Synthesis of 8-(1,2,3-triazol-1-yl)-7-deazapurine nucleosides by azide-alkyne click reactions and direct C-H bond functionalization

Abstract

Treatment of toyocamycin or sangivamycin with 1,3-dibromo-5,5-dimethylhydantoin in MeOH (r.t./30 min) gave 8-bromotoyocamycin and 8-bromosangivamycin in good yields. Nucleophilic aromatic substitution of 8-bromotoyocamycin with sodium azide provided novel 8-azidotoyocamycin. Strain promoted click reactions of the latter with cyclooctynes resulted in the formation of the 1,2,3-triazole products. Iodine-mediated direct C8-H bond functionalization of tubercidin with benzotriazoles in the presence of tert-butyl hydroperoxide gave the corresponding 8-benzotriazolyltubercidin derivatives. The 8-(1,2,3-triazol-1-yl)-7-deazapurine derivatives showed moderate quantum yields and a large Stokes shifts of ~ 100 nm.

Graphical Abstract

Synthetic transformations, biological activities and physicochemical properties of 7-deazapurine derivatives (mainly tubercidin, toyocamycin, and sangivamycin antibiotics) have been extensively studied^1–9 and are subject of the excellent reviews.^10–12 In recent years 7-deazapurine nucleosides have been explored as substrates for 1,4-regioselective copper-catalyzed azide/alkyne cycloaddition (CuAAC), which resulted in developing fluorogenic DNA probes.^13–16 These studies have mainly concentrated on the application of 7-alkynyl derivatives of 7-deazapurines and 7-deazanucleosides for the preparation of the fluorescent 1,2,3-triazol-4-yl probes.^4,17–18 Studies with 7-azido-7-deazapurine nucleosides as well as 8-azido- or 8-alkynyl-7-deazapurine nucleosides have received much less attention due to the lack of availability of the convenient synthetic protocols for their synthesis.^17,19 This is despite the fact that azidonucleosides^20–22 including 8-azido as well as 8-alkynyl purine23 nucleosides have been used as convenient substrates for CuAAC and strain-promoted azide-alkyne cycloaddition (SPAAC).^24–26 This approach is rapidly growing field with various applications, including in vivo imaging, of azido/alkyne-modified nucleoside/nucleotides^26–27 or DNA/RNA fragment click adducts.^28–33

Herein, we report two independent methods for the synthesis of novel 8-triazol-1-yl derivatives of 7-deazapurine nucleosides. One protocol involves strain-promoted click chemistry between 8-azido-7-deazapurine nucleosides and cyclooctynes whereas the second approach explores direct C8-H bond functionalization of 7-deazapurine nucleosides and coupling with benzotriazoles.

The synthesis of the 8-azido-7-deazapurines (3a–c) was attempted from 8-halo-7-deaza substrates. Thus, bromination of 2',3',5'-tri-O-acetyltubercidin 1a using recently reported protocols for the C-8 bromination of purine nucleosides,³⁴ employing 1,3-dibromo-5,5-dimethylhydantoin (DBH, 0.65 eq.) in CH₂Cl₂ at 0 °C for 40 min, provided 7-bromo 2a (32%) in addition to 7,8-dibromo 2b (10%, TLC; Scheme 1). Deacetylation of 2a and 2b with methanolic ammonia gave 7-bromotubercidin 2c (80%) and 7,8-dibromotubercidin 2d (57%). Treatment of tubercidin 1b with N-bromosuccinimide (NBS, 2 eq.) in presence of KOAc in DMF yielded 8-bromotubercidin³⁵ 2e (34%), whereas attempted bromination of 1b with DBH in DMF gave a complex reaction mixture.

Scheme 1.

Synthesis of 8-bromo- and 8-azido-7-deazapurine nucleosides.

The 8-bromination of toyocamycin 1c with 0.75 equiv. of DBH in MeOH (30 min.) proceeded efficiently to give after crystallization 2f (85%). Treatment of 1c with NBS (1.1 eq.) in DMF at r.t. for 2.5 h also yielded 2f (45%) after column chromatography. Replacement of DMF with MeOH gave 2f (64%) after crystallization, although reaction took 4 h for completion. The 8-bromotoyocamycin (2f) has been previously prepared either by coupling of the corresponding 8-bromo-7-deazapurine with the ribose precursor³⁶ or by bromination of toyocamycin 1c with Br₂/H₂O (61%).³⁷ Thus the DBH/MeOH combination is the most efficient for synthesizing 2f in terms of yield, reaction time, and product isolation.

