sartorelli 1977

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Tetramisole Analogues

Ta b le 111.

E D , ( m g / k g )

of

Penicillin Derivativesa

Gram posi tiveb Gram nega tiveb

N o. S.a.

S.p.

S.S. E.c.

J o u r n a l

of

Medicinal Chemistry, 1977

Vol.

20, No. 4 563

mixture was stirred at -25 C for 30 min. A chilled solution of

bis(Me3-6 -APA) [from 4.3 g (0.02 mol) of 6-APA an d hexa-

methyldisilazine] in CHCl:, (50mL) was added and th e mixture

was stirred a t -20 C for

1

h and allowed to come to room

temperature. Dioxane (100mL) was added, and the mixture was

fil tered. Water (0.2 mL) was added to th e fil trate; the mixture

was stirred for 30 min an d filtered free of unreacted 6-APA. Te n

milliliters of a 2 N butano l solution of sodium 2-ethylhexanoate

a n d 1L of ether were added to the filtrate. T he precipitated

product was filtered off, dissolved in methanol (100 mL) , a n d

reprecip itated by th e addition of ether. T he yield was 3.6 g

of

white powder, which decomposed over a 20 C range beginning

at 120 C:

R

Kl3r) 1780,1660,1610,1580,1540,1400,1320, and

765 cm-'; NM R (D2 0-DS S) 6 1.60 (d,J

=

6 Hz, 6 H ), 4.25 s, 1

H) , 4.8

(s,

2 H), 5.57

(s,

2 H), 6.4-6.8 (m ,

2

H ), 7.5-7.9 (m, 2 H);

iodine ti tration 80.3%.

6-[2-[2-0xo-1(2H)-quinoxalinyl]acetamido]-3,3-di-

methyl-7-oxo-4-thia-l-azabicyclo[

.2.01 heptane-2-carboxylic

A c i d S o d i u m Salt (24) (Method

C .

A solution of 1,2-di-

hydro-2-oxoquinoxaline-1-aceticcid (3.3 g, 0.016 mol) in D M F

(20

mL)

was chiued

to

-10 C and carb ony ldiim idaz ole 2.6 g, 0.016

mol) was added. Th e mixture was s t i r red a t

0

C under an N2

atmosphere for 20 min, warmed to room temperature, and placed

under a vacuum for 5 min (to remove the COz liberated during

th e imidazo lide formation ). A solution of 6-APA (3.5 g, 0.016 mol)

and triethylamine (3.2 g, 0.032 mol) in CHC13 (100 mL ) was adde d

and the reaction mixture was stirred for

18

h under an N2 a t -

mosphere. Eigh t milliliters of a 2 N bu tanol solution of sodium

2-ethylhexanoate and

1

L

of

ether were added. Th e mixture was

filtered and the product was reprecipitated from methanol with

ether to give 3.5 g of whi te powder: m p 227-229 C dec;

IR

KBr)

1770,1660,1620, and 760 cm-' NM R (DzO-DSS) 1.58 (d,J

5 Hz, 6 H ), 4.3

(s,

1H), 5.05

s, 2

H) , 5.55

s,

2 H) , 7.15-8.9 (m ,

4 H ), 8.2 a, 1 H); iodine ti tration 98%.

R e f e r e n c e s and Notes

1) J. H. Dewar and G. Shaw, J. Chem. Soc., 3254 (1961).

(2) M. R. Atkinson, G. Shaw, and R.

N.

Warrener,

J.

Chem.

Soc., 4118 (1956).

(3) K. E. Price, A. Gourevitch, and L. C. Cheney, Antimicrob.

Agents Chemother., 670 (1966).

(4) Agar dilution assay, described in M. L. Edwards, R. E.

Bambury, and H.

W.

Ritter, J.Med. C hem., 19,330 (1976).

(5)

Prepared from chlorotrimethylsilane, triethylamine, and

4-hydroxypyridine in toluene (reflux,

18

h).

