Herbicidal properties of antihypertensive drugs: calcium channel blockers

Herbicide resistance is a worldwide problem in weed control. This prompts researchers to look for new modes of action to slow down the evolution of herbicide-resistant weeds. This research aims to determine the herbicidal action of thiazolo[3,2-a]pyrimidines derivatives, which are well known as antihypertensive drugs. The phytotoxic effects of ten compounds were investigated using leaf disc discoloration test and seed germination bioassay. At concentrations of 125 to 250 mg/L, the 5-(3-Fluoro-phenyl)-7-methyl-5H-thiazolo[3,2-a]pyrimidine-6-carboxylic acid ethyl ester (c) was highly active against Oldenlandia verticillata and Eleusine indica. At application rates of 1.25 to 2.5 kg ai/ha, formulated c demonstrated selective post-emergence and pre-emergence herbicidal activity against O. verticillata, E. indica and Cyperus iria. In the crop tolerance test, formulated c outperformed the commercial herbicide diuron, with aerobic Oryza sativa being the most tolerant, followed by Zea mays, and Brassica rapa. The addition of calcium chloride partially nullified compound c's inhibitory effects on weed shoot growth, indicating that it has potential as a calcium channel blocker. Compound c acted by triggering electrolyte leakage without affecting photosystem II. These findings imply that c could be explored further as a template for developing new herbicides with novel modes of action.


Introduction
Over the past, some pharmaceuticals, which have been credited for saving millions of lives, were found to possess the property of pesticide 1,2 . Both pesticides and drugs are following similar regulations in terms of design and production 3,4 . A well-known example is fluconazole, a pharmaceutical fungicide product that is now patented as an agricultural product 1 . The endothall herbicide, which is effective against aquatic and terrestrial plants, is now listed as a chemical scaffold for developing new antimalaria lead 5 .
Calcium channel blockers are important drug targets among pharmacologists due to their ultimate role in treating hypertension 6,7 . Basically, the calcium (Ca 2+ ) channel family can be subdivided into three categories: L-type (Cav1), P/Q, N and R-types (Cav2) and T-type (Cav3) 8 . Along these lines, numerous studies have revealed that different subtypes of calcium channels are associated with cardiovascular 6,9 and neuropsychiatric 7,10 disorders and cancer 8,11 . At a therapeutic dose, the calcium channel blockers decrease the elevated blood pressure of hypertensive patients. However, they do not alter the blood pressure of normotensive individuals as was found in animals. Several major families of calcium channel blockers are verapamil, nifedipine, diltiazem, cinnarizine, bepridil and mibefradil 12 .
In plant stress adaptation and growth processes, calcium ion (Ca 2+ ) has been identified to constitute the key transducer and regulator 13 . Hence, the presence of calcium channel blockers could inhibit the influx and increase the efflux of calcium ions from plant cells 14,15 . Studies have shown that verapamil inhibits the influx of Ca 2+ into the root apex of rice and wheat, while nifedipine prevents the development of root hair and pollen grain germination of Arabidopsis 16 . The calcium antagonist properties are displayed by a broad number of pure natural compounds, primarily coumarins, which have been identified and isolated from plants 14,17 .
Thiazolopyrimidine, an example of calcium channel blocker, has been of interest due to their wellknown biological and medicinal activities [18][19][20][21][22][23][24][25] . Despite their wide range of pharmacological activities, there is a lack of information regarding the phytotoxic behaviour of these fused pyrimidine derivatives.
For the past 30 years, there are no novel herbicides with new sites of action being introduced into the market, making growers highly dependent on existing herbicides, thereby leading to evolution of herbicide resistance in weed, which indirectly imposed new challenges for weed management. Hence, this study aimed to screen for the promising thiazolopyrimidine derivatives and evaluate the pre-and post-herbicidal activity of selected thiazolopyrimidine derivative in several weeds and crop species.
The effects of the selected thiazolopyrimidine derivative on quantum yield and ion leakage were carried out to reveal its potential mode of action, while the role of metal chloride to antagonize herbicidal action of the selected thiazolopyrimidine was also determined.

