N-Edited Guanine Isosteres

Guanine is one out of five endogenous nucleobases and of key interest in drug discovery and chemical biology. Hitherto, the synthesis of guanine derivatives involves lengthy multistep sequential synthesis of low overall diversity, resulting in the quest for innovation. Using a “single-atom skeletal editing” approach, we designed 2-aminoimidazo[2,1-f][1,2,4]triazin-4(3H)-one as a guanine isostere, conserving the biologically important HBA–HBD–HBD (HBA = hydrogen bond acceptor; HBD = hydrogen bond donor) substructure. We realized our design by a simple one-pot two-step method combining the Groebke-Blackburn-Bienaymé reaction (GBB-3CR) and a deprotection reaction to assemble the innovative guanine isosteres in moderate to good yields. Our innovative, diverse, short, and reliable multicomponent reaction synthesis will add to the toolbox of guanine isostere syntheses.


■ INTRODUCTION
Guanine (2-amino-1,9-dihydro-6H-purin-6-one) was first reported in 1844 by the German chemist Julius Bodo Unger and later structurally elucidated by Emil Fischer. 1 Guanine is a purine derivative, consisting of a fused planar pyrimidineimidazole ring system. As a substructure of guanosine, it plays an outstanding role in the propagation of genetic information in living organisms by RNA and DNA ( Figure 1A). 2 Moreover, guanine is a part of several cofactors, for example, cGMP or GDP. Numerous diseases, e.g., cancer and K-RAS, are associated with malfunctioning guanine-dependent proteins and/or guanine catabolism. 3 Several approved drugs are based on guanine moieties such as the anti-herpes simplex acyclovir or the antineoplastic 8-azaguanine ( Figure 1B). 4 Guanine derivatives are typically synthetically accessed by a sequential multistep synthesis from heterocyclic guanine precursors. 5 Thus, there is an urgent need for convergent short and diverse syntheses of novel guanine derivatives. Analysis of the guanine binding interaction in DNA, RNA, and proteins reveals that key pharmacophoric elements include a flat heterocyclic 5−6 ring system and a hydrogen-bonding triade HBA−HBD−HBD (HBA = hydrogen bond acceptor; HBD = hydrogen bond donor) of an acceptor carbonyl-O, an adjacent NH, and an exocyclic amino group ( Figure 1C). Therefore, a guanine bioisostere should be composed of a flat heterocyclic ring system incorporating the essential HBA−HBD−HBD triade. Based on our interest in multicomponent reaction (MCR) chemistry, we reasoned that a generalized scaffold obeying the pharmacophore requirements of guanine would be accessible by an unprecedented atypical Groebke-Blackburn-Bienaymeŕ eaction (GBB-3CR) of a heterocyclic amidine, an aldehyde, and an isocyanide ( Figure 1C). 6 In the spirit of the emerging research area 'single-atom skeletal editing', the imidazo-N-9 of the purine would shift into the next bridgehead 4-position. The resulting scaffold indeed would closely resemble guanine: the key hydrogen-bonding triade is identical, the scaffold consists of a flat hetero 5−6 ring system, and the chemistry would allow substitution at the 6 and 7 positions.
The 7-position corresponds to the (deoxy)ribose position of guanine and biologically relevant derivatives. Due to the logic of the herein used chemistry, a bridgehead N is shifted in the new scaffold, which corresponds to the neighboring 9-position in guanine. In principle, the new heterocyclic ring system could result in a differential distribution of tautomeric microspecies which is important for biological activity. Indeed, analyzing the tautomeric microspecies using the ChemAxon Tautomerizer in water at room temperature revealed that the major species is identical with the guanine major tautomer (Supporting Information). 7 Interestingly, the major tautomer species is present over a broad pH range from 2.5 until 9. Taken together, our design and the predicted properties made us confident to investigate and optimize the GBB-3CR reaction to access a new class of guanine bioisosteres.