2 1

3 .5 4 7 l o o l o o

2 2

2.5 5.2

> l o 0

> l o 0

2 3

1 . 2 6 . 0 > l o 0 > l o 0

2 4

<1 0 3.2 100 >lo0

2 5

< 1 . 0 5 . 3 11 3 0

2 6

1 9 1 9 5 2 > l o 0

2 1 6.7 6.8

100 > lo0

2 8 <1 0 10 6.5 > l o 0

2 9

4.4 37

l o o

30 3 2 3 .6 2 9 > l o 0

31

1 4 2 . 8 1 9 31

3 2

1.0

5 . 6 1 6 > l o 0

33

1 . 0 5 . 3 3 6

> l o 0

3 4

<1.0

2 .4 2 8 3 6

3 5

<1.0 5.2 44 6 8

3 6 <1.0 4 . 3 1 9 l o o

31

1.1 6 . 7 1 6

100

38

<1.0

10 4 8 l o o

3 9

<10 1 9 . 3

> l o 0

> l o 0

Penic il l in G 1 . 0 1 0

Ampicill in

25

Ch lo ro my c e t in

5 0

a

Ea c h c o m p o u n d w a s g iv en t o mic e i n fe c t ed w i th a f a t a l

infec t ion .

Doses of 1 , 10, or 100 mg/kg were g iven in fou r

d o ses a t -1 , 1, 1 9 , a n d 2 5 h .

The challenge was given

a t

0

h. ED,, ca lcula ted according t o L. J. Re e d a n d

H.

Mu e n c h , A m.

J.

H y g ., 2 1 , 4 9 3 , 4 9 7 (1 9 3 8 ) .

S t a p h y lo c o c c u s a u re u s ; S.p .

=

St re p to c o c c u s p n e u mo n ia e ;

S.S.= Salmonel la schot tm uel le r i ; E.c . = Escherichia coli .

S.a. =

dissolved in methanol (150 mL, som e insoluble material, filtered

off) and reprecipitated with ether: yield of reprecipitated product

6.8 g; m p 219-221 C dec; IR (KBr) 1760, 1670, 1620, an d 770

cm-'; NMR (DzO-DSS) S 1.58 (d,J 4 Hz, 6 H) , 4.27 s ,

1

H),

4.88 (s,

2

H) , 5.62 s, 2 H ), 7.5-8.3 (m , 5 H); iodine titration 92.2%.

3,3-Dimet hyl-6-[2-[2-oxo-1(2H)-pyridyl]acetamido]-3,3-

d i m e t hyl-7-0~0-4-thia-l-azabicyclo[. 2 . 0 l h e p t a n e - 2 -

c a r b o x y l i c A c id S o d i u m

Salt

(25) (Method

B). A

solution

of 1 2-dihydro-2-oxopyridine-l-aceticcid (3.1 g, 0.02 mol) a nd

triethylamine (2 g, 0.02 mol) in D MF

(50

mL) was chilled

to

-25

C.

Ethyl chloroformate (2.17 g, 0.02 mol) was added and the

Tetramisole Analogues as Inhibitors of Alkaline Phosphatase, an Enzyme Involved

in the Resistance of Neoplastic Cells to 6-Thiopurines

Kuldeep K. Bhargava, Men

H.

Lee, ' Yow-Mei Huan g, Linda S. Cunningham, Krishna C. Agrawal,

and Alan C. Sartorelli'

D e p a r tme n t of Pharmacology a nd Section of Developmental The rapeu tics, Comprehensive Cancer Center, Yale University

School

of

Medicine, New Haven, Connecticut 06510. Received July

19 1976

A series of tetramisole derivatives was synthesized and tested for inhibitory activity against alkaline phosphatase

which was partially purified from a murine ascitic neoplasm resistant to 6-thiopurines (Sarcoma 180/T G). Thes e

agents included derivatives sub stituted with halogens, CH3,

or

NO1 groups a t e i ther the meta or para position of

the phenyl ring of tetramisole an d 2,3-dehydrotetramisole. T he phenyl ring of tetramisole an d 2,3-dehydrotetramisole

was also replaced by a naph thyl ring, and t he phenyl ring of 2,3-dehydrotetramisole was su bstitu ted by a thienyl

ring system. T he presence of both the thiazolidine and dihydroimidazole rings of tetramisole was found

to

be essential

for enzyme inhibitory activity. Sub stitutio n of

a

naphthyl for the phenyl group and dehydrogena tion a t th e 2 ,3

position of the thiazolidine ring were found to significantly enhan ce inhibitory activity for alkaline phosphatase.

