Tripeptide probes for tripeptidyl protease I production via gene transfer

Article abstract of DOI:10.1021/jm020525x

Tripeptides derived from 5-chloroanthraquinone hydrazide and anthraquinone hydrazide have been prepared as potential reagents to probe cellular expression of tripeptidyl protease I (TPP-I). Attempted chemical synthesis of Gly-L-Pro-L-Ala-chloroanthraquino

Full text of DOI:10.1021/jm020525x

J . Med. Chem. 2003, 46, 1603-1608
1603
Tr ip ep tid e P r obes for Tr ip ep tid yl P r otea se I P r od u ction via Gen e Tr a n sfer
MeeKyoung Kim,† Qinwen Mao,‡ Beverly L. Davidson,‡,§,| and David F. Wiemer*,†
Department of Chemistry, University of Iowa, Iowa City, Iowa 52242-1294 and Program in Gene Therapy,
Departments of Internal Medicine, Neurology, Physiology & Biophysics, University of Iowa College of Medicine,
Iowa City, Iowa 52242-1294
Received November 19, 2002
Tripeptides derived from 5-chloroanthraquinone hydrazide and anthraquinone hydrazide have
been prepared as potential reagents to probe cellular expression of tripeptidyl protease I (TPP-
I). Attempted chemical synthesis of Gly-L-Pro-L-Ala-chloroanthraquinone hydrazide, a compound
that had been reported to serve as a substrate for this enzyme, was complicated by formation
of a pyrazoloquinone. In contrast, formation of pyrazoloquinones was not observed during
coupling reactions with anthraquinone hydrazide, and several tripeptide derivatives of this
compound were prepared. The most attractive probe for TPP-I activity in tests with mouse
kidney tissue sections proved to be Gly-L-Pro-L-Ser anthraquinone hydrazide.
In tr od u ction
One strategy for histochemical analysis of TPP-I
activity in tissue sections was recently reported by
Dikov et al.7 These investigators reported preparation
of tripeptides linked to various aromatic moieties through
hydrazide linkages. For example, 5-chloroanthraquin-
one hydrazide (1) reportedly was converted to the
alanine derivative 2 through DCC-mediated coupling
with tBOC-L-alanine, and the product was converted to
the tripeptide derivative 3 through a sequence of
coupling and deprotection steps (Scheme 1). This tri-
peptide also was reported to serve as a TPP-I substrate,
presumably yielding an intermediate hydrazine that
was converted to a solid, colored hydrazone in tissue
sections through reaction with an aromatic aldehyde.
To assess the utility of new CLN2 gene transfer vectors,
we investigated both similar and novel strategies for
detection of TPP-I activity. In this paper, we report the
preparation and characterization of several tripeptidyl
anthraquinone derivatives and histochemical analyses
of these compounds in mouse tissue sections known to
harbor high levels of endogenous enzyme activity.
Lesions in the CNL2 gene result in neuronal ceroid
lipofuscinoses (NCL’s), a group of rare but devastating
neurodegenerative diseases.1 One type of NCL, known
variously as classical late-infantile neuronal ceroid
lipofuscinosis (LINCL), J ansky-Bielschowsky disease,
CLN2 deficiency, or pepinase deficiency, presents as an
acute seizure disorder at 4-6 years of age and includes
rapid loss of visual, motor, and cognitive functions.2
Recent studies have shown that this disease is charac-
terized by a deficiency in tripeptidyl protease I (TPP-
I), a soluble lysosomal protease,3 and that inactivating
mutations result in accumulation of storage material
in the lysosome.4 Despite the increased understanding
of the molecular basis of this disease, current therapy
for LINCL can only relieve the seizure disorder and
cannot address the underlying cause or the progression
of the disease.1,3 TPP-I is a nonmembrane bound lyso-
somal enzyme that can be secreted from cells overex-
pressing the enzyme and taken up by deficient cells.5
This characteristic, together with the fact that only
partial enzyme replacement will likely ameliorate the
storage deficit, makes TPP-I an attractive candidate for
the development of gene-based therapies.
A major limitation in the development of gene-based
therapies for neurodegenerative diseases, and more
specifically TPP-I deficiency, is an assay to assess the
distribution of the secreted enzyme product. At present,
CLN2 activity in the CNS is monitored by a fluorometric
assay using Ala-Ala-Phe 7-amido-4-methlycoumarin in
tissue lysates.6 This assay allows evaluation of overall
levels of enzyme in dissected regions of the brain but is
somewhat insensitive in that it does not yield specific
information on enzyme distribution within sections of
brain.
Ch em ica l Syn th esis. 5-Chloroanthraquinone hy-
drazide (1) was readily prepared from commercial 1,5-
dichloroanthraquinone (4) through reaction with hy-
drazine as previously described (Scheme 2).8 Coupling
of this hydrazine with tBOC-L-Ala was first conducted
through reaction with DCC, apparently as described by
Dikov et al.,7 but in our hands this procedure gave a
coupled product only in low yield. The same material
was obtained in much better yield through a coupling
mediated with the DCC substitute 1-(3-dimethylami-
nopropyl)-3-ethylcarbodiimide hydrochloride (EDC‚
HCl).9 The product initially was assumed to be the
amide 2 as previously described.7 However, careful
analysis of the NMR data for this series of compounds
did not support assignment of the structures previously
proposed for the coupled products. Of special signifi-
cance is the 13C NMR data. The symmetrical an-
thraquinone 4 shows a single resonance at 180.9 ppm
that can be assigned to the quinone carbonyl carbons.
In the hydrazine derivative 1 this symmetry is lost, and
two quinone resonances are observed at 183.8 and 182.5
* To whom correspondence should be addressed. E-mail: david-
wiemer@uiwoa.edu.
† Department of Chemistry, University of Iowa.
‡ Department of Internal Medicine, University of Iowa College of
Medicine.
§ Department of Neurology, University of Iowa College of Medicine.
| Department of Physiology & Biophysics, University of Iowa College
of Medicine.
10.1021/jm020525x CCC: $25.00 © 2003 American Chemical Society
Published on Web 03/29/2003

1604 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9
Kim et al.
Sch em e 1
Sch em e 3a
Sch em e 2a
a
Reagents and conditions: (a) 6 N HCl/dioxane; (b) N-tBOC-
Gly-L-Pro, EDC‚HCl, HOBt, NMM, THF; (c) 30% HBr/AcOH.
