Chemical Structure and Growth Regulating Activity Relationship of Some Piperidinoacetanilides

Bioehem. Physiol. Pflanzen 175,140-147 (1980)

Chemical Structure and Growth Regulating Activity Relationship of Some Piperidinoacetanilides H. Inhibition of Chlorophyll Degradation in Detached Leaves and the Influence on Chlorophyllase Activity E.

KARANOV1),

R.

VUNKOVA1),

A.

BARTH 2)

and E.

POGONCHEVA1)

1) Institute of Plant Physiology of the Bulgarian Academy of Scienees, Sofia and 2) Sektion of Bio-Seiences of the Martin-Luther-University HaUe, GDR K e y Ter m In d ex: Chlorophyll, chlorophyJlase activity, senescence, detached leaves, piperidinoacetanilide derivatives ; llaphanus sativus, Hordeum vulgare.

Summary Piperidinoacetanilide hydrochlorides and N-methyl-piperidinoacetanilide iodides retard the degradation of chlorophyll in the process of senescence of detached radish leaves. N-Methyl-piperidinoaeetanilide iodides possess a strong activity for retardation of chlorophyll degradation in detached bartey leaves. The selectivity of action of this compounds on retardation of chlorophyll degradation is determined by the nature of the substituents and their position in the anilide ring. Piperidinoacetanilide hydrochlorides inhibit the chlorophyllase activity in radish leaves. Theinhibitory activity is expressed more strongly in in vitro experiments than in experiments in vivo.

Introdnction

After applying retardants to some plants the inhibition of growth is very often accompanied by a more intensive green coloration of the plants (CATHEY 1964). This effect of retardants on some plants is connected with an increase in the chlorophyll content and an inhibition of chlorophyllase activity (SIVCEN and DONDO 1977). On the other hand, when retardants are applied to detached leaves or other plant organs they retal'd the process of senescence (HALEVY and WITTWER 1965). According to HALEVI (1969) the action oi retardants on the senescenee of detaehed leaves is brought about by a meehanism analogieal to that of eytokinim. KULAEVA (1973) has presented the opposite opinion, that the action of retardants and cytokinins on the processes of seneseenee is different on prineiple. The retardants inhibit the total metabolie aetivity of leaves, which is connected with the inhibition of chlorophyll degradation, but cytokinins act by me ans of stimulation of RNA- and protein synthesis, i.e. the retardants act only as conservants. The inhibition effect of re1ardants on the processes of ageing of detaehed leaves and other plant organs has also a practical meaning, beeause after application of retardants it is possiblc to prolong the storage period after picking of some flowers (carnation, rose, gladiolus and othen:), some leaf vegetables, mushroosms and others (KARANOV 1978). In a previous paper (KARANOV et al. 1980) it wa3 shown that piperidinoacetanilide derivatives possess a growth retarding activity, which is connected with the inhibition of the choline esterase activity, but as to their action on

Structure-Activity-Relationship oi Piperidinoacetanilides, H.

141

the senescence of detached leaves there is not information. In connection with thig the present study is concerned with the action of piperidinoacetanilide derivatives on the degradation of chlorophyll and with chlorophyllase activity (see SIVCEV and DONDO 1977) during the process of aging of detached leaves. Material and Methods The experiments on the influence of piperidinoacetttnilide derivatives were developed with disks from mature radish leaves (Raphanus sativa) after incubation in the dark for 96 h at a temperature of 24 oe in soltltions containing the different concentrations oi the compounds studied. Experiments with radish disks were carried out according to the method of BRUCE et al. (1965). The derivatives of N-methyl-piperidinoacetanilide iodides were investigated also on the chlorophyll degradation in seetors horn barley (Horrleum vulgare) leaves using the analogieal ineubation in the dark according to the method of KENDE (1964). The quantity of chlorophylls was determined by means of spectrophotometry. The influcnec of so me piperidinoacetanilide hydroehlorides on the activity of chlorophyllase from radish leaves was studied using the method of SUDYNA et al. (1972). In one set of experiments the influence of the compounds mentioned on chlorophyllase activity was investigated in vivo, in thc other set thc influence of compounds was studied in vitro using a erude enzyme. AU derivatiYcs of piperidinoacetanilide hydrochlorides and N-methyl-piperidinoacetanilide iodides in experiments with radish leavc disks were tested at eoncentrations of 10 -5, 10-4 and 10- 3 M and in experiments with barley leave sections the second groups of derivatives were tested at concentrations of 10-6 , 10-5 , 10- 1 and 10-3 M. In some experiments on cblorophyllase activity th e action of somc piperidinoacetanilide hydrochlorides was compared with the action of kinetin. All fiurlings were obtained from an average of 3 - 4 experiments and are presented as percentages of contro!.

