Preparation of two D2 partial agonists in C-14 and stable isotope labeled forms

Article abstract of DOI:10.1002/jlcr.2951

In support of a program to develop a treatment for diseases resulting from an imbalance in dopamine levels, two drug candidates, 1 and 2, were prepared in stable isotope and C-14 labeled forms. Both compounds contained an aminothiazole ring, and the C-14 label was introduced into the ring using KS¹⁴CN. However, the use of KS¹⁴CN to form [¹⁴C]-2 gave poor results; therefore, benzoyl [¹⁴C]isothiocyanate was used instead, which led to a much improved yield. The stable isotope labeled forms were prepared with the label in the side chain from [²H₅]ethyl iodide for [²H₅]-1 and from 1-[¹³C]methyl [¹⁵N₂]pyrazole[¹³C]carboxaldehyde, which was prepared in turn from [carbonyl-¹³C]DMF, [¹⁵N ₂]hydrazine sulfate, and [¹³C]methyliodide for [ ¹³C₂, ¹⁵N₂]-2. Copyright

Full text of DOI:10.1002/jlcr.2951

Research Article
Received 18 June 2012,
Accepted 06 July 2012
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.2951
Preparation of two D2 partial agonists in C-14
and stable isotope labeled forms
b
b,c
b,c
d
Charles S. Elmore,a,b Mark E. Powell, Hui Xiong, Ye Wu, Lijuan Wang,
*
David Saunders,d and Rebecca A. Urbanekb
In support of a program to develop a treatment for diseases resulting from an imbalance in dopamine levels, two drug candidates,
1 and 2, were prepared in stable isotope and C-14 labeled forms. Both compounds contained an aminothiazole ring, and the C-14
label was introduced into the ring using KS14CN. However, the use of KS14CN to form [14C]-2 gave poor results; therefore, benzoyl
[14C]isothiocyanate was used instead, which led to a much improved yield. The stable isotope labeled forms were prepared with the
label in the side chain from [2H5]ethyl iodide for [2H5]-1 and from 1-[13C]methyl [15N2]pyrazole[13C]carboxaldehyde, which was
prepared in turn from [carbonyl-13C]DMF, [15N2]hydrazine sulfate, and [13C]methyliodide for [13C2, 15N2]-2.
surprisingly, we obtained a 54% yield of 4 and 4b in a ratio of 4:1.
Introduction
The mixture was deprotected in 82% yield to give a mixture of
14C]-1 and [14C]-1b. Unfortunately, the separation of [14C]-1
Dopamine plays many important physiological roles in the
[
and [14C]-1b was difficult, and after two attempted purifications
by preparative HPLC, the benzylcarbamate of [14C]-1 and [14C]-1b
was reformed. The two isomers were then readily separated by
preparative HPLC. Deprotection of the carbamate again proceeded
smoothly, and the target amine was isolated in excellent yield and
high purity without need of further purification.
central nervous system, such as sensory, motor, behavior, and
cognition. Abnormal dopamine neuron functions have been
linked to a wide variety of psychiatric and neurological diseases
and treatments, which alter dopaminergic activities that are
widely used clinically. For example, D2 functional antagonists
are widely utilized as antipsychotics.1 The approval of aripiprazole
(AbilifyW), a D2 partial agonist with activities at multiple 5-HT
receptors, prompted active research and development of various
D2 partial agonists, on the basis of the hypothesis that D2 partial
agonists with an appropriate level of intrinsic activity at D2
receptors can regulate dopamine activity to normal levels in
neurons in either hyperdopaminergic or hypodopaminergic
states, while minimally disturbing other normal dopaminergic
pathways, leading to antipsychotic efficacy and a potentially
superior side effects profile.2–7 Compounds 1 and 2 (Figure 1)
were identified as partial D2 receptor agonists and were required
in labeled form to further probe their biological activity.8
Benzazepine 2 was also required labeled with C-14 for DMPK stu-
dies. In this case, medicinal chemistry used benzoyl isothiocyanate in
place of Potassium isothiocyanate (KSCN) to form the aminothiazole
ring, but we opted to attempt the ring formation with KS14CN to
eliminate two radiochemical steps (Scheme 2). A variety of reaction
conditions were probed using non-radioactive material including
multiple oxidizing reagents, different reaction times, slow addition
of the oxidizing reagent, and higher temperatures; but in all cases,
we obtained results that were unacceptable for radiochemical use
(Table 1). The conversion of starting material 5 to product was poor
and more forcing conditions resulted in decomposition of the start-
ing material. Additionally, the separation of isomers 2 and 2b was
extremely difficult by HPLC. Supercritical fluid chromatography
was effective at separating the isomers but was not available for
use with radioactive compounds.
