Coumarin derivatives as protease-sensitive prodrugs

Article abstract of DOI:10.1002/1521-4184(200106)334:6<209::AID-ARDP209>3.0.CO;2-Y

To overcome the lack of selectivity of anticancer drugs toward malignant cells, the development of prodrugs, which could be activated selectively by tumour-specific proteases is the goal of these studies. In this work tripartate prodrugs have been evaluated consisting of a carrier unit and a spacer group, which allows for intramolecular cyclisation while releasing the third component, the compound attached to the carboxylic acid moiety of the spacer group. As carrier units amino acids or peptides have been used, which are required for recognition by the protease. As the spacer unit the "trimethyl-lock"-spacer has been applied; as a model leaving group p-anisidine was attached to the carboxylic acid moiety. It was intended to test the compounds for their releasing rate of p-anisidine. Two of the evaluated compounds, 9b and 9h, were degraded with half-lives of 46 min at room temperature. However, the poor solubility in aqueous solutions proved the major disadvantage of the TML-based prodrugs.

Full text of DOI:10.1002/1521-4184(200106)334:6<209::AID-ARDP209>3.0.CO;2-Y

Protease-Sensitive Prodrugs
209
Coumarin Derivatives as Protease-Sensitive Prodrugs✩
Karin Achilles
Institute of Pharmacy, Ernst-Moritz-Arndt University Greifswald, Pharmaceutical/Medicinal Chemistry, Friedrich-Ludwig-Jahn-Str. 17,
D-17487 Greifswald, Germany
Key Words: Coumarin-based prodrugs; intramolecular lactonization; trimethyl lock; elastase
the progress of the disease. Although this increased enzyme
occurrence is well known, no PMN-E-sensitive prodrugs
have been described until now.
Protease-cleavable prodrugs need to fulfil two main crite-
ria: On the one hand they have to contain the peptide sequence
required for the recognition by the protease (carrier unit). On
the other hand the release of the attached drug has to be
ensured. Sterical hindrance during the release of the attached
drug could be prevented by introducing a spacer unit between
carrier unit and drug (tripartate prodrug, Figure 1).
Summary
To overcome the lack of selectivity of anticancer drugs toward
malignant cells, the development of prodrugs, which could be
activated selectively by tumour-specific proteases is the goal of
these studies. In this work tripartate prodrugs have been evaluated
consisting of a carrier unit and a spacer group, which allows for
intramolecular cyclisation while releasing the third component, the
compound attached to the carboxylic acid moiety of the spacer
group. As carrier units amino acids or peptides have been used,
which are required for recognition by the protease. As the spacer
unit the “trimethyl-lock”-spacer has been applied; as a model
leaving group p-anisidine was attached to the carboxylic acid
moiety. It was intended to test the compounds for their releasing
rate of p-anisidine. Two of the evaluated compounds, 9b and 9h,
were degraded with half-lives of 46 min at room temperature.
However, thepoor solubility in aqueous solutions provedthemajor
disadvantage of the TML-based prodrugs.
Carrier
Spacer
Drug
Figure 1. Tripartate prodrugs.
The suitability of coumarin acids and its derivatives for the
design of esterase-cleavable prodrugs has been shown in
several studies . Upon unmasking the hydroxyl group the
ring-opened coumarin derivatives usually undergo spontane-
ous lactonisation, thereby releasing the compounds attached
to the carboxylic acid moiety (Scheme 1). In case of I the
lactonisation is the result of the “trimethyl lock”, which was
determined to strongly increase the rate of the lactonisation
Introduction
[11]
One of the current problems in cancer therapy is the poor
selectivity of the presentanticancer drugs toward cancer cells.
A possible approach to overcoming this lack of selectivity is
the design of anticancer prodrugs. Recently tumour-selective
properties have been identified that make it possible to evalu-
ate prodrugs that can be activated in the vicinity of the tumour
reaction due to a restriction of rotational freedom, termed
[1]
due to tumour-selective activation mechanisms . For exam-
[12]
stereopopulation control
. As a result of this intramolecu-
ple, elevated levels of proteases are described for malignant
tumours. Increased levels of proteases can be caused either
by the tendency of the tumour to spread through the extracel-
lular matrix (tumour invasion and metastasis) or by liberation
from infiltrating cells as lytic enzymes against the tumour.
The activation of prodrugs by the serine proteases plasmin
and chymotrypsin and the cysteine protease cathepsin B
has been described . It has been well established that several
lar strain, intermediate II shows a half-life of approximately
100 s in aqueous solutions.
In this paper the synthesis of prodrugs based on the
“trimethyl lock” system as the spacer unit is described. Since
PMN-E has a substrate specificity for amino acids with short
[4]
aliphatic side chains in P , different peptide sequences con-
1
[5]
taining alanine and valine in P will be introduced. For our
[6]
1
investigations we chose p-anisidine as a model leaving group.
proteases, especially cathepsin B are expressed tumour-spe-
cifically, which would predestine them for the tumour-selec-
tive activation of prodrugs by proteases.
O
O
O
R
O
R`
O
R`
Polymorphonuclear elastase (PMN-E) is a serine protease,
which has been found to occur in a number of malignant
diseases in significantly increased levels. Examples are
enzymatically
slowly
[7]
[8]
[9]
lung , breast , and colon cancer , but also inflammatory
[10]
diseases like rheumatoid arthritis . In case of breast cancer,
PMN-E has even been used as a marker for the recurrence or
II
I
O
O
chemically
fast
+
R`H
———
Correspondence: Dr. Karin Achilles, Institute of Pharmacy, Ernst-Moritz-
Arndt University Greifswald, Pharmaceutical/Medicinal Chemistry, Frie-
drich-Ludwig-Jahn-Str. 17, D-17487 Greifswald, Germany.
E-Mail: achilles@mail.uni-greifswald.de
III
Fax; +49 3834 864874
Scheme 1
Arch. Pharm. Pharm. Med. Chem.
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0365-6233/01/0606/0209 $17.50 +.50/0

210
Achilles
The evaluated compounds were tested for their cleavage by
porcine pancreatic elastase, which was used as a model
enzyme.
O-Acylation of 4 with amino acids activated as p-nitrophe-
nol esters, as described by Wang et al., did not work in our
hands, even under varied conditions (reaction time, tempera-
ture, solvent, equivalents of base, etc.). However, the condi-
tions described by Greenwald and co-workers, who used
EDC or DIPC as coupling reagents and up to 7 eq DMAP as
base yielded the desired compounds. O-Acylated compounds
Results and Discussion
To prevent the facile lactonisation of the “trimethyl lock”
system, a multistep synthesis is required: the first step is the
reduction of the carboxylic acid to the alcohol, followed by
the introduction of an O-protective group to selectively acy-
late the phenolic hydroxyl group. The attached peptide now
functions as a “protective group” for the phenolic hydroxyl
group and thus allows the re-oxidation of the alcoholic func-
tion to the acid.
3-(2-Hydroxy-4,6-dimethylphenyl)-3,3-dimethylpropanol
(3) was synthesised by reacting commercially available 3,5-
dimethylphenol and methyl 3,3-dimethylacrylate (1) in re-
fluxing benzene under catalysis of conc. sulfuric acid and
5a, b, and d were obtained in acceptable yields
[11c]
(Scheme 3)
.
In order to synthesise the oligopeptide derivatives, two
strategies can be applied: 4 can be directly acylated with di-
or tripeptides or the peptide chain can be build up in a
stepwise fashion. The direct acylation of 4 with different
tripeptides either activated as p-nitrophenyl esters or by using
different coupling reagents was unsuccessful, however. For
the stepwise synthesis strategy it is necessary to selectively
deblock the amino terminus. Therefore, we tried to remove
the N-terminal BOC-protective group of compound 5a. Un-
fortunately the parallel cleavage of the TBDMS-protective
group occurred instead, probably due to its lability under
strong acidic and basic conditions. An alternative to generate
the free amino terminus is the introduction of Z as N-protec-
tive group (5c), which can be cleaved during hydrogenolysis
by using Pd-C as catalyst. Under these conditions, the N-pro-
tective group was cleaved selectively, so that the N-terminal
unprotected compounds could be obtained and reacted with
BOC- and Z-amino acid-N-hydroxysuccinimide esters with-
out further purification. The dipeptide-spacer compounds
6a–f were obtained (Scheme 3). N-Hydroxysuccinimide es-
ters were prepared from the N-protected amino acid by react-
ing with 2.1 eq N-hydroxysuccinimide and 1.1 eq DCC
following standard coupling procedures. Repeated hydro-
genolysis of compounds 6d–f and subsequent reaction with
BOC-amino acid-NHS gave the tripeptide-spacer derivatives
7a–c (Scheme 3).
subsequent reduction of the coumarin derivative 2 with LAH
[11a]
in THF
. The TBDMS-protective group was introduced
following the procedure described by Wang et al. (Scheme 2)
[11b]
(4)
.
