Allosteric modulation of the dopamine receptor by conformationally constrained type VI β-turn peptidomimetics of Pro-Leu-Gly-NH2

Article abstract of DOI:10.1021/jm070895r

A peptidomimetic of Pro-Leu-Pro-NH₂, 7, possessing an indolizidinone type VI β-turn mimic was synthesized via improved high-yielding protocols for the preparation and Cbz protection of α-allylproline. Bicyclic peptidomimetic 7 and spirobicylic

Full text of DOI:10.1021/jm070895r

J. Med. Chem. 2007, 50, 6725–6729
6725
Allosteric Modulation of the Dopamine Receptor by Conformationally Constrained Type VI
ꢀ-Turn Peptidomimetics of Pro-Leu-Gly-NH2
Ashish P. Vartak,† Kevin Skoblenick,‡ Nancy Thomas,‡ Ram K. Mishra,‡ and Rodney L. Johnson*,†
Department of Medicinal Chemistry, UniVersity of Minnesota, 308 HarVard Street SE, Minneapolis, Minnesota 55455-0343, and Department of
Psychiatry and BehaVioral Neuroscience, McMaster UniVersity, 1200 Main Street West, Hamilton, Ontario L8N 2Z5, Canada
ReceiVed July 24, 2007
A peptidomimetic of Pro-Leu-Pro-NH2, 7, possessing an indolizidinone type VI ꢀ-turn mimic was synthesized
via improved high-yielding protocols for the preparation and Cbz protection of R-allylproline. Bicyclic
peptidomimetic 7 and spirobicylic peptidomimetic 8 enhanced the binding of [3H]N-propylnorapomorphine
to dopamine receptors indicating that a type VI ꢀ-turn is a possible bioactive conformation of the homochiral
Pro-Leu-Pro-NH2 and Pro-Pro-Pro-NH2 analogues of Pro-Leu-Gly-NH2 at the dopamine receptor allosteric
regulatory site.
Introduction
D-Pro-NH2 (4), Pro-Pro-Pro-NH2 (5), and Pro-Pro-D-Pro-NH2
(6). Because of the effect that the proline pyrrolidine ring has
on the φ torsion angle, those PLG analogues that contained a
D-prolyl residue in place of the glycinamide residue are able to
attain a type II ꢀ-turn, while those possessing a L-prolyl residue
at the C-terminal end are not able to attain such a conformation.
The neuropeptide prolyl-leucyl-glycinamide (1, PLG) has
been shown to modulate dopaminergic neurotransmission as
illustrated by its ability to enhance amphetamine- and apomor-
phine-dependent rotational behavior in 6-hydroxydopamine-
lesioned rats.1–3 Evidence supports the hypothesis that PLG acts
as an allosteric modulator of the dopamine receptor.4 PLG and
its peptidomimetics have been shown to increase the binding
affinity of agonists to the high-affinity state of the dopamine
receptor and to shift the ratio of high- and low-affinity states of
the dopamine receptor in favor of the G-protein-coupled high-
affinity state.5,6 The actions of PLG appear to be specific toward
dopamine receptors because PLG does not interact with other
aminergic receptors such as R-adrenergic,5,7 GABA-ergic,8 or
serotonergic receptors.9 Furthermore, studies carried out in cell
lines transfected with human dopamine receptor subtypes have
shown that PLG and a PLG peptidomimetic enhance agonist
binding to the D2S, D2L, and D4 subtypes, whereas the D1 and
D3 subtypes are unaffected.7
Surprisingly, when these prolyl PLG analogues were evalu-
ated in in vitro and in vivo assays for dopamine receptor
modulation activity, the homochiral set of analogues were found
to be as active or even more active, depending on the assay,
than the heterochiral set of analogues.15,16 The activity of the
homochiral set of prolyl PLG analogues, which could not adopt
a type II ꢀ-turn, was inconsistent with the hypothesis, strongly
supported by the good activity of the highly constrained PLG
peptidomimetics like 2, that the bioactive conformation of PLG
is a type II ꢀ-turn. To explain this inconsistency, we speculated
that the homochiral prolyl PLG analogues are able to adopt a
conformation other than a type II ꢀ-turn that places the key
pharmacophores in the same relative topological space in which
these same key pharmacophores exist when PLG assumes a type
II ꢀ-turn conformation.
