Synthesis and dopamine receptor modulating activity of novel peptidomimetics of L-prolyl-L-leucyl-glycinamide featuring α,α- disubstituted amino acids

Article abstract of DOI:10.1021/jm980656r

In the present study, L-prolyl-L-leucyl-glycinamide (1) peptidomimetics 3a-3d and 4a-4d were synthesized utilizing α,α-disubstituted amino acids. These analogues were designed to explore the conformational effects of constraints at the φ3 and ψ3 torsion angles. Constrained conformations were verified by the use of X-ray crystallography and circular dichroism. The effects of Pro-Leu-Gly-NH₂ analognes 3a-3d and 4a-4d on enhancing rotational behavior induced by apomorphine in the 6-hydroxydopamine-lesioned animal models of Parkinson's disease were studied. The ability of these peptidomimetics to increase the binding of agonist N-propylnorapomorphine (NPA) to the dopamine D₂ receptor was also examined. Extended analogue Pro- Leu-Deg-NH₂ was the most active compound of this series. It was 10 times more potent and almost 2 times more effective than 1 in increasing apomorphine-induced rotations (56 ± 15% at 1.0 mg/kg ip) and in enhancing [³H]NPA specific binding (40%).

Full text of DOI:10.1021/jm980656r

J . Med. Chem. 1999, 42, 1441-1447
1441
Syn th esis a n d Dop a m in e Recep tor Mod u la tin g Activity of Novel
P ep tid om im etics of L-P r olyl-L-leu cyl-glycin a m id e F ea tu r in g r,r-Disu bstitu ted
Am in o Acid s
Margaret C. Evans,† Ashish Pradhan,§ Shankar Venkatraman,† William H. Ojala,‡ William B. Gleason,‡
Ram K. Mishra,§ and Rodney L. J ohnson*,†
Department of Medicinal Chemistry, The Biomedical Engineering Center, and Department of Laboratory Medicine and
Pathology, University of Minnesota, Minneapolis, Minnesota 55455-0343, and Department of Psychiatry and Behavioural
Neuroscience, McMaster University, Hamilton, Ontario L8N 3Z5, Canada
Received November 20, 1998
In the present study, L-prolyl-L-leucyl-glycinamide (1) peptidomimetics 3a -3d and 4a -4d were
synthesized utilizing R,R-disubstituted amino acids. These analogues were designed to explore
the conformational effects of constraints at the φ3 and ψ3 torsion angles. Constrained
conformations were verified by the use of X-ray crystallography and circular dichroism. The
effects of Pro-Leu-Gly-NH2 analogues 3a -3d and 4a -4d on enhancing rotational behavior
induced by apomorphine in the 6-hydroxydopamine-lesioned animal models of Parkinson’s
disease were studied. The ability of these peptidomimetics to increase the binding of agonist
N-propylnorapomorphine (NPA) to the dopamine D2 receptor was also examined. Extended
analogue Pro-Leu-Deg-NH2 was the most active compound of this series. It was 10 times more
potent and almost 2 times more effective than 1 in increasing apomorphine-induced rotations
(56 ( 15% at 1.0 mg/kg ip) and in enhancing [3H]NPA specific binding (40%).
In tr od u ction
L-Prolyl-L-leucyl-glycinamide (PLG, 1) is a tripeptide
found in the central nervous system that modulates
dopaminergic neurotransmission.1 Studies have shown
that 1 does not modulate dopaminergic neurotransmis-
sion by affecting either dopamine synthesis, uptake, or
metabolism.2-5 Rather, biochemical and pharmacologi-
cal studies indicate that this modulation is brought
about by a mechanism in which 1 renders the dopamine
receptor more responsive to agonists.6,7 Due to this
activity, 1 and its analogues have been postulated to
have therapeutic potential for the treatment of extrapy-
ramidal motor disorders and depression.8
Proton NMR spectroscopy data9 and X-ray structural
analysis10 have shown 1 to exist in a type II â-turn.
Previously, the peptidomimetic 2-oxo-3(R)-[(2(S)-pyrro-
lidinylcarbonyl)amino]-1-pyrrolidineacetamide (2) was
synthesized by Yu et al.11 This peptidomimetic of 1,
which utilizes the Freidinger γ-lactam to restrict the
ψ2 torsion angle, was found to be 1 000-10 000 times
more potent than 1 in a number of pharmacological
assay systems.11-14 NMR spectroscopy studies15 sug-
gested this analogue existed in a turn conformation in
solution, whereas X-ray analysis16 showed an extended
conformation in the solid state.
The series of compounds 3a -3d and 4a -4d were
synthesized in order to investigate the effects of con-
formationally constraining 1 and peptidomimetic 2. R,R-
Dialkylated glycyl residues were utilized in designing
these eight novel mimetics, since work done previously
with constraints of this type have shown that the
presence of two groups on the R-carbon imposes a
marked restriction on the available φ3 and ψ3 space.17
Syn th eses
The series of eight peptidomimetics 3a -3d and 4a -
4d were synthesized in a similar manner beginning with
available R,R-disubstituted amino acids. As shown in
Scheme 1, the N-tert-butoxycarbonyl (Boc)-protected
R,R-disubstituted amino acids 5a -5d were converted to
amides 6a -6d in excellent yields with ammonia and
the coupling reagent 1-(3-dimethylaminopropyl)-3-eth-
ylcarbodiimide (EDC‚HCl).18 Poorer yields (50%) of the
amides were obtained when bromotris(pyrrolidino)-
phosphonium hexafluorophosphate (PyBroP)18 was used
as the coupling reagent. Compounds 6a -6d were depro-
tected with HCl/dioxane to afford the corresponding HCl
salts of the amines. The free amines of 6b -6d were
† Department of Medicinal Chemistry.