Tests employing

(S)-(-)-6-(4-bromophenyl)-2,3,5,6-tetrahydroimidazo[2,1-b]thiazole

xalate in combination with

6-thioguanine demon strated t ha t the inhibitor of alkaline phosphatase was capable of increasing the toxicity of

6-thioguanine to Sarcom a 180/T G cells in t issue culture.

W e h av e p ro vi de d e v i d e n ~ e ~ - ~hich demonstrates th at

the acquisition of insensitivity to the antileukemic 6-

thiopurines (Le., 6-mercaptopurine an d 6-thioguanine) by

the murine neoplasm Sarcoma 180/TG nd by acute

lymphocytic leukemia cells of man is at least in part due

to an increase n the activity of a particulate-bound alkaline

phosphatase(s) which causes an elevation in the rate of

degradation of the active tumor-inhibitory nucleotide



564

Table I.

Journal of Medic ina l Chemist ry , 1977, Vol. 20, N o . 4

Sartorel l i e t

al .

In t e rme d ia t e s in t h e P re p a ra t io n of Analogues of 2 ,3 -D e h y d ro te t r a miso l e a n d Te t ra miso le

___-

~ _

1 -9 1 0 -1 4

C o m p d R R’ Method Mp, “C

15-18 1 9 , 2 0

so lv e n t 72 Formula Analyses

Kecrystn Yield,

1

2

3

4

5

6

7

8

9

1 0

11

1 2

13

1 4

1 5

16

1 7

18

1 9

20

H

H

H

H

COCH,

COCH,

COCH,

COCH,

COCH,

COCH,

COCH,

COCH,

COCH,

COCH,

H

H

COCH,

COCH,

COCH,

COCH,

A

A

A

A

B

B

B

B

B

C

C

C

C

C

D

D

B

B

C

C

1 4 4 - 1 4 5

1 2 6 - 1 2 8

1 6 5 - 1 6 6

1 7 3 -1 7 4

2 3 4 -2 3 6

1 4 3 - 1 4 5

1 6 4 - 1 6 5

1 7 8 - 1 8 0

1 6 8 - 1 6 9

2 3 4 - 2 3 5

1 3 0 - 1 3 2

1 3 9 - 1 4 1

1 9 7 - 1 9 8

5 5 -5 6

2 8 8 -2 9 0

1 1 5 -1 1 7

1 3 0 - 1 3 2

1 2 7 - 1 2 9

1 1 2 - 1 1 4

290-dec

EtO H 9 5 C. ,H ,BrN ,O S C, H, N

E t O H

E t O H

CH,C,H,

CH, C,H

~

E t O H

E t O H

CH,C,H,

CH,C,H,

Et OH-H, 0

CH3

‘ 5

CH3C6H5

C H 3 C 6 H 5

CH,Cf,H,

EtOH

CH , , H 5

CH,C,H

C H

c 6 H ,

CH,C,H,

EtOH-H,O

9 6

9 8

9 2

80

76

7 9

7 2

7 8

8 4

18

8 0

81

7 8

9 2

9 1

8 0

1 4

8 4

8 3

C:

H, N

C,

H ,

N

c , H, N

C, H, N

c,

H,

N

C, H, N

C , H , N

C ,

H,

N , S

c, H, N

C, H, N

C, H, N

C, H, N

C,

H,

N , S

c, H, N

C, H, N, S

C, H, N

C , H, N , S

C, H,

N

C,

H, N,

S

a 2 -N a p h th y l .

form (s) of the 6-thio purines. Th e availability of a potent

inhibitor of alkaline phosphatase would appear to have

clinical utility in these situations, since in theory such an

agent might restore sensitivity to the 6-thiopurines.