Results and discussion
Aminothiazole 1 was required labeled with C-14 in a metaboli-
cally stable position for DMPK assays. All sites of the molecule
were deemed acceptable for C-14 labeling with the exception
of the ethyl group. There was no in vitro evidence that the
aminothiazole ring was opened or cleaved although this has
been observed in other aminothiazoles9; therefore, we targeted
label incorporation into the carbon of the thiazole ring. This
route utilized the chemistry previously developed by the
medicinal chemistry program and made use of available advanced
intermediates that precluded additional non-radioactive synthetic
work.
After convincing ourselves that direct reaction of KS14CN with 5
was not the best option for the preparation of 2, we prepared ben-
zoyl [14C]isothiocyanate (6) by reacting potassium [14C]thiocyanate
with benzoyl chloride in acetonitrile in nearly quantitative yield
aIsotope Chemistry, DMPK, AstraZeneca Pharmaceuticals LP, 43183 Mölndal Sweden
bCNS-Chemistry, AstraZeneca Pharmaceuticals LP, Wilmington, DE 19850, USA
cAstraZeneca Pharmaceuticals LP, Waltham, MA 02451, USA
dIsoSciences, LLC, King of Prussia, PA 19406, USA
The preparation started with the reaction of KS14CN10 with
aniline 3 in acetic acid followed immediately by a bromine-
induced cyclization (Scheme 1). The cyclization could potentially
produce the linear product 4 or the angular product 4b and not
*Charles S. Elmore, Isotope Chemistry, DMPK, AstraZeneca Pharmaceuticals LP,
43183 Mölndal Sweden.
E-mail: chad_elmore@yahoo.com
J. Label Compd. Radiopharm 2012
Copyright © 2012 John Wiley & Sons, Ltd.

C. S. Elmore et al.
The synthesis of stable isotope labeled 1 started with an
alkylation of amide 11 with [2H5]iodoethane to give a near quan-
titative yield of 12 (Scheme 4). Buchwald–Hartwig amination15,16
followed by cleavage of the imine of 13 afforded an excellent
yield of aniline [2H5]-3, which was converted to thiazole 14 with
benzoyl isothiocyanate in 51% yield. HPLC analysis detected
approximately 5% of the bent isomer present as a contaminant
which is indicative of a successful crystallization and not an overly
selective reaction. Deprotection of the benzyl carbamate pro-
Figure 1. Two partial D2 receptor agonists.
(Scheme 3).11,12 HPLC anaylsis of the product gave two peaks in ceeded smoothly in 90% yield to give 15 and was immediately
varying ratios for the same sample. We suspect that this reflects followed by the deprotection of the benzoyl group. Unfortu-
the reaction of benzoyl [14C]isothiocyanate with the HPLC medium. nately, purification of [2H5]-1 proved problematic. An initial
The benzoyl [14C]isothiocyanate was then reacted with aniline 5 crystallization removed the remaining bent isomer, but several
without further isolation to give thiourea 7 after 15 min, and conver- unidentified impurities eluted close to the target compound on
sion to the thiazole was effected using N-bromosuccinimide in tri- HPLC and two preparative HPLC purifications were required
fluoroacetic acid (TFA).13,14 The crude reaction mixture consisted to enrich the UV area percent purity of the compound to
of 41% of 8, 15% of 8b, and 24.5% of 9. Urea 9 was not present 98.8%.
to a significant extent in the starting material; therefore, there is
likely a competition for the activated intermediate between adven-
titious water and the cyclization. No special drying protocol was
used for the TFA and likely, the water contained in the solvent led
to this by-product which was also observed in the medicinal
chemistry route but at much lower levels. Preparative HPLC
separated the linear and bent forms (8 and 8b), but 8 was contami-
nated with approximately 9% of urea 9. The overall yield of 8 from
benzoyl [14C]isothiocyanate was 21% after preparative HPLC
(corrected for 9 content). The subsequent debenzoylation and
purification by preparative HPLC provided the final compound in
21% yield and 99.3% radiochemical purity. The final step was run
only once and on small scale so it is likely that the yield could be
substantially improved.
