Auxiliary agents for the peroral administration of peptide and protein drugs: Synthesis and evaluation of novel pepstatin analogues

Article abstract of DOI:10.1021/jm980015w

The peroral administration of (poly)peptide drugs requires the development of delivery systems, which provide a protective effect toward a gastrointestinal enzymatic attack. A promising strategy for such systems represents polymer-enzyme inhibitor conjuga

Full text of DOI:10.1021/jm980015w

J . Med. Chem. 1998, 41, 2339-2344
2339
Au xilia r y Agen ts for th e P er or a l Ad m in istr a tion of P ep tid e a n d P r otein Dr u gs:
Syn th esis a n d Eva lu a tion of Novel P ep sta tin An a logu es
Martin Kratzel,*,† Romana Hiessbo¨ck,† and Andreas Bernkop-Schnu¨rch‡
Institute of Pharmaceutical Chemistry, Center of Pharmacy, Althanstrasse 14, A-1090 Vienna, Austria, and
Institute of Pharmaceutical Technology, Center of Pharmacy, Althanstrasse 14, A-1090 Vienna, Austria
Received J anuary 12, 1998
The peroral administration of (poly)peptide drugs requires the development of delivery systems,
which provide a protective effect toward a gastrointestinal enzymatic attack. A promising
strategy for such systems represents polymer-enzyme inhibitor conjugates in which the
embedded therapeutic agent is protected. However, the practical use of polymer-inhibitor
conjugates has so far been limited by high production costs of these auxiliary agents. To solve
this problem for delivery systems shielding from pepsinic degradation, structurally simplified
analogues of the pepsin inhibitor pepstatin A have been synthesized. The synthesis of tripeptide
analogues, described by McConnell et al., led us to pursue further modifications varying the
C-terminus. Our target to attach a spacer moietysenabling the free access of pepsin to the
inhibitorsshould be combined with an attractive synthetic approach providing low production
costs in large-scale preparation. Structure modifications comprised either the side chain of
the third amino acid which served as starting compound designing the C-terminus (L-leucine,
L-isoleucine, L-norvaline) as the length of the spacer link, simulated by a linear alkyl group
(n-butyl, n-hexyl, and n-octyl). The inhibitory activities which have been evaluated by an
enzyme assay were significantly dependent on the nature of the side chain, whereas the length
of the spacer had no influence on the inhibitory effect. Analogues bearing the isobutyl or
n-propyl moiety as side chain displayed a strong inhibitory effect which was comparable to
that pepstatin A. These congeners represent promising auxiliary agents for the peroral
administration of (poly)peptide drugs.
In tr od u ction
already generated a suitable bioadhesive drug delivery
system for its peroral administration.7 Thereby, pep-
statin was covalently attached to a bioadhesive polymer
in which the polypeptide drug was subsequently embed-
ded. The bioadhesive polymer, on one hand, provides
an intimate and prolonged contact with the gastric
mucosa as well as a controlled and sustained drug
release. On the other hand, the inhibitor guarantees a
protective effect toward pepsinic degradation. To keep
it concentrated on the delivery system and to avoid
systemic toxic side effects caused by the inhibition of
physiological essential, but pepstatin A-sensitive en-
zymes,8-10 it was immobilized to the bioadhesive poly-
mer sodium carboxymethylcellulose (f 2). The covalent
attachment of pepstatin was thereby provided via a C8-
spacer supplying the free availability of the inhibitor
for the enzyme.
For the efficient delivery of peptide and protein drugs
by the peroral route, innovative and novel strategies are
needed to overcome the enzymatic barrier of the gas-
trointestinal (GI) tract. Within these strategies, the
combination of bioadhesive polymers with enzyme in-
hibitors has turned out to be a very promising ap-
proach.1-3 Among such enzyme inhibitors, pepstatin A
(1), a slow and tight-binding inhibitor of aspartyl pro-
teinases,4 has recently gained considerable interest as
auxiliary agent for the peroral administration of epi-
dermal growth factor (EGF). Itoh and Matsuo demon-
strated in a double-blind controlled clinical study the
enhanced healing of gastric ulcers after oral administra-
tion of EGF.5 However, this effect is drastically reduced
by the pepsinic degradation of this therapeutic polypep-
tide in the stomach.6 To avoid the proteolysis of EGF
after oral dosing, Bernkop-Schnu¨rch and Dundalek have
However, although this system turned out to be very
successful in various studies,7 its practical use is
strongly limited by the extensive costs of pepstatin.
Therefore, it was the aim of this study to synthesize
simplified pepstatin analogues bearing already an ap-
propriate spacer moiety to provide the basis for linkage
to suitable bioadhesive polymers. The development of
easily accessible analogues should open the door to the
* To whom correspondence should be addressed.
† Institute of Pharmaceutical Chemistry.
‡ Institute of Pharmaceutical Technology.
S0022-2623(98)00015-6 CCC: $15.00 © 1998 American Chemical Society
Published on Web 05/23/1998

2340 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13
Kratzel et al.
Sch em e 1a
As starting compounds benzyloxycarbonyl (Cbz) pro-
tected amino acids (4) were prepared which were
converted to the corresponding Weinreb amides (5) by
reaction with N-methoxy-N-methylhydroxylamine hy-
drochloride after activation with isobutyl chlorofor-
mate.12 The chosen amino acids comprised L-isoleucine
(vide infra) (R1 ) sec-butyl), L-leucine (R1 ) isobutyl),
and L-norvaline as amino acid with a linear side chain
(R1 ) n-propyl) structurally comparable to the strongest
inhibitor synthesized by McConnell et al.11
The following reductive alkylation was performed
using Grignard reagents of different chain length [n-
butylmagnesium bromide (R2 ) n-butyl), n-hexylmag-
nesium bromide (R2 ) n-hexyl), n-octylmagnesium
bromide (R2 ) n-octyl)], affording the ketones 6 in good
to excellent yields. Reduction with sodium borohydride
resulted in a mixture of the diastereoisomeric amino
alcohols 7 in a ratio between 1:2 and 1:4. Taking the
steric hindrances caused by the side chain and the alkyl
spacer into account, we suggest the predominant forma-
tion of the 4S,5S-amino alcohol. Related data concern-
ing the synthesis of pepstatin congeners and vic-amino
alcohols with defined relative stereochemistry can be
found in the literature.13 The last two steps consisted
in deprotection of the amino alcohol by catalytic hydro-
genation to 8 and final coupling with Boc-Val-Val-OH,
yielding the target peptides 9.
a
Isobutyl chloroformate, N-methylmorpholine, N-methoxy-N-
methylhydroxylamine hydrochloride, CH2Cl2, -15 °C to room
temperature; (b) R2MgBr, Et2O, 0 °C; (c) NaBH4, MeOH; (d) Pd/
C/H2, MeOH; (e) EEDQ, THF; R1 ) n-propyl, isobutyl, or sec-butyl;
R2 ) n-butyl, n-hexyl, or n-octyl.
practical use of pepsin inhibitor-polymer conjugates as
vehicles for the peroral administration of EGF as well
as of further peptide and protein drugs which so far
could not be administered by this route of application.
