VOL.

XXIX

NO.

3

THE

ANTITUMOR

JOURNAL

OF

ACTIVITY

ANTIBIOTICS

OF

DERIVATIVES

MYCOPHENOLIC

by

SUZUKI,

SAKAE TAKAKU

Research

Laboratories, Chugai Pharmaceutical Toshima-ku, Tokyo, Japan

for publication

One hundred

and eight derivatives

modifications

at

the

phenolic

OF

ACID

SEIKICHI

(Received

275

and

TAKASHI

September

and/or

Co.,

Ltd.

27, 1975)

of mycophenolic

hydroxyl

MORI

acid (MA) have been prepared

carboxyl

sites.

compounds was as effective as MA in suppressing cell growth whereas several compounds with changes at both the hydroxyl were more effective than MA against EHRLICH solid carcinoma

None

of

these

of L-5178Y cell in vitro, and carboxyl groups and L-1210 leukemia

in mice.

Mycophenolic

acid, a metabolite

of Penicillium

first isolated

in 1896, has been shown

by us and

other several groups to have antiviral, antitumor, antifungal and immunosuppressive properties. Studies on these activities of MA by our co-workers have been reported in this journal. 1,2,3,4) MA has been reported This antibiotic against

this

form

activity

against

unique

vatives

is relatively

the ascitic

increased

active against

antitumor

inactive

several

against

murine

murine

leukemias

of the WALKER 256 tumor. murine

agent.

leukemia Previous

have not been successful.O,T)

may

solid tumors and WALKER 256 in rat.2,s,3)

result

attempts

in an

by others

We now

report

Materials

and

and ascites

Chemical

several

tumors

transformation

but it is active to a product

increase

in clinical

to synthesize

more

derivatives

with

usefulness

active

with increased

of

MA deriactivities.

Methods

Agents All the agents tested in this paper were synthesized in our laboratories. For administration to mice, compounds were dissolved or suspended in saline or saline containing 0.25 ,°o carboxymethylcellulose. Antitumor activity against experimental tumor To develop solid EHRLICH tumor (ESC), ddy/S male mice (4 weeks old) were inoculated subcutaneously with 5 x 108 EHRLICH ascites cells. L-1210 leukemia cells (1 X 10°) were Infected intraperitoneally into (C3H/He x DBA/2) hybrid mice. Minced X-5563 myeloma tumor (5 mg) was implanted subcutaneously into C3H/He mice. Eighteen hours after inoculation with tumor cells intraperitoneal treatment was initiated and continued for 5 consecutive days with various dose levels of compound. Antitumor activity was evaluated by comparison of the tumor weights of control and treated mice with ESC and X-5563 tumors (10th and 12th days after implantation respectively) and the life-span in L-1210 leukemia mice. Inhibition L-5178Y

of the cells

growth

were

grown

of

L-5178Y at 37°C

cells in

in vitro

CO,-incubator

of calf serum. L-5178Y cells (4.0 x 10' cells growth were preincubated in glass tubes for of derivatives were added per tube, and these cells were determined with hemocytometer. agent was obtained graphically.

in RPMI-1640

medium

containing

10

in 0.9 ml medium) in the exponential phase of 20 minutes. After preincubation, 0.1 ml aliquots were incubated for 48 hours. The number of TCID68 (50 % tissue culture inhibitory dose) of

276

THE

Rf value

of thin-layer

JOURNAL

OF

ANTIBIOTICS

MAR.

chromatogram

To obtain some information about the correlation between the biological derivatives and their lipophilic character, Rf values of thin-layer chromatograms by the method of HUSSAIN and LIEN,8) using Eastman chromatogram silica gel and n-BuOH - AcOH - H2O (4:2:1) as developing solvent. After development, the compounds were visualized by means of complexation with 12 and Rf values

Results

MA has a low order to enhance

inhibitory

the activity

activity

effect. was lost

oxidation resulted

nucleus

part, into

against

all features

of

by

methylol

in disappearance

the

MA

structure

of

MA

of methoxyl

to convert

are

essential

We attempted

for

study revealed

structure. group,

For

oxidation

or shortening

in antitumor

1).

product.

preliminary

and lengthening

we attempted

group into enzymatically original forms in vivo.

of this natural

conversion

of or decrease

In this investigation

both L-1210 and ESC (Table

and our

demethylation

activities of the were determined thin-layer sheets the locations of were calculated.