The preparation of 2 in stable isotope labeled form proved
to be less of a challenge (Scheme 5). The required carboxalde-
hyde 16 was prepared in three steps using a modification of
literature routes.17–19 [15N2]Pyrazole was prepared by conden-
sing [15N2]hydrazine with a protected form of malonaldehyde,
and the resulting pyrazole was methylated with 13CH3I. Finally,
a Vilsmeier reaction using [carbonyl-13C]DMF provided 16 in
modest yield after Kugelrohr distillation. Reductive amination
with amine 10 proceeded smoothly, and column chromatography
on basic alumina and SFC purification gave [13C2, 15N2]-2 in a
77% yield.
We next turned our attention to preparing the stable isotope
labeled versions of both 1 and 2. A minimum mass increase of
4 was required for both compounds with no detectable unla-
beled material (<0.5%). The side chains of each compound
seemed a logical site for label incorporation although that would
entail a lengthy synthesis for stable isotope labeled 1 because of
the limited availability of unlabeled starting materials. For
compound 2, the route was potentially shorter as amide 10
was available.
Scheme 2. Attempted conversion of 5 to 2.
Scheme 1. Synthesis of [14C]-1 from KS14CN.
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J. Label Compd. Radiopharm 2012

C. S. Elmore et al.
amine (5), 5-bromo-2,3-dihydro-1H-inden-2-ylcarbamate (11), and 6,7,8,9-
Tetrahydro-5H-1-thia-3,7-diaza-cyclohepta[f]inden-2-ylamine 2HCl (10)
were provided by AstraZeneca CNS-Chemistry, Wilmington, DE. All other
reagents were obtained from Acros, Sigma-Aldrich, Fisher and Strem
and were used without purification.
Experimental
General: KS14CN was purchased from American Radiolabeled Chemicals,
Inc. and [2H5]iodoethane, [15N2] Hydrazine sulfate and [carbonyl-13C] N,
N-dimethylformamidewere were purchased from Isotec, Sigma-Aldrich.
(S)-benzyl 5-amino-2,3-dihydro-1H-inden-2-yl(ethyl)carbamate (3), 3-((1-
methyl-1H-pyrazol-4-yl)methyl)-2,3,4,5-tetrahydro-1H-benzo[d]azepin-7-
1H NMR spectra were recorded on Bruker DPX 300, DRX 400, Avance
500 and DRX 600 spectrometers (manufactured by Bruker Corporation)
Table 1. Investigation of the use of different reaction conditions to convert 5 to 2 and 2b (or 8 and 8b when PhCONCS was used).
All reactions were run at 70 ꢀC for 2 h except for the reaction with PhCONCS, which used the conditions described for the prepara-
tion of compound 8
Thiocyanate
Oxidant
Solvent
Crude yield (ratio of 2–2b or 8–8b)
KSCN
KSCN
KSCN
NH4SCN
PhCONCS
Br2
CuCl2
CuSO4
N-bromosucinimide
N-bromosucinimide
AcOH
MeOH
MeOH
MeCN
Trace
20% (2:1)
20% (2:1)
Trace
CH2Cl2/TFA
56% (2.7:1)
Scheme 3. Preparation of [14C]-2 from KS14CN.
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C. S. Elmore et al.
Scheme 4. Preparation of [2H5]-1 from [2H5]iodoethane.
Scheme 5. Preparation of [13C2, 15N2]-2.
2
in H6-DMSO, or C2H3O2H at 30 ꢀC and were referenced to the residual performed on a 21.2 Â 250 mm column and a flow rate of 15 mL/min
solvent peak. LC/MS analyses were performed on an HP MSD-1100 using using the following methods: method (Phenomenex Synergi Polar-RP,
a 4.6 Â 100 mm Phenomenex Luna-C18(2) column, with a 10–100% gra- 30–90% MeOH-0.1% formic acid over 30 min), Method b (Phenomenex
dient of MeOH-0.1% aq formic acid over 10 min and electrospray
ionization.
