Synthesis and evaluation of novel pyridine based PLG tripeptidomimetics

Article abstract of DOI:10.1039/b718058f

Analogues of the pyridine based PLG (Pro-Leu-Gly-NH₂) peptidomimetic 1 were synthesized and evaluated as dopamine modulating agents. Modifications in the position corresponding to the leucine side chain in PLG afforded derivatives 2, 3 and 4, substituted with H, Me and Bn instead of the isobutyl group, respectively. Changes in the proline residue produced derivative 5, substituted with a symmetrical piperidine ring instead of the pyrrolidine ring and 6, in which the pyrrolidine ring is connected to the pyridine ring via a hydroxymethyl group instead of a keto function. The peptidomimetics were tested for their ability to enhance the maximal effect of N-propylapomorphine (NPA) at dopamine D2 receptors in the functional cell-based R-SAT assay. Compounds 2, 3, and 4, produced a statistically significant increase in the maximal NPA response at 10 nM (117 ± 6%, 118 ± 6%, and 116 ± 3%, respectively), which is similar to the effect of PLG in this assay, whereas 5 was able to potentiate the response to a similar extent at 1 nM concentration (115 ± 5%). All derivatives produced a bell-shaped dose-response curve and none of the compounds were active at the D2 receptor alone, which indicates that the mechanism behind the activity of both the pyridine based mimetics 1-6 and PLG is the same. Interestingly, l-Pro-d-Leu-Gly-NH₂ was found to be more potent than PLG and produced a 119 ± 1% increase in the NPA response at 1 nM. The Royal Society of Chemistry 2008.

Full text of DOI:10.1039/b718058f

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and evaluation of novel pyridine based PLG tripeptidomimetics†
Stina Saitton,‡a,b Andria L. Del Tredici,c Maria Saxin,a,b Tobias Stenstro¨m,b Jan Kihlberg‡b and
Kristina Luthman*a
Received 21st November 2007, Accepted 11th February 2008
First published as an Advance Article on the web 17th March 2008
DOI: 10.1039/b718058f
Analogues of the pyridine based PLG (Pro-Leu-Gly-NH2) peptidomimetic 1 were synthesized and
evaluated as dopamine modulating agents. Modifications in the position corresponding to the leucine
side chain in PLG afforded derivatives 2, 3 and 4, substituted with H, Me and Bn instead of the isobutyl
group, respectively. Changes in the proline residue produced derivative 5, substituted with a
symmetrical piperidine ring instead of the pyrrolidine ring and 6, in which the pyrrolidine ring is
connected to the pyridine ring via a hydroxymethyl group instead of a keto function. The
peptidomimetics were tested for their ability to enhance the maximal effect of N-propylapomorphine
(NPA) at dopamine D2 receptors in the functional cell-based R-SAT assay. Compounds 2, 3, and 4,
produced a statistically significant increase in the maximal NPA response at 10 nM (117 6%, 118
6%, and 116 3%, respectively), which is similar to the effect of PLG in this assay, whereas 5 was able
to potentiate the response to a similar extent at 1 nM concentration (115 5%). All derivatives
produced a bell-shaped dose–response curve and none of the compounds were active at the D2 receptor
alone, which indicates that the mechanism behind the activity of both the pyridine based mimetics 1–6
and PLG is the same. Interestingly, L-Pro-D-Leu-Gly-NH2 was found to be more potent than PLG and
produced a 119 1% increase in the NPA response at 1 nM.
ability.8,18,21 Therefore, the synthesis and evaluation of compounds
Introduction
2, 3 and 4, in which the isobutyl side chain of leucine was replaced
PLG (Pro-Leu-Gly-NH2) is an endogenous peptide found in the
with a hydrogen, a methyl or a benzyl group, respectively, was
central nervous system.1 It is known to exert its pharmacological
undertaken. Modifications in the proline residue of PLG have
effect(s) through the modulation of dopamine D2 receptors.2,3
Previous studies have shown that PLG enhances the effect of
dopamine agonists at D2 receptors in a biphasic dose-dependent
manner to give a bell-shaped dose–response curve.4 A number
of active PLG mimetics, ranging from peptide analogues5–12 to
constrained peptidomimetics,5,13–23 have been presented in the
literature. Recently, we reported the development of a 2,3,4-
substituted pyridine scaffold for tripeptidomimetics and its use in
the synthesis of 1, a potent PLG mimetic as found in the functional
R-SAT assay (Fig. 1).24
also been shown to have a significant influence on the activity
of PLG analogues.7 Two analogues of 1 with modified proline
residues were investigated, the achiral piperidine derivative 5 and
the alcohol 6.
In this study, peptidomimetics 2–6 were designed in order to
further investigate the structural requirements for the dopamine
modulating effects of compounds based on the pyridine scaffold
Fig. 1 PLG and the pyridine based peptidomimetic 1.
(Fig. 2). Studies using peptide analogues and peptidomimetics
of PLG have shown that variations in the side chain of the
leucine residue can have a profound effect on the mimicking
aGo¨teborg University, Department of Chemistry, Medicinal Chemistry, SE-
412 96, Go¨teborg, Sweden. E-mail: luthman@chem.gu.se; Fax: +46-31-772
3840; Tel: +46-31-772 2894
bUmea˚ University, Department of Chemistry, Organic Chemistry, SE-901
87, Umea˚, Sweden
cACADIA Pharmaceuticals Inc., 3911 Sorrento Valley Boulevard, San
Diego, CA, 92121, USA
† Electronic supplementary information (ESI) available: NMR-spectra
of biologically evaluated compounds, and synthetic procedures and
characterization data for compounds 3, 5, 6, 9b, 10b, 11b, 12b, 15, 16,
18, 19, 20, 21 and 22. See DOI: 10.1039/b718058f
‡ Present address: AstraZeneca R&D Mo¨lndal, SE-431 83 Mo¨lndal,
Sweden.
Fig. 2 Synthesized and evaluated analogues of 1.
Org. Biomol. Chem., 2008, 6, 1647–1654 | 1647
This journal is
The Royal Society of Chemistry 2008
©

View Article Online
derivatives 2 and 3. Thus, in the synthesis of 8a and 8b, the isomeric
mixtures were not separated and the subsequent four reaction steps
were performed on the diastereomeric mixtures.
Results and discussion
Synthesis
Protection of the secondary alcohols as benzyl ethers was
accomplished by reaction with sodium hydride and benzyl bro-
mide to provide 9a–c in 77–87% yield. Introduction of the C-
terminal glycolamide◦moiety was performed using KH as the
base in DMSO at 55 C, in a nucleophilic aromatic substitution
reaction. The reaction proceeded in good to moderate yield for the
methyl- and benzyl-derivatives to afford 10b and 10c (80 and 53%,
respectively). A different reactivity was observed for 9a which lacks
a substituent ortho to the site of substitution. Here the reaction
with glycolamide was found to be complete after only two hours, as
compared to days for the other derivatives, and two products, the
wanted 10a and the byproduct 13 were isolated in almost equal
amounts, 43% and 48%, respectively. Attempts to improve the
selectivity by performing the reaction at a lower temperature (room
temperature) were not successful. The formation of a byproduct
resulting from the nucleophilic attack of the amide instead of
the alcohol functionality has previously been reported for the
corresponding 3-methallyl derivative when the reaction was run
at a higher temperature (80 ◦C).24 However, the selectivity barrier
for 9a seems to be considerably lower than for the 3-substituted
derivatives.
