Synthesis and cytotoxic evaluation of cycloheximide derivatives as potential inhibitors of FKBP12 with neuroregenerative properties

Article abstract of DOI:10.1021/jm991038t

On the basis of the new finding that the protein synthesis inhibitor cycloheximide (1, 4-[2-(3,5-dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]-2,6- piperidinedione) is able to competitively inhibit hFKBP12 (K₁ = 3.4 μM) and homologous enzymes, a se

Full text of DOI:10.1021/jm991038t

J . Med. Chem. 1999, 42, 3615-3622
3615
Syn th esis a n d Cytotoxic Eva lu a tion of Cycloh exim id e Der iva tives a s P oten tia l
In h ibitor s of F KBP 12 w ith Neu r or egen er a tive P r op er ties∇
Claudia Christner,† Ralf Wyrwa,‡ Silvia Marsch,§ Gerhard Ku¨llertz,† Ralf Thiericke,‡ Susanne Grabley,‡
Dieter Schumann,§ and Gunter Fischer*,†
Max-Planck Research Unit, Enzymology of Protein Folding, Weinbergweg 22, D-06120 Halle/ Saale, Germany,
Hans-Kno¨ll-Institute of Natural Products Research, Beutenbergstrasse 11, D-07745 J ena, Germany, and Department of
Maxillo-Facial and Plastic Surgery, Friedrich-Schiller-University, Bachstrasse 18, D-07743 J ena, Germany
Received March 16, 1999
On the basis of the new finding that the protein synthesis inhibitor cycloheximide (1, 4-[2-
(3,5-dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]-2,6-piperidinedione) is able to competitively
inhibit hFKBP12 (Ki ) 3.4 µM) and homologous enzymes, a series of derivatives has been
synthesized. The effect of the compounds on the activity of hFKBP12 and their cytotoxicity
against eukaryotic cell lines (mouse L-929 fibroblasts, K-562 leukemic cells) were determined.
As a result, several less toxic or nontoxic cycloheximide derivatives were identified by
N-substitution of the glutarimide moiety and exhibit IC50 values in the range of 22.0-4.4 µM
for inhibition of hFKBP12. Among these compounds cycloheximide-N-(ethyl ethanoate) (10, Ki
) 4.1 µM), which exerted FKBP12 inhibition to an extent comparable to that of cycloheximide
(1), was found to cause an approximately 1000-fold weaker inhibitory effect on eukaryotic protein
synthesis (IC50 ) 115 µM). Cycloheximide-N-(ethyl ethanoate) (10) was able to significantly
speed nerve regeneration in a rat sciatic nerve neurotomy model at dosages of 30 mg/kg.
In tr od u ction
by FKBP12, whereas FKBP12.6 is associated with
cardiac RyR2.11 Using a knockout model, Shou et al.12
were able to show that FKBP12-deficient mice display
severe dilated cardiomyopathy and ventricular septal
defects mimicking human congenital heart disorders.
Peptidyl-prolyl cis/ trans isomerases (PPIases; E.C.