Next, in order to produce the target 4-aza-9-deaza-guanine isosteres, we needed to deprotect the benzyl groups in the previously obtained GBB-3CR intermediates in the following step. For this, we screened 20 deprotection conditions (Table S1, Supporting Information) and found that both trifluoroacetic acid (TFA) 9 and trifluoromethansulfonic acid (TfOH) 10 could cleave the benzyl groups, but the final deprotected products induced by those two acids are slightly different. While TFA can only deprotect the benzyl group (Scheme 3), the superacid TfOH cleaves simultaneously the benzyl group and the R 3 group (Scheme 4).
In the TFA-assisted one-pot deprotection, we found that the deprotected products 6a−6j formed well when R 3 was a cyclohexyl group, achieving yields from 33 to 58%. GBB-3CR intermediates derived from electron-donating-groups-(6a− 6d) and electron-withdrawing-groups-(6e−6i) substituted benzaldehydes both proceeded successfully in the one-pot benzyl deprotection step. It is noteworthy that the one-pot yields of all deprotected products 6a−6j are on average 14% higher than those generated by the two separate steps procedure. Moreover, we also ran the deprotection reactions with all purified GBB-3CR intermediates and summarized the yields in Scheme 3. It is noteworthy that the yield of 6a is much higher when generated from 5l (69%) than 5f (26%), and 5n (66%) can obtain 6d in a higher yield than 5g (59%) as well. Those two examples suggest that the 3,4,5-trimethoxy group in R 1 is easier to cleave than the 2,4-dimethoxy substituent. In addition, on replacing R 3 with the tert-butyl group, the GBB-3CR intermediate 5e failed to provide the target compound 6k, while 5a gave the unexpected trifluoroacetylation product 6l in a 45% yield.
The TfOH-promoted one-pot double deprotection reactions worked well with the benzaldehydes-derived GBB intermediates, affording products 6m−6p in 33−44% yields. The yields of 6m−6p achieved by the one-pot method were found to be superior to those from the two separate steps procedure by an average of 11%. Surprisingly, the GBB product 5n generated from 2,4-dimethoxy benzaldehyde provided the monodeprotected compound 6q (29%) rather than the doubledeprotected product. The distinct yields difference from singlestep deprotection in the synthesis of 6m from 5f (14%) or 5l (72%) demonstrated that the 3,4,5-trimethoxy group in R 1 is easier to deprotect TfOH-assisted, in accordance with its Scheme 3. One-Pot Benzyl Deprotection with TFA a a Reaction conditions of Step II: Ugi reaction crude (0.2−0.5 mmol), 0.1 M TFA, 80°C, 12 h, conventional heating; a yields from the one-pot procedure without isolation of GBB intermediates; b total yields calculated over the two-step procedure with isolated GBB intermediates; c yields from only the deprotection step II with purified GBB intermediates.
higher ring electron density. However, GBB-3CR intermediates constructed with thiophene-2-carbaldehyde (5k) and cyclopropane carbaldehyde (5t) did not afford the desired products 6r or 6s. This may be due to instability of the thiophene and cyclopropane rings in TfOH.
To further fortify the usefulness of our new synthesis, we carried out the control experiment and scale−up reaction (Scheme 5). It turned out that the mono-deprotected compound 6a could be further deprotected in the presence of TfOH to provide 6m in an 88% yield, which indicated that TfOH could not only achieve double deprotection of benzyl and cyclohexyl groups but also cleave the cyclohexyl alone. Our attempt to figure out whether TFA or TfOH could simultaneously cleave two benzyl groups failed; compound 5w could not yield either double-deprotected 6m or singledeprotected 6t. The scale-up reactions of our one-pot procedures were performed on a 4 mmol scale, providing 6a and 6m in 46 and 30% yields, respectively. The D 2 O exchange NMR experiments of 6a and 6m were done to prove the mono-or double-deprotection ( Figures S3 and S4, Supporting Information).
