Synthesis of [14C] - and [13C6]-labeled tipranavir and its potential hydroxyl metabolite and the glucuronide conjugate

Article abstract of DOI:10.1002/jlcr.1528

Tipranavir or Aptivus is a non-peptidic protease inhibitor approved for the combination treatment with ritonavir of HIV infection. Tipranavir labeled with radioactive and stable isotopes of carbon was required for drug metabolism (excretion, distribution,

Full text of DOI:10.1002/jlcr.1528

Research Article
Received 5 March 2008,
Revised 27 May 2008,
Accepted 2 June 2008
Published online in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1528
Synthesis of [14C] - and [13C6]-labeled
tipranavir and its potential hydroxyl
metabolite and the glucuronide conjugate
Ã
Bachir Latli,a Matt Hrapchak,a John A. Easter,b Wayne T. Stolle,c Karl
Grozinger,a Dhileepkumar Krishnamurthy,a and Chris H. Senanayakea
Tipranavir or Aptivuss is a non-peptidic protease inhibitor approved for the combination treatment with ritonavir of HIV
infection. Tipranavir labeled with radioactive and stable isotopes of carbon was required for drug metabolism (excretion,
distribution, and absorption) studies and to develop bioanalytical methods needed for the support of clinical studies.
[7-14C]-Benzoic acid and uniformly labeled benzoic acid (ring-13C6 99 at% 13C) were used to prepare [14C]- and [13C6]-labeled
tipranavir, respectively. Radioactively labeled tipranavir was prepared with a specific activity of 54 mCi/mmol (2GBq/mmol);
it was necessary to dilute its specific activity with unlabeled tipranavir to 28 mCi/mmol (46.45 mCi/mg) because of its
instability. The N-hydroxyl metabolite (12) and the glucuronide conjugate (13), the most abundant metabolites of tipranavir
(when administered in conjunction with ritonavir) were also synthesized.
Keywords: HIV–AIDS; tipranavir; carbon14; carbon13; radiosynthesis
received tipranavir/ritonavir (500 mg/200 mg). The most abun-
dant fecal metabolite was potentially identified as a hydroxyl
metabolite, and the most abundant urinary metabolite was a
Introduction
Tipranavir (Aptivuss, N-[3-{1(R)- (5,6-dihydro-4-hydroxy-2-oxo-
6(R)-phenethyl-6-propyl-2H-pyran-3-yl)propyl}phenyl]-5-trifluor-
omethylpyridine-2-sulfonamide, PNU-140690, Figure 1), ac-
quired from Pharmacia & Upjohn in 2000 and approved for
use in combination with Abbott’s ritonavir by the FDA in June
2005, is the first non-peptidic protease inhibitor drug available
in the US for the treatment of HIV and AIDS.1–4 This is
Boehringer’s second antiretroviral drug after nevirapine (Vir-
amunes).5,6 Tipranavir is a very potent inhibitor of HIV protease
with a Ki of 8.0 pmol.7,8 This compound is a sulfonamide-
containing dihydropyrone with two chiral centers resulting in
four diastereomers. All diastereomers were active as protease
inhibitors. Their inhibition potency in a tandem HIV assay,
ranges from 18 pmol for (1R, 6S) to 220 pmol for tipranavir’s
enantiomer (1S, 6S).7,8
glucuronide conjugate of tipranavir, albeit in small percen-
tages,11 see Figure 2. More importantly, patients taking
tipranavir twice a day with ritonavir have a significant reduction
of their viral load and the regiment also suppressed both wild-
type and protease inhibition-resistant virus.1,2
Results and discussion
The goal of this work was to prepare [14C]- and [13C6]-labeled
tipranavir and its major metabolites to support research and
DMPK studies (Schemes 1 and 2). Initially [7-14C]benzoic acid
was converted to [7-14C]benzoylchloride using thionyl chloride
in refluxing benzene. 1-Phenyl-1-[1-14C]-propanone (3) was then
obtained either from diethyl cadmium, prepared in situ from
cadmium chloride and ethyl magnesium bromide or as
According to 2007 UNAIDS–WHO report, annually there are
2.5 million newly infected people with HIV ‘ca 6800 persons
daily’.9 Over 5700 people die daily from AIDS, according to the
same study mainly because of inadequate access to HIV
aDepartment of Chemical Development, Boehringer Ingelheim Pharmaceuticals,
prevention and treatment. Even when treatment is available, Research and Development Center 900 Ridgebury Road, P.O. Box 368, Ridgefield,
CT 06877-0368, USA
HIV-infected patients develop resistance to their HIV therapy.
bBristol-Myers Squibb, 5 Research Parkway Mail Stop 4BC-316, Wallingford, CT
06492, USA
Thus, combination of two or more drugs is now the standard
therapy to combat this resistance. Tipranavir is particularly
potent and has excellent activity on viral strains resistant to
other protease inhibitors and is metabolized by P450 3A4.10 Its
pharmacokinetic parameters were enhanced when combined
with ritonavir; the metabolism in the presence of 200 mg of
ritonavir was minimal. Only a few metabolites were found in
plasma after administration of [14C]-tipranavir to subjects that
cPfizer Global Research and Development, Eastern Point Road, M.S.8118D-4037
Groton, CT 06340, USA
*Correspondence to: Bachir Latli, Department of Chemical Development,
Boehringer Ingelheim Pharmaceuticals, Research and Development Center 900
Ridgebury Road, P.O. Box 368, Ridgefield, CT 06877-0368, USA.
