The synthesis of carbon-14 labeled (R)-4-(dipropylamino)-1,3,4,5- tetrahydrobenz[cd]indole-6-carboxamide hippurate. A partial ergoline with 5-HT1A agonist activity and an 125I-labeled analog

Article abstract of DOI:10.1002/jlcr.907

Partial ergoline agonists such as (R)-4-(dipropylamino)-1,3,4,5- tetrahydrobenz[cd]-indole-6-carboxamide (LY228729, 1a) mimic a locked conformational analog of serotonin and in fact possess potent in vitro activity as agonists of the 5-HT_1A receptor. In the course of pre-clinical investigation of la for potential use as an anxiolytic agent, 1b was prepared in a five step synthesis from K¹⁴CN. In addition, an ¹²⁵I-analog of 1a was prepared for aid in the development of a radioimmunoassay (RIA). Copyright

Full text of DOI:10.1002/jlcr.907

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2005; 48: 139–148.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.907
Research Article
The synthesis of carbon-14 labeled (R)-4-
(dipropylamino)-1,3,4,5-tetrahydrobenz[cd]indole-6-
carboxamide hippurate. A partial ergoline with 5-HT1A
agonist activity and an 125I-labeled analog
William J. Wheeler*, Steven P. Swanson, David L. Varie and Douglas D.
O’Bannon
Lilly Research Laboratories, A Division of Eli Lilly and Company, Lilly Corporate
Center, DC 1543, Indianapolis, IN 46285, USA
Summary
Partial ergoline agonists such as (R)-4-(dipropylamino)-1,3,4,5-tetrahydrobenz[cd]-
indole-6-carboxamide (LY228729, 1a) mimic a locked conformational analog of
serotonin and in fact possess potent in vitro activity as agonists of the 5-HT1A
receptor. In the course of pre-clinical investigation of 1a for potential use as an
anxiolytic agent, 1b was prepared in a five step synthesis from K14CN. In addition, an
125I-analog of 1a was prepared for aid in the development of a radioimmunoassay
(RIA). Copyright # 2005 John Wiley & Sons, Ltd.
Key Words: 5HT1A; carbon-14; radioiodinated; radioimmunoassay
Introduction
Flaugh et al. reported on the synthesis and pharmacological evaluation of a
number of racemic analogs of 1a.1 Martinelli et al. accomplished the first
synthesis of 1a, based on the diastereoselective epoxidation of N-benzoyl-
1,2,2a,3-tetrahydrobenzo[cd]indole followed by the regioselective ring opening
with S-phenethylamine.2 Mitsunobu reaction of the individual optically pure
aminoalcohols followed by hydrogenolysis and bromination yielded 2 (and its
enantiomer). Varie later reported on an elegant synthesis of 2 from readily
available l-tryptophan.3 Foreman et al. found 1a to be a potent and selective
5-HT1A receptor agonist both in vitro and in vivo.4 In the course of pre-clinical
development, 1b was required for drug disposition studies in laboratory
animals.5 LY228729 has been evaluated clinically for the treatment of anxiety
*Correspondence to: W. J. Wheeler, Eli Lilly and Company, Lilly Corporate Center, DC 1543, Indianapolis,
IN 46285, USA. E-mail: wheeler william joe dr@lilly.com
Copyright # 2005 John Wiley & Sons, Ltd.
Received 21 September 2004
Revised 11 October 2004
Accepted 21 October 2004

140
W. J. WHEELER ET AL.
and depression. The low doses administered during phase I studies, required a
sensitive method for bioanalysis.6 For use in the development of a sensitive
radioimmunoassay (RIA) (6-iodo-1,3,4,5-tetrahydrobenz[cd]indol-4-yl)dipro-
pylamine-[125I] (3c) was required. Preliminary reports of the synthesis and use
of 3c have been published;6ꢀ8 further details of the syntheses of 1b and 3c are
reported herein.
