Strategies to target kyotorphin analogues to the brain

Article abstract of DOI:10.1021/jm970715l

The design, synthesis, and pharmacological evaluation of brain-targeted chemical delivery systems (CDS) for a kyotorphin analogue (Tyr-Lys) are described. The brain-targeted compound contains the active peptide in a packaged, disguised form, flanked betwe

Full text of DOI:10.1021/jm970715l

J . Med. Chem. 1998, 41, 3773-3781
3773
Str a tegies To Ta r get Kyotor p h in An a logu es to th e Br a in
Pei Chen, Nicholas Bodor,* Whei-Mei Wu, and Laszlo Prokai
Center for Drug Discovery, College of Pharmacy, University of Florida, Box 100497 J HMHC, Gainesville, Florida 32610-0497
Received October 20, 1997
The design, synthesis, and pharmacological evaluation of brain-targeted chemical delivery
systems (CDS) for a kyotorphin analogue (Tyr-Lys) are described. The brain-targeted compound
contains the active peptide in a packaged, disguised form, flanked between the lipophilic
cholesteryl ester on the C-terminus and the 1,4-dihydrotrigonellyl redox targetor, attached to
the N-terminus through strategically selected L-amino acid(s) spacer. It was found that for
successful brain targeting, the ꢀ-amine of Lys needs to be also converted to a lipophilic function.
Through sequential enzymatic bioactivation, the Tyr-Lys dipeptide is released in a sustained
manner, producing significant and prolonged analgesic activity as demonstrated by the rat
tail latency test. An alternate strategy was also employed. Lys was replaced by a redox amino
acid pair, Nys+ T Nys, the nicotinamide T 1,4-dihydronicotinamide analogues of Lys (Nys+
is 2-amino-6-(3-carbamoyl-1-pyridiniumyl)hexanoic acid). The Nys form is lipophilic and
facilitates delivery in addition to the C- and N-terminal lipophilic functions. Enzymatic
oxidation to Nys+ provides the lock-in, followed by removal of the lipophilic groups, releasing
Tyr-Nys+ from the brain-targeted analogue (BTRA). Nys+ was shown to be an effective
substitution for Arg or Lys. The activities of the CDS and BTRA, respectively, were antagonized
by naloxone, supporting the designed brain-targeted processes. The most potent compound is
the two-proline spacer containing CDS (CDS-PP), followed by the BTRA.
In tr od u ction
a sequential bioactivation of a specifically designed
packaged peptide construct, which allows passive trans-
port of the original lipophilic precursor to the brain,
followed by “lock-in” produced by the redox targetor and
subsequent enzymatic liberation of the active peptide.
High and prolonged analgesic activity was thus pro-
duced. It was also demonstrated7 that the observed
activity was exclusively centrally mediated, as it can
be fully antagonized by naloxone but not by methyl-
naloxonium salts.
Kyotorphin (KTP, L-tyrosyl-L-arginine) is an endog-
enous neuropeptide which exhibits significant analgesic
action indirectly, through induction of the release of
endogenous enkephalin from nerve terminals inside the
brain.1,2 The name kyotorphin was given to reflect an
endorphin-like substance discovered in Kyoto, J apan.
The analgesic potency of kyotorphin introduced directly
to the brain is about 4.2 times that of Met-enkephalin.2
However, when administered systemically, it shows
activity only briefly, at a high dose of 200 mg/kg. The
general major obstacle for the development of centrally
active peptides is the blood-brain barrier (BBB), the
brain capillary walls formed from tightly joined endot-
helial cells, which allows only compounds with sufficient
lipophilicity to penetrate this barrier.3 The BBB is also
distinct from the peripheral capillary system in that it
contains high concentrations of various lytic enzymes
which by facile metabolic degradation also prevent the
uptake of blood-borne neurotransmitters and neuro-
modulators.4 Finally, many compounds lipophilic enough
to penetrate the BBB are good substrates of P-glyco-
proteins found in the BBB, which promptly transport
these molecules back to the blood. These actually work
as “microscopic wormholes”, and compounds such as
many lipophilic peptides which are substrates of P-
glycoproteins are taken out through these faster than
they enter the brain.5
Briefly, the active target peptide is modified by
introducing a large lipophilic function on the C-terminal,
such as cholesteryl or adamantylethyl esters, while the
N-terminal is converted via strategically chosen L-amino
acid spacers to the dihydrotrigonellyl targetor. The 1,4-
dihydrotrigonellyl function is enzymatically converted
to the quaternary trigonellyl moiety, which provides
about 5 log unit change in the partition properties of
the molecular construct, producing the important brain
“lock-in” process and the facilitated peripheral excretion.
Subsequently, the lipophilic protector is cleaved, and
then endopeptidases will split the locked-in precursor
at the designed linkage between the spacer and the
active peptide. An alternate approach applied for brain
targeting of TRH analogues,8 in addition to the de-
scribed functions, uses a glycine spacer between the
C-terminal and the lipophilic function, which resulted
in producing the C-terminal amide of amino acid linked
to this glycine, by the R-amidating monooxygenase
(PAM) enzyme.
Kyotorphin suffers primarily delivery problems and
not facile efflux. Due to the presence of arginine, which
has a pKa of 13.2, kyotorphin is virtually completely
ionized at all times, which clearly prevents it from
passing the BBB.
