Carbon-14-and carbon-13-labeled phosphoric acid [2-4-(4-cyanophenyl)- thiazol-2-yl]-(2,4-difluorophenyl)-1-[1,2,4]triazol-4-yl-methylpropoxymethyl] monoester dilysine salt, a prodrug of ravuconazole

Article abstract of DOI:10.1002/jlcr.1879

4-Bromobenzoic acid [carboxyl-¹⁴C] and 4-(2-bromoacetyl) [Ar-¹³C₆]benzonitrile were transformed into the title compounds containing [ring¹⁴C-thiazol-4-yl] and [Ar- ¹³C₆-benzonitrile].

Full text of DOI:10.1002/jlcr.1879

Research Article
Received 15 September 2010,
Revised 24 December 2010,
Accepted 29 January 2011
Published online 26 April 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1879
Carbon-14- and carbon-13-labeled phosphoric
acid [2-4-(4-cyanophenyl)-thiazol-2-yl]-
(2,4-difluorophenyl)-1-[1,2,4]triazol-4-yl-
methylpropoxymethyl] monoester dilysine
salt, a prodrug of ravuconazole
Ã
I. Victor Ekhato and J. Kent Rinehart
4-Bromobenzoic acid [carboxyl-14C] and 4-(2-bromoacetyl) [Ar-13C6]benzonitrile were transformed into the title compounds
containing [ring14C-thiazol-4-yl] and [Ar-13C6-benzonitrile]. 14C-Ravuconazole was prepared in 37% yield and Purity 499%.
13C6-Ravuconazole was made in 56% overall yield and Purity of 498%. Each labeled compound was converted by
additional reaction steps to the corresponding labeled prodrug.
Keywords: antifungal agents; 14a-demethylase; ravuconazole; prodrug; phosphoric acid dilysine salt
a rare metabolic conversion of ravuconazole nitrile functionality
into a thiocyanate.6 The incorporation of carbon-14 in the
Introduction
Phosphoric acid [2-4-(4-cyanophenyl)-thiazol-2-yl]-(2,4-difluoro-
phenyl)-1-[1,2,4]triazol-4-yl-methylpropoxymethyl] monoester
dilysine salt 1 (BMS-379224) in Figure 1 is a prodrug of
ravuconazole.1 Ravuconazole 2 (BMS-207147), licensed to BMS
from Eisai Co. Ltd. (ER-30346), is an inhibitor of lanosterol 14a-
demethylase activity, a P-450-dependent monooxygenase and a
key enzyme in the biosynthesis of ergosterol.2 Ergosterol is
essential in fungi for cell viability and proliferation. Ravucona-
zole has been demonstrated by in vivo and animal model
experiments to be more potent against aspergillosis, candidiasis
and cryptococcosis than clinically useful amphotericin B, and the
newer azole antifungals like fluconazole and intraconazole.3 It is
orally active, shows broad spectrum activity and has a generally
favorable safety profile. BMS is further studying this agent to
develop intravenous (iv) formulation for the treatment of
systemic fungal infection. For neutropenic and immunocom-
promised patients who become seriously ill and are not able to
tolerate oral administration, the parenteral administration of an
antifungal agent is critical. In light of the superior antifungal
activity, successful clinical development of an iv formulation of
ravuconazole is highly desirable for this class of patients. A
dilysine salt of the phosphoric acid prodrug is attractive since
lysine and phosphate residues are readily cleared, unlike with
cyclodextrins as carrier4 and would not accumulate in the
presence of renal insufficiency. We have made the radio-isotope
thiazole moiety is intended to avert loss of the label by this rare
metabolic route. After examining the synthetic literature on
ravuconazole we decided that the thiazole unit could be
constructed from readily accessible labeled reagents.7 To make
the stable labeled prodrug a more advanced intermediate
bearing [Ar-13C6]benzonitrile was preferred since it would afford
species with a sufficient molecular mass increase that overlap
with other ions resulting by isotopic mass distribution is
avoided. Once obtained, labeled ravuconazole could be taken
through some additional steps to provide the radio and stable
labeled prodrug targets. In the following section we wish to
describe the synthesis of labeled versions of the dilysine salt of
phosphoric acid prodrug of ravuconazole.
