Synthesis of a novel esterase-sensitive cyclic prodrug of a hexapeptide using an (acyloxy)alkoxy promoiety

Article abstract of DOI:10.1021/jo961696a

Synthetic methodology for preparing novel esterase-sensitive cyclic prodrugs of peptides with increased protease stability and cell membrane permeability compared to linear peptides is described. Cyclic prodrug 1 of the hexapeptide H-Trp-Ala-Gly-Gly-Asp-Ala-OH linked by the N-terminal amino group to the C-terminal carboxyl group via an (acyloxy)alkoxy promoiety was synthesized. A convergent synthetic approach involving Boc[[(alaninyloxy)methyl]carbonyl]-N-tryptophan (2) and H-Ala-Gly-Gly-Asp(OBzl)-OTce (3) was used. The key fragment 2 has the promoiety inserted between the Ala and the Trp residues. Fragment 3 was synthesized by a solution-phase approach using standard Boc-amino acid chemistry. These fragments were coupled to produce the protected linear hexapeptide, which after deprotection was cyclized using standard high-dilution techniques to yield cyclic prodrug 1. In pH 7.4 buffer (HBSS) at 37°C, cyclic prodrug 1 was shown to degrade quantitatively to the hexapeptide (t( 1/4 ) = 206 ± 11 min). The rate of hydrolysis of cyclic prodrug 1 was significantly faster in human blood (t( 1/4 ) = 132 ± 4 min) than in HBSS. Paraoxon, a known inhibitor of esterases, slowed this hydrolysis of cyclic prodrug 1 to a value (t( 1/4 ) = 198 ± 9 min) comparable to the chemical stability. In human blood, cyclic prodrug 1 was shown to be 25-fold more stable than the linear hexapeptide.

Full text of DOI:10.1021/jo961696a

1356
J . Org. Chem. 1997, 62, 1356-1362
Syn th esis of a Novel Ester a se-Sen sitive Cyclic P r od r u g of a
Hexa p ep tid e Usin g a n (Acyloxy)a lk oxy P r om oiety
Sanjeev Gangwar,† Giovanni M. Pauletti, Teruna J . Siahaan, Valentino J . Stella, and
Ronald T. Borchardt*
Department of Pharmaceutical Chemistry, 2095 Constant Avenue, The University of Kansas,
Lawrence, Kansas 66047
Received September 4, 1996X
Synthetic methodology for preparing novel esterase-sensitive cyclic prodrugs of peptides with
increased protease stability and cell membrane permeability compared to linear peptides is
described. Cyclic prodrug 1 of the hexapeptide H-Trp-Ala-Gly-Gly-Asp-Ala-OH linked by the
N-terminal amino group to the C-terminal carboxyl group via an (acyloxy)alkoxy promoiety was
synthesized. A convergent synthetic approach involving Boc[[(alaninyloxy)methyl]carbonyl]-N-
tryptophan (2) and H-Ala-Gly-Gly-Asp(OBzl)-OTce (3) was used. The key fragment 2 has the
promoiety inserted between the Ala and the Trp residues. Fragment 3 was synthesized by a
solution-phase approach using standard Boc-amino acid chemistry. These fragments were coupled
to produce the protected linear hexapeptide, which after deprotection was cyclized using standard
high-dilution techniques to yield cyclic prodrug 1. In pH 7.4 buffer (HBSS) at 37 °C, cyclic prodrug
1 was shown to degrade quantitatively to the hexapeptide (t1/2 ) 206 ( 11 min). The rate of
hydrolysis of cyclic prodrug 1 was significantly faster in human blood (t1/2 ) 132 ( 4 min) than in
HBSS. Paraoxon, a known inhibitor of esterases, slowed this hydrolysis of cyclic prodrug 1 to a
value (t1/2 ) 198 ( 9 min) comparable to the chemical stability. In human blood, cyclic prodrug 1
was shown to be 25-fold more stable than the linear hexapeptide.
In tr od u ction
carboxyl group, and side-chain carboxyl, amino, and
hydroxyl groups that bestow upon the molecule affinity
and specificity for its pharmacological receptor, severely
restrict its ability to permeate biological barriers and
become a substrate for peptidases. With small organic-
based drugs, which exhibit similar structural features
and similar unfavorable physicochemical characteristics,
prodrug (bioreversibly derivatives of drug) strategies
have been successfully employed to transiently (e.g.,
bioreversibly) alter the drug’s physicochemical charac-
teristics and/or its lability to metabolism.15-25 Unfortu-
nately, the synthesis of peptide prodrugs has been limited
due to their structural complexity and the lack of novel
methodology.26
In this paper, we describe a methodology for the
synthesis of novel esterase-sensitive cyclic prodrugs of
linear peptides using an (acyloxy)alkoxy promoiety.
These cyclic prodrugs were designed to be susceptible to
esterase metabolism (slow step), leading to a cascade of
chemical reactions and resulting in generation of the
linear peptide (Scheme 1). To illustrate the synthetic
methodology used to prepare this type of cyclic prodrug,
we describe here the preparation of a cyclic prodrug of a
model hexapeptide27 by linking the N-terminal amino
group to the C-terminal carboxyl group via an (acyloxy)-
alkoxy promoiety. [(Acyloxy)alkoxy]carbamate deriva-
The clinical development of orally active peptide drugs
has been limited by their unfavorable physicochemical
characteristics (e.g., charge, hydrogen-bonding potential,
size), which prevent them from permeating biological
barriers like the intestinal mucosa, and their lack of
stability against enzymatic degradation.1-13 Even when
peptides are administered via the parenteral route (e.g.,
intravenously), they tend not to gain access to important
target areas such as the brain due to their unfavorable
physicochemical characteristics and their metabolic labil-
ity.14
Unfortunately, many of the structural features of a
peptide, such as the N-terminal amino group, C-terminal
* To whom correspondence should be addressed. Phone: (913) 864-
3427. Fax: (913) 864-5736. E-mail: Borchardt@smissman.hbc.ukans.edu.
† Current address: Prolinx, Inc., Bothwell, WA 98021.
X Abstract published in Advance ACS Abstracts, February 15, 1997.
(1) Amidon, G. L.; Lee, H. J . Annu. Rev. Pharmacol. Toxicol. 1994,
34, 321.
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1991, 7, 365.
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Maggiora, L. L. J . Controlled Release 1992, 19, 87.
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S0022-3263(96)01696-9 CCC: $14.00 © 1997 American Chemical Society

Synthesis of a Cyclic Prodrug of a Hexapeptide
J . Org. Chem., Vol. 62, No. 5, 1997 1357
Sch em e 1
convergent approach to the synthesis of the cyclic prodrug
1 involving fragments 2 and 3, as shown in Scheme 2.