Here we report a modified version of this approach
for brain targeting of L-tyrosyl-L-lysine kyotorphin
analogues. It was found that in kyotorphin, arginine
can be replaced with lysine and the resulting L-tyrosyl-
L-lysine (YK) is an analogue which exhibits the same
analgesic effect as kyotorphin when administered icv.9
Efficient, brain-targeted delivery of analgesic en-
kephalin analogues was recently described,6 based on
* To whom correspondence should be addressed.
S0022-2623(97)00715-2 CCC: $15.00 © 1998 American Chemical Society
Published on Web 09/05/1998

3774 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20
Chen et al.
F igu r e 2. Isoelectronic/isosteric analogy of Lys and Nys.
oyl-1-pyridiniumyl)hexanoic acid) mimics the protona-
tion process. Accordingly, the oxidized, quaternary
pyridinium form might produce an active dipeptide,
while the corresponding 1,4-dihydro form could replace
the targetor moiety of CDS. In this construct, the use
of spacers is not needed, but we assume that the free
amine of tyrosine needs to be made lipophilic in a
similar way, by Boc. Thus, replacing Lys by the Nys-
Nys+ redox system, as shown in Figure 1, could be an
effective way to produce centrally mediated analgesic
activity.
Figure 3 is a composite summary of the two alternate
approaches. After administration to the general circu-
latory system, both the CDS and BTRA, respectively,
would undergo passive distribution to the brain, fol-
lowed by oxidation of the dihydrotrigonellyl function in
CDS or that of Nys in the BTRA. Using a recently
developed general and highly reliable method to esti-
mate partition properties of various compounds, includ-
ing peptides,14,15 the dramatic change in partition
followed by introduction of the permanent positive
charge in either system was clearly demonstrated.
Thus, it was found consistently that the 1,4-dihydropy-
ridine to pyridinium salt conversion dramatically re-
duces the log P values in both the CDS and BTRA by
about 5 log units.
F igu r e 1. Chemical delivery system (CDS) and brain-targeted
redox analogue (BTRA) for tyrosyl-lysine (spacer ) Pro, Pro-
Pro, Pro-Ala).
Design
The lysine analogue of kyotorphin (YK)9 was chosen
as the target peptide. It was, however, obvious that the
already established approaches for brain targeting of
peptides cannot be simply applied, since the basic amino
group in lysine (pKa ) 10.3) would prevent passive
transport of a packaged dipeptide. In order to solve this
problem, we have applied two different strategies. First,
the dipeptide was packaged similar to enkephalin;
however, the free ꢀ-amine function in lysine was made
lipophilic by coupling to a Boc moiety. It was assumed
that Boc is biodegradable, if the corresponding precursor
is locked in the brain. Accordingly, the chemical
delivery system (YK-CDS) is shown in Figure 1. For
the final release, the proline endopeptidase10,11 or dipep-
tidyl peptidase12 was the target enzyme, and accordingly
the spacer used was either proline or alanine or a
combination of these two amino acids. The ꢀ-amine
function is derivatized by Boc.
Ch em istr y
The synthesis of the CDS and BTRA is described in
Schemes 1 and 2, respectively. The use of the Fmoc-
Lys(Boc)-OH as the starting material allows selective
manipulation of the two amino functions. Alternately,
for the synthesis of the BTRA, we start with the Boc-
Lys(Fmoc)-OH. The key component, Nys+, is generated
in the final step, before the reduction, by applying the
“Zincke reaction”, using the Zincke reagent from nico-
tinamide,16,17 N-(2,4-dinitrophenyl)nicotinamide chlo-
ride. The cholesteryl esters were prepared using DCC
(dicyclohexylcarbodiimide) as the coupling agent. The
final step, reduction of the quaternary pyridinium-3-
amides, was accomplished using sodium dithionite as
the reducing agent.
However, a basically different strategy was also
tested. Recognizing that highly basic amino acids, such
as arginine and lysine, will essentially be protonated
all the time at biological pHs, it was considered that an
isoelectronic-isosteric replacement of the amino acids
could accomplish dual roles. As shown in Figure 2, a
“redox amino acid” pair13 (2-amino-6-(3-carbamoyl-1,4-
dihydropyridinyl)hexanoic acid T 2-amino-6-(3-carbam-
P h a r m a cologica l Stu d ies
Tail-flick latency, an index of spinal cord-mediated
analgesia,18 was used to evaluate the analgesic effects
in rats of the brain-targeted delivery systems for the
kyotorphin analogues. Each compound, at each dose,
was tested on six animals. As shown in Figure 4, the
simplest CDS, 7a , with a single proline as the spacer,

Targeting Kyotorphin Analogues to the Brain
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20 3775
F igu r e 3. Sequential metabolism of brain-targeted delivery systems of Tyr-Lys and brain-targeted redox analogue (Tyr-Nys+).
Sch em e 1. Synthesis of Tyr-Lys-CDSs 7a -ca
F igu r e 4. Dose-response of CDS-P (7a ) after iv administra-
tion in rat. Data represent mean ( SE of 6 rats for each group.
activity as compared to systemically administered kyo-
torphin which is required to be given at about 200 mg/
kg to observe analgesia.
