Desigualdad

3-amino-benzylidene group was chosen to replace the benzyloxy-substiuent. The substitution of the oxygen linker with an amino-linker might show improved activity, possibly indicating hydrogen bond donor activity of this moiety. On the other hand the amino-benzylidene group is in theory easily introduced by simple nucleophilic substitution of the corresponding 2-halogen-pyridine analogues.

Initially, the introduction of the amino-benzylidene substituent should occur after Knoeve-nagel condensation with the appropriate 2-substituted pyridine aldehyde. A good leaving group in ortho-position next to the pyridine nitrogen was thought to facilitate nucleophilic aromatic substitution reactions with amino-benzylidene derivatives (Scheme 14). Therefore,

rhodanine-3 Rhodanine-N-acetic acid derivatives

Scheme 14: Synthesis of heterocyclic derivatives 22a as replacement for the 3-benzyloxygroup; (i) R=Et (14), X=Cl (20a), NaOAc, 80^◦C, EtOH; (ii) neat ben-zylamine, 190^◦C, see Table 9.

Table 9: Synthesis of heterocyclic modified rhodanine-N-acetic acid derivatives; of R, X for the synthesis of21a–c; yield and chemical shift of CH-signal, see Scheme 14.

# R X yield [%] δ_DMSO−d6

21a Et Cl 20 7.88

21b H Cl no reaction 21c Et Br decomposition 22a Et NHBn no reaction

N-acetic ethyl ester (R=Et, 14) was subjected to Knoevenagel condensation condition with 2-chloro substituted isonicotinaldehyde 20a (Scheme 14). The reaction yielded the desired Knoevenagel product21a in moderate yields of 20 % after simple filtration. The chemical shift of δH 7.88 ppm of the CH-signal identified the product as the Z-isomer. Attempts to synthesise the free carboxylic analogue 21b failed and no reaction was observed. Also the attempt to install the 2-bromo-leaving group on the pyridine moiety failed and only decomposition was observed in the synthesis of derivative 21c. After having installed the chloro-leaving group on the pyridine moiety of the derivative21a, the compound was subjected to aromatic substi-tution conditions in neat benzylamine solution. The reaction mixture was refluxed at 190^◦C.

However, no reaction between the amino-benzylidene group and the 2-chloro-aminopyridine 21a was observed. The lack of reactivity is most likely explained by the poor chloro leaving group compared to a bromo-substituent in 2-position of the pyridine ring. In order to repeat the reaction with the better bromo-leaving group, the Knoevenagel reaction with the 2-bromo-pyridine aldehyde20b was carried out at room temperature and under a nitrogen gas protec-tion atmosphere to prevent decomposiprotec-tion of the aldehyde. None of these efforts resulted in the formation of the bromo-derivative of21a. In order to overcome the decomposition of the starting aldehyde 20b under Knoevenagel condensation conditions, a different strategy had

N Br

Scheme 15: Synthesis of amino-substituted aldehydes 25a–g; (i) TFA, EtOH, CHCl₃, 80^◦C, 2 h; (ii) NH₂-R (neat), reflux (150-190^◦C);(iii) Cs₂CO₃, DMF, NH₂-R; (iv) Cs₂CO₃, ACN, NH2-R; (v) NH2-R, NaOtBu; (vi) Pd(OAc)2, NaOtBu, DPPF, dioxane/toluene (1:1); (vii) NH2-R, CuI, CsOAc, toluene, 110^◦C; (viii) H2O, TFA, rt; for substituents of n,R¹-R⁴ see Table 10.

to employed. Analysing the aldedhyde20b in closer detail identified the aldehyde functional group as the most sensitive functional group. For this reason the aldehyde functional group was protected as its acetal (Scheme 15). By applying this strategy the order of the reaction could be reversed. The nucleophilic aromatic substitution reaction could be carried out prior to the Knoevenagel reaction.

The acetylation of the aldehyde functional group was carried out in ethanol in the presence of TFA. The reaction mixture was heated to reflux to form the full acetal in quantitative yields.