Discussion

modification

references6.8)

or decreased

of olefinic

to benzene

that

Foregoing

and

activity

by chemical

It is of significance inhibitory

1976

retaining

example,

reduction

of methyl

of the

the

that the inhibitory or

group attached

terpene

side

chain

all

activity.

the phenolic

hydroxyl

group

labile functional groups, that could probably That is, we intended to take a course of drug

and/or the carboxyl

be converted to their latentiation but not of

drug design. Table against

1 indicates

ESC

37 mg/kg

and

carboxyl

acylation

the

unchanged.

hydroxyl

the anti-ESC in decrease

of 150 mg/kg

against

L-5178Y

or in several

activity

of the hydroxyl

anti-L-1210

activity

Compounds is intact.

by modification

Compounds

group

had

cells

exceptions

against

group.

and

T/C

at dosages

product

1).

As

with

21 to 30 are carboxyl

acylation,

showed group

or lower group

hetero-atom(s),

activity

against

modifications. did not show

Because

some

compounds

ESC.

Alkyl

values of 75 or

any improved obtained

products

tendency

effect against

On

did

not

acid

improve

the phenolic

reverse

alkyl

alkanoic

the

ESC.

MA against

is the

the

but sometimes

8 to 20 are carbamoyl

in which

over

leaving

the whole,

higher

effects against

24 to 26, in which

by acylation

with

carbamoylation

activity

group

activity,

Compounds

satisfactory

modified

This

esters

products.

Acylation

both L-1210 and ESC.

but some compounds

hydroxyl

on the anti-L-1210

Simple alkyl esters 21 to 23 had increased

had equal

of the phenolic

1 to 7 are acylated

no influence

(acetylation

of activity

derivatives

hydroxyl

of 108 derivatives

dosages

1 to 20 were prepared group

of

enhanced resulted

at

of test product.

Compounds the

TCIDbo values

L-1210

of

groups

hydroxyl

L-1210, that

but they

observed

are substituted

in by

L-1210.

or carbamoylation

of the phenolic

hydroxyl

group have more intense activity against ESC than MA and in some cases the esterification of the carboxyl group enhanced the anti-L-1210 activity, we synthesized compounds 31 to 108 in which

both

the hydroxyl

As was expected, was

converted

Similarly, favorable

into

simultaneous

and carboxyl

compound methyl

are derivatized.

32 (the hydroxyl

ester)

showed

introduction

in case of modification

groups

of

of the

the

group

enhanced functional

hydroxyl

was acetylated

activity

group

groups alone

against which or

the

and the carboxyl both has

L-1210 been

carboxyl

and

proven group

group ESC. to be alone,

VOL.

XXIX

NO.

respectively,

THE

3

showed

improved

JOURNAL

OF

ANTIBIOTICS

277

activity against both of the two tumor types as in the case or

compound 32. On the whole, simple alkyl esters enhanced activity but introduction atoms into the alkyl groups (enhancement particularly against L-1210. group was modified. It appears carbamoyl

that

groups

aliphatic

acids

of activity

acetic

and

aliphatic

butyric

are structural

of the hydroxyl

acids)

acids (36 to 45).

both of R1 and R2 are hydrogen

61

to 63

showed

decreased

limitations group.

is advantageous

the activity,

on

the

Acylation

but

not

acyl

or

with simple

with

aromatic

or

compounds

Carbamoylated

in

(46 to 48) or one of them is hydrogen and another

phenyl, benzyl or cyclohexyl exhibited increased activity. compound

character)

with observations when only the carboxyl

there

are used for modification

(formic,

hetero-atom-containing

which

This is in agreement

for optimization which

of hydrophilic

of hetero-

no

Disubstituted

carbamates other

is

than

effect.

As seen in Table 1, none of these compounds was as effective as MA in suppressing growth of L-5178Y cells in vitro.

However,

L-1210 than

The correlations

MA in vivo. Table

1.

Antitumor

some compounds among

activity

showed three

better

biological

of mycophenolic

acid

activity activities

R

R'

No.

