Synthesis and dopamine receptor modulating activity of 3-substituted γ-lactam peptidomimetics of L-prolyl-L-leucyl-glycinamide

Article abstract of DOI:10.1021/jm020441o

γ-Lactam peptidomimetic 2 of Pro-Leu-Gly-NH₂ (PLG) was substituted at the 3-position with isobutyl, butyl, and benzyl moieties to give the PLG peptidomimetics 3-5, respectively. These compounds were synthesized to test the hypothesis that attac

Full text of DOI:10.1021/jm020441o

J . Med. Chem. 2003, 46, 727-733
727
Syn th esis a n d Dop a m in e Recep tor Mod u la tin g Activity of 3-Su bstitu ted
γ-La cta m P ep tid om im etics of L-P r olyl-L-leu cyl-glycin a m id e
Kristine Dolbeare,† Giuseppe F. Pontoriero,§ Suresh K. Gupta,§ Ram K. Mishra,§ and Rodney L. J ohnson*,†
Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455 and Department of Psychiatry
and Behavioral Neurosciences, McMaster University, Hamilton, Ontario L8N 3Z5, Canada
Received October 4, 2002
γ-Lactam peptidomimetic 2 of Pro-Leu-Gly-NH2 (PLG) was substituted at the 3-position with
isobutyl, butyl, and benzyl moieties to give the PLG peptidomimetics 3-5, respectively. These
compounds were synthesized to test the hypothesis that attaching a hydrophobic moiety to
the lactam ring to mimic the isobutyl side chain of the leucyl residue of PLG would increase
the dopamine receptor modulating activity of such peptidomimetics. These peptidomimetics
were tested for their ability to enhance the binding of [3H]-N-propylnorapomorphine to dopamine
receptors isolated from bovine striatal membranes. The rank order of effectiveness of the
3-substituent was benzyl > n-butyl > isobutyl > H.
Sch em e 1
In tr od u ction
The tripeptide L-prolyl-L-leucyl-glycinamide (1, PLG)
has the unique ability to modulate D2 dopamine recep-
tors within the CNS.1-7 Numerous peptide analogues
and peptidomimetics of PLG have been synthesized and
tested.8-16 One of the most potent PLG peptidomimetics
to be made is the γ-lactam 2.11 PLG and 2 induce an
increase in agonist binding to the D2 dopamine receptor
by increasing the affinity of the receptor for agonists
and by increasing the proportion of D2 receptors existing
in the high affinity state.5,17 Both 1 and 2 also have been
shown to inhibit dopamine-stimulated adenylyl cyclase
activity and to enhance N-propylnorapomorphine (NPA)-
stimulated low Km GTPase activity in rat striatal mem-
branes.18
In the rat nigrostriatal 6-hydroxydopamine lesion
model of Parkinson’s disease,19 1 and 2 have been shown
to potentiate the contralateral rotational behavior in-
duced by apomorphine.2,5,20,21 These two compounds also
protect C57 BL/6 mice against MPTP-induced dopam-
inergic degeneration22 and they attenuate haloperidol-
induced c-fos and Fos expression.23
In the original design of 2, the isobutyl side chain that
would correspond to the leucyl side chain was left off
the molecule for synthetic ease. Early SAR studies with
linear tripeptide analogues of 1 modified at the leucine
position showed that a hydrophobic residue at this
position was important for activity.10 Thus, we postu-
lated that incorporation of a hydrophobic side chain into
the structure of 2 would enhance the activity of this
peptidomimetic. To test this hypothesis, the synthesis
of the 3-isobutyl γ-lactam peptidomimetic 3 was under-
taken. In addition, the 3-butyl and 3-benzyl derivatives
also were synthesized to give peptidomimetics 4 and 5,
respectively. These latter two analogues were made
since SAR studies with linear tripeptide analogues of 1
showed that replacing the leucyl residue with either the
norleucine or phenylalanine residues provided ana-
logues equal in potency and efficacy to that of PLG.
Syn th esis
The synthesis of the 3-substituted γ-lactam PLG
peptidomimetics began with the asymmetric synthesis
of the R-allylated oxazolidinones 6a -c through meth-
odology reported previously.24,25 In each case as shown
in Scheme 1, basic hydrolysis of the oxazolidinone
afforded an acid that was immediately converted to its
methyl ester (7a -c). Since the allyloxycarbonyl (Alloc)
protecting group was expected to be a problem with
* To whom correspondence should be addressed. Address: Depart-
ment of Medicinal Chemistry, University of Minnesota, 308 Harvard
St. SE, Minneapolis, MN 55455-0343. Telephone: 612-624-7997. Fax:
612-624-0139. E-mail: johns022@umn.edu.
† University of Minnesota.
§ McMaster University.
10.1021/jm020441o CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/18/2003

728 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Dolbeare et al.
Sch em e 2
F igu r e 1. Stimulation of [3H]NPA binding to bovine striatal
membranes by the PLG peptidomimetics 2-5. Data represent
the percent increase in specific [3H]NPA binding over the
control value when the indicated concentration of peptidomi-
metic was added directly to the assay buffer. Results are the
means ( SEM of 3-4 separate experiments carried out in
triplicate. * Significantly different (p < 0.01) from control
value; ** significantly different (p < 0.001) from control value.
Sch em e 3
some of the subsequent reaction conditions, it was
removed from 7a -c with tetrakis(triphenylphosphine)-
palladium(0) and dimedone to give the free amines 8a -
c. Protection of the amines with Boc2O in THF under
reflux conditions gave the R-allyl derivatives of the
amino acids leucine, norleucine, and phenylalanine,
compounds 9a -c, respectively.
Conversion of the R-allyl amino acids 9a -c to the
corresponding 3-substituted γ-lactams 12a -c is out-
lined in Scheme 2. Cleavage of the terminal alkene in
9a -c by ozonolysis gave moderate to very good yields
of aldehydes 10a -c. Alternatively, the use of osmium
tetroxide and sodium periodate in this instance gave
yields that generally were quite poor. The aldehydes
obtained after silica gel chromatography were treated
with glycine methyl ester hydrochloride under reductive
amination conditions with sodium cyanoborohydride,
sodium acetate, and activated 4 Å molecular sieves in
methanol. It was found that a portion of the amine
formed in this reductive amination process immediately
cyclized to the desired 3-substituted γ-lactam. Thus the
amines 11a -c were not isolated, but rather the crude
mixture of products was heated under reflux conditions
for 2 days to give the desired γ-lactams 12a -c.
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hy-
drochloride (EDC‚HCl) coupling of the hydrochloride
salt of the γ-lactams with Boc-L-Pro-OH occurred in
excellent yields in a mixture of DMF/CH2Cl2 with
triethylamine and 1-hydroxybenzotriazole (HOBt)
(Scheme 3). Aminolysis with ammonia gave tripeptide
amides 14a -c in moderate yields after crystallization
from ethyl acetate/hexanes. Removal of the tert-butoxy-
carbonyl group with trifluoracetic acid in methanol gave
final products 3-5 as very hygroscopic trifluoracetate
salts.
binding for 2-5 was as follows: 2, 36 ( 1%; 3, 59 (
5%; 4, 89 ( 13%; 5, 116 ( 24%. At this concentration,
the general trend was clearly one in which the ability
of a compound to enhance the binding of the dopamine
receptor agonist to dopamine receptors was increased
by the presence of a lipophilic R-substituent. The rank
order of effectiveness was the benzyl > n-butyl >
isobutyl > H.