Br
X
R
N(n-Pr)2
N(n-Pr)2
N(n-Pr)2
H
N
HN
HN
Bz
3a; X = Br
3b; X = I
3c; X = 125
1a; R = CONH2
1b; R = 14CONH2
2
I
Discussion
In an effort to prepare 1a in one step, 3a was deprotonated with KH/Et2O,
reacted with t-BuLi and then quenched by the addition of trimethylsilyliso-
cyanate to yield 1a (23%) after chromatography. Owing to the poor yield in
this reaction, coupled with the absence of a ready source of C-14-labeled
trimethylsilylisocyanate, we looked to alternative routes. Reaction of 3a
(Scheme 1) with NaCN/Pd(Ph3P)4 in refluxing THF or CuCN/ Pd(OAc)2 in
refluxing DMF at 1108C was unsuccessful. Reaction of 3a with CuCN in
CONH2
Br
1. KH, Et2O
2. t-BuLi, -78oC
N(n-Pr)2
N(n-Pr)2
3. TMS-NCO
4. H3O+, 23%
HN
HN
1a
3a
KCN, Pd(Ph3P)4
THF or
CuCN, Pd(OAc)2
DMF
CN
N(n-Pr)2
HN
4
Scheme 1.
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THE SYNTHESIS OF
141
N-methyl-pyrrolidinone at 1108C yielded some of the desired material 4, but
was very messy with multiple spots seen on TLC. Martinelli et al. had
significantly better results in the reaction of 2 with CuCN (1.2 equivalents) at
2008C to yield 5a in 76%.2 Sakakibara et al. have reported success with
Rosemund–von Braun-type reactions using KCN and NiBr2(Ph3P)2/Zn/Ph3P
in lower boiling solvents (THF or CH3CN).9 In our hands, the best results in
the reaction of 2 with NiBr2(Ph3P)2/Zn/Ph3P to yield 5a were obtained using
CH3CN as the solvent (Scheme 2). Reaction of 2 with K14CN in the presence
of NiBr2(Ph3P)2/ Zn/Ph3P in refluxing acetonitrile gave 5b in 51% yield. The
N-benzoyl protecting group was removed by reaction with n-BuLi/THF at
ꢀ788C, followed by an acetic acid quench to yield 6 (56%). Aromatization was
effected by reaction of 6 with MnO2/HOAc/CH2Cl2 to yield 7 in 58% yield.
The nitrile 7 was added to neat polyphosphoric acid which was pre-heated to
78–828C and heated for 2 h. The reaction mixture was quenched with water to
afford 1b in 74% yield after chromatography. An acetone solution of 1b was
added dropwise to an acetone solution of hippuric acid at 558C. Upon cooling
X
Br
K14CN, Ph3P
N(n-Pr)2
N(n-Pr)2
Zn, NiBr2(Ph3P)2
CH3CN, heat
51%
H
H
N
N
Bz
Bz
5a; R = CN
5b; R = 14CN
2
1. n-BuLi, THF
-78oC
2. HOAc, 56%
14CN
14CN
MnO2, HOAc
N(n-Pr)2
N(n-Pr)2
CH2Cl2, 0oC
58%
H
HN
HN
7
6
1. PPA
2. H2O, 74%
3. hippuric acid. acetone
62%
14CONH2
N(n-Pr)2
HN
1b (as the hippurate salt)
Scheme 2.
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W. J. WHEELER ET AL.
to room temperature, a white solid crystallized, which was collected by
filtration and dried in vacuo to yield 1b hippurate (85%). A mixture of 1a
hippurate and 1b hippurate was re-crystallized twice from acetone to yield 1b
hippurate (62%); specific activity=13.79 mCi/mg (6.63 mCi/mmol).
