Synthesis of substituted thieno[2,3-d ]isothiazoles as potential plant activators

Thieno[2,3-d ]isothiazoles were synthesized as potential plant activators. Based on previously reported thienothiadiazole plant activators, one ring nitrogen was eliminated from the bicyclic motif in order to extend structure activity relationship information. Thieno[2,3-d ]isothiazoles bearing different functional groups were prepared starting from readily available 3,4-dibromothiophene. Introduction of an oxime and a methylthio group in positions 1 and 2 of the thiophene system set the stage for cyclization, which occurred spontaneously after converting the oxime into the more reactive oxime ester in the presence of methanesulfonyl chloride.

Within this study, we further investigated deletions of nitrogen from the parent scaffold.Synthesis of the isomeric thieno [2,3-d]isothiazole system and various carboxylic acids as well as esters (Figure 2, 10) became of interest in connection with their potential as plant activators.

Results and Discussion
Little is known about the synthesis of the thieno [2,3-d]isothiazole system.Paulmier and coworkers 33 synthesized the title compound 12 by cyclization of 3-(methylthio)thiophene-2carbaldehyde oxime 11 with phosphorus pentoxide in phosphoric acid (Scheme 1, upper part).The group of Scrowston 34 performed the cyclization using ketoximes 13a and 13b as starting materials in acetic acid and acetic anhydride leading to compound 14.Moreover, they reported a cyclization method using 4-nitrobenzoic acid ester as a leaving group on the oxime (Scheme 1 lower part).To synthesize thieno [2,3-d]isothiazoles carrying carboxylic acid functionalities in positions 5 or 6 a starting material was required to enable subsequent introduction of the acid functionality.During the planning phase of our approach, it was important to find a substrate which allowed functionalization in both positions 5 and 6 in order to develop a modular synthesis for the design of a focused compound library at a later stage.3,4-Dibromothiophene 15 was identified as suitable starting material which is additionally cost efficient and easily available.According to our synthetic strategy (Scheme 2) one bromine is used to install the sulfur center of the isothiazole; after cyclization the remaining bromine can either react in a metal-halogen exchange reaction to give a carboxylic acid in position 6 (after quenching with CO2) or it can act as directing group for metalation into position 5 followed by CO2H introduction.

Scheme 2. Synthetic strategy.
Starting from 3,4-dibromothiophene 15 a methylthio group was introduced in excellent yield using two equivalents of n-butyllithium and two equivalents of dimethylsulfide according to a procedure of Bäuerle et al. (Scheme 3). 35Next, an oxime has to be introduced in position 5 of the thiophene ring which initially requires introduction of a ketone or aldehyde function in this position.This can be carried out via a Friedel-Crafts acylation since the methylthio group in 4 position selectively activates the adjacent 5 position due to its +M effect.To obtain the methyl ketone 18a, 1.2 equivalents acetylchloride and 1.2 equivalents of AlCl3 were used in dry dichloromethane 36 and 18a was obtained in 63% yield (Scheme 3).Compound 18b was obtained via the same protocol using benzoylchloride as the acylation reagent in 62% yield.The synthesis of the aldehyde 18c was less efficient under Friedel-Crafts conditions 37 and led only to 44% isolated yield after some optimization efforts.Additionally, the reaction workup was tedious owing to the formation of an emulsion caused by the metal salts formed in the workup process.

