Synthesis of 9-deoxo-9a-aza-9a-homoerythromycin A 11,12-hydrogen borate and azithromycin 11,12-hydrogen borate. A new procedure to obtain azithromycin dihydrate

Full text of DOI:10.1021/jo970776r

J . Org. Chem. 1997, 62, 7479-7481
7479
Syn th esis of
-d eoxo-9a -a za -9a -h om oer yth r om ycin A
1,12-Hyd r ogen Bor a te a n d Azith r om ycin
Sch em e 1
9
1
1
1,12-Hyd r ogen Bor a te. A New P r oced u r e
to Obta in Azith r om ycin Dih yd r a te
Miguel Bayod-J asanada,*,† Rodrigo J . Carbajo and
‡
Fernando L o´ pez-Ortiz*,
‡,§
Astur Pharma S. A., Pol ı´ gono Industrial Silvota, 33192
Silvota, Asturias, Spain, and Departamento de Qu ´ı mica
Org a´ nica e Inorg a´ nica, Universidad de Oviedo, 33071
Oviedo, Spain
Received May 1, 1997
In tr od u ction
1
Azithromycin 1 is an azalide antibiotic derived from
erythromycin A with improved biological and pharma-
codynamic properties over the parent compound.2 The
structural modification consists of the insertion of an
N-methyl group between the carbon atoms C9 and C10
of the lactone ring of erythromycin A, affording a 15-
membered aza macrolide. It has been shown that their
antibacterial activity begins by binding to bacterial
3
ribosome and that in the bound state both compounds
4
adopt a similar conformation of the macrocyclic ring. The
synthesis of azithromycin5 is based on the Beckmann
rearrangement of erythromycin A oxime 3 to yield the
imino ether 4.5d This is reduced to 9-deoxo-9a-aza-9a-
homoerythromycin A (5), which is finally converted in
azithromycin5 1 by reductive methylation of the amine
nitrogen with formaldehyde and formic acid (Scheme 1).
Three methods have been reported to achieve the
reduction of imino ether 4: (i) catalytic hydrogenation5
and quality of azaerithromycin 5. Thus, it is known that
treatment of azithromycin with mineral acids hydrolyzes
the cladinose and desosamyne sugars, affording the
e,f
5d
aglycon macrocycle. The hydrolysis is particularly easy
d,f
for the cladinose ring and the desomaynilazithromycin
is a known contaminant of azithromycin. On the other
hand, we have found that azithromycin 1 obtained by this
2
over PtO ; (ii) electrochemically, in an electrolytical cell
2
6
with a current of 1-2 A/dm under an inert atmosphere;
and (iii) through the use of sodium borohydride in
methanol.5d The first two methods are of limited value
for the large scale production of azithromycin due to the
use of relatively expensive catalysts and equipment to
carry out the electrolysis. Alternatively, the reduction
with sodium borohydride under the reaction conditions
described (temperature range of 4-25 °C and aqueous
work up at pH 2.5) has some negative effects on the yield
route was always contaminated with a minor compound,
7a
6
(TLC and HPLC control ), not characterized in the
literature, and this byproduct is also N-methylated,
yielding 7 in the last step of the synthesis outlined in
Scheme 1, so that the contamination is transferred to the
final antibiotic. Careful column chromatography allowed
us to isolate both contaminants, and here we report their
synthesis and structural identification. Furthermore,
intermediates 6 and 7 allowed us to devise a new route
†
Astur Pharma S.A.
Universidad de Oviedo.
7b
‡
to obtain pure azithromycin dihydrate (2) in excellent
§
yields.
Present Address: Facultad de Ciencias Experimentales, AÄ rea de
Qu ´ı mica Org a´ nica, Universidad de Almer ´ı a, 04121 Almer ´ı a, Spain.
1) Bright, G. M.; Nagel, A. A.; Bordner, J .; Desai, K. A.; Dibrino, J .
(
N.; Nowakowska, J .; Vincent, L.; Watrous, R. M.; Sciavolino, F. C.;
English, A. R.; Retsema, J . A.; Anderson, M. R.; Brennan, L. A.;
Borovoy, R. J .; Cimochowski, C. R.; Faiella, J . A.; Girard, A. E.; Girard,
D.; Herbert, C.; Manousos,M.; Mason, R. J . Antibiot. 1988, 41, 1029.
