11â,17r-Dih ydr oxy-9r-flu or o-16r-m eth yl-20-oxo-1,4-pr eg-
n a d ien -21-oic Acid (2). To a solution of 1 (methyl hemiacetal,
5.80 g, 13.7 mmol) in 45 mL of t-BuOH was added a 2.0 M
solution of 2-methyl-2-butene in THF (45 mL, 90 mmol) and 30
mL of saturated aqueous KH2PO4 (pH 4.5). A solution of NaClO2
(80%, 1.74 g, 15.5 mmol) in 15 mL of water was added, and the
two-phase mixture was stirred rapidly. Within 5 min, the
temperature had increased from 25 to 40 °C. A water bath was
applied to cool the mixture to 25 °C. After 16 h, the mixture
was diluted with EtOAc and extracted with water (three times).
(The organic solution retains any unreacted 1 plus side products,
including ca. 5% each of 3 and 4.) The combined aqueous solution
(pH 5) was acidified to pH 2 with 1 M aqueous NaHSO4. The
precipitate was extracted into EtOAc, and this solution was
washed twice with water and brine, dried (MgSO4), filtered, and
concentrated. The product was triturated with 25 mL of EtOAc
yielding a white solid, which was dried in vacuo to give 2.90 g
The characteristically large coupling constant, J ) 231
Hz, between the formate C and H of 4 was measured from
the 13C satellites of the well-isolated proton signal and
is reflected in the DEPT spectra as well. A correlation
between C-17 and the formate H was also observed in
the long-range HETCOR spectrum.
Characterization of synthetic 4 by HPLC/MS using
atmospheric pressure chemical ionization (APCI) yielded
[MH]+ at m/z 407 as the base peak. Under negative ion
conditions, the base peak occurred at m/z 405, consistent
with the presence of the carboxylate function. The
positive ion MS2 (MS/MS) spectrum obtained following
collisionally induced dissociation (CID) of the 407 ion was
dominated by an m/z 387 fragment, indicating the facile
loss of HF. Subsequent CID of the 387 ion yielded a key
MS3 spectrum with structurally informative fragments
at 369 (-H2O), 341 (-HCOOH), 323 (-HCOOH - H2O),
313 (-HCOOH - CO), and 295 (-HCOOH - H2O - CO).
Through a comprehensive series of MSn experiments, it
was confirmed that the 323, 313, and 295 ions are
products of multiple eliminations.
Characterization of Degradant F by HPLC/MSn yielded
spectra indistinguishable from those of 4 and distinct
from those of 2. When a sample of Degradant F (1 mL
HPLC fraction) was treated with 2-3 mg of sodium
carbonate and allowed to stand at 23 °C for several hours,
it underwent conversion to 3. A sample of synthetic 4
behaved likewise.
(52%) of 2: mp 204-206 °C dec; [R]23 +66.0° (c 0.46, MeOH);
D
IR (KBr) ν 3600 (s), 3520 (s), 3455 (s), 2950, 1748, 1709, 1658,
1596, 1578, 1390, 895 cm-1 1H and 13C NMR spectra are
;
tabulated in Supporting Information; HPLC/MS (APCI) m/z 407
(MH+). Compound 2 retained 15 mol % EtOAc (by 1H NMR) after
extended drying in vacuo at 50 °C. Anal. Calcd for C22H27O6F‚
0.15 EtOAc: C, 64.68; H, 6.77. Found: C, 64.56; H, 6.78;
unchanged after further drying in vacuo for 24 h at 100 °C.
11â,17r-Dih yd r oxy-9r-flu or o-16r-m et h yl-1,4-a n d r ost a -
d ien -17â-ca r boxylic Acid (3).6 H5IO6 (3.92 g, 17.2 mmol) was
added to a stirred suspension of dexamethasone (6.00 g, 15.3
mmol) in 60 mL of EtOH and 24 mL of water. After 30 min, a
clear solution resulted, which was stirred for an additional 16
h, forming a white solid. Water (300 mL) was added, and stirring
was continued for 1 h. The suspension was filtered, and the solid
was washed well with water and dried in vacuo at 70 °C to yield
5.62 g (97%) of 3: mp 274-277 °C dec (lit.6 >258 °C dec), [R]23
These observations provide compelling evidence for a
decomposition pathway leading from dexamethasone to
formate 4 via the generation and isomerization of mixed
anhydride 5 in an aqueous medium. The concurrent
formation of 3, an established degradant type,2,3a can be
explained as the consequence of competing intermolecu-
lar reaction of 5 with water or other nucleophilic com-
ponents in solution. These results bear upon the oxidative
behavior of corticosteroids in general and reasonably
extend to the doxorubicin group of antitumor antibiotics
and to other substances having a similarly constituted
side chain.
