Antimicrobial effects of novel siderophores linked to β-lactam antibiotics

Bioorganic & Medicinal Chemistry 8 (2000) 73±93

Antimicrobial Eects of Novel Siderophores Linked to

ꢀ-Lactam Antibiotics

T. Kline,* M. Fromhold, T. E. McKennon, S. Cai, J. Treiberg,^yN. Ihle,^{

D. Sherman,^xW. Schwan,^kM. J. Hickey, P. Warrener, P. R. Witte, L. L. Brody,

L. Goltry, L. M. Barker, S. U. Anderson, S. K. Tanaka, R. M. Shawar, L. Y. Nguyen,

M. Langhorne, A. Bigelow, L. Embuscado^{and E. Naeemi**

PathoGenesis Corporation, 201 Elliott Avenue West, Seattle, WA, 98119, USA

Received 2 August 1999; accepted 26 August 1999

AbstractÐAs a strategy to increase the penetration of antibiotic drugs through the outer membrane of Gram-negative pathogens,

facilitated transport through siderophore receptors has been frequently exploited. Hydroxamic acids, catechols, or very close iso-

steres of catechols, which are mimics of naturally occurring siderophores, have been used successfully as covalently linked escorting

moieties, but a much wider diversity of iron binding motifs exists. This observation, coupled to the relative lack of speci®city of

siderophore receptors, prompted us to initiate a program to identify novel, noncatechol siderophoric structures. We screened over

300 compounds for their ability to (1) support growth in low iron medium of a Pseudomonas aeruginosa siderophore biosynthesis

deletion mutant, or (2) compete with a bactericidal siderophore-antibiotic conjugate for siderophore receptor access. From these

assays we identi®ed a set of small molecules that ful®lled one or both of these criteria. We then synthesized these compounds with

functional groups suitable for attachment to both monobactam and cephalosporin core structures. Siderophore-b-lactam con-

jugates then were tested against a panel of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus strains. Although

several of the resultant chimeric compounds had antimicrobial activity approaching that of ceftazidime, and most compounds

demonstrated very potent activity against their cellular targets, only a single compound was obtained that had enhanced, side-

rophore-mediated antibacterial activity. Results with tonB mutants frequently showed increased rather than decreased suscept-

ibilities, suggesting that multiple factors in¯uenced the intracellular concentration of the drugs. # 2000 Elsevier Science Ltd. All

rights reserved.

Introduction

receptors, a class of iron regulated outer membrane

proteins. The siderophore/siderophore receptor systems,

thus, capture and escort iron through the outer mem-

brane and deliver it into the periplasm. Iron anity

constants for the Pseudomonas aeruginosa siderophores

Bacteria must acquire iron by competing with environ-

mental chelation. One mechanism for bacterial iron

acquisition utilizes siderophores, small-molecule chelat-

ing agents that have evolved in parallel with siderophore

range from as weak as 10^-6M for pyochelin to as strong

32

as 10

M for pyoverdin.¹In Escherichia coli and sev-

*Corresponding author. Tel.: +1-206-505-6914; fax: +1-206-282-

5065; e-mail: tkline@pathogenesis.com

eral Pseudomonas species, uptake of ferrisderophores

requires tonB, an essential protein that couples cyto-

plasmic membrane promotive force to active transport

across the outer membrane.¹Following subsequent

delivery of the ferrisiderophore through the inner mem-

brane, the iron is reductively released in the cytoplasm,

and the siderophore is recycled.²While many side-

rophores are associated with a cognate receptor, there

exists a high degree of promiscuity among siderophore

receptors, allowing cross-recognition of a variety of

siderophores beyond the genus.³The siderophores and

receptors for several Gram-negative species, particularly

E. coli^2,4and P. aeruginosa¹have been reviewed.

^yPresent address: ICOS Corporation, 22021 20th Ave. S.E., Bothell,

WA 98021, USA.

^{Present address: Celltech Chiroscience, 1725 220th St. S.E., Bothell,

WA 98021, USA.

^xPresent address: Department of Pathobiology, School of Public

Health and Community Medicine, University of Washington, Seattle,

WA 98195, USA.

^kPresent address: Department of Biology and Microbiology, 3027

Cowley Hall, University of Wisconsin±La Crosse, La Crosse, WI

54601, USA.

^{Present address: Fred Hutchinson Cancer Research Center, 1100

Fairview Ave. N., Seattle, WA 98109, USA.

**Present address: Department of Bioengineering, University of

Washington, Seattle, WA 98195, USA.

PII: S0968-0896(99)00261-8

74

T. Kline et al. / Bioorg. Med. Chem. 8 (2000) 73±93

Gram-negative bacteria in general, and P. aeruginosa in

particular, present a formidable physical barrier to

antibiotics by means of an outer membrane traversed by

restrictive porins.⁵Alternative access through the outer

membrane via siderophore receptors has been exploited

as a strategy for ``illicit transport'' of antibiotics cova-

lently linked to microbial siderophores.^6±11Mimicry of

naturally occurring siderophores led to use of hydro-

xamic acids,^10±13catechols,^6,8,9,14±21or very close iso-

steres of catechols,^6,16as the escorting moiety. Because

their target is periplasmic, b-lactams have been the most

commonly selected antibiotic core structures for this

strategy. Many cephalosporins and monobactams

showed increased activity against Gram-negative patho-

gens, notably E. coli and P. aeruginosa, when covalently

linked to a siderophoric moiety.^15,18An attempt to

extend this strategy to macrolides failed to decrease the

resultant MIC values against P. aeruginosa or other

Gram-negative organisms.²²Recently, Miller has descri-

bed the application of this strategy to nucleoside anti-

fungal and antiviral agents as well.²³

for the lack of endogenous siderophores in a side-

rophore-de®cient mutant. We constructed PGO 2812, a

genetically de®ned P. aeruginosa mutant de®cient in the

production of pyochelin and pyoverdin. Growth of

PGO 2812 in the medium in which the iron is bound by

dipyridyl re¯ects the ability of the exogenously added

compounds to compete the iron away and transport it

though the bacterial membrane.

In parallel to this assay, we developed an assay that

would measure the competition at siderophore receptors

between a siderophore-antibiotic and a panel of small

molecule siderophore candidates. Attenuation of the

bactericidal activity of the siderophore-antibiotic could

be interpreted as a measure of the ability of the side-

rophore to interfere with the antibiotic conjugate's

binding and uptake via the siderophore receptor.^27,28

This assay thus obviates the need to identify the actual

receptor(s) involved in illicit transport, while at the

same time facilitates the discovery of compounds that

access these receptors.

A variety of structural motifs bind iron; this observa-

tion, coupled with the relative lack of speci®city of

siderophore receptors, prompted us to initiate a pro-

gram to identify novel, noncatechol siderophoric struc-

tures. These structures would function as escorting

agents for b-lactams through the outer membrane of

Gram-negative bacteria. Herein, we report the results of

this study.

For the competition assay, we ®rst synthesized (Scheme 1)

a panel of catechol monobactams in order to obtain an

antibiotic ligand with improved Gram-negative activity

that could be abrogated in a tonB mutant of P. aeruginosa.

Whereas 1, 2, 3 and 4 showed no activity against P.

aeruginosa, and very limited activity against other Gram-

negative species, 5, a catechol monobactam analogue of

piperacillin, was active against both E. coli and P. aer-

uginosa (Table 1). The MIC values of 5, particularly in

P. aeruginosa, were increased signi®cantly in the absence

of tonB.

Results

Assays for siderophores have been described.^24±26We

established two independent determinants of side-

rophore activity and developed assays for each of these.

In the ®rst, referred to as the growth promotion assay,

we measured the ability of compounds to compensate

With 5 in hand, we proceeded to screen approximately

300 compounds obtained from commercial sources. Our

selection criteria for these compounds was driven by

either structural homology to a catechol, e.g. 31, or

Scheme 1. (a) Thiourea, K₃Fe(CN)₆, NaOAc (b) (i) NaOH, H₂O (ii) BrCH₂CO₂^tBu (c) TFA, CH₂Cl₂(d) (i) NHS, DCC, THF (ii) 10, NEt₃, H₂O (e)

allyl chloroformate, DIEA, CH₂Cl₂(f) TFA (g) 11, HBTU, DMF, NMM (h) (i) NaOH (ii) H⁺(i) (Ph₃P)₄Pd, 2-ethyl octanoic acid, Ph₃P (j) (i)

[(Me₃N)₂Si]₂, Me₃SiCl (ii) 12, CH₂Cl₂.

T. Kline et al. / Bioorg. Med. Chem. 8 (2000) 73±93

75

Table 2. Siderophore eects, as determined by the two assays, for

catechols and selected noncatechols^a

Table 1. Antibacterial activity of catechol monobactams in wild-type

and tonB mutants of E. coli and P. aeruginosa. Details of the assay are

given in Experimental

Compound

Growth in

low iron

Competition

with 5

Compound

E. coli E. coli P. aeruginosa P. aeruginosa

ATCC 25922 Á tonB Á tonB

PAO-1

13

+

++

1

2

3

4

5

>50

6

>50

12

>50

6.25

ND

>50

ND

>50

14

15

16

+

++

0.4

1.56

metal-binding capability described in another biological

context (e.g. 19^29,30, 29³¹). The screening results for key

compounds, including catechols and noncatechols, are

shown in Table 2. Of the compounds screened, 69 out of

352, 20%, were able to support growth of PGO 2812 in

dipyridyl-containing media. In competition with 5, 48

out of 310, 15%, were able to protect PA01 from the

bactericidal eects of the drug when the drug was pre-

sent at its MIC value. Of these two classes, 25 out of

310, 8%, were active in both assays.

++

17

18

19

+

++

20

21

Although diverse in structure, and even more diverse in

biochemical application, nearly all of the noncatechol

compounds in Table 2 have in common a salicylimine

motif that may serve as a general metal binding func-

tion. Presumably recognition at the siderophore recep-

tor(s) is determined by other distinguishing features of

these compounds. To better understand the predictive

value of the assays, we selected structural motifs from

compounds that were either active in one assay alone or

active in both assays. These structural motifs were then

covalently incorporated into b-lactam antibiotics, and

the minimal inhibitory concentration (MIC) values

against a panel of selected organisms was determined.

+

22

+

23

24

25

26

27

+

Our expectation was that a b-lactam linked to a func-

tional siderophore would have cidal properties against

wild type E. coli and P. aeruginosa, but be far less

eective in tonB mutants, since facilitated transport

would be unavailable to these mutant strains. Use of the

hypersusceptible ATCC 35151 strain of P. aeruginosa

would allow us to measure the intrinsic eect of that

compound at the molecular target, the penicillin-binding

proteins (PBPs) since in ATC 35151 there is a sub-

stantially reduced permeability barrier. Activity against

S. aureus, a prototypical Gram-positive species, would

also be a measure of the activity of the compound

against the PBPs in the absence of an outer membrane

barrier.

++

28

29

30

+

++

+

++

31

+

Chemistry

Synthesis of ArCH₂Br moieties

^aValues are considered+for growth in low iron if the compound is

able to stimulate sucient Pseudomonas aeruginosa PGO 2812 to give

an optical density (OD₆₀₀) of ꢀ 0.5 A.U. at ꢁ 300 mg/mL. Values are

considered+for competition if the bactericidal activity at compound

concentrations of ꢁ 300 mg/ml, as measured by an OD₆₀₀of ꢀ 0.50±

070. Values are ++ if the OD₆₀₀ꢀ 0.71. Details of the assays are

given in Experimental.

While a few of the siderophores in Table 2 had func-

tional groups that could be recruited for direct attach-

ment to an antibiotic core, the remainder were

synthesized as suitably functionalized derivatives,

particularly halomethyl compounds (Scheme 2) that

76

T. Kline et al. / Bioorg. Med. Chem. 8 (2000) 73±93

could be readily appended onto b-lactams at various

positions.

simply protection of the phenolic hydroxyl and radical

bromination. Synthesis of the complementary 2-(2⁰-

(methoxymethyloxy)) phenyl 5-bromomethyl benzthi-

azole 37 followed the procedure of Stevens et al.³²

The two regiomeric ring closure products 36A and B,

produced in approximately 4:1 para:ortho ratio, were

carried forward as a mixture throughout the pre-

paration of the bromomethyl derivatives. Although

regiomeric enrichments could be achieved at each

stage of synthesis, separation was most conveniently

carried out at the oxime ether intermediate 81 shown in

Scheme 5.

For benzthiazole 20, two alternative attachment sites

were investigated. The synthesis of 2-(4⁰-bromomethyl-

2⁰-(methoxymethyloxy))phenylbenzthiazole 33 from 2-

(4⁰-methyl-2⁰-methoxy) phenylbenzthiazole required

N-protected 2- and 3-bromomethyl anilines 40 and 41

were synthesized as shown from commercially available

anilines.

The key O-protected bromomethylsalicylaldehyde 44

was prepared in 6 steps from 4-methylsalicylic acid, and

converted to the desired acylhydrazones 45±49 either

before or after alkylation of the thiopyridyl inter-

mediate 89 in Scheme 7.

Oxadiazole 52 was prepared by acylation and cyclode-

hydration of salicylic amide,³³followed by protection of

the phenolic hydroxyl and benzylic bromination.

Flavones 59, 60 and 61 were synthesized from the cor-

responding ortho hydroxy acetophenones and sub-

stituted benzoyl chlorides.^34,35

Synthesis of antibiotic siderophore conjugates Schemes

3, 4, 5, 6, and 7

(S)-Dihydroaeruginoic acid 19 is the biogenic precursor

to the Pseudomonas siderophore pyochelin,³⁶and was

one of the most potent siderophores identi®ed in our

screens. We ®rst explored direct acylation of a mono-

bactam by both epimeric dihydroaeruginoic acids in a

linkage conceptually analogous to the siderophore-

monobactam relationship of 5.

Dihydroaeruginoic acids 18 and 19 were synthesized

according to Ankenbauer et al.,³⁷and used to acylate

directly (3S)-trans-3-amino 4-methyl 2-oxoazetidine-1-

sulfonic acid 10³⁸(Scheme 3).

The same structural motif was also used in the con-

struction of 70 (Scheme 4) where a 3-thioether linkage

was used at the attachment site.

A frequently used mode of linkage for siderophores to

both monobactams and to cephalosporins is the oxime

ether a to the amide bond between 2-aminothiazole

acetic acid and the b-lactam.^39±44

Scheme 2. A.(a) MOMCI, DBU, THF 0 ^ꢂC to rt (b) NBS, AIBN, Á,

CCl₄(c) Lawesson's reagent (d) NaOH, K₃Fe(CN)₆(e) BBr₃, CH₂Cl₂,

78 ^ꢂC. B.(a) MeOH, DCC, Et₂O (b) MOMCl, DIEA, CH₃CN (c) (i)

1N NaOH, EtOH (ii) H⁺(d) (i) MsCl, pyridine (ii) NH₃, MsCl(e)

NBS, AIBN, Á CCl₄(f) DIBAL, toluene (g) RCONHNH₂, AcOH,

EtOH (h) BuNHNH₂. C.(a) (i) pyridine, 0 ^ꢂC (ii) p-CH₃C₄H₄COCl

t

(b) 200 ^ꢂC (c) HCl.NH₂OH, NaOAc, EtOH (d) NaH, MOMCl,

CH₂Cl₂(e) NBS, AIBN, Á CCl₄. D.(a) pyridine, R₂R₃C₆H₃COCl (b)

KOH, pyridine (c) AcOH, H₂SO₄(d) (i) PhI(OAc)₂, KOH, MeOH (ii)

HCl, acetone (e) MOMCl, NaH, CH₂Cl₂(f) NBS, AIBN, Á CCl₄.

Scheme 3. (a) NHS, DCC (b) 10, DBU (c) (Bu)₄N⁺SO₃H .

T. Kline et al. / Bioorg. Med. Chem. 8 (2000) 73±93

77

Scheme 6. (a) DCC, HOAt, 82, THF (b) 5:1 TFA-anisole, 0 ^ꢂC to rt

(c) (i) CF₃CON(Me)₃, CH₂Cl₂(ii) (Me)₃Sil (iii) THF (iv) 29 (for 87) or

30 (for 88) CH₃CN.

Scheme 4. (a) NaH, p-MeOC₄H₄CH₂SH, DMF, Nal (b) (i) NaOH,

H₂O/THF (ii) H⁺(c) BnNH₂, EDCl, Et₃N, CH₂Cl₂(d) (i) Tf₂O, pyr-

idine, 50±0 ^ꢂC (ii) l-Cysteine methyl ester, 30 ^ꢂC (iii) NaOH, H₂O/

THF (iv) H⁺(e) (i) TFA, anisole (ii) 69, K₂CO₃, DMF, Nal (iii) TFA,

anisole.

Scheme 5. A(a)^tbutyl carbazate, DIEA (b) N-hydroxypthalimide,

K₂CO₃, Nal (c) H₂NNH₂, EtOH (d) 74, EtOH, AcOH (e) 50%

NaOH, Á (f) 10, DCC, HOAt, DMAP, DMF (g) TFA (h) salicy-

laldehyde, EtOH, AcOH. B(a) allyl alchohol, NaOEt, 60 ^ꢂC (b) 33,

K₂CO₃, KI, DMF, rt (c) (i) (Ph₃P)₄Pd, (Bu)₃SnH, THF (ii) AcOH,

CH₃CN (d) DCC, HOAt, 80 , Et₃N, DMAP, DMF, rt (e) 3N HCl,

THF-H₂O, rt (f) 37, K₂CO₃, Kl, KMF, rt (g) 82, POCl₃, pyr, 0 ^ꢂC (h)

TFA, anisole, CH₂Cl₂(i) 84 POCl₃, pyr, Et₃N, CH₂Cl₂(j) pyr, Nal, THF.

Scheme 7. (a)(i) 1 N NaOH (ii) 4-mercaptopyridine (b) ref. 13 (c)

Ar(MOM)CH₂X (d) 5:1 TFA-anisole.

78

T. Kline et al. / Bioorg. Med. Chem. 8 (2000) 73±93

Bromomethyl derivatives of acylhydrazone 26 and

benzthiazole 20 were incorporated as oximinoethers

into, respectively, the trans (77) and cis (79) mono-

bactams; 20 was additionally incorporated into the

cephalosporins 83 and 85 (Scheme 5).

hypersusceptible P. aeruginosa ATCC 35151 strain that

is considered to be membrane-permeable, all three

compounds were still over an order of magnitude more

active than in the wild type PAO-1. This indicated that

for these compounds permeability remained an obstacle

to high activity.

The key intermediate, aminothiazole derivative 78, was

prepared by transesteri®cation of the commercially

available ethyl ester to the allyl ester, followed by alky-

lation of the oxime with bromide 33, and ®nally Pd[0]-

promoted deallylation. Acid 78 was coupled to 80 to

provide monobactam 79. The cephalosporin synthesis

began with the corresponding transesterication and

alkylation reactions. Facile separation of the alkylation

products 81A and 81B on silica gel provided the indivi-

dual regiomeric benzthiazole derivatives in a 1:10 ratio.

Only the major isomer 81B was carried forward to

cephalosporins 83 and 85.