Tripeptidomimetics 2, 3, 4 and 5 were synthesized according to
a previously published strategy for 1.24 The synthesis is based
on a functionalized pyridine scaffold with the side-chain of the
second amino acid of the tripeptide attached to the 3-position of
the pyridine ring, and with fluorine and iodine as handles in the
neighboring 2- and 4-positions, respectively (Fig. 3). The N- and
C-terminal residues are attached to the functionalized scaffold via
a Grignard reaction utilizing the iodine handle, and a nucleophilic
substitution reaction of fluorine, respectively.
Fig. 3 Building blocks for assembly of the pyridine based peptidomimet-
ics 2, 3 and 4.
The synthesis of the PLG mimetics with modifications in the
Leu-residue, compounds 2, 3 and 4, is described in Scheme 1.
The functionalized scaffolds 7a–c were synthesized from 2-fluoro-
pyridine in 71–89% yield as previously described.25 Attachment
of Boc-protected proline aldehyde via a Grignard reaction using
iPrMgCl to accomplish an iodine–magnesium exchange in the 4-
position of the pyridine ring proceeded in 80–86% yield. To allow
careful control of each reaction step using NMR-spectroscopy
the diastereomeric alcohols of 8c (dr 1 : 9, calculated on isolated
yields) were separated, and the reaction sequence continued with
the major isomer. As no problems were identified in the synthesis
of ketone 4 it was considered most efficient to avoid separation
of the diastereomeric alcohols in the synthesis of the H- and Me-
The secondary alcohols 10a–c were deprotected by catalytic
hydrogenation with Pd/C to afford 11a–c in 74–98% yield. Oxi-
dation of the secondary alcohols using Dess–Martin periodinane
in CH2Cl2 provided 12a–c in 61–86% yield. Also in this step, the
unsubstituted derivative 12a was found to differ from the others.
When allowing the reaction to run overnight, 12a was isolated
in a poor 21% yield from a mixture with the 2-pyridone 14. The
formation of this byproduct could however be avoided simply by
shortening the reaction time to one hour, whereby the yield of
12a was improved to 61%. In the final step, Boc-deprotection of
the pyrrolidine nitrogen was accomplished by treatment with 25%
trifluoroacetic acid in CH2Cl2 for 5–7 minutes, which allowed 2, 3
and 4 to be isolated in 53–83% yield after purification by reversed-
phase preparative HPLC. During the characterization of the TFA
salt of 2 by NMR spectroscopy in MeOH-d4, it was found to
exist in equilibrium between the keto and enol tautomers. The
1
formation of the enol could actually be followed during the H
NMR spectral analysis, where the peaks corresponding to the
enol form increased in intensity over time at the expense of the
keto isomer, until an approximately 1 : 1 ratio was obtained.
By analysis of the free amine in CDCl3, no keto isomer could
be detected, instead an equilibrium between the E- and Z-enol
isomers was observed. This finding was also supported by the lack
of optical activity of 2. The reason for this unusual behavior of
the unsubstituted derivative has not been studied in more detail,
but a possible explanation could be that the lack of a substituent
at the 3-position of the pyridine ring permits coplanarity between
the keto–enol functionality and the aromatic ring which favors
formation of an enol. The 3-substituted derivatives 3 and 4 did
not show the same tautomerization in a polar solvent. The optical
Scheme 1 Synthesis of mimetics 2, 3 and 4 modified at the residue corre-
sponding to leucine. Reagents and conditions: (i) iPrMgCl, Boc-Pro-CHO,
THF, rt; (ii) BnBr, NaH, cat. Bu4NI, THF, rt; (iii) glycolamide, KH,
DMSO, 55 ◦C; (iv) H2, cat. Pd/C, THF or EtOAc, rt; (v) Dess–Martin
periodinane, CH2Cl2, rt; (vi) TFA, CH2Cl2, rt.
1648 | Org. Biomol. Chem., 2008, 6, 1647–1654
This journal is
The Royal Society of Chemistry 2008
©

View Article Online
activity of the methyl derivative 3 was followed over time in MeOH,
and it was found to be stable over 3 days at [a]D = −37.
Aldehyde 16, required for the synthesis of peptidomimetic 5,
was obtained from N-Boc-isonipecotic acid by reduction to the
alcohol 15 (81% yield) followed by oxidation with Dess–Martin
periodinane (82% yield) (Scheme 2). The aldehyde was used
directly in the subsequent Grignard reaction. The synthesis of
5 was accomplished via a route similar to that described for 2–4
above (Scheme 3).
6% at 10 nM).24 Compound 5 was found to produce its highest
effect at 1 nM (115 5%), whereas the response by compound 6
was not statistically significant due to its large variability (114
7%; p >0.05). In addition to the synthesized compounds, the
peptide L-Pro-D-Leu-Gly-NH2 (23) was evaluated as a dopamine
potentiator and found to produce a significant enhancement in the
NPA response at both 0.1 nM (116 3%) and 1 nM (119 1%)
(Fig. 4).
Scheme 2 Synthesis of N-Boc-isonipecotic aldehyde 16. Reagents and
conditions: (i) isobutylchloroformate, N-methylmorpholine, NaBH4, THF,
−20 ◦C; (ii) Dess–Martin periodinane, CH2Cl2, rt.
Fig. 4 Potentiation of the NPA response at the D2 receptor by mimetics
2, 3, 4, 5, 6 and 23. Data represent the percentage of the maximum NPA
response for NPA alone, achieved in the presence of the respective mimetic.
Shown is the average and standard error of between three and ten separate
experiments, each carried out 12 times. Paired t-tests indicate significant
differences from the values for NPA alone (* = p <0.05; ** = p <0.01). #
n = 1.
Compounds 1–6 were also tested for agonist activity at the
D2 receptor. The dopamine agonist NPA was used as a positive
control. None of the peptidomimetics were active at the D2
receptor at concentrations between 0.01 and 10 lM. The results
for mimetics 1, 5 and 6 are shown in Fig. 5.
Scheme 3 Synthesis of mimetic 5 with a modified residue corresponding
to proline. Reagents and conditions: (i) iPrMgCl, 16, THF, rt; (ii) BnBr,
NaH, cat. Bu4NI, THF, rt; (iii) glycolamide, KH, DMSO, 55 ◦C; (iv) H2,
cat. Pd/C, rt; (v) Dess–Martin periodinane, CH2Cl2, rt; (vi) TFA, CH2Cl2,
rt.
Peptidomimetic 6 was synthesized in 56% yield from the
corresponding Boc-protected alcohol24 by treatment with TFA–
CH2Cl2 as described above.
