Synthesis and biological activity of novel neuroprotective diketopiperazines.

Article abstract of DOI:10.1016/S0968-0896(02)00132-3

The cyclic dipeptide cyclo[His-Pro] (CHP) is synthesized endogenously de novo and as a breakdown product of thyrotropin-releasing hormone (TRH), a tripeptide with known neuroprotective activity. We synthesized two isomeric compounds based on the structure

Full text of DOI:10.1016/S0968-0896(02)00132-3

Bioorganic & Medicinal Chemistry 10 (2002) 3043–3048
Synthesis and Biological Activity of Novel Neuroprotective
Diketopiperazines
K. R. C. Prakash,a Y. Tang,a Alan P. Kozikowski,a,* Judith L. Flippen-Anderson,b
Susan M. Knoblachc and Alan I. Fadenc
aDrug Discovery Program, Department of Neurology, Georgetown University Medical Center, 3900 Reservoir Road, N.W,.
Washington, DC 20007-2197, USA
bLaboratory for the Structure of Matter, Code 6030, Naval Research Laboratory, 4555 Overlook Ave., S. W.,
Washington, DC 20375-5000, USA
cDepartment of Neuroscience, Georgetown University Medical Center, 3900 Reservoir Road, N.W,.
Washington, DC 20007, USA
Received 22 October 2001; accepted 5 February 2002
Abstract—The cyclic dipeptide cyclo[His-Pro] (CHP) is synthesized endogenously de novo and as a breakdown product of thyro-
tropin-releasing hormone (TRH), a tripeptide with known neuroprotective activity. We synthesized two isomeric compounds based
on the structure of CHP, in which the histidine residue was replaced by 3,5-di-tert-butyltyrosine (DBT), a phenolic amino acid that
traps reactive oxygen species. These novel diketopiperazines prevented neuronal death in an in vitro model of traumatic injury. In
addition, they dose-dependently prevented death caused by the direct induction of free radicals, and by calcium mobilization
through an agent that evokes rapid, necrotic death. The drugs showed activity in the latter system at picomolar concentrations. The
neuroprotective profile of these compounds suggests that they may be useful as treatments for neuronal degeneration in vivo,
potentially through several different mechanisms. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
related to reversal of pathophysiological secondary
events (i.e., release of glutamate, generation of free
radicals) that are initiated by the insult and evolve from
minutes to days later. For example, TRH or TRH ana-
logues stabilize levels of catecholamines, restore cationic
imbalances, and antagonize the pathophysiological
effects of endogenous opioids, leukotrienes and platelet-
activating factor that occur after injury.7
Thyrotropin-releasing hormone (TRH)1 is a tripeptide
present in the central nervous system (CNS) as well as in
the periphery. Its best-described role is that of a hypo-
thalamic neuroendocrine hormone. Exogenous admin-
istration of TRH elicits a variety of behavioral
responses. Among these are changes in sleep/arousal,2
temperature,3 autonomic outflow,4 and interactions
with many physiological effects of opioids/opiates.5
Whether synthesized endogenously or injected, TRH is
rapidly metabolized in blood and CNS tissue. The cyclic
dipeptide cyclo[His-Pro] (CHP) is generated by primary
cleavage of the pyroglutamyl residue from TRH by the
abundant and relatively non-specific enzyme pyr-
oglutamyl aminopeptidase.8 CHP is also synthesized de
novo endogenously, where it is both detectable in CNS
tissue by radioimmunoassay and has demonstrable
bioactivity. Because CHP and TRH share similarities in
biological activity, and exogenous administration of
TRH can increase CHP, it has been hypothesized that
at least some of the effects of TRH administration may
occur through CHP.9 Therefore, we designed a novel
TRH and its analogues have been investigated as
potential treatments for a variety of disorders, including
CNS trauma, where they showed substantial beneficial
effects. Continuous infusion of TRH or bolus injections
of analogues with intact C-termini and longer half-lives
improved recovery across species in models of traumatic
injury to the brain or spinal cord.6 Such benefits may be
*Corresponding author. Tel.: +1-202-687-0686; fax: +1-202-687-
5065; e-mail: kozikowa@georgetown.edu
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00132-3

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K.R.C.Prakash et al./ Bioorg.Med.Chem.10 (2002) 3043–3048
i
Scheme 1. Reagents and conditions: (a) (BOC)2O, Pr2NEt, rt; (b) DCC, HOBt, N-(tert-butoxycarbonyl)-l-proline; (c) (i) H2/Pd/C; (ii) l-prolina-
mide, DCC, HOBt; (d) (i) CF3COOH, CH2Cl2 (ii) NaCN, MeOH; (e) (i) CF3COOH, CH2Cl2 (ii) Et3N, CH2Cl2.
cyclic dipeptide related to CHP in which the imidazole
moiety is replaced by a di-tert-butylphenol (i.e., the his-
tidine residue is replaced by 3,5-di-tert-butyltyrosine).