Unexpected results were obtained when we tested our

conjugates against organisms intended to serve as con-

trols. When tested against the tonB mutants of both

E. coli and P. aeruginosa, several compounds showed

increased rather than decreased susceptibilities. How-

ever, similar results have been reported for the b-lac-

tams carbenicillin and cefepime, and can be rationalized

by considering mechanisms for eux as well as in¯ux of

drugs through the membrane.^45±49Upregulation of the

MexAB-OprM eux pump system is a signi®cant

mechanism of P. aeruginosa resistance to b-lactam

drugs. While tonB is not essential for the expression of

MexAB-OprM resistance, this energy-coupling protein

nonetheless facilitates MexAB-OprM function. Mutants

de®cient in tonB have been shown to be orders of mag-

nitude more sensitive to drugs from several structural

classes. This sensitivity has been attributed to loss of

TonB enhancement of pump activity.⁵⁰Thus the eect

of increased drug concentration (from compromised

eux pumps) might have oset the eect of decreased

penetration (from loss of siderophore receptor activity)

to give rise to the MIC values measured for many of the

conjugates in Table 3. A second unexpected result was

the robust antistaphylococcal activity of compounds 79,

83 and 85, since Gram-positive activity in b-lactams is

often inferior to Gram-negative activity.⁵¹Again, there

is some precedent for the S. aureus susceptibility: the

benzthiazole nucleus has been reported to impart

improved activity to cephalosporins,⁵¹carbapenems,²⁵

and carbacephems against both methicillin sensitive

(MSSA) and resistant (MRSA) S. aureus.⁵³We did not

observe any signi®cant activity in MRSA strains, how-

ever, suggesting that the mechanism of methicillin resis-

tance was still maintained against these compounds.

Ceftazidime, as well as a variety of siderophore±cepha-

losporin conjugates, exploit the bene®cial properties of

a 3-heterocyclic onium group for enhancing Gram-

negative activity.^35,45±49Isonicotinyl hydrazones 87 and

88 were synthesized by direct quaternization of the 3-

acetoxy cephalosporin derivative 86, by the method of

Brown et al.⁴⁷(Scheme 6).

For the majority of the bromomethyl derivatives,

attachment to the cephalosporin nucleus at the 3-meth-

ylene could be achieved by a convergent route from

intermediate 89 (Scheme 7). We also synthesized 90, 91

and 92 as control analogues lacking the siderophore,

and 93 as a positive control.^12,13

Antimicrobial activity

Siderophores 24 and 25 that were active as determined

by growth promotion alone were able to give rise to

antibiotics (104 and 105 respectively) having anti-

pseudomonal activity. Several additional analogues in

this class had modest activity against E. coli strains,

although this activity was tonB-insensitive. In general,

we observed the E. coli strains to be more susceptible

than P. aeruginosa strains to all our novel compounds.

We next directed our attention to the nature of the

membrane transport of the siderophores when cova-

lently bound to the antibiotic core. We focused on three

representative compounds that, despite attachment to

promising siderophores, were inactive (63), weakly

active (70), or tonB insensitive (70 and 104). The results

are shown in Figure 1.

Mixed results were obtained from siderophores deter-

mined to be positive in both assays. Chimeric com-

pounds in this class included inactive (62, 63, 102) as

well as active (77, 87, 88, 101, 106) analogues against

P. aeruginosa and E. coli. Of these, isonicotinyl

hydrazone 87 alone gave the expected pro®le for tonB-

sensitive transport. Two additional compounds in this

class, 70 and 94, were active only against E. coli.

Siderophore 19 was re-examined as its conjugate 63 in

both siderophore assays. Compound 63 neither sup-

ported growth in low iron nor competed with 5,

although the conjugate was present at concentrations

(50±100 mg/mL) above those required for the side-

rophoric eects of 19 (30 mg/mL). At 50 mg/mL, 63 had

no detectable bactericidal eects. Attachment of 24

(active in the growth promotion assay at 30 mg/mL) to a

cephalosporin (104) abrogated the siderophoric proper-

ties of the acylhydrazone. This observation con®rmed

and explained our observation that the Gram-negative

activity of 104 was insensitive to tonB. More complex

results were obtained with 70 in which we had attached

Hydrazone 23, the associated siderophore of 103 was

inactive in both assays, yet the antibiotic conjugate

showed Gram-negative activity comparable to com-

pounds (e.g. 87, 101) with strong siderophores.

The MIC values of the most active antipseudomonal

compounds, 104 and 106, approached the MIC value

for ceftazidime, but were independent of any in¯uence

of the conjugate siderophores. When tested against the

T. Kline et al. / Bioorg. Med. Chem. 8 (2000) 73±93

79

Table 3. Antibacterial activity of siderophore-b-lactam conjugates in wild-type and tonB mutants of E. coli and P. aeruginosa, hypersusceptible

P. aeruginosa ATCC 35151, and S. Aureus. Details of the assay are given in Experimental

Strains, MIC in mg/mL

Category

Compound

E. coli

ATCC 25922

E. coli

Á tonB

P.aeruginosa

PAO-1

P. aeruginosa

Á tonB

P. aeruginosa

ATCC 35151

S. aureus

ATCC 29213

Positive in growth assay

79

83

85

94

96

97

98

99

12.5

>50

6.25

12.5

6.25

25

12.5

1.56

3.13

6.25

3.13

>50

6.25

12.5

6.25

12.5

0.78

3.13

>50

50

>50

50

>50

50

3.13

12.5

>50

25

>50

25

3.13

25

>50

3.13

6.25

>50

0.4

6.25

0.78

1.56

0.4

3.13

6.25

50

3.13

0.2

0.4

<0.1

1.56

<0.1

100

104

105

12.5

6.25

12.5

Positive in both assays

62^a

63^a

70

77

87

88

94

101

102

106

>5 0

12.5

3.13

12.5

3.13

>50

3.13

>5 0

6.25

25

12.5

3.13

>50

3.13

>5 0

50

12.5

>50

12.5

>50

6.25

N D ^b

N D

50

25

>50

25

ND

N D

<0.1

0.2

<0.1

0.2

0.78

<0.1

25

>50

>5 0

>50

6.25

50

25

1.56

12.5

>50

12.5

>50

0.78

<0.1

Negative in both assays

Controls

103

3.13

1.56

25

6.25

<0.1

6.25

90

91

92

93

1.56

0.78

1.56

0.2

0.78

0.4

0.78

1.56

0.13

25

0.78

2±4

12.5

6.25

12.5

0.25

<0.1

0.03

3.13

6.25

12.5

8

Ceftazidime

0.13±0.25

^aMicrobroth dilution MIC.

^bND=not done.

membrane, not ligated with sucient iron to support

growth, or both.

Conclusions

By the use of our assays, novel noncatechols have been

identi®ed that can function as siderophores in Gram-

negative bacteria. Most of these compounds have an

embedded salicylimine motif within their structure. In

general, more active conjugate compounds were

obtained using siderophores that had shown activity in

both assays than were obtained from siderophores

active in growth promotion alone. Signi®cant bacter-

icidal activity was not, however, generally attributable

to facilitated transport. The nature of the linkage of the

siderophore to the antibiotic core did not appear to

in¯uence the activity of the conjugate.

Figure 1. Comparison, in both indices of activity, of the siderophoric

eects of 19 and 24, ®rst as discrete compounds and then after cova-

lent attachment to antibiotic core structures. The growth in low iron

data assay data represent a single experiment; competition assays were

performed in triplicate. Details of the assay are given in Experimental.

Thus, our screens identi®ed a wide variety of novel

siderophores, but did not predict which of these com-

pounds would serve as escorting agents for b-lactam

antibiotics. Indeed, many of the most eective com-

pounds lost their siderophoric capabilities when

covalently linked to the b-lactam. In most cases the

siderophore-b-lactam conjugates retained good activity

against the hypersusceptible ATCC 35151 strain. Activ-

ity against S. aureus ranged from modest to very good.

Taken together, the ATC 35151 and S. aureus results

thiazoline siderophore 19 to a cephalosporin at C-3, a

position others have used for catechol siderophores:

Although 70 no longer supported the growth of PGO

2812, it did aord modest protection against 5. The

siderophore was apparently recognized at the cognate

receptor(s), but either not transported across the outer

80

T. Kline et al. / Bioorg. Med. Chem. 8 (2000) 73±93

suggest that the direct eects of the antibiotic moieties

at their molecular targets, the penicillin-binding proteins

(PBPs), were unimpaired by the siderophores. The fact

that antibiotic potencies were largely retained, or even

improved, in the tonB mutants of both E. coli and P.

aeruginosa raised the possibility that the MexAB/OprM

eux system may have contributed to the activities of

the siderophore-antibiotic conjugates. These intriguing

possibilities are being investigated.

6.83±6.84 (1H, bs). To a solution of 6 (8.77 g, 40 mmol),

stirred in 144 mL of 1 N NaOH for 15 min, was added

t-butyl bromoacetate (5.81 mL, 40 mmol), and the reac-

tion mixture stirred for 3 h, then extracted four times

with ethyl acetate. The combined extracts were washed

with NaCl, dried, and concentrated in vacuo to a resi-

due that was puri®ed by silica gel chromatography

(10:1, hexane:ethyl acetate) to give 5.67 g (62%) of 7.

t-Butyl ester 7 (123 mg, 0.53 mmol) was treated with

1 mL of TFA for 1.5 h. The reaction mixture was con-

centrated in vacuo, and the residue dissolved in 2 mL of

THF. To this solution, DCC (119 mg, 0.58 mmol) and

NHS (67 mg, 0.58 mmol) were added. After stirring for

2 h, the precipitate was removed by ®ltration, and the

®ltrate was added to a solution of monobactam 10

(81 mg, 0.45 mmol) and triethylamine (82 ml, 59 mmol)

in 1 mL water. The reaction mixture was stirred at room

temperature for 3 h, then concentrated in vacuo, and the

residue puri®ed by chromatography on a Sephadex

Experimental

Chemistry

General. Unless otherwise stated, all reactions were run

under a nitrogen atmosphere. THF was distilled from

benzophenone ketyl; all other anhydrous solvents were

obtained from Aldrich in sure-seal¹bottles and stored

under a nitrogen atmosphere. Commercially-obtained

reagents were of the highest available purity. NMR

spectra were taken on a GE QE-300 MHz or Bruker 400

MHz spectrometer; with proton chemical shifts reported

in ppm referenced to tetramethylsilane. Mass spectra

were taken on a Finnigan SSQ-3000 (low resolution

electrospray), Micromass Quattro II Tandem Quadru-

pole (low resolution electrospray), Micromass 70 SEQ

(EI, CI, low and high resolution FAB), or Perceptive

Biosystems Mariner Electrospray TOF (high resolution

electrospray) instrument. Organic solutions were dried

over anhydrous MgSO₄unless otherwise stated. Vola-

tiles were removed in vacuo on a rotary evaporator at

ca. 1±10 Torr. HPLC puri®cations on the Beckman

Gold Nouveau system were performed on a 250Â21 mm

10 mm 330 A Phenomenex Jupiter octadecylsilane

(ODS) column; HPLC puri®cations on the Gilson sys-

tem were performed on a 250Â21 mm 10 mm 300 A

Phenomenex Jupiter ODS column. Analytical HPLC

data were obtained on the Beckman system using a

Phenomenex Columbus 250Â4.6 mm 5 mm 100 A ODS

column. HPLC eluant A was 0.05% TFA in water;

HPLC eluant B was 0.05% TFA in CH₃CN. Pre-

parative silica gel chromatography was performed

under medium-pressure ¯ash conditions⁵⁴with Merck

40±63 mm silica or on a Biotage system using pre-packed

32±63 mm 60 A silica cartridges.

1

LH-20 column (MeOH) to give 124 mg (59%) of 1. H

NMR (300 MHz, CD₃OD) d 1.28 (9H, t, J=7.0 Hz),

1.48 (3H, d, J=6.0 Hz), 3.18 (6H, q, J=7.0 Hz), 3.44

(2H, s), 3.82±3.84 (1H, m), 4.38 (1H, d, J=3.0 Hz),

6.71±6.72 (1H, m), 6.82±6.84 (1H, m), 6.91±6.92 (1H, m).

HRMS, m/z for C₁₂H₁₄N₂O₇S₂calculated, 362.2117,

found, 361.0181(MH ).

trans-3-[2-(3,4-Dihydroxyphenylcarbonyl)amino-2-(amino-

thiazol-4-yl)acetyl]-4-methyl-2-oxoazetidine-1-sulfonic

acid (2). Ethyl 2-amino-a-(N-(tbutoxycarbonyl)amino)-

4-thiazole acetate (1.23 g, 4.0 mmol) and diisopropyl-

ethylamine (1.96 mL, 11.2 mmol) were stirred in 12 mL

CH₂Cl₂, and allyl chloroformate (1.62 mL, 15 mmol) in

2 mL CH₂Cl₂was added slowly. The mixture was stirred

at room temperature for 3 h. The crude product was

isolated and puri®ed by silica gel chromatography (8:1

1

hexane:ethyl acetate) to give 1.66 g (75%) of 107. H

NMR (300 MHz, CDCl₃) d 1.20 (3H, t, J=7.3 Hz), 1.44

(9H, s), 4.16 (2H, q, J=7.3 Hz), 4.70 (2H, d, J=5.0 Hz),

4.88 (2H, s), 5.26 (1H, m), 5.32±5.34 (1H, m), 5.88±6.12

(1H, m), 7.00 (1H, s).

The 2-allyloxycarbonyl amine 107 (143 mg, 0.37 mmol)

was stirred with 0.5 mL TFA for 20 min. Excess acid

was removed in vacuo, and the residue was dissolved in

ethyl acetate and washed with sodium bicarbonate, dried,

and concentrated in vacuo to give the crude a-amine

(99 mg, 94%) as a residue that was dissolved in 1 mL

DMF. To this solution 3,4-di-(methoxymethyloxy)-

benzoic acid 11 (102 mg, 0.42 mmol), HBTU (158 mg,

0.42 mmol) and N-methylmorpholine (46 ml, 0.42 mmol)

were added. The reaction mixture was stirred overnight,

then concentrated in vacuo. The crude product was

puri®ed by silica gel (4:1 hexane:ethyl acetate) to give

145 mg (77%) of 9. ¹H NMR (300 MHz, CDCl₃) d 1.20±

1.28 (3H, m), 3.44 (3H, s), 3.48 (3H, s), 4.06±4.20 (2H,

m), 4.70 (2H, d, J=5.0 Hz), 5.20 (2H, s), 5.28 (2H, s),

5.20±5.44 (2H, m), 5.86±5.98 (2H, m), 6.99 (1H, s), 7.10

(1H, d, J=10.0 Hz), 7.32±7.36 (1H, m), 7.51±7.53 (1H,

m), 7.62 (1H, d, J=2.0 Hz), 9.00 (1H, bs).

trans-3-[2-(3,4-Dihydroxyphenylthioacetyl)amino]-4-meth-

yl-2-oxoazetidine-1-sulfonic acid triethylammonium salt

(1). Catechol (2.6 g, 110 mmol) was dissolved in 40 mL

of water. To it thiourea (1.52 g, 20 mmol) was added at

room temperature. A solution of potassium ferricyanide

(13.0 g, 40 mmol) and sodium acetate (20.0 g, 240 mmol)

in 60 mL of water was added to the solution, and addi-

tional sodium acetate (20 g) was added to the mixture.

After stirring overnight, the reaction mixture was

dissolved in 1N HCl, and the insoluble material was ®l-

tered o. To the aqueous ®ltrate were added sodium

acetate (20.0 g) and water (40 mL). The mixture was

stirred to precipitate a salt that was ®ltered and dried in

vacuo to give 2.67 g. The reaction was repeated twice

more, for a total of 8.77 g (11%) of 6. ¹H NMR

(300 MHz, CDCl₃) d 1.77 (3H, s), 6.78±6.82 (2H, m),

Compound 9 (173 mg, 0.34 mmol) was re¯uxed in 2 mL

0.5N NaOH for 1 h. After cooling, the reaction mixture

T. Kline et al. / Bioorg. Med. Chem. 8 (2000) 73±93

81

was acidi®ed to pH ꢃ2 with 0.1 N HCl and extracted

into ethyl acetate. The organic layer was washed with

water, dried, and concentrated in vacuo to give 141 mg

(87%) of the free acid that was coupled to trans b-lactam

10 as described for 1. To the crude amide (125 mg,

0.17 mmol) in 0.5 mL CH₂Cl₂was added triphenylpho-

sphine (2 mg), tetrakis(triphenylphosphine) palladium

(2 mg), and then 2-ethyl octanoic acid (28 mg,

0.19 mmol), and the reaction mixture stirred for 2 h at

room temperature. The reaction mixture was con-

centrated in vacuo, and the residue puri®ed on Sepha-

dex LH-20 column (CH₃OH) to give 50 mg (45%) of

108. ¹H NMR (300MHz, CDCl₃) 1.30 (9H, t, J=7.3Hz),

1.50 (3H, d, J=8.5 Hz), 3.10 (6H, q, J=7.3 Hz), 3.46

(6H, two s), 4.00±4.06 (1H, m), 4.36±4.42 (1H, m), 5.20

(4H, two s) 5.60±5.70 (1H, m), 6.40 (2H, bs), 6.48±5.02

(1H, m), 7.08±7.12 (1H, m), 7.54±7.60 (1H, m), 7.66±

7.68 (1H, m), 7.94±8.04 (1H, m), 8.54±8.60 (1H, m).

trans-3-[ꢁ-(4-Ethyl-2,3-dioxopiperazine-1-carbonyl)-3-

(3,4-dihydroxyphenyl)-^D-alanine]amino-4-methyl-2-oxo-

azetidine-1-sulfonic acid triethylammonium salt (4). d-

DOPA (500 mg, 2.4 mmol) was converted to 129 mg

(50%) of 4 according to the procedure described for 3.

¹H NMR (400 MHz, CD₃OD) d 1.16 (3H, t. J=7.5 Hz),

1.24 (9H, t, J=7.3 Hz), 1.46 (3H, d, J=8.5 Hz), 2.94±

2.96 (2H, m), 3.18 (6H, q, J=7.3 Hz), 3.48 (2H, q,

7.5 Hz), 3.58±3.62 (2H, m), 3.85±3.86 (1H, m), 3.38±3.40

(2H, m), 4.32±4.39 (1H, m), 4.49±4.51 (1H, m), 6.53±

6.54 (1H, m), 6.65±6.66 (2H, m). HRMS, m/z for

C₂₆H₂₅N₅O₁₀S calculated, 527.2255, found, 526.1246

(M ).

trans-3-[ꢁ-(4-Ethyl-2,3-dioxopiperazine-1-carbonyl)-3,4-

D,L -dihydroxyphenylglycine]amino-4-methyl-2-oxoaze-

tidine-1-sulfonic acid triethylammonium salt (5). a-

Amino-(3,4-dihydroxyphenyl)acetic acid HBr⁵⁵(73 mg,

0.28 mmol) was converted to 61 mg, (36%) of 5 follow-

ing the same procedures described for 3. ¹H NMR

(400 MHz, CD₃OD) d 1.31 (3H, t, J=7.2 Hz), 1.41 (9H,

t, J=7.3Hz), 1.60±1.64 (3H, m), 3.32 (6H, q, J=7.3 Hz),

3.64 (2H, q, J=7.2 Hz), 3.75±3.76 (2H, m), 3.93±4.06

(1H, m), 4.14±4.15 (2H, m), 4.56±4.57 (1H, m), 5.35±

5.36 (1H, m), 6.86±6.88 (2H, m), 6.97±6.99 (1H, m).

HRMS, m/z for C₁₉H₂₃N₅O₉S calculated, 513.2099,

found, 512.1088 (M ).