Sch em e 4a
a
Reagents and conditions: (a) NH2NH2‚H2O, Pyr, reflux; (b)
N-tBOC-L-Ala, EDC‚HCl, HOBt, NMM, THF; (c) NH2NH2‚H2O,
iPr2NEt, THF, reflux.
ppm. However, in the product of the DCC and EDC‚
HCl coupling reactions with tBOC-L-Ala, only one
carbonyl resonance is apparent (181.1 ppm) and, be-
cause the expected total of 16 aromatic or carbonyl
resonances is observed, it is not possible that an
accidental overlap of two resonances has occurred.
Instead, it appears probable that cyclization to a pyra-
zole derivative occurred under these conditions, as
previously observed with alkyl-substituted hydrazines
derived from related anthraquinones.10 Further coupling
with tBOC-L-Ala might afford an N-acylated derivative
such as compound 5 or its regioisomeric N-acylated
product 6. To explore the possibility of pyrazole forma-
tion, quinone 4 was treated with hydrazine for an
extended period, and the pyrazole 710 was obtained. As
might be expected,11 only a single carbonyl resonance
was observed in the 13C spectrum of this product.
Because the previous report7 contained no spectral
data and only a very brief summary of the reactions
employed, it is not possible to establish whether the
reported biological evaluations were based on a tripep-
tide derived from a quinone or a pyrazole. In this study,
the pyrazole derivative 5 (or 6) was carried on to the
corresponding tripeptide (Scheme 3). After hydrolysis
of the tBOC group gave the free amine 8, an EDC‚HCl-
mediated coupling with tBOC-Gly-L-Pro afforded the
protected tripeptide 9 and final cleavage of the tBOC
group gave the tripeptide 10.
a
Reagents and conditions: (a) NH2NH2‚H2O, pyr, reflux; (b)
N-tBOC-L-Ala, EDC‚HCl, HOBt, NMM, THF; (c) N-tBOC-O-
benzyl-L-Ser, EDC‚HCl, HOBt, NMM, THF; (d) 6 N HCl/dioxane;
(e) N-tBOC-Gly-L-Pro, EDC‚HCl, HOBt, NMM, THF; (f) 30% HBr/
AcOH.
product, as evidenced by observation of two quinone
carbons at ∼180 ppm. Hydrolysis of the tBOC group
gave the desired amine 14 in good yield. Standard EDC‚
HCl coupling with tBOC-Gly-L-Pro and subsequent
reaction with HBr to cleave the tBOC group gave the
desired tripeptide 16. A third tripeptide was prepared
through use of tBOC-O-Bz-L-Ser in the first coupling
reaction to obtain the protected amino acid hydrazide
17. A parallel reaction sequence involving hydrolysis of
the tBOC group to afford the amino acid 18, coupling
with tBOC-Gly-L-Pro to obtain the protected tripeptide
19, and final deprotection gave the tripeptide 20.
A recent report indicated that L-Ala-L-Ala-L-Phe
derivatives were particularly good substrates for TPP-
I.12 Therefore the hydrazine 12 was treated with tBOC-
L-Phe to obtain the coupled product 21 (Scheme 5). After
removal of the tBOC group gave the free amine 22,
coupling with tBOC-L-Ala gave the protected dipeptide
23. In this case, deprotection was accomplished in good
yield by reaction with HBr, but partial racemization
occurred to give a 3:1 mixture of two diastereomers. The
major diastereomer (24) was allowed to react with
tBOC-L-Ala to obtain the protected tripeptide 26, and
final deprotection gave the desired target compound 27.
Additional tripeptides for these studies were derived
from commercial 1-chloroanthraquinone 11 (Scheme 4).
Treatment of compound 11 with hydrazine gave the
expected hydrazine derivative 12.8 Treatment of com-
pound 12 with EDC‚HCl and tBOC-L-Ala clearly gave
the protected peptide 13 rather than a pyrazole type

Tripeptide Probes for Tripeptidyl Protease I Production
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9 1605
Sch em e 5a
and 27, the aldehydes p-NBA and p-AA were more
effective than benzaldehyde itself as regards precipitate
density and color. The combination of tripeptide 20 and
p-AA was most sensitive, resulting in purple precipi-
tates in areas of high endogenous enzyme activity (e.g.,
kidney; Figure 1). Together the data support that
compounds 10, 20, and 27 were specific for TPP-I, with
low to no background (sections incubated with tripep-
tides 16 and 31). This combination of reagents was used
to assess CLN2 expression in murine brain following
viral-mediated gene transfer. Recombinant protein was
readily detectable in areas of low endogenous activity,
demonstrating the utility of histochemical evaluation
of CNS therapies for TPP-I deficiency (data not shown).
Con clu sion s
Five tripeptide derivatives of anthraquinone have
been prepared and tested as histochemical reagents for
detection of TPP-I activity. One, derived from 5-chloro-
anthraquinone hydrazide, was identified as a tetracyclic
pyrazoanthrane based on analysis of 13C NMR data,
while the other four were tripeptide derivatives of the
tricyclic anthraquinone hydrazide. Of these compounds,
the tripeptide Gly-L-Pro-L-Ser anthraquinone hydrazide
was the most effective histochemical reagent, particu-
larly when combined with p-anisaldehyde.
a
Reagents and conditions: (a) N-tBOC-L-Phe, EDC‚HCl, HOBt,
NMM, THF; (b) N-tBOC-D-Phe, EDC‚HCl, HOBt, NMM, THF; (c)
6 N HCl/dioxane; (d) N-tBOC-L-Ala, EDC‚HCl, HOBt, NMM, THF;
(e) 30% HBr/AcOH (f) N-tBOC-L-Ala-L-Ala, EDC‚HCl, HOBt,
NMM, THF.
Exp er im en ta l Section
THF and diethyl ether were freshly distilled from sodium/
benzophenone, while pyridine was freshly distilled from
calcium hydride. All reactions in nonaqueous solvents were
conducted in oven-dried or flame-dried glassware, under a
positive pressure of argon, with magnetic stirring. Flash
column chromatography was carried out on ICN silica gel with
40 µm average particle diameter. NMR spectra were recorded
on either a Brucker AC-300 or a DPX-300 instrument (1H at
300 MHz and 13C at 75 MHz) or on a Brucker DPX-400 MHz
instrument (1H at 400 MHz and 13C at 100 MHz). Spectra were
obtained in CDCl3 solution unless otherwise noted, with (CH3)4-
Si (1H, 0.0 ppm) or CDCl3 (13C, 77.0 ppm) as internal
standards. Low resolution electron impact (EI) mass spectra
Ta ble 1. Intensity and Color of the Final Reaction Product of
TPP-I Histochemical Staining with Tripeptides
tripeptide p-anisaldehyde p-nitrobenzaldehyde
benzaldehyde
10
+++
yellow-green
autofluorescent
-
+++
yellow-green
autofluorescent
-
++
yellow-green
autofluorescent
16
20
-
+++
+++
+
brown
++
dark purple
+++
yellow-green
-
brown
+++
yellow-green
-
27
31
yellow-green
-
were recorded with
a Hewlett-Packard 6890 instrument
operating at 70 eV; only selected ions are reported here. High-
resolution mass spectra were obtained at the University of
Iowa Mass Spectrometry Facility. Elemental analyses were
performed by Atlantic Microlab Inc. (Norcross, GA). All
reagents that are not otherwise described were obtained from
commercial sources and used without further purification.
