1. Synthesis and characterization
Crystallization of the azithromycin 11,12-hydrogenborate. Can we have the control?
J.M. Montejo-Bernardo, S. García-Granda, M.S. Bayod*.
X-Ray Group. Department of Physical and Analytical Chemistry.
Faculty of Chemistry. University of Oviedo. (Asturias, Spain)
(*) Astur-Pharma, S.A. Department of Research and Development. (Silvota, Asturias, Spain)
Synthesis and characterization
The azithromycin 11,12-hydrogenborate [4] is a precursor of the azalide azithromycin (obtained by its hydrolysis at pH 2), an antibiotic with high activity against Gram-positive and Gram-negative bacteria. It is obtained(1) by reductive methylation of intermediate [3] with formaldehyde and formic acid under refluxing chloroform.Compound [3] is synthesized by reduction of the imino ether [2] (obtained in turn from the erythromycin A [1]) with sodium borohydride in methanol at temperatures between 0 and -10 ºC in the absence of mineral acids.
It is know that chemical nature of intermediate borate compounds can be dependent on reduction conditions. The structural assignment of [4] was based on their IR, MS, TLC and NMR spectral data (see Table 1)(2,3).
This boron-containing azalide is a potential antibiotic, but its in vitro activity against Gram-positive and Gram-negative bacteria is significantly lower than that of azithromycin.
[1]
[2]
NaBH4
0/-10 ºC
[4]
[3]
IR (KBr) νmax(cm-1) = 3500, 1730, 1470, 1390, 1090, 1070
1H-NMR (CDCl3) (partial) δ(ppm) = 2.00 (NMe2), 2.30 (NMe), 3.37 (OMe)
13C-NMR (CDCl3) (partial) δ(ppm) = (C=O), 79,40 (C11), (C12), 68, 84 (C9),
64, 08 (C10), (OMe) (NMe2), 34, 39 (NMe)
11B-NMR (CDCl3) δ(ppm) = ω½ = 180 Hz
m/e M+ = 775.5
TLC rf = 0.38 (petroleum ether : ethyl acetate : diethylamine
75:25:10) developer: ethanol/vanillin (sulphuric acid)
H2CO
HCO2H
Figure 1. Synthesis of azithromycin hydrogenborate
Crystallization
Table 1. Data of azithromycin hydrogenborate
The azithromycin 11,12-hydrogenborate was obtained in solid state in two different experimental conditions, giving two crystalline products that have different external features (Figure 2) and different structural parameters (Figure 3).
The first product (labelled as “a”) was obtained in thermodynamics conditions. It was crystallized in acetone (or ethyl acetate) by concentration. The solution borate/solvent is allowed to vanish slowly to dryness by a gradual drop of the temperature. The final solid is washed with ethyl ether to eliminate the free azithromycin residual. The solid resulting is formed by transparent microcrystals with whitish color, prismatic appearance, and with an average size approximate of 0.1 x 0.01 x 0.01mm (Figure 2a). In future, we will call this solid as the thermodynamic product.
The second product (labelled as “b”) was recrystallized in kinetic conditions by a change of the polarity of the solution It was initially solved in acetone, and water is added to be enough a proportion 1:2. The mixture is taken to pH = 10 with sodium hydroxide (at 40%). The residue is collected by filtration and dried. The solid is white and presents a fibrous appearance (Figure 2b, right), forming conglomerates with a radius of 1mm approximately (Figure 2b, left). In the future, we will call this solid as the kinetic product.
The X-ray powder diffraction patterns shown that the thermodynamic product is totally crystalline (Figure 3, left) whereas the kinetic product includes a percentage of amorphous material (Figure 3, right).
By other hand, attempts to crystallize heating and cooling (in reflux) to 0 ºC only provide amorphous solid. a a b b
Figure 2. Images of crystals of hydrogenborate.
a) Termodynamic product. b) Kinetic product.
Kinetic product
Thermodynamic product
a = (17.23) Å
b = (22.54) Å
c = (24.99) Å
α = β = γ = 90.0 º
Resolution
Figure 3. X-ray powder diffraction patterns of both crystalline forms of the hydrogenborate. The kinetic product one is scaled for clarity.
At this moment, the results are not very impressive! For the kinetic product we could not select a crystal useful to X-ray single crystal diffraction, and the indexation of its X-ray powder diffraction pattern does not yield a unique value for the unit cell (the parameters obtained depend on the peaks selected).
For the thermodynamic product, we got a suitable crystal to X-ray diffraction, but the data were not very good to solve the structure and we only have the unit cell parameters. These values agree with the obtained by indexing the X-ray powder diffraction pattern (in brackets, Figure 3 left). The value of the volume (≈ 9700 Å3) indicates that in the unit cell there are eight molecules of the hydrogenborate, and possibly several molecules of solvent.
References
Bayod-Jasanada, M; Carbajo, R.J. López-Ortiz, F. J. Org. Chem. 1997, 62,
Bayod-Jasanada, M; Carbajo, R.J.López-Ortiz, F. Magn. Reson. Chem. 1998, 36,
Bayod-Jasanada, M; Llorente-García, I; Fernandez-Mari, F. EP
Acknowledgments: We thank the Spanish MCyT (BQU ) and AsturPharma S.A. (FUO-EM ) for finnantial support