Structure-activity relationships on phenylalanine-containing inhibitors of histone deacetylase: In vitro enzyme inhibition, induction of differentiation, and inhibition of proliferation in friend leukemic cells

Article abstract of DOI:10.1021/jm0208119

Inhibitors of histone deacetylases (HDACs) are a new class of anticancer agents that affect gene regulation. We had previously reported the first simple synthetic HDAC inhibitors with in vitro activity at submicromolar concentrations. Here, we present structure-activity data on modifications of a phenylalanine-containing lead compound including amino acid amides as well as variations of the amino acid part. The compounds were tested for inhibition of maize HD-2, rat liver HDAC, and for the induction of terminal cell differentiation and inhibition of proliferation in Friend leukemic cells. In the amide series, in vitro inhibition was potentiated up to 15-fold, but the potential to induce cell differentiation decreased. Interestingly, an HDAC class selectivity was indicated among some of these amides. In the amino acid methyl ester series, a biphenylalanine derivative was identified as a good enzyme inhibitor, which blocks proliferation in the submicromolar range and is also a potent inducer of terminal cell differentiation.

Full text of DOI:10.1021/jm0208119

3296
J . Med. Chem. 2002, 45, 3296-3309
Str u ctu r e-Activity Rela tion sh ip s on P h en yla la n in e-Con ta in in g In h ibitor s of
Histon e Dea cetyla se: In Vitr o En zym e In h ibition , In d u ction of Differ en tia tion ,
a n d In h ibition of P r olifer a tion in F r ien d Leu k em ic Cells
Sybille Wittich,† Hans Scherf,‡ Changping Xie,‡ Gerald Brosch,§ Peter Loidl,§ Clarissa Gerha¨user,‡ and
Manfred J ung*,†
Department of Pharmaceutical and Medicinal Chemistry, Westfa¨lische Wilhelms-Universita¨t Mu¨nster,
Hittorfstrasse 58-62, 48149 Mu¨nster, Germany, Toxicology and Cancer Risk Factors, Deutsches Krebsforschungszentrum,
Im Neuenheimer Feld 280, 69120 Heidelberg, Germany, and Department of Molecular Biology, Medical School, University of
Innsbruck, Fritz-Pregl-Strasse 3, 6020 Innsbruck, Austria
Received J anuary 3, 2002
Inhibitors of histone deacetylases (HDACs) are a new class of anticancer agents that affect
gene regulation. We had previously reported the first simple synthetic HDAC inhibitors with
in vitro activity at submicromolar concentrations. Here, we present structure-activity data
on modifications of a phenylalanine-containing lead compound including amino acid amides
as well as variations of the amino acid part. The compounds were tested for inhibition of maize
HD-2, rat liver HDAC, and for the induction of terminal cell differentiation and inhibition of
proliferation in Friend leukemic cells. In the amide series, in vitro inhibition was potentiated
up to 15-fold, but the potential to induce cell differentiation decreased. Interestingly, an HDAC
class selectivity was indicated among some of these amides. In the amino acid methyl ester
series, a biphenylalanine derivative was identified as a good enzyme inhibitor, which blocks
proliferation in the submicromolar range and is also a potent inducer of terminal cell
differentiation.
In tr od u ction
such as SAHA (6a )15 and pyroxamide (6b).16 This class
of compounds had already been known as inducers of
differentiation17 before the development of our lead
compounds and was later identified as HDAC inhibitors.
Compounds 6a ,b have now entered phase I clinical
trials (Chart 1). We reported on structure-activity data
of analogues of 4a with the most potent compound being
its homologue M344 (4b), which induced differentiation
and inhibited proliferation at 500 nM in Friend leuke-
mic cells.18 Several interesting compounds from other
research laboratories have been added to the group of
HDAC inhibitors over the years, but there is still
comparatively little structure-activity data available.
There are reports on pyrrole analogues of 2,19 which are
less potent and a series of benzamides with an orally
active compound called MS27520,21 as the most interest-
ing member. Extensive structure-activity data have
been reported on analogues of the tetrapeptide apicidin
that also displays antiprotozoal activity.22-27 New re-
ports that aim toward subtype selective HDAC inhibi-
tors have been presented as well.28,29
Histone deacetylase (HDAC) inhibitors are a rela-
tively new class of potential drugs for the treatment of
hyperproliferative diseases.1-3 They induce hyperacety-
lation of chromatin, which in turn usually leads to the
relief of transcriptional repression for a certain subset
of genes.4,5 Among these are genes encoding for proteins
that are crucial for the regulation of cell proliferation
or differentiation, e.g., the cyclin-dependent kinase
inhibitor protein p21/WAF1/CIP1.6,7 The aberrant re-
cruitment of HDACs by oncogenic fusion proteins seems
to be a general mechanism for the pathogenesis of
leukemia.8,9 HDAC inhibitors have demonstrated po-
tential for the prevention and treatment of cancer in
numerous cell culture10 and animal models,11 and first
promising data on humans are available as well.12
Therefore, HDACs have emerged as an attractive target
for new anticancer drugs and there is a great demand
for new inhibitors.
J ust five years ago, only a few hydrophobic cyclotet-
rapeptides such as trapoxin B (1) and the antifungal
antibiotic trichostatin A (2) were known as potent
inhibitors of HDAC.13 We had proposed a general model
for HDAC inhibitors 3 and have presented the first
simple synthetic compounds MD85 (4a ) and M232 (5)
that were designed to be potent inhibitors of HDAC
according to this model.14 Our compounds are structur-
ally related to the so-called hybrid polar compounds
So far, there is only limited structural information on
HDACs available. X-ray structures of a bacterial en-
zyme called HDLP with 2 and 6a and without an
inhibitor have been solved,30 and two reports on nico-
tinamide adenine dinucleotide (NAD+)-dependent de-
acetylases, which are not affected by the classical HDAC
inhibitors, are available as well.31,32 HDLP is a homo-
logue of both mammal class enzyme I (homologues of
the yeast transcriptional regulator and HDAC rpd3) and
mammal class enzyme II (homologues of the yeast
transcriptional regulator and HDAC HDA1). The HDLP
structure is useful only on the region surrounding the
* To whom correspondence should be addressed. Tel: +49-251-83-
33335. Fax: +49-251-83-32144. E-mail: jungm@uni-muenster.de.
† Westfa¨lische Wilhelms-Universita¨t Mu¨nster.
‡ Deutsches Krebsforschungszentrum.
§ University of Innsbruck.
10.1021/jm0208119 CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/20/2002

Phenylalanine-Containing Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15 3297
Ch a r t 1. General Model 3 and Examples of Inhibitors of HDAC (1, 2, and 4-6); Proposal for Structural Modifications
7
spacer part of the inhibitors and the catalytic site, which
are conserved in mammal class I and II enzymes. Thus,
further development is still strongly driven by ligand
modification. Here, we report structural modifications
of our lead compound 5 and their effect on the inhibition
of maize HD-2 and a rat liver HDAC preparation as well
as on the inhibition of proliferation and induction of
terminal cell differentiation in Friend leukemic cells.
Maize HD-2 had shown a good predictive value in a
qualitative and also semiquantitative fashion in the
series of amide analogues 4 of trichostatin A (2).18 This
had not necessarily to be the case as maize HD-2 is
structurally quite different from mammalian HDACs33
and has been attributed to a HDAC class of its own,
which is called class III.5 The latter term has also been
used for the newly discovered class of the NAD+-
dependent Sir family.34 Maize HD-2 is a nucleolar
phosphoprotein with sequence homology to other phos-
phoproteins such as B23 or FKBPs,5 and its active site
might be different from the HDLP homologues from
classes I and II. Still, it served as a good model in
previous studies and we wanted to compare the data
from mammal and maize enzymes on a larger series of
compounds. The investigations presented here led to an
improved inhibitor 11l with a biphenylalanine moiety,
which in turn is a promising starting point for further
modifications.
modifications of 4a supported that hypothesis,18 but
confirmation was only achieved by the X-ray structure
of HDLP.30 Some of the potent inhibitors that were
discovered afterward were also hydroxamic acids. The
Merck group has shown elegantly in the apicidin series
that weak chelators such as carboxylic acids or ketones
retain high potency if the rest of the molecule is
optimized for binding.26 A modification of the spacer was
not undertaken at this point as it is known from the
SAHA series that the suberoyl compounds display the
peak of activity.17 We focused on the region responsible
for selective binding to the enzyme, and we have
modified the amino acid portion of the molecule as well
as the functionality at the “C terminus”. As we postulate
a second lipophilic binding site that is occupied by the
second phenylalanine in trapoxin B (1), we set out to
synthesize amide analogues of 5 in order to increase
lipophilic contacts with the enzyme as depicted by the
general structure 7 (see Chart 1). We also intended to
vary the amino acid part of the molecule and to analyze
the influence of the stereochemistry on the activity in
selected cases.
The synthesis followed the original procedure14 and
in some instances a modified version35 starting from
monomethyl suberate (8), which is converted to the
protected monohydroxamates 9. As the target hydrox-
amic acid 11 is very polar, the synthesis was run in a
fashion that the penultimate compound 10 of the
synthetic sequence was purified by chromatography and
deprotected so that only recrystallization was necessary
afterward. Benzyl hydroxylamine and hydrogenation or
trityl hydroxylamine and acidic removal were used to
generate the desired hydroxamate 11 (see Scheme 1).
The amino acids were introduced as the methyl esters
and amide hydrochlorides or tosylates. The amino acid
amides were synthesized from suitably N-protected
amino acids by amide coupling and deprotection. Cbz
Ch em istr y
Among our lead compounds 4a and 5, the phenyla-
lanine inhibitor 5 was less potent but it displayed an
encouraging profile as an inducer of differentiation. We
set out to modify this compound as well. As an enzyme-
inhibiting group, we continued to use the hydroxamate
function. HDAC is a zinc-dependent amidohydrolase,
which was only a working hypothesis when we began
our first studies. Structure-activity data from our

