Synthesis of 2H- and 3H-labelled iptakalim hydrochloride

Article abstract of DOI:10.1002/jlcr.844

Iptakalim hydrochloride (N-(1-methylethyl)-1,1,2-trimethyl-propylamine hydrochloride) is a promising antihypertensive drug discovered and developed by Beijing Institute of Pharmacology and Toxicology. Deuterium- and tritium-labelled compounds were designed and synthesized for pharmacokinetic and pharmacological mechanism studies. A derivatization method has been employed in the synthesis procedure. Copyright

Full text of DOI:10.1002/jlcr.844

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2004; 47: 583–590.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.844
Research Article
2
3
Synthesis of H- and H-labelled iptakalim
hydrochloride
Cheng Zhang*, Rifang Yang, Lanfu Chen, Bohua Zhong, Liuhong Yun
and Hai Wang
Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China
Summary
Iptakalim hydrochloride (N-(1-methylethyl)-1,1,2-trimethyl-propylamine hydrochlor-
ide) is a promising antihypertensive drug discovered and developed by Beijing
Institute of Pharmacology and Toxicology. Deuterium- and tritium-labelled
compounds were designed and synthesized for pharmacokinetic and pharmacological
mechanism studies. A derivatization method has been employed in the synthesis
procedure. Copyright # 2004 John Wiley & Sons, Ltd.
Key Words: iptakalim hydrochloride; tritium labelled; deuterium labelled; synthesis
Introduction
Iptakalim hydrochloride (N- (1-methylethyl) -1,1,2-trimethyl- propylamine
hydrochloride) (Figure 1) is a new chemical entity with potent anti-hypertension
activity. It cannot only decrease blood pressure in a dose-dependent manner,
but also reverse the hypertensive cardiovascular remodelling. The molecular
mechanisms underlying its antihypertensive action include ATP-sensitive
1
potassium channel activation and endothelin antagonism. The compound is
now moving from preclinical study into clinical investigation.
Iptakalim hydrochloride is a small molecular compound. The free base is
volatile and lacks a chromophore. Moreover, the steric effect of the multiple
methyl groups hinders derivatisation. These features make it very difficult to
carry out pharmacokinetic and pharmacological mechanism studies by HPLC
or GC. To overcome these problems we designed and synthesized deuterium-
and tritium-labelled isotopomers of iptakalim hydrochloride, bearing in mind
the available synthetic routes and also the likely metabolite pathways of the
compound (Scheme 1). These compounds provided convenient and useful
*Correspondence to: C. Zhang, Beijing Institute of Pharmacology and toxicology, 27 Taiping Road,
Beijing 100850, China. E-mail: zhangch927076@yahoo.com.cn
Copyright # 2004 John Wiley & Sons, Ltd.
Received 5 February 2004
Revised 15 April 2004
Accepted 26 April 2004

5
84
C. ZHANG ET AL.
CH3
CH3
H C H
3
.HCl
C
C
N
CH
H
H C
3
CH3
CH3
Figure 1. Structure of iptakalim hydrochloride
CH3
CH3
C
CH3
CH3
H C
3
H C
CH3
3
H C
3
HC
H C
C
N
CH
CH3
HC
NH2
HC
C
N
C
OH
H
H
H C
CH3
3
CH3
H C
3
3
CH3
CH3
CH3
O
H C
CH CH3
3
3
CH3
CH3
CH
CH3
H C
3
HC
C
N
+
CH
CH3
HO
C
C
N
O
H
H C
H C
3
3
CH3
CH CH3
3
CH3
H C
3
H C
H C
3
3
HC
H C
C
OH
N
OH
O
H C
H C
3
CH3
3
3
Scheme 1. Possible metabolic pathways of iptakalim hydrochloride
method for the pharmacokinetics and pharmacological mechanism studies of
2
iptakalim hydrochloride.
Results and discussion
The deuterium-labelled iptakalim hydrochloride was obtained by the
2
condensation of 2,3-dimethyl-2-butamine with [ H ] 2-bromopropane in an
7
autoclave. The synthetic route is shown in Scheme 2.
2
Preparation of [ H ]-iptakalim hydrochloride was starting from 2,3- dimethyl-
7
2
-butene, which was reacted with urea in an acid-catalyzed Ritter-type reaction
to give 1. Compound 1 was hydrolyzed under alkaline condition to give 2, which
2
was condensed with [ H ] 2-bromopropane to afford 3. It is difficult to purify 3
7
by chromatographic methods and HPLC because of the similar properties of 2
and 3 and of their hydrochlorides. We applied a derivatisation strategy for the
purification of 3 in the experiment. Since iptakalim does not react with benzoyl
chloride, excessive benzoyl chloride was added to the reaction mixture in order
2
to convert 2 to its amide completely. So [ H ]-iptakalim was then isolated from
7
the reaction mixture as its hydrochloride with general method.
The most important step for the synthesis of the tritium-labelled iptakalim
hydrochloride is to construct a carbon–carbon or carbon–nitrogen double
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 583–590