Initial attempts to prepare a suitable tin or borate precursor for the
preparation of 3c from (6-iodo-1,3,4,5-tetrahydrobenz[cd]indol-4-yl)dipropyl-
aminey (3b) were unsuccessful (reaction of 3b with hexamethyldistannane/
Pd(Ph3P)4/THF, n-BuLi/(CH3)3SnCl, n- or t-BuLi/(i-PrO)3B). Reaction of 3b
with KH/THF followed by reaction of the resulting anion with TIPS triflate
yielded 8 (Scheme 3). Treatment of 8 with t-BuLi/PhCH3/ TMEDA at ꢀ708C
followed by reaction with chlorotrimethylstannane ((CH3)3SnCl) yielded 9
(57%). Faced with the unlikely prospect of removing the TIPS protecting
group in 9 without affecting the (CH3)3Sn- moiety, 3b was reacted with 10
equivalents of t-BuLi/TMEDA/PhCH3 at ꢀ708C, followed by reaction of the
resulting lithiated species with (CH3)3SnCl to afford 10 in 68% yield after
chromatography and crystallization from CH3OH/H2O.
Treatment of 10 with I2/CH2Cl2 afforded 3b as a single spot on TLC (silica
gel, pentane/Et2O/Et3N, 70:30:1) (Scheme 4). Reaction of 10 with Na125I/
EtOH in the presence of chloramine T yielded 3c after purification on HPLC
(specific activity 1800 Ci/mmol).z
I
Sn(CH3)3
Sn(CH3)3
1. t-BuLi, PhCH3
TMEDA, -70oC
1. KH, THF
N(n-Pr)2
N(n-Pr)2
N(n-Pr)2
2. TIPS triflate
2. (CH3)3SnCl
3. t-BuLi, PhCH3
TMEDA, -78oC
4. (CH3)3SnCl
HN
N
HN
TIPS
3b
10
9
Scheme 3.
125I
Sn(CH3)3
I
N(n-Pr)2
N(n-Pr)2
N(n-Pr)2
Na125I, EtOH
chloramine T
I2, CH2Cl2
HN
HN
HN
11
6
8
Scheme 4.
y The (S)-dipropyl-(6-iodo-1,3,4,5-tetrahydrobenzo[cd]indol-4-yl)amine (8) was a generous gift from Dr
Michael E. Flaugh of the Lilly Research Laboratories, A Division of Eli Lilly and Company, Indianapolis,
IN 46285.
z The preparation and purification of (S)-dipropyl-(6-iodo-1,3,4,5-tetrahydrobenzo[cd]indol-4-yl)amine-
[
125I] (3c) was conducted at Amersham International plc, Whitchurch, Cardiff, Wales, UK using methods
supplied by the authors.
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THE SYNTHESIS OF
143
Results
C-14-labeled 1b (as its hippurate salt) was prepared in five radiochemical steps
in 7.6% overall yield; K14CN was the source of the radioactivity. The specific
activity was 13.79 mCi/mg (6.63 mCi/mmol); the radiochemical purity was
98.3% by HPLC and 98–99.3% by TLC. This material co-eluted with
authentic 1a in three TLC systems and by HPLC. A method has been
developed for the radioiodination of 10; 3c was prepared with a specific
activity of 1800 Ci/mmol; the radiochemical purity was 98% (with 0.3%
iodine). In support of phase I clinical trials of 1a, which was evaluated for use
in the treatment of depression and anxiety, the use of 3c enabled optimal
sensitivity in the RIA of 50 pg/ml. The specificity of the RIA was maximized
by the use of a rabbit anti-LY228729 antiserum that displayed low cross
reactivity with mono-N-des-propyl-LY228729 (a metabolite found in the
plasma).6
Experimental
NMR spectra were obtained on a Varian Mercury Plus 400 mHz nuclear
magnetic resonance spectrometer. Chemical shifts are reported in parts per
million (ppm) downfield from tetramethylsilane. Electrospray mass spectro-
metry was conducted on a Waters Micromass ZQ single quadropole mass
spectrometer. Flash chromatography was performed on silica gel. Radio-
chemical purity (RCP) was assessed by HPLC with radiochemical detection.