Scheme 3. Synthesis of carbonyl substrates 18a-d.
Hence, a lithiation strategy was investigated for 18c and 18d. 38,39Again taking advantage of the aryl activation by the SMe group, 16 was brominated with N-bromosuccinimide to give 2,4dibromo-3-(methylthio)thiophene 17. 38 Then, metal-halogen-exchange was performed using nbutyllithium, followed by quenching with dimethylformamide as electrophile.Unfortunately, the desired aldehyde 18c was obtained in only 45% yield also via this route.However, the workup procedure of this two-step process was much more convenient and fast compared to the Friedel-Crafts acylation reaction.Trifluoromethyl ketone 18d was synthesized also via this sequence using N,N-diethyl-trifluoroacetamide as electrophile; in this case a high yield of 85% was obtained for 18d (Scheme 3).
The carbonyl functions of all analogs were converted to the corresponding oximes using hydroxylamine hydrochloride and sodium acetate as a base in dry methanol at room temperature. 40Starting from the methyl ketone 18a, the oxime compound 19a could be obtained with 89% yield (Scheme 4).According to NMR data and melting point (only 2 °C interval) only one stereoisomer was formed.To determine the structure of this product, single crystal X-raycrystallography was used.It was revealed that the hydroxyl group of the oxime is trans to the thiophene ring representing the E-isomer.Moreover, it could be shown that the oxime nitrogen is close to the thiophene sulfur atom but is not coplanar with the ring (Figure 3).This is in accordance with work from Farell and coworkers. 41The formation of the aldoxime 19c could be performed with 83% yield via the same protocol.This time, two stereoisomers were detected in a 1:1 mixture by NMR spectroscopy.The two compounds could not be separated entirely but pure amounts of both of them were obtained.The compound with the larger Rf value in a light petroleum/ethyl acetate mixture was analyzed by Xray crystallography and identified as the E-isomer (Figure 4).As for the methyl ketoxime 19a, the oxime group points to the sulfur atom of the thiophene ring.The molecule is mostly planar with only the S-methyl group pointing out of the plane.The mixture of the two isomers could be isomerized to the Z-isomer by heating in acetic acid for one hour.Isomerization occurs also during storage at room temperature after a few days.

Scheme 4. Oxime formation and cyclization.
The synthesis of the phenyl ketoxime 19b took longer owing to deactivation at the carbonyl center; the product was formed in 75% in a 1:5 mixture of E/Z isomers (Scheme 4).To decrease the reaction time (3 days), the reaction was performed in dry ethanol under reflux in the presence of 2.5 equivalents of base to inhibit isomerization to the Z-isomer.With this protocol, complete conversion was achieved overnight and the product obtained with 68% yield in an E/Z mixture of 1:2.5 (not shown).The reaction with the trifluoromethyl ketone 19d at room temperature showed no conversion overnight.Therefore, the reaction mixture was also heated to reflux and after two days conversion was completed.The reason for the long reaction time might be that the initial intermediate formed from the nucleophilic attack of the hydroxyl amine at the carbonyl group is quite stable due to the electron withdrawing effect of the trifluoromethyl group.Hence, elimination of water is hindered.Nevertheless the product was isolated in 71% yield with an E/Z mixture of 1:1 (Scheme 4).Regarding the nomenclature of the isomers it should be considered, that due to the higher priority of the trifluoromethyl group, the thiophene substituent is cis to the hydroxyl group in the E-isomer.
For the cyclization to the thienoisothiazoles, the oximes 19 were first converted to the corresponding oxime esters by reaction with p-nitrobenzoyl chloride or methanesulfonyl chloride.These activated oxime esters were not isolated since they were expected to spontaneously cyclize to the desired products, provided that the correct stereochemistry is established.Attack of the sulfur nucleophile can only occur if the leaving group X adopts a trans configuration relative to the thiophene ring; otherwise, the leaving group impedes the nucleophilic attack on the nitrogen due to steric hindrance (Figure 5).As a result, E-oximes are preferred for cyclization reactions.In a first experiment oxime 19a was reacted with p-nitrobenzoyl chloride in the presence of K2CO3 in dry dichloromethane.After workup the cyclization product was isolated in 55% yield.To further facilitate the process, the reaction was performed using methanesulfonyl chloride and Et3N as base again in dry dichloromethane as solvent.After 30 minutes stirring at room temperature 20a was isolated in an improved 68% yield.Since X-ray analysis of 19a revealed that the oxime nitrogen points towards the thiophene sulfur, rotation around the thiophene-oxime bond seems possible in solution and cyclization can indeed take place.