Gen er a l P r oced u r e for P r ep a r a tion of Hyd r a zin oa n -
th r a qu in on es 1 a n d 12. To a stirred solution of the an-
thraquinone (4 or 11, 19.8 mmol) in pyridine (90 mL) was
added hydrazine monohydrate (59.9 mmol) dropwise, and the
reaction mixture was heated at reflux for 1-3 h. After the
reaction mixture had cooled to room temperature, it was
concentrated in vacuo. To obtain compound 12, this residue
was purified by column chromatography (CHCl3). Compound
1 was purified by crystallization from xylene followed by
column chromatography (CHCl3).
Gen er a l P r oced u r e for P ep tid e Bon d F or m a tion w ith
EDC‚HCl. A solution of 1-hydroxybenzotriazole (365 mg, 2.7
mmol), an N-tBOC-protected amino acid (2.7 mmol), and EDC‚
HCl (537 mg, 2.8 mmol) in THF (11 mL) was cooled in an ice
bath and stirred for 10 min. To this reaction mixture, hydrazi-
noanthraquinone (643 mg, 2.7 mmol) and N-methylmorpholine
(NMM, 273 mg, 2.7 mmol) were added, and the resulting
mixture was stirred for 1 h at 0 °C. After removal of the ice
bath, the reaction mixture was allowed to stand at room
temperature for 14-19 h, then washed with water and
extracted with chloroform. After the combined organic layers
were dried (MgSO4 or Na2SO4), concentration in vacuo and
Instead of conducting a parallel sequence with the minor
diastereomer 25, tBOC-D-Phe was coupled with hydra-
zine 12 to obtain the R isomer 28. After deprotection to
the free amine 29, EDC‚HCl-mediated coupling with
tBOC-L-Ala-L-Ala gave the protected tripeptide 30, and
final cleavage of the tBOC group gave the tripeptide
derivative 31.
Biologica l Eva lu a tion . Five different substrates,
compounds 10, 16, 20, 27, and 31 were tested his-
tochemically on frozen sections of murine tissues (see
the Experimental Section for the preparation method).
The sections were incubated in substrate solutions
containing 1 mM of the substrate and 1 mg/mL of
benzaldehyde (BA), p-anisaldehyde (p-AA), or p-ni-
trobenzaldehyde (p-NBA) in 0.1 M acetate buffer (pH
4.5) at 37 °C for 4 h. The substrates were systematically
compared for sensitivity to TPP-I activity using condi-
tions to control the rate of crystalline formation and
diffusion of product, and the results are summarized in
Table 1. Among the five substrates, compounds 10, 20,
and 27 were readily hydrolyzed by endogenous TPP-I
in murine tissues, revealing lysosomal accumulation of
precipitates. There was no evidence of hydrolysis of
tripeptides 16 and 31 by TPP-I. For compounds 10, 20,

1606 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9
Kim et al.
F igu r e 1. Murine kidney sections treated with a tripeptide conjugate and an aldehyde.
purification of the residue by flash column chromatography
(CHCl3 or CHCl3:EtOAc:MeOH 47:50:3 ) gave the desired
product.
1H), 7.87 (dd, J ) 7 Hz, 86, 7.75, 1H), 7.67 (t, J ) 8.11 Hz,
1H), 7.53 (d, J ) 6.44 Hz, 1H), 7.36 (d, J ) 8.53 Hz, 1H), 7.20
(d, J ) 6.61 Hz, 1H, exchanges with D2O), 4.12-4.02 (m, 1H),
1.42 (s, 9H), 1.29 (d, J ) 7.23 Hz, 3H); 13C NMR (DMSO) δ
183.2, 181.4, 172.6, 155.4, 151.4, 136.9, 136.9, 136.7, 135.7,
135.0, 134.5, 133.1, 128.7, 126.4, 118.1, 117.4, 112.5, 79.2, 48.8,
28.2 (3C), 17.4; HRMS (FAB) calcd for C22H23N3O5Cl (M + H)+
444.1326, found 444.1315.
Gen er a l P r oced u r e for Rem ova l of N-tBOC Gr ou p s.13
a . Hyd r och lor ic Acid in Dioxa n e. The N-tBOC-amino-
anthraquinonylhydrazide (1.2 mmol) was placed into a round-
bottom flask and 6 N HCl in dioxane (5 mL) was added. The
suspension was stirred for 1 h at room temperature. After
removal of solvent by evaporation, the residue was crystallized
from ethanol/ether. b. Hyd r obr om ic Acid in Acetic Acid .
The N-tBOC-tripeptidyl-anthraquinonylhydrazide (1.18 mmol)
was dissolved in a 30% solution of HBr in acetic acid (3 mL)
and stirred for 1 h at room temperature. After the reaction
mixture was diluted with ether, the precipitate was collected
by filtration, washed with ether, and finally purified by column
chromatography (CHCl3:EtOAc:MeOH, 45:50:5) or crystalliza-
tion from ethanol/ether.
N-t BOC-Ala -5-ch lor o-1-a n t h r a [1,9-cd ]p yr ozol-6(2H )-
on e (5 or isom er 6). A solution of 1-hydroxybenzotriazole (50
mg, 0.37 mmol), N-tBOC-L-Ala (69 mg, 0.37 mmol), and EDC‚
HCl (74 mg, 0.37 mmol) in THF (1.7 mL) was cooled in an ice
bath and stirred for 10 min. To this reaction mixture were
added compound 1 (100 mg, 0.37 mmol) and N-methylmor-
pholine (0.04 mL, 0.37 mmol), and the resulting mixture was
stirred for 1 h at 0 °C. After removal of the ice bath, the
reaction mixture was allowed to stand at room temperature
for 14 h, then washed with water and extracted with chloro-
form. After the combined organic layers were dried (MgSO4
or Na2SO4), concentration in vacuo and purification of the
residue by flash column chromatography (CHCl3) gave com-
pound 5/6 as a dark red solid (101 mg, 65%): 1H δ 8.39 (d, J
) 8.1 Hz, 1H), 8.24 (dd, J ) 6.6, 2.2 Hz, 1H), 8.04 (d, J ) 7.3
Hz, 1H), 7.77 (t, J ) 7.8 Hz, 1H), 7.64-7.57 (m, 2H), 5.76-
5.66 (m, 1H), 5.44 (d, J ) 8.1 Hz, 1H), 1.65 (d, J ) 6.9 Hz,
3H), 1.49 (s, 9H); 13C NMR δ 181.1, 173.6, 155.1, 144.1, 137.9,
137.3, 135.0, 133.2, 132.2, 131.6, 129.6, 126.4, 124.6, 123.6,
123.1, 120.7, 80.1, 49.2, 28.2 (3C), 19.2; HRMS (FAB) calcd
for C22H21N3O4Cl (M + H)+ 426.1221, found 426.1226.