Resu lts a n d Discu ssion
8
The procedure is as follows: 9-deoxo-9a-aza-9a-homo-
erythromycin A 11,12-hydrogen borate (6) is quantita-
tively obtained by reduction of imino ether 4 with sodium
borohydride in methanol at temperatures between 0 and
(2) (a) Retsema, J .; Girard, A.; Schelkly, W.; Manousus, M.; Ander-
son, M.; Bright, G.; Borovoy, R.; Brennan, L.; Mason, R. Antimicrob.
Agents Chemother. 1987, 31, 1939. (b) Kirst, H. A.; Sides, G. D.;
Antimicrob. Agents Chemother. 1989, 33, 1419.
-10 °C, and the aqueous workup is performed in the
(
3) Brennan, R. J .; Awan, A.; Barber, J .; Hunt, E.; Kennedy, K. L.;
Sadegholnejat, S. J . Chem. Soc., Chem. Commun. 1994, 1615.
4) Awan, A.; Brennan, R. J .; Regan, A. C.; Barber, J . J . Chem. Soc.,
Chem. Commun. 1995, 1653.
5) (a) US Patent 4,517,359 to Pliva. (b) US Patent 4,474,768 to
Pfizer. (c) Azitrhomycin (Zitromax) received FDA approval on Nov 1,
991. (d) Djokic, S.; Kobrehel, G.; Lazarevski, G.; Lopotar, N.; Tam-
absence of mineral acids. Compound 6 is converted into
azithromycin 11,12-hydrogenborate (7) by reductive me-
thylation with formaldehyde and formic acid under
refluxing chloroform. Hydrolysis of 7 keeping the pH
value at 2 affords azithromycin in its hygroscopic form,
(
(
1
burasev, Z.; Kamenar, B.; Nagl, A.; Vickovic, I. J . Chem. Soc., Perkin
Trans. 1 1986, 1881. (e) Djokic, S.; Kobrehel, G.; Lopotar, N.; Nagl,
A.; Mrvos, D. J . Chem. Res., Synop. 1988, 152, J . Chem. Res., Miniprint
(7) (a) Analytical protocol according to USP XXIII, p 152. (b)
Azithromycin dihydrate is the preferred form for the preparation of
formulations of therapeutical use; Allen, D. J . Eur. Patent 0,298,650.
(8) (a) Bayod, M.; Fern a´ ndez, J . R. Patent ES P9601561 to Astur
Pharma. (b) Ibid. Eur.Patent 97500120.7.
1
988, 1239. (f) Yang, B. V.; Goldsmith, M.; Rizzi, J . P. Tetrahedron
Lett. 1994, 35, 3025.
6) Vajtner, Z.; Lopotar, N.; Kobrehel, G.; Djokic, S. AT 387 391 B
to Pliva.
(
S0022-3263(97)00776-7 CCC: $14.00 © 1997 American Chemical Society

7
480 J . Org. Chem., Vol. 62, No. 21, 1997
Notes
1
6
i.e. monohydrate, which is transformed in the final step
of 1 have been previously assigned in CDCl
3
as well as
7
b
17
to therapeutic agent azithromycin dihydrate 2 by re-
crystallization in a mixture of acetone-water (Scheme
in buffered D
2
O and DMSO-d
6
.
As expected, the carbon
atoms C11 and C12 in 7 are deshielded related to those
of azithromycin (1) by 6.6 and 3.5 ppm, respectively. This
fact allowed us to identify the link of the boron atom in
7 to the oxygens O11 and O12 of the macrocycle.
The formation of the hydrogen borate 6 is not too
surprising if one considers the ability of boron to act as
1
).
The structural assignment of compounds 6 and 7 is
based on their MS and NMR spectral data. The FABMS
spectrum of compound 6 shows the molecular ion at m/ z
7
61, which corresponds to an increase of 26 units related
to the molecular weight of 5 (C37 12) due to the
molecular fragment BOH present in the new compound
13). The calculated isotopic pattern cor-
1
8
H
70
N
2
O
a protective group of 1,2-diols and the favorable ar-
rangement19 of the hydroxy groups OH11 and OH12 in
2
0
6
(C37
H
69BN
2
O
the conformation of azithromycin in chloroform.
relates well with the molecular formula proposed (Sup-
porting Information). Moreover, the 11B NMR spectrum
of 6 shows a broad signal at 10.0 ppm (W1/2 198 Hz), in
the expected range for a boron nucleus bonded to three
oxygen atoms with a trigonal geometry.9
The positions of binding of the boron atom to the
molecular framework was determined by identifying the
carbon atoms whose chemical shift was affected by the
proximity of the boron moiety related to the non-boron-
Compound 7 is a new boron-containing azalide and
therefore a potential antibiotic. Consequently, its in vitro
microbiological activity was screened against Gram-
positive and Gram-negative organisms (Supporting In-
formation). Unfortunately, in all cases tested the activity
of 7 was significantly lower than that of azithromycin.