Our rationale was that the known structure of CHP,
combined with the ability of certain phenols to act as
scavengers for reactive oxygen species (ROS) might
yield compounds with novel neuroprotective potential.
We report the route of synthesis of the two diaster-
eomeric cyclic dipeptides, cyclo[(di-tert-Bu)Tyr-Pro],
together with details of their action in several different
in vitro models of neuronal injury. These molecules
appear to provide an interesting profile of action that
warrants further study.
the diketopiperazines 5 and 6 in good yield. Chromato-
graphic purification of the crude diastereomeric mixture
gave equal amounts of 5 and 6. The structures of com-
pounds 5 and 6 were established by NMR analysis, and
additionally, a single-crystal X-ray analysis was carried
out on 6 (Fig. 1). The second route is the more efficient
one of the two, as only three synthetic steps are required
from the starting amino acid 1b.
Biological studies — activity in a model of CNS injury
To evaluate whether 5 and 6 showed neuroprotective
activity we tested them in several models of cell death.
3,5-Di-tert-butyltyrosine (DBT) and cyclo(Gly-Pro)
(CGP),11 the components of the new drugs 5 and 6,
were used as controls. The in vitro mechanical punch
model has been used by our group and others to mimic
both acute cell destruction and secondary loss asso-
ciated with traumatic injury in vivo.12 In this model,
neuronal-glial co-cultures are subjected to physical
Chemistry
The diketopiperazines 5 and 6 were prepared in opti-
cally pure form starting from the benzyl or ethyl ester of
30,50-di-tert-butyltyrosine (1a/1b)10 in two different ways
(Scheme 1). Compound 1a was prepared in racemic
form by reaction of the anion of benzyl N-(diphenyl-
methylene)glycinate with 4-(bromomethyl)-2,6-di-tert-
butylphenol followed by HCl workup. Next, the free
amine 1a was treated with (BOC)2O in the presence of
diisopropylethylamine to afford the amino-protected
tyrosine derivative 2. The benzyl ester group of 2 was
cleaved by hydrogenolysis over Pd/C to obtain the free
acid, which was then coupled with l-prolinamide in
presence of HOBt and DCC to give 4 as a diaster-
eomeric mixture. Compound 4 was further converted to
5 and 6 by treatment with trifluoroacetic acid followed
by triethylamine. The two diastereomers (5 and 6) were
separated by column chromatography.
As an alternative and shorter route to the same cyclic
peptides, the ethyl ester 1b was coupled with the N-(tert-
butoxycarbonyl)-l-proline in the presence of HOBt and
DCC in CH2Cl2 to yield the dipeptide 3 in 80% yield.
This intermediate was then treated with TFA in meth-
ylene chloride followed by NaCN in MeOH to provide
Figure 1. Single-crystal X-ray structure of the diketopiperazine 6.

K.R.C.Prakash et al./ Bioorg.Med.Chem.10 (2002) 3043–3048
3045
disruption with a device that rapidly deploys a series of
28 parallel blades to the culture surface. Cells under the
blades are destroyed on contact, and the cultures are
then washed to remove resultant debris. However, over
the course of the ensuing 24 h after impact, neurons
throughout the culture slowly begin to die; calcein
staining reveals that the highest proportion of cell death
occurs at and near the site of impact, with progressively
lower and more delayed cell death/injury taking place as
the distance from impact increases. Because this injury
is both delayed and indirect, it serves as a useful model
of secondary injury mechanisms that contribute to
impairment in vivo.12 In our co-culture system, activity
of the enzyme lactate dehydrogenase in the culture
media correlates with total neuronal death. Treatment
with either 5 or 6 reduced cell death in this model (Fig. 2);
this activity followed an inverted U-shaped dose-
response curve. Neither DBT nor CGP prevented cell
death in this model, suggesting that neither the 3,5-di-
tert-butyltyrosine nor the cyclo(Gly-Pro) components of
5 and 6 were singularly involved in their neuroprotective
activity.
some neuroprotection in this model, but it was 4-fold
less than that provided by equal concentrations of 5 or
6. These data suggest that both the DBT and CGP
moieties of 5 and 6 may each contribute, albeit mini-
mally, to the observed neuroprotective properties of 5
and 6 against cell death induced by maitotoxin. MK-801
showed no activity.