‡ The Biomedical Engineering Center and Department of Laboratory
Medicine and Pathology.
§ Department of Psychiatry and Behavioural Neuroscience.
10.1021/jm980656r CCC: $18.00 © 1999 American Chemical Society
Published on Web 03/20/1999

1442 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 8
Evans et al.
Sch em e 1
Sch em e 2
coupled to N-benzyloxycarbonyl-(Cbz)-Pro-Leu-OH to
give the protected tripeptides 8b-8d , respectively, while
the deprotected derivative of 6a was coupled to Boc-Leu-
OH to give dipeptide 7a . Coupling reagents EDC‚HCl
and PyBroP were both utilized in the above coupling
reactions to afford either the di- or tripeptides in greater
than 50% yields. Dipeptide 7a was deprotected with
HCl/dioxane, and the product obtained was coupled with
the pentafluorophenyl ester of Boc-Pro-OH19 to afford
tripeptide 8a in greater than 80% yields. The Cbz group
of 8b-8d and the Boc group of 8a were removed by
hydrogenolysis and HCl/dioxane, respectively, to give
the final tripeptides 3a -3d .
Ta ble 1. Physical Properties of the Synthetic Intermediates of
3a -3d and 4a -4d
yield,
%
compd
mp, °C [M + H]+ a
[R]D, deg
formulab
6a
7a
8b
8c
8d
9b
9c
9d
10a
10b
10c
10d
11a
11b
11c
11d
14b
92 159-163
63 214-218
50 oil
50 111-113
50 nd
60 108-110
50 109-111
61 98-100
58 86-90
43 126-128
35 192-194
60 132-134
66 118-122
81 155-157
50 92-94
231
345
nd
473
nd
347
382
374
314
299
312
343
411
396
443
457
415
C11H22N2O3
-20.6 (c 0.8, MeOH) C17H33N3O4
-79.2 (c 0.4, MeOH) nd
C25H36N4O5
-71.3 (c 1.0, MeOH) nd
-84.3 (c 0.2, MeOH)
-28.2 (c 1.0, CH2Cl2) C15H26N2O5S
-24.6 (c 1.0, CH2Cl2) C16H29N3O4S
-24.6 (c 1.0, CH2Cl2) nd
Scheme 2 illustrates the synthetic route to peptido-
mimetics 4a -4d . Boc-protected amides 6a , 6c, and 6d
and methyl ester 6b were deprotected with HCl/dioxane,
and the corresponding HCl salts were coupled to Boc-
D-Met-OH to afford dipeptides 9a -9d . Freidinger lac-
tam formation was accomplished by stirring these
dipeptides in neat iodomethane followed by treatment
with NaH.11,20 The cyclized products 10a -10d were
deprotected and then coupled to either Cbz-Pro-OH or
Boc-Pro-OH to afford the tripeptides 11a -11d and 12b.
Amidation to afford 13b or 14b was accomplished by
stirring 11b or 12b in a 50% NH3/MeOH solution for
11 days at 50 °C. Final Pro-Leu-Gly-NH2 analogues 4a -
4d were obtained after either hydrogenolysis or HCl/
dioxane treatment of the respective protected precur-
sors.The physicalproperties ofthe syntheticintermediates
of 3a -3d and 4a -4d are given in Table 1.
+16.1 (c 0.4, MeOH)
C15H27N3O4
+39.7 (c 0.8, MeOH) C14H22N2O5
-8.0 (c 1.0, CH2Cl2) C15H25N3O4
-15.0 (c 1.0, CH2Cl2) C16H27N3O4
-34.7 (c 0.63, MeOH) nd
-19.3 (c 0.7, MeOH) C19H29N3O6
-48.0 (c 1.0, MeOH) C23H30N4O5
-48.0 (c 1.0, MeOH) C24H32N4O5
-66.2 (c 1.0, MeOH) C21H26N4O5
50 72-74
50 132-136
a
b
FAB MS m/z. CHN analyses were found to be within (0.4%
of the calculated data, unless otherwise indicated. nd, not deter-
mined.
X-Ra y Cr ysta llogr a p h y
The X-ray crystal structures of 8a and 14b were
solved and are depicted in Figures 1 and 2, respec-
tively.21 In the crystals of 8a there were two unique,
but similar, molecules found which defined the unit of

Peptidomimetics of L-Prolyl-L-leucyl-glycinamide
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 8 1443
F igu r e 3. CD spectra of PLG (1) and the R,R-dialkylated
glycyl analogues 3a and 3c.
F igu r e 1. ORTEP drawing of 8a A with crystallographic
numbering system.
F igu r e 4. CD spectra of PLG lactam-containing peptidomi-
metics 2, 4a , 4b, and 4d .
respectively. Compounds 1, 3c, and 4b exhibited curves
of ideal type II â-turns, with broad minima at 230 nm
and strong maxima at 208 nm.22,23 Extended analogue
3a produced a spectrum with no significant CD signal,
indicative of a flexible molecule which exhibits several
conformations.24 The lactam analogues 2, 4a , and 4d
produced curves which were shifted from those of the
ideal type II â-turn (with maxima around 217 nm).