Scheme 3 shows the synthesis of the key intermediate
2 in which the promoiety has been inserted between Boc-
Ala and Trp. The synthesis of 2 was begun by reacting
1-chloromethyl chloroformate with p-nitrophenol in the
presence of N-methylmorpholine (NMM) to afford 1-(chlo-
romethyl)-p-nitrophenyl carbonate (4) in 84% yield.
Substitution of the chloride in 4 with iodide was achieved
by reaction with NaI to give the iodo compound 5 in 94%
yield. Compound 5 was then reacted with the cesium
salt of Boc-Ala in DMF to give a mixture of the desired
product Boc-(alaninyloxy)methyl p-nitrophenyl carbonate
(6) in 70% yield and the side product p-nitrophenyl ester
of Boc-alanine (Boc-Ala-OpNP) in 30% yield as deter-
mined by NMR. This reaction was shown to afford better
yields of 6 when the cesium salt of Boc-Ala rather than
the sodium or potassium salts of this amino acid was
used. This mixture was difficult to separate, and it was
used directly in the next step without further purification.
The mixture of compound 6 and Boc-Ala-OpNP was
coupled with Trp-OBzl in the presence of NMM and
1-hydroxybenzotriazole (HOBT) in hexamethylphosphor-
amide (HMPA) to afford the Boc-[[(alaninyloxy)methyl]-
carbonyl]-N-tryptophan benzyl ester (7) and the side
product Boc-Ala-Trp-OBzl. Removal of the benzyl group
from compound 7 and Boc-Ala-Trp-OBzl was achieved by
hydrogenolysis using 10% Pd/C as a catalyst under an
H2 atmosphere in EtOH to give 2 and Boc-Ala-Trp-OH
in 96% yield. Compound 2 was purified from Boc-Ala-
Trp-OH by preparative reversed-phase HPLC.
Scheme 4 illustrates the solution-phase synthesis of
tetrapeptide 3 using standard Boc-amino acid chemis-
try.40 However, in using the solution-phase approach it
was necessary to temporarily protect the terminal R-car-
boxyl group during the synthesis of linear tetrapeptide
3. The key to this solution-phase approach was the
selective protection of the R- and â-carboxyl groups of the
Asp residue. The selective deprotection of the R-carboxyl
group of Asp must be carried out under conditions that
do not affect the protecting group on the â-carboxyl group
or the ester bond of the promoiety. Initially, we tried to
protect the R-carboxyl group of the Asp residue as the
2,4-dimethoxybenzyl (Dmb) ester41 and the â-carboxyl
group as a benzyl ester. However, during removal of the
Dmb protecting group in the final step of synthesis of
the hexapeptide, the indole ring of the Trp residue was
alkylated by the 2,4-dimethoxylbenzyl cation in almost
quantitative yield. Different scavengers were used,
without success, to suppress this alkylation reaction.42
Eventually, we successfully used the trichloroethyl (Tce)
ester protecting group for the R-carboxyl group of Asp
residue, which is quite stable to acidic conditions and can
be removed by zinc in AcOH.43,44 Compounds 9 and 13
were, therefore, used in the synthesis of 3. Treatment
of Boc-Asp(OBzl)-OH with 2,2,2-trichloroethanol in the
presence of 1-[3-(dimethylamino)propyl]-3-ethylcarbodi-
imide hydrochloride (EDC), HOBT, and NMM gave Boc-
Asp(OBzl)-OTce (8) in 83% yield. Compound 8 was
tives of primary and secondary amines as prodrugs have
previously been described in the literature and shown
to degrade by a similar mechanism.28-35 However, to our
knowledge, this is the first demonstration of the way in
which this promoiety can be used to prepare a cyclic
prodrug of a peptide. In separate study,36 we have shown
that cyclic prodrug 1 has enhanced cell membrane
permeability and protease stability compared to the
linear peptide. In addition, we have been able to show
that the enhanced cellular permeability is related to the
structural features of the cyclic prodrug.37
Resu lts a n d Discu ssion
Syn th esis of th e Cyclic P r od r u gs. The synthesis
of cyclic prodrug 1 of the linear peptide H-Trp-Ala-Gly-
Gly-Asp-Ala-OH was attempted by two different ap-
proaches. The first approach involved the synthesis of
the linear hexapeptide on Wang resin using standard
Fmoc-amino acid chemistry.38,39 The amino terminal of
this peptide was coupled with 1-chloromethyl chlorofor-
mate to give the ClCH2OCO-Trp-Ala-Gly-Gly-Asp-Ala-
resin. This peptide was cleaved from the resin to give
the precursor ClCH2OCO-Trp-Ala-Gly-Gly-Asp-Ala-OH
for the final cyclization reaction. However, cyclization
of ClCH2OCO-Trp-Ala-Gly-Gly-Asp-Ala-OH under vari-
ous basic conditions did not yield the desired cyclic
prodrug, resulting only in degradation of the precursor
peptide.
The alternative approach, which was then pursued,
involved insertion of the (acyloxy)alkoxy promoiety be-
tween two amino acids in this sequence before the final
cyclization step. In the model hexapeptide used in these
studies, we decided to insert the promoiety between the
Ala and the Trp residues. Therefore, we devised a
(27) Gray, R. A.; Vander Velde, D. G.; Burke, C. J .; Manning, M.
C.; Middaugh, C. R.; Borchardt, R. T. Biochemistry 1994, 33, 1323.
(28) Folkmann, M.; Lund, F. J . Synthesis 1990, 1159.
(29) Gogate, U. S.; Repta, A. J .; Alexander, J . Int. J . Pharm. 1987,
40, 235.
(30) Gogate, U. S.; Repta, A. J . Int. J . Pharm. 1987, 40, 249.
(31) Nishimura, T.; Yoshimura, Y.; Miyake, A.; Yamoka, M.; Ta-
kanohashi, K.; Hamaguchi, N.; Hirai, S.; Yashiki, T.; Numata, M. J .
Antibiot. 1987, 1, 81.
(32) Svahn, C. M.; Merenyi, F.; Karlson, L.; Widlund, L.; Gralls, M.
J . Med. Chem. 1986, 29, 448.
(33) Swaan, P. W.; Stehouwer, M. C.; Blok, R. I. C.; Tukker, J . J .
Pharm. Res. 1993, 10, S-295.
(34) Alexander, J .; Cargill, R.; Michelson, S. R.; Schwan, H. J . Med.
Chem. 1988, 31, 318.
(35) Alexander, J .; Fromtling, R. A.; Bland, J . A.; Pelak, B. A.;
Gilfillan, E. C. J . Med. Chem. 1991, 34, 78.