Cleavage of the TBDMS-group in a mixture of glacial
acetic acid, THF and water followed by stepwise oxidation
of the alcohol to the appropriate aldehyde with pyridinium-
chlorochromate and then to the carboxylic acid by using
sodium chlorite as mild oxidising reagents led to the com-
pounds 8a–i (Scheme 4). Attachment of the p-anisidine resi-
due was performed with DCC/HOBt as coupling reagents and
led to compounds 9a–h (Scheme 4). The coupling of com-
pound 8i was not successful.
Scheme 2
OTBDMS
AA1
To determine whether the synthesised prodrugs could be
activated by elastase, the respective compounds were dis-
solved in acetonitrile and then diluted with tris-buffer (0.1 M,
AA1
O
5a:
5b:
5c:
5d:
BOC-Ala
BOC-Val
Z-Val
EDC, 7 eq DMAP
4
(BOC-Pro:
BOC-Pro
DIPC, 3 eq DMAP)
OTBDMS
O
AA3
AA2
AA2 AA1
AA1
R
O
O
AA2
BOC-Ala
6b: BOC-Val
6c: BOC-Pro
AA1
Val
Val
Val
Val
Val
Val
1. HAc, 2. PCC
5a,b,d, 6a-c, 7a-c
1. H2/Pd-C
6a:
3. NaClO2, 4. p-Anisidin
5c
2. AA-NHS
6d:
6e:
6f:
Z-Ala
Z-Val
Z-Pro
R = OH AA3
AA2
AA1
R = p-anisidine
8a
8b
8c
8d
8e
8f
8g
8h
8i
BOC-Ala
BOC-Val
BOC-Pro
Val
Val
Val
Val
Val
Val
9a
9b
9c
9d
9e
9f
9g
9h
-
OTBDMS
AA3
AA2
AA1
BOC-Ala
BOC-Val
BOC-Pro
Ala
Val
Pro
O
1. H2/Pd-C
2. AA-NHS
6d-f
AA3
AA2 AA1
BOC-Ala
BOC-Ala
BOC-Ala
7a: BOC-Ala Ala Val
7b: BOC-Ala Val Val
7c BOC-Ala Pro Val
Scheme 3
Scheme 4
Arch. Pharm. Pharm. Med. Chem. 334, 209–215 (2001)

Protease-Sensitive Prodrugs
211
pH 8.0). All compounds were only poorly soluble in aqueous of the synthesised compounds gave log p values between 4
and 6, which is in good accordance with the poor solubility
in aqueous solutions observed before. If in these calculations
the N-terminal BOC-group is replaced by a MeOSuc- or an
Ac-group, the water solubility increases about a factor of 50.
Replacement of the p-anisidine residue by the more polar
sulfanilic acid did not further increase the water solubility.
The O-acylation of compound 4 with MeOSuc- or Ac-amino
acids, however, did not proceed in acceptable yields, so that
the theoretical results could not be confirmed.
The results demonstrate the general feasibility of the con-
cept to activate TML-prodrugs by elastase. Further experi-
ments are planned in order to increase the water solubility of
these compounds by a suitable derivatisation strategy.
solvents. Up to 15% of acetonitrile had to be used to prepare
solutions with concentrations between 0.05 and 0.1 mM. It
has been described that many enzymes are still active in
organic solvents, but the enzyme activity decreases with
[13]
increasing amounts of organic solvent
. Our investigations
show that PPE losses two third of its activity after an incuba-
tion of 2 h in buffered solution. Additionally, the enzyme
activity decreases by 50% with every 10% increase in ace-
tonitrile concentration. The use of surfactants (Pluronic F
127, Tween 80, Chremophor EL, Triton X 100; 1% or 10%,
respectively) failed to increase the solubility. Despite these
solubility problems, the stability of compounds 9a–h was
investigated by incubating them with purified PPE (Tris,
0.1 M, pH 8.0). At various times 20 µl aliquots were with-
drawn and immediately analysed by RP-HPLC (detection
Acknowledgement
wavelength: 250 nm, solvent: KH PO (20 mM, pH 3.3)/ace-
2
4
Financial support from the Fonds der Chemischen Industrie is gratefully
acknowledged. The author would like to thank Prof. P.J. Bednarski for
critical reading of the manuscript.
tonitrile = 30/70). In case of compounds 9a and 9c–g, no
hydrolysis was observed during the course of the experiment.
On the other hand, compounds 9b and 9h were degraded with
half-lives of 46 min each, as judged by HPLC-peak area
(Figure 1). Running a linear gradient (30 min 95/5 to 20/80
Experimental
KH PO (20 mM, pH 3.3)/acetonitrile) BOC-Val(OH) and
2
4
General: Mp: not corrected, Linström apparatus.– IR spectra (cm–1):
Perkin-Elmer 1600 series FTIR; in KBr, if not noted otherwise.– NMR
spectra: Bruker DPX 200 (200 MHz) and Bruker DPX 200 (300 MHz); δ in
ppm; 1H values from spectra in CDCl3, if not noted otherwise; standard
reference for all spectra in CDCl3 and d4-MeOH was TMS (δ = 0 ppm) or
(in cases of silyl groups containing compounds) thesignal of theundeuterated
solvent in D2O.– Elemental analysis: Perkin-Elmer elemental analyser 2400
CHN.– Mass spectra: Intectra AMD 402/3.– Chromatography: cc: Merck
silica gel 60 (7734); tlc: Merck Alufolien, silica gel 60 F254.– HPLC: Merck
Hitachi, LaChrom; Pump: L-7100; Detector: DAD L-7450.
Solvents were dried/purified following standard laboratory procedures
(e.g. DCM by refluxing over P4O10 for 2 h and storing the distilled solvent
over 4Å molecular sieve) or were purchased commercially. Unless noted
otherwise, moisture and air sensitive reactions were conducted under a dry
nitrogen atmosphere. All amino acids used in this work are L-amino acids.
PPE was purchased from Serva (20929).
BOC-Ala-Val-Val(OH) could be detected as the primary
cleavage products. Cleavage occurred only in presence of the
active enzyme, without enzyme and with enzyme denaturised
by heating no hydrolysis was observed. Nevertheless, it was
not possible to quantify these compounds and their cleavage
products within one HPLC-experiment because all com-
pounds show immense differences in polarity and solubility
in aqueous solution. Therefore, further investigations in order
to determine the rate of p-anisidine release were not per-
formed.
1
2
3
4
Abbreviations: Ac: Acetyl; BOC: tert-butyloxycarbonyl; DCC: dicyclo-
hexyl carbodiimide; DCM: dichloromethane; DIPC: diisopropyl carbodi-
imide; DMAP: dimethylaminopyridine; EDC: N-(3-dimethylaminopropyl)-
N′-ethylcarbodiimide hydrochloride; LAH: lithiumaluminium hydride;
MeOSuc: Methoxysuccinyl; NHS: N-hydroxysuccinimide ester; HOBt: 1-
hydroxybenzotriazol hydrate; PCC: pyridinium chlorochromate; PE: petrol
ether; PPE: porcine pancreatic elastase; TEA: triethyl amine; TBDMSCl:
tert-butyldimethylsilyl chloride, THF: tetrahydrofuran; TML: trimethyl-
lock; TMSCl: trimethyl chlorosilane; Z: benzyloxycarbonyl.
6
8
10
12
14
16
Retention time [min]
Figure 2. HPLC chromatogram showing the degradation of 9b catalysed by
PPE, at time 0, PPE was added. At various times (1: 0 min; 2: 20 min; 3:
40 min; 4: 60 min) aliquots were withdrawn and immediately loaded onto
the HPLC column.
Methyl-3,3-dimethylacrylate (1)[11a]
Yield: 14.4 g (0.13 mol, 63%) of a colourless liquid which was directly
used for the next step without further purification.– C6H10O2 (114.14).