3298 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15
Wittich et al.
Ta ble 1. Inhibition of In Vitro Enzyme Activities of Selected HDACs and Proliferation of Cultured Friend Leukemic Cells
IC50 ( SD (nM),
maize HD-2
(maize HD-1)
IC50 ( SD (nM),
rat liver HDAC
IC50 (µM), proliferation
of Friend leukemic cells
no.
R′
R′′
R′′′
2
6a
6c
5
3 ( 0.1a
12 ( 1b
165 ( 25b
1150 ( 55
800 ( 130e
840 ( 95
790 ( 70
460 ( 30
260 ( 20
390 ( 25
1010 ( 90
330 ( 40
880 ( 85
1540 ( 80
800 ( 90
1340 ( 140
290 ( 25
380 ( 30
130 ( 15
510 ( 40
410 ( 60
320 ( 20
310 ( 10
0.04c
0.99
NDd
15
64
15
69
72
76
58
>100
28
>100
11
35
0.84
6.2
22
5.2
5.9
13
1000 ( 80a
320 ( 20
H3CO-
H2N-
PhNH-
BnNH-
Ph(CH2)2NH-
Ph(CH2)2NH-
Ph(CH2)3NH-
pyrrolyl-
H3CO-
Bn-
Bn-
Bn-
Bn-
Bn-
H-
Bn-
Bn-
H-
H-
H-
H-
H-
H-
Bn-
H-
H-
Bn-
Bn-
H-
H-
H-
H-
H-
H-
H-
H-
H-
500 ( 5a
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
11m
11n
11o
11p
11q
11r
1100 ( 35
80 ( 5 (120 ( 10)
140 ( 5 (40 ( 5)
330 ( 15 (25 ( 5)
340 ( 10
380 ( 10
520 ( 35
760 ( 15
HO-f
H3CO-
H3CO-
H3CO-
H3CO-
H3CO-
H3CO-
H3CO-
H-
750 ( 15
4-MeOBn-
4-NO2Bn-
4-PhBn-
iPr-
1-naphthyl-methyl-
2-naphthyl-methyl-
2-thenyl-
3-indolyl-methyl-
1-naphthyl-methyl-
250 ( 15
850 ( 45
210 ( 5
900 ( 10
360 ( 20
40 ( 2
700 ( 35
H3CO-
Ph(CH2)2NH-
120 ( 10
35 ( 5
15
a
b
d
Taken from literature.37 Taken from literature.52 c Taken form literature.18 Not determined. e Taken from literature.48 f As the
dicyclohexylamine salt.
Sch em e 1. Synthetic Route to HDAC Inhibitor 11a
human (HeLa) and protozoal (Eimeria sp.) enzyme
inhibition in the apicidin series, this is the only other
report on a larger series of inhibitors with HDACs from
different organisms or classes. Standard radiolabeled
histones from chicken reticulocytes were used as a
substrate for maize enzymes whereas the fluorogenic
substrate developed in our group38 was used with the
rat liver deacetylase. Both systems have previously been
shown to lead to similar results.37
First, we have compared the impact of exchanging the
methyl ester substituent in our lead structure 5 by an
amide function on the deacetylase inhibitory properties.
A Phe-Phe derivative was virtually inactive in both
systems (data not shown), which prompted us to test
the effect of simple amines on the activity. With regard
to maize HD-2, only the primary amide 11a is signifi-
cantly less active than the methyl ester 5. The secondary
amides show a steady decrease in enzyme inhibition
with an increased number of methylene groups between
the phenyl ring and the amide nitrogen. Thus, the
anilide 11b is the most potent phenylalanine amide with
an IC50 of 80 nM. The primary amide 11a is less active
than 5 in the rat system as well. The trend of activity
in 11b-f is altered with the rat liver HDAC preparation
as there is an initial activity increase with the number
of methylene groups between the phenyl ring and the
amide group with a peak at the phenethylamide 11d .
So, we tested the amides 11b-d also for their inhibition
of maize HD-1, which is a homologue of the yeast
deacetylase rpd3 and thus mammal class I enzymes.
Therefore, it should resemble the rat liver preparation
more than maize HD-2 in its inhibitor recognition. The
benzylamide 11c and the phenethylamide 11d are very
similar in their activities, but both are more potent than
the anilide 11b. So, this indicates a reversed structure-
activity relationship with respect to maize HD-1 inhibi-
tion as compared to maize HD-2 and a similarity to the
closer related rat HDAC preparation. The pyrrolidide
11g as a tertiary amide displays an activity comparable
a
Reagents: (i) BOP-Cl, DIPEA, H3NOBn+Cl-, respectively,
H2NOTrt, CH2Cl2, 95% (R ) Bn), 78% (R ) Trt). (ii) LiOH, H2O,
THF, and then HCl, 68% (R ) Bn), 80% (R ) Trt). (iii) iBuOCOCl,
NMM, H3NR+X- (X ) Cl, Tos), THF, 30-86%. (iv) BOP-Cl,
DIPEA, H3NR+X- (X ) Cl, Tos), CH2Cl2, 33-69%. (v) H2, Pd/C,
MeOH for R ) Bn, 34-91%; TFA, Et3SiH, CH2Cl2 for R ) Trt,
42-93%.
or Boc protection were employed, and again, hydrogena-
tion or acidic hydrolysis led to the desired amino acid
amide building blocks (see Supporting Information for
details). The amino acid methyl esters were prepared
from the acids by treatment with thionyl chloride in
methanol. To obtain the (R)-phenylalanine-derived acid
11i, the ester function was cleaved with lithium hy-
droxide in the benzyl-protected precursor 10h and the
resulting acid 11i was then hydrogenated as with other
NHO-benzyl compounds.
Resu lts a n d Discu ssion
In Vitr o En zym e In h ibition . As a source of enzy-
matic activity, we used a highly purified maize HDAC
HD-236 and a partially purified rat liver HDAC prepara-
tion.37,38 We have also studied the inhibition of the rpd3
homologue maize HD-139 (which is a homologue of
mammalian class I HDACs) in selected cases (Table 1).
In addition to the large set of comparative data on

Phenylalanine-Containing Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15 3299
to the methyl ester 5 with maize HD-2, but it is more
active in inhibiting rat liver HDAC preparation. Inter-
estingly, the potency of the (R)- and (S)-phenethyla-
mides (11d ,e, respectively) is almost identical with
maize HD-2 and similar with the rat liver HDAC
preparation while both the (R)-acid 11i and -methyl
ester 11h are less active than their enantiomers in both
assays (data for (S)-acid not shown). Although we have
used standard peptide coupling methodology, which
should lead only to a minimum of racemization, we
nevertheless wanted to rule out that these results stem
from complete racemization. We have evaluated several
chiral high-performance liquid chromatography (HPLC)
stationary phases but could not achieve a complete
baseline separation of the two enantiomers. The best
separation was monitored on a (S)-valine-(R)-1-(R-
naphthyl)ethylamide phase and led to a limit of quan-
titation of around 5% of 11e in 11d vs 10% of 11d in
11e. No sign of the undesired enantiomer was detected
in both cases (see Supporting Information for details);
therefore, it was proven that the amides 11d ,e display
similar HDAC inhibitory properties despite an opposite
stereochemistry. As the other compounds in this report
are synthesized with similar methods, we do not expect
significant racemization in the other inhibitors as well.
HDAC) for 6a while for 6c values of 320 (maize HD-2)
and 1150 nM (rat liver HDAC) were obtained. Com-
pound 6a has been reported to inhibit immunoprecipi-
tated human HDACs 1 and 3 at much lower concentra-
tions (10-20 nM for 6a and 250-300 nM for 6c15); so,
our compounds might display lower values for isolated
enzymes as well. On the other hand, the predictive value
of those isolated HDAC systems does not seem to be as
good as ours for 6a and the cellular activity is registered
in the low micromolar region for both the hybrid polar
compounds17 and our structures (see below).
In h ibition of P r olifer a tion a n d In d u ction of
Ter m in a l Cell Differ en tia tion . For in vivo investiga-
tions, Friend leukemic cells were used. They have been
found to be a suitable model for induction of differentia-
tion by HDAC inhibitors in many studies.18,40-42 Friend
cell differentiation is accompanied by an accumulation
of hemoglobin, which is easily visualized by benzidine
staining. Screening tests were performed at 10 and 50
µM, and dose-response studies were done according to
those initial results. If the compounds were highly toxic
at 50 µM (>95%), dose-response studies were not
extended to that concentration (Figure 1).
By comparing their profiles for inhibition of prolifera-
tion and induction of terminal differentiation, the
inhibitors could be divided into three classes. The first
group consists of the pyrrolidide 11g, the (R)-phenyla-
lanine acid 11i, the nitrophenylalanine compound 11k ,
and the valine derivative 11m . These compounds do not
lead to an induction of differentiation. While 11g,i are
also poor inhibitors of proliferation, 11k ,m do inhibit
Friend cell proliferation in a dose-dependent manner.
The largest group is characterized by a concentration-
dependent inhibition of proliferation and also a dose-
dependent induction of terminal differentiation that
does not exceed 25% of the surviving cells. These are
the inhibitors benzylamide 11c, phenethylamides 11d ,e,
phenylpropylamide 11f, methoxyalanine 11j, 2-naph-
thylalanine 11o, tryptophan 11q, and naphthylalanine
amide 11r . The third group is formed by the compounds
that lead to a pronounced induction of terminal dif-
ferentiation in Friend leukemic cells (40% or more of
the surviving cells) and are also inhibitors of prolifera-
tion. These are the standards trichostatin A (2) and
SAHA (6a ), the lead compound 5, its enantiomer 11h ,
the primary amide 11a , the anilide 11b, the 1-naph-
thylalanine methyl ester 11n , the biphenylalanine
compound 11l, and the thienyl congener 11p . They show
more or less steep dose-response curves with regard
to inhibition of proliferation. Generally, a strong induc-
tion of differentiation is only detected at concentrations
where also marked inhibition of proliferation is encoun-
tered. The notable exception to this is the lead com-
pound 5 with 45% induction of differentiation at only
20% inhibition of proliferation (at 10 µM) and to some
extent its enantiomer 11h . The cellular activity in the
amide series is rather disappointing whereas especially
with the more lipophilic and sterically demanding
substituents a good induction of differentiation and/or
inhibition of proliferation is observed in the amino acid
methyl ester series. Maybe a poor uptake of the amides
by the cells is a reason for this although their lipophi-
licity is similar to that of the esters (e.g., logP ) 3.39
for 11d , 3.78 for 11f, 3.01 for 11n , and 3.69 for 11l).
Next, we have modified the amino acid substituent
in the phenylalanine methyl ester lead structure 5.
While the methoxyphenylalanine 11j is more active
against HD-2, it is less active in inhibiting the rat liver
deacetylase preparation. With the thienyl analogue 11p
and the aliphatic valine derivative 11m , this order is
reversed. The 4-nitrophenylalanine compound 11k is
less active in both systems while the bulky biphenyla-
lanine congener 11l is about 2-fold more active than the
unsubstituted compound in both series. The impact of
benzoannellation of the phenyl ring in the naphthyl
compounds 11n ,o is dependent on the position of the
naphthyl ring. While the hydroxamate derived from
1-naphthylalanine 11n is more active in both systems
and is the best inhibitor in the rat liver deacetylase
assay, its regioisomer 11o is similar in its activity to 5
with rat liver HDAC. Compound 11o is far more
effective on the maize enzyme with an IC50 of 40 nM.
Finally, the heteroaromatic tryptophan derivative 11q
is also a good inhibitor of maize HD-2 with an IC50 of
120 nM and it does show an increased rat liver deacety-
lase inhibition as well. The indole nucleus or suitable
replacements in that position have previously been
shown to be an important part of potent HDAC inhibi-
tors in the apicidin series.22 To see whether the combi-
nation of the two most successful structural modifica-
tions among the amide and the methyl ester series with
regard to rat liver HDAC inhibition does lead to a
further increase in inhibitory potency, we have synthe-
sized the phenethylamide of 1-naphthylalanine and
have converted it to the requisite suberoylamide hy-
droxamate 11r . However, it does not reach the activity
of either the parent phenethylamide 11d or the naph-
thylalanine 11n in the inhibition of the rat enzyme
preparation whereas it is indeed the best inhibitor of
maize HD-2 (35 nM). Besides trichostatin A (2), the
hybrid polar compounds SAHA (6a ) and SBHA (6c)
were included as a comparison. We have measured IC50
values of 1000 (maize HD-2) and 165 nM (rat liver

3300 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15
Wittich et al.