2
SYNTHESIS OF H- AND H-LABELLED
3
585
CH3
H C
CH3
H C
3
3
a
b
HC
C
NHCONH2
H C
CH3
3
H C
3
CH3
1
CH3
CH3
H C
CD3
3
H C
3
c
HC
H C
C
NH2
HC
C
N
CD
.
HCl
H
CD3
3
CH3
H C
CH3
3
2
3
CH3
O
H C
3
d
HC
H C
C
N
H
C
3
CH3
2
Scheme 2. Synthesis of [ H ]-iptakalim hydrochloride. Reagents and conditions:
7
(
(
(
a) Urea, mix of 98% sulfuric acid and glacial acetic acid (1:1,v/v), 9.5 h at 528C;
2
b) 50% Sodium hydroxide, glycol, 8 h at 1208C; (c) [ H ] 2-Bromopropane
7
98% D, Aldrich), glycol, toluene, 1708C, 17 h; (d) 12% Sodium hydroxide,
benzoyl chloride, 508C, 2.5 h
CH3
CH2
CH3
CH3
H3C
H3C
H3C
H3C
CH3
H3C
CH3
C
H
C
N
H
C
.HCl
C
H
C
N
C
.HCl
CH3
C
C
N C
H H
.HC
CH3
HC
2
CH3
CH3
CH3
II
CH3
III
I
Figure 2. Structures of I, II and III
bond in the core structure of the compound. There are three sites that can be
used to construct a double bond (Figure 2 I–III).
At first, we attempted to synthesize the compounds I and II by condensing of
2,3-dimethyl-2-butylamine with acetone or 2-brompropene. Unfortunately, none
of the desired intermediates were obtained because of the poor reactivity of 2-
bromopropene and difficulty of isolating the reactive intermediate II. So we
chose III (2,3-dimethyl-N- (1-methylethyl)-3-ene-2-butamine) as the precursor to
introduce tritium via a catalytic tritiation of the double bond. The synthetic route
3
of compound III and [ H ]-iptakalim hydrochoride are shown in Scheme 3.
2
In this procedure, 2,3-dimethyl-2-butene was treated with cyanamide and N-
bromosuccinimide to get 4, and then stirred in a solution of ethanol containing
anhydrous HCl to get 5. The product was pure enough to be used directly for the
next step. After treatment with KI and K CO in 1,4-dioxane, 6, a mixture of three
2
3
3,4
compounds was obtained as reported previously. All these three compounds can
be converted to 7 by alkaline hydrolysis, so the mixture was subjected to alkaline
hydrolysis to provide 2,3-dimethyl-3-butene-2-amine in good yield.
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 583–590