The best results were obtained against Bacillus sp.
(inhibitory minimal concentration (IMC) 50 µg/mL),
which corresponds to a 125-fold decrease in activity vs
that of azithomycin.
1
0
13
containing product. The assignment of the C spectra
was based mainly on the study of the 2D heteronuclear
1
1
multiple-quantum coherence (HMQC) and heteronu-
clear multiple-bond coherence (HMBC)12 spectra (Sup-
porting Information). The strategy of this work was to
establish in the HMBC spectrum the connectivity of a
Exp er im en ta l Section
Gen er a l. Imino ether 4 was prepared according to literature
methods.5d Fast atom bombardment (FAB) mass spectra were
1
13
obtained in a Finnigan Mat (Mat95) spectrometer. H-, C-,
known carbon, e.g. the lactone carbon of the macrolide
and 11B-NMR spectra were measured in a Bruker AMX-400 at
ring, with protons separated by two or three bonds13 and
4
00, 100.61, and 128.38 MHz, in CDCl
3
solutions. NMR chemi-
then to found out in the HMQC spectrum the direct
1
13
cal shifts are expressed in ppm upfield from TMS ( H and C)
and pure BF
1
response ( J CH) of these protons with the corresponding
11
3
2
‚OEt ( B). 2D HMQC and HMBC spectra were
carbon nuclei. A full analysis will be published else-
where.14 Significantly, the largest C chemical shifts
differences between 5 and 6 were observed for C11 and
C12. In the boron derivative, C11 is deshielded by 7.68
ppm and C12 by 3.83 ppm relative to the azaerhytromy-
cin A 5 and both signals were slightly broadened. The
chemical shift variations for all other carbons were lower
than 1.6 ppm. Furthermore, this limiting value cor-
responded to C10, which may be also affected by the
proximity of the boron substituent. Similar â-deshielding
effects have been found in the azaerythromycin A 11,12-
acquired and processed using standard Bruker software. An-
tibiograms to determine the in vitro biological activity of
compound 7 were performed following standard protocols.
Aza er ith r om ycin Hyd r ogen obor a te (6). To a stirred
solution of imino ether 4 (89 g, 121 mmol) in methanol (450 mL)
was added sodium borohydride (35 g, 921 mmol) over a period
of 4 h at a temperature below -5 °C. The mixture was stirred
during two additional hours under the same reaction conditions
and then it was left at room temperature for 20 h. Solvent
evaporation affords a crude product which was redisolved in a
13
mixture of CH
2
Cl
2
2
-H O. The organic layer was separated and
dried over anhydrous sodium sulfate. Filtration and solvent
evaporation yielded 85 g (92.4%) of azaerithromycin hydro-
genoborate 6.
5
c
cyclic carbonate and erythromycin A 11,12-methylene
acetal.15 Consequently, these results clearly established
the link of the boron atom in 6 to the 11,12-hydroxy
groups of the aglycon moiety of 5.