The experiments described above demonstrate that the poor
solubility is a major drawback of the TML-based prodrugs.
At the time we planned the syntheses we were aware, that
hydrophobicity might lead to a poor solubility of these com-
pounds in aqueous solvents. However, synthesis and enzy-
matic activation of coumarin-based prodrugs have been
described in several publications – in no cases were solubility
problems reported. As a measure for hydrophobicity of or-
ganic compounds the octanol-water partition coefficient p is
4,4,5,7-Tetramethyl-3,4-dihydrocoumarin (2)[11a]
Yield: 14.9 g (0.073 mol, 87%) of a colourless liquid which was directly
used for the next step without further purification.– H NMR: δ = 1.44 (s,
6H, CH3), 2.27 (s, 3H, CH3), 2.46 (s, 3H, CH3), 2.59 (s, 2H, CH2), 6.74 (m,
2H, Ar-H).– C13H16O2 (204.27).
1
3-(2-Hydroxy-4,6-dimethylphenyl)-3,3-dimethylpropan-1-ol (3)[11a]
Yield: 13.2 g (63 mmol, 92%) of a colourless solid, which was directly
used for the next step without further purification.– H NMR: δ = 1.52 (s,
6H, CH3), 2.13 (s, 3H, CH3), 2.23 (t, J = 7.2 Hz, 2H, CH2), 2.45 (s, 3H, CH3),
3.58 (t, J = 7.2 Hz, 2H, CH2), 6.31, 6.65 (s, 1H, Ar-H).– C13H20O2 (208.30).
1
commonly used. In order to obtain comparable values, the log
[14]
p-values were estimated by computational methods:
most
Arch. Pharm. Pharm. Med. Chem. 334, 209–215 (2001)

212
Achilles
1-O-(tert-Butyldimethylsilyl)-3-(2-hydroxy-4,6-dimethylphenyl)-
complete (tlc-control, ca. 2.5 h). The catalyst was filtered off and the solution
concentrated in vacuo. The residue was directly used for the next step without
further purification and characterisation.
The desired N-unprotected compound (1 eq) was dissolved in dry DCM
and the appropriate N-protected amino acid-NHS (2 eq) was added. The
solution was stirred until the reaction was complete (tlc), the solvent was
evaporated, and the residue taken up in ethyl acetate. The organic phase was
washed with 1% NaHCO3-solution (3×) and 1 M HCl and dried with
Na2SO4. The solvent was evaporated in vacuo and the residue purified by cc
(cyclohexane/ethyl acetate = 7/3).
3,3-dimethylpropanol (4)[11b]
Yield: 18.0 g (60.0 mmol, 86%) of a colourless solid.– 1H NMR: δ = 0.07
(s, 6H, Si-CH3), 0.87 (s, 9H, Si(CH3)3), 1.54 (s, 6H, CH3), 2.11 (t, J = 7.0 Hz,
2H, CH2), 2.18 (s, 3H, CH3), 2.45 (s, 3H, CH3), 3.59 (t, J = 7.0 Hz, 2H, CH2),
5.66 (s, 1H, OH), 6.40, 6.48 (s, 1H, Ar-H).– C13H34O2Si (250.50).
General Procedure for the O-Acylation of 4 with N-Protected Amino Acids
N-Protected amino acid (2 eq), 4 (1 eq), and DMAP (7 eq) were dissolved
in dry DCM and the solution cooled to 0 °C. To the solution was added EDC
(2.9 eq) and the mixture was subsequently stirred at r.t. overnight. It was
diluted with DCM, and washed three times with 1% NaHCO3 and 1 N HCl.
After drying with Na2SO4 the solvent was removed in vacuo. After cc (10%
ethyl acetate in cyclohexane) 5 was obtained.
1-O-(tert-Butyldimethylsilyl)-3-[2-N-(BOC-alanylvalyloxy)-4,6-dimethyl-
phenyl]-3,3-dimethylpropanol (6a)
Starting from 5c (983 mg, 1.77 mmol), 6a (228 mg, 0.39 mmol, 22%) was
obtained as a colourless liquid.– 1H NMR: δ = –0.03 (s, 6H, SiMe2), 0.87 (s,
9H, t-Bu), 1.05, 1.08 [d, J = 6.9 Hz, 3H, CH3(Val)], 1.37 [d, J = 7.3 Hz, 3H,
CH3(Ala)], 1.46 (s, 9H, t-Bu; 6H, CH3), 2.03 (m, 2H, CH2), 2.23 (s, 3H,
CH3), 2.46 [m, 1H, β-H(Val)], 2.53 (s, 3H, CH3), 3.47 [t, J = 7.4 Hz, 2H,
CH2), 4.21 [m, 1H, α-H(Ala)], 4.78 [dd, J = 4.4 and 9.0 Hz, 1H, α-H(Val)],
5.02 (m, 1H, NH), 6.49 (s, 1H, Ar-H), 6.73 (m, 1H, NH), 6.83 (s, 1H, Ar-H).–
IR: ν = 3450, 2994, 1770, 1758, 1635, 1245 cm–1.– C31H56N2O6Si (580.39).
1-O-(tert-Butyldimethylsilyl)-3-[2-N-(BOC-alanyloxy)-4,6-dimethyl-
phenyl]-3,3-dimethylpropanol (5a)[11b]
Starting from BOC-alanine (500 mg, 2.64 mmol), 4 (425 mg, 1.32 mmol),
DMAP (1130 mg, 9.24 mmol), and EDC (585 mg, 3.79 mmol), 5a (470 mg,
0.96 mmol, 36%) was obtained as a colourless liquid.– 1H NMR: δ = –0.03
(s, 6H, SiMe2), 0.87 (s, 9H, t-Bu), 1.46 (s, 6H, CH3; s, 9H, t-Bu), 1.59 [d,
J = 7.3 Hz, 3H, CH3(Ala)], 2.05 (m, 2H, CH2), 2.25 (s, 3H, CH3), 2.55 (s,
3H, CH3), 3.52 (m, 2H, CH2), 4.55 [m, 1H, α-H(Ala)], 5.16 (m, 1H, NH),
6.54, 6.85 (s, 1H, Ar-H).– IR: ν = 3454, 1770, 1758, 1246 cm–1.–
C27H47NO5Si (493.76).
1-O-(tert-Butyldimethylsilyl)-3-[2-N-(BOC-valylvalyloxy)-4,6-dimethyl-
phenyl]-3,3-dimethylpropanol (6b)
Starting from 5c (727 mg, 1.30 mmol), 6b (290 mg, 0.47 mmol, 36%) was
obtained as a colourless liquid.– 1H NMR: δ = –0.03 (s, 6H, SiMe2), 0.84 (s,
9H, t-Bu), 0.94, 0.96 [2d, J = 6.6 Hz, 6H, CH3(Val)], 1.44 (s, 9H, t-Bu), 1.45
(s, 6H, CH3), 2.05 [m, 2H, CH2; 1H, β-H(Val)], 2.23 (s, 3H, CH3), 2.45 [m,
1H, β-H(Val)], 2.53 (s, 3H, CH3), 3.47 (m, 2H, CH2), 3.93 [dd, J = 6.5 and
8.8 Hz, 1H, α-H(Val)], 4.78 [dd, J = 4.4 and 8.8 Hz, 1H, α-H(Val)], 5.11 (m,
1H, NH), 6.40 (m, 1H, NH), 6.48, 6.83 (s, 1H, Ar-H).– IR: ν = 3324, 2961,
1769, 1758, 1246, 1051 cm–1.– C34H60N2O6Si (620.95).
1-O-(tert-Butyldimethylsilyl)-3-[2-N-(BOC-valyloxy)-4,6-dimethylphenyl]-
3,3-dimethylpropanol (5b)
Starting from BOC-valine (575 mg, 2.64 mmol), 4 (425 mg, 1.32 mmol),
DMAP (1130 mg, 9.24 mmol) and EDC (585 mg, 3.79 mmol), 5b (570 mg,
1.09 mmol, 41%) was obtained as a colourless liquid.– 1H NMR: δ = –0.03
(s, 6H, SiMe2), 0.87 (s, 9H, t-Bu), 1.06, 1.12 [d, J = 6.9 Hz, 3H, CH3(Val)],
1.50 (s, 6H, CH3; 9H, t-Bu), 2.03 (m, 2H, CH2), 2.26 (s, 3H, CH3), 2.41 [m,
1H, β-H(Val)], 2.56 (s, 3H, CH3), 3.51 (m, 2H, CH2), 4.48 [m, 1H, α-
H(Val)], 5.14 (m, 1H, NH), 6.51, 6.85 (s, 1H, Ar-H).– IR: ν = 3458, 2976,
1757, 1715, 1246, 1174 cm–1.– C29H51NO5Si (521.82).