5.2.1.8) are enzymes that catalyze the cis/ trans isomer-
ization of peptidyl-prolyl amide bonds in peptides and
unfolded proteins.1,2 Until today, three distinct PPIase
families have been identified according to amino acid
sequence homology and to the characteristics of inhibi-
tion by drugs of microbial origin: cyclophilins, FK506-
binding proteins (FKBPs),3 and parvulins.4 Members of
each of these families are evolutionarily conserved from
prokaryotic to eukaryotic organisms and are localized
in virtually all cell types.5,6
PPIases are reported to play an important role in a
range of biological processes. Beside the cyclophilins,
various FKBPs were shown to have regulatory functions
as stable or dynamic part of heterooligomeric complexes
containing physiologically relevant proteins.7 As an
example, FKBP52 seems to be functionally associated
with untransformed steroid hormone receptors.8 Re-
cently it was found that FKBP12 binds to and modulates
the properties of transforming growth factor â-type 1
(TGF-â1) receptors9 and inhibits epidermal growth
factor (EGF) receptor tyrosine autophosphorylation.10
Several studies have demonstrated the coordination of
gating of major intracellular Ca2+ release channels, e.g.,
IP3 receptors and ryanodine receptors (RyR) of the
sarcoplasmic reticulum of skeletal and cardiac muscles,
Initially published reports of inhibitors of FKBPs were
focused on the macrolactone-type agents FK50613 and
rapamycin.14 These compounds originally attracted
scientific and clinical interest because of their immu-
nosuppressive properties.15 It is believed that complexes
of FK506-FKBP12 bind to and thus inactivate the
Ca2+-activated protein serine/threonine phosphatase
calcineurin, leading to a block of Ca2+-dependent T- and
B-lymphocyte responses,16 whereas rapamycin-FKBP12
targets homologues of the yeast TOR molecules includ-
ing the human RAFT/FRAP.17 In addition to FK506 and
rapamycin, various small molecule FKBP12 inhibitors
devoid of immunosuppressive activity were described,
containing the R-dicarbonyl amide key structural ele-
ment of the FKBP binding domain, which is supposed
to interact with the active site of the PPIase.18 Among
these compounds, a number of pipecolate and N-(gly-
oxyl)prolyl FKBP12 ligands were characterized as caus-
ing submicromolar FKBP12 inhibition (Ki,app ) 1-100
nM).18b,d Extremly potent inhibitors of this type carry
bulky hydrophobic alkyl groups such as 1,1-dimethyl-
propyl and 3,4,5-trimethoxyphenyl as substituents for
the pyranose ring region of FK506 as well as simple
alkyl or alkylaryl esters instead of the lead cyclohexyl-
ethyl moiety. However, based on results obtained by free
energy perturbation techniques in Monte Carlo statisti-
cal mechanics simulations, Lamb and J orgensen18e
recently brought into question reported binding data for
some of these R-dicarbonyl amides, especially those
containing a 3-(3-pyridyl)propyl moiety such as GPI-
* Author for correspondence: Prof. G. Fischer. Tel: +49345 5522801.
Fax: +49345 5511972. E-mail: fischer@enzyme-halle.mpg.de.
† Max-Planck Research Unit.
‡ Hans-Kno¨ll-Institute of National Products Research.
§ Friedrich-Schiller-University.
∇ Abbreviations: EDL, extensor digitorum longus muscles; FKBP,
FK506-binding protein; pNA, 4-nitroanilide; rhCyp18, recombinant
human cytosolic cyclophilin with a molecular mass of 18 kDa; IP3,
inositol 1,4,5-trisphosphate; PPIase, peptidyl-prolyl cis/ trans isomerase.
10.1021/jm991038t CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/14/1999

3616 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Christner et al.
1046. Replacement of the diketo portion by other func-
tionalities led to less potent peptidic19 as well as
sulfonamide20 and urea linked21 FKBP12 inhibitors with
generally micromolar affinity for the enzyme.
Most recently, FK506 and FKBP12 ligands such as
V-10,36722 were shown to increase axonal regeneration
in neuronal cell cultures and different animal models
via a yet unknown calcineurin-independent mech-
anism.18d,23 Furthermore, FKBP inhibitors were proven
to exert powerful antineurodegenerative effects on dam-
aged central and peripheral neurons.18d,24,25 General
neuroprotective and neurotrophic properties of GPI-
1046, published by Steiner and Hamilton,25 are, how-
ever, controversially discussed as Harper and co-
workers26 failed to reproduce corresponding in vitro and
in vivo activities.
Taking into account the clinical potential of selectively
acting nonimmunosuppressive FKBP inhibitors, espe-
cially for the treatment of human nerve injuries and
neurodegenerative disorders, such as Parkinson’s and
Alzheimer’s Diseases, we performed a screening of
effectors of the enzyme activity of hFKBP12. A collection
of structurally diverse, pure secondary metabolites was
used to identify new lead structures. Surprisingly,
cycloheximide (1)27 was found to specifically inactivate
the PPIase activity of hFKBP12. The glutarimide an-
tibiotic cycloheximide (1), which is produced by several
strains of Streptomyces, is exclusively applied in bio-
chemical research as a potent inhibitor of eukaryotic
protein synthesis.28 We therefore became interested in
the synthesis of cycloheximide derivatives which would
allow to discriminate between the contributions of the
inhibition of eukaryotic translation and of FKBP by 1
to observed biological effects. Since neuroprotective
properties of both inhibitors of FKBP22-25 and subtoxic
dosages of cycloheximide (1)29 have been shown, we
chose the rat sciatic nerve neurotomy model30 as a
representative system to evaluate the possible role of
FKBP inhibition by active cycloheximide derivatives,
which do not affect protein synthesis, for neuronal
regeneration.