Compound 108 (50 mg, 0.08 mmol) was stirred with 1:1

TFA:CH₂Cl₂at room temperature for 30 min. and con-

centrated in vacuo. The residue was puri®ed on a

Sephadex LH-20 column (CH₃OH) to give 8 mg (22%)

of 2. ¹H NMR (300 MHz, CD₃OD) d 1.52 (3H, d,

J=8.5 Hz), 4.16±4.22 (1H, m), 4.38±4.44 (1H, m), 4.80

(1H, s), 5.78 (1H, s), 6.78±6.80 (1H, m), 7.30±7.40 (2H,

m). MS, m/z 470.4 (M ).

trans-3-[ꢁ-(4-Ethyl-2,3-dioxopiperazine-1-carbonyl)-3-

(3,4-dihydroxyphenyl)-^L-alanine]amino-4-mehtyl-2-oxo-

azetidine-1-sulfonic acid (3). l-DOPA (0.99 g, 5.0 mmol)

was re¯uxed in 1,1,1,3,3,3-hexamethyldisilazane (8 mL)

and trimethylsilyl chloride (2 mL) for 2 h. After cooling,

excess reagent was removed by concentration in vacuo.

The residue was dissolved in dry CH₂Cl₂(10 mL) and

cooled to 0 ^ꢂC. A solution of 4-ethyl-2, 3-dioxopiper-

azine-1-carbonyl chloride 12 (1.12 g, 5.5 mmol) in 8 mL

CH₂Cl₂was added dropwise. The mixture was stirred

for 1.5 h at room temperature, and concentrated in

vacuo. The residue was dissolved in 2:1 acetone:water

(30 mL) at pH 1ꢃ2 with vigorous stirring for 18 min.

After the removal of acetone, the aqueous solution was

extracted with 1:1 ethyl acetate:n-butanol. The aqueous

phase was saturated with NaCl and extracted again with

1:1 ethyl acetate:n-butanol. The combined extracts were

dried and concentrated in vacuo to give 0.85 g (49%)

of 109. ¹H NMR (400 MHz, CD₃OD) 1.19 (3H, t,

J=7.2 Hz), 2.96 (1H, dd, J=6.8, 7.2 Hz), 3.08 (1H, dd,

J=4.9, 8.1 Hz), 3.50 (2H, q, J=7.2 Hz), 3.59±3.62 (2H,

m), 3.98±4.02 (2H, m), 4.56±4.60 (1H, m), 6.56 (1H,

dd, J=2.1, 5.9 Hz), 6.67±6.69 (1H, m), 9.29 (1H, d,

J=7.1 Hz).

2-(2-Methoxymethyloxy-4-methyphenyl) benzthiazole (32).

To an icebath-cooled solution of 2-(2-hydroxy-4-meth-

ylphenyl) benzthiazole (50.0 g, 20.7 mmol) in 50 mL

THF was added 1,8-diazabicyclo [5.4.0] undec-7-ene

(DBU, 3.1 mL, 20.7 mmol) followed by a solution of

chloromethyl methyl ether (1.75 g, 21.7 mmol) in 10 mL

THF. The reaction mixture was allowed to equilibrate

to ambient temperature and was stirred overnight. The

THF was removed in vacuo and the residue dissolved in

CH₂Cl₂, washed successively with saturated NH₄Cl,

NaHCO₃, NaCl, dried, and concentrated in vacuo to

give 5.78 g (99%) of 32 as a waxy solid that was homo-

genous by tlc (R_F=0.25 in 100% hexane). ¹H NMR

(400 MHz, CDCl₃) d 2.40 (3H, s), 3.57 (3H, s), 5.51 (2H,

s), 6.99 (1H, d, J=8.1 Hz), 7.07 (1H, s), 7.46±7.48 (2H,

m), 7.91 (2H, dd, J=8.1, 9.7 Hz), 8.41 (1H, d, J=

8.1 Hz). MS m/z 286.1 (MH⁺)

2-(2-Methoxymethyloxy-4-bromomethylphenyl) benzthia-

zole (33). To a solution of 32 (5.78 g, 20 mmol) in

200 mL CCl₄was added N-bromosuccinimide (3.98 g,

22.3 mmol) followed by 2,2-azobisisobutyronitrile

(AIBN, 333 mg). The solution was re¯uxed while being

illuminated by a 150 W halogen light for 16 h, and

additional AIBN was added at 6 and 12 h. The reaction

mixture was washed with water, the aqueous phase re-

extracted with CH₂Cl₂, and the combined organic pha-

ses washed with NaCl, dried, and concentrated in vacuo

to an oily residue. The product was puri®ed on ¯ash

silica (99:2 hexane:ethyl acetate) to give 1.1 g of recov-

ered starting material, followed by elution with 96:5

hexane:ethyl acetate to give 3.47 g. (58%) of 33. ¹H

NMR (400 MHz, CDCl₃) d 3.59 (3H, s), 4.52 (2H, s),

5.45 (2H, s), 7.26 (1H, d, J=16.2 Hz), 7.38±7.40 (1H,

m), 7.50 (2H, d, J=16.0 Hz), 8.06 (2H, dd, J=8.1,

Coupling of 109 to trans b-lactam 10 as described for 1

gave 3 in 84% yield. ¹H NMR (400MHz, DMSO) d 1.06

(3H, t, J=7.1 Hz), 1.17 (9H, t, J=7.3 Hz), 1.35 (3H, d,

J=6.1 Hz), 2.82±2.83 (1H, m), 2.86±2.87 (1H, m), 3.09

(6H, q, J=7.3 Hz), 3.37 (2H, q, J=7.1 Hz), 3.51±3.58

(3H, m), 3.85±3.88 (2H, m), 4.26±4.28 (1H, m), 4.40±

4.42 (1H, m), 6.43±6.45 (1H, m), 6.57±6.61 (2H, m) 8.71

(1H, s), 8.74 (1H, s), 8.93 (1H, d, J=7.8 Hz), 9.08 (1H,

d, J=7.4 Hz). HRMS, m/z C₂₀H₂₅N₅O₁₀S calculated,

527.2255, found, 526.1246 (M ).

82

T. Kline et al. / Bioorg. Med. Chem. 8 (2000) 73±93

8.8 Hz), 8.51 (1H, d, J=8.1 Hz). MS, m/z 363.9/365.9

(MH⁺).

7.14 (1H, s), 7.22 (2H, d, J=3.5 Hz), 7.40 (1H, dd,

J=1.6, 1.9 Hz), 7.95 (1H, d, J=8.2 Hz), 8.53 (1H, d,

J=2.8 Hz). MS, m/z 286.0 (MH⁺), 308.2 (MNa⁺).

2-(2-Methoxymethyloxyphenyl)-2-methyl-benzthiazole

(36A) and 2-(2-Methoxylmethyloxyphenyl)-4-methyl-

benzthiazole (36B). To an icebath-cooled solution of

m-toluidine (6.37 mL, 58.8 mmol) and triethylamine

(8.20 mL, 58.8 mmol) in 20 mL dry CH₂Cl₂, anisoyl

chloride (10.0 mL, 58.8 mmol) was added dropwise. The

reaction mixture was allowed to equilibrate to ambient

temperature overnight, and washed successively with

1 N HCl, saturated NaHCO₃, and NaCl, dried, and

concentrated in vacuo to give 13.7 g (99%) of 34 as an

o-white solid. MS, m/z 242.0 (MH⁺).

2-(2-Methoxymethyloxyphenyl)-2-bromomethylbenzthia-

zole (37A) and 2-(2-))-4-methoxy-methyloxyphenyl bro-

momethylbenzthiazole (37B). Radical bromination of 36

(1.84 g, 6.4 mmol) was performed as described for 33.

Chromatography on silica gel using successively 99:2,

then 96:5, hexane:ethyl acetate gave 2.2 g (94%) of 37.

¹H NMR (400 MHz, CDCl₃) d 3.54 and 3.56 (3H, two s,

1:3 ratio), 4.62 and 4.71 (2H, two s 1:3 ratio), 5.37±5.40

(2H, two s), 7.15 (1H, s), 7.25 (2H, dd, J=1.6, 8.3 Hz),

7.37±7.42 (2H, m), 7.93±7.96 (1H,m), 8.53 (1H, d,

J=2.8 Hz).

Amide 34 was combined with Lawesson's reagent

(13.2 g, 32.6 mmol) in 100 mL of toluene, and re¯uxed

for 11 h. After cooling, a mixture of 98:3 hexane:ethyl

acetate was added to the solution, and the product pre-

cipitated upon standing at room temperature for several

hours. Recrystallization from EtOH±hexane gave 7.4 g

(53%) of the thioamide 35. MS, m/z 258.0 (MH⁺).

1-(2-[N-tert-Butoxycarbonyl]aminophenyl)methyl bromide

(40). To a rapidly stirring solution of 2-aminobenzyl

alcohol (5.00 g, 40.6 mmol) in CH₂Cl₂(100 mL) was

added di-tbutyldicarbonate (9.75 g, 44.6 mmol) and

DIEA (7.8 mL, 44.6 mmol). After 18 h the solution was

washed with aqueous HCl (1 M, 3Â35 mL) and water

(25 mL). The organic phase was dried and con-

centrated to give 10.9 g of a brown oil. Chromato-

graphy (80:20 hexane:ethyl acetate) aorded 9.1 g

(quantitative) of 38 as pale yellow oil. To a solution of

this material (3.18 g, 14.2 mmol) in THF (50 mL) at

20 ^ꢂC was added triphenylphosphine (4.49g, 17.1 mmol),

followed by N-bromosuccinimide (3.04 g, 17.1 mmol).

After 3 h the solvent was removed in vacuo, and the

residue chromatographed (90:10 hexane:ethyl acetate)

To a suspension of 35 (7.4 g, 28.9 mmol) in 20 mL EtOH

was added a solution of 7.5 M NaOH (30.8 mL) fol-

lowed by an additional 62.5 mL of water. This suspen-

sion was added in 1 mL aliquots to a 90 ^ꢂC solution of

K₃Fe(CN)₆(38 g, 115.6 mmol) in 200 mL of water. The

reaction mixture was stirred an additional 90 min fol-

lowing complete addition, then cooled to room tem-

perature, and the product ®ltered o. Recrystallization

from MeOH gave 5.6 g (90%) of 110 as light brown

1

to provide 3.14 g (77%) of 40 as a tan oil. H NMR

1

crystals. H NMR (400 MHz, CDCl₃) d 2.51 and 2.62

(400 MHz, CDCl₃) d 1.56 (9H, s), 4.53 (2H, s), 6.70 (1H,

bs), 7.08 (1H, dt, J=0.9, 7.5 Hz), 7.30 (1H, dd, J=1.4,

7.6 Hz), 7.36 (1H, dt, J=1.5, 7.8 Hz), 7.85 (1H, d,

J=8.1 Hz).

(3H, two s, 1:2 ratio), 4.02 and 4.06 (3H, two s), 6.99±

7.25 (3H, m), 7.39±7.51 (2H, m), 7.77 (1H, d, J=16.0 Hz),

7.98 (1H, t, J=15.5 Hz), 8.49±8.52 (1H, m). MS, m/z

256.0 (MH⁺).

1-(3-[N-tert-Butoxycarbonyl]aminophenyl)methyl bromide

(41). Intermediate 39 was prepared as described for 38

from 3-aminobenzyl alcohol (5.00 g, 40.6 mmol) to give

3.6 g (40%) of 39. Bromination of 39 (230 mg,

1.03 mmol) as described for 40 provided 257 mg (87%)

To a 78 ^ꢂC solution of 110 (3.45 g, 13.5 mmol) in

15 mL CH₂Cl₂a solution of 1 M BBr₃in CH₂Cl₂

(70 mL) was added dropwise over 1 h. The reaction

mixture was allowed to equilibrate to ambient tempera-

ture overnight, then re-cooled to 78 ^ꢂC and treated

dropwise with MeOH until cessation of eervescence.

The quenched reaction mixture was poured into 8%

aqueous NaOH (50 mL), and the organic and aqueous

phases separated. The aqueous phase was acidi®ed with

5 N HCl and extracted with 4:1 CH₂Cl₂:MeOH. The

combined organic phases were concentrated in vacuo to

a bright yellow solid that was recrystallized from ethyl

acetate±hexane to give 2.54 g (83%) of 111 that was ca.

1

of 41 as an orange oil that solidi®ed upon standing. H

NMR (400 MHz, CDCl₃) d 1.54 (9H, s), 4.47 (2H, s),

6.63 (1H, bs), 7.07 (1H, d, J=7.1 Hz), 7.23±7.28 (2H,

m), 7.53 (1H, s).

4-Bromomethyl-2-(methoxymethyloxy)benzaldehyde (44).

4-Methyl salicylic acid (1.86 g, 12.2 mmol) was dis-

solved in 20 mL diethyl ether. To it EtOH (5 mL,

122 mmol) was added, the ¯ask was cooled in an ice-

bath. DCC (3.02 g, 14.6 mmol) was added in portions.

After the addition the mixture was stirred at room tem-

perature overnight. Solid was removed by ®ltration, the

®ltrate was washed with water and dried to give a crude

methyl ester. The ester was dissolved in a solution of

20 mL dry CH₃CN and diisopropylethylamine (4.7 mL,

27.0 mmol). Chloromethyl methyl ether (2 mL,

26.3 mmol) in CH₃CN solution was added dropwise.

The mixture was re¯uxed overnight. Solvents were

removed in vacuo, and the residue was puri®ed by silica

gel column (80:10 hexane:ethyl acetate) to give 2.26 g

(88%) of 106.

1

4:1 by integration of the H NMR methyl signals at d

2.5 and 2.6. H NMR (400 MHz, CDCl₃) 2.51 and 2.62

(3H, two s, 1:4), 6.89±7.90 (7H, m).

1

HPLC (60 to 90% B in A over 40 min): T_R22 min and

23 min. MS, m/z 242.1 (MH⁺).

The phenolic group of 111 (2.54 g, 11.2 mmol) was pro-

tected as described for 32. Chromatography on silica gel

1

(98:2 hexane:ethyl acetate) gave 1.84 g (58%) of 36. H

NMR (400 MHz, CDCl₃) d 2.50 and 2.61 (3H, two s,

1:2), 3.54 and 3.56 (3H, two s, 1:2 ratio), 5.41 (2H, s),

T. Kline et al. / Bioorg. Med. Chem. 8 (2000) 73±93

83

¹H NMR (400 MHz, CDCl₃) d 2.36 (3H, s), 3.52 (3H, s),

3.87 (3H, s), 5.24 (2H, s), 6.85 (1H, d, J=7.9 Hz), 7.00

(1H, s), 7.70 (1H, d, J=7.9 Hz).

30 min then allowed to stir at room temperature for 1 h.

The resulting mixture was washed with 1N HCl, H₂O,

NaCl, dried, and concentrated in vacuo. The resulting

oil was puri®ed on silica gel (50:50 hexane:ethyl acetate)

1

Methyl ester 106 (13.4 g, 63.7 mmol) was re¯uxed with

1N NaOH (95 mL) and EtOH (95 mL) for 2 h. The

mixture was cooled to room temperature. EtOH was

removed in vacuo, and the aqueous solution was

washed with CH₂Cl₂, then acidi®ed to pH 1 with 0.1N

HCl layered with ethyl acetate. The organic layer was

separated and dried to give 11.24 g (90%) of 42 as a

white solid. ¹H NMR (400 MHz, CDCl₃) d 2.41 (3H, s),

3.56 (3H, s), 5.41 (3H, s), 6.99 (1H, d, J=8.0 Hz), 7.07

(1H, s), 8.06 (1H, d, J=8.0 Hz).

to give 8.1 g (87%) of 50 as a white solid. H NMR

(400 MHz, CDCl₃) d 2.46 (3H, s), 5.77 (1H, bs), 6.38

(1H, bs), 7.23 (1H, d, J=8.1 Hz), 7.32±7.38 (3H, m),

7.54 (1H, dt, J=1.7, 7.8 Hz), 7.95 (1H, dd, J=1.7,

7.8 Hz), 8.09 (2H, d, J=8.2 Hz).

2-(4-Methylphenyl)-1,3-benzoxazin-4-one (51). Inter-

mediate 50 (3.00 g 11.7 mmol) was heated in a heating

mantle over its melting point (ꢃ200 ^ꢂC) for 1 h. The

reaction was cooled, and the resulting mass slurried in

CH₂Cl₂(100 mL), then extracted with water, NaCl,

dried, and concentrated in vacuo. The resulting residue

was puri®ed on silica gel (80:20 hexanes:ethyl acetate) to

give 1.21 g (43%) of 51 as a white solid. ¹H NMR

(400 MHz, CDCl₃) d 2.43 (3H, s), 7.35 (1H, d,

J=8.0 Hz), 7.47±7.49 (2H, m), 7.74±7.78 (1H, m), 7.99

(1H, d, J=8.1 Hz), 8.21±8.23 (1H, m), 8.29±8.31 (2H,

m). MS, m/z 238.0 (MH⁺).

Acid 42 (5.03 g, 25.7 mmol) was dissolved in 65 mL

pyridine. The ¯ask was cooled in an ice-bath. To it

methanesulfonyl chloride (2.2 mL, 28.3 mmol) was

added dropwise. The mixture was allowed to stir for 1 h.

Ammonia gas was bubbled for 2 min. An additional

13 mL of methanesulfonyl chloride was added slowly,

and the reaction mixture stirred overnight. After con-

centration in vacuo, the residue was partitioned between

0.01N HCl and ethyl acetate. The organic phase was

washed successively with saturated CuSO₄and water,

dried, and concentrated in vacuo. The residue was pur-

i®ed on silica gel (90:10 hexane:ethyl acetate) to give

3-(4-Methylphenyl)-5-(2-hydroxyphenyl)-1,2,4-oxadiazole

(21). Intermediate 51 (1.20 g, 5.06 mmol) was suspended

in absolute EtOH (25 mL). Sodium acetate (0.50 g,

6.07 mmol) and hydroxylamine hydrochloride (0.42g,

6.07mmol) were added to the suspension at ambient tem-

perature. The reaction was stirred for 16h, diluted with

CH₂Cl₂(75 mL), and washed with water, NaCl, dried, and

concentrated in vacuo. The resulting solid was puri®ed via

chromatography (90:10 hexane:ethyl acetate) to give

1

3.4 g (75%) of 43. H NMR (400 MHz, CDCl₃) d 2.39

(3H, s), 3.52 (3H, s), 5.27 (2H, s), 6.86 (1H, d, J=

8.0 Hz), 7.03 (1H, s), 7.44 (1H, d, J=8.0 Hz).

Nitrile 43 (3.4 g, 19.2 mmol) was brominated as descri-

bed for 33. Puri®cation on silica gel (96:5 then 90:10

hexane:ethyl acetate) gave 2.57 g (52%) of bromide 112.

¹H NMR (400 MHz, CDCl₃) d 3.54 (3H, s), 4.43 (2H, s),

5.31 (2H, s), 7.10 (1H, q, J=1.5, 7.9 Hz), 7.26 (1H, d,

J=1.5 Hz), 7.54 (1H, d, J=7.9 Hz).

1

0.77g (64%) of 52 as a white, ¯occulant solid. H NMR

(400MHz, CDCl₃) d 2.45 (3H, s), 7.04 (1H, dt, J=0.7,

7.9 Hz), 7.15 (1H, d, J=8.4Hz), 7.34 (2H, d, J=8.0 Hz),

7.52 (1H, dt, J=1.6, 7.9 Hz), 7.99±8.02 (3H, m), 10.57

(1H, s). HRMS, m/z for C₁₅H₁₃N₂O₂calculated,

253.0977, found, 253.0977 (MH⁺).

