Synthesis of a doxycycline-[13CD3] standard

Article abstract of DOI:10.1002/jlcr.3397

A stable isotope labelled mass spectrometry internal standard of the antibiotic doxycycline was prepared to assist in pharmacokinetic analyses. Our approach was to first N-demethylate doxycycline using a non-classical Polonovski reaction and then re-methylate using methyl-[¹³CD₃] iodide, which gave doxycycline-[¹³CD₃] with an isotopic purity of 99%.

Full text of DOI:10.1002/jlcr.3397

Research article
Received 07 January 2016,
Revised 12 February 2016,
Accepted 04 March 2016
Published online 6 April 2016 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3397
13
Synthesis of a doxycycline-[ CD3] standard
*
James E. Bupp and Mary J. Tanga
A stable isotope labelled mass spectrometry internal standard of the antibiotic doxycycline was prepared to assist in
pharmacokinetic analyses. Our approach was to first N-demethylate doxycycline using a non-classical Polonovski reaction
and then re-methylate using methyl-[13CD3] iodide, which gave doxycycline-[13CD3] with an isotopic purity of 99%.
Keywords: MS internal standard; doxycycline; antibiotic; demethylation
Purification with ethylenediaminetetraacetic acid to remove the
iron salts, followed by reverse phase flash chromatography and
crystallization gave 99% pure N-desmethyldoxycycline 3 with 0.6%
doxycycline 1 in 9% overall yield.
We determined that the best approach for methylation of N-
desmethyldoxycycline 3, a secondary amine, with labelled methyl
Introduction
The tetracycline family, which was discovered as a result of the
screening of soil samples for antibiotics, has been effectively
used for decades. This drug class is recognized as having
interesting pleiotropic properties,1 and currently, there are over
200 clinical trials underway.2 Based on this renewed interest,
the study of potential uses of tetracyclines is expanding. As a
member of the tetracycline antibiotic class, doxycycline is used
for the treatment of a variety of infections, including sinusitis,
pelvic inflammatory disease, acne and rosacea. Prophylactically,
it is used against malaria, by preventing the development of
parasites in the blood that cause malaria.3,4 Doxycycline is also
used prophylactically and therapeutically for the prevention
and treatment of anthrax and plague, and for the treatment of
the tick borne diseases such as Lyme5 and Rocky Mountain
spotted fever.6
iodide-[13CD3] was to use polymer-supported triphenylphosphine
and diisopropyl azodicarboxylate in tetrahydrofuran as shown in
Scheme 2. These reaction conditions provided the corresponding
tertiary amine product 4. With this method, only trace amounts of
the quaternization product were observed even in the presence
of a large excess of methyl iodide (three equivalents).9 Purification
using reverse phase chromatography followed by crystallization
from ethanol/water and then treatment with HCl provided highly
pure doxycycline-[13CD3] hyclate with an isotopic purity of 99%
(33% yield).
The therapeutic potential and applications of tetracyclines are
being redefined and expanded, and as such, stable isotope
labelled doxycycline is a valuable research tool for studying its
biodistribution and mechanism of action.
Conclusion
This approach for the synthesis of stable isotope labelled
doxycycline 4 by first N-demethylation and then re-methylation
with stable isotope labelled iodomethane provides an efficient
synthesis of doxycycline-[13CD3] 4, which is not readily accessible
by other routes.
Results and discussion
A mass spectroscopy (MS) analytical standard of doxycycline with
M + 4 isotopic incorporation was prepared as a chemical probe to
assist in pharmacokinetic analyses. Our approach for the efficient
synthesis of stable isotope labelled doxycycline-[13CD3] 4 was to
first N-demethylate doxycycline and then re-methylate using
iodomethane-[13CD3].
This work extends the utility of the non-classical Polonovski
reaction from alkaloids to tetracyclines.7,8 Formation of the
demethylated tetracyclines is an important intermediate for the
synthesis of novel tetracyclines as well as labelled standards.
Utilizing a modified Polonovski reaction in a two-step process as
shown in Scheme 1, the free base of doxycycline 1 was readily
converted to the N-oxide 2 using 3-chloroperoxybenzoic acid
(m-CPBA). The N-oxide 2 was 95% pure by LC/MS (liquid
Experimental
General methods
All reactions were carried out under inert argon atmosphere using
commercial reagents of the highest grade and without further purification.
Doxycycline hyclate was purchased from Sigma-Aldrich (St. Louis, MO).
Methyl iodide-[13CD3] was purchased from Cambridge Isotope Laboratories
(Tewksbury, MA). Intermediates were analysed by nuclear magnetic
resonance (NMR) (Varian Mercury VX 300 MHz or Varian 400 MHz). All
chemical shifts are reported in parts per million (δ) downfield from
chromatography mass spectrometry). After conversion of the N- Biosciences Division, SRI International, 333 Ravenswood Ave, Menlo Park, CA
94025, USA
oxide 2 to the hydrochloride, treatment with iron (Fe(0)/FeCl3)
formed N-desmethyl product 3 in 79% yield by LC/MS. A side
reaction gave 21% doxycycline 1 as a byproduct by LC/MS, which
was readily removed during the workup and purification procedure.
*Correspondence to: Mary J. Tanga, SRI International, Biosciences Division, 333
Ravenswood Ave, Menlo Park, CA 94025, USA.
E-mail: mary.tanga@sri.com
J. Label Compd. Radiopharm 2016, 59 291–293
Copyright © 2016 John Wiley & Sons, Ltd.