F igu r e 1. Lineweaver-Burk plot for the inhibition of
hFKBP12 by cycloheximide (1). The initial velocity v of the
PPIase-catalyzed reaction was determined by means of the
protease-coupled PPIase assay as described in the Experimen-
tal Section using R-chymotrypsin (410 µg/mL) as a protease.
hFKBP12 (14.2 nM) was incubated with concentrations of
compound 1 of 0.0 (b), 0.9 (0), 1.8 (2), 3.6 (O), and 7.2 (9) µM
in 35 mM HEPES (pH 7.8) for 10 min. Remaining PPIase
activity was monitored immediately after reactions were
started by addition of 0.023-1.0 mM Suc-Ala-Leu-Pro-Phe-
pNA.
K2CO3.33 Under the used conditions N-1 and C-10
reactions cannot change the sterochemistry of the four
stereocenters of cycloheximide (1). Similarly, the con-
figuration on C-8 was not altered during synthesis of
1
the acetyl derivative 3 as confirmed by H NMR (dd, J
) 8.2, 3.2 Hz, 1H, 8-H).
Resu lts a n d Discu ssion
In the course of screening for new inhibitors of
hFKBP12, using the protease-coupled PPIase assay
developed by Fischer et al.,1 a striking inhibitory activity
of cycloheximide (1) was observed. As cycloheximide (1)
differs completely from known FKBP12 ligands in terms
of structural composition, it was chosen as a novel lead
structure for detailed binding studies as well as for the
investigation of structure-activity relationships in a
series of synthesized cycloheximide derivatives in rela-
tion to cytotoxicity against eukaryotic cells.
Ch em istr y
A number of synthetic approaches were pursued to
create cycloheximide derivatives. To obtain the oxime
2, cycloheximide (1) was treated with hydroxylamine in
pyridine. The acetyl derivative 3 of cycloheximide (1)
was prepared by selective acetylation of the hydroxyl
group with acetic acid anhydride under basic condi-
tions.31
In the literature there is precedence for the partial
alkylation of cycloheximide analogues to N-alkylcyclo-
heximide derivatives under basic conditions32 starting
from alkyl halides. This method proved to be a facile
route to provide N-alkylated cycloheximide derivatives
with various alkyl substitutions at the nitrogen atom
of the glutarimide moiety. A number of useful alkyl
halides containing several functionalities are available
commercially. The synthesis of all the N-alkyl deriva-
tives, except 5, was achieved in a straightforward
manner starting from bromoalkanes in the presence of
K2CO3 and catalytic amounts of 18-crown-6. The deriva-
tive 5 containing a methyl group on the ring nitrogen
was prepared from methyl iodide in the presence of
On the basis of standard gel permeation chromatog-
raphy methods,34 the reversible character of the cyclo-
heximide-hFKBP12 interaction was demonstrated.
Evaluation of the inhibition kinetics by the Lineweaver-
Burk plot revealed a competitive mode of inhibition of
hFKBP12 by 1 (Figure 1). Using the formula of a
competitive inhibitor model, a respecting Ki value of 3.4
µM was calculated (Table 1). To characterize whether
enzyme inhibition by cycloheximide (1) expresses speci-
ficity with regard to different PPIases, we incubated
members of three PPIase families with compound 1
(Table 1). Compared to the effect on hFKBP12, cyclo-
heximide (1) caused a 2-60-fold weaker inhibition of
the activity of further members of the FKBP family of
PPIases such as E. coli FKBP26 (Ki ) 10.3 µM), rabbit
FKBP52 (Ki ) 24.2 µM), FKBP22 from Photobacterium
sp. (Ki ) 76.2 µM), and L. pneumophila FKBP25 (Mip)
(Ki ) 124.0 µM). Except for a slight inhibitory effect on
E. coli parvulin (Ki ) 187.0 µM), no deterioration of the
PPIase activity of tested FKBP homologous enzymes E.
