Design, synthesis, and dopamine receptor modulating activity of diketopiperazine peptidomimetics of L-prolyl-L-leucylglycinamide

Article abstract of DOI:10.1021/jm970328b

The diketopiperazine 'C5' conformational mimic has been incorporated into the L-prolyl-L-leucylglycinamide (PLG, 1) structure and into the bicyclic lactam PLG peptidomimetic structure 3 to give compounds 5 and 6, respectively. These analogues were designed to explore the idea that the N- terminal 'C5' conformation, which was found in the crystal structure of 2 and which was mimicked in 4 by the diketopiperazine function, was a factor in the high potency of these two agents. Through the use of the [³H]spiroperidol/N- propylnorapomorphine (NPA) D₂ receptor competitive binding assay, both 5 and 6 were found to increase the affinity of the dopamine receptor for agonists and both were found to increase the percentage of D₂ receptors which existed in the high-affinity state. These effects were observed when Gpp(NH)p was either absent or present, and they were analogous to the effects observed previously for PLG and the PLG peptidemimetics 2 and 4. However, the potency seen with 5 and 6 was less than that seen for 2 and 4, suggesting that while the N-terminal 'C5' conformation may play a role in the potency of the γ- lactam peptidomimetics of PLG, it does not appear to be the primary factor. In the 6-hydroxydopamine-lesioned animal model of Parkinson's disease, 5 altered apomorphine-induced rotational behavior in a dose-dependent manner. The maximum effect occurred at a dose of 0.01 mg/kg ip and resulted in a 52.27 ± 13.96% (p < 0.001, n = 7) increase in rotations compared to apomorphine administered alone.

Full text of DOI:10.1021/jm970328b

3594
J . Med. Chem. 1997, 40, 3594-3600
Design , Syn th esis, a n d Dop a m in e Recep tor Mod u la tin g Activity of
Dik etop ip er a zin e P ep tid om im etics of L-P r olyl-L-leu cylglycin a m id e
Paul W. Baures,† William H. Ojala,‡ Willard J . Costain,§ Michael C. Ott,§ Ashish Pradhan,§
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, and Department of Psychiatry and Biomedical Sciences,
McMaster University, Hamilton, Ontario L8N 3Z5, Canada
Received May 19, 1997X
The diketopiperazine “C5” conformational mimic has been incorporated into the L-prolyl-L-
leucylglycinamide (PLG, 1) structure and into the bicyclic lactam PLG peptidomimetic structure
3 to give compounds 5 and 6, respectively. These analogues were designed to explore the idea
that the N-terminal “C5” conformation, which was found in the crystal structure of 2 and which
was mimicked in 4 by the diketopiperazine function, was a factor in the high potency of these
two agents. Through the use of the [3H]spiroperidol/N-propylnorapomorphine (NPA) D2 receptor
competitive binding assay, both 5 and 6 were found to increase the affinity of the dopamine
receptor for agonists and both were found to increase the percentage of D2 receptors which
existed in the high-affinity state. These effects were observed when Gpp(NH)p was either
absent or present, and they were analogous to the effects observed previously for PLG and the
PLG peptidomimetics 2 and 4. However, the potency seen with 5 and 6 was less than that
seen for 2 and 4, suggesting that while the N-terminal “C5” conformation may play a role in
the potency of the γ-lactam peptidomimetics of PLG, it does not appear to be the primary factor.
In the 6-hydroxydopamine-lesioned animal model of Parkinson’s disease, 5 altered apomorphine-
induced rotational behavior in a dose-dependent manner. The maximum effect occurred at a
dose of 0.01 mg/kg ip and resulted in a 52.27 ( 13.96% (p < 0.001, n ) 7) increase in rotations
compared to apomorphine administered alone.
In tr od u ction
propylnorapomorphine competition assay, thereby sug-
gesting that an N-terminal “C5” conformation might be
a factor in the potency of peptidomimetics 2 and 4. In
order to explore this issue further, diketopiperazines 5
and 6 have been designed. In 5, the diketopiperazine
“C5” conformational mimic has been incorporated into
the PLG structure, while in 6 this structural component
has been incorporated into the structure of the bicyclic
lactam PLG analogue 3.
L-Prolyl-L-leucylglycinamide (PLG, 1) is able to modu-
late the dopamine D2 receptor by increasing the affinity
of the receptor for agonists1,2 and by increasing the
percentage of D2 receptors that exist in the high-affinity
state.2 This modulatory action on dopamine D2 recep-
tors also has been observed for several PLG peptidomi-
metics that have been synthesized.3-8 In earlier work,
PLG peptidomimetics 2 and 3, which contain the γ-lac-
tam and bicyclic lactam conformational constraints,
respectively, were designed to mimic the postulated
bioactive type II â-turn conformation of PLG.3,6 Pepti-
domimetic 2 was found to be substantially more potent
than PLG in a number of pharmacological assay sys-
tems.3,4 In contrast, peptidomimetic 3 was found to be
about 5 times more effective than PLG in enhancing the
binding of the dopamine receptor agonist 2-amino-6,7-
dihydroxy-1,2,3,4-tetrahydronaphthalene (ADTN) to the
dopamine receptor.6
In an attempt to discern the structural basis behind
the high potency of 2, the diketopiperazine peptidomi-
metic 4 was synthesized.8 This analogue was designed
to mimic an N-terminal “C5” conformation involving an
intramolecular hydrogen bond between the prolyl ni-
trogen and the lactam NH, which had been found in the
crystal structure of 2.9 Peptidomimetic 4, like 2, was
found to be quite potent in the [3H]spiroperidol/N-
† Department of Medicinal Chemistry.
‡ The Biomedical Engineering Center and Department of Laboratory
Medicine and Pathology.
§ Department of Psychiatry and Biomedical Sciences.
X Abstract published in Advance ACS Abstracts, October 1, 1997.
S0022-2623(97)00328-2 CCC: $14.00 © 1997 American Chemical Society

Diketopiperazine Peptidomimetics of PLG
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22 3595
Sch em e 1
Sch em e 2
Ch em istr y
In the synthesis of 5 (Scheme 1), the N-chloroacetyl
derivative 7 was reacted with L-leucine benzyl ester to
give 8, using reaction conditions previously reported in
the literature to make diketopiperazines.10 Conversion
of 8 to the desired PLG diketopiperazine analogue 5 was
accomplished as shown in Scheme 1.
A similar approach to that above was used to generate
the diketopiperazine bicyclic lactam analogue 6 (Scheme
2). The N-(chloroacetyl)proline derivative 10 was con-
verted to its R-iodo derivative 11. This material was
immediately reacted with bicyclic lactam 13a , which
had been obtained from the previously described pro-
tected bicyclic lactam 12a ,6 to give 14a in a 61% yield
after chromatographic purification. Deprotection of 14a
followed by cyclization of the resulting intermediate
gave the desired diketopiperazine product 15 in a 36%
yield. Treatment of 15 with NH3/MeOH gave primarily
the desired PLG analogue 6 along with two of its
diastereoisomers (16 and 17). Separation of 17 from 6
and 16 was accomplished by preparative TLC. Com-
pounds 6 and 16 then were separated from one another
by preparative HPLC. Subsequently, it was found that
formation of the epimerization products 16 and 17 could
be avoided by using bicyclic lactam amide 13b in the
reaction with 11, as the product of this condensation
reaction, 14b, could be cleanly and directly converted
to 6.