5
86
C. ZHANG ET AL.
H C
Br
C
H C
3
CH3
H C
CH3
3
Br
C
CH3 NH
3
a
b
C
NHCN
CH3
C
NH
C
OCH CH3.HCl
2
H C
H C
H C
CH
3
3
3
CH3
3
4
5
CH3
O
H C
3
C
C
NHCNH2
H C
2
CH3
CH3
CH3
CH3
H C
3
C
C
CH H C
C
C
H C
CH3
c
3
3
3
d
H C
N
CH2
6
C
C
.
NH2
HCl
2
HN
C
N
C
H C
2
CH3
OH
CH3
OCH CH3
7
2
H C
O
3
C
C
NHC
OCH CH3
2
H C
2
CH3
H C
CH
3
CH
3
CH3
3
e
f
3
C
C
N
H
CH
.
HCl
C H H
11
2
NH
C
.
HCl
6
H
CH3
H C
CH3
2
CH3
8
III
9
3
[ H ]-iptakalim hydrochloride
2
3
Scheme 3. Synthesis of [ H ]-iptakalim hydrochloride. Reagents and conditions:
2
(a) NBS, H NCN, CH Cl , room temperature, 72 h; (b) HCl/EtOH, room
2 2 2
temperature, 6 h; (c) KI, K CO , 1,4-dioxane, 908C, 24 h; (d) KOH, EtOH,
2
3
H O, reflux, 35 h; (e) 2-Bromopropane, K CO , NMP, 1708C, 17 h; 12%
2
2
3
3
sodium hydroxide, benzoyl chloride, 508C, 6 h; (f) [ H ], PtO , EtOH, 1,4-
2
2
dioxane, room temperature, 19 h
The condensation of 7 with 2-bromopropane was carried out smoothly in an
autoclave. Because of the similarity of 7 and 8 and their hydrochlorides, the
same procedure as used in the purification of 3 was adopted here to scavenge
the excess 7 and allow isolation of 8 as its hydrochloride.
The catalytic tritiation of 8 was carried out under the atmospheric pressure.
The reaction was modelled several times with hydrogen to ensure the high
purity of the desired product and the reliability of the reaction condition
before tritium was used. Since our laboratory cannot offer the structural
3
3
characterisation such as H NMR and H MS for the tritium-labelled
compound, we synthesized a deuterium-labelled iptakalim hydrochloride with
1
the same method. The H NMR and MS showed that two deuterium atoms
can be introduced to both the original double bond and the adjacent three
methyl groups. This result suggested that the tritium labelling would not limit
to the original double bond. Since this kind of isotopic scrambling is likewise
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 583–590

2
SYNTHESIS OF H- AND H-LABELLED
3
587
not a problem for the pharmacokinetics and pharmacological mechanism
studies, we decided to introduce two tritium atoms into the structure of
iptakalim hydrochloride with this method. The specific radioactivity of 9
achieved to 53.3 Ci mmolꢀ and its radiochemical purity was 98%.
1
Materials and methods
Melting points were determined in open capillary tubes and are uncorrected.
IR spectra were recorded as KBr discs on a Nicolet Magna-IR 550
spectrophotometer. Mass spectra were recorded on a Zebspec mass spectro-
1
meter. The H NMR spectra were recorded at 400 MHz on a JNM-GX400,
and the chemical shifts were reported in ppm(d) relative to trimethylsilane
(
0.0 ppm). A FJ-2107P liquid scintillation counter was used to determine radio-
2
activity in liquid samples. [ H ] 2-bromopropane (98 at% D) was obtained
7
from Aldrich. Tritium gas was purchased from China Institute of Atomic
Energy (CIAE). All other reagents were ACS grade and were obtained from
commercial sources.
Experimental
2
; 3-Dimethyl-2-buturea; 1
To a suspension of glacial acetic acid (6.0 ml) and urea (8.0 g, 0.143 mol) in a
0 ml three-necked flask were added 2,3-dimethyl-2-butene (5.6 g, 0.060 mol).
5
A mix of 98% sulfuric acid and glacial acetic acid (1:1, v/v, 17.5 ml) was added
dropwise at such a rate that the temperature did not exceed 508C. The
resulting mixture was stirred for 9.5 h at 528C. The reaction mixture was then
poured into an ice-cold solution of 50% sodium hydroxide (180 ml). The
mixture was then filtered, washed with cold water (5 ꢁ 10 ml). The filtrate was
dried under reduced pressure to give 3.06 g (35.4%) 1 as pale yellow solid. m.p.
ꢀ
1
1
1
67–1698C. IR (KBr)cm : 3454, 3360, 3216, 2972, 1654, 1612, 1560. H
NMR(CDCl ) d: 0.88–0.90 (d, 6 H, J=6.71 Hz), 1.26(s, 6 H), 4.20–4.50(2 ꢁ br,
3
+
3
H). MS(FAB) m=z: 145.2 [M +H].
2; 3-Dimethyl-2-butamine; 2
5
0% Sodium hydroxide (120 ml) was added to a solution of 1 (196 g, 1.36 mol)
in glycol (392 ml). The mixture was stirred for 8 h at 1208C (oil bath
temperature). 88.5 g of 2 was obtained by distillation (95–1028C) as colorless
1
liquid. H NMR(CDCl ) d: 0.88(d, 6 H), 1.04(s, 6 H), 1.53(m, 3 H). MS(FAB)
3
+
m=z: 102.1 [M +H].
2
[
H ]-iptakalim hydrochloride, 3
7
2
(8.0 g, 0.079 mol), [ H ] 2-bromopropane (5.0 g, 0.038 mol), glycol (5.7 ml)
2
7
and toluene (40 ml) were added to an autoclave. The mixture was heated at
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 583–590