coli SlyD and E. coli trigger factor, the cyclophilin

Cycloheximide Derivatives as Inhibitors of FKBP12
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3617
Ta ble 1. Specificity of Cycloheximide (1) and Its Effect on
Different PPIases
obtained by substitution of the N-1 hydrogen atom of 1
by ethyl butanoate (9). A further reduction of the
hFKBP IC50 to a value of 4.4 µM was observed for
cycloheximide-N-(ethyl ethanoate) (10). Thus, ethyl
ethanoate 10, which caused at least an ∼150-fold
weaker cytotoxic effect against the eukaryotic cell lines
L-929 and K-562 compared to the lead structure cyclo-
heximide (1) but had a similar Ki value of 4.1 µM, was
selected for further investigations.
PPIase
family
PPIasea
Ki (µM)b
FKBPs
hFKBP12
3.4 ( 0.7
10.3 ( 1.8
24.2 ( 2.2
76.2 ( 6.2
124.0 ( 10.1
>200
E. coli FKBP26
rabbit FKBP52
Photobacterium sp. FKBP22
L. pneumophila FKBP25 (Mip)
E. coli trigger factor
E. coli SlyD
hCyp18
hPin1
E. coli parvulin
>200
Direct evidence for the profound deterioration of the
ability of cycloheximide-N-(ethyl ethanoate) (10) to
inhibit protein translation was obtained as a result of
comparative studies of the influence of 1 and 10 on
protein synthesis of the R-1 mating pheromone (R-factor)
from S. cerevisiae by means of a rabbit reticulocyte type
I translation assay. As illustrated in Figure 2, both
compounds differ significantly in their inhibitory activ-
ity on protein translation by 3 orders of magnitude. The
discrepancy in the calculated IC50 values for cyclohex-
imide (1, 0.1 µM) and cycloheximide-N-(ethyl ethanoate)
(10, 115.0 µM) confirms the above-described difference
in cytotoxicity against eukaryotic cells.
cyclophilins
parvulins
>200
>200
187.0 ( 12.4
a
Concentrations of enzymes were 14 nM, 52 nM, 1 µM, 41 nM,
40 nM, 12 nM, 2 µM, 2 nM, 4 nM, and 6 nM for hFKBP12, rabbit
FKBP52, E. coli FKBP26, Photobacterium sp. FKBP22, L. pneu-
mophila FKBP25 (Mip), E. coli trigger factor, E. coli SlyD, hCyp18,
b
hPin1, and E. coli parvulin, respectively. Inhibition constants Ki
for the inhibition of PPIase activity by compound 1 were deter-
mined according to the Experimental Section. Using these meth-
ods, a Ki value of 0.5 ( 0.3 nM was measured for FKBP12
inhibition by FK506 in agreement with previous reports.15b,21,38
Data are reported as mean ( SD for three determinations.
hCyp18, and the parvulin hPin1 was observed (IC50
>
On the basis of these results, cycloheximide-N-(ethyl
ethanoate) (10), which satisfied required criteria for a
differential cycloheximide-derived FKBP inhibitor, was
selected for in vivo evaluation of neuroregenerative
efficacy. The neurotrophic effect of compound 10 was
studied employing the rat sciatic nerve neurotomy
model30 using FK506 as a positive control. Thereby the
influence of both compounds on axonal regeneration was
indirectly approached by assessment of walking behav-
ior following neurotomy for 8 and 10 weeks. Addition-
ally, the weight differences between the afterward
prepared, sciatic nerve-innervated extensor digitorum
longus muscles (EDL) of the left operated hind limbs
and the corresponding right unlesioned legs were mea-
sured. The established animal model allowed us to
reproduce the published enhancement in rate of axonal
regeneration in rats treated with 1 mg/kg FK506
compared to placebo-treated controls (Table 3). Analysis
of the walking behavior according to the criteria given
in the Experimental Section showed a significant earlier
onset of normal walking, reflected by a 100 ( 48%
increased rate of functional recovery. On the basis of
the evaluation of the differences of muscle weights, an
18 ( 12% positive regenerative tendency was seen for
FK506-treated rats.
