 6.51 (s, 1H), 9.71 (s, 1H), 10.27 (d, 1H, J ) 8); MS

(ISN) 412.3 (M - H)^-. Anal. (C₁₈H₂₃NO₆S₂) C, H, N, S.

Meth yl (R)-3,4,5,6,7,8,9,10,11,13-d eca h yd r o-1H-17-h y-

d r oxy-15-m eth oxy-14-m eth yl-13-oxo-6-th ioxo-12,2,5-ben -

zoxa th ia a za cyclop en ta d ecin e-4-ca r boxyla te (87e): white

solid (from t-BuOMe/hexane), mp 185-188 °C; ¹H NMR

(DMSO-d₆) δ 1.28-1.84 (m, 6H), 1.89 (s, 3H), 2.52-2.73 (m,

2H), 2.85 (dd, 1H, J ) 14, 11), 3.20 (dd, 1H, J ) 14, 4), 3.59/

3.67 (2 d, 2 × 1H, J ) 11), 3.68 (s, 3H), 3.73 (s, 3H), 4.10-

4.21/4.23-4.35 (2 m, 2 × 1H), 5.18-5.30 (m, 1H), 6.52 (s, 1H),

9.76 (s, 1H), 10.31 (d, 1H, J ) 8); MS (ISP) 440.2 (M + H)⁺.

Anal. (C₂₀H₂₇NO₆S₂) C, H, N.

1

Et₂O), mp 202-204 °C; H NMR (DMSO-d₆) δ 1.32-1.80 (m,

6H), 1.90 (s, 3H), 2.04-2.24 (m, 2H), 2.66 (dd, 1H, J ) 14,

11), 3.08 (dd, 1H, J ) 14, 4), 3.63 (s, 2H), 3.64 (s, 3H), 3.73 (s,

3H), 4.08-4.20/4.30-4.42 (2 m, 2 × 1H) 4.42-4.54 (m, 1H),

6.52 (s, 1H), 8.30 (d, 1H, J ) 8.5), 9.74 (s, 1H); MS (ISN) 424.3

(M - H)^-. Anal. (C₂₀H₂₇NO₇S) C, H, N, S.

Meth yl (R)-1,3,4,5,6,7,8,9,10,11,12,14-d od eca h yd r o-18-

h ydr oxy-16-m eth oxy-15-m eth yl-6,14-dioxo-13,2,5-ben zoxa-

th ia a za cycloh exa d ecin e-4-ca r boxyla te (86f) was prepared

starting from 76 and 82f: white solid (from EtOAc/hexane),

mp 200-202 °C; ¹H NMR (DMSO-d₆) δ 1.18-1.76 (m, 8H), 1.93

(s, 3H), 1.95-2.25 (m, 2H), 2.73 (dd, 1H, J ) 13, 11), 2.97 (dd,

1H, J ) 13, 4), 3.62 (s, 3H), 3.67 (d, 1H, J ) 11), 3.73 (s, 3H),

3.77 (d, 1H, J ) 11), 4.02-4.16/4.25-4.29 (2 m, 2 × 1H), 4.29-

4.41 (m, 1H), 6.51 (s, 1H), 8.23 (d, 1H, J ) 8), 9.72 (s, 1H); MS

(ISN) 438.2 (M - 1)^-. Anal. (C₂₁H₂₉NO₇S) C, H, N, S.

Meth yl (R)-1,3,4,5,6,7,8,9,10,11,12,14-d od eca h yd r o-18-

h yd r oxy-16-m et h oxy-15-m et h yl-14-oxo-6-t h ioxo-13,2,5-

ben zoxath iaazacycloh exadecin e-4-car boxylate (87f): white

solid (from EtOAc/hexane), mp 100-103 °C; ¹H NMR (DMSO-

d₆) δ 1.14-1.42 (m, 4H), 1.46-1.76 (m, 4H), 1.93 (s, 3H), 2.43-

2.59/2.63-2.78 (2 m, 2 × 1H), 2.90 (dd, 1H, J ) 14, 12), 3.10

(dd, 1H, J ) 14, 3), 3.66 (s, 3H), 3.67 (d, 1H, J ) 11), 3.73 (s,

3H), 3.82 (d, 1H, J ) 11), 4.00-4.13/4.30-4.43 (2 m, 2 × 1H),

5.04-5.16 (m, 1H), 6.52 (s, 1H), 9.75 (s, 1H), 10.18 (d, 1H, J )

7); HRMS calcd for (C₂₁H₂₉NO₆S₂Na⁺) 478.1334, found 478.1334.

Methyl (R)-1,3,4,5,6,7,8,9,10,11,12,13,14,16-tetradecahydro-

20-h y d r o x y -18-m e t h o x y -17-m e t h y l-16-o x o -6-t h io x o -

15,2,5-b e n zoxa t h ia a za cyclooct a d e cin e -4-ca r b oxyla t e

(87g): white solid (from Et₂O/hexane), mp 151-53 °C; ¹H NMR

(DMSO-d₆) δ 1.05-1.88 (m, 12H), 1.92 (s, 3H), 2.45-2.75 (m,

2H), 3.07 (dd, 1H, J ) 14, 12), 3.35 (dd, 1H, J ) 14, 4), 3.55

(d, 1H, J ) 13), 3.68 (s, 3H), 3.73 (s, 3H), 3.75 (d, 1H, J ) 13),

4.15-4.32 (m, 2H), 5.12-5.24 (m, 1H), 6.53 (s, 1H), 9.89 (s,

1H), 10.27 (d, 1H, J ) 7); HRMS calcd for (C₂₃H₃₃NO₆S₂Na⁺)

506.1647, found 506.1650.

Meth yl (R)-1,3,4,5,6,7,8,9,10,11,12,13,14,16-tetr a d eca h y-

d r o-20-h yd r oxy-18-m eth oxy-17-m eth yl-6,16-d ioxo-15,2,5-

ben zoxa th ia a za cycloocta d ecin e-4-ca r boxyla te (86g) was

prepared starting from 76 and 82g: white solid (from Et₂O),

1

mp 170-174 °C; H NMR (DMSO-d₆) δ 1.10-1.72 (m, 12H),

1.92 (s, 3H), 2.00-2.20 (m, 2H), 2.87 (dd, 1H, J ) 13, 11), 3.16

(dd, 1H, J ) 13, 4), 3.52 (d, 1H, J ) 13), 3.65 (s, 3H), 3.71 (d,

1H, J ) 13), 3.73 (s, 3H), 4.25 (t, 2H, J ) 5), 4.48-4.60 (m,

1H), 6.53 (s, 1H), 8.30 (d, 1H, J ) 8), 9.82 (s, 1H); MS (ISP)

468.5 (M + H) ⁺. Anal. (C₂₃H₃₃NO₇S) C, H, N, S.

Meth od L. Syn th esis of 6-Th ioxo La cton es. Meth yl (R)-

1,3,4,5,6,7,8,9,10,12-Deca h yd r o-16-h yd r oxy-14-m et h oxy-

13-m eth yl-12-oxo-6-th ioxo-11,2,5-ben zoxath iaazacyclotet-

r a d ecin e-4-ca r boxyla te (87d ). A mixture of 85d (3.32 g, 6.0

mmol) and Lawesson’s reagent [2,4-bis(4-methoxyphenyl)-2,4-

dithioxo-1,3,2,4-dithiaphosphetane] (2.70 g, 6.6 mmol) in

toluene (80 mL) was heated to 80 °C for 0.5 h. The mixture

was cooled and evaporated, and the residue was chromato-

graphed (SiO₂; DCM, then EtOAc/hexane 1:1). The collected

product [silyl-protected-87d , 2.9 g; TLC R_f) 0.72 (EtOAc/

hexane 1:1)] was deprotected using method A-2 (86d ) to give

87d (2.07 g, 80%): white crystals (from MeOH/Et₂O/hexane),

mp 141-143 °C; TLC R_f) 0.26 (EtOAc/hexane 1:1); ¹H NMR

(DMSO-d₆) δ 1.60-2.00 (m, 4H) superimposed by 1.90 (s, 3H),

2.55-2.68/2.85-2.98 (2 m, 2 × 1H), 2.88 (dd, 1H, J ) 14, 12),

3.16 (dd, 1H, J ) 14, 4), 3.62 (d, 1H, J ) 10), 3.66 (s, 3H), 3.73

(m, 1H), 3.81 (d, 1H, J ) 10), 4.02-4.12/4.44-4.54/4.85-4.96

(3 m, 3 × 1H), 6.51(s, 1H), 9.76 (s, 1H), 10.28 (d, 1H, J ) 7);

MS (ISN) 426.5 (M - H)^-. Anal. (C₁₉H₂₅NO₆S2) C, H, N, S.