Excitotoxic cell death
The next objective was to determine whether 5 and 6
prevented excitotoxic cell death induced by a glutamate
pulse. Neither compound prevented this type of cell
death. Higher concentrations of these drugs also had no
effect (data not shown). MK-801, a selective inhibitor of
the NMDA subtype of glutamate receptors, served as a
positive control.
Apoptotic death
Staurosporine induces apoptotic death and activation of
enzymes (caspases) specifically associated with this type
of cell self-destruction in cultured neurons.14 Therefore
we examined whether 5 and 6 could prevent this type of
cell death in our culture system. The drugs had no effect
on death induced by 0.1 mM staurosporine at con-
centrations of 0.1–100 mM (data not shown). MK-801
was also ineffective in this model.
Multiple factors have been implicated in the secondary
injury response. They include free radicals, glutamate,
influx and cellular dysregulation of sodium and calcium,
breakdown of phospholipids with the generation of
toxic by-products, activation of inflammatory cascades
and alterations in signal transduction (i.e., protein
kinase C and others), and induction of programmed
(apoptotic)13,14 cell death cascades. Therefore, to char-
acterize the neuroprotective activity of 5 and 6 further,
we evaluated whether they would prevent cell death
induced by several of these injury-associated factors/
mechanisms.
Calcium-induced necrosis
Maitotoxin is a marine, algal, polyether type neurotoxin
that induces cell death via external calcium influx and
mobilization of intracellular calcium stores.15 It pro-
duces rapid deterioration and necrotic morphology, as
well as activation of enzymes associated with necrotic
cell death.15 Compounds 5 and 6 were very effective at
preventing death induced by a maitotoxin pulse (Fig. 3).
High concentrations of DBT and CGP also showed
Figure 3. Bars represent the meanꢀSEM for n=40–46 wells per con-
dition. {p <0.01, p <0.0001 for t-test with Bonferroni correction.
Figure 4. Bars represent the meanꢀSEM for n=40–46 wells per con-
dition. +p<0.05, {p <0.01, ?p <0.0001 for t-test with Bonferroni
correction.
Figure 2. Bars represent the meanꢀSEM for n=40–46 wells per con-
dition. {p <0.01, ?p <0.0001 for t-test with Bonferroni correction.

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K.R.C.Prakash et al./ Bioorg.Med.Chem.10 (2002) 3043–3048
Free-radical-induced death
high as 100 mM, these compounds failed to show any
cyototoxicity. Therefore, these agents may have poten-
tial utility as treatments for acute CNS injuries such as
stroke and trauma.
As anticipated based upon the presence of the di-tert-
butylphenol group in these cyclic dipeptides, both com-
pounds 5 and 6 increased survival in a model of free
radical induced cell death16 initiated by FeSO4 (Fig. 4).
However, in contrast to the results seen with the
mechanical punch, the neuroprotective efficacy in this
model followed a dose-dependent activity curve. DBT
and CGP showed neuroprotective activity as well, with
DBT and 6 providing the best protection at the highest
concentrations tested. As glycine itself is known in the
literature17 to serve as an antioxidant, it was not sur-
prising to find that CGP, a cyclic derivative of glycine,
shows significant neuroprotection even at a concentra-
tion of 0.1 mM in this model. These data indicate that
not only does the DBT moiety contribute to the free-
radical scavenging properties of 5 and 6, but that the
CGP moiety may do so as well. Vitamin E, a well-
described anti-oxidant, was utilized as a positive control.
Experimental
Chemical methods
General. Starting materials were obtained from the
Aldrich Chemical Co. or from other commercial sup-
pliers. Solvents were purified as follows: diethyl ether
was distilled from phosphorus pentoxide; THF was
freshly distilled under nitrogen from sodium/benzophe-
1
none. H and 13C NMR spectra were obtained with a
Varian Unity Inova instrument at 300 and 75.46 MHz,
1
respectively. H chemical shifts (d) are reported in ppm
downfield from internal TMS. 13C chemical shifts are
referenced to CDCl3 (central peak, d=77.0 ppm). NMR
assignments were made with the help of COSY,
NOESY, DEPT, and HETCOR experiments. Melting
points were determined in Pyrex capillaries with a Tho-
mas-Hoover Unimelt apparatus and are uncorrected.