En zym e Assa y
The enzyme inhibitory activity of all compounds (3-
9) was determined using horseradish peroxidase as the
pepsin sensitive protein which is in its native form able
to oxidize o-phenylenediamine, leading to an orange-
colored stain. Pepsin leads to a degradation of this
enzyme. The remaining activity of peroxidase can
therefore be set in correlation with the inhibition of
pepsin effected by the tested compounds.7
Ch em istr y
It was a clue to us that McConnell et al. have reported
on tripeptide analogues 3 of pepstatin A, which can act
as pepsin inhibitors.11 In comparison with pepstatin A,
these compounds contain the first three amino acid
residues and therefore only one statine unit.
Resu lts a n d Discu ssion
Concerning a straight synthetic approach, we sub-
stitued the terminal statine unit of the simplified
tripeptide analogues of pepstatin A, described by Mc-
Connell et al., by various amino alcohol residues.
Whereas all compounds without the Boc-Val-Val moiety
(3-8) did not show any inhibitory activity in the enzyme
assay, all target analogues 9 displayed a strong inhibi-
tory effect toward pepsin. Furthermore, we could
demonstrate that the inhibitory activity is determined
by the nature of the alkyl side chain on C-4, which is in
good accordance with results obtained by McConnell et
al.11 The series derived from L-isoleucine as the third
amino acid exhibited the significantly lowest inhibitory
activities. In contrast, analogues with a linear side
chain on C-4 (derived from L-norvaline) or with a
isobutyl group on C-4 (derived from L-leucine) displayed
a comparably stronger inhibitory activity. The variation
of the spacer, represented by a linear alkyl group on
C-5, had no significant influence on the inhibitory effect
of all tested target compounds (9). Hence, the im-
mobilization of these novel pepstatin analogues to
bioadhesive polymers via spacers bound at C-5 should
have no influence on their inhibitory potency. Results
of inhibition studies are listed in Table 1. Pepstatin A
derivatives, as described here, should therefore allow
The activity of these analogues depends on the side
chain of the terminally located statine. When tested,
eight analogues exhibited a comparably high inhibitory
activity for compounds with a side chain derived from
isoleucinesmatching the structure of naturally occur-
ring statineswhich could even be exceeded by derivative
3 (R ) C4H9) carrying a linear n-butyl side chain. The
N-benzyloxycarbonyl group (instead of the isovaleryl
moiety of pepstatin A) had no considerable influence on
the inhibitory activity.
Regarding our target to immobilize the inhibitor on
the matrix system, an additional modification either of
the N-terminally located group or of the C-terminus
became necessary. Although the C-terminus represents
the sensible part of the molecule, we decided to perform
a simplification of the statine unit, considering a simple
and efficient synthetic approach. Our strategy is out-
lined in Scheme 1.

Novel Pepstatin Analogues
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13 2341
Ta ble 1. Inhibitory Effect of Pepstatin and Analogues toward
the Pepsin Hydrolysis of Horseradish Peroxidase
5R-γ (5 mmol) in dry tetrahydrofuran (20 mL) was added at 0
°C under an argon atmosphere the selected Grignard reagent
(10 mmol of a ethereal solution). The mixture was stirred for
3 h (TLC control) and then quenched with HCl (1 N, 5 mL).
The organic layer was separated, followed by extraction of the
aqueous layer with diethyl ether (2 × 20 mL). The combined
organic extracts were washed with brine, dried, evaporated,
and purified by flash chromatography (ethyl acetate-hexane,
1:1) to afford 6a -i as colorless oils or solids, respectively.
diastereoisomeric
ratio 4S,5S:4S,5R
IC50 (M) ( SD
(n ) 3)
compound
R1
R2
pepstatin A
1:0
2.5:1
3:1
3:1
2:1
2.5:1
2:1
4.5:1
4:1
(5.9 ( 2.5) × 10-8
(9.1 ( 3.2) × 10-7
(7.0 ( 3.5) × 10-7
(10.7 ( 8.5) × 10-7
(12.4 ( 0.3) × 10-7
(8.3 ( 3.6) × 10-7
(7.9 ( 2.7) × 10-7
(3.3 ( 1.4) × 10-5
(2.7 ( 1.2) × 10-5
(1.5 ( 0.9) × 10-5
9a
9b
9c
9d
9e
9f
9g
9h
9i
n-propyl n-butyl
n-propyl n-hexyl
n-propyl n-octyl
isobutyl n-butyl
isobutyl n-hexyl
isobutyl n-octyl
sec-butyl n-butyl
sec-butyl n-hexyl
sec-butyl n-octyl
4-[(Ben zyloxyca r bon yl)a m in o]n on a n -5-on e (6a ). This
compound was synthesized from L-norvaline N-methoxy-N-
methylamide (5R) and n-butylmagnesium bromide as the
Grignard reagent: colorless solid; yield 74%; mp 40-42 °C;
IR (KBr/liquid film, cm-1) 3295 (NH), 1719 (N-CO-O), 1690
4:1
1
(CdO); H NMR (CDCl3) δ 0.83-0.95, 1.15-1.35, 1.36-1.57,
1.66-1.82 (each m, 6H, 4H, 3H, 1H, aliph H), 2.46 (m, 2H,
6-H), 4.37 (m, 1H, 4-H), 5.07 (s, 2H, benzyl-H), 5.50 (br d, 1H,
J ) 6.6 Hz, NH), 7.32 (s, 5H, arom H); 13C NMR (CDCl3) δ
13.7, 13.8 (1-C, 9-C), 18.2, 22.2, 25.5, 33.8 (2-C, 3-C, 7-C, 8-C),
39.4 (6-C), 59.4 (4-C), 66.8 (benzyl-C), 128.0, 128.1, 128.4 (arom
CH), 136.3 (arom C), 155.9 (N-CO-O), 209.2 (CdO); MS m/z
291.2 (M+).
the production of very effective polymer-inhibitor con-
jugates for a reasonable price. Delivery systems based
on such modified polymers are of high practical rel-
evance, representing a useful tool for the peroral
administration of mainly pepsinic degraded peptide and
protein drugs.