However, such curves have been shown in the literature
to represent turn type conformations.25,26 Incorporation
of the lactam constraint changes the amide group
environment and takes a secondary amide to a tertiary
amide. It can therefore be postulated that the CD
spectrum of a lactam would be shifted from that of a
typical amide. Circular dichroism previously done on
peptides containing R,R-disubstituted amino acids yielded
spectra which were suggestive of type II â-turn confor-
mations.27 These spectra were similar to those obtained
in this study.
F igu r e 2. ORTEP drawing of 14b with crystallographic
numbering system.
Ta ble 2. φ and ψ Torsion Angles Observed in the Crystal
Structures of 8a and 14b
torsion angle, deg
φ2
ψ2
φ3
ψ3
-4
-176
+177
0
14b
8a (A)
8a (B)
ideal type II â-turn
-49
-136
-124
-60
+134
+139
+145
120
+85
-176
-177
80
pattern. As summarized in Table 2, both solid-state
forms for 8a (A and B) were found to exist in an
extended conformation. In contrast, for peptidomimetic
14b, the φ, ψ torsion angles were close to those for an
ideal type II â-turn.
P h a r m a cology
The effects of Pro-Leu-Gly-NH2 analogues 3a -3d and
4a -4d in enhancing rotational behavior induced by
apomorphine in the 6-hydroxydopamine-lesioned animal
model of Parkinson’s disease13,28 are summarized in
Table 3. The analogues were tested at 0.01, 0.1, 1.0, and
CD Sp ectr oscop y
Shown in Figures 3 and 4 are the CD spectra of the
leucine-containing compounds 1, 3a , and 3c and the CD
spectra of the lactam analogues 2, 4a , 4b, and 4d ,

1444 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 8
Evans et al.
Ta ble 3. Effects of PLG Analogues 3a -3d and 4a -4d on
Apomorphine-Induced Rotational Behavior in Rats with
Unilateral 6-Hydroxydopamine Substantia Nigra Lesionsa
% change from control, mean ( SEM
compd
0.01 µg/kg
0.10 µg/kg
1.0 µg/kg
10.0 µg/kg
2b
20
75
191
1 ( 6
3a
3b
3c
3d
4a
4b
4c
4d
6 ( 5
15 ( 10
-15 ( 6
-2 ( 4
2 ( 3
31 ( 11
18 ( 6
-1 ( 8
14 ( 13
-3 ( 7
10 ( 4
-5 ( 2
1 ( 4
56 ( 15**
8 ( 5
-7 ( 5
-26 ( 5
-25 ( 6**
8 ( 14
4 ( 4
5 ( 2
-2 ( 3
17 ( 8
-8 ( 5
19 ( 8*
19 ( 12
-6 ( 1
4 ( 6
-4 ( 4
-2 ( 6
7 ( 4
a
Apomorphine dose ) 0.25 mg/kg ip. Number of contralateral
rotations in 10 min expressed as a percentage change from control
rotational response (n ) 5). Statistical difference from the respec-
tive control group is indicated as follows: *p < 0.05, **p < 0.01.
Maximal response observed upon PLG administration was found
F igu r e 6. Dose-response effect for 3a on [3H]-N-propylnor-
apomorphine ([3H]NPA) binding to the dopamine D2 receptor.
*p < 0.05, **p < 0.01 from control.
b
to be 32% at 1 mg/kg.13,28 Data is from Mishra et al.13 and is
the number of contralateral rotations in 30 min expressed as a
percentage change from control rotational response (n ) 5).
) 180°). In contrast, the 1-aminocyclopropanecarboxylic
(Ac3c) residue favors φ and ψ torsion angles of 90° and
0°, respectively and thus promotes a turn conformation.
An increase in ring size to the five-membered (Ac5c) or
six-membered (Ac6c) rings yields residues which pro-
mote either turn (φ ) 90°, ψ ) 0°) or helix (φ ) -60°, ψ
) -30°) type structures.
Low doses of either 3a or 4c significantly potentiated
apomorphine-induced rotations (Table 3). The maximal
effect observed upon administration of 3a (56% at 1.0
µg/kg) was about 1.5 times greater than the previously
reported maximal response observed upon administra-
tion of 1 (32% at 1 mg/kg)13,28 and occurred at a 1000-
fold lower dose. Analogue 4c (19% at 1.0 µg/kg) was also
shown to be more potent, but it produced only about
one-half the maximal response of that seen with 1.
Neither analogue 3a nor 4c potentiated apomorphine-
induced rotations to the degree observed upon admin-
istration of peptidomimetic 2, which showed percentage
increases of 191% and 75% at doses of 10 and 1.0 µg/
kg, respectively.13
Analogues 3a , 3d , 4c, and 4d produced equal, or
greater, percentage increases in [3H]NPA specific bind-
ing (Figure 5) compared to that of 1 (24% at 1 µM),6,7
but at a 10-fold lower dose. In contrast, analogues 3b,
3c, 4a , and 4b were found to be less active than 1 at 1
µM (data not shown) in increasing the agonist binding
to dopamine receptors. Thus, 3a was the most active
compound in increasing apomorphine-induced rotations
and [3H]NPA specific binding. In a separate study, 3a
was also found to be the most active of this series in
decreasing haloperidol-induced catalepsy.29 Compound
3d proved to be an interesting analogue in that at low
doses it increased [3H]NPA specific binding and apo-
morphine-induced rotations, although the latter effect
was not shown to be statistically significant. In contrast
to the other analogues which lost their modulating
activity at high doses, however, 3d significantly de-
creased apomorphine-induced rotations suggesting that
it may induce an unfavorable conformational change in
the D2 receptor resulting in decreased apomorphine
binding.