The analogues were compared for their activity as
shown in Figure 5. The comparison was made at a
single dose of 0.0223 mmol/kg, equivalent to 7.5 mg/kg
kyotorphin. The highest activity was achieved when
two L-proline moieties are used as the spacer. This is
followed by one proline, then the Nys containing the
novel BTRA, and CDS containing the L-proline-alanine
spacer. The time-response relationships in all cases
show remarkably sustained effects (Figure 5). (Two-
factor ANOVA with replication, p < 0.05 in all cases as
a
compared to kyotorphin or the vehicle.)
The proline spacer is replaced with Pro-Pro and Pro-Ala in
7b,c, respectively, by extending 2 with a Pro or Ala before coupling
with 3.
All possible partial derivatizations were also tested.
As shown in Figure 6, the lipophilic intermediate
containing cholesteryl and Boc, in addition to the two
proline spacers, but without the dihydrotrigonellyl
targetor (4a ) does not produce any activity. The posi-
tively charged trigonellyl targetor does not lead to brain
delivery (6b), or when the dihydrotrigonellyl-prolyl-
prolyl-Tyr-Lys (DPPYK) is administered, the lack of the
increased the rat tail-flick latency periods significantly
at all doses, compared to the vehicle. A dose-dependent
response was observed at doses of 0.003-0.0223 mmol/
kg (equivalent to 1.0, 2.5, 5.0, and 7.5 mg/kg kyotorphin,
respectively; single factor for the maximum responses,
p < 0.01). This represents a dramatic increase in

3776 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20
Chen et al.
F igu r e 5. Rat tail-flick latency periods of Tyr-Lys-CDSs and Tyr-Nys-BTRA after iv administration at doses of 0.0223 mmol/kg
(equivalent to 7.5 mg/kg kyotorphin). Data represent mean ( SE of 6 rats for each group; CDS-PA, 7c; BTRA, 12; CDS-P, 7a ;
CDS-PP, 7b.
Sch em e 2. Synthesis of Tyr-Nys-BTRA (12)

Targeting Kyotorphin Analogues to the Brain
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20 3777
F igu r e 6. Rat tail-flick latency periods after iv administration of CDS-PP and its major intermediates at doses of 0.0223 mg/kg
(equivalent to 7.5 mg/kg kyotorphin). Data represent mean ( SE of 6 rats for each group; PPYK(B)C, 4a ; TPPYK(B)C, 6b; DPPYK,
7b without cholesteryl ester and Boc ester moieties; DPPYKC, 7b without the Boc ester moiety; CDS-PP, 7b.
F igu r e 7. Rat tail-flick latency periods after iv administration of BTRA and its major intermediates at doses of 0.0223 mmol/kg
(equivalent to 7.5 mg/kg kyotorphin). Data represent mean ( SE of 6 rats for each group; BYKC, 9; BYNC, 11; YN, Tyr-Nys;
YNC, Tyr-Nys cholesteryl ester; BTRA, 12.
lipophilic cholesteryl function again prevents activity.
Finally, the fully packaged YK, but without the Boc
protection (DPPYKC) of the ꢀ-amine, does not show
activity. Only the described constructs, represented by
CDS-PP (7b), showed a dramatic increase in the rat tail-
flick latency.
Similar studies on the BTRA intermediates (Figure
7) demonstrate, again, that the fully protected dihydro
form of the BTRA shows the desired analgesic activity.
In this case, the lipophilic cholesteryl ester of the Nys
analogue (YNC) shows minimal activity, similarly to the
fully packaged CDS without the Boc derivatization
(DPPYKC, Figure 6). The results suggest that both the
lipophilic cholesteryl and Boc groups are needed in
addition to the dihydropyridine form of the redox
targetor in both cases for facilitating delivery. The
pyridinium salt form(s) then provide the “lock-in” mech-
anism. Finally, the central mediation of the observed
analgesia was demonstrated by administering naloxone
at 30 min after administration of BTRA and CDSPP,
respectively. In both cases the analgesia was fully
antagonized, as shown in Figure 8.
Con clu sion s
The 1,4-dihydropyridine T pyridinium redox targetor
was applied to differential and prolonged brain targeting
of enkephalin-releasing, analgesic dipeptide analogues
of kyotorphin. Incorporating this brain-targeting moiety
into various lipophilic constructs indicated the release
of active peptides via the predicted sequential metabo-

3778 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20
Chen et al.
F igu r e 8. Effects of naloxone on CDS-PP- and BTRA-induced rat tail-flick latency period increases. Data represent mean ( SE
of 6 rats for each group. The tail-flick latency periods for CDS-PP and BTRA were not recorded at 0.75 and 1.5 h; CDS-PP, 7b;
BTRA, 12.
26,27), 1.00 (3H, -CH3 19), 1.40 (9H, Boc), 0.80-2.00 (36H, H
on cholesterol ring and â,γ,δ-CH2- of Lys), 3.10 (2H, ꢀ-CH2 of
Lys), 3.35 (1H, R-CH of Lys), 4.60 (2H, R-NH2 of Lys), 5.38
(1H, ꢀ-NH- of Lys). Anal. (C38H66O4N2) C, H, N.
lism. The lysine ꢀ-amino function, however, was re-
quired to be converted to a lipophilic Boc derivative.
Alternatively, a novel redox amino acid pair (Nys+
T
Nys) provides both targeting and an effective replace-
ment for Arg or Lys.