5
88
C. ZHANG ET AL.
1
708C for 17 h. After cooling to room temperature, a solution of 40% sodium
hydroxide (20 ml) was added to dissolve the insoluble solid. The resulting
solution was extracted with toluene (3 ꢁ 15 ml). The combined organic extracts
were washed with water (3 ꢁ 10 ml). Then 12% sodium hydroxide (20 ml) and
benzoyl chloride (2.0 ml in 4.0 ml toluene) were added to the toluene layer. The
mixture was stirred at 508C for 2.5 h. The toluene layer was then washed with
2
5% hydrochloride acid (3 ꢁ 10 ml) and water (2 ꢁ 10 ml). The aqueous layer
was combined and washed with ether, and then the pH level was adjusted to
2–13 by addition of 40% sodium hydroxide, and the layer extracted with
ether (3 ꢁ 15 ml) and dried over anhydrous K CO . After treatment with a
1
2
3
ꢀ1
solution of anhydrous HCl in ether (2.5 mol l , 10 ml), the precipitation was
washed with ether to give a white solid. Recrystallization from 2-propanol/
ether afforded 1.39 g (19.4%) of 3 as white crystals. m.p. 228–2308C. IR
ꢀ
1
(
KBr)cm : 3436, 2969, 2827, 2755, 2426, 2233, 1588, 1473, 1401, 1155, 1052.
1
H NMR(D O) d: 0.96–0.98(d, 6 H), 1.32(s, 6 H), 2.0–2.1(m, 1 H). MS(FAB)
m=z: 151.2 [M -Cl].
2
+
(
2-Bromo-1,1,2-trimethyl-propyl) cyanamide, 4
N-bromosuccinimide(25.0 g, 0.140 mol) was added to a stirred, cooled (5–
08C) solution of the 2,3-dimethyl-2-butene (10.7 g, 0.127 mol) and cyanamide
21.3 g, 0.508 mol) in methylene chloride (70 ml). The resulting reaction
1
(
mixture was stirred for 1 h at 5–108C and then stirred for 72 h at room
temperature. The methylene chloride layer was washed with water
(
6 ꢁ 100 ml), dried over Na SO , and concentrated in vacuo. The residue was
2
4
treated with cyclohexane and methylene chloride to give 3.5 g (13.4%) of 4 as
white powder. m.p. 109–1118C.
Ethyl ((2-bromo-1,1,2-trimethyl-n-propyl) amino) carboximidate, 5
A solution containing 1.3 g of anhydrous HCl in ethanol (30 ml) was added to
a solution of 4 (13.0 g, 0.063 mol) in ethanol (30 ml) at 5–108C, and another
3
0 ml of ethanol was added. The resulting solution was stirred for 6 h at room
temperature and then concentrated in vacuo. The resulting solid was washed
several times with ether and dried to yield 17.0 g (93.8%) of 5 as white solid.
ꢀ
1
1
m.p. 119–1218C. IR (KBr)cm : 3242, 3149, 1676, 1558, 1385, 1085. H
NMR(CDCl ) d: 1.42–1.46 (t, 3 H, J=6.94 Hz), 1.54 (s, 6 H), 1.85 (s, 6 H),
3
4
.55–4.61 (q, 2 H, J=6.94 Hz), 8.31 (s, 1 H), 8.71–9.12 (d, 2 H). MS(FAB) m=z:
+
50.9[M -HCl].
2
2; 3-Dimethyl-3-butene-2-aminehydrochloride; 7
KI (9.1 g, 0.055 mol) and anhydrous K CO (12.0 g, 0.087 mol) were added to
2
3
a solution of 5 (8.0 g, 0.028 mol) in 1,4-dioxane (220 ml). The resulting reaction
Copyright # 2004 John Wiley & Sons, Ltd.
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2
SYNTHESIS OF H- AND H-LABELLED
3
589
mixture was stirred at 908C for 24 h. The insoluble material in the 1,4-dioxane
was separated and washed with 1,4-dioxane, and the filtrate was concentrated
in vacuo. The residue was treated with cyclohexane to give 3.0 g of 6 as white
solid. The mixture of 6 was then mixed with KOH (3.50 g), ethanol (25 ml) and
water (10 ml), refluxed for 35 h, and distilled to collect the volatile component
ꢀ
1