Luna C18(2), 30–70% MeOH-0.1% formic acid over 40 min), or Method
Ɣ (Phenomenex Synergi Polar-RP, 2–20% MeOH-0.1% Formic acid over
HPLC analyses were performed using a Agilent 1100 series HPLC 20 min), or method d (Phenomenex Luna C18(2), 2–10% MeCN-0.1%
system using one of the following methods: method A (Phenomenex TFA over 20 min). Silica gel purifications were performed on a Teledyne
Luna C-18(2), 40–60% MeOH-0.1% Formic acid), method B (Phenomenex ISCO CombiFlash Companion using gradient elution.
Synergi Polar-RP, 2–10% MeOH-0.1% TFA), method C (Phenomenex Luna
(R)-benzyl [2-14C]-(2-amino-6,7-dihydro-5H-indeno[5,6-d]thiazol-
C-18(2), 30–60% MeOH-0.1% Formic acid), method D (Phenomenex
Synergi Polar-RP, 2–20% MeOH-0.1% Formic acid) or method E (Phenom-
6-yl(ethyl)carbamate (4)
enex Luna C-18(2), 5–95% MeCN-0.1% Formic acid),
Gradients were run over 20 min and were followed by a 5 min wash
with 100% organic mobile phase. Preparative HPLC purifications were KSCN and 222 mg (0.72 mmol) of (S)-benzyl 5-amino-2,3-dihydro-1H-
Method A: A solution of 65 mg (35 mCi, 53 mCi/mmol, 0.66 mmol) of [14C]
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J. Label Compd. Radiopharm 2012

C. S. Elmore et al.
[2-14C]-6,7,8,9-tetrahydro-2-amino-7-((1-methyl-1H-pyrazol-
inden-2-yl(ethyl)carbamate (3) in 3 mL of acetic acid was stirred for 1 h. A
solution of 37 mL (0.71 mmol) of Br2 in 3 mL of acetic acid was added
dropwise over 30 min and the solution was stirred overnight. The
reaction mixture was diluted with 15 mL of ethyl acetate and 10 mL of
1N NaOH and the pH of the solution was adjusted to approximately 14
with 50% NaOH. The layers were separated, and the aqueous layer was
extracted with 15 mL of EtOAc. The combined organic layers were dried
(Na2SO4) and filtered, and the filtrate was concentrated to dryness to give
18.8 mCi as a yellow oil. HPLC analysis using method A showed 80%
radiochemical purity with a single 20% radiolabeled impurity. This impur-
ity co-eluted on HPLC with (S)-benzyl [2-14C]-(2-amino-7,8-dihydro-6H-
indeno[5,4-d]thiazol-7-yl(ethyl)carbamate (4b).
4-yl)methyl)-5H-Thiazolo[4,5-h][3]benzazepine ([14C]-2)
A solution of 2.8 mCi (52 mmol) of 8 in 2 mL of 48% aq HBr was heated at
reflux for 7 h in a sealed vial. The solution was cooled to room tempera-
ture and carefully diluted to pH 10 with 50% NaOH, and the resulting
solution was extracted with 3 Â 20 mL of EtOAc. The combined organic
layers were dried (Na2SO4), and the drying agent was removed by
filtration. The solvent was evaporated and the residue was purified by
preparative HPLC using method Ɣ. The resulting product containing
fractions were each assayed by HPLC to insure their purity and were then
combined, basified with NaHCO3 and extracted with EtOAc. The EtOAc
solution was concentrated to dryness and the residue was taken up in
0.6 mL of EtOH to give 0.6 mCi of [14C]-2 at a radiochemical purity of
Method B: A solution of 9.5 mCi (0.17 mmol) of [14C]-1 and [14C]-1b (in
a 4:1 ratio), 73 mL (0.52 mmol) of NEt3 and 52 mg (0.18 mmol) of dibenzyl-
dicarbonate in 1.7 mL of 50% aq THF was stirred at room temperature
overnight. The solution was then diluted with 3 mL of CH2Cl2 and 2 mL
of water, and the layers were separated. The aqueous layer was extracted
with another 3 mL of CH2Cl2, and the combined organic layers were dried
(Na2SO4). The solid was removed by filtration, and the filtrate concen-
trated to dryness. The residue was purified by preparative HPLC using
method. The product containing fractions were combined, basified with
NaHCO3, and extracted with CH2Cl2. The combined organic layers were
dried (Na2SO4) and filtered and the filtrate concentrated to dryness to
give 4.1 mCi with a radiochemical purity of 96.9% (method A).