1-O-(tert-Butyldimethylsilyl)-3-[2-N-(BOC-prolylvalyloxy)-4,6-dimethyl-
phenyl]-3,3-dimethylpropanol (6c)
Starting from 5c (654 mg, 1.18 mmol), 6c (407 mg, 0.66 mmol, 56%) was
obtained as a colourless liquid.– 1H NMR: δ = –0.03 (s, 6H, SiMe2), 0.87 (s,
9H, t-Bu), 1.04, 1.05 [d, J = 6.9 Hz, 3H, CH3(Val)], 1.44 (s, 9H, t-Bu; 6H,
CH3), 1.97 (m, 2H, CH2; 4H, 3′-H, 4′-H (Pro)], 2.23 (s, 3H, CH3), 2.45 [m,
1H, β-H(Val)], 2.53 (s, 3H, CH3), 3.45 [m, 2H, CH2; 2H, 5′-H(Pro)], 4.37
[m, 1H, -H(Pro)], 4.76 [m, 1H, α-H(Val)], 6.48 (m, 1H, Ar-H; 1H, NH), 6.82
(s, 1H, Ar-H).– IR: ν = 3456, 2958, 2856, 1757, 1691, 1394, 1248, 1167,
1090 cm–1.– C34H58N2O6Si (618.93).
1-O-(tert-Butyldimethylsilyl)-3-[2-N-(Z-valyloxy)-4,6-dimethylphenyl]-
3,3- dimethylpropanol (5c)
Starting from Z-valine (727 mg, 2.89 mmol), 4 (465 mg, 1.45 mmol),
DMAP (1240 mg, 10.12 mmol), and EDC (804 mg, 4.19 mmol), 5c (522 mg,
0.94 mmol, 65%) was obtained as a colourless liquid.– 1H NMR: δ = -0.03
(s, 6H, SiMe2), 0.87 (s, 9H, t-Bu), 1.08, 1.15 [d, J = 6.9 Hz, 3H, CH3(Val)],
1.48 (s, 6H, CH3), 2.05 (m, 2H, CH2), 2.27 (s, 3H, CH3), 2.47 [m, 1H,
β-H(Val)], 2.57 (s, 3H, CH3), 3.51 (m, 2H, CH2), 4.57 [m, 1H, α-H(Val)],
5.18 (s, 2H, CH2), 5.40 (m, 1H, NH), 6.51, 6.86 (s, 1H, Ar-H), 7.40 (m, 5H,
Ar-H).– IR: ν = 3433, 2994, 1769, 1758, 1374, 1246, 1056 cm–1.–
1-O-(tert-Butyldimethylsilyl)-3-[2-N-(Z-alanylvalyloxy)-4,6-dimethyl-
phenyl]-3,3-dimethylpropanol (6d)
Starting from 5c (867 mg, 1.56 mmol), 6d (539 mg, 0.91 mmol, 58%) was
obtained as a colourless liquid.– 1H NMR: δ = –0.03 (s, 6H, SiMe2), 0.87 (s,
9H, t-Bu), 1.06 [m, 6H, CH3(Val)], 1.47 [m, 6H, CH3; 3H, CH3(Ala)], 2.04
(m, 2H, CH2), 2.22 (s, 3H, CH3), 2.49 [m, 1H, β-H(Val)], 2.56 (s, 3H, CH3),
3.51 (t, J = 7.5 Hz, 2H, CH2), 4.34 [m, 1H, α-H(Ala)], 4.79 [dd, J = 4.6 and
9.0 Hz, 1H, α-H(Val)], 5.15 (s, 2H, CH2), 5.75 (m, 1H, NH), 6.52 (s, 1H,
Ar-H), 6.61 (m, 1H, NH), 6.86 (s, 1H, Ar-H), 7.35 (m, 5H, Ar-H).– IR:
ν = 3308, 2958, 1769, 1758, 1246, 1051 cm–1.– C32H54N2O6Si (590.88).
C
32H49NO5Si (555.83).
O-Acylation of 4 with BOC-Proline
1-O-(tert-Butyldimethylsilyl)-3-[2-N-(BOC-prolyloxy)-4,6-dimethyl-
phenyl]-3,3-dimethylpropanol (5d)[11c]
Yield: 100 mg (0.10 mmol, 84%) of a colourless liquid.– 1H NMR: δ =
–0.03 (s, 6H, SiMe2), 0.87 (s, 9H, t-Bu), 1.65 (s, 6H, CH3; 9H, t-Bu), 2.01
[m, 2H, CH2; 4H, 3′-H, 4′-H (Pro)], 2.26 (s, 3H, CH3), 2.55 (s, 3H, CH3),
3.54 [m, 2H, CH2; 2H, 5‘-H (Pro)], 4.56 [m, 1H, α-H(Pro)], 6.59, 6.85 (s,
1H, Ar-H).– IR: ν = 2976, 1762, 1701, 1397, 1256, 1142 cm–1.–
C29H49NO5Si (519.80).
1-O-(tert-Butyldimethylsilyl)-3-[2-N-(Z-valylvalyloxy)-4,6-dimethyl-
phenyl]-3,3-dimethylpropanol (6e)
Starting from 5c (653 mg, 1.18 mmol), 6e (431 mg, 0.69 mmol, 58%) was
obtained as a colourless liquid.– 1H NMR: δ = –0.03 (s, 6H, SiMe2), 0.84 (s,
9H, t-Bu), 0.94, 0.97, 1.05, 1.07 [d, J = 6.9 Hz, 3H, CH3(Val)], 1.44 (s, 6H,
CH3), 2.05 [m, 2H, CH2; 1H, β-H(Val)], 2.24 (s, 3H, CH3), 2.43 [m, 1H,
β-H(Val)], 2.53 (s, 3H, CH3), 3.47 (t, J = 7.5 Hz, 2H, CH2), 4.05 [dd, J = 6.5
and 8.6 Hz, 1H, α-H(Val)], 4.78 [dd, J = 4.4 and 8.9 Hz, 1H, α-H(Val)], 5.12
(s, 2H, CH2), 5.41 (d, J = 8.6 Hz, 1H, NH), 6.35 (d, J = 9.0 Hz, 1H, NH),
6.48, 6.83 (s, 1H, Ar-H), 7.35 (m, 5H, Ar-H).– IR: ν = 2959, 1757, 1691,
1395, 1248, 1167 cm–1.– C37H58N2O6Si (654.97).
General Procedure for the Lengthening of the Peptide Chain with
N-Protected Amino Acid N-Hydroxysuccinimide Esters
The appropriate Z-protected compound was dissolved in dry ethanol
(50 ml) and Pd-C (100 mg) was added. The solution was degassed and
subsequently stirred in an hydrogen atmosphere until the reaction was
Arch. Pharm. Pharm. Med. Chem. 334, 209–215 (2001)

Protease-Sensitive Prodrugs
213
1-O-(tert-Butyldimethylsilyl)-3-[2-N-(Z-prolylvalyloxy)-4,6-dimethyl-
phenyl]-3,3-dimethylpropanol (6f)
allowed to warm to r.t. One equivalent of Na2SO3 was added to decompose
HOCl and H2O2. The pH was adjusted to 2 with 1 N HCl and the product
extracted with ethyl acetate (3 × 20 ml). The combined organic phases were
dried with Na2SO4 and the solvent was removed under reduced pressure. The
crude product was purified by cc (5% MeOH in DCM).
