Synthesis of a Pseudodisaccharide α-C-Glycosidically Linked to an 8-Alkylated Guanine

The synthesis of stable guanofosfocin analogues has attracted considerable attention in the past 15 years. Several guanofosfocin analogues mimicking the three constitutional elements of mannose, ribose, and guanine were designed and synthesized. Interest in ether-linked pseudodisaccharides and 8-alkylated guanines is increasing, due to their potential applications in life science. In this article, a novel guanofosfocin analogue 6, an ether-linked pseudodisaccharide connected α-C-glycosidically to an 8-alkylated guanine, was synthesized in a 10-longest linear step sequence from known diol 13, resulting in an overall yield of 26%. The key steps involve the ring-opening of cyclic sulfate 8 by alkoxide generated from 7 and a reductive cyclization of 4-N-acyl-2,4-diamino-5-nitrosopyrimidine 19 to form compound 6.


OPEN ACCESS
Guanofosfocin B (1, Figure 1) is one of the three guanofosfocins which were isolated in 1996 by Nippon Roche [7] from the fermentation broth of Streptmyces sp. AB 2570 and Trichoderma sp. FD 5372. Guanofosfocins are of interest as strong inhibitors of chitin synthases (IC 50 : 1-10 nM). Detailed investigations of their biological activity were, however, hampered by their rapid decomposition. The instability, not surprising considering the two activated acetal moieties found in 1, was addressed by several groups by synthesizing stable open chain analogues of 1 while potentially retaining the promising biological properties. Sugimura et al. designed several analogues such as 2 ( Figure 1) that maintained the alkoxy substituent attached to the C(8) of guanine [8][9][10]. Later on, they replaced the mannosyl moiety by a carba-mannosyl unit, and synthesized analogue 3 ( Figure 1) [11]. Vasella et al. aimed at C-mannosides, replacing the anomeric oxygen by a methylene group, and prepared analogues 4 [12] and 5 [13] (Figure 1). We also considered the guanofosfocin analogue 6 of interest, in analogy to other, stable ether-linked pseudo-disaccharides [14][15][16] and report a synthesis of this ether-linked pseudo-disaccharide containing an 8-alkylated guanine.

Results and Discussion
The structure of 6 is characterized by an ether-linked pseudo-disaccharide with the α-C-mannopyranosyl unit linked to C(8) of a guanine via a methylene group. Accordingly, we had to incorporate a methylene group between the guanyl and mannosyl moieties and install the ether bond between the secondary C(3)-OH group of mannose and the C(6)-OH group of an allofuranose. Retrosynthetically (Scheme 1), the ether bond could be formed by ring-opening cyclic sulfate 8 by the alkoxy anion corresponding to alcohol 7 [17], and the 8-substituted guanine could be formed by regioselective 4-N-acylation of 2,4-diamino-5-nitrosopyrimidine 9, followed by reductive cyclization, using a procedure developed by Vasella et al. [18]. Our synthesis started with the preparation of the protected α-allofuranose-diol 13, following known procedures as outlined in Scheme 2 [19][20][21][22]. The secondary alcohol 7 was synthesized from commercially available methyl α-D-mannoside in 36% overall yield using the three-step sequence reported by Vasella et al. [13] Reagents and conditions: (a) Ph 3 P, DIAD, toluene, 66%; (b) 7, NaH, DMF, 39%.
To improve the yield of the etherification, we prepared the cyclic sulfate 8 (Scheme 3). It was obtained in 69% from diol 13 by treatment with thionyl chloride and subsequent oxidation with NaIO 4 in the presence of catalytic Ru(II)Cl 2 ·xH 2 O [24]. The cyclic sulfate 8 was a colorless solid that darkened upon storage, even in the refrigerator (0-5 °C) and so was used immediately. The reaction between the cyclic sulfate 8 and the oxyanion derived from alcohol 7 in HMPA/THF took place smoothly to furnish the ether-linked pseudo-disaccharide 15 in a yield of 86% upon acidic aqueous work-up. The polar solvent proved crucial for the high yield. Alcohol 15 was acetylated and converted to the carboxylic acid 18 in a yield of 88% by dihydroxylation of 16 by OsO 4 /NMO, cleavage of the resulting diol by NaIO 4 , and oxidation of the resulting aldehyde by NaH 2 PO 4 /NaClO 2 . Treatment of acid 18 with oxalyl chloride in the presence of catalytic DMF furnished the acid chloride. Though the reaction was carried out for 1 h, the conversion was completed within 5 min, as observed by monitoring the reaction mixture by IR spectroscopy. The acid chloride was stable enough to allow routine characterization (IR, 1 H-NMR and 13 C-NMR). It reacted with 6-(benzyloxy)-5-nitrosopyrimidine-2,4diamine (9) to afford amide 19, accompanied by a color change from purple to blue-green. As purification of amide 19 by chromatography led to yields below 50%, presumably caused by a strong absorption of amide 19 on silica gel, the crude amide 19 was treated directly with triphenylphosphine in xylene under reflux to furnish guanine 6 in an overall yield of 50% from 18 (Scheme 3). The reductive cyclisation was accompanied by a color change from blue-green to brown. With guanine 6 in hand, we performed a few scouting reactions to test its macrocyclization reactions via direct intra-molecular N-glycosylation. The first results showed that acetonide in 6 was not a good glycosyl donor for N-glycosylation. We then hydrolysed 6 to its corresponding vicinal diol. The macrocyclization is under investigation and the results will be reported in due course.