Mass spectra were measured in the EI mode at an
ionization potential of 70 eV. TLC was performed on
Merck silica gel 60F254 glass plates; column chromato-
graphy was performed using Merck silica gel (60–200
mesh). Abbreviations: THF, tetrahydrofuran; LDA,
lithium diisopropylamide; DCC, dicyclohexylcarbo-
diimide; DMF, dimethylformamide; HOBt, 1-hydroxy-
benzotriazole.
Conclusion
In summary, the novel cyclic dipeptides reported herein
show neuroprotective properties in a neuronal-glial
model of trauma, which involves activation of multiple
secondary injury factors. They are also able to block
calcium-induced necrotic cell death initiated by maito-
toxin as well as death induced by FeSO4, a free radical
generator. Secondary tissue damage and associated cell
death after acute CNS insults thought to reflect a mul-
tifactorial biochemical process that together cause cel-
lular necrosis or apoptosis.18 Development of
compounds 5 and 6, like other diketopiperazines or
related tripeptides,19 was based upon the concept that
effective treatment of CNS injury requires either use of
multiple neuroprotective agents with complementary
mechanisms or use of single compounds (like TRH)
with multifactorial actions. In the present studies, com-
pounds 5 and 6 attenuated both trauma and maitotoxin
induced cell death in vitro; each of these models causes
almost exclusively necrotic cell death. Data from the
DBT and CGP controls suggest that each of these
components contributes to the neuroprotective effects of
5 and 6. In contrast, the compounds did not inhibit
glutamate induced cell death, which can cause either
necrotic or apoptotic cell death, depending upon con-
centration, duration of treatment or age of cultures.
They also did not inhibit a classical apoptotic cell death
model. Free radicals may cause either necrotic or apop-
totic cell death through different mechanisms.20 Taken
together, the ability of compounds 5 or 6 to modify
three different in vitro models, all of which may be
associated with cellular necrosis, suggests that the pro-
tective actions reflect antinecrotic properties. Yet their
inability to modify glutamate induced changes indicates
that other pro-necrotic factors may be involved, such as
free radical induced lipid peroxidation20 or effects of
calpains.15 Thus, the unique combination of these moi-
eties yields compounds with a pharmacological profile
that indicates an action through several different second-
ary injury mechanisms. When tested at concentrations as
30,50-Di-tert-butyltyrosine benzyl ester (1a).10 To a solu-
tion of benzyl N-(diphenylmethylene)glycinate18 (1.3 g,
4.0 mmol) in THF (20 mL) at ꢁ78 ꢂC was added under
a N2 atmosphere LiHMDS (5.0 mL, 1.0 M solution in
THF, 5.0 mmol). After stirring for 15 min at ꢁ78 ꢂC, 4-
(bromomethyl)-2,6-di-tert-butylphenol (1.2 g, 4.0 mmol)
was added dropwise, and the resulting mixture was stir-
red for an additional 8 h. The solvent was removed
under reduced pressure, and the residue was stirred with
ether/1 N HCl (20 mL, 1:1 mixture) overnight at rt. The
reaction mixture was neutralized to pH 7.0 and extrac-
ted with ether (3 ꢃ 100 mL). The combined ether layers
were washed with brine, dried (Na2SO4), and con-
centrated. The residue on purification by flash chroma-
tography (hexane/ethyl acetate 1:1) yielded the ester 1a
(0.84 g) in 54% yield: 1H NMR (CDCl3) d 1.43 (s, 18H),
2.88 (dd, J=7.5, 13.5 Hz, 1H), 3.05 (dd, J=5.7, 13.5
Hz, 1H), 3.79 (dd, J=5.7, 7.2 Hz, 1H), 5.13 and 5.19
(ABq, J=12.3 Hz, 2H), 7.00 (s, 2H), 7.33 (m, 5H). 13C
NMR (CDCl3) d 30.51, 34.47, 41.27, 56.29, 66.83,
125.99, 127.70, 128.28, 128.49, 128.77, 135.75, 136.20,
152.90, 175.40. MS (EI) m/z 383 (M+, 3%), 219
(100%).