1
99.3% (method D) and a specific activity of 53 mCi/mmol. H NMR (2H6-
DMSO, 300 MHz): d 7.52 (s, 1H), 7.33 (s, 1H), 7.27 (s, 1H), 7.24 (br, 2H),
7.07 (s, 1H), 3.77 (s, 3H), 3.46 (s, 2H), 2.81 (m, 4H), 2.49 (m, 4H). LC/MS:
316 (100%), 317 (27%), 314 (16%)
(S)-benzyl 5-bromo-2,3-dihydro-1H-inden-2-yl([2H5]ethyl)
carbamate (12)
A solution of 494 mg (1.43 mmol) of (S)-benzyl 5-bromo-2,3-dihydro-1H-
inden-2-ylcarbamate (11) in 3 mL of DMF was cooled at 0 ꢀC as 86 mg
(2.1 mmol) of NaH was added portion-wise. After stirring for 20 min,
137 mL (1.71 mmol) of [2H5]iodoethane was added and the reaction
was stirred at 0 ꢀC for 10 min and was then warmed to room temperature
and stirred for 30 min. The reaction was cooled to 0 ꢀC and diluted with
3 mL of water. The aqueous layer was extracted with 3 Â 15 mL of hexane
and the combined organic extracts were dried (MgSO4) and filtered. The
filtrate was concentrated to give 526 mg of a colorless oil. HPLC analysis
(method E) showed the compound to be >95 UV area percent pure.
LC/MS: 381 (100%), 379 (98%), 380 (23%), 382 (20%).
[2-14C]-(R)-N6-ethyl-6,7-dihydro-5H-indeno[5,6-d]thiazole-2,6-
diamine ([14C]-1)
A sealed solution of 4 mCi (36 mmol) of 4 and 0.23 mL (0.11 mmol) of trii-
sopropylsilane in 0.30 mL of 33% HBr in HOAc was stirred for 1 h and was
then diluted with 20 mL of ethyl acetate that resulted in the formation of
a white solid. The solid was removed by filtration, dissolved in 5 mL of
water, and filtered. The solution was basified with 1 M NaOH and
extracted with 40 mL of ethyl acetate. The organic layer was dried
(MgSO4) and filtered, and the filtrate was concentrated to dryness to give
3.7 mCi of [14C]-1 as a white solid. HPLC analysis using method B showed
a radiochemical purity of 99.6%. 1H NMR (C2H3O2H, 500 MHz): d 7.34 (s,
1H), 7.21 (s, 1H), 3.61 (m, 1H), 3.31 (m, 2H), 3.19 (m, J = 7, 15.5 Hz, 2H),
2.79 (m, 2H), 2.72 (q, J = 7 Hz, 2H), 1.16 (t, J = 7 Hz, 3H).
(S)-benzyl 5-amino-2,3-dihydro-1H-inden-2-yl([2H5]ethyl)
carbamate ([2H5]-3)
A solution of 51.7 mg (56 mmol) of Pd2(dba)3, 53.8 mg (0.11 mmol) of dicy-
clohexyl(2’,4’,6’-triisopropylbiphenyl-2-yl)phosphine, 434 mg (4.51 mmol)
NaOt-Bu, 0.42 mL (2.48 mmol) of benzophenone imine, and 856 mg
(2.26 mmol) of 12 in 4.5 mL of PhCH3 was sparged with N2 and then
heated at 70 ꢀC for 2 h. The reaction mixture was passed through a
bed of celite and was concentrated to dryness to give 1.45 g of a
brown oil containing 13. The oil was taken up in 9 mL of methanol
and the resulting solution was stirred as 235 mg (3.38 mmol) of
hydroxylamine hydrochloride and 370 mg (4.51 mmol) of NaOAc were
added; stirring was continued overnight. The solution was then fil-
tered, diluted with 10 mL of CH2Cl2, and filtered again. The filtrate
was concentrated to dryness and the residue was purified by silica
gel chromatography (10–30% EtOAc in hexane). The product contain-
ing fractions were combined and concentrated to give 0.7 g of [2H5]-
3 as a yellow oil. HPLC analysis (method E) showed a UV area percent
purity of >95.
benzoyl [14C]isothiocyanate (6)
The synthesis of benzoyl [14C]isothiocyanate used a modification of a
literature method.11,12 A slurry of 194 mg (110 mCi, 1.96 mmol) of [14C]
potassium thiocyanate in 10 mL of acetonitrile was stirred as 0.23 mL
(2.0 mmol) of benzoyl chloride was added and the reaction mixture was
heated at 70 ꢀC for 1 h. The solution was filtered and the solvent removed
to give 104 mCi of a yellow oil. The compound was used directly in the
next reaction.