General
Commercially available reagents were used without further purification. Water-free solvents were dried: THF was distilled from Na/benzophenone; toluene from Na; CH 2 Cl 2 , MeCN, MeOH, pyridine, and triethylamine from CaH 2 ; acetone, and chloroform were dried over 4 Å molecular sieves. Technical solvents were distilled: AcOEt, CH 2 Cl 2 from K 2 CO 3 ; Et 2 O from FeSO 4 ·7 H 2 O; cyclohexane, hexane, MeOH, and toluene without any other additive. The reactions were carried out in oven-dried glassware, under an N 2 or Ar atmosphere, unless stated otherwise. Qualitative TLC: precoated silica-gel plates (Merck silica gel 60 F 254 ); detection by heating with 'mostain' (400 mL of 10% H 2 SO 4 soln., 20 g of (NH 4 ) 6  NMR-spectra were recorded on Bruker magnetic resonance spectrometer ( 1 H at 300 MHz, 13 C at 75 MHz): chemical shifts in ppm relative to a residual undeuterated solvent peak. MS spectra were recorded on an IONSPEC Ultima ESI-FT-ICR spectrometer at 4.7 T. (8). A solution of thionyl chloride (0.11 mL, 1.5 mmol) in CH 2 Cl 2 (0.85 mL) was added dropwise to an ice-cooled solution of diol 13 (230 mg, 0.74 mmol) in CH 2 Cl 2 (5 mL) and pyridine (0.24 mL, 3 mmol). The mixture was stirred for 5 min, when TLC revealed the disappearance of starting material. The mixture was diluted with CH 2 Cl 2 and washed with water. The combined aqueous layers were extracted with CH 2 Cl 2 . The combined organic layers were dried (Na 2 SO 4 ) and concentrated. The residue was dissolved in CH 2 Cl 2 /MeCN/H 2 O (2/2/3), to which was added NaIO 4 (320 mg, 1.5 mmol) followed by Ru(II)Cl 2 ·xH 2 O (10 mg). After 10 min, the mixture was diluted with CH 2 Cl 2 , the organic layer was separated, the water layer was extracted with CH 2 Cl 2 . The combined organic layers were dried (Na 2 SO 4 ) and concentrated in vacuo. The residue was purified by Flash Column Chromatography (FCC, EtOAc/cyclohexane, 1/3→1/1) to afford the cyclic sulfate 8 as a white solid (191 mg, 69%). m.p. 120-122 °C (dec.) (EtOAc/hexane). 1 13 (14). To a solution of diol 13 (305 mg, 0.98 mmol) and triphenylphosphine (310 mg, 1.18 mmol) in dry toluene (6 mL) was added diisopropyl azodicarboxylate (0.25 mL, 94% pure, 1.18 mmol) dropwise at room temperature. The mixture was stirred under reflux overnight. The solvent was removed in vacuo, the residue was purified by FCC  (15). To a mixture of NaH (60% in oil, 93 mg, 2.32 mmol) in HMPA (3 mL) and THF (1 mL) was added alcohol 7 (1.10 g, 2.32 mmol) in THF (15 mL (16). Triethylamine (0.32 mL, 2.29 mmol) and DMAP (61 mg, 0.50 mmol) in dry CH 2 Cl 2 (2 mL), were added dropwise at 0 °C to a solution of alcohol 15 (0.88 g, 1.15 mmol) and acetic anhydride (0.21 mL, 2.29 mmol) in dry CH 2 Cl 2 (3 mL). The resulting mixture was stirred at room temperature overnight. The reaction was quenched by adding water (1 mL), and the solvent was removed in vacuo. A solution of the residue in EtOAc (20 mL) was washed with water and 0.1 N HCl. The aqueous layer was extracted with EtOAc, the combined organic layers were dried (MgSO 4 ) and concentrated to give the