Phenylalanine-Containing Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15 3301

3302 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15
Wittich et al.
F igu r e 1. Induction of differentiation in Friend leukemic cells by HDAC inhibitors. Circles and dotted lines represent percent
proliferation, and squares and straight lines represent percent induction of differentiation in surviving cells. Data for 2 was
taken from the literature.18
Histon e Hyp er a cetyla tion . We had already dem-
onstrated that the lead compound 5 induces histone
hyperacetylation in Friend cells at concentrations where
cellular activity is observed as well.18 To determine
whether the mechanism of action is identical with the
novel inhibitors, we investigated the influence of the
biphenylalanine compound 11l on the acetylation status
of Friend cell histone H4 by employing standard acid-
urea-triton (AUT) gels as described in the litera-
ture.43,44 Indeed, a clear time-dependent shift to hyper-
acetylated species of histone H4 is observed in cells
treated with 5 µM 11l. The peak of hyperacetylation is
F igu r e 2. Time-dependent acetylation status of histone H4
monitored after 12 h (Figure 2).
in Friend leukemic cells treated with 5 µM of 11l. A +
represents treated cells, and a - represents solvent control.
Con clu sion
Ac0-4, number of acetylated lysines; M, marker (Cytochrom
Structural modifications of our phenylalanine methyl
ester lead compound 5 have resulted in a potentiation
of the enzyme inhibitory properties in both maize HD-2
and rat liver HDAC preparation. While the inhibition
of maize HD-2 was especially pronounced in the amino
acid amide series, resulting in an up to 15-fold increase
c). See the Experimental Section for details.
in inhibitory potential, an increase of the size of the
amino acid substituent led to the best results in the rat
liver system. The rise in activity in this assay was
limited to a factor of three. The results from the

Phenylalanine-Containing Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15 3303
Knauer) column was used in the assay. Trichostatin A (2) and
SAHA (6a ) were purchased from Calbiochem, and SBHA (6c)
was from Aldrich. Phenylalanine pyrrolidide was purchased
from Bachem. Amino acids were obtained from Calbiochem,
and other chemicals were from Fluka and Aldrich. Compounds
5 and 9a were synthesized according to the literature.35
Dulbecco’s modified Eagle’s medium (DMEM), penicillin G
sodium, and streptomycin sulfate were purchased from Life
Technologies. LogP values were calculated using HyperChem
Pro 6.02.
Syn th esis of In h ibitor s. Am in o Acid Bu ild in g Block s.
The amino acid methyl esters were prepared from the com-
mercially available acids using methanol/thionyl chloride
according to standard procedures. The amino acid amide salts
were prepared from the amines and commercially available
Boc- or Cbz-protected amino acids via mixed anhydride
coupling and subsequent hydrogenation (Cbz) or acidic removal
of the protecting group (trifluoroacetic acid, Boc). The liberated
bases were treated with HCl in ether or p-toluenesulfonic acid
in ethyl acetate to precipitate the salts. See Supporting
Information for details and spectroscopic data.
Meth od A. Am id e F or m a tion Usin g BOP -Cl. To a
suspension or solution of the acid in dry CH2Cl2 (5 mL/mmol)
was added diisopropyl ethylamine (DIPEA, 1 equiv) under
nitrogen, and the mixture was stirred for 10 min. Then, BOP-
Cl (1 equiv), the R-amino acid methyl ester hydrochloride (or
appropriately protected hydroxylamine) (1.1 equiv) and again
DIPEA (2 equiv) were added. After it was stirred overnight,
most of the CH2Cl2 was removed under reduced pressure and
ethyl acetate (100 mL) was added. The solution was washed
three times with 5% NaHCO3 solution and once consecutively
with water, 2 M HCl solution, waterm and saturated brine
(50 mL each). The organic layer was dried over Na2SO4, and
the solvent was evaporated.
Meth od B. Am id e F or m a tion Usin g Mixed An h yd r id e.
To a solution of the acid in THF (10 mL/mmol) was added
N-methyl morpholine (NMM, 1 equiv) under nitrogen, and the
solution was stirred for 5 min. The solution was cooled to -15
°C and stirred for another 5 min. Then, isobutyl chloroformate
(1 equiv) was added dropwise, and the mixture was stirred
for 10 min. The amine (or the R-amino acid methyl ester
hydrochloride) (1 equiv) and again NMM (2 equiv) were added,
and the suspension was stirred for 15 min at -15 °C and 2 h
at room temperature. The mixture was then poured in 50 mL
of 2 M HCl solution and extracted three times with ethyl
acetate (each 50 mL). The organic layers were then washed
with 5% NaHCO3 solution, water, and saturated brine (each
50 mL). The organic phase was dried over Na2SO4, most of
the solvent was evaporated, and the crude product was
precipitated with hexane.
phenylalanine amides 11b-d (with the additional data
on maize HD-1) indicate a HDAC subtype or class
selectivity, but certainly tests with pure mammalian
HDAC subtypes are necessary to substantiate this. Both
models were able to identify inhibitors of proliferation
and inducers of differentiation in leukemic cells in a
primary in vitro screen. Thus, the importance of HDAC
assays as a tool for drug development is further em-
phasized. The semiquantitative prediction of cellular
activity was not as consistent in this series as compared
to the amide analogues of trichostatin A (2).18 The
amides are mostly moderately active in the Friend
leukemic cells, but better predictive results are obtained
in the ester series with the good inhibitors 11l,n being
also among the most active compounds in cell culture.
While the difference in cellular activity cannot be
attributed to a simple difference in lipophilicity, still a
differential uptake of these compounds might be a
reason for the observed results. Additionally, a different
selectivity in HDAC subtype inhibition might account
for the varying cellular activity profiles in seemingly
very similar compounds. Such alternative binding modes
even for simple homologues have been shown on a
homology model of human HDAC1 that is based on the
HDLP X-ray structure.45 So, the observed IC50 in the
rat liver system might be a result of a differential
inhibition of various HDAC subtypes. Still, the rat liver
HDAC is a convenient and accessible screening tool and
the example of 6a shows that highly purified HDACs
are not necessarily better in quantitative predictions of
cellular activity. Another explanation for the observed
discrepancies might be the involvement of additional
mechanisms apart from histone hyperacetylation by
different hydroxamic acids in other pathways. The
hydroxamic acid moiety is able to chelate iron and zinc
ions and might interact with other enzymes, but at least
for the lead compound 5, it was shown specifically that
it does not inhibit a number of matrix metalloproteases
in concentrations where histone hyperacetylation occurs
(unpublished results). Generally, the different profiles
of cellular activity in structurally similar compounds
(inhibition of proliferation vs induction of differentia-
tion) should stimulate further work.
Su m m a r y. The best compound from a series of
structural modifications of a lead HDAC inhibitor with
a phenylalanine substructure was the biphenylalanine
methyl ester derivative 11l with an IC50 below 1 µM
causing inhibition of proliferation and induction of
terminal cell differentiation in Friend leukemic cells.
This compound is also inducing histone hyperacetylation
at low micromolar concentrations and opens up new
routes for further systematic improvement, e.g., by
palladium-mediated biaryl coupling.
Meth od C. Ester Clea va ge. The ester was dissolved in
THF (2 mL/mmol), and an aqueous solution of LiOH (0.5 M, 2
equiv) was added. The solution was stirred overnight at room
temperature. To the mixture was then added water (20 mL)
and ethyl acetate (50 mL). The aqueous layer was acidified
with 6 M HCl dropwise (to pH 3) and consecutively extracted
with ethyl acetate (3 × 50 mL). The organic layer was washed
with water and saturated brine and dried over Na2SO4, and
the solvent was evaporated.
Meth od D. Hyd r ogen a tion . The N-benzyl precursor was
dissolved in methanol (10 mL/mmol), and 10% palladium on
charcoal (10%, w/w) was added. The mixture was treated with
hydrogen under atmospheric pressure for 4 h and was filtered
subsequently. Then, the solvent was evaporated. The hydrox-
amic acids were redissolved in methanol and precipitated with
diethyl ether.
Meth od E. Rem ova l of Tr t P r otectin g Gr ou p s. The
trityl precursor was dissolved in CH2Cl2, and trifluoracetic
acid/CH2Cl2 (1:1, v/v; 2 mL/mg) was added. The resulting
yellow solution was treated dropwise with triethylsilane until
the color disappeared. The mixture was then stirred for 2 h,
and the solvent was evaporated afterward. The residue was
redissolved with methanol, and the solvent was removed under
reduced pressure again. This process was repeated three times,
Exp er im en ta l Section
Melting points are uncorrected. Elemental analysis was
performed on a Foss-Heraeus CHN-O-Rapid. IR spectra were
recorded on a Shimadzu 470 in KBr or a Biorad FTS 135 in
KBr. 1H nuclear magnetic resonance (NMR) was done on a
Varian Gemini 200 (200 MHz) and 13C NMR on the same
instrument (50.29 MHz). MS spectrometry was done on a
Finnigan MAT 312 (EI). Flash chromatography was performed
using silica gel 60, 230-400 mesh (Merck). Dichloromethane
and tetrahydrofuran (THF) were dried over molecular sieves
(3 Å). Shimadzu RF 535 was used as fluorescence detector for
HPLC and a LiChrosorb RP 18 5 µm (125 mm × 3 mm,