Results
Leaf disc discolouration test. Results have shown that compound c at a concentration of 500 mg/L exhibited the most phytotoxic effect in which it displayed a score of 4 even on day 1 of treatment followed by compound d, e, b, f, h and i on day 2, and lastly compound a on day 4 of treatment ( Fig.   1). The complete diminish in the green colour of the diuron-exerted Oldenlandia verticillata leaf was recorded on day 6. Of the ten compounds, however, compound g and j did not achieve a score of 4 within 7 days of treatment which implies they are less phytotoxic compared to other compounds.
Compound c was further undergone leaf disc discolouration test at concentrations of 0, 62.5, 125, 250 and 500 mg/L (Fig. 2), and apparently, 125 mg/L is the lowest concentration for it to exhibit the full phytotoxicity within 7 days of incubation.
Germination test. At 500 mg/L concentration, compound b, c, e, f, g, j and diuron provided complete inhibition of Eleusine indica germination and root growth (Table 1). Visibly, all compounds suggested a lower reduction of shoot length than that of the root, where the shoot lengths were more than 50% of control except for compound c, in which seed germination and seedling growth were inhibited entirely. Figure 3 shows the inhibition on seed germination, shoot and root growth of bioassay species when compound c applied at concentrations of 62.5, 125, 250, and 500 mg/L. This effect was most noticeable with an increasing concentration of c, where full inhibition was observed at a concentration of as low as 125 mg/L. On the contrary, the shoot growth of E. indica was less suppressed at lower concentrations of 31.25 and 62.5 mg/L. The minimum concentration for compound c to show completion inhibition is 125 mg/L. This corresponds to the leaf disc test result, which showed a complete diminish in green colour at the similar concentration on day 5. The concentration of compound c, which gives 50% inhibition (ED50) on E. indica was described in Table   2, showing the most inhibitory effect on root growth (9 mg/L), followed by germination (39 mg/L) and shoot growth (63 mg/L).
Pre-emergence application. There was a significant interaction (p<0.05) between the tested compound and application rate on shoot growth, emergence and root growth of test weeds and crops.
The increased dose of compound c and diuron decreased the shoot dry weight of weeds. At 2.5 kg ai/ha, compound c inhibited the weeds by 40 to 60%, whereas diuron provided complete weed inhibition at a similar application rate (Fig. 6). Likewise, weed seedling emergence was reduced by diuron and compound c in a dose-dependent manner, but more significant herbicidal activity was exhibited by diuron than compound c (Fig. 7). Application of compound c at 2.5 kg ai/ha was able to suppress 20 to 40% weed emergence, whereas complete inhibition was evident for diuron at a similar dose. On the other hand, compound c had less inhibition on root growth of weeds, with C. iria being the least susceptible, followed by E. indica and O. verticillata when increasing the dose. By contrast, complete inhibition of weed root growth was observed at 1.25 kg ai/ha diuron (Fig. 8).
The shoot growths of test crops were not significantly (p>0.05) affected by compound c with increasing dose even at the maximum application rate of 5.0 kg ai/ha ( Fig. 9). Similarly, the increased dose of compound c did not inhibit the root growths of the crops significantly (p>0.05), with Z. mays being the most tolerant, followed by aerobic O. sativa and B. rapa (Fig. 10). On the contrary, diuron demonstrated excellent inhibitory property by reducing the shoot dry weight (Fig. 9) and root length ( Fig. 10) of the crops by 100%, even at the lowest application rate of 1.25 kg ai/ha.

Electrolyte leakage determination.
In this study, diuron did not cause significant electrolyte leakage in O. verticillata at concentrations ranging from 100 to 300 mg/L during the 24-hour incubation period (Table 3) verticillata was incubated for 24 hours. However, this is not the case for compound c, where it did not exert any effects on quantum yield (Table 4). Table 5, compound c at 125 mg/L indicated higher inhibitory effects than diuron either on E. indica seed germination, shoot or root growths. Moreover, at the similar concentration, the treatment of diuron solely and the combination of metal chloride solutions with diuron portraited a lower inhibitory effect than compound c. With the supplementation of Ca 2+ to c, only the shoot length of E. indica was recovered.