Gen er a l P r oced u r e for P ep tid e Ch a in Elon ga tion
(Com p ou n d s 2-4, 9). Peptide cholesteryl ester in CH2Cl2
or Pro-OtBu ester (for 3) was stirred at 0 °C, and Fmoc-
protected (2, 4) or Boc-protected (9) amino acid in CH2Cl2 or
nicotinic acid (3) (equimolar to the peptide ester) in DMF was
added, which was followed immediately by DCC (1.2:1 molar
ratio to the peptide ester) in CH2Cl2 and HOBt (1.4:1 molar
to the peptide ester) dissolved in DMF. The mixture was
stirred for 30 min at 0 °C and then 24 h at room temperature.
The DCU yielded was filtered, and the solution was washed
with 1 N HCI (3 × 100 mL), 5% NaHCO3 (3 × 100 mL), and
saturated NaCl solution (100 mL), respectively. The organic
phase was dried over anhydrous Na2SO4 and evaporated. The
protected peptide ester was deprotected with piperidine/CH2-
Cl2 (1:3) or TFA/H2O (3; 19:1), and the solvent was removed
in vacuo. The products were purified by silica-gel column
chromatography. Purification procedure and analytical data
of the compounds prepared are given below.
Exp er im en ta l Section
All chemicals used were reagent grade or peptide synthesis
grade. All solvents used were ACS reagent grade, from Fisher
Scientific. Melting points were taken on a Fisher-J ones
melting point apparatus and are uncorrected. All synthesized
products were characterized by FAB (fast atom bombardment)
mass spectrometry by using a Kratos MS80RFA mass spec-
trometer (Manchester, U.K.). 1H NMR spectra were recorded
on a 300 MHz instrument. UV spectrophotometry was done
on a Carry 210 UV-visible spectrometer (Varian, Walnut
Creek, CA). Elemental analyses of compounds synthesized
were performed by Atlantic Microlab, Inc. (Necroses, GA), and
the Department of Chemistry, University of Florida (Gaines-
ville, FL). TLC (thin layer chromatography) was carried out
on silica-gel-coated aluminum plates (Merck DC-Alufolien
Kiesegel 60 F254). Column chromatography was performed by
using silica gel, neutral alumina, and Sephadex-LH 20,
respectively.
Lys(Boc) Ch olester yl Ester (1). A solution of N-R-Fmoc-
Lys(Boc)-OH (4.68 g, 10.0 mmol) in CH2Cl2 was stirred at 0
°C in an ice bath. Cholesterol (3.88 g, 10.0 mmol) in 75 mL
CH2Cl2 was added, which was followed immediately by DCC
(2.44 g, 11.0 mmol) in 25 mL CH2Cl2 and DMAP (1.68 g, 14.0
mmol) in 25 mL CH2Cl2. The mixture was stirred for 30 min
at 0 °C and 48 h at room temperature. The DCU yielded was
filtered, and the solvent was removed in vacuo to give the solid
N-R-Fmoc-Lys(Boc) cholesteryl ester. The product was dis-
solved in 60 mL piperidine/CH2Cl2 (1:3) and was stirred at
room temperature for 0.5 h. The solvent was then removed
in vacuo. The crude material was purified by silica-gel column
chromatography (7% CH3OH and 10% Et2O in CH2Cl2) to
afford a white solid (4.71 g, 76.4%): TLC Rf ) 0.65, CH3OH/
CH2Cl2 (1:9); MS-FAB m/z ) 637, (M + Na)+; mp 79-80 °C;
1H NMR (300 MHz) δ 0.65 (3H, -CH3 18), 0.85 (6H, CH-(CH3)2
Tyr -Lys(Boc) Ch olester yl Ester (2). Silica-gel column
chromatography (5% CH3OH and 10% Et2O in CH2Cl2) af-
forded a white solid (yield 72.14%): TLC Rf ) 0.58, CH3OH/
CH2Cl2 (1:9); MS-FAB m/z ) 801, (M + Na)+; mp 103-104
1
°C; H NMR (300 MHz) δ 0.65 (3H, -CH3 18), 0.85 (6H, CH-
(CH3)2 26,27), 1.00 (3H, -CH3 19), 1.40 (9H, Boc), 0.80-2.80
(38H, H on cholesterol ring, â,γ,δ-CH2- of Lys, â-CH2- of Tyr),
3.10 (2H, ꢀ-CH2- of Lys), 3.35 (1H, R-CH of Lys), 4.60 (2H,
R-NH2 of Tyr), 4.75 (1H, R-NH- of Lys), 5.38 (1H, ꢀ-NH- of Lys),
6.95 (4H, -CH) of aromatic ring of Tyr, J ) 6 Hz), 7.80 (1H,
-OH of Tyr, J ) 6 Hz). Anal. (C47H75O6N3) C, H, N.
Nicotin yl-P r o-OH (3). Precipitation from ethyl ether gave
a white solid (yield 62.29%): TLC Rf ) 0.12, CH3OH/CH2Cl2
(1:9); MS-FAB m/z ) 221, (M + H)+; mp 176-177 °C; 1H NMR
(300 MHz) δ 1.8 (2H, δ-CH2 of Pro), 2.2 (2H, â-CH2 of Pro),
3.5 (2H, δ-CH2 of Pro), 4.4 (1H, R-CH of Pro), 7.2 (1H, 5 CH
of pyridine ring), 7.8 (1H, 4 CH of pyridine ring), 8.6 (2H,
2, 5 CH of pyridine ring), 8.8 (1H, -COOH of Pro). Anal.
(C11H12N2O3) C, H, N.