The full acetate23 was obtained after simple basic extraction as clear oil. The crude was suf-ficiently pure (NMR >95 %) for the following substitution reaction. Different reaction conditions have been explored for the nucleophilic aromatic substitution of the 2-bromo-pyridine derivative 23 (Scheme 15, Table 10). At the beginning, the amination was carried out in neat benzylamine solution at boiling point of the corresponding amine (100-190^◦C) (method (ii) in Scheme 15).

However, the desired nucleophilic substitution did not occur and the starting material could be recovered. Next, bases were added to increase the nucleophilicity of the benzylamine deriva-tives. Here, caesium carbonate was used in dimethylformamide or acetonitrile, however the substitution product was not observed and the starting materials were recovered after column chromatography (methods (iii-iv), Scheme 15). Using sodium tert-butoxide as a strong base, resulted in the successful formation of the desired 2-amino-pyridine24i and 24j in moderate yields of 21-36 % (method (v), Scheme 15). But the trifluoromethyl substituted analogue24b could not be synthesised under these reaction conditions. Different conditions for the substitu-tion were explored in order to improve the nucleophilicity of the amines, but reacsubstitu-tion yields did not improve. A different approach for the substitution reaction was the palladium catalyst am-ination reaction using Pd(OAc)₂ as palladium source (method (vi), Scheme 15). The reaction proceeded to the desired product 24k, but the yields were not satisfactory (18 %),

reflect-3 Rhodanine-N-acetic acid derivatives

Table 10: Synthesis of amino-substituted aldehydes, yield and method; n, R¹-R⁴ for amino-substituted acetals 24a–n and aldehydes 25a–g; reaction conditions used and yields; see Scheme 15.

aldehyde

# n R¹ R² R³ R⁴ method yield [%] # yield [%]

24a 1 H H H CH₃ (ii) no reaction

24b 1 H H H CF3 (ii) no reaction

24c 1 H H H H (ii) no reaction

24d 1 CH₃ H H H (ii) no reaction

24e 2 H H H H (ii) no reaction

24f 2 H H H OH (ii) no reaction

24g 1 H H H CH₃ (iii) no reaction

24h 1 H H H CH3 (iv) no reaction

24b 1 H H H CF₃ (v) no reaction

24i 2 H H H H (v) 21-32 25a 44-100

24j 1 H H H CH3 (v) 36 25b 100

24k 1 H H H H (vi) 18 25c 100

24l 1 H H H CH₃ (vii) 43 25f 43

24m 1 H H H CF3 (vii) n.a. 25d 100

24n 1 H H H H (vii) 65 n.a. n.a.

ing the poor reactivity of substituted aldehydes/ acetals from type 20a–b with benzylamine derivatives.^[175] In order to overcome the lack of reactivity an Ullman-type coupling reaction with Cu(I) has been used for the formation of the acetals 24l, 24m, and 24n (Method (d) in Scheme 15).^[175]Therefore, a modified version of the amination protocol of Frey et al. has been applied to the acetal 23.^[175] Copper iodide has been used as source for Cu(I) and caesium acetate served as the base. The reaction proceeded in excellent yields (43-65 %) and has been chosen as the preferred approach for the synthesis of derivatives of the type24. In all cases, the desired 2-amino aldehydes25a, 25b, 25c, 24l, and 25d were obtained after acidic hydrolysis of the acetal with TFA at room temperature. Compound 26a was chosen for the further Knoevenagel reaction with the ethyl ester of14 (Scheme 16). The reaction proceeded in good yields of 75 %. In addition to these heterocyclic modification several other pyridine-carboxaldehydes, furanyl-aldehydes as well as cinnamonyl-aldehydes were chosen for the Knoevenagel reaction with rhodanine-N-acetic acid 2 and thiazolidine-2,4-dione derivatives and their ethyl-ester analogues7 and 14 (Scheme 16). The yields of the reactions varied from 14-100 % and the chemical shift of the CH-signal in¹H-NMR experiments showed only forma-tion of the thermodynamically more stable Z-isomer.^[160] The chemical shift for the CH-signal in DMSO-d₆ (7.05-7.96 ppm) were observed further downfield than for CDCl₃(7.29-7.75 ppm).