J. E. Bupp and M. J. Tanga
Scheme 1. N-demethylation of doxycycline
Scheme 2. Synthesis of stable isotope labelled doxycycline
tetramethylsilane. High resolution mass spectra (HRMS) were run using a Doxycycline-[13CD3] hyclate [(4S,4aR,5S,5aR,6R,12aS)-4-(dimethylamino)-
Thermo LTQ-Orbitrap XL hybrid ion-trap mass spectrometer. LC/MS data
were obtained using a Thermo LCQ Fleet and Finnigan Surveyor System.
Reverse phase chromatography was performed using Silicycle C18 silica gel
(40–63 μ) on a Biotage Isolera IV preparative flash chromatography system.
3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,12a-oc
tahydrotetracene-2-carboxamide] (4)
A suspension of triphenylphosphine, polymer bound (200–400 mesh,
~3.0 mmol/g, 174.3 mg, 0.523 mmol, 1.5 equiv) in THF (20 mL) was stirred
for 1 h at RT. Next, diisopropyl azodicarboxylate (DIAD) (103.1 L, d 1.027,
N-desmethyldoxycycline [(4S,4aR,5S,5aR,6R,12aS)-3,5,10,12,12a- 105.8 mg, 0.523 mmol, 1.5 equiv), N-desmethyldoxycycline (150 mg,
0.349 mmol) and iodomethane-13CD3 (13C, 99%; D3, 99%, stabilized with
copper wire) (0.065 mL, d 2.34, 152.3 mg, 1.044 mmol, 3 equiv) were
added. The reaction mixture was stirred at RT for 4 days and then diluted
with ethyl ether (20 mL) and extracted with water (3 × 20 mL). The
combined aqueous phases were washed with ethyl ether (20 mL),
evaporated in vacuo to remove residual volatile organic solvent and then
lyophilized to give crude product (133 mg). This material was filtered
through a plug of Silicycle C18 (700 mg) eluting with CH3CN/water (4/6).
pentahydroxy-6-methyl-4-(methylamino)-1,11-dioxo-1,4,4a,5,5a,
6,11,12a-octahydrotetracene-2-carboxamide] (3)
Doxycycline hyclate (4.08 g, 7.95 mmol) was free based in methanol with
sodium hydroxide (1 equiv) and evaporated in vacuo. Doxycycline (free
base) 1 plus NaCl was suspended in CHCl3/isopropanol (3:1) (240 mL)
and then warmed to 50 °C to maximize solubility. The resulting mixture
was cooled to À78 °C and then treated with m-CPBA (97%, 1.72 g, 1.25
equiv) in one portion. The resulting suspension was stirred at À20 °C to
Evaporation gave 91 mg of a solid, which was acidified with 1 N HCl
À10 °C over 45 min to form the N-oxide 2. The formation of the N-oxide
(0.2 mL) and crystallized from EtOH/water (1/1) (4 × 0.33 mL) at 0 °C.
2 was monitored by MS. The N-oxide mixture was acidified with 1.23 M
Further evaporation in vacuo gave the hyclate product (67 mg). This
ethereal HCl (6.46 mL, 7.95 mmol). The mixture at À20 °C was treated
material was triturated with hexane (2 × 1 mL) and ether (2 × 1 mL) and
with iron powder (66.6 mg, 325 mesh, 97%, 0.15 equiv catalyst) and a
evaporated in vacuo giving 60 mg of pure product 4 (33% yield) with
an isotopic purity of 99%. 1H NMR: (400 MHz, CD3OD) δ 7.49 (t, 1H,
solution of FeCl3•6 H2O (64.5 mg, 0.03 equiv catalyst) in isopropanol
(1.5 mL). The stirred mixture was allowed to warm to RT (room
J = 8.2 Hz), 6.95 (d, 1H, J = 8.2 Hz), 6.84 (d, 1H, J = 8.2 Hz), 4.42 (d, 1H,
temperature) over 2 h. The LC/MS showed conversion to N-desmethyl