F igu r e 5. Effect of PLG analogues (0.1 µM) on [3H]-N-
propylnorapomorphine ([3H]NPA) binding to the dopamine D2
receptor. *p < 0.05 from control.
10.0 µg/kg, ip. Most of the analogues exhibited a bell-
shaped dose-response profile, which is characteristic
of 1 and its analogues.6,13 Compounds 3a and 4c
produced a statistically significant increase in apomor-
phine-induced rotational behavior at a dose of 1.0 µg/
kg. The percent increase for 3a was 56%, while that for
4c was 19%. Interestingly, analogue 3d produced a
significant decrease (-25%) in apomorphine-induced
rotation at a dose of 10.0 µg/kg. Compound 3c also
produced a decrease at this dose level, but it was not
found to be significant.
Analogues 3a -3d and 4a -4d were tested for their
ability to increase the binding of agonist N-propylnor-
apomorphine (NPA) to the dopamine D2 receptor (Figure
5). All of the analogues, except 3c (∼5%), were shown
to increase [3H]NPA specific binding by at least 15% at
0.1 µM. The rank order potency of analogues which
exhibited a statistically significant increase in agonist
binding was found to be 3a > 4c > 4d > 3d . Analogue
3a was the most active compound, increasing [3H]NPA
specific binding by 40%. The full dose-response curve
for 3a is shown in Figure 6.
Discu ssion
R,R-Disubstituted amino acids have demonstrated
great versatility as peptide constraints because of the
restrictions placed on the residues’ φ and ψ torsion
angles.17 For example, the diethylglycine (Deg) residue
favors formation of a fully extended structure (φ and ψ
Our previous studies with conformationally con-
strained analogues of 1 suggested that its bioactive
conformation was a type II â-turn.11,19,30 Thus, the good
activity of 3a in the tripeptide series 3a -3d was

Peptidomimetics of L-Prolyl-L-leucyl-glycinamide
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 8 1445
δ 4.30-4.40 (m, 2 H, Pro R-CH, Leu R-CH), 3.30-3.41 (m, 2
H, Pro δ-CH2), 2.39-2.47 (m, 1 H, Pro â-CH), 1.96-2.10 (m, 5
H, Pro â-CH and γ-CH2, Leu â-CH2), 1.72-1.87 (m, 2 H, Deg
â-CH2), 1.53-1.66 (m, 3 H, Deg â-CH2, Leu γ-CH), 0.91 (d, J
) 4.8 Hz, 3 H, Leu δ-CH3), 0.79 (d, J ) 4.8 Hz, 3 H, Leu δ-CH3),
0.72 (t, J ) 7.5 Hz, 6 H, Deg-CH3); 13C NMR (CDCl3) δ 7.7
(Deg γ-C), 20.8 (Leu δ-C), 22.7 (Pro γ-C), 24.1 (Leu γ-C), 25.3
(Pro â-C), 28.4 (Leu â-C), 30.5 (Deg â-C), 46.6 (Pro δ-C), 54.0
(Leu R-C), 60.2 (Pro R-C), 65.4 (Deg R-C), 169.9 (CONH), 172.0
(CONH), 176.7 (CONH2); FAB MS m/z 341 [M + H]+. Anal.
(C17H33N4O3Cl‚2H2O) C, H, N.
2-[3(R)-[(2(S)-P yr r olid in ylca r bon yl)a m in o]-2-oxop yr -
r olid in -1-yl]-2-eth ylbu ta n a m id e Hyd r och lor id e (4a ). Pre-
cipitation from MeOH/Et2O provided 0.11 g (33%) of 4a : mp
136-141 °C; [R]D +6.9 (c 0.42, MeOH); 1H NMR (D2O) δ 4.56-
4.63 (m, 1 H, Pro R-CH), 4.33-4.38 (m, 1 H, lactam 3-CH),
3.51-3.72 (m, 2 H, lactam 5-CH2), 3.41-3.45 (m, 2 H, Pro
δ-CH2), 2.41-2.49 (m, 2 H, lactam 4-CH2), 1.88-2.12 (m, 6 H,
Pro γ-CH2 and â-CH2, Deg â-CH2), 1.72-1.83 (m, 2 H, Deg
â-CH2), 0.77-0.85 (m, 6 H, Deg γ-CH3); 13C NMR (D2O) δ 7.5,
7.7 (Deg γ-C), 24.5, 24.6 (Deg â-C), 24.9 (Pro γ-C), 25.9 (lactam
4-C), 30.3 (Pro â-C), 44.3 (Pro δ-C), 47.2 (Deg R-C), 52.8 (lactam
5-C), 170.3 (CONH), 174.2 (lactam-CO), 178.7 (CONH2); FAB
MS m/z 311 [M + H]+. Anal. (C15H27N4O3Cl‚2H2O) C, H, N.
surprising, since it was predicted that the presence of
the diethylglycine residue would promote an extended
conformation. Indeed, an X-ray structure of a derivative
of 3a , 8a , indicated the predicted extended conforma-
tion. However, the CD spectrum of 3a revealed no
defined structure indicating that the analogue was quite
flexible. This lack of constraint could allow the dieth-
ylglycine analogue to adopt the bioactive conformation
of 1 at the receptor. What is more difficult to reconcile
is the relative inactivity of 3b-3d in comparison to 3a ,
since on the basis of the restrictions placed on the φ and
ψ torsion angles by the cyclic R,R-disubstituted residues
one would expect these analogues to be capable of
mimicking the postulated bioactive conformation of 1.