(40) Bodanszky, M.; Bodanszky, A. The Practice of Peptide Synthesis,
2nd ed.; Springer-Verlag: Berlin, 1994; p 75.
(41) Kim, C. U.; Misco, P. F. Tetrahedron Lett. 1985, 26, 2027.
(42) Lundt, B. F.; J ohansen, N. L.; Volund, A.; Markussen, J . Int.
J . Pept. Protein Res. 1978, 12, 258.
(43) Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic
Synthesis; 2nd ed.; J ohn Wiley & Sons, Inc.: New York, 1991; p 241.
(44) Hamada, Y.; Kondo, Y.; Shibata, M.; Shioiri, T. J . Am. Chem.
Soc. 1989, 111, 669.
(36) Pauletti, G. M.; Gangwar, S.; Okumu, F. W.; Siahaan, T. J .;
Stella, V. J .; Borchardt, R. T. Pharm. Res. 1996, 13, 1615.
(37) Gangwar, S.; J ois, S. D. S.; Vander Velde, D. G.; Siahaan, T.
J .; Stella, V. J .; Borchardt, R. T. Pharm. Res. 1996, 13, 1657.
(38) Atherton, E.; Sheppard, R. T. In Solid Phase Peptide Synthe-
sis: A Practical Approach, 2nd ed.; IRL Press: Oxford, 1989; pp 25.
(39) Wang, S. S. J . Am. Chem. Soc. 1973, 95, 1328.

1358 J . Org. Chem., Vol. 62, No. 5, 1997
Gangwar et al.
Sch em e 2
Sch em e 3
Sch em e 4
treated with 50% TFA in DCM to give 9 in quantitative
yield. The tripeptide Boc-Ala-Gly-Gly-OH (13) was syn-
thesized using standard Boc-amino acid chemistry with
EDC and HOBT as coupling reagents. Coupling between
the tripeptide 13 and Tce ester 9 in the presence of EDC
and HOBT gave the tetrapeptide 14 in 86% yield; it was
then treated with 50% TFA in DCM to yield 3 in 95%
yield.
Scheme 5 shows the assembly of fragments 2 and 3
for the synthesis of cyclic prodrug 1. Tetrapeptide 3 was
reacted with Boc-[[(alaninyloxy)methyl]carbonyl]-N-tryp-
tophan (2) in the presence of EDC, HOBT, and NMM to
give the fully protected linear hexapeptide 15 in 70%
yield. Both of the protecting groups on 15 were removed
by treatment first with zinc in AcOH to remove the Tce
protecting group and then with 50% TFA in DCM to
remove the Boc protecting group, affording 16 in 60%
overall yield. The linear hexapeptide 16 was purified by
preparative reversed-phase HPLC. Cyclization of 16
under high dilution conditions using EDC and HOBT as
condensation reagents did not yield the desired cyclic
prodrug 17. Cyclization was then accomplished by the
standard high-dilution technique using N,N-bis(2-oxo-3-
oxazolidinyl)phosphinic chloride (BOP-Cl) as an activat-
ing reagent45 in the presence of NMM and 4-(N,N-
(dimethylamino)pyridine (DMAP) to give cyclic peptide
17 in 20% yield after HPLC purification. The low yield
for the cyclization reaction may be due to the possible
formation of oligomers even under high dilution condi-
tions and the low recovery during purification by pre-
parative HPLC. Hydrogenolysis of cyclic peptide 17 to
remove the Bzl protecting group from the Asp residue
was achieved with 10% Pd/C as a catalyst under an H2
atmosphere in EtOH to yield the desired cyclic prodrug
1 in quantitative yield. The crude cyclic prodrug 1 was
(45) Diago-Meseguer.; Palomo-Coll, A. L.; Fernandez-Lizarbe, J . R.;
Zugaza-Bilbabo, A. Synthesis 1980, 547.

Synthesis of a Cyclic Prodrug of a Hexapeptide
J . Org. Chem., Vol. 62, No. 5, 1997 1359
Sch em e 5
F igu r e 1. Stability of the linear hexapeptide (4) and cyclic
prodrug 1 in human blood (in the presence (x) and absence
(b) of paraoxon) and in Hanks’ balanced salt solution (HBSS),
pH 7.4 (0). Disappearance of the cyclic prodrug was monitored
by HPLC over a time period of 180 min. Data points are
averages ( SD of triplicate.
prodrug 1 was designed to undergo esterase-mediated
hydrolysis of the ester bond of the carbamate moiety
followed by two fast chemical degradation steps to release
the linear hexapeptide (Scheme 1). The stability of cyclic
prodrug 1 was evaluated in human blood and was
compared to its chemical degradation in Hanks’ balanced
salt solution (HBSS), pH 7.4. In this physiological buffer
system, cyclic prodrug 1 degraded quantitatively to the
linear hexapeptide with an apparent half-life (t1/2) of 206
( 11 min. The rate of hydrolysis of cyclic prodrug 1 was
significantly faster in human blood (t1/2 ) 132 ( 4 min)
than in HBSS (Figure 1). This facilitated hydrolysis in
human blood was inhibited (t1/2 ) 198 ( 9 min) when
paraoxon, a known inhibitor of serine-dependent es-
terases, was included in the incubation mixture. In
human blood, cyclic prodrug 1 was 25-fold more stable
than the linear hexapeptide. These data suggest that
cyclic prodrug 1 is converted to the linear hexapeptide
by an esterase-mediated reaction in human blood.
In separate studies,36 we evaluated the relative cellular
permeability properties of cyclic prodrug 1 and the linear
hexapeptide. Transport studies were conducted using
monolayers of Caco-2 cells, an in vitro cell culture model
of the intestinal mucosa.47 In comparison with the linear
hexapeptide, cyclic prodrug 1 was found to be at least
76 times more able to permeate this model of the
intestinal mucosa.
purified by preparative reversed-phase HPLC and was
analyzed by analytical reversed-phase HPLC.
Using the same methodology as described above, we
also synthesized cyclic prodrug 18 of the hexapeptide
H-Trp-Ala-Gly-Gly-Asp-Ala-OH by linking the N-termi-
nal amino group of the Trp residue to a side-chain
C-terminal carboxyl group of the Asp residue via the
(acyloxy)alkoxy linker. However, cyclic prodrug 18 was
found to be very unstable and, therefore, could not be
evaluated in human blood stability studies and cellular
permeability studies.46
In separate studies,37 we determined the major solution
conformation of cyclic prodrug 1 using NMR, CD, and
molecular dynamic simulations that indicate that the
cyclic prodrug 1 exhibits one major conformer in solution
and has a well-defined secondary structure. The solution
conformation of cyclic prodrug 1 appears to be compact
due to intramolecular hydrogen bonding and the presence
of a â-turn. The increased ability of cyclic prodrug 1 to
permeate membranes could be due to reductions in the
average hydrodynamic radius of the molecule, thus
increasing paracellular flux, and/or to an increase in
passive diffusion via the transcellular route because of a
reduction in the hydrogen-bonding potential of the cyclic
prodrug.