Starting from 5c (727 mg, 1.31 mmol), 6f (446 mg, 0.73 mmol, 56%) was
obtained as a colourless liquid.– 1H NMR: δ = –0.03 (s, 6H, SiMe2), 0.87 (s,
9H, t-Bu), 1.01 [m, 6H, CH3(Val)], 1.46 (s, 6H, CH3), 2.06 [m, 2H, CH2; 4H,
3′-H, 4é-H (Pro)], 2.27 (s, 3H, CH3), 2.42 [m, 1H, β-H(Val)], 2.56 (s, 3H,
CH3), 3.51 [m, 2H, CH2; 2H, 5′-H (Pro)], 4.44 [m, 1H, α-H(Pro)], 4.73 [dd,
J = 4.4 and 9.0 Hz, 1H, α-H(Val)], 5.21 (s, 2H, CH2), 6.51 (m, 1H, Ar-H;
1H, NH), 6.85 (s, 1H, Ar-H), 7.35 (m, 5H, Ar-H).– IR: ν = 2994, 1770, 1758,
1246, 1055 cm–1.– C37H56N2O6Si (652.95).
3-[2-N-(BOC-alanyloxy)-4,6-dimethylphenyl]-3,3-dimethylpropionic Acid
(8a)[11b]
Starting from 5a (496 mg, 1.00 mmol), a colourless liquid (103 mg,
0.26 mmol, 26%) was obtained.– 1H NMR: δ = 1.45 (s, 9H, t-Bu), 1.57 [m,
6H, CH3; 3H, CH3(Ala)], 2.22, 2.54 (s, 3H, CH3), 2.82 (s, 2H, CH2), 4.51
[m, 1H, α-H(Ala)], 5.12 (m, 1H, NH), 6.52, 6.82 (s, 1H, Ar-H).– IR: = 3354,
2994, 1769, 1758, 1246, 1057 cm–1.– C21H31NO6 (393.48).
1-O-(tert-Butyldimethylsilyl)-3-[2-N-(BOC-alanylalanylvalyloxy)-
4,6-dimethylphenyl]-3,3-dimethylpropanol (7a)
Starting from 6d (533 mg, 0.90 mmol), 7a (200 mg, 0.30 mmol, 33%) was
obtained as a colourless solid.– Mp: 67 °C.– 1H NMR: δ = 0.03 (s, 6H,
SiMe2), 0.87 (s, 9H, t-Bu), 1.07, 1.09 [d, J = 6.9 Hz, 3H, CH3(Val)], 1.39,
1.43 [d, J = 7.0 Hz, 3H, CH3(Ala)], 1.43 (s, 9H, t-Bu; 3H, CH3), 1.60 (s, 3H,
CH3), 2.07 (m, 2H, CH2), 2.26 (s, 3H, CH3), 2.49 [m, 1H, β-H(Val)], 2.56
(s, 3H, CH3), 3.50 [t, J = 7.4 Hz, 2H, CH2), 4.18, 4.53 [m, 1H, α-H(Ala)],
4.77 [dd, J = 4.4 and 9.0 Hz, 1H, α-H(Val)], 4.97 (m, 1H, NH), 6.51 (s, 1H,
Ar-H), 6.64, 6.68 (m, 1H, NH), 6.85 (s, 1H, Ar-H).– IR: ν = 3302, 2952,
2934, 1758, 1693, 1644, 1517, 1472, 1396, 1247, 1167 cm–1.–
3-[2-N-(BOC-valyloxy)-4,6-dimethylphenyl]-3,3-dimethylpropionic Acid
(8b)
Starting from 5b (896 mg, 1.72 mmol), a viscous yellowish liquid
(204 mg, 0.49 mmol, 29%) was obtained.– 1H NMR: δ = 1.04, 1.10 [d,
J = 6.8 Hz, 3H, CH3(Val)], 1.44 (s, 9H, t-Bu), 1.55 (s, 3H, CH3), 1.61 (s, 3H,
CH3), 2.22 (s, 3H, CH3), 2.35 (m, 1H, β-H), 2.54 (s, 3H, CH3), 2.84 (m, 2H,
CH2), 4.41 [m, 1H, α-H(Val)], 5.11 (m, 1H, NH), 6.52, 6.82 (s, 1H, Ar-H).–
IR: = 3358, 2976, 1757, 1715, 1369, 1246, 1174 cm-1.– MS (FAB):
m/z = 444.1 [M+Na]+.– C23H35NO6 (412.54).
C
35H61N3O7Si (663.98).
3-[2-N-(BOC-prolyloxy)-4,6-dimethylphenyl]-3,3-dimethylpropionic Acid
1-O-(tert-Butyldimethylsilyl)-3-[2-N-(BOC-alanylvalylvalyloxy)-
4,6-dimethylphenyl]-3,3-dimethylpropanol (7b)
(8c)[11c]
Starting from 5d (369 mg, 0.71 mmol), a viscous liquid (166 mg,
0.40 mmol, 56%) was obtained.– 1H NMR: δ = 1.46 (s, 9H, t-Bu), 1.65, 1.68
(s, 3H, CH3), 2.00, 2.31 [m, 4H, 3′-H, 4′-H (Pro)], 2.23 (s, 3H, CH3), 2.56
(s, 3H, CH3), 2.84 (s, 2H, CH2), 3.54 [m, 2H, 5′-H(Pro)], 4.56 [m, 1H,
α-H(Pro)], 6.56, 6.82 (s, 1H, Ar-H).– IR: ν = 3433, 2976, 1762, 1701, 1397,
1142 cm–1.– C23H33NO6 (419.52). C, H, N (*0.5 H2O).
Starting from 6e (449 mg, 0.69 mmol), 7b (201 mg, 0.29 mmol, 42%) was
obtained as a colourless solid.– Mp: 85 °C.– H NMR: δ = –0.03 (s, 6H,
1
SiMe2), 0.87 (s, 9H, t-Bu), 1.06, 1.12 [2d, J = 6.9 Hz, 6H, CH3(Val)], 1.38
(d, J = 7.0 Hz, 3H, CH3(Ala)], 1.48 (s, 9H, t-Bu; 6H, CH3), 2.13 [m, 2H,
CH2; 1H, β-H(Val)], 2.26 (s, 3H, CH3), 2.46 [m, 1H, β-H(Val)], 2.56 (s, 3H,
CH3), 3.50 (t, J = 7.4 Hz, 2H, CH2), 4.25 [m, 1H, α-H(Ala)], 4.39 [m, 1H,
α-H(Val)], 4.80 [dd, J = 4.6 and 8.9 Hz, 1H, α-H(Val)], 5.23 (d, J = 7.5 Hz,
1H, NH), 6.52, 6.86 (s, 1H, Ar-H), 6.78 (d, J = 8.8 Hz, 1H, NH), 6.93 (d,
J = 8.7Hz, 1H, NH).– IR: ν = 3306, 2959, 2928, 1755, 1691, 1648, 1514,
1471, 1390, 1366, 1253, 1169 cm–1.– C37H65N3O7Si (692.03).
3-[2-N-(BOC-alanylvalyloxy)-4,6-dimethylphenyl]-3,3-dimethylpropionic
Acid (8d)
Startingfrom6a (280 mg, 0.48 mmol), a viscous yellowish liquid (139 mg,
0.28 mmol, 59%) was obtained.– 1H NMR: δ = 1.06 [m, 6H, CH3(Val)], 1.33
[d, J = 7.3 Hz, 3H, CH3(Ala)], 1.55, 1.60 (s, 3H, CH3), 1.43 (s, 9H, t-Bu),
2.21 (s, 3H, CH3), 2.39 [m, 1H, β-H(Val)], 2.54 (s, 3H, CH3), 2.80 (m, 2H,
CH2), 4.21 [m, 1H, α-H(Ala)], 4.68 [m, 1H, α-H(Val)], 5.09 (m, 1H, NH),
6.50, 6.82 (s, 1H, Ar-H), 7.04 (m, 1H, NH).– IR: ν = 3427, 2973, 1709, 1529,
1392, 1368, 1173 cm–1.– MS (EI): 492 (0.6), 297 (8), 241 (34), 241 (12), 215
(68), 204 (13), 187 (38), 72 (100).– MS (FAB): m/z = 515.3 [M+Na]+.–
C26H40N2O7 (492.62).