Although a previous study showed that cyclic imino
acids are tolerated at the C-terminal position of 1,31 it
is clear that cyclic R,R-disubstituted residues have a
detrimental effect. It may be that ethyl side chains of
3a , which are free to rotate, are able to access hydro-
phobic binding sites that the cyclic R,R-disubstituted
residues cannot.
1-[3(R)-[(2(S)-P yr r olid in ylca r bon yl)a m in o]-2-oxop yr -
r olid in -1-yl]cyclop r op a n e-1-ca r boxa m id e Hyd r och lor id e
(4b). Precipitation from MeOH/Et2O provided 190 mg (79%)
of 4b: mp 170-174 °C; [R]D -7.1 (c 0.58, MeOH); 1H NMR
(D2O) δ 4.36-4.42 (m, 2 H, Pro R-CH, lactam 3-CH), 3.35-
3.61 (m, 4 H, Pro δ-CH2, lactam 5-CH2), 2.02-2.52 (m, 2 H,
lactam 4-CH2), 2.02-2.18 (m, 4 H, Pro γ-CH2 and â-CH2),
1.44-1.55 (m, 2 H, Ac3c â-CH2), 1.26-1.35 (m, 2 H, Ac3c
â-CH2); 13C NMR (D2O) δ 16.1, 17.5 (Ac3c â-C), 24.4 (Pro γ-C),
25.1 (lactam 4-C), 30.4 (Pro â-C), 38.0 (Pro δ-C), 46.0 (Ac3c
R-C), 47.1 (lactam 5-C), 53.1 (lactam 3-C), 59.1 (Pro R-C), 170.4
(CONH), 176.2 (lactam-CO), 176.3 (CONH2); FAB MS m/z 281
[M + H]+. Anal. (C13H22N4O3Cl) C, H, N.
Gen er a l Dep r ot ect ion P r oced u r e for t h e N-Ben zyl-
oxyca r bon yl Gr ou p . Compounds 3b-3d , 4c, and 4d were
made by a general procedure in which a solution of the
N-benzyloxycarbonyl-protected tripeptide (1.0 mmol) in MeOH
(10 mL) containing 5% Pd/C (1 mg) was hydrogenated at 30
psi for 3 h. The solution was filtered through Celite, and
solvent was removed from the filtrate under aspirator pressure
to yield product which was crystallized from EtOAc/MeOH.
L-P r olyl-L-leu cyl-1-a m in ocyclop r op a n eca r b oxa m id e
(3b). Yield ) 20 mg (60%): mp 142-144 °C; [R]D +82.1 (c 0.6,
MeOH); 1H NMR (D2O) δ 4.31-4.35 (m, 1 H, Pro R-CH), 4.16
(t, J ) 7.8 Hz, 1 H, Leu R-CH), 3.29-3.37 (m, 2 H, Pro δ-CH2),
2.36-2.40 (m, 1 H, Pro â-CH), 1.90-2.03 (m, 3 H, Pro â-CH,
Leu â-CH2), 1.47-1.60 (m, 4 H, Pro γ-CH2, Leu γ-CH, Ac3c
â-CH), 1.49-1.75 (m, 1 H, Ac3c â-CH), 1.02-1.13 (m, 2 H, Ac3c
â-CH), 0.86, 0.83 (d, J ) 4.8 Hz, Leu δ-CH3); 13C NMR (D2O)
δ 17.4 (Ac3c â-C), 21.8, 22.4 (Leu δ-C), 24.3 (Ac3c â-C) 30.6
(Leu γ-C), 37.5 (Pro γ-C), 34.6 (Pro â-C), 39.2 (Leu â-C), 47.2
(Pro δ-C), 54.2 (Leu R-C), 60.1 (Pro R-C), 78.2 (Ac3c R-C), 170.9,
176.8 (CONH), 177.6 (CONH2); FAB HRMS m/z 311.2107
(C15H26N4O3 + H+ requires 311.2085); HPLC (CHCl3/MeOH,
3:1) tR ) 3.9 min.
L-P r olyl-L-leu cyl-1-a m in ocyclop en t a n eca r b oxa m id e
(3c). Yield ) 30 mg (50%): mp 142-144 °C; [R]D -74.2 (c 0.6,
MeOH); 1H NMR (D2O) δ 4.23-4.30 (m, 1 H, Pro R-CH), 4.12-
4.16 (m, 1 H, Leu R-CH), 3.11-3.25 (m, 2 H, Pro δ-CH2), 2.27-
2.33 (m, 1 H, Pro â-CH), 2.09-2.17 (m, 1 H, Pro â-CH), 1.44-
1.91 (m, 13 H, Ac5c-CH2, Pro γ-CH2, Leu γ-CH and â-CH2),
0.92, 0.86 (d, J ) 5.4 Hz, 6 H, Leu δ-CH3); 13C NMR (D2O) δ
24.5 (Ac5c γ-C), 24.9, 25.1 (Leu δ-C), 26.6, 26.7 (Ac5c â-C), 30.8
(Pro γ-C), 38.1 (Leu γ-C), 40.3 (Leu â-C), 46.8 (Pro â-C), 47.2
(Pro δ-C), 53.3 (Leu R-C), 60.4 (Pro R-C), 67.6 (Ac5c R-C), 170.0,
174.3 (CONH), 180.4 (CONH2); FAB HRMS m/z 339.2394
(C17H30N4O3 + H+ requires 339.2398); HPLC (CHCl3/MeOH,
3:1) tR ) 4.2 min.