Treatment of sangivamycin 1d with DBH (1.1 eq.) in MeOH (r.t., 30 min) yielded 2g (63%), whereas bromination of 1d with NBS (1.5 eq.) in DMF for 1.5 h gave 2g in 40% yield in addition to unidentified byproducts. The synthesis of 8-bromosangivamycin 2g is usually achieved by the hydrolysis of 8-bromotoyocamycin 2f with a concentrated NH₄OH and 50% H₂O₂ solution.³⁶

Attempts to synthesize 8-azidotubercidin 3a from 8-bromotubercidin 2e [NaN₃ (1–5 eq)/DMF/rt-153 °C; NaN₃ (3 eq)/TsOH (3 eq)/EtOH/reflux) were unsuccessful. The higher electron density of π-excessive pyrrole ring most probably prevented the nucleophilic substitution reaction. However, treatment of 8-bromotoyocamycin 2f with NaN₃ in DMF at room temperature for 16 h produced 8-azidotoyocamycin 3b in 46% isolated yield (70%, based on TLC and ¹H NMR of the crude reaction mixture). Although this azidation reaction was light- and heat-sensitive and required the reaction, purification, and characterization to be carried out in the dark, the isolated 8-azido product 3b was stable for days when stored in refrigerator at 4 °C.^23,38 The presence of the EWG on the 7-position of the pyrrole ring in toyocamycin, as compared to tubercidin, could be the reason for successful substitution of bromide by azide in 2f. Treatment of 8-bromosangivamycin 2g with NaN₃ in DMF (as well as in DMSO or MeOH) at r.t or 70 °C failed to produce the desired 8-azidosangivamycin 3c.

SPAAC reaction of 8-azidotoyocamycin 3b with symmetrically fused cyclopropyl cyclooctyne 4 (OCT) in an aqueous solution of acetonitrile (ACN) at ambient temperature (4 h) gave the 8-(1,2,3-triazol-1-yl) product 5 as mixture of two isomers (60%, Scheme 2). Analogous reaction (4 h) of 3b with a strain modulated dibenzylcyclooctyne 6 (DBCO) produced triazole 7 as mixture of isomers (47%) after RP-HPLC purification. The resulting triazolyl products have "light-up" (inherited) fluorescent properties (Table 1), due to increased conjugation in the 7-deazapurine ring, and can therefore be used for fluorescent imaging in cancer cells, as recently reported with 8-triazolyl purine and 5-triazolyl pyrimidine nucleosides.^26–27 CuAAC reaction of 8-azido 3b with phenylacetylene in aqueous MeOH (4 days/r.t.) gave 8-(4-phenyl-1H-1,2,3-triazol-1-yl)toyocamycin 9 (65%) after silica column purification.

Scheme 2.

Click reactions between 8-azidotoyocamycin and cyclooctynes 4, 6 and phenylacetylene 8.

Table 1.

Photophysical data for 8-(1,2,3-triazol-1-yl)-7-deazapurine nucleosides.

	5	7	9	10	11a	12a
εmax (M−1 cm−1)	12200	12600	16000	16700	17100	17700
λmax (abs)b (nm)	287	290	295	278	278	276
λmax (exc)b (nm)	295	299	301	321	322	343
λmax (emi)b (nm)	380	434	412	416	419	480
Stokes Shift (nm)	85	135	111	95	97	137
ϕFb (%)	1.4	0.5	0.4	0.2	0.7	0.8

^aAs mixture of 5- and 6-isomers.

^bIn MeOH

Our attempts to prepare 8-alkynyl-7-dezapurine derivatives, which could serve as substrates for the synthes0is of the underdeveloped 8-(1,2,3-triazol-4-yl) adducts via click reactions with organoazides were unsuccessful. Thus, treatment of 8-bromotubercidin (2e), 8-bromotoyocamycin (2f), 8-bromo-2',3',5'-tri-O-acetyltoyocamycin,³⁹ or 8-iodotoyocamycin [prepared by treatment of the silylated toyocamycin with LDA (5 equiv.) and iodine (I₂, 1.5 equiv.) in THF (16%)] with trimethylsilylacetylene (TMSA) in the presence of TEA and (PPh₃)₂PdCl₂/CuI in anhydrous DMF showed mainly unchanged starting material under various reaction conditions. These results are in sharp contrast to 7-halo-7-deazapurines that undergo smooth Sonagashira cross-coupling providing 7-alkynyl analogues, which after further modifications provide derivatives with interesting photophysical and biological properties.^4,17

Since syntheses of 8-azidotubercidin 3a for the click reactions were unsuccessful, the possibility to prepare 8-triazolyl adducts of tubercidin 1b, using a direct C8-H activation of the 7-deazapurine ring, was explored next. Direct C-H fuctionalization of purines and purine nucleosides has recently gained much attention.^19,40–43 The examples include regioselective Pd-catalyzed direct C8-H arylation of the 6-phenyl-7-deazapurines with aryl halides to provide 8-arylated products albeit in low to moderate yields.⁴⁴ A regioselective direct C-H amination of the 7-deazapurines to give access to the 8-amino-,⁴⁵ or 7-amino-7-deazapurine analogues has been also reported.⁴⁶ We found that treatment of tubercidin 1b with benzotriazole (2 equiv.) in the presence of I₂ (0.4 equiv.) and tert-butylhydroperoxide (TBHP; 5–6 M/decane, 2 equiv.)⁴⁷ in anhydrous DMF at 35 °C for 96 h showed ~30% conversion (TLC) to 8-(benzotriazol-1-yl)tubercidin 10 which was isolated in 18% yield after column chromatography and HPLC (Scheme 3). Increasing the amount of iodine from catalytic to stoichiometric (3 equiv.) afforded 10 in 35% yield after 16 h. ¹H NMR data showed that the 7.60 ppm doublet for H7 in 1b collapsed to a singlet at 7.04 ppm in 10.