P h a r m a cology
Discu ssion
The substituted γ-lactam PLG peptidomimetics 3-5
were evaluated in a functional in vitro assay utilizing
bovine striatal membranes.6,26 These analogues along
with 2 were tested for their ability to enhance the
binding of [3H]NPA at three concentrations: 1 nM, 10
nM, and 100 nM. The results are shown in Figure 1. At
a concentration of 100 nM, the percent increase in NPA
Earlier work on the structure-activity relationships
of PLG showed that replacing the leucyl isobutyl side
chain with smaller alkyl moieties gave PLG analogues
that possessed little or no ability to enhance the binding
of dopamine receptor agonists such as ADTN (2-amino-
6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene) to dopam-

L-Prolyl-L-leucyl-glycinamide Peptidomimetics
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 729
were visualized by UV, I2, ninhydrin spray (amines), 2,6-
dichlorophenol indophenol spray (acids), and 2,4-dinitrophe-
nylhydrazine (aldehydes). Chromatographic purification on
silica gel (Merck, grade 60, 240-400 mesh, 60 Å) was done by
flash or gravity methods. Optical rotations were measured on
a Rudolph Research Autopol III polarimeter at the 589 nm
Na D-Line. Melting points were obtained on a Thomas-Hoover
melting point apparatus and are uncorrected. Elemental
analyses were performed by M-H-W Laboratories, Phoenix,
Arizona.
(R)-N-Allyloxyca r b on yl-r-a llyl-leu cin e Met h yl E st er
(7a ). Allyl oxazolidinone 6a 25 (11.9 g, 36.8 mmol) was dissolved
in 184 mL of 1 N NaOH and 185 mL of MeOH and the solution
was heated at reflux for 2 days. The solution was concentrated
in vacuo and the residue partitioned between H2O and EtOAc.
NaHSO4 (10% solution) was added to the aqueous solution
until the pH ) 1, and then the solution was extracted with
EtOAc (6×). The organic layer was washed with H2O followed
by saturated NaCl solution and then dried (MgSO4) and
filtered. The filtrate was concentrated in vacuo to give the free
acid as a yellow oil.
ine receptors. However, the PLG analogues possessing
either a butyl or benzyl group in place of the isobutyl
side chain had about the same activity as PLG in
enhancing the binding of ADTN to dopamine recep-
tors.10
The incorporation of conformational constraints into
the PLG structure such as the lactam moiety in 2 and
the bicyclic thiazolidine lactam moiety in 15 yielded
PLG peptidomimetics that were more active than PLG
itself in modulating dopamine receptors even though
these peptidomimetics lacked the isobutyl side chain
found in PLG.11,13 We postulated that the placement of
lipophilic side chains on the lactam and bicyclic thia-
zolidine lactam scaffolds would enhance the activity of
these PLG peptidomimetics by virtue of their ability to
access the lipophilic binding pocket with which the
leucyl isobutyl side chain interacts.
Previously, we reported on the dopamine receptor
modulating activity of the substituted bicyclic lactams
16-18.25 These PLG peptidomimetics were evaluated
in the rat nigrostriatal 6-hydroxydopamine lesion rota-
tional model. Compound 16 maximally enhanced the
rotations produced by apomorphine by 44% at a dose of
1 µg/kg ip. For compound 17, the maximal percent
increase in rotations was 56% at a dose of 0.1 µg/kg ip.
while for 18 it was 30% at 1 µg/kg ip. In comparison,
the unsubstituted bicyclic lactam 15 only produced a
23% increase in rotations at a dose of 0.1 µg/kg ip. The
results of this previous study showed that incorporation
of a lipophilic substituent on the bicyclic thiazolidine
lactam scaffold enhanced the dopamine receptor modu-
lating activity of the PLG peptidomimetics.
The acid was dissolved in 50 mL of DMF and 10 g of K2CO3
was added. The reaction was cooled to 0 °C and MeI (5.3 mL,
85 mmol) was added dropwise. The reaction was stirred at 0
°C under Ar for 1.5 h and then at room temperature for 4 h.
Solvent was removed from the reaction via high vacuum and
the residue dissolved in H2O and extracted with EtOAc (5×).
The organic layer was washed with H2O, saturated NaHCO3
solution, and saturated NaCl solution successively and then
dried (MgSO4), filtered, and concentrated in vacuo to give a
yellow oil. Silica gel chromatography (4 × 40 cm) with 5%
EtOAc/hexanes as the eluting solvent yielded 9.88 g (99%) of
1
7a as a clear oil. [R]D -5.6 (c 1.70, MeOH). H NMR (CDCl3)
δ 0.76 (d, 3 H, J ) 7.2 Hz), 0.89 (d, 3 H, J ) 6.0 Hz), 1.54-
1.70 (m, 2 H), 2.33-2.44 (m, 2 H), 3.11 (dd, 1 H, J ) 7.5, 13.5
Hz), 3.74 (s, 3 H), 4.53 (d, 2 H, J ) 6.0 Hz), 5.00-5.06 (m, 2
H), 5.18-5.33 (m, 2 H), 5.62-5.50 (m, 1 H), 5.84-5.97 (m, 2
H); 13C NMR (CDCl3) δ 22.4, 23.7, 24.5, 40.7, 43.7, 52.5, 63.5,
65.0, 117.4, 118.8, 132.2, 132.9, 153.8, 174.4. FAB MS (glycerol
matrix) m/z: 270 [M + H]+. Anal. (C14H23NO4) C, H, N.
(R)-N-Allyloxyca r b on yl-r-a llyl-n or leu cin e
Met h yl
Ester (7b). Allyl oxazolidinone 6b25 (7.29 g, 22.5 mmol) was
converted to 7b by the same procedure as described above for
7a . The product was obtained as a clear oil in a yield 4.56 g
1
(75%). [R]D 7.4 (c 1.81, MeOH). H NMR (CDCl3) δ 0.85 (t, 3
H, J ) 7.1 Hz), 0.90-1.00 (m, 1 H), 1.20-1.33 (m, 3 H), 1.69-
1.78 (m, 1 H), 2.23-2.30 (br t, 1 H), 2.48 (dd, 1 H, J ) 7.4,
14.1 Hz), 3.05 (dd, 1 H, J ) 7.4, 13.8 Hz), 3.73 (s, 3H), 4.51 (d,
2 H, J ) 5.1 Hz), 5.01-5.06 (m, 2 H), 5.16-5.31 (m, 2 H), 5.52-
5.66 (m, 1 H), 5.83-5.95 (m, 1 H); 13C NMR (CDCl3) δ 14.0,
22.6, 26.3, 35.1, 39.8, 52.8, 64.1, 65.2, 117.4, 118.9, 132.5, 133.0,
154.0, 173.9. FAB MS (glycerol matrix) m/z: 270 [M + H]+.
Anal. (C14H23NO4) C, H, N.
The present study demonstrates that the dopamine
receptor modulating activity of the lactam PLG pepti-
domimetics also is enhanced by the placement of a
lipophilic substituent off the lactam scaffold. Interest-
ingly, in this case, the benzyl derivative provided the
greatest enhancement followed by the n-butyl and
isobutyl derivatives. This is a different rank order than
that seen with the bicyclic lactam scaffold where it was
n-butyl > isobutyl > benzyl. Although this difference
could be due to the fact that a different assay system
was used in the bicyclic lactam series than that in the
lactam series, there is ample experience in the literature
that previously demonstrates a good correlation between
the activity in these two model systems.16,20 It seems
more likely that either we are seeing differences in
transport into the brain coming into play for the bicyclic
thiazolidine lactams or that the different scaffolds
induce a subtle change in the interaction of the lipophilic
side chain with the PLG binding site.