Stereochemical assignments of the bicyclic ring ste-
reocenters of 6 and 17 were made from their X-ray
crystal structures.11 The crystal structure of 6 is shown
in Figure 1.12 The bicyclic ring stereocenters of 6
possessed the desired 2S,5R,7R configurations, while
the configurations of the bicyclic ring stereocenters of
17 were 2S,5R,7S. The stereochemistry of the bicyclic
ring stereocenters of 16 could not be assigned because
of the inability to obtain a crystal structure and because
the very small amount of material which was obtained
precluded extensive spectroscopic studies from being
carried out.
torsion angle which defines the “C5” conformation is the
ψ1 torsion angle. From the crystal structure of 6 (Figure
1), the ψ1 torsion angle (N3-C8-C9-N4) was found to
have a value of 21.1(6)°. This is similar to the value of
31.2(2)° observed in 48 and to the value of -0.8(2)° seen
in the crystal structure of 2.9
P h a r m a cology
The ability of the PLG peptidomimetics 5 and 6 to
modulate dopamine receptors was determined by mea-
suring the activity of these compounds in a [3H]spiro-
peridol/N-propylnorapomorphine (NPA) D2 receptor
competitive binding assay in the presence or absence
of 5′-guanylylimidodiphosphate (Gpp(NH)p), a nonhy-
drolyzable analogue of GTP. The graphical presenta-
tion of the competition experiment for 5 is illustrated
in Figure 2. A similar set of curves was obtained for
compound 6. Computer analysis of the competition
curves yielded kinetic data for 5 and 6, which are
summarized in Table 1. Also included in Table 1 for
comparison are the data obtained previously for PLG
(1)2 and the lactam diketopiperazine PLG peptidomi-
metic 4.8
The diketopiperazine moiety was incorporated into 5
and 6 in order to mimic a “C5” conformation. The
Peptidomimetics 5 and 6 showed the same ability to
increase the affinity of the dopamine receptor for

3596 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22
Baures et al.
(52%) in the high-affinity receptor state agonist dis-
sociation constant was seen upon treatment with 6,
where the dissociation constant went from a control
value of 0.14 nM to a value of 0.067 nM. No significant
change in the dissociation constant of NPA for the low-
affinity state of the receptor was observed.
Peptidomimetics 5 and 6 also increased the percent-
age of D2 receptors that existed in the high-affinity state.
This effect was seen in both the presence and absence
of Gpp(NH)p. In the absence of Gpp(NH)p, treatment
with 5 increased the ratio of the percentage of receptors
in the high- and low-affinity states (RH/RL) from 1.35
to 1.97. Similarly, treatment with 6 resulted in an
increase in RH/RL from 0.96 to 1.36. As can be seen in
Table 1, the RH/RL ratio was significantly altered by the
presence of Gpp(NH)p. Both 5 and 6, however, were
able to attenuate significantly the Gpp(NH)p-induced
shift to the low-affinity state. Thus, treatment with 5
resulted in a change in the ratio from 0.257 to 1.47,
while in the case of 6 the change in the ratio was from
0.225 to 0.985. What is significant is that even with
Gpp(NH)p present, 5 and 6 were able to return the RH/
RL ratios to values observed in the respective controls
where Gpp(NH)p was absent.
F igu r e 1. ORTEP drawing of 6 with crystallographic num-
bering system. The non-hydrogen atoms are shown at the 50%
probability level.
Since peptidomimetic 6 had been found previously to
be effective in enhancing rotational behavior induced
by apomorphine in the 6-hydroxydopamine-lesioned
animal model of Parkinson’s disease,13 peptidomimetic
5 was tested for its effects on apomorphine-induced
rotational behavior in 6-hydroxydopamine-lesioned rats.
Administration of apomorphine (0.25 mg/kg ip) in the
absence of 5 resulted in a mean response of 75.0 ( 5.96
contralateral rotations over a 10 min observation period.
Besides producing contralateral rotations, the admin-
istration of apomorphine produced stereotypical behav-
iors in the 6-hydroxydopamine-lesioned rats (e.g., groom-
ing of the contralateral side). When administered in
the absence of apomorphine, 5 did not produce any
observable change in behavior, and it did not result in
any contralateral rotations. Peptidomimetic 5, however,
altered apomorphine-induced rotational behavior in a
dose-dependent manner displaying a bell-shaped dose-
response relationship, which is typical of PLG and its
analogues (Figure 3). The maximal effects of 5 occurred
at a dose of 0.01 mg/kg ip, resulting in a 52.27 ( 13.96%
(p < 0.001, n ) 7) increase in rotations compared to
apomorphine administered alone. In comparison, PLG
and 6 previously had been shown to produce their
maximum increase of approximately 30% at doses of 1
F igu r e 2. Competition curves of [3H]spiroperidol/NPA in
control as well as in membranes treated with 5 and the effect
of Gpp(NH)p: (O) control minus Gpp(NH)p, (b) 5 minus Gpp-
(NH)p, (0) control plus Gpp(NH)p, and (9) 5 plus Gpp(NH)p.
The concentration of [3H]spiroperidol in the assay was 0.2 nM.
Gpp(NH)p concentration was 100 µM, and the concentration
of 5 was 100 nM. Computer analysis of the curves revealed
two sites, high- and low-affinity sites of agonist binding with
binding parameters as described in Table 1. Each value is an
average of four separate experiments ( SEM.
agonists in the [3H]spiroperidol/N-propylnorapomor-
phine D2 receptor competitive binding assay as that
found previously for PLG and the PLG peptidomimetics
2 and 4.2,4,8 The only difference was the concentration
at which the effects were observed. For both 5 and 6,
this concentration was 100 nM. In comparison, it had
been shown previously that the concentration at which
the effects were observed for PLG was 1 µM, while that
for 2 and 4 was 1 nM. As has been demonstrated
previously for PLG and its peptidomimetics,2,4,8 both 5
and 6 decreased the dissociation constant of the high-
affinity state of the dopamine receptor for the agonist
NPA. This was seen when Gpp(NH)p was either absent
or present, although in these particular experiments
statistical significance was clear only in the case where
Gpp(NH)p was present. In the presence of Gpp(NH)p,
compound 5 decreased the high-affinity receptor state
agonist dissociation constant from a control value of 0.12
nM to a value of 0.08 nM. An even greater decrease
mg/kg ip and 0.1 mg/kg, respectively,
13 while 2 increased
apomorphine-induced rotations by 191 ( 15% at 0.01
mg/kg ip.14
The above data show that peptidomimetics 5 and 6
possess the same ability to modulate dopamine recep-
tors as PLG and the previously synthesized PLG pep-
tidomimetics 2-4. In the [3H]spiroperidol/N-propyl-
norapomorphine D2 receptor competitive binding assay,
the potency of 5 and 6 was greater than that seen for 1
(PLG) but not as great as that observed for 2 or the
analogue of 2 which possesses the diketopiperazine
moiety, compound 4. A slightly different profile was
seen in the assay measuring apomorphine-induced
rotational behavior in 6-hydroxydopamine-lesioned rats.