Targeting Kyotorphin Analogues to the Brain
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20 3779
P r o-Tyr -Lys(Boc) Ch olester yl Ester (4a ). Silica-gel col-
umn chromatography (5% CH3OH and 10% Et2O in CH2Cl2)
afforded a white solid (yield 58.22%): TLC Rf ) 0.51, CH3-
OH/CH2Cl2 (1:9); MS-FAB m/z ) 898, (M + Na)+; mp 97-99
Lys, δ-CH2- of Pro), 4.40-4.60 (4H, R-CH of Pro, R-NH of Ala,
Tyr and Lys), 5.38 (1H, ꢀ-NH- of Lys), 6.95 (4H, -CH) of
aromatic ring of Tyr, J ) 6 Hz), 7.20 (1H, H5 of pyridine ring),
7.40-7.60 (2H, -OH of Tyr, H4 of pyridine ring), 8.6 (2H, H2,6
of pyridine ring). Anal. (C61H90O9N6) C, H, N.
1
°C; H NMR (300 MHz) δ 0.65 (3H, -CH3 18), 0.85 (6H, CH-
(CH3)2 26,27), 1.00 (3H, -CH3 19), 1.40 (9H, Boc), 0.80-3.00
(43H, H on cholesterol ring, â,γ,δ-CH2- of Lys, â-CH2- of Tyr,
â,δ-CH2-, R-NH of Pro), 3.10 (2H, ꢀ-CH2- of Lys), 3.35 (1H,
R-CH of Lys), 3.5 (2H, δ-CH2 of Pro), 4.4 (1H, R-CH of Pro),
4.60 (2H, R-NH- of Tyr and Lys), 5.38 (1H, ꢀ-NH- of Lys), 6.95
(4H, -CH) of aromatic ring of Tyr, J ) 6 Hz), 7.80 (1H, -OH
of Tyr). Anal. (C52H82O7N4‚H2O) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of Tr igon ellyl
P ep tid e Ch olester yl Ester s 6a -c. Nicotinoyl-spacer-Tyr-
Lys(Boc) cholesteryl ester in 50 mL ethyl ether was stirred at
0 °C, and dimethyl sulfate (10:1 molar ratio to the nicotinyl
peptide ester) was added. The mixture was stirred overnight
at room temperature. The product yielded was filtered and
washed with ethyl ether several times to afford white solids.
Tr igon ellyl-P r o-Tyr -Lys(Boc) ch olester yl ester (6a ):
yield 91.84%; MS-FAB m/z ) 996, M+; mp 153-155 °C; 1H
NMR (CDCI3) 8 0.65 (3H, -CH3 18), 0.85 (6H, CH-(CH3)2 26,-
27), 1.00 (3H, -CH3 19), 1.40 (9H, Boc), 0.80-3.00 (50H, H on
cholesterol ring, â,γ,δ-CH2- of Lys, â-CH2- of Tyr, 2H, â-CH3,
â,γ-CH2- of 2 Pro), 3.40 (3H, CH3-N+ on pyridine ring), 3.1-
3.6 (7H, ꢀ-CH2- of Lys, R-CH of Lys, δ-CH2- of 2 Pro), 4.40-
4.60 (4H, R-CH of 2 Pro, R-NH of Tyr and Lys), 5.38 (1H, ꢀ-NH-
of Lys), 6.90 (4H, -CH) of aromatic ring of Tyr, J ) 6 Hz),
7.60 (1H, H5 of pyridine ring), 7.90-8.10 (2H, -OH of Tyr, H4
of pyridine ring), 8.6 (1H, H6 of pyridine ring), 9.0 (1H, H2 of
pyridine ring). Anal. (C59H88O8N5‚HSO4-‚H2O) C, H, N.
Tr igon ellyl-P r o-P r o-Tyr -Lys(Boc) ch olester yl ester (6b):
Ala -Tyr -Lys(Boc) Ch olester yl Ester (4b). Silica-gel col-
umn chromatography (5% CH3OH and 10% Et2O in CH2Cl2)
afforded a white solid (yield 73.0%): TLC Rf ) 0.50, CH3OH/
CH2Cl2 (1:9); MS-FAB m/z ) 872, (M + Na)+; mp 103-104
1
°C; H NMR (300 MHz) δ 0.65 (3H, -CH3 18), 0.85 (6H, CH-
(CH3)2 26,27), 1.00 (3H, -CH3 19), 1.40 (9H, Boc), 0.80-3.00
(39H, H on cholesterol ring, â,γ,δ-CH2- of Lys, â-CH2- of Tyr,
2H, R-CH of Ala), 2.90 (3H, â-CH3 of Ala), 3.10 (2H, ꢀ-CH2- of
Lys), 3.35 (1H, R-CH of Lys), 4.20 (2H, R-NH2 of Ala), 4.60
(2H, R-NH- of Tyr and Lys), 5.38 (1H, ꢀ-NH- of Lys), 6.95 (4H,
-CH) of aromatic ring of Tyr, J ) 6 Hz), 7.80 (1H, -OH of
Tyr). Anal. (C50H82O7N4) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of Nicotin oyl
P ep tid e Ch olester yl Ester s 5a-c. Peptide-cholesteryl ester
in CH2Cl2 was stirred at 0 °C. Nicotinoyl-Pro-OH (equimolar
to the peptide ester) in DMF was added, which was followed
immediately by DCC (1.2:1 molar to the peptide ester) in CH2-
Cl2 and HOBt (1.4:1 molar to the peptide ester) dissolved in
DMF. The mixture was stirred for 30 min at 0 °C and then
96 h at room temperature. The DCU yielded was filtered, and
the solution was washed with 1 N HCl (3 × 100 mL), 5%
NaHCO3 (3 × 100 mL), and saturated NaCl solution (100 mL),
respectively. The organic phase was dried over anhydrous Na2-
SO4 and evaporated. The products were purified by silica-gel
column chromatography. Purification procedure and analyti-
cal data of the compounds prepared are given below.