7-Ch lor op yr a zoloa n th r on e (7). To a stirred suspension
of 1,5-dichloroanthraquinone (343 mg, 1.2 mmol) and iPr2NEt
(240 mg, 1.9 mmol) in THF (5.5 mL) was added hydrazine
monohydrate (186 mg, 3.7 mmol) dropwise, and the reaction
mixture was heated at reflux for 2 h. After cooling, the mixture
was washed with water and extracted with chloroform. The
combined organic layers were washed with brine and dried
over MgSO4. Then the residue was separated by column
chromatography (silica gel) with chloroform/EtOAc (1/1) as an
eluent to give 7-chloropyrazoloanthrone (7) as a brown solid
(57 mg, 18%); 1H NMR (DMSO) δ 8.23 (dd, J ) 7.11, 1.86 Hz,
1H), 7.88-7.82 (m, 2H), 7.78-7.62 (m, 2H), 7.34 (dd, J ) 7.25,
5-Ch lor o-1-a n th r a qu in on ylh yd r a zin e (1). Purple solid;
1H NMR δ 8.25 (dd, J ) 7.7, 1.3 Hz, 1H), 7.99 (dd, J ) 8.5, 1.3
Hz, 1H), 7.70-7.55 (m, 4H); 13C NMR δ 183.8, 182.5, 150.1,
148.4, 137.4, 136.9, 135.3, 135.1, 134.9, 133.5, 129.5, 126.5,
119.5, 118.3; EIMS, m/z (relative intensity) 274 ([M + 2]+, 3),
272 (M+, 8), 257 (10), 242 (8), 229 (20), 187 (4), 173 (8), 159
(17), 145 (11), 132 (100), 119 (41), 105 (24), 95 (41), 81 (17), 69
(16), 55 (17).
N-tBOC-Ala -5-ch lor o-1-a n th r a qu in on ylh yd r a zid e (2).
HOBt (28 mg, 0.21 mmol), EDC‚HCl (42 mg, 0.22 mmol), and
N-tBOC-L-Ala (40 mg, 0.21 mmol) were suspended in THF (0.9
mL) and stirred for 15 min at 0 °C. 7-Chloropyrazoloanthrone
(7, 54 mg, 0.2 mmol) and N-methylmorpholine (23 µL) were
added, and the reaction mixture was stirred for 2 h at 0 °C.
After removal of the ice bath, the reaction mixture was allowed
to warm to room temperature and stirred for 14 h. The reaction
mixture was diluted with water and extracted with chloroform.
The combined organic layers were washed with brine and dried
over MgSO4. Final purification by column chromatography
(silica gel, 9:1, chloroform/EtOAc) gave compound 2 (48 mg,
0.11 mmol, 51%) as a dark red solid and compound 5/6 (2.2
mg, 5.2 µmol, 2%) as a dark red solid: 1H (DMSO) δ 10.35 (s,
1H, exchanges with D2O), 10.28 (s, 1H, exchanges with D2O),
8.25 (dd, J ) 7.59, 1.69 Hz, 1H), 7.92 (dd, J ) 8.1, 1.49 Hz,

Tripeptide Probes for Tripeptidyl Protease I Production
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9 1607
3.53 (m, 4H), 2.39-1.95 (m, 4H), 1.54 (d, J ) 7.1 Hz, 3H); 13
NMR (CD3OD) δ 186.7, 184.6, 175.1, 175.1, 174.8, 153.1, 136.6,
135.8, 135.7, 135.6, 135.0, 134.3, 128.1, 127.8, 119.9, 119.3,
115.7, 67.1, 61.9, 60.0, 47.9, 30.9, 25.8, 17.6; HRMS (FAB) calcd
for C24H26N5O5 (M - Br)+ 464.1934, found 464.1950.
0.69 Hz, 1H); 13C NMR (DMSO) δ 181.7, 153.7, 137.1, 136.3,
135.5, 134.7, 134.3, 133.0 (2C), 128.6, 126.2, 118.2, 115.2,
110.0.
C
Ala -5-ch lor o-1-a n th r a [1,9-cd ]p yr ozol-6(2H)-on e (8). Red
1
solid; H NMR (CD3OD) δ 8.24 (d, J ) 8.1 Hz, 1H), 8.04 (dd,
J ) 3.2, 6.0 Hz, 1H), 7.82 (d, J ) 7.4 Hz, 1H), 7.70 (t, J ) 8.0
Hz, 1H), 7.55-7.49 (m, 2H) 5.21 (q, J ) 7.1 Hz, 1H), 1.80 (d,
J ) 7.1 Hz, 3H); 13C NMR (CD3OD) δ 181.9, 170.9, 146.3, 139.1,
138.2, 136.7, 134.9, 133.5, 132.9, 130.7, 127.7, 125.8, 124.8,
124.4, 121.4, 50.7, 17.3; HRMS (FAB) calcd for C17H13N3O2Cl
(M - Cl)+ 326.0696, found 326.0721.
N-t BOC-O-Ben zyl-L-Ser -1-a n t h r a q u in on ylh yd r a zid e
1
(17). Dark red solid; H NMR δ 10.54 (s, 1H, exchanges with
D2O), 8.31-8.21 (m, 3H), 7.78-7.71 (m, 3H), 7.42-7.30 (m,
5H), 7.23 (d, J ) 8.5 Hz, 1H), 5.46 (d, J ) 6.7 Hz, 1H), 4.64 (d,
J ) 11.7 Hz, 1H), 4.59 (d, J ) 11.7 Hz, 1H), 4.51-4.48 (m,
1H), 4.00 (dd, J ) 8.8, 3.6 Hz, 1H), 3.70 (dd, J ) 9.0, 6.4 Hz,
1H), 1.49 (s, 9H); 13C NMR δ 185.3, 182.9, 170.4, 155.5, 151.0,
137.1, 135.1, 134.1, 134.0, 133.8, 133.3, 132.6, 128.5 (2C),
128.0, 127.9 (2C), 126.8, 126.6, 118.4, 118.0, 114.4, 80.7, 73.7,
69.7, 53.4, 28.3 (3C); HRMS (FAB) calcd for C29H30N3O6 (M +
H)+ 516.2135, found 516.2129.