In considering potential conformations that the prolyl homo-
chiral peptides could assume, we noted that the presence of a
prolyl residue in the i + 2 position introduces the possibility of
a cis-amide bond with a resulting type VI ꢀ-turn conformation.17
A comparison of this turn with the type II ꢀ-turn reveals several
similarities, like the key amide NH2 group projecting in the same
relative space in both turn types (Figure 1). The major
topological difference between the two turn conformations is a
nearly 109° difference in the projection of the carboxamide
carbonyl groups. This suggested to us the possibility that the
homochiral peptides 3 and 5 might assume a type VI ꢀ-turn
when interacting with the PLG allosteric binding site. To test
this hypothesis, the design, synthesis, and evaluation of ana-
logues of 3 and 5 constrained in a type VI ꢀ-turn conformation
were undertaken. Although the restriction of the ω dihedral angle
to a value of 0° can be achieved in a number of ways, we viewed
the indolizidinone approach of Germanas18 as the most apt, since
the two-carbon bridge between the R-carbons of the i + 1 and
i + 2 residues of the turn provides minimal steric deviation.
Incorporation of this mimic into the prolyl tripeptides 3 and 5
Investigations in our laboratory have centered on the synthesis
and evaluation of conformationally constrained analogues of
PLG designed to test the hypothesis that the bioactive confor-
mation of PLG at the allosteric site is a type II ꢀ-turn
conformation.10 This approach led to the successful development
of numerous PLG peptidomimetics,11–13 one example of which
was the highly rigid spiro bicyclic analogue 2.14 These pepti-
domimetics supported the proposition that the bioactive con-
formation of PLG is a type II ꢀ-turn.
In other studies, a set of PLG analogues possessing a more
flexible framework was examined in which prolyl residues were
substituted for the leucyl and glycinamide residues of PLG.15,16
These analogues consisted of Pro-Leu-Pro-NH2 (3), Pro-Leu-
* To whom correspondence should be addressed. Phone: +1-612-624-
7997. Fax: +1-612-624-0139. E-mail: johns022@umn.edu.
†
University of Minnesota.
McMaster University.
‡
10.1021/jm070895r CCC: $37.00
2007 American Chemical Society
Published on Web 12/01/2007

6726 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Brief Articles
Scheme 1
Figure 1. Pro-Leu-Gly-NH2 (1, PLG) in a type-II ꢀ-turn conformation
(gray) overlaid with Pro-Leu-Pro-NH2 (3) in a type-VI ꢀ-turn
conformation.
Figure 2. Type-VI ꢀ-turn mimics of Pro-Leu-Pro-NH2 (3) and Pro-
Pro-Pro-NH2 (5), compounds 7 and 8, respectively.
afforded the bicyclic peptidomimetic 7 and the spirobicyclic
peptidomimetic 8, respectively (Figure 2).
protection of 9 led to rapid decomposition of Cbz-Cl and a low
yield (∼20%) of 10. However, we found that the reaction of
the tetramethylammonium salt of 9 with 2 equiv of Cbz-Cl in
the presence of 1 equiv of Et3N led to the formation of a stable
mixed-anhydride, which provided 10 in 60% yield upon
saponification. The reaction could be made quantitative by the
addition of another equivalent of Cbz-Cl, followed by saponi-
fication with aqueous KOH. This quantitative Cbz protection
was reproducible on a scale as large as 15 g and suggests that
this protocol may be potentially useful for many such substrates
where steric hindrance around the nitrogen prevents efficient
Cbz protection.
Treatment of 10 with diazomethane followed by oxidative
cleavage of the terminal olefin of the resulting ester provided
aldehyde 11. The subsequent olefination of 11 required the
glycine anion equivalent 12. Traditionally, this phosphonate is
prepared through an Arbuzov reaction between trimethyl
phosphite and a Cbz-protected R-bromoglycine ester, followed
by replacement of the Cbz group with a Boc group.22 We used
an alternative method in which a photochemical electrophilic
bromination of Boc-Gly-OEt by NBS23 was carried out in
hexanes instead of the typical hazardous and expensive CCl4
solvent to generate an R-bromoglycine intermediate that could
be converted to 12 through an Arbuzov reaction.