In this case, 5 was 10 times more potent than 6, which
in turn was 10 times more potent than 1 (PLG).

Diketopiperazine Peptidomimetics of PLG
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22 3597
Ta ble 1. Modulation of [3H]Spiroperidol/N-Propylnorapomorphine Binding Competition by 5 and 6a
binding parameters
experiment
control for 5
-Gpp(NH)p
+Gpp(NH)p
pretreatment with 5 (100 nM)
-Gpp(NH)p
+Gpp(NH)p
control for 6
KH (nM)
KL (nM)
RH (%)
RL (%)
RH/RL
0.080 ( 0.001
0.120 ( 0.009
120 ( 5
172 ( 7
56.25 ( 3.52
18.75 ( 3.09
43.75 ( 3.52
81.25 ( 3.09
1.35 ( 0.18
0.257 ( 0.023
0.068 ( 0.007
0.080 ( 0.010b
91 ( 6
104 ( 2
63.75 ( 3.43
59.25 ( 2.28e
36.25 ( 3.42
40.75 ( 2.29e
1.97 ( 0.12b
1.47 ( 0.137c
-Gpp(NH)p
+Gpp(NH)p
0.098 ( 0.009
0.140 ( 0.064
171 ( 18
183 ( 7
49.0 ( 2.12
18.50 ( 1.71
51.0 ( 2.12
81.5 ( 1.71
0.96 ( 0.081
0.225 ( 0.026
pretreatment with 6 (100 nM)
-Gpp(NH)p
0.066 ( 0.019
0.067 ( 0.007c
122 ( 15
106 ( 5
57.25 ( 2.59b
49.25 ( 2.72e
42.75 ( 2.59b
50.75 ( 2.72e
1.363 ( 0.14b
0.985 ( 0.104c
+Gpp(NH)p
control for 1f
-Gpp(NH)p
0.09 ( 0.005
0.13 ( 0.01
73.2 ( 5.5
60.0 ( 8.6
49 ( 5
19 ( 2
51 ( 3
82 ( 5
0.96 ( 0.05
0.23 ( 0.01
+Gpp(NH)p
pretreatment with 1 (1 µM)f
-Gpp(NH)p
0.04 ( 0.002c
0.06 ( 0.006c
60.1 ( 5.4
78.0 ( 9.9
61 ( 4b
32 ( 3c
41 ( 2
72 ( 6
1.48 ( 0.06c
0.44 ( 0.03c
+Gpp(NH)p
control for 4g
-Gpp(NH)p
0.08 ( 0.005
0.19 ( 0.02
69 ( 3.5
50 ( 3
46 ( 1
55 ( 1.8
78 ( 1.2
0.83 ( 0.001
0.28 ( 0.002
+Gpp(NH)p
22 ( 0.8
pretreatment with 4 (1 nM)g
-Gpp(NH)p
0.02 ( 0.003e
0.05 ( 0.006e
56 ( 2.5
64 ( 3.5
65 ( 4d
42 ( 3d
33 ( 3.8
59 ( 1.8
1.97 ( 0.007e
0.71 ( 0.003e
+Gpp(NH)p
a
Competition data were computer-analyzed as described in the Experimental Section. KH and KL represent the inhibitor constant (Ki)
of agonist calculated for the high- and low-affinity components of the [3H]spiroperidol binding, respectively. RH and RL are the percentage
of receptors in the high- and low-affinity form for the agonist, respectively. Values for each preparation are the mean ( SEM of three to
four separate experiments with each experiment carried out in duplicate or triplicate. Concentration of Gpp(NH)p was 100 µM. Statistical
c
e
difference from the respective control group is indicated as follows: bp < 0.05; p < 0.01; dp < 0.005; p < 0.001. f Data for 1 from ref 2.
g
Data for 4 from ref 8.
graphic purification on silica gel (Merck, grade 60, 240-400
mesh, 60 Å) was done by flash or gravity methods, and
preparative HPLC was done with a Waters Associates 25 ×
100 mm PrepPak cartridge. 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, AZ.
1H and 13C NMR spectra were measured in CDCl3 at 300 and
75.5 MHz, respectively, with CDCl3 as the internal reference
for 1H (δ 7.26) and 13C (δ 77.06).
(2S )-1-(Ch lor oa ce t yl)-2-(m e t h oxyca r b on yl)p yr r oli-
d in e (7). L-Proline (10 g, 7.0 mmol) was suspended in
anhydous MeOH, and HCl gas was bubbled into the suspen-
sion until all the solid was dissolved and the solution became
warm. The reaction mixture was stirred for 15 min before it
F igu r e 3. Effect of 5 (dosage ranging from 0.000 01 to 1 mg/
kg ip) on contralateral rotational behavior of apomorphine
(0.25 mg/kg ip) in rats with unilateral 6-hydroxydopamine
substantia nigra lesions (n ) 7). Number of contralateral
rotations in 10 min are expressed as a percentage change from
control rotational response. Each point represents the mean
percentage change ( SEM. Statistical significance is expressed
as *p < 0.01, **p < 0.001.
was concentrated to a viscous oil. The resulting Pro-OMe‚-
HCl was partitioned between CH2Cl2 and 1 M NaHCO3. The
organic layer was dried (MgSO4) and then concentrated under
reduced pressure while keeping the temperature below 20 °C.
Anhydrous benzene was added to this oil followed by the
dropwise addition of chloroacetyl chloride (6.9 mL, 7.0 mmol).
The solution was refluxed for 2 h while it was stirred
vigorously. The solution was then concentrated under aspira-
tor pressure and the resulting residue partitoned between CH2-
Cl2 and H2O. The organic phase was washed with 10% citric
acid, 1 M NaHCO3, H2O, and saturated NaCl. The organic
phase was dried (MgSO4) and then stripped of solvent to give
a light golden oil. Passage of this material through a pad of
silica gel with EtOAc/hexanes (1:1) as the eluant removed final
traces of impurities and provided the product in a yield of 5.6
g (24%): [R]D ) -91.4 (c 2.25, MeOH); FAB MS m/ z 206 [M
+ H]+.
Peptidomimetic 5 was as potent as 2 in this in vivo
assay system; however, 2 produced a much greater
percent increase in apomorphine-induced rotations than
did 5. These results suggest that while an N-terminal
“C5” conformation may play a part in the potency of the
γ-lactam peptidomimetics of PLG, it does not appear to
be the primary factor.
Ben zyl (2S)-[(8a S)-Hexa h yd r o-1,4-d ioxop yr r olo[1,2-a ]-
p yr a zin -2(1H)-yl]-4-m eth ylp en ta n oa te (8). (2S)-1-(Chlo-
roacetyl)-2-(methoxycarbonyl)pyrrolidine (7; 0.70 g, 3.40 mmol)
and L-leucine benzyl ester (0.90 g, 3.76 mmol) were dissolved
in 20 mL of 2-ethoxyethanol. To this solution was added Et3N
(0.47 mL, 3.40 mmol) dropwise. The solution was refluxed for
60 h before the solvent was removed under vacuum. The
residue was partitioned between CH2Cl2 and 10% citric acid.