In summary, compared to pepstatin A, analogues
generated within this study display following advan-
tages: (i) The presented inhibitors show a strong
inhibitory activity, even by varying the spacer unit. (ii)
The spacer moiety will allow an easy attachment of the
inhibitor to the bioadhesive polymer. (iii) These deriva-
tives can be easily produced under comparably low
production costs.
4-[(Ben zyloxycar bon yl)am in o]u n decan -5-on e (6b). This
compound was synthesized from L-norvaline N-methoxy-N-
methylamide (5R) and n-hexylmagnesium bromide as the
Grignard reagent: colorless solid; yield 68%; mp 47-50 °C;
IR (KBr/liquid film, cm-1) 3295 (NH), 1718 (N-CO-O), 1690
1
(CdO); H NMR (CDCl3) δ 0.79-0.95, 1.17-1.60, 1.68-1.83
(each m, 6H, 11H, 1H, aliph. H), 2.42 (m, 2H, 6-H), 4.33 (m,
1H, 4-H), 5.03 (s, 2H, benzyl-H), 5.77 (br d, 1H, J ) 6.6 Hz,
NH), 7.26 (s, 5H, arom H); 13C NMR (CDCl3) δ 13.5, 13.7 (1-C,
11-C), 18.1, 22.2, 23.1, 28.5, 31.2, 33.3 (2-C, 3-C, 7-C, 8-C, 9-C,
10-C), 39.3 (6-C), 59.2 (4-C), 66.4 (benzyl-C), 127.6, 127.7, 128.1
(arom CH), 136.2 (arom C), 155.8 (N-CO-O), 209.0 (CdO);
MS m/z 319.2 (M+).
Exp er im en ta l Section
Melting points were determined on a Kofler hot plate
apparatus and are uncorrected. Infrared spectra were re-
corded on a Perkin-Elmer 1600 FTIR spectrophotometer. 1H
and 13C NMR spectra were measured on a Varian Unity Plus
300 spectrometer. 1H spectra were referenced to tetrameth-
ylsilane (δ 0.0); in 13C spectroscopy CDCl3 served as the
internal standard (δ 77.0). MS spectra were measured on
Shimadzu QP 5000, Finnigan 8230, and Finnigan MAT 900S
instruments. For inhibition studies a microtitration plate
reader (Anthos Reader 2001) was used.
Flash chromatography was performed on MERCK silica gel
60, TLC on plastic sheets (MERCK silica gel 60 F254). Rf values
were determined using ethyl acetate-hexane (1:1) as the
mobile phase. Tetrahydrofuran was dried over sodium/ben-
zophenone; dichloromethane over phosphorus pentoxide.
4-[(Ben zyloxycar bon yl)am in o]tr idecan -5-on e (6c). This
compound was synthesized from L-norvaline N-methoxy-N-
methylamide (5R) and n-octylmagnesium bromide as the
Grignard reagent: colorless solid; yield 76%; mp 55-58 °C;
IR (KBr/liquid film, cm-1) 3293 (NH), 1718 (N-CO-O), 1693
1
(CdO); H NMR (CDCl3) δ 0.85-0.95, 1.17-1.45, 1.45-1.70,
1.74-1.90 (each m, 6H, 12H, 3H, 1H, aliph H), 2.46 (m, 2H,
6-H), 4.38 (m, 1H, 4-H), 5.07 (s, 2H, benzyl-H), 5.56 (br d, 1H,
J ) 6.6 Hz, NH), 7.32 (s, 5H, arom H); 13C NMR (CDCl3) 13.5,
13.9 (1-C, 13-C), 18.3, 22.5, 23.5, 29.0, 29.3, 29.4, 31.7, 33.7
(2-C, 3-C, 7-C, 8-C, 9-C, 10-C, 11-C, 12-C), 39.7 (6-C), 59.4 (4-
C), 66.8 (benzyl-C), 127.9, 128.0, 128.4 (arom CH), 136.3 (arom
C), 155.9 (N-CO-O), 209.1 (CdO); MS m/z 348.2 (M+ + 1).
4-[(Ben zyloxyca r b on yl)a m in o]-2-m et h yln on a n -5-on e
(6d ). This compound was synthesized from L-leucine N-
methoxy-N-methylamide (5â) and n-butylmagnesium bromide
as the Grignard reagent: colorless oil; yield 71%; IR (KBr/
liquid film, cm-1) 3335 (NH), 1721 (N-CO-O), 1702 (CdO);
1H NMR (CDCl3) δ 0.80-1.08, 1.25-1.43, 1.56, 1.70 (each m,
11H, 2H, 2H, 1H, aliph H), 2.48 (m, 2H, 6-H), 4.39 (m, 1H,
4-H), 5.08 (s, 2H, benzyl-H), 5.36 (br d, 1H, J ) 7.8 Hz, NH),
7.33 (s, 5H, arom H); 13C NMR (CDCl3) δ 13.7 (9-C), 21.7, 23.3,
24.8 (1-C, 2-C, 2-CH3), 22.2, 25.6 (7-C, 8-C), 39.5, 40.8 (3-C,
6-C), 58.2 (4-C), 66.8 (benzyl-C), 127.9, 128.0, 128.4 (arom CH),
136.3 (arom C), 156.1 (N-CO-O), 209.7 (CdO); MS m/z 305.2
(M+).
N-(Ben zyloxyca r b on yl)-L-n or va lin e
N-Met h oxy-N-
m eth yl a m id e (5r). A solution of N-(benzyloxycarbonyl)-L-
norvaline (61.75 mmol, 15.5 g) and N-methylmorpholine (123.5
mmol, 13.6 mL) in dry dichloromethane (150 mL), containing
2 g of molecular sieve (4 Å) was cooled to -15 °C prior to the
slow addition of isobutyl chloroformate (8.0 mL). After 15 min
N-methoxy-N-methylhydroxylamine hydrochloride (64 mmol,
6.22 g) was added. The reaction mixture was stirred for 1 h
at -15 °C, and then the cooling bath was removed and the
solution stirred for a further 3 h. Finally, the solution was
washed with HCl (1 N, 2 × 20 mL), saturated aqueous
NaHCO3 solution (2 × 20 mL), and brine (2 × 20 mL). The
organic layer was dried (Na2SO4) and evaporated, giving a
yellowish oil which was purified by flash chromatography
(ethyl acetate-hexane, 1:1) to afford 16.5 g (91%) of 5R as a
colorless oil: IR (KBr/liquid film, cm-1) 1655 (CO-N(CH3)-
OCH3), 1718 (N-CO-O); 1H NMR (CDCl3: δ 0.89 (br, 3H,
CH3), 1.36, 1.53, 1.65 (each m, 2H, 1H, 1H, CH2CH2), 3.15 (s,
3H, NCH3), 3.73 (s, 3H, OCH3), 4.72 (m, 1H, R-H), 5.05, 5.11
(AB system, 2H, J ) 12.0 Hz, benzyl-H), 5.52 (br, 1H, NH),
7.30 (s, 5H, arom H); 13C NMR (CDCl3) δ 13.4 (CH3), 18.4, 34.5
(CH2CH2), 50.4 (NCH3), 61.2 (OCH3), 66.4 (benzyl-C), 127.6,
127.7, 128.2 (arom CH), 136.2 (arom C), 155.9 (N-CO-O); MS
m/z 294.15 (M+).
