A convenient synthesis of 13C4-leflunomide and its primary metabolite 13C4-A77 1726

Article abstract of DOI:10.1002/jlcr.701

A convenient synthesis of leflunomide and its primary pharmacologically active metabolite A77 1726, each labeled with four ¹³C atoms has been achieved. Starting from ¹³C₄-ethyl acetoacetate, each step of the synthesis proc

Full text of DOI:10.1002/jlcr.701

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 613–622.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.701
Research Article
A convenient synthesis of 13C4-Leflunomide and its
primary metabolite C4-A77 1726
13
Robert J. Faragher1, John. M. Motto1, Maciej A. Kaminski2 and
Adrian L. Schwan1,*
1 Department of Chemistry and Biochemistry, University of Guelph, Guelph, ON,
Canada, N1G 2W1
2 Health and Pharmaceutical Services International, P.O. Box 21012 R.P.O.
Meadowvale, Mississauga, ON, Canada, L5N 6A2
Summary
A convenient synthesis of leflunomide and its primary pharmacologically
active metabolite A77 1726, each labeled with four 13C atoms has been
achieved. Starting from 13C4-ethyl acetoacetate, each step of the synthesis
proceeds in 60–82% yield. Analysis of the mass spectra of the labeled and
unlabeled materials demonstrates that the compounds prepared herein will be
suitable as analytical standards for bioavailability studies. Copyright # 2003
John Wiley & Sons, Ltd.
Key Words: leflunomide; metabolite; A77 1726; stable isotopes; synthesis; 13C
labeling
Introduction
Leflunomide is a recently approved disease modifying antirheumatic
drug (DMARD) for the treatment of rheumatoid arthritis.1–6 The
drug’s primary metabolite A77 1726 is believed to be the active
ingredient7–9 which possesses a mode of action involving the inhibition
of pyrimidine synthesis and the consequent suppression of inflamma-
*Correspondence to: A. L. Schwan, Department of Chemistry and Biochemistry, University of
Guelph, Guelph, ON, Canada, N1G 2W1. E-mail: schwan@uoguelph.ca
Copyright # 2003 John Wiley & Sons, Ltd.
Received 14 October 2002
Revised 8 January 2003
Accepted 31 January 2003

614
R. J. FARAGHER ET AL.
tion. Leflunomide’s clinical efficacy rivals that of methotrexate and
salazopyrene yet elicits a faster onset of response.10 Furthermore, unlike
methotrexate,11,12 leflunomide does not result in bone marrow
toxicity.2,4 The adverse effects of leflunomide have been summarized
as mild to moderate and can be easily resolved.3,13
This paper outlines the preparation of leflunomide and its active
metabolite A77 1726, each labeled with four 13C atoms. The use of 13C
labeled drugs is well established as a reliable method for establishing
drug pharmacokinetics.14–16 Moreover, in this particular instance, the
incorporation of an additional four mass units in analytical standards of
the labeled drug and its metabolite ensures sufficient mass differentia-
tion when compared with unlabeled material, thus establishing its
usefulness for HPLC/MS analysis in bioavailability studies.
Results and discussion
Given the common synthesis of leflunomide,17,18 the choice of 13C
atoms as the stable isotope labels is the preferred option as many of the
available sites for deuterium labels are enolizable at one or more steps
during the synthesis. The use of 13C atoms precludes this problematic
issue and since ethyl acetoacetate, a common precursor to the isoxazole
ring of leflunomide17,18 is commercially available with four 13C atoms,
the principal requirement for the chosen synthesis is conveniently in
place.
As shown in Scheme 1, the 13C4-ethyl acetoacetate (1) was treated
with ethyl orthoformate to install the ethoxymethylene unit of 2 (79%).
The distilled material (2) was then reacted with hydroxylamine
hydrochloride which was neutralized in situ. Instead of using crude
isoxazole 3 directly in the subsequent step, purification by distillation
was performed. Even though this distillation was performed only on a
small (3–4 g) scale using a short-path apparatus equipped with a 10 cm
Vigreux column, isoxazole 3 was still isolated in 77% yield. Acidic
hydrolysis of the ester component of 3 was essentially quantitative
affording crude acid 4. Toluene recrystallization gave pure 4 in 60%
yield; some decomposition of the material during the recrystallization
process was observed.