The organic fraction was washed with 1 M NaHCO3 and
Exp er im en ta l Section
Gen er a l Asp ects. All apparatus were oven-dried and
cooled in a dessicator. THF and CH2Cl2 were distilled from
Na/benzophenone and CaH2, respectively. Thin-layer chro-
matography was performed on Analtech 250 µm silica gel HLF
Uniplates and visualized by UV, I2, ninhydrin spray (amines),
and 2,6-dichlorophenol indophenol spray (acids). Chromato-

3598 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22
Baures et al.
saturated NaCl before it was dried over MgSO4. This solution
was stripped of solvent to give an oil which was purified by
column chromatography on silica gel with EtOAc/i-PrOH (25:
1) as the eluant. The material obtained from the column was
dissolved in CH2Cl2 and this solvent then removed in vacuo.
This process was carried out several times to remove traces
of the eluting solvents. The product was obtained as 740 mg
(64%) of a clear oil: [R]D ) -78.4 (c 1.15, MeOH); TLC Rf
(EtOAc/i-PrOH, 25:1) ) 0.43; 1H NMR (CDCl3) δ 7.26-7.36
(m, 5 H, Ph-H), 5.26-5.31 (m, 1 H, 2-CH), 5.04-5.18 (m, 2 H,
PhCH2), 4.09-4.12 (m, 1 H, 8a-CH), 4.09 (d, J ) 15.9 Hz, 1 H,
NCH2CO), 3.72 (d, J ) 15.9 Hz, 1 H, NCH2CO), 3.46-3.62
(m, 2 H, 6-CH2), 2.31-2.41 (m, 1 H, 8-CH2), 1.78-2.11 (m, 3
H, 7- and 8-CH2), 1.64-1.80 (m, 2 H, 3-CH2), 1.35-1.45 (m, 1
H, 4-CH), 0.90 (d, J ) 7.2 Hz, 3 H, CH3), 0.85 (d, J ) 7.5 Hz,
3 H, CH3); 13C NMR (DEPT, CDCl3) δ 171.6, 169.6, 164.2 (CO),
135.8, 129.3, 129.2, 129.1, 129.0, 128.9 (Ph C), 67.9 (CH2Ph),
59.8, 54.1 (2- and 8a-C), 48.1 (NCH2CO), 45.7 (6-C), 37.1, 29.3
(3- and 8-C), 25.4 (4-C), 23.7 (7-C), 23.4, 21.7 (CH3); FAB MS
m/ z 359 [M + H]+. Anal. (C20H26N2O4‚0.25CH2Cl2) C, H, N.
Met h yl [N-[(2S)-[(8a S)-H exa h yd r o-1,4-d ioxop yr r olo-
[1,2-a ]p yr a zin -2(1H)-yl]-4-m eth ylp en ta n oyl]a m in o]a ce-
ta te (9). Compound 8 (400 mg, 1.18 mmol) was dissolved in
MeOH, and this solution then was added to a suspension of
40 mg of Pd/C in MeOH. This mixture was hydrogenated in
a Parr vessel for 8 h. The Pd/C was collected by gravity
filtration through a paper filter, and the MeOH was removed
under aspirator pressure. The resultant oil was azeotroped
(2×) from CH2Cl2 before finally being dissolved in CH2Cl2.
1-Hydroxybenzotriazole monohydrate (160 mg, 1.18 mmol) and
glycine methyl ester hydrochloride (163 mg, 1.30 mmol) were
added all at once to this solution. Triethylamine (181 µL, 1.30
mmol) was added dropwise followed by the addition of dicy-
clohexylcarbodiimide (268 mg, 1.30 mmol) all at once. The
solution was stirred for 20 h at room temperature. The
precipitated dicyclohexylurea was removed by filtration, and
the CH2Cl2 filtrate was washed with 10% citric acid, 1 M
NaHCO3, and saturated NaCl before it was dried over MgSO4.
This solution was stripped of solvent to give an oil which was
purified by column chromatography on silica gel with EtOAc/
MeOH (9:1) as the eluant. A yield of 162 mg (37%) was
obtained: [R]D ) -30.3 (c 1.5, MeOH); TLC Rf (EtOAc/MeOH,
9:1) ) 0.34; 1H NMR (CDCl3) δ 6.70 (t, J ) 6.0 Hz, 1 H, NH),
5.07 (t, J ) 8.0 Hz, 1 H, 2-CH), 3.71-4.15 (m, 5 H, 8a-CH,
NCH2CON), 3.67 (s, 3 H, OCH3), 3.48-3.61 (m, 2 H, 6-CH2),
2.30-2.40 (m, 1 H, 8-CH2), 1.84-2.10 (m, 3 H, 7- and 8-CH2),
1.56-1.75 (m, 2 H, 3-CH2), 1.38-1.47 (m, 1 H, 4-CH), 0.89 (d,
J ) 6.0 Hz, 3 H, CH3), 0.84 (d, J ) 6.0 Hz, 3 H, CH3); 13C
NMR (DEPT, CDCl3) δ 170.9, 170.6, 170.6, 164.1 (CO), 59.9,
53.9 (2- and 8a-C), 53.0 (OCH3), 47.7, 41.5 (NCH2CON,
NHCH2CO), 45.6 (6-C), 36.1, 29.3 (3- and 8-C), 25.3 (4-C), 23.2
(7-C), 23.4, 22.6 (CH3); FAB MS m/ z 340 [M + H]+. Anal.
(C16H25N3O5) C, H, N.
mL of CH2Cl2. This solution was cooled to -30 °C, after which
NEt3 (3.81 mL, 27.3 mmol) was added slowly followed by the
dropwise addition of chloroacetyl chloride (2.39 mL, 30 mmol)
as a solution in 10 mL of CH2Cl2. After 2 h at room
temperature, 75 mL of a 10% citric acid solution was added,
and the layers were separated. The organic layer was washed
with 75 mL each of 1 M NaHCO3 and saturated NaCl. The
organic layer was dried over MgSO4 and then concentrated to
an oil, which was purified by chromatography on silica gel with
EtOAc/hexane (1:1) as the eluant. The purified oil was
obtained in a yield of 4.9 g (72%): [R]D ) -97.8 (c 0.55, MeOH);
TLC Rf (EtOAc/hexane, 1:1) ) 0.39; 1H NMR (shows rotamers
about the amide bond present; CDCl3) δ 4.18-4.21 (m, 1 H,
R-CH), 3.82-3.95 (m, 2 H, CH2Cl), 3.38-3.56 (m, 2 H, δ-CH2),
1.97-2.07 (m, 1 H, â-CH2), 1.77-1.93 (m, 3 H, â- and γ-CH2),
1.25-1.31 (m, 9 H, C(CH3)3); 13C NMR (of major rotamer;
CDCl3) δ 171.3, 165.4 (CO), 81.9 (C(CH3)3), 60.6 (R-C), 47.6
(δ-C), 42.4 (CH2Cl), 29.6 (â-C), 28.4 (C(CH3)3), 25.3 (γ-C); FAB
MS m/ z 248 [M + H]+. Anal. (C11H18NO3Cl‚0.8H2O) C, H,
N, Cl.