The X-ray crystal structures of 4d (Supporting Information) and 5l were obtained, demonstrating the solid-state structures of the scaffold. Interestingly, the G-analogue 5l exhibits a trifurcated hydrogen-bonding pattern in a circular tetrameric macrocyclic conformation ( Figure 2). This closely mimics the G-binding pattern found in all GTP/GDP-protein structures, suggesting that our heterocyclic G-mimic indeed could act as a bioisosteric G-mimic.
In summary, an innovative GBB-3CR-based one-pot twostep synthesis of novel 4-aza-9-deaza-guanine isosteres has been developed using a 'single-atom skeletal editing' strategy. Generally, most of our two-step syntheses are not high yielding, still they are superior to previous multistep synthesis protocols and will help to enrich the toolbox of guanine isosteres by an unprecedented new member scaffold. Combining GBB-3CR reaction and subsequent TFA-or TfOH-assisted deprotection reaction, mono-deprotected guanine isosteres and double deprotected guanine isosteres can be achieved, separately. Having the same hydrogen-binding pattern as guanine, our 9-deaza-guanine isosteres may also form similar interactions with biological receptors. Currently, biological evaluation of our G-analogues is ongoing and will be reported in due course. ■ EXPERIMENTAL SECTION General Information. Reagents were available from commercial suppliers and used without any purification unless otherwise noted. All isocyanides were made in-house via the Ugi procedure. 11 Other reagents were purchased from Sigma-Aldrich, ABCR, Acros, Fluorochem, AK Scientific, Combiblocks, or A2B and were used without further purification. Nuclear magnetic resonance spectra were recorded on a Bruker Avance 500 spectrometer. Chemical shifts for 1 H NMR were reported relative to TMS (δ 0 ppm) or internal solvent peak (CDCl 3 δ 7.26 ppm, CD 3 OD δ 3.31 ppm, or D 2 O δ 4.79 ppm), and coupling constants were in hertz (Hz). The following abbreviations were used for spin multiplicity: s = singlet, d = doublet, t = triplet, dt = double triplet, ddd = doublet of double doublet, m = multiplet, and br = broad. Chemical shifts for 13 C NMR were reported in ppm relative to the solvent peak (CDCl 3 δ 77.23 ppm, DMSO δ 39.52 ppm, and CD 3 OD δ 49.00 ppm). Flash chromatography was performed on a Grace Reveleris X2 system using Grace Reveleris Silica columns (12 g), and a gradient of petroleum ether/ethyl acetate (0−100%) or dichloromethane/ methanol (0−20%) was applied. Thin layer chromatography was performed on Fluka precoated silica gel plates (0.20 mm thick, particle size 25 μm). Mass spectra were measured on a Waters Investigator Supercritical Fluid Chromatograph with a 3100 MS Scheme 4. One-Pot Double Deprotection with TfOH a a Reaction conditions of Step III: Ugi reaction crude (0.1−0.5 mmol), 0.1 M TfOH, 55°C, 4 h, conventional heating. a yields from the one-pot procedure without isolation of GBB intermediates; b total yields calculated over the two-step procedure with isolated GBB intermediates; c yields from only the deprotection step III with purified GBB intermediates.
Detector (ESI) using a solvent system of methanol and CO 2 on a Viridis silica gel column (4.6 × 250 mm, 5 μm particle size) and reported as (m/z). High-resolution mass spectra (HRMS) were recorded using an LTQ-Orbitrap-XL (Thermo Fisher Scientific; ESI pos. mode) at a resolution of 60,000@m/z 400. All microwave irradiation reactions were carried out in a Biotage Initiator microwave synthesizer. Melting points were obtained on a melting point apparatus and were uncorrected. The yields given refer to chromatographically purified compounds unless otherwise stated.