N-t BOC-Gly-L-P r o-L-Ala -5-ch lor o-1-a n t h r a [1,9-cd ]p y-
r ozol-6(2H)-on e (9). Yellow solid; 1H NMR δ 8.42 (d, J ) 8.1
Hz, 1H), 8.28 (dd, J ) 7.1, 1.8 Hz, 1H), 8.08 (d, J ) 7.3 Hz,
1H), 7.80 (t, J ) 7.8 Hz, 1H), 7.67-7.59 (m, 2H), 5.87-5.82
(m, 1H), 5.52 (s, 1H), 4.70 (dd, J ) 6.1, 1.6 Hz, 1H), 4.01 (dd,
J ) 5.0, 5.0 Hz, 2H), 3.64-3.57 (m, 1H), 3.50-3.41 (m, 1H),
2.42-1.91 (m, 4H), 1.67 (d, J ) 7.0 Hz, 3H), 1.47 (s, 9H); 13C
NMR δ 181.3, 172.7, 170.6, 168.7, 155.8, 144.3, 138.0, 137.4,
135.1, 133.2, 132.3, 131.7, 129.8, 126.5, 124.7, 123.8, 123.2,
120.8, 79.8, 60.6, 48.5, 46.4, 43.1, 28.3 (3C), 27.6, 24.9, 18.5;
HRMS (FAB) calcd for C29H31N5O6Cl (M + H)+ 580.1963, found
580.1949.
O-Ben zyl-L-Ser -1-a n th r a qu in on ylh yd r a zid e (18). Red
solid; 1H NMR (CD3OD) δ 8.18 (dd, J ) 8.9, 3.3 Hz, 1H), 8.10
(dd, J ) 7.3, 1.8 Hz, 1H), 7.88-7.67 (m, 3H), 7.60 (dd, J ) 7.5,
1.1 Hz, 1H), 7.42-7.29 (m, 5H), 7.21 (dd, J ) 8.5, 1.3 Hz, 1H),
4.67 (d, J ) 11.9 Hz, 1H), 4.61 (d, J ) 11.9 Hz, 1H), 4.30 (t, J
) 4.4 Hz, 1 H), 4.01-3.93 (m, 2H); 13C NMR (CD3OD) δ 186.8,
184.4, 168.4, 152.5, 138.5, 136.6, 135.8, 135.7, 135.5, 135.0,
134.2, 129.8 (2C), 129.4 (3C), 128.1, 127.8, 119.5, 119.5, 115.9,
74.9, 69.1, 53.8; HRMS (FAB) calcd for C24H22N3O4 (M - Cl)+
416.1610, found 416.1607.
N-t BOC-Gly-L-P r o-O-b en zyl-L-Ser -1-a n t h r a q u in on yl-
h yd r a zid e (19). Dark red solid; 1H NMR δ 10.56 (s, exchanges
with D2O), 8.84 (s, exchanges with D2O), 8.29-8.16 (m, 2H),
7.79-7.70 (m, 4H), 7.47-7.22 (m, 6H), 4.76-4.52 (m, 3H), 4.09
(dd, J ) 9.4, 3.6 Hz, 1H), 4.00-3.90 (m, 2H), 3.72 (dd, J )
10.0, 4.9 Hz, 1H), 3.69-3.61 (m, 1H), 3.53-3.48 (m, 2H), 2.25-
2.01 (m, 4H), 1.43 (s, 9H); 13C NMR δ 186.8, 183.3, 171.1, 169.6,
169.2, 156.8, 151.4, 137.3, 135.4, 134.4, 134.3, 133.9, 133.4,
132.9, 128.6 (2C), 128.1, 127.7 (2C), 127.0, 126.8, 118.5, 118.3,
114.6, 80.1, 73.5, 68.9, 61.2, 52.8, 46.8, 43.2, 28.5, 28.2 (3C),
24.9; HRMS (FAB) calcd for C36H39N5O8Na (M + Na)+ 692.2696,
found 692.2693.
Gly-L-P r o-L-Ala-5-ch lor o-1-an th r a[1,9-cd]pyr ozol-6(2H)-
1
on e (10). Yellow solid; H NMR (CD3OD) δ 8.28 (d, J ) 7.9
Hz, 1H), 8.19 (dd, J ) 5.4, 1.6 Hz, 1H), 7.92 (d, J ) 7.4 Hz,
1H), 7.74 (t, J ) 7.6 Hz, 1H), 7.63-7.59 (m, 2H) 5.63 (q, J )
6.9 Hz, 1H), 4.51 (dd, J ) 8.2, 3.7 Hz, 1H), 3.78 (s, 2H), 3.51-
3.45 (m, 2H), 2.26-1.88 (m, 4H), 1.59 (d, J ) 7.3 Hz, 3H); 13
C
NMR ((CD3)2SO) δ 180.7, 172.7, 171.4, 164.3, 143.3, 137.6,
136.0, 135.1, 134.3, 132.3, 132.0, 129.3, 126.2, 124.1, 123.4,
123.2, 120.5, 59.0, 47.8, 47.7, 45.9, 29.4, 24.1, 16.8; HRMS
(FAB) calcd for C24H23N5O4Cl (M - Cl)+ 480.1439, found
480.14.
1-Hyd r a zin oa n th r a qu in on e (12).9 Dark brown solid; 1H
NMR δ 10.41 (s, 1H, exchanges with D2O), 8.26-8.22 (m, 2H),
7.82-7.67 (m, 3H), 7.63-7.54 (m, 2H); 13C NMR δ 184.9, 183.7,
154.3, 135.2, 134.7, 134.2, 133.9, 133.0, 133.0, 126.8 (2), 118.3
(2), 116.5.