(S)-N-Allyloxyca r bon yl-r-a llyl-p h en yla la n in e Meth yl
Ester (7c). Allyl oxazolidinone 6c24 (9.64 g, 27 mmol) was
converted to 7c by the same procedure as described above for
7a . The product was obtained as a clear oil in a yield 7.55 g
1
(92%). [R]D 7.3 (c 1.81, MeOH). H NMR (CDCl3) δ 2.64 (dd, 1
H, J ) 7.5, 13.5 Hz), 3.14 (d, 1 H, J ) 13.5 Hz), 3.25 (dd, 1 H,
J ) 6.6, 13.8 Hz), 3.63 (d, 1 H, J ) 16.2 Hz), 3.76 (s, 3 H),
4.54-4.67 (m, 2 H), 5.14-5.09 (m, 2 H), 5.23-5.36 (m, 2 H),
5.58-5.72 (m, 2 H), 5.88-6.01 (m, 1H), 7.02-7.29 (m, 5 H);
13C NMR (CD3CN) δ 40.5, 41.3, 53.5, 65.7, 66.1, 117.9, 120.1,
128.3, 129.6, 131.4, 133.7, 134.8, 137.6, 155.6, 174.0. FAB MS
(glycerol matrix) m/z: 304 [M + H]+. Anal. (C17H21NO4) C, H,
N.
(R)-r-Allyl-leu cin e Meth yl Ester (8a ). Methyl ester 7a
(2.70 g, 10 mmol) was dissolved in 50 mL of dry THF.
Dimedone (8.43 g, 60 mmol) and 0.092 g of Pd(PPh3)4 (0.8%)
were added, and the solution was stirred under N2 overnight.
The solution was diluted with Et2O and extracted with 1 N
HCl (6 × 75 mL). The acid solution was made basic by the
addition of solid K2CO3 until the solution reached a pH of 12.
Extraction of the basic aqueous solution with EtOAc (4 × 100
Exp er im en ta l Section
Gen er a l Asp ects. Thin-layer chromatography was per-
formed on Analtech 250 µm silica gel GHLF Uniplates which

730 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Dolbeare et al.
mL) was followed by washing the organic layer with saturated
NaHCO3 solution and saturated NaCl solution. The organic
layer was dried (MgSO4), filtered, and concentrated in vacuo
to a yellow oil. Kugelrohr distillation at reduced pressure
yielded 1.19 g (64%) of 8a as a clear oil. [R]D 41.3 (c 1.02,
Hz), 3.75 (s, 3 H), 5.09-5.15 (m, 2 H), 5.35 (s, 1 H), 5.61-5.70
(m, 1 H), 7.06-7.34 (m, 5 H); 13C NMR (CDCl3) δ 28.3, 39.8,
40.6, 52.4, 64.8, 79.1, 118.9, 126.8, 128.1, 129.7, 132.3, 136.3,
153.9, 172.9. FAB MS (glycerol matrix) m/z: 320 [M + H]+.
Anal. (C18H25NO4) C, H, N.
1
MeOH). H NMR (CDCl3) δ 0.72 (d, 3 H, J ) 6.0 Hz), 0.84 (d,
(R)-N-(ter t-Bu t oxyca r b on yl)-r-for m ylm et h yl-leu cin e
Meth yl Ester (10a ). R-Allyl amino acid 9a (6.73 g, 23.6 mmol)
was dissolved in 150 mL of dry CH2Cl2, and MeOH (1 mL)
was added. The solution was cooled to -78 °C, and ozone was
bubbled into the solution until a blue color persisted. Nitrogen
was bubbled into the solution, and then dimethyl sulfide (5.2
mL, 0.7 mol) was added dropwise via syringe to quench the
reaction. The solution initially was stirred at -78 °C and then
allowed to warm to room-temperature overnight. Concentra-
tion in vacuo of the solution provided a clear oil that was
chromatographed twice on a 4 × 10 cm silica gel column using
5f20% EtOAc/hexane as the eluent. Final yield was 4.02 g
(59%) of 10a as a clear oil that was used immediately in the
next reaction. [R]D 42.3 (c 0.62, MeOH). 1H NMR (CDCl3) δ
0.79 (d, 3 H, J ) 6.0 Hz), 0.89 (d, 3 H, J ) 6.0 Hz), 1.40 (s, 9
H), 1.48-1.61 (m, 2 H), 2.31-2.35 (m, 1 H), 2.88 (d, 1 H, J )
18.3 Hz), 3.63-3.72 (m, 1 H), 3.75 (s, 3 H), 5.78 (s, 1 H), 9.62
(s, 1 H); 13C NMR (CDCl3) δ 23.2, 23.6, 23.8, 28.2, 43.9, 49.7,
52.8, 59.3, 79.6, 153.9, 173.6, 199.44.
3 H, J ) 6.0 Hz), 1.40-1.48 (m, 1 H), 1.56 (s, 2 H), 1.60-1.70
(m, 2 H), 2.11 (dd, 1 H, J ) 8.4, 13.5 Hz), 2.44 (dd, 1 H, J )
6.0, 13.5 Hz), 3.60 (s, 3 H), 5.03 (d, 2 H), 5.62-5.49 (m, 1 H);
13C NMR (CDCl3) δ 23.2, 24.8, 25.2, 46.3, 49.2, 52.4, 60.8, 120.1,
133.2, 178.4. FAB MS (glycerol matrix) m/z: 186 [M + H]+.
Anal. (C10H19NO2) C, H, N.
(R)-r-Allyl-n or leu cin e Meth yl Ester (8b). Methyl ester
7b (4.05 g, 15 mmol) was deprotected under the same condi-
tions as described above for 8a . Kugelrohr distillation at 65
°C under reduced pressure yielded 2.39 g (86%) of 8b as a clear
1
oil. [R]D 20.3 (c 1.38, MeOH). H NMR (CDCl3) δ 0.87 (t, 3 H,
J ) 7.2 Hz), 1.03-1.14 (m, 1 H), 1.22-1.37 (m, 3 H), 1.52 (dt,
1 H, J ) 4.5, 12.6 Hz), 1.68-1.78 (m, 3 H), 2.23 (dd, 1 H, J )
8.4, 13.5 Hz), 2.54 (dd, 1 H, J ) 6.0, 13.2 Hz), 3.69 (s, 3 H),
5.09-5.14 (m, 2 H), 5.60-5.73 (m, 1 H); 13C NMR (CDCl3) δ
14.1, 23.1, 26.3, 39.9, 44.4, 52.2, 61.0, 119.6, 132.9, 177.5. FAB
MS (glycerol matrix) m/z: 186 [M + H]+. Anal. (C10H19NO2)
C, H, N.
(S)-r-Allyl-p h en yla la n in e Meth yl Ester (8c). Methyl
ester 7c (7.10 g, 23.4 mmol) was deprotected under the same
conditions as described above for 8a . Kugelrohr distillation at
100-102 °C at 0.1 mmHg yielded 4.59 g (89%) of 8c as a clear
(R)-N-(ter t-Bu t oxyca r b on yl)-r-for m ylm et h yl-n or leu -
cin e Meth yl Ester (10b). R-Allyl amino acid 9b (2.74 g, 9.60
mmol) was oxidatively cleaved by the same procedure used
for 10a . The product was obtained as a clear oil (0.7 g, 25%)
after chromatographic purification on a 4 × 10 cm silica gel
column with 10% EtOAc/hexane as the eluent. [R]D 4.5 (c 1.47,
MeOH). 1H NMR (CDCl3) δ 0.84 (t, 3 H, J ) 7.2 Hz), 1.00-
1.09 (m, 1 H), 1.18-1.30 (m, 3 H), 1.39 (s, 9 H), 1.59-1.69 (m,
1 H), 2.16-2.25 (m, 1 H), 2.93 (d, 1 H, J ) 17.1 Hz), 3.50 (d,
1
oil. [R]D 4.8 (c 1.68, MeOH). H NMR (CDCl3) δ 1.54 (s, 2 H),
2.28 (dd, 1 H, J ) 8.4, 13.2 Hz), 2.68 (dd, 1 H, J ) 7.2, 13.2
Hz), 2.75 (d, 1 H, J ) 13.5 Hz), 3.14 (d, 1 H, J ) 13.5 Hz), 3.65
(s, 3 H), 5.10-5.17 (m, 2 H), 5.61-5.75 (m, 1 H), 7.10-7.26
(m, 5 H); 13C NMR (CDCl3) δ 44.1, 45.5, 51.5, 61.5, 119.2, 126.6,
128.0, 129.5, 132.2, 135.8, 176.0. FAB MS (glycerol matrix)
m/z: 220 [M + H]+. Anal. (C13H17NO2) C, H, N.