Biologica l Resu lts
To be effective, a prodrug should degrade chemically
and/or enzymatically to the parent drug in vivo. Cyclic
(46) Gangwar, S.; Pauletti, G. M.; Siahaan, T. J .; Stella, V. J .;
Borchardt, R. T. 210th Annual Meeting of the American Chemical
Society, Chicago, Aug 20-24, 1995.
(47) Hidalgo, I. J .; Raub, T. J .; Borchardt, R. T. Gastroenterology
1989, 96, 736.

1360 J . Org. Chem., Vol. 62, No. 5, 1997
Gangwar et al.
methyl carbonate (4) (6.9 g, 30 mmol) in acetone (50 mL), and
the reaction mixture was stirred at 50 °C for 24 h. After being
stirred overnight, the solvent was evaporated and the residue
was redissolved in Et2O (100 mL). The Et2O layer was
successively washed with 10% aqueous Na2SO3 (2 × 20 mL),
H2O (2 × 50 mL), and saturated aqueous NaCl (20 mL) and
dried over anhydrous Na2SO4. The solvent was removed under
reduced pressure to afford 1-iodomethyl p-nitrophenyl carbon-
ate (5) (9.1 g, 94%) as a pure yellow oil. The purity of
compound 5 was determined by 13C-NMR (see the Supporting
Information). 1H-NMR (CDCl3, δ): 6.03 (2H, s), 7.39 (2H, d,
J ) 9 Hz), 8.24 (2H, d, J ) 9 Hz). The NMR spectrum of 5 is
identical to that reported in the literature.48
Boc-[[(a la n in yloxy)m et h yl]ca r b on yl]-N-t r yp t op h a n
Ben zyl Ester (7). The cesium salt of Boc-Ala was prepared
by reacting Boc-Ala (2 g, 10 mmol) with Cs2CO3 (1.7 g, 5.2
mmol) in CH3OH (30 mL). After the reaction mixture was
stirred for 1 h, the solvent was removed under reduced
pressure to afford the cesium salt of Boc-Ala as a white powder,
which was used directly in the next reaction. A solution of
the cesium salt of Boc-Ala (0.96 g, 3 mmol) in DMF (20 mL)
was added slowly to an ice-cold stirred solution of iodomethyl
p-nitrophenyl carbonate (5) (1 g, 3 mmol) in DMF (50 mL)
followed by addition of Boc-Ala (2 g, 10 mmol) over a period of
2 h. After the reaction mixture was stirred for 24 h at room
temperature, the solvent was removed under reduced pressure
to afford the oily residue, which was then dissolved in EtOAc
(100 mL) and washed with 10% NaHCO3 (2 × 20 mL), H2O (2
× 50 mL), and saturated aqueous NaCl (20 mL). The organic
layer was dried over anhydrous Na2SO4 and the solid was
decanted out. The EtOAc was evaporated under reduced
pressure to give a mixture of Boc-(alaninyloxy)methyl p-
nitrophenyl carbonate (6) (70% yield) and the side product Boc-
Ala-OpNP (30% yield) as determined by NMR. This mixture
was difficult to separate and was used directly in the next step
without any further purification. 1H of compound 6 (CDCl3,
δ): 1.28-1.30 (12H, br), 4.23 (1H, br), 5.43 (1H, d, J ) 7.4
Hz), 5.81 and 5.74 (2H, dd, J ) 5.5 Hz), 7.27 (2H, d, J ) 9
Hz), 8.08 (2H, d, J ) 7.3 Hz).
NMM (0.12 mL, 1.05 mmol) was added to a stirred solution
of a mixture of Boc-(alaninyloxy)methyl p-nitrophenyl carbon-
ate (6) (0.4 g, 1.04 mmol) Boc-Ala-OpNP (0.096 g, 0.31 mmol),
TrpOBzl‚HCl (0.35 g, 1.05 mmol), and HOBT (0.142 g, 1.05
mmol) in HMPA (50 mL). After being stirred for 24 h at room
temperature, the reaction mixture was diluted with CH2Cl2
(200 mL) and washed with cold 10% NaOH (2 × 50 mL) and
H2O (2 × 100 mL). The CH2Cl2 layer was separated, dried
over anhydrous Na2SO4, and concentrated under reduced
pressure to yield a mixture (pale yellow oil) of Boc-[[(alanin-
yloxy)methyl]carbonyl]-N-tryptophan benzyl ester (7) (75%
yield) and the side product Boc-Ala-Trp-OBzl (25% yield). This
mixture was used in the next step because of the difficulty of
separating the components. 1H-NMR of compound 7 (CDCl3,
δ): 1.35-1.43 (3H, br), 1.44 (9H, s), 3.30 (2H, d, J ) 5.4 Hz),
4.70 (1H, br), 4.89-4.98 (1H, br), 5.04 (2H, s), 5.69 (2H, br),
6.82 (2H, br),7.07-7.31 (8H, br), 7.50 (1H, d, J ) 7.4 Hz); MS
(FAB) m/ z 539 (M+ + 1).
Con clu sion
In conclusion, we have described a methodology for
preparation of an (acyloxy)alkoxy-linked cyclic prodrug
of a model hexapeptide via the N- and C-terminal ends.
It should be feasible to use this methodology to cyclize
other biologically active peptides by linking the C-
terminal carboxyl group to a side-chain amino (e.g., Lys,
Arg) or hydroxyl (e.g., Ser, Thr, Tyr) group or by linking
a side-chain carboxyl group (e.g., Asp, Glu) to a side-chain
amino (e.g., Lys, Arg) or hydroxyl (e.g., Ser, Thr, Tyr)
group. The cyclic prodrug prepared in this study was
shown to degrade chemically to the linear hexapeptide.
Human blood increased the rate of hydrolysis of the cyclic
prodrug to the hexapeptide, suggesting the presence of
an esterase in blood capable of hydrolyzing the [(acyloxy)-
alkoxy]carbamate linkage. In separate studies,36 we have
demonstrated that this cyclic prodrug showed improved
peptidase stability and a greater ability to permeate cell
membranes as compared to the parent peptide. The
application of this methodology to biologically active
peptides (e.g., opioid peptides) is currently under inves-
tigation in our laboratory.