Bromide 112 (175 mg, 0.68 mmol) was dissolved in 1 mL

dry toluene under nitrogen. To it DIBAL (1.5 M in tolu-

ene, 680 ml) was introduced at 0 ^ꢂC. After the addition, the

mixture was stirred at room temperature for 2 h, then 1 N

HCl was added to acidify the mixture to pH 2, and stir-

ring was continued for 15 min. The reaction mixture was

extracted with ethyl acetate, washed with water, dried,

and concentrated in vacuo to give 126 mg (71%) of

aldehyde 44. ¹H NMR (400 MHz, CDCl₃) d 3.53 (3H, s),

4.45 (2H, s), 5.32 (2H, s), 7.10 (1H, d, J=8.0 Hz), 7.25

(1H, d, J=1.3 Hz), 7.81 (1H, d, J=8.0 z), 10.45 (1H, s).

3-(4-Bromomethylphenyl)-5-(2-methoxymethyloxyphenyl)-

1,2,4-oxadiazole (52). To a solution of 21 (0.76 g,

3.16 mmol) in CH₂Cl₂(25 mL) at 0 ^ꢂC was added NaH

(0.15 g, 3.80 mmol). The reaction was stirred at 0 ^ꢂC

for 30 min, then chloromethyl methyl ether (0.28 g,

3.50 mmol) was added. The reaction was warmed to

ambient temperature and stirred for 2 h. The reaction

was quenched with water and washed with NaCl, dried,

and concentrated in vacuo to give 0.78 g (83%) of 113 as

1

a white solid which required no further puri®cation. H

NMR (400 MHz, CDCl₃) d 2.43 (3H, s), 3.56 (3H, s),

5.36 (2H, s), 7.16 (1H, t, J=7.6 Hz), 7.29±7.32 (3H, m),

7.51±7.54 (1H, m), 8.07 (2H, d, J=8.1 Hz), 8.16 (1H,

dd, J=1.6, 7.8 Hz). MS, m/z 297.1 (MH⁺). Intermediate

113 (0.5 g, 1.69 mmol) was brominated according to the

general procedure described for 33. Chromatography

(90:10 hexane:ethyl acetate) gave 200 mg (32%) of 52 as

Benzoyl (4-bromomethyl) salicylhydrazone (50). Alde-

hyde 44 (126 mg, 0.48 mmol) was stirred at ambient

temperature with benzoyl hydrazide (65 mg, 0.48 mmol)

in EtOH for 2 h, and 112 mg (43%) of 45 was collected

by ®ltration. H NMR (400 MHz, CDCl₃) d 3.53 (3H,

s), 4.45 (2H, s), 5.32 (2H, s), 7.10 (1H, d, J=7.9 Hz),

7.25 (1H, s), 7.81 (1H, d, J=7.9 Hz), 10.45 (1H, s).

1

an o white solid. H NMR (400 MHz, CDCl₃) d 3.56

(3H, s), 4.55 (2H, s), 5.36 (2H, s), 7.17 (1H, dt, J=0.9,

8.0 Hz), 7.31 (1H, d, J=8.5 Hz), 7.52±7.56 (3H, m),

8.14±8.17 (3H, m).

N-(4-Methyl)-benzoylsalicylamide (50). To a solution of

salicylamide (5.00 g, 36.4 mmol) in CH₂Cl₂(100 mL)

was added pyridine (3.12 g, 40.0 mmol). The reaction

was cooled to 0 ^ꢂC and p-toluoyl chloride (5.63 g,

36.4 mmol) added. The reaction was stirred at 0 ^ꢂC for

6-Methyl-3-methoxymethyloxy¯avone (57). To a solu-

tion of 55³¹(500 mg, 2.0 mmol) in CH₂Cl₂(20 mL) at

84

T. Kline et al. / Bioorg. Med. Chem. 8 (2000) 73±93

0 ^ꢂC was added NaH (90 mg of a 60% suspension,

2.2 mmol) followed by chloromethylmethyl ether (180 L,

2.4 mmol). The solution was allowed to slowly warm to

23 ^ꢂC over 2.5 h. The reaction mixture was diluted with

NH₄Cl and ethyl acetate. The phases were separated,

and the organic phase dried and concentrated in vacuo

to give a colorless oil that was puri®ed by chromato-

graphy (80:20 hexane:ethyl acetate) to give 500 mg

(85%) of 57 as a white solid. R_f=0.17 in 90:10 hex-

s), 7.33±7.37 (1H, m), 7.45±7.49 (3H, m), 7.60±7.65 (2H,

m), 7.98±8.00 (2H, m), 8.19 (1H, dd, J=1.5, 8.0 Hz).

HRMS, m/z for C₁₈H₁₆O₄Br calculated, 375.0232,

found, 375.0236 (MH⁺).

N-(4R)-[[2-[2-(2-Hydroxy)phenyl]thiazolin-4-yl]carbonyl]-

(3S,4S)-trans-3-amino-4-methyl-2-azetidinone-1-sulfonic

acid, tetrabutylammonium salt (59). To a rapidly stir-

ring suspension of [R]-sodium dihydroaeruginoate 18³⁶

(75 mg, 0.3 mmol) in THF (3 mL) in a ¯ask under N₂,

was added DCC (63 mg, 0.3 mmol) and NHS (53 mg,

0.46 mmol). The reaction mixture was stirred for 2 h and

®ltered. In a separate ¯ask under N₂, DBU (46 mL,

0.3 mmol) was added to a suspension of 10 in CH₂Cl₂

(2 mL) for 5 min until a clear solution was obtained. The

above ®ltrate was added to this solution, and the reac-

tion mixture allowed to stir at ambient temperature for

18 h. The reaction was diluted with CH₂Cl₂to 20 mL

and was extracted into water (4Â10 mL). The solution

was neutralized to pH 7 with dilute aqueous NaOH,

and tetrabutylammonium hydrogen sulfate (170 mg,

0.5 mmol) was added. The product was extracted with

CH₂Cl₂(3Â15 mL) and concentrated in vacuo to pro-

vide 89 mg (46%) of 62 as the tetrabutylammonium salt.

¹H NMR (400 MHz, CDCl₃) d 0.98 (12H, t, J=7.3 Hz),

1.41 (8H, sext., J=7.3 Hz), 1.58±1.66 (11H, m), 3.21±

3.26 (8H, m), 3.60±3.74 (2H, m), 3.87±3.99 (1H, m),

4.55±4.63 (1H, m), 5.34 (1H, t, J=8.9 Hz), 6.87±6.92

(1H, m), 6.98 (1H, d, J=8.4 Hz), 7.35±7.47 (3H, m),

11.84 (1H, bs). HRMS, m/z for C₁₄H₁₄N₃O₆S₂calcu-

lated, 384.0324, found, 384.0339 (MH⁺).

1

ane:ethyl acetate. H NMR (400 MHz, CDCl₃) d 2.39

(3H, s), 3.02 (3H, s), 5.12 (2H, s), 7.40±7.44 (5H, m),

7.96±7.98 (3H, m).

6-Bromomethyl-3-methoxymethyoxyl¯avone (59). A solu-

tion of 57 (500 mg, 1.69 mmol) in CCl₄(17 mL) was

brominated with NBS (330 mg, 1.86 mmol) and AIBN

(28 mg, 0.17 mmol) as described for 33 to give, puri®ca-

tion on silica gel (90:10 hexane:ethyl acetate), 400 mg

(63%) of 59 as a white solid. R_f=0.20 in 90:10 hex-

1

ane:ethyl acetate; H NMR (400 MHz, CDCl₃) d 3.01

(3H, s), 4.48 (2H, s), 5.11 (2H, s), 7.42±7.45 (5H, m),

7.64 (1H, dd, J=2.4, 8.6 Hz), 7.94±7.97 (2H, m), 8.16

(1H, d, J=2.3 Hz). HRMS, m/z for C₁₈H₁₆O₄Br calcu-

lated, 375.0232, found, 375.0235 (MH⁺).

3-Methoxymethyloxy-3⁰-methyl¯avone (58A). Inter-

mediate 58A was prepared from 56A³³(500 mg,

2.0 mmol) as described for 57 to give, after chromato-

graphy (80:20 hexane:ethyl acetate) 540 mg (92%) of

58A as a white solid: R_f=0.12 in 90:10 hexane:ethyl

acetate. ¹H NMR (400 MHz, CDCl₃) d 2.48 (3H, s),

3.15 (3H, s), 5.22 (2H, s), 7.34 (1H, d, J=7.4 Hz), 7.43

(2H, t, J=7.7 Hz), 7.56 (1H, d, J=8.4 Hz), 7.64±7.74

(1H, m), 7.87±7.89 (2H, m), 8.28 (1H, d, J=8.0 Hz).

N-(4S)-[[2-[2-(2-Hydroxy)phenyl]thiazolin-4-yl]carbonyl]-

(3S,4S)-trans-3-amino-4-methyl-2-azetidinone-1-sulfonic

acid, tetrabutylammonium salt (63). The epimeric amide

was prepared in an analogous manner using (S)-sodium

dihydroaeruginoate³⁶19 (47 mg, 0.19 mmol) and [S]-

trans-3-amino-4-methyl-2-oxoazetidine-1-sulfonic acid³⁷

(32 mg, 0.18 mmol) to give 86 mg (80%) of 63 after

chromatography (90:10 CH₂Cl₂:MeOH). ¹H NMR

(400 MHz, CDCl₃) d 0.98 (12H, t, J=7.3 Hz), 1.41 (8H,

sext., J=7.3 Hz), 1.60±1.64 (11H, m), 3.19±3.24 (8H,

m), 3.63±3.70 (1H, m), 3.89±4.00 (1H, m), 4.55±4.63

(1H, m), 5.34 (1H, t, J=8.9 Hz), 6.87±6.92 (1H, m), 6.99

(1H, d, J=8.3 Hz), 7.31±7.40 (2H, m), 7.43±7.46 (1H,

m). HRMS, m/z for C₁₄H₁₄N₃O₆S₂calculated, 384.0324,

found, 384.0335 (MH⁺).

3-Methoxymethyloxy-3⁰-bromomethyl¯avone (60). Inter-

mediate 60 was prepared from 58A (540 mg, 1.82 mmol)

as described for 59 to give, after chromatography (90:10

hexane:ethyl acetate), 420 mg (62%) of 60 as a white

solid. R_f=0.18 in (90:10 hexane:ethyl acetate). ¹H NMR

(400 MHz, CDCl₃) d 3.14 (3H, s), 4.60 (2H, s), 5.25 (2H,

s), 7.43±7.45 (1H, m), 7.53±7.57 (3H, m), 7.68±7.71 (1H,

m), 8.00±8.03 (1H, m), 8.12 (1H, s), 8.28 (1H, dd,

J=1.6, 8.0 Hz). HRMS, m/z for C₁₈H₁₆O₄Br calculated,

375.0232, found, 375.0227(MH⁺).

3-Methoxymethyloxy-4⁰-methyl¯avone (58B). Inter-

mediate 58B was prepared from 56B³³(500 mg,

2.0 mmol) as described for 58A to give, after chromato-

graphy (80:20 hexane:ethyl acetate) 460 mg (78%) of

58B as a white solid. R_f=0.24 (90:10 hexane:ethyl ace-

Methyl (4-((4-methoxyphenyl)methylthio)methyl)-3-(meth-

oxymethyloxy) benzoate (65). Ester 64 was brominated

as described for 33, and the product (1.00 g, 3.5 mmol)

was dissolved in DMF (5 mL), and to it NaH (0.15 g,

3.8 mmol) was added under nitrogen. The reaction

was stirred for 20 min, then 4-methoxy-a-toluenethiol

(0.59 g, 3.8 mmol) added followed by NaI (catalytic).

The reaction was stirred for 2 h, H₂O added (20 mL),

and the aqueous layer extracted with ethyl acetate

(3Â20 mL). The organic phases were combined, washed

with H₂O, brine, dried, and concentrated in vacuo.

Puri®cation on silica gel (80:20 hexane:ethyl acetate)

1

tate). H NMR (400 MHz, CDCl₃) d 2.55 (3H, s), 7.09

(1H, s), 7.45 (2H, d, J=8.1 Hz), 7.50±7.54 (1H, m),

7.68±7.83 (2H, m), 8.25±8.28 (2H, m), 8.35 (1H, dd,

J=1.6, 8.0 Hz).

3-Methoxymethyl-4⁰-bromomethyl¯avone (61). Inter-

mediate 61 was prepared from 57C (460 mg, 1.55 mmol),

NBS (304mg, 1.71mmol) and AIBN (26 mg, 0.15mmol)

as described for 59 to give, after chromatography (90:10

hexane-ethyl acetate) 300 mg (51%) of 61 as a white

1

gave 0.43 g (34%) of 65 as an o white solid. H NMR

(400 MHz, CDCl₃) d 3.53 (3H, s), 3.55 (4H, s), 3.79 (3H,

s), 3.88 (3H, s), 5.25 (2H, s), 6.84 (2H, d, J=8.6 Hz),

1

solid. R_f=0.20 (90:10 hexane:ethyl acetate). H NMR

(400 MHz, CDCl₃) d 3.07 (3H, s), 4.48 (2H, s), 5.16 (2H,

T. Kline et al. / Bioorg. Med. Chem. 8 (2000) 73±93

85

6.99 (1H, dd, J=1.4, 8.0 Hz), 7.1 (1H, d, J=1.3 Hz),

7.18 (2H, d, J=8.6 Hz), 7.74 (1H, d, J=8.0 Hz). MS,

m/z 385 (MNa⁺)

5.26±5.30 (3H, m), 6.83 (2H, d, J=8.5 Hz), 6.99 (1H, d,

J=8.1 Hz), 7.10 (1H, s), 7.17 (2H, d, J=8.5 Hz), 7.87

(1H, d, J=8.0 Hz), 10.12 (1H, bs). HRMS, m/z for

C₂₂H₂₆NO₅S₂calculated, 448.1252, found, 448.1241

(MH⁺).

4-((4-Methoxyphenyl)methylthio)methyl)-3-(methoxymeth-

yloxy)benzoic acid (66). 65 (0.42 g, 1.2 mmol) was dis-

solved in a solution of 1:1 THF-1N NaOH (10 mL) and

EtOH (2 mL). The reaction was stirred at ambient tem-

perature for 2 h, then extracted with Et₂O (10 mL). The

aqueous layer was acidi®ed with 1 N HCl to pH 3. The

aqueous layer was extracted with CH₂Cl₂(2Â15 mL),

the organic layers combined, dried, and concentrated in

vacuo to give 0.37 g (90%) of 66 as a pale yellow solid

which required no further puri®cation. ¹H NMR

(400 MHz, CDCl₃) d 3.58 (7H, s), 3.80 (3H, s), 5.41 (2H,

s), 6.84 (2H, d, J=8.7 Hz), 7.07 (1H, dd, J=1.4,

8.1 Hz), 7.17±7.19 (2H, m), 8.11 (1H, d, J=8.1 Hz).

(6R,7R)-7-[2-Aminothiazol-4-yl)-2-[(Z)-[1-(carboxymeth-

ylethyloxy]imino]acetamido]-3-[4(3-hydroxy-4-[4-carboxy-

2-thiazolinyl]phenylmethyl]thio]methyl]-8-oxo-1-aza-5-

oct-2-ene-2-carboxylic acid (70). Acid 68 (0.05 g,

0.11 mmol) was dissolved in TFA (1 mL) and anisole

(0.1 mL) added. This was stirred for 2 h, then the TFA

and anisole removed in vacuo, and the crude thiol

dissolved in DMF (1 mL). Diphenylmethyl-protected

cephalosporin 69⁵⁶(0.81 g, 0.11 mmol), K₂CO₃(0.017 g,

0.12 mmol) were added, followed by NaI (catalytic). The

reaction was stirred under N₂at ambient temperature for

16h. The protected intermediate 115 was precipitated by

addition of HO (5mL), collected via ®ltration, and

washed further with HO and hexanes. Intermediate 115

was deprotected by treatment with 5:1 TFA:anisole

(0.6 mL). The product was precipitated by addition of

Et₂O, collected via ®ltration, further washed with Et₂O,

and dried in vacuo to give the 5 mg (8%) of 70 as a brown

N-Benzyl(4-((4-methoxyphenyl)methylthio)methyl)-3-

(methoxymethyloxy) benzamide (67). Acid 66 (0.36 g,

1.0 mmol) was dissolved in CH₂Cl₂(5 mL) and BnNH₂

(0.12 g, 1.1 mmol) added followed by EDCl (0.21 g,

1.1 mmol) and Et₃N (0.11 g, 1.1 mmol). The reaction

was stirred at ambient temperature for 16 h, then

washed with H₂O, brine, dried, and concentrated in

vacuo. The resulting residue was puri®ed on silica gel

(70:30 hexane:ethyl acetate) to give 0.28 g (62%) of 67

1

solid. H NMR (400MHz, DMSO-d₆) d 1.28±1.31 (6H,

m), 3.40±3.65 (9H, m), 5.13±5.19 (1H, m), 5.52±5.64 (1H,

m), 6.72±6.95 (4H, m), 7.32±7.52 (3H, m), 9.29±9.33 (2H,

m). HRMS, m/z for C₂₈H₂₉N₆O₁₀S₄calculated, 737.0828,

found, 737.0837 (MH⁺).

1

as a pale yellow solid. H NMR (400 MHz, CDCl₃) d

3.41 (3H, s), 3.56 (4H, d, J=5.4 Hz), 3.80 (3H, s), 4.69

(2H, d, J=5.6 Hz), 5.25 (2H, s), 6.83±6.85 (2H, m),

7.02±7.06 (3H, m), 7.17±7.19 (2H, m), 7.26±7.28 (1H,

m), 7.33±7.38 (3H, m), 8.07 (1H, bs), 8.17 (1H, d,

J=8.0 Hz). MS, m/z 438.3 (MH⁺).

4-[[2-(tert-Butoxycarbonyl)hydrazino]carbonyl]benzyl-

chloride (71). 4-Chloromethyl benzoyl chloride (10.0 g,

53 mmol) and t-butyl carbazate (8.38 g, 63.5 mmol) were

stirred in 200 mL CH₂Cl₂and cooled in an icebath.

Diisopropylethylamine (9.2 mL, 53 mmol) was added

slowly, and the reaction mixture stirred at room tem-

perature for 2 h, diluted with another 200 mL of

CH₂Cl₂, washed with water, dried, ®ltered, and con-

centrated in vacuo. The residue was recrystallized from

[R] 2-(2-(Methoxymethyloxy)-(4-(4-methoxyphenyl)meth-

ylthiomethyl)phenyl)) thiazoline-4-carboxylic acid (68).

Amide 67 (0.27 g, 0.62 mmol), was dissolved in anhy-

drous CH₂Cl₂(25 mL) and cooled to 50 ^ꢂC under an

atmosphere of N₂. Pyridine (49 mg, 0.68 mmol) was

added, followed by slow addition of Tf₂O (0.18 g,

0.68 mmol). After complete addition of Tf₂O the reac-

tion was allowed to warm to 0 ^ꢂC and stirred at that

temperature for 4 h. The reaction was cooled to 30 ^ꢂC

and pyridine (49 mg, 0.68 mmol) added followed by l-

cysteine methyl ester hydrochloride (0.12 g, 0.68 mmol).