Combining the γ-lactam constraint with the R,R-
disubstituted glycine constraints provided peptidomi-
metics 4a -4d which showed activity, but none came
close to approaching the potency seen with the simple
γ-lactam peptidomimetic 2. Clearly this combination of
constraints was detrimental to their activity even
though X-ray crystallography and CD spectroscopy
indicated that 4a -4d were capable of existing in the
postulated type II â-turn bioactive conformation. Inter-
estingly, in this series it was the analogue containing
the C-terminal Ac5c R,R-disubstituted residue, 4c, that
showed the best activity, while the least active com-
pound was 4a with the diethyl substitution. This profile
was opposite of that seen with the R,R-disubstituted
tripeptide series 3a -3d suggesting that the two series
may be interacting with the Pro-Leu-Gly-NH2 modula-
tory site in a different manner. Further studies with a
series of Pro-Leu-Gly-NH2 analogues and lactam pep-
tidomimetics containing flexible, hydrophobic groups at
the C-terminal R-carbon should provide some insight on
this hypothesis.
Exp er im en ta l Section
Gen er a l Asp ects. DMF was purchased in Aldrich Sure-
Seal bottles, and CH2Cl2 was distilled from CaH2. Thin-layer
chromatography was performed on Analtech 250-µm silica gel
GF Uniplates and visualized by UV, I2, and ninhydrin spray
(amines). Chromatographic purification on silica gel (Merck,
grade 60, 240-400 mesh, 60 Å) was done by gravity methods.
Optical rotations were measured on a Rudolph 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, AZ. 1H and 13C NMR spectra
were obtained on a GE Omega 300-MHz instrument.
Gen er a l Dep r otection P r oced u r e for th e N-ter t-Bu -
toxyca r bon yl Gr ou p . Compounds 3a , 4a , and 4b were
prepared by a general procedure in which N-tert-butoxycar-
bonyl-protected tripeptide (1.0 mmol) was dissolved in 4 N HCl/
dioxane (10 mL), and the resulting solution was stirred
overnight at room temperature. Excess HCl and the dioxane
then were removed under aspirator pressure by forming an
azeotrope with CH2Cl2 (2×).
L-P r olyl-L-leu cyl-d iet h ylglycin a m id e H yd r och lor id e
(3a ). Crystallization from EtOH provided 102 mg (74%) of
3a : mp 158-162 °C; [R]D -57.8 (c 1.25, MeOH); 1H NMR (D2O)
L-P r olyl-L-leu cyl-1-am in ocycloh exan ecar boxam ide (3d).
Yield ) 30 mg (50%): mp 111-116 °C; [R]D -69.0 (c 0.3,

1446 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 8
Evans et al.
1
MeOH); H NMR (D2O) δ 4.33-4.42 (m, 2 H, Pro R-CH, Leu
mg (88%) of crystals: mp 219-222 °C; [R]D -67.7 (c 0.51,
R-CH), 3.31-3.40 (m, 2 H, Pro δ-CH2), 2.38-2.42 (m, 1 H, Pro
â-CH), 2.14-2.19 (m, 1 H, Pro â-CH), 1.84-1.97 (m, 3 H, Leu
γ-CH and â-CH2), 1.60-1.70 (m, 9, Ac6c δ-CH2, γ-CH2 and
â-CH, Pro γ-CH2), 1.20-1.29 (m, 3, Ac6c â-CH2), 0.88, 0.92 (d,
J ) 5.4 Hz, 6 H, Leu δ-CH3); 13C NMR (D2O) δ 21.6 (Leu δ-C),
21.6 (Ac6c δ-C), 21.7 (Leu δ-C), 22.4, 24.3 (Ac6c γ-C), 25.0, 25.2
(Ac6c â-C), 30.2 (Pro γ-C), 30.6 (Leu γ-C), 33.9 (Leu â-C), 40.2
(Pro â-C), 47.2 (Pro δ-C), 53.7 (Leu R-C), 60.1 (Pro R-C), 60.9
(Ac6c R-C), 170.3, 174.6 (CONH), 180.7 (CONH2); FAB HRMS
m/z 353.2545 (C16H32N4O3 + H+ requires 353.2554); HPLC
(CHCl3/MeOH, 3:1) tR ) 3.7 min.