Meth yl (2S,5R,7R)-1-Aza -7-[[[[(2S)-(ter t-bu toxyca r bo-
n yl)p yr r olid in -1-yl]ca r b on yl]m e t h yl]a m in o]-8-oxo-4-
th ia bicyclo[3.3.0]octa n e-2-ca r boxyla te (14a ). Compound
10 (332 mg, 1.34 mmol) was dissolved in 10 mL of spectroscopic
grade acetone, and NaI (201 mg, 1.34 mmol) was added all at
once to this solution. This solution was refluxed for 15 min
after which the precipitate was removed by filtration and the
golden-colored filtrate was concentrated under vacuum to give
(2S)-1-(iodoacetyl)-2-(tert-butoxycarbonyl)pyrrolidine (11) as an
oil. This material was dissolved in 2 mL of DMF, and the
solution was added dropwise to a solution of methyl (2S,5R,7R)-
1-aza-7-amino-8-oxo-4-thiabicyclo[3.3.0]octane-2-carboxylate
(13a ) in DMF [This solution was prepared by reacting methyl
(2S,5R,7R)-1-aza-7-[(tert-butoxycarbonyl)amino]-8-oxo-4-
thiabicyclo[3.3.0]octane-2-carboxylate (12a )6 (385 mg, 1.22
mmol) with 4 N HCl in dioxane for 1 h. The dioxane and
excess HCl were removed under vacuum, and the resultant
hydrochloride salt was azeotroped from CH2Cl2 (3×). The
hydrochloride salt was dissolved in 2 mL of DMF at 0 °C, and
NEt3 (356 µL, 2.56 mmol) was added dropwise]. The reaction
mixture was stirred for 12 h at room temperature before the
solution was concentrated under vacuum to a golden solid. The
solid was partitioned between 15 mL of 1 M NaHCO3 and 50
mL of EtOAc. The aqueous phase was again extracted with
50 mL of EtOAc. The combined organic fractions were dried
(MgSO4), filtered, and concentrated to give a crude oil which
was purified by silica gel chromatography with CH2Cl2/MeOH
(10:1) as the eluant. This provided 316 mg (61%) of a light-
gold oil: [R]D ) +103.3 (c 0.3, MeOH); TLC Rf (CH2Cl2/MeOH,
10:1) ) 0.56; 1H NMR (of major rotamer; CDCl3) δ 5.18-5.21
(m, 1 H, 5-CH), 4.99-5.04 (m, 1 H, 2-CH), 4.36-4.40 (m, 1 H,
7-CH), 3.74 (s, 3 H, OCH3), 3.52-3.63 (m, 4 H, NCH2CO,
pyrrolidine 2-CH, pyrrolidine 5-CH2), 3.29-3.46 (m, 3 H,
NCH2CO, 3-CH2), 2.61 (bs, 1 H, NH), 2.43-2.52 (m, 1 H,
6-CH2), 2.30-2.38 (m, 1 H, 6-CH2), 1.85-2.18 (m, 4 H,
pyrrolidine 3- and 4-CH2), 1.43 (s, 9 H, C(CH3)3); 13C NMR (of
the two rotamers; CDCl3) δ 176.6, 176.5, 171.9, 170.8, 170.8,
170.4, 169.9 (CO), 83.0, 82.0 (C(CH3)3), 65.1, 65.0 (5-C), 60.3,
60.0, 59.9, 59.2, 58.5, 58.4 (2- and 7-C, pyrrolidine 2-C), 53.5
(OCH3), 49.7, 49.2 (NCH2CO), 47.2, 46.6 (pyrrolidine 5-C), 37.3
(CH2S), 33.3, 33.0 (6-C), 32.1, 29.7 (pyrrolidine 3-C), 28.6, 28.6
(C(CH3)3), 25.1, 22.9 (pyrrolidine 4-C); FAB MS m/ z 428 [M
+ H]+. Anal. (C19H29N3O6S) C, H, N.
[N-[(2S)-[(8a S)-Hexa h yd r o-1,4-d ioxop yr r olo[1,2-a ]p y-
r a zin -2(1H)-yl]-4-m eth ylp en ta n oyl]a m in o]a ceta m id e (5).
Compound 9 (117 mg, 0.34 mmol) was dissolved in MeOH
saturated with NH3. The solution was stirred at room tem-
perature for 12 h. Removal of the MeOH and NH3 from the
solution provided 5 as a foam in quantitative yield: [R]D
)
1
-76.9 (c 0.9, MeOH); H NMR (CDCl3) δ 7.54 (t, J ) 6.0 Hz,
1 H, CONH), 6.77 (bs, 1 H, CONH2), 6.51 (bs, 1 H, CONH2),
5.06 (t, J ) 8.4 Hz, 1 H, 2-CH), 3.67-4.17 (m, 7 H, 8a-CH,
NHCH2CO, NCH2CO), 3.44-3.58 (m, 2 H, 6-CH2), 2.28-2.35
(m, 1 H, 8-CH2), 1.80-2.06 (m, 3 H, 7- and 8-CH2), 1.64-1.69
(m, 2 H, 3-CH2), 1.31-1.45 (m, 1 H, 4-CH), 0.89 (d, J ) 6.0
Hz, 3 H, CH3), 0.82 (d, J ) 6.0 Hz, 3 H, CH3); 13C NMR (CDCl3)
δ 172.6, 171.6, 170.3, 164.4 (CO), 59.9, 54.8 (2 and 8a-C), 48.4,
43.2 (NCH2CO, NHCH2CO), 45.7 (6-C), 37.1, 29.3 (3- and 8-C),
25.4 (4-C), 23.4 (7-C), 23.7, 22.3 (CH3); FAB MS m/ z 325 [M
+ H]+. Anal. (C15H24N4O4) C, H, N.
(2S,5R,7R)-1-Aza -7-[[[[(2S)-(ter t-b u t oxyca r b on yl)p yr -
r olidin -1-yl]car bon yl]m eth yl]am in o]-8-oxo-4-th iabicyclo-
[3.3.0]octa n e-2-ca r boxa m id e (14b). This material was
prepared in the same manner as that described above for 14a .