Conversion of acid 4 to its acid chloride was performed in a typical
manner by reaction with thionyl chloride. At this stage the acid chloride
is usually isolated and purified.19, 20 In this study however, it was found
Copyright # 2003 John Wiley & Sons, Ltd.
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13C4-LEFLUNOMIDE AND 13C4-A77 1726
615
that direct reaction with 4-trifluoromethylaniline (6) proved most
efficient. Thus the reaction of crude acid chloride 5 with aniline 6 and
sodium bicarbonate in a two-phase mixture of toluene and water21 was
carried out. The wet, white solid which precipitated from the reaction
mixture was filtered out and dissolved in ethyl acetate. The resulting
solution was dried with magnesium sulfate and evaporated to dryness.
Recrystallization from toluene afforded labeled leflunomide (7) in 60%
yield starting from the acid. The purity of the labeled leflunomide was
established by HPLC to be 99.8%.
Conversion of leflunomide to its metabolite A77 1726 was performed
by treating leflunomide 7 with methanolic sodium hydroxide
solution. Stirring the reaction mixture for 2 h at room temperature
resulted in the complete conversion of 7 to the ring opened metabolite 8.
Acidification of the mixture yielded a white precipitate. Recrystalliza-
tion of this material from toluene afforded labeled A77 1726 in 82%
yield. The metabolite was established to be 99.8% pure by HPLC
analysis.
The identity of all the compounds in the reaction scheme was
confirmed by spectral analysis and comparison of their physical
properties with published data. 13C NMR spectroscopy proved to be
particularly diagnostic. For instance, carbon chemical shifts were fully
consistent with expectations. Most telling was the chemical shift
migration of carbon-4 of 7 at 112 ppm to 81 ppm in 8, a change
consistent with the loss of isoxazole aromaticity and the resulting
increase of electron donation from the hydroxy group to shield carbon-
4. The 13C,13C coupling constants were also consistent with predictions.
13
The 13Csp3
,
C
coupling constant involving the labeled methyl group
sp2
was consistently in the 43–50 Hz range. Also, once the isoxazole ring
was formed, the two internal carbons of the four carbon isotope linkage
13
13
uniformly exhibited a
C
,
sp2
C
sp2
coupling constant of 70–71 Hz.
Overall the consistency of the 13C–13C coupling constants for the
whole set of compounds proves that the backbone structure of the four
labeled carbon atoms remained intact through the entire series of
reactions, as presented in Scheme 1. It should be noted that the four 13
C
labels and particularly the presence of one of the labels on a methyl
group opens the possibility that compounds 7 and 8 may prove suitable
for the investigation of pharmacokinetic problems using NMR
spectroscopy.22
To establish the value of the isotopically labeled compounds for
bioavailability studies, their mass spectra must be compared to those of
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 613–622

616
R. J. FARAGHER ET AL.
Scheme 1.
the unlabeled analogues. Commercial leflunomide was converted to its
active metabolite, i.e. the unlabeled A77 1726 using conditions identical
to those described for the synthesis of the labeled material. The mass
spectra of each of these materials were recorded and are presented in the
experimental section. A close inspection of the mass spectra of the
unlabeled compounds revealed that there are no significant peaks
present at m/z>272. To complement this, compounds 7 and 8 do not
exhibit peaks in the m/z=268–272 range that are distinguishable from
the spectrum noise. Thus the labeled compounds 7 and 8 meet all
the necessary criteria to be excellent candidates for bioavailability
studies.
Copyright # 2003 John Wiley & Sons, Ltd.
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13C4-LEFLUNOMIDE AND 13C4-A77 1726
617
Conclusions
A simple, multistep synthesis of 13C4-leflunomide (7) is presented.
