Synthesis of stable isotopically labelled versions of Lamotrigine and its methylated metabolite

Article abstract of DOI:10.1002/jlcr.590

Lamotrigine is a sodium channel antagonist used for the treatment of epilepsy. Synthesis of stable isotopically labelled (SIL) [M + 7] versions of Lamotrigine (1) and its N-methylated metabolite (2) are described. The routes to prepare these compounds used [M + 5] labelled [¹³C, ¹⁵N₄]-aminoguanidine (obtained from labelled thiourea). The overall yield for the metabolite (2) was 34% from [M + 3] labelled [¹³C, ¹⁵N₂]-thiourea. Copyright

Full text of DOI:10.1002/jlcr.590

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 611–618.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.590
Research Article
Synthesis of stable isotopically labelled
versions of lamotrigine and its methylated
metabolite
Calvin O Manning*, Alan H Wadsworth and Ian Fellows
Chemical Development, GlaxoSmithKline Research and Development,
Stevenage, Hertfordshire, SG1 2NY, UK
Summary
Lamotrigine is a sodium channel antagonist used for the treatment of epilepsy.
Synthesis of stable isotopically labelled (SIL) [M+7] versions of Lamotrigine
(1) and its N-methylated metabolite (2) are described. The routes to prepare
these compounds used [M+5] labelled [13C, 15N4]-aminoguanidine (obtained
from labelled thiourea). The overall yield for the metabolite (2) was 34% from
[M+3] labelled [13C, 15N2]-thiourea. Copyright #2002 JohnWiley& Sons, Ltd.
Key Words: lamotrigine; mass label; aminoguanidine
Introduction
Cl
Cl
Cl
Cl
N
N
N
N
CH3
NH2
N
N
H2N
NH2
HN
Lamotrigine (1)
(2)
*Correspondence to: C. O. Manning, Chemical Development, GlaxoSmithKline Research,
Stevenage, Herts, SG1 2NY, UK. E-mail: cm64908@gsk.com
Copyright # 2002 John Wiley & Sons, Ltd.
Received 26 February 2002
Accepted 11 March 2002

612
C. O. MANNING ET AL.
Lamotrigine (1) is a sodium channel antagonist marketed as an
anticonvulsive agent for the treatment of epilepsy. To enable the
quantification of unlabelled parent compound (1) and its N-2
methylated metabolite (2) in biological fluids, mass labelled versions
were required for use as internal standards in the mass spectrometric
assays. The two chlorine atoms present in the compounds under study
meant that an additional seven mass units were required to be
incorporated into the SIL versions. This was to ensure absence of
interfering molecular ions arising from the natural isotope distribution
pattern.
Results and discussion
The SIL versions of Lamotrigine (1) and its N-2 methylated metabolite
(2) were synthesized as outlined in Schemes 1, 2 and 3. This allowed
both compounds to be prepared using the same route and labelled
starting materials.
Commercially available 2,3-dichlorobenzoic acid (3) was converted in
to acid chloride (4) and further reacted without isolation with copper
[13C, 15N]-cyanide (11) to yield [M+2]-2,3-dichlorobenzoylcyanide (5).
The key step involved a condensation of [M+5]-aminoguanidine (14)
with [M+2]-2,3-dichlorobenzoylcyanide (5). In order to maximise
incorporation of the more valuable [M+5]-aminoguanidine sulfate
(14) four equivalents of the [M+2]-benzoyl cyanide (5) were used to one
of [M+5]-aminoguanidine sulfate (14). The resulting product (Z)-2-
(2,3-dichlorophenyl)-2-(guanidinoimino)acetonitrile (6) was subse-
quently cyclized in refluxing propan-1-ol to obtain [M+7]-Lamotrigine
(7). There was a small quantity (3% by HPLC) of the (E)-isomer of (6)
present prior to the cyclization. This does not cyclize and remained
unchanged but was readily removed by chromatography over silica gel.
(M+7)-Lamotrigine (7) was methylated with iodomethane to yield the
metabolite. The resulting iodide salt was neutralised by slurrying the
solid in concentrated aqueous ammonia solution, followed by filtration
to give the free base (8), which was converted into the desired mesylate
salt (9).
Copper (I) [13C, 15N]-cyanide (11) was prepared as shown in Scheme 2
and was obtained from [13C, 15N]-potassium cyanide (10) using copper
sulfate and sodium metabisulfite as described by Young.1
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 611–618