Thus, 10 (633 mg, 2.56 mmol) was converted to its iodo
derivative 11 with NaI (383 mg, 2.56 mmol) which was then
reacted with (2S,5R,7R)-1-aza-7-amino-8-oxo-4-thiabicyclo-
[3.3.0]octane-2-carboxamide (13b) that had been prepared from
the deprotection of (2S,5R,7R)-1-aza-7-[(tert-butoxycarbonyl)-
amino]-8-oxo-4-thiabicyclo[3.3.0]octane-2-carboxamide (12b)6
(770 mg, 2.56 mmol). Product was obtained as an oil in a 70%
(770 mg) yield: [R]D ) +77.9 (c 1.35, MeOH); 1H NMR (of major
rotamer; CDCl3) δ 7.57 (s, 1 H, CONH), 5.58 (s, 1 H, CONH),
(2S)-1-(Ch lor oa cetyl)-2-(ter t-bu toxyca r bon yl)p yr r oli-
d in e (10). Z-Pro-t-OBu (8.35 g, 27.3 mmol) was hydrogenated
at 40 psi of H2 in the presence of 500 mg of Pd/C for 4 h. The
mixture was filtered through filter paper and the filtrate
concentrated under vacuum. The residue was azeotroped from
CH2Cl2 (2×), and the oil which remained was dissolved in 75

Diketopiperazine Peptidomimetics of PLG
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22 3599
5.23 (t, J ) 6.0 Hz, 1 H, 5-CH), 4.73-4.77 (m, 1 H, 2-CH),
4.23-4.27 (m, 1 H, 7-CH), 3.33-3.67 (m, 7 H, NCH2CO, 3-CH2,
pyrrolidine 2-CH, pyrrolidine 5-CH2), 2.51 (bs, 1 H, NH), 2.38-
2.42 (m, 1 H, 6-CH2), 2.10-2.19 (m, 1 H, pyrrolidine 3-CH2),
1.86-2.03 (m, 3 H, pyrrolidine 3- and 4-CH2), 1.41 (s, 9 H,
C(CH3)3); 13C NMR (of major rotamer; CDCl3) δ 176.0, 172.0,
171.8, 171.0 (CO), 82.1 (C(CH3)3), 64.8 (5-C), 62.3, 60.4, 59.0
(2- and 7-C, pyrrolidine 2-C), 49.2 (NCH2CO), 46.6 (pyrrolidine
5-C), 41.6 (CH2S), 34.6 (6-C), 29.6 (pyrrolidine 3-C), 28.6
(C(CH3)3), 25.2 (pyrrolidine 4-C); FAB MS m/ z 413 [M + H]+.
Meth yl (2S,5R,7R)-1-Aza -7-[(8a S)-h exa h yd r o-1,4-d iox-
opyr r olo[1,2-a ]pyr azin -2(1H)-yl]-8-oxo-4-th iabicyclo[3.3.0]-
octa n e-2-ca r boxyla te (15). Compound 14a (316 mg, 0.74
mmol) was dissolved in 4 N HCl in dioxane after which the
solution was stirred for 3 h at room temperature. The dioxane
and excess HCl were removed under vacuum, and the residue
was azeotroped from CH2Cl2 (2×). The product was dried
under high vacuum for 3 h before being dissolved in 30 mL of
CH2Cl2. The solution was cooled to -20 °C before 1-hydroxy-
benzotriazole monohydrate (100 mg, 0.74 mmol) and NEt3 (206
µL, 1.48 mmol) were added consecutively. The ice bath was
removed, and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide
hydrochloride (283 mg, 1.48 mmol) was added in four portions
over a 3 min period. This solution then was stirred for 16 h
at room temperature. An additional 50 mL of CH2Cl2 was
added to the solution before it was washed consecutively with
50 mL each of 1 N HCl, 1 M NaHCO3, and a saturated NaCl
solution. The organic layer was dried over MgSO4, filtered,
and concentrated to a light-gold oil. Final purification was
done by silica gel chromatography with EtOAc/MeOH (9:1). A
yield of 95 mg (36%) was obtained. A portion of this material
was transferred to a vial as a solution in CH2Cl2 in order to
obtain a sample for elemental analysis: [R]D ) +140.7 (c 0.6,
MeOH); TLC Rf (EtOAc/MeOH, 9:1) ) 0.29; 1H NMR (CDCl3)
δ 5.23 (dd, J ) 2.4, 6.0 Hz, 1 H, 5-CH), 5.07 (dd, J ) 3.6, 8.7
Hz, 1 H, 2-CH), 4.78 (t, J ) 8.7 Hz, 1 H, 7-CH), 4.25 (d, J )
17.1 Hz, 1 H, NCH2CO), 4.12 (t, J ) 8.7 Hz, 1 H, 8a-CH),
3.80 (d, J ) 17.1 Hz, 1 H, NCH2CO), 3.78 (s, 3 H, OCH3),
3.51-3.68 (m, 2 H, NCH2), 3.35-3.50 (m, 2 H, SCH2), 2.48-
2.56 (m, 2 H, 6-CH2), 2.34-2.46 (m, 1 H, 8-CH2), 1.87-2.16
(m, 3 H, 7- and 8-CH2); 13C NMR (CDCl3) δ 173.6, 170.6, 168.5,
163.0 (CO), 64.4 (5-C), 59.6, 59.0, 57.3 (2-, 7-, and 8a-C), 53.7
(OCH3), 51.7 (NCH2C), 46.0 (NCH2), 37.5 (CH2S), 29.4 (8-C),
26.9 (6-C), 23.2 (7-C); FAB MS m/ z 354 [M + H]+. Anal.
(C15H19N3O5S‚CH2Cl2) C, H, N.
(2S,5R,7R)-1-Aza -7-[(8a S)-h exa h yd r o-1,4-d ioxop yr r olo-
[1,2-a ]p yr a zin -2(1H)-yl]-8-oxo-4-th ia bicyclo[3.3.0]octa n e-
2-ca r boxa m id e (6). Meth od A. Ester 15 (70 mg, 0.20 mmol)
was dissolved in cold NH3 in MeOH, and this solution was
stirred for 5 h at room temperature. The solvents were
removed, and the resulting oil was purified by using normal
phase preparatory TLC. Two bands were obtained with an
Rf (CH2Cl2/MeOH, 10:1) of 0.49 and 0.41, respectively. The
top band contained the two diastereoisomers, 6 and 16, while
the bottom band consisted of diastereoisomer 17, as deter-
mined from X-ray crystallography. Diastereoisomers 6 and
16 were separated by HPLC with EtOAc/MeOH (9:1) as the
eluant. Retention times for 6 and 16 were 18.4 and 15.6 min,
respectively.