1
yield 87.75%; MS-FAB m/z ) 1094, M+; mp 169-170 °C; H
NMR (CDCl3) δ 0.65 (3H, -CH3 18), 0.85 (6H, CH-(CH3)2 26,-
27), 1.00 (3H, -CH3 19), 1.40 (9H, Boc), 0.80-3.00 (50H, H on
cholesterol ring, â,γ,δ-CH2- of Lys, â-CH2- of Tyr, 2H, â-CH3,
â,γ-CH2- of 2 Pro), 3.40 (3H, CH3-N+ on pyridine ring), 3.1-
3.6 (7H, ꢀ-CH2- of Lys, R-CH of Lys, δ-CH2- of 2 Pro), 4.40-
4.60 (4H, R-CH of 2 Pro, R-NH of Tyr and Lys), 5.38 (1H, ꢀ-NH-
of Lys), 6.95 (4H, -CH) of aromatic ring of Tyr, J ) 6 Hz),
7.80 (1H, H5 of pyridine ring), 8.19-8.30 (2H, -OH of Tyr, H4
of pyridine ring), 8.60 (1H, H6 of pyridine ring), 9.20 (1H, H2
of pyridine ring). Anal. (C63H92O9N6‚HSO4-‚H2O) C, H, N.
Tr igon ellyl-P r o-Ala-Tyr -Lys(Boc) ch olester yl ester (6c):
1
yield 91.76%; MS-FAB m/z ) 1068, M+; mp 185-187 °C; H
NMR (CDC13) δ 0.65 (3H, -CH3 18), 0.85 (6H, CH-(CH3)2 26,-
27), 1.00 (3H, -CH3 19), 1.40 (9H,Boc), 0.80-3.00 (47H, H on
cholesterol ring, â,γ,δ-CH2- of Lys, â-CH2- of Tyr, 2H, â-CH3,
R-CH of Ala, â,γ-CH2-, R-NH of Pro), 2.90 (3H, â-CH3 of Ala),
3.40 (3H, CH3-N+ on pyridine ring), 3.1-3.6 (5H, ꢀ-CH2- of Lys,
R-CH of Lys, δ-CH2- of Pro), 4.40-4.60 (4H, R-CH of Pro, R-NH
of Ala, Tyr and Lys), 5.38 (1H, ꢀ-NH- of Lys), 6.95 (4H, -CH)
of aromatic ring of Tyr, J ) 6 Hz), 7.70 (1H, H5 of pyridine
ring), 7.00-8.20 (2H, -OH of Tyr, H4 of pyridine ring), 8.60
(1H, H6 of pyridine ring), 9.20 (1H, H2 of pyridine ring). Anal.
(C62H93O9N6‚HSO4-‚2H2O) C, H, N.
Gen er a l P r oced u r e for t h e Dit h ion it e R ed u ct ion
(7a -c, 12). A trigonellyl peptide ester in deaerated CH3OH/
H2O (1:1) was stirred at 0 °C under argon. NaHCO3 and
Na2S2O4 (10:1 molar ratio to the trigonellyl peptide ester) was
then added in small portions. The mixture was stirred at 0
°C under argon for 1 h. Then 35 mL saturated NaCl solution
was added to dilute the solution which was extracted with 20
mL CH2Cl2. The organic phase was dried over anhydrous Na2-
SO4 and evaporated. Natural aluminum oxide column chro-
matography (5% CH3OH in CH2Cl2) afforded a yellow solid.
1,4-Dih yd r ot r igon ellyl-P r o-Tyr -Lys(Boc) ch olest er yl
ester (7a ): yield 25.0%; UVmax (MeOH) ) 348 nm; TLC Rf )
0.33, CH3OH/CH2Cl2 (3:97); mp dec. Anal. (C59H89O8N5‚2H2O)
C, H, N.
Nicot in oyl-P r o-Tyr -Lys(Boc) Ch olest er yl E st er (5a ).
Silica-gel column chromatography (5% CH3OH and 10% Et2O
in CH2Cl2) afforded a white solid (yield 67.28%): TLC Rf )
0.47, CH3OH/CH2Cl2 (1:9); 1H NMR (CDCl3) δ 0.65 (3H, -CH3
18), 0.85 (6H, CH-(CH3)2 26,27), 1.00 (3H, -CH3 19), 1.40 (9H,
Boc), 0.80-3.00 (42H, H on cholesterol ring, â,γ,δ-CH2- of Lys,
â-CH2- of Tyr, â,γ-CH2- of Pro), 3.1-3.6 (5H, ꢀ-CH2- of Lys,
R-CH of Lys, δ-CH2- of Pro), 4.40-4.60 (3H, R-CH of Pro, R-NH
of Tyr and Lys), 5.38 (1H, ꢀ-NH- of Lys), 6.95 (4H, -CH) of
aromatic ring of Tyr, J ) 6 Hz), 7.20 (1H, H5 of pyridine ring),
7.40-7.60 (2H, -OH of Tyr, H4 of pyridine ring), 8.6 (2H, H2,6
of pyridine ring); MS-FAB m/z ) 981, (M + H)+; mp 121-123
°C. Anal. (C58H85O8N5‚H2O) C, H, N.