E-mail: blatli@rdg.boehringer-ingelheim.com
J. Label Compd. Radiopharm 2008, 51 314–320
Copyright r 2008 John Wiley & Sons, Ltd.

B. Latli et al.
O
N
H
N
CF3
N
OH
O
H
N
S
N
N
O
O
O
Aptivus® (Tipranavir)
Viramune® (Nevirapine)
O
O
H
N
N
N
N
H
O
S
N
H
N
O
OH
S
Norvir® (Ritonavir)
Figure 1. Structures of three HIV inhibitors.
HO
O
HO
O
HO
CF3
CF3
N
O
OH
N
N
OH
H
HO
N
S
S
O
O
O
O
O
O
O
O
(12)
(13)
Figure 2. Tipranavir metabolites.
previously reported by Arisawa et al. from diethyl zinc and with a specific activity of 54 mCi/mmol or isotopic enrichment of
aluminum chloride in 60–70% yield.12 Nitration with 90% nitric 86.5%. It was found that this material was not stable even when
acid at À451C gave 1-[3-nitrophenyl]-1-[1-14C]-propanone (4) in stored at À801C. To minimize the radiolysis it was diluted with
56% yield. (R)-4-Hydroxy-6-phenethyl-6-propyl-5,6-dihydro-2- unlabeled tipranavir to a specific activity of about 28 mCi/mmol.
pyran-2-one (5)13–16 and the propanone (4) were subjected to The rate of radiolysis was slower when the compound is kept as
a Kneovenagel type condensation to give compound (6) in 65% a solution of absolute ethanol at À801C. The chemical identity of
yield. The other chiral center was introduced via an asymmetric this compound was established by comparing its 1H and 13C
hydrogenation of the double bond in compound (6) using the NMR and its chromatographic retention time with that of
chiral rhodium catalyst, (-)-1,2-bis((2R,5R)-2,5-dimethyphophola- authentic tipranavir sample. The preparation of labeled 5-
no)benzene(cyclooctadiene)-rhodium
(I)
tetrafluoroborate trifluoromethylpyridine-2-sulfonyl chloride was also considered.
([((R,R)-Me-DuPHOS)Rh(Cod)]BF4) at 601C under hydrogen in [14C]-2-Chloro-5-trifluoromethylpyridine can be purchased from
methanol and in the presence of sodium carbonate.17,18 This commercial suppliers and then converted in two steps to 5-
hydrogenation gave a product in 94% or more enantiomeric trifluoromethylpyridine-2-sulfonyl chloride via a thiol-intermedi-
excess as judged from chiral HPLC. Reduction of the nitro group ate as seen in Scheme 1. The possibility that sulfonamides may
using palladium on carbon gave the aryl amine (8) in 97% yield. undergo a glutathione-S-transferase-catalyzed cleavage has
Coupling to 5-trifluoromethyl-2-pyridinesulfonyl chloride (10), precluded us from pursuing this route.20
which was prepared by bubbling chlorine gas in a mixture of
[13C6]-Tipranavir was obtained using the same route starting
aqueous HCl solution (1.0 N) and 5-(trifluoromethyl)pyridine-2- from [13C6]-benzoic acid with 99 at% 13C.
thiol (9),19 gave labeled tipranvir in 43% yield. The final step was
Tipranavir metabolites (12) and (13) were prepared as
modified from the reported procedure,13,16 instead of using described in Scheme 2. In the synthesis of (12), the nitro group
21
pyridine to couple the sulfonyl chloride derivative to (8), N,N- in (7) was reduced to the hydroxylamine with KBH4/BiCl3 and
dimethylaniline and BHT (butylated hydroxytoluene) were used then coupled with 5-trifluoromethylpyridine-2-sulfonyl chloride
to give a clean reaction. [14C]-Tipranavir was obtained initially (10) in the presence of pyridine in methanol to give the desired
J. Label Compd. Radiopharm 2008, 51 314–320
Copyright r 2008 John Wiley & Sons, Ltd.
www.jlcr.org

B. Latli et al.
O
O
O
O
TiCl4, pyridine, THF
OH
SOCl2
C6H6
Cl
Cd(Et)2
C6H6
90% HNO3
#
OH
#
*
#
*
#
*
*
100%
56-80%
60-80%
NO2
14C]-(2)
13C6]-(2)
[
14C]-(1)
[
[
[
14C]-(3)
13C6]-(3)
[
14C]-(4)
13C6]-(4)
[
13C6]-(1)
O
[
O
[
(5)
OH
OH
NO2
NO2
H2, MeOH
#
[((R,R)-MeDuPHOS)Rh(cod)]BF4
*
#
*
10% Pd/C
O
O
H2, 80 PSI, 57 oC, MeOH
O
O
81-90%
14C]-(7)
13C6]-(7)
65-70%
[
[
14C]-(6)
[
13C6]-(6)
CF3
[
CF3
N
OH
O
OH
H
N
(10)
SO2Cl
N
NH2
S
#
O
*
#
O
*
N,N-Dimethylaniline
BHT, CH2Cl2
O
O
O
90-97%
14C]-(8)
13C6]-(8)
43-65%
14C]-Tipranavir
13C6]-Tipranavir
[
[
[
[
CF3
Cl2
CF3
N
SO2Cl
aq. HCl
N
SH
* : 14
C
89%
(10)
# : 13C6
(9)
Scheme 1. Synthesis of [14C]- and [13C6]-tipranavir.