Gly-L-P r o-L-Ser -1-a n th r a qu in on ylh yd r a zid e (20). Dark
1
red solid; H NMR (CD3OD) δ 8.32 (dd, J ) 8.9, 3.3 Hz, 1H),
N-tBOC-Ala -1-a n th r a qu in on ylh yd r a zid e (13). Dark red
1
8.24 (dd, J ) 7.3, 1.8 Hz, 1H), 7.88-7.81 (m, 2H), 7.76-768
(m, 2H), 7.53 (dd, J ) 7.5, 1.1 Hz, 1H), 4.61-4.53 (m, 2H),
solid; H NMR δ 10.48 (s, 1H, exchanges with D2O), 8.77 (s,
1H, exchanges with D2O), 8.12-8.08 (m, 2H), 7.66-7.58 (m,
3H), 7.43 (t, J ) 8.2 Hz, 1H), 7.17 (d, J ) 8.4 Hz, 1H), 5.37 (d,
J ) 7.6 Hz, 1H), 4.22-4.38 (m, 1H), 1.49 (s, 9H), 1.46 (d, J )
7.2 Hz, 3H); 13C NMR δ 185.1, 182.7, 172.7, 155.9, 150.9, 135.0,
134.1, 133.9, 133.7, 133.2, 132.5, 126.7, 126.5, 118.3, 117.6,
114.2, 80.5, 48.6, 28.3 (3C), 17.7; HRMS (FAB) calcd for
3.69-3.54 (m, 4H), 2.37-2.32 (m, 2H, 2.14-2.06 (m, 2H); 13
C
NMR (CD3OD) δ 186.8, 184.6, 167.7, 166.8, 166.4, 153.2, 136.6,
135.8, 135.8, 135.6, 135.0, 134.3, 128.1, 127.8, 120.1, 119.3,
113.4, 62.1, 60.7, 47.5, 41.6, 30.3, 26.1, 20.9; HRMS (FAB) calcd
for C24H27N5O6 (M - Br + H)+ 481.1961, found 481.1961.
N-t BOC-L-P h e-1-a n t h r a q u in on ylh yd r a zid e (21) a n d
N-tBOC-D-P h e-1-a n th r a qu in on ylh yd r a zid e (28). Golden
orange solid; 1H NMR δ 10.47 (s, 1H), 8.27-8.21 (m, 2H), 7.76-
7.70 (m, 3H), 7.45-7.21 (m, 6H), 6.78 (d, J ) 8.25 Hz, 1H),
5.16 (br, 1H), 4.53-4.44 (m, 1H), 3.14 (d, J ) 7.31 Hz, 2H),
1.48 (s, 9H); 13C NMR δ 185.6, 183.2, 171.0, 151.1, 136.2, 135.3,
134.4, 134.3, 134.0, 133.5, 132.9, 129.4 (3C), 129.0 (2C), 127.2,
127.0, 126.9, 118.7, 118.0, 114.6 81.9, 54.6, 37.8, 28.3 (3C); 21,
HRMS (FAB) calcd for C28H28N3O5 (M + H)+ 486.2029, found
486.2036; 28, HRMS (FAB) calcd for C28H28N3O5 (M + H)+
486.2029, found 486.2032.
C
22H24N3O5 (M + H)+ 410.1716, found 410.1731. Anal. Calcd
for C22H23O5N3: C, 64.52; H, 5.67; N, 10.27. Found: C, 64.45;
H, 5.71; N, 10.21.
Ala -1-a n th r a qu in on ylh yd r a zid e (14). Red solid; 1H NMR
(CD3OD) δ 8.25 (dd, J ) 6.8, 2.0 Hz, 1H), 8.17 (dd, J ) 7.4,
1.7 Hz, 1H), 7.87-7.65 (m, 4H), 7.36 (dd, J ) 7.4, 1.2 Hz, 1H),
4.24-4.17 (m, 1H), 1.69 (d, J ) 6.9 Hz, 3H); 13C NMR (CD3-
OD) δ 186.7, 184.4, 171.2, 152.6, 136.7, 135.8, 135.7, 135.5,
135.0, 134.2, 128.1, 127.8, 119.5, 119.3, 115.9, 64.4, 17.6;
HRMS (FAB) calcd for C17H16N3O3 (M - Cl)+ 310.1192, found
310.1206.
N-t BOC-Gly-L-P r o-L-Ala -1-a n t h r a q u in on ylh yd r a zid e
(15). Dark red solid; H NMR δ 10.51 (s, 1H, exchanges with
L-P h e-1-a n th r a qu in on ylh yd r a zid e (22) a n d D-P h e-1-
a n th r a qu in on ylh yd r a zid e (29). Dark orange solid; 1H NMR
(CD3OD) δ 8.23 (dd, J ) 7.2, 1.3 Hz, 1H), 8.15 (dd, J ) 7.2,
1.9 Hz, 1H), 7.82-7.74 (m, 2H), 7.63 (dd, J ) 7.4, 1.0 Hz, 1H),
7.45-7.31 (m, 6H), 6.53 (dd, J ) 8.5, 1.0 Hz, 1H), ?3.98 (t, J
) 7.2 Hz, 1H), 3.13 (d, J ) 1.5 Hz, 1H), 3.11 (d, J ) 2.9 Hz,
1H); 13C NMR δ 186.7, 184.5, 173.7, 152.4, 137.6, 136.6, 135.8,
135.6, 135.5, 134.9, 134.3, 130.8 (2C), 130.2 (2C), 128.7, 128.1,
127.7, 119.6, 119.2, 115.6, 56.0, 41.1; 22, HRMS (FAB) calcd
for C23H20N3O3 (M + H)+ 386.1505, found 386.1509; 29 HRMS
(FAB) calcd for C23H20N3O3 (M + H)+ 386.1505, found 386.1504.
N-tBOC-L-Ala -L-P h e-1-a n th r a qu in on ylh yd r a zid e (23).
Dark orange solid; 1H NMR δ 10.40 (s, 1H), 8.13 (dd, J ) 5.3,
5.3 Hz, 2H), 7.76-7.57 (m, 4H), 7.32-7.29 (m, 6H), 6.49 (d, J
) 6.6 Hz, 1H), 5.71 (d, J ) 7.6 Hz, 1H), 4.89-4.87 (m, 1H),
4.19-4.14 (m, 1H), 3.18 (br, 2H), 1.42 (s, 9H), 1.24 (d, J )
1
D2O), 9.06 (s, 1H, exchanges with D2O), 8.23-8.17 (m, 2H),
7.75-7.66 (m, 3H), 7.55 (t, J ) 8.0 Hz, 1H), 7.26 (d, J ) 8.7
Hz, 1H), 7.20 (d, J ) 7.3 Hz, 1H, exchanges with D2O), 5.45
(s, 1H), 4.65-4.52 (m, 2H), 4.02 (dd, J ) 16.6, 4.7 Hz, 1H),
3.78-3.71 (m, 2H), 3.55-3.47 (m, 1H), 2.22-1.98 (m, 4H), 1.50
(d, J ) 7.3 Hz, 3H), 1.46 (s, 9H); 13C NMR δ 185.3, 183.3, 171.7,
171.6, 169.8, 156.4, 151.6, 135.4, 134.4, 134.2, 133.9 (2C),
133.3, 132.8, 127.0, 126.7, 118.5, 118.4, 80.5, 61.2, 48.5, 47.1,
43.4, 28.8, 28.3 (3C), 24.9, 16.9; HRMS (FAB) calcd for
C
29H33N5O7Na (M + Na)+ 586.2278, found 586.2277.
