Synthesis of pyrazole ligandoside

The synthesis of pyrazole ligandoside was started from the commercially available reagent 4-bromo-2-methoxyaniline (3) as depicted in Scheme 4-1.

Scheme 4-1 Synthesis of the protected ligand building block 6. a) 1) NaNO2, SnCl2, HCl, 0 ºC, 66%, 2) 1,1,3,3-tetramethoxypropane, EtOH, HCl, reflux, 85%; b) Et2O-BBr3, DCM, -78 ºC, 68%; c) TIPSOTf, DIPEA, DCM, 0 ºC, 99%. TIPS = triisopropylsilyl.

Diazotization of compound 3 with sodium nitrite gave the diazonium salt (Scheme 4-2),²⁹⁴ followed by reduction with stannous chloride afforded the corresponding substituted phenyl hydrazine hydrochlorides intermediate.

Scheme 4-2 Mechanism of diazonium salt formation and the hydrazine intermediate.

Without further purification, the hydrazine intermediate went on to cyclize with excessive malonaldehyde bis(dimethyl acetal) in ethanol to form the pyrazole ring in 4.²⁹⁵ Cleavage of the methyl group with Lewis acid borane at -78°C provided the desired phenol 5. The demethylation reaction proceeds via a bimolecular mechanism involving two Et2O-BBr3 adducts with the formation of phenol and bromomethane.²⁹⁶ (Scheme 4-4) Low temperature is necessary because of the high energy of the B-O bond.

After this project was finished, H. Batchu et al. reported that the ortho-hydroxy group is accessible by N-phenylpyrazoles using lead(II) acetate as catalyst.²⁹⁷ In a plausible mechanism, Pd(II) activates the ortho-H to form a five-membered cyclopalladium intermediate. After oxidation and elimination, a C-O bond can be formed. In our case, Cu(I) was applied when coupling the base building block with the sugar moiety, thus, a similar cyclo intermediate may form with H or OH and the N on the pyrazole rings (Scheme 4-3). This explains why the hydroxyl group should be protected before coupling to the sugar and why N‑phenylpyrazole and other base building blocks in Figure 4-2 failed to generate the desired product when reacted with the sugar.

Scheme 4-3 Proposed mechanism of ortho-hydroxylation of N-phenylpyrazole.

Subsequent reaction of 5 with triisopropylsilyl triflate provided the key intermediate 6.

Because the electronic density of phenyl in 1-phenyl-1H-pyrazole is higher than 2-phenyl-1,3-dioxane in the case of salen base 7, the hydroxyl group here is much more acidic. As a result, the reaction rate of 5 is faster than the salen base building block 7.

With the key building block 6 in hand, we started working on the sugar moiety. The Hoffer’s α-chloro sugar (8) was synthesized from 2-deoxyribose (9). (Scheme 4-5) First, the ribose was methylated at 1’-hydroxyl group (10), followed by

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4-methylbenzoyl protection giving 11. Then, chloride substitution of the methoxyl group furnished the sugar building block 8 after precipitation in anhydrous ether.

Scheme 4-4 Biomolecular mechanism of BBr3-assisted cleavage of methyl ethers.

Scheme 4-5 Synthesis of the protected ligand building block 8. a) AcCl, MeOH, Ag2CO3, rt; b) 4-methylbenzoyl chloride, pyridine, 45 ºC, 87% for 2 steps; c) AcCl, HCl, AcOH, 0 ºC, 55%. Tol = 4-methylbenzoyl.

Due to their insolubility or failure to form a copper complex with the substrates, the base building blocks of benzene, pyridine, fluorobenzene and anisole (Figure 4-3) all failed to form a C-C bond with the sugar. The Corey-House synthesis was achieved with intermediate 6.^{298, 299} The reaction occurred in three steps (Scheme 4-6a). First, bromobenzene was treated with t-butyllithium to convert it into a phenyl lithium species. 6 was reluctant to undergo a lithium halogen exchange. In practice, neither n- nor s-butyllithium could generate the organic lithium species efficiently. So an excessive and sufficiently strong base, tert-butyllithium, was used. Because the product t-BuBr can react with t-BuLi, 2 equivalents t-BuLi must be used (Scheme 4-6b). Second, the phenyl lithium species was treated with copper bromide to create a lithium diphenyl cuprate intermediate, called “Gilman reagent” in honor of H.