Fig. 5 Agonist activity of 1, 5 and 6 and the reference compound NPA
at the D2 receptor.
All of the evaluated pyridine based PLG mimetics produced
bell-shaped dose–response curves, and none were active as agonists
at the dopamine D2 receptor. These findings suggest that the
mechanism behind the activity of peptidomimetics 1–6 and PLG
is the same. However, the functional assay that has been used in
this study gives no information regarding the binding site(s), so it
is not known whether peptidomimetics 1–6 bind to the same site
at the D2 receptor as PLG, or not.
Earlier studies with peptide analogues of PLG modified at the
leucine residue have shown that a hydrophobic residue in this
position is a prerequisite for activity.8 Substitution of the isobutyl
group of PLG with benzyl or n-butyl afforded derivatives with
activities comparable to that of PLG in enhancing the binding
Pharmacology
The PLG mimetics synthesized were evaluated in the R-SAT
assay, a pharmacologically predictive cell-based functional assay
based on the ligand-dependent transformation of NIH/3T3 cells
as previously described.24,26 The compounds were tested for their
ability to enhance the maximum response of the dopamine agonist
N-propylapomorphine (NPA) at the human D2 receptor (Fig. 4).
All derivatives showed a bell-shaped dose–response curve. A
statistically significant enhancement of the NPA response was
produced by compounds 2 (117
(116 3%), all at 10 nM. These activities were comparable to
the enhancement produced by PLG in the R-SAT assay (115
6%), 3 (118
5%) and 4
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 1647–1654 | 1649
©

View Article Online
of the dopamine agonist ADTN to dopamine receptors, whereas
substitution with smaller alkyl moieties, propyl or ethyl, yielded
inactive analogues. This trend cannot be found in our series of
pyridine based PLG mimetics. The differences among 1, 2, 3, and
4 are too small to enable a ranking based on their activities, and
they are therefore considered to be of equal potency. It can be
speculated that the pyridine scaffold itself might be able to interact
with a hydrophobic pocket in the receptor, thereby reducing the
importance of such an interaction by the side chain moiety.
However, attention should also be given to the racemization of
2 via keto–enol tautomerism and the possibility that the two
enantiomers differ in activity.7
Conclusion
Five novel PLG mimetics based on a 2,3,4-trisubstituted pyridine
scaffold have been synthesized and four of them, plus the peptide
L-Pro-D-Leu-Gly-NH2, were found to have dopamine modulatory
activity in the R-SAT assay comparable to (2, 3, and 4), or
higher (5 and 23), than PLG. None of the mimetics showed any
agonistic activity at the D2 receptor, and all produced bell-shaped
dose–response curves similar to that of PLG. These findings
suggest a similar mechanism behind the activity of both PLG and
the pyridine based peptidomimetics 1–6 and provide additional
information towards the elucidation of the bioactive conformation
of PLG.
Earlier studies have shown that the proline residue of PLG
can be quite sensitive to structural modifications. For example, a
peptide analogue with a thiazolidine-4-carbonyl group instead of a
proline residue was found to be inactive, whereas the pyroglutamyl
derivative of PLG showed an activity comparable to that of
the parent peptide.7 In our series, the activity of the piperidine
derivative 5 is considered most interesting since it shows that
significant changes in the proline moiety can be accepted in the
pyridine based PLG mimetics. It is also the only pyridine based
PLG mimetic that is active at 1 nM, where the increase in the
NPA response was similar to that produced by PLG at 10 nM. In
analogue 5, the symmetrical piperidine ring makes the compound
achiral, which simplifies synthetic and stability considerations.
Also, the nitrogen atom in the 4-substituted piperidine ring
of 5 is positioned quite differently from the nitrogen atom in
the 2-substituted pyrrolidine ring of 1. The acceptance of this
replacement by the PLG binding site adds interesting information
to the discussion about the bioactive conformation of PLG, which
has been postulated in several studies to be a type II b-turn.13–17
In the previous study of mimetic 1, we concluded that 1 could
not adopt a type II b-turn conformation, and speculated about
the possibility of an extended conformation as a second bioactive
conformation of PLG.24 This hypothesis has been suggested by
others,5 and is supported by the activity of 5, which is predicted
to adopt extended conformations to an even greater extent than
1, due to the 180◦ constraint imposed by the para-relationship
of the peptide backbone through the piperidine ring. Alcohol
6 produced an enhancement of the NPA response that was not
statistically significant and no conclusions about the outcome of
the bioisosteric replacement of the keto-functionality of 1 with an
alcohol in 6 can therefore be drawn.
In the design of the pyridine scaffold, it was noticed that the
direction of the side chain moiety that is attached to the 3-position
of the pyridine ring, was close to the direction of a D-amino acid in
the corresponding position of the parent peptide. The established
ability of mimetic 1 to mimic the action of PLG therefore suggested
that the PLG diastereomer L-Pro-D-Leu-Gly-NH2 (23) could be
active as a dopamine modulator. Although several tripeptide
derivatives have been evaluated as PLG mimetics, most with an L-
configuration in the second amino acid moiety,8 to our knowledge
no 2D-isomer of an 2L-dopamine modulating active tripeptide has
been reported. The hypothesis of the pyridine scaffold as an L-D-L
tripeptidomimetic, was therefore challenged by the inclusion of
L-Pro-D-Leu-Gly-NH2 (23) in our test series. Most interestingly,
23 was found to be more potent than PLG, and produced a
similar response at 1 nM as PLG did at a 10 nM concentration
(Fig. 4).
Experimental section
Chemistry. General data
All chemical reagents were commercially available. THF was
distilled from potassium/sodium. TLC analysis was performed
on silica gel F254 (Merck) and detection was carried out by
examination under UV light and staining with phosphomolybdic
acid. After work-up, all organic phases were dried with MgSO4.
Flash column chromatography was performed on silica gel with
solvent of HPLC grade or analytical grade. Analytical reversed-
phase HPLC was performed on a Beckman System Gold HPLC
equipped with a Kromasil C-8 column (250 × 4.6 mm) using
acetonitrile (0.1% TFA) in H2O (0.1% TFA), 0–100% linear
gradient over 60 minutes. A flow-rate of 1.5 mL min−1 was used
and detection was at 254 nm. Preparative reversed-phase HPLC
was performed on a Kromasil C-8 column (250 × 20 mm) using
the same eluent, a flow rate of 11 mL min−1 and detection
at 254 nm. Chiral HPLC chromatography was performed on
a Pirkle Covalent (S,S)-Whelk-O 1 10/100 Krom Fec column
using CH2Cl2–iPrOH–heptane (48 : 4 : 48) as the eluent, a flow
rate of 1.0 mL min−1 and detection at 254 nm. Melting points
were determined on a Bu¨chi melting point apparatus and are
uncorrected. Specific rotations were measured on a Perkin-Elmer
model 343 polarimeter. 1H and 13C NMR spectra were recorded on
a Bruker DRX-400 spectrometer at 400 and 100 MHz, respectively,
and calibrated using the residual peak of solvent as an internal
reference (CDCl3 [CHCl3 dH 7.26, CDCl3 dC 77.0] or CD3OD
[CD2HOD dH 3.34, CD3OD dC 49.0]) or DMSO-d6 [dH 2.50, dC
39.52]. Infrared spectra were recorded using an ATI Mattson
Infinity Series FTIR spectrometer. High-resolution mass spectral
analysis was performed at Instrumentstationen, Lund University,
Sweden. Compounds 7a,27 7b,27 7c,25 8b25 and 1725 were synthesized
as reported earlier. The tripeptide 23 was commercially available
(Bachem). Data on compounds 3, 5, 6, 9b, 10b, 11b, 12b, 15, 16,
18, 19, 20, 21, and 22 and selected NMR-spectra are presented in
the ESI†.