3304 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15
Wittich et al.
and the product was finally precipitated with diethyl ether
from the solution in methanol.
(0.114 mL, 105 mg, 1.04 mmol), isobutyl chloroformate (0.136
mL, 142 mg, 1.04 mmol), (S)-2-amino-N-benzyl-3-phenyl-
propionamide hydrochloride (302 mg, 1.04 mmol), and NMM
(0.228 mL, 210 mg, 2.08 mmol); yield 410 mg (59%); mp 156
7-Tr it yloxyca r b a m oyl-h ep t a n oic Acid Met h yl E st er .
This was synthesized by method A from monomethyl suberate
(8) (2.5 g, 13.3 mmol), DIPEA (2.30 mL, 1.72 g, 13.3 mmol),
O-tritylhydroxylamine (4.0 g, 14.6 mmol), and DIPEA (4.60
mL, 3.44 g, 26.6 mmol). The crude product was chromato-
graphed using ethyl acetate/hexane (2:1); yield 4.6 g (78%);
1
°C. IR: 1656, 1544. H NMR (CDCl3): δ 7.80-7.03 (m, 25H),
4.67 (m, 1H), 4.41-4.19 (m, 2H), 3.15-3.03 (m, 2H), 2.11-
2.04 (m, 2H), 1.57-1.38 (m, 4H), 1.25-1.08 (m, 6H). 13C NMR
(CDCl3): δ 173.09, 170.89, 137.68, 136.81, 129.36, 129.14,
128.74, 128.69, 128.14, 127.71, 127.52, 127.03, 54.72, 43.60,
38.61, 36.39, 28.68, 25.29, 23.20. MS (EI): m/z 243 (Trt+).
1
mp 96 °C. IR: 1740, 1665. H NMR (CDCl3): δ 7.34-7.26 (m,
15H), 3.66 (s, 3H), 2.26 (t, J ) 7.5 Hz, 2H), 1.62-1.47 (m, 4H),
1.39-0.85 (m, 6H). 13C NMR (CDCl3): δ 174.15, 141.50, 129.13,
128.13, 128.03, 51.42, 34.06, 31.37, 28.79, 28.75, 24.77, 23.46.
MS (EI): m/z 243 (Trt+).
(S)-Octa n ed ioic Acid (1-Ben zylca r ba m oyl-2-p h en yl-
eth yl)a m id e Hyd r oxya m id e (11c). Compound 11c was
synthesized by method E from 10c (200 mg, 0.299 mmol); yield
85 mg (67%); mp 168 °C. IR: 1638, 1546. 1H NMR (DMSO-
d6): δ 10.32 (s, 1H), 8.66 (s, 1H), 8.46 (t, J ) 5.8 Hz, 1H), 8.05
(d, J ) 8.3 Hz, 1H), 7.28-7.12 (m, 10H), 4.55-4.53 (m, 1H),
4.25 (d, J ) 5.8 Hz, 2H), 3.04-2.95 (m, 1H), 2.82-2.70 (m,
1H), 2.06-1.86 (m, 4H), 1.40-1.34 (m, 4H), 1.10 (bs, 4H). 13C
NMR (DMSO-d6): δ 171.94, 171.19, 169.05, 139.11, 137.89,
129.03, 128.08, 127.88, 126.96, 126.56, 126.08, 53.88, 41.96,
37.68, 35.11, 32.19, 28.29, 28.13, 24.98, 24.89. MS (EI): m/z
425 (M+). Anal. (C24H31N3O4) C, H, N.
(S)-Octa n ed ioic Acid Ben zyloxy-a m id e (1-P h en eth yl-
ca r ba m oyl-2-p h en yl-eth yl)a m id e (10d ). Compound 10d
was synthesized by method B from 9a (300 mg, 1.07 mmol),
NMM (0.117 mL, 108 mg, 1.07 mmol), isobutyl chloroformate
(0.140 mL, 137 mg, 1.07 mmol), (S)-2-amino-N-phenethyl-3-
phenyl-propionamide tosylate (471 mg, 1.07 mmol), and NMM
(0.234 mL, 216 mg, 2.14 mmol); yield 420 mg (74%); mp 160
°C. IR: 1638, 1545. 1H NMR (CD3OD): δ 7.39-7.13 (m, 15H),
4.83 (s, 2H), 4.55 (dd, J ) 9.10/6.10 Hz, 1H), 3.41-3.26 (m,
2H), 3.09-2.99 (m, 1H,), 2.86-2.74 (m, 1H), 2.69 (t, J ) 7.45
Hz, 2H), 2.12 (t, J ) 7.30 Hz, 2H), 2.00 (t, J ) 7.30 Hz, 2H),
1.54-1.37 (m, 4H), 1.28-1.16 (m, 4H). 13C NMR (CD3OD): δ
175.85, 173.56, 140.37, 138.55, 130.27, 129.80, 129.61, 129.49,
129.43, 127.76, 78.95, 56.02, 41.94, 39.14, 36.14, 36.75, 36.43,
33.70, 29.66, 29.56, 26.36. MS (EI): m/z 529 (M+).
7-Tr ityloxyca r ba m oyl-h ep ta n oic Acid (9b). Compound
9b was synthesized by method C from 7-trityloxycarbamoyl-
heptanoic acid methyl ester (3.9 g, 8.7 mmol); yield 3.0 g (80%);
mp 150 °C. IR: 1739, 1679. 1H NMR (CDCl3): δ 7.34-7.21
(m, 15H), 2.27 (t, J ) 7.5 Hz, 2H), 1.57-1.46 (m, 4H), 1.30-
1.07 (m, 6H). 13C NMR (CDCl3): δ 178.57, 141.45, 129.14,
128.16, 109.49, 33.85, 28.70, 24.53. MS (EI): m/z 243 (Trt+),
165 (C13H9+).
(S)-Octa n ed ioic Acid Ben zyloxy-a m id e (1-Ca r ba m oyl-
2-p h en yl-eth yl)a m id e (10a ). Compound 10a was synthe-
sized by method A from 9a (300 mg, 1.07 mmol), DIPEA (0.185
mL, 138.3 mg, 1.07 mmol), BOP-Cl (272 mg, 1.07 mmol),
L-phenylalanine amide hydrochloride (236 mg, 1.18 mmol), and
DIPEA (0.555 mL, 415 mg, 3.21 mmol); yield 148 mg (33%);
mp 173 °C. IR: 1674, 1637, 1543. 1H NMR (DMSO-d6): δ 10.93
(s, 1H), 7.92 (d, J ) 8.49 Hz, 1H), 7.42-7.12 (m, 10H), 7.03 (s,
2H), 4.76 (s, 2H), 4.47-4.40 (m, 1H), 3.03-2.94 (m, 1H), 2.77-
2.65 (m, 1H), 2.03-1.86 (m, 4H), 1.40-1.25 (m, 4H), 1.07-
0.99 (m, 4H). 13C NMR (DMSO-d6): δ 173.34, 171.93, 169.37,
136.08, 138.18, 129.09, 128.72, 128.25, 128.17, 127.92, 126.10,
76.72, 53.58,37.62, 35.18, 32.21, 28.25, 28.15, 25.05, 24.79. MS
(EI): m/z 425 (M+).
(S)-Octa n ed ioic Acid (1-Ca r ba m oyl-2-p h en yl-eth yl)-
a m id e Hyd r oxya m id e (11a ). Compound 11a was synthe-
sized by method D from 10a (150 mg, 0.352 mmol) and 10%
Pd on charcoal (15 mg); yield 40 mg (34%); mp 138 °C. IR:
1675, 1630. 1H NMR (DMSO-d6): δ 10.33 (s, 1H), 8.66 (s, 1H),
7.92 (d, J ) 8.38 Hz, 1H), 7.22-7.15 (m, 5H), 7.03 (s, 2H),
4.43-4.38 (m, 1H), 3.02-2.93 (m, 1H), 2.76-2.68 (m, 1H),
(S)-Octa n ed ioic Acid Hyd r oxya m id e (1-P h en eth ylca r -
ba m oyl-2-p h en yl-eth yl)a m id e (11d ). Compound 11d was
synthesized by method D from 10d (360 mg, 0.680 mmol) and
10% Pd on charcoal (36 mg); yield 180 mg (60%); mp 119 °C.
1
IR: 1637, 1542. H NMR (DMSO-d6): δ 10.33 (s, 1H), 8.66 (s,
2.03-1.85 (m, 4H), 1.43-1.29 (m, 4H), 1.11-1.04 (m, 4H). 13
C
1H), 8.05-7.95 (m, 2H), 7.30-7.15 (m, 10H), 4.47-4.38 (m,
1H), 3.36-3.19 (m, 2H), 2.93-2.84 (m, 1H), 2.73-2.61 (m, 3H),
NMR (DMSO-d6): δ 173.23, 171.85, 169.05, 138.11, 128.99,
127.85, 126.03, 53.51, 37.54, 35.11, 32.17, 28.29, 28.11, 24.96.
MS (EI): m/z 120 (C8H10N+). Anal. (C17H25N3O4) C, H, N.
(S)-Octa n ed ioic Acid Ben zyloxya m id e (2-P h en yl-1-
p h en ylca r ba m oyl-eth yl)a m id e (10b). Compound 10b was
synthesized by method B from 9a (300 mg, 1.07 mmol), NMM
(0.117 mL, 108 mg, 1.07 mmol), isobutyl chloroformate (0.140
mL, 137 mg, 1.07 mmol), (S)-2-amino-3N-diphenyl-propiona-
mide tosylate (441 mg, 1.07 mmol), and NMM (0.234 mL, 216
mg, 2.14 mmol); yield 160 mg (30%); mp 165 °C. IR: 1655,
1641, 1536. 1H NMR (CD3OD): δ 7.48-7.08 (m, 15H), 4.82 (s,
2H), 4.82-4.70 (m, 1H), 3.21-3.11 (m, 1H), 3.01-2.90 (m, 1H),
2.18 (t, J ) 7.2 Hz, 2H), 2.00 (t, J ) 7.1 Hz, 2H), 1.51-1.48
(m, 4H), 1.28-1.17 (m, 4H). 13C NMR (CD3OD): δ 176.11,
138.29, 130.34, 129.76, 129.65, 129.45, 127.83, 125.50, 121.59,
78.92, 56.68, 39.21, 36.66, 33.66, 29.58, 26.64, 26.40. MS (EI):
120 (C8H10O+), 105 (C8H9+).
2.03-1.86 (m, 4H), 1.40-1.29 (m, 4H), 1.11-1.04 (m, 4H). 13
C
NMR (DMSO-d6): δ 171.85, 171.07, 169.06, 139.28, 137.97,
128.99, 128.52, 128.18, 127.85, 126.05, 125.96, 53.79, 40.05,
37.72, 35.10, 34.95, 32.19, 28.27, 28.11, 24.96, 24.89. MS (EI):
m/z 439 (M+). Anal. (C25H33N3O4) C, H, N.
(R)-Octa n ed ioic Acid Ben zyloxy-a m id e (1-P h en eth yl-
ca r ba m oyl-2-p h en yl-eth yl)a m id e (10e). Compound 10e
was synthesized by method B from 9a (300 mg, 1.07 mmol),
NMM (0.117 mL, 108 mg, 1.07 mmol), isobutyl chlorformate
(0.140 mL, 146 mg, 1.07 mmol), (R)-2-amino-N-phenethyl-3-
phenyl-propionamide tosylate (471 mg, 1.07 mmol), and NMM
(0.234 mL, 216 mg, 2.14 mmol); yield 340 mg (60%); mp 164
1
°C. IR 1638, 1546. H NMR (CD3OD): δ 7.41-7.13 (m, 15H),
4.83 (s, 2H), 4.55 (dd, J ) 9.15/6.10 Hz, 1H), 3.40-3.26 (m,
2H), 3.09-2.99 (m, 1H), 2.86-2.74 (m, 1H), 2.69 (t, J ) 7.45
Hz, 2H), 2.12 (t, J ) 7.38 Hz, 2H), 2.00 (t, J ) 7.25 Hz, 2H),
1.54-1.37 (m, 4H), 1.18-1.10 (m, 4H). 13C NMR (CD3OD): δ
175.83, 173.56, 140.35, 138.53, 130.25, 129.78, 129.60, 129.47,
129.41, 127.74, 127.34, 78.93, 56.01, 41.92, 39.12, 36.74, 36.41,
33.66, 29.60, 26.55, 26.34. MS (EI): m/z 529 (M+).
(S)-Octa n ed ioic Acid Hyd r oxya m id e (2-P h en yl-1-p h e-
n ylca r ba m oyl-eth yl)a m id e (11b). Compound 11b was syn-
thesized by method D from 10b (100 mg, 0.20 mmol) and 10%
Pd on charcoal (10 mg); yield 50 mg (61%); mp 141 °C. IR:
1
1642, 1534. H NMR (DMSO-d6): δ 10.14 (s, 1H), 8.23 (d, J )
(R)-Octa n ed ioic Acid Hyd r oxya m id e (1-P h en eth ylca r -
b a m oyl-2-p h en yl-et h yl)a m id e (11e). Compound 11e was
synthesized by method D from 10e (300 mg, 0.566 mmol) and
10% Pd on charcoal (30 mg); yield 190 mg (76%); mp 126 °C.
8.2 Hz, 1H), 7.61-7.05 (m, 10H), 4.78-4.55 (m, 1H), 3.09-
2.84 (m, 2H), 2.09-1.88 (m, 4H), 1.45-1.26 (m, 4H), 1.14-
1.06 (m, 4H). 13C NMR (DMSO-d6): δ 172.17, 170.30, 169.09,
138.78, 137.66, 129.08, 128.58, 127.92, 126.20, 123.29, 119.34,
54.64, 37.66, 35.04, 32.18, 28.29, 28.13, 25.01, 24.92. MS (EI):
m/z 120 (C8H10N+). Anal. (C23H29N3O4) C, H, N.
1
IR: 1640, 1549. H NMR (DMSO-d6): δ 10.32 (s, 1H), 8.66 (s,
1H), 8.04-7.95 (m, 2H), 7.30-7.15 (m, 10H), 4.46-4.39 (m,
1H), 3.36-3.19 (m, 2H), 2.93-2.84 (m, 1H), 2.73-2.61 (m, 3H),
2.03-1.85 (m, 4H), 1.43-1.25 (m, 4H), 1.09 (bs, 4H). 13C NMR
(DMSO-d6): δ 171.85, 171.07, 169.06, 139.28, 137.97, 128.99,
128.52, 128.18, 127.85, 126.05, 125.96, 53.79, 40.05, 37.72,
(S)-Octa n ed ioic Acid (1-Ben zylca r ba m oyl-2-p h en yl-
eth yl)a m id e Tr ityloxy-a m id e (10c). Compound 10c was
synthesized by method B from 9b (450 mg, 1.04 mmol), NMM