OH
NH
OH
OH
NO2
BiCl3, KBH4
O
O
O
O
25% H2O/EtOH
71%
(7)
(11)
CF3
OH
N
N
OH
O
S
O
O
Pyridine, MeOH
CF3
HO
O
O
HO
35%
O
HO
N
(10)
SO2Cl
CF3
N
O
(12)
H
N
S
HO
OAc
O
O
CF3 AcO
OAc
1. Ag2O, MeCN
2. aq. NaOH
N
OH
O
O
O
H
+
N
O
S
Br
O
O
O
12%
(13)
O
O
Tipranavir
Scheme 2. Synthesis of the hydroxyl-metabolite and the glucuronide conjugate.
adduct in 25% overall yield in two steps after preparative
HPLC purification. The glucuronide conjugate was prepared
by reacting tipranavir with the acetobromo-a-D-glucuronic acid
methyl ester in the presence of silver oxide followed by basic
hydrolysis. Preparative HPLC purification gave the desired (13) in
12% yield. No attempts were made to separate the a/b
epimers.
Experimental procedures
Materials and methods
Liquid scintillation counting was accomplished using a Beckman
LS5000TA and ready safeTM cocktail (Beckman, Fullerton, CA).
Radio-TLC was carried out on a BIOSCAN System 200 imaging
www.jlcr.org
Copyright r 2008 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2008, 51 314–320

B. Latli et al.
scanner using an auto change 1000 and WinScan software were washed with water (10 mL), aqueous KOH (2 N, 15 mL),
version 2.1a (Bioscan Inc., Washington, DC). The quantification of brine, dried over Na2SO4, filtered, and concentrated in vacuo.
the HPLC chromatograms was carried out using an HPLC system The residue was mixed with 2 mL of absolute ethanol and
comprised of a Radiomatic A515 Flo-one\Beta radioactivity flow cooled to À151C for 1 h. The resulting solid was filtered, washed
detector (Packard Instrument Company, Meriden, CT), two with cold ethanol, and dried under high vacuum at room
pumps (HITACHI L-6200A intelligent pump), a linear UVIS 200, temperature to give 307 mg of the desired product. A second
Ultima FloTM AP coctail (Packard, Meriden, CT), and radiomatic crystallization from the mother liquor gave 19 mg of material.
500TR V 3.60 software for data evaluation.
The analytical HPLC purity verification was carried out on a
Total yield 58%, 102 mCi.
(6R)-4-Hydroxy-3-[(E)-1-(3-nitophenyl)-1-[1-14C]propenyl]-6-phe-
Zorbax SB300-C8 column, particle size 5 mm, (4.6 Â 250 mm) nethyl-6-propyl-5,6-dihydro-2H-pyran-2-one ([14C]-6): To a stirred
fitted with an OPTI-Guard (1 mm C8) column guard. UV solution of the above compound (321 mg, 1.79 mmol, 102 mCi)
detection was at 220 nm. The column heater was kept at 351C. and (5) (547 mg, 2.1 mmol) in dry THF (5 mL) and pyridine
Mobile phase: A (water), B (acetonitrile), both 10 mM TFA. (0.34 mL, 4.2 mmol) under nitrogen atmosphere cooled to
Gradient: 20–100%B in 30 min, then back to 20%B in 5 min, and À101C was added a solution of TiCl4 in toluene (4 mL, 1 M).
hold for 2 min. For enantiomeric purity, the mobile phase The resulting dark red solution was stirred at À101C for 30 min,
consisted of isocratic acetonitrile:triethylamine: acetic acid then warmed to room temperature and stirred for 21 h. The
(1000:1:6 v/v/v). UV detection at 254 nm and using Astec reaction was quenched by the addition of water (10 mL), and
Cyclobond I 2000 column (5 mm, 4.6 Â 250 mm). Mass spectra THF was removed in vacuo. The aqueous was extracted with
were acquired by a Hewlett-Packard auto sampler Series 1150, CH2Cl2 (2 Â 15 mL). The combined extracts were washed with
connected to a Micromass LCZ mass spectrometer in the ES aqueous HCl (2 N, 20 mL), water (20 mL), and brine (20 mL), dried
mode. NMR spectra were recorded with a Brucker 400 MHz over Na2SO4, filtered and concentrated in vacuo. The residue
DPXB and 500 MHz spectrometers using deuterated methanol, was dissolved in toluene (10 mL) and heated with stirring to
chloroform, or dimethyl sulfoxide as a solvent and tetramethyl 601C for 3 h to equilibrate the geometric isomers. After cooling
silane as the internal standard (Cambridge Isotope Laboratories, to room temperature, toluene was removed and the residue was
Andover, MA). Pre-coated TLC sheets (silica gel 60 F254) were purified by silica gel chromatography using 70% EtOAc:hexane
obtained from EM Science (Gibbstown, NJ). [7-14C]benzoic was to give 543 mg of product in 72% yield, total activity 70.58 mCi.