The reaction was slowly allowed to warm to ambient

temperature and stirred for 16 h. The reaction was

washed with H₂O, brine, dried, and concentrated in

vacuo. The resulting residue was puri®ed on silica gel

(70:30 hexane:ethyl acetate) to give 80 mg (30%) of 114

as a yellow oil. ¹H NMR (400 MHz, CDCl₃) d 3.53±3.63

(9H, m), 3.79 (3H, s), 3.83 (3H, s), 5.16 (1H, t,

J=9.3 Hz), 5.25 (2H, s), 6.83 (2H, d, J=8.6 Hz), 6.99

(1H, dd, J=1.4, 8.1 Hz), 7.08±7.11 (1H, d, J=1.3 Hz),

7.18 (2H, d, J=8.7 Hz), 7.94 (1H, d, J=8.0 Hz). Ester

114 (80 mg, 0.18 mmol) was dissolved in a mixture of 1:1

THF:1 M NaOH (5 mL). The reaction was stirred for

1 h, washed with Et₂O (5 mL), and the aqueous layer

acidi®ed with 1N HCl to pH 3. This was extracted with

CH₂Cl₂(2Â10 mL), the organic layers combined, dried,

and concentrated in vacuo to give 60 mg (75%) of 68 as

a yellow solid which was used without further puri®ca-

tion. ¹H NMR (400 MHz, CDCl₃) d 3.52±3.78 (12H, m),

1

ethyl acetate:hexane to give 12 g (80%) of 71. H NMR

(400 MHz, CDCl₃) d 1.42 (9H, s), 4.50 (2H,s), 6.74 (1H,

bs), 7.33 (2H, d, J=8.0 Hz), 7.71 (2H, d, J=8.0 Hz),

8.43 (1H, bs).

4-[[2-(tert-Butoxycarbonyl)hydrazino]carbonyl]benzyloxy

phthallimide (72). Intermediate 71 (3.58 g, 12.6 mmol)

and N-hydroxyphthallimide (2.47 g, 15.1 mmol), were

dissolved in 15 mL DMF, and K₂CO₃(189 mg,

1.26 mmol) and NaI (catalytic) were added. The reac-

tion was stirred at 80 ^ꢂC for 30 min, ®ltered, and the ®l-

trate poured into 0.1 N HCl (500 mL). The precipitate

was collected by ®ltration, washed with water, dried,

and recrystallized from ethyl acetate to give 3.36 g

1

(62%) of 72. H NMR (400 MHz, CDCl₃) d 1.40 (9H,

s), 5.16 (2H, s), 6.83 (1H, s). 7.52 (2H, d, J=8.0 Hz),

7.66 (2H, dd, J=3.0, 6.0 Hz), 7.72±7.76 (5H, m), 8.34

(1H, bs).

4-[[2-(tert-Butoxycarbonyl)hydrazino]carbonyl]benzylhy-

droxylamine (73). An ethanolic solution of compound

72 was treated with hydrazine and re¯uxed for 2 h. After

cooling the precipitate was ®ltered o, and the ®ltrate

was dried and concentrated in vacuo to give a crude

86

T. Kline et al. / Bioorg. Med. Chem. 8 (2000) 73±93

benzyloxyamine 73 which was used directly in the next

1

(1H, m), 4.47 (1H, m), 5.23 (2H, s), 6.93 (3H, m), 7.22

(1H, m), 7.30 (1H, m), 7.52 (2H, d, J=8.3 Hz), 7.93

(2H, d, J=8.3 Hz), 8.64 (1H, s). MS, m/z 602 (MH⁺).

reaction. H NMR (400 MHz, CDCl₃) d 1.49 (9H, s),

4.86 (2H, s), 7.46 (2H, d, J=8.0 Hz), 7.84 (2H, d,

J=8.0 Hz).

2-(Tritylamino)-ꢁ-((3-methoxymethyloxy-4-(2-benzthi-

azolyl)benzyloxy)imino)-4-thiazole acetic acid (78). The

free base (obtained by treatment of ethyl-2-(trityl-

2-(Butoxycarbonylamino)-2-[[(Z)-4-[[2-butoxycarbonyl)-

hydrazino]carbonyl]benzylhydroxyl]imino] - 4 - thiazole

acetic acid (75). Benzyloxyamine 73 (2.14 g, 7.67 mmol)

and ethyl 2-N-Boc-amino-4-thiazole glyoxalate 74

(2.09 g, 6.79 mmol) were re¯uxed in 40 mL EtOH with a

catalytic amount of acetic acid for 4 h. After concentra-

tion in vacuo the residue was puri®ed by silica gel col-

umn (using a step gradient of 70:30, then 60:40, then

40:60 hexane:ethyl acetate) to give 2.1 g (53%) of oxime

amino)-a-(hydroxylimino)-4-thiazolyleacetate

mono-

hydrochloride (6.8 g, 15.5 mmol) with NaHCO₃and

extraction into CH₂Cl₂) was suspended in 86 mL allyl

alcohol and warmed to 60 ^ꢂC. Sodium ethoxide (420 mg,

6.2 mmol) was dissolved in 2 mL allyl alcohol and added

to the reaction. Stirring was maintained at 60 ^ꢂC for 4 h,

then allowed to continue at room temperature over-

night. After adjusting the pH to 7 with 2 N HCl, the

reaction mixture was concentrated in vacuo, and the

crude allyl ester precipitated with methyl-t-butyl ether.

The product was dissolved in CHCl₃, washed with NaCl,

dried, and concentrated in vacuo to give 4.8 g (70%) of

allyl ester 116 suciently pure to carry on in the sub-

sequent reaction. ¹H NMR (400 MHz, CDCl₃) 4.83 (2H,

dd, J=0.9, 5.0 Hz), 5.39 (2H, dd, J=10.4, 17.1 Hz),

5.93±6.02 (1H, m), 6.48 (1H, s), 6.96 (1H, bs), 7.26±7.52

(15H, m), 8. 56 (1H, bs). MS, m/z 470.4 (MH⁺).

1

115. H NMR (400 MHz, CDCl₃) d 1.33 (3H, t, J=

7.1 Hz), 1.45 (9H, s), 1.51 (9H, s), 4.42 (2H, q, J=

7.1 Hz), 5.15 (2H, s), 7.46 (3H, m), 7.84 (2H, d, J=

7.6 Hz), 9.74 (1H, bs.), 10.33 (1H, bs).

Oxime 115 was re¯uxed with 3.5 mL 2N NaOH and

3.5 mL EtOH for 2 h. After cooling, the reaction mix-

ture was acidi®ed with 0.1 N HCl to pHꢃ3, and

extracted with ethyl acetate. The extract was con-

1

centrated in vacuo to give 1.12 g (97%) of acid 75. H

NMR (400 MHz, CDCl₃) d 1.51 (9H, s,), 1.53 (9H, s),

5.06 (2H, s), 7.09 (2H, d, J=7.5 Hz), 7.17 (1H, s), 7.47

(2H, d, J=7.5 Hz), 7.59 (1H, s), 9.79 (1H, s).

A mixture of bromomethyl intermediate 33 (1.65 g,

4.5 mmol) and allyl ester 116 (2.15 g, 5 mmol) was stir-

red at room temperature overnight in 10 mL dry DMF.

The reaction mixture was diluted with CHCl₃, washed

with NaCl, dried, and concentrated in vacuo to an oil

that was puri®ed on silica gel (80:20 hexane:ethyl ace-

trans-3-[4-(2-tert-Butoxycarbonylaminothiazol-4-yl)-2-

[(Z)-4-butoxycarbonyl)hydrazino]carbonyl])benzlhydroxyl]-

imino]acetamido]-4-methyl-2-oxoazetidine-1-sulfonic acid

triethylammonium salt (76). Acid 75 (200 mg,

0.374 mmol) was stirred with HOAt (50 mg,

0.374 mmol), DCC (77 mg, 0.374 mmol), and DMAP

(12 mg, catalytic amount) in 2 mL DMF for 1 h. Simul-

taneously 10 (67 mg, 0.374 mmol) was stirred in 0.5 mL

DMF with triethylamine (52 mL. 0.374 mmol) for 1 h,

then added to the activated acid solution. The mixture

was stirred at room temperature overnight. DMF was

removed in vacuo. The residue was puri®ed by Sepha-

dex LH-20 column (MeOH) to give 160 mg (53%) of

1

tate) to give 2.1 g (61%) of 117. H NMR (400 MHz,

CDCl₃) d 3.56 (3H, s), 4.82 (2H, d, J=5.7 Hz), 5.33 (2H,

dd, J=3.7, 17.5 Hz), 5.35 (2H, s), 5.43 (2H, s), 5.92±5.99

(1H, m), 6.53(1H, s), 6.99±7.50(19H, m), 7.93 (1H, d,

J=7.8 Hz), 8.09 (1H, d, J=8.0 Hz), 8.49 (1H, d,

J=8.1 Hz). MS, m/z 753.2 (MH⁺).

A solution of 117 (1.05 g, 1.4 mmol) in dry THF was

added under Argon via canula to a solution of tetra-

kis(triphenylphospine)palladium (generated in situ from

palladium acetate (31.4 mg, 0.14 mmol) and triphenyl-

phosphine (183 mg, 0.7 mmol) in 4 mL dry THF).

After 15 min of stirring, tributyltin hydride (420 mL,

1.54 mmol) was added, and the reaction stirred at room

temperature for 5 h. After concentration in vacuo, the

residue was applied to a silica column, and the tributyl-

stannate ester eluted with 93:8 CHCl₃:MeOH. The elut-

ing solvent was removed, and the residue was dissolved

in CH₃CN and washed with hexane. The CH₃CN phase

was treated with a 5:1 CH₃CN-AcOH solution (pH 3) at

room temperature for 2 h to hydrolyze the tributyl-

stannate ester. After washing the reaction mixture with

hexane, the CH₃CN was removed in vacuo, the product

dissolved in CHCl₃, washed with water, dried, and the

solvent removed in vacuo to give 660 mg (66%) of 78

that was >90% free acid, <10% tributylstannate ester

by integration of the ¹H NMR signals. ¹HNMR

(400 MHz, CDCl₃) d 0.84±0.92 (1H, m), 1.31±1.39 (2H

m), 1.60±2.17 (1H, m), 3.45 (3H, s), 5.25 (2H, s), 5.42

(2H, s), 6.57 (1H, s), 7.10±7.69 (19H, m), 7.89 (1H, d,

J=7.9 Hz), 8.07 (1H, d, J=7.9 Hz), 8.43 (1H, d,

J=7.9 Hz). MS, m/z 713.3 (MH⁺).

1

amide 76. H NMR (400 MHz, CD₃OD) d 1.28 (9H, t,

J=7.3 Hz), 1.34 (9H, s), 1.51±1.58 (12H, m), 3.19 (6H,

q, J=7.3 Hz), 4.07 (1H, m), 4.61 (1H, d, J=2.7 Hz),

5.30 (2H, s), 7.37 (1H, s), 7.52 (2H, d, J=7.9 Hz), 7.86

(2H, d, J=7.9 Hz).

trans-3-[4-(2-Aminothiazol-4-yl)-2-[(Z)-4-hydroxybenzyl-

idenehydrazide)benzlhydroxyl] imino]acetamido]-4-meth-

yl-2-oxoazetidine-1-sulfonic acid triethylammonium salt

(77). Amide 76 (25 mg, 0.03 mmol) was treated with

2 mL 1:1 TFA:CH₂Cl₂for 15 min at room temperature.

The reaction mixture was neutralized with triethyla-

mine, concentrated in vacuo, and the residue puri®ed by

Sephadex LH-20 column (MeOH). The hydrazide was

dissolved in 1 mL EtOH. To it salicylaldehyde (5 mL,

0.047 mmol) and one drop of acetic acid were added,

and the mixture was re¯uxed for 30 min, then stirred at

room temperature an additional 30 min. After con-

centration in vacuo the residue was puri®ed on Sepha-

dex LH-20 (MeOH) to give 8 mg (36% for two steps) of

77. ¹H NMR (400 MHz, DMSO-d₆) d 1.15 (9H, t,

J=7.5 Hz), 1.40 (3H, d, J=6.1 Hz), 3.04 (6H, m), 3.70

T. Kline et al. / Bioorg. Med. Chem. 8 (2000) 73±93

87

cis-3-[2-(2-Aminothiazol-4-yl)-2-((Z)-(3-hydroxy-4-(2-thi-

azolyl)benzyl)oxyimino)acetamido]-4-methyl-2-azetidinone-

1-sulfonic acid (67). A solution of acid 78 (660 mg,

0.93 mmol), DCC (209 mg, 1.10 mmol), HOAt (125 mg,

0.93 mmol), and DMAP (35 mg) in 2.5 mL dry DMF

was stirred at room temperature for 30 min. Simulta-

neously, a solution of the cis b-lactam 80³⁴(166 mg,

0.93 mmol) and NEt₃(128 mL, 0.93 mmol) in 1 mL dry

DMF was stirred at room temperature. The amine

solution was added to the activated ester solution, and

the reaction mixture was stirred overnight. After

removing the solvent in vacuo, the residue was puri®ed

on silica gel (95.5:5:0.5 CHCl₃:-MeOH:NEt₃) to give

430 mg (54%) of 118. ¹H NMR (400 MHz, DMSO-d₆) d

1.17 (3H, t, J=6.2 Hz), 3.49 (3H, s), 3.94±4.00 (1H, m),

5.06 (1H, d, J=4.4 Hz), 5.19 (2H, s), 5.52 (2H, s), 6.73

(1H, s), 7.00±7.52 (19H, m), 8.11 (1H, dd, J=8.1,

16.6 Hz), 8.05 (1H, d, J=7.9, 16.6 Hz), 8.46 (1H, d,

J=8.2 Hz), 8.83±8.84 (1H, bs). MS, m/z 875.3 (MH⁺),

873.4 (MH ).

s), 5.46 (2H, s), 5.57 (2H, s) 6.55 (1H, s), 6.92±7.83

(20H, m), 8.05 (1H, d, J=8.0 Hz), 8.53 (1H, d, J=

7.9 Hz).

Diphenylmethyl ester 82 was prepared by reaction of

7-amino cephalosporonic acid (5.0 g, 18.4 mmol) with

p-toluenesulfonic acid (3.85 g, 20.2 mmol) and diphe-

nyldiazomethane (prepared fresh from diphenylhy-

drazine (5.9 g, 30 mmol) according to Holton and

Shechter⁵⁷) in 150 mL 2:1 CH₃CN:H₂O for 48 h. Excess

diphenyldiazomethane was quenched by dropwise addi-

tion of AcOH until the deep purple reaction mixture

had decolorized. The reaction mixture was neutralized

with NaHCO₃and extracted with CH₂Cl₂. The organic

phase was washed with NaCl, dried, and concentrated

in vacuo to give a residue. Puri®cation on silica gel

(60:40 hexane:ethyl acetate, then 100% ethyl acetate)

1

gave 4.0 g (50%) of 82. H NMR (400 MHz,CDCl₃) d

2.00 (3H, s), 3.46 (2H, dd, J=18.4, 18.5 Hz), 4.76 (1H,

d, J=17.6 Hz), 4.78 (2H, d, J=4.6 Hz), 5.00 (1H, d,

J=16.6 Hz), 6.98 (1H, s), 7.26±7.45 (10H, m). MS m/z

439.1 (MH⁺).

A solution of 118 (165 mg, 0.19 mmol) in 1 mL THF was

cooled to 0 ^ꢂC and treated with 1 mL 6 N aqueous HCl

dropwise. The icebath was removed, and the reaction

mixture stirred at room temperature overnight. After

lyophilization, the residue was puri®ed on reverse phase

HPLC (linear gradient of 20 to 35% B in A over 40 min)

To a 0 ^ꢂC solution of 82 (76 mg, 0.17 mmol) in 2 mL

CH₂Cl₂was added pyridine (67 mL, 0.85 mmol), then

119 (136 mg, 0.19 mmol) in 1 mL CH₂Cl₂, followed by

POCl₃(20 mL, 0.22 mmol). After 100 min of stirring at

0 ^ꢂ, the reaction mixture was diluted with CH₂Cl₂,

washed successively with 0.02 N HCl, then NaCl, dried,

and concentrated in vacuo. The crude product was

puri®ed on silica gel (75:25, then 60:40, then 90:10 hex-

1

to give 7 mg (6%) of 79. H NMR (400 MHz, DMSO-

d₆) d 1.29±1.32 (3H, s), 4.00±4.11 (1H, m), 5.09±5.13

(1H, m), 5.17 (2H, s), 6.80 (1H, s), 7.03±7.68 (4H, m),

8.07 (1H, d, J=8.1 Hz), 8.14 (1H, d, J=7.9 Hz), 9.29±

9.30 (1H, s). MS m/z 589.1(MH⁺), 587.0 (MH ).

HRMS, m/z for C₂₃H₂₁N₆O₇S₃calculated, 589.0634,

found, 589.0650 (MH⁺).

1

ane:ethyl acetate) to give 69 mg (36%) of pure 120. H

NMR (400 MHz, CDCl₃) d 1.99 (3H, s), 3.03 (2H, dd,

J=13.2, 18.6 Hz), 3.49 (3H, s), 4.78 (1H, d, J=5.0 Hz),

4.80 (2H, d, J=13.6 Hz), 5.38 (2H, d, J=6.2 Hz), 5.65

(2H, dd, J=11.7, 14.7 Hz), 5.83 (1H, dd, J=4.4,

4.9 Hz), 6.84 (1H, s), 6.94 (1H, d, J=12.2 Hz) 7.03±7.52

(30H, m), 8.04 (1H, d, J=7.9 Hz), 8.50 (1H, d,

J=1.7 Hz). MS, m/z 1133.4 (MH⁺).

Allyl-2-(tritylamino)-ꢁ-methoxymethyloxy)phenyl)benz-

thiazolyl)methyloxy)imino)-4-thiazole acetate (81) and

allyl-2-(tritylamino)methoxymethyloxy)phenyl)benzthia-

zolyl)methyloxy)imino)-4-thiazole acetate (81B). Alkyla-

tion of 116 (1.47 g, 3.4 mmol) with the regiomeric

bromides 37 (1.13 g, 3.1 mmol) in 10 mL dry DMF was

performed as for 117. Chromatography on silica gel

(90:10 hexane:ethyl acetate) gave 1.13 (49%) of 81B. ¹H

NMR (400 MHz, CDCl₃) d 3.52 (3H, s) 4.72±4.73 (2H,

d, J=5.1 Hz), 5.05±5.38 (2H, m), 5.42±5.46 (2H, m),

5.57 (2H, s), 5.76±5.81 (1H, m), 6.54 (1H, s), 6.54±7.46

(20H, m), 8.03 (1H, d, J=7.9 Hz), 8.53 (1H, d,

J=8.0 Hz). MS, m/z 753.1 (MH⁺).

Intermediate 120 was treated with 5:1 TFA-anisole for

2.5 h, the product precipitated with diisopropyl ether,

and the crude precipitate lyophilized from 50:50

CH₃CN:H₂O (0.05% TFA) to give 24 mg (70%) of 83.

¹H NMR (DMSO-d₆) d 2.02 (3H, s), 3.33±3.61 (2H, m),

4.97 (1H, dd, J=3.8, 4.5 Hz), 5.14 (1H, dd, J=4.8,

5.8 Hz), 5.39±5.54 (2H, m), 5.74 (1H, dd, J=3.1,

4.7 Hz), 5.83 (1H, dd, J=4.8, 4.9 Hz), 6.75 (1H, s), 6.99±

7.64 (4H, m), 8.04 (1H, d, J=7.9 Hz), 8.18 (1H, d,

J=2.3 Hz), 9.69-9.71 (1H, m), HRMS, m/z for C₂₉H₂₄

N₆O₈S₃calculated, 681.0899, found, 681.0882 (MH⁺).