Gly-L-P r o-L-Ala -1-a n th r a qu in on ylh yd r a zid e (16). Dark
1
red solid; H NMR (CD3OD) δ 8.31 (dd, J ) 7.5, 1.5 Hz, 1H),
8.34 (dd, J ) 7.0, 1.8 Hz, 1H), 7.90-7.80 (m, 2H), 7.75-7.64
(m, 2H), 7.43 (d, J ) 8.1 Hz, 1H), 4.53-4.44 (m, 2H), 3.78-

1608 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9
Kim et al.
16.4 Hz, 3H); 13C NMR δ 185.1, 183.1, 173.5, 170.8, 155.7,
150.8, 150.6, 136.0, 135.0, 134.0, 133.8, 133.2, 132.5, 129.3
(2C), 128.3 (2C), 127.0, 126.7, 126.6, 118.2, 117.9, 113.9, 80.3,
52.7, 50.1, 37.7, 28.1 (3C), 17.7; HRMS calcd for C31H33N4O6
(M + H)+ 557.2400, found 557.2404.
L-Ala -L-P h e-1-a n th r a qu in on ylh yd r a zid e (24). Dark or-
ange solid; 1H NMR (CD3OD) δ 8.26-8.15 (m, 2H), 7.86-7.62
(m, 3H), 7.52-7.36 (m, 6H), 6.59 (d, J ) 8.5 Hz, 1H), 4.39-
4.33 (m, 1H), 4.24-4.21 (m, 1H), 3.29 (d, J ) 8.0 Hz, 2H), 1.56
(dd, J ) 10.0, 7.3 Hz, 3H); 13C NMR (CD3OD) δ 186.7, 184.4,
173.1, 169.8, 152.6, 152.6, 137.8, 136.7, 135.6, 135.5, 134.9,
134.2, 130.9 (2C), 130.4 (2C), 128.4, 128.1, 127.8, 119.5, 119.3,
115.7, 55.4, 54.7, 38.6, 17.8; HRMS (FAB) calcd for C26H25N4O4
(M+) 457.1876, found 457.1887.
J ackson Laboratory (Bar Harbor, ME). All procedures were
approved by the Animal Care and Use Review Committee of
University of Iowa. The mice were sacrificed with an overdose
of anesthetic and then transcardially perfused with 0.9% (w/
v) saline solution, followed by 2% paraformaldehyde in 0.2 M
phosphate buffer. The tissues were removed and postfixed for
2 h. After fixation, the tissues were cryoprotected by immersion
in 25% sucrose and frozen in OCT compound (Sakura Finetek
U.S.A., Inc, Torrance, CA). Sections (10 µm) were cut with a
cryostat and mounted onto gelatin-coated slides. Sections were
embedded in 0.5% celloidin, followed by incubation in substrate
solutions containing 1 mM of the substrate, 1 mg/mL of
benzaldehyde, p-anisaldehyde, or p-nitrobenzaldehyde in 0.1
M acetate buffer (pH 4.5) at 37 °C for 4 h. Slides were rinsed
and cover-slipped before viewing on a Leica DM RBE micro-
scope equipped with a Spot-RT Digital Camera and associated
software. For all experiments, tissue staining was repeated
on a minimum of 5 sections/animal per compound. A minimum
of three individual animals was tested to confirm consistency
of the results.
N-tBOC-L-Ala -L-Ala -L-P h e-1-a n th r a qu in on ylh yd r a zid e
1
(26). Orange solid; H NMR δ 8.47 (d, J ) 7.4 Hz, 1H), 8.45
(d, J ) 8.0 Hz, 1H), 8.33 (d, J ) 7.8 Hz, 1H), 8.17 (d, J ) 7.4
Hz, 1H), 7.85-7.79 (m, 2H), 7.75-7.67 (m, 2H), 7.29-7.10 (m,
5H), 6.72-6.94 (m, 1H), 6.24-6.18 (m, 1H), 4.97-4.94 (m, 1H),
4.55-4.50 (m, 1H), 4.14-4.04 (m, 1H), 3.49 (dd, J ) 14.2, 5.5
Hz, 1H), 3.29 (dd, J ) 13.6, 7.2 Hz, 1H), 1.46 (s, 9H), 1.38 (d,
J ) 6.9 Hz, 3H), 1.34 (d, J ) 7.2 Hz, 3H); 13C NMR δ 184.4,
182.6, 172.5, 171.6, 171.2, 155.0, 144.6, 138.2, 135.6, 134.2,
133.6, 131.5, 131.0, 129.4, 129.3, 129.2 (2C), 128.5 (2C), 127.0,
124.2, 123.8, 121.0, 115.7, 80.5, 53.4, 48.9, 48.9, 38.7, 28.3 (3C),
18.0, 17.9; HRMS calcd for C34H38N5O7 (M + H)+ 628.2771,
found 628.2774.
Ack n ow led gm en t. We thank Ronald E. Haskell for
his technical assistance in the early stages of this
project. Financial support from the Batten Disease
Support & Research Association is gratefully acknowl-
edged.
L-Ala-L-Ala-L-P h e-1-an th r aqu in on ylh ydr azide (27). Dark
orange solid; 1H NMR (CD3OD) δ 8.31 (dd, J ) 6.7, 1.7 Hz,
1H), 8.23 (dd, J ) 7.0, 1.3 Hz, 1H), 7.88 (dt, J ) 7.4, 1.7 Hz,
2H), 7.70 (dd, J ) 7.5, 0.9 Hz, 1H), 7.52 (dd, J ) 8.4, 7.7 Hz,
1H), 7.40-7.22 (m, 5H), 6.78 (dd, J ) 8.6, 0.8 Hz, 1H), 4.72 (t,
J ) 8.0 Hz, 1H), 4.39 (dd, J ) 7.1, 2.6 Hz, 1H), 3.58 (dd, J )
7.4, 7.4 Hz, 1H), 3.18 (d, J ) 12.7 Hz, 2H), 1.51 (d, J ) 7.1 Hz,
3H), 1.39 (d, J ) 7.2 Hz, 3H); 13C NMR (CD3OD) δ 186.7, 184.5,
174.7, 173.3, 170.9, 152.7, 138.0, 136.6, 135.8, 135.6, 135.5,
135.0, 134.3, 130.7 (2C), 130.0 (2C), 128.3, 128.1, 127.8, 119.8,
119.2, 115.4, 55.5, 55.4, 55.2, 38.8, 18.3, 17.8; HRMS (FAB)
calcd for C29H30N5O5 (M - Br)+ 528.2247, found 528.2253.