purchased from Amersham (GE HealthCare). [13C6]-benzoic acid
(6R)-4-Hydroxy-3-[(E)-1-(3-nitophenyl)-1-[1-14C]propyl]-6-phe-
was purchased from Isotec (St. Louis, MO). 5-(Trifluromethyl)pyr- nethyl-6-propyl-5,6-dihydro-2H-pyran-2-one ([14C]-7): A mixture of
idine-2-thiol was obtained from Ryan Scientific, Inc. (Mt. the above compound (534 mg, 1.29 mmol), K2CO3 (40 mg,
Pleasant, SC). The catalyst [((R,R)-Me-DuPHOS)Rh(COD)]BF4 was 0.23 mmol) in nitrogen purged methanol (1.5 mL) was placed
obtained from Strem (Newburyport, MA).
in a stainless steel Parr bomb apparatus. The pressure bomb was
purged with hydrogen, and then charged to 70 psi and heated
with stirring to 571C for 20 min. The pressure was released, and
14 mg of [((R,R)-Me-DuPHOS)Rh(COD)]BF4 was added as a
solution in nitrogen purged methanol (1 mL). The Parr was then
purged with hydrogen and charged to 80 psi and heated with
stirring at 571C for 4 h. The pressure was released, and the
contents of the flask were transferred to a round bottom flask
using methanol. Methanol was removed under vacuum and the
residue was partitioned between CH2Cl2 (25 mL) and aqueous
HCl (2 N, 25 mL), and the organic layer was washed with water
(20 mL), brine (20 mL), dried over Na2SO4, filtered and concen-
trated in vacuo. The residue was purified by silica gel
chromatography using 1% THF/CH2Cl2 to give 466 mg of the
desired product in 85% yield, 60.1 mCi.
(6R)-3-[(1R)-1(3-aminophenyl)-1-[1-14C]propyl]-4-hydroxy-6-phe-
nethyl-6-propyl-5,6-dihydro-2H-pyran-2-one ([14C]-8): A solution of
the above compound (466 mg, 1.1 mmol) and 10% Pd/C
(150 mg) in nitrogen purged methanol (5 mL) was stirred under
hydrogen at atmospheric pressure at room temperature. On
completion, the reaction contents were filtered through a short
pad of Celites and eluted with methanol. The filtrate was
concentrated in vacuo to give 397 mg of material in 99.9% yield,
which was used without further purification.
Synthesis
1-Phenyl-1-[1-14C]-propanone ([14C]-3): To a solution of [7-14C]-
benzoic acid (536 mg, 4.39 mmol, 250 mCi) in benzene (5 mL)
was added thionyl chloride (1.7 mL, 23.3 mmol) and refluxed for
3 h. Excess thionyl chloride and benzene were then removed in
vacuo to a stirrable volume. More benzene was added (10 mL)
and distilled off to remove excess of thionyl chloride. Fresh
solution of diethylcadmium was prepared from a solution of
ethyl magnesium bromide (3.8 mL, 1.42 M) in diethyl ether at
01C and cadmium chloride (521 mg, 2.84 mmol), which was
added via cannulation over 10 min to the ethyl magnesium
bromide. The resulting mixture was warmed to room tempera-
ture and then heated to reflux for 90 min. Diethyl ether was
distilled off and swapped with benzene. This freshly prepared
diethylcadmium was then mixed with [1-14C]benzoyl chloride in
benzene and stirred for 14 h at room temperature. The reaction
was cooled in an ice bath and 6 mL of 20% v/v H2SO4/H2O (cold)
was added. The mixture was extracted with pentane (2 Â 15 mL).
The combined extracts were washed with water (10 mL),
saturated NaHCO3 (15 mL), brine (20 mL), dried over Na2SO4,
filtered, and concentrated in vacuo to 4 mL. Purification by silica
gel chromatography packed and eluted with 96:4 pentane:ether
gave 412 mg, 175 mCi of product.
5-trifluoromethylpyridine-2-sulfonyl chloride (10): A suspension
of 5-(trifluoromethyl)pyridine-2-thiol (9) (1.5 g, 8.0 mmol) in HCl
solution (1.0 N, 40 .0 mL) was stirred at 01C in an ice bath.