Using the procedures of the synthesis of 87d , the following

6-thioxo lactones were prepared starting from the silyl-

protected lactones 87a -c and 87e-g, respectively.

Meth yl 2-For m yl-5-m eth oxy-6-m eth yl-3-[dim eth yl(th ex-

yl)silyloxy]ben zoa te (88). Using method D (21a ), 64 (11.2

g, 50.0 mmol) was silylated with (thexyl)Me₂SiCl (10.8 mL,

55 mmol) to give 88 (16.5 g, 90%): white crystals (from

1

hexane), mp 68-69 °C; H NMR (CDCl₃) δ 0.32 (s, 6H), 0.94

(d, 6H, J ) 7), 0.98 (s, 6H), 1.68-1.84 (m, 1H), 2.04 (s, 3H),

3.85 (s, 3H), 3.95 (s, 3H), 6.31 (s, 1H), 10.24 (s, 1H).

Meth yl (R)-2-[2-a cetyla m in o-2-(m eth oxyca r bon yl)eth -

ylsu lfa n ylm et h yl]-3-[d im et h yl(t h exyl)silyloxy]-5-m et h -

oxy-6-m eth ylben zoa te (89) was obtained by using method

I-1 (76), but replacing ^L-Cys-OMe‚HCl by Ac-L-Cys-OMe (0.18

g, 1.0 mol), and aldehyde 67 by 88 (0.37 g, 1.0 mol): colorless

foam (0.36 g, 69%); TLC R_f) 0.22 (EtOAc/hexane 1:1); ¹H

NMR (CDCl₃) δ 0.27/0.30 (2 s, 2 × 3H), 0.94 (d, 6H, J ) 7),

0.99 (s, 6H), 1.68-1.84 (m, 1H), 1.94 (s, 3H), 2.08 (s, 3H), 2.74

(dd, 1H, J ) 14, 4), 3.08 (dd, 1H, J ) 14, 5), 3.70 (d, 1H, J )

13), 3.71 (s, 3H), 3.77 (s, 3H), 3.89 (d, 1H, J ) 13), 3.94 (s,

3H), 4.70-4.80 (m, 1H), 6.37 (s, 1H), 6.40 (d, 1H, J ) 8). In

addition, the side product methyl 2,6-dimethyl-3-[dimethyl-

(thexyl)silanyloxy]-5-methoxybenzoate (0.08 g, 23%) was iso-

Met h yl (R)-3,4,5,6,7,9-h exa h yd r o-1H -13-h yd r oxy-11-

m eth oxy-10-m eth yl-9-oxo-6-th ioxo-8,2,5-ben zoxa th ia a za -

cyclou n d ecin e-4-ca r boxyla te (87a ): white crystals (from

t-BuOMe/hexane), mp 164-166 °C; ¹H NMR (DMSO-d₆) δ 1.99

(s, 3H), 3.05 (dd, 1H, J ) 15, 6), 3.15 (d, 1H, J ) 11), 3.22 (dd,

1H, J ) 15, 4), 3.65 (s, 3H), 3.74 (s, 3H), 4.17 (d, 1H, J ) 11),

1

lated as colorless oil: TLC R_f) 0.55 (EtOAc/hexane 1:1); H

NMR (CDCl₃) δ 0.11 (s, 6H), 0.83 (d, 6H, J ) 7), 0.85 (s, 6H),

Antibacterial Agents from Cyclothialidine

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1509

1.58-1.75 (m, 1H), 1.93/1.94 (2 s, 2 × 3H), 3.63 (s, 3H), 3.77

(s, 3H), 6.24 (s, 1H).

with EtOAc and washed successively with 0.5 N aq HCl, 5%

aq NaHCO₃, and brine. The organic layer was dried and

evaporated to give 99 (4.31 g, 81%), colorless oil: ¹NMR

(CDCl₃) δ 0.26/0.30 (2 s, 2 × 3H), 0.94 (d, 6H, J ) 7), 0.98 (s,

6H), 1.73 (m, 1H), 2.09 (s, 3H), 2.36 (s, 3H), 2.65 (m, 2H), 2.83

(dd, 1H, J ) 14, 5), 3.21 (dd, 1H, J ) 14, 6), 3.63 (d, 1H, J )

12), 3.78 (s, 3H), 3.80 (s, 3H), 3.87 (d, 1H, J ) 12), 4.48 (t, 1H,

J ) 5), 4.85 (d, 2H, J ) 6), 5.33 (d, 1H, J ) 6), 5.34-5.42 (m,

1H), 5.45 (d, 1H, J ) 10), 5.95-6.12 (m, 1H), 6.38 (s, 1H), 6.88

(d, 1H, J ) 8).

Meth yl (4R,8S)-1,3,4,5,6,7,8,10-Octa h yd r o-14-[d im eth -

yl(t h exyl)silyloxy]-12-m et h oxy-11-m et h yl-4-(3-m et h yl-

1,2,4-oxa d ia zol-5-yl)-6,10-d ioxo-9,2,5-oxa t h ia a za b en zo-

cyclod od ecin e-8-ca r boxyla te (101). The allyl ester 99 (4.0

g, 6.0 mmol) was cleaved using method K (84d ) and the

resulting hydroxy acid 100 (3.6 g, 5.7 mmol) was cyclized using

method H-2 (85d ), to give 101 (2.25 g, 64%): amorphous solid;

¹H NMR (CDCl₃) δ 0.26/0.31 (2 s, 2 × 3H), 0.94 (d, 6H, J ) 7),

1.00 (s, 6H), 1.77 (m, 1H), 2.19 (s, 3H), 2.38 (s, 3H), 2.86 (dd,

1H, J ) 14, 10), 2.89 (dd, 1H, J ) 14, 12.6), 3.08 (dd, 1H, J )

14, 3), 3.26 (d, 1H, J ) 11), 3.28 (dd, 1H, J ) 14, 4), 3.78 (s,

3H), 3.84 (s, 3H), 4.09 (d, 1H, J ) 11), 5.51-5.61 (m, 1H), 5.92

(dd, 1H, J ) 12.6, 3), 6.39 (s, 1H), 6.75 (d, 1H, J ) 8.5).

Meth yl (4R,8S)-1,3,4,5,6,7,8,10-Octa h yd r o-14-[d im eth -

yl(t h exyl)silyloxy]-12-m et h oxy-11-m et h yl-4-(3-m et h yl-

1,2,4-oxadiazol-5-yl)-10-oxo-6-th ioxo-9,2,5-oxath iaazaben -

zocyclod od ecin e-8-ca r boxyla te (102). Using method K

(87d ), 101 (2.08 g, 3.4 mmol) was reacted with Lawesson’s

reagent to give 102 (1.66 g, 78%): white solid (from EtOAc/

hexane), mp 132-135 °C; ¹H NMR (CDCl₃) δ 0.25/0.31 (2 s, 2

× 3H), 0.94 (d, 6H, J ) 7), 1.00 (s, 6H), 1.76 (m, 1H), 2.19 (s,

3H), 2.38 (s, 3H), 3.12 (d, 1H, J ) 11), 3.13 (dd, 1H, J ) 14,

6), 3.28 (dd, 1H, J ) 14, 12), 3.49 (dd, 1H, J ) 14, 5), 3.54 (dd,

1H, J ) 14, 2.5), 3.77 (s, 3H), 3.86 (s, 3H), 4.13 (d, 1H, J )

11), 5.94 (dd, 1H, J ) 12, 2.5), 6.00 (dd, 1H, J ) 6, 5), 6.39 (s,

1H), 8.45 (d, 1H, J ) 8.5).

(4R ,8S )-1,3,4,5,6,7,8,10-Oct a h yd r o-14-h yd r oxy-8-h y-

d r oxym et h yl-12-m et h oxy-11-m et h yl-4-(3-m et h yl-1,2,4-

oxa d ia zol-5-yl)-6-th ioxo-9,2,5-ben zoxa th ia a za cyclod od e-

cin -10-on e (103). To a solution of 102 (0.63 g, 1.0 mmol) in

MeOH/THF (1:1, 10 mL) was added at 0 °C portionwise, over

1 h, NaBH₄(0.19 g, 5 mmol). The reaction mixture was diluted

with ice-cold 1 N aq HCl and then extracted with EtOAc. The

organic layer was washed with 5% NaHCO₃and brine, dried,

and evaporated. The residue was chromatographed (SiO₂;

EtOAc-hexane 1:1), and the silyl group of the purified product

was subsequently cleaved off using method A-2 (86d ), to give

103 (0.32 g, 70%): white crystals (from MeOH/Et₂O), mp 160

°C (dec); [R]_D) +14.0° (c ) 0.8, EtOAc); ¹H NMR (DMSO-d₆)

δ 1.94 (s, 3H), 2.35 (s, 3H), 2.93 (dd, 1H, J ) 14, 2), 3.00 (dd,

1H, J ) 14, 10), 3.20 (dd, 1H, J ) 14, 2), 3.37 (d, 1H, J ) 11),

3.42 (dd, 1H, J ) 14, 4), 3.50-3.71 (m, 2H), 3.72 (s, 3H), 4.09

(d, 1H, J ) 11), 5.16 (t, 1H, J ) 7), 5.83-5.95 (m, 1H), 6.00-

6.12 (m, 1H), 6.50 (s, 1H), 9.74 (s, 1H), 10.85 (d, 1H, J ) 8);

MS (ISN) 452.3 (M - H)^-. Anal. (C₁₉H₂₃N₃O₆S₂) C, H, N, S.