1-O-(tert-Butyldimethylsilyl)-3-[2-N-(BOC-alanylprolylvalyloxy)-
4,6-dimethylphenyl]-3,3-dimethylpropanol (7c)
Starting from 6f (475 mg, 0.73 mmol), 7c (150 mg, 0.23 mmol, 32%) was
obtained as a colourless liquid.– 1H NMR: δ = –0.03 (s, 6H, SiMe2), 0.87 (s,
9H, t-Bu), 1.12 [m, 6H, CH3(Val)], 1.38 [d, J = 6.8 Hz, 3H, CH3(Ala)], 1.47
(s, 9H, t-Bu; 6H, CH3), 2.06 (m, 2H, CH2; 4H, 3′-H, 4′-H (Pro)], 2.27 (s, 3H,
CH3), 2.47 [m, 1H, β-H(Val)], 2.56 (s, 3H, CH3), 3.51 [t, J = 7.4 Hz, 2H,
CH2), 3.70 (m, 2H, 5′-H(Pro)], 4.55 [m, 1H, α-H(Ala)], 4.76 [m, 1H,
α-H(Pro); 1H, α-H(Val)], 5.43 (m, 1H, NH), 6.54, 6.84 (s, 1H, Ar-H), 7.47
(m, 1H, NH).– IR: ν = 2964, 2933, 1751, 1688, 1641, 1512, 1366, 1249,
1172 cm–1.– C37H63N3O7Si (690.01).
3-[2-N-(BOC-valylvalyloxy)-4,6-dimethylphenyl]-3,3-dimethylpropionic
Acid (8e)
Starting from 6b (367 mg, 0.59 mmol), a viscous yellowish liquid was
obtained (242 mg, 0.46 mmol, 79%).– 1H NMR: δ = 0.96, 1.11 [2d,
J = 6.8 Hz, 6H, CH3(Val)], 1.42 (s, 9H, t-Bu), 1.55, 1.62 (s, 3H, CH3), 2.15
[m, 1H, β-H(Val)], 2.21 (s, 3H, CH3), 2.40 [m, 1H, β-H(Val)], 2.54 (s, 3H,
CH3), 2.80 (m, 2H, CH2), 3.95, 4.70 [m, 1H, α-H(Val)], 5.16 (m, 1H, NH),
6.50 (s, 1H, Ar-H), 6.82 (s, 1H, Ar-H; m, 1H, NH), 8.02 (s, 1H, COOH).–
IR: ν = 3308, 2966, 1757, 1709, 1664, 1525, 1389, 1367, 1175 cm–1.– MS
(EI): 299 (18), 248 (30), 215 (12), 116 (10), 72 (100).– MS (FAB):
m/z = 543.4 [M+Na]+.– C28H44N2O7 (520.67).
General Procedure for the Cleavage of the TBDMS-Protective Group and
the Oxidation of 5–7 to the Carboxylic Acids
Either 5, 6, or 7 (1 mmol) was dissolved in THF (2 ml), water (2 ml), and
glacial acetic acid (6 ml). The solution was stirred for 1 h at r.t. and the
solvent was subsequently removed in vacuo. The residue was used for the
next step without further purification.
PCC (2 eq) were dissolved in dry DCM. To this solution was added a
solution of the appropriate alcohol (1 eq) in dry DCM. During the addition
the colour of the mixture turned to black. The reaction was completed while
stirring overnight. The solvent was evaporated, the residue taken up in a
minimum of DCM and filtrated over a short silica gel column (4 cm, ether).
The eluent was evaporated to dryness and the residue was used for the next
step without further purification.
3-[2-N-(BOC-prolylvalyloxy)-4,6-dimethylphenyl]-3,3-dimethylpropionic
Acid (8f)
Starting from 6c (481 mg, 0.78 mmol), a viscous yellowish liquid was
obtained (182 mg, 0.35 mmol, 45%).– 1H NMR: δ = 1.05, 1.09 [d,
J = 6.8 Hz, 3H, CH3(Val)], 1.46 (s, 9H, t-Bu), 1.58, 1.62 (s, 3H, CH3), 1.90
(m, 4H, 3′-H, 4′-H (Pro)], 2.22 (s, 3H, CH3), 2.41 [m, 1H, β-H(Val)], 2.54
(s, 3H, CH3), 2.82 (m, 2H, CH2), 3.43 (m, 2H, 5′-H(Pro)], 4.40 [m, 1H,
α-H(Pro)], 4.64 [m, 1H, α-H(Val)], 6.51, 6.82 (s, 1H, Ar-H), 7.69 (m,
A solution of the appropriate aldehyde (1 eq) and NaH2PO4 (0.6 eq) in
water (1.4 ml) and ACN (3.9 ml) was cooled to 0 °C by an ice-salt bath. To
this solution, was added dropwise a solution of NaClO2 (2.5 eq, 80%) in
water (5 ml). The reaction mixture was stirred for 1 h at 0 °C, then it was
Arch. Pharm. Pharm. Med. Chem. 334, 209–215 (2001)

214
Achilles
N-(4-Methoxyphenyl)-3-[2-N-(BOC-valyloxy)-4,6-dimethylphenyl]-
3,3-dimethylpropionamide (9b)
1H, NH), 8.02 (s, 1H, COOH).– IR: ν = 3422, 2968, 2930, 1668, 1392,
1166 cm–1.– MS (EI): 297 (45), 241 (66), 213 (50), 183 (12), 114 (43), 70
(100).– MS (FAB): m/z = 541.3 [M+Na]+.– C28H42N2O7 (518.67).
Starting from 8b (191 mg, 0.45 mmol), DCC (112 mg, 0.54 mmol), HOBt
(76 mg, 0.50 mmol), p-anisidine (61 mg, 0.50 mmol), and DMAP (11 mg,
0.09 mmol), 9b (137 mg, 0.26 mmol, 58%) was obtained as a colourless
solid.– Mp: 141 °C.– 1H NMR: δ = 1.09, 1.15 [d, J = 6.9 Hz, 3H, CH3(Val)],
1.43 (s, 9H, t-Bu), 1.70 (s, 6H, CH3), 2.23, 2.47 (s, 3H, CH3), 2.42 [m, 1H,
β-H(Val)], 2.55, 2.65 (s, 1H, CH2), 3.75 (s, 3H, OCH3), 4.10 [dd, J = 4.9 and
8.9 Hz, 1H, α-H(Val)], 4.99 (d, J = 8.9 Hz, 1H, NH), 6.54, 6.80 (s, 1H,
Ar-H), 6.74, 7.21 (d, J = 9.0 Hz, 2H, Ar-H), 7.73 (s, 1H, NH).– IR: ν = 3334,
2966, 1745, 1537, 1512, 1244 cm–1.– C30H42N2O6 (526.68). C, H, N.
3-[2-N-(BOC-alanylalanylvalyloxy)-4,6-dimethylphenyl]-3,3-dimethyl-
propionic Acid (8g)
Starting from 7a (189 mg, 0.28 mmol), a viscous yellowish liquid (90 mg,
0.16 mmol, 57%) was obtained.– 1H NMR: δ = 1.04 [m, 6H, CH3(Val)], 1.28
[m, 6H, CH3(Ala)], 1.42 (s, 9H, t-Bu), 1.55, 1.59 (s, 3H, CH3), 2.20 (s, 3H,
CH3), 2.41 [m, 1H, β-H(Val)], 2.54 (s, 3H, CH3), 2.79 (m, 2H, CH2), 4.16,
4.53, 4.68 (m, 1H, α-H), 5.14 (m, 1H, NH), 6.49, 6.81 (s, 1H, Ar-H), 7.05
(d, J = 7.4 Hz, 1H, NH), 7.21 (d, J = 8.8 Hz, 1H, NH).– IR: ν = 3303, 2970,
2932, 1760, 1715, 1650, 1540, 1392, 1248, 1174 cm-1.– MS (FAB):
m/z = 586.5 [M+Na]+.– C29H45N3O8 (563.69).
N-(4-Methoxyphenyl)-3-[2-N-(BOC-prolyloxy)-4,6-dimethylphenyl]-
3,3-dimethylpropionamide (9c)
Starting from 8c (146 mg, 0.35 mmol), DCC (86 mg, 0.42 mmol), HOBt
(59 mg, 0.38 mmol), p-anisidine (47 mg, 0.38 mmol), and DMAP (9 mg,
0.07 mmol), 9c (125 mg, 0.24 mmol, 68%) was obtained as a colourless
solid.– Mp: 84 °C.– 1H NMR: δ = 1.43 (s, 9H, t-Bu), 1.69, 1.75 (s, 3H, CH3),
2.44 [m, 4H, 3′,4′-H(Pro)], 2.22, 2.57 (s, 3H, CH3), 2.73, 2.80 (s, 1H, CH2),
3.54 [m, 2H, 5′-H (Pro)], 3.74 (s, 3H, OCH3), 4.61 [m, 1H, α-H(Pro)], 6.58,
6.78 (s, 1H, Ar-H), 6.74, 7.22 (d, J = 9.0 Hz, 2H, Ar-H), 8.05 (s, 1H, NH).–
IR: ν = 3349, 2975, 1763, 1675, 1537, 1512, 1408, 1146 cm–1.– C30H40N2O6
(524.66). C, H, N.