Chlorine gas was then bubbled slowly for 90 min. The resulting
white suspension was extracted with methylene chloride. The
combined extracts were washed with a saturated solution of
NaHCO3, dried over MgSO4, filtered and concentrated in vacuo
at room temperature to give 1.83 g of a white solid in 89% yield,
1-[3-Nitrophenyl]-1-[1-14C]propanone ([14C]-4): The above com-
pound (412 mg, 3.07 mmol, 175 mCi) was cooled to À451C and
concentrated HNO3 (4 mL) was added. The resulting yellow
solution was stirred for 1 h at À45 to À351C. The reaction was
then mixed with ice chips and warmed to room temperature
and extracted with ether (2 Â 15 mL). The combined extracts
J. Label Compd. Radiopharm 2008, 51 314–320
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B. Latli et al.
which was kept at À801C. H NMR (CDCl3): d 8.26(d, J = 8.22 Hz, analysis indicated that [14C]-tipranavir contained about 4% of
1
1H), 8.32(dd, J = 2.16 Hz, 8.22 Hz, 1H), 9.08(d, J = 2.16 Hz, 1H).
N-[3-{1(R)-(5,6-dihydro-4-hydroxy-2-oxo-6(R)-phenethyl-6-pro-
pyl-
dine-2-sulfonamide ([14C]-tipranavir): A solution of (6R)-3-[(1R)-1- 9.83 min for the (1S, 6S)-enantiomer (PNU141275). The diaster-
(3-aminophenyl)-1-[1-14C]-propyl]-4-hydroxy-6-phenyl-6-propyl-
eomer (1R, 6S) and (1S, 6S) have the same retention times
the (1S, 6R)-diastereomer, Rt = 8.17 min, and negligible amounts
of the other diastereomers. The enantiomer (1S, 6S) was easily
-2H-pyran-3-yl)-[1-14C]propyl}phenyl]-5-trifluoromethylpyri- separated from tipranavir, Rt = 7.17 min for tipranavir and
5,6-dihydro-2H-pyran-2-one (30 mCi, specific activity = 54 mCi/ (9.83 min).
mmol, 0.55 mmol) N,N-dimethylaniline (80.0 mL, 0.628 mmol),
2,6-di-tert-butyl-4-methylphenol (25.0 mg, 0.112 mmol) in
Synthesis of [13C6]-tipranavir
CH2Cl2 (3.0 mL), and water (40.0 mL) were stirred at 01C in
an ice bath. 5-Trifluoromethyl-2-pyridinesulfonyl chloride
(155.5 mg, 0.63 mmol) was then added in one portion and the
reaction was stirred at this temperature until deemed complete
by TLC. After the workup the ethyl acetate extracts were dried
over MgSO4, filtered, and concentrated in vacuo. The residue
was purified by flash chromatography silica gel (packed in
hexane) and eluted with ethyl acetate. The product was isolated
in 67% radiochemical yield (225 mg, 20.6 mCi) as a fluffy white
solid. A specific activity of 54 mCi/mmol was calculated by
weighing 736 mg of material into a vial and dissolving it in
1.22 mL of absolute ethanol to make a 1.0 mM solution. Aliquots
of 1, 2 and 5 mL were then taken, diluted in 5.0 mL of LSC Ready
Safe cocktail, and counted. The total and specific activities were
calculated from the average radioactivity per mL. For radioactive
TLC, an aliquot of 2.0 mL of this 1.0 mM solution was spotted on
a 20 cm TLC plate and developed using 30% EtOAc:Hexanes or
5% MeOH/CHCl3. UV visualization showed the labeled tipranavir
and the reference standard co-migrated. The plate was dried,
coated with an acrylic clear film, and re-dried under a stream of
nitrogen. The plate was then scanned using Bioscan. HPLC: Rad-
detection, Rt = 19.30 min (98.1%); UV detection, Rt = 19.10 min
(98.98%). Because of the instability of this material, it was mixed
with 181.74 mg of cold tipranavir in ethyl acetate (5.0 mL). Most
of the solvent was then removed under a stream of nitrogen
and the residue was purified by flash chromatography to give
329 mg of a white solid, Rf = 0.25 in 40% EtOAc:Hexanes. The
specific activity was counted from a solution of 0.538 mg of
[14C]-tipranavir in 0.892 mL of ethanol. The new specific activity
was 28.86 mCi/mmol. For 1H NMR, a sample of 13 mg was taken
and dissolved in deuterated methanol (300 mL). The solution was
placed into a Teflon tube liner (Wilmad, catalog. No. 6005-7). The
tube was sealed with a Teflon plug and then inserted into a
screw-cap NMR tube (Wilmad catalog No. 507-TR-8, OD
5.0 mm)). This double encapsulation was necessary to avoid
contamination from the accidental breakage of the NMR tube.
1H NMR (MeOH-d4): d: 0.82(t, J = 7.32 Hz, 3H), 0.88(t, J = 7.25 Hz,
3H), 1.28–137 (m, 2H), 1.61–1.98(m, 5H), 2.10–2.16(m, 1H),
2.50–2.68(m, 4H), 3.90(dd, J = 7.12, 9.45 Hz, 1H), 6.96–6.93(m,
1H), 6.97–7.02(m, 2H), 7.03–7.07(m, 2H), 7.10–7.15(m, 1H), 7.15-
7.25(m, 3H), 8.01(d, J = 8.21 Hz, 1H), 8.20(dd, J = 2.5, 8.21 Hz, 1H),
8.94(d, J = 2.5 Hz, 1H). 13C NMR (MeOH-d4): d:13.28, 14.67, 17.88
25.75, 30.86, 37.87, 40.49, 40.85, 43.62, 81.81, 106.19, 120.87,
122.57, 124.12, 125.63, 126.09, 129.24, 129.51, 137.00, 137.71,
142.83, 147.61, 148.06, 161.59, 166.88, 169.87.