General procedure for the Grignard coupling reactions to produce
8a, 8c and 18. The pyridine derivative (1.0 eq.) was dissolved in
freshly distilled THF and isopropyl magnesium chloride (1.0 eq.)
was added as a 2.0 M solution in THF. The reaction was allowed
to stir for 1–2 h at room temperature under a nitrogen atmosphere,
before the aldehyde (1.0 eq.) was added as a solution in THF. The
reaction was stirred overnight before it was quenched by the
addition of H2O and extracted with diethyl ether. The combined
1650 | Org. Biomol. Chem., 2008, 6, 1647–1654
This journal is
The Royal Society of Chemistry 2008
©

View Article Online
organic phases were washed with brine, dried and concentrated to
yield a crude oil, which was purified as specified below.
(mixture of isomers) d 164.20 (d, J = 239 Hz), 163.99 (d, J =
239 Hz), 155.99, 154.94, 154.65, 154.37, 153.99, 147.81, 147.63,
147.47, 147.16, 137.78, 137.46, 128.43, 127.96, 127.75, 127.46,
120.25 (d, J = 3 Hz), 119.38 (d, J = 3 Hz), 108.08 (d, J =
37 Hz), 107.15 (d, J = 38 Hz), 79.99, 79.76, 79.45, 79.35, 78.54,
72.55, 72.33, 72.05, 71.59, 62.38, 60.49, 60.26, 47.42, 47.19, 46.96,
46.81, 28.46, 26.29, 25.58, 25.32, 24.55, 24.42, 23.59, 22.91; IR
3065, 3031, 2974, 2880, 1692, 1611, 1567, 1453, 1369, 1273,
1172, 1085 cm−1; HRMS (FAB) calcd for C22H28FN2O3 [M + H]+
387.2084, found 387.2087.
(1R,1S)-[(2S)-1-tert-Butoxycarbonylpyrrolidin-2-yl]-1-(2-fluoro-
pyridin-4-yl)-methanol (8a). The general procedure applied to
pyridine derivative 7a (0.50 g, 2.24 mmol) and Boc-Pro-CHO in
THF (5 mL, total volume) yielded a crude oil, which was purified
by flash chromatography using CH2Cl2–MeOH–heptane (5 : 1:
10) as the eluent to afford 8a (0.53 g, 80%) as a colorless oil:
1H NMR (CDCl3) (mixture of isomers) d 8.17–8.12 (m, 1H),
7.17–7.11 (m, 1H), 6.97–6.91 (m, 1H), 6.12 (br s, 0.8H), 5.44
(br s, 0.2H), 5.24–4.51 (m, 1H), 4.30–3.79 (m, 1H), 3.69–3.18 (m,
2H), 2.06–1.57 (m, 4H), 1.60–1.35 (m, 9H); 13C NMR (CDCl3)
(mixture of isomers) d 163.79 (d, J = 239 Hz), 157.75 (br), 157.40
(d, J = 7 Hz), 147.20 (d, J = 14 Hz), 119.98, 107.89 (d, J =
39 Hz), 80.98, 76.37 (br), 63.21, 62.88, 47.59, 28.85, 28.48, 28.27,
27.84, 23.70; IR (neat) 3391 (br), 2974, 2938, 2887, 1690, 1668,
1613, 1409 cm−1; HRMS (FAB) calcd for C15H22FN2O3 [M + H]+
297.1614, found 297.1612.
3-Benzyl-4-[benzyloxy-1-((2S)-1-tert-butoxycarbonyl-pyrrolidin-
2-yl)-methyl]-2-fluoro-pyridine (9c). The general procedure
applied to alcohol 8c major (0.75 g, 1.9 mmol) in THF (5 mL)
yielded a crude oil, which was purified by flash chromatography
using EtOAc–heptane (1 : 4) as the eluent to afford 9c (0.62 g,
1
87%) as a brownish viscous oil: H NMR (CDCl3) (rotamers) d
8.18–8.04 (m, 1H), 7.44 (s, 1H), 7.39–7.02 (m, 10H), 5.11 (br s,
0.5H), 4.78 (br s, 0.5H), 4.48–3.89 (m, 5H), 3.59–2.92 (m, 2H),
2.11–1.75 (m, 2H), 1.76–1.04 (m, 11H); 13C NMR (CDCl3) d
162.51 (d, J = 240 Hz), 155.32, 152.94, 145.48, 145.29, 145.05,
144.89, 138.80, 138.35, 137.73, 137.41, 128.62, 128.30, 127.52,
126.45, 122.08, 120.61, 79.73, 78.15, 71.41, 60.72, 46.89, 30.14,
28.47, 28.15, 25.94, 24.01, 23.33; IR (neat) 2957, 2923, 2855, 1690,
1389, 1258, 1091, 1014 cm−1; [a]D −20 (c 1.0, CHCl3); HRMS
(FAB) calcd for C29H33FN2O3 [M + H]+ 477.2553, found 477.2554.
(1R,1S)-(3-Benzyl-2-fluoro-pyridin-4-yl)-1-[(2S)-1-tert-butoxy-
carbonylpyrrolidin-2-yl]-methanol (8c major and 8c minor). The
general procedure applied to pyridine derivative 7c (0.94 g,
3.0 mmol) and Boc-Pro-CHO in THF (15 mL, total volume)
yielded a crude oil, which was purified by flash chromatography us-
ing heptane–EtOAc (6 : 1) as the eluent to afford the diastereomers
8c major (0.57 g, 74%) and 8c minor (62 mg, 8%) as pale yellow oils:
8c major: 1H NMR (CDCl3) d 8.11 (d, 1H, J = 5.0 Hz), 7.33–7.11
(m, 6H), 6.23 (br s, 1H), 4.84 (dd, 1H, J = 9.0, 2.6 Hz), 4.23–
4.03 (m, 3H), 3.46–3.33 (m, 1H), 3.27–3.18 (m, 1H), 1.90–1.44 (m,
4H), 1.52 (s, 9H); 13C NMR (CDCl3); d 162.60 (d, J = 239 Hz),
159.67, 154.90 (br), 145.68 (d, J = 16 Hz), 138.64, 128.64, 128.24,
126.49, 120.51, 120.32 (d, J = 30 Hz), 81.29, 76.41, 73.94, 63.42,
47.62, 30.64, 28.69, 23.76; IR (neat) 3317, 2976, 2932, 2882, 1658,
1606, 1404, 1163; HRMS (FAB) calcd for C22H28FN2O3 [M + H]+
387.2084, found 387.2084; [a]D +22 (c 1.0, CHCl3). 8c minor: IR
(neat) 3356, 2976, 2881, 2360, 1679, 1607, 1401, 1256, 1164, 1114;
[a]D −48 (c 1.0, CHCl3); HRMS (FAB) calcd for C22H28FN2O3
[M + H]+ 387.2084, found 387.2083.