[2-14C]-6,7,8,9-tetrahydro-2-benzamido-7-((1-methyl-1H-
pyrazol-4-yl)methyl)-5H-Thiazolo[4,5-h][3]benzazepine (8)
A
solution of 503 mg (1.96 mmol) of 3-((1-methyl-1H-pyrazol-4-yl)
methyl)-2,3,4,5-tetrahydro-1H-benzo[d]azepin-7-amine (5) in 5 mL of
CH2Cl2 was stirred as a solution of 104 mCi (1.86 mmol) of benzoyl [14C]
isothiocyanate in 2 mL of CH2Cl2 was added. After 15 min, 2.4 mL of
TFA and 349 mg (1.96 mmol) of N-bromosuccinimide in 13.5 mL of CH2Cl2
were added sequentially. The reaction mixture was stirred for 30 min and
was then diluted with 10 mL of 1 M NaOH. The mixture was then
extracted with CH2Cl2, and the combined organic extracts were dried
(Na2SO4). The drying agent was removed and the solvent was evapo-
(R)-benzyl 2-benzamido-6,7-dihydro-5H-indeno[5,6-d]
thiazol-6-yl([2H5]ethyl)carbamate (14)
A solution of 600 mg (1.9 mmol) of [2H5]-3 in 5 mL of CH2Cl2 was stirred as
260 mL (2.0 mmol) of benzoyl isothiocyanate in 2 mL of CH2Cl2 was added.
After 45 min, 349 mg (1.96 mmol) of N-bromosuccinimide in 12 mL of
rated to give 76 mCi of 8 with a radiochemical purity of 41% (method CH2Cl2 was added dropwise over 20 min. The solution was stirred for
C). The major impurities consisted of 15% of bent isomer 8b and 24.5% 30 min and was then diluted with 10 mL of MeCN, and the solvent
of urea 9. Purification by HPLC using method b gave 24.6 mCi of 8 as a removed. An additional 10 mL of MeCN was added, and the resulting solid
white solid (radiochemical purity of 91% with a 9% impurity of urea 9 was collected to give 456 mg of 16 as a light yellow solid. HPLC analysis
using method C).
(method E) showed a UV area percent purity of >95.
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C. S. Elmore et al.
(R)-N-(6-([2H5]ethylamino)-6,7-dihydro-5H-indeno[5,6-d]
thiazol-2-yl)benzamide (15)
and was diluted with saturated NaHCO3 until no longer acidic. The mixture
was extracted with 4Â 50 mL of ethyl acetate, and the combined organic
extracts were washed with brine and then dried (Na2SO4). The organic layer
was filtered, and the filtrate concentrated to dryness to give 2.02 g of oil that
was purified by Kugelrohr distillation to give 1.03 g of 1-[13C]methyl [15N2]
pyrazole[13C]carboxaldehyde.
A slurry of 530 mL (2.6 mmol) of triisopropylsilane, 456 mg (0.96 mmol) of
14, and 7.9 mL of 33% HBr in HOAc was stirred for 2 h and was then
concentrated to remove any volatiles. The slurry was diluted with 8 mL
of Et2O, and the resulting white solid was removed to give 300 mg of
15 after air drying. HPLC analysis (method E) showed a UV area percent
purity of >90 with a 5% impurity of the bent isomer present.