Further elution with 85:15 hexane:ethyl acetate gave

1

121 mg (5%) of 81A. H NMR. (400 MHz, CDCl₃) d

3.52 (3H, s), 4.73 (2H, d, J=5.1 Hz), 5.05±5.38 (2H, m),

5.42 (2H, s), 5.44 (2H, s), 5.76±5.81 (1H, m), 6.54 (1H,

s), 6.54±7.46 (19H, m), 7.98 (1H, s), 8.04 (1H, s), 8.53

(1H, s).

(6R,7R)-7-[2-Aminothiazol-4-yl)-2-[(Z)-[2-(2-hydroxy)-

phenyl-5-benthiazolyl)methyloxy]imino]acetamido]-3-pyr-

idiniummethyl)-4-yl]thio]methyl]-8-oxo-1-aza-5-thiabi-

cyclo[4.2.0]oct-2-ene-2-carboxylic acid (85). An ice-

cooled solution of p-methoxybenzyl-[[[7-amino-3-chloro-

methyl-4-yl]thio]methyl]-8-oxo-1-aza-5-thiabicyclo[4.2.0]-

oct-2-ene-2-carboxylate hydrochloride (84) (100 mg,

0.25 mmol), NEt₃(35 mL, 0.25 mmol), 119 (180 mg,

0.25 mmol), and pyridine (88 L, 1.25 mmol) in 10 mL

dry CH₂Cl₂was stirred for 1 h, then further cooled to

10 ^ꢂ. POCl₃(28 mL, 0.31 mmol) was added via syringe,

(6R,7R)-7-[2-Aminothiazol-4-yl)-2-[(Z)-[2-(2-hydroxy)-

phenyl-5-benthiazolyl)methyloxy]imino]acetamido]-3-ace-

toxymethyl)-4-yl]thio]methyl]-8-oxo-1-aza-5-thiabicyclo-

[4.2.0]oct-2-ene-2-carboxylic acid (83). Allyl ester 81B

(1.03 g, 1.37 mmol) was treated with [Ph₃P]₄Pd and n-

Bu₃SnH as described for 117 to give 595 mg (61%) of

1

free acid 119. H NMR (400 MHz, CDCl₃) d 3.52 (3H,

88

T. Kline et al. / Bioorg. Med. Chem. 8 (2000) 73±93

and the reaction mixture stirred for 80 min at 10 ^ꢂ,

then concentrated in vacuo and dissolved in ethyl acetate.

The organic phase was washed with NaCl, dried, and

concentrated in vacuo to give a residue that was puri®ed

on silica gel (75:25, then 65:35 hexane:ethyl acetate) to

brie¯y to 40 ^ꢂ, then cooled to room temperature. Tri-

methylsilyliodide (TMSI, 193 mL, 2.25 mmol) was

added and the reaction stirred at room temperature for

30 min, then concentrated in vacuo, and the residue

dissolved in 1 mL CH₃CN. THF (250 mL) was added to

consume excess TMSI, followed by addition of a sus-

pension of 2-hydroxyphenylisonicotinylhydrazide (29)

in 3 mL CH₃CN. A clear solution was obtained within

30 min, and the reaction stirred for 3 h at room tem-

perature, then poured into a solution of 96:5 acetone-

MeOH. The precipitate was collected and puri®ed on

reverse phase HPLC (linear gradient 10 to 35% B in A

over 40 min) to give 7 mg (2%) of 87. ¹H NMR

(400 MHz, DMSO-d₆) d 1.42 (3H, s), 1.43 (3H, s), 3.45±

3.83 (2H, m), 5.24 (1H, d, J=5.5 Hz), 5.65 (2H, dd,

J=14.7, 17.4 Hz), 5.97 (1H, bd, J=5.7 Hz), 6.73 (1H, s),

6.95±6.99 (1H, m), 7.33±7.62 (2H, m), 7.69 (1H, d,

J=7.6 Hz), 8.39±8.84 (2H, m), 9.17 (1H, d, J=5.8 Hz),

9.48 (1H, d, J=7.7 Hz), 10.86 (1H, bs). HPLC (3 to

70% B in A over 30 min) T_R=15.7 min. MS, m/z 709.2

(MH⁺). HRMS, m/z for C₃₀H₂₉N₈O₉S₂calculated,

709.1499, found, 709.1479 (MH⁺).

1

give 36.6 mg (14%) of 121 as a yellow solid. H NMR

(400 MHz, CDCl₃) d 2.89±3.33 (2H, m), 3.50 (3H, s),

3.80 (3H, s), 4.20±4.49 (2H, m), 4.75 (1H, d, J=5.0 Hz),

5.11 (2H, s), 5.39 (2H, d, J=8.0 Hz), 5.63 (2H, dd,

J=11.6, 14.8 Hz), 5.78 (1H, dd, J=4.3, 4.9 Hz), 6.80

(1H, s), 6.86±7.49 (24H, m), 8.05 (1H, d, J=8.1 Hz),

8.52 (1H, d, J=9.6 Hz). MS m/z 1063.3. (MH⁺).

To a solution of intermediate 121 (36 mg, 0.04 mmol) in

350 mL THF was added NaI (5.6 mg, 0.04 mmol) and

dry pyridine (50 mL, 0.6 mmol). The reaction mixture

was stirred for 5 h at room temperature, concentrated in

vacuo, and the product precipitated with diisopropyl

ether and lyophilized from 50:50 CH₃CN:H₂O (0.05%

TFA) to give a tan solid that was homogeneous by

HPLC (3 to 70% B in A over 30 min) T_R=33.5 min.

MS m/z 1106.4 (MH⁺).

Treatment with 5:1 TFA-anisole gave, upon precipita-

tion from diisopropyl ether, 5 mg (18%) of 85. ¹H NMR

(400 MHz, DMSO-d₆) d 3.18±3.48 (2H, m), 3.63 (2H, s),

5.10 (1H, d, J=5.0 Hz), 5.32 (2H, s), 5.86 (1H, dd,

J=3.0, 5.0 Hz), 6.68 (1H, s), 6.72±8.24 (9H, m), 8.62±

8.66 (1H, m), 8.99 (1H, d, J=5.6 Hz), 9.68 (1H, d,

J=8.0 Hz). HPLC (3 to 70% B in A over 30 min)

T_R=19.1 min. MS, m/z 700.2 (MH⁺).

(Z)-2-Amino-ꢁ-[1-(carboxy-1-methylethyloxyimino]-4-

acetamido]-3-(4-(2,4-dihydroxyphenyl)hydrazido-1-pyri-

dylmethyl)-4-yl]thio]methyl]-8-oxo-1-aza-5-thiabicyclo-

[4.2.0]oct-2-ene-2-carboxylic acid (88). Compound 88

was synthesized from 30 (90 mg, 0.35 mmol) and 86

(185 mg, 0.35 mmol) as described for 87 to give, after

HPLC puri®cation on a linear gradient of 3±30% B in a

over 30 min, 2 mg (1%) of 88. ¹H NMR (400 MHz,

DMSO-d₆) d 1.34 (3H, s), 1.35 (3H, s), 3.28±3.74 (2H,

m), 5.14 (1H, d, J=5.0 Hz), 5.53±5.58 (2H, m), 5.88

(1H, dd, J=3.1, 5.0 Hz), 6.64 (1H, s), 7.39±7.47 (2H,

m), 7.46 (1H, d, J=16.6 Hz), 8.51 (2H, dd, J=6.7,

16.7 Hz), 9.15±9.41 (2H, m), 9.86 (1H, s), 10.83 (2H, bs),

10.89 (2H, bs). HPLC (3 to 70% B in A over 30 min)

T_R=15.8 min. MS, m/z 725.1 (MH⁺).

(Z)-2-Aminoa[1-(carboxy)-1-methylethyloxyimino]-4-ace-

tamido]-3-(4-(2-hydrazido-1-pyridylmethyl)-4-yl]thio]meth-

yl]-8-oxo-1-aza-5-thiabicyclo[4.2.0]oct-2-ene-2-carboxylic

acid (87). To a 0 ^ꢂsolution of (Z)-2-amino a[1-(t-butoxy-

carbonyl)-1-methylethyloxyimino]-4-acetic acid (905mg,

2.73 mmol) and HOAt (375 mg, 2.75 mmol) in 20 mL

THF was added DCC (570 mg, 2.75 mmol), and the

reaction stirred for 1h at room temperature. To the

activated ester solution was added 82 in 10 mL THF,

and the reaction stirred overnight. The solvent was

removed in vacuo, and the residue dissolved in CHCl₃,

washed with 0.1 N citric acid, saturated NaHCO₃,

NaCl, dried, and concentrated in vacuo. Puri®cation on

silica gel (60:40 hexane:ethyl acetate) gave 1.36 g (66%)

of 122. ¹H NMR (400 MHz, CDCl₃) d 1.43 (9H, s), 1.59

(3H, s), 1.60 (3H, s), 2.02 (3H, s), 3.37±3.59 (2H, m),

4.82 (1H, d, J=13.8 Hz), 5.07 (2H, dd, J=4.2, 5.0 Hz),

6.04 (1H, dd, J=4.0, 4.9 Hz), 6.50 (2H, bs), 6.89 (1H, s),

6.95 (1H, s), 7.32±7.65 (10H, m).

(6R,7R)-7-[2-Aminothiazol-4-yl)-2-[(Z)-[1-(carboxymeth-

ylethyloxy]imino]acetamido]-3-[[[1-[N-((3-hydroxy-4-(2-

benzthiazolyl)benzyl)carbamoylmethyl]pyridinium - 4 - yl]-

thio]methyl]-8-oxo-1-aza-5-thiabicyclo[4.2.0]oct-2-ene-2-

carboxylic acid (94).

A

solution of 89¹³(482 mg,

0.64 mmol) and 33 (717 mg, 1.99 mmol) in 4 mL dry

DMF was stirred at room temperature for 24 h. Tri-

turation with 50:50 diethyl ether:hexane, followed by

50:50 diisopropyl ether:hexane, followed by diisopropyl

ether, gave a precipitate that was collected and puri®ed

on a column of silica gel (CHCl₃, followed by 10:90

1

MeOH:CHCl₃,) to give 470 mg (71%) of 123. H NMR

(400 MHz, CDCl₃) d 1.37 (9H, s), 1.53 (3H, s), 1.56

(3H,s), 3.52 (3H, s), 3.73 (3H, s), 3.48±3.82 (4H, m),

4.11±4.43 (2H, m), 5.08±5.16 (1H, m), 5.16 (2H, s), 5.51

(2H, s), 5.84±5.95 (3H, m), 6.78±7.89 (13H, m), 8.04

(1H, d, J=8.1 Hz), 8.49 (1H, d, J=7.7 Hz), 8.96 (1H, d,

J=7.9 Hz), 9.05±9.09 (2H, bs). MS m/z 1038.3 (MH⁺).

Protected cephalosporin 122 (1.0 g, 1.34 mmol) was

treated with 5:1 TFA-anisole for 2.5 h to give 743 mg

(quant) of 86. H NMR (400 MHz, DMSO-d₆) d 1.46

(6H, s), 2.03 (3H, s) 3.48±3.76 (2H m), 5.00 (2H, d,

J=12.9 Hz), 5.19 (1H, d, J=4.9 Hz), 5.87 (1H, dd, J=

3.5, 4.9 Hz), 6.77 (1H, s) 9.45 (1H, d, J=7.8 Hz). MS,

m/z 528 (MH⁺).

1

A solution of 123 (280 mg, 0.25 mmol) in 2 mL CH₂Cl₂

was cooled on ice, and a mixture of 5:1 TFA-anisole

(2.4 mL) was added. After 30 min the icebath was

removed, and the reaction mixture allowed to stir for an

additional 2.5 h. Trituration with diisopropyl ether

Acetate 86 (264 mg, 0.5 mmol) was suspended in CH₂Cl₂

and treated with tri¯uoromethyl(N-methyl N-trimethyl-

silyl)acetamide (MSTFA, 620 L, 3.5 mmol), warmed

T. Kline et al. / Bioorg. Med. Chem. 8 (2000) 73±93

89

gave, upon cooling, 240 mg of crude product. Puri®ca-

tion on HPLC (linear gradient of 30±40% B in A over

40 min, B=0.05%TFA in CH₃CN, A=0.05% TFA in

H₂O) gave 8 mg (4%) of 94. ¹H NMR (400 MHz,

DMSO-d₆) d 1.37 (3H, s), 1.38 (3H, s), 3.45±3.72 (2H,

m), 4.32 (2H, s), 5.16 (1H, d, J=4.9 Hz), 5.65 (2H, s),

5.79 (1H, dd, J=4.8, 4.9 Hz), 6.67 (1H, s), 7.04 (2H, dd,

J=1.4, 6.8 Hz), 7.38±7.51 (2H, m), 8.00 (2H, d,

J=7.0 Hz), 8.09 (1H, d, J=7.8 Hz), 8.19 (1H, d,

J=8.1 Hz), 8.84 (2H, d, J=7.0 Hz), 9.40 (1H, d,

J=8.1 Hz). HPLC (3±70% B in A over 30 min) T_R=

20.4 min. HRMS, m/z for C₃₆H₃₂N₇O₃S₄calculated,

818.1195, found, 818.1221 (MH⁺).

[4.2.0]oct-2-ene-2-carboxylate (92). A solution of 126 in

2 mL of CH₂Cl₂(58 mg, 0.06 mmol) was reacted with

10:1 TFA:anisole as described for 91 to give 37 mg

1

(71%) of 92 as a light yellow powder. H NMR (400

MHz, DMSO-d₆) d 1.45 (6H, t, J=5.5 Hz), 3.56±3.84

(1H, m), 4.19±4.41 (3H, m), 4.43 (2H, s), 5.08 (1H, s),

5.26 (1H, m), 5.60±5.63 (1H, m), 6.29 (2H, d,

J=8.1 Hz), 6.77 (1H, d, J=11.6 Hz), 7.67 (2H, t,

J=7.6 Hz), 7.80 (1H, t, J=7.6 Hz), 8.04±8.13 (5H, m),

8.67 (2H, dd, J=7.1, 9.4 Hz), 9.50 (1H, d, J=7.7 Hz).

HRMS, m/z for C₃₀H₂₉N₆O₈S₃calculated, 697.1209,

found, 697.1216 (M⁺).

(6S,7S)-7-[2-(2-Aminothiazol-4-yl)-2-[(Z)-(1-carboxy-1-

methylethoxy)imino]acetamido]-3-[[[1-[N-(3-hydroxy-3⁰-

methyl¯avone)]pyridinium-4-yl]thio]methyl]-8-oxo-1-aza-

4-thiabicyclo[4.2.0]oct-2-ene-2-carboxylate (96). Bromo-

methyl intermediate 59 (55 mg, 0.15 mmol) was reacted

with 89 (100 mg, 0.14 mmol) in 2 mL DMF as described

for 123 to give 73 mg (50%) of 127 that was homo-

geneous by TLC. R_f=0.18 (90:10 CH₂Cl₂:MeOH). A

solution of 127 (43 mg, 0.04 mmol) in 2 mL of CH₂Cl₂

was treated with 10:1 TFA:anisole for 3.5 h. An addi-

tional 10:1 TFA:anisole was added, and the reaction

continued at 0 ^ꢂC for 2 h, then precipitated as for 91 to

give 25 mg (66%) of 96 as a light brown solid. ¹H NMR

(400 MHz, DMSO-d₆) d 1.47±1.55 (6H, m), 3.61±3.84

(4H, m), 4.43 (2H, s), 5.27±5.28 (1H, m), 5.87±5.90 (3H,

m), 6.77 (1H, s), 7.38 (1H, s), 7.54±7.58 (1H, m), 7.64±

7.71 (2H, m), 7.80±7.82 (1H, m), 7.89±7.91 (1H, m), 8.10

(2H, d, J=7.1 Hz), 8.19±8.21 (1H, m), 8.33 (2H, d,

J=7.8 Hz), 8.39 (1H, s), 9.00 (2H, d, J=7.0 Hz), 9.50

(1H, d, J=8.3 Hz), 9.86 (1H, s). HRMS, m/z for C₃₈H₃₃

N₆O₁₀S₃calculated, 829.1420, found, 829.1433 (M⁺).

(6R,7R)-7-[2-Aminothiazol-4-yl)-2-[(Z)-[1-(carboxymethyl-

ethyloxy]imino]acetamido]-3-[[pyridinium-4-yl]thio]meth-

yl]-8-oxo-1-aza-5-thiabicyclo[4.2.0]oct-2-ene-2-carboxylic

acid (90). Thiopyridine 90 was prepared by TFA

deprotection of 89 (210 mg, 0.28 mmol) as described for

94. HPLC puri®cation (5±25% B in A over 40 min) gave

10.1 mg. (6%) 90. ¹H NMR (400 MHz, DMSO-d₆) d

1.53 (3H, s), 1.55 (3H, s), 3.61±3.87(2H, m), 4.43 (2H,

dd, J=11.0, 12.8 Hz), 5.31 (1H, d, J=4.9 Hz), 5.96 (1H,

d, J=4.9 Hz), 6.83 (1H, s), 7.83 (2H, d, J=6.7), 8.67

(2H, d, J=6.5 Hz), 9.54 (1H, d, J=8.3 Hz). HPLC (3±70%

B in A over 30 min) T_R=12.2 min. HRMS, m/z C₂₂H₂₃

N₆O₇S₃calculated, 579.0790 (MH⁺), found, 579.0789.

(6R,7R)-7-[2-Aminothiazol-4-yl)-2-[(Z)-[1-(carboxymeth-

ylethyloxy]imino]acetamido]-[3-[4-methylphenyl)-5-(2-hy-

droxyphenyl)-1,2,4-oxadiazolyl]pyridinium-4-yl]]-thio-

methyl]-8-oxo-1-aza-5-thiabicyclo[4.2.0]oct-2-ene-2-carb-

oxylic acid (95). Bromomethyl intermediate 52 was

converted to 124 (68 mg, 0.0065 mmol), which was

deprotected according to the procedure described for 94

to give 15 mg (28%) of 95. ¹H NMR (400 MHz, DMSO

d₆) d 1.33±1.36 (6H, m), 3.62±3.71 (2H, m), 4.07±4.11

(1H, m), 4.28±4.33 (1H, m), 5.13±5.16 (1H, m), 5.69±

5.78 (3H, m), 6.79 (1H, s), 6.99 (1H, t, J=7.5 Hz), 7.06

(1H, d, J=8.3 Hz), 7.47 (1H, dt, J=1.6, 8.6 Hz), 7.59±

7.62 (2H, m), 7.93±7.98 (3H, m), 8.07±8.10 (2H, m),

8.77±8.82 (2H, m).

(6S,7S)-7-[2-(2-Aminothiazol-4-yl)-2-[(Z)-(1-carboxy-1-

methylethoxy)imino]acetamido]-3-[[[1-[N-(3-hydroxy-4⁰-

methyl¯avone)]pyridinium-4-yl]thio]methyl]-8-oxo-1-aza-4-

thiabicyclo[4.2.0]oct-2-ene-2-carboxylate (97). The pro-

tected intermediate was prepared from 60 (55 mg,

0.15 mmol) and 89 (100 mg, 0.14 mmol) as described for

127, to give 80 mg (54%) of 128 that was homogeneous

by tlc. R_f=0.18 (90:10 CH₂Cl₂:MeOH). A solution of

128 (50 mg, 0.04 mmol) in 2 mL CH₂Cl₂was treated

with 10:1 TFA:anisole as described for 96 to give 30 mg

(6S,7S)-7-[2-(2-Aminothiazol-4-yl)-2-[(Z)-(1-carboxy-1-

methylethoxy)imino]acetamido]-3-[[[1-[N-benzyl]pyridi-

nium-4-yl]thio]methyl]-8-oxo-1-aza-4-thiabicyclo[4.2.0]-

oct-2-ene-2-carboxylate (91). A solution of 125 (50 mg,

0.06 mmol) in 2 mL of CH₂Cl₂was treated with anisole

(200 L), cooled to 0 ^ꢂC, and neat TFA (2.0 mL) added.