Results and Discussion

The results of the experiments on retardation of chlorophyll degradation in radish leave disks after application of piperidinoacetanilide hydrochlorides (Fig. 1) show that almost all compounds investigc1ted possess an activity which depends on the nature of substitucnts and their position in the aromatic ring system. Tbe orders of activity of the substituents depending on their nature and position in the anilide ring are as follows:

ortho: Cl> OC 20 5 ;;;:: CFa > N0 2 > CHa ;;;:: H > F meta: Cl> Br;;;:: CFa ;;;:: J > N02 > F> OC2 H5 > OCHa ;;;:: CHa ;;;:: H para: Cl> BI';;;:: CFa ;;;:: F > NO z > OC2H5 > H> CHa The established orders of activity of the substituents show that the introduction of all substituents, investigated increase the aciivity of the unsubstituted compound, with the exception of the introduction of the fluorine atom in ortho position and the methyl group in para position. The orders of the activity of isomers depending on the nature of the substituents in the anilide ring are as folIows: F, Cl, CFa and NO z: CHa and OC2 H5 : Br: 10'

meta > para > ortho ortho > meta > para para > meta

142

E.

KARANOV

et al.

The established orders of activities of the mbstituents and isomers show that in order to express high activity in inhibiting chlorophyll degradation in radish leaf disks piperidinoacetanilide hydrochloride derivatives must contain substituents in meta or para position. For high activity the substituents must possess predominantly electron attracting propertieil. The results from testing N-methyl-piperidinoacetanilide iodide derivatives (Fig. 2) for retardation of chlorophyll degradation in radish leaf disks show that all compounds possess a very well expressed activity. The orders of activity of substituents depending on their nature and position in the aromatic ring are as follows: ortho: N0 2 > H> CH3 > OCH3 meta: Cl > N0 2 > CH3 > H > OCH3 para: Cl > N0 2 > CH3 > H> OCH3 The orders of activity of isomers depending on the nature of substituents in the anilide ring system are as follows: CH3 and OCH3 : Cl and N0 2 :

~

250

meta > para > ortho para> meta > ortho

lqeH2:0NH{tl er

____________________

~A~

______________________

-F

-H

-CI

-Br

~

-NO 2

o 200

..c ~ 150 o

100 250

0

L -I-'

C 0

u

4-

0

200 0

~ 150

E

~ 0

100 250

o 200 L

iI 150 100

~-J

-- -- ---543543543543543543543543543543 -~--

-----~

n

concentrations 10-n M Fig. 1. Influence of some piperidinoacetanilide hydrochlorides for retardation of chlorophyll degradalir;rt in detached radish leaves.

Structure-Activity- Relationship of Piperidinoacetanilides, 11.

143

established orders of activity of 3ubstituents and isomers of N-methyl-piperidinoacetanilide iodides show, that in order to obtain a strong effect on the retardation of chlorophyll degradation in radish feaf disks, as in the case with piperidinoacetanilide hydrochlorides, it is necessary to introduce substituents in meta or para position of the anilide ring. The substituent must also possess predominantly electron attracting properiies. Whcn comparing the activities of N-methyl-piperidinoacetanilide iodide derivatives with the activity of piperidinoacetanilide hydrochlorides it could be seen that the first group of derivatives produces a stronger action. The results from experiments of N-methyl-piperidinoacetanilide iodides for retarding of chlorophyll degradation in barley leaf sectors (Fig. 3) show that some of the derivatives studied possess a very high activity, for example, N-methyl-piperidinoacetanilide iodide at a concentration of 10-6 M retards chlorophyll degradation up to 148% approximately alld N-methyl-piperidinoacet-3-methyl-allilide iodide at the same concentratioll shows an effect of 184% (control 100%). The estabJished activity of meta methyl substituted derivatives on chlorophyll degradation is so high that it is comparable with the activity of some cytokinins, like kinetin, benzyladenine and others. Th~

la~H,coNH-(~t1 T

R

(

200 0

:5 150 L

0

--' 0

200

L

+-'

c

0

0

"-

100 250

0

+-' Q)

E

150

0

100

~

250

0

o 200 L

~ 150 100

-H

-eH3

A

-oC~

-el

-N°2

.J~_J

JJJJJ JJ

TI3 543 5T3 543 5T3 n concentrations 1cf1M

Fig.2. I nflu ence of some N-methyl-piperidinoacetanilide iodides for retardation of chlorophyll degradation in detached radish leaves.

144

E.

KARANOV

et al.