Discussion
Compared with chlorine and bromine derivatives, compounds containing fluorine atom demonstrated a faster colour change on leaf discs, such as c, b and d (Fig. 1). One plausible explanation is that they are highly dissolved in water as they can easily form hydrogen bonds with water molecules and react with the leaf tissues. This can be seen where many commercial synthetic herbicides contain fluorine elements, for instance, dithiopyr, ethoxyfen, butafenacil, metflurazon flufenpyr and fluridone 26 .
As seeds grow, they not only depend on the roots for water and nutrient uptake but also it is their shoots that perform photosynthesis which enables the seedling to grow healthier. In this in vitro germination experiment, compounds were added to the filter papers in the Petri dish, where the seedling roots develop. Thus, it is reasonable that compound c exerted greater inhibition on the roots than shoots (Table 1), as shown by the study of Bourgou et al. 27 , where greater inhibitory effects of the phenolic compounds were observed on roots if compared to shoots.
The outcomes from germination bioassays implied that compound c is a strong root and germination inhibitor. It is the most phytotoxic among all the derivatives of thiazolopyrimidine, and this finding is consistent with previously performed leaf disc assays. Research done by Abe and Kameya 28 discovered that 430 μM (approximately 100.23 mg/L) of diuron inhibited asparagus seed germination by 65%. Unlike asparagus, complete inhibition of E. indica germination by diuron was found at 500 mg/L.
During post-emergence application on the weeds, generally, the inhibitory effect of c on shoot growth was varied with application levels, with increased suppression at higher rates. In our study, the optimum rate of c to fully suppress the shoot growth of E. indica was 2.5 kg ai/ha ( Fig. 4) whilst the early post-emergence study of rice herbicide, propanil combined with thiobencarb at 3.6 kg ai/ha conducted by Norhafizah et al. 29 was found to decrease the shoot fresh weight of E. indica greater than 90%. Although diuron fully inhibited the shoot growth of O. verticillata at the beginning first week of all treatments, the seedlings treated by compound c was still able to grow (data not shown). However, they gradually wilted and died after 3 weeks of treatment. These findings indicate that when introduced to O. verticillata seedlings, the phytotoxicity of c is comparable to that of diuron ( Fig. 4).
A good selective herbicide helps with weed control but does not harm the crops at the same time.
In this research, compound c is not suitable to be applied as a post-emergence herbicide in B. rapa and Z. mays since complete inhibition of crop growth is evident irrespective of any application rate.
Nonetheless, the treatment on aerobic O. sativa yielded promising results because no significant effect was observed when treated with c at even 5.0 kg ai/ha (Fig. 5). In an experiment by Mahmoudi et al. 30 , the application of thiobencarb at the rate of 3.16 kg ai/ha resulted in minor rice injury, and they proposed that 3.16 kg ai/ha thiobencarb should be applied in Iranian paddy fields. This rate is slightly higher than the effective rate of c (2.5 ka ai/ha) for weed control, suggesting that compound c is a potential alternative of thiobencarb for weed control in O. sativa.
Post-emergence herbicide usually requires a higher application rate than pre-emergence herbicide as the latter is applied to kill weeds before emerging from the soil. Nonetheless, the findings from this research indicated that post-emergence treatment has an overall lower effective application rate than pre-emergence treatment. These can be explained by the fact that soil pH determines the adsorption and desorption of herbicide in soil which in turn can affect its bioavailability to the target plant 31,32 . In this study, being a weak acid compound such as c, the low soil pH of 4.7 may have increased the binding of compound c to soil particle, thus leading to lower herbicidal activity when applied as preemergence control.
Time-course studies measuring the electrical conductivity of the bathing medium during an initial 18-hour dark incubation followed by 6-hour light exposure may distinguish compounds with the lightdependent mode of action from light-independent compounds 33 . In our study, diuron suggested a negligible ions leakage (Table 3). This result is consistent with that reported by Dayan (Table 4). In other words, diuron has contributed to a rapid reduction of quantum yields irrespective of any concentration and incubation period. This is in line with the outcome of Dayan and Zaccaro 34 , in which diuron acted drastically, blocking nearly 100% of electron flow within 3 hours of dark incubation and retaining this degree of inhibition throughout the experiment when cucumber cotyledon discs were exposed to diuron at 100 μM. On the contrary, compound c showed a negligible effect on the photosynthetic electron transport system. It did not induce any inhibition of electron flow during the whole incubation cycle, suggesting that c does not target the electron transport system of the plant.
The key concern with calcium ion (Ca 2+ ), potassium ion (K + ) and sodium ion (Na + ) selection is attributed to their essential roles and transport for optimum plant growth 35 . At the onset of this study, no example of the thiazolo[3,2-a]pyrimidine derivatives was reported as calcium channel blocker in the plant. We were, thus, intrigued by the possibility of thiazolopyrimidine compound as plants' calcium channel antagonist despite its well-known properties in animals 20 .
Since diuron is not a calcium channel blocker, adding calcium ions does not affect the diuron's inhibitory effect (Table 5). At 125 mg/L, the phytotoxic effect of c was higher than that of diuron. This importance. More research should also be conducted to elucidate this compound's mechanism action as a calcium channel blocker.

Materials and methods
The following experiments on plants were performed in accordance with relevant guidelines and regulations and had been approved by Universiti Malaysia Terengganu (UMT), Malaysia.