Chemistry
For the synthesis of 7, we followed Germanas’ general route
to the indolizidinone framework18 but made several improve-
ments and modifications. The key starting material for the
indolizidinone synthesis was (R)-R-allyl proline (9, Scheme 1),
of which substantial quantities were required. This amino acid
is traditionally prepared through Seebach’s “self-regeneration
of chirality” approach where the acid-catalyzed condensation
of proline with 6 equiv of pivalaldehyde in pentane with
continuous removal of water yields a highly moisture-sensitive
oxazolidinone.19 Alkylation of the lithium enolate of this ox-
azolidinone with allyl bromide yields a stable allylated oxazo-
lidinone, which when hydrolyzed provides 9. There are several
shortcomings to this approach including the high cost of
pivalaldehyde, the large excess of pivalaldehyde required, and
the duration of the condensation reaction (typically 3–4 days).
Modifications made to the condensation and hydrolysis reaction
steps of this sequence provided 9 in overall yields of 70–80%
on a 20 g scale (see Supporting Information).
Cbz protection of 9 has been a low-yielding process, as
documented in the past. Repeated additions of Cbz-Cl under
Schotten-Baumann conditions over 3 days produce excellent
yields of 9 on a small scale,20 but we found this approach to be
not scalable. We previously reported a procedure for the Boc
protection of 9 that involved its solubilization in acetonitrile
by forming a salt with tetramethylammonium hydroxide
(TMAH).21 Application of these same conditions to the Cbz
In our hands, attempts at affecting the olefination between
the lithium enolate of 12 and aldehyde 11 in THF led to low
yields of the desired alkene 13. Fortunately, a protocol by Zhang,
et al.24 that uses a DBU enolate in CH2Cl2 at room temperature
worked well in this case and gave alkene 13 in excellent yield
with the Z-isomer predominating.

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6727
Scheme 2
mixed anhydride reagent isobutyl chloroformate in the presence
or absence of DMAP. The main byproduct in these reactions
was the diketopiperazine 20 (Scheme 3). We observed an
identical result during the synthesis of spirobicyclic lactam 8.25
The coupling reagents bis(2-oxo-3-oxazolidinyl)phosphinic
chloride (BOP-Cl) and 1,3-dicyclohexylcarbodiimide (DCC)
were comparable to EDC. On the other hand, the coupling
behavior of 2-chloro-1-methylpyridinium iodide (Mukaiyama’s
reagent, CMPI) was radically different depending on whether
DMAP was present. With CMPI alone, the yield of 21 was only
∼30% after 12 h. When DMAP was combined with CMPI, an
exothermic reaction was observed that yielded 21 in a 60–75%
yield within 1–2 h, averting formation of 20.
The direct amidation of 21 to give the primary carboxamide
derivative 23 was not attainable by the following conditions:
heating a solution of 21 in methanolic ammonia at 100 °C in a
sealed stainless steel vessel, heating a solution of 21 in
methanolic ammonia in the presence of DMAP, NaCN, or LiI,
and Wienreb’s amidation with NH3 in the presence of an
equivalent of trimethylalane. However, 21 could be hydrolyzed
quite easily to the acid 22 with LiOH in a mixture of THF and
HMPA at reflux. EDC-mediated coupling of the free carboxylic
acid 22 with ammonia then afforded carboxamide 23. Subse-
quent removal of the Boc protecting group from 23 gave the
target molecule 7 as its HCl salt.
We also attempted to incorporate the primary carboxamide
function prior to the coupling stage. While we could form
carboxamide 24 upon treating 16 with methanolic ammonia,
diketopiperazine 20 also formed. Attempts to couple 24 to Boc-
Pro-OH with CMPI failed. Instead, diketopiperazine 20 and Boc-
prolinamide were isolated, suggesting that the primary amide
function of 24 initially gets acylated, thereby forming 25.
Nucleophilic attack of the free amine on the imide carbonyl
then ejects Boc-prolinamide and forms diketopiperazine 20.