Meth yl (R)-2-[2-Acetyla m in o-2-(m eth oxyca r bon yl)-eth -

ylsu lfa n ylm et h yl]-3-h yd r oxy-5-m et h oxy-6-m et h ylb en -

zoa te (90). Using method A-2 (86d ), 89 (360 mg, 0.69 mol)

was converted to 90 (255 mg, 96%): white foam; TLC R_f)

1

0.41 (EtOAc); H NMR (DMSO-d₆) δ 1.93/1.96 (2 s, 2 × 3H),

2.68 (dd, 1H, J ) 14, 8), 2.80 (dd, 1H, J ) 14, 6), 3.61 (d, 1H,

J ) 13), 3.62 (s, 3H), 3.68 (d, 1H, J ) 13), 3.74/3.80 (2 s, 2 ×

3H), 4.40-4.52 (m, 1H), 6.53 (s, 1H), 8.27 (d, 1H, J ) 8), 9.78

(s, 1H). Anal. (C₁₇H₂₃NO₇S) C, H, N, S.

Meth yl (R)-3-Hyd r oxy-5-m eth oxy-2-[2-(m eth oxyca r bo-

n yl)-2-th ioa cetyla m in o-eth ylsu lf a n ylm eth yl]-6-m eth yl-

ben zoa te (91). Reacting 89 (276 mg, 0.5 mol) according to

methods L (87d ) and A-2 (86d ) afforded 91 (160 mg, 78%):

1

light-yellow foam; TLC R_f) 0.60 (EtOAc); H NMR (DMSO-

d₆) δ 1.93/2.50 (2 s, 2 × 3H), 2.84 (dd, 1H, J ) 14, 9), 2.94 (dd,

1H, J ) 14, 5), 3.62 (d, 1H, J ) 13), 3.64 (s, 3H), 3.71 (d, 1H,

J ) 13), 3.73/3.80 (2 s, 2 × 3H), 5.04-5.16 (m, 1H), 6.53 (s,

1H), 9.81 (s, 1H), 10.28 (d, 1H, J ) 7); MS (ISP) 402.4 (M +

H)⁺. Anal. (C₁₇H₂₃NO₆S₂) C, H, N, S.

Using the procedures of the reaction sequence 76 f 83d f

84d f 85d f 86d /87d , but replacing 76 by 77, and 82d by

82b or 82d , the lactones 93, 94, and 96, 97 were prepared,

respectively.

(R)-1,3,4,5,6,7,8,10-Octa h yd r o-14-h yd r oxy-12-m eth oxy-

11-m eth yl-4-(3-m eth yl-1,2,4-oxadiazol-5-yl)-9,2,5-ben zoxa-

th ia a za cyclod od ecin e-6,10-d ion e (93): white crystals (from

1

MeOH), mp 220-223 °C; H NMR (DMSO-d₆) δ 1.91 (s, 3H),

2.34 (s, 3H), 2.50 (m, 1H), 2.70 (dd, 1H, J ) 15, 12), 2.90 (m,

1H), 3.27 (dd, 1H, J ) 15, 4), 3.72 (s, 3H), 3.75/3.82 (2 d, 2 ×

1H, J ) 11), 4.50-4.72 (m, 2H), 5.33-5.47 (m, 1H), 6.50 (s,

1H), 8.87 (d, 1H, J ) 9), 9.73 (s, 1H); MS (ISN) 406.3 (M - H)

^-. Anal. (C₁₈H₂₁N₃O₆S) C, H, N, S.

(R)-1,3,4,5,6,7,8,10-Octa h yd r o-14-h yd r oxy-12-m eth oxy-

11-m eth yl-4-(3-m eth yl-1,2,4-oxa d ia zol-5-yl)-6-th ioxo-9,2,5-

ben zoxa th ia a za cyclod od ecin e-10-on e (94): white solid

(from EtOAc/hexane), mp 191-194 °C; ¹H NMR (DMSO-d₆) δ

1.91 (s, 3H), 2.36 (s, 3H), 2.84-2.96 (m, 1H), 3.03 (dd, 1H, J )

14, 11), 3.40 (dd, 1H, J ) 14, 4), 3.38-3,50 (m, 1H), 3.72 (s,

3H), 3.72/3.79 (2 d, 2 × 1H, J ) 10.5), 4.61-4.73 (m, 1H), 4.79-

4.93 (m, 1H), 6.80-6.92 (m, 1H), 6.50 (s, 1H), 9.75 (s, 1H),

10.87 (d, 1H, J ) 8.5); MS (ISN) 422.4 (M - H) ^-. Anal.

(C₁₈H₂₁N₃O₅S₂) C, H, N, S.

(R)-1,3,4,5,6,7,8,9,10,12-Decah ydr o-16-h ydr oxy-14-m eth -

oxy-13-m et h yl-4-(3-m et h yl-1,2,4-oxa d ia zol-5-yl)-11,2,5-

ben zoxa th ia a za cyclotetr a d ecin e-6,12-d ion e (96): white

crystals (from MeOH/Et₂O), mp 191-192 °C; [R]_D) -15.0° (c

) 0.5, EtOAc); ¹H NMR (DMSO-d₆) δ 1.58-1.90 (m, 5H), 1.91

(s, 3H), 1.93-2.08/2.31-2.48 (2 m, 2 × 1H), 2.34 (s, 3H), 2.88

(dd, 1H, J ) 14, 11.5), 3.25 (dd, 1H, J ) 14, 4), 3.70 (d, 1H, J

) 11), 3.73 (s, 3H), 3.86 (d, 1H, J ) 11), 4.02-4.16/4.50-4.62/

5.10-5.22 (3 m, 3 × 1H), 6.52 (s, 1H), 8.64 (d, 1H, J ) 8), 9.74

(s, 1H); MS (ISP) 436.4 (M + H) ⁺. Anal. (C₂₀H₂₅N₃O₆S) C, H,

N, S.

(R)-1,3,4,5,6,7,8,9,10,12-Decah ydr o-16-h ydr oxy-14-m eth -

oxy-13-m eth yl-4-(3-m eth yl-1,2,4-oxa d ia zol-5-yl)-6-th ioxo-

11,2,5-ben zoxa th ia a za cyclotetr a d ecin e-12-on e (97): white

crystals (from acetone/hexane), mp 196-197°C; [R]_D) -26.2°

(c ) 0.5, EtOAc); ¹H NMR (DMSO-d₆) δ 1.60-2.05 (m, 4H)

superimposed by 1.91 (s, 3H), 2.34 (s, 3H), 2.56-2.72/2.84-

3.00 (2 m, 2 × 1H), 3.03 (dd, 1H, J ) 14, 11.5), 3.40 (dd, 1H,

J ) 14, 4), 3.69 (d, 1H, J ) 11), 3.73 (s, 3H), 3.89 (d, 1H, J )

11), 4.04-4.18/4.48-4.62/5.68-5.80 (3 m, 3 × 1H), 6.52 (s, 1H),

9.77 (s, 1H), 10.60 (d, 1H, J ) 8); MS (ISP) 452.3 (M + H)⁺.

Anal. (C₂₀H₂₅N₃O₅S₂) C, H, N, S.