Exp er im en ta l Section
Gen er a l Meth od s. 1H NMR spectra were recorded on 500
and 300 MHz instruments. Chemical shifts are expressed in
parts per million (δ) relative to tetramethylsilane (TMS) with
either TMS or residual solvent as an internal reference.
Abbreviations are as follows: s, singlet; d, doublet; t, triplet;
q, quartet; b, broad. High-resolution mass spectra (HRMS)
were obtained using VG Analytical ZAB double-focusing
spectrometer. All other starting materials were purchased
from Aldrich Chemical Co., Sigma Chemical Co., Fluka
Chemicals, or Bachem Bioscience, Inc. and used as received.
HPLC was conducted using a Rainin HPLX pumping system
with a Dynamax UV detector. Data are reported as follows:
column type, eluent, flow rate, and retention time. The desired
product was purified by a preparative reversed-phase HPLC
system using a C-18 column (12 µm, 300 Å, 25 cm × 21.4 mm
i.d., flow rate 5 mL/min) eluting with a gradient of solvent A,
0.1%TFA/H2O:5%ACN, and solvent B, ACN. The gradient
method used for the preparative HPLC was started from 20%
of solvent B and ended with 100% of solvent B over 65 min.
The desired peptide was analyzed by analytical reversed-phase
HPLC using a C-18 column (5 µm, 300 Å, 25 cm × 4.6 mm
i.d., flow rate 1 mL/min) eluting with a gradient of solvent A,
0.1%TFA/H2O:5%ACN, and solvent B, ACN. The total HPLC
analysis run was 18 min. The solvent gradient used for the
analytical HPLC was started with a gradient change of solvent
B from 10% to 50% for over 14 min; then, the composition of
solvent B was increased to 100% in 2 min followed by elution
at 100% solvent B for 2 min. Finally, a gradient change back
to 10% solvent B was done over 2 min.
1-Ch lor om eth yl p-Nitr op h en yl Ca r bon a te (4). A solu-
tion of 1-chloromethyl chloroformate (3.2 mL, 36 mmol) was
added dropwise into an ice-cold reaction mixture of p-nitro-
phenol (5 g, 36 mmol) and N-methylmorpholine (3.6 mL, 36
mmol) in CHCl3 (50 mL). The reaction mixture was stirred
at 0 °C for 1 h and at ambient temperature for 24 h. After
being stirred overnight, the reaction mixture was successively
washed with 10% aqueous citric acid (2 × 20 mL), H2O (2 ×
50 mL), saturated aqueous NaHCO3 (2 × 40 mL), H2O (2 ×
50 mL), and saturated aqueous NaCl (20 mL). The CHCl3
layer was dried over anhydrous Na2SO4, decanted, and evapo-
rated to give pure 1-chloromethyl p-nitrophenyl carbonate (4)
(6.9 g, 84%), which is a light yellow oil. The purity of
compound 4 was determined by 1H- and 13C-NMR (see the
Supporting Information). 1H-NMR (CDCl3, δ): 5.85 (2H, s),
7.42 (2H, d, J ) 8 Hz), 8.30 (2H, d, J ) 9 Hz). The NMR
spectrum of 4 is identical to that reported in the literature.48
1-Iod om eth yl p-Nitr op h en yl Ca r bon a te (5). NaI (5 g,
30 mmol) was added in one portion to a solution of 1-chloro-
Boc-[[(alan in yloxy)m eth yl]car bon yl]-N-tr yptoph an (2).
A mixture of Boc-[[(alaninyloxy)methyl]carbonyl]-N-tryp-
tophan benzyl ester (7) (0.2 g, 0.37 mmol) and Boc-Ala-Trp-
OBzl (0.043 g, 0.09 mmol) was dissolved in absolute EtOH (25
mL), and 10% Pd-C (0.03 g) was added. The reaction mixture
was stirred under an H2 atmosphere using a hydrogen balloon
on the top of the round-bottom flask. After 10 h, the reaction
mixture was filtered and the filtrate was concentrated under
reduced pressure to yield Boc-[[(alaninyloxy)methyl]carbonyl]-
N-tryptophan (2) and Boc-Ala-Trp-OH, both in 96% yield. The
desired product 2 was purified from the side product Boc-Ala-
Trp-OH by a preparative reversed-phase HPLC system using
a C-18 column (12 µm, 300 Å, 25 cm × 21.4 mm i.d., flow rate
5 mL/min). The peptide 2 was analyzed by analytical reversed-
phase HPLC using a C-18 column (5 µm, 300 Å, 25 cm × 4.6
mm i.d., flow rate 1 mL/min) with a retention time of 15.26
(48) J ose, A. European Pat. Appl. 0167451, 1985.

Synthesis of a Cyclic Prodrug of a Hexapeptide
J . Org. Chem., Vol. 62, No. 5, 1997 1361
min. 1H-NMR ((CD3)2CO, δ): 1.30 (3H, d, J ) 6.9 Hz), 1.39
(9H, s), 3.19-3.43 (2H, m), 4.16 (1H, d, J ) 6.2 Hz), 4.53-
4.54 (1H, br), 4.78 (1H, br), 5.62-5.74 (2H, br), 7.03 (1H, t, J
) 6.8 Hz), 7.10 (1H, t, J ) 6.8 Hz), 7.38 (1H, d, J ) 7.5 Hz).7.63
(1H, d, J ) 7.8 Hz); MS (FAB) m/ z 449 (M+ + 1). Anal. Calcd
for C21H27N3O8: C, 56.13; H, 6.01; N, 9.35. Found: C, 56.19;
H, 6.09; N, 9.29. HRMS: calcd for C21H27N3O8 449.1798, found
449.1804.
residue was dissolved in EtOAc (100 mL). The EtOAc solution
was successively washed with 10% aqueous citric acid (2 × 30
mL), H2O (50 mL), saturated NaHCO3 (2 × 40 mL), H2O (50
mL), and saturated aqueous NaCl (50 mL). The EtOAc layer
was dried over anhydrous Na2SO4 and concentrated under
reduced pressure to furnish Boc-Ala-Gly-Gly-OBzl (12) (0.71
g, 90%) as a yellow oil. Compound 4 was determined to be
1
pure by H-NMR (see the Supporting Information). 1H-NMR
(CDCl3, δ): 1.28 (3H, d, J ) 7.2 Hz), 1.31 (9H, s), 3.85-4.15
(5H, m), 5.06 (2H, s), 5.45 (1H, br), 7.25 (5H, br), 7.41 (1H,
br). MS (FAB) m/ z: 394 (M+ + 1). HRMS: calcd for
C19H27N3O6 394.1960, found 394.1978.
