studies on anti-helicobacter pylori agents. Part 2: New cephem derivatives

Bioorganic & Medicinal Chemistry 8 (2000) 2317±2335

Studies on Anti-Helicobacter pylori Agents.

Part 2: New Cephem Derivatives

Yoshiki Yoshida, ^a,* Keiji Matsuda, ^aHiroshi Sasaki, ^aYoshimi Matsumoto, ^b

Satoru Matsumoto, ^bShuichi Tawara ^band Hisashi Takasugi ^a

^aMedicinal Chemistry Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., 2-1-6 Kashima, Yodogawa-ku, Osaka 532-8514, Japan

^bMedicinal Biology Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., 2-1-6 Kashima, Yodogawa-ku, Osaka 532-8514, Japan

Received 8 May 2000; accepted 6 June 2000

AbstractÐThe synthesis and optimization of the anti-Helicobacter pylori activity of a novel series of cephem derivatives are

described. Introduction of thio-heterocyclic groups containing N- and S-atoms to the 3-position and phenyl or thienyl acetamido

groups to the 7-position of the cephem nucleus dramatically improved the activity. From this series of derivatives, compound 13i

was found to have extremely potent in vitro anti-H. pylori activity, superior therapeutic ecacy compared to AMPC and CAM, no

cross-resistance between CAM or MNZ and low potential for causing diarrhea due to instability to b-lactamase. # 2000 Elsevier

Introduction

In a previous paper,¹⁷we reported a series of benzyloxy-

isoquinoline derivatives which show good in vitro anti-

H. pylori activity, but which failed to show therapeutic

ecacy, and a typical compound 1 is shown in Figure 1.

Therefore, as part of a further screening program of

various compounds, we were drawn to antibiotic com-

pounds, especially cephem derivatives; even though

generally it has been believed that the anti-H. pylori

activity of cephem derivatives is rather poor and weaker

than that of penicillin derivatives such as AMPC, we

found in our preliminary study that cephem derivatives

display high potential for anti-H. pylori activity. More-

over, they are fundamentally stable under acidic condi-

tions and are less susceptible to degradation in the

stomach compared to AMPC and, furthermore, much

information is available on mechanism, bactericidal

activity and toxicity. Furthermore, cephem compounds

containing a non-oxime structure at the 7-position have

low potential for causing diarrhea, a major side eect

of AMPC, due to low stability towards b-

lactamase,^{18 20}and thereby reduced potential for dis-

ruption of intestinal microbial ¯ora, due to rapid

deactivation by b-lactamase in intestine. Therefore,

using this design concept we searched for a compound

having a non-oxime structure at the 7-position for low

stability towards b-lactamase, along with potent anti-

H. pylori activity, and investigated the preparation of

novel cephem analogues substituted at the 3- and/or 7-

position of the cephem nucleus. In this paper, we

describe the synthesis and biological evaluation of

Since its discovery in the gastric mucosa of humans,¹the

clinical importance of eradication of Helicobacter pylori

(H. pylori) has increased signi®cantly^{2 6}due to its rela-

tionship to diseases such as chronic gastritis, peptic

ulcer, certain malignant peptic complications and the

like. To date only a small number of multi-drug therapy

regimens containing antibacterial agents, for example

amoxicillin (AMPC) and clarithromycin (CAM), with a

proton pump inhibitor such as omeprazole or lansopra-

zole, antiprotozoal agent or bismuth salt, are used for

eradication of H. pylori.⁷Although these therapies have

shown a reasonable response, they are not entirely suc-

cessful, and furthermore, there remain problems such as

drug resistance,^8,9side eects^10,11and non-compliance.¹²

However, whilst new anti-H. pylori agents have been

studied by many investigators,^{13 16}there are currently

no new anti-H. pylori compounds that show superior

therapeutic eradication ecacy to AMPC and CAM. As

a result, the need for alternative and novel structural

types is evident, and has stimulated the search for novel

agents that have potent therapeutic ecacy against H.

pylori and resolve the problems with current treatment.

*Corresponding author. Present address. Intellectual Property Division,

Fujisawa Pharmaceutical Co., Ltd., 2-1-6 Kashima, Yodogawa-ku,

Osaka 532-8514, Japan. Tel.: +81-6-6390-1226; fax: +81-6-6304-

1264; e-mail: yoshiki_yoshida@po.fujisawa.co.jp

PII: S0968-0896(00)00163-2

2318

Y. Yoshida et al. / Bioorg. Med. Chem. 8 (2000) 2317±2335

cephem compounds with excellent in vitro activity and

therapeutic ecacy.²¹

Chemistry

Cephem derivatives having various substituents at the 7-

position and 1,2,4-triazol-3-ylthio moiety at the 3-position

were synthesized by the typical route shown in Scheme 1.

Treatment of 3-mesylated compound 3 having a benz-

hydryl ester at the 4-position and formamido at the 7-

position with 3-mercapto-1,2,4-triazole using i-Pr₂NEt

as a base gave triazolylthio derivative 4, which was

deformylated with c-HCl to yield 7-amino compound 5.

Acylation of 5 with various activated carboxy deriva-

tives using PCl₅or the Vilsmeier method gave 7-acy-

lated compounds 6b±i. Deprotection of the benzhydryl

ester with TFA/anisole then aorded 7b±i in good yield.

In the case of a compound having a reactive amino

moiety at the 7-position (6a), the acylation was per-

formed using a formamido protected amino group. Sub-

sequently, after deformylation of 6a, deprotection of the

benzhydryl ester of 8 yielded 7a. Moreover, acylation of 5

with bromoacetyl bromide gave 6j and subsequent reac-

tion with 2-mercapto-5-methyl-1,3,4-thiadiazole gave 9;

deprotection of the benzhydryl ester then yielded 7j.

Figure 1. Anti-H. pylori agents.

Scheme 1. Reagents: (a) 3-mercapto-1,2,4-triazole, N,N-diisopropylethylamine (DIPEA), DMF; (b) c-HCl, MeOH±THF; (c) RCO₂H, PCl₅, MSA,

CH₂Cl₂±THF; (d) RCOX, MSA, THF; (e) RCO₂H, POCl₃, DMF, MSA, AcOEt±THF; (f) TFA, anisole, CH₂Cl₂; (g) t-BuOK, 2-mercapto-5-

methyl-1,3,4-thiadiazole, THF±DME.

Y. Yoshida et al. / Bioorg. Med. Chem. 8 (2000) 2317±2335

2319

Scheme 2. Reagents: (a) MsCl, K₂CO₃, DMF; (b) RSH, t-BuOK, THF±DME; (c) RSNa, THF±DME; (d) RSH, DIPEA, DMF; (e) TFA, anisole,

CH₂Cl₂; (f) KSCN, NaOCH₃, Br₂, MeOH, HCl; (g) HBr, Cu, NaNO₂, thiourea; (h) benzyl disul®de, n-BuLi; (i) Na, EtOH; (j) H₂NNH₂, CS₂,

MeOH, (k) pyridine.

Preparation of 7-phenylacetoamido derivatives having

various heterocyclic thio moieties at the 3-position was

performed according to the route shown in Scheme 2.

3-Mesylated compound 11 was obtained by mesylation

(MsCl±K₂CO₃/DMF, 30 ^ꢀC) of the corresponding 3-

hydroxy cephem derivative 10²²in good yield, while

mesylation using i-Pr₂NEt as a base did not aord good

results. Subsequent treatment of 11 with various hetero-

cyclic mercaptans or their salts using various methods

(such as t-BuOK or i-Pr₂NEt as a base and DMF or

THF±DME as a solvent) gave 12a±m, followed by

deprotection of the benzhydryl ester to aord 13a±m.

These mercapto derivatives, except for 16, 19 and 22,

were commercially available. Thioles 16, 19 and 22 were

prepared as shown in Scheme 2. Aminothiadiazole 15

was obtained by cyclization of amidine with potassium

thiocyanate and bromine. Treatment of 15 with HBr

and NaNO₂gave bromo derivative, followed by addi-

tion of thiourea to yield 5-mercapto-1,2,4-thiadiazole 16.

After coupling of 4-bromopyrazole 17 and benzyl disul-

®de using n-BuLi, reaction with Na aorded 4-mercap-

topyrazole 19. Treatment of AcOEt with hydrazine and

carbon disul®de gave potassium acetylhydrazino-

dithioate 21, which was cyclized using pyridine to yield

oxadiazole derivative 22.

ation of 12e,i using PCl₅/Py conditions. Treatment of

23a,b with activated carboxylic acids gave the corre-

sponding acylated compounds 24a±e, which were

deprotected to aord ®nal compounds 25a±e. In the case

of compounds having a reactive amino moiety at the 7-

position 24d,e, the acylation was performed using a

compound having a Boc or formyl protective group,

respectively. After acylation of 23a with d-2-(Boc-

amino)-2-(4-hydroxyphenyl)acetic acid, simultaneous

deprotection of the Boc and benzhydryl groups aorded

25d. 25e was prepared by a similar method to com-

pound 7a in Scheme 1. Next, concerning compounds

30a,b, after de-formylation of 7-formamido derivative

3, acylation with carboxylic acids using PCl₅aorded 3-

mesyl-7-acylated compounds 28a,b. Treatment of 28a,b

with heterocyclic mercaptans using i-Pr₂NEt or t-BuOK

as the base, respectively, followed by deprotection of the

resulting benzhydryl ester 29a,b, yielded 30a,b.

Compounds having dierent spacer moieties between

the cephem nucleus and the heterocyclic moiety 32, 35,

40 and 44 were prepared as shown in Scheme 4. Com-

pound 32 which has a natural type partial structure at

the 3-position was obtained by phenylacetylation of

31.²³Treatment of 3-mesylated compound 11 with

5-methylthiadiazol-2-ylmethyl thiobenzoate 33 using

NaOMe to hydrolyze the thioester gave 5-methylthia-

diazol-2-ylmethylthio derivative 34, which was depro-

tected at the benzhydryl group to aord 35 having the

opposite thiomethylene spacer part, compared to the

natural type 32. 2-Chloromethylthiothiazole 37, a starting

material for 40 having a thiomethylenethio spacer

Compounds 25a±e and 30a,b were prepared by the

routes shown in Scheme 3, though these compounds

could also be prepared by the same method as shown in

Scheme 1. Regarding preparation of 25a±e, 7-amino

compounds having a 1,3,4-thiadiazol-2-ylthio moiety at

the 3-position (23a,b) were obtained by de-phenylacetyl-

2320

Y. Yoshida et al. / Bioorg. Med. Chem. 8 (2000) 2317±2335

Scheme 3. Reagents: (a) PCl₅, Py, CH₂Cl₂; (b) PCl₅, MSA, CH₂Cl₂±THF; (c) DCC, CH₂Cl₂; (d) TFA, anisole, CH₂Cl₂; (e) c-HCl, MeOH±THF;

(f) DIPEA, DMF; (g) t-BuOK, THF±DME.

moiety, was obtained by reaction of bromo-

chloromethane 36 with 2-mercaptothiazole. Treatment

of mesylate 11 with NaSH gave 3-mercapto derivative

38; subsequent addition of 37 yielded 39, which was

deprotected at the benzhydryl group to yield 40. Com-

pound 42 was obtained by coupling reaction of 3-vinyl-

tosylate 41 with 2-mercapto-5-methylthiadiazole. After

deprotection of the Boc and benzhydryl protective

groups at the 7- and 4-positions, respectively, using

TFA/anisole conditions, acylation of 43 with phenyl-

acetyl chloride aorded 44 having a vinylthio spacer

moiety. Preparation of the cephamycin-type compound

49 is also shown in Scheme 4. Treatment of imino pro-

tected compound 45 with DDQ/MeOH gave 46 having

a methoxy group at the 7a-position, which was depro-

tected with Girard's Reagent T to yield 7-amino com-

pound 47. After acylation with phenylacetyl chloride,

deprotection aorded 7a-methoxy cephamycin-type

compound 49. Regarding compounds having a hydro-

xyimino structure at the 7-position (2, 2⁰), treatment of

3-mesylated compound 50²⁴with triazolylmercaptan

using i-Pr₂NEt as a base gave a mixture of 51 and its Á2

isomer 51⁰, after isolation, which were deprotected with

HCO₂H/c-HCl to yield 2 and 2⁰, respectively.

Results and Discussion

In the search for novel compounds with anti-H. pylori

activity, we initiated a random screening eort and

found in our preliminary study that cephem derivatives

display some potential for anti-H. pylori activity, even

though generally it has been believed that the anti-H.

pylori activity of cephem derivatives is rather poor and

inferior to penicillin derivatives. Subsequent intentional

screening of the Fujisawa cephem library uncovered

compound 2 having a 1,2,4-triazol-3-ylthio moiety at

the 3-position of the cephem nucleus, which displayed

good anti-H. pylori activity. Its activity was not only

superior to that of CFDN²⁵and FK041²⁴having dif-

ferent partial structures at the 3-position and the same

structure at the 7-position, but also was good compared

Y. Yoshida et al. / Bioorg. Med. Chem. 8 (2000) 2317±2335

2321

Scheme 4. Reagents: (a) PhCH₂COCl, Et₃N, acetone±H₂O; (b) NaOMe, MeOH±THF±DMF; (c) TFA, anisole, CH₂Cl₂; (d) t-BoOK, DMF;

(e) NaSH, DIPEA, DMF; (f) NaI, DIPEA, DMF; (g) 2-mercapto-5-methyl-1,3,4-thiadiazole, DIPEA, DMF; (h) PhCH₂COCl, MSA, THF;

(i) DDQ, MeOH; (j) Girard T, AcOEt±MeOH; (k) 3-mercapto-1,2,4-triazole, DIPEA, DMF; (l) HCO₂H, c-HCl.

with AMPC. The Á2 isomer 2⁰had much weaker activ-

ity, as is the case generally for antibacterial cephalo-

sporins. Consequently, we adopted compound 2 with a

characteristic heterocyclic thio moiety at the 3-position

as a seed compound in order to investigate the potency

of cephem compounds. We ®rst prepared and evaluated

the anti-H. pylori activity of a series of analogues.

