Synthesis of 2-(6-Acetamidobenzothiazolethio)acetic Acid Esters as Photosynthesis Inhibitors

Abstract

The synthesis and photosynthesis-inhibiting activity of 13 new 2-(6-acetamidobenzothiazolethio)acetic acid esters are reported. The new compounds were prepared by acetylation of 2-(alkoxycarbonylmethylthio)-6-aminobenzothiazoles with acetic anhydride. The structure of the compounds was verified by ¹H NMR spectra. The compounds inhibit photosynthetic electron transfer in spinach chloroplasts. The structure - activity relation was studied. Lipophilicity was found to influence substantially photosynthetic electron transfer.

Introduction

2-Alkylthio-6-aminobenzothiazoles have shown good antimycobacterial [1], antiyeast [2,3,4], anticandidous [5], and antialgal [6] activities, as well as inhibition of photosynthetic electron transport in spinach chloroplasts [7]. Their N-formyl [3, 4, 8] and N-acetyl [9, 10] derivatives have manifested antimycobacterial [8, 9], antifungal, anticandidous [10], antialgal and photosynthesis inhibiting [11] activities too.

Another antimicrobially active group of compounds, 2-(alkoxycarbonylmethylthio)-6-aminobenzothiazoles [12] have shown good antiyeast activity against Saccharomyces cerevisae and several derivatives exhibited good anticandidous activity against the clinical pathogen Candida Cruzei, the best of them being the n-hexyl and benzyl derivatives [12, 13]. The antialgal efficiency of these compounds is low, but they have manifested interesting inhibition of photosynthetic electron transport in spinach chloroplasts - the inhibitory activity increases with the increasing lipophilicity of the molecules [13, 14].

Results and Discussion

Based on the above experience with biologically active benzothiazole derivatives, thirteen new 2-(6-acetamidobenzothiazolethio)acetic acid esters (

Table 1. Characterization of the prepared 2-(6-acetamidobenzothiazolethio)acetic acid esters.

	Comp. R	Formula Mr	C	Wi(calc.) %/ Wi(found) % H N S			Yield %	M.p. (°C)
1	-CH3	C12H12N2O3S2	48.63	4.08	9.45	21.64	94.5	154.5-155.5
		296.37	48.40	4.02	9.26	21.65
2	-C2H5	C13H14N2O3S2	50.30	4.55	9.03	20.66	99.8	131.0-132.5
		310.40	50.53	4.59	9.04	20.80
3	-(CH2)2CH3	C14H16N2O3S2	51.83	4.97	8.63	19.77	92.5	111.5-112.5
		324.42	51.65	5.03	8.63	19.99
4	-CH2CH=CH2	C14H14N2O3S2	52.16	4.38	8.69	19.89	86.8	119-120
		322.41	52.49	4.36	8.43	20.19
5	-CH2C≡CH	C14H12N2O3S2	52.48	3.78	8.74	20.02	90.5	123.0-124.5
		320.39	52.29	3.66	8.77	19.73
6	-(CH2)3CH3	C15H18N2O3S2	53.23	5.36	8.28	18.95	74.0	113-114
		338.45	53.42	5.37	8.34	18.97
7	-CH(CH3)C2H5	C15H18N2O3S2	53.23	5.36	8.28	18.95	88.6	117-118
		338.45	53.02	5.41	8.16	18.83
8	-(CH2)4CH3	C16H20N2O3S2	54.52	5.72	7.95	18.19	85.2	116.5-118.5
		352.48	54.55	5.74	7.92	18.09
9	-(CH2)5CH3	C17H22N2O3S2	55.71	6.05	7.64	17.49	79.1	77-79
		366.50	56.03	6.21	7.64	17.44
10	-(CH2)6CH3	C18H24N2O3S2	56.81	6.36	7.36	16.85	47.4	82.5-84.5
		380.53	57.04	6.44	7.31	16.88
11	-(CH2)7CH3	C19H26N2O3S2	57.84	6.64	7.10	16.25	38.0	71.5-73.5
		394.56	58.06	6.74	7.07	16.25
12	-(CH2)8CH3	C20H28N2O3S2	58.79	6.91	6.86	15.70	39.2	80-81
		408.59	58.50	6.92	6.76	15.63
13	-CH2-C6H5	C18H16N2O3S2	58.04	4.33	7.52	17.22	99.3	122.5-124.0
		372.47	58.09	4.42	7.48	17.43

) have been synthesized by acetylation of 2-(alkoxycarbonylmethylthio)-6-amino-benzothiazoles [12] with acetic anhydride (Scheme 1).