Bioassay species.
A total of three weed species, namely Eleusine indica (grass), Oldenlandia verticillata (broadleaf) and Cyperus iria (sedge) and three crops, Oryza sativa (paddy), Zea mays (corn) and Brassica rapa (mustard), were used as bioassays. These weed species were selected as bioassay species due to their invasiveness and high abundance in aerobic rice fields, oil palm plantations, corn  (Table S1) were respectively dissolved in ethyl acetate to produce 1 ml of solution at 500 mg/L and evaporated to dryness in 3-cm diameter Petri dishes under a fume hood.
Then, 1 mL of distilled water was added to all compounds, and the Petri dishes were left overnight.
Leaf discs of O. verticillata with 5 mm diameter were punched from fully developed leaves. One leaf disc was dipped into each Petri dish containing the solution in a growth chamber at 30/20°C with a 12 h photoperiod. The photosynthetic photon flux density of the fluorescent lamps in the growth chamber was maintained at 100 μm m -2 s -1 . Ethyl acetate was used as control negative and evaporated to dryness, and 1 mL of distilled water was added as described above. Leaf disc colour retention was scored daily for 7 days by referring to the modified method of Itoh et al. 38 (Table S2) Pre-emergence herbicidal test. Formulated compound c and diuron were applied at rates of 0, 1.25, 2.5, and 5.0 kg ai/ha, respectively, into a plastic cup containing 40 g of loam soil. Non-treated cup (control negative) was applied with acetone combined with carriers of kaolinite, silica oxide and gibbsite and evaporated to dryness. Then, pre-germinated weed species (10 seeds per replicate) and crop species (1 seed per replicate) were placed uniformly above the treated soil. There were 5 replicates for each treatment, and all the cups were placed at the glasshouse and moistened to provide an optimum condition for seedling growth. The emergence, shoot dry weight, and root length of each bioassay species were recorded after two weeks of treatment, and the data were expressed in percentage with respect to control negative.

Mode of action studies
Quantum yield determination. The study of quantum yield was referred to a modified method by Dayan and Zaccaro 34  The leaf discs, which incubated in the dark for 3-and 18-hour, followed by 6-hour light incubation in the previous section of quantum yield, were reused to measure their electrical conductivity. Handheld electrical conductivity meter (Eutech Instruments) was used to measure the electrical conductivity of the buffer medium (EC1) before boiling. The content from each petri dish (leaf disc and buffer solution) was poured into a test tube and immersed in a hot water bath at a temperature of 95 °C for 20 min.
After boiling, the buffer solution was left to cool before the second reading (EC2) was taken. The electrolyte leakage is expressed as percentage control using the following formula:

Statistical analysis
Leaf disc discolouration test. Treatments were arranged in a completely randomized design with 5 replicates. No statistical analysis was carried out for the leaf disc discolouration test because the colour change of each replicate within the similar treatment was identical.
Germination test. Treatments were arranged in a complete randomized design with 5 replicates.
All the percentage data for the germination test of c were fitted into the logistic function 43 , as below: where Y is the percentage of germination, shoot length or root length, d is the coefficients corresponding to the upper asymptotes, x is the concentration of compound c, x0 is the concentration of compound c, which inhibits the germination, shoot length and root length by 50% for untreated seeds and b is the line gradient.
Pre-and post-emergence herbicidal test. Treatments were arranged as factorial in a complete randomized design with 5 replications where factor one is the type of phytotoxic compound, whereas factor two is the application rate. The percentages of data were checked for normality and homogeneity of variance before subjected to two-way ANOVA analysis. Tukey test was used to compare the mean between treatments at 5% of the significance level.
Mode of action determination. Each treatment was arranged in a completely randomized design with 6 replicates. All data for quantum yield and electrolyte leakage determination were tested for normality and homogeneity of variance before performing one-way ANOVA. For calcium antagonist determination, arcsine square root transformation was performed on data of diuron and compound c with or without the addition of metal chloride before subjected to one-way ANOVA. The Tukey test was used to compare the means between groups at 5% of the significance level.        Oldenlandia verticillata throughout 10 days of incubation period. Each score representing different colour retention of leaf disc. Score 1: Green -The leaf disc surface is completely green; Score 2: Green brownish -10-30% of leaf disc surface is dark brown in colour; Score 3: Brown greenish -50-80% of leaf disc surface is dark brown in colour; Score 4: Dark brown -More than 90% of the leaf disc surface, in aggregate, is dark brown.       Oryza sativa (B) and Zea mays (C) 2 weeks after pre-emergence application.