Thirty minute fractions were collected, diluted with scintillation fluid and
counted on a Packard Scintillation Spectrometer. RCP was further assessed by
TLC-autoradiography. The radioactivity was detected on X-ray film, the lanes
of the TLC were cut, mixed with methanol and scintillation fluid and counted.
The following TLC systems were used: A (CHCl3/EtOH/NH4OH, 90:10:10
drops), B (CHCl3/acetone/CH3OH/NH4OH, 63:27:7:2) and C (CHCl3/
CH3OH/HOAc, 18:6:1).
(-)-4-(R)-(dipropylamino-1,3,4,5-tetrahydrobenz[cd]indole-6-carboxamide, 1a
Potassium hydride (25% mineral oil dispersion, 0.057 g, 0.35 mmol) was
added to a flask under argon and washed with hexanes (3 ꢁ 10 ml) to
remove the oil. Ether (8 ml) was added and the resulting suspension cooled to
08C and then treated with 3a (0.100 g, 0.298 mmol) in Et2O (1.5 ml). The
reaction mixture was stirred for 15 min and then allowed to warm to room
temperature and stirred for an additional 2 h. The mixture was then
chilled to ꢀ788C and treated by the dropwise addition of t-BuLi (1.6 M,
0.475 ml, 0.76 mmol). Stirring was continued at ꢀ788C for 15 min, then at
ꢀ408C for 2 h. After cooling back to ꢀ788C, the mixture was treated
with TMS-isocyanate (0.058 ml, 0.358 mmol), allowed to warm to ꢀ108C
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W. J. WHEELER ET AL.
and stirred for 2 h. Water was added and after a 15 min stir, the layers were
separated. The aqueous layer was extracted with Et2O and the combined
organic extracts were concentrated in vacuo. The residual brown oil was
dissolved in 3 N HCl (4 ml) and washed with Et2O; the pH of the aqueous layer
was adjusted to 10 with 5 N NaOH and extracted with Et2O (4 ꢁ 20 ml).
The combined Et2O extracts were dried (anhydrous Na2SO4) and concentrated
in vacuo. The brown semi-solid residue was purified by chromatography
over silica gel, eluting with PhCH3/ EtOAc/CH3OH/NH4OH (50:50:2:0.5)
to yield 1a (0.022 g, 25%) after crystallization from Et2O. CI-MS (NH3):
M+, m/z=299; 1H-NMR (CDCl3) d 0.91 (t, 6H, CH3), 1.48 (dt, 4H,
CH2Me), 2.57 (t,1H, CH2Et), 2.80 (t, 1H, 3a-H), 2.98 (qt, 1H, 3b-H), 3.02
(t, 1H, 5a-H), 3.22 (m, 1H, 4b-H), 3.54 (qt, 1H, 5b-H), 5.72 (bs, 2H, NH2),
6.90 (s, 1H, 2-H), 7.15 (d, 1H, 8-H), 7.45 (d, 1H, 7-H), and 8.04 (s, 1H, indole-
NH).