SYNTHESIS OF STABLE ISOTOPICALLY LABELLED
Cl
613
Cl
Cl
Cu13C15N (11),
KI, Xylene
144oC
Cl
O
Cl
O
Cl
(COCl)2
OH
13C 15N
Cl
CH2Cl2, DMF
76% (two steps)
O
(4)
(5)
(3)
15NH2
H2SO4, H2O,
MeCN
H15
N
.0.5 H2SO4
13C
H15
N
15NH2
77%
(14)
Cl
Cl
Cl
Cl
15N
15N
Reflux
60%
15N
13C
15N
13C
13C
13C
H125
N
15N
15N
15NH2
15NH2
H125
(6)
N
(7)
(1) MeI, Acetone
(2) aq NH3
Cl
Cl
Cl
Cl
MeSO3H,
MeOH, EtOAc
15N
15N
15N
CH3
CH3
15N
13C
15N
13C
77%
(two steps)
13C
13C
15N
H15
N
H15
N
15NH2
15NH2
.MeSO3H
(9)
(8)
Scheme 1. Synthesis of SIL (Lamotrigine)
[13C, 15N4]-Aminoguanidine (14) was prepared as shown in Scheme 3.
[13C, 15N2]-Thiourea (12) was treated with 0.5 equivalents of dimethyl-
sulfate in water at reflux to obtain [M+3]-S-methyl isothiouronium
sulfate (13).2 Further reaction with [15N2]-hydrazine liberated methane-
thiol and gave [M+5]-aminoguanidine (14).3 The product was isolated
by removal of volatile material under vacuum to give the crude sulfate
salt (14), contaminated with sodium sulfate. The yield was essentially
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 611–618

614
C. O. MANNING ET AL.
CuSO4, Na2S2O5
K13C15N
Cu13C15N
(11)
(10)
94%
Scheme 2. Preparation of (M+2) copper cyanide
15NH2
Me
H15N
13C
S
S
H215N15NH2
15NH2
100%
(0.5eq) Me2SO4, H2O
95%
13C
13C
H125N
H15N
15NH2
H15N
15NH2
.0.5 H2SO4
.0.5 H2SO4
(13)
(14)
(12)
Scheme 3. Synthesis of (M+5) aminoguanidine
quantitative (no S-methyl isothiourea detected in the mass spectrum)
and the salt (14) was used directly in the next step.
Conclusion
Syntheses of [M+7] SIL labelled versions of Lamotrigine and the N2-
methylated metabolite of lamotrigine were developed for their use as
internal standards in mass spectrometric assays. During this work, a
procedure for preparing SIL [13C, 15N4]-aminoguanidine sulfate was
developed. This procedure could be modified by using the more
expensive [15N2]-hydrazine hydrate (rather than the sulfate) to obtain
[13C, 15N4]-aminoguanidine free from sodium sulfate.
Experimental
1H NMR spectra were recorded on a Varian Unity 400 spectrometer.
15N NMR spectra were measured on a Varian 400 spectrometer at 258C,
chemical shifts were referenced to CH3NO2 at 0 ppm via external
benzamide at À278.4 ppm.4 To reduce 15N relaxation times, chromium
acetylacetonate was added to solutions in DMSO-d6 and chromium
nitrate was added to D2O solutions. Mass spectra were recorded on a
Micromass Platform II mass spectrometer and accurate masses were
carried out on a Microman Q-ToF mass spectrometer. Thin layer
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 611–618