1-[3(R)-[(2(S)-P yr r olid in ylca r bon yl)a m in o]-2-oxop yr -
r olid in -1-yl]cyclop en ta n e-1-ca r boxa m id e (4c). Yield ) 6
mg (85%): mp 142-144 °C; [R]D -80.5 (c 0.6, MeOH); 1H NMR
(D2O) δ 4.38-4.65 (m, 1 H, Pro R-CH), 4.06-4.17 (m, 1 H,
lactam 3-CH), 3.55-3.79 (m, 3 H, Pro δ-CH2, lactam 5-CH),
3.28-3.38 (m, 1 H, lactam 5-CH), 3.14-3.23 (m, 1 H, Pro
â-CH), 2.29-2.57 (m, 2 H, Pro â-CH, lactam 4-CH), 2.16-2.23
(m, 4 H, Pro γ-CH2, lactam 4-CH, Ac5c â-CH), 1.93-2.10 (m,
3 H, Ac5c â-CH and γ-CH2), 1.64-1.77 (m, 4 H, Ac5c γ-CH2);
13C NMR (D2O) δ 24.3, 24.5 (Ac5c γ-C), 24.5, 25.0 (Ac5c â-C),
30.5 (Pro γ-C), 36.0 (Pro â-C), 38.3 (lactam 4-C), 40.0 (Pro δ-C),
47.1 (lactam 5-C), 53.5 (lactam 3-C), 60.1 (Pro R-C), 67.6 (Ac5c
R-C), 170.3 (CONH), 174.7 (lactam-CO), 179.9 (CONH2); FAB
MS m/z 309 [M + H]+. Anal. (C15H24N4O3) C, H, N.
1-[3(R)-[(2(S)-P yr r olid in ylca r bon yl)a m in o]-2-oxop yr -
r olid in -1-yl]cycloh exa n e-1-ca r boxa m id e (4d ). Yield ) 30
mg (86%): mp 111-115 °C; [R]D -50.5 (c 1.0, MeOH); 1H NMR
(D2O) δ 4.95-5.12 (m, 1 H, Pro R-CH), 4.37-4.43 (m, 1 H,
lactam 3-CH), 3.44-3.80 (m, 4 H, Pro δ-CH2, lactam 5-CH2),
2.84-2.98 (m, 2 H, Pro â-CH2), 1.76-2.38 (m, 10 H, Pro γ-CH2,
lactam 4-CH2, Ac6c δ-CH2 and γ-CH2), 1.42-1.54 (m, 4 H, Ac6c
â-CH2); 13C NMR (D2O) δ 22.4 (Ac6c δ-C), 22.6, 25.2 (Ac6c γ-C),
25.4, 25.6 (Ac6c â-C), 31.0 (Pro γ-C), 31.7 (lactam 4-C), 32.5
(Pro â-C), 43.8 (lactam 5-C), 47.0 (Pro δ-C), 52.9 (lactam 3-C),
60.6 (Pro R-C), 64.5 (Ac6c R-C), 175.3 (CONH); 176.4 (lactam-
CO), 179.3 (CONH2); FAB MS m/z 323 [M + H]+. Anal.
(C16H26N4O3) C, H, N.
MeOH). Anal. (C22H40N4O5) C, H, N.
Gen er a l Cou p lin g P r oced u r e w ith Br om otr is(p yr r o-
lid in o)p h osp h on iu m Hexa flu or op h osp h a te (P yBr oP ).
Compounds 8b-8d , 9b-9d , 11c, and 11d were prepared by
the following general procedure. In a dry flask, the N-tert-
butoxycarbonyl-protected amino acid (1.10 mmol) and the HCl
salt of either a C-terminal protected amide or ester (1.0 mmol)
were dissolved in CH2Cl2. PyBroP (1.0 mmol) and DMAP (0.01
mmol) then were added to the solution. The mixture was cooled
in an ice bath to 0 °C, and then diisopropylethylamine (3.0
mmol) was added. The reaction was allowed to proceed for 2
days, after the solution to came to room temperature. The
solution was washed with 10% citric acid, 1 M NaHCO3, water,
and brine. The organic layer was dried over MgSO4, filtered,
and concentrated under aspirator pressure whereupon the
crude product was purified by either crystallization (8c, EtOAc/
hexanes; 9c, EtOAc/hexanes; 9d , EtOAc/hexanes; 11c, EtOAc/
hexanes; 11d , EtOAc/hexanes) or silica gel column chroma-
tography (8b, CH2Cl2/MeOH, 10:1; 8d , CH2Cl2/MeOH, 10:1;
9b, EtOAc/hexanes, 1:1).
Gen er a l Cycliza tion P r oced u r e for F or m a tion of th e
F r eid in ger La cta m Rin g. Lactams 10a -10d were synthe-
sized by the following general procedure. In a dry flask, MeI
(10 mL) and DMF (2 mL) were added to a Boc-D-methionine
dipeptide (9a -9d ; 1.0 mmol). The solution was stirred for 1
day whereupon the excess MeI was completely removed under
aspirator pressure by formation of an azeotrope with CH2Cl2
(3×). To the sulfonium iodide in DMF was added CH2Cl2 to
double the volume. The solution was cooled in an ice bath to
0 °C, and NaH (60% dispersion in mineral oil, 2.0 mmol) was
added in one portion. The reaction was stirred at 0 °C for 2 h,
and then the mixture was allowed to warm to room temper-
ature and react overnight. The solvents were removed under
reduced pressure, and the residue remaining was dissolved
in EtOAc. Solid citric acid (2.0 mmol) was added, and the
mixture was allowed to stir for 10 min. The solution was
washed with 10% citric acid, 1 M NaHCO3, water, and brine.
The organic layer was dried over MgSO4, filtered, and con-
centrated under aspirator pressure after which the crude
product was purified by crystallization (10a , EtOAc/MeOH;
10b-10d , EtOAc/hexanes).
