Nociones b´ asicas de la Investigaci´ on biom´ edica

Studies began with d-glucose (182), a readily available sugar with good prece- dence as a cyclopropane substrate, as demonstrated by Nagarajan.45 _{Conversion of}

d-glucose into d-glucal involved the use of methodology pioneered by Fischer93–95 and refined by others.89,96

Synthesis began with peracetylation of d-glucose (182) using acetic anhydride and catalytic perchloric acid (Scheme 2.2); α-bromoglycoside 210 was obtained through addition of hydrobromic acid. Upon isolation of crude gly- cosyl halide 210, reaction in the presence of zinc dust provided tri-O-acetyl-d-glucal (179), the first potential substrate for cyclopropanation. In the interests of incor- porating more stable protecting groups, 179 was deprotected using triethylamine in aqueous methanol. The resulting d-glucal (185) was purified in good yield (66% from d-glucose), by precipitation of impurities from a methanol/acetone/diethyl ether solvent mixture. d-Glucal (185) was then perbenzylated upon treatment with an excess of sodium hydride, followed by addition of benzyl bromide. Thus, tri- O-benzyl-d-glucal (188) was obtained in very good yield (84%). Both protected glycals 179 and 188 were used as substrates for cyclopropanation.

O OH OH O OAc Br O OAc O OBn O OH

i), ii) iii)

iv) v) HO AcO AcO HO BnO HO AcO AcO BnO HO OH OAc 182 210 179 185 188

Reagents and Conditions: i) Ac2O, HClO4, 40–50 ◦C, 30 min; ii) HBr, AcOH, r.t., 90 min;

iii) Zn, AcOH(aq), 0◦C to r.t., overnight; iv) NEt3, MeOH(aq), r.t., 90 min, 66% from d-glucose;

v) BnBr, NaH, DMF, 0◦_{C to r.t., overnight, 84%.}

Scheme 2.2 Synthesis of acetyl glucal (179) and benzyl glucal (188) The original procedure for M¸akosza cyclopropanation involves a biphasic system, in which the aqueous phase is comprised of a caustic solution of 50% (w/w) sodium hydroxide in water.12 _{Nagarajan’s synthesis of cyclopropane 97 from glycal 188}

utilised a subsequent modification by M¸akosza, wherein the aqueous phase employs a comparatively low concentration of sodium hydroxide. A high concentration of potassium fluoride is employed, to ensure the water activity of the solution is com- parable to 50% (w/w) sodium hydroxide in water.97 _{This modification allows for}

carbene addition to substrates that would normally not survive the highly basic conditions of standard M¸akosza cyclopropanation. With this in mind, attempts to synthesise gem-dihalocyclopropanated carbohydrates began with tri-O-acetyl-d- glucal (179) (Scheme 2.3). This material was subjected to M¸akosza cyclopropa- nation, using the modified M¸akosza conditions employed by Nagarajan for the cy- clopropanation of glycal 188 [6% (w/w) sodium hydroxide, 47% (w/w) potassium fluoride, 47% (w/w) water].45 _{Unfortunately, glycal 179 was not stable under these}

conditions, and only degraded material was recovered after extraction with diethyl ether. This, together with TLC and NMR evidence, indicated significant loss of the acetate protecting groups. The use of ultrasound has been reported to improve cyclopropanations,9,98_{providing much faster reaction rates. It was thought that ul-}

trasonicating the reaction of 179 with bromoform and 50% (w/w) aqueous sodium hydroxide might promote carbene addition before significant deprotection occurs. These conditions led to consumption of starting material in approximately 2 hours, but isolated yields of cyclopropanes 211 and 212 were poor, ranging from 5–10%, presumably again due to loss of the acetate groups and subsequent retention of the resulting polar material in the aqueous phase upon workup.

O OAc AcO AcO O OAc AcO AcO O OAc AcO AcO H H H H Br Br Br Br i) 179 211 212

Reagents and Conditions: i) CHBr3, TEBAC, NaOH/KF(aq), r.t.