In CDCl3, the primary carboxamide hydrogens of 23 appeared
in a typical hydrogen-bonded pattern with chemical shifts of
5.7 and 8.3 ppm. Further support for a hydrogen bond was
obtained by the addition of CD3OD to the NMR sample of 23,
which led to complete exchange of the non-H-bonded, upfield
hydrogen while the H-bonded downfield hydrogen required 30
min for complete exchange. Similar behavior was seen with a
solution of 7 in CD3OD, in which case the downfield carboxa-
mide hydrogen resonance at 7.87 ppm diminishes more slowly
than the upfield carboxamide hydrogen resonance at 7.42 ppm.
Such behavior was observed previously with 8 and its Boc-
protected precursor.25 The results for 7 and 8 are consistent with
a hydrogen bond between the carboxamide NH and the prolyl
carbonyl group that one would expect in a type VIa ꢀ-turn
conformation.
Reduction of the olefin and hydrogenolysis of the Cbz group
of 13 was accomplished simultaneously with H2 over Pd/C.
Heating the resulting product for 5–6 h at reflux in MeOH in
the presence of sodium metal resulted in complete cyclization
to give 14 in a 2:1 ratio of trans to cis isomers. After removal
of the Boc group from 14 by TFA, the free amine was reacted
with benzaldehyde to form a Schiff base. Initial attempts at
alkylating the lithium enolate of the Schiff base with isobutyl
bromide in THF at temperatures from -78 °C to the reflux
temperature were met with failure, even though the enolate was
found to be quite stable to heat. The use of isobutyl iodide in
place of the bromide, solvation of the enolate by HMPA, and
heating the reaction to reflux in THF, on the other hand, resulted
in a complete reaction within about 20 min to give the alkylated
product 15 as a single diastereoisomer. Breakdown of the Schiff
base with aqueous TFA provided bicyclic amine 16.
We also explored the use of bis-Boc protection in place of
the Schiff base for the alkylation reaction (Scheme 2). When
the 2:1 trans/cis diastereoisomeric mixture of lactam 14 was
treated with 10 equiv of Boc2O and DMAP in CH3CN, a
diastereoisomeric mixture of the bis-Boc lactam 17 was obtained
in an 88% yield. Surprisingly, the coupling constants of the
lactam R-proton in this diastereoisomeric mixture (8.1 and 10.2
Hz for the major isomer and 9.9 and 3.6 Hz for the minor
isomer) indicated that the trans/cis ratio was now 4:9. The
precise mechanism for this inversion is not known, although it
is known that epimerization does takes place when indolizidi-
nones of this relative stereochemistry are exposed to alkoxide.
On the basis of the observation that DMAP causes a vigorous
bubbling of CO2 when added to a solution of Boc2O, we believe
that the transient tert-butoxide liberated in this process may be
responsible for the deprotonation of the R-proton. Reprotonation
from the less sterically hindered si face leads to the preferential
formation of a cis-lactam.
Deprotonation of diastereoisomeric mixture 17 by LDA
followed by addition of isobutyl iodide led to the formation of
lactam 18 instead of the expected alkylated product, a net N f
C shift. This product formed even without isobutyl iodide being
present. An NOE between the carbamate NH and the methyl
ester hydrogens served to support the expected R configuration
about the newly formed quaternary carbon. In contrast, depro-
tonation of 17 with KHMDS in the presence of 2 equiv of allyl
bromide afforded a single diastereoisomer of the R-allylated
bicyclic lactam 19. An NOE between the tert-butyl hydrogens of
the Boc group and the hydrogens of the methyl ester suggested
that the allylation had occurred trans to the methyl ester function.