Experimental
All reactions were carried out under an atmosphere of argon
unless otherwise specified. Solvents were commercial grade and
used without purification or drying. Column chromatography
was carried out on Merck Kiesegel 60 (230 m) silica gel. Flash
chromatographic separations were performed on a Biotage
Flash System using pre-packed silica gel cartridges. TLC
visualization reagents included (10% iodine plus 10% AcOH) in
and stable labeled versions of the prodrug 1 BMS 379224 Department of Chemical Synthesis, Bristol-Myer Squibb Company, Rt. 206/
Provinceline Rd, Princeton, NJ 08374, USA
to support further preclinical pharmacokinetics characteri-
zation. Our plan called for the radio-label to be placed in
*Correspondence to: I. Victor Ekhato, Department of Chemical Synthesis, Bristol-
Myer Squibb Company, Rt. 206/Provinceline Rd, Princeton, NJ 08374, USA.
the thiazole ring instead of making [benzonitrile-14CN] or
f[U-14C]benzonitrileg variant.5 Studies in our laboratory showed E-mail: ihoezo.ekhato@bms.com
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Copyright r 2011 John Wiley & Sons, Ltd.

I. V. Ekhato and J. K. Rinehart
Figure 1. Prodrug and parent ravuconazole, targets of the labelling sequence.
40% aqueous KI, and cerium sulfate in 10% sulfuric acid. 1 H NMR 50 mL round bottom flask equipped with a reflux condenser and
spectra were recorded at 300, 400 or 500 MHz. Chemical shifts are argon inlet was added 15 mL of anhydrous dimethylformamide.
given in ppm relative to tetramethylsilane (TMS). Agilent 1100 The reaction vessel was repeatedly evacuated and filled with
HPLC System including solvent degasser, pump, automated argon to eliminate air. A solution of 4 (1.0 g, 5.02 mmol) in DMF
injector, and a variable UV detector connected to IN/US BetaRam (5 mL) was added to make a total reaction volume of 20 mL, and
model 4 Flow Detector with a 0.25 mL detector cell was used in the the degassing process was repeated. The mixture was stirred at
analyses of compounds. The HPLC column was Waters Xterra RP18, 961C overnight under argon atmosphere, cooled to room
3m, 4.6ꢀ 150 mm, PN 18600004. Elution Solvent System A = Water temperature and concentrated to a residue. The residue was
10.05% TFA and B = Acetonitrile10.05% TFA and compound diluted with toluene (30 mL), and the solution was washed with
eluted under gradient condition with detection by UV at 254 nm 3 N ammonium hydroxide solution (2 ꢀ 20 mL), brine (20 mL)
and 280 nm. LC/MS analysis was performed on a Finnigan LXQ and dried over MgSO4. The solution was evaporated to provide
Mass Spectrometer System, LC/MS method: Generic-B20 (1p ESI 5 (680 mg, 93 mCi, 93%). 1H-NMR (400 MHz, CDCl3),d 2.63 (s, 3 H),
full mass). Only the molecular ion and/or base peaks in MS are 7.76 (d, J = 8.5 Hz, 2H), 8.03 (d, J = 8.5 Hz, 2H). HPLC with Waters
given. 4-Bromobenzoic acid [14C-carboxyl] was purchased from Xterra 4.6 ꢀ 150 mm column RP18 3.5 micron, elution solvent
ViTrax, Placentia, CA, USA and was diluted with ‘cold’ 4- A = Water10.05 TFA; B = acetonitrile10.05% TFA, 0–12 min 10%
bromobenzoic acid from Aldrich Chemical Company before it B, 12–17 min 90%, 17–20 min 10% B, Flow rate 1 mL/min, at UV
was used, and 40-bromoaceto[Ar-13C6]phenone was custom 254 and 270 nm gave retention time of 4.4 min.
synthesized by Isotec Inc., and supplied through Aldrich Chemicals.