Scheme 3.

Iodine-mediated oxidative cross-coupling of tubercidin with benzotriazole.

The C8 regioselectivity for the oxidative cross-coupling product 10 was established by a X-ray crystal structure determination (Figure 1).⁴⁸ It is noteworthy that the 7-deazapurine ring and the benzotriazole ring are twisted to each other with an angle of 67.9° between the planes, which does not favor intermolecular π-π interactions. The glycosyl torsion angle C4–N9–C1′–O4' is 59.8° (syn conformation) and the furanose pseudorotation angle⁴⁹ is 162.9° (²E conformation). The C3'–C4'–C5'–O5′ torsion angle is 54.1° and is in g+/gg range.

Figure 1.

X-ray crystal structure of 10 showing atom labeling. Solvent molecules and H-atoms are omitted for clarity.

Treatment of 1b with 5-methylbenzotriazole [I₂ (0.2 equiv.)/TBHP/DMF/rt/5 days] produced a 3:2 mixture of 5-methyl- and 6-methylbenzotriazol-1-yl adducts 11a/11b (6%). Reaction with stoichiometric amount of iodine (3 equiv.) resulted in the efficient conversion to 11a/11b (48%; ~80% based on TLC after 15 min.), showing generality of the iodine-mediated direct activation of tubercidin ring at C8. Analogous treatment of 1b with 5-chlorobenzotriazole (3 equiv.) produced 5/6-chlorobenzotriazol-1-yl adducts 12a/12b (1:1, 21%; ~60% conversion on TLC). Because 7-deazapurine is structurally similar to indole, the coupling of 1b with benzotriazole might have occurred via initial iodination of the pyrrole ring of 7-deazapurine followed by trapping of the resulting iodonium, or 7-iodo iminium intermediates, with nucleophilic triazole. Subsequent elimination of HI, as proposed for the iodine-catalyzed direct activation of indoles with azoles mediated by TBHP,⁴⁷ should yield products 10–12. Subjection of the sangivamycin 1c and toyocamycin 1b to direct C8-H functionalization with benzotriazole (r.t. to 80 °C/48 h) gave unchanged substrate or resulted in rather complex reaction mixture. These results reiterate the literature report that the iodine catalyzed C-H activation of indoles with azoles works rather sluggishly with electron deficient indole substrates.⁴⁷

The photophysical properties of 8-(1,2,3-triazol-1-yl)-7-deazapurine adducts 5, 7, 9 and 10–12 were determined in MeOH. All triazol derivatives have similar absorption and excitation spectra with an absorption maximum between 276 nm and 295 nm and comparable extinction coefficient values (Table 1 and Figure S2). Moderate quantum yields and a large Stokes shifts of ~ 100 nm observed here are analogous to the fluorescence properties of aliphatic and benzylic derivatives of 1-(purin-8-yl)triazoles.²³ The emission maximum of triazol 5 (380 nm) is blue shifted compared to other derivatives (412–480 nm), likely due to the absence of an aromatic substituent (Figure 2). Emission spectrum of chloro substituted benzotriazole 12 is red-shifted compared to benzotriazoles 10 and 11, suggesting a resonance conjugation between the aromatic ring and Cl electrons.

Figure 2.

Emission spectra for 8-(1,2,3-triazol-1-yl)-7-deazapurine nucleosides.

Emission properties of the triazole 10 were also investigated in aqueous solutions. Compound 10 is fluorescent at neutral and basic pH (7.0 to 12.0) and becomes non-fluorescent at acidic pH. Interestingly, compounds 10 and 11 seem to be highly sensitive to UV light, as an increase in emission intensity (8.1 and 2.4 fold) upon irradiation with 280 nm light was observed (Figure S3). Triazole 10 has an average lifetime about 3.8 ns with two well-defined components 1 ns (10%) and 4.1 ns (90%).

In summary, bromination of 7-deazapurine nucleosides with DBH/MeOH gave 8-bromotoyocamycin and 8-bromosangivamycin in high yields. Treatment of the 8-bromotoyocamycin with sodium azide yielded 8-azidotoyocamycin. Strain promoted click chemistry of 8-azidotoyocamycin with cyclooctynes provided the corresponding 8-triazol-1-yl products. Iodine-mediated direct C-H arylation of tubercidin with benzotriazoles gave the corresponding 8-(benzotriazol-1-yl)tubercidin products. The 8-(1,2,3-triazol-1-yl)-7-deazapurine derivatives showed moderate quantum yields and a large Stokes shifts of ~ 100 nm.

Highlights.

• Efficient bromination of 7-deazapurine nucleosides with DBH in MeOH

• Click reactions of 8-azidotoyocamycin with alkynes gave fluorescent triazoles

• Direct C8-H bond functionalization of tubercidin with benzotriazoles