First, we investigated various 7-acylated compounds

having the 1,2,4-triazol-3-ylthio moiety at the 3-position

of cephem nucleus. Table 1 shows the minimum inhibitory

concentration values (MIC, mg/mL) against three strains

of H. pylori including the somewhat insensitive strain H.

pylori 9005. First of all, we evaluated compound 7a hav-

ing no hydroxyimino group at the 7-position, which thus

2322

Y. Yoshida et al. / Bioorg. Med. Chem. 8 (2000) 2317±2335

Table 1. Structure±activity relationships (1): 7-position optimization

novel derivatives having a non-oxime structure at the 7-

position. Regarding other heterocyclic groups, the 1,2,5-

thiadiazolyl compound 7b did not give improved activity

especially against H. pylori 9005 and 2-thienyl compound

7c, which is rather lipophilic, showed slightly improved

activity. On the other hand, benzyl derivative 13a showed

good activity, in particular against H. pylori 9005. A ben-

zamide derivative at the 7-position (7d) showed greatly

decreased activity, and phenoxyacetamide derivative 7e

gave poor activity compared to the seed compound 2.

On the other hand, a compound having the phenyl-

thioacetamide partial structure (30a) showed the same

activity compared with seed compound 2. Thus we exam-

ined compounds having other heterocyclic moieties in

place of the phenylthio ring at the 7-position; however,

compound 7j did not show increased activity. Concerning

non-cyclic derivatives at the 7-position, while 7g showed

decreased activity, cyanovinylthio derivative 7f had good

and nearly equipotent activity with 13a. We also exam-

ined substituents on the phenyl ring, and as a result, 3-

chlorophenyl compound 7h showed the same activity as

13a, but the 3-nitro derivative 7i gave decreased activity.

MIC (mg/mL)^a

Compound no.

R₁

R₂

Helicobacter pylori

9005 13001 FP1757

CFDN

0.2

0.1

0.39

0.1

0.2

0.1

FK041

2

0.00625 0.00625

2⁰(Á2 isomer)

3.13 0.78

0.78

Next, we examined various 3-heterocyclic thio derivatives

having a phenylacetamido moiety as the best partial

structure at the 7-position (Table 2). Regarding only N-

atom-containing heterocyclic compounds, 1,2,3-triazol-

4-yl derivative 13b showed improved activity against all

strains while pyrazole and tetrazole derivatives (13c,d)

did not give improved activity. Subsequently, we intro-

duced heterocyclic compounds containing N- and S-

atoms (13e-i), and were surprised to ®nd that all thia-

diazole and thiazole derivatives had remarkably potent

anti-H. pylori activity, especially against H. pylori 9005,

which is a somewhat insensitive strain even with 13a

(MIC; 0.05 mg/mL). The in vitro activity of these com-

pounds was superior to that of the reference com-

pounds: 10-fold improved compared to AMPC and

about 50-fold relative to CAM, against all strains tested.

On the other hand, N- and O-atom-containing hetero-

cyclic compounds, for example oxadiazole derivative

13j and thiophene derivative 13k, showed dramatically

decreased activity. A pyridine derivative 13l gave some-

what decreased activity compared with 13e. We also

examined a fused ring compound (13m); however, even

though this fused cyclic compound had a thiazole partial

ring structure, it showed poor activity. Therefore, these

results showed that the anti-H. pylori activity of this

type of compound is fairly speci®c for mono-hetero-

cyclic rings containing N- and S-atoms.

7a

7b

7c

13a

7d

7e

30a

7j

0.05 0.00156 0.00313

0.39 0.00156 0.00313

0.05 0.00156 0.00156

0.025 0.00313 0.00156

3.13 0.1

0.78

0.2

0.1

0.2

0.0125 0.025

0.00625 0.00625

0.00313 0.00313

7f

0.025 0.00313 0.00313

7g

7h

7i

3.13 0.39

0.39

0.025 0.00313 0.00156

0.1

0.00625 0.00625

Subsequently, we investigated the spacer group between

the cephem nucleus and the heterocyclic moiety at the

3-position, a cephamycin type derivative, and com-

pounds having other partial structures at the 7-position,

together with a 1,3,4-thiadiazol-2-ylthio group at the 3-

position (Table 3). Regarding spacer group, we eval-

uated several compounds having dierent spacer struc-

tures between the cephem nucleus and the 5-methyl-

1,3,4-thiadiazol-2-yl or 2-thiazolyl moieties as typical

heterocyclic groups. All prepared compounds (methyl-

ene-S 32, S-methylene 35, S-methylene-S 40, vinyl-S 44)

gave decreased activity, though S-methylene derivative

AMPC

CAM

0.05 0.025

0.2 0.05

0.0125

0.1

^aMIC (mg/mL), brucella agar+7% horse blood, 37 ^ꢀC, 72 h, 10%

CO₂, stamp method.

has low potential for causing diarrhea, a major side eect

of AMPC, due to low stability towards b-lactamase.

Encouragingly, 7a showed increased anti-H. pylori

activity compared to 2, which has a hydroxyimino

moiety at the 7-position, and we thus decided to prepare

Y. Yoshida et al. / Bioorg. Med. Chem. 8 (2000) 2317±2335

2323

Table 2. Structure±activity relationships (2): 3-position optimization

expected, these compounds showed excellent in vitro

anti-H. pylori activity against all strains tested.

In the next phase, we examined the therapeutic eect of

typical compounds in a mouse infection model (Table

4). A dosage regimen of two times per day for 4 days at

0.32 and 0.1 mg/kg, in H. pylori FP1757 infected mouse,

followed by assessment of eradication was employed. It

was noted that nearly all compounds tested showed ther-

apeutic ecacy in parallel with the in vitro activity.

Though compound 13b showed an eradication eect at a

dosage of 0.32mg/kg in ®ve out of seven mice, 25e showed

eradication in only one out of eight mice, and this eradi-

cation eect of 25e was the same as AMPC. Compound

25a having the most potent in vitro activity showed a good

eradication eect at a dosage of 0.1 mg/kg in 6/8 mice but

did not show complete eradication even at a dosage of

0.32 mg/kg, suggesting 25a may have unfavorable prop-

erties such as poor solubility or stability. Though 30b

and 13g showed decreased therapeutic eects compared

with 25a, 13h showed excellent eect at a dosage of

0.32 mg/kg, but the eect did not extend to the dosage

of 0.1 mg/kg. Compound 13i showed superior thera-

peutic eect, re¯ecting the in vitro activity, yet 13i did

not necessarily give the best in vitro activity against the

test strain. This compound showed eradication in all

mice at a dosage of 0.32 mg/kg and in 5 out of 8 mice at

a dosage of 0.1 mg/kg. This eect is superior to that of

other test compounds and reference compounds. These

results show that there are many unknown factors

concerning therapeutic eect, namely permeation into

mucosa, stability in mucosa, oral absorption and the like.

MIC (mg/mL)^a

Helicobacter pylori

Compound no.

R₂

9005

13001

FP1757

13a

13b

13c

13d

13e

13f

13g

13h

13i

0.05

0.00313

0.00156

0.0125

0.05

0.00156

0.00078

0.0125

0.00078

0.00156

0.1

0.0125

0.05

0.00625

0.00313

0.00156

0.00625

0.78

0.00313

0.00078

0.00156

0.00078

0.1

13j

Additionally, we examined the time-dependence of the

bactericidal activity (Fig. 2), because in a previous study

we uncovered benzyloxyisoquinoline derivatives which

show excellent in vitro anti-H. pylori activity, but no

therapeutic ecacy.¹⁷With these compounds, we con-

cluded that the poor bactericidal activity at early contact

times may be the cause of poor in vivo eect. From the

data shown in Figure 2, 13i showed good bactericidal

activity at 6 h even at 1MIC concentration and the

bactericidal activity was slightly superior to AMPC,

although benzyloxyisoquinoline derivative 1 expresses

poor bactericidal activity at 6 h even at 16MIC. Conse-

quently, the importance of bactericidal activity at early

contact times was suggested more clearly.

13k

13l

0.39

0.2

0.025

0.00313

0.025

0.00313

0.0125

13m

0.05

^aMIC (mg/mL), brucella agar+7% horse blood, 37 ^ꢀC, 72 h, 10%

CO₂, stamp method.

35 was better than the other compounds. From these

data it is clear that the anti-H. pylori activity of this

class of compounds is speci®c in connection with the

thioheterocyclic structure at the 3-position of the

cephem nucleus. Moreover, cephamycin type derivative

49 which was expected to have good activity was infer-

ior to compound 13i. Next, we prepared compounds

having other partial structures at the 7-position together

with the 1,3,4-thiadiazol-2-ylthio group at the 3-position.

Thienylvinyl derivative 25b showed moderate activity

against H. pylori 13001 and FP1757, although it was

reasonable against H. pylori 9005, and tetrazolylmethyl

compound 25c had reasonable activity. Compound

25d, having a d-2-amino-2-(4-hydroxyphenyl)acetamido

moiety which is the 6-position structure of AMPC,

showed dramatically decreased activity. We also esti-

mated various combination compounds having 3- and

7-partial structures which had displayed good activity

in the earlier compounds (Table 4, 25a,e, 30b). As

Furthermore, we estimated the in vitro eect against

CAM and metronidazole (MNZ) resistant strains (Table

5). 13i showed excellent activity against H. pylori 16021

and 16043, which are highly resistant strains in the clinical

environment, and there was no cross-resistance with

CAM and MNZ.

In the next phase, we evaluated stability towards b-lacta-

mase of 13i in order to estimate the potential for causing

diarrhea, a major side eect of AMPC (Table 5). The

relative values of 13i and AMPC against cephaloridine

(CER), a typical unstable drug towards cephalosporinase,

are shown. 13i was 2-fold more unstable than CER

towards B. fragilis FP784 (cephalosporinase) and 40-fold

more unstable than AMPC. Towards TEM (penicillinase)

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Y. Yoshida et al. / Bioorg. Med. Chem. 8 (2000) 2317±2335

Table 3. Structure±activity relationships (3): spacer optimization and other derivatives

MIC (mg/mL)^a

Helicobacter pylori

13001

Compound no.

R₁

R₂

R₃

9005

0.39

FP1757

0.05

32

H

0.1

35

0.05

0.025

0.2

0.00625

0.05

40

H

0.1

44

H

0.025

0.00625

0.05

0.78

0.39

13i

49

H

0.00078

0.0125

0.05

0.00156

0.0125

0.05

OCH₃

H

25b

25c

25d

0.0125

0.05

H

0.00156

0.39

0.00156

0.39

H

0.39

^aMIC (mg/mL), brucella agar+7% horse blood, 37 ^ꢀC, 72 h, 10% CO₂, stamp method.

Table 4. Structure±activity relationships: therapeutic ecacy

MIC (mg/mL)^a

Therapeutic ecacy^b

(eradication ratio)

Compound no.

R₁

R₂

Helicobacter pylori

Dose (mg/kg)

9005

13001

FP1757

0.00078

0.1

0/8

0.32

13b

25e

25a

30b

13g

13h

0.0125

0.00156

0.00039

0.00078

0.00313

0.00078

0.00156

5/7

1/8

6/8

3/5

5/8

8/8

0.00313

0.00156

0.00625

0.00156

0.00625

0.00078

0.00039

0.00078

0.00156

0/7

6/8

1/5

0/8

1/8

13i

0.00625

0.05

0.00078

0.025

0.05

0.00156

0.0125

0.1

5/8

0/8

8/8

1/8

0/8

AMPC

CAM

0.2

NT^c

^aMIC (mg/mL), brucella agar+7% horse blood, 37 ^ꢀC, 72 h, 10% CO₂, stamp method.

^bMouse, PO, infection; H. pylori FP1757, therapy; 2/dayÂ4 days, termination; 2 weeks after ®nal therapy.

^cNT=not tested.

Y. Yoshida et al. / Bioorg. Med. Chem. 8 (2000) 2317±2335

2325

Figure 2. Bactericidal activity against H. pylori FP1757.

Table 5. MIC against clarithromycin- and metronidazole-resistant strains and stability towards b-lactamase

MIC (mg/mL)

Stability towards b-lactamase^b

Compound

Helicobacter pylori

B. fraglis FP784

TEM

V_max

16021^a

16043^a

K_m(mg/ml)

V_max

V_max/K_m

K_m(mg/ml)

V_max/K_m

c

13i

0.0016

0.025

50

NT

0.0016

0.05

50

NT

14.5

27.3

NT^d

NT

0.351

0.0175

NT

1.0

1.98

0.0521

NT

1.0

47.4

22.0

NT

276

3.61

2.94

NT

1.0

6.33

11.1

NT

1.0

AMPC

CAM

MNZ

CER

31.7

^aClarithromycin- and metronidazole-resistant strain; underlined values indicate resistant strains.

^bB. fragilis FP784, cephalosporinnase, TEM; penicillinase.

^cRelative value (CER=1.0), V_max/K_m; larger values mean more unstable.

^dNT=not tested.

13i was 6-fold more unstable than CER though only

2-fold more stable compared to AMPC. From this result,

13i showed considerable lability towards b-lactamase

including cephalosporinase and penicillinase, so it was

expected that this compound has low potential for

causing diarrhea due to reduced potential for disruption

of intestinal microbial ¯ora.

as in vitro activity, and a study of factors that in¯uence

therapeutic ecacy.

Experimental

Melting points were determined using a Thomas-Hoover

capillary melting apparatus and are uncorrected. IR

spectra were recorded on a Horiba Spectradesk FT-210

spectrometer as KBr disks or Nujol as indicated. NMR

spectra were measured on a Bruker AC200P (¹H,

200 MHz) and chemical shifts are expressed in d ppm

with TMS as internal standard. Mass spectra were mea-

sured on a Hitachi M-1000H mass spectrometer (APCI)

or a Finnigan MAT TSQ-70 (FAB). Elemental analyses

were carried out on a Perkin Elmer 2400 CHN Ele-

mental Analyzer. Reagents and solvents were used as

obtained from commercial suppliers without further

puri®cation. Column chromatography was performed

using silica-gel or non-ionic adsorption resin, Diaion

HP-20, and reaction progress was determined by TLC

analysis on silica-gel coated glass plates. Visualization

was with UV light (254 nm) or iodine.