Meth od B. A solution of 4 N HCl in dioxane was added to
14b (700 mg, 1.7 mmol). This solution was stirred for 3 h,
after which time the dioxane and excess HCl were removed
under aspirator pressure to give an oily residue. This material
was azeotroped from a mixture of CH2Cl2/MeOH (2×) and then
left under vacuum to remove all traces of solvents. The
resulting material was dissolved in 30 mL of dry DMF. To
this stirred solution at 0 °C were added consecutively NEt3
(249 µL, 1.7 mmol), 1-hydroxybenzotriazole monohydrate (230
mg, 1.7 mmol), and 1-[3-(dimethylamino)propyl]-3-ethylcar-
bodiimide hydrochloride (650 mg, 3.4 mmol). The reaction
mixture was stirred at room temperature for 48 h. The DMF
was removed under vacuum, and the resulting residue was
purified by chromatography with EtOAc/MeOH (9:1) as the
eluant. Final traces of impurities were removed by prepara-
tive HPLC on silica gel with EtOAc/MeOH (9:1) to give a
homogeneous white solid. Crystallization from CH2Cl2/MeOH
provided 75 mg (13%) of 6: mp 249-252 °C; [R]D ) +66.0 (c
1.0, MeOH); TLC Rf (CH2Cl2/MeOH, 10:1) ) 0.25; TLC Rf
(EtOAc/MeOH, 9:1) ) 0.10; 1H NMR (CDCl3/MeOH-d4) δ 7.14
(s, 1 H, CONH), 6.01 (s, 1 H, CONH), 5.12 (dd, J ) 2.4, 3.6
Hz, 1 H, 5-CH), 4.79 (dd, J ) 4.2, 4.8 Hz, 1 H, 2-CH), 4.38 (d,
J ) 15.6 Hz, 1 H, NCH2CO), 4.23 (dd, J ) 2.4, 7.5 Hz, 1 H,
7-CH), 4.12-4.17 (m, 1 H, 8a-CH), 3.82 (d, J ) 15.6 Hz, 1 H,
NCH2CO), 3.39-3.63 (m, 4 H, NCH2, SCH2), 2.45-2.57 (m, 2
H, 6-CH2), 2.29-2.39 (m, 1 H, 8-CH), 1.83-2.04 (m, 3 H, 7-
and 8-CH2); 13C NMR (CDCl3/MeOH-d4) δ 173.1, 172.3, 172.3,
163.5 (CO), 64.5 (5-C), 61.1, 60.5, 59.6, 59.4 (2-, 7-, and 8a-C,
NCH2CO), 46.0 (NCH2), 36.8 (CH2S), 29.1 (6-C), 28.8 (8-C), 23.2
(7-C); FAB MS m/ z 339 [M + H]+. Anal. (C14H18N4O4S‚H2O)
C, H, N, S.
Isom er 16: TLC Rf (CH2Cl2/MeOH, 10:1) ) 0.25; [R]D
)
+139.5 (c 1.0, MeOH); 1H NMR (CDCl3) δ 6.77 (s, 1 H, CONH),
5.53 (s, 1 H, CONH), 5.15 (dd, J ) 3.6, 5.1 Hz, 1 H, 5-CH),
4.83 (dd, J ) 4.8, 8.7 Hz, 1 H, 2-CH), 4.58-4.65 (m, 1 H, 7-CH),
4.29 (d, J ) 15.6 Hz, 1 H, NCH2CO), 4.13-4.18 (m, 1 H, 8a-
CH), 3.88 (d, J ) 15.6 Hz, 1 H, NCH2CO), 3.45-3.72 (m, 4 H,
NCH2, SCH2), 2.58-2.66, 2.33-2.48 (m, 3 H, 6- and 8-CH2),
1.89-2.16 (m, 3 H, 7- and 8-CH2).
Isom er (2R,5R,7R)-17: TLC Rf (CH2Cl2/MeOH, 10:1) )
1
0.18; [R]D ) +60.7 (c 1.0, MeOH); H NMR (CDCl3) δ 6.62 (s,
1 H, CONH), 5.58 (s, 1 H, CONH), 5.66 (t, J ) 9.9 Hz, 1 H,
5-CH), 5.03-5.07 (m, 1 H, 7-CH), 4.80-4.85 (m, 1 H, 2-CH),
4.18-4.23 (m, 2 H, 8a-CH, NCH2CO), 3.70-3.79 (m, 2 H,
NCH2CO, SCH2), 3.52-3.62 (m, 2 H, NCH2), 3.38 (dd, J ) 3.6,
8.4 Hz, 1 H, SCH2), 2.89-2.99 (m, 1 H, 6-CH2), 2.35-2.46 (m,
1 H, 8-CH2), 1.81-2.21 (m, 4 H, 6-, 7-, and 8-CH2).
X-r a y Diffr a ct ion . Colorless crystals of 6 monohydrate
and 17 were grown from chloroform/methanol. All measure-
ments were made on a Rigaku AFC6S diffractometer with
graphite monochromated Cu KR radiation (λ ) 1.541 78 Å)
using the ω-2θ scan mode at 173(1) K by using a Molecular
Structure Corp. low-temperature device. Intensities were
corrected for Lorentz and polarization effects. Equivalent
reflections were merged, and absorption effects were corrected
using the ψ-scan method.15 The structures were solved by
direct methods using SHELXS8616 and refined using the
TEXSAN structure analysis package.17 All non-hydrogen
atoms were refined anisotropically. Hydrogens bonded to
carbon atoms were placed in calculated positions (0.95 Å) and
were not refined. Hydrogens bonded to heteroatoms (excluding
the water atom) were located in difference Fourier maps, and
their positional parameters were refined.
[3H]Sp ir op er id ol/N-P r op yln or a p om or p h in e Bin d in g
Com p etition Assa y. Bovine striatal membranes were pre-
pared essentially as described in previous reports.2,18 The
tissues were homogenized in 10 volumes of 0.25 M sucrose in
a Potter-Elvehjem homogenizer and centrifuged at 1000g for
15 min. The supernatant was then centrifuged for 1 h at
100000g. The resulting pellet was resuspended in 50 mM Tris-
HCl-1 mM EDTA buffer (pH 7.4) and stored at -80 °C in
small aliquots. On the day of use, the membrane preparations
were thawed and diluted with Tris-HCl buffer containing 1
mM EDTA, 5 mM MgCl2, 0.1 mM dithiothreitol, 0.1 mM
phenylmethanesulfonyl fluoride, 0.1 mM benzamide, and 1 mg/
mL soybean trypsin inhibitor before using for the ligand
binding assay.
The [3H]spiroperidol/N-propylnorapomorphine binding com-
petition assays were performed using 150-200 µg of the above
membrane protein preparation in a total volume of 1.0 mL of
buffer. [3H]Spiroperidol (0.2 nM) and varying concentrations
of N-propylnorapomorphine were added to the reaction mix-
ture in the absence and presence of 100 µM Gpp(NH)p and
the PLG peptidomimetics 5 and 6; 50 nM ketanserin was
included in the [3H]spiroperidol binding assays to occlude the
presence of serotonergic sites. Incubation of membranes with
ligands was carried out for 1 h at 22 °C. At the end of the
incubation the bound and free ligands were separated by rapid
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 Model 1780 scintillation
counter. Nonspecific binding was determined in parallel
assays in the presence of 1.0 µM (+)-butaclamol.

3600 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22
Baures et al.
The binding data were analyzed as previously described.19
In brief, curves were analyzed using the weighted nonlinear
curve-fitting Prism program (Graph Pad Software, San Diego,
CA). Data were subjected to analysis for either one site or
multiple binding sites together with statistical analysis com-
paring “goodness of fit” between one or two affinity state
models. A two-site model was selected only if a statistically
significant improvement of the fit data was obtained over a
one-site model. The IC50 values obtained from the competition
curves were converted to Ki values via the Prism software
program, which employs the Cheng and Prusoff equation.20
Rota tion a l Beh a vior Assa y. Male Sprague-Dawley rats
weighing 250-300 g (Charles River, St. Constant, Quebec)
were individually housed under constant temperature and
humidity with a 12 h light/12 h dark cycle and were allowed
free access to food and water. 6-Hydroxydopamine lesions of
the substantia nigra were made in these rats in the following
manner. The rats were pretreated with desipramine (15 mg/
kg ip) 30 min prior to surgery, in order to spare noradrenergic
pathways from the neurotoxic effects of 6-hydroxydopamine.