3-[2-N-(BOC-alanylvalylvalyloxy)-4,6-dimethylphenyl]-3,3-dimethyl-
propionic Acid (8h)
Starting from 7b (242 mg, 0.35 mmol), a yellowish solid (134 mg,
0.23 mmol, 65%) was obtained.– Mp: 109° C.– 1H NMR: δ = 0.89, 1.05 [m,
6H, CH3(Val)], 1.27 (m, 3H, CH3(Ala)], 1.42 (s, 9H, t-Bu), 1.53, 1.61 (s, 3H,
CH3), 2.16 [m, 1H, β-H(Val)], 2.20 (s, 3H, CH3), 2.34 [m, 1H, β-H(Val)],
2.54 (s, 3H, CH3), 2.80 (m, 2H, CH2), 4.18, 4.33, 4.70 (m, 1H, α-H), 5.23
(m, 1H, NH), 6.48, 6.80 (s, 1H, Ar-H), 7.14, 7.18 (m, 1H, NH).– IR: ν = 3292,
2994, 1770, 1758, 1374, 1246, 1053 cm–1.– MS (ESI): m/z = 614.5
[M+Na]+.– C31H49N3O8 (591.75).
N-(4-Methoxyphenyl)-3-[2-N-(BOC-alanylvalyloxy)-4,6-dimethylphenyl]-
3,3-dimethylpropionamide (9d)
Starting from 8d (80 mg, 0.16 mmol), DCC (40 mg, 0.19 mmol), HOBt
(27 mg, 0.18 mmol), p-anisidine (22 mg, 0.18 mmol), and DMAP (4 mg,
0.03 mmol), 9d (24 mg, 0.04 mmol, 25%) was obtained as a colourless
solid.– Mp: 103° C.– 1H NMR: δ = 1.09, 1.11 [d, J = 6.8 Hz, 3H, CH3(Val)],
1.32 [d, J = 7.1 Hz, 3H, CH3(Ala)], 1.43 (s, 9H, t-Bu), 1.68 (s, 6H, CH3),
2.22 (s, 3H, CH3), 2.41 [m, 1H, β-H(Val)], 2.59 (s, 3H, CH3), 2.54, 2.66 (s,
1H, CH2), 3.74 (s, 3H, OCH3), 4.22 [dq, J = 7.1 Hz, 1H, α-H(Ala)], 4.62 [dd,
J = 5.0 and 8.4 Hz, 1H, α-H(Val)], 4.93 (d, J = 8.4 Hz, 1H, NH), 6.52 (s, 1H,
Ar-H), 6.75 (d, J = 9.2 Hz, 2H, Ar-H), 6.79 (s, 1H, Ar-H), 7.05 (m, 1H, NH),
7.26 (d, J = 9.2 Hz, 2H, Ar-H), 7.99 (s, 1H, NH).– IR: ν = 3314, 2973, 2930,
1759, 1708, 1652, 1512, 1365, 1243, 1173 cm–1.– C33H47N3O7 (597.76). C,
H, N.
3-[2-N-(BOC-alanylprolylvalyloxy)-4,6-dimethylphenyl]-3,3-dimethyl-
propionic Acid (8i)
Starting from 7c (138 mg, 0.20 mmol), a viscous yellowish liquid (58 mg,
0.09 mmol, 45%) was obtained.– 1H NMR: δ = 1.05 [m, 6H, CH3(Val)], 1.31
[m, 3H, CH3(Ala)], 1.40 (s, 9H, t-Bu), 1.54, 1.57 (s, 3H, CH3), 1.98 (m, 3H,
3′-H, 4′-Hs (Pro); 1H, β-H(Val)], 2.19 (s, 3H, CH3), 2.30 [m, 1H, 3′-H(Pro)],
2.52 (s, 3H, CH3), 2.79 (m, 2H, CH2), 3.58 (m, 2H, 5′-H(Pro)], 4.56 (m, 3H,
α-H), 6.48, 6.79 (s, 1H, Ar-H), 7.44 (d, J = 8.4 Hz, 1H, NH), 7.55 (d,
J = 8.2 Hz, 1H, NH).– IR: ν = 3220, 2962, 2930, 1759, 1691, 1640, 1531,
1453, 1366, 1254, 1169, 1092 cm–1.– MS (ESI): m/z = 612.4 [M+Na]+;
(FAB): m/z = 612.4 [M+Na]+.– C31H47N3O8 (589.73).
N-(4-Methoxyphenyl)-3-[2-N-(BOC-valylvalyloxy)-4,6-dimethylphenyl]-
3,3-dimethylpropionamide (9e)
General Procedure for the Attachment of 8 with p-Anisidine
Starting from 8e (160 mg, 0.31 mmol), DCC (77 mg, 0.37 mmol), HOBt
(52 mg, 0.34 mmol), p-anisidine (42 mg, 0.34 mmol), and DMAP (8 mg,
0.06 mmol), 9e (43 mg, 0.07 mmol, 22%) was obtained as a colourless
solid.– Mp: 153° C.– 1H NMR: δ = 0.90, 0.91 [d, J = 6.6 Hz, 3H, CH3(Val)],
1.09, 1.12 [d, J = 6.6 Hz, 3H, CH3(Val)], 1.41 (s, 9H, t-Bu), 1.68 (s, 6H,
CH3), 2.03 [m, 1H, β-H(Val)], 2.21 (s, 3H, CH3), 2.39 [m, 1H, β-H(Val)],
2.48 (s, 3H, CH3), 2.66, 2.73 (s, 1H, CH2), 3.73 (s, 3H, OCH3), 3.94, 4.62
[m, 1H, α-H(Val)], 5.03 (d, J = 8.6 Hz, 1H, NH), 6.50 (s, 1H, Ar-H), 6.69
(d, J = 8.0 Hz, 1H, NH), 6.75 (d, J = 9.0 Hz, 2H, Ar-H), 6.79 (s, 1H, Ar-H),
7.26 (d, J = 9.0 Hz, 2H, Ar-H), 8.22 (s, 1H, NH).– IR: ν = 3332, 2960, 2929,
1762, 1687, 1654, 1514, 1245, 1172 cm–1.– C35H51N3O7 (625.81). C, H, N.
To a solution of the appropriate starting compound (1 eq) in dry DCM
(3 ml), was added DCC (1.2 eq) at 0 °C and the resulting mixture was stirred
for 10 min. Then HOBt (1.1 eq) was added and the mixture was stirred for
10 min, followed by the addition of p-anisidine (1.1 eq) and DMAP (0.2 eq).
The reaction mixture was stirred for 1 h at 0 °C and stirring was continued
at r.t. overnight. The solvent was evaporated, the residue taken up in ethyl
acetate, and the precipitated urea was separated by filtration. The organic
phase was washed with sat. NaHCO3-solution (3×20 ml), 10% citric acid-
solution (2×20 ml), and water (2×20 ml) and dried with Na2SO4. The solvent
was removedin vacuo andthecrude productpurifiedbycc(ethylacetate/PE).
N-(4-Methoxyphenyl)-3-[2-N-(BOC-prolylvalyloxy)-4,6-dimethylphenyl]-
N-(4-Methoxyphenyl)-3-[2-N-(BOC-alanyloxy)-4,6-dimethylphenyl]-
3,3-dimethylpropionamide (9f)
3,3-dimethylpropionamide (9a)
Starting from 8f (120 mg, 0.23 mmol), DCC (57 mg, 0.28 mmol), HOBt
(39 mg, 0.25 mmol), p-anisidine (31 mg, 0.25 mmol), and DMAP (6 mg,
0.05 mmol), 9f (30 mg, 0.05 mmol, 21%) a colourless solid was obtained.