The reaction was allowed to stir at 0 ^ꢂC for 2 h followed

by slow warming to rt over 30 min. Addition of ethyl

ether (20 mL) to the reaction mixture gave 35 mg (81%)

of 91 as brown solid. ¹H NMR (400 MHz, DMSO-d₆) d

1.50 (3H, s), 1.51 (3H, s), 3.57±3.84 (2H, m), 4.44 (2H,

d, J=1.7 Hz), 5.29 (1H, t, J=5.4 Hz), 5.74 (2H, s), 5.93

(1H, dd, J=4.9, 8.3 Hz), 6.80 (1H, s), 7.48±7.58 (7H,

m), 8.05±8.10 (4H, m), 8.95 (2H, d, J=7.0 Hz), 9.51

(1H, d, J=8.4 Hz). HRMS, m/z for C₂₉H₂₉N₆O₇S₃cal-

culated, 669.1260, found, 669.1255 (M⁺).

1

(72%) 97 as a light brown solid. H NMR (400 MHz,

DMSO-d₆) d 1.43 (6H, m), 3.52±3.56 (1H, m), 3.72±3.79

(1H, m), 4.16±4.20 (1H, m), 4.28±4.42 (2H, m), 5.77±

5.78 (2H, m), 5.83±5.88 (1H, m), 6.71±6.77 (1H, m),

7.47±7.53 (2H, m), 7.67±7.70 (2H, m), 7.72±7.77 (2H,

m), 7.79±7.85 (2H, m), 8.04 (2H, dd, J=7.0, 12.4 Hz),

8.13 (2H, dd, J=1.4, 8.0 Hz), 8.22±8.28 (2H, m), 8.89±

8.94 (2H, m), 9.43±9.46 (1H, m), 9.79 (1H, s); HRMS,

m/z calcd. for C₃₈H₃₃N₆O₁₀S₃829.1420, found,

829.1408 (M⁺).

(6S,7S)-7-[2-(2-Aminothiazol-4-yl)-2-[(Z)-(1-carboxy-1-

methylethoxy)imino]acetamido]-3-[[[1-[N-(3-hydroxy-6-

methyl¯avone)]pyridinium-4-yl]thio]methyl]-8-oxo-1-aza-

4-thiabicyclo[4.2.0]oct-2-ene-2-carboxylate(98). The pro-

tected intermediate was prepared from 61 (55 mg,

0.15 mmol) and 89 (100 mg, 0.14 mmol) as described for

127, to give 85 mg (57%) of 129 that was homogeneous

(6S,7S)-7-[2-(2-Aminothiazol-4-yl)-2-[(Z)-(1-carboxy-1-

methylethoxy)imino] acetamido]-3-[[[1-[N-acetophenone]-

pyridinium-4-yl]thio]methyl]-8-oxo-1-aza-4-thiabicyclo-

90

T. Kline et al. / Bioorg. Med. Chem. 8 (2000) 73±93

by TLC. R_f=0.18 (90:10 CH₂Cl₂:MeOH). A solution of

129 (50 mg, 0.04 mmol) in 2 mL of CH₂Cl₂was treated

with 10:1 TFA:anisole for 3.5 h, and the product pre-

cipitated as for 96 to give 20 mg (48%) of 98 as a light

C₂₉H₃₀N₇O₇S₃calculated, 684.1368, found, 684.1348

(M⁺).

5-Bromosalicylaldehyde (200 mg, 1.0 mmol) was added

to a solution of 132 (24 mg, 0.037 mmol) in DMF

(0.5 mL) and the resulting mixture was allowed to stir at

room temperature for 72 h, diluted with CH₂Cl₂, and

®ltered. The ®ltrate was concentrated in vacuo, sus-

pended in warm EtOAc, and allowed to stand for 1 h to

1

brown solid. H NMR (400 MHz, DMSO-d₆) d 1.41±

1.44 (6H, m), 3.51±3.55 (2H, m), 3.70±3.78 (2H), 4.16±

4.19 (2H, m), 5.03 (2H, s), 5.21±5.24 (2H, m), 5.59±5.62

(1H, dd, J=3.9, 8.0 Hz), 5.83±5.88 (2H, m), 6.87 (1H,

s), 7.50±7.64 (2H, m), 7.85±7.88 (1H, m), 7.93±7.98 (1H,

m), 8.01±8.08 (2H, m), 8.20±8.23 (2H, m), 8.38±8.41

(1H, m), 8.94±8.99 (2H, m), 9.43±9.46 (1H, m), 9.79

1

give 16 mg (50%) of 100 as a yellow solid. H NMR

(400 MHz, DMSO-d₆) d 1.43 (3H, d, J=5.0 Hz), 1.48

(3H, d, J=5.5 Hz), 3.72±3.79 (2H, m), 4.37 (2H, s),

5.19±5.25 (2H, m), 5.71 (2H, s), 6.97 (2H, d, J=7.4 Hz),

7.26±7.29 (1H, m), 7.45 (2H, d, J=7.6 Hz), 7.53±7.60

(2H, m), 7.86±7.89 (1H, m), 8.01±8.06 (2H, m), 8.90±

8.92 (3H, m), 12.68 (1H, bs). HRMS, m/z for C₃₆H₃₃

N₇O₈S₃Br calculated, 866.0736, found, 866.0750 (M⁺).

10

(1H, s). HRMS, m/z for C₃₈H₃₃N₆O S₃calculated,

829.1420, found, 829.1429 (M⁺).

(6R,7R)-7-[2-(2-Aminothiazol-4-yl)-2-[(Z)-(1-carboxy-1-

methylethoxy)imino]acetamido]-3-[[[1-[N-2-[(2-hydroxy)-

benzylidene] aminobenzyl] pyridinium-4-yl]thio]methyl]-8-

oxo-1-aza-5-thiabicyclo[4.2.0]oct-2-ene-2-carboxylate

(99). Bromomethyl intermediate 40 (76 mg, 0.27 mmol)

was reacted with 89 (200 mg, 0.27 mmol) as described

for 123 to give 217 mg (79%) of a brown glassy residue,

of which 101 mg, (0.097 mmol.) was treated with 5:1

TFA:anisole at 10 ^ꢂfor 4 h. Precipitation with diethyl

ether gave 86 mg (79%) of 130 as a beige solid. ¹H

NMR (400 MHz, CD₃OD) d 1.48 (3H, s), 1.49 (3H, s),

3.52±3.58 (2H, m), 4.32±4.36 (2H, m), 5.08 (1H, d,

J=4.8 Hz), 5.45 (2H, s), 5.76 (1H, d, J=4.8 Hz), 6.64

(1H, t, J=7.3 Hz), 6.70 (1H, d, J=7.8 Hz), 6.96 (1H, s),

7.10 (2H, d, J=7.7 Hz), 7.76 (2H, d, J=7.0 Hz), 8.35

(2H, d, J=6.8 Hz). HRMS, m/z for C₂₉H₃₀N₇O₇S₃cal-

culated, 684.1368, found, 684.1352 (M⁺). A suspension

of 130 (25 mg, 0.036 mmol) in EtOH (1.0 mL) was trea-

ted with salicylaldehyde (39 mL, 0.36 mmol). After 48 h

the reaction was diluted with CH₂Cl₂and ®ltered. The

resulting ®ltrate was concentrated in vacuo, suspended

in CH₂Cl₂, and ®ltered to aord 15 mg (53%) of 99 as

(6R,7R)-7-[2-(2-Aminothiazol-4-yl)-2-[(Z)-[1-carbonyl-1-

(methylethyloxy)imino]acetamido]-3-[[[1-[N-(3-hydroxy-4-

(benzoylhydrazonomethyl)benzyl)]pyridinium-4-yl]thio]-

methyl]-8-oxo-1-aza-5-thiabicyclo[4.2.0]oct-2-ene-2-car-

boxylate (101). Cephalosporin 89 (7.5 mg, 0.01 mmol)

was reacted with bromomethyl intermediate 45 (11 mg,

0.01 mmol) to give, after 5:1 TFA:anisole deprotection,

5 mg (59%) of 101. ¹H NMR (400 MHz, CD₃OD) d

1.50 (3H, s), 1.51 (3H, s), 3.47±3.51 (1H, m), 3.64±3.68

(1H, m), 4.30 (1H, d, J=13.7 Hz), 4.49 (1H, d, J=

12.8 Hz), 5.10 (1H, d, J=4.7 Hz), 5.47±5.54 (2H, m),

5.78 (1H, d, J=4.7 Hz), 6.86±6.88 (2H, m), 6.98 (1H, s),

7.41±7.43 (3H, m), 7.50±7.52 (1H, m), 7.83±7.85 (4H,

m), 8.42 (1H, s), 8.56 (2H, d, J=6.6 Hz). HRMS, m/z

for C₃₇H₃₆N₈O₉S₃calculated, 832.4577 found, 831.1689

(M ).

(6R,7R)-7-[2-(2-Aminothiazol-4-yl)-2-[(Z)-[1-carbonyl-1-

(methylethyloxy)[N-(3-hydroxy-4-[(2-hydroxy-3-methyl-

benzoylhydrazonomethyl)benzyl)]pyridinium - 4-yl]thio] -

methyl]-8-oxo-1-aza-5-thiabicyclo[4.2.0]oct-2-ene-2-car-

boxylate (102). Cephalosporin 89 (91 mg, 0.12 mmol)

was reacted with bromomethyl intermediate 46 (49 mg,

0.12 mmol) in dry DMF to give, after 5:1 TFA:anisole

1

an amber solid. H NMR (400 MHz, DMSO-d₆) d 1.43

(3H, d, J=6.0 Hz), 1.49 (3H, d, J=6.5 Hz), 3.63±3.79

(2H, m), 4.30±4.39 (2H, m), 5.17±5.30 (1H, m), 5.46

(1H, s), 5.82±5.90 (2H, m), 6.99 (2H, d, J=6.2 Hz), 7.35

(2H, d, J=7.0 Hz), 7.40±7.54 (3H, m), 7.76 (1H, d,

J=6.5 Hz), 7.84 (1H, d, J=5.8 Hz), 7.94±8.01 (1H, m),

8.69±8.75 (1H, m), 8.80±8.87 (2H, m), 11.62 (1H, s),

11.90 (1H, s), 12.74 (1H, bs). HRMS, m/z for C₃₆H₃₄

N₇O₈S₃calculated, 788.1631, found, 788.1632 (M⁺).

1

deprotection, 27 mg (26%) of 102. H NMR (400 MHz,

CD₃OD) d 1.25 (6H, s), 2.05 (3H, s), 3.38±3.42 (1H, m),

3.57±3.59 (1H, m), 4.09±4.12 (1H, m), 4.21±4.22 (1H,

m), 5.01±5.03 (1H, m), 5.46±5.47 (2H, m), 5.71 (1H, d,

J=4.8 Hz), 6.61 (2H, d, J=8.6 Hz), 6.80±6.82 (1H, m),

7.12 (2H, d, J=8.6 Hz), 7.50±7.52 (1H, m), 7.64±7.66

(2H, m), 8.00 (1H, d, J=8.1 Hz), 8.45±8.47 (2H, m),

8.63±8.65 (1H, m). MS, m/z 861.5 (M ).

(6R,7R)-7-[2-(2-Aminothiazol-4-yl)-2-[(Z)-(1-carboxy-1-

methylethoxy)imino]acetamido]-3-[[[1-[N-3-[(2-hydroxy-5-

bromo)benzylidene]aminobenzyl]pyridinium-4-yl]thio]-

methyl]-8-oxo-1-aza-5-thiabicyclo [4.2.0]oct-2-ene-2-car-

boxylate (100). Bromomethyl intermediate 41 (225 mg,

0.79 mmol) was reacted with 89 (230 mg, 0.31 mmol) as

described for 123 to give 217 mg (68%) of quaternary

intermediate 131. The crude product (217 mg, 0.21

mmol) was treated with 10:1 TFA:anisole to give, upon

precipitation, 190 mg of a peach solid. HPLC puri®ca-

tion of 50 mg (30±70% B in A over 30 min.) provided

(6R,7R)-7-[2-(2-Aminothiazol-4-yl)-2-[(Z)-[1-carbonyl-1-

(methylethyloxy)imino]acetamido]-3-[[[1[N-(3-hydroxy-4-

hydrazonomethyl)benzyl)]pyridinium-4-yl]thio]methyl]-8-

oxo-1-aza-5-thiabicyclo[4.2.0]oct-2-ene-2-carboxylate (103).

Cephaolosporin 89 (87 mg, 0.115 mmol) was reacted

with 49 (43 mg, 0.115 mmol) in dry DMF to give, after

5:1 TFA:anisole deprotection, 5 mg (6%) of 103. ¹H

NMR (400 MHz, DMSO-d₆) d 1.42 (3H, s), 1.43 (3H, s),

3.55 (5H, bs), 3.74 (1H, d, J=5.3 Hz), 4.37±4.38 (2H,

m), 5.21±5.22 (1H, m), 5.67±5.68 (2H, m), 5.85±5.87

(1H, m), 6.71 (1H, s), 7.01±7.04 (2H, m), 7.28±7.29

(2H, m), 7.74 (1H, d, J=5.1 Hz), 8.02±8.03 (2H, m),

1

24 mg (64%) of 132. H NMR (400 MHz, CD₃OD) d

1.60 (3H, s), 1.61 (3H, s), 3.65±3.69 (2H, m), 4.46±4.50

(2H, m), 5.24 (1H, d, J=4.9 Hz), 5.54 (2H, s), 5.88 (1H,

d, J=4.9 Hz), 6.97±7.03 (2H, m), 7.09 (1H, s), 7.27±7.35

(1H, m), 7.33 (1H, t, J=8.0 Hz), 7.93 (2H, d,

J=7.2 Hz), 8.63 (2H, d, J=7.1 Hz). HRMS, m/z

T. Kline et al. / Bioorg. Med. Chem. 8 (2000) 73±93

91

8.87±8.88 (2H, m), 8.99 (1H, s), 9.44 (1H, s), 11.26 (1H,

s). HRMS, m/z for C₃₀H₃₂N₈O₈S₃calculated, 728.4316,

found, 727.1447 (M⁺).

6.83±6.86 (2H, m), 7.18±7.59 (5H, m), 7.96±8.00 (2H,

m), 8.28±8.33 (2H, m), 8.83±8.96 (3H, m), 9.44 (1H, d,

J=8.3 Hz), 12.14 (1H, s). MS, m/z 1052.3 (M⁺).

(6R,7R)-7-[2-(2-Aminothiazol-4-yl)-2-[(Z)-[1-carbonyl-1-

(methylethyloxy)imino]acetamido]-3-[[[1[N-(3-hydroxy-4-

acetylhydrazonomethyl)benzyl]pyridinium-4-yl]thio]meth-

yl]-8-oxo-1-aza-5-thiabicyclo[4.2.0]oct-2-ene-2-carboxy-

late (104). Cephalosporin 89 (271 mg, 0.36 mmol) was

reacted with 47 (115 mg, 0.36 mmol) in dry DMF to

give, after 5:1 TFA:anisole deprotection, 30 mg (26%)

The entire crude hydrazide product 134 was stirred with

5:1 TFA:anisole in CH₂Cl₂for 3 h, and the product

precipitated with diisopropyl ether to give 4 mg (8%

1

from 133) of 106. H NMR (400 MHz, DMSO-d₆) d

1.37 (3H, s), 1.38 (3H, s), 3.38±3.72 (4H, m), 4.31 (2H,

s), 5.16±5.17 (1H, d, J=2.9 Hz), 5.63 (2H, s), 5.80 (1H,

bs), 6.66 (1H, s), 6.88±7.00 (2H, m), 7.62±7.64 (1H, m),

7.99±8.00 (2H, m), 8.79±8.84 (2H, m), 9.39±9.40 (1H, d,

J=7.4 Hz), 10.19 (1H, s). HPLC (15 to 25% B in A over

40 min) T_R17.6 in. HRMS, m/z for C₃₆H₃₄N₉O₉S₃cal-

culated, 832.1642, found, 832.1646 (M⁺).

1

of 104. H NMR (400 MHz, CD₃OD) d 1.52 (3H, s),

1.53 (3H, s), 1.99 (3H, s), 3.51 (1H, d, J=8.0 Hz), 3.69±

3.71 (1H, m), 4.30±4.34 (1H, m), 4.50±4.52 (1H, m), 5.12

(1H, d, J=4.9 Hz), 5.48±5.51 (2H, m), 5.80±5.81 (1H,

m), 6.86 (2H, d, J=5.6 Hz), 7.03 (1H, s), 7.39±7.41 (1H,

m), 7.80±7.81 (2H, m), 8.17 (1H, s), 8.56±8.57 (2H, m).

MS, m/z 770.0 (M⁺).

Biology

(6R,7R)-7-[2-(2-Aminothiazol-4-yl)-2-[(Z)-[1-carbonyl-1-

(methylethyloxy)imino]acetamido]-3-[[[1[N-(3-hydroxy-4-

[(2 - hydroxy - 4 - butyrylhydrazonomethyl)benzyl)]pyridin-

ium-4-yl]thio]methyl]-8-oxo-1-aza-5-thiabicyclo[4.2.0]oct-

2-ene-2-carboxylate (105). Cephalosporin 89 (157 mg,

0.2 mmol) was reacted with bromomethyl intermediate

48 (151 mg, 0.2 mmol) in dry DMF to give, after 5:1

TFA:anisole deprotection, 20 mg (12%) of 105. ¹H

NMR (400 MHz, CDOD) d 1.00±1.02 (3H, m), 1.61±

1.75 (8H, m), 2.30±2.32 (2H, m), 3.72±3.75 (1H, m),

3.79±3.82 (1H, m), 4.39±4.42 (1H, m), 4.58±4.61 (1H,

m), 5.22 (1H, d, J=4.7 Hz), 5.60±5.63 (2H, m), 5.88±

5.89 (1H, m), 6.78±6.80 (1H, m), 6.96±6.97 (1H, m), 7.18

(1H, d, J=12 Hz), 7.48±7.50 (1H, m), 7.95 (2H, d, J=

6.9 Hz), 8.17 (1H, d, J=2.3 Hz), 8.31 (2H, d, J=3.6 Hz).

MS, m/z 797.2 (M⁺).

Construction of siderophore-de®cient P. aeruginosa

strains

Gene replacements disrupting pyochelin and pyoverdin

synthesis were performed essentially as described.^58,59

Brie¯y, pMJ19, a derivative of vector pEX18T (Gen-

Bank #AF004910) with an insert consisting of the 5⁰

portion of the pchD open reading frame (ORF) from

PAO1, a gentamicin-resistance (GmR) marker, and the

3⁰portion of the pchA ORF, was mobilized into strain

PAO1 by conjugation. Transconjugants were selected

on L agar containing 200 mg/mL Gm and patched onto

L agar with 500 mg/mL carbenicillin (Cb) to identify and

eliminate merodiploid organisms. DNA from clones

with the appropriate Gm^R, Cb^Sphenotype was puri®ed

and the alteration of the pch locus (truncation of pchD

and pchA and the deletion of pchCB ORFs) was con-

®rmed by polymerase chain reaction (PCR) and South-

ern blotting using previously described hybridization

conditions.⁶⁰This pyochelin-de®cient strain was desig-

nated PAO1Ápch.