Furthermore, labeled leflunomide 7 can readily be converted to its
labeled metabolite 13C4-A77 1726 (8). The procedure affords gram
quantities of these substances in >99.8% purity. The mass spectra of
the labeled standards demonstrate that the introduction of the
additional four mass units is sufficient to ensure their proper
differentiation from their unlabeled analogs during the HPLC/MS
analysis stage of bioavailability studies.
Experimental
Melting points were measured with a Thomas Hoover melting point
apparatus and are uncorrected. IR spectra were recorded on a Bomem
1
FT-IR spectrometer. H NMR (400 MHz) and 13C NMR (100.6 MHz)
spectra were recorded on a Bruker Avance 400 MHz NMR Spectro-
1
meter. H NMR spectra are reported relative to TMS. 13C NMR
spectra which were run in CDCl3 were referenced to the solvent peak at
77.0 ppm. 13C NMR spectra which were run in DMSO-d6 were
referenced to the solvent peak at 39.7 ppm. Mass spectra were obtained
with a VG ZAB-R instrument located at the McMaster Regional Center
for Mass Spectrometry. HPLC analyses were performed on a Waters
400 instrument equipped with a Jones Chromatography Ltd. Genesis
4.6 ꢀ 250 mm C18 reversed phase column. The mobile phase consisting
of methanol:water in the ratio of 80:20 was pumped at a rate of 1 ml/
min. A UV detector set at 254 nm was used. [1,2,3,4-13C4]-Ethyl
acetoacetate (99% 13C) was purchased from Isotec, Inc. All other
chemicals and reagents were purchased from Fisher or Aldrich.
Reactions were carried out under dry nitrogen.
13C4-Ethyl 2-(ethoxymethylene)-3-oxo-butanoate (2)
To a flame dried, 25 ml round-bottomed flask equipped with a reflux
condenser, were added ethyl acetoacetate (5.08 g, 37.9 mmol), ethyl
orthoformate (6.30 ml, 37.9 mmol) and freshly distilled acetic anhydride
(7.14 ml, 75.7 mmol) via syringe. The flask was placed into an oil bath
and refluxed for 60 min. After cooling to r.t., the solution was
concentrated under vacuum and the product was distilled to afford
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R. J. FARAGHER ET AL.
5.68 g (79%) of a mixture of E/Z isomers of 2 as an orange oil
possessing ca. 90% purity (by NMR); bp: 60–668C (0.3 mm). Spectral
data for 2: 1H NMR (400 MHz, CDCl3), mixture of isomers, d: 7.50, (m,
1 H), 4.13–4.02 (m, 4 H), 2.38–1.95 (m, 3 H), 1.24–1.09 (m, 6 H); 13C
NMR (100.6 MHz, CDCl3), major isomer, d: 194.3 (ddd,
1J
¼ 56:4 Hz, 1J
¼ 43:8 Hz, 2J
13C;13C
164.5 (dd, J
13C;13C
13C;13C
¼ 3:4 Hz, ketone C),
1
2
13C;13C
13C;13C
¼ 3:4 Hz, ester C), 114.3 (ddd,
¼ 76:2 Hz, J
1J
¼ 76:2 Hz, J
¼ 56:4 Hz, J
1
2
13C;13C
carbonyls), 72.2 (s, CH2), 59.8 (s, CH2), 27.6 (dd, J
13C;13C
13C;13C
¼ 13:8 Hz, C between
1
13C;13C
¼ 43:8 Hz,
2J
¼ 13:8 Hz, CH3C(O)), 14.8 (s, CH2CH3), 13.7 (s, CH2CH3),
13C;13C
anticipated peak near 164 ppm obscured by labeled carbons; 13C NMR
1
13C;13C
(100.6 MHz, CDCl3), minor isomer, d: 196.1 (dd, J
¼ 54:6 Hz,
1J
¼ 43:1 Hz, (anticipated J
¼ 1:9 Hz not resolved), ketone
2
13C;13C
13C;13C
1
2
13C;13C
13C;13C
¼ 1:9 Hz, ester C), 113.1
C), 164.8 (dd, J
(ddd, 1J
¼ 77:6 Hz, J
¼ 77:6 Hz, 1J
between carbonyls), 72.2 (s, CH2), 59.9 (s, CH2), 31.1 (dd,
¼ 54:6 Hz, 2J
¼ 13:7 Hz, C
13C;13C
13C;13C
13C;13C
1J
¼ 43:1 Hz, J
¼ 13:7 Hz, CH3C(O)), 14.7 (s, CH2CH3),
13C;13C
2
13C;13C
13.7 (s, CH2CH3); anticipated peak near 164 ppm obscured by labeled
carbons.