SYNTHESIS OF STABLE ISOTOPICALLY LABELLED
615
chromatography was carried out using Polygram SIL G/UV254 plastic
sheets. Chromatography was carried out using Merck 9385 silica gel.
HPLC analysis was carried out on a Prodigy ODS3 5m (150 Â 4.6 mm2)
column eluted with a gradient. Eluent A=0.1% aqueous trifluroacetic
acid, eluent B=95% acetonitrile, 5% water, 0.1% trifluroacetic acid,
gradient=10–80% B over 20 min; UV detection at 235 nm.
The isotopically labelled thiourea and hydrazine sulfate were
obtained from Aldrich and the labelled potassium cyanide from
Cambridge Isotope Laboratories.
Copper [13C, 15N]-cyanide (11)
Copper sulfate pentahydrate (8.38 g, 33.6 mmol) and water (20 ml) were
stirred at 458C. The resulting solution at 458C was treated with a
solution of sodium metabisulfite (1.73 g, 9.1 mmol) in water (10 ml). A
solution of potassium hydroxide (0.775 g, 13.8 mmol) and potassium
cyanide (1.8 g, 26.9 mmol) in water (8.5 ml) was added dropwise over
5 min.
The resulting suspension was stirred at for 1 h at 458C, followed by
30 min at 238C.
The suspension was filtered, washed with water (4 Â 30 ml), ethanol
(2 Â 30 ml), and ether (2 Â 10 ml) and was dried under vacuum to give
the title compound (11) as a white solid (2.30 g, 94%).
S-Methyl [13C, 15N2]-isothiouronium sulfate (13)
A stirred suspension of [13C, 15N2]-thiourea (12) (0.7 g, 8.86 mmol) and
water (3 ml) was treated with dimethylsulfate (0.4 2 ml, 4.43 mmol) and
heated at reflux for 20 h. The mixture was cooled and concentrated to
dryness under vacuum to give the title compound (13) as a white solid
(1.192 g, 95%); m.p. 2438C ; m/z 94 (MH+, 100%).
[13C, 15N4]-Aminoguanidine sulfate (14)
A stirred suspension of S-methyl [13C, 15N2] isothiouronium sulfate (13)
(1.05 g, 7.39 mmol), [15N2] hydrazine sulfate (0.98 g, 7.42 mmol) and
water (10 ml) was treated with 2 M aqueous sodium hydroxide (7.8 ml,
15.6 mmol). The resulting mixture was stirred for 16 h at 208C and then
purged with a stream of nitrogen for 30 min to remove the majority of
the volatile methanethiol. Two molar aqueous sulfuric acid was added
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C. O. MANNING ET AL.
to obtain pH 2. The volatiles were removed under reduced pressure to
give the title compound (14) as a white solid (2.272 g); m/z 80.0571
(calculated 80.0585); d15N(400 MHz, DMSO-d6) À286.6 (1N, d, JNÀN
=25.1 Hz), À314.0 (1N, m), 329.4 (2N, t, J =4 Hz).
2,3-Dichlorobenzoyl [13C, 15N]-cyanide (5)
2,3-Dichlorobenzoic acid (3.85 g, 20.16 mmol) was stirred in dichloro-
methane (50 ml). One drop of N,N-dimethylformamide was added
followed by the addition of oxalyl chloride (2.64 ml, 30.26 mmol). The
solution was left to stir for 4 h before concentrating under reduced
pressure.
The resulting acid chloride was dissolved in anhydrous xylene (60 ml)
and treated with copper [13C, 15N]-cyanide (2.3 g, 25.14 mmol) and
potassium iodide (4.02 g, 24.22 mmol). The mixture was heated to reflux
for 24 h followed by removal of the solvent under reduced pressure. The
residue was triturated with isohexane (15 ml), filtered and dried under
vacuum at 238C to give a cream solid (3.075 g, 76%); dH (400 MHz,
CDCl3) 8.08 (1 H,dd), 7.84 (1 H,dd), 7.49 (1 H, t).
(Z)-2-(2,3–Dichlorophenyl)-2-([13C,15N4]-guanidinoimino)
aceto [13C, 15N]-nitrile (6)
Crude labelled aminoguanidine sulfate (14) (1.294 g, ꢀ3.8 mmol) was
dissolved in concentrated sulfuric acid (80 g) and water (45 ml). A