4-[(Ben zyloxyca r b on yl)a m in o]-2-m et h ylu n d eca n -5-
on e (6e). This compound was synthesized from L-leucine
N-methoxy-N-methylamide (5â) and n-hexylmagnesium bro-
mide as the Grignard reagent: colorless oil; yield 73%; IR
(KBr/liquid film, cm-1) 3330 (NH), 1718 (N-CO-O), 1705
1
(CdO); H NMR (CDCl3) δ 0.80-1.08, 1.20-1.43, 1.45-1.64,
1.70 (each m, 11H, 6H, 2H, 1H, aliph H), 2.48 (m, 2H, 6-H),
4.39 (m, 1H, 4-H), 5.07 (s, 2H, benzyl-H), 5.37 (br d, 1H, J )
7.8, NH), 7.32 (s, 5H, arom H); 13C NMR (CDCl3) δ 13.9
(11-C), 21.7, 23.3, 24.8 (1-C, 2-C, 2-CH3), 22.4, 23.4, 28.8, 31.5
(7-C, 8-C, 9-C, 10-C), 39.8, 40.8 (3-C, 6-C), 58.2 (4-C), 66.8
(benzyl-C), 127.9, 128.0, 128.4 (arom CH), 136.3 (arom C),
156.1 (N-CO-O), 209.6 (CdO); MS m/z 333.5 (M+).
Gen er a l P r oced u r e for th e P r ep a r a tion of Keton es 6.
To a stirred solution of amino acid N-methoxy-N-methylamide

2342 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13
Kratzel et al.
4-[(Be n zyloxyca r b on yl)a m in o]-2-m e t h ylt r id e ca n -5-
on e (6f). This compound was synthesized from L-leucine
N-methoxy-N-methylamide (5â) and n-octylmagnesium bro-
mide as the Grignard reagent: colorless oil; yield 78%; IR
(KBr/liquid film, cm-1) 3436 (NH), 1711 (N-CO-O), 1699
1H NMR (CDCl3) δ 0.85-0.97, 1.20-1.52 (each m, 6H, 14H,
aliph H), 2.18 (br d, 1H, J ) 4.8 Hz, OH), 3.65 (m, 2H, 4-H,
5-H), 4.89 (br d, 1H, J ) 7.8 Hz, NH), 5.09 (s, 2H, benzyl-H),
7.34 (s, 5H, arom H); 13C NMR (CDCl3) δ 13.9, 14.0 (1-C, 11-
C), 19.4, 22.6, 26.0, 29.3, 31.2, 31.7, 33.1 (2-C, 3-C, 6-C, 7-C,
8-C, 9-C, 10-C), 55.6 (4-C), 66.8 (benzyl-C), 74.6 (5-C), 128.0,
128.1, 128.5 (arom CH), 136.5 (arom C), 156.8 (N-CO-O); MS
m/z 322.2 (M+ + 1).
4-[(Ben zyloxyca r bon yl)a m in o]tr id eca n -5-ol (7c): yield
90%; IR (KBr/liquid film, cm-1) 3317 (OH), 1686 (N-CO-O);
1H NMR (CDCl3) δ 0.83-0.95, 1.20-1.50 (each m, 6H, 18H,
aliph H), 2.24 (br d, 1H, OH), 3.63 (m, 2H, 4-H, 5-H), 4.92 (br
d, 1H, J ) 7.8 Hz, NH), 5.09 (s, 2H, benzyl-H), 7.33 (s, 5H,
arom H); 13C NMR (CDCl3) δ 13.9, 14.0 (1-C, 13-C), 19.4, 22.6,
26.0, 29.2, 29.5, 29.6, 31.2, 31.8, 33.1 (2-C, 3-C, 6-C, 7-C, 8-C,
9-C, 10-C, 11-C, 12-C), 55.6 (4-C), 66.8 (benzyl-C), 74.6 (5-C),
127.9, 128.0, 128.5 (arom CH), 136.5 (arom C), 156.8 (N-CO-
O); MS m/z 350.2 (M+ + 1).
1
(CdO); H NMR (CDCl3) δ 0.82-1.05, 1.20-1.38, 1.48-1.65,
1.70 (each m, 11H, 10H, 2H, 1H, aliph H), 2.49 (t, 2H, J ) 4.5
Hz, 6-H), 4.41 (m, 1H, 4-H), 5.08 (s, 2H, benzyl-H), 5.39 (br d,
1H, J ) 7.5 Hz, NH), 7.33 (s, 5H, arom H); 13C NMR (CDCl3)
δ 13.9 (13-C), 21.7, 23.3, 24.8 (1-C, 2-C, 2-CH3), 22.4, 23.4, 28.8,
29.6, 30.2, 31.9 (7-C, 8-C, 9-C, 10-C, 11-C, 12-C), 39.8, 40.8
(3-C, 6-C), 58.2 (4-C), 66.8 (benzyl-C), 127.9, 128.0, 128.4 (arom
CH), 136.3 (arom C), 156.1 (N-CO-O), 209.8 (CdO); MS m/z
362.3 (M+ + 1).
4-[(Ben zyloxyca r b on yl)a m in o]-3-m et h yln on a n -5-on e
(6g). This compound was synthesized from L-isoleucine N-
methoxy-N-methylamide (5γ) and n-butylmagnesium bromide
as the Grignard reagent: colorless solid; yield 75%; mp 40-
43 °C; IR (KBr/liquid film, cm-1) 3340 (NH), 1728 (N-CO-
O), 1713 (CdO); 1H NMR (CDCl3) δ 0.84-1.09, 1.29, 1.55, 1.90
(each m, 10H, 3H, 2H, 1H, aliph H), 2.47 (t, 2H, J ) 7.2 Hz,
6-H), 4.34 (m, 1H, 4-H), 5.07 (s, 2H, benzyl-H), 5.46 (br d, 1H,
J ) 7.2 Hz, NH), 7.32 (s, 5H, arom H); 13C NMR (CDCl3) δ
11.5, 13.7 (1-C, 9-C), 16.0 (3-CH3), 22.1, 23.9, 25.4 (2-C, 7-C,
8-C), 37.0 (3-C), 40.7 (6-C), 64.2 (4-C), 66.8 (benzyl-C), 127.9,
128.0, 128.4 (arom CH), 136.2 (arom C), 156.3 (N-CO-O),
210.0 (CdO); MS m/z 306.2 (M+ + 1).