Comparing the activity of the substituents depending on their nature and position in the aromatic ring of N-methyl-piperidinoacetanilide iodides the following orders of activity of substituellt3 and isomers were established:

ortho: CH3 > H > OCH3 > Cl ~ N0 2 meta: CH3 > H> OCH3 > Cl > N02 para: CR3 > H > OCH3 ~ Cl > N02 CHs : meta> para ~ ortho OCHs and Cl: para > ortho > meta NO;!: pam > meta> ortho Tbe established orders of activity show that for high activity it is necessary (as in the case mentioned above) to introduce substituents in meta or para position oi the anilide ring of N-methyl-piperidinoacetanilide iodide. But with this test object only the introduction of a methyl group possessing electron decreasing properties leads to an increase in activity. The introduction oi a methoxy group, a chlorine atom or a nitro group result in a decrease in the activity oi a unsubstituted compound, independently of their position in the anilide ring. Besides, the substituents with a strongcr electron attracting property in meta position, cause a stronger decrease in thc activity oi the unsubstituted compoulld. It is interesting to compare thc orders of activities oi substituents in the anilide ring of N-methyl-piperidinoacetanilide iodides on two test objects used (dicotyledonous - radish and monocotyledonous - barley). It could be seen that thc orders of activity are quite different. This leads us to the assumption that

o

200

-H

---'o ::5 150 L

L.

0

+-'

g U

'+-

o

~15O ~

OE o

;;;.

100 200

100

~

H 2(IJ

100

~::

,~ ~~~d'bF

65436543654365436543n n concentrotions 1O- M Fig. fl. Influence of so me N-methyl-piperidinoacetanilide iodides for retardation of chlorophyll degradation in detached barley leaves.

Structure-Activity-Relationship of Piperidinoacetanilides, 11.

145

the selectivity of action of N-methyl-piperidinoacetanilide iodides on retardation of chlorophyll degradation is mainly determined by the position of the substituents in the anilide ring and their nature (of course only valid by the given basic structure of this biologie active compounds). Results from experiments on the influence of so me piperidinoacetanilide hydrochlorides on chlorophyllase activity are presented in Tables 1 to 4.

of piperidinoaeet-3-methyl-anilide hydroehloride and leinetin on ihe activity ehlorophyllase in mdish leaves

Table 1. 1nfluence

of

In experiments in vivo chlorophyllase was isolated after 9611 incubation of leaves in the dark at 24°C on solutions containing the test compounds. T11e activity of chlorophyllase for control is 5.6 mg per 10 min Compounds

Concentration in moleil

Inhibition of the chlorophyllase activity in % (control = 100 %) in vivo

in vitro

31

70

pi peridinoacet-3-meth yl-anilide hydrochloride

10-4

kinetin

10-4

26

62

kinetin

10- 5

60

73

of piperidinoaeeianilide hydroehloride and the methyl substituted derivatives on the acti'City of chlorophyllase from radish leaves in 'Citra.

Table 2. 1nfluenee

T11e activity of chlorophyllase for control is 6.4 mg per 10 min Compounds

Concentration in moleil

Inhibition in % (control = 100%)

piperidinoacetanilide hydro chloride

10- 3

59

pi peridinoacet-2-meth yl-anilid e hydroc11loride

10- 3

69

piperidinoaeet-3-methyl-anilide hydrochloride

10- 3

70

pi peridinoacet-4-meth yl-anilide hydrochloride

10- 3

70

From the data in Table 1 it be comes evident that piperidinoacet-3-methyl-anilide hydrochloride inhibits the activity of chlorophyllase in experiments in vivo and in vitro. But the inhibitory action in experiments in vitro is expressed twice as strong. The action of kinetin is analogical and its inhibitory effect at a concentration of 10-4 M is approximately equal to the effect of piperidinoacet-3-methyl-anilide hydro chloride. From the data presented in Table 2 it can be seen that the introduction of a methyl group in the aromatic ring of piperidinoacetanilide hydrochloride is increasing the inhibitory activity about up to 10%, independently of the position of the substituents.

146

E.

KARANOV

et al.

Table 3. Influence of chloro substituted derivatives of piperidinoacetanilide hydrochlorides on the activity of chlorophyllase from radish leaves in vitro. The activity of chlorophyllase for control is 4.1 mg per 10 min Compounds

Concentration in molejl

piperidinoacet-2-chloro-anilide hydro chloride

Inhibition in % (control = 100%) 42

piperidinoacet-3-chloro-anilide hydro chloride

10- 3

65

piperidinoacet-4-chloro-anilide hydrochloride

10-3

39

Table 4. I nfluence of nitro substituted derivatives of piperidinoacetanilide hydrochlorides on the activity of chlorophyllase from radish leaves in vitro. The activity of chlorophyllase for control is 5.0 mg per 10 min Compounds