: 1H NMR δ 5.15 (d, 1H), 4.89 (dd, 1H), 4.59 (dd, 1H), 4.40
6
(
d, 1H), 4.11 (dq, 1H), 3.61 (d, 1H), 3.48 (ddq, 1H), 3.34 (s, 3H),
3.27 (d, 1H), 3.22 (dd, 1H), 3.02 (d, 1H), 3.00 (dd, 1H), 2.72 (dq,
1
1
H), 2.42 (ddd, 1H), 2.33 (d, 1H), 2.28 (s, 6H), 2.25 (dq, 1H),
.94 (ddq, 1H), 1.86 (ddq, 1H), 1.71 (m, 2H), 1.65 (d, 1H), 1.65
The structural determination of compound 7 was
achieved in an analogous way. The FABMS spectrum
of azaerythromycin derivative 7 showed the molecular
ion at 775, m/ z and again the difference of 26 units with
the molecular weight of 1 (MW 749) is accounted for by
the formation of its hydrogen borate. The 11B NMR
spectrum yields a broad singlet at 10.0 ppm (W1/2 159
Hz). Here, the NMR data have to be compared with
(dd, 1H), 1.57 (dd, 1H), 1.45 (ddq, 1H), 1.39 (dd, 1H), 1.36 (d,
3
H), 1.27 (s, 3H), 1.25 (s, 3H), 1.24 (m, 1H), 1.22 (d, 3H), 1.18
(
d, 3H), 1.16 (d, 3H), 1.05 (s, 3H), 1.02 (d, 3H), 0.91 (d, 3H), 0.88
(t, 3H); 13C NMR δ 180.12, 103.01, 94.47, 82.99, 80.60, 79.66,
78.14, 77.45, 73.58, 72.86, 70.77, 68.78, 65.85, 65.68, 58.65, 57.12,
4
2
9.51, 45.54, 42.44, 41.68, 40.34, 34.55, 29.46, 28.74, 27.38, 21.82,
1
1
1.53, 21.36, 21.32, 18.20, 15.43, 14.70, 14.45, 11.53, 8.90;
B
+
NMR δ 10.0; FABMS m/ z 761 [M] .
1
13
those of azithromycin (1). The H and C NMR spectra
(
16) Barber, J . Magn. Reson. Chem. 1991, 29, 740.
(
(
9) Wrackmeyer, B. Annu. Rep. NMR Spectrosc. 1988, 20, 61.
10) Kalinowski, H. O.; Berger, S.; Braun, S. 13C-NMR Spektroskopie;
(17) Brennan, R. J .; Barber, J . J . Magn. Reson. Chem. 1992, 30, 327.
(
18) Ferrier, R. J . Adv. Carbohydr. Chem. Biochem. 1978, 35, 31.
George Thieme: Stuttgart, 1984, p 77.
11) Bax, A. Griffey, R. H.; Hawkins, B. I. J . Magn. Reson. 1983,
5, 301.
(19) Up to date four boron-containing natural macrodiolides isolated
(
from microorganisms are known where the conformation of the
macrocycle favors the binding of four oxygens to a boron atom in a
Boeseken complex. (a) Boromycin: Dunitz, J . D.; Hawley, D. H.; Miklos,
D.; White, D. N. J .; Berlin, Y.; Marusic, R.; Prelog, V. Helv. Chim. Acta
5
(
(
12) Bax, A.; Summers, M. F. J . Am. Chem. Soc. 1986, 108, 2093.
13) Three criteria were applied to distinguish between correlations
2
2
derived from
bonded to the carbon as deduced from the DEPT and heteronuclear
spin-echo experiments (APT), (ii) C chemical shifts, and (iii) cor-
relations with other vecinal protons.
J CH or J CH for a given proton: (i) number of protons
1
971, 54, 1709. (b) Aplasmomycin: Nakamura, H.; Iitaka, Y.; Kitahara,
1
3
T.; Okazaki, T.; Okami, Y. J . Antibiot. 1977, 31, 714. (c) Borophycin:
Hemscheidt, T.; Puglisi, M. P.; Larsen, L. K.; Patterson, G. M. L.;
Moore, R. E.; Rios, J . L.; Clardy, J . J . Org. Chem. 1994, 59, 3467. (d)
Tartrolon B: Schummer, D.; Schomburg, D.; Irschik, H.; Reichenbach,
H.; H o¨ fle, G. Liebigs Ann. 1996, 965.
(
14) Carbajo-Mart ´ı nez, R. J .; Bayod-J asanada, M.; L o´ pez-Ortiz, F.
Magn. Reson. Chem., submitted.
15) Hunt, E.; Knowles, D. J . C.; Shillingford, C.; Wilson, J . M.;
Zomaya, I. I. J . Antibiot. 1989, 41, 1029.
(
(
20) Lazarevski, G.; Vinkovic, M.; Kobrehel, G.; Dokic, S. Tetrahe-
dron 1993, 49, 721.