1 H, J ) 16.8 Hz), 3.73 (s, 3 H), 5.56 (s, 1 H), 9.64 (s, 1H); 13
C
NMR (CDCl3) δ 14.3, 22.9, 26.1, 28.8, 36.5, 49.5, 53.3, 60.6,
80.4, 154.7, 173.7, 199.8.
(R)-N-(ter t-Bu t oxyca r b on yl)-r-a llyl-leu cin e Met h yl
Ester (9a ). Amine methyl ester 8a (4.80 g, 18.4 mmol) was
dissolved in 125 mL of dry THF. Boc2O (4.84 g, 18.4 mmol)
was added, and the solution was refluxed under Ar for 2 days.
H2O (50 mL) was added along with 50 mg of DMAP to catalyze
any excess Boc2O breakdown, and the solution was stirred for
1 day. The solution was extracted with EtOAc (5×), and then
the organic layer was washed successively with 10% NaHSO4
solution, saturated NaHCO3 solution, and saturated NaCl
solution. The organic layer was dried (MgSO4), filtered, and
concentrated in vacuo to yield a clear oil, which was chro-
matographed on a 4 × 40 cm silica gel column. Elution with
3% EtOAc in hexanes gave a quantitative yield of 9a as a clear
(R )-N-(ter t-b u t oxyca r b on yl)-r-for m ylm et h yl-p h en yl-
a la n in e Meth yl Ester (10c). R-Allyl amino acid 9c (1.03 g,
3.22 mmol) was oxidatively cleaved by the same procedure
used for 10a . The desired product was obtained as a clear oil
(0.83 g, 80%) that was used immediately in the next reaction.
[R]D -4.2 (c 1.24, MeOH). 1H NMR (CDCl3) δ 1.44 (s, 9 H),
2.97 (d, 1 H, J ) 9.2 Hz), 3.06 (d, 1 H, J ) 9.2 Hz), 3.61 (d, 1
H, J ) 13.5 Hz), 3.73 (s, 3 H), 3.78-3.87 (m, 1 H), 5.57 (s, 1
H), 6.99-7.28 (m, 5 H), 9.67 (s, 1H); 13C NMR (CDCl3) δ 28.0,
41.1, 48.4, 52.4, 60.6, 79.4, 127.0, 128.0, 129.5, 134.6, 153.9,
171.9, 198.8.
Meth yl 3(R)-[N-(ter t-Bu toxyca r bon yl)a m in o]-3-(2-m e-
th ylp r op yl)-2-oxo-1-p yr r olid in ea ceta te (12a ). Aldehyde
10a (2.42 g, 8.4 mmol) was dissolved in 34 mL of dry MeOH,
and the solution was stirred at room temperature under
nitrogen. Glycine methyl ester hydrochloride (1.06 g, 8.4
mmol), sodium acetate (2.07 g, 25.6 mmol), and 9 g of 4 Å
molecular sieves were added, and the reaction was stirred at
room temperature for 1.5 h. Sodium cyanoborohydride (1.06
g, 16.8 mmol) was added, and the reaction was stirred for 2
days. The reaction was acidified with 10% citric acid solution
to pH 4 and then made basic (pH 10) by the addition of
saturated NaHCO3 solution. The aqueous solution was ex-
tracted 6 × with EtOAc. The organic layer was washed 2×
with saturated NaCl solution, then dried (MgSO4), filtered, and
concentrated in vacuo to a yellow oil. This oil was dissolved in
toluene and heated at reflux for 2 days. The solvent was
removed, and the yellow oil was chromatographed on a 3 ×
45 cm silica gel column using 25% EtOAc/hexanes as the
eluent to give 1.09 g (39%) of 12a as a clear oil. [R]D -24.9 (c
1.10, MeOH). 1H NMR (CDCl3) δ 0.79 (d, 3 H, J ) 7.5 Hz),
0.84 (d, 3 H, J ) 6.3 Hz), 1.30 (s, 9 H), 1.46-1.52 (m, 1 H),
1.65-1.76 (m, 2 H), 2.26-2.33 (m, 2 H), 3.20 (dt, 1 H, J ) 2.5,
8.6 Hz), 3.32-3.41 (m, 1 H), 3.60 (s, 3 H), 3.66 (d, 1 H, J )
17.1 Hz), 4.24 (d, 1 H, J ) 17.1 Hz), 5.05 (s, 1 H); 13C NMR
(CDCl3) δ 23.8, 24.1, 24.7, 28.4, 32.7, 43.1, 44.3, 44.7, 52.2,
60.1, 79.4, 154.6, 168.8, 174.9. FAB MS (glycerol matrix) m/z:
329 [M + H]+. Anal. (C16H28N2O5) C, H, N.
1
oil. [R]D -6.2 (c 1.02, MeOH). H NMR (CDCl3) δ 0.74 (d, 3 H,
J ) 7.2 Hz), 0.87 (d, 3 H, J ) 6.3 Hz), 1.40 (s, 9 H), 1.53-1.65
(m, 2 H), 2.28-2.40 (m, 2 H), 3.08 (dd, 1 H, J ) 7.2, 13.5 Hz),
3.70 (s, 3 H), 4.98-5.03 (m, 2 H), 5.51-5.60 (m, 2 H); 13C NMR
(CDCl3) δ 22.7, 23.6, 24.4, 28.3, 40.7, 43.8, 52.1, 63.3, 78.9,
118.4, 132.5, 153.7, 174.3. FAB MS (glycerol matrix) m/z: 286
[M + H]+. Anal. (C15H27NO4) C, H, N.
(R)-N-(ter t-Bu toxyca r bon yl)-r-a llyl-n or leu cin e Meth yl
Ester (9b). Amine methyl ester 8b (2.04 g, 11 mmol) was
protected with the tert-butoxycarbonyl group in the same
manner as described for 9a to yield 2.85 g (91%) of 9b as a
1
white solid. [R]D 7.5 (c 0.94, MeOH). H NMR (CDCl3) δ 0.87
(t, 3 H, J ) 7.2 Hz), 0.97-1.07 (m, 1 H), 1.26-1.34 (m, 3 H),
1.43 (s, 9 H), 1.68-1.78 (m, 1 H), 2.21 (br t, 1 H, J ) 11.7 Hz),
2.51 (dd, 1 H, J ) 7.2, 13.5 Hz), 2.96-3.02 (m, 1 H), 3.73 (s, 3
H), 5.03-5.07 (m, 2 H), 5.37 (br s, 1 H), 5.56-5.69 (m, 1 H);
13C NMR (CDCl3) δ 14.1, 22.6, 26.3, 28.5, 35.1, 39.8, 52.7, 63.8,
79.3, 118.8, 132.8, 154.0, 174.2. FAB MS (glycerol matrix)
m/z: 286 [M + H]+. Anal. (C15H27NO4) C, H, N.