30,50-Di-tert-butyltyrosine ethyl ester (1b)..10 To a solu-
tion of ethyl N-(diphenylmethylene)glycinate18 (1.3 g,
3.9 mmol) in THF (20 mL) at ꢁ78 ꢂC was added LDA
(5.0 mL, 1.0 M solution in THF, 5.0 mmol) under a N2
atmosphere. After stirring for 15 min at ꢁ78 ꢂC, a solu-
tion of 4-(bromomethyl)-2,6-di-tert-butylphenol (1.2 g,

K.R.C.Prakash et al./ Bioorg.Med.Chem.10 (2002) 3043–3048
3047
4.0 mmol) in THF (5 mL) was added. The resulting
mixture was stirred for an additional 5 h at ꢁ78 ꢂC. The
solvent was removed. To the resulting residue was
added ether (10 mL) followed by HCl (1 N, 10 mL), and
the mixture was stirred at room temperature overnight.
The reaction mixture was neutralized to pH 7.0 and
extracted with ether (3 ꢃ 100 mL). The combined ether
layers were washed with brine, dried (Na2SO4), and
concentrated. The residue was purified by column
chromatography on silica gel (EtOAc/MeOH 15:1) to
the solution was diluted with CH2Cl2 (500 mL), washed
with water, dried (Na2SO4), and concentrated. The
residue was passed through a short column of silica gel to
give the crude product 4 (470 mg, 97%). This material
was dissolved in CH2Cl2/CF3COOH (10:1, 5 mL) at 0 C,
and the solution was stirred for 2 h at the same tempera-
ture. The solvent was removed under reduced pressure,
the residue was dissolved in CH2Cl2/triethylamine (5:1, 10
mL), and the resulting mixture was stirred overnight. The
reaction mixture was diluted with CH2Cl2, washed with
saturated NaHCO3, dried (Na2SO4), and concentrated to
give an oil. Purification by column chromatography on
silica gel [CHCl3/MeOH (9:1)] gave the pure compounds 5
(80 mg, 21% yield) and 6 (121 mg, 32% yield).
1
give the ester 1b. Yield: 1.0 g (65%). H NMR (CDCl3)
d 1.27 (t, J=7.2 Hz, 3H), 1.43 (s, 18H), 2.84 (dd, J=7.8,
13.8 Hz, 1H), 3.02 (dd, J=5.4, 13.8 Hz, 1H), 3.70 (dd,
J=5.4, 7.5 Hz, 1H), 4.18 (q, J=7.2 Hz, 2H), 7.00 (s,
2H). 13C NMR (CDCl3) d 14.37, 30.46, 34.43, 41.16,
56.12, 60.98, 125.95, 127.75, 136.13, 152.80, 175.43. MS
(EI) m/z 321 (M+, 1%), 219 (100%).
From compound 3. A solution of compound 3 (16 mg,
0.03 mmol) in CH2Cl2/CF3COOH (10:1, 5 mL) was
stirred for 2 h at 0 ꢂC. The reaction mixture was diluted
with CH2Cl2, washed with saturated NaHCO3, dried
(Na2SO4), and concentrated to give an oil. The residue
was dissolved in MeOH (5 mL), NaCN (2 mg) was
added, and the resulting mixture was stirred at 45 ꢂC
overnight. The solvent was removed under reduced
pressure, and the residue was purified by column chro-
matography on silica gel [CHCl3/MeOH (9:1)] to give
the compounds 5 and 6 (5 mg, 43%, and 5.2 mg, 45%,
respectively).
N-(tert-Butoxycarbonyl)- 30,50-di-tert-butyltyrosine benzyl
ester (2). To a solution of compound 1a (0.42 g, 1.09
mmol) in CH3CN (10 mL) was added at room tem-
perature under N2, iPr2NEt (0.18 g, 1.4 mmol), followed
by (BOC)2O (0.305 g, 1.4 mmol). The mixture was stir-
red overnight. The solvent was removed under reduced
pressure, and the residue was purified by flash chroma-
tography (hexane/ethyl acetate 15:1) to give the pure
1
product (2, 0.48 g) in 90% yield: H NMR (CDCl3) d
1.44 (s, 18H), 1.47 (s, 9H), 3.06 (m, 2H), 4.65 (dd,
J=6.3, 14.1 Hz, 1H), 5.13 (m, 4H), 6.95 (s, 2H), 7.27
(m, 2H), 7.35 (m, 3H). 13C NMR (CDCl3) d 28.52,
30.48, 34.43, 54.82, 67.08, 79.94, 126.04, 126.54, 128.10,
128.47, 128.74, 135.39, 136.11, 153.01, 155.17, 172.24.