1-[13C6]Phenyl-1-propanone ([13C6]-3): To a solution of benzoic
acid-13C6 (1.0 g, 7.8 mmol) in benzene (5 mL) was added thionyl
chloride (3.1 mL, mmol) and the resulting was stirred under
reflux for 5 h. After cooling to room temperature, the solution
was concentrated in vacuo and the residue was dissolved again
in benzene (5 mL) and concentrated under reduced pressure to
chase excess of thionyl chloride. The remaining colorless oil was
diluted in anhydrous methylene chloride (5 mL) and added at
À501C to a suspension of AlCl3 (1.04 g, 7.8 mmol) in methylene
chloride (7 mL). The resulting was warmed gradually to À301C in
1 h period. A solution of diethyl zinc in toluene (4.3 mL, 1.1 M)
was added dropwise in 45 min period and the resulting mixture
was warmed to room temperature and stirred for 2 h. Water
(30 mL) was added and the mixture was extracted with
methylene chloride. The combined extracts were dried over
MgSO4, filtered, and concentrated in vacuo to give 3.0 g of a
colorless oil. This oil was purified by flash chromatography to
give 0.88 g of colorless oil in 81% yield from benzoic acid. MS:
MH1 = 141 as the only peak.
1-[13C6][3-Nitrophenyl]-1-propanone ([13C6]-4): To the above
compound (627 mg, 4.48 mmol) was added cold 90% nitric acid
(7 mL) at À251C. The resulting yellow solution was warmed to
À101C and stirred for 30 min. Ice water (15 mL) was then added
dropwise. The resulting solid was filtered, washed with cold
water, and dried under high vacuum at room temperature to
give 450 mg of the desired product in 56% yield. MS ESI1): MH1
= 186 (100%).
(6R)-4-Hydroxy-3-[(E)-1-[13C6]-(3-nitophenyl)-1-propenyl]-6-phe-
nethyl-6-propyl-5,6-dihydro-2H-pyran-2-one ([13C6]-6): To a stirred
solution of the above compound (450 mg, 2.51 mmol) and (5)
(850 mg, 3.26 mmol) in dry THF (10 mL) and pyridine (0.5 mL,
6.28 mmol) under nitrogen atmosphere cooled to À101C was
added a solution of TiCl4 in toluene (1 M , 5 mL). The resulting
dark red solution was stirred at À101C for 30 min, then warmed
to room temperature and stirred for 16 h. The reaction mixture
was cooled to 01C and water (10 mL) was added dropwise. The
mixture was partitioned with ethyl acetate (10 mL), and the
aqueous was extracted with ethyl acetate (20 mL). The
combined organic extracts were washed with aqueous HCl
(2 N, 20 mL), water (20 mL), brine (20 mL), dried over Na2SO4,
filtered, and concentrated in vacuo. The residue was dissolved in
toluene (10 mL) and heated with stirring for 3 h at 601C to
equilibrate the geometric isomers. Toluene was then distilled off
and the residue was purified by silica gel chromatography to
give 695 mg of the desired product in 65% yield. MS (ESI): MH1
= 428.3 (100%).
Stereochemical purity of [14C]-tipranavir
(6R)-4-Hydroxy-3-[(E)-1-[13C6]-(3-nitophenyl)-1-propyl]-6-phe-
The three other diastereomers were obtained from the nethyl-6-propyl-5,6-dihydro-2H-pyran-2-one ([13C6]-7): A mixture
Analytical department for HPLC comparison. Solutions of of the above material (695 mg, 1.65 mmol) and K2CO3 (40 mg,
1.0 mg/mL of each compound were prepared using the mobile 0.29 mmol) in nitrogen purged methanol (3 mL) was placed in a
phase as the solvent (acetonitrile: triethylamine: acetic acid in 100 mL stainless steel Parr bomb apparatus and securely closed.
1000:1:6 v/v/v) for better separations of the diasteromers. HPLC The Parr was purged with hydrogen, and then charged to 70 psi
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Copyright r 2008 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2008, 51 314–320

B. Latli et al.
and heated with stirring to 571C for 20 min. The pressure was crude residue was purified using the Combiflash Companion
released and 14 mg of [((R,R)-Me-DuPHOS)Rh(COD)]BF4 was (40 g column, 2% MeOH/ CH2Cl2) giving 700 mg, 71% yield as
added via the addition port as a solution in nitrogen purged beige foam.