N S

Scheme 16: Synthesis of heterocyclic derivatives 26a–j; X=S,O; R=H,Et, R¹=25f, 2-pyridine, 3-pyridine, 4-pyridine, furan, cinnamonaldehyde (i) NaOAc, EtOH, 80^◦C, see Ta-ble 11.

Table 11: Synthesis of heterocyclic rhodanine-N-acetic acid/ ester derivatives; R¹-R² and X of heterocyclic compounds 26a–j; yield and chemical shift of CH-signal, see Scheme 16.

yield NMR shift of CH [ppm]

# R¹ X R² [%] δ_solvent δ_H δ_C

26a Et S 25f 75 CDCl₃ 7.53 131.6

26b H S 2-pyridinyl 54 DMSO-d6 7.92 129.4

26c H S 3-pyridinyl 74 DMSO-d₆ 7.96 130.6

26d H S 4-pyridinyl 82 DMSO-d₆ 7.90 n.a.

26e H S phenylallylidene 20 DMSO-d6 7.71-7.75 n.a.

26f Et S 2-pyridinyl 100 CDCl₃ 7.67 128.6

26g Et S 3-pyridinyl 14 CDCl₃ 7.75 129.9

26h Et S 4-pyridinyl 28 DMSO-d6 7.05 n.a.

26i Et O 4-pyridinyl 21 CDCl₃ 7.29 n.a.

26j Et S 2-furanyl 38 CDCl₃ 7.51 119.1-119.2

3.2.11 Reduction of the exo-cyclic double bond in rhodanine-N-acetic acid derivatives The exo-cyclic double bond within the rhodanine or thiazolidine-2,4-dione derivatives are part of a Michael acceptor system. The double bond is conjugated to a carbonyl group, making it potentially prone to 1,4-nucleophilic attack from biological nucleophiles such as gluthathione.^[82,83]

Previous examples of nucleophilic attacks to the Michael acceptor system in rhodanine and thiazolidine-2,4-dione derivatives have been discussed in section 3.2.2 (Scheme 3). In or-der to study the effect of the double bond on the activity against various protozoa, a range of saturated analogues have been synthesised. There are essentially three methods to re-duce the double bond in rhodanine-like derivatives.[104,176,177] Derivatives with an exo-cyclic sulphur group can be reduced by LiBH₄ in the presence of pyridine or via the Hantzsch-ester method.[104,176,177]The reported yields for the LiBH4 in pyridine reduction for rhodanine

deriva-3 Rhodanine-N-acetic acid derivatives

Scheme 17: Synthesis of reduced analogues 27a–c; for X=S, R^1,2=OH,H (i) Hantzsch ester 28, activated SiO₂ gel, toluene, 85^◦C, dark; for X=O, R^1,2=OH,H (i) Pd/C, H₂, dioxane, see Table 12.

Table 12: Reduction of rhodanine-N-acetic ester analogues; X and R¹-R² of reduced ana-logues27a–c; yield, chemical shift of CH-signal, see Scheme 17.

# X R1 R2 method yield

27a S H H (i) 67

27b O H H (ii) 98

27c O OH OH (ii) 100

tives were only 26 %.^[161]For this reason, the second approach, the Hantzsch-ester reduction, was chosen for the reduction of rhodanine derivatives. The Hantzsch ester is a milder reduc-ing agent compared to LiBH₄.^[177]Compounds with an exo-cyclic carbonyl group were reduced by catalytic hydrogenation with Pd/C and H₂ (Scheme 17). The Hantzsch ester reduction of the exo-cyclic double bond in27a proceeded in good yields of 67 %. The reduced analogue 27a was easily purified by direct application of the crude silica to column chromatography.

The hydrogenation of the compounds from the type7 worked very well, yielding the products 27b and 27c after simple filtration over celite.

3.2.12 Synthesis of photo-affinity label for identification of target proteins