(6R,7R)-7-[2-Aminothiazol-4-yl)-2-[(Z)-[1-(t-butoxycarb-

onyl)-methylethyloxy]imino]acetamido]-3-[[[1-[N-(3-meth-

oxymethyloxy-4-formyl)benzyl)methyl]pyridinium-4-yl]-

thio]methyl]-8-oxo-1-aza-5-thiabicyclo[4.2.0]oct-2-ene-2-

carboxylic acid (106). Cephalosporin 89 (518 mg, 0.67

mmol) was reacted with bromomethyl aldehyde 44

(380 mg, 1.46 mol) in 3 mL dry DMF to give inter-

mediate 133 that was used directly in the following

Interruption of a gene required for pyoverdin synthesis

was accomplished by inserting the pvdD ORF from

PAO1 into pEX100T⁵⁵and cloning a tetracycline-resis-

tance (Tc^R) marker into the unique Pst I site of that

insert, creating a vector designated pWS92-29. Intro-

duction of this construct into PAO1Ápch was again

performed by conjugation and the selection of organ-

isms required initial plating on media containing 100 mg/

mL Tc, 200 mg/mL Gm, 5% sucrose, and 5 mM FeCl₃.

The identity of the resultant strain, PAO1(Ápch,

pvdD::Tc), assigned PGO 2812, was con®rmed by PCR

and Southern blotting as described above.

1

reaction. H NMR (400 MHz, CDCl₃) d 1.41 (9H, s),

1.44±1.46 (6H, two s), 3.45±3.55 (2H, m), 3.45 (3H, s),

3.71 (3H, s), 4.32 (2H, m), 5.20 (2H, s), 5.27±5.37(1H,

m), 5.40 (2H, s), 5.75±6.00 (3H, m), 6.84±7.31 (4H, m),

7.32±7.33 (4H, m), 7.96±7.99 (2H, m), 8.92±9.00 (2H,

m), 10.22 (1H, s). MS, m/z 933.2 (M⁺).

To a solution of aldehyde 133 (56 mg, 0.06 mmol) in

3 mL MeOH was added nicotinoyl hydrazide (84 mg,

0.6 mmol) and the reaction mixture stirred at room

temperature for 6 h. Additional hydrazide (0.8 mmol)

was added, and the reaction stirred overnight. After

concentration in vacuo, intermediate 134 was puri®ed

away from unreacted nicotinyl hydrazide and aldehyde

Growth in low iron assay

The in¯uence of the various siderophore-like com-

pounds on bacterial growth in low iron conditions

was studied using the P. aeruginosa strain PGO 2812

described above. Cells from a saturated overnight cul-

ture were diluted to approximately 0.01 OD₆₀₀in LB

(Luria Broth Base, Miller; Gibco #0414-05-5) containing

2 mM 2,2-dipyridyl (Sigma #D-7505). The LB-dipyridyl

1

on silica gel (9:1 CHCl₃:MeOH). H NMR (400 MHz,

DMSO-d₆) d 1.38 (9H, s), 1.41 (3H, s), 1.42 (3H, s), 3.45

(3H, s), 3.52±3.81 (2H, m), 3.71 (3H, s), 4.32 (2H, s),

5.21 (2H, s), 5.24 (1H, d, J=4.9 Hz), 5.36 (2H, s), 5.72

(2H, s), 5.87±5.90 (1H, dd, J=3.3, 4.9 Hz), 6.71 (1H, s),

92

T. Kline et al. / Bioorg. Med. Chem. 8 (2000) 73±93

media was prepared at least 24 h in advance. Each assay

contained 4 dilutions of compound at ®nal concentra-

tions of 11, 33, 100, 300 mg/mL. Each sample contained

compound (resuspended in DMSO), DMSO to 3%

total volume, bacteria and LB in a ®nal volume of 1 mL.

After shaking overnight at 37 ^ꢂC, the OD₆₀₀was mea-

sured and compared to controls. Compounds were

scored as positive if cultures had OD600 values ꢀ0.5.

used to spot the plates. The MIC was de®ned as the

lowest drug concentration that inhibited bacterial

growth. Tobramycin was included in all assays for

quality control.

Acknowledgements

The authors would like to thank Dr. David Holub for

his assistance in puri®cation of several target com-

pounds. The insights and comments of Dr. David

Ryckman and Dr. Alice Erwin are also acknowledged.

We thank Sharon Steele, Julie Bukowski, and the mass

spectrometry facility at the University of Washington

for the mass spectra, and Kathie Shapiro, Jennifer Ring,

and Michelle Babcock for their assistance in preparing

the manuscript. The generous contributions from

Otsuka Chemical Company of the cephalosporin inter-

mediate 84, and from Takeda Chemical Company of

the gift of dihydroxyphenylglycine 14, are very much

appreciated.

Competition assay

Overnight cultures of P. aeruginosa strain PA01 were

diluted to an OD₄₉₂of 0.0003 in LB and compound 5

was added to a concentration of 6.25 mg/mL (approxi-

mately the MIC value). Candidate siderophore com-

pounds (in DMSO) were added to 1 mL aliquots to a

®nal concentration of 50 mg/mL. Cultures were grown

with shaking for 20 h at 37 ^ꢂin 96-well culture blocks

(AGTC, Gaithersburg, MD) and turbidities were

measured with an HT7000 Bio Assay Reader (Perkin±

Elmer, Foster City, CA). Compounds resulting in cul-

tures with OD492 <0.5 were considered negative; 0.5 to

0.7 were considered positive and above 0.7 were con-

sidered strongly positive.

References

Determination of MIC values

1. Meyer, J-M.; Stintzi, A. In Pseudomonas; Montie, J., Ed.;

Plenum Press: New York, 1998; pp 201±243.

Screening panel. For the screening panel, bacterial iso-

lates were cultivated from 70 ^ꢂfrozen stocks by 2

consecutive overnight passages (P1, P2) at 37 ^ꢂon 5%

blood agar (Remel, Lenexa, KS). The E. coli BR158

tonB mutant was received from Klaus Hantke, Tubin-

gen, Germany. The P. aeruginosa PAO1 was received

from Steven Lory, Seattle, WA. The P. aeruginosa PAO1

Cox ÁtonB was created at PathoGenesis for this study

by William Schwan. All other strains were obtained

from the American Type Culture Collection (ATCC;

Rockville, MD) and are designated S. aureus ATCC

29213, E. coli ATCC 25922 and P. aeruginosa ATCC

35151.

2. Earhart, C. F. In Escherichia coli and Salmonella, Cellular

and Molecular Biology; Neidhardt, F. C., Ed.; ASM Press:

Washington, D.C., 1996; Vol. I. pp 1075±1090.

3. Nielands, J. B. Ann. Rev. Micobiol. 1982, 36, 285.

4. van der Helm, D. Met. Ions Biol. Syst. 1998, 35, 355.

5. Nikaido, H. Drug Resistance Updates 1998, 1, 94.

6. Nikaido, H.; Rosenberg, E. Y. J. Bacteriol. 1990, 172, 1361.

7. Diarra, M. S.; Lavoie, M. C.; Jacques, M.; Darwish, I.;

Dolence, E. K.; Dolence, J. A.; Ghosh, A.; Ghosh, M.; Miller,

M. J.; Malouin, F. Antimicrob. Agents Chemother. 1996, 40,

2610.

8. Curtis, N. A. C.; Eisenstadt, R. L.; East, S. J.; Cornform, R.

J.; Walker, L. A.; White, A. J. Antimicrob. Agents Chemother.

1988, 32, 1879.

9. Yamano, Y.; Nishikawa, T.; Komatsu, Y. Appl. Micobiol.

Biotechol. 1994, 40, 892.

10. Minnick, A. A.; McKee, J. A.; Dolence, K.; Miller, M. J.

Antimicrob. Agents Chemother. 1992, 36, 840.

Compounds. All compounds for screening were provided

at a concentration of 2.5 mg/mL in DMSO (Sigma).

Any further dilutions were carried out in DMSO.

11. Brochu, A.; Brochu, N.; Nicas, T. I.; Parr, Jr. T. R.; Min-

nick, Jr., A. A.; Dolence, E. K.; McKee, J. A.; Miller, M. J.;

Lavoie, M. C.; Malouin, F. Antimicrob. Agents Chemother.

1992, 2166.

12. McKee, J. A.; Miller, M. J. Bioorg. Med. Chem. Lett. 1991,

1, 513.

13. Miller, M. J.; McKee, J. A.; Minnick, A. A.; Dolence, E.

K. Biol Metals. 1991, 4, 62.

14. Kinzel, O.; Tappe, R.; Gerius, I.; Budzikiewicz, H. J.

Antibiot. 1998, 51, 499.

Susceptibility testing broth microdilution. Broth micro-

dilution susceptibility testing was performed in accor-

dance with National Committee for Clinical Laboratory

Standards (NCCLS) standards by broth microdilu-

tion.⁶¹Bacterial inoculum (ꢃ5Â105 CFU/mL ®nal

concentration) was added to the cation-adjusted Muel-

ler Hinton (MH) broth (Remel), and microdilution

trays were incubated at 37 ^ꢂovernight. The MIC was

de®ned as the lowest drug concentration that inhibited

visible bacterial growth. Tobramycin was included in all

assays for quality control.

15. Tsuji, K.; Tubouchi, H.; Yasumur, K.; Matsumoto, M.;

Ishikawa, H. Bioorg. Med. Chem. 1996, 4, 2135.

16. Munoz-Bellido, J. L.; Garcõa-Rodriguez, J. A. Infectious

Disease Clin. Practice 1996, 5, 232.

17. Choi, K. I.; Cha, J. H.; Pae, A. N.; Cho, Y. S.; Kang, H-

Y.; Koh, H. Y.; Change, M. H. J. Antibiot. 1995, 48, 1375.

18. Shibihara, S.; Okonogi, T.; Yoshida, T.; Murai, Y.Kudo.;

Tosohaki, K.; Inouye, S.; Dondo, S. J. Antibiot. 1990, 43, 62.

19. Aplincourt, M.; Gerard, C.; Hugel, R. P.; Pierrard, J. C.;

Rimbault, J.; Bertrandie, A.; Magny, B.; Siret, P. J. Poly-

hedron 1992, 11, 1161.

Agar dilution. Agar dilution susceptibility testing was

performed in accordance with NCCLS standards by

agar dilution.⁶²Bacterial inoculum (ꢃ1Â104 CFU per

spot) was applied onto the MH agar (Remel), and the

plates were incubated at 37 ^ꢂC overnight. A Multipoint

Inoculator (MAST Group LTD., Merseyside, UK) was

T. Kline et al. / Bioorg. Med. Chem. 8 (2000) 73±93

93

20. Hashizume, T.; Sanada, M.; Nakagawa, S.; Tanaka, N. J.

Antibiot. 1990, 43, 1617.

43. Silley, P.; Griths, J. W.; Monsey, D.; Harris, A. M.

Antimicrob. Agents. Chemother. 1990, 34, 1806.

21. Arnould, J. C.; Bertrandie, A.; Bird, T. G. C.; Coucherot,

D.; Jung, F.; Lohmann, J. J.; Olivier, A.; Bailey, J. P.; Bell,

W.; Davies, G. M. J. Med. Chem 1992, 35, 2631. See also refs.

34±36.

22. Poras, H.; Kunesch, G.; Barriere, J-C.; Berthaud, N.;

Andremont, A. J. Antibiot. 1998, 51, 786.

44. Mochizuki, H.; Yajada, H.; Oikawa, Y.; Murakami, K.;

Ishiguro, J.; Kosuzume, H.; Aizawa, N.; Mochida, E. Anti-

microb. Agents. Chemother. 1988, 32, 1648.

45. Walker, D. G.; Brodhuehrer, P. R.; Brundidge, S. P.; Shih,

K. M.; Sapino, Jr., C. J. Org.Chem. 1988, 53, 983.

46. Arimoto, M.; Yokohama, S.; Sudou, M.; Ichikawa, Y.;

Hayano, T.; Tagawa, H.; Furukawa, M. J. Antibiot. 1988, 41,

1795.

23. Ghosh, A.; Miller, M. J.; DeClercq, E.; Balzarini, J.

Nucleosides & Nucleotides 1999, 18, 217.

24. Growth in low-iron medium: Ambrosi, H-D; Hartmann,

V.; Pistoris, D.; Reissbrodt, R.; Trowitzsch-Kienast, W. Eur.

J. Org. Chem. 1998, 541.

25. Rabsch, W.; Winkelman, G. Biol. Metals 1991, 4, 244±250.

26. Transport of ⁵⁹Fe: Thulasiraman, P.; Newton, S. M. C.;

Xu, J.; Raymond, K. N.; Mai, C.; Hall, A.; Montague, M. A.;

Klebba, P. J. Bacteriol. 1998, 180, 6689.

47. Brown, R. F.; Kinnick, M. D.; Morin, J. M., Jr.; Vasile,

R. T.; Counter, F. T.; Davidson, E. O.; Ensminer, P. W.;

Eudaly, J. A.; Kasher, J. S.; Katner, A. S.; Koehler, R. E.;

Kurz, K. D.; Lidstrom, T. D.; Lunn, W. H. W.; Preson, D. A.;

Ott, J. L.; Quay, J. F.; Shadle, J. K.; Steinerg, M. I.; Stucky, J. F.;

Swartzendruber, J. K.; Turner, J. R.; Webber, J. A.; Wright, W.

E.; Zimmerman, K. M. J. Med. Chem. 1990, 33, 2114.

48. Naito, T.; Aburaki, S.; Kamachi, H.; Narita, Y.; Oka-

mura, J.; Kawaguchi, H. J. Antibiot. 1986, 39, 1092.

49. Miyake, A.; Yoshimura, Y.; Yamaoka, M.; Nishimura, T.;

Hashimoto, N.; Imada, A. J. Antibiot. 1992, 45, 709 .

50. Zhao, Q.; Li, X-Z.; Mistry, A.; Srikurmar, R.; Zhang, L.;

Lomovskaya, O.; Poole, K. Antimicrob. Agents. Chemother.

1998, 42, 2225.

27. Zahner, H.; Hunter, R.; Bachman, E. Arch. Mikrobiol.

1960, 36, 325.

28. Mollmann, U.; Ghosh, A.; Dolence, E. K.; Dolence, J. A.;

Ghosh, A.; Miller, M. J.; Reissbrod, R. Biometals 1998, 11, 1.

29. Elliott, G. T.; Nagle, W. A.; Kelly, K. F.; McCollough, D.;

Bona, R. L.; Burns, E. R. J. Med. Chem. 1989, 32, 1039.

30. Bergeron, R. J.; Wiegand, J.; McManis, J. S.; McCosar, B.

H.; Weimar, W. R.; Brittenham, G. M.; Smith, R. E. J. Med.

Chem. 1999, 42, 2432.

51. Tsushima, M.; Iwamatsu, K.; Tamura, A.; Shibahara, S.

Bioorg. Med. Chem. 1998, 6, 1009±1017.

31. Richardson, D. R.; Tran, E. H.; Ponka, P. Blood 1995, 86,

4295 .

52. Ternansky, R. J.; Draheim, S. E.; Pike, A. J.; Bell, F. W.;

West, S. J.; Jordan, C. L.; Wu, C. Y. E.; Preseton, D. A.;

Alborn, Jr., W.; Kasher, J. S.; Hawkins, B. L. J. Med. Chem.

1992, 36, 1971.

53. Shinagawa, H.; Yamaga, H.; Houchigai, H.; Sumita, Y.;

Sunagawa, M. Bioorg. Med. Chem. 1997, 5, 601±621.

54. Still, W. C.; Kahn, M.; Milton, M. J. Org Chem. 1978, 43,

2923.

32. Stevens, M. F. G.; McCall, C. J.; Lelieveld, P.; Alexander,

P.; Richter, A; Davies, D. E. J. Med. Chem. 1994, 37, 1689.

33. Vikrishchuk, N. I.; Suzkalev, K. F.; Korol'chenko, G. A.;

Ryabukhin, Y. I. Khim.-Farm. Zh. 1995, 29, 30.

34. Moriarty, R. M.; Prakash, O.; Musallam, H. A. J. Hetero.

Chem. 1985, 22, 583.

35. Wheeler, T. S.; Rabjohn, H. Organic Syntheses; John

Wiley & Sons: New York, 1963; Collection Vol. IV, p 478.

36. Serino, L.; Reimmann, C.; Visca, P.; Beyeler, M.; Chiesa,

V. D.; Haas, D. J. Bacteriol. 1998, 179, 248.

37. Ankenbauer, R. G.; Toyokuni, T.; Staley, A.; Rinehart, K.

L.; Cox, C. D. J. Bacteriol. 1998, 170, 5344.

38. Floyd, D. M.; Fritz, A. W.; Pluscec, J.; Weaver, E. R.;

Cimarusti, C. M. J. Org. Chem. 1982, 47, 5160.

39. Koh, H. Y.; Choi, K. I.; Cho, Y. S.; Cha, J. H.; Pai, A. N.;

Kang, H-Y.; Change, M. H. Korean J. Med. Chem. 1996, 6,

333.

40. Kim, Y-Z.; Lim, J-C.; Yeo, J-H.; Bang, C-S.; Kim, S-S.;

Lee, T. H.; Oh, S. H.; Moon, Y-C.; Lee, C-S. J. Antibiot. 1996,

49, 496.

41. Kim, M. G.; Jung, J. C.; Sung, M. J.; Choi, Y. K.; An, S.

G.; Lee, S-J.; Yoon, G-J.; Park, M. H. Bioorg. Med. Chem

Lett. 1996, 6, 2077.

42. Arisawa, M.; Sekine, Y.; Shimizu, S.; Takano, H.;

Angehrn, P.; Then, R. L. Antimicrob. Agents Chemother. 1991,

35, 653.

55. Manickam, S.; Sandie, T.; Han-Li, S. Arch. Biochem. Bio-

phys. 1996, 329, 175.

56. Naito, T.; Aburaki, S.; Kamachi, H.; Narita, Y.; Oku-

mura, J.; Kawaguchi, H. J. Antibiot. 1986, 39, 1092.

57. Holton, T. H.; Shechter, H. J. Org. Chem. 1995, 60, 4725.

58. Schweizer, H. P.; Hoang, T. T. Gene 1995, 158, 15.

59. Schweizer, H. P. Mol. Microbiol. 1992, 6, 1195.

60. Schwan, W. R.; Coulter, S. N.; Ng, E. Y.; Langhorne, M. H.;

Ritchie, H. D.; Brody, L. L.; Westbrock-Wadman, S.; Bayer,

A. S.; Folger, K. R.; Stover, C. K. Infect. Immun. 1998, 66, 567.

61. National Committee for Clinical Laboratory Standards.

In Methods for Dilution Antimicrobial Susceptibility Tests for

Bacteria That Grow Aerobically; 4th Ed.; Villanova, Ed.;

Approved Standard Document M7-A4, 1997; Vol. 17, No. 2,

pp 10±15.

62. National Committee for Clinical Laboratory Standards.

In Methods for Dilution Antimicrobial Susceptibility Tests for

Bacteria That Grow Aerobically; 4th Ed.; Villanova, Ed.;

Approved Standard Document M7-A4, 1997; Vol. 17, No. 2,

pp. 6±10.

Article Doi

Antimicrobial effects of novel siderophores linked to β-lactam antibiotics

DOI: 10.1016/S0968-0896(99)00261-8

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