(+)-(2aR,4S)-1-benzoyl-4-(dipropylamino)-1,2,2a, 3,4,5,-hexahydrobenz-[cd]-
indole-6-carbonitrile-[nitrile-14C], 5
In a flame-dried flask, filled with argon, (+)-1-benzoyl-6-bromo-N,N-
dipropyl-1,2,2a,3,4,5-hexahydrobenz-[cd]-indol-4-amine (2, 2.03 g, 4.61 mmol)
was mixed with potassium cyanide-[14C] (100 mCi, 21.7 mCi/mmol,
4.61 mmol), potassium cyanide (0.1498 g, 2.31 mmol), nickel (II) bromide-
bis-triphenylphosphine (0.341 g, 0.461 mmol), zinc (0.092 g, 1.38 mga), and
triphenylphosphine (0.2398 g, 0.922 mmol). The mixture was diluted with
acetonitrile (25 ml) and the resulting emerald green solution was stirred under
reflux for 7 h (the color gradually changed to orange-brown). TLC (hexanes/
EtOAc/Et3N 65:35:5) indicated that the reaction was not complete, so stirring
under reflux was continued overnight. Additional nickel (II) bromide-bis-
triphenylphosphine (0.341 g, 0.461 mmol), zinc (0.092 g, 1.38 mga), and
triphenylphosphine (0.2398 g, 0.922 mmol) were added and stirring at reflux
was continued for an additional 24 h. TLC still showed a small amount of
unreacted 2. The solvent was removed in vacuo and the residue was partitioned
between ethyl ether (60 ml) and concentrated NH4OH (25 ml) which was
diluted with ice. The aqueous layer was re-extracted with ether (25 ml) and the
combined ether extracts were washed successively with NH4OH and brine
(2 ꢁ 25 ml). The ether was dried (anhydrous MgSO4) and concentrated
in vacuo. The residue was purified by flash chromatography on silica gel 60,
eluting with 20 ml fractions of hexanes/EtOAc/Et3N 65:35:5. Fractions
14–19 were combined and concentrated to yield 0.908 g, (51%) of 5b as a
yellow oil, which crystallized upon standing. This material was identical to 5a
by TLC.
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THE SYNTHESIS OF
145
(-)-(2aR,4S)-(dipropylamino)-1,2,2a,3,4,5-hexahydro-benz-[cd]-indole-6-car-
bonitrile -[nitrile -14C], 6
A THF solution (10 ml) of 5b (0.908 g, 2.35 mmol) was cooled to ꢀ728C (dry
ice/2-propanol) under argon and treated dropwise with n-butyllithium (1 M,
2.35 ml, 2.35 mmol). Stirring was continued for 15 min (no starting material
remained by TLC, hexanes/EtOAc/Et3N 65:35:5) and the excess n-butyl-
lithium was quenched by the careful dropwise addition of acetic acid (0.25 ml).
The mixture was allowed to warm to room temperature and then extracted
with 1 N HCl (10 ml, then 5 ml additional) and water (2 ꢁ 10 ml). The
combined aqueous extracts were made basic with 1 N NaOH and extracted
with methylene chloride (2 ꢁ 50 ml, 2 ꢁ 15 ml). The combined organic extracts
were washed with brine (25 ml), dried (anhydrous Na2SO4), and concentrated
in vacuo. The crude residue was crystallized from ethanol/water (15 ml/5 ml),
then concentrated to 10 ml) to yield 6 as a white crystalline solid (0.3749 g,
56.4%) which co-migrated as a single spot with an authentic sample of
unlabeled 6 on TLC (hexanes/EtOAc/Et3N 65:35:5).
(-)-4-(S)-(dipropylamino)-1,3,4,5-tetra-hydrobenz-[cd]-indole-6-carbonitrile-
[nitrile-14C], 7
A methylene chloride solution (2.5 ml) of 6 (0.3749 g, 1.325 mmol) was diluted
with glacial acetic acid (2.5 ml) and chilled to ꢀ5 to 08C in an ice bath. To the
stirred solution was added manganese dioxide (0.173 g, 1.987 mmol) and
stirring was continued for 5 h. TLC (hexanes/EtOAc/Et3N 65:35:5) showed
unreacted starting material remaining; an additional 0.025 g of manganese
dioxide was added and the mixture was stirred for two more hours. Starting
material still remained by TLC, so the mixture was stored at ꢀ208C overnight.
The reaction mixture was diluted with methylene chloride (5 ml) and
filtered through a bed of talc. The filter cake was washed with methylene
chloride (5 ꢁ 10 ml). The combined filtrates were diluted with water
(10 ml) and the aqueous made basic by the dropwise addition of 5 N
NaOH (15 ml). The aqueous layer was washed with methylene chloride
(2 ꢁ 25 ml) and the combined methylene chloride extracts were washed
with brine (50 ml) and dried (anhydrous Na2SO4). Concentration in vacuo
yielded a crude product which was purified by flash chromatography.