as colorless liquid. A solution of anhydrous HCl in ether (2.5 mol l , 20 ml)
was then added to give the desired product as white solid. Recrystallization
from ether/ethanol gave 1.1 g (29.2%) of 7 as white crystals. m.p. >3008C. IR
ꢀ1
1
(
KBr)cm : 3435, 2986, 2912, 1596, 914. H NMR (CDCl ) d 1.58 (s, 6 H),
3
1
.88 (s, 3 H), 5.00 (s, 1 H), 5.14 (s, 1 H) 8.6–8.8 (br, 3 H). MS (FAB) m=z:
+
00.1[M -Cl].
1
2; 3-Dimethyl-N-ð1-methylethylÞ-3-butene-2-amine hydrochloride; 8
7
(1.1 g, 0.0081 mol), 2-bromopropane (2.0 g, 0.016 mol), anhydrous K CO
2
3
(2.5 g, 0.018 mol) and N-methyl-pyrrolidone (20 ml) were added to an
autoclave, the mixture was stirred at 1708C for 17 h. After cooling to room
temperature, 50% sodium hydroxide (15 ml) was added and the reaction
mixture was extracted with toluene (3 ꢁ 25 ml). The toluene layer was washed
with water and then 12% sodium hydroxide (4.5 ml) and benzoyl chloride
(2.0 ml in 4 ml toluene) were added. The mixture was stirred at 508C for 6 h.
The toluene layer was then washed with 25% hydrochloric acid (3 ꢁ 10 ml) and
water (2 ꢁ 10 ml). The aqueous layers were combined and washed with ether,
and then the pH level was adjusted to 12–13 by addition of 50% sodium
hydroxide, extracted with ether (3 ꢁ 15 ml). The ether layer was washed with
water (30 ml), dried over K CO , treated with a solution of anhydrous HCl in
2
3
ꢀ1
ether (2.5 mol l , 10 ml), and the solvent was removed in vacuo. The crude
product was purified by silica gel column chromatography with chloroform,
petroleum ether (b.p. 60–908C) and methanol (3:4:1, v/v/v) as eluent to give 8
ꢀ
1
(
Rf 0.42) as a white solid (300 mg, 20.8%). m.p. 199–2018C. IR (KBr)cm :
1
3
408, 2974, 2760, 1587, 932. H NMR(CDCl ) d: 1.47–1.50 (d, 6 H,
3
J=6.36 Hz), 1.70 (s, 6 H), 2.01 (s, 3 H), 3.20–3.30 (m, 1 H), 5.14 (s, 1 H),
+
5
.18 (s, 1 H), 9.10–9.30 (br, 2 H). MS(FAB) m=z: 142.2[M -Cl].
3
1,2- H ]-N- (1-Methylethyl)-1,1,2-trimethyl-propylamine hydrochloride ([ H ]-
3
[
2
2
iptakalim hydrochloride), 9
Drops of ethanol were added to a mixture of 8 (12.4 mg, 0.070 mmol), PtO2
7.4 mg, 81%Pt, Fluka) and 1,4-dioxane (4.0 ml). The resulting solution was
stirred under a tritium atmosphere (4.7 ml, 12.0 Ci) at room temperature for
9 h when the uptake of gas had stopped. Upon completion of the reaction, the
5
(
1
solution was filtered to remove catalyst and the residue was washed with
chloroform. The solvent was removed in vacuo. The residue was re-dissolved in
ethanol and was lyophilized to remove volatile tritium. This procedure was
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 583–590

5
90
C. ZHANG ET AL.
repeated three times. Then the residue was evaporated to dryness under
reduced pressure to yield 10 mg of 9 as white solid which was then re-dissolved
in 75% ethanol to 500 ml. The total radioactivity of 9 was determined to be
3
.0 Ci. The specific radioactivity of 53.3 Ci mmolꢀ1 was calculated from liquid
scintillation counting. The radiochemical purity was shown to be 98%, the
active material co-eluting with the inactive reference.
Acknowledgements
We thank Mr Decun Shen and Ms Sha Liao for invaluable assistance in
catalytic tritiation.
References
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4
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 583–590