acetate 16 as a slightly yellow oil (0.94 g, 100%). IR (cm  13 (17). To a mixture of alkene 16 (0.850 g, 1.05 mmol) and N-methylmorpholine N-oxide (NMO) (213 mg, 1.58 mmol) in acetone (6 mL) and water (2 mL) was added osmium tetroxide (0.2 w% in water, 2.6 mL, 0.021 mmol, 0.02 eq.) at 0 °C. The resulting mixture was stirred for 45 h when TLC revealed the disappearance of the alkene 16. Upon addition of sodium sulfite (1.12 g) the yellow suspension turned into a slightly yellow two-layered solution. The upper organic layer was separated, the aqueous layer was extracted with EtOAc, the combined organic layers were dried (MgSO 4 ) and concentrated to give the crude diol (802 mg, 91%) as a mixture of two epimers in a ratio of ca. 0.75:1 (based on the integrals in the 1   To the diol (630 mg, 0.747 mmol) in MeOH (3 mL) and H 2 O (5 mL) was added sodium periodate (190 mg, 0.897 mmol) in H 2 O (3 mL) at 0 °C, the mixture was stirred for 60 min, and then extracted with EtOAc. The combined organic layers were dried (Mg 2 SO 4 ) and concentrated to give the aldehyde 17 as a slightly yellow oil which was pure according to the NMR spectrum. IR (cm   (18). The crude aldehyde 17 in acetonitrile (6 mL) was treated with NaH 2 PO 4 (34 mg, 0.25 mmol) in H 2 O (2 mL) and H 2 O 2 (30%, 0.12 mL, 1.12 mmol), respectively, at 0 °C, followed by addition NaClO 2 (0.17 g, 80%, 1.49 mmol) in H 2 O (2 mL) at 0 °C. The resulting mixture was stirred overnight, brought to pH 2.0 with 1N HCl, and extracted with EtOAc. The combined organic layers were dried (MgSO 4 ) and concentrated to give acid 18 as a colorless gum   (19). To a solution of acid 18 (0.470 g, 0.57 mmol) in CH 2 Cl 2 (4 mL) was added oxalyl chloride (0.15 mL, 1.7 mmol) followed by a drop of DMF at 0 °C. The resulting mixture was stirred for 1 h and concentrated in vacuo. The crude acid chloride was pure according to its NMR spectra and used directly for next step.  . To a solution of 2,4-diamino-5-nitrosopyrimidine 9 (0.21 g, 0.86 mmol) and pyridine (0.07 mL) in dry THF (12 mL) was added the above acid chloride (0.47 g, 0.57 mmol) in THF (7 mL) at 0 °C dropwise. Once the acid chloride was added, the deep-blue solution turned green. After stirring for 1 h, the purple solid was filtered off, and the filtrate was concentrated in vacuo to afford the amide 19 (600 mg).    30 (s, 3 H). 13

Conclusions
In summary, a new linear analogue of guanofosfocin 6 was synthesized in 10 steps from the known sugar diol 13 in 26% overall yield. Key steps were the ring-opening of cyclic sulfate 8 by a sugar sec-alkoxide and the reductive cyclization of 4-N-acyl-2,4-diamino-5-nitrosopyrimidine 19. The robustness of the above etherification and of the reductive cyclization will find more applications in synthetic organic chemistry. The structure of 6 was characterized as an ether-linked pseudodisaccharide α-C-glycosidically linked to an 8-alkylated guanine. This compound, together with previously synthesized analogues of guanofosfocins, allows expansion of the relevant research in this field of medicinal chemistry and chemical biology.