Phenylalanine-Containing Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15 3305
35.10, 34.95, 32.19, 28.27, 28.11, 24.96, 24.89. MS (EI): m/z
(s, 1H), 8.25 (d, J ) 7.92 Hz, 1H), 7.26-7.18 (m, 5H), 4.46-
4.43 (m, 1H), 3.58 (s, 3H), 2.99-2.85 (m, 2H), 1.86-2.05 (m,
4H), 1.39-1.36 (m, 4H), 1.13-1.12(m, 4H). 13C NMR (DMSO-
d6): δ 172.19, 169.06, 137.27, 128.95, 128.11, 126.41, 53.31,
51.70, 34.89, 32.21, 28.23, 28.11, 24.97. MS (EI): m/z 350 (M+).
Anal. (C18H26N2O5) C, H, N.
439 (M+). Anal. (C25H33N3O4) C, H, N.
(S)-Octa n ed ioic Acid Ben zyloxy-a m id e [2-P h en yl-1-(3-
p h en yl-p r op ylca r ba m oyl)eth yl]a m id e (10f). Compound
10f was synthesized by method A from 9a (300 mg, 1.07 mmol),
DIPEA (0.185 mL, 138 mg, 1.07 mL), BOP-Cl (272 mg, 1.07
mmol), (S)-2-amino-3-phenyl-N-(3-phenyl-propyl)propionamide
tosylate (536 mg, 1.18 mmol), and DIPEA (0.555 mL, 414 mg,
3.21 mmol); yield 300 mg (52%); mp 159 °C. 1H NMR (CD3-
OD): δ 7.39-7.11 (m, 15H), 4.60 (s, 2H), 4.60-4.53 (m, 1H),
3.15-3.02 (m, 3H), 2.92-2.80 (m, 1H), 2.54-2.47 (m, 2H),
2.18-2.11 (m, 2H), 2.03-1.96 (m, 2H), 1.73-1.65 (m, 2H)
1.60-1.33 (m, 4H), 1.34-1.02 (m, 4H). 13C NMR (CD3OD): δ
175.95, 173.54, 172.89, 142.95, 138.48, 136.98, 130.29, 129.61,
129.43, 129.34, 127.78, 126.85, 78.93, 56.15, 39.97, 39.14,
36.72, 34.01, 33.66, 32.04, 29.60, 26.62, 26.36. MS (EI): m/z
543 (M+).
(S)-Octa n ed ioic Acid Hyd r oxya m id e [2-P h en yl-1-(3-
p h en yl-p r op ylca r ba m oyl)eth yl]a m id e (11f). Compound
11f was synthesized by method D from 10f (250 mg, 0.450
mmol) and 10% Pd on charcoal (25 mg); yield 190 mg (91%);
mp 115 °C. IR: 1761, 1635, 1545. 1H NMR (DMSO-d6): δ 10.36
(s, 1H), 8.68 (s, 1H), 8.05-7.99 (m, 2H), 7.30-7.15 (m, 10H),
4.48-4.45 (m, 1H), 3.04-2.68 (m, 4H), 2.53-2.45 (m, 2H),
2.05-1.86 (m, 4H), 1.70-1.59 (m, 2H), 1.40-1.31 (m, 4H),
1.10-1.09 (m, 4H). 13C NMR (DMSO-d6): δ 171.90, 171.07,
169.10, 141.66, 137.95, 129.03, 128.19, 128.14, 127.87, 126.06,
125.59, 56.02, 38.04,: 37.81, 35.13, 32.29, 32.19, 30.67, 28.29,
28.15, 25.02, 24.91. MS (EI): m/z 453 (M+). Anal. (C26H35N3O4)
C, H, N.
(R)-2-(7-Ben zyloxycar bam oyl-h eptan oylam in o)-3-ph en -
yl-p r op ion ic Acid (10i). Compound 10i was synthesized by
method C from 10h (510 mg, 19.5 mmol); yield 410 mg (83%);
1
mp 115 °C. IR: 1742, 1641, 1541. H NMR (CD3OD): δ 7.44-
7.18 (m, 10H), 4.83 (s, 2H), 4.70-4.63 (m, 1H), 3.27-3.17 (m,
1H) 2.97-2.85 (m, 1H), 2.16-1.97 (m, 4H), 1.49-1.43 (m, 4H),
1.24-1.12 (m, 4H). 13C NMR (CD3OD): δ 176.01, 174.85,
138.59, 130.32, 130.23, 129.69, 129.42, 127.78, 78.94, 54.86,
38.4, 36.66, 33.70, 29.69, 29.59, 26.63, 26.42. MS (EI): m/z 426
(M+).
(R)-2-(7-Hyd r oxyca r ba m oyl-h ep ta n oyla m in o)-3-p h en -
yl-p r op ion ic Acid (11i). Compound 11i was synthesized by
method D from 10i (200 mg, 0.45 mmol) and 10% Pd on
charcoal (20 mg); yield 65 mg (41%) of viscous oil. The acid
was precipitated from a solution in methanol with an excess
of dicylohexylamine (dropwise addition of a solution in diethyl
ether). IR: 2937, 2858, 1631, 1395. 1H NMR (free acid, DMSO-
d6): δ 10.32 (s, 1H), 8.65 (s, 1H), 8.09 (d, J ) 8.00 Hz, 1H),
7.29-7.17 (m, 5H), 4.45-4.34 (m, 1H), 3.09-2.99 (m, 1H),
2.86-2.75 (m, 1H), 2.04-1.86 (m, 4H), 1.37 (bs, 4H), 1.12-
1.04 (m, 4H). 13C NMR (free acid, DMSO-d6): δ 173.25, 172.16,
169.16, 137.78, 129.05, 128.10, 126.34, 53.29, 36.77, 35.06,
32.28, 28.39, 28.19, 25.11, 25.04. MS (EI): m/z 336 (M+). Anal.
(C29H47N3O5) C, H, N.
(S)-Oct a n ed ioic Acid (1-Ben zyl-2-oxo-2-p yr r olid in -1-
yl-eth yl)a m id e Tr ityloxy-a m id e (10g). Compound 10g was
synthesized by method B from 9b (500 mg, 1.16 mmol), NMM
(0.128 mL, 117 mg, 1.16 mmol), isobutyl chlorformate (0.151
mL, 158 mg, 1.16 mmol), l-phenylalanine-pyrrolidide (253 mg,
1.16 mmol), and NMM (0.255 mL, 234 mg, 2.32 mmol). The
crude product was chromatographed using ethyl acetate/
methanol (25:1); yield 320 mg (44%); mp 90 °C. IR: 1630, 1449.
1H NMR (CDCl3): δ 7.77 (bs, 1H), 7.17-7.45 (m, 20H), 6.40
(d, J ) 8.2 Hz, 1H), 4.84-4.96 (m, 1H), 3.29-3.38 (m, 3H),
2.93-3.01 (m, 2H), 2.54-2.59 (m, 1H), 2.09-2.16 (m, 2H),
0.83-1.71 (m, 14H). 13C NMR (CDCl3): δ 172.40, 169.78,
136.46, 129.51, 129.13, 128.43, 127.03, 52.24, 46.40, 45.82,
39.81, 36.50, 28.84, 24.07, 25.82, 25.35. MS (EI): m/z 243
(Trt+).
(S)-Oct a n ed ioic Acid (1-Ben zyl-2-oxo-2-p yr r olid in -1-
yl-eth yl)a m id e Hyd r oxya m id e (11g). Compound 11g was
synthesized by method E from 10g (300 mg, 0.48 mmol); yield
110 mg (59%) viscous oil. IR: 1625, 1455. 1H NMR (CDCl3):
δ 7.42-7.38 (m, 5H), 5.09-4.85 (m, 1H), 3.69-3.47 (m, 3H),
3.20-3.18 (m, 2H), 2.87-2.78 (m, 1H), 2.42-2-31 (m, 3H),
1.93-1.75 (m, 6H), 1.45 (bs, 3H). 13C NMR (CDCl3): δ 173.84,
171.03, 136.74, 129.72, 128.79,127.37, 53.09, 46.88, 46.35,
39.11, 35.86, 32.53, 28.02, 26.08, 25.10, 25.40, 24.34. MS (EI):
m/z 120 (C8H10O+), 201 (C13H15NO+). Anal. (C21H31N3O4‚
0.5H2O) C, H, N.
(S)-3-(4-Meth oxy-p h en yl)-2-(7-tr ityloxyca r ba m oyl-h ep -
ta n oyla m in o)p r op ion ic Acid Meth yl Ester (10j). Com-
pound 10j was synthesized by method B from 9b (500 mg, 1.16
mmol), NMM (0.128 mL, 117 mg, 1.16 mmol), isobutyl chlo-
roformate (0.151 mL, 158 mg, 1.16 mmol), O-methyl-L-tyrosine
methyl ester hydrochloride (285 mg, 1.16 mmol), and NMM
(0.255 mL, 234 mg, 2.32 mmol). The crude product was
chromatographed using ethyl acetate/hexane (3:1); yield 550
mg (75%); mp 146 °C. IR: 1750, 1669, 1644. 1H NMR
(CDCl3): δ 7.95 (bs, 1H), 7.41-7.72 (m, 15H), 7.17 (d, J ) 8.7
Hz, 2H), 6.99 (d, J ) 8.8 Hz, 2H), 6.03 (d, J ) 8.0 Hz, 1H),
4.98-5.05 (m, 1H), 3.95 (s, 3H), 3.89 (s, 3H), 3.16-3.29 (m,
2H), 2.26-2.34 (m, 2H), 1.013-1.812 (m, 10H). MS (EI): m/z
243 (Trt+).
(S)-3-(4-Meth oxy-p h en yl)-2-(7-h yd r oxyca r ba m oyl-h ep -
ta n oyla m in o)p r op ion ic Acid Meth yl Ester (11j). Com-
pound 11j was synthesized by method E from 10j (150 mg,
0.240 mmol); yield 85 mg (93%); mp 103 °C. IR: 1739, 1666.
1H NMR (DMSO-d6): δ 10.48 (s, 1H), 8.80 (s, 1H), 8.35 (d, J
) 7.8 Hz, 1H), 7.32 (d, J ) 8.5 Hz, 2H), 7.03 (d, J ) 8.6 Hz,
2H), 4.65-4.58 (m, 1H), 3.91 (s, 3H), 3.79 (s, 3H), 3.18-3.12
(m, 1H), 3.04-2.96 (m, 1H), 2.24 (t, J ) 7.4 Hz, 2H), 2.12 (t,
J ) 7.5 Hz, 2H), 1.66-1.57 (m, 4H), 1.40-1.32 (m, 4H). 13C
NMR (CDCl3): δ 172.64, 158.84, 130.31, 128.00, 114.11, 112.
42, 55.30, 53.17, 52.33, 37.18, 36.23, 28.57, 25.29. MS (EI):
m/z 380 (M+). Anal. (C19H28N2O6) C, H, N.
(R)-2-(7-Ben zyloxycar bam oyl-h eptan oylam in o)-3-ph en -
yl-p r op ion ic Acid Meth yl Ester (10h ). Compound 10h was
synthesized by method A from 9a (670 mg, 2.4 mmol), DIPEA
(0.415 mL, 310 mg, 2.40 mmol), BOP-Cl (611 mg, 2.40 mmol),
D-phenylalanine methyl ester hydrochloride (569 mg, 2.64
mmol), and DIPEA (1.25 mL, 931 mg, 7.20 mmol); yield 730
(S)-3-(4-Nit r o-p h en yl)-2-(7-t r it yloxyca r b a m oyl-h ep -
ta n oyla m in o)p r op ion ic Acid Meth yl Ester (10k ). Com-
pound 10k was synthesized by method B from 9b (630 mg,
1.46 mmol), NMM (0.161 mL, 148 mg, 1.46 mmol), isobutyl
chloroformate (0.191 mL, 199 mg, 1.46 mmol), p-nitro-L-
phenylalanine methyl ester hydrochloride (381 mg, 1.46 mmol),
and NMM (0.322 mL, 296 mg, 2.92 mmol). The crude product
was chromatographed using ethyl acetate/hexane (1:2); yield
1
mg (69%); mp 89 °C. IR: 1733, 1641, 1521. H NMR (CDCl3):
δ 8.28 (bs, 1H) 7,38-7.06 (m, 10H), 5.87 (d, J ) 7.64 Hz, 1H),
4.91-4.83 (m, 3H), 3.72 (s, 3H), 3.14-3.04 (m, 2H), 2.18-2.00
(m, 4H), 1.62-1.50 (m, 4H), 1.29-1.26 (m, 4H). 13C NMR
(CDCl3): δ 172.64, 172,5, 135.97, 129.30, 128.87, 128.68,
127.24, 53.04, 52.40, 37.99, 36.27, 28.44, 25.22. MS (EI): m/z
162 (C10H10O2+).
(R)-2-(7-Hyd r oxyca r ba m oyl-h ep ta n oyla m in o)-3-p h en -
yl-p r op ion ic Acid Meth yl Ester (11h ). Compound 11h was
synthesized by method D from 10h (200 mg, 0.45 mmol) and
10% Pd on charcoal (20 mg); yield 110 mg (70%); mp 109 °C.
IR: 1739,1662, 1626. 1H NMR (DMSO-d6): δ 10.32 (s,1H), 8.66
1
470 mg (50%); mp 166 °C. IR: 1748, 1646. H NMR (CDCl3):
δ 8.15 (d, J ) 8.8 Hz, 2H), 7.76 (bs, 1H), 7.45-7.27 (m, 17H),
6.09 (d, J ) 6.9 Hz, 1H), 4.97-4.87 (m, 1H), 3.74 (s, 3H), 3.24-
3.10 (m, 2H), 2.17-2.09 (m, 2H), 1.72-1.44 (m, 4H), 1.30-
1.15 (m, 6H). 13C NMR (CDCl3): δ 173.10, 172.02, 147.59,
144.41, 141.58, 130.56, 129.43, 128.45, 124.03, 53.12, 52.95,
38.31, 36.59, 31.57, 28.97, 25.56. MS (EI): m/z 165 (C13H9+).
(S)-2-(7-Hyd r oxyca r ba m oyl-h ep ta n oyla m in o)-3-(4-n i-
tr o-p h en yl)p r op ion ic Acid Meth yl Ester (11k ). Compound
11k was synthesized by method E from 10k (400 mg, 0.627