The coupling reaction of 16 with Boc-proline to give 21 was
sluggish with reagents that produce relatively stable esters (e.g.,
HOBt ester of Boc-proline via treatment with 1-(3-dimethyl-
aminopropyl)-3-ethylcarbodimide (EDC)), as well as with the
Pharmacology
Bicyclic peptidomimetic 7 and spirobicyclic peptidomimetic
8, the synthesis of which was carried out as reported by us
previously,25 were examined for their ability to potentiate the
binding of tritiated N-propylnorapomorphine ([3H]NPA) to
bovine striatal membranes.5,26 The results are shown in Figure
3. Compound 7 exhibited a bell-shaped dose response curve
that is typical of the allosteric modulation of the dopamine
receptor by PLG and its analogues. Statistically significant
increases were observed at the following concentrations of 7:
10 nM, 100 nM, and 1 µM. The maximum effect for 7 was
19.5 ( 3.75% at 100 nM. For spirobicyclic 8, a bell-shaped
dose response curve was also seen but only at 10 nM was a
significant increase in [3H]NPA binding observed (11.4 (

6728 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Brief Articles
Scheme 3
3.3%). At 100 nM, PLG peptidomimetic 2 and the triproline
analogue 5 enhance NPA binding to dopamine receptors by 38.2
( 11.9% and 34.0 ( 13.1%, respectively.
mimetics that assume a type II ꢀ-turn bioactive conformation.
In this regard, the data indicate that the amide NH2 group may
be a more important pharmacophore group than the carboxamide
carbonyl group in activating the D2 receptor allosteric modula-
tory site, as the major difference between a type II ꢀ-turn
conformation and a type VI ꢀ-turn conformation is the nearly
109° difference in orientation of the carboxamide carbonyl
groups. The binding conformation of the homochiral prolyl PLG
analogues 3 and 5 perhaps possesses a greater capability of
projecting key pharmacophores in the correct topological space
when compared to the heterochiral prolyl analogues 4 and 6
that are relatively unconstrained when compared to the active
highly rigid type-II ꢀ-turn PLG peptidomimetics, such as 2.
Clearly, in terms of allosteric modulation of the dopamine
receptor, more than one secondary structure is able to project
pharmacophores in an orientation that satisfies the pharmacoph-
ore requirements for bioactivity.
Discussion
The greater activity of bicyclic peptidomimetic 7 compared
to the spirobicyclic peptidomimetic 8 may be because the greater
rigidity of the spirobicyclic scaffold prevents the optimal
interaction of 8 with the allosteric binding site. It also may be
that the isobutyl side chain of 7 interacts more effectively with
the hydrophobic pocket in the allosteric binding site than does
the propylene-bridging unit of 8. Previous work has shown that
PLG analogues and peptidomimetics with a hydrophobic moiety
placed in this topological region possess enhanced dopamine
receptor modulating activity.26–28
The activity of 7 and 8 supports the hypothesis that the
homochiral prolyl peptides 3 and 5 may assume a type VI ꢀ-turn
as their bioactive conformation and that such a conformation is
able to initiate a modulatory response because it can present
the necessary topological arrangement of important pharma-
cophore moieties in a manner similar to that of PLG peptido-
Experimental Section
(6S,8aR,2′S)-6-N-(tert-Butoxycarbonyl-2′-pyrrolidinylcarbo-
nyl)amino-6-(2-methylpropyl)-8a-carboxamidoindolizidin-5-
one (23). To the crude carboxylic acid 22 (480 mg, 1.38 mmol)
were added CH2Cl2 (5 mL), DMF (5 mL), and HATU (279 mg,
1.05 equiv). The mixture was stirred for 15 min. The flask was
evacuated (vacuum needle line) for 1 min, and excess NH3 was
introduced through a balloon. This resulted in an exothermic
reaction and the precipitation of a canary-yellow solid. The mixture
was stirred for 24 h, diluted with CH2Cl2 (50 mL), and filtered.
The filtrate was concentrated under reduced pressure, and the
resulting residue was mixed with xylenes (50 mL). The solvent
was removed, and the crude yellow oil that was obtained was
dissolved in EtOAc. This solution was loaded onto a plug of silica
gel (30 g) with the aid of EtOAc. The plug was washed with ether
(300 mL), which removed most of the HMPA, and then eluted with
EtOAc (100 mL), EtOAc/MeOH (20:1, 100 mL), EtOAc/MeOH
(15:1, 200 mL), and EtOAc/MeOH (10:1, 100 mL). The product
was found in the last 80 mL of the eluent. The relevant fractions
were concentrated under reduced pressure to yield a white foam
that was triturated with Et2O and then dissolved in EtOAc/CH2Cl2/
Et2O (1:1:40). The solution was allowed to stand uncovered to give
a white powder that was filtered to give 23 (430 mg, 87%). [R]D
-43.6 (c 1.0, CDCl3); Rf ) 0.4 (EtOAc/MeOH, 9:1); 1H NMR
Figure 3. Enhancement of [3H]NPA binding to bovine striatal membranes
by 7 and 8. Data represent the percent increase in specific [3H]NPA binding
over the control value when the indicated concentration of compound was
added directly to the assay buffer. Results are the mean ( SEM of three
to four separate experiments carried out in triplicate. The data were analyzed
by a one way analysis of variance followed by Dunnett’s posthoc test: (/)
significantly different (p < 0.01) from control value; (//) significantly
different (p < 0.001) from control value.