Gilman’s contribution to the field.³⁰⁰ In the Gilman reagent, the cuprate and lithium ions directly link to the carbon atom, forming an 8-membered ring with two lithium and two cuprate ions coordinating between four phenyl groups (Scheme 4-6c),³⁰¹ thus negative charges accumulate on the coordinate carbon atom and make the C-Cu and

C-Li more reactive. The reaction has to be conducted in ethyl ether to avoid side reactions. Finally, the diphenyl cuprate was reacted with the protected chloride ribose 8 to furnish the cross-product ligandoside. However, the coupling of the Gilman reagent with bromobenzene resulted in the undesired dibenzene by-product (Scheme 4-6b). β- and α-conformers are very close to each other on TLC, but they were still separable by column chromatography.

Scheme 4-6 Mechanism of Corey-House synthesis: a) reaction steps towards nucleotide 12. PhBr represents 6. R-Cl represents Hoffer’s α-chloro sugar 8. b) side reaction in the Corey-House synthesis;

c) structure of 8-membered ring Gilman reagent.

The yield of 12 depended on several factors: 1) Moisture and oxygen can degrade the organolithium intermediate or oxidize the Cu(I) complex. 2) Insufficient reaction time and improper temperature control for the second step can leave a certain amount of organolithium unreacted. In practice, increasing the temperature rapidly at the final step generated the undesired α-conformer product. 3) The hydrochloric acid generated from the chloride ribose can destroy the Gilman reagent.

57 Scheme 4-7 Synthesis of the building block 15 and of the nucleoside 16. a) 1) t-BuLi, Et2O, -78 ^oC; 2) CuBr-Me2S, -30 ^oC; 3) 8, rt, 53%; b) K2CO3, MeOH, rt, 65%; c) DMTr-Cl, DIPEA, DCM, rt, 74%; d) P(OCH2CH2CN) (NiPr2)2, diisopropylammonium tetrazolide, DCM, rt, quant.; e) TBAF, THF, rt, 87%.

Figure 4-4 Pentose region of the 400 MHz NOESY spectrum of 12 in CDCl3.

Next, deprotection of 12 to 13 was achieved with potassium carbonate in methanol (Scheme 4-7). Appropriate reaction time and concentration must be attained to ensure that only the 4-methylbenzoyl but not TIPS group is cleaved. NOESY spectra for both conformers were measured to confirm the ligandoside configuration. NOESY allows us to correlate nuclei through space smaller than 5Å. From the spectrum of 12 (Figure 4-4), it can be seen that in the β-ligandoside the 1’-H (5.25 ppm) is correlated with the 2’-H bottom (2.58 ppm). 2’-H top (2.20 ppm) is coupled with 3’-H (5.61 ppm). 3’-H is

further correlated with 4’-H (4.57 ppm) and 5’-H (4.60 ppm). No correlation between 1’-H and 3’-H can be observed. In contrast, α-conformer shows a correlation peak between the 1’-H and the 3’-H. The 3’-H is coupled to 2’-H top; 2’-H bottom correlates with the aromatic protons. Based on the data, the configuration of the obtained ligandoside is verified.

Finally, 13 was protected with dimethoxytrityl chloride (DMTr-Cl) at the 5’-hydroxyl group to give 14 and 14 was further reacted with the phosphoramidite reagent to provide 15, needed for oligonucleotide solid-phase synthesis.

Figure 4-5 X-ray crystal structure of nucleoside 16: a) crystal structure of single molecular; b) neighboring interaction in a unit cell.

Completely deprotected 13 furnished the pyrazole ligandoside 16. Small crystals of 16 were attained by slow evaporation of the unpolar ethyl acetate solution. As presented in Figure 4-5, the result of an X-ray diffraction analysis further proved the β-configuration. In the X-ray crystal, the phenyl ring and the pyrazole ring are slightly tilted against each other by 23.9°~24.5°. The dihedral angle between two planes is narrow which facilitates copper chelating with the nitrogen and oxygen atom.

Unexpectedly, the nitrogen atom (N2) and the hydroxyl group (O4) of the pyrazole are not on the same side. In the unit cell (Figure 4-5b), an interactions between N2 and O2ⁱ of one adjacent sugar, and O4 and O3ⁱⁱ of another adjacent sugar are observed, which could be responsible for the “anti” conformation around the anti anti bond.

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Next, the UV-Vis spectrum of ligandoside 16 was measured. The highest peak is at 246 nm; the second highest peak is found at 284 nm, similar to the methylated monomer 19 (see Chapter 4.3.1.2) resulting from the benzyl-pyrazole bicycle system. As canonical bases have an average peak at 260 nm, the molar absorbance of 16 was determined at 260 nm, to calculate the concentration of the oligonucleotides.