3306 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15
Wittich et al.
mmol); yield 156 mg (63%); mp 158 °C. IR: 1737, 1642. 1H
NMR (DMSO-d6): δ 10.32 (s, 1H), 8.66 (s, 1H), 8.32 (d, J )
7.9 Hz, 1H), 8.14 (d, J ) 8.6 Hz, 2H,), 7.80 (d, J ) 8.4 Hz,
2H), 4.62-4.53 (m, 1H), 3.61 (s, 3H), 3.23-2.94 (m, 2H), 2.04-
1.85 (m, 4H), 1.38-1.35 (m, 4H), 1.11 (m, 4H). 13C NMR
(DMSO-d6): δ 172.21, 171.65, 169.05, 146.28, 145.68, 130.40,
123.12, 52.58, 51.86, 36.28, 34.86, 32.15, 28.22, 28.05, 24.89.
MS (EI): m/z 395 (M+). Anal. (C18H25N3O7) C, H, N.
(S)-2-(7-Hyd r oxyca r ba m oyl-h ep ta n oyla m in o)-3-n a p h -
th a len -1-yl-p r op ion ic Acid Meth yl Ester (11n ). Compound
11n was synthesized by method D from 10n (300 mg, 0.45
mmol) and 10% Pd on charcoal (30 mg); yield 115 mg (64%);
1
mp 148 °C. IR: 1722, 1641. H NMR (DMSO-d6): δ 10.33 (s,
1H), 8.67 (s, 1H), 8.38 (d, J ) 7.7 Hz, 1H), 8.08-7.37 (m, 7H),
4.63-4.57 (m, 1H), 3.58 (s, 3H), 3.52-3.20 (m, 2H), 2.04-1.86
(m, 4H), 1.40-1.33 (m, 4H), 1.24-0.90 (m, 4H). 13C NMR
(DMSO-d6): δ 172.21, 172.12, 169.05, 133.33, 133.16, 131.29,
128.61, 127.25, 127.19, 126.14, 125.52, 123.06, 52.62, 51.73,
34.86, 33.95, 32.17, 28.24, 28.05, 24.87. MS (EI): m/z 400 (M+).
Anal. (C22H28N2O5) C, H, N.
(S)-2-(7-Ben zyloxyca r b a m oyl-h ep t a n oyla m in o)-3-b i-
p h en yl-4-yl-p r op ion ic Acid Meth yl Ester (10l). Compound
10l was synthesized by method B from 9a (300 mg, 1.07 mmol),
NMM (0.117 mL, 108 mg, 1.07 mmol), isobutyl chlorformate
(0.140 mL, 146 mg, 1.07 mmol), l-biphenylalanine methyl ester
hydrochloride (312 mg, 1.07 mmol), and NMM (0.234 mL, 216
mg, 1.07 mmol). The crude product was chromatographed
using dichloromethane/methanol (25:1); yield 250 mg (45%);
mp 143 °C. IR: 1739, 1654. 1H NMR (CD3OD): δ 7.59-7.26
(m, 14H), 4.88 (s, 2H), 4.76-4.69 (m, 1H), 3.71 (s, 3H), 3.26-
3.16 (m, 1H), 3.02-2.90 (m, 1H), 2.14 (t, J ) 7.3 Hz, 2H), 1.95
(S)-2-(7-Ben zyloxycar bam oyl-h eptan oylam in o)-3-n aph -
th a len -2-yl-p r op ion ic Acid Meth yl Ester (10o). Compound
10o was synthesized by method B from 9a (300 mg, 1.07
mmol), NMM (0.118 mL, 108 mg, 1.07 mmol), isobutyl chlo-
roformate (0.140 mL, 1.07 mmol), â-(2-naphthyl)-L-alanine
methylester hydrochloride (284 mg, 1.07 mmol), and NMM
(0.236 mL, 216 mg, 2.14 mmol); yield 450 mg (86%); mp 103
(t, J ) 7.3 Hz, 2H), 1.66-1.33 (m, 4H), 1.33-0.97 (m, 4H). 13
C
1
°C. IR: 3306, 1758, 1644, 1539. H NMR (CDCl3): δ 8.35 (bs,
NMR (CD3OD): δ 176.07, 173.61, 142.04, 141.04, 137.40,
130.69, 130.29, 129.83, 78.93, 52.69, 38.05, 36.59, 33.62, 29.58,
26.53, 26.34. MS (EI): m/z 516 (M+).
1H), 7.19-7.83 (m, 12H), 5.93 (d, J ) 7.8 Hz, 1H), 4.91-5.01
(m, 3H), 3.73 (s, 3H), 3.19-3.73 (m, 2H), 2.13 (t, J ) 7.3 Hz,
2H), 1.97 (bs, 2H), 1.55 (bs, 4H), 1.21 (bs, 4H). 13C NMR
(CDCl3): δ 172.74, 172.40, 133.46, 132.55, 129.27, 128.82,
128.69, 128.38, 128.09, 127.76, 127.61, 127.25, 126.32, 125.89,
78.21, 53.04, 53.043, 38.10, 36.21, 32.84, 28.30, 25.22, 24.87.
MS (EI): m/z 490 (M+).
(S)-3-Bip h en yl-4-yl-2-(7-h yd r oxyca r ba m oyl-h ep ta n oy-
la m in o)p r op ion ic Acid Meth yl Ester (11l). Compound 11l
was synthesized using method D from 10l (200 mg, 0.390
mmol) and 10% Pd on charcoal (20 mg); yield 114 mg (69%);
1
mp 136 °C. IR: 1739, 1647. H NMR (DMSO-d6): δ 10.31 (s,
(S)-2-(7-Hyd r oxyca r ba m oyl-h ep ta n oyla m in o)-3-n a p h -
th a len -2-yl-p r op ion ic Acid Meth yl Ester (11o). Compound
11o was synthesized by method D from 10o (400 mg, 0.82
mmol) and 10% Pd on charcoal (40 mg); yield 265 mg (81%);
mp 124 °C. IR: 1739, 1646, 1539. 1H NMR (DMSO-d6): δ 10.25
(bs, 1H), 8.77 (bs, 1H), 8.18 (d, J ) 8.1 Hz, 1H), 7.80-7.63 (m,
4H), 7.43-7.29 (m, 3H), 4.52 (m, 1H), 3.53 (s, 3H), 3.16-2.93
(m, 2H), 1.97-1.78 (m, 4H), 1.33-1.23 (m, 4H), 1.02-1.00 (m,
4H). 13C NMR (DMSO-d6): δ 172.30, 172.25, 169.06, 135.04,
132.94, 131.87, 127.47, 127.41, 126.05, 125.56, 53.37, 51.87,
36.84, 34.97, 32.24, 28.33, 28.16, 25.07, 24.96. MS (EI): m/z
400 (M+). Anal. (C22H28N2O5) C, H, N.
1H), 8.65 (s, 1H), 8.30 (d, J ) 7.9 Hz, 1H), 7.65-7.27 (m, 9H),
4.55-4.46 (m, 1H), 3.61 (s, 3H), 3.11-2.84 (m, 2H), 2.08-1.84
(m, 4H), 1.41-1.38 (m, 4H), 1.30-1.00 (m, 4H). 13C NMR
(DMSO-d6): δ 172.19, 172.05, 169.01, 139.77, 138.24, 136.55,
129.52, 128.78, 127.14, 126.36, 53.22, 51.67, 47.03, 36.21,
34.88, 32.13, 28.24, 28.09, 24.91. MS (EI): m/z 426 (M+). Anal.
(C24H30N2O5) C, H, N.
(S)-2-(7-Ben zyloxycar bam oyl-h eptan oylam in o)-3-m eth -
yl-bu tyr ic Acid Meth yl Ester (10m ). Compound 10m was
synthesized by method A from 9a (300 mg, 1.07 mmol), DIPEA
(0.185 mL, 138 mg, 1.07 mmol), BOP-Cl (272 mg, 1.07 mmol),
L-valine methyl ester hydrochloride (198 mg, 1.18 mmol), and
DIPEA (0.555 mL, 414 mg, 3.21 mmol); yield 270 mg (64%);
mp 60 °C. IR: 1739, 1646, 1538. 1H NMR (CDCl3): δ 8.79 (bs,
1H), 7.37-7.26 (m, 5H), 6.07 (d, J ) 8.69 Hz, 1H), 4.89 (s,
2H), 4.54 (dd, J ) 8.64/4.93 Hz, 1H), 3.71 (s, 3H), 2.26-1.99
(m, 5H), 1.64-1.54 (m, 4H), 1.30-1.25 (m, 4H), 0.98-0.87 (m,
6H). 13C NMR (CDCl3): δ 173.13, 172.86, 129.24, 128.78,
128.67, 57.06, 52.15, 36.42, 31.30, 28.55, 25.40, 24.86, 24.77,
19.01, 17.96, 17.85. MS (EI): m/z 392 (M+).
(S)-3-Th ioph en -2-yl-2-(7-tr ityloxyca r ba m oyl-h ep ta n oy-
la m in o)p r op ion ic Acid Meth yl Ester (10p ). Compound 10p
was synthesized by method B from 9b (610 mg, 1.41 mmol),
NMM (0.155 mL, 143 mg, 1.41 mmol), isobutyl chloroformate
(0.184 mL, 193 mg, 1.41 mmol), â-(2-thienyl)-L-alanine methyl
ester hydrochloride (313 mg, 1.41 mmol), and NMM (0.310 mL,
286 mg, 2.82 mmol); yield 670 mg (79%); mp 130 °C. IR: 1747,
1
1651. H NMR (CDCl3): δ 7.74 (bs, 1H), 7.34-7.27 (m, 15H),
7.17 (d, J ) 5.2 Hz, 1H), 6.95-6.91 (m, 1H), 6.76 (d, J ) 3.0
Hz, 1H), 6.07 (d, J ) 7.3 Hz, 1H), 4.93-4.84 (m, 1H), 3.76 (s,
3H), 3.38 (d, J ) 4.8 Hz, 2H), 2.21-2.13 (m, 2H), 1.85-1.48
(m, 4H), 1.18-0.95 (m, 6H). 13C NMR (CDCl3): δ 172.62,
171.62, 137.46, 129.14, 128.16, 127.09, 126.78, 124.92, 53.01,
52.53, 36.50, 32.15, 28.84, 25.33. MS (EI): m/z 243 (Trt+), 165
(C13H9+), 126 (C6H8NS+).
(S)-2-(7-Hyd r oxyca r ba m oyl-h ep ta n oyla m in o)-3-m eth -
yl-bu tyr ic Acid Meth yl Ester (11m ). Compound 11m was
synthesized by method D from 10m (170 mg, 0.43 mmol) and
10% Pd on charcoal (17 mg); yield 77 mg (59%) viscous oil. IR:
1742, 1652. 1H NMR (DMSO-d6): δ 10.33 (s, 1H), 8.66 (s, 1H),
8.07 (d, J ) 8.08 Hz, 1H), 4.17-4.10 (m, 1H), 3.60 (s, 3H),
2.16-1.87 (m, 5H), 1.45 (m, 4H), 1.20 (m, 4H), 0.87-0.82 (m,
6H). 13C NMR (DMSO-d6): δ 172.72, 172.30, 169.12, 57.37,
51.58, 35.11, 34.86, 32.28, 29.80, 28.40, 25.25, 25.07, 19.01,
18.34. MS (EI): m/z 302 (M+), 72 (C4H10N+). Anal. (C14H26N2O5‚
0.5H2O) C, H, N.
(S)-2-(7-Ben zyloxycar bam oyl-h eptan oylam in o)-3-n aph -
th a len -1-yl-p r op ion ic Acid Meth yl Ester (10n ). Compound
10n was synthesized by method B from 9a (300 mg, 1.07
mmol), NMM (0.118 mL, 108 mg, 1.07 mmol), isobutyl chlo-
roformate (0.140 mL, 146 mg, 1.07 mmol), â-(1-naphthyl)-L-
alanine methyl ester hydrochloride (284 mg, 1.07 mmol), and
NMM (0.236 mL, 216 mg, 2.14 mmol); yield 360 mg (69%);
mp 80 °C. IR: 1744, 1648, 1539. 1H NMR (CDCl3): δ 8.58 (bs,
1H), 8.09-7.22 (m, 12H), 6.03 (d, J ) 7.6 Hz, 1H), 5.03-4.93
(m, 1H), 4.89 (s, 2H), 3.62 (s, 3H), 3.58-3.51 (m, 2H), 2.11-
1.99 (m, 4H), 1.61-1.47 (m, 4H), 1.39-1.04 (m, 4H). 13C NMR
(CDCl3): δ 172.77, 172.61, 134.05, 132.05, 132.53, 132.37,
129.27, 128.97, 128.82, 128.71, 127.46, 126.44, 125.90, 125.34,
123.62, 78.47, 53.20, 52.31, 36.20, 35.27, 32.74, 28.46, 25.11,
24.77. MS (EI): m/z 490 (M+).
(S)-2-(7-H yd r oxyca r b a m oyl-h ep t a n oyla m in o)-3-t h io-
p h en -2-yl-p r op ion ic Acid Meth yl Ester (11p ). Compound
11p was synthesized by method E from 10p (500 mg, 0.835
mmol); yield 120 mg (42%); mp 85 °C. IR: 1735, 1672. 1H NMR
(DMSO-d6): δ 10.33 (s, 1H), 8.67 (s, 1H), 8.32 (d, J ) 7.7 Hz,
1H), 7.34 (d, J ) 4.9 Hz, 1H), 6.95-6.89 (m, 2H), 4.50-4.42
(m, 1H), 3.61 (s, 3H), 3.30-3.04 (m, 2H), 2.07 (t, J ) 6.96 Hz,
2H), 1.91 (t, J ) 7.14, 2H), 1.43 (bs, 4H), 1.17 (m, 4H). 13C
NMR (DMSO-d6): δ 172.29, 171.54, 169.08, 139.00, 126.72,
126.25, 124.65, 53.38, 51.84, 34.93, 32.20, 30.89, 28.29, 28.15,
24.94. MS (EI): m/z 356 (M+). Anal. (C16H24N2O5S) C, H, N.
(S)-3-(1H-In dol-3-yl)-2-(7-tr ityloxycar bam oyl-h eptan oy-
la m in o)p r op ion ic Acid Meth yl Ester (10q). Compound 10q
was synthesized by method B from 9b (500 mg, 1.16 mmol),
NMM (0.128 mL, 117 mg, 1.16 mmol), isobutyl chloroformate
(0.151 mL, 158 mg, 1.16 mmol), l-tryptophane methyl ester
hydrochloride (293 mg, 1.16 mmol), and NMM (0.255 mL, 234
mg, 2.32 mmol). The crude product was chromatographed
using ethyl acetate/hexane (2:1); yield 280 mg (39%); mp 112