(S)-N-(ter t-Bu toxycar bon yl)-r-allyl-ph en ylalan in e Meth -
yl Ester (9c). Amine methyl ester 8c (4.29 g, 19.6 mmol) was
protected with the tert-butoxycarbonyl group in the same
manner as described for 9a to give a quantitative yield of 9c
as a clear oil. [R]D 7.6 (c 0.85, MeOH). 1H NMR (CDCl3) δ 1.48
(s, 9 H), 2.60 (dd, 1H, J ) 7.2, 13.2 Hz), 3.13 (d, 1 H, J ) 13.5
Hz), 3.22 (dd, 1 H, J ) 6.3, 13.5 Hz), 3.62 (d, 1 H, J ) 14.7

L-Prolyl-L-leucyl-glycinamide Peptidomimetics
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 731
Meth yl 3(R)-[N-(ter t-Bu toxyca r bon yl)a m in o]-3-bu tyl-
2-oxo-1-p yr r olid in ea ceta te (12b). Aldehyde 10b (0.70 g, 2.43
mmol) was converted to lactam 12b by the same method as
that described above for 12a . The product was obtained as a
clear oil (0.31 g, 39%) after chromatography on a 3 × 45 cm
silica gel column using 10% EtOAc/hexanes followed by 60%
Meth yl 3(R)-[[[1-(ter t-Bu toxyca r bon yl)-2(S)-p yr r olid i-
n yl]ca r b on yl]a m in o]-3-b en zyl-2-oxo-1-p yr r olid in ea ce-
ta te (13c). Lactam 12c (0.84 g, 2.32 mmol) was converted to
the title compound by the same procedures that were used
above for 13a . The material was obtained as a clear oil (0.97
g, 91%) after chromatography on a 3 × 45 cm silica gel column
that was eluted with 10% MeOH/EtOAc. [R]D 37.7 (c 1.01,
1
EtOAc/hexanes as the eluents. [R]D -40.9 (c 0.71, MeOH). H
1
MeOH). H NMR (CDCl3) δ 1.33 (s, 9 H), 1.82-1.86 (m, 2 H),
NMR (CDCl3) δ 0.85 (t, 3 H, J ) 7.5 Hz), 1.20-1.33 (m, 4 H),
1.37 (s, 9 H), 1.58-1.81 (m, 2 H), 2.25-2.32 (m, 1 H), 2.39-
2.50 (m, 1 H), 3.22-3.31 (m, 1 H), 3.46-3.78 (m, 1 H), 3.67 (s,
3 H), 3.67-3.73 (buried d, 1 H), 4.34 (d, 1 H, J ) 16.8 Hz),
5.03 (s, 1 H); 13C NMR (CDCl3) δ 14.0, 23.0, 25.5, 28.5, 31.1,
35.8, 44.4, 44.7, 52.3, 60.2, 79.6, 154.7, 169.0, 174.9. FAB MS
(glycerol matrix) m/z: 329 [M + H]+. Anal. (C16H28N2O5) C,
H, N.
2.00-2.05 (br m, 1 H), 2.16-2.20 (br m, 1 H), 2.36-2.47 (m, 1
H), 2.62-2.69 (m, 1 H), 2.74-2.82 (m, 1 H), 3.09-3.26 (m, 3
H), 3.38-3.42 (m, 2 H), 3.72 (s, 3 H), 3.98 (s, 2 H), 4.20-4.27
(br m, 1 H), 7.18-7.32 (s, 6 H); 13C NMR (DMSO-d6) δ 23.1,
28.0, 28.1, 30.8, 40.9, 43.6, 44.1, 46.5, 51.9, 59.0, 60.6, 78.4,
126.9, 128.1, 130.1, 135.1, 153.2, 168.8, 172.2, 173.1. FAB MS
(glycerol matrix) m/z: 460 [M + H]+. Anal. (C24H33N3O6) C,
H, N.
Meth yl 3(R)-[N-(ter t-Bu toxyca r bon yl)a m in o]-3-ben zyl-
2-oxo-1-p yr r olid in ea ceta te (12c). Aldehyde 10c (0.261 g, 0.8
mmol) was converted to lactam 12c by the same method as
that described above for 12a . The product was obtained as a
clear oil (0.17 g, 58%) after chromatography on a 3 × 45 cm
silica gel column using 20% EtOAc/hexanes as the eluent. [R]D
3(R)-[[[1-(ter t-Bu toxyca r bon yl)-2(S)-p yr r olid in yl]ca r -
bon yl]a m in o]-3-(2-m eth ylp r op yl)-2-oxo-1-p yr r olid in ea c-
eta m id e (14a ). Methyl ester 13a (0.94 g, 2.21 mmol) was
placed in 50 mL of a concentrated solution of methanolic
ammonia overnight. Solvent was removed in vacuo, and for a
total of three times the oily residue was dissolved in CH2Cl2
and then evaporated to dryness. The oil was chromatographed
on a 3 cm × 45 cm silica gel column with 10% MeOH/EtOAc
as the eluting solvent to yield a white solid. Crystallizaton from
EtOAc/hexanes afforded 0.46 g (51%) of product. mp 171-172
1
22.6 (c 0.71, MeOH). H NMR (CDCl3) δ 1.44 (s, 9 H), 2.42-
2.50 (m, 1 H), 2.54-2.61 (m, 1 H), 2.69-2.78 (m, 1 H), 3.11 (s,
2 H), 3.20 (t, 1 H, J ) 9.2 Hz), 3.71 (s, 3 H), 3.90 (d, 1 H, J )
17.1 Hz), 3.99 (d, 1 H, J ) 17.1 Hz), 5.19 (s, 1 H), 7.16-7.29
(m, 5 H); 13C NMR (CDCl3) δ 28.2, 31.3, 41.2, 44.0, 44.5, 52.1,
60.6, 79.5, 126.9, 128.1, 130.1, 135.1, 154.5, 168.5, 174.0. FAB
MS (glycerol matrix) m/z: 363 [M + H]+. Anal. (C19H26N2O5)
C, H, N.
1
°C; [R]D -35.0 (c 0.98, MeOH). H NMR (DMSO-d6, rotamers
present) δ 0.82 (d, 3 H, J ) 6.5 Hz), 0.89 (d, 3 H, J ) 7.0 Hz),
1.30 and 1.34 (s, 9 H), 1.51-1.61 (m, 2 H), 1.70-1.75 (m, 4
H), 2.05-2.07 (m, 1 H), 2.14-2.18 (m, 1 H), 2.29-2.35 (m, 1
H), 3.21-3.54 (m, 4 H), 3.53 (d, 1 H, J ) 17.0 Hz), 3.87 (d, 1
H, J ) 16.5 Hz), 4.10-4.15 (m, 1 H), 7.18 (s, 1 H), 7.28 (s, 1
H), 7.94 (s, 1 H); 13C NMR (DMSO-d6) δ 23.0, 23.2, 23.3, 24.6,
28.0, 29.3, 30.8, 44.0, 44.4, 45.9, 46.6, 58.7, 59.7, 78.4, 153.2,
169.5, 172.8, 173.0. FAB MS (glycerol matrix) m/z: 411 [M +
H]+. Anal. (C20H34N4O5) C, H, N.
Meth yl 3(R)-[[[1-(ter t-Bu toxyca r bon yl)-2(S)-p yr r olid i-
n yl]ca r bon yl]a m in o]-3-(2-m eth ylp r op yl)-2-oxo-1-p yr r o-
lid in ea ceta te (13a ). Lactam 12a (0.78 g, 2.37 mmol) was
treated with excess HCl (4 N in dioxane, 10 mL) overnight at
room temperature. The excess HCl and dioxane were removed
in vacuo, and for a total of three times the residue was
dissolved in CH2Cl2 and the solution evaporated to dryness.