40-(2-Bromoacetyl) [carbony ꢁ14 C]-benzonitrile 6
1-(4-bromophenyl) [carbonyl ꢁ14 C]ethanone 4
Catalytic AlCl3, 5 mg, was added to a solution of 4-acetylbenzoni-
4-Bromobenzoic acid [14C-carboxyl] 3 (1.005 g, 5 mmol, 100 mCi)
was stirred in anhydrous toluene (20 mL), oxalyl chloride (1.308 mL,
15 mmol) followed by catalytic DMF were added. After 1 h the
reaction solution was concentrated under reduced pressure, and
further co-evaporated with toluene to give a solid product. A
solution of this material in CH2Cl2 (20 mL) was added at ꢁ51C to
N,O-dimethylhydroxylamine hydrochloride (634 mg, 6.3 mmol) in
dichloromethane (30 mL) containing pyridine (1.158 mL,
14.31 mmol), and stirred for 1 h allowing it to warm to room
temperature. The reaction mixture was diluted with chloroform,
(30 mL), and washed with water, (30 mL), 1 N hydrochloric acid,
(30 mL), brine (20 mL) and dried. Evaporation gave an amide
(1.260 g, 5 mmol), and to a solution of this material in dry THF
20 mL at ꢁ51C was added 3.0 M solution of methylmagnesium
iodide (5 mL, 15 mmol). The reaction was stirred at room
temperature overnight, quenched with diluted hydrochloric acid,
1.0N, 20mL and extracted with ether (3ꢀ 30 mL). The ethereal
extract was washed with 1 N HCl (2 ꢀ 20 mL), brine, (20 mL) and
dried over anhydrous sodium sulfate. The solution was evaporated
to yield 4 (1.0 g, Equant). 1H-NMR (400 MHz, CDCl3), d 2.57 (s, 3H),
7.57 (d, J= 8.5Hz, 2H), 7.79 (d, J=8.5Hz, 2H). 13C-NMR (100.61 MHz,
CDCl3) 196.9, 135.8, 131.8, 129.8, 128.2 and 26.
trile [carbonyl-14C] 5 (680mg, 4.68mmol) in ethyl acetate (20 mL)
at 51C. Bromine (239mL, 4.68mmol) in ethyl acetate was added
under an atmosphere of argon, and the reaction was stirred for a
further 30min at room temperature. HPLC indicated 65:35 ratio of
the desired compound to putative dibromo contaminant. After
the reaction was quenched with 10% solution of sodium
thiosulfate, the solution was washed with 20mL of water,
20mL of brine and dried on sodium sulfate. A solution of the
crude product in dichloromethane was applied to a column of
silica gel, and the unwanted dibromo compound was eluted with
10% ether in pet ether. Further elution of the column with 25%
ether in pet ether gave 6 (516mg, 49.2%). 1H-NMR (400MHz,
CDCl3), d 4.42 (s, 3H), 7.99 (d, J = 8.5 Hz, 2H), 8.07 (d, J = 8.5 Hz, 2H).
13C-NMR (100.61 MHz, CDCl3) 190.0, 136.9, 132.6, 129.4, 117.6,
117.1 and 30. HPLC with Waters Xterra 4.6 ꢀ 150 mm column RP
18; elution solvent A = water10.05% TFA, B = Acetonitrile10.05
TFA, gradient condition, 0–12min 30–90% B, 12–16min, 90–30%
B, Flow rate mL/min at UV detection wavelength of 254 and
270 nm to give retention time of 6.9 min.
4-{2-[2-(2,4-Difluoro-phenyl)-2-hydroxy-1-methyl-3-
[1,2,4]triazol-4-yl-propyl]-}-[14C]thiazol-4-yl-benzonitrile,
7
14C BMS-207147
4-Acetyl[carbonyl ꢁ14 C]benzonitrile 5
A mixture of 4-(2-bromoacetyl)benzenenitrile [carbonyl-14C]) 4
(516 mg, 2.30 mmol) and BMS-226630 (768.6 mg, 2.46 mmol) in
anhydrous methanol (10 mL) was refluxed for 1.5 h under argon
To
0.201 mmol) and zinc cyanide (353.72 mg, 3.01 mmol) in a
tetrakis(triphenylphospine)palladium(0)
(232.28 mg,
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Copyright r 2011 John Wiley & Sons, Ltd.