4-(Be n zyloxyca r b on yla m in o)-3-m e t h yl-u n d e ca n -5-
on e (6h ). This compound was synthesized from L-isoleucine
N-methoxy-N-methylamide (5γ) and n-hexylmagnesium bro-
mide as the Grignard reagent: colorless oil; yield 70%; IR
(KBr/liquid film, cm-1) 3340 (NH), 1721 (N-CO-O), 1710
(CdO); 1H NMR (CDCl3) δ 0.83-1.15, 1.18-1.38, 1.57, 1.91
(each m, 10H, 7H, 2H, 1H, aliph. H), 2.47 (m, 2H, 6-H), 4.36
(m, 1H, 4-H), 5.09 (s, 2H, benzyl-H), 5.40 (br d, 1H, J ) 7.8
Hz, NH), 7.34 (s, 5H, arom H); 13C NMR (CDCl3) δ 11.6, 14.0
(1-C, 11-C), 16.2 (3-CH3), 22.4, 23.4, 24.0, 28.8, 31.5 (2-C, 7-C,
8-C, 9-C, 10-C), 37.1 (3-C), 41.1 (6-C), 64.2 (4-C), 66.9 (benzyl-
C), 128.0, 128.1, 128.5 (arom CH), 136.3 (arom C), 156.4 (N-
CO-O), 210.0 (CdO); MS m/z 333.3 (M+).
4-[(Be n zyloxyca r b on yl)a m in o]-3-m e t h ylt r id e ca n -5-
on e (6i). This compound was synthesized from L-isoleucine
N-methoxy-N-methylamide (5γ) and n-octylmagnesium bro-
mide as the Grignard reagent: colorless solid; yield 74%; mp
32-37 °C; IR (KBr/liquid film, cm-1) 3305 (NH), 1715 (N-
CO-O), 1687 (CdO); 1H NMR (CDCl3) δ 0.82-1.05, 1.15-1.38,
1.55, 1.90 (each m, 10H, 11H, 2H, 1H, aliph H), 2.48 (t, 2H, J
) 7.5 Hz, 6-H), 4.34 (m, 1H, 4-H), 5.08 (s, 2H, benzyl-H), 5.43
(br d, 1H, J ) 8.4 Hz, NH), 7.33 (s, 5H, arom H); 13C NMR
(CDCl3) δ 11.6, 14.0 (1-C, 13-C), 16.1 (3-CH3), 22.6, 23.4, 23.9,
28.8, 29.0, 29.6, 31.8 (2-C, 7-C, 8-C, 9-C, 10-C, 11-C, 12-C),
37.1 (3-C), 41.1 (6-C), 64.2 (4-C), 66.9 (benzyl-C), 128.0, 128.1,
128.4 (arom CH), 136.1 (arom C), 156.4 (N-CO-O), 209.4
(CdO); MS m/z 254.1 (M+ - 107).
4-[(Ben zyloxycar bon yl)am in o]-2-m eth yln on an -5-ol (7d):
yield 91%; IR (KBr/liquid film, cm-1) 3336 (OH), 1687 (N-
1
CO-O); H NMR (CDCl3) δ 0.82-0.98, 1.18-1.56, 1.58-1.72
(each m, 9H, 8H, 1H, aliph H), 2.18 (br, 1H, OH), 3.50-3.80
(m, 2H, 4-H, 5-H), 4.86 (br d, 1H, J ) 7.2 Hz, NH), 5.09 (s,
2H, benzyl-H), 7.33 (s, 5H, arom H); 13C NMR (CDCl3) δ 14.0
(9-C), 21.6, 23.7, 24.7 (1-C, 2-C, 2-CH3), 22.7, 28.2, 32.7, 38.0
(3-C, 6-C, 7-C, 8-C), 53.9 (4-C), 66.8 (benzyl-C), 74.8 (5-C),
128.0, 128.1, 128.5 (arom CH), 136.4 (arom C), 156.7 (N-CO-
O); MS m/z 308.2 (M+ + 1).
4-[(Ben zyloxyca r b on yl)a m in o]-2-m et h ylu n d eca n -5-
ol (7e): yield 93%; IR (KBr/liquid film, cm-1) 3334 (OH), 1687
1
(N-CO-O); H NMR (CDCl3) δ 0.82-0.98, 1.15-1.54, 1.56-
1.70 (each m, 9H, 12H, 1H, aliph H), 2.30 (br, 1H, OH), 3.50-
3.78 (m, 2H, 4-H, 5-H), 4.98 (br d, 1H, J ) 7.8 Hz, NH), 5.07
(s, 2H, benzyl-H), 7.32 (s, 5H, arom H); 13C NMR (CDCl3) δ
14.0 (11-C), 22.1, 23.1, 24.7 (1-C, 2-C, 2-CH3), 22.5, 25.7, 29.2,
31.7, 34.2, 41.7 (3-C, 6-C, 7-C, 8-C, 9-C, 10-C), 52.8 (4-C), 66.6
(benzyl-C), 73.7 (5-C), 127.8, 127.9, 128.4 (arom CH), 136.6
(arom C), 156.7 (N-CO-O); MS m/z 336.2 (M+ + 1).
4-[(Be n zyloxyca r b on yl)a m in o]-2-m e t h ylt r id e ca n -5-
ol (7f): yield 92%; IR (KBr/liquid film, cm-1) 3339 (OH), 1685
1
(N-CO-O); H NMR (CDCl3) δ 0.80-0.98, 1.15-1.55, 1.57-
1.72 (each m, 9H, 16H, 1H, aliph H), 2.19 (br, 1H, OH), 3.54-
3.80 (m, 2H, 4-H, 5-H), 4.86 (br d, 1H, J ) 8.4 Hz, NH), 5.09
(s, 2H, benzyl-H), 7.34 (s, 5H, arom H); 13C NMR (CDCl3) δ
14.1 (13-C), 22.1, 23.2, 24.7 (1-C, 2-C, 2-CH3), 22.6, 26.0, 29.2,
29.5, 29.7, 31.8, 33.1, 38.1 (3-C, 6-C, 7-C, 8-C, 9-C, 10-C,11-C,
12-C), 53.9 (4-C), 66.8 (benzyl-C), 74.8 (5-C), 128.0, 128.1, 128.5
(arom CH), 136.5 (arom C), 156.7 (N-CO-O); MS m/z 364.3
(M+ + 1).