D
+52.4° (c 0.48, MeOH); lit.6 [R]D +46.6° (dioxane); IR (KBr) ν
1
3534 (s), 3000-2300 (br), 1689, 1655, 1598, 1264 cm-1; H and
13C NMR spectra are tabulated in Supporting Information;
HPLC/MS (APCI) m/z 379 (MH+). Anal. Calcd for C21H27O5F:
C, 66.64; H, 7.19. Found: C, 66.67; H, 7.29.
17r-F or m yloxy-11â-h yd r oxy-9r-flu or o-16r-m et h yl-1,4-
a n d r osta d ien -17â-ca r boxylic Acid (4). CDI (97% pure by 1H
NMR, 5.05 g, 30 mmol) was added over 5 min to a stirred
solution of 3 (10.4 g, 27.5 mmol) in anhydrous THF (64 mL) at
20-25 °C under N2 (CO2 evolved). After 1 h, the solution was
cooled in ice, and formic acid (96%, 4% water, 3.2 mL, 82 mmol)
was added at <15 °C. Triethylamine (11.6 mL, 83 mmol) was
then added at <25 °C. The solution was stirred at 25 °C for 16
h. Water (800 mL) was added, and the solution (pH 8.5) was
washed with EtOAc (discarded), acidified to pH 3 with 150 mL
of 1 M aqueous NaHSO4, and extracted twice with EtOAc. These
extracts were combined and washed with water and brine, dried
(Na2SO4), filtered, and concentrated in vacuo to give 11.1 g
(100%) of an 88:12 mixture of 4 and 3. This material was
dissolved in 110 mL of boiling 2:1 (v/v) EtOH-H2O. The solution
was allowed to cool to 25 °C, and seed crystals of 4 (10 mg) were
added to initiate crystallization. Crystallization was completed
at -10 °C. The solid was collected by filtration and dried in vacuo
Exp er im en ta l Section
Gen er a l Meth od s. HPLC analyses were performed using a
Waters Symmetry C8 column (3.9 × 150 mm) with a mobile
phase of 10 mM NH4OAc in H2O/MeOH (7:3 to 2:8). Excitation
energies in CID/MSn experiments were selected manually to
reduce the selected ion intensity to 1-10% of the base fragment
intensity. NMR spectra were obtained at 600 MHz (1H) and 150
MHz (13C). Melting points are uncorrected. Elemental analyses
were performed by Atlantic Microlab, Norcross, GA.
at 50 °C to afford 7.25 g (65%) of 4: mp 228-232 °C dec; [R]23
D
+27.6° (c 0.47, MeOH); IR (KBr) ν 3500-2400 (br), 1741, 1707,
11â,17r-Dih ydr oxy-9r-flu or o-16r-m eth yl-20-oxo-1,4-pr eg-
n a d ien -21-a l (1)1 Meth yl Hem ia ceta l. The method of Lewbart
and Mattox was used4 with a modified workup procedure. Cu-
(OAc)2‚H2O (0.24 g, 1.20 mmol) was added to a suspension of
dexamethasone (6.00 g, 15.3 mmol) in MeOH (120 mL). The
mixture was stirred vigorously, open to the air, giving at first a
purple-blue color. After 1.5 h, the clear blue-green solution was
diluted with 240 mL of EtOAc and filtered through a Florisil
pad, eluting with another 240 mL of EtOAc. Concentration in
vacuo afforded 6.76 g of 1 as a foam. 1H NMR (CDCl3) showed
10% of the aldehyde (δ 9.5, s, 1H) and 70% of the methyl
hemiacetal (δ 3.5, two s, 3H): HPLC/MS (APCI) m/z 391 (MH+
of aldehyde, major), 423 (MH+ of methyl hemiacetal, minor). This
material was used in the following experiment without purifica-
tion.
1663, 1596, 1225, 1202 cm-1 1H and 13C NMR spectra are
;
tabulated in Supporting Information; HPLC assay (dried basis)
4 98.5%, 3 1.5%; HPLC/MS (APCI) m/z 407 (MH+). Compound
4 proved to be hygroscopic, as varying microanalytical values
indicated hydration ranging up to 2 mol of H2O per mol of 4. A
sample was dried in vacuo for 24 h at 100 °C. Anal. Calcd for
C
22H27O6F: C, 65.01; H, 6.70. Found: C: 64.57; H, 6.79.
Su p p or tin g In for m a tion Ava ila ble: Tabulated 1H and
13C NMR spectra of 2-4 with peak assignments and MS3 of 4
and Degradant F. This material is available free of charge via
the Internet at http://pubs.acs.org.
J O020282G
6836 J . Org. Chem., Vol. 67, No. 19, 2002