MS (EI) m/z 483 (M+, 5%), 219 (100%).
5: [a]2D5ꢁ137ꢂ (c 0.93, CHCl3). Mp 194–197 ꢂC. 1H
NMR (CDCl3) d 1.45 (s, 18H), 1.98 (m, 3H), 2.37 (m,
1H), 2.69 (dd, J=10.8, 14.4 Hz, 1H), 3.59 (m, 3H), 4.12
(t, J=7.2 Hz, 1H), 4.27 (d, J=8.7 Hz, 1H), 5.21 (s, 1H),
5.68 (s, 1H), 7.00 (s, 2H). 13C NMR (CDCl3) d 22.70,
28.62, 30.41, 34.52, 37.12, 45.53, 56.58, 59.34, 125.73,
126.37, 136.94, 153.33, 165.46, 169.48. Anal. calcd for
C22H32N2O3: C, 70.92; H, 8.66; N, 7.52. Found: C,
71.13; H, 8.50; N, 7.63.
N-[N-(tert-Butoxycarbonyl)prolyl]-30,50-di-tert-butyltyrosine
ethyl ester (3). To a solution of N-(tert-butoxy-
carbonyl)proline (53 mg, 0.24 mmol) in DMF (3 mL)
was added HOBt (35 mg, 0.26 mmol), followed by DCC
(50 mg, 0.24 mmol). The mixture was stirred for 2 h,
then a solution of 3,5-di-tert-butyltyrosine ethyl ester
(1b) (80 mg, 0.29 mmol) and triethylamine (0.3 mL) in
DMF (5 mL) was added, and the resulting mixture was
stirred for 12 h at room temperature. The solvent was
pumped off, and the residue was purified by flash chro-
matography on silica gel (hexane/ethyl acetate 1:1) to
give 3 (125 mg, 97%) as a white foam. 1H NMR (CDCl3)
d 1.10–1.14 (dt, J=7.2 Hz, 3H), 1.39 (s, 27H), 1.90 (m,
4H), 3.00 (m, 2H), 3.36 (m, 3H), 4.18 (m, 2H), 4.76 (m,
1H), 5.16 (s, 1H), 6.36 (br s, 1H), 6.95 (s, 1H), 7.00 (s, 1H).
6: [a]2D5 ꢁ23ꢂ (c 1.07, CHCl3). Mp 216–218 ꢂC. 1H NMR
(CDCl3) d 1.44 (s, 18H), 1.76 (m, 2H), 1.95 (m, 1H),
2.16 (m, 1H), 2.64 (dd, J=6.6, 10.5 Hz, 1H), 2.93 (dd,
J=3.9, 13.8 Hz, 1H), 3.13 (dd, J=5.7, 13.5 Hz, 1H),
3.42 (m, 1H), 3.63 (m, 1H), 4.23 (q, J=5.1 Hz, 1H), 5.23
(s, 1H), 5.80 (s, 1H), 6.99 (s, 2H). 13C NMR (CDCl3) d
21.95, 29.11, 30.49, 34.43, 40.54, 45.27, 57.90, 59.43,
125.55, 126.83, 136.65, 153.50, 165.21, 169.39. Anal.
calcd for C22H32N2O3: C, 70.92; H, 8.66; N, 7.52.
Found: C, 70.66; H, 8.45; N, 7.65.
Cyclo[(R)-30,50-di-tert-butyl-Tyr-L-Pro] (5) and cyclo[(S)-
30,50-di-tert-butyl-Tyr-L-Pro] (6). From compound 2. To
a solution of compound 2 (480 mg, 1.0 mmol) in tert-
butyl alcohol (20 mL) was added 10% Pd/C (40 mg).
The resulting mixture was stirred at room temperature
under an H2 atmosphere for 2 h, and the catalyst was
filtered off. After removal of the solvent, the residue was
dissolved in DMF/EtOAc (1:5, 12 mL), and to this
solution was added HOBt (162 mg, 1.2 mmol), followed
by DCC (254 mg, 1.23 mmol). The resulting solution
was stirred for 2 h, and then prolinamide (124 mg, 1.08
mmol) was added. The mixture was stirred overnight at
room temperature. The precipitate was filtered off, and
Acknowledgements
We are indebted to the Department of Defense (DAMD
17–99–2-9007) for the support of this work.
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