The structures of compounds 1-13 were verified by ¹H NMR spectra. The chemical shifts of the hydrogen atoms of the benzothiazole skeleton are not influenced by the alkyl substituents.

The synthesised compounds were tested for photosynthesis inhibiting activity, they inhibited the oxygen evolution rate (OER) in spinach chloroplasts. The photosynthesis - inhibiting activity was expressed by IC₅₀ values, i.e. by molar concentrations causing a  50% decrease of OER with respect to the untreated control sample (

Table 2. Experimental values of IC₅₀ of the studied compounds concerning OER inhibition in spinach chloroplasts and calculated logP.

Compound	logP	IC50 (μmol dm-3)
1	0.86	-
2	1.20	857
3	1.67	514
4	1.60	411
5	1.14	350
6	2.07	83
7	2.09	-
8	2.46	56
9	2.86	47
10	3.26	106
11	3.65	430
12	4.05	1879
13	2.64	622

OER = oxygen evolution rate.

IC₅₀ = molar concentration of the inhibitor causing 50 % decrease of activity against the control.

). The dependence of photosynthesis inhibiting activity on the lipophilicity of the derivatives with R = n-alkyl and allyl showed a quasi-parabolic course with a maximum activity with the hexyl derivative ( IC₅₀ = 47 μmol dm^-3). Whereas the biologically active compounds, in order to reach their site of action, must penetrate through several compartments of thylakoid membranes (e.g. a series of lipid bilayers separated by aqueous layers), the highest biological effect is shown by molecules with a suitable lipophilicity, which enables them to cross both the above mentioned compartments. The passage of the short-chain compounds through the thylakoid membranes is limited due to their too low partition coefficient, resulting in an insufficient number of inhibitors reaching the site of action in proteins situated on the inner side of thylakoid membranes. On the other hand, the long-chain compounds due to their strong interaction with membrane lipids, remain predominantly incorporated in the lipid part of the membrane without reaching and damaging the corresponding membrane proteins. Similar results were also obtained for the dependence of photosynthesis inhibiting activity on the lipophilicity of 2-alkylthio-6-aminobenzothiazoles [7], 6-acetamido-2- alkylthiobenzothiazoles [11] and 2-(alkoxycarbonyl- methylthio)-6-aminobenzothiazoles [13]. Based on the results obtained by EPR spectroscopy it was shown that the site of the above benzothiazole derivatives in the photosynthetic apparatus of spinach chloroplasts is the donor side of the photosystem 2, upstream of the site of diphenylcarbazide action, i.e. in the oxygen evolving complex [14, 15].

The influence of quantum chemical parameters obtained by the AM1 method [16] as well as of the calculated values of lipophilicity [17] upon inhibition of oxygen evolution in spinach chloroplasts caused by 2-(6-acetamidobenzothiazolethio)acetic acid esters was studied.

The various alkyl substituents do not substantially influence the distribution of the molecular electrostatic potential [17] or charge density at the atoms. For this reason the studied biological activity of compounds 1-13 does not depend upon the obtained quantum chemical parameters. The biological activity is substantially influenced by lipophilicity. Its parabolic dependence on the biological activity has high statistical significance.

log(1/IC₅₀) = (3.3700±0.4596)logP - (0.7102±0.0868)log²P - (0.5934±0.5506)
f = 0.958 s = 0.1874 F = 33.6 n = 9

Using the bilinear model [18, 19] gave better results, which were statistically more significant. For this model, the best value of the lipophilicity (logP₀) was also calculated.

log(1/IC₅₀) = (1.3755±0.1251)logP - (3.6472±0.3030)log(β+1) +(1.3108±0.2326)
f = 0.9799, s = 0.13040, F = 72.6,
n = 9, logP0 = 2.71, β= 1.1690.10-3

The F-test value is statistically significant at the 99 % level of probability. From these facts, it results and it can also be shown by the distribution of the lipophilicity on the Van der Waals surface of the molecules (Fig. 1), that the biological efficiency of the molecule decreases when the value of logP is higher or lower than the value calculated for logP₀. The compound 9 (R = n-hexyl) with the best biological activity has the largest green area of lipophilicity (Fig. 1).

Experimental

General

The starting 2-(alkoxycarbonylmethylthio)-6-aminobezothiazoles were prepared and purified according to [12]. Melting points were determined on a Kofler hotstage apparatus and are uncorrected. ¹H NMR spectra were obtained on a TESLA BS 587 spectrometer (80 MHz) in deuterated dimethyl sulfoxide (DMSO) solution. Tetramethylsilane was used as internal standard.