methanol (0.5 mL). The bomb was again purged with hydrogen
5-Trifluoromethyl-pyridine-2-sulfonic acid hydroxy-{3-[(R)-1-((R)-
and then charged to 80 psi and heated with stirring at 571C for 4-hydroxy-2-oxo-6-phenethyl-6-propyl-5,6-dihydro-2H-pyran-3-yl)-
4 h. The pressure was released and the contents of the flask propyl]-phenyl}-amide (12): 5-Trifluoromethyl-pyridine-2-sulfonyl
were transferred to a round bottom flask using methanol. The chloride (30 mg, 0.12 mmol) in MeOH (2 mL) was added to the
methanol was then removed under reduced pressure and the above compound (50 mg, 0.12 mmol) and pyridine (50 mL) in
residue was partitioned between CH2Cl2 (25 mL) and aqueous MeOH (2 mL) at 01C. The mixture was stirred for 1 h then
HCl (2 N, 25 mL). The CH2Cl2 layer was removed and washed concentrated. The mixture was diluted with CH2Cl2, quenched by
with water (20 mL), brine (20 mL), dried over Na2SO4, filtered, addition of 0.5 N HCl, extracted with CH2Cl2, dried over Na2SO4
and concentrated in vacuo to give 715 mg in 81% yield. MS (ESI): and concentrated giving a colorless oil. The crude product was
MH1 = 430.3 (100%).
purified by preparative HPLC (Waters Masslynx, 15 to 100%
(6R)-3-[(1R)-1-[13C6]-(3-Aminophenyl)-1-propyl]-4-hydroxy-6-phe- MeCN/H2O in 10 min). Collected only m/z 619, the combined
nethyl-6-propyl-5,6-dihydro-2H-pyran-2-one ([13C6]-8): A stirred fractions were concentrated with an E-Z evaporator giving (12)
solution of the above compound (715 mg, 1.65 mmol) and (23 mg, 35%) as an off white solid. LRMS m/z 619 (M1). H NMR
1
10% Pd–C (20 mg) in nitrogen purged methanol (5 mL) was (d6-DMSO) d: 0.76 (t, J = 7.5, 3H), 0.84 (t, J = 7.5, 3H), 1.25 (m, 4H),
stirred under hydrogen at atmospheric pressure at room 1.74 (m, 1H), 1.63 (m, 2H), 1.86 (m, 2H), 1.99 (m, 1H), 2.57 (m, 2H),
temperature. On completion the mixture was filtered through 3.82 (dd, J = 6.5, 9, 1H), 7.00 (m, 1H), 7.08 (s, 1H), 7.15 (m, 5H), 7.26
a short pad of Celites and washed with methanol. The filtrate (m, 2H), 8.01 (d, J = 8.01 Hz, 1H), 8.52 (dd, J = 2.5, 8.01 Hz, 1H), 9.08
13
was concentrated under reduced pressure to give 649 mg of (s, 1H), 10.73 (s, 1H), 11.23 (s, 1H). C NMR (d6-DMSO) a: 12.7,
¨
material in 100% yield, which was used without further 14.3, 16.4, 24.3, 29.2, 36.8, 41.4, 79.5, 120.7, 122.1, 125.7, 125.8,
purification. MS (ESI): MH1 = 400.3 (100%).
[N-(3-((1R)-1-[(6R)-4-Hydroxy-2-oxo-6-phenethyl]-6-propyl-5,6-di- 165.9.
126.9, 127.5, 128.1, 128.4, 136.1, 141.6, 145.5, 146.7, 155.3, 164.9,
hydroxy-2H-pyran-3-yl]propyl}[13C6]phenyl)-5-trifluormethyl)-2-pyr-
(2S,3S,4S,5R)-3,4,5-Trihydroxy-6-((R)-6-oxo-2-phenethyl-2-propyl-
idiesufonamide ([13C6]-tipranavir): To a stirred solution of the 5-{ (R)-1-[3-(5-trifluoromethyl-pyridine-2-sulfonylamino)-phenyl]-
above material (649 mg, 1.65 mmol) in dry CH2Cl2 (10 mL) and propyl}-3,6-dihydro-2H-pyran-4-yloxy)-tetrahydro-pyran-2-carboxylic
pyridine (307 uL, 3.88 mmol) under nitrogen at À101C was acid (13): A mixture of acetobromo-a-D-glucuronic acid methyl
added 5-trifluoromethyl-2-pyridinesulfonyl chloride (467 mg, ester (1.0 g, 2.5 mmol), tipranavir (2.3 g, 3.8 mmol), and Ag2O
1.9 mmol). The reaction was allowed to warm to room (880 mg, 3.8 mmol) in MeCN (15 mL) was stirred over night at
temperature and stirred for 3 h. The reaction was then quenched room temperature. Then 1 N NaOH (15 mL) was added and the
by the addition of aqueous HCl (2 N, 0.4 mL), and extracted with mixture was stirred for 1 h. The mixture was filtered (Celites),
CH2Cl2 (2 Â 25 mL). The combined extracts were washed with then acidified with 3 N HCl. The mixture was extracted with
brine (25 mL), dried over Na2SO4, filtered, and concentrated in EtOAc, dried over Na2SO4 and concentrated giving yellow oil.