Meth od J -2. Acyla tion of Am in e 77. Allyl 3-[Dim eth yl-

(th exyl)silyloxy]-2-[(R)-2-((R)-3-h yd r oxy-3-(m eth oxyca r -

b on yl)-p r op ion yla m in o)-2-(3-m et h yl-1,2,4-oxa d ia zol-5-

yl)-et h ylsu lfa n ylm et h yl]-5-m et h oxy-6-m et h ylb en zoa t e

(99). To a solution of 77 (4.29 g, 8.0 mmol) and (R)-2-

hydroxysuccinic acid 1-methyl ester (98)³⁰(1.78 g, 12.0 mmol)

in MeCN (60 mL) was added EDC (2.3 g, 12.0 mmol), and the

mixture was stirred at 0 °C for 4 h. The mixture was diluted

(R)-3-(2,2-Dim eth yl-1,3-d ioxola n -4-yl)p r op ion ic Acid

(105a ). A mixture of ethyl (R)-3-(2,2-dimethyl-1,3-dioxolan-4-

yl)acrylate³¹(104, 4.0 g, 20 mmol) and 5% Pd-C (0.4 g) in

EtOAc (50 mL) was hydrogenated for 1 h at atmospheric

pressure. The catalyst was filtered off and the solvent was

evaporated. The residual oil was subjected to bulb-to-bulb

distillation to afford ethyl (R)-3-(2,2-dimethyl-1,3-dioxolan-4-

yl)propionate (2.9 g, 72%): colorless oil, bp ∼130 °C/0.1 mbar;

¹H NMR (CDCl₃) δ 1.26 (t, 3H, J ) 7), 1.35/1.41 (2 s, 2 × 3H),

1.78-1.98 (m, 2H), 2.32-2.54 (m, 2H), 3.55 (dd, 1H, J ) 8, 7),

4.02-4.20 (m, 4H). A mixture of this material (2.8 g, 14.0

mmol) and KOH (1.18 g, 21.0 mmol) in MeOH (14 mL) was

heated to 40 °C for 1.5 h. The solution was concentrated and

partitioned between EtOAc and H₂O. The aqueous layer was

acidified to pH 3 by the addition of 3 N aq HCl and then

extracted with EtOAc. The organic layer was dried and

evaporated to yield 105a (1.97 g, 82%): colorless oil; ¹H NMR

(CDCl₃) δ 1.35/1.41(2 s, 2 × 3H), 1.75-1.99 (m, 2H), 2.38-

2.59 (m, 2H), 3.57 (dd, 1H, J ) 8, 6.5), 4.02-4.20 (m, 2H).

1510 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6

Angehrn et al.

Allyl 2-[(R)-2-[(R)-3-(2,2-Dim et h yl-1,3-d ioxola n -4-yl)-

p r op ion yla m in o]-2-(3-m eth yl-1,2,4-oxa d ia zol-5-yl)eth yl-

su lfa n ylm eth yl]-3-[d im eth yl(th exyl)silyloxy]-5-m eth oxy-

6-m eth ylben zoa te (106a ). Using Method J -2 (99), 77 (37.8

g, 70.6 mmol) was reacted with 105a (18.7 g, 106 mmol) to

give, after chromatographic purification (SiO₂; EtOAc/hexane

112 was obtained. For the cleavage of the allyl ester, the

following procedure³³was used, causing concomitant cleavage

of the N-allyloxycarbonyl protecting group, which subsequently

was reintroduced: To a solution of the allyl ester intermediate

(3.7 g, 5.0 mol) in DCM (50 mL) were added at 20 °C

4-trimethylsilyl-morpholine (2.66 mL, 15 mmol) and trimeth-

ylsilyl acetate (2.26 mL, 15 mmol), and the mixture was stirred

for 5 min. Pd(PPh₃)₄(115 mg, 0.1 mmol) was added and

stirring was continued for 2 h. Solvents were evaporated, the

residual oil was dissolved in MeOH (50 mL), the solution was

evaporated again after standing for 0.5 h at 20 °C, and the

remaining oil was evaporated once more from toluene (50 mL).

A mixture of the residual oil, allyl chloroformate (0.79 mL,

7.5 mmol), and N-methylmorpholine (1.65 mL, 15 mmol) in

DCM (50 mL) was stirred at 0 °C for 2 h and then evaporated.

The residue was dissolved in EtOAc and the solution was

washed with 1 N aq HCl and brine, dried, and evaporated.

The crude N-allyloxycarbonyl-ω-hydroxycarboxylic acid inter-

mediate (3.82 g) was further processed to afford 112: pale-

1

1:1), 106a (38.4 g, 79%): pale-yellow oil; H NMR (CDCl₃) δ

0.26/0.30 (2 s, 2 × 3H), 0.94 (d, 6H, J ) 7), 0.98 (s, 6H), 1.35/

1.40 (2 s, 2 × 3H), 1.65-1.95 (m, 3H), 2.10 (s, 3H), 2.30 (t, 2H,

J ) 7), 2.36 (s, 3H), 2.84 (dd, 1H, J ) 14, 4), 3.20 (dd, 1H, J )

14, 5), 3.52 (t, 1H, J ) 6), 3.63 (d, 1H, J ) 12), 3.78 (s, 3H),

3.87 (d, 1H, J ) 12), 4.00-4.16 (m, 2H), 4.83 (d, 2H, J ) 6),

5.22 (d, 1H, J ) 10), 5.34-5.43 (m, 1H), 5.44 (d, 1H, J ) 18),

5.95-6.14 (m, 1H), 6.38 (s, 1H), 6.62 (d, 1H, J ) 8).

Allyl 3-[Dim et h yl(t h exyl)silyloxy]-2-[(R)-2-((R)-4-h y-

d r oxy-5-tr ityloxy-p en ta n oyla m in o)-2-(3-m eth yl-1,2,4-ox-

a d ia zol-5-yl)-eth ylsu lfa n ylm eth yl]-5-m eth oxy-6-m eth yl-

ben zoa te (108a ). A solution of 106a (9.7 g, 14 mmol) in 80%

aq AcOH (56 mL) was heated to 60 °C for 20 min. The solvents

were evaporated, and the solution of the residual oil in EtOAc

was washed with aq NaHCO₃and brine, dried, and evaporated

to give crude 107a (9.9 g) as a pale-yellow oil. A solution of

this material and (Ph)₃CCl (4.3 g, 15.4 mmol) in pyridine (7

mL) was stirred at 20 °C for 21 h. The mixture was evaporated,

the residue was dissolved in EtOAc, and the solution was

washed with 1 N aq HCl and brine, dried, and evaporated.

The crude product was chromatographed (SiO₂; EtOAc/hexane

1

yellow foam; H NMR (CDCl₃) δ 0.27/0.28 (2 s, 2 × 3H), 0.93

(d, 6H, J ) 7), 0.98 (s, 6H), 1.65-2.00 (m, 5H), 2.08 (s, 3H),

2.85-3.13 (m, 2H) superimposed by 3.02 (dd, 1H, J ) 13, 4),

3.20 (dd, 1H, J ) 13, 5), 3.63 (d, 1H, J ) 11), 3.78 (s, 3H), 3.94

(d, 1H, J ) 11), 4.30-4.43 (m, 1H), 4.50 (d, 2H, J ) 6), 4.60

(d, 2H, J ) 6), 5.18-5.37 (m, 3H), 5.81-6.02 (m, 2H), 6.38 (s,

1H), 8.48 (d, 1H, J ) 7).

(R)-4-(3-Am in om eth yl-1,2,4-oxa d ia zol-5-yl)-16-[d im eth -

yl(th exyl)silyloxy]-1,3,4,5,6,7,8,9,10,12-decah ydr o-14-m eth -

oxy-13-m et h yl-6-t h ioxo-11,2,5-b en zoxa t h ia a za cyclot et -

r a d ecin -12-on e (113). To a solution of 112 (0.97 g, 1.4 mmol)

in DCM (19 mL) were added dimethylaminotrimethylsilane

(1.32 mL, 8.4 mol) and trimethylsilyl trifluoroacetate (1.45 mL,

8.4 mmol). The solution was stirred at 20 °C for 10 min, and

then Pd(PPh₃)₄(97 mg, 0.084 mmol) was added and stirring

was continued for 2.5 h. The mixture was evaporated and the

residual oil was dissolved in EtOAc (50 mL). The solution was

washed with 10% aq NaHCO₃and brine, dried, and evapo-

rated. The residue was chromatographed (SiO₂; EtOAc/hexane

1:2) to give 113 (0.67 g, 78%): foam; TLC R_f) 0.27 (EtOAc);

¹H NMR (CDCl₃) δ 0.27/0.28 (2 s, 2 × 3H), 0.93 (d, 6H, J ) 7),

0.98 (s, 6H), 1.58 (s, 2H), 1.70-2.10 (m, 5H), 2.08 (s, 3H), 2.87-

3.19 (m, 2H) superimposed by 3.04 (dd, 1H, J ) 13, 4), 3.24

(dd, 1H, J ) 13, 5), 3.62 (d, 1H, J ) 11), 3.78 (s, 3H), 3.94 (s,

2H), 3.95 (d, 1H, J ) 11), 4.32-4.43/4.76-4.89/5.96-6.05 (3

m, 3 × 1H), 6.38 (s, 1H), 8.48 (d, 1H, J ) 8).