MA

TCIDsoa' (mcg/ml)

of these

ESC and/or derivatives

derivatives

Antitumor Compound

against

activity

(T/C)

ESCb) (T/C)

L-1210b)

-OH

-COON

0.10

1.24

0.34

1

-OCOCH

-COOH

0.17

1.33,

0.13

2

-0CO

-COON

0.51

1.27

0.47

3

-OCO(CH2)6CH3

-COON

0.73

1.31*

0.47*

4

-OCO(CH2)10CH3

- COON

7.63

1.11*

1.00*

5

-OCOCH2CI

-000H

0.23

1.28

0.20

6

-OCOCH=CHCH

-COON

0.54

1.14

0.54

7

- OSO2 CH3

-COON

1.20

1.28

0.65

8

-OCONH2

-COOH

0.11

1.01

0.44

9

-COONa

0.18

0.97

0.44

10

-COOH

0.40

1.27

0.20

3

(CH2)3CH3

3

II

-COON

0.15

1.37

0.12

12

-COON

0.27

1.36

0.18

THE

278

JOURNAL

Table

OF

1.

MAR.

ANTIBIOTICS

(continued) Antitumor

Compound

R

No.

R'

TCIDso (mcg/ml)

13

-COON

14

-COOH

15

-COON

-COON

16

17

-COOH

18

-COOH

19

-COON

20

-OCONHNHCSNH2

21

- OH

22

-OH

23

-COON COOCH3 -COOC

-OH

activity

2H5

- C OOC4H9

24

-OH

25

-OH

26

-OH

-COOC

27

-OH

- COOCH

28

-OH

2H40H

29

-OH

-CONHCH

30

-OH

-CH2OH

31

-OCHO

-COOCH3

32

-OCOCH3

-COOCH3

2CH20CONH2

2OOOH

L-1210 (T/C)

ESC (T/C)

1976

VOL.

XXIX

NO.

3

THE

JOURNAL

Table

OF

1.

279

ANTIBIOTICS

(continued) Antitumor

Compound

R

activity

R'

No.

TCID,, (mcg/ml)

L-1210 (T/C)

ESC (T/C)

0.34

1.37

0.32

0.55

1.33

0.55

0.23

1.18

1.27

0.69

1.19

0.69

-COOCH2CI-y0H

0.75

1.00

0.69

38

-000C2H5

0.29

1.08*

0.27*

39

-COOC2H5

0.32

1.16

0.34

-COOC2H5

9.74

1.22*

1.00*

0.85*

1.00*

33

-OCOCH3

-COOC

34

-000C7H15

2H5

35 -COOCH3

36

37

-000CH2NH2-H

CI

40

41

-OCO(CH2)5NH2•HCI

42

-OCOCH2NH2•HCI

-000C3H7

8.40

0.80

0.92

43

-OCOCH2NH2

-COOC2H5

0.24

1.18

0.24

1.92

1.16

0.54

•HC1

-COOCZH5

44

14.0

-COOC2H5 45

-OCO(CH2)4C00No

-COOC2H5

0.92

1.07

0.68

46

-OCONH

-COOCH3

0.20

1.56

0.20

47

-OCONH2

-COOC2H5

0.29

1.47

0.10

48

-OCONH2

- C OOC4H9

0.54

1.45

0.14

49

-OCONH2

-COOC2H40H

0.18

1.18

0.23

50

-OCONH2

0.26

1.18

0.23

51

-OCONH2

1.8

1.01

1.12

2

52

-COOCH3

0.17

1.15

0.27

53

-COOC2H5

0.12

1.23

0.17

280

THE

JOURNAL

OF

ANTIBIOTICS

MAR.

Antitumor Compound

R

R'

activity

TCIDs0 (mcg/ml)

L-1210 (T/C)

ESC (T/C)

0.34

1.15

0.33

0.19

1.57

0.13

0.31

1.37

0.20

0.25

1.23

0.47

0.70

1.14*

0.70*

0.24

1.18

0.21

-COOC2H5

9.8

0.97

0.82

61

-COOCH3

1.11

0.78

1.00

62

-COOCH3

3.32

1.16

0.94

50

0.95

1.43

0.43

1.46

0.21

No.