The oxygen evolution rate (OER) in spinach chloroplasts was determined spectrophotometrically (Specord UV VIS, Carl Zeiss, Jena, Germany) by the Hill reaction. The measurements were carried out in phosphate buffer (20 mmol, pH=7.2) containing sucrose (0.4 mol dm^-3), MgCl₂ (5 mmol dm^-3) and NaCl (15 mmol dm^-3) using 2, 6-dichlorophenolindophenol as electron acceptor. All samples contained the same amount of chlorophyll (30 μg cm^-3) and they were irradiated (~100 W m^-2) from 10cm distance with a halogen lamp (250 W) using a water filter to exclude warming of the samples (suspension temperature 22 °C) [20]. The compounds were dissolved in DMSO because of their too low water solubility. The applied DMSO concentration (up to 5%) did not affect the OER.

Results of ¹H NMR analysis (80 MHz, deuterated DMSO)

6-Acetamidobenzothiazole skeleton

δ10.15 (NH, s, 1H); f. 39 (H-4, d, J=1.6 Hz, 1H); 7.75 (H-7, d, J=8.8Hz, 1H); 7.50 (H-6, dd, J=8.8 and 1.6 Hz, 1H); 2.09 (COCH₃, s, 3H). -S-CH₂COOR substituents

1 δ: 4.31 (SCH₂, s, 2H); 3.71 (OCH₃, s, 3H).

2 δ: 4.29 (SCH₂, s, 2H); 4.16 (OCH₂, q, 2H); 1.20 (CH₃, t, 3H).

3 δ: 4.29 (SCH₂, s, 2H); 4.08 (OCH₂, t, 2H); 1.60 (CH₂, sx, 2H); 0.86 (CH₃, t, 3H)

4 δ: 4.34 (SCH₂, s, 2H); 4.65 (OCH₂, d, J=5.1 Hz, 2H); 5.9 (=CH, m, 1H); 5.2 (=CH₂, m, 2H)

5 δ: 4.35 (SCH₂, s, 2H); 4.81 (OCH₂, d, J=2.4 Hz, 2H); 3.58 (≡CH, t, J´2.4 Hz, 1H)

6 δ: 4.29 (SCH₂, s, 2H); 4.12 (OCH₂, t, 2H); 1.4 (CH₂CH₂, m, 4H); 0.82 (CH₃, t, 3H)

7 δ: 4.25 (SCH₂, s, 2H); 4.81 (CH, sx, 1H); 1.54 (CH₂, qi, 2H); 1.17 (CH₃, d, 3H); 0.83 (CH₃, t, 3H).

8 δ: 4.28 (SCH₂, s, 2H); 4.10 (OCH₂, t, 2H); 1.5 (CH₂, m, 2H); 1.3 (CH₂, m, 4H); 1.19 (CH₃, t, 3H).

9 δ: 4.27 (SCH₂, s, 2H); 4.10 (OCH₂, t, 2H); 1.5 (CH₂, m, 2H); 1.2 (CH₂, m, 6H); 0.80 (CH₃, t, 3H).

10 δ: 4.27 (SCH₂, s, 2H); 4.10 (OCH₂, t, 2H); 1.5 (CH₂, m, 2H); 1.2 (CH₂, m, 8H); 0.82 (CH₃, t, 3H).

11 δ: 4.27 (SCH₂, s, 2H); 4.10 (OCH₂, t, 2H); 1.5 (CH₂, m, 2H); 1.2 (CH₂, m, 10H); 0.83 (CH₃, t, 3H).

12 δ: 4.27 (SCH₂, s, 2H); 4.09 (OCH₂, t, 2H); 1.5 (CH₂, m, 2H); 1.1 (CH₃, t, 12H) ); 0.84 (CH₃, t, 3H).

13 δ: 4.36 (SCH₂, s, 2H); 5.20 (OCH₂Ph, s, 2H); 7.33 (Ph, bs, 5H).

2-(6-Acetamidobenzothiazolethio)acetic Acid Esters 1 -13

Acetic anhydride (0.01 mol, 1.0 g) was added to 2-(alkoxycarbonylmethylthio)-6-aminobenzothiazoles [12] (5 mmol). After solidifying, the reaction mixture was boiled for 10 min with water, filtered off and washed with hot water.

Samples for analysis and testing were crystallised from acetone-water (4:1-5:1) and from methanol (derivatives 7-12) using charcoal.