solution of labelled 2,3-dichlorobenzoyl cyanide (5) (3.075 g,
15.22 mmol) in acetonitrile (17 ml) was added dropwise. The resulting
suspension was rapidly stirred for 5 days before adding saturated
aqueous sodium hydrogen carbonate (1500 ml). The mixture was
extracted three times with ethyl acetate (260 ml and 2 Â 100 ml). The
combined ethyl acetate portions were dried (MgSO4), and concentrated
under reduced pressure to give compound (6) as a yellow solid (0.767 g,
77%), which was used immediately.
6-(2,3-Dichlorophenyl)-1,2,4-[3,5-13C2,1,2,4-15N3]triazine-
3,5-[15N2]diamine (7)
Compound (6) (0.767 g) was heated at reflux for 1 h. After concentration
under reduced pressure, the crude product was purified by chromato-
graphy over silica gel (100 g) eluting with dichloromethane–methanol
Copyright # 2002 John Wiley & Sons, Ltd.
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SYNTHESIS OF STABLE ISOTOPICALLY LABELLED
617
(95:5). The fractions containing product were combined and evaporated
to dryness to give a white solid (0.603 g). The solid was crystallized from
1-propanol (7 ml) to give [M+7]-lamotrigine (7) as a white solid
(0.462 g, 46% from aminoguanidine);
HPLC chemical purity >99% a/a; m/z 263.0074 (calculated
263.0071); dH(400 MHz, DMSO-d6) 7.70(d of d, J=8 Hz, J=1.5 Hz,
1 H, aromatic CH); 7.45(t, J=8 Hz, 1 H, aromatic CH); 7.36(d of d,
J=8 Hz, J=1.5 Hz, 1 H, aromatic CH); 6.42(d, J=89 Hz, 2 H, NH2)
6–7.2(broad singlet, 2 H, NH2). d15N(400 MHz, DMSO-d6) À305.7 (1N,
t of d, JH–N=89.6 Hz, JC–N=24.5 Hz), À296.6 (1N, t of d,
JH–N=86.4 Hz, JC–N=20.0 Hz), À177.3 (1N, m), À97.5 (1N, d,
JN–N=21.5 Hz), 27.0 (1N, d, JN–N=21.5 Hz).
6-(2,3-Dichlorophenyl)-5-[13C,15N]imino-2-methyl-,2,5-dihydro-
[3-13C,15N3]1,2,4-triazin-3-[15N]amine methanesulfonate (9)
To a stirred mixture of [M+7]-lamotrigene (7) (0.2 g, 0.760 mmol) and
acetone (30 ml) was added iodomethane (0.2 ml, 3.21 mmol) and left to
stir for 24 h. The mixture was concentrated to dryness and stirred with
0.880 ammonia (4 ml) for 4 h (to remove HI). The resulting slurry was
filtered and dried under vacuum at 408C.
The cream solid was stirred with methanol (0.5 ml) and methane-
sulfonic acid (50 ml). The resulting solution was treated by dropwise
addition of ethyl acetate (5.1 ml) and then cooled to 0–58C for 30 min,
filtered and dried under vacuum. This gave the title compound (9) as a
white solid (0.2182, 77%); HPLC chemical purity 99.3%; m/z 277.0227
(calculated 277.0232); dH (400 MHz, DMSO-d6) 9.11(d, J=90 Hz, 1 H,
NH2); 8.15(d of d of t, J=91 Hz, J=7 Hz, J=3.5 Hz, 1 H, NH2);
7.9–9.0(2 broad doublets, J=89 Hz, 2 H, NH2); 7.86(d of d, J=8 Hz,
J=1.5 Hz, 1 H, aromatic CH), 7.55(t, J=8 Hz, 1 H, aromatic CH);
7.50(d of d, J=8 Hz, J=1.5 Hz, 1 H, aromatic CH); 3.75(apparent
quartet, 3 H, N–CH3); 2.30(s, 3 H, CH3–SO3H); d15N(400 MHz, DMSO-
d6) À290.8 (1N, t of d, JH–N=91.1 Hz, JC–N=23.4 Hz), À272.9 (1N, t of
d, JH–N=86.8 Hz, JC–N=25.1 Hz), À218.8 (1N, m), À177.8 (1N, m),
30.9 (1N, d, JN–N =12.9 Hz).
Acknowledgements
The authors thank Peter Moore, Mark Wipperman and Vicky Sinclair
1
for H and 15N NMR spectra and Alec Simpson for accurate mass
spectra.
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References
1. Young RC. J Med Chem 1988; 31: 670.
2. Cheung AHT, Cheu DD, Lacey E. J Label Compd Radiopharm 1987; 24:
879–893.
3. Smith GBL, Anzelmi E. J Am Chem Soc 1935; 57: 2730.
4. Brownlee RTC, Sadek M. Magn Res Chem 1986; 24: 821.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 611–618