4-[(Ben zyloxycar bon yl)am in o]-3-m eth yln on an -5-ol (7g):
yield 90%; IR (KBr/liquid film, cm-1) 3337 (OH), 1688 (N-
1
CO-O); H NMR (CDCl3) δ 0.80-0.97, 0.98-1.18, 1.20-1.64
(each m, 9H, 1H, 8H, aliph H), 2.30 (br, 1H, OH), 3.55-3.62,
3.62-3.68 (each m, 1H, 1H, 4-H, 5-H), 4.74 (br d, 1H, J ) 8.7
Hz, NH), 5.09 (s, 2H, benzyl-H), 7.34 (s, 5H, arom H); 13C NMR
(CDCl3) δ 11.3, 14.0, 16.2 (1-C, 3-CH3, 9-C), 22.7, 24.6, 28.1,
31.9 (2-C, 6-C, 7-C, 8-C) 35.4 (3-C), 60.5 (4-C), 66.9 (benzyl-
C), 72.3 (5-C), 128.0, 128.1, 128.5 (arom CH), 136.4 (arom C),
157.3 (N-CO-O); MS m/z 308.2 (M+ + 1).
Gen er a l P r oced u r e for th e P r ep a r a tion of Alcoh ols 7.
To a solution of ketone 6 (5 mmol) in methanol (25 mL) was
added at room temperature 250 mg of sodium borohydride.
After 30 min of stirring (TLC control), the solution was
neutralized with HCl (2 N), concentrated, and partitioned
between water (50 mL) and dichloromethane (20 mL). After
extraction of the aqueous phase with dichloromethane (2 ×
20 mL), the organic layers were combined, dried (Na2SO4), and
brought to dryness, yielding alcohols 7 as white solids.
4-[(Ben zyloxycar bon yl)am in o]n on an -5-ol (7a): yield 85%;
IR (KBr/liquid film, cm-1) 3302 (OH), 1687 (N-CO-O); 1H
NMR (CDCl3): δ 0.82-0.95, 1.20-1.55 (each m, 6H, 10H, aliph
H), 2.16 (br, 1H, OH), 3.54-3.72 (m, 2H, 4-H, 5-H), 4.89 (br d,
1H, J ) 7.8 Hz, NH), 5.09 (s, 2H, benzyl-H), 7.34 (s, 5H, arom
H); 13C NMR (CDCl3) δ 13.9, 14.0 (1-C, 9-C), 19.4, 22.7 (2-C,
8-C), 27.9, 28.2, 31.2 (3-C, 6-C, 7-C), 54.6/55.6 (4-C), 66.8
(benzyl-C), 73.3/74.5 (5-C), 128.0, 128.1, 128.5 (arom CH), 136.5
(arom C), 156.8 (N-CO-O); MS m/z 294.3 (M+ + 1).
4-[(Ben zyloxyca r b on yl)a m in o]-3-m et h ylu n d eca n -5-
ol (7h ): yield 94%; IR (KBr/liquid film, cm-1) 3335 (OH), 1688
1
(N-CO-O); H NMR (CDCl3) δ 0.80-0.98, 0.98-1.18, 1.18-
1.65 (each m, 9H, 1H, 12H, aliph H), 2.14 (br, 1H, OH), 3.55-
3.62, 3.65-3.75 (each m, 1H, 1H, 4-H, 5-H), 4.69 (br d, 1H, J
) 9.3 Hz, NH), 5.10 (s, 2H, benzyl-H), 7.34 (s, 5H, arom H);
13C NMR (CDCl3) δ 11.3, 14.0, 16.2 (1-C, 3-CH3, 11-C), 22.6,
24.7, 25.9, 29.3, 31.8, 32.2 (2-C, 6-C, 7-C, 8-C, 9-C, 10-C), 35.4
(3-C), 60.5 (4-C), 66.9 (benzyl-C), 72.4 (5-C), 128.0, 128.1, 128.5
(arom CH), 136.4 (arom C), 157.3 (N-CO-O); MS m/z 336.2
(M+ + 1).
4-[(Be n zyloxyca r b on yl)a m in o]-3-m e t h ylt r id e ca n -5-
ol (7i): yield 90%; IR (KBr/liquid film, cm-1) 3337 (OH), 1687
1
4-[(Ben zyloxyca r bon yl)a m in o]u n d eca n -5-ol (7b): yield
(N-CO-O); H NMR (CDCl3) δ 0.81-0.98, 0.98-1.18, 1.18-
92%; IR (KBr/liquid film, cm-1) 3314 (OH), 1686 (N-CO-O);
1.63 (each m, 9H, 1H, 16H, aliph H), 2.22 (br, 1H, OH), 3.55-

Novel Pepstatin Analogues
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13 2343
3.62, 3.63-3.73 (each m, 1H, 1H, 4-H, 5-H), 4.72 (br d, 1H, J
) 9.6 Hz, NH), 5.09 (s, 2H, benzyl-H), 7.34 (s, 5H, arom H);
13C NMR (CDCl3) δ 11.3, 14.0, 16.2 (1-C, 3-CH3, 13-C), 22.6,
24.6, 26.0, 29.2, 29.5, 29.7, 31.8, 32.2 (2-C, 6-C, 7-C, 8-C, 9-C,
10-C, 11-C, 12-C), 35.4 (3-C), 60.5 (4-C), 66.9 (benzyl-C), 72.3
(5-C), 128.0, 128.1, 128.5 (arom CH), 136.4 (arom C), 157.3
(N-CO-O); MS m/z 364.2 (M+ + 1).
Gen er a l P r oced u r e for th e P r ep a r a tion of Am in o
Alcoh ols 8. The N-Cbz-protected amino alcohol 7 (3 mmol)
was dissolved in methanol (20 mL, with addition of 0.1 mL of
acetic acid) and hydrogenated with Pd/C (80 mg, 10%) as
catalyst overnight. After filtration the solution was concen-
trated in vacuo, redissolved in dichloromethane, and washed
with Na2CO3 solution (2 N, 2 × 20 mL) and brine (2 × 20 mL).
Then, the solution was dried (Na2SO4) and evaporated under
reduced pressure, yielding amino alcohols 8 as white solids.