General procedure for the nucleophilic substitution reactions to
produce 10a, 10b, 10c and 20. Glycolamide (5.0 eq.) was dissolved
1
2
˚
in DMSO (pre-dried with 4 A molecular sieves, of the total
volume) and KH (5.0 eq.) was added carefully. After a few minutes,
the evolution of gas stopped and the pyridine derivative (1.0 eq.)
was added as a solution in DMSO (12 of the total volume). The
reaction was stirred at the temperature, and for the time, specified
below before it was quenched by the addition of NH4Cl (aq., sat.)
and extracted with CH2Cl2. The combined organic phases were
washed with brine, dried and concentrated to yield a crude oil,
which was purified as specified below.
2-{4-[(1R,1S)-Benzyloxy-1-((2S)-1-tert-butoxycarbonyl-pyrro-
lidin-2-yl)-methyl]-pyridin-2-yloxy}-acetamide (10a). The gen-
eral procedure was applied to pyridine derivative 9a (0.33 g,
0.85 mmol) in DMSO (2.5 mL total volume). The reaction was
stirred at 55 ◦C for 2 hours. The work-up procedure yielded a crude
oil, which was purified by flash chromatography using EtOAc–
heptane (1: 1, then 2 : 1) as the eluent to afford the diastereomers
10a (0.16 g, 43%) and the byproduct 13 (0.18 g, 48%) as colorless
oils: 10a: 1H NMR (CDCl3) (mixture of isomers and rotamers) d
8.15–8.07 (m, 1H), 7.39–7.23 (m, 5H), 7.06–6.97 (m, 0.2H), 6.94–
6.77 (m, 1.8H), 6.42 (br s, 1H), 5.96 (br s, 1H), 5.13–4.95 (m, 0.5H),
4.83 (s, 2H), 4.79–4.70 (m, 0.5H), 4.68–3.82 (m, 3H), 3.59–2.78 (m,
2H), 2.11–1.66 (m, 3H), 1.58–1.32 (m, 9H), 1.11–0.90 (m, 1H);
13C NMR (CDCl3) (mixture of isomers and rotamers) d 171.70,
162.32, 162.14, 154.73, 154.46, 154.31, 153.90, 153.14, 152.95,
152.14, 151.83, 146.90, 146.75, 146.56, 146.30, 138.04, 137.91,
137.55, 128.31, 128.22, 127.66, 127.45, 127.30, 127.13, 117.07,
116.13, 109.12, 108.20, 79.96, 79.70, 79.42, 79.17, 78.65, 72.11,
71.91, 71.62, 71.21, 64.39, 62.29, 62.17, 60.37, 60.15, 47.27, 46.97,
46.81, 46.59, 28.35, 26.14, 25.43, 25.27, 24.40, 24.29, 23.52, 23.43,
22.79; IR 3332, 3061, 2976, 2881, 1682, 1613, 1560, 1417, 1306,
General procedure for the benzyl protections to produce 9a, 9b,
9c and 19. The alcohol (1.0 eq.) was dissolved in THF and
stirred while NaH (1.5 eq.) was added carefully, followed by benzyl
bromide (1.5 eq.) and tetrabutylammonium iodide (0.05 eq.). The
reaction was stirred at room temperature overnight before it was
quenched by the addition of NH4Cl (aq., sat.) and extracted with
diethyl ether. The combined organic phases were washed with
brine, dried and concentrated to yield a crude oil, which was
purified as specified below.
4-[(1R,1S)-Benzyloxy-1-((2S)-1-tert-butoxycarbonyl-pyrrolidin-
2-yl)-methyl]-2-fluoro-pyridine (9a). The general procedure
applied to alcohol 8a (0.37 g, 1.24 mmol) in THF (5 mL) yielded
a crude oil, which was purified by flash chromatography using
EtOAc–heptane (1 : 10) as the eluent to afford 9a (0.37 g, 77%,
diastereomeric mixture) as a colorless oil: 1H NMR (CDCl3)
(mixture of isomers) d 8.22–8.13 (m, 1H), 7.38–7.20 (m, 5H),
7.16–7.04 (m, 1H), 7.02–6.88 (m, 1H), 5.20–5.07 (m, 0.5H),
4.87–4.75 (m, 0.5H), 4.68–4.75 (m, 2.7H), 3.99–3.82 (m, 0.3H),
3.62–3.17 (m, 1.3H), 2.96–2.77 (m, 0.7H), 2.12–1.85 (m, 2H),
1.85–1.31 (m, 10H), 1.07–0.87 (m, 1H); 13C NMR (CDCl3)
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 1647–1654 | 1651
©

View Article Online
1164, 1119, 1061 cm−1; HRMS (FAB) calcd for C24H32N3O5 [M +
H]+ 442.2342, found 442.2345. 13: 1H NMR (CDCl3) (mixture of
isomers and rotamers) d 9.51–9.39 (m, 1H), 8.41–8.27 (m, 1H),
8.26–8.16 (m, 1H), 7.39–7.25 (m, 5H), 7.19–6.99 (m, 1H), 6.41–
5.75 (br s, 1H), 5.10–3.84 (m, 6H), 3.57–2.83 (m, 2H), 2.12–1.81 (m,
2H), 1.79–1.29 (m, 10H), 1.14–0.96 (m, 1H); 13C NMR (CDCl3)
(mixture of isomers and rotamers) d 170.89, 170.65, 154.91, 154.54,
154.13, 153.06, 152.36, 151.84, 150.95, 150.88, 150.79, 147.38,
147.04, 146.88, 146.72, 137.92, 137.64, 128.43, 128.33, 128.22,
127.77, 127.59, 127.47, 119.00, 117.90, 113.14, 112.15, 112.06,
80.34, 79.99, 79.82, 79.63, 79.31, 79.15, 72.26, 72.05, 71.81, 71.39,
62.46, 62.28, 62.11, 60.61, 47.37, 46.95, 46.69, 28.45, 28.37, 26.35,
25.62, 25.35, 24.75, 24.29, 23.65, 22.93; IR 3370, 3063, 2976, 2881,
1682, 1566, 1519, 1403, 1169, 1080 cm−1; HRMS (FAB) calcd for
C24H32N3O5 [M + H]+ 442.2342, found 442.2342.