Concentration in moleil

Inhibition in % (control = 100%)

piperidinoacet-2-nitro-anilide hydro chloride

10-3

72

piperidinoacet-3-nitro-anilide hydrochloride

10- 3

74

piperidinoacet-4-nitro-anilide hydro chloride

10-3

The introduction of a chlorine atom in ortho or para position in the anilide ring does not change the activity of the unsubstituted compound, but the introduction of a chlorine atom in meta position increased the inhibitory potency on in vitro enzyme activity up to 29% approximately (Table 3). Contrary to this is the influence of a nitro group on the activity of the unsubstituted compound (Table 4). Independently of the position of introduction into the anilide ring the nitro group decreases the inhibitory potency by about 10%. The findings concerning the influence of the substituents in the anilide ring on the inhibitory activity of piperidinoacetanilide hydrochlorides for the inhibition of chlorophyllase activity in vitro do not correlate with the established dependencies in experiments on the retardation of chlorophyll degradation in radish leaf disks. This leads us to the conclusion that the action of piperidinoacetanilide hydrochlorides for retardation of chlorophyll degradation during the senescence of detached leaves is not directly connected with the inhibition of chlorophyllase activity, perhaps the inhibitory action is brought about by another process. The established inhibitory action of piperidinoacetanilide hydro chloride derivatives on the activity of chlorophyllase could only be one point on the possible sites of their action on the metabolil'lm of detached leaves.

Structure-Activity- Relationship of Piperidinoacetanilides, Il.

147

The results of the experiments from the presented investigations permit the following conclusions:

1. Piperidinoacetanilide hydrochlorides and N-methyl-piperidinoacetanilide iodides retard the degradation of chlorophyll in the pro ces!! of senescence of detached radish leaves. The introduction of 8ubstituents in meta or para position of the aromatic ring system increases the activity of the substances. 2. N-methyl-piperidinoacetanilide iodides possess a strong activity for retardation of chlorophyll degradation in detached barley leaves. The introduction of electron donating substituents in meta position of the anilide ring increases the activity, whereas the introduction of electron attracting substituents decreases the activity. 3. The selectivity of action of N-methyl-piperidinoacetanilide iodides on retardation oI chlorophyll degradation in detached leaves is determined by the nature of the substituents and their position in the anilide ring.

4. Piperidinoacetanilide hydrochlorides inhibit the chlorophyllase activity in radish leaves. The inhibitory activity is expressed more strongly in vitro experiments than in experiments in vivo. References BRUCE, M., ZWAR, J., and KEFFOllD, N.: Chemical Strueture and Plant Kinin Aetivity - The Aetivity of Urea and Thiourea Derivatives. Life Sei. 4, 461-466 (1965). CATHEY, H. M.: Physiology of Growth Retarding Chemieals. Ann. Rev. Plant Physiol. 1;), 271-301 (1964). HALEVY, A. H.: Eifeet of Growth Retardants on Drought Resistance and Longevity of Various Plants. Proe. 17th Intern. Hort. Congress 3, 277 -283 (1967). - and WrTTwER, S. H.: Chemieal Regulation of Leaf Seneseenee. :yrich. Agr. Exp. Sta. Quart. Bull. 48,30-35 (196ö). KARANov, E. N.: Ph. D. Thesis, Sofia 1978. - BARTH, A., CIlHISTOVA, L., and IVAxovA, V. R: Chemical Structure and Growth Regulating Activity Relationship of Some Piperidinoacetanilides - I. Growth Retarding Activity and the Influence on Choline Esterase. Biochem. Physiol. Pflanzen 17;), 130-139 (1980). KENDE, H.: Preservation of Chlorophyll in Leaf Sections by Substances Obtained from Root Exudate. Science 145, 1066-1067 (1964). KULAEVA, O. N.: Citokininy ich struktura i funkcijn. Moskva 197". SrvcEv, M. 1., and DONDO, V. V.: Korreljllcia dinamiki soderscanija chlorophylla i aktivnosti ehlorophyllasy v listjach rastenyi. Izvestja AN SSSR, biologija 2, 186-193 (1977). SUDYNA, E. G., DOVBYZ, E. F., BABENKO, E. 1., and Fo~nZrNA, R. N.: Kinetika chlorophyllaznoi reakcyi pri razlicnych koncentracijach enzima. Ukrainskyi botanieeskyi shurnal 29, 419 -424 (1972).

Received September 15, 1979. Authors' addresses: Dr. EMANUIL N. KARANOV, Bulgarian Academy of Seiences, Institute of Plant Physiology, Sofia 13, 36 Street, bloek 6, Bulgarüt; Prof. Dr. ALFREDBARTH, Universität Halle, Sektion Biowissenschaften, DDR - 4020 Halle (Saale), Domplatz 1.