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I. V. Ekhato and J. K. Rinehart
atmosphere. After cooling to room temperature, the reaction rate of mL/min gave retention time 8.2 min, chemical purity
mixture was diluted with 36 mL of dichloromethane, and 146 mg 498.0% and radiochemical purity499%.
of NaHCO3 followed by 20 mL of water. The aqueous portion
was separated, and was further extracted with dichloromethane 40-Acetyl [Ar ꢁ13 C6]benzonitrile 11
(2 ꢀ 15 mL). The combined organic extract was washed with
Zn(CN)2 (1.768 g, 15.06 mmol) and tetrakis(triphenylphospine)-
20 mL of water, 20 mL of brine, dried over MgSO4 and
palladium(0) (1.610 g, 1.34 mmol), 4-(2-bromoaceto[Ar-13C6])-
evaporated to a residue. The residue was purified by column
phenone 10 (5.0 g, 25.11 mmol) under the reaction conditions
described for 3 gave crystaline 11 (3.5 g, 95%). 1H-NMR
chromatography on silica gel. Elution of the column with 1–2%
methanol in dichloromethane gave pure fractions that were
combined, evaporated to a solid which crystalized from ethyl
(400 MHz, CDCl3), d 8.04 (dm, JC–H = 164 Hz, JC–C = 13.0 Hz, 2H),
7.75 (dm,
J
C–H = 164 Hz, JC–C = 13.0 Hz, 2 H). 13C-NMR
acetate-hexane to give 7 14C-BMS-207147, (634.6 mg, 63%,
36.83 mCi, specific activity 58.04 mCi/mg or 25 mCi/mmol). 1H-
NMR (400 MHz, CDCl3), d 8.01 (d, J = 8.2 Hz, 2H), 7.83 (s, 1H), 7.73
(d, J = 8.2 Hz, 1H), 7.68 (s, 1H), 7.63 (s, 1H), 7.50 (m, J = 6.4 Hz,
J = 6.4 Hz, J = 3.1 Hz, 1H), 6.80 (m, 1H), 5.72 (s, 1H), 4.90 (d,
J = 14.4 Hz, 1H ), 4.25 (d, J = 14.4 Hz, 1H), 4.07 (q, J = 7.3 Hz, 1H),
1.22 (d, J = 7.3 Hz, 2H). Chemical purity 499% and radiochemical
purity 499.8% at retention time 5.7 min as authentic BMS-
207147-01.
(125.77 MHz, CDCl3) 116.4 (td, 1JC–C = 1JC–C = 60 Hz, 2JC–C = 6.0 Hz,
1
2
CCN), 128.8 (td, JC–C = 1JC–C = 65.8 Hz, JC–C = 6.6 Hz, 2CH), 132.7
(td, 1JC–C = 1JC–C = 65 Hz, 2JC–C = 6.6 Hz, 2CH), 140.1 (td,
1JC–C = 1JC–C = 58 Hz, JC–C = 8.6 Hz, CCO). HPLC (condition as in
5) gave retention time 4.4 min.
2
4-(2-Bromoacetyl) [Ar ꢁ13 C6]benzonitrile 12
Phenyltrimethylammonium tribromide (8.27 g, 22.0 mmol) was
added portion-wise over 10min to a stirred solution of 11 (3.20g,
21.17 mmol) in 20 mL of dry THF. The pale solution was stirred
over 20min during which a heavy precipitate was formed. Ice-cold
water (30mL) was added and the solid was separated by filtration,
washed with water and dried under high vacuum overnight. It was
crystalized from hexane to give 12 (3.32 g, 68.1%). 1H-NMR
(400MHz, CDCl3), d 8.10 (dm, JC–H = 158.9Hz, JC–C = 13.0 Hz, 2H),
7.82 (dm, JC–H = 161.5 Hz, JC–C = 13.0 Hz, 2H). 13C-NMR (125.77 MHz,
Phosphoric acid mono-[2-4-(4-cyano-phenyl)-[14C]-thiazol-2-
yl]-1-(2,4-difluoro-phenyl)-1-[1,2,4]triazol-4-ylmethyl-pro-
poxymethyl]ester dilysine 12, BMS-379224-04
14C-BMS-207147 7 (466 mg, 27.04 mCi, 1.05 mmol)) in dry THF
(5 mL) was added slowly to a suspension of sodium hydride
(60% dispersion, 130 mg, 3.2 mmol) in dry THF (6 mL). Next, a
solution of iodine (134 mg, 0.529 mmol, 0.5 eq) in dry THF (3 mL)
was added, and stirred at 171C for 30 min. BMS-371000 (330 mL,
1.26 mmol) was added neat, and the mixture was stirred at room
temperature overnight. After the reaction mixture was cooled in
ice-bath, 4 mL of water was added followed by toluene, (15 mL),
stirred briefly and allowed to settle. The aqueous phase was
discarded and the organic portion was washed repeatedly with
5 mL portions of brine until it became a clear solution. The
1
2
CDCl3) 117.10 (td, JC–C = 1JC–C = 60 Hz, JC–C = 9.6Hz, CCN), 128.8
(td, JC–C = 1JC–C =58.2Hz, JC–C = 8.3 Hz, 2CH), 132.7 (td, JC–C
=
1
2
1
1JC–C = 56.2 Hz, 2JC–C = 6.6Hz, 2CH), 140.1 (td, 1JC–C = 1JC–C = 58.2 Hz,
2JC–C = 9.5Hz, CCO). HPLC (condition as in 6) retention time
6.9 min.