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6729
(7) Verma, V.; Mann, A.; Costain, W.; Pontoriero, G.; Castellano, J. M.;
Skoblenick, K.; Gupta, S. K.; Pristupa, Z.; Niznik, H. B.; Johnson,
R. L.; Nair, V. D.; Mishra, R. K. Modulation of Agonist Binding to
Human Dopamine Receptor Subtypes by L-Prolyl-L-Leucyl-glycina-
mide and a Peptidomimetic Analogue. J. Pharmacol. Exp. Ther. 2005,
3151228–1236.
(8) Miller, L.; Kastin, A. J. MIF-1 and Tyr-MIF-1 Do Not Alter GABA
Binding on the GABAA Receptor. Brain Res. Bull. 1990, 25, 917–919.
(9) Gulati, A.; Bhargava, H. N. Effect of Melanotropin Release Inhibiting
Factor on Changes by Haloperidol and Centbutindole in Cerebral Cortical
5-Hydroxytryptamine Receptors. Pharmacology 1990, 41, 98–106.
(10) Yu, K.-L.; Rajakumar, G.; Srivastava, L. K.; Mishra, R. K.; Johnson,
R. L. Dopamine Receptor Modulation by Conformationally Con-
strained Analogues of Pro-Leu-Gly-NH2. J. Med. Chem. 1988, 31,
1430–1436.
(11) Sreenivasan, U.; Mishra, R. K.; Johnson, R. L. Synthesis and Dopamine
Receptor Modulating Activity of Lactam Conformationally Constrained
Analogues of Pro-Leu-Gly-NH2. J. Med. Chem. 1993, 36, 256–263.
(12) Subasinghe, N. L.; Bontems, R. J.; McIntee, E.; Mishra, R. K.; Johnson,
R. L. Bicyclic Thiazolidine Lactam Peptidomimetics of the Dopamine
Receptor Modulating Peptide Pro-Leu-Gly-NH2. J. Med. Chem. 1993,
36, 2356–2361.
(13) Genin, M. J.; Mishra, R. K.; Johnson, R. L. Dopamine Receptor
Modulation by a Highly Rigid Spiro Bicyclic Peptidomimetic of Pro-
Leu-Gly-NH2. J. Med. Chem. 1993, 36, 3481–3483.
(CDCl3, 300 MHz, rotamers present about the N-CO2t-Bu bond)
δ 8.26 (s, 1H, CONH2), 7.68 and 6.43 (2s, 1H, CONH), 5.68 (s,
1H, CONH2), 4.26 (br s, Pro R-CH), 3.73–3.29 (m, 4H, δ-CH2
and 3-CH2), 2.56 (dd, J1 ) 11.7 Hz, J2 ) 6.0 Hz, 1H, ꢀ-CH2),
2.35–1.60 (m, 14H, Pro ꢀ-CH2, 1-CH2, Pro γ-CH2, 2-CH2, 7-CH2,
8-CH2, CH2CH(CH3)2), 1.45 (s, 9H, t-Bu), 0.97 (dd, J1 ) 12 Hz,
J2 ) 7.2 Hz, 6H, CH(CH3)2); 13C NMR (CDCl3, 75 MHz, rotamers
present about the N-CO2t-Bu bond) δ 176.2 (CONH2), 171.2
(CONH), 170.0 (5-C), 80.8 (C(CH3)3), 71.0 (8a-C), 60.7, 59.8 (Pro
R-C), 58.6 (6-C), 47.4 (Pro δ-C or 3-C), 45.9 (CH2CH(CH3)2), 45.0
(Pro δ-C or 3-C), 38.4 (1-C), 31.2 and 30.0 (Pro ꢀ-C), 30.3 (7-C),
29.3 (8-C), 28.1 (t-Bu), 27.5 (Pro γ-C), 24.9 (CH2CH(CH3)2), 24.3
(CH2CH(CH3)2), 21.4 (8-C). ESI-HRMS m/z 472.2752 (M + Na)+,
C23H38N4O5 + Na+ requires 472.2740. Anal. (C23H38N4O5) C, H, N.