Th e anthelmintic tetramisole [ f)-2,3,5,6-tetrahydro-

6-phenylimidazo[2,1- lthiazole hydrochloride] and c ertain

of its analogue s have recently been rep orted to be relatively

potent stereospecific inhibitors of nonspecific alkaline

phosphatase activity (orthophosphoric monoester phos-

phohy drolase, E .C. 3.1.3.1.).5-9Th is class of compounds

is inhibitory toward the alkaline phosphatases of most

mam malian tissues; the in testin al enzyme, however, is an

exception, being resistant to the action of these agents6

Since this class of agen ts appeared to have po tential as

inhibito rs of th e en zym e(s) of neopla stic cells, we examine d

the inhibitory poten tial of several available tetramisole

derivatives using a partially purified particulate-bound

alkaline phosphatase

of

Sarcoma 180/TG, the 6-thio-

guanine insensitive variant of Sarcoma 180 which appears

to have achieved resistance through elevation of alkaline

p ho sp ha ta se (s ) a ~ t i v i t y . ~he most pote nt agent tested

in this initial study was

(*)-6-(m-bromophenyl)-5,6-di-

hydroimidazo

[

2 , l -b] thiazole oxalate (R8231). T h e

mechanism of inhibition of alkaline phosphatase from

Sarc om a 180/T G caused by this class of comp ounds was

of

the uncompetitive type , a finding which corroborated

an earlier report6 documenting this type of inhibition w ith

other enzy me systems.

In the present study th e relationship between structure

and inh ibitory potency against alkaline phosphatase for

derivativ es of this class was expande d by the syn thesis and

testing of a series of analogues for inhibitory activity

toward partially purified alkaline phosphatase from

Sarcoma 180/TG ascites cells.

Chemistry. A

series of meta- and para-substituted

phenyl derivatives of (*)-2,3-dehydrotetramisole and

(&)-tetram isole was synthesized following th e proc edures

described by Raeym aekers e t al.1° In th e case of 2,3-

dehydrotetramisole the para substituen ts were

Br,

CH3,

or NOL

and in one instance both the meta and para

sub stitu ent s were CH, groups. In the tetramisole series

Scheme

I

A C 2 0

p y r i d i n e

SOC I 2 / A C p O

J

R<NYs)

the m eta substitu ents were CH3 or NOz and the para

substituen ts were CH3, Br, C1,

or

F.

In addition, the

phenyl group was replaced by a nap hthy l ring system in

an at tempt to examine the effect of increased bulk a t this

position on enzyme inhibitory potency. T he phenyl ring

of 2,3-dehydrotetramisolewas

also

substituted by a thienyl

ring according to the synthetic procedure of Raeymaekers

et a1.l0 T he proce dure employed for th e syn thesis of

2,3-dehydrotetramisole analogues involved condensation

of a bromomethyl aryl ketone an d 2-aminothiazole, fol-

lowed by acetylation, sodium borohydride reduction, and

ring closure with thionyl chloride and acetic an hydride

(Scheme

I).

A similar sequence

of

reactions w as utilized

to obtain the tetramisole analogues except tha t m ethod

D

was employed for the condensation of th e bromomethyl

aryl ketone with 2-aminothiazoline. T he newly synthesized

intermediates in this sequence of reactions are listed in



Tetramisole Analogues

Table 11. Subs t i tu ted 2 ,3-D ehydro te t ramis oles a n d Te t ra miso le s

Journal of Medic inal Chemistry

1977, Vol.20 No.

4

565

C o m p d R

2 1 - 23 2 4 , 2 5

Yield,

MP, C Form ula Analyses

2 -N a p h th y l .

Scheme I1

H

H

/ e t h y l e n e o x i de /

N oO H

I

CHZCHZOH

Table

I

and the new analogues of tetramisole

or

2,3-

dehydrotetramisole are lis ted in Table

11.

T o determine th e necessity for

an

intac t thiazolidine ring

system in the tetram isole analogues for inhibition of th e

activity of alkaline phosphatase, we have synthesized

1-8-

ydroxyethyl-2-mercaptomethyl-4-phenylimidazole

(27). Compound 27 was obtained by th e procedu re shown

in Scheme

11;

this involved initially reacting 2-mercap-

to-4(5)-phenylimidazole with methyl iodide to yield th e

corresponding -SCH3 derivative 26, which was then

converted to the desired product (27) using ethylene oxide

in th e presence of a catalytic am ount of NaOH.I2

Bio log ica l Re s u l t s an d D is cus s ion . T he concen-

trat ion s of analogues of 2,3-dehy drotetramiso le and t et-

ramisole required to produ ce 50% inhibition of the activity

of a particulate bou nd alkaline phosp hatase from Sarco ma

180 /TG ascites cells are shown in Table

111.