4-{2-[2-(2,4-Difluoro-phenyl)-2-hydroxy-1-methyl-3-[1,2,4]
triazol-4-yl-propyl]-thiazol-4-yl}-[Ar ꢁ13 C6]benzonitrile, 13
solution was concentrated under reduced pressure at o301C to 13C6 BMS-207147
give 8 as a creamy white solid. The material 8 was taken up in
Compound 12 (2.72 g, 11.8 mmol) and BMS-226630 (3.98 g,
CH2Cl2 (8 mL), cooled in ice bath and trifluoroacetic acid 2.4 mL
was added. After 10 min at room temperature, the solution was
concentrated under reduced pressure, and further co-evapo-
rated with toluene at o201C to afford the crude product. In a
solution of minimum methanol, the compound was applied to a
C18 silica gel column that had been equilibrated phosphate
buffer pH 7.0. The column was eluted first with 10% methanol in
water, and then by step gradient elution with 20, 40, 60%
methanol in water. Fractions containing the pure desired
compound were combined and concentrated by rotary eva-
porator to give a solid. To the material in warm (551C) methanol
(4 mL) was slowly added with stirring, a solution of lysine (2 eq)
in water/MeOH, 2 mL (0.5/1.5). The reaction was cooled to
ꢁ101C and the solid which separated was collected by filtration,
washed with 2% water in methanol and dried at 501C under
vacuum overnight to give 9 (395 mg, 43.8%, 6.7 mCi, 17 mCi/mg,
14.2 mCi/mmol based on Mol Wt 839.85). 1H-NMR (400 MHz,
DMSO-d6), d 9.12 (s, 1), 8.0 (d, 2H), 7.94 (s, 1H), 7.83 (s, 1H), 7.72
(d, 1H), 7.22 (q, 1H), 6.91 (m, 1H), 6.79 (m, 1H), 5.42 (tr, 1H), 5.35
(q, 1H ), 5.24 (tr, 1H), 3.76 (q, 1H), 3.46 (m, 3H), 2.86 (tr, 6H), 1.83
(m, 3H), 1.75 (m, 3H), 1.63 (m, 6H), 1.49 (m, 8H). HPLC with
Waters Xterra MS C8 4.6 ꢀ 150 mm column, gradient condition
14.8 mmol) as described for 5, gave after crystalization from
ethyl acetate-hexane, 13 (892 mg, 53%). 1H-NMR (400 MHz,
CDCl3), d 8.20 (m, 1H), 7.95 (m, 1H), 7.83 (s, 1H), 7.80 (m, 1H), 7.68
(s, 1H), 7.63 (d, 1H), 7.51 (m, 2H), 6.80 (m, 2H), 5.72 (s, 1H), 4.91
(d, 1H), 4.24 (d, 1H), 4.07 (q, 1H) and 1.22 (d, 3H). MS (EI) m/z
444.2 [M1H]1, 375, 224. 13C-NMR (125.77 MHz, CDCl3) 111.6
(td, 1JC–C = 1JC–C = 60.1 Hz, 2JC–C = 10.5 Hz, CCN), 126.7 (td, 1JC–C
=
=
2
1
1JC–C = 59.1 Hz, JC–C = 8.5 Hz, 2CH), 132.69 (td, JC–C = 1JC–C
60.1 Hz, JC–C = 8.6 Hz, 2CH), 137.8 (td, JC–C = 1JC–C = 58.2 Hz,
2
1
2JC–C = 10.5 Hz, CC = C). HRMS (EI) [M1H]1 444.1390
(12C C6H17F2N5OS requires 444.1401). Isotopic distribution [M1
13
16
6] 98 13C6-atom% for 12C C6H17F2N5OS; [M15] 2.0 13C5 atom% for
13
16
12 12
16
C C13C5H17F2N5OS; [M10]o0.01 atom% for C22H17F2N5OS.