Also, in order to decrease respiratory distress, atropine (0.06
mg/kg ip) was administered prior to anesthesia with sodium
pentobarbital (50 mg/kg ip). 6-Hydroxydopamine (8 µg in 4
µL of 0.9% saline with 0.1% ascorbic acid) was infused directly
into the substantia nigra pars compacta (coordinates -4.8 A,
+1.9 L, -7.5 D to bregma, according to the atlas of Paxinos
and Watson21) at a rate of 1 µL/min using a 30 gauge Hamilton
syringe. Each rat was given 10 mL of sterilized saline (sc) to
limit dehydration and 0.2 mL (0.03 mg/mL sc) of Temgesic to
limit pain after the surgery.
(3) 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.
Chem. 1988, 31, 1430-1436.
(4) Mishra, R. K.; Srivastava, L. K.; J ohnson, R. L. Modulation of
High-Affinity CNS Dopamine D2 Receptor by L-Pro-L-Leu-
Glycinamide (PLG) Analogue 3(R)-(N-L-Prolylamino)-2-oxo-1-
pyrrolidineacetamide. Prog. Neuro-Psychopharmacol. Biol. Psy-
chiatr. 1990, 14, 821-827.
(5) 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.
(6) 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.
(7) Genin, M. J .; Mishra, R. K.; J ohnson, R. L. Dopamine Receptor
Modulation by a Highly Rigid Spiro Bicyclic Peptidomimetic of
Pro-Leu-Gly-NH2. J . Med. Chem. 1993, 36, 3481-3483.
(8) 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.
(9) Valle, G.; Crisma, M.; Toniolo, C.; Yu, K.-L.; J ohnson, R. L.
Crystal-State Structural Analysis of Two γ-Lactam-Restricted
Analogs of Pro-Leu-Gly-NH2. Int. J . Pept. Protein Res. 1989, 33,
181-190.
(10) Diafi, L.; Couquelet, J .; Tronche, P.; Gardette, D.; Gramain, J .-
C. 8-Hydroxylated Derivatives of Diazabicyclo[4.3.0]nonanes,
2,5-Dioxodiazabicyclo[4.3.0]nonanes and Some Related Esters.
J . Heterocycl. Chem. 1990, 27, 2181-2187.
Ten days postoperatively all animals were tested for rota-
tional behavior by administering only apomorphine (0.25 mg/
kg ip) for a total of three times with a 10 day washout period
between tests. Only those rats exhibiting a minimum of 40
contralateral rotations over a 10 min period were considered
to be lesioned successfully and were included in the study. The
animals were observed in a clear round flat-bottom plastic bowl
to which they were allowed to acclimate for 10 min before
testing was started. Rotational counts were taken manually
at 5 min intervals after a 5 min latency period from the point
of drug administration to allow for distribution of drug.
The ability of 5 to modify the rotational response to
apomorphine (0.25 mg/kg ip) was tested at several doses
(Figure 3) with a 2 day washout period between testing.
Compound 5 was given at the same time as apomorphine
through the ip route of administration. Solutions of 5 were
made by dissolving the compound in MeOH and then diluting
the solution with 0.9% saline containing 0.1% ascorbic acid.
Control experiments were carried out with vehicle (0.9% saline
solution containing 0.1% ascorbic acid). A Latin square design
was used for the administration of drugs in order to rule out
any potential enhanced sensitivity due to increasing drug
doses. The rotational counts were compared to baseline
apomorphine-induced rotations for each animal and analyzed
using repeated measures ANOVA. Results are expressed as
the percent change compared to control.
(11) The authors have deposited atomic coordinates for these struc-
tures with the Cambridge Crystallographic Data Centre. The
coordinates can be obtained, upon request, from the Director,
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, U.K.
(12) The crystal structure of 6, to our knowledge, provides the first
reported crystal structure of a compound containing the 5,5-
bicyclic thiazolidine lactam â-turn mimic, which restricts the ψ2
and φ3 torsional angles of a â-turn. In 6, the ψ2 (N2-C7-C6-
N3) and φ3 (C1-C2-N2-C7) torsional angles possess values of
142.7(4)° and 120.5(4)°, respectively. These can be compared to
the corresponding values for an ideal type II â-turn, which are
ψ2 ) 120° and φ3 ) 80°.
(13) 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.
(14) Mishra, R. K.; Marcotte, E. R.; Chugh, A.; Barlas, C.; Whan, D.;
J ohnson, R. L. Modulation of Dopamine Receptor Agonist-
Induced Rotational Behavior in 6-OHDA-Lesioned Rats by a
Peptidomimetic Analogue of Pro-Leu-Gly-NH2 (PLG). Peptides
1997, in press.
(15) North, A. C. T.; Phillips, D. C.; Mathews, F. S. A Semi-empirical
Method of Absorption Correction. Acta Crystallogr. 1968, 24A,
351-359.
(16) Sheldrick, G. M. SHELXS86. Program for the Solution of Crystal
Structures; University of Go¨ttingen: Germany, 1986.
(17) TEXSAN-TEXRAY Structure Analysis Package, Molecular Struc-
ture Corp., 3200 Research Forest Dr., The Woodlands, TX 77381;
1985.
(18) Kazmi, S. M. I.; Ramwami, J .; Srivistava, L. K.; Rajakumar, G.;
Ross, G. M.; Cullen, M.; Mishra, R. K. Characterization of High-
Affinity Dopamine D2 Receptors and Modulation of Affinity
States by Guanine Nucleotides in Cholate-Solubilized Bovine
Striatal Preparations. J . Neurochem. 1986, 47, 1493-1502.
(19) Kazmi, S. M. I.; Mishra, R. K. Comparative Pharmacological
Properties and Functional Coupling of µ and δ Opioid Receptor
Sites in Human Neuroblastoma SH-SY5Y Cells. Mol. Pharmacol.
1987, 32, 109-118.
(20) Cheng, Y. C.; Prusoff, W. H. Relationship Between the Inhibition
Constant (KI) and the Concentration of Inhibitor Which Causes
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Ack n ow led gm en t. This work was supported in part
by an NIH grant (NS20036) to R.L.J . and an NIH
predoctoral traineeship (GM07994) to P.W.B. The au-
thors thank Tom Crick for mass spectral analysis.
Su p p or tin g In for m a tion Ava ila ble: X-ray crystallo-
graphic data including tables of positional parameters, bond
distances, and bond angles for 6 and 17 (20 pages). Ordering
information is given on any current masthead page.
Refer en ces
(1) Chiu, S.; Paulose, C. S.; Mishra, R. K. Effect of L-Prolyl-L-Leucyl-
Glycinamide (PLG) on Neuroleptic-Induced Catalepsy and Dopam-
ine/Neuroleptic Receptor Bindings. Peptides 1981, 2, 105-111.
(2) 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-
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(21) Paxinos, G.; Watson, C. The Rat Brain in Stereotaxic Coordi-
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J M970328B