Scheme 5. Nitrile formation from Z-19c.
The cyclization reaction of the aldoxime 19c was repeated using methanesulfonyl chloride as activating agent.Using the pure E-isomer of 19c, 75% of the cyclized product 20c was isolated accompanied by 10% of nitrile 21 formed upon elimination of water from the oxime.On the other hand, when the pure Z-oxime of 19c was submitted to the same reaction conditions only formation of the nitrile 21 (Scheme 5, 78% isolated yield) was observed which is in accordance to literature. 42When the 1:1 mixture of isomers was converted at room temperature overnight, the main product was again nitrile 21 and only traces of cyclization product were detected.When the experiment was repeated at -10 °C, 15% of the cyclized product 20c was isolated but still, the nitrile was the main product.
For the formation of the phenyl analog 20b, the 1:2.5 mixture of isomers 19b was used.Only 21% of cyclization product was isolated.Considering the amount of E-isomer, which is the only one that can cyclize, this represents an effective yield of 53%.
The protocol was also applied on the 1:1 isomeric mixture of the trifluoromethyl ketoxime 19d.The reaction yielded thienothiazole product 20d in 11%.Moreover, 70% of oxime ester 22 was isolated and analyzed by X-ray crystallography (Figure 6).It revealed to be the Z-isomer which is the unreactive isomer towards cyclization.The low yield may be attributed to isomerisation of the E-to the Z-isomer in solution or upon storage.The last step of the synthesis was the introduction of an ester group on the ring system in position 6.First, a metal-halogen-exchange with n-butyllithium in dry diethylether at -70 °C was performed and then dry carbon dioxide was used as an electrophile at -100 °C (Scheme 6).The reaction resulted in good yields for the thienoisothiazole 23c and its methyl and phenyl analogs 23a and 23b.The electrophilic attack of carbon dioxide on the trifluoromethyl compound 20d resulted in decomposition of the ring system.Even lower temperatures did not inhibit this reaction.The resulting carboxylic acids 23a-c were esterified using concentrated sulfuric acid in dry methanol under reflux.All three compounds 24a-c were obtained in good yields (Scheme 6).To access the isomeric isothiazolo-5-carboxylate, substrate 20a was lithiated with LDA in tetrahydrofuran at -50 °C followed by quenching with dry carbon dioxide at -100 °C.The carboxylic acid 25 was esterified with sulfuric acid in dry methanol to give 26 in 78% yield (Scheme 7).

Scheme 7. Ester introduction in position 5 of 20a.
In an alternative approach towards the non-halogenated 5-carboxylate, unsubstituted 3methylthienoisothiazole 27 was synthesized starting from 6-bromo-3-methylthienoisothiazole 20a.A metal-halogen-exchange was performed with n-butyllithium and water was added as electrophile.The carboxy group was introduced in 5 position in the same manner as above (LDA, then CO2) to give 28 in 88% yield.Finally, esterification in dry methanol with sulfuric acid gave 29 in 77% (Scheme 8).Scheme 8. Debromination and ester introduction on 20a.

Conclusions
A series of thieno [2,3-d]isothiazoles was synthesized from a common, cheap and readily available 3,4-dibromothiophene. It was demonstrated that only the E-isomers can cyclize to the target compounds.The Z-isomer is either converted to a nitrile such as 21, or can be isolated as oxime ester intermediate.X-ray analysis was carried out on key compounds to confirm their structure and to corroborate the observed reactivities.Finally, methods to introduce an acid or ester functionality in 5 and 6 positions of the isothiazole core were developed.The compounds are submitted to biological testing and the results will be reported in due course.

Experimental Section
General.Flash column chromatography was performed on silica gel 60 (40-63 µm), obtained from Merck.Melting points were determined using a Kofler type Leica Galen III micro hot stage microscope and are uncorrected.NMR spectra were recorded on a Bruker AC 200 spectrometer and chemical shifts are reported in parts per million, using TMS as internal standard.Combustion analysis was carried out at the Microanalytic Laboratory, University of Vienna.