Phenylalanine-Containing Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15 3307
1
°C. IR: 1740, 1657. H NMR (CDCl3): δ 7.67 (bs, 1H), 6.96-
scintillation cocktail. The compounds were tested in a starting
concentration of 40 µM, and active substances were diluted
further.
7.53 (m, 20H), 5.92 (d, J ) 8.1 Hz, 1H), 4.91-4.95 (m, 1H),
3.72 (s, 3H), 3.30-3.34 (m, 2H), 2.09 (t, J ) 1.9 Hz, 2H), 0.89-
1.83 (m, 10H). 13C NMR (CDCl3): δ 172.65, 129.11, 128.22,
123.03, 122.19, 119.61, 118.55, 111.49, 52.63, 52.46, 36.50,
32.90, 31.24, 28.71, 27.68, 25.35. MS (EI): m/z 165 (C13H9+),
243 (Trt+).
Rat liver HDAC was purified on Q-sepharose with an
increasing gradient of sodium chloride. The detailed procedure
is described elsewhere,
37 and this preparation is commercially
available as well (Calbiochem). Our nonisotopic coumarin-
labeled acetyllysine, which can be obtained from Calbiochem
(Nr. 382155), was employed as a substrate.38 We use a
modification of the original procedure that involves the use of
an internal standard in order to ensure accuracy and preci-
sion.48 The latter method was slightly modified with an
increased starting concentration of the substrate, which leads
to higher reproducibility of the IC50 values (data not shown).
In both assays, 2 and 6a ,c were used as reference compounds
while blank solvents and the O-benzyl hydroxamate precursor
of 535 served as negative controls. The HPLC assay was
performed on the LiChrosorb column with acetonitrile/water
(40/60, v/v) as mobile phase at a flow rate of 0.6 mL/min.
Retention time of substrate is 3.47 min, and that of the
internal standard 7-hydroxycoumarin is 2.17 min. Excitation
wavelength is 330 nm, and emission wavelength is 395 nm.
Results are taken from duplicate determinations on six levels
of inhibitor concentration.
Stock solutions of the inhibitors were made at a concentra-
tion of 12 mM in DMSO and 1 mg/mL in ethanol for TSA and
were further diluted with enzyme buffer (15 mM tris-HCl, pH
7.9; 0.25 mM EDTA; 10 mM NaCl; 10% (v/v) glycerol; 10 mM
2-mercaptoethanol). A substrate stock solution was prepared
using an aliquot of 12 µL of a solution of the fluorogenic
substrate (4.682 mg/mL in ethanol), 24 µL of a solution of the
standard 7-hydroxycoumarin (3.66 mg/mL in DMSO), and
enzyme buffer to a total volume of 1 mL.
(S)-2-(7-H yd r oxyca r b a m oyl-h ep t a n oyla m in o)-3-(1H -
in d ol-3-yl)p r op ion ic Acid Meth yl Ester (11q). Compound
11q was synthesized by method E from 10q; yield 110 mg
(59%); mp 118 °C. IR: 1739, 1658, 1604, 1548. 1H NMR
(DMSO-d6): δ 10.85 (s, 1H), 10.36, (s, 1H), 8.70 (s, 1H), 8.23
(d, J ) 7.6 Hz, 1H), 6.96-7.49 (m, 5H), 4.46-4.49 (m, 1H),
3.56 (s, 3H), 3.00-3.45 (m, 2H), 1.87-2.08 (m, 4H), 1.38-1.42
(m, 4H),1.11-1.14 (m, 4H). 13C NMR (DMSO-d6): δ 172.69,
172.49, 169.32, 136.15, 127.10, 123.70, 121.60, 118.49, 118.06,
111.51, 109.65, 53.09, 51.86, 35.02, 32.33, 28.42, 28.31, 27.11,
25.11. MS (EI): m/z 253. Anal. (C20H27N3O5) C, H, N.
(S)-Octa n ed ioic Acid Ben zyloxy-a m id e (2-Na p h th a len -
1-yl-1-p h en eth lca r ba m oyl-eth yl)a m id e (10r ). Compound
10r was synthesized by method B from 9a (300 mg, 1.07
mmol), NMM (0.118 mL, 108 mg, 1.07 mmol), isobutyl chlo-
roformate (0.140 mL, 146 mg, 1.07 mmol), (S)-2-amino-3-
naphthalen-1-yl-N-phenethyl-propionamide tosylate (525 mg,
1.07 mmol), and NMM (0.236 mL, 216 mg, 2.14 mmol); yield
1
400 mg (67%); mp 130 °C. IR: 1639, 1546. H NMR (CDCl3):
δ 6.95-8.08 (m, 17H), 4.73 (s, 2H), 4.62-4.60 (m, 1H), 3.39-
3.46 (m, 1H), 3.07-3.35 (m, 3H), 2.55-2.40 (m, 2H), 2.04-
1.88 (m, 4H), 1.43-1.29 (m, 4H), 1.15-0.95 (m, 4H). 13C NMR
(CDCl3): δ 175.85, 173.41, 140.30, 135.42, 134.42, 133.38,
130.31, 129.85, 129.76, 129.61, 129.45, 128.69, 127.30, 127.25,
126.70, 126.37, 124.74, 78.90, 55.55, 41.90, 36.70, 36.32, 33.64,
29.58, 26.53, 26.36. MS (EI): m/z 302 (C21H20NO+).
A 10 µL amount of the substrate/standard stock solution
was added to a mixture of 100 µL of rat enzyme preparation
(at 4 °C) and 10 µL of inhibitor dilution. After 15 min at 4 °C,
the mixture was then incubated for 90 min at 37 °C. After
this time, the reaction was stopped by the addition of 72 µL of
1 M HCl/0.4 M sodium acetate and 800 µL of ethyl acetate.
After it was centrifuged (10 000g, 5 min), an aliquot of 200 µL
of the upper phase was taken and the solvent was removed
by a stream of nitrogen. The residue was dissolved in 600 µL
of the chromatography eluent, and 20 µL was injected via
autosampler onto the HPLC system. The amount of remaining
substrate is calculated relative to the substrate control without
enzyme (each as quotient of the peak area of the substrate
divided by the peak area of the internal standard).
In d u ction of Ter m in a l Cell Differ en tia tion . Friend
leukemic cells (MEL DS19 murine erythroleukemia cells) were
maintained in DMEM containing 100 units/mL penicillin G
sodium and 100 µg/mL streptomycin sulfate supplemented
with 10% fetal bovine serum (Greiner) at 37 °C in a 5% CO2
atmosphere. To test compounds for potential to induce cell
differentiation, log-phase cells with a population doubling time
of 11-13 h were used. Serial dilutions of compounds were
prepared in 24 well plates (Falcon) using 1 mL DMEM/well.
If compounds were dissolved in DMSO, control wells contained
the same amount of solvent (generally 2 µL/mL medium, 0.1%
final concentration). Subsequently, the cell suspension was
added to the wells (1 mL/well, 8 × 104 cells/mL; final cell
concentration 4 × 104 cells/well). After 72 h, the experiment
was evaluated. Cell numbers were counted using a Casy 1 TTC
flow cytometer (Scha¨rfe System). The proliferation of treated
cells was expressed as percent proliferation in comparison with
the solvent control.
(S)-Octa n ed ed ioic Acid Hyd r oxya m id e (2-Na p h th a len -
1-yl-1-p h en eth ylca r ba m oyleth yl)a m id e (11r ). Compound
11r was synthesized by method D from 10r (320 mg, 0.580
mmol) and 10% Pd on charcoal (32 mg); yield 155 mg (54%);
mp 137 °C. IR: 1640, 1543, 1456. 1H NMR (DMSO-d6): δ 10.44
(s, 1H), 8.76 (s, 1H), 8.34 (d, J ) 8.4 Hz, 1H), 8.19-7.29 (m,
12H), 4.77-4.69 (m, 1H), 3.62-3.26 (m, 2H), 2.76-2.85 (m,
2H), 2.04-2.20 (m, 4H), 1.45-1.59 (m, 4H), 1.20-1.32 (m, 4H).
13C NMR (DMSO-d6): δ 172.37, 171.37, 169.48, 139.73, 134.27,
133.73, 132.04, 128.97, 128.88, 128.61, 127.70, 127.31, 126.37,
125,84, 125.60, 124.18, 53.74, 35.53, 35.43, 35.35, 32.64, 28.73,
28.60, 25.36. MS (EI): m/z 489 (M+). Anal. (C29H35N3O4‚
0.5H2O) C, H, N.
An a lysis of th e Op tica l P u r ity of 11d ,e. A Chirex 3014
column (Phenomenex, 250 mm × 4.6 mm) was used for the
determination of enantiomeric purity. Stock solutions of the
compounds (5 mg/mL in methanol) were diluted with the
chromatography eluent (isohexane/dichloromethane/methanol,
60/40/7.5, v/v). Retention time was 8.96 min for 11e and 9.76
min for 11d .
En zym e In h ibition . For partial purification of maize HD-
1, maize embryos were extracted and a crude chromatin
preparation was employed.46 The chromatin extract was ap-
plied to Q-Sepharose chromatography, and HD-1 enzyme
fractions were pooled and concentrated by ammonium sulfate
precipitation (20-45%). The precipitate was subjected to
Sephacryl Hi-Prep S-200, and peak fractions were used for
HDAC inhibition assays.39 For purification of maize HD-2, the
chromatin fraction of maize embryos was separated on Q-
sepharose.36 Radioactively labeled chicken core histones are
used as the enzyme substrate according to established meth-
odology.47 The enzymes are liberating tritiated acetic acid from
the substrate, which is quantitated by scintillation counting.
IC50 values are results of triple determinations. An amount of
50 µL of maize enzyme (at 30 °C) was incubated (30 min) with
10 µL of total [3H]acetate prelabeled chicken reticulocyte
histones (1 mg/mL). Reaction was stopped by addition of 36
µL of 1 M HCl/0.4 M acetate and 800 µL of ethyl acetate. After
it was centrifuged (10 000g, 5 min), an aliquot of 600 µL of
the upper phase was counted for radioactivity in 3 mL of liquid
Differentiated Friend leukemic cells accumulate hemoglo-
bin. Therefore, the induction of cell differentiation was deter-
mined by benzidine staining according to the literature.49 To
100 µL of cells suspension, 10 µL of a 0.4% solution of benzidine
in 12% acetic acid containing 2% H2O2 was added. Within 5
min, hemoglobin-containing cells stain blue. Benzidine-positive
and -negative cells were counted under the microscope in a
hemocytometer, and the percentage of positive cells was
calculated. All compounds were first tested at 10 and 50 µM