Intermediates

providing a series of structural mo difications on either t he

thiazoline, thiazolidine , or the imidazole rings were also

tested for their capacities to inhibit alkaline phosphatase

but are not included in Table 111, since none of these

derivatives, listed in Table I, nor compounds

26

and

27

were found t o possess significant inhibitory activity u p to

a concentration of a t least 5 X

M.

These findings

suppo rt the concept tha t both the thiazolidine and di-

hydroimidazole rings are required for inhibitory potency

toward alkaline phosphatase. Th e data of Table

I11

demo nstrate th at substitution of either

an

electronegative

group at the para position of the p henyl ring

or

the bulky

nap hthy l ring system in place of the phenyl group increases

th e inhibitory activity of these com pounds relative to the

unsu bstitute d derivative. Dehydrogenation of the thia-

zolidine ring of compound

25

a t positions 2,3 to give

23

increased inh ibitory activity; however, dehydro genation

of other analogues (Le., p-B rPh and p-CH sPh derivatives)

did not markedly alter potency. Compound

23

was the

most active agent tested as an inhibitor of alkaline

Tab le 111. Co nce ntra tion s of Analogues of

2 ,3 -D e h y d ro te t r a miso l e a n d Te t ra miso le Re q u i re d fo r

50

Inhib i t ion of the Act iv i ty of Part icu la te Bound Alka l ine

Ph o sp h a ta se f ro m Sa rc o ma 1 8 0 / TG A sc i te s Ce l ls

150

Co rn ud a S t ru c tu re R R ' uMb

2 1

2 2

R 8 2 3 1

2 3 5 ,6 -D ih y d ro -6 -

(2-naphthy1)imidazo-

[

2 , 1 4 1 h ia z ol e

(2- th ieny1)imidazo-

[ 2 ,1 4 1 h i a z o le

5 ,6-Dihydro-6-

R

R b N y S

24

25 2 ,3 ,5 ,6-Te trahydro-6-

(2-naphthy1)imidazo-

[2 ,1 4 1 h i a z o le

H H 4 5 .0

Br H 1.3

8 .3O, H

CH, CH,

11.0

6.6H , H

H Br 2 .0

1 . 0

50.0

H H 3 8 . 0

Br H 2.6

C1 H 4. 5

F

H 1 6 . 0

CH , H 8.1

H CH , 4 0 .0

H NO; 38 .0

9.0

Com poun ds which a re no t numbere d were synthesized-

fo r s tu d i e s of st ruc ture -ac t iv i ty re la t ionships according to

p u b l i sh e d p ro c e d u res . A l l c o m p o u n d s w e re t e s t e d as

oxala te sa l ts . The

I,,

i s the con centra t ion of drug re -

quired to r e d u c e b y 50 the observed ac t iv i ty of t h e e n -

zyme. The ac t iv i ty of a lka line phospha tase u sed in these

e x p e r ime n t s w a s 4 3 6 0 u n i t s lmL; t h e a c tiv i ty u n i t s fo r t h e

e n z y me w e re d e f in e d p rev iou s ly . Fo r e a c h e x p e r ime n t

50

p L o f e n z y m e w a s u se d .

phosp hatase, requiring only one-half th e conc entration of

R8231, which was previously re rte d to be th e most active

of enzyme activity. T o determine wh ether inhib itors of

alkaline phosphatase of this class have the potential to

restore the sensitivity of Sarcoma 180/TG to 6-thio-

guanine, the effects of (S)-(-)-6-(4-bromophenyl)-

2,3,5,6-tetrahydroimidazo[2,1-b]thiazole xalate L-p-

bromotetramisole) on t he growth-inhibitory properties of

6-thioguanine were measured in tissue culture.

L-P-

age nt of th e tetramisole series,6o produce

50

inhibition



566

J o u r n a l of Medicinal Ch emis try,

1977, Vol. 20, No. 4

Table IV.

of

S arcom a l 80 /T G Ce ll s in Cul tu re

Effec t

of

12-p-Brom ote t ram iso le n the Gro wth

________ _

____

R a t i o

of

Sartore l l i e t a l .

was stirred at room tem perature for 2 h, solvent was removed

under vacuum, and the residue was suspended in water and

extracted with chloroform. Th e chloroform layer was dried

(anhydrous Na2 S04 ) nd t he solvent was removed

to

leave a solid

which was crystallized from an appropriate solvent.