200 µM). Thus, cycloheximide (1) exhibits a certain
inhibitory specificity for FKBP-like PPIases, which have
been differentiated previously by their ability to bind
FK506.
To identify the critical functional groups in the
pharmacophore of cycloheximide (1) for hFKBP12 in-
hibition, it was necessary to prepare derivatives with
modifications in positions R1, R2, and R3 (Table 2). In
addition, the effect of selected derivatives on the eu-
karyotic cell lines L-929 and K-562 was investigated by
means of standard proliferation assays.35 As expected,
cycloheximide (1) was revealed to be extremely cytotoxic
against both cell lines, thus indicating a relatively low
selectivity for hFKBP12 inhibition (Table 2).
It was found that the oxime derivative 2, obtained by
modification of the C-10 carbonyl group, shows a ∼260-
fold lower cytotoxicity than 1 but a dramatically reduced
inhibitory activity on hFKBP12. Inability to inhibit
hFKBP12 was observed in the case of the O-acetyl
derivative 3, which displayed only a slightly decreased
cytotoxic effect on L-929 and K-562 cells compared to
1, and for the nontoxic O,N-substituted compound 4.
These results indicate a possible involvement of the C-10
carbonyl and the C-8 hydroxyl group in the binding of
cycloheximide (1) to the active site of hFKBP12. In
contrast, the calculated inhibitor constants for several
of the N-substituted derivatives are in the same order
of magnitude as the IC50 value of 1. Therefore, the
structural composition of position R3 is presumably less
important for inhibitory activity on hFKBP12.
Exceptions are compounds with hydrophobic substit-
uents in position R3 such as methyl (5) and benzyl (6),
reflected by low values of hFKBP12 IC50 of 124.1 and
116.7 µM, respectively. Changing the functional group
at R3 to 4-cyanobenzyl (7) yielded a compound with
measurable inhibitory activity on hFKBP12 in the
middle micromolar range that considerably affected the
proliferation of K-562 and L-929 cells. Similar toxic
properties but a ∼3-fold lower hFKBP IC50 value of 21.6
µM were demonstrated by the acetic acid amide 8. A
derivative with a comparable inhibitor constant but a
∼26-fold lower cytotoxicity against eukaryotic cells was
Before neuroregenerative testing of cycloheximide-N-
(ethyl ethanoate) (10), the acute toxicity on 15-day-old
embryonized hen’s eggs was determined according to
Nishigori et al.,36 to get a crude measure for in vivo
tolerated dosages. The obtained value of the maximal
tolerated dosage (MTD) of compound 10 indicated an
in vivo digestibility for dosages up to 50 mg/kg. There-
fore, subtoxic cycloheximide-N-(ethyl ethanoate) (10)
dosages of 30 mg/kg were administered to the site of
the sciatic nerve lesion to get an analyzable effect.
Assessment of walking behavior for a time interval of 8
weeks following neurotomy revealed an 46 ( 20%
increase of functional recovery in cycloheximide-N-(ethyl
ethanoate)-treated rats, whereas a less marked effect
was obvious after 10 weeks (14 ( 7%). These results of
behavioral studies agreed well with the 20 ( 1% and
13 ( 3% faster rebuilding of degenerated EDL muscles

3618 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Christner et al.