4-Am in on on a n -5-ol (8a ): yield 85%; 1H NMR (CDCl3) δ
0.80-0.95 (m, 6H, CH3), 1.15-1.40 (m, 10H, CH2), 2.42/2.57
(m, 1H, 4-H), 3.15/3.33 (m, 1H, 5-H), 3.80 (br, 3H, NH2, OH);
13C NMR (CDCl3 + MeOD-d3) δ 13.6, 13.7 (1-C, 9-C), 19.3, 22.4,
28.1, 31.0, 33.5 (2-C, 3-C, 6-C, 7-C, 8-C), 54.9 (4-C), 74.1 (5-
C); MS m/z 160.2 (M+ + 1).
9-C, 10-C, 11-C, 12-C), 38.5 (3-C), 61.2 (4-C), 72.3 (5-C); MS
m/z 230.2 (M+ + 1).
Gen er a l P r oced u r e for Am id e F or m a tion yield in g
Com p ou n d s 9. A mixture of N-deprotected amino alcohol 8
(1 mmol) and Boc-Val-Val-OH (1 mmol, 320 mg) in a solvent
mixture of tetrahydrofuran and dichloromethane was treated
with EEDQ (2-ethoxy-1-(ethoxycarbonyl)-1,2-dihydroquinoline)
(1.2 mmol, 300 mg) and stirred at room temperature for 24 h.
Then, the solution was evaporated, redissolved in ethyl acetate,
and washed with HCl (1 N, 2 × 10 mL), saturated aqueous
NaHCO3 solution (2 × 20 mL), and brine (2 × 20 mL). The
organic phase was dried (Na2SO4) and evaporated. Flash
chromatography of the residue (ethyl acetate-hexane; 1:1)
afforded the pure products.
4-[N -[(t er t -Bu t oxyca r b on yl)-L-va lyl-L-va lyl]a m in o]-
n on a n -5-ol (9a ): yield 63%; Rf 0.45; HRMS 457.352 (M+), calcd
457.352 for C24H47N3O5.
4-[N-[(ter t-Bu toxyca r bon yl)-L-va lyl-L-va lyl]a m in o]u n -
d eca n -5-ol (9b): yield 60%; Rf 0.45; IR (KBr/liquid film, cm-1
)
1
3328 (NH, OH), 1716 (N-CO-O), 1660 (CO-NH); H NMR
(CDCl3 + MeOD-d3) δ 0.80-0.92, 1.05-1.15, 1.15-1.40 (m,
32H, chain-CH2, CH3), 1.42 [s, 9H, C(CH3)3], 2.13, 2.18 [each
m, 1H, 1H, CH(CH3)2], 3.52, 4.08, 4.25, 4.90 (each m, R-H, 4-H,
5-H), 5.25, 6.85, 7.30 (3 × br d, NH, OH); HRMS 485.383 (M+),
calcd 485.383 for C26H51N3O5.
4-Am in ou n d eca n -5-ol (8b): yield 88%; 1H NMR (CDCl3 +
MeOD-d3) δ 0.64-0.80 (m, 6H, CH3), 1.00-1.35 (m, 14H, CH2),
2.38/2.53 (m, 1H, 4-H), 3.22/3.30 (m, 1H, 5-H), 4.18 (br, 3H,
NH2, OH); 13C NMR (CDCl3 + MeOD-d3) δ 13.5, 13.6 (1-C, 11-
C), 19.2, 22.2, 25.8, 29.0, 31.2, 31.5, 33.2 (2-C, 3-C, 6-C, 7-C,
8-C, 9-C, 10-C), 54.9 (4-C), 73.9 (5-C); MS m/z 188.2 (M+ + 1).
4-Am in otr id eca n -5-ol (8c): yield 82%; 1H NMR (CDCl3 +
MeOD-d3) δ 0.62-0.78 (m, 6H, CH3), 1.05-1.38 (m, 18H, CH2),
2.40/2.51 (m, 1H, 4-H), 3.24/3.35 (m, 1H, 5-H), 4.10 (br, 3H,
NH2, OH); 13C NMR (CDCl3 + MeOD-d3) δ 13.5, 13.6 (1-C, 13-
C), 19.2, 22.2, 25.8, 29.0, 29.3, 29.8, 31.2, 31.5, 33.2 (2-C, 3-C,
6-C, 7-C, 8-C, 9-C, 10-C, 11-C, 12-C), 54.8 (4-C), 74.0 (5-C);
MS m/z 216.2 (M+ + 1).
4-[N -[(t er t -Bu t oxyca r b on yl)-L -va lyl-L -va lyl]a m in o]
tr id eca n -5-ol (9c): yield 68%; Rf 0.50; HMRS 513.414 (M+),
calcd 513.414 for C28H55N3O5.
4-[N-[(ter t-Bu t oxyca r b on yl)-L-va lyl-L-va lyl]a m in o]-2-
m eth yln on a n -5-ol (9d ): yield 65%; Rf 0.45; HMRS 471.367
(M+), calcd 471.367 for C25H49N3O5.
4-[N-[(ter t-Bu t oxyca r b on yl)-L-va lyl-L-va lyl]a m in o]-2-
m eth ylu n d eca n -5-ol (9e): yield 68%; Rf 0.55; IR (KBr/liquid
film, cm-1) 3370 (NH, OH), 1720 (N-CO-O), 1670 (CO-NH);
1H NMR (CDCl3) δ 0.80-0,95 (m, 21H, CH3), 1.15-1.48 (m,
12H, chain-CH2), 1.41 [s, 9H, C(CH3)3], 2.19, 2.38 [each m, 1H,
1H, CH(CH3)2], 3.33, 3.48, 3.95, 4.37 (each m, R-H, 4-H, 5-H),
5.40, 6.90, 7.20 (3 × br d, NH, OH); HRMS m/z 499.399 (M+),
calcd 499.399 for C27H53N3O5.
4-Am in o-2-m eth yln on a n -5-ol (8d ): yield 89%; 1H NMR
(CDCl3 + MeOD-d3) δ 0.60-0.85 (m, 9H, CH3), 0.95-1.15, 1.22,
1.43 (each m, 7H, 1H, 1H, CH, CH2), 2.43/2.58 (m, 1H, 4-H),
3.08/3.28 (m, 1H, 5-H), 4.18 (br, 3H, NH2, OH); 13C NMR
(CDCl3 + MeOD-d3) δ 13.3 (9-C), 21.0, 23.0, 24.1 (1-C, 2-C,
2-CH3), 22.2, 27.9, 30.8, 33.7 (3-C, 6-C, 7-C, 8-C), 52.8 (4-C),
73.6 (5-C); MS m/z 173.2 (M+).
4-[N-[(ter t-Bu t oxyca r b on yl)-L-va lyl-L-va lyl]a m in o]-2-
m eth yltr id eca n -5-ol (9f): yield 70%; Rf 0.60; HRMS m/z
527.430 (M+), calcd 527.430 for C29H57N3O5.