Me th od D. 2-Aminothiazoline (0.01 mol) was dissolved in 20

mL of acetonitrile and was added in small portions to

0.01

mol

of bromomethyl aryl ketone. Th e mixture was stirred at room

temperature for

30

min and filtered. Th e precipitate was dried

and m acerated with a

10

Na2 C0 3 olution. Th e free base was

the n crystallized from ethanol.

C y c l i z a t i o n R e a c t i o n

for

T e t r a m i s o l e a n d 2 ,a - De h yd r o-

te t ram iso le Der iva t ives . The

2-acetylimino-3-(2-hydroxy-

arylethyl) d erivatives

(0.005

mol) obtained from method C were

added

in

small portions to

3.0

mL of thionyl chloride at 5 C over

a period of 30 min. Th e mixture was stirred a t room temperature

for

1

h, AczO (15 mL) was added, and the acetyl chloride which

formed was removed under vacuum. The m ixture was refluxed

for another

0.5

h an d the excess Ac20was removed under vacuum.

The residue was dissolved

in

dilute HC1 an d filtered. Th e filtrate

was made alkaline (pH 8.5) and extracted with chloroform. Th e

chloroform layer was dried (anhydrous N a2S0 4) and f lash

evaporated. Th e residue was dissolved in a minimum am ount

of

2-propanol and oxalate

d t s

were prepared by addin g a solution

of oxalic acid in 2-propanol. Th e final produc ts as oxalates were

recrystallized from ethanol.

2-Mercaptomethy1-4(5)-phenylimidazole.

solution of 0.352

g (0.002 mol) of

2-mercapto-4(5)-phenylimidazole1'

nd methyl

iodide (0.3 g,

0.003

mol) in 20 mL of methanol w as stirred for

4

h a t room temperature. Solvent was removed by flash evaporation

and th e residue was macerated with 10% N aH C0 3 solution and

crystallized from ethyl alcohol-ethyl ether as white prisms

to

yield

0.27 g (75%), m p 131-133 C.

Acknowledgment. This research

was

upported in part

by U.S. Public Health Service Grants CA-02817 and

CA-16359 from the National Cancer Institute.

Dr.

Robert

Legendre of Janssen

R

D, Inc., generously supplied

(S)-(-)-6-(4-bromophenyl)-2,3,5,6-tetrahydroimidazo-

[2,1-b]thiazole oxalate.

References and Notes

(1)

Special Fellow of the Le ukem ia Society of America.

(2) M.

K.

Wolpert,

S.

P.

Damle, J.

E.

Brown, E. Sznycer,

K.

C.

Agraw al, an d A. C. Sartorelli, Canc er Res., 31,16 20 (1971).

(3)

M. Rosman, M. H. Lee, W. A. Creasey, and A. C. Sartorelli,

Cancer Res.,

34,

1952 (1974).

(4)

A, C. Sartorelli, M. H. Lee,

M.

Rosman, and K. C. Agrawal,

Pharmacological Basis of Cancer Chemotherapy , W illiams

and Wilkins, Baltimore, Md., 1975, p 643.

(5) D.

C, I. Thienpont , 0. F.

J.

Vanparijs,

A.

H. M. Raey-

maekers, J. Vandenberk, P. A.

J.

Demoen, F . N. Allewijn,

R. P. H. Marsboom, C.

J.

E. Niemegeers,

K. H. L.

Schel-

lekens, and

P. A. J.

Janssen, Natu re (London),

209,

1084

(1966).

(6)

H.

Van Belle, Biochim. Biophys. Acta, 289,

158

(1972).

(7) M. Borgers,

J .

Histochem. Cytochern., 21,

812

(1973).

(8) M. Borgers and F. Tho ne, Histoche mistry , 44, 273 (1975).

(9) M. H. Lee, Y. M. Huang,

K.

C. Aga wal, and

A.

C. Sartorelli,

Biochem. Pha rma col., 24, 1175 (1975).

(10)

A.

H. M. Raeymaekers,

F. T.

N. Allewijn,

J.

Vandenberk,

P. J.

A. Demoen,

T. T.

T. Van offenvert , and P .

A.

J.

Janssen,

J.

M e d .

Chem. 9

545 (1966).

11)

G.

R.

Clemo,

T.