General Experimental Procedure and Characterization. General Procedure A: Synthesis of 1,2,. Hydrazinecarbothioamide hydroiodide (20 mmol, 1.0 equiv) was suspended in 20 mL of 2-propanol (20 mL, 1.0 M), then benzylamine (21 mmol, 1.05 equiv) was added. The reaction was heated at 40°C for 10 h, and then the reaction mixture was kept stirring at room temperature for 2 more days. The solid was filtered and the solvents were evaporated in vacuum. The remaining crude was recrystallized with DCM and diethyl ether to give white solid amino-3benzylguanidine hydroiodide. To a mixture of 1-amino-3-benzylguanidine hydroiodide (5.0 mmol, 1.0 equiv) and K 2 CO 3 (5.1 mmol, 1.02 equiv) in DMSO (10 mL, 0.5 M), ethyl-2-amino-2-thioxoacetate (5.5 mmol, 1.1 equiv) was added and kept for 2.5 h at 75°C in an oil bath. After heating, the gray mixture was poured under vigorous stirring into 70 mL of ice water and stirred for another 18 h to yield a yellow crystalline precipitate. The precipitate was filtration and washed with water (3 × 20 mL) and then ethylacetate/ether (1:1, 3 × 20 mL). The solid was recrystallized from methanol or purified by silica chromatography to give 4a−4d.
General Procedure B. Corresponding 6-amino-3-(benzylamino)-1,2,4-triazin-5(4H)-one (0.2−1 mmol, 1.0 equiv) and aldehyde (0.24−1.4 mmol, 1.2 equiv) were dissolved in MeOH (0.8−4 mL, 0.25 M) in a microwave tube, the mixture was stirred at room Scheme 5. Control Experiment and Scale-Up Reaction Figure 2. X-ray structure of G-analogue 5l (CCDC 2190420) in solid state. 2D structure and 3D structure of the tetrameric macrocyclic assembly exhibiting a dense hydrogen-bonding network (dotted lines). For clarity, one molecule is shown in golden sticks, including the important trifurcated hydrogen-bonding pattern (blue dotted lines). temperature for 10 min, then isocyanide (0.24−1.4 mmol, 1.2 equiv) and Sc(OTf) 3 (10 mmol %, 0.1 equiv) were added, and the tube was sealed. Then the mixture was heated at 100°C under microwave in a sealed tube for 2 h. During the reaction, the temperature was monitored by the temperature−time profile on the screen of the microwave machine. After the reaction, the mixture was purified by silica gel column chromatography (MeOH/DCM = 1−5%) to give compounds 5a−5u.
General Procedure C: Benzyl Deprotection with TFA. GBB intermediate (0.2−0.5 mmol) was dissolved in TFA (2−5 mL, 0.1 M). The reaction mixture was stirred at 80°C overnight in a sealed tube. After reaction, the reaction mixture was diluted with 20 mL of DCM, and then the solvents were removed under reduced pressure. Then, the residue was diluted with EA (50 mL) and washed with sat· NaHCO 3 (50 mL × 3). Then the organic layer was dried over MgSO 4 , filtered, and the solvent was removed under reduced pressure. Then the crude compound was purified by silica gel column chromatography (MeOH/DCM = 2−10%) to get the deprotected products 6a−6l.
General Procedure D: Deprotection with TfOH. GBB intermediate (0.1−0.5 mmol) was treated with triflic acid (1−5 mL, 0.1 M), then heated at 55°C for 4 h. After the reaction, the mixture was quenched with water and neutralized with sat.NaHCO 3 . The aqueous layer was extracted with EA and the combined organic layer was washed with brine, dried, and concentrated under vacuum. Then the crude compounds were purified by silica gel column chromatography (MeOH/DCM = 2−10%) to get the deprotected products 6m−6q.
General Procedure E: One-Pot Synthesis. First, GBB reactions were carried out according to procedure B; after the reaction, the solvent was removed directly and the reaction mixture underwent in situ deprotection reaction following procedure C or D. Then the crude compounds were purified by silica gel column chromatography (MeOH/DCM = 2−10%) to get the deprotected products 6m−6q.

* sı Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.joc.3c00467. Experimental procedure and 1 H and 13 C{ 1 H} NMR spectra for all compounds along with the X-ray crystallographic data for 4d and 5l (PDF)