1

1:3) to give 108a (8.5 g, 68%): colorless oil; H NMR (CDCl₃)

δ 0.25/0.29 (2 s, 2 × 3H), 0.94 (d, 6H, J ) 7), 0.98 (s, 6H),

1.60-1.88 (m, 3H), 2.08 (s, 3H), 2.17-2.46 (m, 2H) superim-

posed by 2.34 (s, 3H), 2.84 (dd, 1H, J ) 14, 5), 3.05-3.17 (m,

2H), 3.23 (dd, 1H, J ) 14, 6), 3.64 (d, 1H, J ) 12), 3.75 (s,

3H), 3.75-3.82 (m, 1H), 3.85 (d, 1H, J ) 12), 4.82 (d, 2H, J )

6), 5.27-5.47 (m, 3H), 5.96-6.10 (m, 1H), 6.37 (s, 1H), 6.68

(d, 1H, J ) 8), 7.19-7.36 (m, 10H), 7.40-7.47 (m, 5H).

(4R,9S)-3,4,5,6,7,8,9,11-Octa h yd r o-1H-15-h yd r oxy-9-h y-

d r oxym et h yl-13-m et h oxy-12-m et h yl-4-(3-m et h yl-1,2,4-

oxadiazol-5-yl)-6-th ioxo-10,2,5-ben zoxath iaazacyclotr ide-

cin -11-on e (110). Subjecting 108a (4.3 g, 5.0 mmol) consecu-

tively to methods K (84d ), H-2 (85d ), and L (87d ) afforded

109a (0.62 g) as a foam. A solution of this material (0.61 g,

1.0 mmol) and pTsOH‚H₂O (0.21 g, 1.1 mmol) in MeOH (16

mL) was heated for 25 min to 60 °C. The cooled mixture was

diluted with EtOAc, washed with 5% aq NaHCO₃and brine,

dried, and evaporated. The residue was subjected to method

A-2 (86d ) to give 110 (0.22 g, 47%): white crystals (from

( R ) -4 -( 3 -A m i n o m e t h y l -1 , 2 , 4 -o x a d i a z o l -5 -y l ) -

1,3,4,5,6,7,8,9,10,12-d eca h yd r o-16-h yd r oxy-14-m et h oxy-

13-m eth yl-6-th ioxo-11,2,5-ben zoxath iaazacyclotetr adecin -

12-on e Hyd r och lor id e (114). A mixture of 113 (0.3 g, 0.5

mmol) and NH₄F (0.1 g) in MeOH (10 mL) was stirred for 0.5

h at 20 °C and then partitioned between EtOAc and 5% aq

NaCl. The organic layer was dried and evaporated. The

solution of the residue (0.3 g) in EtOAc (20 mL) was filtered

and then treated at 0 °C with 3 N HCl-Et₂O (0.17 mL). The

precipitate that formed was collected by filtration, washed with

Et₂O, and dried to give 114 (0.22 g, 94%): white solid; ¹H NMR

(DMSO-d₆) δ 1.60-2.08 (m, 5H) superimposed by 1.91 (s, 3H),

2.58-2.74/2.85-3.00 (2 m, 2 × 1H), 3.08 (dd, 1H, J ) 14, 11),

3.38 (dd, 1H, J ) 14, 4), 3.71 (d, 1H, J ) 11), 3.73 (s, 3H), 3.91

(d, 1H, J ) 11), 4.06-4.20 (m, 1H), 4.30 (s, 2H), 4.50-4.62

(m, 1H), 5.68-5.80 (m, 1H), 6.56 (s, 1H), 8.64 (br s, 3H), 9.87

(s, 1H), 10.74 (d, 1H, J ) 8); HRMS calcd for (C₂₀H₂₆N₄O₅S₂-

Na⁺) 467.1423, found 467.1423.

(R)-N-[5-(1,3,4,5,6,7,8,9,10,12-Deca h yd r o-16-h yd r oxy-

14-m eth oxy-13-m eth yl-12-oxo-6-th ioxo-11,2,5-ben zoxath ia-

azacyclotetradecin-4-yl)-1,2,4-oxadiazol-3-ylmethyl]acetamide

(115). A mixture of 113 (30 mg, 0.05 mmol), Ac₂O (0.5 mL),

and pyridine (0.01 mL) was heated to 60 °C for 1 h. The

solvents were evaporated, and the residue was chromato-

graphed (SiO₂; EtOAc/hexane 1:1 and EtOAc). The silyl

protecting group of the purified product [29 mg, TLC R_f) 0.40

(EtOAc)] was cleaved off according to method A-2 (86d ) to give

115 (19 mg, 75%): white solid; TLC R_f) 0.23 (EtOAc); ¹H

NMR (DMSO-d₆) δ 1.60-2.08 (m, 4H) superimposed by 1.86/

1

MeOH), mp 198-199 °C; H NMR (DMSO-d₆) δ 1.95 (s, 3H),

2.14-2.28 (m, 2H), 2.34 (s, 3H), 2.64-2.80 (m, 1H), 2.84-2.98

(m, 1H), 2.96 (dd, 1H, J ) 14, 11), 3.34 (dd, 1H, J ) 14, 4),

3.53-3.61 (m, 2H), 3.68 (d, 1H, J ) 11), 3.72 (s, 3H), 3.94 (d,

1H, J ) 11), 4.97 (t, 1H, J ) 5), 5.36-5.47 (m, 1H), 5.88-6.00

(m, 1H), 6.50 (s, 1H), 9.71 (s, 1H), 10.55 (d, 1H, J ) 8); MS

(ISP) 468.1 (M + H)⁺. Anal. (C₂₀H₂₅N₃O₆S₂) C, H, N, S.

(4R,10S)-1,3,4,5,6,7,8,9,10,12-Deca h yd r o-16-h yd r oxy-10-

h yd r oxym eth yl-14-m eth oxy-13-m eth yl-4-(3-m eth yl-1,2,4-

oxa d ia zol-5-yl)-6-th ioxo-11,2,5-ben zoxa th ia a za cyclotet-

r a d ecin -12-on e (111) was prepared from 77 in analogous

manner as 110, but replacing 105a by (R)-4-(2,2-dimethyl-1,3-

dioxolan-4-yl)butyric acid³²(105b): white solid (from EtOAc/

hexane), mp 180-83 °C; ¹H NMR (DMSO-d₆) δ 1.68-2.00 (m,

4H) superimposed by 1.96 (s, 3H), 2.34 (s, 3H), 2.56-2.71/

2.73-2.88 (2 m, 2 × 1H), 3.03 (dd, 1H, J ) 14, 10), 3.34 (dd,

1H, J ) 14, 4), 3.44-3.66 (m, 2H), 3.68 (d, 1H, J ) 11), 3.73

(s, 3H), 3.95 (d, 1H, J ) 11), 4.86 (t, 1H, J ) 5), 4.98-5.09 (m,

1H), 5.73-5.85 (m, 1H), 6.51 (s, 1H), 9.74 (s, 1H), 10.54 (d,

1H, J ) 8); MS (ISP) 482.4 (M + H)⁺. Anal. (C₂₁H₂₇N₃O₆S₂‚

0.2H₂O) C, H, N.

(R)-4-(3-Allyloxycar bon ylam in om eth yl-1,2,4-oxadiazol-

5-yl)-1,3,4,5,6,7,8,9,10,12-d eca h yd r o-16-[d im eth yl(th exyl)-

silyloxy]-14-m eth oxy-13-m eth yl-6-th ioxo-11,2,5-ben zoxa -

th ia a za cyclotetr a d ecin -12-on e (112). Using the procedures

of the reaction sequence 76 f 83d f 84d f 85d , but replacing

76 by 78 and subsequently treating the resulting product with

Lawesson’s reagent as described in method L (87d ), the lactone

Antibacterial Agents from Cyclothialidine

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1511

1.91 (2 s, 2 × 3H), 2.58-2.72/2.84-2.98 (2 m, 2 × 1H), 3.04

(dd, 1H, J ) 14, 11), 3.38 (dd, 1H, J ) 14, 4), 3.71 (d, 1H, J )

11), 3.73 (s, 3H), 3.89 (d, 1H, J ) 11), 4.04-4.17 (m, 1H), 4.37

(d, 2H, J ) 6), 4.48-4.60 (m, 1H), 5.70-5.82 (m, 1H), 6.52 (s,

1H), 8.50 (t, 1H, J ) 6), 9.77 (s, 1H), 10.60 (d, 1H, J ) 7);

HRMS calcd for (C₂₂H₂₈N₄O₆S₂Na⁺) 531.1348, found 531.1346.

DNA Gyr a se In h ibition . A DNA supercoiling assay¹²was

used to assess the DNA gyrase inhibitory activity. DNA gyrase

A₂B₂complex was reconstituted by incubation of equimolar

quantities of its subunits at 25 °C for 0.5 h.⁴⁴As described

previously, relaxed pUC18 DNA was incubated with DNA

gyrase at 37 °C for 0.5 h with the compound to be tested

present at concentrations between 0.001 and 100 µg/mL.³⁵

Samples were analyzed by 0.8% agarose gel electophoresis, and

the inhibiting activity of the compound tested was assessed

by determining the MNEC, i.e., the highest inhibitor concen-

tration not yet affecting the complete supercoiling of the

plasmid. The MNEC, determined visually, was found to be 2-5

times lower than the IC₅₀of the supercoiling reaction.