HPLC with Xterra 4.6ꢀ 150 mm column, gradient condition and
detection at UV wavelength of 254 and 270nm, flow rate of 1 mL/
min gave same retention time 13.1min as authentic BMS-207147-
01 (070D3U-001-01A) and chemical purity of 499.2%.
Phosphoric acid [2-4-(4-cyano-[Ar ꢁ13 C6]phenyl)-thiazol-2-
yl]-1-(2,4-difluoro-phenyl)-1-[1,2,4]triazol-4-ylmethyl-
propoxymethyl] monoester dilysine salt 15 13C6 BMS-379224
0–12 min, 25–85% B, and 12–18 min, 85–25% B, where A = 0.1 M From sodium hydride 60% dispersion in oil (137.15 mg,
aq NH4OAc/0.01 M NH4OAc in MeOH (80:20) and B = 0.1 mmol 3.42 mmol), 13 (500 mg, 1.14 mmol), iodine (145.04 mg,
aq NH4OAc/0.01 M NH4OAc in MeOH/MeCN (5:20:70) at a Flow 0.571 mmol), BMS-371000 (353.87 mg, 354 uL, 1.36 mmol), lysine
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I. V. Ekhato and J. K. Rinehart
(340.15 mg, 2.28 mmol), as in experiment 9 gave compound 15 prodrug, and Scheme 3 summarizes an analogous preparation
1
(650 mg, 67.8%). H-NMR 400 MHz, DMSO-d6), d 9.12 (s, 1), 8.19 of 13C6-ravuconazole and the corresponding prodrug. 4-
(m, 1H), 7.95 (s, 1H), 7.88 (m, 1H), 7.83 (s, 1H), 7.58 (q, 1H), 7.26 Bromobenzoic acid [14C-carboxy] 3 is our starting reagent for
(q, 1H), 6.91 (m, 1H), 6.81 (m, 1H), 5.46 (tr, 1H), 5.35 (q, 2H ), 5.28 the radiolabeled ravuconazole. We decided based on earlier
(tr, 1H), 3.43 (tr, 3H), 2.85 (tr, 6H), 1.83 (m, 3H), 1.75 (m, 3H), 1.63 work7 to go through the sequence that includes the reaction of
(m, 6H), 1.49 (m, 8H). H PLC with Waters Xterra MS C8 4-(2-bromoacetyl[14C-carbonyl])benzonitrile 6 with BMS-226630
4.6 ꢀ 250 mm column, gradient 0–16 min, 25–85% B, and to form 4-{2-[2,4-difluoro-phenyl)-2-hydroxy-1-methyl-3-[1,2,4]
16–18 min, 85–25% B, where A = 0.1 M aq NH4OAc/0.01 M triazol-4-yl-propyl]-[14C]thiazol-4-yl-benzonitrile 7. The com-
NH4OAc in MeOH (80:20) and B = 0.1 mmol aq NH4OAc/0.01 M pound 6 was prepared from 3 by DMF-catalyzed exchange
NH4OAc in MeOH/MeCN (5:20:70) at a flow rate of mL/min gave reaction with oxalyl chloride to make 4-bromo[14C-carboxy]-
retention Time of 8.9 min and chemical purity 498.7%.
benzoyl chloride. 4-Bromo-N-methoxy-N-methylbenzamide was
generated from it according to the Weinreb’s protocol,8 and
subsequently treated with methylmagnesium iodide to give
4-bromoaceto[carboxyl 2-14C]phenone 4. Palladium-catalyzed
Results and discussion
Scheme 1 describes the preparation of 14C-ravuconazole, cyanation of 4 with Zn(CN)2 at 951C under argon atmosphere9
Scheme illustrates its conversion to carbon-14-labeled overnight gave 5 in 86% yield from 4-bromobenzoic acid.