Conclusion

In summary, we have prepared a novel series of cephem

derivatives and evaluated them as anti-H. pylori com-

pounds. The SAR study in this series of compounds

revealed the following main features. (1) Concerning the

3-position, various thio-heterocyclic groups showed

extremely potent activity, and especially mono-hetero-

cyclic compounds containing N- and S-atoms gave

excellent activity. (2) Regarding the 7-position groups,

phenyl or thienyl acetamido groups showed good results

and the presence of an oxime part gave decreased

activity. (3) Compounds having good in vitro activity

showed clear therapeutic ecacy, and 13i containing a

(5-methyl-1,3,4-thiadiazol-2-yl)thio moiety at the 3-

position and a phenylacetamido group at the 7-position

gave the best ecacy, superior to AMPC and CAM. (4)

There was no cross-resistance between 13i and CAM or

MNZ. (5) 13i has low potential for causing diarrhea due to

instability to b-lactamase. Consequently, 13i (FR182024)

having excellent therapeutic ecacy was selected as a can-

didate compound for further development. Future pub-

lications will report optimized therapeutic ecacy as well

Benzhydryl 7ꢀ-formamido-3-(1,2,4-triazol-3-yl)thio-3-ce-

phem-4-carboxylate (4). To a solution of 3-mercapto-

1,2,4-triazole (6.76 g, 66.6mmol) in DMF (200mL) was

added N,N-diisopropylethylamine (DIPEA, 6.62g, 51.2

mmol) and the mixture was stirred at room temperature

for one hour. To a solution of benzhydryl 7b-formamido-

3-methanesulfonyloxy-3-cephem-4-carboxylate (25.0 g,

51.2 mmol) in DMF (100 mL) was added the thiol

2326

Y. Yoshida et al. / Bioorg. Med. Chem. 8 (2000) 2317±2335

solution obtained above at 30 ^ꢀC. After stirring under

ice-cooling for 4 h, the reaction mixture was poured into

a mixture of AcOEt and water, and the mixture was

adjusted to pH 5.0 with 1N HCl. The separated organic

layer was washed with brine (3Â), dried over magne-

sium sulfate and evaporated under reduced pressure.

The residue was chromatographed over silica-gel (elu-

2.71 (s, 3H), 3.67 and 3.98 (ABq, 2H, J=17.6 Hz), 5.17

(d, 1H, J=4.9 Hz), 5.57 (dd, 1H, J=4.9, 9.0 Hz), 6.96 (s,

1H), 7.01 (d, 1H, J=15.6 Hz), 7.25 (d, 1H, J=15.6 Hz),

7.3±7.6 (m, 10H), 8.08 (d, 1H, J=9.0 Hz).

(Z)-Benzhydryl 7ꢀ-[2-(2-aminothiazol-4-yl)-2-trityloxy-

iminoacetoamido]-3-(1,2,4-triazol-3-yl)thio-3-cephem-4-

carboxylate (51). Yield 13%; IR (KBr) 1788, 1685,

1531cm ¹; ¹H NMR (DMSO-d₆) d 3.47 (s, 2H), 5.38 (d,

1H, J=5.0 Hz), 5.99 (dd, 1H, J=5.0, 8.4 Hz), 6.63 (s, 1H),

6.96 (s, 1H), 7.2±7.6 (m, 27H), 8.75 (s, 1H), 9.90 (d, 1H,

J=8.4 Hz), 14.6 (brs, 1H).

1

ent AcOEt:hexane, 2:1) to give 13.2 g of 4 (52%): H

NMR (CDCl₃) d 2.92 (d, 2H, J=15.3 Hz), 4.94 (d, 1H,

J=4.9 Hz), 5.88 (dd, 1H, J=4.9, 9.0 Hz), 6.93 (s, 1H),

7.1±7.5 (m, 11H), 8.19 (s, 1H); APCI-MS m/z 494

(MH⁺).

Preparation of 12c, 12f, 12j±k, 12m, 29a, 42, 51 and 51⁰

was carried out by a similar method to that described

for 4.

(Z)-Benzhydryl 7ꢀ-[2-(2-aminothiazol-4-yl)-2-trityloxy-

iminoacetoamido]-3-(1,2,4-triazol-3-yl)thio-3-cephem-4-

carboxylate D2-isomer (51⁰). Yield 14%; IR (KBr) 1784,

1

1680, 1533 cm ¹; H NMR (DMSO-d₆) d 5.29 (d, 1H,

Benzhydryl 7ꢀ-(2-phenylacetamido)-3-(pyrazol-4-yl)thio-

3-cephem-4-carboxylate (12c). Yield 45%; ¹H NMR

(DMSO-d₆) d 3.13 (d, 2H, J=4.0 Hz), 3.53 (d, 2H,

J=2.6 Hz), 5.12 (d, 1H, J=4.6 Hz), 5.65 (dd, 1H, J=4.6,

8.3 Hz), 6.92 (s, 1H), 7.1±7.7 (m, 16H), 8.06 (s, 1H), 9.09

(d, 1H, J=8.3 Hz), 13.39 (s, 1H).

J=5.0 Hz), 5.55 (s, 1H), 5.83 (dd, 1H, J=5.0, 8.4 Hz),

6.68 (s, 1H), 6.84 (s, 1H), 7.2±7.5 (m, 27H), 8.64 (s, 1H),

10.01 (d, 1H, J=8.4 Hz), 14.3 (brs, 1H).

Benzhydryl 7ꢀ-amino-3-(1,2,4-triazol-3-yl)thio-3-cephem-

4-carboxylate (5). To a solution of 4 (13.1 g, 0.30 mol)

in a mixture of MeOH (65 mL) and THF (10 mL) was

added concd HCl (11 mL). After stirring at room tem-

perature for 1.5 h, the reaction mixture was poured into

a mixture of AcOEt and water, and the mixture was

adjusted to pH 6.5 with an aqueous sodium hydrogen car-

bonate solution. The separated organic layer was washed

with brine, dried over magnesium sulfate and evaporated

under reduced pressure. The residue was chromatographed

over silica-gel (eluent AcOEt) to give 1.79g of 5 (15%): ¹H

NMR (CDCl₃) d 3.44 and 3.78 (ABq, 2H, J=17.9 Hz),

4.73 (d, 1H, J=5.0 Hz), 4.91 (d, 1H, J=5.0 Hz), 6.96 (s,

1H), 7.1±7.5 (m, 10H), 8.14 (s, 1H).

Benzhydryl 7ꢀ-(2-phenylacetamido)-3-(1,2,4-thiadiazol-

1

5-yl)thio-3-cephem-4-carboxylate (12f). Yield 56%; H

NMR (CDCl₃) d 3.65 (d, 2H, J=2.7 Hz), 3.47 and 3.85

(ABq, 2H, J=18.1Hz), 5.08 (d, 1H, J=5.1 Hz), 5.93 (dd,

1H, J=5.1, 9.0 Hz), 6.15 (d, 1H, J=9.0 Hz), 6.97 (s, 1H),

7.1±7.5 (m, 15H), 8.48 (s, 1H); FAB±MS m/z 601.1

(MH⁺).

Benzhydryl 7ꢀ-(2-phenylacetamido)-3-(5-methyl-1,3,4-oxa-

diazol-2-yl)thio-3-cephem-4-carboxylate (12j). Yield 49%;

¹H NMR (DMSO-d₆) d 3.32 (s, 3H), 3.54 (s, 2H), 3.76 and

3.99 (ABq, 2H, J=18.1 Hz), 5.26 (d, 1H, J=4.9 Hz), 5.82

(dd, 1H, J=4.9, 8.4 Hz), 6.92 (s, 1H), 7.2±7.6 (m, 15H),

9.21 (d, 1H, J=8.4 Hz).

Preparation of 26 and 27 was carried out by a similar

method to that described for 5.

Benzhydryl 7ꢀ-(2-phenylacetamido)-3-(thiophen-2-yl)thio-

3-cephem-4-carboxylate (12k). Yield 61%; ¹H NMR

(DMSO-d₆) d 3.32 (s, 2H), 3.53 (d, 2H, J=2.7 Hz), 5.18

(d, 1H, J=4.7 Hz), 5.70 (dd, 1H, J=4.7, 8.3 Hz), 6.95 (s,

1H), 7.1±7.6 (m, 17H), 7.88 (d, 1H, J=4.1 Hz), 9.12 (d,

1H, J=8.3 Hz).

Benzhydryl 7ꢀ-[2-(2-aminothiazol-4-yl)acetamido]-3-(5-

methyl-1,3,4-thiadiazol-2-yl)thio-3-cephem-4-carboxylate

(26). Yield 69%; H NMR (DMSO-d₆) d 2.68 (s, 3H),

3.38 (s, 2H), 3.56 and 3.79 (ABq, 2H, J=17.8 Hz), 5.27

(d, 1H, J=5.0 Hz), 5.87 (dd, 1H, J=5.0, 8.5 Hz), 6.25 (s,

1H), 6.88 (s, 2H), 6.97 (s, 1H), 7.2±7.5 (m, 10H), 9.05 (d,

1H, J=8.5 Hz).

1

Benzhydryl 7ꢀ-(2-phenylacetamido)-3-(benzothiazol-2-yl)-

thio-3-cephem-4-carboxylate (12m). Yield 70%; ¹H NMR

(DMSO-d₆) d 3.55 (d, 2H, J=4.1 Hz), 3.75 and 4.02 (ABq,

2H, J=17.9 Hz), 5.33 (d, 1H, J=5.1 Hz), 5.89 (dd, 1H,

J=5.1, 8.3 Hz), 6.96 (s, 1H), 7.1±8.1 (m, 19H), 9.31 (d,

1H, J=8.3 Hz).

Benzhydryl 7ꢀ-amino-3-methanesulfonyloxy-3-cephem-4-

1

carboxylate (27). Yield 27%; H NMR (CDCl₃) d 2.73

(s, 3H), 3.70 and 3.89 (ABq, 2H, J=18.5 Hz), 4.79 (d,

1H, J=5.1 Hz), 5.01 (d, 1H, J=5.1 Hz), 6.97 (s, 1H),

7.2±7.5 (m, 10H).

Benzhydryl 7ꢀ-[2-(phenythio)acetamido]-3-(1,2,4-triazol-

1

Benzhydryl 7ꢀ-[2-(2-aminothiazol-4-yl)acetamido]-3-(1,2,

4-triazol-3-yl)thio-3-cephem-4-carboxylate (8). To a solu-

tion of phosphorus pentachloride (738 mg, 3.54 mmol)

in CH₂Cl₂(8 mL) was added (2-formylaminothiazol-4-

yl)acetic acid (601 mg, 3.23 mmol) at 15 ^ꢀC and the

mixture was stirred under ice-cooling for 30 minutes. To

a solution of 5 (1.50 g, 3.23 mmol) in THF (23 mL) was

added N-trimethylsilylacetamide (1.27 g, 9.67 mmol)

under ice-cooling, and the mixture was stirred at the

same temperature for 30 min. Thereto was dropwise

3-yl)thio-3-cephem-4-carboxylate (29a). Yield 39%; H

NMR (CDCl₃) d 3.39 and 3.76 (ABq, 2H, J=18.0 Hz),

3.59 (d, 2H, J=4.4 Hz), 4.94 (d, 1H, J=4.9 Hz), 5.81 (dd,

1H, J=4.9, 9.0 Hz), 6.92 (s, 1H), 7.1±7.4 (m, 15H), 7.68

(d, 1H, J=9.0 Hz), 8.12 (s, 1H).

(E)-Benzhydryl 7ꢀ-t-butoxycarbonylamino-3-[2-(5-methyl-

1,3,4-thiadiazol-2-yl)thio]vinyl-3-cephem-4-carboxylate

1

(42). Yield 42%; H NMR (DMSO-d₆) d 1.41 (s, 9H),

Y. Yoshida et al. / Bioorg. Med. Chem. 8 (2000) 2317±2335

2327

added the acid chloride solution obtained above under

ice-cooling and the mixture was stirred at the same

temperature for one hour. The reaction mixture was

poured into a mixture of aqueous sodium hydrogen car-

bonate solution and AcOEt with stirring. The separated

organic layer was washed with brine, dried over mag-

nesium sulfate and evaporated under reduced pressure.

The residue (crude 6a) was dissolved in a mixture of

MeOH (10 mL) and THF (2 mL) and concd HCl (1.3 mL)

was added to the solution. After stirring at room tem-

perature for 1.5 h, the reaction mixture was poured into a

mixture of AcOEt and water, and the mixture was adjus-

ted to pH 6.5 with an aqueous sodium hydrogen carbon-

ate solution. The separated organic layer was washed with

brine, dried over magnesium sulfate and evaporated under

reduced pressure. The residue was chromatographed

over silica-gel (eluent AcOEt) to give 639 mg of 8 (33%):

¹H NMR (DMSO-d₆) d 3.34 (s, 2H), 3.47 and 3.77 (ABq,

2H, J=17.3 Hz), 5.24 (d, 1H, J=4.8 Hz), 5.77 (dd, 1H,

J=4.8, 8.6 Hz), 6.25 (s, 1H), 6.8±7.6 (m, 12H), 8.73 (brs,

1H), 8.98 (d, 1H, J=8.6 Hz).

J=4.8, 8.2 Hz), 8.72 (brs, 1H), 8.75 (s, 1H), 9.31 (d,

1H, J=8.2 Hz), 14.5 (brs, 1H); FAB±MS m/z 425.9

(MH⁺).

Preparation of 7c and 7i was carried out by a similar

method to that described for 7b.

7ꢀ-(2-Thienyl)acetamido-3-(1,2,4-triazol-3-yl)thio-3-ce-

phem-4-carboxylic acid (7c). Yield 53%; ¹H NMR

(DMSO-d₆) d 3.45 and 3.79 (ABq, 2H, J=17.3 Hz),

3.75 (s, 1H), 5.18 (d, 1H, J=4.8 Hz), 5.67 (dd, 1H,

J=4.8, 8.3 Hz), 6.90±6.96 (m, 2H), 7.35 (dd, 1H, J=1.6,

4.8 Hz), 8.67 (brs, 1H), 9.15 (d, 1H, J=8.3 Hz), 14.4 (brs,

1H).

7ꢀ-[2-(3-Nitrophenyl)acetamido]-3-(1,2,4-triazol-3-yl)-

thio-3-cephem-4-carboxylic acid (7i). Yield 40%; ¹H

NMR (DMSO-d₆) d 3.44 and 3.79 (ABq, 2H, J=17.2 Hz),

3.72 (s, 2H), 5.17 (d, 1H, J=4.8 Hz), 5.66 (dd, 1H, J=4.8,

8.2 Hz), 7.55±7.75 (m, 2H), 8.05±8.20 (m, 2H), 8.70 (brs,

1H), 9.26 (d, 1H, J=8.2 Hz), 14.5 (brs, 1H); FAB±MS m/z

463.0 (MH⁺).