J = 2.8 Hz), 3.63–3.54 (m, 2H), 3.00–2.89 (m, 3H), 2.82–2.70 (m, 2H), 2.61–
product 3 in 79% yield with 21% doxycycline 1 byproduct contaminant.
2.54 (m, 1H), 1.54 (d, 3H, J = 6.8 Hz), 1.17 (t, 1.5H, J = 7.0 Hz, hyclate
ethanol methyl peak). 13C NMR: (75 MHz, DMSO-d6) δ 192.54, 173.89,
The resulting solution was cooled in an ice bath and then poured into
10 mM ethylenediaminetetraacetic acid tetrasodium salt (pH 10)
171.71, 161.10, 147.85, 136.75, 115.93, 115.66, 115.49, 107.23, 95.15,
(400 mL), stirring for 15 min at RT lowered the pH to 3. The phases were
73.11, 67.99, 64.50, 63.04, 55.97, 53.81, 45.25, 44.00–40.00 (m, 4C),
separated, and the aqueous phase was washed with CHCl3 (2 × 50 mL).
18.52, 15.85. HRMS: (ESI, Full-scan) m/z (relative intensity) calculated,
The aqueous solution was adjusted to pH 7 with 1 M NaOH (14 mL) and
449.18273; found, 449.18266 (M + H, 100). FTIR: (Solid Phase ATR) 3205,
2529, 2160, 1977, 1574, 1455, 1243, 1002, 885, 710 cmÀ1. UV: (MeOH)
then filtered through a plug of reverse phase C18 silica gel (25 g) rinsing
with water. Lyophilization gave a crude solid (4 g), which was dissolved in
water and injected onto a column of 50 g of Silicycle C18 eluting with a
gradient of 100% water to 100% CH3CN giving 560 mg of crude product.
λmax 355.0 nm (ε14,746), 270.1 (14,888). Optical Rotation: (MeOH)
c = 0.099, [α] = À100°. HPLC: Varian Pursuit C18 (3 μ), 50 × 2 mm; 0.1%
formic acid in water/0.1% formic acid in CH3CN 100/0 to 60/40 over
This material was crystallized from ethanol/water (1/1) leaving 302 mg
5 min then hold; 360 nm; Rt = 5.80 min; UV purity >99%.
(8.8% yield) of 99.4% pure N-desmethyldoxycycline
3 with 0.6%
doxycycline 1. 1H NMR: (300 MHz, CD3OD) δ 7.46 (t, 1H, J = 8.2 Hz), 6.93
(d, 1H, J = 8.2 Hz), 6.82 (d, 1H, J = 8.2 Hz), 3.76–3.65 (m, 2H), 2.82–2.66
(m, 1H), 2.77 (s, 3H), 2.50–2.40 (m, 2H), 1.51 (d, 3H, J = 6.9 Hz). FTIR: (Solid
Acknowledgements
Phase ATR) 3205, 2529, 2160, 1977, 1567, 1455, 1394, 1323, 1244, 1220, This project has been funded in whole or in part with Federal
1114, 1044, 1001, 933, 886, 858, 809, 709 cmÀ1. UV: (MeOH) λmax funds from the National Institute of Allergy and Infectious
360.0 nm (ε14,335), 274.9 (14,663). Optical rotation: (MeOH) c = 0.051, Diseases, National Institutes of Health, US Department of Health
[α] = À174°. Elemental analysis: calculated for C21H22N2O8•2.7 H2O: C, and Human Services, under Contract No. HHSN272201100022l.
52.65; H, 5.76; N, 5.85; found: C, 52.76; H, 5.76; N, 5.78. MS: (ESI) m/z
(relative intensity) 431 (M + H, 100), 445 (0.62). HPLC: Varian Pursuit C18
(3 μ), 50 × 2 mm; 0.1% formic acid in water/0.1% formic acid in CH3CN
Conflict of interest
100/0 to 60/40 over 5 min then hold; 350 nm; Rt = 5.60 min; purity >99%. The authors did not report any conflict of interest.
www.jlcr.org
Copyright © 2016 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2016, 59 291–293

J. E. Bupp and M. J. Tanga
[5] D. Bremell, C. Säll, M. Gisslén, L. Hagberg, J. Med. Case Rep. 2011, 5,
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