Notes
Azith r om ycin Hyd r ogen obor a te (7). To a solution of
J . Org. Chem., Vol. 62, No. 21, 1997 7481
product was dissolved in ethanol and precipitated by addition
of water. The solid was filtered off and dried under vacuum at
40 °C affording 15 g (70.5%) of azithromicyn (1). The compound
thus obtained matched the USP standard7a for azithromycin.
azaerithromycin hydrogenoborate (6) (50 g, 65.7 mmol) in
chloroform (500 mL) was added a mixture of formic acid (5.5
mL, mmol) and 35% aqueous formaldehyde (11.75 mL, mmol).
The reaction was refluxed for 14 h and then cooled to room
temperature. Water was added (500 mL) and the mixture was
then acidified to pH <4 with mineral acid. The organic layer
was discarded. Conventional workup of the aqueous layer with
methylene dichloride (350 mL) at pH >9 gave a crude product,
which when washed with diethyl ether yielded the pure title
compound 7 (29 g, 57%).
Azith r om ycin Dih yd r a te (2). To a solution of azithromicyn
1) (51 g, 68.1 mmol) in acetone was added water over 30 min.
(
The solution was stirred for 24 h at room temperature. The solid
formed was filtered off and dried under vacuum at 40 °C,
affording 45 g (84.2%) of pure azithromycin dihydrate according
7a
to the USP standard.
7
: 1H NMR δ 10.31 (s, 1H), 5.23 (d, 1H), 4.91 (dd, 1H), 4.53
dd, 1H), 4.41 (d, 1H), 4.11 (dq, 1H), 3.58 (d, 1H), 3.54 (bs, 1H),
.49 (ddq, 1H), 3.35 (s, 3H), 3.23 (dd, 1H), 2.99 (dd, 1H), 2.72
dq, 1H), 2.46 (ddd, 1H), 2.46 (d, 1H), 2.36 (dq, 1H), 2.31 (s, 3H),
Ack n ow led gm en t. We are indebted to Prof. J . E.
Su a´ rez-Fern a´ ndez and P. Garc ´ı a (Departamento de
Microbiolog ´ı a, Universidad de Oviedo) for performing
the biological assays. R.J .C. wishs to thank the Min-
isterio de Educaci o´ n y Cultura for a predoctoral fellow-
ship. The “Servicio de Resonancia Magn e´ tica Nuclear”
of the University of Oviedo is acknowledged for allocat-
ing spectrometer time.
(
3
(
2
1
1
1
3
1
7
4
2
.30 (d, 1H), 2.29 (s, 6H), 2.15 (d, 1H), 1.98 (m, 1H), 1.97 (m,
H), 1.95 (ddq, 1H), 1.70 (d, 1H), 1.66 (ddd, 1H), 1.57 (dd, 1H),
.48 (ddq, 1H), 1.37 (d, 3H), 1.30 (s, 3H), 1.29 (m, 1H), 1.26 (ddd,
H), 1.25 (s, 3H), 1.23 (d, 3H), 1.18 (d, 3H), 1.13 (d, 3H), 1.07 (s,
1
3
H), 1.02 (d, 3H), 0.87 (d, 3H), 0.75 (t, 3H); C NMR δ 180.16,
03.10, 94.26, 83.37, 80.40, 78.34, 78.26, 78.07, 77.33, 73.52,
2.86, 70.74, 69.05, 68.83, 65.92, 65.57, 64.31, 49.55, 45.56, 42.52,
1.75, 40.37, 35.85, 34.58, 28.75, 27.69, 26.44, 21.98, 21.60, 21.37,
Su ppor tin g In for m ation Available: Copies of the FABMS,
1H- C-, DEPT, HMQC, and HMBC spectra of compounds 6
and 7 and a table showing the results of the biological
screening of 7 (15 page). This material is contained in libraries
on microfiche, immediately follows this article in the microfilm
version of the journal, and can be ordered from the ACS; see
any current masthead page for ordering information.
1
1
13
1.36, 18.45, 15.59, 14.65, 11.01, 8.75, 7.09; B NMR δ 10.0;
+
FABMS m/z 775 [M] .
Azith r om ycin (1). A solution of azithromycin hydrogenobo-
rate (7) (22 g, 28.4 mmol) in a mixture of acetonitrile (250 mL)
and water (125 mL) was acidified to pH 2 and stirred for 30
min at a temperature of 15 °C. Then, a mixture of CH
O was added and the pH increased to 9. The organic layer
was filtered over Zeolite and evaporated to dryness. The crude
2 2
Cl -
H
2
J O970776R