(br s, 0.1H), 4.99–4.89 (m, 0.3H), 4.83–4.71 (m, 2H), 4.65–4.45
(m, 0.6H), 4.23–3.85 (m, 1H), 3.59–2.91 (m, 2H), 2.08–1.13 (m,
13H); 13C NMR (CDCl3) (mixture of isomers) d 171.80, 171.76,
162.21, 157.97, 154.83, 146.74, 146.42, 116.96, 116.49, 109.13,
108.60, 80.98, 77.20, 74.35, 64.44, 63.35, 62.89, 47.84, 47.62, 28.34,
28.12, 26.62, 23.78; IR 3350 (br), 2976, 2882, 1674, 1613, 1408,
1167, 1061 cm−1; HRMS (FAB) calcd for C17H26N3O5 [M + H]+
352.1872, found 352.1870.
2-{3-Benzyl-4-[1-((2S)-1-tert-butoxycarbonyl-pyrrolidin-2-yl)-
hydroxy-methyl]-pyridin-2-yloxy}-acetamide (11c). The general
procedure was applied to 10c (0.11 g, 0.21 mmol) and Pd/C
(51 mg) in THF (5 mL). The reaction was stirred for two hours.
Work-up, including purification by flash chromatography
(CH2Cl2–MeOH–heptane 4 : 1 : 10), afforded 11c (56 mg, 74%) as
a white foam: [a]D +6.0 (c 0.2, CHCl3); 1H NMR (CDCl3) d 8.08 (d,
1H, J = 5.0 Hz), 7.30–7.07 (m, 6H), 6.18 (br s, 1H), 5.46–5.31 (m,
2H), 4.93 (d, 1H, J = 16 Hz), 4.87 (d, 1H, J = 8.7 Hz), 4.65 (d, 1H,
J = 16 Hz), 4.28–4.04 (m, 3H), 3.49–3.25 (m, 2H), 1.74–1.17 (m,
13H); 13C NMR (CDCl3) d 171.45, 160.23, 158.35, 152.08, 144.97,
139.76, 128.70, 127.95, 126.38, 120.39, 117.14, 81.16, 74.71, 64.21,
63.40, 47.62, 31.40, 30.27, 28.52, 28.36, 23.85; HRMS (FAB) calcd
for C24H32N3O5 [M + H]+ 442.2342, found 442.2350.
2-{3-Benzyl-4-[benzyloxy-1-((2S)-1-tert-butoxycarbonyl-pyrro-
lidin-2-yl)-methyl]-pyridin-2-yloxy}-acetamide (10c). The gen-
eral procedure was applied to pyridine derivative 9c (0.42 g,
0.88 mmol) in DMSO (12 mL total volume). The reaction was
stirred at room temperature for one week and at 55 ◦C for one day.
An additional 5 eq. of pre-reacted glycolamide and KH were
added and the reaction was stirred for 5 days at 55 ◦C. The
work-up procedure described above yielded a crude oil, which
was purified by flash chromatography using EtOAc–heptane (1 :
1) as the eluent to afford recovered starting material (0.10 g, 24%),
and 10c (0.25 g, 53%) as a white foam: [a]D −10 (c 1.0, CHCl3);
1H NMR (CDCl3) (rotamers); d 8.14–8.04 (m, 1H), 7.36–7.11 (m,
9H), 7.06–6.98 (m, 2H), 5.55–5.25 (m, 2H), 5.21–4.80 (m, 2H),
4.63–4.15 (m, 4H), 4.15–3.93 (m, 2H), 3.50–2.95 (m, 2H), 2.14–
1.78 (m, 2H), 1.73–1.04 (m, 11H); 13C NMR (CDCl3) (rotamers)
d 171.49, 160.13, 155.22,154.63, 149.86, 144.66, 144.37, 140.16,
139.65, 137.83, 137.57, 128.71, 128.32, 127.55, 126.30, 121.61,
121.09, 117.00, 79.52, 77.64, 71.51, 71.33, 64.16, 60.86, 46.87,
46.53, 30.89, 28.41, 28.15, 27.39, 25.92, 23.95, 23.12; IR (neat)
3020, 1215 cm−1; HRMS (FAB) calcd for C31H38N3O5 [M + H]+
532.2811, found 532.2814.
General procedure for the oxidation reactions to produce 12a, 12b,
12c and 22. The alcohol (1.0 eq.) was dissolved in CH2Cl2 and
Dess–Martin periodinane (15 wt% in CH2Cl2, 0.5 M, 2.0 eq.) was
added. The reaction was stirred at room temperature for the time
specified below, before it was quenched by the addition of Na2S2O5
(12 eq.) in NaHCO3 (aq., sat.) and extracted with CH2Cl2. The
combined organic phases were washed with NaHCO3 (aq., sat.)
and brine, dried and concentrated to yield a crude oil, which was
purified as specified below.
2-{4-[((2S)-1-tert-Butoxycarbonyl-pyrrolidine-2-yl)-carbonyl]-
pyridin-2-yloxy}-acetamide (12a). The general procedure was
applied to alcohol 11a (55 mg, 0.16 mmol) for 1 hour. Purification
by flash chromatography (EtOAc–heptane 4 : 1) afforded 12a
1
(33 mg, 61%) as a colorless oil: [a]D −17.1 (c 0.7, CH2Cl2); H
General procedure for the catalytic hydrogenation reactions to
produce 11a, 11b, 11c and 21. The benzyl ether (1.0 eq.) was
dissolved in the solvent specified below and Pd/C (10 wt%) was
added. The reaction was stirred under H2 at room temperature for
the time specified below, before the catalyst was filtered off using
NMR (CDCl3) (rotamers) d 8.34 (d, 0.5H, J = 5.2 Hz), 8.30 (d,
0.5H, J = 5.2 Hz), 7.41–7.36 (m, 1H), 7.33 (s, 0.5H), 7.27 (s, 0.5H),
6.34 (br s, 1H), 5.88–5.65 (m, 1H), 5.17 (dd, 0.5H, J = 9.2, 3.9 Hz),
5.09–5.03 (m, 0.5H), 4.90–4.86 (m, 2H), 3.71–3.43 (m, 2H), 2.40–
2.21 (m, 1H), 2.01–1.82 (m, 3H), 1.45 (s, 4.5H), 1.28 (s, 4.5H); 13
C
R
a plug of Celiteꢀ and the filtrate was concentrated to afford an
NMR (CDCl3) (two rotamers of equal intensity) d 198.18, 197.94,
170.93, 170.76, 162.90, 162.80, 154.42, 153.48, 148.42, 148.21,
145.09, 144.88, 115.78, 115.54, 109.67, 109.32, 80.21, 80.09, 64.94,
64.86, 61.84, 61.38, 46.78, 46.58, 30.54, 29.44, 28.41, 28.21, 24.31,
23.51; IR 3337, 3057, 2977, 2882, 1681, 1557, 1410, 1163 cm−1;
HRMS (FAB) calcd for C17H24N3O5 [M + H]+ 350.1716, found
350.1723. Byproduct 14: 1H NMR (CDCl3) (rotamers) d 7.45 (d,
0.55H, J = 6.7 Hz), 7.40 (d, 0.45H, J = 6.7 Hz), 7.07 (s, 0.45H),
7.04 (s, 0.55H), 6.75–6.67 (m, 1H), 5.12–5.06 (m, 0.45H), 5.03–4.97
(m, 0.55H), 3.70–3.40 (m, 3H), 2.38–2.23 (m, 1H), 1.99–1.53 (m,
3H), 1.45 (s, 4H), 1.31 (s, 5H); HRMS (FAB) calcd for C15H21N2O4
[M + H]+ 293.1501, found 293.1504.
oil, which was either pure or was purified as specified below.
