CuAAC reaction study

In a prospective study, we optimized the CuAAC reaction of salan Ti(IV)-bis-chelate 19a and phenylacetylene 29a as well as with the more “drug-like” 27/28 with (azidomethyl)benzene 31 as model reaction under copper mediated CuAAC reaction condition as shown in Figure 39. Both

reactions proceed smoothly and give the corresponding product 30 and 32. However, the CuAAC reaction between 19a and 27/28 turned out to be a challenge. The solubility of the formed products were extremely poor in polar (DMF, DMSO, MeCN), nonploar (Toluene, Hexane) and protic polar solvents (H2O, EtOH, MeOH, AcOH) even at elevated temperature. Hence, purification of product 38a and 38b was impossible since the reaction mixture did not dissolve as needed.

Figure 39. CuAAC reaction study of “drug-like” 27/28 with azido functionalized salan Ti(IV)-bis-chelate 19a.

Starting from pteridine 24, the coupling reaction with 4-ethynylaniline 36 was then investigated.

4-ethynylaniline 36 was prepared from 4-iodoaniline 33 by Sonogashira coupling with trimethylsilylacetylene 34 and concomitant silyl deprotection. After pteridine 24 reacted with 4-ethynylaniline 36 in DMA for 24 h, the coupling product 37 was obtained in 38 % yield. The following selective hydrolysis of one amino functionality to give alkynyl functionalized pteridione 40 was carried out with aqueous sodium hydroxide under anaerobic conditions according to Seeger et al.^[107]

or with aqueous hydrochloride acid. ^[108-109] Both reactions did not result in any detectable amount of the aspired 2-amino-6-((4-ethynylphenylamino)methyl)-pteridin-4(4αH)-one 40. (Figure 40)

Figure 40. Attempted synthesis of pteridione precursor 40.

In an alternative approach the pteridine 24 was converted to pteridione 39 by treatment with 33 % hydro bromic acid in acetic acid solution (Table 11).^[110] Next the coupling reaction of pteridione 39 with 4-amino-phenylacetylene 36 under different reaction conditions was studied as illustrated in Table 11. After the reaction had finished, the crude product was obtained in poor yield and purity. In contrast to pteridine 24, pteridione 39 was found to be stable in [D6]-DMSO during NMR measurement. After the coupling reaction with the alkyne 36, the raw NMR showed no remaining 39 but no product 40 was formed either. The reaction was also monitored via HPLC, the disappearance of starting material 39 was optimized without the formation of a new major peak. This fact might be based on either extremely low solubility or a complete lack of reaction. Since the consumption of the starting material was detected by HPLC it was either consumed by decomposition or by reaction. The analysis of the formed precipitate was unsuccessful. Purification of the precipitate by dissolving in aqueous sodium hydroxide, and subsequent reprecipitation by adding hydrochloride acid did not improve purity; also, precipitation in H2O at 0 ^oC did not give the final product.

We also tried the coupling reaction of pteridione 39 with other aromatic nucleophiles containing an alkynyl group, such as 4-ethynylbenzoic acid (26), also no target product was obtained, and instead the potassium salt of 26 was isolated. The coupling with prop-2-ynyl-4-aminobenzoate (25) did not give the target product either, which may result from the poor nucleophilicity of the amino group.

However, complete degradation of the starting material 39 was observed after prolonged reaction time. (Figure 41)

Table 11. Reaction optimization of pteridione 39 and alkyne 36.

Entry Time (h) Temp (^oC) Work-up procedure

1 12 r.t. Aqueous sodium hydroxide was added to the reaction, then the crude precipitated by adding aqueous hydrochloride acid.

2 12 r.t. After solvent evaporation, the crude residue was taken up in [D6]-DMSO. A ¹H NMR recorded no product was observed.

3 12 r.t.

H2O was added to the reaction, kept the mixture at 0 ^oC for 24 h.

Precipitate was filtered off and a ¹H NMR spectrum was recorded in [D6]-DMSO, no main product was observed.