(6S,8aR)-6-N-(2′-Pyrrolidinylcarbonyl)amino-6-(2-methylpro-
pyl)-8a-carboxamidoindolizidine-5-one Hydrochloride (7·HCl).
To 23 (100 mg, 0.22 mmol) was added 4 N HCl in dioxane (5
mL) under a stream of argon. A white solid precipitated from the
solution within 15–20 s. The suspension was stirred for an extra
15 min, and a vacuum line was inserted in the septum to remove
excess HCl. The residue was then evaporated under reduced
pressure to afford a white gum, which was dissolved in CH2Cl2,
and the solvent was then removed (5×). The resulting white gum
was dissolved in CH2Cl2/MeOH (10:1), and this solution was poured
into ether, upon which a white precipitate formed. The solvents
were decanted and the residue was dried for 2 days under high
vacuum to yield 74 mg of 1 as a hygroscopic white solid. Rf ) 0.7
(14) Khalil, E. M.; Ojala, W. H.; Pradhan, A.; Nair, V. D.; Gleason, W. B.;
Mishra, R. K.; Johnson, R. L. Design, Synthesis, and Dopamine
Receptor Modulating Activity of Spiro Bicyclic Peptidomimetics of
L-Prolyl-L-leucyl-glycinamide. J. Med. Chem. 1999, 42, 628–637.
(15) Johnson, R. L.; Rajakumar, G.; Mishra, R. K. Dopamine Receptor
Modulation by Pro-Leu-Gly-NH2 Analogues Possessing Cyclic Amino
Acid Residues at the C-Terminal Position. J. Med. Chem. 1986, 29,
2100–2104.
1
(EtOH/NH4OH, 4:1); [R]D -24.8 (c 0.5, CD3OD); H NMR (300
MHz, CD3OD, acquired within 5 min of mixing) δ 7.87 and 7.42
(2s, 2H, CONH2, these resonances disappear within 30 min of
mixing), 4.31 (app dd, J1 ) 6.2 Hz, J2 ) 6.6 Hz, 1H, Pro R-CH),
3.67–3.29 (m, 4H, Pro δ-CH2 and 3-CH2), 2.67 (dd, J1 ) 5.7 Hz,
J2 ) 12.0 Hz, 1H, Pro ꢀ-CH2), 2.46–1.66 (m, 14H, Pro ꢀ-CH2,
Pro γ-CH2, 2-CH2, 1-CH2, CH2CH(CH3)2, 7-CH2, 8-CH2), 0.98 (dd,
J1 ) 11.4 Hz, J2 ) 6.3 Hz, 6H, CH(CH3)2). 13C NMR (CDCl3, 75
MHz) δ 179.5 (CONH2), 170.1 (CONH), 173.6 (5-C), 70.1 (8a-
C), 61.2 (Pro R-C), 58.6 (6-C), 48.0 (Pro δ-C or 3-C), 44.0
(CH2CH(CH3)2), 43.8 (Pro δ-C or 3-C), 38.1 (1-C), 32.1 (Pro ꢀ-C),
31.4 (7-C), 29.3 (8-C), 24.9 (Pro γ-C), 24.5 (CH(CH3)2), 24.3
(CH(CH3)2), 21.4 (8-C). ESI-HRMS m/z 351.2358 (M)+,
(C18H31N4O3)+ requires 351.2364. Anal. (C18H31ClN4O3 ·1/3H2O)
C, H, N.
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Acknowledgment. This work was supported in part by NIH
grant NS20036 to R.L.J. We thank Dr. David Rusterholz for helpful
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Supporting Information Available: Experimental details for
the synthesis of 9–14 and 16–22. This material is available free of
charge via the Internet at http://pubs.acs.org.
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