(S)-2-Am in o-N-[5-[(R)-1,3,4,5,6,7,8,9,10,12-d eca h yd r o-

16-h ydr oxy-14-m eth oxy-13-m eth yl-12-oxo-6-th ioxo-11,2,5-

b en zoxa t h ia a za cyclot et r a d ecin -4-yl]-1,2,4-oxa d ia zol-3-

ylm eth yl]pr opion am ide Hydr och lor ide (116). Using meth-

od J -2 (99), 113 (0.1 g, 0.16 mmol) was coupled with N-

allyloxycarbonyl-^L-alanine³⁴(30 mg, 0.18 mmol) to give N-

allyloxycarbonyl-^L-alaninyl-116 (102 mg, TLC R_f) 0.36 (EtOAc/

hexane 2:1)) as a foam. For the removal of the protecting

groups and the formation of the HCl salt, this material was

subjected to the procedures described for the preparation of

114 from 112. The solution of the resulting oily product in H₂O

(2 mL) was lyophilized to give 116 (24 mg, 26%): white solid;

¹H NMR (DMSO-d₆) δ 1.36 (d, 3H, J ) 7), 1.62-2.10 (m, 4H)

superimposed by 1.91 (s, 3H), 2.58-2.74/2.85-3.00 (2 m, 2 ×

1H), 3.08 (dd, 1H, J ) 14, 11.5), 3.38 (dd, 1H, J ) 14, 4), 3.72

(d, 1H, J ) 11), 3.73 (s, 3H), 3.83-3.96 (m, 1H), 3.90 (d, 1H,

J ) 11), 4.04-4.17 (m, 1H), 4.41-4.64 (m, 3H), 5.67-5.79 (m,

1H), 6.55 (s, 1H), 8.20 (br s, 3H), 9.13 (t, 1H, J ) 6), 9.84 (s,

1H), 10.70 (d, 1H, J ) 7); HRMS calcd for (C₂₃H₃₁N₅O₆S₂H⁺)

538.1794, found 538.1794.

In Vitr o An tiba cter ia l Activity. Minimum inhibitory

concentrations (MICs) for the test compounds against the test

strains were determined by an agar dilution method as

recommended by the National Committee for Clinical Labora-

tory Standards (NCCLS) (National Committee for Clinical

Laboratory Standards. Methods for dilution antimicrobial

susceptibility tests for bacteria that grow aerobically, 3rd ed.;

National Committee for Clinical Laboratory Standards: Vil-

lanova, PA, 1993; Vol. 13, no. 25, M7-A3.). Test medium 1, for

test strains N. meningititis 69480, S. aureus 887, S. pyogenes

b15, and M. luteus ATCC8340 was BB2 + 1% Isovitalex + 7.5%

sheep blood + menadione. Test medium 2 for test strains E.

coli 25922, Xanthomonas maltophilia AC 739, S. aureus 25923,

S. aureus Smith, S. epidermidis 16/2, S. pyogenes b15, S.

faecalis 6 was Mueller-Hinton agar. The compounds were

incorporated into a melted medium of 50 °C just prior to the

pouring and use of the plates. The inoculum of approximately

10⁴colony-forming units (CFU) was prepared from appropi-

ately diluted overnight cultures and applied to the agar surface

with a multipoint inoculating device (Denley A400 multipoint

inoculator). The lowest concentration of the compound that

prevented the macroscopic growth of a culture after 18 h of

incubation at 35 °C was taken as the minimal inhibitory

concentration (MIC).

(R)-1,3,4,5,6,7,8,9,10,12-Decah ydr o-16-h ydr oxy-4-[3-(iso-

p r op yla m in o)-m et h yl-1,2,4-oxa d ia zol-5-yl]-14-m et h oxy-

13-m eth yl-6-th ioxo-11,2,5-ben zoxath iaazacyclotetr adecin -

12-on e Hyd r och lor id e (117). To a mixture of 113 (61 mg,

0.1 mmol), acetone (0.1 mL), NaOAc (16 mg), AcOH (0.08 mL),

and H₂O (0.25 mL) in THF (0.5 mL) was added at 0 °C

portionwise, over 30 min, NaBH₄(20 mg, 0.5 mmol). Stirring

was continued for 10 min, and the mixture was then diluted

with EtOAc and washed with aq NaHCO₃and brine. The

organic layer was dried and evaporated. The residue was

chromatographed (SiO₂; EtOAc/hexane 1:2) to yield a minor

reaction product [8 mg, TLC R_f) 0.66 (EtOAc/hexane 1:2);

diisopropyl derivative] and a major product [40 mg, TLC R_f)

0.36 (EtOAc/hexane 1:2)]. For the removal of the protecting

group and the formation of the HCl salt, the latter material

was subjected to the procedures described for the preparation

of 114 from 113, and the solution of the resulting oily product

in H₂O (2 mL) was lyophilized to give 117 (33 mg, 61%): white

In Vivo An tiba cter ia l Effica cy. The in vivo efficacy was

determined in a septicaemia mice model. Septicaemia was

induced in outbred Swiss albino mice (J bm MoRo, weight, 16-

20 g). Mice were infected by intraperitoneal injection of a

diluted overnight culture of the test organism, S. aureus Smith,

which was injected as a suspension in 4% gastric mucin.

Bacterial challenge doses were about 4 times the number of

organisms required to kill 50% of infected but unmedicated

animals within 48 h. The test compounds were formulated as

microsuspensions in gelatine and administered intravenously

1 and 3 h after bacterial challenge. Control and treatment

groups at each dose were composed of five mice each. The 50%

effective dose (ED₅₀, in milligrams per kilogram of body weight)

was calculated by probit analysis as described by Finney

(Finney, D. J . Statistical methods in biological assay, 3rd ed.;

Charles Griffin & Co., Ltd.: London, 1978.) from the survival

rates on day 4 after infection).

1

solid; H NMR (DMSO-d₆) δ 1.26 (d, 6H, J ) 6.5), 1.62-2.08

(m, 4H) superimposed by 1.91 (s, 3H), 2.60-2.74/2.86-3.00

(2 m, 2 × 1H), 3.08 (dd, 1H, J ) 14, 11), 3.38 (dd, 1H, J ) 14,

4), 3.72 (d, 1H, J ) 11), 3.73 (s, 3H), 3.92 (d, 1H, J ) 11),

4.04-4.18 (m, 1H), 4.44 (s, 2H), 4.44-4.60 (m, 1H), 5.66-5.78

(m, 1H), 6.55 (s, 1H), 9.31 (br s, 2H), 9.85 (s, 1H), 10.71 (d,

1H, J ) 7); HRMS calcd for (C₂₃H₃₂N₄O₅S₂H⁺) 509.1892, found

509.1890.

(R)-1,3,4,5,6,7,8,9,10,12-Deca h yd r o-16-h yd r oxy-4-(3-h y-

d r oxym eth yl-1,2,4-oxa d ia zol-5-yl)-14-m eth oxy-13-m eth -

yl-6-t h ioxo-11,2,5-b e n zoxa t h ia a za cyclot e t r a d e cin -12-

on e (118). To a solution of 113 (1.0 g, 1.64 mmol) in 25% aq

AcOH (34 mL) was added at 0 °C NaNO₂(0.67 g, 8.5 mmol).

The mixture was stirred at 0 °C for 1 h. After setting the pH

to 8 by the addition of 14% aq NaOH, the mixture was

extracted with EtOAc. The organic layer was washed with aq

NaHCO₃and brine, dried, and evaporated, and the residue

was chromatographed (SiO₂; EtOAc/hexane 1:1). The material

isolated from the most polar fraction [20 mg; TLC R_f) 0.24

(EtOAc/hexane 1:1)] was subjected to method A-2 (86d ) to give

118 (79 mg, 10%): white solid (from EtOAc/hexane); ¹H NMR

(DMSO-d₆) δ 1.60-2.06 (m, 4H) superimposed by 1.91 (s, 3H),

2.56-2.71/2.84-2.98 (2 m, 2 × 1H), 3.04 (dd, 1H, J ) 14, 11.5),

3.38 (dd, 1H, J ) 14, 4), 3.71 (d, 1H, J ) 11), 3.73 (s, 3H), 3.91

(d, 1H, J ) 11), 4.04-4.16 (m, 1H), 4.47-4.62 (m, 1H)

superimposed by 4.54 (d, 2H, J ) 6), 5.68-5.84 (m, 1H)

superimposed by 5.73 (t, 1H, J ) 6), 6.52 (s, 1H), 9.79 (s, 1H),

10.61 (d, 1H, J ) 7); HRMS calcd for (C₂₀H₂₅N₃O₆S₂Na⁺)

490.1082, found 490.1081.