3308 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15
Wittich et al.
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final concentration. According to activity/toxicity profiles, a
range of concentration was chosen for a dose-response analy-
sis. In selected cases, dose-response experiments were re-
peated at the same concentrations and deviations were below
5%. So, we have not included error bars and the figures are
representations of one experiment and not a mean. Trichos-
tatin A (2) and SAHA (6a ), known inducers of cell differentia-
tion in Friend leukemic cells, were included as positive
controls.
Histon e Hyp er a cetyla tion . Friend leukemic cells (1 × 105
cells/ml in 20 mL) were incubated in DMEM for 24 h and then
treated with 5 µM 11l for various time periods as indicated.
Histones were isolated as described previously50 with slight
modifications. Briefly, 5 × 106 cells were harvested by cen-
trifugation and washed twice with cold phosphate-buffered
saline (PBS). Cells were lysed with 1 mL of lysis buffer (10
mM tris HCl, 50 mM sodium bisulfite, 1% Triton X-100, 10
mM MgCl2, 8.6% sucrose, pH 6.5), and nuclei were collected
by centrifugation. Cell pellets were washed with 10 mM tris
HCl, pH 7.4, containing 13 mM EDTA and suspended in 0.1
mL of ice-cold water. HCl was added until a final concentration
of 0.4 M, and the mixtures were incubated on ice for 1 h. Acid
soluble nuclear proteins were obtained by centrifugation at
10 000g for 10 min. Supernatants were mixed with 300 µL of
cold ethanol and incubated at -20 °C overnight. Histone-
containing pellets were obtained by centrifugation, air-dried,
and dissolved in 50 µL of water. Protein was quantified
according to Bradford.51 The samples were subjected to AUT-
PAGE analysis as described before.43,44
Ack n ow led gm en t. We thank the Fonds der Che-
mischen Industrie, the Fo¨rderergesellschaft der West-
fa¨lischen Wilhelms-Universita¨t, the DPhG-Stiftung fu¨r
den wissenschaftlichen Nachwuchs im Stifterverband
fu¨r die Deutsche Wissenschaft, the Deutsche Fors-
chungsgemeinschaft, the Austrian Science Foundation
(Grant P13620 to P.L.), the Austrian Academy of
Sciences (APART-fellowship to G.B.), and the Verein zur
Fo¨rderung der Krebsforschung in Deutschland e.V.
(C.G.) for support. We also thank Dr. Kevin Nash (Bayer
AG, Stoke Court, G.B.) for performing the tests for MMP
inhibition.
Su p p or tin g In for m a tion Ava ila ble: Experimental and
spectral data for amino acid building blocks and calibration
curves and HPLC chromatograms for chiral separation of
10d ,e. This material is available free of charge via the Internet
at http://pubs.acs.org.
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