4 14 days r.t. The reaction was monitored by HPLC (Elute: MeOH-H2O), no main product was observed.

Figure 41. Attempted coupling of pteridione 39 with other aromatic nucleophiles 25 and 26.

To enhance the nucleophilicity of the alkynyl aniline 36, its N-methylated counterpart 42 was synthesized. Starting from 4-iodoaniline 33, a sequence of N-methylation, Sonogashira coupling with TMSA, deprotection gave 42 in an overall yield of 80 %. It was further coupled with pteridione 39 in DMA under basic conditions (K2CO3, Na2CO3, Cs2CO3, NaOH and KOH). The reaction failed to give the desired compound 43. Instead, the starting material 39 and some unknown byproduct were obtained. (Figure 42)

Figure 42. Reaction of pteridione 39 with secondary amine 42.

We next used a strongly nucleophilic secondary amine instead of 42 to react with pteridione 39.

When pteridione 39 reacted with N-methyl aniline 44 in DMA,^[111] the mixture was stirred at r.t. for 24 hours, and then was monitored via HPLC. We noticed the disappearance of starting materials, and formation of trace amounts of 45 identified by LC-MS, and unknown byproducts. (Figure 43)

Figure 43. Reaction of pteridione 39 with N-methyl aniline 44.

3.2.5.4 Discussion

Based on the retrosynthetic analysis, we have successfully synthesized two folate like precursors 27 and 28, respectively. Salan Ti(IV)-bis-chelate containing azido motif 19a was also been prepared according to previous research. We tried different bi-functional compounds containing alkynyl group and amino group as the linker. Pteridine 24 successfully coupled with the linker, but CuAAC approach with the Ti(IV) complex failed, decomposition of the starting material and unknown byproduct were observed. When pteridine 24 was converted to pteridione 39, the stability and solubility improved.

While the coupling reaction with substituted anilines did not result in a conversion or formation of any coupling products, the reaction with the more nucleophilic aliphatic amines like octylamine (data not shown) resulted in the formation of less than 5% substitution product. This further underpins the extremely low electrophilicity of pteridione 39. (Figure 44)

Figure 44. Summary on the reaction of pteridine 24 and pteridione 39 with different nucleophiles.

Due to either the poor solubility of the pteridine-2,4-diamine derivatives or the low reactivity of pteridione 39, various methods failed to yield a uniform product. Recently, Schibli’s group reported a method for preparing ¹⁸F labeled folate-probes for PET imaging. Parts of the synthetic approach are quite similar to ours, in their work, they encountered the same difficulties on the low reactivity and solubility.^[112] (Figure 45)

Figure 45. Reaction of pteridine 24 and pteridione 39 reported by Schibli and co-workers.

In an effort to tackle above mentioned problems, we then used an ester bond to connect the folate via the γ-carboxylic acid with the salan Ti(IV)-bis-chelates.^[113] We successfully prepared the tosylated Ti(IV) complex 51, which was esterificated in DMF with sodium salt of folic acid 50 to obtain the folate-conjugated Ti(IV) complex 52. However, only some precipitates which has extremely low solubility were obtained. (Figure 46)

Figure 46. Attempted esterification of folic acid 50 with tosylated salan Ti(IV)-bis-chelate 51.

Schibli’s group solved the challenge by redesigning the synthetic route, starting from 4-amino-2-nitrobenzoic acid (53), after protection with 9-fluorenylmethoxycarbonyl, the given product was coupled with L-glutamic acid di-tert-butyl ester. After removal of the Fmoc group, the given compound 54 was coupled to pteridione 39, the N² amino group of the pteridione protected as N,N-dimethyl formamidine to enhance solubility, to afford the intermediate 55 in 14 % yield. In the last two steps, deprotection of the amidine and ¹⁸Fsubstitution, only 4 % of the final targeting compound was obtained.^[112] (Figure 47) 

Figure 47. Synthesis of ¹⁸F based folic acid probe by Schibli and co-workers. ^[112]