( 6-chloroimidazo [ 2 , 1-b ] thiazol-5-yl ) prop-2-enyl ]-5 , 6-dimethoxy-3-methyl-1 , 4-benzoquinone : a new inhibitor of NADH dehydrogenase with antitumor activity 1

In this work we describe the synthesis and the biological activity of 2-[(E)-3-(6chloroimidazo[2,1-b]thiazol-5-yl)prop-2-enyl]-5,6-dimethoxy-3-methyl-1,4-benzoquinone i.e. an imidazothiazole derivative connected to the benzoquinone ring of Q0. This compound was tested as specific inhibitor of the NADH:ubiquinone (UBQ) reductase activity of NADH dehydrogenase in mitochondrial membranes. Binding of the imidazothiazole moiety to the quinone site normally occupied by the isoprenoid lateral side chain increases the inhibitory effect (with an IC50 for NADH-Q1 activity of 0.24 μM) whereas the benzoquinone moiety seems to lose the capability as electron acceptor from Complex I. The new compound was also tested as potential antitumor agent at the National Cancer Institute.


Introduction
Mitochondrial NADH-CoQ oxidoreductase, also known as Complex I, is the most complicated and the least understood of the respiratory chain complexes.Despite recent progress in structural studies, there is no structural information about Complex I that is comparable with the crystallographic data available for other respiratory complexes.This lack of information is principally due to its complexity (the mammalian enzyme consists of at least 46 subunits).Another characteristic feature of Complex I is related to the high number of its inhibitors: over sixty different families of compounds are known to inhibit Complex I, most of which are commonly used as pesticides, especially acaricides and anthelmintics.At the cellular level, Complex I function is particularly crucial in tissues such as neurons and pancreatic islet beta cells that rely heavily upon mitochondrial ATP and NAD-linked pathways. 2,3The potent cytotoxic effect of inhibitors in cancer cell cultures suggests that Complex I activity is particularly essential also in transformed cells. 4,5Moreover in the last decades the interest in this NADH-ubiquinone reductase is increasing due to its possible involvement in the pathogenesis of human neurodegenerative diseases such as Alzheimer's, Parkinson's, diabetes and in aging.All physiological quinones present a common moiety: the 2,3-dimethoxy-5-methyl-1,4benzoquinone (Q 0 see Figure 1) and a long isoprenoid chain (from 6 to 10 isoprenoid units) in the 6 position of the ring.
Because of their high hydrophobicity, these long chain quinones are not useful for "in vitro" determination of NADH-ubiquinone reductase and ubiquinol-cytochrome c reductase activity; on the other hand short chain quinones (with 5-10 carbon atoms either in a isoprenoid or in a saturated side chain) are the choice molecules for the enzymatic assay. 6,7In particular Q 1 (see Figure 1) is the most widely used as electron acceptor from Complex I, whereas Q 2 (see Figure 1) shows an activity that is 25-30% with respect to NADH-Q 1 reductase activity.This difference is due to an inhibition effect of Q 2 on Complex I.The identification and characterization of specific quinone features involved on the inhibition of Complex I represent a significant advance in understanding the functional mechanism of the complex and also contribute to the development of drugs specifically acting on mitochondria.In a previous study it was shown that thienylimidazo[2,1-b]thiazoles 8,9 were able to inhibit Complex I acting in a non competitive way with the ubiquinone substrate and interact with a site which is mutually exclusive with that of rotenone but not exclusive with that of piericidin and several inhibitors of NADH dehydrogenase.Founded on these data we planned the synthesis of an imidazothiazole derivative connected to the benzoquinone ring of CoQ 0 by means of a short chain resembling the isoprenoid side chain of Q 1 in order to evaluate the effect on Complex I of the modified CoQ 0 and to elucidate the ambiguous quinone specificity in the interaction with the enzyme.We have also tested the new derivative as potential antitumor agent.

Chemistry
The synthesis of the designed compound is outlined in the Scheme 1.

Scheme 1
The esters 2a and 2b were obtained by a Wittig reaction from the appropriate ylide and the aldehyde 1.The two isomers were purified by flash chromatography and the structures were assigned unambiguously by 1 H-NMR.The double bond protons in 2a (trans) are doublets at 6.50 and 7.67 ppm with a high coupling constant (J=16.2Hz) whereas the same protons in 2b (cis) are at 6.12 and 7.07 ppm with a coupling constant of 12 Hz.The esters 2a and 2b were reduced to the corresponding vinyl alcohol 3a and 3b using diisobutylaluminiumhydride.2,3-Dimethoxy-5-methyl-1,4-benzoquinone Q 0 was converted to 2,3-dimethoxy-5methylhydroquinone 4 by a conventional reductive step employing sodium dithionite as the reducing agent.In the subsequent step, compound 4 was reacted with the alcohol, trans isomer, 3a.The condensation was carried out in dioxane at room temperature and under nitrogen in order to prevent oxidation of the hydroquinone to the corresponding benzoquinone.Boron trifluoride etherate was used as the acidic condensing agent. 10,11The hydroquinone 5 was not characterized but was subjected as such to mild oxidation with silver(I) oxide to afford the substituted benzoquinone 6.When the reaction was performed with the alcohol, cis isomer, 3b did not react with the hydroquinone 4.