Boc-Asp(OBzl)-OTce (8). Boc-Asp(OBzl)-OH (1 g, 3 mmol),
2,2,2-trichloroethanol (0.4 mL, 3 mmol), and DMAP (0.18 g,
1.5 mmol) were dissolved in CH2Cl2 (30 mL) and cooled to 0
°C. To this cooled solution was added EDC (0.57 g, 3 mmol),
and the reaction mixture was stirred at 0 °C for 3 h and at
ambient temperature for 21 h. The precipitate was filtered
out, and the filtrate was diluted with EtOAc (100 mL). The
EtOAc layer was successively washed with saturated NaHCO3
(2 × 20 mL), H2O (2 × 50 mL), and saturated aqueous NaCl
(20 mL). The EtOAc layer was dried over anhydrous Na2SO4
and concentrated under reduced pressure to yield Boc-Asp-
(OBzl)-OTce (8) (1.13 g, 83%) as a yellow oil. 1H-NMR (CDCl3,
δ): 1.45 (9H, s), 2.93 and 3.15 (2H, dd, J ) 17, 4.5 Hz), 4.67
and 4.75 (2H, dd, J ) 12.3 Hz), 4.7-4.75 (1H, m), 5.13 (2H,
s), 5.56 (1H, d, J ) 9 Hz), 7.34-7.38 (5H, m); MS (FAB) m/ z
454 (M+ + 1). Anal. Calcd for C18H22NO6Cl3: C, 47.68; H,
4.86; N, 3.09. Found: C, 48.20; H, 4.70; N, 3.20. HMRS: calcd
for C18H22NO6Cl3 454.0591, found: 454.0594.
Boc-Ala -Gly-Gly-OH (13). To a solution of Boc-Ala-Gly-
Gly-OBzl (12) (0.5 g, 1.3 mmol) in 50 mL of absolute EtOH
was added in one portion 10% Pd-C (50 mg). The reaction
mixture was stirred under a H2 atmosphere for 24 h. After
being stirred for 24 h, the reaction mixture was filtered to
remove the Pd-C, and the solvent was removed under reduced
pressure to give Boc-Ala-Gly-Gly-OH (13) (0.37 g, 95%). 1H-
NMR (CDCl3, δ): 1.39 (3H, d, J ) 7.2 Hz), 1.43 (9H, s), 3.99-
4.15 (5H, m), 5.03 (1H, br), 6.91-7.03 (2H, br); MS (FAB) m/ z
325 (M++Na). HRMS: calcd for C12H21N3O6 304.1533, found
304.1509. Anal. Calcd for C12H21N3O6: C, 47.52; H, 6.93; N,
13.86. Found: C, 47.18; H, 6.58; N, 13.48.
Boc-Ala -Gly-Gly-Asp (OBzl)-OTce (14). To a cooled (0 °C)
stirred solution of Boc-Ala-Gly-Gly-OH (13) (1.6 g, 5.13 mmol),
TFA‚Asp(OBzl)-OTce (9) (2.4 g, 5.13 mmol), HOBT (0.69 g, 5.13
mmol), and NMM (0.5 mL, 5.13 mmol) in CH2Cl2 (100 mL)
was added in one portion EDC (0.99 g, 5.16 mmol). The
reaction mixture was stirred at 0 °C for 4 h and at ambient
temperature for 24 h. The reaction mixture was diluted with
CH2Cl2 (250 mL) and washed with 10% aqueous citric acid (2
× 50 mL), H2O (100 mL), saturated NaHCO3 (2 × 50 mL),
H2O (100 mL), and saturated aqueous NaCl (50 mL). The
organic layer was dried over anhydrous Na2SO4 and concen-
trated under reduced pressure to furnish Boc-Ala-Gly-Gly-Asp-
(OBzl)-OTce (14) (2.8 g, 86%) as a pale yellow oil. 1H-NMR
(DMSO-d6, δ): 1.17 (3H, d, J ) 7.2 Hz), 1.37 (9H, s), 2.85 and
2.97 (2H, dd, J ) 17 Hz and 6.2 Hz), 3.71-3.79 (3H, br), 3.98
(2H, br), 4.86 (2H, d, J ) 4.3 Hz), 5.12 (2H, s), 7.01 (1H, d, J
) 6.5 Hz), 7.33-7.37 (5H, br), 8.07 (2H, d, J ) 6 Hz), 8.54
(1H, d, J ) 7.8 Hz). MS (FAB) m/ z: 645 (M+ + Li), 661 (M+
+ Na). Anal. Calcd for C25H32N5O7Cl3: C, 48.47; H, 5.17; N,
11.31. Found: C, 48.45; H, 5.09; N, 11.43.
TF A‚H-Asp (OBzl)-OTce (9). TFA (5 mL) was added to a
stirred solution of Boc-Asp(OBzl)-OTce (8) (1 g, 2.2 mmol) in
CH2Cl2 (5 mL). The reaction mixture was stirred at room
temperature for 45 min. Volatile compounds in the reaction
mixture were removed by rotary evaporation under vacuum.
The residue was triturated and washed with anhydrous Et2O,
and the solid was isolated by decantation. The solid TFA‚H-
Asp(OBzl)-OTce (9) (0.78 g, 100%) was dried under vacuum
to remove the residual Et2O and was used in the next step
without further purification. MS (FAB) m/ z 354 (M+ + 1).
Boc-Ala -Gly-OBzl (10). EDC (1 g, 5.3 mmol) was added
to a cold stirred solution (0 °C) of Boc-alanine (1 g, 5.3 mmol),
HCl‚Gly-OBzl (1.07 g, 5.3 mmol), and 0.71 g of HOBT (0.71 g,
5.3 mmol) in 50 mL of CH2Cl2. To this solution was added
NMM (0.5 mL, 5.3 mmol), and the reaction mixture was stirred
at 0 °C for 4 h and at ambient temperature for 24 h. The
reaction mixture was concentrated under reduced pressure,
and the residue was dissolved in EtOAc (100 mL). The EtOAc
layer was successively washed with 10% aqueous citric acid
(2 × 20 mL), H2O (40 mL), saturated NaHCO3 (2 × 20 mL),
H2O (40 mL), and saturated aqueous NaCl (25 mL). The
EtOAc layer was dried over anhydrous Na2SO4 and concen-
trated in vacuo. The residue was recrystallized from EtOAC/
petroleum ether to give a colorless solid of Boc-Ala-Gly-OBzl
TF A‚H-Ala -Gly-Gly-Asp (OBzl)-OTce (3). Boc-Ala-Gly-
Gly-Asp(OBzl)-OTce (14) (0.29 g, 0.44 mmol) was dissolved in
CH2Cl2 (5 mL), and the solution was cooled to 0 °C. To this
clear solution was added TFA (5 mL), and the reaction mixture
was stirred at room temperature for 1 h. Volatile compounds
in the reaction mixture were removed by rotary evaporation
under vacuum. The residue was triturated and washed with
anhydrous Et2O, and the solid was isolated by decantation.