2
Scheme 1. (i) a. (COCI)2, cat. DMF, Toluene, 231, 1 h; b. Me(MeO)NH. HCI, pyr, CHCI3 0 to 231C, 2 h; c. MeMgl, THF, ꢁ781C, 18 h, quant; (ii) 4 mol % Pd(PPh3)4, Zn(CN)2 DMF,
801C, 18 h, 93 %; (iii) cat, AICI3, Br2, EtOAc, 0.5 h, 231C, 49%; (iv) BMS-226630, MeoH, reflux, 1.5 h, 63%.
Scheme 2. (v) NaH, I2, THF, 171C, 30 min, BMS-37100, 231C, o.n; (vi) CH2CI2, TFA, 10 min 231C, C18 Column Isolation, 43.8% 2 steps.
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I. V. Ekhato and J. K. Rinehart
Scheme 3. (vii) 4 mol % Pd(PPh3)4, Zn(CN)2, 801C, DMF, 95 %; (viii) (C6H5N1(CH3)3Brꢁ3 ), THF, 20 min 231C, 89%, (ix) BMS-226630, MeoH, Reflux, 53 %; (v) and (viii) see
Schemes 1 & 2.
We found the bromination of 5, catalyzed by AlCl3, to be
problematic. An unexpectedly high amount (35%) of 40-(2,2-
Conclusion
dibromoacetyl)-benzonitrile was formed along with the desired
4-(2-bromoacetyl)-benzonitrile 6. While a major weakness in
the radio-labeling sequence, the impurity is easily removed by
flash chromatography on silica gel column. Compound 6
was refluxed in methanol containing BMS-226630 to give 7 in
63% purified yield. Owing to the variable yield we observed
during the method development we changed the method for
transforming BMS-207147 into BMS-379224 to achieve en-
hanced and reproducible yield. Accordingly, the sodium salt
of 7 was reacted with BMS-371000, in the presence of I2
overnight, to make trialkyl phosphonate ester, 8.1 A non-
extraction workup was adopted to isolate the monoester in the
subsequent de-protection step. After the solvent was evapo-
rated the residue was subjected to a step gradient reverse
phase chromatography. This procedure gave reproducible yields
of the acid monoester. Formation of the dilysine salt of the
monoester was completed by adding aqueous solution of lysine
in methanol at 551C to give 9 14C BMS-379224 in 64% yield.
The problematic bromination step encountered during the
carbon-14 synthesis was solved in the stable label synthesis by
substituting, as a superior brominating agent, phenyltrimethyl-
In summary, we have successfully prepared a version of
ravuconazole containing [14C]-thiazole moiety in four steps
from 4-bromo[carboxy-14C]benzoic acid. By a similar sequence, a
version of ravuconazole bearing [Ar-13C6]benzonitrile was made.
These radio-isotope and stable labeled ravuconazole analogs
were respectively transformed into the dilysine salt of the
phosphoric acid monoester prodrug. Our decision to incorporate
carbon-14 in the thiazole ring system was based on considera-
tions of metabolic stability and cost.
Acknowledgements
I. V. E. gratefully acknowledges the Department of Chemical
Syntheses for support and helpful resources; Albert DelMonte,
Chien K. Chen, and Eric D. Dowdy, Chem. Process; BMS for
the supply of BMS-226630-02 and BMS-371000; and Yasutsugu
Ueda (BMS, Wallingford, CT) for useful discussions about the
synthesis.
References
ammonium tribromide (C6H5N1(CH3)3Brꢁ).10 Beginning from
3
4-(2-bromoaceto-[Ar-13C6])phenone 10 the nitrile 11 was made
in495% yield. This was followed by bromination with
phenyltrimethylammonium tribromide to afford 12 in 89%
yield. The putative dibromide was formed in about 10%, leading
overall, to an improved yield of 13 13C6-BMS 207147. In a
sequence of reactions similar to the radiosynthetic route, the
compound 13 was treated with BMS-371000 to make the
trialkylphosphate, deprotected with TFA to the phosphoric acid
monoester. It was isolated in the same manner and converted to
the dilysine salt 15 13C6 BMS-379224 in 68% yield. Where
possible these compounds were matched with authentic
reference samples by HPLC and TLC techniques and gave
analytical results consistent with the assigned structures.
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J. Label Compd. Radiopharm 2011, 54 357–362