The hydrochloride salt was dried under vacuum for 3 h and
then dissolved in 30 mL of dry CH2Cl2 and 10 mL of dry DMF.
HOBt‚H2O (0.39 g, 2.85 mmol) and Boc-Pro-OH (0.61 g, 2.85
mmol) were added, and the solution was cooled to -78 °C.
Triethylamine (0.8 mL, 5.7 mmol) and EDC‚HCl (0.55 g, 2.85
mmol) were added, and the reaction was allowed to warm to
room-temperature overnight. The reaction was stirred under
nitrogen at room temperature for 3 days. Solvent was removed
in vacuo and the residue dissolved in CH2Cl2. This solution
was washed with 1 M NaHCO3, 10% citric acid, and saturated
NaCl solution. The organic layer was dried (MgSO4), filtered,
and concentrated in vacuo to a yellow oil. This oil was
chromatographed on a 3 × 45 cm silica gel column that was
eluted with 5% MeOH/EtOAc. The product was obtained in a
yield of 0.95 g (94%) as a clear oil. [R]D -92.3 (c 1.34, MeOH).
1H NMR (DMSO-d6) δ 0.85 (d, 6 H, J ) 7.2 Hz), 1.33 (s, 9 H),
1.53-1.59 (m, 2 H), 1.70-1.81 (m, 4 H), 1.97-2.01 (br m, 1
H), 2.09-2.17 (m, 1 H), 2.35-2.26 (br m, 1 H), 3.14-3.39 (m,
4 H), 3.61 (s, 3 H), 3.80 (d, 1 H, J ) 17.1 Hz), 4.10-4.13 (m,
1 H), 4.20 (d, 1 H, J ) 18.3 Hz), 7.42 (s, 1 H); 13C NMR
(DMSO-d6) δ 23.5, 23.8, 24.2, 24.8, 28.5, 30.4, 31.2, 44.1, 44.2,
44.4, 47.0, 52.3, 59.4, 60.1, 78.8, 153.7, 169.5, 172.4, 173.8. FAB
MS (glycerol matrix) m/z: 426 [M + H]+. Anal. (C21H35N3O6)
C, H, N.
Meth yl 3(R)-[[[1-(ter t-Bu toxyca r bon yl)-2(S)-p yr r olid i-
n yl]ca r b on yl]a m in o]-3-b u t yl-2-oxo-1-p yr r olid in e a ce -
ta te (13b). Lactam 12b (0.29 g, 0.88 mmol) was converted to
the title compound by the same procedures that were used
above for 13a . The material was obtained as a clear oil in a
yield of 0.37 g (98%). [R]D -91.2 (c 1.22, MeOH). 1H NMR
(DMSO-d6) δ 0.78 (t, 3 H, J ) 7.4 Hz), 1.16-1.27 (m, 4 H),
1.36 (s, 9 H), 1.57-1.90 (m, 5 H), 2.07-2.14 (br m, 1 H), 2.23-
2.39 (m, 2 H), 3.23-3.42 (m, 4 H), 3.61 (s, 3 H), 3.61-3.67
(buried d, 1 H), 4.09-4.15 (br m, 1 H), 4.29 (d, 1 H, J ) 17.1
Hz), 7.85 (s, 1 H); 13C NMR (DMSO-d6)) δ 14.3, 23.0, 23.5, 25.0,
28.5, 29.2, 31.3, 36.0, 44.1, 44.4, 47.0, 52.3, 59.1, 60.1, 78.7,
153.6, 169.6, 172.6, 173.9. FAB MS (glycerol matrix) m/z: 426
[M + H]+. Anal. (C21H35N3O6) C, H, N.
Meth yl 3(R)-[[[1-(ter t-Bu toxyca r bon yl)-2(S)-p yr r olid i-
n yl]ca r b on yl]a m in o]-3-b u t yl-2-oxo-1-p yr r olid in ea cet a -
m id e (14b). Methyl ester 13b (0.28 g, 0.66 mmol) was
converted to the amide by the same procedure as that used to
make 14a . Crystallizaton from EtOAc/hexanes afforded 0.19
g (70%) of product. mp 174-175 °C; [R]D -35.2 (c 0.84, MeOH).
1H NMR (DMSO-d6, rotamers present) δ 0.86 (t, 3 H, J ) 7.5
Hz), 1.16-1.30 (m, 4 H), 1.33 (s, 9 H), 1.54-1.58 (m, 2 H),
1.68-1.77 (m, 3 H), 2.05-2.11 (m, 2 H), 2.27-2.30 (m, 1 H),
3.23-3.33 (m, 4 H), 3.53 (d, 1 H, J ) 17.0 Hz), 3.87 (d, 1 H, J
) 17.0 Hz), 4.09-4.11 (m, 1 H), 7.21 (s, 1 H), 7.30 (s, 1 H),
8.15 (s, 1 H); 13C NMR (DMSO-d6) δ 13.8, 22.5, 23.1, 24.5, 28.0,
28.6, 30.8, 35.9, 43.9, 45.8, 46.4, 58.6, 59.8, 78.4, 153.1, 169.6,
172.9, 173.0. FAB MS (glycerol matrix) m/z: 411 [M + H]+.
Anal. (C20H34N4O5) C, H, N.
Meth yl 3(R)-[[[1-(ter t-Bu toxyca r bon yl)-2(S)-p yr r olid i-
n yl]ca r bon yl]a m in o]-3-ben zyl-2-oxo-1-p yr r olid in ea ceta -
m id e (14c). Methyl ester 13c (0.50 g, 1.09 mmol) was
converted to the amide by the same procedure as that used to
make 14a . Crystallizaton from EtOAc/hexanes afforded 0.30
g (62%) of product. mp 239-240 °C; [R]D 20.2 (c 0.91, MeOH).
1H NMR (DMSO-d6, rotamers present) δ 1.33 and 1.40 (s, 9
H), 1.65-1.85 (m, 3 H), 2.10-2.38 (m, 4 H), 2.88-2.97 (m, 2
H), 3.06-3.15 (m, 2 H), 3.29-3.34 (m, 2 H), 3.90 (d, 1 H, J )
16.8 Hz), 4.13-4.16 (m, 1 H), 7.18-7.29 (m, 7 H), 8.33 (s, 1
H); 13C NMR (DMSO-d6) δ 23.1, 28.0, 28.1, 30.9, 41.3, 43.8,
45.8, 46.6, 58.9, 60.8, 78.6, 127.1, 128.1, 130.1, 134.6, 159.2,
169.4, 172.6, 172.9. FAB MS (glycerol matrix) m/z: 445 [M +
H]+. Anal. (C23H32N4O5) C, H, N.
3(R)-[[[2(S)-P yr r olid in yl]ca r bon yl]a m in o]-3-(2-m et h -
ylp r op yl)-2-oxo-1-p yr r olid in ea cet a m id e Tr iflu or oa ce-
ta te (3‚CF 3CO2H). Protected lactam 14a (93.7 mg, 0.23 mmol)
was dissolved in 20 mL of dry CH2Cl2 and 349 µL TFA (4.56
mmol) was added to this solution. The solution was stirred at
room-temperature overnight, and then the solvent was re-
moved in vacuo. For three times, the residue was dissolved in
CH2Cl2 and the solvent then evaporated. The clear oil which
was obtained was chromatographed on a 1 × 20 cm silica gel

732 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Dolbeare et al.
column eluting with 10% MeOH/CHCl3 to give a white solid.