TFA‚H-Ala-Gly-Gly-Asp(OBzl)-OTce (3) (0.27 g, 95%) was
dried under vacuum to remove the residual Et2O and was used
in the next step without further purification. The purity of
1
(10) (1.48 g, 83%), which was determined by H-NMR to have
high purity. 1H-NMR (CDCl3, δ): 1.35 (3H, d, J ) 6 Hz), 1.42
(9H, s), 4.05 (2H, d, J ) 5 Hz), 4.25 (1H, m), 5.15 (2H, s), 5.28
(1H, d, J ) 5 Hz), 7.02 (1H, br), 7.33 (5H, s). MS (FAB) m/ z:
337 (M+ + 1). HRMS: calcd for C17H24N2O5 337.1774, found
337.1763.
1
this compound was determined by H-NMR (see the Support-
Boc-Ala -Gly-OH (11). To a solution of Boc-Ala-Gly-OBzl
(10) (1g, 3 mmol) in absolute EtOH (50 mL) was added 10%
Pd-C (100 mg) in one portion. The reaction mixture was
stirred for 24 h under a H2 atmosphere using a balloon on the
top of the round-bottom flask. After being stirred for 24 h,
the reaction mixture was filtered to remove the Pd-C, and
the solvent was removed under reduced pressure to give Boc-
Ala-Gly-OH (11) (0.74 g, 95%). The purity of compound 11
was determined by 1H-NMR (see the Supporting Information).
1H-NMR (CDCl3, δ): 1.33 (3H, d, J ) 7 Hz), 1.41 (9H, s), 3.97
(2H, d, J ) 5.6 Hz), 4.2 (1H, m), 6.18 (1H, br), 7.50 (1H, br).
MS (FAB) m/ z: 247 (M+ + 1). HRMS: calcd for C10H18N2O5
247.1277, found 247.1294.
Boc-Ala -Gly-Gly-OBzl (12). Boc-Ala-Gly-OH (11) (0.5 g,
2 mmol), HCl‚Gly-OBzl (0.4 g, 2 mmol), and HOBT (0.27 g, 2
mmol) were dissolved in CH2Cl2 (40 mL) and cooled to 0 °C.
To this solution were added EDC (0.39 g, 2 mmol) and NMM
(0.2 mL, 2 mmol), and the reaction mixture was stirred for 2
h at 0 °C and at ambient temperature for 24 h. The reaction
mixture was concentrated under reduced pressure, and the
ing Information). 1H-NMR (DMSO-d6, δ): 1.36 (3H, d, J )
7.2 Hz), 2.85 and 2.98 (2H, dd, J ) 17.0, 6.0 Hz), 3.70-4.01
(6H, m), 4.86 (2H, d, J ) 4.1 Hz), 5.12 (2H, s), 7.36-7.37 (5H,
br), 8.09 (2H, br), 8.24 (1H, br), 8.64 (1H, d, J ) 7.2 Hz). MS
(FAB) m/ z: 528 (M+ + 1).
B o c -Ala -(O C H 2 O C O )-T r p -Ala -G ly -G ly -As p (O B zl)-
OTce (15). EDC (0.09 g, 0.44 mmol) was added to a cooled (0
°C) stirred solution of Boc-[[(alaninyloxy)methyl]carbonyl]-N-
tryptophan (2) (0.2 g, 0.44 mmol), Ala-Gly-Gly-Asp(OBzl)-OTce
(3) (0.29 g, 0.44 mmol), HOBT (0.6 g, 0.44 mmol), and NMM
(0.09 mL, 0.89 mmol) in CH2Cl2 (50 mL). The mixture was
stirred for 2 h at 0 °C and for 30 h at ambient temperature.
The reaction mixture was diluted with CH2Cl2 (250 mL) and
washed with 10% aqueous citric acid (2 × 50 mL), H2O (100
mL), saturated NaHCO3 (2 × 50 mL), H2O (100 mL), and
saturated aqueous NaCl (50 mL). The organic layer was dried
over anhydrous Na2SO4 and concentrated under reduced
pressure to furnish an oil of Boc-Ala-(OCH2OCO)-Trp-Ala-Gly-
Gly-Asp(OBzl)-OTce (15) (0.3 g, 70%). 1H-NMR (DMSO-d6,
δ): 1.15 (3H, d, J ) 7.7 Hz), 1.23 (3H, d, J ) 6.8 Hz), 1.35

1362 J . Org. Chem., Vol. 62, No. 5, 1997
Gangwar et al.
(9H, s), 2.80-3.2 (4H, m), 4.85 (2H, s), 5.11 (2H, s), 5.48-5.63
(2H, m), 6.93-6.99 (1H, m), 7.02-7.07 (1H, m), 7.14 (1H, s),
7.29-7.40 (7H, m), 7.66 (1H, d, J ) 7.5 Hz), 7.79 (1H, m),
8.02-8.10 (2H, m), 8.28 (1H, d, J ) 6.6 Hz), 8.56 (1H, d, J )
7.5 Hz), 10.78 (1H, s). MS (FAB) m/ z: 994 (M+ + Na).
HRMS: calcd for C41H51N7O14Cl3 970.2560, found 970.2539.
TF A‚H -Ala -(OCH 2OCO)-Tr p -Ala -Gly-Gly-Asp (OBzl)-
OH (16). To a stirred solution of Boc-Ala-(OCH2OCO)-Trp-
Ala-Gly-Gly-Asp(OBzl)-OTce (15) (0.2 g, 0.2 mmol) in AcOH
(50 mL) was added Zn dust (1 g) over 1 h. After the reaction
mixture was stirred for 24 h at room temperature, the
insoluble material was filtered out and the filtrate was
concentrated under reduced pressure to give an oily residue.
The resulting residue was dissolved in CH2Cl2 (10 mL), and
the solution was cooled to 0 °C. To this clear solution were
added TFA (5 mL), phenol (2 g), and ethanedithiol (0.2 mL).
After the solution was strirred at room temperature for 2 h,
CH2Cl2 was evaporated, and the residue was triturated with
anhydrous Et2O. The solid was washed with anhydrous Et2O,
isolated by decantation, and dried under reduced pressure to
give TFA‚H-Ala-(OCH2OCO)-Trp-Ala-Gly-Gly-Asp(OBzl)-OH
(16) (0.10 g, 60%). The product was further purified by
preparative reversed-phase HPLC using a C-18 column (12
µm, 300 Å, 25 cm × 21.4 mm i.d., flow rate 5 mL/min). Peptide
16 was analyzed by analytical reversed-phase HPLC using a
C-18 column (5 µm, 300 Å, 25 cm × 4.6 mm i.d., flow rate 1
mL/min); the retention time of linear peptide 16 is 13.03 min.