7ꢀ-[2-(1,2,5-Thiadiazol-3-yl)acetamido]-3-(1,2,4-triazol-

3-yl)thio-3-cephem-4-carboxylic acid (7b). To a solution

of phosphorus pentachloride (738 mg, 3.54 mmol) in a

mixture of CH₂Cl₂(8 mL) and THF (4 mL) was added

(1,2,5-thiadiazol-3-yl)acetic acid (465 mg, 3.23 mmol) at

15 ^ꢀC and the mixture was stirred under ice-cooling

for 30 min. To a solution of 5 (1.5 g, 3.23 mmol) in THF

(23 mL) was added N-trimethylsilylacetamide (1.27 g,

9.67 mmol) under ice-cooling, and the mixture was

stirred at the same temperature for 30 min. Thereto was

dropwise added the acid chloride solution obtained

above under ice-cooling and the mixture was stirred at

the same temperature for 1 h. The reaction mixture was

poured into a mixture of aqueous sodium hydrogen

carbonate solution and AcOEt with stirring. The

separated organic layer was washed with brine, dried

over magnesium sulfate and evaporated under reduced

pressure. To a solution of the residue (crude 6b) in a

mixture of CH₂Cl₂(6 mL) and anisole (2 mL) was added

tri¯uoroacetic acid (4 mL) with stirring under ice-cool-

ing. The mixture was stirred at the same temperature

for one hour. The reaction mixture was poured into

diisopropyl ether (IPE, 120 mL) and the resulting

precipitate was collected by ®ltration and dried in

vacuo. The precipitate was dissolved in a mixture of

a sodium hydrogen carbonate solution and THF,

adjusted to pH 5.0 with 1N HCl with stirring and

evaporated under reduced pressure to remove the

organic solvent. The resulting solution was subjected to

column chromatography on Diaion HP-20 (eluent 20%

aqueous isopropyl alcohol). The desired fractions were

evaporated under reduced pressure to remove the

organic solvent and the residue was adjusted to pH 2.0

with 1N HCl and extracted twice with a mixture of

THF and AcOEt. The organic layer was dried over

magnesium sulfate and evaporated, and the resulting

precipitate was collected by ®ltration and washed

Benzhydryl 7ꢀ-benzoylamino-3-(1,2,4-triazol-3-yl)thio-3-

cephem-4-carboxylate (6d). To a solution of 5 (1.0 g,

2.15 mmol) in THF (15 mL) was added N-trimethylsilyl-

acetamide (846 mg, 6.46 mmol) at ambient temperature,

and the mixture was stirred for 30 min. Thereto was

dropwise added a solution of benzoyl chloride (0.26 ml,

2.23 mmol) in CH₂Cl₂(1 mL) under ice-cooling and the

mixture was stirred for 1 h. The reaction mixture was

poured into a mixture of aqueous sodium hydrogen

carbonate solution, THF and AcOEt with stirring, and

adjusted to pH 2.0 with 1N HCl. The separated organic

layer was washed with brine, dried over magnesium sul-

fate and evaporated under reduced pressure to give 1.30 g

1

of 6d (100%): H NMR (CDCl₃) d 3.17 and 3.67 (ABq,

2H, J=18.2 Hz), 5.01 (d, 1H, J=4.9 Hz), 6.16 (dd, 1H,

J=4.9, 9.2 Hz), 6.76 (s, 1H), 7.1±7.5 (m, 15H), 7.98 (d,

1H, J=9.2 Hz), 8.17 (s, 1H).

Preparation of 6j, 32, 44 and 48 was carried out by a

similar method to that described for 6d.

Benzhydryl 7ꢀ-(2-bromoacetamido)-3-(1,2,4-triazol-3-yl)-

1

thio-3-cephem-4-carboxylate (6j). Yield 66%; H NMR

(CDCl₃) d 3.42 and 3.75 (ABq, 2H, J=17.9 Hz), 3.71 (s,

2H), 4.96 (d, 1H, J=4.7 Hz), 5.82 (dd, 1H, J=4.7,

9.0 Hz), 6.91 (s, 1H), 7.10±7.55 (m, 10H), 7.74 (d, 1H,

J=9.0 Hz), 8.27 (s, 1H).

7ꢀ-(2-Phenylacetamido)-3-(5-methyl-1,3,4-thiadiazol-2-yl)

thiomethyl-3-cephem-4-carboxylic acid (32). Yield 42%;

1

IR (Nujol) 1768, 1712, 1659, 1533 cm

.

(E)-7ꢀ-(2-Phenylacetamido)-3-[2-(5-methyl-1,3,4-thiadia-

zol-2-yl)thio]vinyl-3-cephem-4-carboxylic acid (44). Yield

1

62%; IR (KBr) 1776, 1664, 1537 cm

;

¹H NMR

(DMSO-d₆) d 2.72 (s, 3H), 3.54 (d, 2H, J=5.0 Hz), 3.67

and 3.96 (ABq, 2H, J=17.4 Hz), 5.14 (d, 1H, J=4.9 Hz),

5.70 (dd, 1H, J=4.9, 8.2 Hz), 7.13 (d, 1H, J=15.6 Hz),

7.17 (d, 1H, J=15.6 Hz), 7.2±7.6 (m, 5H), 9.16 (d, 1H,

J=8.2 Hz); FAB±MS m/z 475.0 (MH⁺).

with AcOEt to give 577 mg of 7b (42%): IR (Nujol)

1

3263, 1757, 1660, 1554, 1298, 1232 cm

;

¹H NMR

(DMSO-d₆) d 3.45 and 3.81 (ABq, 2H, J=17.2 Hz),

4.02 (s, 2H), 5.19 (d, 1H, J=4.8 Hz), 5.69 (dd, 1H,

2328

Y. Yoshida et al. / Bioorg. Med. Chem. 8 (2000) 2317±2335

7ꢁ-Methoxy-7ꢀ-(2-phenylacetamido)-3-(5-methyl-1,3,4-

thiadiazol-2-yl)thio-3-cephem-4-carboxylic acid (48).

Benzhydryl 7ꢀ-[2-(1-tetrazolyl)acetamido]-3-(1,3,4-thiadia-

zol-2-yl)thio-3-cephem-4-carboxylate (24c). Yield 67%;

¹H NMR (DMSO-d₆) d 3.67 and 3.90 (ABq, 2H,

J=17.8 Hz), 5.32 (d, 1H, J=5.1 Hz), 5.37 (s, 2H), 5.93

(dd, 1H, J=5.1, 8.4 Hz), 6.97 (s, 1H), 7.2±7.5 (m, 10H),

9.36 (s, 1H), 9.64 (s, 1H), 9.66 (d, 1H, J=8.4 Hz).

1

Yield 30%; IR (KBr) 1782, 1738, 1695, 1497 cm ¹; H

NMR (DMSO-d₆) d 2.69 (s, 3H), 3.23 (s, 3H), 3.48 (s,

2H), 3.55 and 3.83 (ABq, 2H, J=17.8Hz), 5.61 (s, 1H),

6.98 (s, 1H), 7.16 (d, 1H, J=3.0 Hz), 7.2±7.7 (m, 15H),

8.54 (s, 1H).

Benzhydryl 7ꢀ-[2-(2-formamidothiazol-4-yl)acetamido]-3-

(5-methyl-1,3,4-thiadiazol-2-yl)thio-3-cephem-4-carboxy-

late (24e). Yield 63%; H NMR (DMSO-d₆) d 2.67 (s,

3H), 3.60 (s, 2H), 3.72 and 3.90 (ABq, 2H, J=17.7 Hz),

5.28 (d, 1H, J=5.1 Hz), 5.88 (dd, 1H, J=5.1, 8.4 Hz), 6.96

(s, 1H), 6.97 (s, 1H), 8.45 (s, 1H), 9.17 (d, 1H, J=8.4 Hz),

12.2 (s, 1H).

Benzhydryl 7ꢀ-(2-phenoxyacetamido)-3-(1,2,4-triazol-3-

yl)thio-3-cephem-4-carboxylate (6e). To a solution of

phosphorus pentachloride (492 mg, 2.36 mmol) in

CH₂Cl₂(5 mL) was added 2-phenoxyacetic acid (343 mg,

2.25 mmol) at 15 ^ꢀC and the mixture was stirred under

ice-cooling for 30 min. To a solution of 5 (1.0 g,

2.15 mmol) in THF (15 mL) was added N-trimethylsilyl-

acetamide (846 mg, 6.46 mmol) under ice-cooling, and

the mixture was stirred at the same temperature for 30

min. Thereto was dropwise added the acid chloride

solution obtained above under ice-cooling and the mix-

ture was stirred for 1 h. The reaction mixture was

poured into a mixture of aqueous sodium hydrogen

carbonate solution and AcOEt with stirring. The sepa-

rated organic layer was washed with brine, dried over

magnesium sulfate and evaporated under reduced pres-

sure. The residue was chromatographed over silica-gel

1

Benzhydryl 7ꢀ-[2-(phenylthio)acetamido]-3-methanesul-

fonyloxy-3-cephem-4-carboxylate (28a). Yield 80%; ¹H

NMR (CDCl₃) d 2.78 (s, 3H), 3.65 and 3.88 (ABq, 2H,

J=18.5 Hz), 3.70 (d, 2H, J=3.9 Hz), 5.02 (d, 1H,

J=5.0 Hz), 5.85 (dd, 1H, J=5.0, 9.3 Hz), 6.95 (s, 1H), 7.2±

7.5 (m, 15H), 7.51 (d, 1H, J=9.3 Hz).

(Z)-Benzhydryl 7ꢀ-[2-(2-cyanoethenylthio)acetamido]-3-

methanesulfonyloxy-3-cephem-4-carboxylate (28b). Yield

1

46%; H NMR (DMSO-d₆) d 3.19 (s, 3H), 3.74 (s, 2H),

1

(eluent AcOEt:hexane) to give 1.26 g of 6e (98%): H

3.77 and 3.90 (ABq, 2H, J=18.0Hz), 5.30 (d, 1H,

J=4.9 Hz), 5.74 (d, 1H, J=10.5Hz), 5.84 (dd, 1H, J=4.9,

8.2 Hz), 6.93 (s, 1H), 7.2±7.5 (m, 10H), 7.66 (d, 1H,

J=10.5 Hz), 9.32 (d, 1H, J=8.2 Hz).

NMR (CDCl₃) d 3.29 and 3.52 (ABq, 2H, J=18.0 Hz),

4.52 (s, 2H), 4.99 (d, 1H, J=4.8 Hz), 5.87 (dd, 1H,

J=4.8, 9.2 Hz), 6.8±7.5 (m, 17H), 8.13 (s, 1H).

Preparation of 6f±g, 24a±c, 24e and 28a,b was carried

out by a similar method to that described for 6e.

Benzhydryl 7ꢀ-[2-(3-chlorophenyl)acetamido]-3-(1,2,4-

triazol-3-yl)thio-3-cephem-4-carboxylate (6h). Phospho-

rus oxychloride (440mg, 2.87mmol) was added dropwise

to a mixture of DMF (220 mg, 2.87 mmol) and AcOEt

(700 mL) under ice-cooling. After stirring for 20 min at

the same temperature, the mixture was cooled until a

precipitate appeared. To the suspension were added

THF (10 mL) and 3-chlorophenylacetic acid (403 mg,

2.36 mmol). The mixture was stirred at the same tem-

perature for 30 min to give an activated acid solution. To

a suspension of 5 (1.0 g, 2.15 mmol) in THF (10 mL) was

added N-trimethylsilylacetamide (845 mg, 6.46 mmol).

The suspension was stirred at room temperature for

30 min to give a clear solution. To this solution was

added the activated acid solution prepared above at

20 ^ꢀC and the mixture was stirred at 25 to 10 ^ꢀC for

an hour. The mixture was poured into aqueous sodium

hydrogen carbonate solution. The extract was washed

with water and brine, dried over magnesium sulfate and

evaporated in vacuo. The residue was triturated with

diethyl ether to give 955 mg of 6h (72%): IR (KBr)

(Z)-Benzhydryl 7ꢀ-[2-(2-cyanoethenylthio)acetamido]-3-

(1,2,4-triazol-3-yl)thio-3-cephem-4-carboxylate (6f). Yield

43%; ¹H NMR (CDCl₃) d 3.25 (s, 2H), 3.43 and 3.82 (ABq,

2H, J=17.9 Hz), 4.99 (d, 1H, J=4.8 Hz), 5.30 (d, 1H,

J=10.4 Hz), 5.82 (dd, 1H, J=4.8, 8.8 Hz), 6.95 (s, 1H),

7.1±7.5 (m, 11H), 7.72 (d, 1H, J=8.8Hz), 8.20 (s, 1H).

(Z)-Benzhydryl 7ꢀ-[2-(2-allylthioethenylthio)acetamido]-3-

(1,2,4-triazol-3-yl)thio-3-cephem-4-carboxylate (6g). Yield

46%; ¹H NMR (CDCl₃) d 3.29 and 3.61 (ABq, 2H,

J=18.0 Hz), 3.30 (d, 2H, J=6.9 Hz), 3.34 (s, 2H), 5.00

(d, 1H, J=4.8 Hz), 5.08 (d, 1H, J=2.8 Hz), 5.14 (d, 1H,

J=9.8 Hz), 5.75 (dd, 1H, J=4.8, 8.9 Hz), 5.7±5.9 (m,

2H), 6.12 (dd, 1H, J=8.2, 40.2 Hz), 6.94 (s, 1H), 7.2±7.5

(m, 10H), 7.63 (d, 1H, J=8.9 Hz), 8.17 (s, 1H).

Benzhydryl 7ꢀ-[2-(3-thienyl)acetamido]-3-(1,3,4-thiadiazol-

2-yl)thio-3-cephem-4-carboxylate (24a). Yield 22%; ¹H

NMR (CDCl₃) d 3.62 and 3.88 (ABq, 2H, J=17.9Hz),

3.67 (s, 2H), 5.04 (d, 1H, J=5.1 Hz), 5.91 (dd, 1H, J=5.1,

9.2 Hz), 6.37 (d, 1H, J=9.2 Hz), 6.9±7.4 (m, 13H), 9.11 (s,

1H).

1

1790, 1730, 1670, 1535 cm ¹; H NMR (DMSO-d₆) d

3.3±3.7 (m, 4H), 5.24 (d, 1H, J=5.0 Hz), 5.74 (dd, 1H,

J=5.0, 8.0 Hz), 6.93 (s, 1H), 7.2±7.6 (m, 14H), 8.70 (brs,

1H), 9.23 (d, 1H, J=8.0 Hz), 14.5 (brs, 1H); APCI-MS

m/z 618 (MH⁺).

(E)-Benzhydryl 7ꢀ-[3-(3-thienyl)acryloylamino]-3-(1,3,4-

thiadiazol-2-yl)thio-3-cephem-4-carboxylate (24b). Yield

59%; ¹H NMR (CDCl₃) d 3.63 and 3.88 (ABq, 2H,

J=18.0Hz), 5.09 (d, 1H, J=5.0 Hz), 6.07 (dd, 1H, J=5.0,

9.0 Hz), 6.34 (d, 1H, J=15.5 Hz), 6.75 (d, 1H, J=9.0 Hz),

6.97 (s, 1H), 7.2±7.5 (m, 13H), 7.78 (d, 1H, J=15.5Hz),

9.12 (s, 1H).