N-tBOC-L-Ala -L-Ala -D-P h e-1-a n th r a qu in on ylh yd r a zid e
Refer en ces
(1) Bennett, M. J .; Hofmann, S. L. The neuronal ceroid-lipofusci-
noses (Batten disease): A new class of lysosomal storage
diseases. J . Inherit. Metab. Dis. 1999, 22, 535-544.
(2) Mole, S. E. Batten disease: Four genes and still counting.
Neurobiol. Dis. 1998, 5, 287-303.
(3) Sleat, D. E.; Donnelly, R. J .; Lackland, H.; Liu, C. G.; Sohar, I.;
Pullarkat, R. K.; Lobel, P. Association of Mutations in
Lysosomal Protein with Classical Late Infantile Neuronal Ceroid
Lipofuscinosis. Science 1997, 277, 1802-1805.
a
(4) Palmer, D. N.; Bayliss, S. L.; Westlake, V. J . Batten Disease
and the ATP Synthase Subunit-C Turnover Pathway - Raising
Antibodies to Subunit-C. Am. J . Med. Genet. 1995, 57, 260-
265.
1
(30). Red-orange solid; H NMR δ 8.44 (d, J ) 7.0 Hz, 1H),
(5) Haskell, R. E.; Huges, S. M.; Chiorini, J . A.; Alisky, J . M.;
Davidson, B. L. Gene Therapy 2002, in press.
8.39 (d, J ) 8.0 Hz, 1H), 8.29 (d, J ) 7.1 Hz, 1H), 8.13 (d, J )
7.0 Hz, 1H), 7.81-7.75 (m, 2H), 7.71-7.60 (m, 2H), 7.32-7.16
(m, 5H), 6.75-6.72 (m, 1H), 6.19-6.16 (m, 1H), 5.07-5.04 (m,
1H), 4.61-4.55 (m, 1H), 4.18-4.13 (m, 1H), 3.50 (dd, J ) 13.3,
5.4 Hz, 1H), 3.24 (dd, J ) 10.1, 7.7 Hz, 1H), 1.44 (s, 9H), 1.36
(d, J ) 6.0 Hz, 3H), 1.30 (d, J ) 6.9 Hz, 3H); 13C NMR δ 184.2,
182.5, 172.6, 171.8, 171.3, 155.6, 145.0, 138.1, 135.9, 134.2,
133.6, 131.4, 130.9, 129.5, 129.3 (2C), 128.7, 128.5 (2C), 127.2,
124.1, 123.7, 120.9, 118.3, 80.4, 53.5, 50.9, 48.8, 38.6, 28.3 (3C),
18.3, 18.0.
(6) Page, A. E.; Fuller, K.; Chambers, T. J .; Warburton, M. J .
Purification and Characterization of a Tripeptidyl Peptidase I
from Human Osteoclastomas: Evidence for Its Role in Bone
Resorption. Arch. Biochem. Biophys. 1993, 306, 354-359.
(7) Dikov, A.; Dimitrova, M.; Ivanov, I.; Krieg, R.; Halbhuber, K. J .
Original Method for the Histochemical Demonstration of Trip-
eptidyl Aminopeptidase I. Cell. Mol. Biol. 2000, 46, 1219-1225.
(8) Moehlau, R. Uber Anthrachinoyl-monohydrazine. Chem. Ber.
1912, 45, 2233-2247.
(9) (a) Sheehan, J . C.; Cruickshank, P. A.; Boshart, G. L.
A
Convenient Synthesis of Water-soluble Carbodiimides. J . Org.
Chem. 1961, 26, 2525-2528. (b) Xu, Y.; Miller, M. J . Total
Syntheses of Mycobactin Analogues as Potent Antimycobacterial
Agents using a Minimal Protecting Group Strategy. J . Org.
Chem. 1998, 63, 4314-4322.
L-Ala-L-Ala-D-P h e-1-an th r aqu in on ylh ydr azide (31). Dark
red solid; 1H NMR (400 MHz, CD3OD) δ 8.31 (dd, J ) 7.7, 1.5
Hz, 1H), 8.23 (dd, J ) 7.0, 1.6 Hz, 1H), 7.86 (dd, J ) 7.4, 1.9
Hz, 1H), 7.84 (dd, J ) 7.5, 1.6 Hz, 1H), 7.71 (dd, J ) 7.2, 0.9
Hz, 1H), 7.58 (dd, J ) 8.7, 7.4 Hz, 1H), 7.39-7.20 (m, 5H),
6.99 (dd, J ) 8.3, 0.8 Hz, 1H), 4.71 (dd, J ) 9.2, 4.0 Hz, 1H),
4.37 (q, J ) 7.2 Hz, 1H), 3.53 (q, J ) 7.2 Hz, 1H), 3.21 (dd, J
) 13.6, 5.2 Hz, 1H), 2.98 (dd, J ) 14.0, 9.2 Hz, 1H), 1.29 (d, J
) 7.2 Hz, 3H), 1.18 (d, J ) 6.8 Hz, 3H); 13C NMR (CD3OD) δ
186.7, 184.5, 174.6, 172.9, 170.7, 155.6, 138.6, 136.7, 135.5,
135.1 (2C), 135.0, 130.6 (2C), 129.6 (2C), 128.2, 128.1, 128.0,
119.8, 119.2, 118.0, 55.6, 55.2, 55.0, 39.4, 18.6, 17.8; HRMS
(10) Bradley, W.; Geddes, K. W. 1,9-Pyrazoloanthrone. I. Replacement
of Halogens in derivatives of 1,9-pyrazoloanthrone. J . Chem. Soc.
1952, 1630-1635.
(11) Zhang, L. H.; Meier, W. E.; Watson, E. J .; Gibson, E. P. Reversal
of the Regioselectivity of Anthrapyrazole Formation - A New
Synthesis of Losoxantrone (DUP941). Tetrahedron Lett. 1994,
35, 3675-3678.
(12) Vines, D.; Warburton, M. J . Purification and characterization
of a tripeptidyl aminopeptidase I from rat spleen. Biochim.
Biophys. Acta 1998, 1384, 233-242.
(13) Bodanszky, M.; Bodanszky, A. The Practice of Peptide Synthesis;
Springer-Verlag: Berlin, New York, 1984; pp 145 and 165.
(FAB) calcd for
528.2242.
Tissu e P r ep a r a tion a n d In cu ba tion w ith Su bstr a te.
C
29H30N5O5 (M - Br)+ 528.2247, found
C57BL/6J mice (6 to 8 weeks old) were purchased from the
J M020525X