The solid was dissolved in H2O, and then the solution was
lyophilized to give 52.8 mg (55%) of product as a hygroscopic
white solid. The product was shown to be pure by analytical
HPLC analysis on a LiChrosorb RP-18 4.6 × 250 mm analyti-
cal column using 40% H2O/CH3CN with 0.1% TFA as the
eluting solvent: tR 3.0 min. HPLC analysis on the RP-18
column eluting with 20% H2O/MeOH and 0.1% TFA gave a tR
triplicate in 1.0 mL of assay buffer containing the indicated
concentrations of radioligand and PLG analogues, and 150-
200 µg of membrane protein to start the reaction. Incubation
of the membranes with ligands was carried out at 25 °C for
60 min in darkness. At the end of the incubation period, the
bound and free ligands were separated by vacuum filtration
on Whatman GF/B filters. The filters were washed with 3 × 5
mL of Tris-EDTA buffer, and the radioactivity was determined
on a Beckman scintillation counter (Model 1780). The percent
increase in NPA binding for compounds 2-5 at the concentra-
tions of 1, 10, and 100 nM was as follows: 1 nM: 2, 8 ( 5%;
3, 12 ( 4%; 4, 32 ( 11%; 5, 26 ( 2%; 10 nM: 2, 45 ( 3%;
3, 9 ( 3%; 4, 43 ( 13%; 5, 24 ( 6%; 100 nM: 2, 36 ( 1%; 3,
59 ( 5%; 4, 89 ( 13%; 5, 116 ( 24%.
1
3.7 min. [R]D 14.2 (c 0.76, MeOH). H NMR (D2O) δ 0.60 (d, 3
H, J ) 6.5 Hz), 0.68 (d, 3 H, J ) 6.5 Hz), 1.40 (dd, 1 H, J )
7.5, 13.0 Hz), 1.48-1.55 (m, 2 H), 1.74-1.83 (m, 3 H), 2.07-
2.11 (m, 1 H), 2.15-2.23 (m, 2 H), 3.09-3.18 (m, 2 H), 3.25-
3.37 (m, 2 H), 3.59 (d, 1 H, J ) 17.0 Hz), 3.93 (d, 1 H, J ) 17.5
Hz), 4.10 (t, 1 H, J ) 8.0 Hz); 13C NMR (D2O) δ 22.2, 22.9,
23.5, 23.9, 29.4, 29.7, 44.0, 45.0, 45.8, 46.3, 59.3, 61.5, 168.4,
172.3, 175.6. FAB HRMS (m-nitrobenzyl alcohol matrix) m/z:
311.2090 (C15H26N4O3 + H+ requires 311.2084).
Ack n ow led gm en t. This work was supported in part
by a NIH grant (NS20036) to R.L.J .
3(R)-[[[2(S)-P yr r olid in yl]ca r b on yl]a m in o]-3-b u t yl-
2-oxo-1-p yr r olid in ea ceta m id e
Tr iflu or oa ceta te
(4‚
CF 3CO2H). The tert-butoxycarbonyl group of 14b (33.7 mg,
0.082 mmol) was removed by the same procedure as that
described above for 3. A yield of 26.2 mg (75%) was obtained.
The product was shown to be pure by analytical HPLC analysis
on a LiChrosorb RP-18 4.6 × 250 mm analytical column using
40% H2O/CH3CN with 0.1% TFA as the eluting solvent: tR
3.0 min. HPLC analysis on the RP-18 column eluting with 20%
H2O/MeOH and 0.1% TFA gave a tR 3.8 min. [R]D 3.0 (c 0.57,
Refer en ces
(1) Mishra, R. K.; Chiu, P.; Mishra, C. P. Pharmacology of L-Prolyl-
L-Leucyl-Glycinamide (PLG): A Review. Methods Find. Exp.
Clin. Pharmacol. 1983, 5, 203-233.
(2) Kostrzewa, R. M.; Kastin, A. J .; Sobrain, S. K. Potentiation of
Apomorphine Action in Rats by L-Prolyl-L-leucyl-glycinamide.
Pharmacol. Biochem. Behav. 1978, 9, 375-378.
(3) Chiu, S.; Paulose, C. S.; Mishra, R. K. Effects of L-Prolyl-L-leucyl-
glycinamide (PLG) on Neuoleptic-induced Catalepsy and Dopam-
ine/Neuroleptic Receptor Bindings. Peptides 1981, 2, 105-111.
(4) Chiu, S.; Paulose, C. S.; Mishra, R. K. Neuroleptic Drug-induced
Dopamine Receptor Supersensitivity: Antagonism by L-Prolyl-
L-leucyl glycinamide. Science 1981, 214, 1261-1262.
(5) Smith, J . R.; Morgan, M. The Effects of PLG on Drug Induced
Rotation in Rats. Gen. Pharmacol. 1982, 13, 203-207.
(6) Srivastava, L. K.; Bajwa, S. B.; J ohnson, R. L.; Mishra, R. K.
Interaction of L-Prolyl-L-Leucyl-Glycinamide with Dopamine D2
Receptor: Evidence for Modulation of Agonist Affinity States
in Bovine Striatal Membranes. J . Neurochem. 1988, 50, 960-
968.
1
MeOH). H NMR (D2O) δ 0.67 (t, 3 H, J ) 7.5 Hz), 0.99-1.05
(m, 1 H), 1.08-1.21 (m, 3 H), 1.54 (dt, 1 H, J ) 4.5, 12.5 Hz),
1.61 (dt, 1 H, J ) 4.5, 12.4 Hz), 1.80-1.92 (m, 3 H), 2.11 (ddd,
1 H, J ) 2.5, 8.5, 13.5 Hz), 2.22-2.28 (m, 2 H), 3.14-3.24 (m,
2 H), 3.30 (dd, 1 H, J ) 8.5, 18.5 Hz), 3.39 (dt, 1 H, J ) 2.0,
10.0 Hz), 3.69 (d, 1 H, J ) 17.0 Hz), 3.96 (d, 1 H, J ) 17.5 Hz),
4.15 (dd, 1 H, J ) 7.0, 8.5 Hz); 13C NMR (D2O) δ 13.0, 22.1,
23.6, 24.3, 29.0, 29.9, 35.6, 45.1, 45.8, 46.4, 59.4, 61.8, 168.7,
172.4, 175.7. FAB HRMS (m-nitrobenzyl alcohol matrix) m/z:
311.2092 (C15H26N4O3 + H+ requires 311.2084).
(7) Bhargava, H. N. Effects of Prolyl-leucyl-glycinamide and Cyclo-
(leucyl-glycine) on the Supersensitivity of Dopamine Receptors
in the Brain Induced by Chronic Administration of Haloperidol
to Rats. Neuropharmacology 1984, 23, 439-444.
(8) J ohnson, R. L.; Rajakumar, G.; Mishra, R. K. Dopamine Receptor
Modulation of Pro-Leu-Gly-NH2 Analogues Possessing Cyclic
Amino Acid Residues at the C-Terminal Position. J . Med. Chem.
1986, 29, 2100-2104.
(9) J ohnson, R. L.; Rajakumar, G.; Yu, K. L.; Mishra, R. K. Synthesis
of Pro-Leu-Gly-NH2 Analogues Modified at the Prolyl Residue
and Evaluation of Their Effects on the Receptor Binding Activity
of the Central Dopamine Receptor Agonist, ADTN. J . Med.
Chem. 1986, 29, 2104-2107.
(10) J ohnson, R. L.; Bontems, R. J .; Yang, K. E.; Mishra, R. K.
Synthesis and Biological Evaluation of Analogues of Pro-Leu-
Gly-NH2 Modified at the Leucyl Residue. J . Med. Chem. 1990,
33, 1828-1832.