The material was eluted with 10 ml fractions of chloroform (fractions 1–27)
and then 20:1 chloroform/methanol (fractions 28–50). Fractions 30–37
were combined and concentrated; the residue was crystallized from
hexane/EtOAc to yield 7 (0.216 g, 58%) as a white solid which was a single
component by TLC (EtOAc/PhCH3 6:4), co-migrating with authentic
unlabeled 7.
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(-)-4-(S)-(dipropylamino-1,3,4,5-tetrahydrobenz[cd]indole-6-carboxamide-
[carbonyl-14C], 1b
Polyphosphoric acid (10 g) was warmed to 788C and stirred while 7 was added
portionwise. The sides of the flask were washed down with toluene (2 ml) and
stirring was continued at 78–828C for 2 h, whereupon the reaction mixture was
poured into ice and made basic with 5 N NaOH. The resulting mixture was
extracted with methylene chloride (5 ꢁ 35 ml) and the combined extracts were
washed with brine, then dried (anhydrous Na2SO4). The crude product was
purified by flash chromatography eluting with 10 ml fractions of 8:2 ethyl
acetate/methanol. Fractions 8–16 were combined and concentrated in vacuo to
yield 1b as an amorphous foam (0.170 g). TLC (9:1 EtOAc/CH3OH) showed a
single spot which co-migrated with 1a.
(-)-4-(S)-(dipropylamino-1,3,4,5-tetrahydro-benz-[cd]-indole-6-carboxamide-
[carbonyl-14C] hippurate salt, 1b hippurate
An acetone solution (1.75 ml) of 1b (0.16992 g, 0.568 mmol) was added
dropwise to a mixture of hippuric acid (0.102 g, 0.568 mmol) and acetone
(5.25 ml) which was stirred at 508C. The mixture was allowed to cool to room
temperature whereupon a white precipitate formed which was collected by
filtration, washed with acetone, and dried to yield 1b hippurate (0.230 g).
A mixture of 1b hippurate (0.17276 g) and 1a hippurate (0.17276 g) was
recrystallized twice from 2-propanol/water (90:10) (3.1 ml) to yield diluted
LY228729-[14C] hippurate (0.21928 g, 50.7%), specific activity 13.79 mCi/mg.
The 1b hippurate co-eluted with 1a hippurate in the following TLC systems;
the radiochemical purity was determined by TLC-autoradiography:
CHC13=EtoH=NH4OH 90 : 10 : 10 drops
CHCl3=acetone=MeOH=NH4OH 63 : 27 : 7 : 2 99:32% rf ¼ 0:752
CHCl3=MeOH=HOAc 18 : 6 : 1 97:98% rf ¼ 0:442
99:33% rf ¼ 0:489
HPLC on a Jones C-8 column (4.6 ꢁ 250 mm, 5 m), eluting at 0.8 ml/min with
CH3CN/0.03 M KH2PO4 (60:40 at pH=3) and fluorescence (290 nm em,
370 nm ex) and UV detection (290 nm) showed a single peak (with the
exception of the hippuric acid) co-eluting with authentic material;
RCP=98.3%, RT=6.5 min.
(S)-dipropyl-(6-trimethylstannanyl-1,3,4,5-tetrahydrobenzo[cd]indol-4-yl)a-
mine, 10
A toluene (13 ml) solution of 3a (0.500 g, 1.31 mmol) under argon was treated
with TMEDA (0.25 ml, 1.71 mmol) and chilled to ꢀ708C. To this solution was
added t-BuLi (1.6 M in pentane, 8.2 ml, 13.12 mmol) and the resulting yellow
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THE SYNTHESIS OF
147
mixture was stirred at ꢀ708C for 15 min. Chlorotrimethylstannane (2.61 g,
13.1 mmol) in toluene (7 ml) was added to the solution dropwise. The solution
was allowed to warm slowly to room temperature and then stirred overnight.
The solution was added to water (5 ml) and extracted with Et2O (3 ꢁ 20 ml).