2-{4-[1-((2S)-1-tert-Butoxycarbonyl-pyrrolidin-2-yl)-(1R,1S)-
hydroxy-methyl]-pyridin-2-yloxy}-acetamide (11a). The general
procedure was applied to benzyl ether 10a (0.15 g, 0.34 mmol)
and Pd/C (75 mg) in THF (4 mL). The reaction was stirred over
two days, with additional Pd/C (75 mg) being added twice. A
precipitate was formed that could not be removed by filtration
R
through Celiteꢀ but had to be separated from the product
either by flash chromatography, or by dissolving the crude brown
mixture in EtOAc and performing the filtration procedure again.
Work-up yielded a brown liquid, which was purified by flash
chromatography EtOAc–heptane (2 : 1 then 4 : 1 then 6 : 1) to
2-{3-Benzyl-4-[((2S)-1-tert-butoxycarbonyl-pyrrolidine-2-yl)-
carbonyl]-pyridin-2-yloxy}-acetamide (12c). The general proce-
dure was applied to alcohol 11c (46 mg, 0.10 mmol) for three hours.
Purification by flash chromatography (EtOAc–heptane 1 : 1)
1
afford 11a (105 mg, 88%) as a colorless oil: H NMR (CDCl3)
(mixture of isomers) d 8.08–8.01 (m, 1H), 6.94–6.78 (m, 2H), 6.51
(br s, 1H), 6.35 (br s, 1H), 6.06 (br s, 0.5H), 5.26 (br s, 0.2H), 5.11
1652 | Org. Biomol. Chem., 2008, 6, 1647–1654
This journal is
The Royal Society of Chemistry 2008
©

View Article Online
afforded 12c (39 mg, 86%) as white crystals: [a]D +32 (c 1.0,
CHCl3); 1H NMR (CDCl3) (rotamers) d 8.22–8.12 (m, 1H), 7.35–
7.09 (m, 6H), 5.39–5.23 (m, 2H), 4.98–4.85 (m, 2H), 4.77–4.68 (m,
1H), 4.16–3.99 (m, 2H), 3.65–3.51 (m, 1H), 3.51–3.36 (m, 1H),
2.15–1.99 (m, 1H), 1.95–1.58 (m, 3H), 1.45 (s, 5H), 1.41 (s, 4H);
13C NMR (CDCl3) (two rotamers, major and minor) d 203.17,
201.50, 171.10, 170.94, 160.84, 160.62, 154.48, 153.59, 148.04,
147.33, 145.33, 145.16, 139.79, 139.38, 128.69, 128.57, 128.33,
128.22, 126.59, 126.39, 120.82, 120.50, 115.81, 115.52, 80.36,
80.01, 64.37, 64.24, 64.01, 63.90, 46.81, 46.92, 32.30, 32.27, 29.12,
28.57, 28.38, 24.17, 22.64; HRMS (FAB) calcd for C24H29N3O5
[M + H]+ 440.2185, found 440.2186.
transfection and frozen at −135 ◦C. The cells were frozen by a
standard method of slow freezing in a cryovial in a Styrofo◦am
◦
container placed at −70 C. After overnight freezing at −70 C,
the cells were moved to a −135 ◦C cryogenic freezer for long-
term storage. These freezers use a proprietary mix of refrigerants
without liquid nitrogen. For each experiment testing potentiation
of the NPA-induced response, cells were thawed, resuspended in
media containing saturating amounts of N-propylapomorphine
(1 lM), (NPA, pEC50 7.6
0.4, n = 7) and added directly
to the compounds at a density of 20 000 cells per well in half-
area 96-well plates. No difference was observed between cells
transfected immediately prior to NPA activation24 and cells frozen
prior to use. After 5 days of incubation, the b-galactosidase
activity was measured by the addition of o-nitrophenyl b-D-
galactopyranoside (in phosphate-buffered saline with 5% Nonidet
P-40 detergent). The resulting colorimetric reaction was measured
in a spectrophotometric plate reader (Titertek, Huntsville, AL)
at 420 nM. For each experiment, compounds were plated in 12
wells for each concentration of compound tested. Each experiment
was performed 3–10 times. Student’s t-test was used to evaluate
the statistical significance of the difference between the averaged
values for treatment with both NPA and compound, and NPA
alone. To test the agonist response at the D2 receptor, the
compounds were plated in triplicate for each concentration of
compound tested.
General procedure for the Boc-deprotection reactions to produce
2, 3, 4, 5 and 6. The carbamate (1.0 eq.) was dissolved in CH2Cl2
3
4
(
of the total volume) and trifluoroacetic acid (14 of the total
volume) was added. The reaction was stirred for 5 to 8 minutes and
then immediately concentrated under reduced pressure without
heating to yield a crude solid, which was purified by reversed-
phase HPLC to afford the products.
2-[4-((2S)-(Pyrrolidine-2-yl)-carbonyl)-pyridin-2-yloxy]-aceta-
mide TFA-salt (2). The general procedure was applied to carba-
mate 12a (15 mg, 0.043 mmol) in CH2Cl2–TFA (4 mL total volume)
for 5 minutes. Freeze-drying allowed isolation of the TFA-salt of 2
(8.2 mg, 53%) as a white solid: [a]D 0 (MeOH); 1H NMR (CD3OD)
(keto : enol 1 : 1) d 8.38 (d, 1H, J = 5.3 Hz), 8.20 (d, 1H, J = 5.3 Hz),
7.51 (d, 1H, J = 5.3 Hz), 7.46 (s, 1H), 7.10 (d, 1H, J = 5.3 Hz), 7.04
(s, 1H), 5.35 (dd, 1H, J = 9.4 and 6.7 Hz), 4.87 (s, 2H), 4.82 (s, 2H),
3.80–3.69 (m, 1H), 3.48–3.40 (m, 2H), 3.34–3.21 (m, 1H), 2.71–
2.59 (m, 1H), 2.21–1.78 (m, 6H), 1.71–1.61 (m, 1H); 13C NMR
(CD3OD) (keto and enol) d 194.65, 164.99, 164.69, 152.36, 149.63,
148.68, 143.69, 117.14, 116.50, 111.67, 110.84, 99.29, 68.79, 65.41,
65.13, 64.79, 47.50, 47.16, 30.30, 26.42, 25.06, 25.02; HRMS (FAB)
calcd for C12H16N3O3 [M + H]+ 250.1192, found 250.1189.