Nicot in oyl-P r o-P r o-Tyr -Lys(Boc) Ch olest er yl E st er
(5b). Silica-gel column chromatography (5% CH3OH and 10%
Et2O in CH2Cl2) afforded a white solid (yield 65.34%): TLC
Rf ) 0.47, CH3OH/CH2Cl2 (1:9); MS-FAB m/z ) 1079, (M +
1
H)+; mp 131-132 °C; H NMR (CDCl3) δ 0.65 (3H, -CH3 18),
0.85 (6H, CH-(CH3)2 26,27), 1.00 (3H, -CH3 19), 1.40 (9H, Boc),
0.80-3.00 (50H, H on cholesterol ring, â,γ,δ-CH2- of Lys,
â-CH2- of Tyr, 2H, â-CH3, â,γ-CH2- of 2 Pro), 3.1-3.6 (7H,
ꢀ-CH2- of Lys, R-CH of Lys, δ-CH2- of 2 Pro), 4.40-4.60 (4H,
R-CH of 2 Pro, R-NH of Tyr and Lys), 5.38 (1H, ꢀ-NH- of Lys),
6.95 (4H, -CH) of aromatic ring of Tyr, J ) 6 Hz), 7.20 (1H,
H5 of pyridine ring), 7.40-7.60 (2H, -OH of Tyr, H4 of pyridine
ring), 8.6 (2H, H2,6 of pyridine ring). Anal. (C61H90O9N6) C,
H, N. Anal. (C63H92O9N6) C, H,N.
1,4-Dih yd r otr igon ellyl-P r o-P r o-Tyr -Lys(Boc) ch oles-
ter yl ester (7b): yield 27.3%; UVmax (MeOH) ) 348 nm; TLC
Rf )0.35, CH3OH/CH2Cl2 (3:97); mp dec. Anal. (C64H96O9N6‚
3H2O) C, H, N.
1,4-Dih yd r otr igon ellyl-P r o-Ala -Tyr -Lys(Boc) ch oles-
ter yl ester (7c): yield 28.6%; UVmax (MeOH) ) 348 nm; TLC
Rf ) 0.38, CH3OH/CH2Cl2 (3:97); mp dec. Anal. (C62H94O9N6‚
3H2O) C, H, N.
Nicotin oyl-P r o-Ala-Tyr -Lys(Boc) Ch olester yl Ester (5c).
Silica-gel column chromatography (5% CH3OH and 10% Et2O
in CH2Cl2) afforded a white solid (yield 72.65%): TLC Rf )
0.46, CH3OH/CH2Cl2 (1:9); MS-FAB m/z ) 1053, (M + H)+;
1
mp 155-156 °C; H NMR (CDCl3) δ 0.65 (3H, -CH3 18), 0.85
(6H, CH-(CH3)2 26,27), 1.00 (3H, -CH3 19), 1.40 (9H, Boc),
0.80-3.00 (47H, H on cholesterol ring, â,γ,δ-CH2- of Lys,
â-CH2- of Tyr, 2H, â-CH3, R-CH of Ala, â,γ-CH2-, R-NH of Pro),
2.90 (3H, â-CH3 of Ala), 3.1-3.6 (5H, ꢀ-CH2- of Lys, R-CH of
Syn th esis of Lys(F m oc) Ch olester yl Ester (8). The
procedure was the same as for 1 except that N-R-Boc-Ly-
(Fmoc)-OH (4.68 g, 10.0 mmol) was used and the deprotection

3780 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 20
Chen et al.
was carried out in TFA/CH2Cl2 (1:3): white solid, 4.31 g
and 7.5 mg/kg kyotorphin) was administered to monitor the
dose-response. Vehicle and 22.3 µmol/kg drugs (equimolar
to 7.5 mg/kg kyotorphin) were also administered to study the
pharmacological activities of the other YK brain-targeted
delivery systems and their important intermediates.
(58.48%); TLC Rf ) 0.35, CH3OH/CH2Cl2 (1:19); MS-FAB m/z
1
) 60, (M + Na)+; mp 74-75 °C; H NMR (CDCl3) δ 0.65 (3H,
-CH3 18), 0.85 (6H, CH-(CH3)2 26,27), 1.00 (3H, -CH3 19), 0.80-
2.00 (36H, H on cholesterol ring and â,γ,δ-CH2- of Lys), 3.10
(2H, ꢀ-CH2 of Lys), 3.35 (1H, R-CH of Lys), 4.60 (2H, R-NH2 of
Lys), 5.38 (1H, ꢀ-NH- of Lys), 6.05 (1H, CH- of Fomc), 7.20-
7.80 (8H, H of Fomc ring). Anal. (C48H68O4N2) C, H, N.