Holmes, an d G. C. Leitch,

J .

Chern.

Soc.,

753 (1938).

(12)

K.

Butler, H. L. How es,

J.

E. Lynch, and

D. K.

Pirie, J . Med.

Chem.,

10,

891 (1967).

(13) M. H. Lee and A. C. Sartorelli, Biochim. Biophys. Acta, 358

69 (1974).

Growt l i (cel l no . /m L

(

guan ine /

x

1 0 ' )

L-o-Brom o-

- _ _ _ _ _ ~ _ ( -

b6- th io -

G' tramisole (-)-6-Thio-

( t

)-B-Thio-

guanine

concn , f iM guanine guanine

x 100

0

2 . 5 2 . 0 80

10

3 . 6 2 . 5

69

5 0 3 . 5 2 .3 6 6

100

1 . 3 0 . 6 4

4

9

Log phase S a rcom a 180 /TG ce l ls were t rea ted w i th the

ind ica ted concen t ra t ions

of

L-p-brom ote t ram iso leoxa la te

in the presence or absence of 6-th ioguan ine

(

96 h. Cells were inoculated a t a conce ntra t ion

of l o

cel ls /mL in F ischer 's m edium supp lemen ted with

10

horse s erum and incuba ted a t 37

C .

bers were determ ined us ing a Model ZBI Coulter coun ter .

M )

f o r

At 9 6 h , cel l num -

Brom otetramisole was selected for such tests since (a) it

is a relatively stable , water-soluble derivative of this class

and (b)

it

is among the mos t potent known inhibitors of

the alkaline phosphatase activity of this neoplasm I5 , ,=

1.8 X M). Tes ts in culture showed that L-p-bromo-

tetramisole required

a

concentration of

lo-*

M

to cause

inhibition of the growth of Sarcoma 18 0/TG (Table IV).

At this level, L-p-brom otetramisole produced s ignificant

enhancement of the toxicity of 6-thioguanine to Sarcom a

180 /TG ; however, the high concentration of the alkaline

phosph atase inhib itor required to accomplish this effect

in vitro indicates the need for an even more efficacious

derivative to achieve restoration of sensitivity of neoplastic

cells to 6-thioguanine in vivo.

Experimental Section

Melting points were determined on a Thomas-Hoover capillary

melting point app arat us and are uncorrected. Analyses were

performed by t he B aron Consulting C ompany, Orange, Conn.

Spectral data were obtained using a Perkin-Elmer grating

IR

spectrophotometer, Model 257, and a Varian T-60A NMR

spectrophotometer. Tetrameth ylsilane was used as an internal

stan dar d for NM R determinations. NM R and IR spectra were

as expected.

Inh ib i t ion

of

Alk al ine P hos phat ase . Alkaline phosphatase

from th e murine ascitic neoplasm Sarcoma 1 80/T G was isolated

and partially purified according to a procedure previously de-

~ c r i b e d ; ' ~he solubilized enzyme (enzyme A) after the ethanol

fractionation step was employed in this investigation. Th e assay

procedure was the same as previously reported.'

S yn thes i s

of

In te rm edia te s of 2,3-Dehydrote tramisole and

Te tra mi sol e. M eth od A. Bromomethyl aryl ketone (0.02 mol)

was refluxed for

1

h with 0.02 mol

of

2-aminothiazole in 25 mL

of 2-propano l. Th e resulting solid, 2-imino-3-aroylmethyl-

thiazoline,

was

filtered, dried, an d macerated with a 10% Na2C 0,,

solution. Th e mixture was filtered and crystallized from an

approp riate solvent as listed in Table I.

M e t h o d

B.

T o a mixture of 2-imino-3-aroylmethylthiazoline

(0.01 mol) and 5

mL

of pyridine in

50mL

of ch loroform was added

acetic anhyd ride (0.02 mol). Th e mixture was refluxed for

1.5

h an d the chloroform was removed by distillation to leave an oil

which was crystallized by maceration with petroleum ether. The

acetate was then recrystallized from an appropr iate solvent.

M e t h o d

C

T o a solution of

2-acetylimino-3-aroylmethyl-

thiazoline

(0.005

mol) in 25 mL of methanol maintained a t

10 O C

was added in small portions 0.0025 mol of NaBH 4. T he solution

sartorelli 1977

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