Biological results (a) Evaluation of activity on mitochondrial NADH dehydrogenase
The biological activity of the new compound 6 was evaluated on mitochondrial NADH dehydrogenase.It was initially tested to evaluate whether it was an electron acceptor itself.The electron acceptors commonly employed as substitutes of the physiological ubiquinones are short chain CoQ homologues (the most commonly used acceptor has been Q 1 ) and analogues (such as decylbenzoquinone).The experiments were conducted using submitochondrial particles from beef heart (SMP).For comparison purpose Q 0 , Q 1 and 3a were also tested.As shown in Table 1, neither 3a nor 6 are electron acceptors; thus we tested if they inhibited the NADH-Q 1 reductase of Complex I. We assayed the effect of 3a and 6 on the NADH-O 2 activity to measure their inhibition of the overall electronic flux and their effect on the NADH-Q 1 activity (NADH Coenzyme Q reductase) using Q 1 as electron acceptor and blocking Complex III (ubiquinol cytochrome c reductase) by antimycin and Complex IV by KCN.Compound 6 (where quinone and imidazothiazole moieties are connected by a propylene chain) shows a good potency as Complex I inhibitor with an IC 50 = 0.25 µM for NADH-Q 1 , IC 50 = 0.12 µM for NADH-O 2 .It has an IC 50 for NADH-O 2 lower than IC 50 for NADH-Q 1 , this induces to think that it is a competitive inhibitor compared to Q 1 .Moreover the derivative 3a lacking the quinone head, is a very weak inhibitor: IC 50 = 57.04µM for NADH-Q 1 , IC 50 = 49.66 µM for NADH-O 2 .These last experimental results suggest that the hydrophilic benzoquinone ring is fundamental to inhibit the NADH-ubiquinone reductase activity of Complex I and probably it locates the imidazothiazole derivative in the hydrophobic core of Complex I. 12 The test was performed by the National Cancer Institute (NCI, Bethesda, MD).According to a primary screening, compound 6 was evaluated for its cytotoxic activity on three human cell lines, such as NCI-H460 lung cancer, MCF7 breast cancer and SF-268 glioma.A compound is considered active when it reduces the growth of any of the cell lines to 32% or less.Compound 6 was active and it was passed on for evaluation in the full panel of sixty cell lines.This panel is organized into subpanels representing leukemia, melanoma and cancers of lung, colon, kidney, ovary, breast, prostate and central nervous system.The tested compound was evaluated using five concentrations at ten-fold dilutions (the highest being 10 -4 M and the others 10 -5 ,10 -6 ,10 -7 ,10 -8 M) taking into consideration the growth inhibitory power (GI 50 ), the cytostatic effect (TGI) and the cytotoxic effect (LC 50 ).The values of GI 50 (expressed as log 10 ) range from -4.65 to -6.40, log 10 TGI from -4.07 to -5.27 and log 10 LC 50 from -4.00 to -4.57. Figure 2 reports only the tumor lines where compound 6 showed GI 50 values < -5.10.In conclusion compound 6 was particularly selective against two subpanels (leukemia, melanoma) and two cell lines: NCI-H23 (non-small-cell lung cancer) and OVCAR-4 (ovarian cancer).-logM .The 1 H-NMR spectra were recorded in (CD 3 ) 2 SO on a Varian Gemini (300 MHz) spectrometer; the chemical shifts (referenced to solvent signal) are expressed in  (ppm) and J in Hz.In this data the abbreviation th = thiazole.Petroleum ether refers to the fraction boiling at 60-80 °C.Compound 1 was synthesized according to the literature. 13All reagents, unless otherwise stated, were supplied by Aldrich Chemical Company Ltd and were used as supplied.