7ꢀ-[2-{(5-Methyl-1,3,4-thiadiazol-2-yl)thio}acetamido]-

3-(1,2,4-triazol-3-yl)thio-3-cephem-4-carboxylic acid (7j).

To a solution of 2-mercapto-5-methyl-1,3,4-thiadiazole

(2.0 g, 15.2 mmol) in a mixture of THF (10 mL) and 1,2-

dimethoxyethane (DME, 10 mL) was added t-BuOK

(1.70 g, 15.2 mmol) at 10 ^ꢀC, and the mixture was

Y. Yoshida et al. / Bioorg. Med. Chem. 8 (2000) 2317±2335

2329

stirred under ice-cooling for 30 min. The resulting pre-

cipitate was collected by ®ltration, washed with DME

and dried in vacuo to give a solid. This solid was added

to a solution of 6j (500 mg, 0.854 mmol) in DME (5 mL)

at a temperature from 0 ^ꢀC to 10 ^ꢀC, and the mixture

was stirred for 1 h. The reaction mixture was poured into a

mixture of AcOEt and 1N HCl with stirring. The organic

layer was separated, washed with brine, dried over

magnesium sulfate and evaporated under reduced

pressure. To a solution of the residue (crude 9) in a

mixture of CH₂Cl₂(1.2 mL) and anisole (0.4 mL) was

added tri¯uoroacetic acid (0.8 mL) with stirring under ice-

cooling. The mixture was stirred at said temperature for

1 h. The reaction mixture was poured into IPE (24 mL)

and the resulting precipitate was collected by ®ltration

and dried in vacuo. The precipitate was dissolved in a

mixture of a sodium hydrogen carbonate solution and

THF, adjusted to pH 5.0 with 1N HCl with stirring and

evaporated under reduced pressure to remove the

organic solvent. The resulting solution was subjected to

column chromatography on Diaion HP-20 (eluent 20%

aqueous isopropyl alcohol). The desired fractions were

evaporated under reduced pressure to remove the

organic solvent and the residue was adjusted to pH 2.0

with 1N HCl and extracted twice with a mixture of

THF and AcOEt. The organic layer was dried over

magnesium sulfate and evaporated, and the result-

ing precipitate was collected by ®ltration and washed

with AcOEt to give 249 mg of 7j (62%): ¹H NMR

(DMSO-d₆) d 3.44 and 3.78 (ABq, 2H, J=17.3 Hz),

4.12 (d, 2H, J=13.5 Hz), 5.19 (d, 1H, J=4.8 Hz), 5.68

(dd, 1H, J=4.8, 8.2 Hz), 8.72 (brs, 1H), 9.28 (d,

1H, J=8.2 Hz), 14.5 (brs, 1H); FAB±MS m/z 471.8

(MH⁺).

7ꢀ-Benzamido-3-(1,2,4-triazol-3-yl)thio-3-cephem-4-car-

1

boxylic acid (7d). Yield 45%; H NMR (DMSO-d₆) d

3.48 (s, 2H), 5.28 (d, 1H, J=4.7 Hz), 5.83 (dd, 1H, J=4.7,

8.0 Hz), 7.4±7.6 (m, 3H), 7.8±8.0 (m, 2H), 8.71 (brs, 1H),

9.42 (d, 1H, J=8.0 Hz); FAB±MS m/z 403.9 (MH⁺).

7ꢀ-(2-Phenoxyacetamido)-3-(1,2,4-triazol-3-yl)thio-3-ce-

phem-4-carboxylic acid (7e). Yield 41%; IR (KBr) 1756,

1

1674, 1462cm ¹; H NMR (DMSO-d₆) d 3.45 (d, 2H,

J=3.6 Hz), 4.61 (d, 2H, J=2.0 Hz), 5.21 (d, 1H,

J=4.6 Hz), 5.68 (dd, 1H, J=4.6, 8.4 Hz), 6.8±7.0 (m, 3H),

7.2±7.4 (m, 2H), 8.72 (brs, 1H), 9.15 (d, 1H, J=8.4 Hz);

FAB±MS m/z 433.9 (MH⁺).

(Z)-7ꢀ-[2-(2-Cyanoethenylthio)acetamido]-3-(1,2,4-triazol-

3-yl)thio-3-cephem-4-carboxylic acid (7f). To a solution of

6f (720 mg, 1.19 mmol) in a mixture of CH₂Cl₂(2.2 mL)

and anisole (0.72 mL) was added tri¯uoroacetic acid

(1.44 mL) with stirring under ice-cooling and the mix-

ture stirred for 1 h. The reaction mixture was poured

into IPE (40 mL) and the resulting precipitate was col-

lected by ®ltration and dried in vacuo. The precipitate

was dissolved in a mixture of a sodium hydrogen car-

bonate solution and THF, adjusted to pH 5.0 with 1N

HCl with stirring and evaporated under reduced pres-

sure to remove the organic solvent. The resulting solu-

tion was subjected to column chromatography on Diaion

HP-20 (eluent 20% aqueous isopropyl alcohol). The

desired fractions were concentrated under reduced

pressure and freeze-dried to give 365 mg of 7f (70%): IR

1

(KBr) 2214, 1772, 1670, 1616 cm ¹; H NMR (D₂O) d

3.47 and 3.82 (ABq, 2H, J=17.4 Hz), 3.77 (s, 2H), 5.20

(d, 1H, J=4.7 Hz), 5.57 (d, 1H, J=10.5 Hz), 5.65 (d,

1H, J=4.7 Hz), 7.48 (d, 1H, J=10.5 Hz), 8.46 (s, 1H);

FAB±MS m/z 425.0 (MH⁺).

7ꢀ-[2-(2-Aminothiazol-4-yl)acetamido]-3-(1,2,4-triazol-3-

yl)thio-3-cephem-4-carboxylic acid (7a). To a solution of

8 (620 mg, 1.02 mmol) in a mixture of CH₂Cl₂(1.8 mL)

and anisole (0.6 mL) was added tri¯uoroacetic acid

(1.2 mL) with stirring under ice-cooling and the mixture

stirred for 1 h. The reaction mixture was poured into IPE

(36 mL) and the resulting precipitate was collected by ®l-

tration and dried in vacuo. The precipitate was dissolved

in a mixture of a sodium hydrogen carbonate solution

and THF, adjusted to pH 5.0 with 1N HCl with stirring

and evaporated under reduced pressure to remove the

organic solvent. The resulting solution was subjected to

column chromatography on Diaion HP-20 (eluent 20%

aqueous isopropyl alcohol). The desired fractions were

evaporated under reduced pressure to remove the

organic solvent and the residue was adjusted to pH 2.0

with 1N HCl and extracted twice with a mixture of THF

and AcOEt. The organic layer was dried over magnesium

sulfate and evaporated, and the resulting precipitate was

collected by ®ltration and washed with AcOEt to give

Preparation of 7g,h, 13c,d, 13f, 13h, 13j±m, 25a±e

and 30a,b was carried out by a similar method to that

described for 7f.

(Z)-7ꢀ-[2-(2-Allylthioethenylthio)acetamido]-3-(1,2,4-tria-

zol-3-yl)thio-3-cephem-4-carboxylic acid (7g). Yield 55%;

IR (KBr) 1770, 1670, 1608, 1508 cm ¹; ¹H NMR (D₂O)

d 3.1±4.1 (m, 6H), 5.02 (m, 1H), 5.05 (d, 1H, J=4.6 Hz),

5.65 (d, 1H, J=4.6 Hz), 5.80 (m, 1H), 6.6±7.3 (m, 3H),

8.45 (s, 1H).

7ꢀ-[2-(3-Chlorophenyl)acetamido]-3-(1,2,4-triazol-3-yl)-

thio-3-cephem-4-carboxylic acid (7h). Yield 68%; IR

1

(KBr) 3270, 1780, 1660, 1550 cm ¹; H NMR (DMSO-

d₆) d 3.37 and 3.58 (ABq, 2H, J=17.0 Hz), 3.56 and

3.58 (total 1H, each s), 5.17 (d, 1H, J=5.0 Hz), 5.66 (dd,

1H, J=5.0, 8.0 Hz), 7.2±7.4 (m, 4H), 8.68 (brs, 1H),

9.19 (d, 1H, J=8.0 Hz), 14.4 (brs, 1H); FAB±MS m/z

451.9 (MH⁺).

1

228 mg of 7a (51%): H NMR (DMSO-d₆) d 3.37 (s,

2H), 3.44 and 3.72 (ABq, 2H, J=17.3 Hz), 5.17 (d, 1H,

J=4.7 Hz), 5.67 (dd, 1H, J=4.7, 8.3 Hz), 6.24 (s, 1H),

6.91 (s, 2H), 8.67 (brs, 1H), 8.93 (d, 1H, J=8.3 Hz);

FAB±MS m/z 439.9 (MH⁺).

7ꢀ-(2-Phenylacetamido)-3-(pyrazol-4-yl)thio-3-cephem-

4-carboxylic acid (13c). Yield 41%; IR (KBr) 1759,

1

1659, 1537cm ¹; H NMR (DMSO-d₆) d 3.27 (d, 2H,

J=2.7 Hz), 3.48 and 3.54 (ABq, 2H, J=13.9Hz), 5.04 (d,

1H, J=4.6 Hz), 5.55 (dd, 1H, J=4.6, 8.2 Hz), 7.1±7.4 (m,

5H), 7.85 (s, 2H), 9.05 (d, 1H, J=8.2 Hz), 13.32 (brs,

1H); FAB±MS m/z 417.0 (MH⁺).

Preparation of 7d,e was carried out by a similar method

to that described for 7a.

2330

Y. Yoshida et al. / Bioorg. Med. Chem. 8 (2000) 2317±2335

7ꢀ-(2-Phenylacetamido)-3-(1-methyltetrazol-5-yl)thio-3-

cephem-4-carboxylic acid (13d). Yield 58%; IR (KBr)

1784, 1666, 1531 cm ¹; ¹H NMR (DMSO-d₆) d 3.42 and

3.62 (ABq, 2H, J=17.4 Hz), 3.52 (d, 2H, J=5.2 Hz), 4.05

(s, 3H), 5.18 (d, 1H, J=4.9 Hz), 5.74 (dd, 1H, J=4.9,

8.3 Hz), 7.1±7.4 (m, 5H), 9.17 (d, 1H, J=8.3 Hz); FAB±

MS m/z 433.0 (MH⁺).

40.90; H, 2.75; N, 12.72. Found: C, 41.16; H, 2.74; N,

12.46.

(E)-7ꢀ-[3-(3-Thienyl)acryloylamino]-3-(1,3,4-thiadiazol-

2-yl)thio-3-cephem-4-carboxylic acid (25b). Yield 49%;

¹H NMR (DMSO-d₆) d 3.75 and 3.97 (ABq, 2H,

J=17.6 Hz), 5.30 (d, 1H, J=5.0 Hz), 5.93 (dd, 1H, J=5.0,

8.4 Hz), 6.55 (d, 1H, J=15.7Hz), 7.33 (d, 1H, J=5.0 Hz),

7.54 (d, 1H, J=15.7 Hz), 7.62 (dd, 1H, J=2.9, 5.0 Hz),

7.86 (d, 1H, J=2.9 Hz), 9.13 (d, 1H, J=8.4 Hz), 9.65 (s,

1H); MS m/z 453.0 (MH⁺).

7ꢀ-(2-Phenylacetamido)-3-(1,2,4-thiadiazol-5-yl)thio-3-

cephem-4-carboxylic acid (13f). Yield 61%; IR (KBr)

1

1790, 1724, 1666, 1531 cm ¹; H NMR (DMSO-d₆) d

3.54 (d, 2H, J=4.5 Hz), 3.69 and 4.01 (ABq, 2H,

J=17.8 Hz), 5.27 (d, 1H, J=5.1 Hz), 5.84 (dd, 1H,

J=5.1, 8.4 Hz), 7.1±7.4 (m, 5H), 8.80 (s, 1H), 9.30 (d,

1H, J=8.4 Hz); FAB±MS m/z 435.0 (MH⁺).

7ꢀ-[2-(1-Tetrazolyl)acetamido]-3-(1,3,4-thiadiazol-2-yl)-

thio-3-cephem-4-carboxylic acid (25c). Yield 32%; IR

1

(KBr) 1778, 1693, 1666, 1542 cm ¹; H NMR (DMSO-

d₆) d 3.61 and 3.89 (ABq, 2H, J=17.6 Hz), 5.28 (d, 1H,

J=5.1 Hz), 5.38 (s, 2H), 5.86 (dd, 1H, J=5.1, 8.3 Hz),

9.38 (s, 1H), 9.64 (d, 1H, J=8.3 Hz), 9.66 (s, 1H); FAB±

MS m/z 427.0 (MH⁺).

7ꢀ-(2-Phenylacetamido)-3-(thiazol-2-yl)thio-3-cephem-4-

carboxylic acid (13h). Yield 52%; IR (KBr) 1782, 1660,

1537cm

1

;

¹H NMR (DMSO-d₆) d 3.52 (d, 2H,

J=4.3 Hz), 3.50 and 3.74 (ABq, 2H, J=17.5 Hz), 5.21 (d,

1H, J=4.9 Hz), 5.75 (dd, 1H, J=4.9, 8.3 Hz), 7.1±7.4 (m,

5H), 7.88 (s, 2H), 9.22 (d, 1H, J=8.3 Hz); FAB±MS m/z

434.0 (MH⁺). Anal. calcd for C₁₈H₁₅N₃O₄S₃: C, 49.87;

H, 3.49; N, 9.69. Found: C, 49.55; H, 3.43; N, 9.56.

(D)-7ꢀ-[2-Amino-2-(4-hydroxyphenyl)acetamido]-3-(1,3,4-

thiadiazol-2-yl)thio-3-cephem-4-carboxylic acid (25d).

1

Yield 36%; IR (KBr) 1770, 1614, 1516cm ¹; H NMR

(DMSO-d₆) d 3.48 and 3.79 (ABq, 2H, J=17.8 Hz), 5.00 (s,

1H), 5.22 (d, 1H, J=4.8 Hz), 5.53 (dd, 1H, J=4.8, 8.5 Hz),

6.74 (d, 2H, J=8.5 Hz), 7.09 (d, 2H, J=8.5 Hz), 8.66 (d,

1H, J=8.5 Hz), 9.43 (s, 1H); FAB±MS m/z 466.0 (MH⁺).