4-Am in o-2-m eth ylu n d eca n -5-ol (8e): yield 87%; 1H NMR
(CDCl3 + MeOD-d3) δ 0.60-0.85 (m, 9H, CH3), 0.90-1.15, 1.25,
1.45 (each m, 11H, 1H, 1H, CH, CH2), 2.45/2.58 (m, 1H, 4-H),
3.05/3.27 (m, 1H, 5-H), 4.20 (br, 3H, NH2, OH); 13C NMR
(CDCl3 + MeOD-d3) δ 13.2 (11-C), 20.9, 23.0, 24.0 (1-C, 2-C,
2-CH3), 22.2, 25.8, 29.0, 31.2, 31.5, 33.2 (3-C, 6-C, 7-C, 8-C,
9-C, 10-C), 52.9 (4-C), 73.8 (5-C); MS m/z 201.2 (M+).
4-Am in o-2-m eth yltr id eca n -5-ol (8f): yield 88%; 1H NMR
(CDCl3) δ 0.78-0.98 (m, 9H, CH3), 1.12-1.36, 1.44, 1.65 (each
m, 15H, 1H, 1H, CH, CH2), 2.58/2.80 (m, 1H, 4-H), 3.24/3.42
4-[N-[(ter t-Bu t oxyca r b on yl)-L-va lyl-L-va lyl]a m in o]-3-
m eth yln on a n -5-ol (9g): yield 58%; Rf 0.55; HRMS m/z
471.367 (M+), calcd 471.367 for C25H49N3O5.
4-[N-[(ter t-Bu t oxyca r b on yl)-L-va lyl-L-va lyl]a m in o]-3-
m eth ylu n d eca n -5-ol (9h ): yield 60%; Rf 0.60; IR (KBr/liquid
film, cm-1) 3436 (NH, OH), 1718 (N-CO-O), 1670 (CO-NH);
1H NMR (CDCl3 + MeOD-d3) δ 0.70-1.05, 1.10-1.28 (m, 32H,
chain-CH2, CH3), 1.42 [s, 9H, C(CH3)3], 2.06, 2.20 [each m, 1H,
1H, CH(CH3)2], 4.00, 4.32, 4.90, 4.99 (each m, R-H, 4-H, 5-H),
7.20 (br d, NH); HRMS m/z 499.398, calcd 499.398 for
(m, 1H, 5-H), 4.18 (br, 3H, NH2, OH); 13C NMR (CDCl3
+
C
27H53N3O5.
MeOD-d3) δ 13.3 (9-C), 20.9, 22.9, 24.1 (1-C, 2-C, 2-CH3), 22.6,
25.7, 29.2, 29.5, 31.2, 31.8, 33.1, 34.3 (3-C, 6-C, 7-C, 8-C, 9-C,
10-C, 11-C, 12-C), 53.0 (4-C), 73.5 (5-C); MS m/z 229.2 (M+).
4-Am in o-3-m eth yln on a n -5-ol (8g): yield 90%; 1H NMR
(CDCl3) δ 0.83-0.90 (m, 9H, CH3), 1.12-1.42, 1.53, 1.65 (each
m, 9H, CH, CH2), 2.38/2.48 (m, 1H, 4-H), 3.45/3.58 (m, 1H,
5-H); 13C NMR (CDCl3) δ 10.8, 14.1, 15.6 (1-C, 2-CH3, 9-C),
22.8, 25.4, 28.4, 29.8 (2-C, 6-C, 7-C, 8-C), 37.7 (3-C), 60.2 (4-
C), 71.4 (5-C); MS m/z 174.2 (M+ + 1).
4-[N-[(ter t-Bu t oxyca r b on yl)-L-va lyl-L-va lyl]a m in o[-3-
m eth yltr id eca n -5-ol (9i): yield 63%; Rf 0.60; HRMS m/z
527.430 (M+), calcd 527.430 for C29H57N3O5.
En zym e Assa y. Pepsin inhibition studies of all compounds
(3-9) were carried out in
a modified way as described
previously by Bernkop-Schnu¨rch and Dundalek.7 First, 1.00
mg of the test compound was dissolved in 500 µL of dimethyl
sulfoxide (DMSO) and the solution diluted in 1:10 (v:v) steps
with the same solvent. The 100 L of an appropriate dilution
was transferred to the first well of a microtitration plate (96-
well, not binding) and diluted in 1:2 (v:v) steps with DMSO in
the following five vertical wells. To each 50 µL of diluted test
compound, 75 µL of a fresh prepared pepsin solution [1 mg of
pepsin (40 units; Sigma, St. Louis, MO) per mL of 0.05 N HCl]
were added. Next, 37.5 µg of horseradish peroxidase (Sigma)
in 75 µL of 0.05 N HCl was transferred to each well, and
reaction mixtures were incubated for 2 h at 37 °C. Thereafter,
samples were diluted in 1:2 (v:v) steps with 0.05 N HCl in the
following eleven horizontal wells. To each 100 µL of diluted
reaction mixture was added 100 µL of the substrate medium
(24 mg of o-phenylenediamine dihydrochloride, 12 mL of 0.2
4-Am in o-3-m eth ylu n d eca n -5-ol (8h ): yield 92%; 1H NMR
(CDCl3) δ 0.85-0.96 (m, 9H, CH3), 1.15-1.45, 1.55, 1.60-1.70
(each m, 13H, CH, CH2), 2.38/2.48 (m, 1H, 4-H), 3.45/3.60 (m,
1H, 5-H); 13C NMR (CDCl3) δ 10.8, 14.1, 15.6 (1-C, 2-CH3, 11-
C), 22.6, 25.5, 26.1, 29.4, 30.1, 31.9 (2-C, 6-C, 7-C, 8-C, 9-C,
10-C), 37.8 (3-C), 60.3 (4-C), 71.3 (5-C); MS m/z 202.2 (M+
1).
+
4-Am in o-3-m eth yltr id eca n -5-ol (8i): yield 91%; 1H NMR
(CDCl3) δ 0.82-0.97 (m, 9H, CH3), 1.10-1.44, 1.55, 1.65 (each
m, 17H, CH, CH2), 2.36/2.48 (m, 1H, 4-H), 3.43/3.60 (m, 1H,
5-H); 13C NMR (CDCl3) δ 10.7, 14.0, 15.6 (1-C, 2-CH3, 13-C),
22.5, 23.8, 25.3, 26.1, 29.2, 29.6, 30.0, 31.8 (2-C, 6-C, 7-C, 8-C,

2344 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13
Kratzel et al.
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