Ta ble 2. Evaluation of the Effect of Cycloheximide (1) and Cycloheximide Derivatives on the Activity of hFKBP12 and Cytotoxicity
Against Eukaryotic Cell Lines L-929 and K-562
cytotoxicity
IC50 (µg/mL)
compd
no.
hFKBP12
IC50 (µM)
R1
R2
R3
L-929
K-562
1
2
3
4
5
6
7
8
9
O
NOH
O
O
O
O
O
O
O
OH
OH
H
H
H
3.6
<0.39
86.9
9.3
<0.39
116.5
1.1
177.0
>200
>200
124.1
116.7
68.2
21.6
22.3
4.4
OC(O)CH3
OC(O)NH-1-adamantane
CH2C(O)OC2H5
CH3
CH2Ph
p-CNPhCH2
CH2C(O)NH2
(CH2)3C(O)OC2H5
CH2C(O)OC2H5
nd
nd
OH
OH
OH
OH
OH
OH
>200
135.1
1.4
5.3
144.9
76.6
132.6
81.6
0.8
4.6
109.2
64.9
10
O
ent time intervals. This will include a complete dose-
dependence study.
Con clu sion
In summary, the present results characterize the
inhibition of FKBP12 by cycloheximide (1), which
represents a new biological property of the compound.
Cycloheximide (1) originally became the subject of
pharmaceutical interest because of its fungicidal ef-
fectiveness, especially against yeasts and parasitic
fungi, as well as its antiviral activity.28a Further studies
were extended to the investigation of the potential
pharmaceutical suitability of cycloheximide (1) and
synthetic analogues for the prevention of proliferative
skin diseases and epileptical disorders.32 Today, exclu-
sively the ability of cycloheximide (1) to inhibit eukary-
otic protein synthesis is utilized in biochemical areas.28
However, cycloheximide (1) concentrations in the range
of 0.1-100 µM routinely used in biological experiments37
may also cause a significant inhibition of FKBPs which
has to be considered for the interpretation of results.
As an example, the neuroregenerative capacity of cy-
cloheximide-N-(ethyl ethanoate) (10) was demonstrated
as a cycloheximide derivative ineffective in terms of
translational inhibition. Thus, a possible contribution
of the FKBP inhibition to neuroprotective effects of
micromolar concentrations of cycloheximide (1) shown
in different animal models29 can be hypothesized.
F igu r e 2. Effect of cycloheximide (1) and cycloheximide-N-
(ethyl ethanoate) (10) on eukaryotic protein translation.
Synthesis of S. cerevisiae R-factor in rabbit reticulocyte lysate
was followed in the presence of 0.0-100 µM 1 (9) and 10 (0),
respectively, by measuring incorporation of [35S]Met for 60 min
at 30 °C as stated in the Experimental Section.
Ta ble 3. Effect of FKBP12 Inhibitors FK506 and 10 on Axonal
Regeneration Following Sciatic Nerve Neurotomy
functional recovery (%)a
b
walking behavior
compd (mg/kg) 8 weeks 10 weeks
∆mEDL
dosage
8 weeks 10 weeks
FK506
10
1
30
nd
+100 ( 48 nd
+18 ( 12
+46 ( 20 +14 ( 7
+20 ( 1 +13 ( 3
a
Neuroregenerative activities were assessed relative to placebo-
caused effects as described in the Experimental Section. Data are
Exp er im en ta l Section
Gen er a l Meth od s. 4-[2-(3,5-Dimethyl-2-oxocyclohexyl)-2-
hydroxyethyl]-2,6-piperidinedione (cycloheximide) (1) and re-
agents were purchased from Aldrich Chemical Co. (Steinheim,
Germany). Mass spectra were obtained on a Hewlett-Packard
MALDI-TOF MS system G2025A. NMR spectra (Bruker
Avance DPX 300, DRX 500; TMS as internal standard; 1H,
300 or 500 MHz; 13C, 75 MHz; CDCl3 unless otherwise noted)
were recorded for all compounds. Melting points were deter-
mined on a Bu¨chi melting point B-545. TLC sheets of silica
gel 60 F254 from Merck (Darmstadt, Germany) were used for
qualitative information. Chromatographical purification was
carried out on silica gel 60 M (Macherey-Nagel GmbH & Co.