13C4-Ethyl 5-methyl-4-isoxazolecarboxylate (4)
To a 25 ml round-bottomed flask were added ester 2 (5.40 g, 28.4 mmol)
and ethanol (6 ml) and the mixture was cooled to 08C. To the reaction
mixture was added, via a dropping funnel a solution of hydroxylamine
hydrochloride (2.17 g, 31.2 mmol) and sodium acetate trihydrate (4.31 g,
31.2 mmol) dissolved in water (8 ml). The mixture was stirred overnight
at 08C and was extracted with EtOAc (50 ml, 2 ꢀ 25 ml) and the
combined organic extracts were dried over Na2SO4. After filtration and
concentration under vacuum, the residue was fractionally distilled
(10 cm Vigreux column) to afford 3.46 g (77%) of ester 3 as a colourless
1
oil; bp: 35–368C (0.2 mm). Spectral data for 3: H NMR (400 MHz,
2
3
CDCl3), d: 8.38 (dd, J
& J
¼ 7:7 & 3.7 Hz, ring H), 4.24 (dq,
13C;H
13C;H
3J
1J
¼ 3:2 Hz;
3JH;H ¼ 7:1 Hz,
CH2CH3),
2.61
(ddd,
13C;H
2
3
¼ 131:3 Hz; J
& J
¼ 7:1 & 2.3 Hz, ring CH3), 1.28
13C;H
13C;H
13C;H
(t, JH;H ¼ 7:1 Hz, CH2CH3); 13C NMR (100.6 MHz), d:172.6 (ddd,
3
1
2
1J
¼ 71:4 Hz; J
¼ 49:9 Hz; J
¼ 89:3 Hz; J
¼ 7:4 Hz, ring C a to
13C;13C
13C;13C
13C;13C
1
2
O), 160.9 (dd, J
¼ 7:4 Hz, ester C), 149.4 (d,
13C;13C
13C;13C
1J
2J
¼ 52:4 Hz), 109.0 (ddd, J
¼ 4:8 Hz, ring C bearing carbonyl), 60.1 (s, CH2CH3), 14.0 (s,
¼ 89:3 Hz; J
¼ 71:4 Hz;
13C;13C
1
1
13C;13C
13C;13C
13C;13C
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13C4-LEFLUNOMIDE AND 13C4-A77 1726
619
CH2CH3), 11.7 (dd, 1J
¼ 49:9 Hz; 2J
13C;13C
13C;13C
¼ 4:8 Hz, CH3 on
ring).
13C4-5-Methylisoxazole-4-carboxylic acid (4)
To a 25 ml round-bottomed flask equipped with a reflux condenser,
were added ester 3 (3.02 g, 19.0 mmol) and 1:1:1 mixture of concentrated
HCl, water and acetic acid (10 ml) and the solution was refluxed for
9.5 h. After cooling, acetone (10 ml) was added and the mixture was
concentrated under vacuum to give a yellow solid. Recrystallization
from toluene yielded 1.49 g (60%) of pure acid 4, mp: 143.5–1458C; lit.23
1
mp of unlabeled analog, 147–1498C. Spectral Data for 4: H NMR
2
3
(400 MHz, DMSO-d6), d: 9.41 (br s, acid H), 8.76 (dd, J
& J
13C;H
13C;H
¼ 7:8 & 3.7 Hz, ring H), 2.62 (ddd, 1J
¼ 131:2 Hz; 2J
&
13C;H
13C;H
3J
¼ 7:1 & 2.4 Hz, CH3); 13C NMR (100.6 MHz, DMSO-d6), d:
13C;H
1
1
2
174.0 (ddd, J
¼ 70:3 Hz; J
¼ 49:4 Hz; J
¼ 7:3 Hz,
13C;13C
13C;13C
13C;13C
ring C a to O), 162.9 (dd, 1J
¼ 85:2 Hz; 2J
¼ 50:3 Hz), 110.1 (ddd, 1J
¼ 7:3 Hz, acid C),
13C;13C
13C;13C
151.0 (d, 1J
¼ 85:3 Hz;
13C;13C
13C;13C
1J
¼ 70:3 Hz; J
¼ 4:7 Hz, ring C bearing carbonyl), 12.3
2
13C;13C
13C;13C
1
1
13C;13C
(dd, J
13C;13C
¼ 4:7 Hz, CH3).