Scheme 2.3 Cyclopropanation of tri-O-acetyl-d-glucal (179)

The use of chloroform to generate the corresponding gem-dichlorocyclopropanes encountered similar difficulties. Incorporation of a variety of cosolvents (dichloro- methane, diethyl ether or toluene) was also explored, but failed to do more than aid stirring of the viscous reaction mixtures. These observations show that the ac- etate groups are too labile under M¸akosza conditions. Therefore, the more robust substrate tri-O-benzyl-d-glucal (188) was subsequently employed for cyclopropana- tions.

Nagarajan previously reported that the original M¸akosza cyclopropanation of tri-O- benzyl-d-glucal (188) was initially unsuccessful, presumably due to the use of 50% (w/w) sodium hydroxide in water.45 _{When this reaction was repeated under ultra-}

of inseparable products was obtained. Therefore, the modified potassium fluoride method of M¸akosza was attempted. This reaction proceeded efficiently, using either ultrasonication or magnetic stirring. Thus, cyclopropane 97 was obtained in 66% yield, along with a minor amount (10%) of isomer 190 (formed due to carbene ad- dition from the top face), previously unreported by Nagarajan (entry 1, Table 2.1). Cyclopropane 97 was obtained as a white solid, which was stable at room temper- ature under air for several months. Cyclopropane 190 was isolated as an unstable yellow oil. It was stored under argon in the freezer, but even under these conditions it degraded into an unidentifiable mixture within a few months.

Table 2.1 Cyclopropanation of tri-O-benzyl-d-glucal (188)a

O OBn BnO BnO O OBn BnO BnO O OBn BnO BnO H H H H Br Br Br Br i) 1 2 3 1 2 3 188 97 190

Reagents and Conditions: i) CHBr3, TEBAC, NaOH/KF(aq), r.t. or 20–30◦C.

Entry Reaction method Reaction time Recovered 188 Yield (97) Yield (190)

1 magnetic stirring 2 d 1% 66% 10% 2 magnetic stirring 2 d 16% 63% 7% 3 ultrasonication 10 x 10 min 42% 35% 5% 4 ultrasonication 8 x 30 min 30% 48% 6% 5 ultrasonication 12 x 30 min 28% 43% 7% 6 ultrasonication 12 x 30 min 30% 48% 8% a

all yields are of isolated products

The major isomer 97 was easily identified by NMR spectroscopy, with a charac- teristic doublet of doublets at 1.87 ppm in the proton spectrum, corresponding to the H-2 methine at the ring junction. This proton coupled to H-1 (J = 7.8 Hz), which was significantly further downfield at 3.94 ppm, due to the attached pyran ring oxygen. A coupling constant of 4.9 Hz was observed between H-2 and H-3. All characterisation data agreed very well with that reported by Nagarajan.

The minor isomer 190 had a similar proton NMR spectrum to that of 97. The H-2 methine appeared as a triplet (J = 8.1 Hz) at 2.30 ppm, due to coupling with H-1 (at 4.07 ppm), and H-3. This indicated all three protons were on the α-face, therefore this product was the β-cyclopropyl product. Conversely, major isomer 97 was determined to be the α-cyclopropyl product, due to the observed coupling constant (4.9 Hz) between H-2 and H-3.

The use of ultrasonication was explored to optimise this reaction (Table 2.1, entries 3–6). Extended periods of sonication caused the sonication bath to heat up, which could have allowed cyclopropane ring–opening reactions to occur, and reduce the yield of cyclopropanes. Therefore, the temperature was maintained between 20–

30 ◦_{C through replacement of the water in the sonication bath at regular intervals.}

Sonication led to improved conversion, as cyclopropanes were generated in good yield after several hours sonication. However, as starting material was usually re- covered upon purification, reversion to the conventionally stirred reaction described by Nagarajan was deemed prudent, requiring a less complicated experimental pro- cedure.

This cyclopropanation was now a very effective means of providing major cyclo- propane 97, though it was occasionally quite temperamental (compare entries 1 and 2, Table 2.1). Typically once all reagents were added, a clear biphasic mixture was observed, which steadily turned dark–brown throughout the course of the reaction. However, several times when this reaction was performed, the mixture would only turn a pale–yellow colour, which usually indicated a lack of any significant reactiv- ity. Presumably, this variable nature between successive reactions is responsible for several discrepancies in yield as shown in Table 2.1.