54

-COOC2H5

-OCONH(CH2)5CH3

-COOC2H5

55

56

-OCONH(CH2)

11CH3

-COOCaH5

-COOC2H5

57

58 - COOC2H5

59 -000C2H5 60

-OCONH(CH

2)5COONa

63

-COOCH3

64

-000C

65

-COOCH3

0.41

1.56

0.41

66

-COOCH3

0.25

1.22

0.26

67

-000C

0.19

1.42*

0.39*

68

-COOC2H5

0.18

1.62

0.26

-COOC2H5

0.11

1.54

0.08

-COOC2H5

0.31

1.62

0.17

0.25

1.38

0.23

0.14

1.56

0.13

0.20

1.57

0.20

0.24

1.64

0.21

69 70

2H5

2H5

71

-000C2H5

72

73 74

-COOC2H5

-COOC2H5

-COOC

2H5

1976

VOL.

XXIX

NO.

3

THE

JOURNAL

OF

ANTIBIOTICS

281

Antitumor Compound

R

R'

No.

75

-COOC2H5

76

-COOC2H

5

77

activity

TCIDeo (mcg/ml)

L-1210 (T/C)

ESC (T/C)

0.21

1.60

0.22

0.15

1.60

0.17

2.7

1.14

0.62

78

-COOC2H5

0.92

1.02

0.78

79

-000C2H5

0.42

1.10

0.41

80

-COOC2H5

0.23

1.45

0.18

81

-COOC3H9

0.18

1.57

0.23

82

-COOC,H.

0.23

1.48

0.21

0.34

1.57

0.31

0.48

1.53

0.42

0.42

1.26

0.43

0.16

1.51

0.20

0.17

1.51

0.19

4.8

1.00

1.02

0.45

1.14

0.42

0.24

0.63

0.15

0.16

1.22

0.19

-COOC2H5

83

-COOC2H5

84

85

-COOC

2H5

86

-COOC2H5

87

-COOC2H5

88 -COOC2H5

89 - COOC2H5

90 -000C2H5 -000C2H5

91 Arginine

salt

92

- C OOC2H5

0.12

1.42

0.10

93

-000C2H5

1.25

1.14

0.61

94

- COOC2H5

2.12

1.14*4

0.79**

95

-C OOC2H5

1.24

1.00

0.88

THE

282

JOURNAL

OF

Table 1.

ANTIBIOTICS

MAR.

(continued) Antitumor

Compound

R

1976

R'

TCIDbo (mcg/ml)

No.

activity

L-1210 (T/C)

ESC (T/C)

96

-COOC2H5

0.33

1.62

0.26

97

-COOC2H5

0.62

1.05

0.26

0.51

0.98

0.51

0.13

1.39

0.08

9.8

1.02

0.95

0.12

1.25

0.45

0.74

1.14

0.77

0.72

1.20

0.63

0.38

1.14

0.45

0.17

1.14

0.72

nt

1.09

0.52

nt

1.18

1.02

1.05

0.98

98 -COOC2H5 99 - COOC2H5 -COOC2H5

too

101

-OCONHOH

-COOCH

102

-OCONHNHCSNH

3

2

103

-COOC2H5

104

-COOCH3

105

-COOCH3

106

a) hl

and

107

-0SO2Na

-COOCH3

108

-OCONH2

-CH2OCONH2

1.6

TCIDa0 value against L-5178Y cells in vitro. The ratio of treated uroun/control erouo in average life-span (L-1210) or average tumor weight after 10 days after tumor inoculation (ESC) at the doses of 150 (not marked), 75 (*) and 37 mg/kg (**), respectively. (These compounds with marks were toxic at the dose of 150 or 75 mg/kg.) their

lipophilic

character

values of these compounds of compounds

are illustrated

were

corresponded

plotted

more

graphically.

against

closely

As shown

T/C values

to activity

in Figs.

of ESC and L-1210.

against

ESC than

that

the activity

vitro, but was under

of compounds

the influence

has the lowest

TCIDbo value

in vivo activity

may be caused

Correlation and 4, values.

between

respectively.

but its activities

against

Rf values

against

and activities all highly

was not observed

L-1210 did not depend

characteristics

by glucuronidation

As expected,

This characteristic

of other

of the agent.

L-1210,

against

excretion

upon

the activity

It is notable

in the activity

against against

that

in MA

This lower

in urine.°'

ESC or L-1210

compounds

i.e.,

suggested for these upon the activity

ESC and L-1210 are moderate.

and rapid

active

TCIDbo values

against

points are scattered more widely in Fig. 2 than in Fig. 1. It is apparently tendencies that the activity of compounds against ESC is mainly dependent in vitro, whereas

1 and 2, TCIDbo

are

shown

L-1210 ESC.

in Figs.

showed

This

fact

3

high Rf suggests

VOL.