Acknowledgements
We thank the Knut and Alice Wallenberg Foundation, the
Norwegian Research Council and the Swedish Research Council
for providing financial support for this project. We also thank
Umea˚ University for providing laboratory facilities.
References
2-[3-Benzyl-4-((2S)-(pyrrolidine-2-yl)-carbonyl)-pyridin-2-
yloxy]-acetamide TFA-salt (4). The general procedure was ap-
plied to carbamate 12c (10 mg, 0.023 mmol) in CH2Cl2–TFA (2 mL
total volume) for 5 minutes. Freeze-drying allowed isolation of the
TFA-salt of 4 (9 mg, 83%) as a white solid: [a]D +12 (c 0.47,
1 R. K. Mishra, S. Chiu, P. Chiu and C. P. Mishra, Meth. Find. Exp. Clin.
Pharmacol., 1983, 5, 203.
2 R. K. Mishra, M. H. Makman, W. J. Costain, V. D. Nair and R. L.
Johnson, Neurosci. Lett., 1999, 269, 21.
3 L. K. Srivastava, S. B. Bajwa, R. L. Johnson and R. K. Mishra,
J. Neurochem., 1988, 50, 960.
4 S. Chiu, C. S. Paulose and R. K. Mishra, Peptides, 1981, 2, 105.
5 M. C. Evans, A. Pradhan, S. Venkatraman, W. H. Ojala, W. B. Gleason,
R. K. Mishra and R. L. Johnson, J. Med. Chem., 1999, 42, 1441.
6 C. Thomas, U. Ohnmacht, M. Niger and P. Gmeiner, Bioorg. Med.
Chem. Lett., 1998, 8, 2885.
7 R. L. Johnson, G. Rajakumar, K. L. Yu and R. K. Mishra, J. Med.
Chem., 1986, 29, 2104.
8 R. L. Johnson, R. J. Bontems, K. E. Yang and R. K. Mishra, J. Med.
Chem., 1990, 33, 1828.
9 W. J. Costain, A. T. Buckley, M. C. Evans, R. K. Mishra and R. L.
Johnson, Peptides, 1999, 20, 761.
1
MeOH); H NMR (CD3OD) d 8.30 (d, 1H, J = 5.3 Hz), 7.37
(d, 1H, J = 5.3 Hz), 7.32–7.24 (m, 2H), 7.22–7.17 (m, 3H), 5.28
(dd, 1H, J = 9.4, 7.2 Hz), 4.93–4.88 (in residual solvent peak,
2H), 4.34 (s, 2H), 3.40–3.33 (m, 1H), 3.31–3.22 (m, 1H), 2.27–
2.16 (m, 1H), 2.01–1.89 (m, 1H), 1.75–1.63 (m, 1H), 1.47–1.37 (m,
1H); 13C NMR (CD3OD) d 197.83, 173.74, 163.06, 146.95, 144.24,
141.10, 129.92, 129.53, 127.44, 124.62, 117.24, 66.21, 65.39, 47.34,
31.78, 28.70, 24.69; HRMS (FAB) calcd for C19H22N3O3 [M + H]+
340.1661, found 340.1664.
10 R. L. Johnson, G. Rajakumar and R. K. Mishra, J. Med. Chem., 1986,
29, 2100.
11 G. Rajakumar, F. Naas, R. L. Johnson, S. Chiu, K. L. Yu and R. K.
Mishra, Peptides, 1987, 8, 855.
12 P. W. Baures, A. Pradhan, W. H. Ojala, W. B. Gleason, R. K. Mishra
and R. L. Johnson, Bioorg. Med. Chem. Lett., 1999, 9, 2349.
13 M. J. Genin, R. K. Mishra and R. L. Johnson, J. Med. Chem., 1993,
36, 3481.
14 U. Sreenivasan, R. K. Mishra and R. L. Johnson, J. Med. Chem., 1993,
36, 256.
Pharmacology. Functional assays
The R-SAT (Receptor Selection and Amplification TechnologyTM
)
assay was performed essentially as previously described.24,26
Briefly, NIH/3T3 cells at 70–80% confluency were transfected,
using Polyfect (Qiagen, Los Angeles, CA) as described in the
manufacturer’s protocols, with DNA encoding the human D2
receptor, Gqi5, and b-galactosidase, at 10 lg, 20 lg, and 50 lg
per roller bottle, respectively. Cells were harvested 1 day after
15 P. W. Baures, W. H. Ojala, W. B. Gleason, R. K. Mishra and R. L.
Johnson, J. Med. Chem., 1994, 37, 3677.
This journal is The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 1647–1654 | 1653
©

View Article Online
16 K. L. Yu, G. Rajakumar, L. K. Srivastava, R. K. Mishra and R. L.
Johnson, J. Med. Chem., 1988, 31, 1430.
17 N. L. Subasinghe, R. J. Bontems, E. McIntee, R. K. Mishra and R. L.
Johnson, J. Med. Chem., 1993, 36, 2356.
22 E. M. Khalil, W. H. Ojala, A. Pradhan, V. D. Nair, W. B. Gleason,
R. K. Mishra and R. L. Johnson, J. Med. Chem., 1999, 42, 628.
23 K. Weber, U. Ohnmacht and P. Gmeiner, J. Org. Chem., 2000, 65,
7406.
18 K. Dolbeare, G. F. Pontoriero, S. K. Gupta, R. K. Mishra and R. L.
Johnson, J. Med. Chem., 2003, 46, 727.
19 K. Dolbeare, G. F. Pontoriero, S. K. Gupta, R. K. Mishra and R. L.
Johnson, Bioorg. Med. Chem., 2003, 11, 4103.
24 S. Saitton, A. L. Del Tredici, N. Mohell, R. C. Vollinga, D. Bostro¨m, J.
Kihlberg and K. Luthman, J. Med. Chem., 2004, 47, 6595.
25 S. Saitton, J. Kihlberg and K. Luthman, Tetrahedron, 2004, 60,
6113.
20 C. Palomo, J. M. Aizpurua, A. Benito, J. I. Miranda, R. M. Fratila, C.
Matute, M. Domercq, F. Gago, S. Martin-Santamaria and A. Linden,
J. Am. Chem. Soc., 2003, 125, 16243.
21 E. M. Khalil, A. Pradhan, W. H. Ojala, W. B. Gleason, R. K. Mishra
and R. L. Johnson, J. Med. Chem., 1999, 42, 2977.
26 T. A. Spalding, C. Trotter, N. Skjærbæk, T. L. Messier, E. A. Currier,
E. S. Burstein, D. Li, U. Hacksell and M. R. Brann, Mol. Pharmacol.,
2002, 61, 1297.
27 P. Rocca, C. Cochennec, F. Marsais, L. Thomas-dit-Dumont, M.
Mallet, A. Godard and G. Que´guiner, J. Org. Chem., 1993, 58, 7832.
1654 | Org. Biomol. Chem., 2008, 6, 1647–1654
This journal is
The Royal Society of Chemistry 2008
©