vacuo. The residue was purified by silica gel chromatography The crude oil was fractionated by filtration through a plug of
using 35% ethyl acetate: hexane. The fraction containing the silica; elution first with (75% EtOAc/Hexane) then with (20%
pure material were combined and concentrated in vacuo. The MeOH/ CH2Cl2) gave 230 mg (12% yields) as white solid. A
residue was crystallized from ethyl acetate/hexane in three days fraction (75 mg) was further purified by preparative HPLC
at À181C and the white solid was filtered, washed with cold (Waters Masslynx, 15–100% MeCN/H2O in 10 min). Collected
hexane, and dried under high vacuum at room temperature to only m/z 778, the combined fractions were concentrated with an
give 426 mg of material with a chemical purity of 97.8% at E–Z evaporator giving (13) (30 mg) as a white solid as a mixture
1
254 nm by HPLC. MS (ESI1): MH1 = 609.1 and MNa1 = 631.38 of a and b epimers. LRMS m/z 779 (M1). H NMR (d6-DMSO) d:
(100%), MS (ESI-): MÀ = 607.2. 1HNMR (d6-DMSO): d: 0.78(t, 0.70 (t, J = 7.5, 3H), 0.78 t, J = 7.5, 3H), 1.24 (m, 4H), 1.58 (m, 2H),
J = 7.32 Hz, 3H), 0.89(t, J = 7.22 Hz, 3H), 1.4(m, 2H), 1.8(m, 4H), 1.81 (m, 3H), 2.00 (m, 1H), 2.48 (m, 1H), 2.78 (d, J = 18, 1H), 2.83
2.01(m, 1H), 2.6(m, 4H), 3.8(m, 1H), 6.55–7.4(m, 9H), 8.2(d, (d, J = 18, 1H), 3.32 (m, 3H), 3.40 (t, J = 9, 1H), 3.87 (d, J = 9.5, 1H),
J = 8.25 Hz, 1H), 8.50(d, J = 8.3 Hz, 1H), 9.02(s, 1H), 10.7(s, 2H). 13
C
3.93 (t, J = 7.5, 1H), 5.09 (d, J = 7.5, 1H), 5.31 (d, J = 4, 1H), 5.50 (d,
NMR (d6-DMSO): d:12.64, 14.19, 16.32, 24.15, 29.17, 35.85, 40.52, J = 5, 1H), 6.91 (m, 1H), 7.06 (m, 4H), 7.12 (m, 1H), 7.16 (m, 1H),
41.85, 79.34, 103.87, 117.35(t, Jc-c = 57 Hz), 120.4(t, Jc-c = 53 Hz), 7.24 (m, 2H), 8.14(d, J = 8.5, 1H), 8.43 (dd, J = 2.1, 8.5, 1H), 9.12 (s,
123.86(t, Jc-c = 48.67 Hz), 128.20(t, Jc-c = 62.5 Hz), 135.81(t, Jc- 1H), 10.61 (brs, 1H). 13C NMR (d6-DMSO) d: 12.6, 14.2, 16.1, 23.9,
c = 55.59 Hz), 146.06(t, Jc-c = 51.18 Hz),160.5, 164.61, 165.84.
29.3, 31.7, 41.5, 71.1, 72.8, 75.3, 76.1, 80.2, 98.4, 110.5, 118.1,
120.4, 121.5, 122.9, 124.2, 124.7, 125.7, 127.5, 127.8, 128.4, 136.6,
141.4, 144.9, 147.2, 159.7, 163.5, 164.7, 169.9.
Synthesis of the metabolites
(R)-4-Hydroxy-3-[(R)-1-(3-hydroxyamino-phenyl)-propyl]-6-phe-
nethyl-6-propyl-5,6-dihydro-pyran-2-one (11): KBH4 (180 mg,
Conclusion
3.5 mmol) was added to a mixture of (7) (1.0 g, 2.4 mmol) and [14C]-Tipranavir was synthesized in seven steps with a specific
BiCl3 (150 mg, 0.48 mmol) in 25% H2O/EtOH at 151C. After activity of 54 mCi/mmol. However, due to its instability the
stirring for 15 min, a second portion of KBH4 (180 mg, 3.5 mmol) specific activity was diluted to half. The synthesis included a
and BiCl3 (150 mg, 0.48 mmol) was added. After stirring an Kneovenagel type condensation of (R)-4-Hydroxy-6-phenethyl-6-
additional 15 min, the mixture was filtered (Celites), quenched propyl-5,6-dihydro-2-pyran-2-one with 1-(3-nitrophenyl-1-pro-
by addition of 1% HCl. The mixture was extracted with CH2Cl2, panone and an asymmetric hydrogenation that gave 94% ee.
dried over Na2SO4 and concentrated giving a yellow oil. The A total of 245 mg of more than 98% chemical and radiochemical
J. Label Compd. Radiopharm 2008, 51 314–320
Copyright r 2008 John Wiley & Sons, Ltd.
www.jlcr.org

B. Latli et al.
pure material were obtained after flash chromatography
purification. Carbon-13 labeled tipranavir was prepared in seven
steps using the same route and starting from [13C6]-benzoic
acid. The two potential metabolites found when tipranavir was
administered with ritonavir, the hydroxyl-metabolite and the
glucuronide conjugate were prepared to help in confirming the
identities of those metabolites.
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Acknowledgement
We thank our colleagues Scott Leonard, Scott Campbell, and
Dr. Fenghe Qiu for help in obtaining NMR and mass spectra.
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J. Label Compd. Radiopharm 2008, 51 314–320