7ꢀ-(2-Phenylacetamido)-3-(5-methyl-1,3,4-oxadiazol-2-yl)-

thio-3-cephem-4-carboxylic acid (13j). Yield 36%; IR

1

(KBr) 1782, 1724, 1659, 1533 cm ¹; H NMR (DMSO-

d₆) d 3.41 (s, 3H), 3.53 (d, 2H, J=5.6 Hz), 3.65 and 3.95

(ABq, 2H, J=18.0 Hz), 5.18 (d, 1H, J=4.9 Hz), 5.72

(dd, 1H, J=4.9, 8.3 Hz), 7.2±7.4 (m, 5H), 9.17 (d, 1H,

J=8.3 Hz); FAB±MS m/z 433.0 (MH⁺).

7ꢀ-[2-(2-Aminothiazol-4-yl)acetamido]-3-(5-methyl-1,3,4-

thiadiazol-2-yl)thio-3-cephem-4-carboxylic acid (25e).

Yield 55%; H NMR (DMSO-d₆) d 2.72 (s, 3H), 3.39 (s,

2H), 3.69 and 3.92 (ABq, 2H, J=17.6 Hz), 5.23 (d, 1H, J=

5.0 Hz), 5.78 (dd, 1H, J=5.0, 8.4 Hz), 6.28 (s, 1H), 7.06

(brs, 2H), 9.02 (d, 1H, J=8.4 Hz); FAB±MS m/z 471.0

(MH⁺).

1

7ꢀ-(2-Phenylacetamido)-3-(2-thienyl)thio-3-cephem-4-car-

boxylic acid (13k). Yield 79%; IR (KBr) 1767, 1632,

1531 cm ¹; ¹H NMR (DMSO-d₆) d 3.37 (s, 2H), 3.51 (d,

2H, J=4.2 Hz), 5.11 (d, 1H, J=4.7 Hz), 5.61 (dd, 1H,

J=4.7, 8.3 Hz), 7.1±7.5 (m, 7H), 7.85 (d, 1H, J=4.1 Hz),

9.08 (d, 1H, J=8.3 Hz); FAB±MS m/z 432.9 (MH⁺).

7ꢀ-[2-(Phenylthio)acetamido]-3-(1,2,4-triazol-3-yl)thio-3-

cephem-4-carboxylic acid (30a). Yield 47%; IR (KBr)

1

1763, 1608, 1540cm ¹; H NMR (D₂O) d 3.35 and 3.83

(ABq, 2H, J=17.3Hz), 3.77 (d, 2H, J=3.5 Hz), 5.09 (d,

1H, J=4.7 Hz), 5.57 (d, 1H, J=4.7 Hz), 7.2±7.5 (m, 5H),

8.46 (s, 1H); FAB±MS m/z 449.8 (MH⁺).

7ꢀ-(2-Phenylacetamido)-3-(2-pyridyl)thio-3-cephem-4-

carboxylic acid (13l). Yield 55%; IR (KBr) 1782, 1660,

1537 cm ¹; ¹H NMR (DMSO-d₆) d 3.53 (s, 2H), 3.51 and

3.88 (ABq, 2H, J=17.5 Hz), 5.26 (d, 1H, J=4.9 Hz), 5.77

(dd, 1H, J=4.9, 8.4 Hz), 7.1±7.4 (m, 7H), 7.75 (dt, 1H,

J=1.8, 7.8 Hz), 8.45 (d, 1H, J=4.0 Hz), 9.26 (d, 1H,

J=8.4 Hz); FAB±MS m/z 427.9 (MH⁺).

(Z)-7ꢀ-[2-(2-Cyanoethenylthio)acetamido]-3-(1,3,4-thia-

diazol-2-yl)thio-3-cephem-4-carboxylic acid (30b). Yield

45%; IR (KBr) 2212, 1770, 1616 cm ¹; ¹H NMR (D₂O)

d 3.70 and 3.97 (ABq, 2H, J=17.5 Hz), 3.80 (s, 2H),

5.29 (d, 1H, J=4.9 Hz), 5.60 (d, 1H, J=10.5 Hz), 5.72

(d, 1H, J=4.9 Hz), 7.51 (d, 1H, J=10.5 Hz), 9.43 (s,

1H); FAB±MS m/z 442.0 (MH⁺).

7ꢀ-(2-Phenylacetamido)-3-(benzothiazol-2-yl)thio-3-ce-

phem-4-carboxylic acid (13m). Yield 32%; IR (KBr)

1

1782, 1659, 1537cm ¹; H NMR (DMSO-d₆) d 3.55 (d,

2H, J=4.2 Hz), 3.68 and 4.00 (ABq, 2H, J=17.8 Hz), 5.28

(d, 1H, J=5.0 Hz), 5.82 (dd, 1H, J=5.0, 8.4 Hz), 7.1±7.6

(m, 7H), 7.91 (d, 1H, J=7.4 Hz), 8.07 (d, 1H, J=7.2 Hz),

9.27 (d, 1H, J=8.4 Hz); FAB±MS m/z 483.8 (MH⁺).

7ꢀ-(2-Phenylacetamido)-3-(1,2,4-triazol-3-yl)thio-3-ce-

phem-4-carboxylic acid (13a). To a solution of 12a

(348mg, 0.597 mmol) in a mixture of CH₂Cl₂(1.5mL) and

anisole (0.5 mL) was added tri¯uoroacetic acid (1.0 mL)

with stirring under ice-cooling and the mixture stirred for 1

h. The reaction mixture was poured into IPE (30 mL) and

the resulting precipitate was collected by ®ltration and

dried in vacuo. The precipitate was dissolved in a mix-

ture of a sodium hydrogen carbonate solution, AcOEt

and THF at pH 7.5, and the aqueous layer was washed

with a mixture of AcOEt and THF. Then AcOEt and

THF were added to the aqueous layer and the mixture

7ꢀ-[2-(3-Thienyl)acetamido]-3-(1,3,4-thiadiazol-2-yl)thio-

1

3-cephem-4-carboxylic acid (25a). Yield 81%; H NMR

(DMSO-d₆) d 3.54 (s, 2H), 3.73 and 3.96 (ABq, 2H,

J=17.6 Hz), 5.24 (d, 1H, J=5.0 Hz), 5.79 (dd, 1H,

J=5.0, 8.3 Hz), 7.01±7.04 (m, 1H), 7.26 (m, 1H), 7.43±

7.48 (m, 1H), 9.19 (d, 1H, J=8.3 Hz), 9.65 (s, 1H); FAB±

MS m/z 441.0 (MH⁺). Anal. calcd for C₁₅H₁₂N₄O₄S₄: C,

Y. Yoshida et al. / Bioorg. Med. Chem. 8 (2000) 2317±2335

2331

was adjusted to pH 2.0 with 1N HCl with stirring. The

organic layer was washed twice with brine, dried over

magnesium sulfate and evaporated under reduced

pressure to give a precipitate. The precipitate was col-

lected by ®ltration, washed with AcOEt and dried in

vacuo to give 154 mg of 13a (62%): IR (Nujol) 3250,

7ꢁ-Methoxy-7ꢀ-(2-phenylacetamido)-3-(5-methyl-1,3,4-

thiadiazol-2-yl)thio-3-cephem-4-carboxylic acid (49). Yield

23%; IR (KBr) 1782, 1664, 1564cm ¹; ¹H NMR (DMSO-

d₆) d 2.71 (s, 3H), 2.35 (s, 3H), 3.59 (d, 2H, J=3.4 Hz),

3.57 and 3.75 (ABq, 2H, J=17.6 Hz), 5.31 (s, 1H), 7.2±7.4

(m, 5H), 9.53 (s, 1H).

1

1755, 1660, 1530 cm ¹; H NMR (DMSO-d₆) d 3.3±3.6

(m, 4H), 5.16 (d, 1H, J= 4.7 Hz), 5.60 (dd, 1H, J=4.7,

8.3 Hz), 7.1±7.3 (m, 5H), 8.69 (brs, 1H), 9.14 (d, 1H,

J=8.3 Hz), 14.4 (brs, 1H).

Benzhydryl 7ꢀ-(2-phenylacetamido)-3-methanesulfonyloxy-

3-cephem-4-carboxylate (11). To a solution of 10 (800 mg,

1.6 mmol) in DMF (5 mL) were added methanesulfonyl

chloride (421 mg, 3.68 mmol) and K₂CO₃(310 mg,

2.24 mmol) at 35 ^ꢀC with stirring and the mixture

was stirred at 20 ^ꢀC for 1 h. The resulting mixture

was poured into a mixture of H₂O and AcOEt and

the organic layer was sequentially washed with brine

(3Â), dried over magnesium sulfate and evaporated

under reduced pressure. The residue was triturated

with a mixture of AcOEt and IPE, and the precipi-

tate was collected by ®ltration to give 850 mg of

11 (92%): ¹H NMR (CDCl₃) d 2.79 (s, 3H), 3.52

and 3.79 (ABq, 2H, J=18.4 Hz), 3.62 (d, 2H,

J=2.4 Hz), 5.02 (d, 1H, J=5.0 Hz), 5.87 (dd, 1H,

J=5.0, 9.2 Hz), 6.19 (d, 1H, J=9.2 Hz), 6.92 (s, 1H),

7.2±7.5 (m, 15H).

Preparation of 13b, 13e, 13g, 13i, 35, 40 and 49 was car-

ried out by a similar method to that described for 13a.

7ꢀ-(2-Phenylacetamido)-3-(1,2,3-triazol-4-yl)thio-3-ce-

phem-4-carboxylic acid (13b). Yield 51%; IR (Nujol)

1

1745, 1695, 1650, 1535 cm ¹; H NMR (DMSO-d₆) d

3.27 (s, 2H), 3.51 and 3.80 (ABq, 2H, J=18.3 Hz), 5.09

(d, 1H, J=4.7 Hz), 5.62 (dd, 1H, J=4.7, 8.2 Hz), 7.1±

7.3 (m, 5H), 8.32 (brs, 1H), 9.11 (d, 1H, J=8.2 Hz);

FAB±MS m/z 417.9 (MH⁺).

7ꢀ-(2-Phenylacetamido)-3-(1,3,4-thiadiazol-2-yl)thio-3-

cephem-4-carboxylic acid (13e). Yield 65%; IR (Nujol)

1

1782, 1693, 1659, 1535 cm ¹; H NMR (DMSO-d₆) d

3.52 (d, 2H, J=4.4 Hz), 3.57 and 3.89 (ABq, 2H,

J=17.0 Hz), 5.23 (d, 1H, J=5.0 Hz), 5.79 (dd, 1H,

J=5.0, 8.3 Hz), 7.2±7.4 (m, 5H), 9.25 (d, 1H, J=8.3 Hz),

9.65 (s, 1H); FAB±MS m/z 434.9 (MH⁺).

Benzhydryl 7ꢀ-(2-phenylacetamido)-3-(1,2,4-triazol-3-yl)

thio-3-cephem-4-carboxylate (12a). To a solution of 3-

mercapto-1,2,4-triazole (490mg, 4.85mmol) in a mix-

ture of THF (5mL) and DME (5 mL) was added t-

BuOK (427mg, 3.81mmol) at 10^ꢀC with stirring and

the mixture was stirred at the same temperature for

20min. A solution of 11 (2.0g, 3.46mmol) in a mixture

of THF (10 mL) and DME (10 mL) was added drop-

wise to the solution obtained above at 20 ^ꢀC, and

the mixture was stirred at a temperature from 10 ^ꢀC

to 5 ^ꢀC for 2 hours. The reaction mixture was poured

into a mixture of 0.1N HCl (20 mL) and AcOEt

(30 mL). The organic layer was sequentially washed

with water, aqueous sodium hydrogen carbonate

solution and brine, and dried over magnesium sulfate

and evaporated under reduced pressure. The residue

was subjected to silica-gel chromatography (eluent n-

7ꢀ-(2-Phenylacetamido)-3-(1,2,3-thiadiazol-5-yl)thio-3-

1

cephem-4-carboxylic acid (13g). Yield 52%; H NMR

(DMSO-d₆) d 3.52 and 3.83 (ABq, 2H, J=18.0Hz), 3.55

(d, 2H, J=14.0 Hz), 5.20 (d, 1H, J=5.0 Hz), 5.79 (dd, 1H,

J=5.0, 8.0 Hz), 7.2±7.3 (m, 5H), 8.92 (s, 1H), 9.24 (d, 1H,

J=8.0 Hz).

7ꢀ-(2-Phenylacetamido)-3-(5-methyl-1,3,4-thiadiazol-2-

yl)thio-3-cephem-4-carboxylic acid (13i). Yield 64%; IR

1

(KBr) 1784, 1663, 1535 cm ¹; H NMR (DMSO-d₆) d

2.73 (s, 3H), 3.52 and 3.62 (ABq, 2H, J=14.0 Hz), 3.55

and 3.85 (ABq, 2H, J=17.6 Hz), 5.22 (d, 1H,

J=5.0 Hz), 5.78 (dd, 1H, J=5.0, 8.3 Hz), 7.15±7.35 (m,

5H), 9.24 (d, 1H, J=8.3 Hz); FAB±MS m/z 448.9

(MH⁺). Anal. calcd for C₁₈H₁₆N₄O₄S₃: C, 48.20; H,

3.60; N, 12.49. Found: C, 48.65; H, 3.68; N, 12.45.

1

hexane:AcOEt) to give 351 mg of 12a (17%): H NMR

(CDCl₃) d 3.21 and 3.59 (ABq, 2H, J=17.8 Hz), 3.47 (s,

2H), 4.90 (d, 1H, J=4.5 Hz), 5.84 (dd, 1H, J=4.5,

9.3 Hz), 6.87 (s, 1H), 7.1±7.4 (m, 16H), 8.34 (s, 1H).

7ꢀ-(2-Phenylacetamido)-3-(5-methyl-1,3,4-thiadiazol-2-

yl)methylthio-3-cephem-4-carboxylic acid (35). Yield

55%; IR (KBr) 1776, 1678, 1540cm ¹; ¹H NMR (DMSO-

d₆) d 2.69 (s, 3H), 3.49 and 3.56 (ABq, 2H, J=15.0 Hz),

3.78 and 3.82 (ABq, 2H, J=17.4 Hz), 4.54 and 4.61 (ABq,

2H, J=15.0 Hz), 5.06 (d, 1H, J=4.9 Hz), 5.61 (dd, 1H,

J=4.9, 8.3 Hz), 7.20±7.35 (m, 5H), 9.13 (d, 1H,

J=8.3Hz); FAB±MS m/z 463.1 (MH⁺).

Preparation of 12e, 12h,i, 12l and 29b was carried out

by a similar method to that described for 12a.