¼ 49:4 Hz; J
Preparation of 13C4-5-methyl-N-[4-(trifluoromethyl)phenyl]-4-Isox-
azolecarboxamide (13C4-leflunomide, 7)
Following a method previously published,21 to a flame dried 10 ml
round-bottomed flask was added acid 4 (1.46 g, 11.2 mmol) and an
excess of freshly distilled thionyl chloride (4.23 ml) and the mixture was
stirred at 508C for 4 h. After cooling the mixture was concentrated in
vacuo (50 mm) until constant mass was achieved, to afford crude acid
chloride 5. To a vigorously stirred mixture of NaHCO3 (0.984 g,
11.7 mmol) and p-trifluoromethylaniline (6) (1.89 g, 11.7 mmol), toluene
(20 ml) and water (4.1 ml) stirring vigorously at 608C was added over a
period of 20 min. neat acid chloride 5. After stirring for 2 h at 608C the
mixture was allowed to cool overnight. The white solid which
precipitated was filtered and was washed with water. The material
was dissolved in EtOAc (50 ml) to make a solution which was washed
with water (15 ml) and dried over MgSO4. Filtration and concentration
under vacuum afforded a crude solid which was recrystallized from
toluene to afford 2.07 g (60%) of 7 as a white solid; mp. 165–1668C;
1
lit.24mp of unlabeled analog, 166–1678C. Spectral data for 7: H NMR
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620
R. J. FARAGHER ET AL.
(400 MHz, CDCl3), d: 10.32 (s, NH), 9.07 (dd, 2J
& 3J
13C;H
13C;H
¼ 8:5 &
3.8 Hz, ring H), 7.90 (d, 3JH;H ¼ 8:6 Hz, Ar H), 7.68 (d, 3JH;H ¼ 8:6 Hz,
1
2
3
13C;H
13C;H
13C;H
¼ 7:1 & 2.1 Hz,
Ar H), 2.62 (ddd, J
¼ 131:2 Hz; J
& J
CH3); 13C NMR (100.6 MHz, DMSO-d6), d: 173.6 (ddd,
1J ¼ 71:4 Hz; 1J ¼ 49:5 Hz; 2J
¼ 6:6 Hz, C-5), 159.8
13C;13C
13C;13C
13C;13C
1J
¼ 77:5 Hz;
2J
C-7),
3
149.2
(d,
13C;13C
13C;13C
¼ 6:6 Hz,
(dd,
1J
13C;13C
13C;F
¼ 49:5 Hz, C-3), 142.3 (s, C-8)), 126.2 (q, J
¼ 3:7 Hz, C-
¼ 22:0 Hz, C-
¼ 77:5 Hz;
10)), 124.6 (q, 1J
¼ 271:3 Hz, C-12)), 124.1 (q, 2J
¼ 1:8 Hz, C-9)), 112.0 (ddd, J
13C;F
13C;F
2
1
13C;F
13C;13C
11), 120.2 (q, J
1J
1J
¼ 71:4 Hz;
¼ 49:5 Hz; 2J
1J
C-4),
12.4
(dd,
13C;13C
13C;13C
13C;13C
¼ 4:2 Hz,
13C;13C
¼ 4:2 Hz, C-6); IR (nujol), cmꢁ1: 3329,
3290, 3069, 1652, 1604, 1538, 1532, 1456, 1329, 1318, 1257, 1163, 1123,
1111, 1063, 854; MS (EI), m/z: 274 (M+, 22), 273 ((M-1)+, 8), 161 (5),
114 (1 0 0), 113 (28), 70 (7). HRMS: Calculated for C183C4H9N2O2F3,
274.0750; Found, 274.0730. HPLC retention time: 6.85 min.