Synthesis of (E) and (Z)-3-(6-chloroimidazo[2,1-b]thiazol-5-yl)prop-2-en-1-ol (3a and 3b).
A solution of 2 mL of DIBAL-H (diisobutylaluminium hydride as 1 M solution in hexane) was added dropwise over a period of 15 min.to a stirred solution of 2a or 2b (0.26 g, 1 mmol) in anhydrous toluene under dry N 2 at -13 °C.The mixture was stirred for 2 h at 0°C and for 18 h at room temperature.The excess reagent was decomposed by careful addition of methanol (30 mL) followed by 5% HCl (50mL).The mixture was adjusted to pH 7 with saturated NaHCO 3 solution and extracted with ethyl acetate (4x30mL).The organic layers were combined, washed with brine (2x20mL) and dried (Na 2 SO 4 ).The solvent was evaporated in vacuo to afford the crude alcohol (3a or 3b).Purification by column chromatography (petroleum ether/acetone 9/1) gave pure 3a as a white solid (66%) or 3b as a white solid (60%).6).2,3-Dimethoxy-6-methyl-1,4-benzoquinone (Q 0 ) (0.26 g, 1.4 mmol) was dissolved in 100 mL of diethyl ether and shaken with sodium dithionite (1.74 g, 10 mmol) in 50 mL of water until the solution become colorless.The mixture was extracted with diethyl ether.The organic phase was dried over anhydrous sodium sulphate, filtered and evaporated.The hydroquinone 4, obtained as colorless crystals, was dissolved in 20 mL of dry dioxane.This solution was added to 3a (0.30 g, 1.4 mmol) dissolved in 20 mL of dry dioxane and stirred in a nitrogen atmosphere at room temperature.The resulting solution was treated with 0.18 mL of boron trifluoride etherate (1.4 mmol) in 10 mL of dry dioxane over 20 min.The mixture was stirred for 12 h at room temperature, treated with 80 mL of water and extracted with diethyl ether (4x20mL).The organic phase containing the crude compound 5 was dried over anhydrous sodium sulfate, treated with silver(I) oxide (0.32 g, 1.4 mmol) and stirred for 6h.The solution was filtered and the filtrate was concentrated under reduced pressure.The residue was purified by column chromatography (petroleum ether/diethyl ether 70/30).Compound 6 was obtained by precipitation using a mixture acetone/petroleum ether as an orange solid (19%).Submitochondrial particles (SMP) were prepared from Beef Heart Mitochondria (BHM) by sonic irradiation of the frozen and thawed BHM, 14 the particles were essentially broken membrane fragments. 15Protein was evaluated by the Biuret method of Gornall et al. 16 with addition of 10% Sodium Deoxicholate and using Bovine Serum Albumin (BSA) as standard.NADH-CoQ reductase was assayed essentially as described by Yagi 17 and modified by Degli Esposti et al. 18 in presence of 2 mM KCN and 2 µM Antimycin A to block Complex IV and III respectively.Determination of the kinetic constants were accomplished following the decrease in absorbance in a double wavelength spectrophotometer at 340 minus 380 nm (extinction coefficient 3.5 mM -1 cm -1 ) due to the oxidation of NADH (75µM) in presence of different ubiquinone concentrations.NADH-O 2 reductase activity was assayed essentially in the same conditions but omitting KCN and Antimycin A from the assaying mixture.

(c) Antitumor activity Three cell panel
Each cell line was inoculated and preincubated on a microtitre plate.Test agents were then added at a single concentration and the culture incubated for 48 h.End-point determinations were made with sulforhodamine B (SRB), a protein-binding dye.Results for each test compound are reported as the percent of growth of the treated cells when compared to the untreated control cells.

Sixty cell panel
Each cell line was inoculated onto microtitre plates, then preincubated for 24-28 h at 37°C for stabilization.Subsequently, test agents were added in five 10-fold dilutions and the culture was incubated for an additional 48 h in 5% CO 2 atmosphere and 100% humidity.For each test agent, a dose-response profile was generated.End-point determinations of cell growth were performed by in situ fixation of cells, followed by staining with SRB which binds to the basic amino acids of cellular macromolecules.The solubilized stain was measured spectrophotometrically in order to determine relative cell growth in treated and untreated cells.A plate reader was used to read the optical densities, and a microcomputer processed the optical densities in the special concentration parameters: GI 50 , TGI and LC 50 .The optical density of the test well after 48 h period of exposure to the test drugs is T, the optical density at time zero is T 0 and the control optical density is C. GI 50 is the concentration of test drug where 100x(T-T 0 )/(C-T 0 )=50 TGI is the concentration of test drug where 100x(T-T 0 )/(C-T 0 )=0 LC 50 is the concentration of test drug where 100x(T-T 0 )/T 0 = -50 (the control optical density is not used in the calculation of LC 50 ).

Figure 2 .
Figure 2. Antitumor activity of compound 6.GI 50 expressed as -log of the molar concentration.

Table 1 .
Activity on mitochondrial NADH dehydrogenase