6,7,8,9-tetrahydro-2-amino-7-((1-[13C]methyl-1H-[13N2]
pyrazol-4-yl)[13C]methyl)-5H-Thiazolo[4,5-h][3]benzazepine
([13C2, 15N2]-2)
(R)-N6-[2H5]ethyl-6,7-dihydro-5H-indeno[5,6-d]thiazole-2,6-
diamine ([2H5]-1)
A mixture of 0.77 g (2.63 mmol) of 6,7,8,9-Tetrahydro-5H-1-thia-3,7-diaza-
cyclohepta[f]inden-2-ylamine 2HCl salt, 300 mg (2.63 mmol) of 1-[13C]
methyl-1H--[15N2]pyrazole-4-[13C]carbaldehyde, 453 mg (5.52 mmol) of
NaOAc, and 963 mL (3.29 mmol) of titanium(IV) isopropoxide in 3 mL of
2-propanol was stirred for 1 h. The mixture was diluted with 12 mL
of MeOH and 330 mg (5.26 mmol) of NaBH3CN (0.330 g, 5.26 mmol)
was added. After stirring overnight, 10 mL of 10N NaOH was added
and the mixture was stirred for 10 min. The mixture was then filtered
through celite, and the filtrate was evaporated. The residue was parti-
tioned between water and CH2Cl2. The organic layer was separated,
and the aqueous layer was extracted again with CH2Cl2. The combined
CH2Cl2 layers were dried (MgSO4) and filtered, and the filtrate was
evaporated. The residue was purified by column chromatography on
basic alumina with MeOH/CH2Cl2 (0–5%) to give 670 mg of a yellow
solid that was further purified by SFC. The product containing fractions
were combined to give 640mg of [13C2, 15N2]-2 as a white solid (77%). 1H
NMR (2H6-DMSO, 500 MHz): d 7.50 (d, J = 4.5Hz, 1H), 7.31 (s, 1H), 7.29 (m, 3H),
7.06 (s, 1H), 3.76 (d, J = 140 Hz, 3H), 3.44 (d, J = 134 Hz, 2H), 2.80 (m, 4H), 2.48
(m, 4H). 13C NMR (2H6-DMSO, 150 MHz): d 166.0, 151.2, 139.6, 139.0,
135.1, 130 (dt, J = 4,5, 13 Hz), 128.0, 120.4, 117.9, 116.6 (dd, J = 52, 5),
54.8, 54.6, 52.5, 38.3 (dd, J = 55.2, 23.4 Hz), 35.6, 35.5. LC/MS, 318
(100%), 317 (6%).
A solution of 153 mg (0.45 mmol) of 15 in 3 mL of 48% aq HBr was heated
at 130 ꢀC for 6 h. The reaction mixture was diluted with 5 mL of MeCN
and was stirred for 10 min. A white solid formed and was collected by
filtration to give 100 mg after air drying. The solid was dissolved in
2 mL of water and was filtered to remove any undissolved solids. The
pH of the solution was adjusted to 10 by the dropwise addition of 10 M
NaOH, and the resulting white solid was collected by filtration. The solid
was washed with 5 mL of water and was then air dried to afford 40 mg of
a white solid. This material was deemed impure (UV area % of 93%,
method E) and was further purified twice by preparative HPLC using
method d. Fractions containing pure material were combined, basified
with aqueous NaHCO3 and extracted into EtOAc. The combined EtOAc
extracts were dried (MgSO4) and filtered, and the filtrate was concen-
trated to give 26 mg of a white solid and a purity of 98.8% UV area %
at 210 nm (method E). 1H NMR (2H6-DMSO, 500 MHz): d 7.40 (s, 1 h),
7.13 (s, 1H), 3.48 (m, 1H), 3.06 (m, 2H), 2.63 (m, 2H) LC/MS 239 (100%),
240 (15.5%), 241 (5.3%), 238 (1.5%).
[
15N2]Pyrazole
The synthesis of [15N2]pyrazole used a modification of a literature pre-
paration.17 A mixture of 21 g (0.16 mmol) of [15N2]hydrazine sulfate,
350 mL of EtOH, and 550 mL of water was stirred and heated at 75 ꢀC
for 1.5 h until complete dissolution occurred, and then 27 g (0.16 mmol)
of 1,1,3,3-tetramethoxypropane was added dropwise over 15 min. The
reaction was heated for a further 2 h and was then stirred at room tem-
perature for 24 h. The ethanol was removed, and the reaction mixture
was diluted with 300 mL of water. The solution was neutralized with
36 g of solid CaCO3 and was extracted with 5 Â 200 mL of ether. The
organic extracts were combined, dried (MgSO4) and filtered, and the fil-
trate was concentrated to dryness to afford 7.58 g of a pale yellow solid.
Acknowledgements
The authors would like to thank Jennifer Van Anda, John McCauley,
and David Coomber for purification support; James E. Hall for NMR
support; and James Hulsizer, Scott Lehr, Gary Moore, and Gary
Steelman for supplying chemical intermediates for the synthesis.
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