Mass Spectrum of unlabelled leflunomide. MS (EI), m/z: 271
((M+1)+, 14), 270 (M+, 26), 161 (5), 110 (1 0 0), 109 (19), 85 (14).
Conversion of 7 to 13C4-2-cyano-3-hydroxy-N-[4-(trifluoromethyl)phe-
nyl]-2-butenamide (13C4-A77 1726, 8)
To a 25 ml round bottomed flask were added labeled leflunomide 7
(1.30 g, 4.81 mmol) and MeOH (50 ml). The stirred solution was cooled
to 108C and an aqueous 1.1 M NaOH solution (5 ml) was added.
Stirring continued for 2 h at which time water (5 ml) was added. The
solution was slowly brought to acidic pH through the slow addition of
concentrated HCl resulting in a formation of a white precipitate. The
precipitate was collected via suction filtration and was recrystallized
from toluene to afford 1.07 g (82%) as a 15:1 (approximately) mixture of
tautomers, mp. 216.5–2188C; lit.25mp of unlabeled analog, 230–2338C.
1
Spectral data for 8: H NMR (400 MHz, DMSO-d6, major (enol)
tautomer), d: 11.00 (s, v br, 1 H), 10.69 (s, 1 H), 7.76 (d, 3JH;H ¼ 8:6 Hz,
3
1 H), 7.65 (d, JH;H ¼ 8:6 Hz, 1 H), 2.25 (ddd, 1J
¼ 129:0 Hz;
13C;H
2J
&
3J
¼ 5:9 Hz & 3.3 Hz, CH3); 13C NMR (100.6 MHz,
13C;H
13C;H
1
1
DMSO-d6), d: 187.1 (dd, J
¼ 73:9 Hz; J
¼ 46:2 Hz, C-5),
13C;13C
13C;13C
1
3
166.6 (dd, J
126.0 (q, 3J
¼ 72:1 Hz; J
¼ 2:8 Hz, C-7), 141.7 (s, C-8),
13C;13C
13C;13C
¼ 3:6 Hz, C-10), 124.5 (q, 1J
¼ 271:3 Hz, C-12), (q,
13C;F
13C;F
2J
1J
1J
¼ 32:0 Hz, C11), 121.3 (q, 2J
¼ 1:7 Hz, C-9), 118.3 (d ,
13C;F
13C;F
1
¼ 87:7 Hz, C-3), 81.1 (ddd (apparent dt), J
¼ 72:1 Hz; 2J
¼ 73:9 Hz;
13C;13C
13C;13C
¼ 6:9 Hz, C-4), 23.1 (ddd, 1J
¼
13C;13C
13C;13C
13C;13C
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13C4-LEFLUNOMIDE AND 13C4-A77 1726
621
46:2 Hz; 2J
¼ 6:9 Hz; 3J
13C;13C
¼ 2:8 Hz, C-6); IR (DMSO-d6,
13C;13C
mixture of tautomers), cmꢁ1: 3459, 3334, 3193, 3041, 2979, 2179,
1596, 1523, 1496, 1411, 1329, 1310, 1258, 1165; MS (EI),
m/z: 274 (M+, 21), 273 ((M-1)+, 5), 161 (1 0 0), 160 (26), 114 (20),
111 (12), 70 (27). HRMS: Calculated for C183C4H9N2O2F3, 274.0750;
Found, 274.0717. HPLC retention time: 3.18 min.
Mass Spectrum of unlabelled A77 1726. MS (EI), m/z: 272 ((M+2)+,
4) 271 ((M+1)+, 21), 270 (M+, 33), 161 (1 0 0), 160 (24), 142 (10), 110
(7), 68(8).
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J Label Compd Radiopharm 2003; 46: 613–622