XXIX

NO.

Fig. 1. Correlation activity against

Fig.

3.

between ESC.

Correlation

tivity

against

THE

3

JOURNAL

TCIDbo

between

Rf

value

value

OF

and

and

ANTIBIOTICS

Fig. 2. Correlation activity against

the lipophilic

is an important tial activity In

character

TCIDbo

value

and

ac-

against

Correlation between against L-1210.

Rf

value

and

ac-

of the compound

factor on exhibiting

the poten-

L-1210 in mice.

conclusion,

observations

between L-1210.

ESC.

Fig. 4. tivity

that

283

these

with other

results,

similar

to

drugs, indicate

the

hydrophilic and lipophilic balance (HLB) which influences

absorption

Esterification character fication

of

the

concerned

ments

on enzymatic

The activity

curative

in a living

carboxyl

appeared

to an appropriate of hydroxyl

subtly

shown

and distribution

in Tables

groups

with

group

extent.

However

absorption

of compound

and stability 46 at various

seems

structural

by HLB

to be a very

important

in increasing

limitations

alone.

in vivo, affinity

and other

2, 3 and 4, respectively.

effects than

some

can not be explained

decomposition,

body

to be advantageous

observed

Changes

to tumor

factor.

the lipophilic on the modi-

in activity

cells, behavior

may

be

of frag-

factors. dosage

levels

In these

tumor

against system,

L-1210,

ESC and X-5563 were

compound

46 showed

better

MA. Acknowledgements

The authors are grateful in preparing the manuscript. Tohoku

University,

to Drs. T. AKIBA and H. OGAWA for their advice during this study and We also thank Prof. Y. HASHIMOTO, Faculty of Pharmaceutical Science,

for his critical

review

of this

manuscript.

THE

284

Table

2.

Comparative

antitumor

Dose* (mg/kg)

Compound

JOURNAL

OF

activity

Av.

survival

150

11.2

1.51

-0 .8

50

9.6

1.30

1.4

15

9.0

1.22

2.2

500

9.6

1.30

0.6

300

9.6

1.30

0.5

150

8.8

1.19

0.4

50

8.6

1.16

1.4

15

8.2

1.11

2.0

7.4

1.00

3.7

daily for 5 days, weight before and at 6 days

antitumor

Dose* (mg/kg)

activity Av.

4.

daily 10 days

Antitumor

for 5 days. after tumor

activity

Av.

weighed

ESC.

Survival

TC

6.0

6,6 6/6

301.0

49.5

6,'6

413.6

67.3

6/6

144.4

23.5

6/6

180.0

29.4

6/6

333.8

54.3

6/6

463.6

75.5

6,6

613.7

100.0

6/6

wt.**

46 and

MA

against

T/C

X-5563. Survival

0

10/10

22.8

1.1

10/10

1,146.6

56.5

10;10

0

150

267.8

13.1

10,10

50

1,708.4

84.2

10,10

2,029.0

100.0

10,10

* Injected intraperitoneally daily for 5 days. were

against

20.3

(mg)

Control

Tumors

MA

36.8

tumor

50

**

46 and

124.8

of compound

150

MA

wt.**

inoculation.

inoculation.

300

46

tumor

(mg)

Dose* (mg/kg)

Compound

after

of compound

tumor

Control

Table

change**

(g) -5 .0 -3 .8

Comparative

intraperitoneally were weighed

wt.

1.65

MA

Injected Tumors

Body

T/C

L-1210.

1.86

46

* **

against

12.2

Infected intraneritoneally Difference in the body

Compound

day

MA

13.8

Control

3.

46 and

300

MA

Table

of compound

MAR.

500

46

* **

ANTIBIOTICS

12 days

after

tumor

inoculation.

1976

VOL.

XXIX

NO.

3

THE

JOURNAL

OF

ANTIBIOTICS

285

References

1) 2)

ANDO, K.;

S. SuzuKI,

Antibiotics

21:

activity

J. Antibiotics

22:

297302,

of mycophenolic

J. Antibiotics

acid.

J.

22:

165-169,

MITSUI, A. & S. SUZUKI:

activity

of mycophenolic

1969

NoTO, T.; M. SAWADA, K. ANDO & K. KOYAMA: Some biological acid.

4)

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