Ack n ow led gm en t. The authors are grateful to

Petra Schmitz, Patrick Schneider, Guy Wassner, and

David Wechsler for the chemical syntheses; Karin

Kuratli, J aqueline Nell, Marie-The´re`se Traendlin, Vero-

nique Schirmer, and Harry Straub for the biological

evaluation; Wolf Arnold, Walther Meister, J osiane

Kohler, and Fernand Kachler for the spectroscopical and

elemental analysis of the new compounds, and Hisao

Shimada for the testing of selected compounds for

cytotoxicity.

Refer en ces

(1) Chopra, I. Antibiotic resistance in Staphylococcus aureus: Con-

cerns, causes and cures. Expert Rev. Anti-Infective Ther. 2003,

1, 45-55.

(2) Wood, M. J . Chemotherapy for Gram-positive nosocomial sepsis.

J . Chemother. 1999, 11, 446-452.

1512 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6

Angehrn et al.

(3) (a) Gellert, M.; Mizuuchi, K.; O’Dea, M. H.; Nash, H. A. DNA

gyrase: An enzyme that introduces superhelical turns into DNA.

Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 3872- 3876. (b) Hooper,

D. C. Bacterial topoisomerases, anti-topoisomerases and anti-

topoisomerase resistance. Clin. Infect. Dis. 1998, 27(Suppl. 1),

S54-S63.

(4) Hooper, D. C.; Wolfson, J . S. Mode of action of new quinolones:

New data. Eur. J . Clin. Microbiol. Infect. Dis. 1991, 10, 223.

(5) Gellert, M.; O’Dea, M. H.; Itoh, T.; Tomizawa, J . Novobiocin and

coumermycin inhibit DNA supercoiling catalysed by DNA gy-

rase. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 4474-4478.

(6) (a) Mizuuchi, K.; O’Dea, M. H.; Gellert, M. DNA gyrase: Subunit

structure and ATPase activity of the purified enzyme. Proc. Natl.

Acad. Sci. U.S.A. 1978, 75, 5960-5963. (b) Reece, R. J .; Maxwell,

A. DNA gyrase: Structure and function. CRC Crit. Rev. Biochem.

Mol. Biol. 1991, 26, 335-375.

(7) (a) Wolfson, J . S. Quinolone antimicrobial agents: Adverse

effects and bacterial resistance. Eur. J . Clin. Microbiol. Infect.

Dis. 1989, 8, 1080-1092. (b) Voss, A.; Milatovic, D.; Wallrauch-

Schwarz, C.; Rosdahl, V. T.; Braveny, I. Methicillin-resistant

Staphylococcus aureus in Europe. Eur. J . Clin. Microbiol. Infect.

Dis. 1994, 13, 50-55.

(8) (a) Kim, O. K.; Ohemeng, K. A. Patents on DNA gyrase

inhibitors: J anuary 1995 to March 1998. Exp. Opin. Ther.

Patents 1998, 8, 959-969. (b) Lambert, H. P.; O’Grady, F. W.

Antibiotics and chemotherapy. In Coumarins, 6th ed.; Lambert,

H. P.; O’Grady, F. W., Eds.; Churchill Livingstone: Edinburgh,

1992; pp 140-141.

(17) (a) Arisawa, M.; Goetschi, E.; Kamiyama, T.; Masciadri, R.;

Shimada, H.; Watanabe, J .; Hebeisen, P.; Link, H. PCT Int. App.

WO 9218490, 1992; Chem. Abstr. 1993, 119, 117285. (b) Geiwiz,

J .; Goetschi, E.; Hebeisen, P.; Link, H.; Luebbers, T. European

Patent App. EP 675122, 1995; Chem. Abstr. 1996, 124, 146213.

(18) Go¨tschi, E.; J enny, Ch-J .; Reindl, P.; Ricklin, F. Total synthesis

of cyclothialidine. Helv. Chim. Acta 1996, 79, 2219-2234.

(19) (a) 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. Pharmac. Ther. 1993, 60,

367-380. (b) Goetschi, E.; Angehrn, P.; Gmuender, H.; Hebeisen,

P.; Link, H.; Masciadri, R.; Reindl P.; Ricklin, F. The DNA gyrase

inhibitor cyclothialidine: Progenitor of a new class of antibacte-

rial agents. In Medicinal Chemistry: Today and Tomorrow;

Yamazaki, M., Ed.; Blackwell Science Ltd.: Oxford, 1996; pp

263-270. (c) Goetschi, E.; Angehrn, P.; Gmuender, H.; Hebeisen,

P.; Kostrewa, D.; Link, H.; Luebbers, T.; Masciadri, R.; Reindl

P.; Ricklin, F.; Theil, F. P. From the DNA gyrase inhibitor

cyclothialidine to a new class of antibacterial agents. In Natural

product chemistry at the turn of the century; Atta-ur-Rahman,

Choudhary, M. I., Khan, K. M., Eds.; Print Arts: Karachi, 2002;

pp 498-497.

(20) Geiwiz, J .; Goetschi, E.; Hebeisen, P. Concise synthesis of

cyclothialidine analogues with ring sizes from 12 to 15: Novel

macrocyclization protocol involving reductive thiolation. Syn-

thesis 2003, 1699-1704.

(21) Richter, L. S.; Marsters, J . C., J r.; Gadek, T. R. Two new

procedures for the introduction of benzyl-type protecting groups

for thiols. Tetrahedron Lett. 1994, 35, 1631.

(22) Kurihara, T.; Nakajima, Y.; Mitsunobu, O. Synthesis of lactones

and cycloalkanes. Cyclization of ω-hydroxy acids and ethyl

R-cyano-ω-hydroxycarboxylates. Tetrahedron Lett. 1976, 28,

2455-2458.

(9) Lesher, G. Y.; Froelich, E. J .; Gruett, M. D.; Baily, J . H.;

Brundage, R. P. 1,8-Naphthyridine-Deivatives: A new class of

chemotherapeutic agents. J . Med. Pharm. Chem. 1962, 5, 1063-

1065.

(10) (a) Hoeksma, H.; J ohnson, J . L.; Hinman, J . W. Structural

studies on streptonivicin, a new antibiotic. J . Am. Chem. Soc.

1955, 77, 6710-6711. (b) Kaczka, E. A.; Wolf, F. J .; Rathe, F.

P.; Folkers, K. Cathomycin. I. Isolation and characterization. J .

Am. Chem. Soc. 1955, 77, 6404-6405. (c) Berger, J .; Schocher,

A. J .; Batcho, A. D.; Pecherer, B.; Keller, O.; Maricq, J .; Karr,

A. E.; Vaterlaus, B. P.; Furlenmeier, A.; Spiegelberg, H. Produc-

tion, isolation and synthesis of the coumermycins (sugordo-

mycins), a new streptomycete antibiotic complex. In Proceedings

of the 5th interscientific conference on antimicrobial agents and

chemotherapy and the 4th international congress of chemo-

therapy, Washington D. C. Antimicrob. Agents Chemother. 1965,

778-785. (d) Kawaguchi, H.; Tsukiura, H.; Okanishi, M.; Miyaki,

T.; Ohmori, T.; Fujisawa, K.; Koshiyama, H. Studies on coumer-

mycin, a new antibiotic: I. Production, isolation and characteri-

sation of coumermycin A1. J . Antibiot. Ser. A 1965, 18, 11-25.

(11) (a) Clements, J . M.; Beckett, R. P.; Brown, A.; Catlin, G.; Lobell,

M.; Palan S.; Thomas, W.; Whittaker, M.; Wood, S.; Salama, S.;

Baker, P. J .; Rodgers, H. F.; Barynin, V.; Rice, D. W.; Hunter,

M. G. Antibiotic activity and characterization of BB-3497, a novel

peptide deformylase inhibitor. Antimicrob. Agents Chemother.

2001, 45, 563-570. (b) J arvest, R. L.; Berge, J . M.; Berry, V.;

Boyd, H. F.; Brown, M. J .; Elder, J . S.; Forrest, A. K.; Fosberry,

A. P.; Gentry, D. R.; Hibbs, M. J .; J aworski, D. D.; O’Hanlon, P.

J .; Pope, A. J .; Rittenhouse, S.; Sheppard, R. J .; Slater-Radosti,

C.; Worby, A. Nanomolar inhibitors of Staphylococcus aureus

methionyl tRNA synthetase with potent antibacterial activity

against Gram-positive pathogens. J . Med. Chem. 2002, 45,

1959-1962.