Benzhydryl 7ꢀ-(2-phenylacetamido)-3-(1,3,4-thiadiazol-2-

yl)thio-3-cephem-4-carboxylate (12e). Yield 40%; ¹H

NMR (CDCl₃) d 3.43 and 3.78 (ABq, 2H, J=18.0 Hz),

3.64 (d, 2H, J=1.9 Hz), 5.03 (d, 1H, J=5.0 Hz), 5.90 (dd,

1H, J=5.0, 9.1 Hz), 6.27 (d, 1H, J=9.1 Hz), 6.97 (s, 1H),

7.2±7.4 (m, 15H), 9.11 (s, 1H).

7ꢀ-(2-Phenylacetamido)-3-(thiazol-2-yl)thiomethylthio-3-

cephem-4-carboxylic acid (40). Yield 66%; IR (KBr)

Benzhydryl 7ꢀ-(2-phenylacetamido)-3-(thiazol-2-yl)thio-

3-cephem-4-carboxylate (12h). Yield 47%; ¹H NMR

(DMSO-d₆) d 3.53 (d, 2H, J=3.8 Hz), 3.54 and 3.77

(ABq, 2H, J=17.7 Hz), 5.26 (d, 1H, J=5.0 Hz), 5.83

(dd, 1H, J=5.0, 8.4 Hz), 6.97 (s, 1H), 7.2±7.5 (m, 15H),

7.90 (s, 2H), 9.26 (d, 1H, J=8.4 Hz).

1

1776, 1660, 1537cm ¹; H NMR (DMSO-d₆) d 3.50 and

3.56 (ABq, 2H, J=13.8 Hz), 3.87 (s, 2H), 4.74 (s, 2H), 5.11

(d, 1H, J=4.8Hz), 5.64 (dd, 1H, J=4.8, 8.2 Hz), 7.2±7.4

(m, 5H), 7.75 (d, 1H, J=3.4 Hz), 7.81 (d, 1H, J=3.4Hz),

9.13 (d, 1H, J=8.2Hz); FAB±MS m/z 480.0 (MH⁺).

2332

Y. Yoshida et al. / Bioorg. Med. Chem. 8 (2000) 2317±2335

Benzhydryl 7ꢀ-(2-phenylacetamido)-3-(5-methyl-1,3,4-thia-

diazol-2-yl)thio-3-cephem-4-carboxylate (12i). Yield 58%;

¹H NMR (DMSO-d₆) d 2.71 (s, 3H), 3.53 (d, 2H,

J=3.7Hz), 3.57 and 3.87 (ABq, 2H, J=17.7 Hz), 5.26 (d,

1H, J=5.0 Hz), 5.85 (dd, 1H, J=5.0, 8.3 Hz), 6.97 (s, 1H),

7.2±7.5 (m, 15H), 9.28 (d, 1H, J=8.3 Hz).

solution at 0 ^ꢀC and then bromine (38.4 g, 0.24 mol) was

added dropwise to the solution at 15 ^ꢀC. After stirring

at 10 ^ꢀC for 30 min, 0 ^ꢀC for 30 min and at the room

temperature for 3 h, MeOH was evaporated in vacuo

and then AcOEt (200 mL) was added to the residue and

the resulting insoluble material was ®ltered o. The

®ltrate was poured into brine and the aqueous layer was

extracted with AcOEt (3Â), dried over magnesium

sulfate and evaporated under reduced pressure. The

residual gum was extracted with Et₂O (4Â) and hydro-

gen chloride gas was passed through the Et₂O solution,

and the resulting precipitate was collected by ®ltration,

washed with Et₂O and dried in vacuo to give 5.34 g of

Benzhydryl 7ꢀ-(2-phenylacetamido)-3-(2-pyridylthio)-3-

cephem-4-carboxylate (12l). Yield 29%; ¹H NMR

(DMSO-d₆) d 3.53 (d, 2H, J=4.0 Hz), 3.56 and 3.89

(ABq, 2H, J=17.6 Hz), 5.29 (d, 1H, J=5.0 Hz), 5.83 (dd,

1H, J=5.0, 8.4 Hz), 6.92 (s, 1H), 7.1±7.4 (m, 7H), 7.77

(dt, 1H, J=1.9, 7.7 Hz), 8.48 (d, 1H, J=3.9 Hz), 9.29 (d,

1H, J=8.4 Hz).

1

15 (16%): H NMR (DMSO-d₆) d 7.97 (brs, 2H), 8.00

(s, 1H).

(Z)-Benzhydryl 7ꢀ-[2-(2-cyanoethenylthio)acetamido]-3-

(1,3,4-thiadiazol-2-yl)thio-3-cephem-4-carboxylate (29b).

Yield 25%; H NMR (DMSO-d₆) d 3.57 (s, 2H), 3.64

and 3.92 (ABq, 2H, J=17.9 Hz), 5.09 (d, 1H, J=5.0 Hz),

5.38 (d, 1H, J=10.3 Hz), 5.87 (dd, 1H, J=5.0, 9.0 Hz),

6.99 (s, 1H), 7.2±7.5 (m, 11H), 7.54 (d, 1H, J=9.0 Hz),

9.15 (s, 1H).

5-Mercapto-1,2,4-thiadiazole (16). To a mixture of 15

(5.33 g, 38.8 mmol) in water (53 mL) were added 47%

hydrobromic acid (64.5 mL) and Cu powder (38 mg),

and the mixture was cool to 15 ^ꢀC, and then sodium

nitrite (5.35 g, 77.6 mmol) in water (7.75 mL) was added

dropwise at 15 ^ꢀC. After stirring at 10 ^ꢀC for 2.5 h

and at 40 ^ꢀC for another hour, the mixture was adjusted

to pH 6.2 with 3N NaOH with stirring at room tem-

perature and poured into Et₂O. The extracted Et₂O

layer was washed with aqueous NaHSO₃solution and

brine, dried over magnesium sulfate and evaporated

under reduced pressure to give crude bromothiadiazole

derivative. To a mixture of this crude bromide in THF

(13.3 mL) and water (4.4 mL) was added thiourea

(2.03 g, 26.7 mmol). After stirring at 65 ^ꢀC for an hour,

THF was evaporated and Et₂O was added to the mix-

ture. The mixture was adjusted to pH 7.0 with NaHCO₃

solution and the separated aqueous layer was adjusted

to pH 3.0 with 1N HCl. The resulting aqueous layer was

extracted with Et₂O (3Â), washed with brine, dried over

magnesium sulfate and evaporated under reduced pres-

sure. To the residue in EtOH (8.3 mL) was added 1N

KOH/EtOH solution (17 mL), and then Et₂O (100 mL)

was added to the solution. The resulting potassium salt

was collected by ®ltration, washed with Et₂O and dried

in vacuo. To a mixture of potassium salt in water

(12 mL) was added Et₂O (16 mL) and the solution was

adjusted to pH 3.3 with 6N HCl. The aqueous layer was

extracted with Et₂O (3Â), washed with brine, dried over

magnesium sulfate and evaporated under reduced pres-

1

Benzhydryl 7ꢀ-(2-phenylacetamido)-3-(1,2,3-triazol-4-yl)-

thio-3-cephem-4-carboxylate (12b). To a solution of 11

(1.5 g, 2.60 mmol) in a mixture of THF (12 mL) and

DME (12 mL) was added dropwise 4-mercapto-1,2,3-

triazole sodium salt (443 mg, 3.60 mmol) with stirring

under ice-cooling. After stirring at the same temperature

for 3 h, to the reaction mixture were added AcOEt

(100 mL) and 1N HCl (20 mL) with stirring. The

organic layer was separated, sequentially washed with

water, aqueous sodium hydrogen carbonate solution

and brine, dried over magnesium sulfate and evaporated

1

under reduced pressure to give 1.47 g of 12b (97%): H

NMR (CDCl₃) d 3.61 (s, 2H), 3.59 and 3.73 (ABq, 2H,

J=17.9 Hz), 4.88 (d, 1H, J=4.6 Hz), 5.75 (dd, 1H,

J=4.6, 8.4 Hz), 6.91 (s, 1H), 7.05±7.35 (m, 15H), 7.45 (d,

1H, J=8.4 Hz), 7.64 (s, 1H).

Preparation of 12d and 12g was carried out by a similar

method to that described for 12b.

Benzhydryl 7ꢀ-(2-phenylacetamido)-3-(1-methyl-tetrazol-

5-yl)thio-3-cephem-4-carboxylate (12d). Yield 77%; ¹H

NMR (CDCl₃) d 3.39 and 3.76 (ABq, 2H, J=18.0 Hz),

3.55 (s, 2H), 3.79 (s, 3H), 5.04 (d, 1H, J=5.0 Hz), 5.91

(dd, 1H, J=5.0, 9.1 Hz), 6.22 (d, 1H, J=9.1 Hz), 6.95 (s,

1H), 7.1±7.4 (m, 15H).

1

sure to give 0.85 g of 16 (19%): H NMR (DMSO-d₆) d

8.61 (s, 1H), 11.93 (brs, 1H).

4-Benzylthiopyrazole (18). To a solution of 4-bromo-

pyrazole (15 g, 102 mmol) in THF (225 mL) was added

dropwise 1.64 M n-BuLi hexane solution (205 mL,

337 mmol) at 0 ^ꢀC. After stirring at room temperature for

1 hour, the solution was cooled to 0 ^ꢀC, benzyl disul®de

(25.1 g, 102 mmol) was added and the resulting solution

was stirred for 2 h at ice cooling. The solution was

poured into a mixture of AcOEt and ice±water, and

adjusted to pH 5.6, and the separated organic layer was

washed with water and brine, dried over magnesium sul-

fate and evaporated under reduced pressure. The residue

was triturated with hexane and the resulting precipitate

was collected by ®ltration, washed with hexane and dried

in vacuo to give 13.0 g of 18 (67%): IR (KBr) 3153, 1493,

Benzhydryl 7ꢀ-(2-phenylacetamido)-3-(1,2,3-thiadiazol-5-

yl)thio-3-cephem-4-carboxylate (12g). Yield 63%; ¹H

NMR (DMSO-d₆) d 3.4±3.6 (m, 2H), 3.50 and 3.85

(ABq, 2H, J=18.0 Hz), 5.24 (d, 1H, J=5.0 Hz), 5.87 (dd,

1H, J=5.0, 8.0 Hz), 6.98 (s, 1H), 7.2±7.4 (m, 15H), 8.84

(s, 1H), 9.28 (d, 1H, J=8.0 Hz).

5-Amino-1,2,4-thiadiazole hydrochloride (15). To a solu-

tion of amidine acetate (25.0 g, 0.24 mol) in MeOH

(250 mL) was added potassium thiocyanate (23.3 g,

0.24 mol). After stirring at the room temperature for

10 min, 28% sodium methoxide in MeOH solution

(92.6 g, 0.48 mol) was added dropwise to the above

Y. Yoshida et al. / Bioorg. Med. Chem. 8 (2000) 2317±2335

2333

1

1452, 1369, 1138 cm ¹; H NMR (DMSO-d₆) d 3.86 (s,

1H), 7.1±7.4 (m, 6H), 7.65 (s, 1H), 12.93 (brs, 1H).

62%; H NMR (DMSO-d₆) d 2.73 (s, 3H), 3.78 (brs,

2H), 5.27 (d, 1H, J=5.0 Hz), 5.39 (d, 1H, J=5.0 Hz),

6.98 (s, 1H), 7.2±7.5 (m, 10H).

4-Mercaptopyrazole (19). To a solution of 18 (1.19 g,

6.25 mmol) in EtOH (24 mL) was added sodium (2.88 g,

125 mmol) under re¯ux. After stirring under re¯ux for

1.5 h, the mixture was poured into a mixture of AcOEt and

water, and the separated aqueous layer was adjusted to pH

5.5. and poured into AcOEt. The separated organic layer

was washed with brine, dried over magnesium sulfate

(D)-Benzhydryl 7ꢀ-[2-(t-butoxycarbonylamino)-2-(4-hy-

droxyphenyl)acetamido]-3-(1,3,4-thiadiazol-2-yl)thio-3-

cephem-4-carboxylate (24d). To a mixture of 23a

(1.93 g, 3.73 mmol), d-2-(t-butoxycarbonylamino)-2-

(4-hydroxyphenyl)acetic acid (1.28 g, 4.79 mmol) and

CH₂Cl₂(40 mL) was added 1,3-dicyclohexyl-

carbodiimide (825 mg, 4.00 mmol) at 10 ^ꢀC. After

stirring at room temperature for 2 hours, AcOEt

was added to the mixture and insoluble material

was ®ltered o. The ®ltrate was adjusted to pH 6.8

and the separated organic layer was washed with brine,

dried over magnesium sulfate and evaporated in vacuo.

The residue was subjected to column chromatography

on silica gel (eluent AcOEt:hexane, 2:1) to give 1.43g of

24d (52%): ¹H NMR (DMSO-d₆) d 1.38 (s, 9H), 3.53 and

3.81 (ABq, 2H, J=17.8 Hz), 5.16 (d, 1H, J=7.7 Hz), 5.21

(d, 1H, J=5.1 Hz), 5.56 (d, 1H, J=7.7Hz), 5.90 (dd, 1H,

J=5.1, 8.5 Hz), 6.66 (d, 2H, J=8.5 Hz), 6.97 (s, 1H), 7.1±

7.6 (m, 12H), 9.23 (d, 1H, J=8.5 Hz), 9.36 (s, 1H), 9.62 (s,

1H).

and evaporated under reduced pressure to give 0.56 g of

1

19 (89%): IR (KBr) 3263, 1709, 1373 cm

.

Potassium acetylhydrazinodithioate (21). A mixture of

AcOEt (25.8 g, 293 mmol) and hydrazine monohydrate

(13.3 g, 266 mmol) was stirred under re¯ux for 5 h and

then cooled to room temperature. To the solution were

added a solution of KOH (17.6 g, 314 mmol) in MeOH

(100mL) and carbon disul®de (22.1 g, 290 mmol). After

stirring at room temperature for 5 h, the resulting pre-

cipitate was collected by ®ltration, washed with MeOH

and dried in vacuo to give 13.0g of 21 (59%): IR (KBr)

1

2910, 1670, 1527, 1259cm ¹; H NMR (DMSO-d₆) d

3.33 (s, 3H), 9.31 (brs, 1H), 9.74 (brs, 1H).