3(R)-[[[2(S)-P yr r olid in yl]ca r b on yl]a m in o]-3-b en zyl-
2-oxo-1-p yr r olid in ea ceta m id e
Tr iflu or oa ceta te
(5‚
CF 3CO2H). The tert-butoxycarbonyl group of 14c (47.2 mg,
0.11 mmol) was removed by the same procedure as that
described above for 3. A yield of 27.6 mg (57%) was obtained.
The product was shown to be pure by analytical HPLC analysis
on a LiChrosorb RP-18 4.6 × 250 mm analytical column using
40% H2O/CH3CN with 0.1% TFA as the eluting solvent: tR
3.3 min. HPLC analysis on the RP-18 column eluting with 20%
H2O/MeOH and 0.1% TFA gave a tR 3.2 min. [R]D 9.3 (c 0.45,
MeOH). 1H NMR (D2O) δ 1.79-1.96 (m, 4 H), 2.20-2.28 (m, 3
H), 2.82 (d, 1 H, J ) 13.5 Hz), 2.88 (d, 1 H, J ) 13.5 Hz), 2.96-
3.00 (m, 1 H), 3.12 (d, 1 H, J ) 17.2), 3.13-3.20 (m, 2 H), 3.83
(d, 1 H, J ) 17.0 Hz), 4.17 (t, 1 H, J ) 7.5 Hz), 7.01-7.13 (m,
5 H); 13C NMR (D2O) δ 23.6, 28.3, 29.8, 41.1, 44.6, 45.7, 46.3,
59.4, 62.5, 127.7, 128.5, 129.8, 132.9, 168.5, 172.1, 174.9. FAB
HRMS (m-nitrobenzyl alcohol matrix) m/z: 345.1922
(C18H24N4O3 + H+ requires 345.1927).
(11) Yu, K.-L.; Rajakumar, G.; Srivastava, L. K.; Mishra, R. K.;
J ohnson, R. L. Dopamine Receptor Modulation by Conforma-
tionally Constrained Analogues of Pro-Leu-Gly-NH2. J . Med.
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(12) Sreenivasan, U.; Mishra, R. K.; J ohnson, R. L. Synthesis and
Dopamine Receptor Modulating Activity of Lactam Conforma-
tionally Constrained Analogues of Pro-Leu-Gly-NH2. J . Med.
Chem. 1993, 36, 256-263.
(13) Subasinghe, N. L.; Bontems, R. J .; McIntee, E.; Mishra, R. K.;
J ohnson, R. L. Bicyclic Thiazolidine Lactam Peptidomimetics of
the Dopamine Receptor Modulating Peptide Pro-Leu-Gly-NH2.
J . Med. Chem. 1993, 36, 2356-2361.
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Modulation by a Highly Rigid Spiro Bicyclic Peptidomimetic of
Pro-Leu-Gly-NH2. J . Med. Chem. 1993, 36, 3481-3483.
(15) Baures, P. W.; Ojala, W. H.; Gleason, W. B.; Mishra, R. K.;
J ohnson, R. L. Design, Synthesis, X-ray Analysis, and Dopamine
Receptor-Modulating Activity of Mimics of the “C5” Hydrogen-
Bonded Conformation in the Peptidomimetic 2-Oxo-3(R)-[(2(S)-
pyrrolidinylcarbonyl)amino]-1-pyrrolidineacetamide. J . Med.
Chem. 1994, 37, 3677-3683.
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Mem br a n e P r ep a r a tion . Bovine striatal membranes were
prepared essentially as described previously.6,26 The tissues
were homogenized in 10 volumes of 0.25 M sucrose in a Potter-
Elvehjem homogenizer and centrifuged at 1000g for 10 min.
The supernatant was saved, and the pellet was resuspended
in 10 volumes of 0.25 M sucrose and centrifuged as described
above. The two supernatants were pooled and centrifuged at
17000g for 20 min. The resulting pellet was resuspended in
50 mM Tris-HCL buffer containing 1 mM EDTA (pH 7.4, Tris-
EDTA buffer). The concentration of protein was determined
using the Bradford method,27 and the membrane preparation
was stored at -70 °C in 1 mL aliquots. On the day of use, the
membrane preparation was thawed and diluted with Tris-
EDTA buffer containing 5 mM MgCl2, 0.1 mM dithiothreitol
(DTT), 0.1 mM PMSF, 100 µg/mL bacitracin, and 5 µg/mL
soybean trypsin inhibitor (assay buffer) before being used for
the binding assays.
Bin d in g Assa ys. The binding of [3H]N-propylnorapomor-
phine (NPA) to the membrane preparation was assayed in

L-Prolyl-L-leucyl-glycinamide Peptidomimetics
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J ohnson, R. L. Modulation of Agonist Stimulated Adenylyl
Cyclase and GTPase Activity by L-Pro-L-Leu-Glycinamide and
Its Peptidomimetic Analogue in Rat Striatal Membranes. Neu-
rosci. Lett. 1999, 261, 21-24.
(19) Ungerstedt, U. Postsynaptic Supersensitivity After 6-Hydroxy-
dopamine Induced Degeneration of the Nigro-striatal Dopamine
System. Acta Physiol. Scand. 1971, 367 Suppl., 69-93.
(20) Ott, M. C.; Mishra, R. K.; J ohnson, R. L. Modulation of
Dopaminergic Neurotransmission in the 6-Hydroxydopamine
Lesioned Rotational Model by Peptidomimetic Analogues of
L-Prolyl-L-leucyl-glycinamide. Brain. Res. 1996, 737, 287-291.
(21) Mishra, R. K.; Marcotte, E. R.; Chugh, A.; Barlas, C.; Whan, D.;
J ohnson, R. L. Modulation of Dopamine Receptor Agonist-
Induced Rotational Behaviour in 6-OHDA-Lesioned Rats by a
Peptidomimetic Analogue of Pro-Leu-Gly-NH2 (PLG). Peptides
1997, 18, 1209-1217.
(23) Ott, M. C.; Costain, W. J .; Mishra, R. K.; J ohnson, R. L. L-Prolyl-
L-leucyl-glycinamide and its Peptidomimetic Analogue 3(R)-[(S)-
Pyrrolidinylcarbonyl]amino]-2-oxo-1-pyrrolidineacetamide (PAO-
PA) Attenuate Haloperidol-Induced c-fos Expression in the
Striatum. Peptides 2000, 21, 301-308.
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P.; Holocomb, R. C.; Wood, J . L.; Carroll, P. J .; Hirschmann, R.
De Novo Design, Synthesis, and X-ray Structures of Pyrrolinone-
Based â-Strand Peptidomimetics. J . Am. Chem. Soc. 1994, 116,
9947-9962.
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R. K.; J ohnson, R. L. Synthesis and Dopamine Receptor Modu-
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(26) Baures, P. W.; Ojala, W. H.; Costain, W. J .; Ott, M. C.; Pradhan,
A.; Gleason, W. B.; Mishra, R. K.; J ohnson, R. L. Design,
Synthesis, and Dopamine Receptor-Modulating Activity of Dike-
topiperazine Peptidomimetics of L-Prolyl-L-leucylglycinamide. J .
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(27) Bradford, M. M. A Rapid Method and Sensitive Method for the
Quantitation of Microgram Quantities of Protein Utilizing the
Principle of Protein-dye Binding. Anal. Biochem. 1976, 72,
248-254.
(22) Marcotte, E. R.; Chugh, A.; Mishra, R. K.; J ohnson, R. L.
Protection Against MPTP Treatment by an Analogue of Pro-Leu-
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J M020441O