1H-NMR (DMSO-d6, δ): 1.24 (3H, d, J ) 7.2 Hz), 1.29 (3H, d,
J ) 7.2 Hz), 2.70-2.95 (2H, m), 3.15-3.19 (2H, m), 3.71-3.82
(4H, m), 4.35 (1H, m), 4.62 (1H, m), 5.10 (2H, s), 5.68 (2H, m),
6.95-7.20 (3H, br), 7.36 (5H, br), 7.68 (1H, br), 7.85 (1H, br),
8.12 (2H, br), 8.31 (4H, br). MS (FAB) m/ z 740 (M+ + 1).
HRMS: calcd for C34H42N7O12 740.2891, found 740.2912.
Cyclic Hexa p ep tid e P r od r u g 1. A solution of linear
peptide 16 (0.138 g, 0.186 mmol) and NMM (0.2 mL, 1.98
mmol) in CH2Cl2 (100 mL) was added dropwise to a cooled (0
°C), stirred solution of BOP-Cl (0.236 g, 0.93 mmol) and DMAP
(0.023 g, 0.186 mmol) in 50 mL of CH2Cl2 over 2 h. After the
reaction mixture was stirred for 3 days at room temperature,
the solvent was evaporated under reduced pressure and the
residue was dissolved in CH2Cl2 (100 mL). The organic layer
was successively washed with 10% aqueous citric acid (2 × 20
mL), H2O (50 mL), saturated NaHCO3 (2 × 20 mL), H2O (50
mL), and saturated aqueous NaCl (20 mL). The organic layer
was dried over anhydrous Na2SO4 and concentrated under
reduced pressure to give crude cyclic peptide prodrug 17. The
crude product was purified by preparative reversed-phase
HPLC using a C-18 column (12 µm, 300 Å, 25 cm × 21.4 mm
i.d., flow rate 5 mL/min). After HPLC purification, compound
17 was isolated in 20% yield (0.027 g). Cyclic peptide 17 was
analyzed by analytical reversed-phase HPLC using a C-18
column (5 µm, 300 Å, 25 cm × 4.6 mm i.d., flow rate 1 mL/
min). The retention time of cyclic peptide 17 is 14.3 min. MS
(FAB) m/ z: 722 (M+ + 1).
atmosphere for 24 h. The reaction mixture was filtered to
remove the Pd-C, and the solvent was then removed under
reduced pressure to give cyclic prodrug 1 (0.023 g, 100%). The
crude product was purified by preparative reversed-phase
HPLC using a C-18 column (12 µm, 300 Å, 25 cm × 21.4 mm
i.d., flow rate 5 mL/min). Cyclic peptide 1 was analyzed by
analytical reversed-phase HPLC using a C-18 column (5 µm,
300 Å, 25 cm × 4.6 mm i.d., flow rate 1 mL/min) with a
retention time of 11.2 min. 1H-NMR (D2O, δ): 0.98 (3H, d, J
) 7.2 Hz), 1.37 (3H, d, J ) 7.2 Hz), 2.65-2.85 (2H, m), 3.10-
3.45 (2H, m), 3.65-4.40 (8H, m), 5.34 (1H, d, J ) 6.3 Hz), 5.95
(1H, d, J ) 6.6 Hz), 7.11 (1H, t, J ) 7.2 Hz) 7.20 (1H, t, J )
7.2 Hz) 7.27 (1H, s), 7.46 (1H, d, J ) 6.3 Hz), 7.53 (1H, d, J )
7.8 Hz). MS (FAB) m/ z: 632 (M+ + 1). HRMS: calcd for
C27H33N7O11 632.2316, found 632.2319.
Blood Hyd r olysis. Cyclic prodrug 1 was incubated with
human blood (Topeka Blood Bank, Topeka, KS) at ∼24 µM
for 4 h in a temperature-controlled (37.0 ( 0.5 °C) shaking
water bath (60 rpm). At various time points, 20 µL samples
were removed and residual enzyme activity quenched by
adding 150 µL of a freshly prepared 6 N guanidinium
hydrochloride solution in Hanks’ balanced salt solution con-
taining 0.01% (v/v) phosphoric acid. A portion (150 µL) of this
mixture, which exhibited a pH-value around pH 3, was
transferred to an Ultrafree-MC 5000 NMWL filter unit (Mil-
lipore, Bedford, MA) and centrifuged at 7500 rpm (5000g) for
60 min (4 °C). Aliquots (50 µL) of the filtrate were diluted
with mobile phase and analyzed by HPLC. Recovery for the
prodrug was g97%. Chromatographic analyses were carried
out on a Shimadzu LC-10A gradient system (Shimadzu, Inc.,
Tokyo, J apan) consisting of LC-10AD pumps, a SCP-6 control-
ler, and a RF-535 fluorescence detector connected to a LCI-
100 integrator (Perkin-Elmer, Norwalk, CT). Samples were
injected on a Dynamax C-18 reversed-phase column (5 µm, 300
Å, 25 cm × 4.6 mm i.d., Rainin Instruments, Woburn, MA)
equipped with a guard column. The fluorescence of the eluent
was monitored at emission λ ) 345 nm (excitation λ ) 285
nm). Gradient elution was performed at a flow rate of 1 mL/
min from 0.1-74.0% (v/v) acetonitrile in water using trifluo-
roacetic acid (0.1%, v/v) as the ion-pairing agent. Under these
conditions, the retention times of the linear model hexapeptide
and the cyclic prodrug 1 were 7.7 and 11.8 min, respectively.
Ack n ow led gm en t. Financial support was provided
by Glaxo Inc., the United States Public Health Service
(DA09315) and the Swiss National Science Foundation.
We would also like to thank Dr. Todd Williams, Mr.
Robert Drake and Ms. Homigol Biesada at the Mass
Spectroscopy Laboratory for their technical support.
Su p p or tin g In for m a tion Ava ila ble: Copies of NMR
spectra (16 pages). This material is contained in libraries on
microfiche, immediately follows this article in the microfilm
version of the journal, and can be ordered from the ACS: see
any current masthead page for ordering information.
To a solution of cyclic hexapeptide 17 (0.027 g, 0.037 mmol)
in absolute EtOH (15 mL) was added in one portion 10% Pd-C
(10 mg). The reaction mixture was stirred under
a
H2
J O961696A