2-Mercapto-5-methyl-1,3,4-oxadiazole (22). A solution

of 21 (29.6 g, 157 mmol) in pyridine (89 mL) was re¯uxed

for 18 h. After cooling to room temperature, pyridine

was evaporated, water was added to the residue, and the

solution was adjusted to pH 1.0 and extracted with

THF/AcOEt (3Â). The combined organic layer was

washed with brine (2Â), dried over magnesium sulfate

and evaporated under reduced pressure. The residue was

triturated with hexane and the resulting precipitate was

collected by ®ltration, washed with hexane and dried in

Benzhydryl 7ꢀ-(2-phenylacetamido)-3-(5-methyl-1,3,4-

thiadiazol-2-yl)methylthio-3-cephem-4-carboxylate (34).

To a solution of (5-methyl-1,3,4-thiadiazol-2-yl)methyl

thiobenzoate 33 (300 mg, 1.2 mmol) in a mixture of THF

(1 mL) and DMF (3 mL) was added 0.5 M sodium

methoxide methanol solution (2.4 mL, 1.2 mmol) at

ice-cooling and the mixture was stirred at the same

temperature for 40 min. The resulting mixture was

cooled to

70 ^ꢀC and a solution of 11 (579 mg,

1.0 mmol) in a mixture of THF (1.2 mL) and DMF

(3.6 mL) was added dropwise. After stirring at the same

temperature for 1.5 h, the solution was poured into a

mixture of AcOEt and water, and adjusted to pH 7.0

with 1N HCl. The separated organic layer was washed

with brine (2Â), dried over magnesium sulfate and

evaporated under reduced pressure. The residue was

chromatographed over silica-gel (eluent AcOEt:hexane,

2:1) to give 420 mg of 34 (67%): ¹H NMR (DMSO-d₆) d

2.66 (s, 3H), 3.50 and 3.58 (ABq, 2H, J=14.0 Hz), 3.84

and 3.92 (ABq, 2H, J=17.2 Hz), 4.61 and 4.66 (ABq, 2H,

J=15.4 Hz), 5.14 (d, 1H, J=4.9Hz), 5.69 (dd, 1H, J=4.9,

8.3 Hz), 6.87 (s, 1H), 7.2±7.6 (m, 15H), 9.17 (d, 1H,

J=8.3Hz).

1

vacuo to give 12.0 g of 22 (66%): H NMR (CDCl₃) d

2.44 (s, 3H), 11.50 (brs, 1H).

Benzhydryl 7ꢀ-amino-3-(1,3,4-thiadiazol-2-yl)thio-3-ce-

phem-4-carboxylate hydrochloride (23a). A suspension of

phosphorus pentachloride (14.2 g, 68.2 mmol) in CH₂Cl₂

(210mL) was stirred at ambient temperature for 30min.

To the mixture was added pyridine (553mL) at 15^ꢀC

with stirring. After stirring at 20^ꢀC to 15^ꢀC for

30minutes, to the resulting mixture was added 12e (27.9 g,

46.5 mmol) at the same temperature. After stirring at 10

to 5 ^ꢀC for 1.5 h, MeOH (28mL) was added dropwise to

the mixture at 20 to 10^ꢀC for 10min. After being stir-

red at ambient temperature for 50min, to the reaction

mixture was added water (56 mL) under ice-cooling, and

the mixture was stirred at the same temperature for 30min.

The separated organic layer was washed with water, dried

over magnesium sulfate and evaporated in vacuo. The

residue was triturated with AcOEt to give 15.3g of 23a

(64%): ¹H NMR (DMSO-d₆) d 3.82 (m, 2H), 5.29 (d, 1H,

J=5.0Hz), 5.40 (d, 1H, J=5.0 Hz), 6.99 (s, 1H), 7.2±7.5

(m, 10H), 9.71 (s, 1H).

2 - (Chloromethylthio)thiazole (37). To

a solution of

t-BuOK (8.98 g, 80 mmol) in DMF (160 mL) was added

2-mercaptothiazole (9.38 g, 80 mmol) under ice-cooling.

After stirring at ambient temperature for 20min, to the

mixture was added a solution of bromochloromethane

(11.4 g, 88mmol) in DMF (5 mL) at 5 ^ꢀC. After 1 h at

the same temperature, the reaction mixture was poured

into a mixture of AcOEt and water, and the separated

organic layer was washed with brine (3Â), dried over

magnesium sulfate and evaporated under reduced pres-

sure. The residue was chromatographed over silica-gel

(eluent AcOEt:hexane, 1:9) to give 9.83g of 37 (68%): ¹H

NMR (CDCl₃) d 5.22 (s, 1H), 7.37 (d, 1H, J=3.4 Hz),

7.82 (d, 1H, J=3.4 Hz).

Preparation of 23b was carried out by a similar method

to that described for 23a.

Benzhydryl 7ꢀ-amino-3-(5-methyl-1,3,4-thiadiazol-2-yl)-

thio-3-cephem-4-carboxylate hydrochloride (23b). Yield

2334

Y. Yoshida et al. / Bioorg. Med. Chem. 8 (2000) 2317±2335

Benzhydryl 7ꢀ-(2-phenylacetamido)-3-(thiazol-2-yl)thio-

methylthio-3-cephem-4-carboxylate (39). To a mixture

of NaSH (62 mg, 1.1 mmol) in DMF (2.3 mL) was

added DIPEA (194 mg, 1.5 mmol) at ambient tempera-

ture. After stirring at the same temperature for 30 min,

this solution was added to a solution of 11 (579 mg,

1.0 mmol) in DMF (5.2 mL) at 5 ^ꢀC and stirred at 0 ^ꢀC

for 1 h. A solution of 37 (215 mg, 1.3 mmol) in acetone

(4.3 mL) was treated with sodium iodide (390 mg,

2.6 mmol) and heated. After stirring at 50 ^ꢀC for 2 h, the

mixture was poured into a mixture of AcOEt and water,

and the separated organic layer was washed with brine

(3Â), dried over magnesium sulfate and evaporated

under reduced pressure. The residue in DMF (2 mL)

was added to the mercapto solution prepared above at

5 ^ꢀC. After stirring at 0 ^ꢀC for 40 min, the reaction

mixture was poured into a mixture of AcOEt and water,

and the separated organic layer was washed with brine,

dried over magnesium sulfate and evaporated under

reduced pressure. The residue was chromatographed

over silica-gel (eluent AcOEt:hexane, 1:1) to give 427 mg

of 39 (66%): IR (KBr) 1782, 1680, 1533 cm ¹; ¹H NMR

(DMSO-d₆) d 3.52 and 3.56 (ABq, 2H, J=14.0 Hz), 3.94

(s, 2H), 4.77 (s, 2H), 5.18 (d, 1H, J=4.6 Hz), 5.71 (dd,

1H, J=4.6, 8.4 Hz), 6.85 (s, 1H), 7.1±7.5 (m, 15H), 7.72

(d, 1H, J=3.4 Hz), 7.80 (d, 1H, J=3.4 Hz), 9.17 (d, 1H,

J=8.4 Hz); APCI-MS m/z 646 (MH⁺).

Benzhydryl 7ꢀ-amino-7ꢁ-methoxy-3-(5-methyl-1,3,4-thia-

diazol-2-yl)thio-3-cephem-4-carboxylate (47). To a solu-

tion of 46 (4.5 g, 6.04 mmol) in AcOEt (45 mL) was

added Girard T (2.03 g, 12.08 mmol) at ambient tem-

perature. After stirring at the same temperature for 8 h,

the mixture was poured into a mixture of AcOEt and

water, and the separated organic layer was washed with

brine (2Â), dried over magnesium sulfate and evapo-

rated under reduced pressure to give 3.18 g of 47

1

(100%): IR (KBr) 1781, 1737, 1697, 1606, 1377 cm

;

¹H NMR (DMSO-d₆) d 2.68 (s, 3H), 3.35 (s, 3H), 3.51

and 3.78 (ABq, 2H, J=17.4 Hz), 5.13 (s, 1H), 6.96 (s,

1H), 7.2±7.5 (m, 10H); FAB±MS m/z 527.0 (MH⁺).

(Z)-7ꢀ-[2-(2-Aminothiazol-4-yl)-2-hydroxyiminoacetoa-

mido]-3-(1,2,4-triazol-3-yl)thio-3-cephem-4-carboxylic acid

(2). A mixture of 51 (720 mg, 0.821 mmol), formic acid

(2.88 mL) and concd HCl (0.21 mL) was stirred at room

temperature for 2 h, then poured into a mixture of

AcOEt:acetone (2:1) and the resulting precipitate was

collected by ®ltration. The precipitate was dissolved in

sodium hydrogen carbonate solution (30 mL) and

adjusted to pH 3.0 with 1N HCl. The resulting solution

was subjected to column chromatography on Diaion

HP-20 (eluent 5% aqueous isopropyl alcohol), and the

desired fractions were evaporated under reduced pres-

sure. The resulting precipitate was collected by ®ltra-

tion, washed with water and dried in vacuo to give 185

1

(E)-7ꢀ-Amino-3-[2-(5-methyl-1,3,4-thiadiazol-2-yl)thio]-

vinyl-3-cephem-4-carboxylic acid (43). To a solution of

42 (2.0 g, 3.21 mmol) in a mixture of CH₂Cl₂(6.0 mL)

and anisole (2.0 mL) was added tri¯uoroacetic acid

(4.0 mL) with stirring under ice-cooling. The mixture

was stirred at the same temperature for 1 h. The reac-

tion mixture was poured into IPE (100 mL) and the

resulting precipitate was collected by ®ltration and dried

in vacuo. The precipitate was dissolved in a mixture of

sodium hydrogen carbonate solution and AcOEt at pH

7.5, and the separated aqueous layer was adjusted to pH

2.5 with 1N HCl with stirring. The resulting precipitate

was collected by ®ltration, washed with water and dried

in vacuo to give 800 mg of 43 (70%): ¹H NMR (DMSO-

d₆) d 2.72 (s, 3H), 3.62 and 3.92 (ABq, 2H, J=17.4 Hz),

4.85 (d, 1H, J=9.2 Hz), 5.06 (d, 1H, J=9.2 Hz), 7.12 (d,

1H, J=15.4 Hz), 7.17 (d, 1H, J=15.4 Hz).

mg of 2 (48%): IR (KBr) 1772, 1664, 1625, 1587 cm

;

¹H NMR (DMSO-d₆) d 3.34 and 3.49 (ABq, 2H,

J=17.0 Hz), 5.21 (d, 1H, J=5.0 Hz), 5.76 (dd, 1H,

J=5.0, 8.3 Hz), 6.64 (s, 1H), 7.10 (s, 2H), 8.68 (brs, 1H),

9.45 (d, 1H, J=8.3 Hz).

Preparation of 2⁰was carried out by a similar method to

that described for 2.

(Z)-7ꢀ-[2-(2-Aminothiazol-4-yl)-2-hydroxyiminoacetoa-

mido]-3-(1,2,4-triazol-3-yl)thio-3-cephem-4-carboxylic acid

D2-isomer (2⁰). Yield 37%; IR (KBr) 1765, 1658, 1633,

1531 cm ¹; ¹H NMR (DMSO-d₆) d 5.19 (s, 1H), 5.24 (d,

1H, J=5.0 Hz), 5.61 (dd, 1H, J=5.0, 8.4 Hz), 6.71 (s,

1H), 7.19 (brs, 2H), 7.26 (s, 1H), 8.58 (brs, 1H), 9.59 (d,

1H, J=8.4 Hz), 11.33 (brs, 1H).

Biological methods

Benzhydryl 7ꢀ-(3,5-di-t-butyl-4-hydroxybenzylidene

amino)-7ꢁ-methoxy-3-(5-methyl-1,3,4-thiadiazol-2-yl)thio-

3-cephem-4-carboxylate (46). To a mixture of benzhydryl

7b-(3,5-di-tert-butyl-4-hydroxybenzylideneamino)-3-(5-

methyl-1,3,4-thiadiazol-2-yl)thio-3-cephem-4-carboxylate

45 (8.1 g, 11.3mmol) in MeOH (325mL) was added 2,3-

dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 2.57g,

11.3 mmol) portionwise at 20 ^ꢀC. After stirring at the

same temperature for 30 min, AcOEt (325 mL) was

added to the mixture and stirred another 30 min at

20 ^ꢀC. The resulting mixture was evaporated and the

residue was chromatographed over silica-gel (eluent

AcOEt:hexane, 1:2) to give 4.58 g of 46 (54%): IR (KBr)

MIC. MICs were determined by the agar dilution

method. H. pylori cell suspensions (0.5 McFarland)

were prepared from cells grown on Brucella agar con-

taining 2% starch and 3% fetal bovine serum (FBS)

under 10% CO₂, 37 ^ꢀC for 72 h. Ten-fold dilutions of

these suspensions were inoculated on Brucella agar

plates containing 7% de®brinated horse blood and

serial dilutions of test compounds. MICs were read after

72 h incubation under 10% CO₂, 37 ^ꢀC.

Therapeutic ecacy in a mouse model. ICR mouse

infected with 10⁸CFU of H. pylori FP1757 were orally

treated with drugs two times per day for 4 days. The

number of viable organisms in the gastric mucosa 2

weeks after ®nal treatment were grown on a Brucella

agar plate containing 3% horse serum, 2% starch,

1

1776, 1737, 1627, 1429, 1373 cm ¹; H NMR (DMSO-

d₆) d 1.40 (s, 18H), 2.69 (s, 3H), 3.60 (s, 3H), 3.55 and

3.83 (ABq, 2H, J=17.4 Hz), 5.60 (s, 1H), 6.98 (s, 1H),

7.2±7.5 (m, 10H), 7.69 (s, 2H), 8.54 (s, 1H).

Y. Yoshida et al. / Bioorg. Med. Chem. 8 (2000) 2317±2335

2335

Skirrow's antibiotics, 10 mg/mL of nalidixic acid and

30 mg/mL of bacitracin.

9. Tomas, K. W. L.; Augustine, F. B. C.; Joseph, J. Y. S.;

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10. Moayyedi, P.; Sahay, P.; Tompkins, D. S.; Axon, A. T. R.

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Stability to ꢀ-lactamase. Stability to b-lactamase was

assayed photometrically.

Bactericidal eect. Bactericidal activity against H. pylori

was evaluated by monitoring viable cell counts in

Brucella broth containing 2% starch and 3% FBS.

14. Dekker, K. A.; Inagaki, T.; Gootz, T. D.; Huang,

L. H.; Kojima, Y.; Kohlbrenner, W. E.; Matsunaga, Y.;

McGuirk, P. R.; Nomura, E.; Sakakibara, T.; Sakemi, S.;

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Acknowledgements

We are grateful to Dr. David Barrett, Medicinal Chem-

istry Research Laboratories, for his help in preparing

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studies on anti-helicobacter pylori agents. Part 2: New cephem derivatives

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