The purity of the compound was 85% (RP-HPLC).– Mp: 83° C.– 1H NMR:
δ = 1.09 [m, 6H, CH3(Val)], 1.43 (s, 9H, t-Bu), 1.69 (s, 6H, CH3), 1.85 (m,
4H, 3′-H, 4′-H (Pro)], 2.21 (s, 3H, CH3), 2.46 [m, 1H, β-H(Val)], 2.53 (s,
3H, CH3), 2.68, 2.74 (s, 1H, CH2), 3.37 (m, 2H, 5′-H(Pro)], 3.73 (s, 3H,
OCH3), 4.37 [m, 1H, α-H(Pro)], 4.57 [m, 1H, α-H(Val)], 6.51 (s, 1H, Ar-H),
6.74 (d, J = 9.0 Hz, 2H, Ar-H), 6.77 (s, 1H, Ar-H), 7.25 (d, J = 9.0 Hz, 2H,
Ar-H), 7.92 (m, 1H, NH), 8.17 (s, 1H, NH).– IR: ν = 3320, 2971, 2931, 1761,
1678, 1511, 1396, 1244, 1171 cm–1.– C35H49N3O7 (623.79).
Starting from 8a (100 mg, 0.25 mmol), DCC (62 mg, 0.30 mmol), HOBt
(42 mg, 0.28 mmol), p-anisidine (34 mg, 0.28 mmol), and DMAP (6 mg,
0.05 mmol), 9a (57 mg, 0.11 mmol, 46%) was obtained as a colourless
solid.– Mp: 85° C.– 1H NMR: δ = 1.40 (s, 9H, t-Bu), 1.57 [d, J = 7.5 Hz, 3H,
CH3(Ala)], 1.68, 1.70 (s, 3H, CH3), 2.21, 2.46 (s, 3H, CH3), 2.53, 2.63 (s,
1H, CH2), 3.73 (s, 3H, OCH3), 4.51 [dq, J = 7.5 Hz, 1H, α-H(Ala)], 5.14 (d,
J = 7.5 Hz, 1H, NH), 6.54 (s, 1H, Ar-H), 6.73 (d, J = 8.8 Hz, 2H, Ar-H), 6.78
(s, 1H, Ar-H), 7.19 (d, J = 8.8 Hz, 2H, Ar-H), 7.77 (s, 1H, NH).– IR:
ν = 3346, 2976, 2927, 1762, 1663, 1512, 1366, 1245, 1160, 1065 cm–1.–
C
28H38N2O6 (498.62). C, H, N.
Arch. Pharm. Pharm. Med. Chem. 334, 209–215 (2001)

Protease-Sensitive Prodrugs
215
N-(4-Methoxyphenyl)-3-[2-N-(BOC-alanylalanylvalyloxy)-4,6-dimethyl-
phenyl]-3,3-dimethylpropionamide (9g)
References
✩
Dedicated to Prof. Dr. Dr. h.c. P. Pflegel on the occasion of his 65th
birthday.
Starting from 8g (78 mg, 0.14 mmol), DCC (35 mg, 0.17 mmol), HOBt
(24 mg, 0.15 mmol), p-anisidine (19 mg, 0.15 mmol), and DMAP (3 mg,
0.03 mmol), 9g (33 mg, 0.05 mmol, 35%) was obtained as a colourless
[1] W.A. Denny, W.R. Wilson, J. Pharm. Pharmacol. 1998, 50, 387–394.
1
solid.– Mp: 99° C.– H NMR: δ = 1.04 [m, 6H, CH3(Val)], 1.28 [m, 6H,
CH3(Ala)], 1.42 (s, 9H, t-Bu), 1.67, 1.70 (s, 3H, CH3), 2.22 (s, 3H, CH3),
2.41 [m, 1H, β-H(Val)], 2.50 (s, 3H, CH3), 2.71, 2.78 (s, 1H, CH2), 3.73 (s,
3H, OCH3), 4.17 (m, 1H, α-H), 4.58 (m, 2H, α-H), 5.28 (m, 1H, NH), 6.55
(s, 1H, Ar-H), 6.75 (d, J = 9.0 Hz, 2H, Ar-H), 6.79 (s, 1H, Ar-H), 7.23 (m,
1H, NH), 7.26 (d, J = 9.0 Hz, 2H, Ar-H), 7.53 (d, J = 9.2 Hz, 1H, NH), 8.11
(s, 1H, NH).– IR: ν = 3320, 2974, 2932, 1655, 1512, 1245, 1170 cm–1.–
[2] L.F. Tietze, M. Newman, T. Mollers, R. Fischer, K.-H. Glusenkamp,
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C
36H52N4O8 (668.84). C, H, N.
[5] A. Bianco, D. Kaiser, G. Jung, Pept. Res. 1999, 54, 544–548.
N-(4-Methoxyphenyl)-3-[2-N-(BOC-alanylvalylvalyloxy)-4,6-dimethyl-
phenyl]-3,3-dimethylpropionamide (9h)
[6] (a) G.M. Dubowchik, R.A. Firestone, Bioorg. Med. Chem. Lett. 1998,
8, 3341–3346. (b) G.M. Dubowchik, K. Mosure, J.O. Knipe, R.A.
Firestone, Bioorg. Med. Chem. Lett. 1998, 8, 3347–3352.
Starting from 8h (71 mg, 0.12 mmol), DCC (30 mg, 0.14 mmol), HOBt
(20 mg, 0.13 mmol), p-anisidine (16 mg, 0.13 mmol), and DMAP (3 mg,
0.03 mmol), 9h (36 mg, 0.05 mmol, 43%) was obtained as a colourless
solid.– Mp: 165° C.– 1H NMR: δ = 0.91, 0.92, 1.09, 1.13 [d, J = 6.8 Hz, 3H,
CH3(Val)], 1.31 (d, J = 7.3 Hz, 3H, CH3(Ala)], 1.43 (s, 9H, t-Bu), 1.68, 1.70
(s, 3H, CH3), 2.16 [m, 1H, β-H(Val)], 2.22 (s, 3H, CH3), 2.39 [m, 1H,
β-H(Val)], 2.50 (s, 3H, CH3), 2.59, 2.78 (s, 1H, CH2), 3.74 (s, 3H, OCH3),
4.14 [m, 1H, α-H(Val)], 4.37 [dq, J = 7.3 Hz, 1H, α-H(Ala)], 4.63 [dd,
J = 5.4 and 7.7 Hz, 1H, α-H(Val)], 5.15 (d, J = 9.6 Hz, 1H, NH), 6.53 (s, 1H,
Ar-H), 6.76 (d, J = 9.0 Hz, 2H, Ar-H), 6.79 (s, 1H, Ar-H), 6.96 (m, 1H, NH),
7.18 (m, 1H, NH), 7.26 (d, J = 9.0 Hz, 2H, Ar-H), 8.11 (m, 1H, NH).– IR:
ν = 3321, 2967, 2932, 1760, 1651, 1512, 1367, 1245, 1171 cm–1.–
[7] (a) J.-I. Yamashita, M. Ogawa, M. Abe, N. Hayashi, Y. Kurusu, K.
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C
38H56N4O8 (696.89). C, H, N.
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HPLC Experiments
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The respective compound was dissolved in acetonitrile and diluted with
tris-buffer [0.1 M, pH 8.0; final concentration acetonitrile: 15%; final con-
centrations of compounds [mM] (retention times, solvent 1): 9a: 0.055
(8.35 min); 9b: 0.040 (12.17 min); 9c: 0.042 (12.37 min); 9d: 0.046
(9.11 min); 9e: 0.043 (12.19 min); 9f: 0.042 (11.93 min); 9g: 0.040
(6.91 min); 9h: 0.038 (8.78 min)]. The assays were performed at room
temperature with a PPE concentration of 75 µg/ml (stock solution of PPE
was prepared in 1 mM acetic acid). At various times 20 µl aliquots were
withdrawn and immediately analysed by HPLC (detection wavelength:
250 nm; column: CC 250/4 Nucleosil 100-5 C18 HD; oven temperature:
30° C; flow: 0.7 ml/min; mobile phase: KH2PO4 (20 mM, pH 3.3)/acetoni-
trile [either 30/70 (solvent 1) or a linear gradient: within 30 min from 95/5
to 20/80 (solvent 2)].
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[13] A. Kibanov, Acc. Chem. Res. 1990, 23, 114–120.
[14] HyperChem Pro 6.0, Hypercube Inc.
Received: March 15, 2001 [FP554]
Arch. Pharm. Pharm. Med. Chem. 334, 209–215 (2001)