Boc-Tyr -Lys Ch olester yl Ester (9). Silica-gel column
chromatography (3% AcOH and 10% CH3OH in CH2Cl2)
afforded a white solid (yield 81.90%): TLC Rf ) 0.05, CH3-
OH/CH2Cl2 (1:10); MS-FAB m/z ) 801, (M + Na)+; mp 121-
Tail-flick latency, an index of spinal cord-mediated analge-
sia,17 was measured to evaluate the analgesic effects of the
brain-targeted delivery systems for YK. Time between pre-
sentation of a focused beam of light and the reflex removal of
the tail from the stimulus was defined as the tail-flick latency
period (T), and the tail-flick latency difference between each
time point and control was defined as the change in tail-flick
latency. In the absence of response, a cut-off period of 1 min
was used. Each drug at each dose was tested on six animals.
Control latency (T0) was obtained 10 min prior to the drug
administration; the test latencies were measured at 15 min,
30 min, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h after the administration
of drugs. The instrument used was a model 33 tail-flick
analgesia meter (Litc, Inc., Landing, NJ ). The beam intensity
was set to achieve 7-8 s as a baseline tail-flick latency.7
Sta tistics. Rat tail-flick latency measurements were evalu-
ated by using spreadsheets (Microsoft, Excel 5.0). The mean,
standard deviation, and standard error were calculated for
each group. The significance of differences between groups
was determined by using ANOVA (analysis of variance).
1
123 °C; H NMR (300 MHz) δ 0.65 (3H, -CH3 18), 0.85 (6H,
CH-(CH3)2 26,27), 1.00 (3H, -CH3 19), 1.40 (9H, Boc), 0.80-
2.80 (38H, H on cholesterol ring, â,γ,δ-CH2- of Lys, â-CH2- of
Tyr), 3.10 (2H, ꢀ-CH2- of Lys), 3.45 (1H, R-CH of Lys), 4.60
(1H, R-NH of Tyr), 4.75 (1H, R-NH- of Lys), 5.38 (2H, ꢀ-NH- of
Lys), 6.95 (4H, -CH) of aromatic ring of Tyr, J ) 6 Hz), 7.80
(1H, -OH of Tyr). Anal. (C47H75O6N3) C, H, N.
N-(2,4-Din itr op h en yl)n icotin a m id e Ch lor id e (10). Nic-
otinamide (2.5 g, 20 mmol) was mixed with 1-chloro-2,4-
dinitrophenol (6.25 g, 30 mmol) in 20 mL DMF and gradually
heated to 100 °C. The mixture was stirred for 1 h at that
temperature and then cooled down to the room temperature.
The material obtained was dissolved in MeOH (30 mL) and
poured into 125 mL vigorously stirred Et2O. The precipitant
was again dissolved in 30 mL MeOH and poured into 125 mL
Et2O. The precipitant was then dissolved in 50 mL MeOH
and stirred with active carbon for 30 min. The active carbon
was filtered off, and the filtrate was evaporated in vacuo.
Sephadex LH-20 column chromatography (MeOH) afforded the
title compound (2.69 g, 46.54%): MS-FAB m/z ) 289, M+; mp
177-179 °C; 1H NMR (300 MHz) δ 3.70 (2H, -NH2), 8.05 (1H,
H6 on bezene ring), 8.40 (1H, H3 on bezene ring), 8.60 (1H, H5
on bezene ring), 8.80 (1H, H5 on pyridine ring), 9.00 (1H, H4
on pyridine ring), 9.35 (1H, H6 on pyridine ring), 9.70 (1H, H2
on pyridine ring). Anal. C, H, N.
Boc-Tyr -Nys+ Ch olester yl Ester (11). Boc-Tyr-Lys cho-
lesteryl ester (1.56g, 2.0 mmol) in CH3OH was stirred at 0 °C,
and N-(2,4-dinitrophenol)nicotinamide chloride (0.65 g, 2.25
mmol) was added. The mixture was stirred overnight at room
temperature. The product was precipitated from ethyl ether,
filtered off, and washed with ethyl ether several times.
Sephadex LH-20 column chromatography (CH3OH) afforded
a yellow solid (1.45 g, 81.92%): MS-FAB m/z ) 885, M+; mp
168-169 °C; 1H NMR (CDCl3) δ 0.65 (3H, -CH3 18), 0.85 (6H,
CH-(CH3)2 26,27), 1.00 (3H, -CH3 19), 1.40 (9H, Boc), 0.80-
2.80 (38H, H on cholesterol ring, â,γ,δ-CH2- of Lys, â-CH2- of
Tyr), 3.10 (2H, ꢀ-CH2- of Lys), 3.45 (1H, R-CH of Lys), 4.60
(1H, R-NH of Tyr), 4.75 (1H, R-NH- of Lys), 5.38 (2H, ꢀ-NH- of
Lys), 6.95 (4H, -CH) of aromatic ring of Tyr, J ) 6 Hz), 7.80
(1H, -OH of Tyr), 8.80 (1H, H5 on pyridine ring), 9.00 (1H, H4
on pyridine ring), 9.35 (1H, H6 on pyridine ring), 9.70 (1H, H2
on pyridine ring). Anal. (C53H79O7N4‚Cl-‚3.5H2O) C, H, N.
Boc-Tyr -Nys ch olester yl ester (12): yield 27.8%; UVmax
(MeOH) ) 348 nm; TLC Rf ) 0.33 CH3OH/CH2Cl2 (3:97); mp
dec. Anal. (C53H80O7N4‚H2O) C, H, N.
Ack n ow led gm en t. This work was supported by a
grant (RO1 DAO9268) from the National Institute on
Drug Abuse (Bethesda, MD).
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