The combined organic layers were washed with brine, dried (anhydrous
MgSO4) and concentrated in vacuo. The crude product was purified by flash
chromatography on silica gel, eluting with 50 ml fractions of pentane/Et2O/
Et3N (70:30:1). Fractions 14–22 were combined and concentrated in vacuo to
yield 10 (0.485 g, 89%). NMR and mass spectrometry indicated contamination
with the reduced product. The solid was re-crystallized from methanol/water
to yield pure 10 as a white crystalline solid (0.157 g, 27%) (TLC (silica gel,
pentane/Et2O/Et3N, 70:30:1) showed a single spot at rf=0.25 (rf(3b)=0.17).
ES-MS: [M+H]+, m/z=421 (significant isotope peaks plus H at 417, 418, 419,
420, 422, 423 and 425); 1H-NMR (CDCl3) d 0.08 (s, 9H, Sn(CH3)3 plus Sn–H
coupling), 0.64 (t, 6H, CH3), 1.22 (m, 4H, CH2Me), 2.31 (m, 4H, CH2Et), 2.52
(t, 1H, 3-H), 2.73 (m, 3H, 3-H and 5-H), 3.02 (m, 1H, 4-H), 6.53 (s, 1H, 2-H),
6.94 (m, 2H, 7-H and 8-H), 7.50 (bs, 1H, NH).
HR-QTOF-MS: Analysis calculated for C20H32N2Sn: 417.1661. Found:
417.1669.
(S)-dipropyl-(6-iodo-1,3,4,5-tetrahydrobenzo[cd]indol-4-yl)amine, 3b
A methylene chloride (5 ml) solution of 10 (0.041 g, 0.1 mmol) was reacted with
iodine (0.025 g, 0.1 mmol) and stirred for 2 h. TLC (silica gel, pentane/Et2O/
Et3N; 70:30:1) showed quantitative conversion to 8. This material was
1
identical to an authentic sample of 3b. ES-MS: [M+H]+, m/z=388; H-
NMR (CDCl3) d 0.92 (t, J=7.05 Hz, 6H, CH3), 1.49 (m, 4H, CH2Me), 2.57
(m, 5H, CH2Et, 3a-H), 2.80-2.97 (m, 2H, 5a-H, 3b-H), 3.01 (m, 1H, 5b-H),
3.24 (m, 1H, 4b-H), 6.85 (s, 1H, 2-H), 6.95 (d, J=8.4 Hz), 1H, 8-H), 7.45 (d,
J=8.4 Hz, 1H, 7-H) and 7.91 (bs, 1H, NH).
(S)-dipropyl-(6-iodo-1,3,4,5-tetrahydrobenzo[cd]indol-4-yl)amine-[125I], 3c
An ethanol (0.1 ml) solution of 10 (0.1 g) was mixed with aqueous NaI-[125I]
(0.1 ml, 10.0 mCi) and treated with Na Chloramine T (0.050 ml, 5 mg/ml in
water). The mixture was allowed to react for 5 min and then treated with
aqueous NaHSO3 (0.05 ml, 10 mg/ml) to terminate the reaction. The crude
reaction mixture was purified on a Aquapore C-18 column (4.6 ꢁ 250 mm) by
gradient elution (40%B to 55%B over 30 min) at a flow rate of 1 ml/min.
Solvent A=0.1 M NH4Oac at pH 5.0, solvent B=CH3OH. The product eluted
at 17 min. The total yield (6.1 mCi) was diluted to 100 mCi/ml and stored at
ꢀ208C. The radiochemical purity was determined by HPLC on a Vydac
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Protein C18 column by gradient elution (30%B to 40%B over 20 min) to yield
3c. Solvent A=water with 0.1% TFA, solvent B=propanol with 0.1% TFA.
Acknowledgements
The authors appreciate the support of Ms Paula Santa (Eli Lilly and
Company) and Mr Steven Gabriel (formerly of Eli Lilly and Company) in the
preparation of this manuscript.
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