(12) Otter, R.; Cozzarelli, N. R. Escherichia coli DNA gyrase. Methods

Enzymol. 1983, 100, 171.

(13) Watanabe, J .; Nakada, N.; Sawairi, S.; Shimada, H.; Ohshima,

S.; Kamiyama, T.; Arisawa, M. Cyclothialidine, a novel DNA

gyrase inhibitor. I. Screening, taxonomy, fermentation and

biological activity. J . Antibiot. 1994, 47, 32-36.

(14) Kamiyama, T.; Shimma, N.; Ohtsuka, T.; Nakayama, N.; Itezono,

Y.; Nakada, N.; Watanabe, J .; Yokose, K. Cyclothialidine, a novel

DNA gyrase inhibitor. II. Isolation, characterization and struc-

ture elucidation. J . Antibiot. 1994, 47, 37-45.

(15) (a) Nakada, N.; Shimada, H.; Hirata, T.; Aoki, Y.; Kamiyama,

T.; Watanabe, J .; Arisawa, M. Biological characterization of

cyclothialidine, a novel DNA gyrase inhibitor. Antimicrob. Agents

Chemother. 1993, 37, 2656-2661. (b) Nakada, N.; Gmuender,

H.; Hirata, T.; Arisawa, M. Mechanism of inhibition of DNA

gyrase by cyclothialidine, a new DNA gyrase inhibitor. Antimi-

crob. Agents Chemother. 1994, 38, 1966-1973.

(23) Mandell, L.; Caine, D.; Kilpatrick, G. E. The synthesis of

dienones related to santonin and ψ-santonin via aryl participa-

tion. J . Am. Chem. Soc. 1961, 83, 4457-4460.

(24) Schoenholzer, P. Private communication.

(25) Wissner, A.; Grudzinskas, C. V. Reaction of tert-butyldimethyl-

silyl esters with oxalyl chloride-dimethylformamide: Preparation

of carboxylic acid chlorides under neutral conditions. J . Org.

Chem. 1978, 43, 3972-3974.

(26) Makara, G. M.; Klubek, K.; Anderson, W. K. An improved

synthesis of 5,7-dimethoxy-4-methyl-phthalide, a key intermedi-

ate in the synthesis of mycophenolic acid. Synth. Commun. 1996,

26, 1935-1942.

(27) Paz Cabal, M.; Coleman, R. S.; Danishefsky, S. J . Total synthesis

of calicheamicinone: A solution to the problem of the elusive

urethane. J . Am. Chem. Soc. 1990, 112, 3253-3255.

(28) Street, L. J .; Baker, R.; Book, T.; Kneen, C. O.; MacLeod, A. M.;

Merchant, K. J .; Showell, G. A.; Saunders, J .; Herbert, R. H.;

Freedman, S. B.; Harley, E. A. Synthesis and biological activity

of 1,2,4-oxadiazole derivatives: Highly potent and efficacious

agonists for cortical muscarinic receptors. J . Med. Chem. 1990,

33, 2690-2697.

(29) Auterhoff, H.; Oettmeier, R. Carbodiimide als Kondensations-

mittel bei der Darstellung von Estern und Aethern des Dantrons.

Arch. Pharm. 1975, 308, 732-736.

(30) Miller, M. J .; Bajwa, J . S.; Mattingly, P. G.; Peterson, K.

Enantioselective syntheses of 3-substituted 4-(alkoxycarbonyl)-

2-azetidinones from malic acid. J . Org. Chem. 1982, 47, 4928-

4933.

(31) Hubschwerlen, C. A convenient synthesis of _{L-(S)-glyceraldehyde}

acetonide from L-ascorbic acid. Synthesis 1986, 11, 962-964.

(32) Hori, K.; Hikage, N.; Inagaki, A.; Mori, S.; Nomura, K.; Yoshii,

E. Total synthesis of tetronomycin. J . Org. Chem. 1992, 57,

2888-2902.

(33) Merzouk, A.; Guibe´, F.; Loffet, A. On the use of silylated

nucleophiles in the palladium catalysed deprotection of allylic

carboxylates and carbamates. Tetrahedron Lett. 1992, 33, 477-

480.

(34) Stevens, C. M.; Watanabe, R. Amino acid derivatives. I. Car-

boallyloxy derivatives of R-amino acids. J . Am. Chem. Soc. 1950,

72, 725-727.

(35) Gmu¨nder, H.; Kuratli, K.; Keck, W. Effect of Pyrimido[1,6-a]-

benzimidazoles, quinolones, and Ca²⁺on the DNA gyrase-

mediated cleavage reaction. Antimicrob. Agents Chemother.

1995, 39, 163-169.

(36) (a) Kostrewa, D.; D’Arcy, A. Unpublished results. (b) Lewis, J .

R.; Singh, O. M. P.; Smith, C. V. Skarzynsky, T.; Maxwell, A.;

Wonacott, A. J .; Wigley, D. B. The nature of inhibition of DNA

gyrase by the coumarins and the cyclothialidines revealed by

X-ray crystallography. EMBO J . 1996, 15, 1412-1420.

(16) (a) Lewis, J . R.; Singh, O. M. P.; Smith, C. V.; Maxwell, A.;

Skarzynsky, T.; Wonacott, A. J .; Wigley, D. B. Crystallization

of inhibitor complexes of an N-terminal 24 kDa fragment of the

DNA gyrase B protein. J . Mol. Biol. 1994, 241, 128-130. (b)

Yamaji, K.; Masubuchi, M.; Kawahara. F.; Nakamura, Y.;

Nishio, A.; Matsukuma, S.; Fujimori, M.; Nakada, N.; Watanaba,

J .; Kamiyama, T. Cyclothialidine analogues, novel DNA gyrase

inhibitors. J . Antibiot. 1997, 50, 402-411.

Antibacterial Agents from Cyclothialidine

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1513

(37) Boehm, H.-J .; Boehringer, M.; Bur, D.; Gmuender, H.; Huber,

W.; Klaus, W.; Kostrewa, D.; Kuehne, H.; Luebbers, T.; Meunier-

Keller, N.; Mueller, F. Novelinhibitors of DNA gyrase: 3D

structure based biased needle screening, hit validation by

biophysical methods, and 3D guided optimization. A promising

alternative to random screening. J . Med. Chem. 2000. 43, 2664-

2674.

clothialidine, coumermycin, and novobiocin. Antimicrob. Agents

Chemother. 1996, 40, 1060-1062.

(40) La´zn´ıcˇek, M.; La´zn´ıcˇkova´, A. The effect of lipophilicity on the

protein binding and blood cell uptake of some acidic drugs. J .

Pharm. Biomed. Anal. 1995, 13, 823-828.

(41) Angehrn, P.; Goetschi, E. Unpublished results.

(42) Radominska-Pandya, A.; Czernik, P. J .; Little, J . M. Structural

and functional studies of UDP-glucuronosyltransferases. Drug

Metab. Rev. 1999, 31, 817-899.

(43) Rutherford, K. G.; Batiste, J . L. H.; Prokipcak, J . M. New

approach to the synthesis of optically active trityl systems. Can.

J . Chem. 1969, 47, 4073-4076.

(44) (a) Mizuuchi K.; Mizuuchi, M.; O’Dea, M. H.; Gellert, M. Cloning

and simplifiedpurification of Escherichia coli DNA gyrase A and

B protein. J . Biol. Chem. 1984, 14, 9199-9201. (b) Hallett, P.;

Grimshaw, A. J .; Wigley, D. B.; Maxwell, A. Cloning of the DNA

gyrase genes under tac promoter control: Overproduction of the

gyrase A and B proteins. Gene 1990, 93, 139-142.

(38) (a) Hebeisen, P.; Angehrn, P.; Gmuender, H.; Goetschi, E.; Link,

H.; Luebbers, T.; Schneider, F. New DNA gyrase inhibitors

related to cyclothialidine: ‘seco’ analogues as potent antibacterial

agents. Poster 042 In Programme and Abstracts of the 20th

International Congress of Chemotherapy; J une 29-J uly 3, 1997,

Sydney, Australia. (b) Rudolph, J .; Theis, H.; Hanke, R.; En-

dermann, R.; J ohannsen, L.; Geschke, F.-U seco-Cyclothia-

lidines: New concise synthesis, inhibitory activity toward

bacterial and human DNA topoisomerases, and antibacterial

properties. J . Med. Chem. 2001, 44, 619-626.

(39) Stieger, M.; Angehrn, P.; Wohlgensinger, B.; Gmuender, H. GyrB

mutations in Staphylococcus aureus strains resistant to cy-

J M0310232

Article Doi

New Antibacterial Agents Derived from the DNA Gyrase Inhibitor Cyclothialidine

DOI: 10.1021/jm0310232

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Full text of DOI:10.1021/jm0310232

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