Gen er a l Am id a tion P r oced u r e. Compounds 13b and 14b
were prepared by reacting the corresponding protected tri-
peptide methyl ester (1.0 mmol) in 50% NH3/MeOH (10 mL)
at 50 °C for 4 days. After the MeOH and excess NH3 were
removed by evaporation, the product was purified by silica gel
column chromatography (CH2Cl2/MeOH, 10:1) in the case of
13b and by crystallization (EtOAc/MeOH) in the case of 14b.
X-r a y Diffr a ction . Yellowish, flat crystals of 8a were grown
from EtOAc/MeOH. All measurements were made on a Rigaku
AFC6S diffractometer with graphite monochromated Cu KR
radiation (λ ) 1.541 78 Å). The data were collected at a
temperature of 173(1) K using ω - 2θ scan technique.
Intensities were corrected for Lorentz and polarization effects.
The structure was solved by direct methods using SHELXS8632
and expanded using Fourier techniques.33 All calculations were
performed using the teXsan crystallographic software pack-
age.34 Due to the fact that the crystal was extremely thin, the
number of observed reflections was too small to support a
complete anisotropic refinement. Some non-hydrogen atoms
were refined anisotropically, while the rest were refined
isotropically. Hydrogen atoms were included but not refined.
Gen er a l Cou p lin g P r oced u r e w ith 1-(3-Dim eth yla m i-
n op r op yl)-3-et h ylca r b od iim id e (E DC‚H Cl). Compounds
6a , 7a , 11a , and 11b were made by the following general
procedure. In a dry flask, the N-tert-butoxycarbonyl-protected
amino acid (1.15 mmol), the HCl salt of either a C-terminal
protected ester or amide (1.0 mmol), and HOBt‚H2O (1.15
mmol) were dissolved in either CH2Cl2 or DMF. The solution
was cooled to -78 °C in a dry ice/acetone bath after which
NEt3 (2.0 mmol) and EDC‚HCl (1.15 mmol) were added. The
mixture was stirred at a -78 °C for 30 min and then warmed
to room temperature whereupon the reaction was allowed to
proceed for 2 days. When DMF was used as the solvent, it was
removed under high vacuum and the residue which remained
was dissolved in EtOAc. The solution was washed with 1 M
NaHCO3, 10% citric acid, water, and brine. The organic layer
was dried over MgSO4, filtered, and concentrated under
aspirator pressure. The crude product was purified by either
crystallization (6a , CH2Cl2/MeOH; 7a , EtOAc/MeOH) or silica
gel column chromatography (11a , CH2Cl2/MeOH, 10:1; 11b,
CH2Cl2/MeOH, 15:1).
N-ter t-Bu toxyca r bon yl-L-p r olyl-L-leu cyl-d ieth ylglyci-
n a m id e (8a ). In a dry flask under nitrogen, the HCl salt of
the C-terminal amide 7a (1.0 mmol) was dissolved in CH2Cl2.
Boc-L-proline pentafluorophenyl ester (1.0 mmol) was added
to the solution whereupon the solution was cooled to -78 °C
with a dry ice/acetone bath. NEt3 (1.0 mmol) then was added
after which the reaction was allowed to warm to room
temperature where it was kept for 2 days. The CH2Cl2 was
removed under aspirator pressure, and the residue obtained
was dissolved in EtOAc. The EtOAc solution was washed
successively with 1 M NaHCO3, 10% citric acid, water, and
brine. The organic layer then was dried over MgSO4, filtered,
and concentrated under aspirator pressure. The crude product
was purified by crystallization from EtOAc/MeOH to give 106
Colorless, prism crystals of 14b were grown from EtOAc/
MeOH. All measurements were made on a Siemens SMART
Platform CCD diffractometer at 173(2) K using the hemisphere
collection technique. The structure was solved and refined by
direct methods using SHELXTL-Plus V5.0.35 All non-hydrogen
atoms were refined with anisotropic displacement parameters.
Hydrogen atoms were placed in ideal positions and refined as
riding atoms with relative isotropic displacement parameters.
Cir cu la r Dich r oism Stu d ies. The peptidomimetics were
dissolved in 50 mM potassium phosphate buffer solution (pH
) 5.4) with the concentrations of the samples studied ranging

Peptidomimetics of L-Prolyl-L-leucyl-glycinamide
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 8 1447
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from 500 to 880 µM. CD spectra were measured on a J ASCO
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P h a r m a cologica l Assa ys. Evaluation of the effects of 3a -
3d and 4a -4d on apomorphine-induced rotational behavior
in rats with unilateral 6-hydroxydopamine lesions in the
substantia nigra was conducted as described previously.13
Measurement of the enhancement of [3H]-N-propylnorapomor-
phine binding to the dopamine D2 receptor was performed as
described by Srivastava et al.7
Ack n ow led gm en t. This work was supported in part
by an NIH grant (NS20036) to R.L.J . The authors thank
Drs. Kevin Mayo and Andrew Cox for use of the
spectropolarimeter, Tom Crick for mass spectral analy-
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deposition number for 8a is CCDC 115703, and that for 14b is
CCDC 115702. The coordinates can be obtained, upon request,
from the Director, Cambridge Crystallographic Data Centre, 12
Union Road, Cambridge CB2 1EZ, U.K., or via the internet at
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leucyl-glycinamide. Brain Res. 1996, 737, 287-291.
(29) Costain, W. J .; Buckley, A. T.; Evans, M. C.; Mishra, R. K.;
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