KG, Du¨ren, Germany). Elemental analysis was performed on
a CHN-O-Rapid from Foss Heraeus.
b
reported as mean ( SD of n determinations as noted. Weight
differences between EDL muscles of axotomized left hind limbs
and corresponding right controls.
of the lesioned hind limbs sampled after 8 and 10 weeks,
respectively, compared to placebo-treated animals. Thus,
cycloheximide-N-(ethyl ethanoate) (10) seems to ef-
fectively accelerate regeneration of the rat sciatic nerve
at dosages of 30 mg/kg. Further experiments are in
progress to determine the neuroregenerative efficacy in
case of additional daily repeated subcutaneous injections
of cycloheximide-N-(ethyl ethanoate) dosages for differ-

Cycloheximide Derivatives as Inhibitors of FKBP12
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3619
compound 6 as a colorless, viscous oil: MALDI-TOF-MS [M
4-[2-(3,5-Dim eth ylcycloh exyl 2-oxim e)-2-h ydr oxyeth yl]-
2,6-p ip er id in ed ion e (2). To a solution of 1.0 g (3.56 mmol)
of cycloheximide (1) in 6.0 mL of pyridine was added 2.0 mL
of a saturated solution of [NH3OH]Cl in water. The reaction
mixture was stirred at 35 °C for 2 h. After concentration in
vacuo and addition of 5.0 mL of water, a white precipitate was
observed, which was filtered, washed twice with water and
CH2Cl2, and dried to yield 863 mg (82.3%) of the oxime 2: mp
) 210-213 °C dec; MALDI-TOF-MS [M + H]+ ) 297.0 m/e;
1H NMR (500 MHz) (DMSO-d6) δ 10.59 (s, 1H), 10.20 (s, 1H),
4.74 (d, 1H, J ) 7.1 Hz), 3.66 (q, 1H, J ) 8.8 Hz), 3.20 (dd,
1H, J ) 9.6, 5.4 Hz), 2.59-2.43 (comp, 3H), 2.30-2.13 (comp,
4H), 2.08 (d, 1H, J ) 13.0 Hz), 1.93 (mult, 1H), 1.53 (d, 1H, J
) 12.5 Hz), 1.44 (mult, 1H), 1.24-1.13 (comp, 2H), 1.01 (d,
3H, J ) 7.6 Hz), 0.92 (d, 3H, J ) 7.5 Hz); 13C NMR (75 MHz)
δ 173.4, 173.2, 160.5, 67.0, 41.2, 38.5, 36.3, 34.7, 34.6, 26.7,
26.5, 22.1, 21.4, 20.1, 17.3. Anal. (C15H24N2O4) C, H, N.
4-[2-(3,5-Dim et h yl-2-oxocycloh exyl)-2-a cet oxyet h yl]-
2,6-p ip er id in ed ion e (3). To a solution of 1.0 g (3.56 mmol)
of cycloheximide (1) in 7.0 mL of pyridine was added 2.5 mL
(26.5 mmol) of acetic acid anhydride. The reaction mixture was
stirred at 25 °C for 1.5 h. After concentration in vacuo the
crude product was purified by recrystallization from ethanol
to yield 947 mg (82.3%) of compound 3 as white crystals: mp
) 144-145 °C; MALDI-TOF-MS [M + H]+ ) 324.5 m/e; 1H
NMR (500 MHz) δ 8.63 (s, 1H), 5.28 (dt, 1H, J ) 8.2, 3.2 Hz),
2.86 (dd, 1H, J ) 17.3, 3.0 Hz), 2.66-2.49 (comp, 3H), 2.35-
2.18 (comp, 2H), 2.12 (mult, 2H), 2.00 (s, 3H), 1.83 (mult, 2H),
1.69-1.52 (comp, 4H), 1.19 (d, 3H, J ) 7.2 Hz), 0.93 (d, 3H, J
) 6.4 Hz); 13C NMR (75 MHz) δ 212.0, 172.2, 172.0, 170.4,
69.4, 49.0, 42.6, 40.6, 38.6, 38.2, 36.8, 36.1, 27.2, 26.6, 20.8,
18.0, 14.0. Anal. (C17H25NO5) C, H, N.
1
+ H]+ ) 372.9 m/e; H NMR (300 MHz) δ 7.37-7.19 (comp,
5H), 4.92 (s, 2H), 4.16 (dt, 1H, J ) 10.8, 2.5 Hz), 2.86 (d, 2H,
J ) 12.7 Hz), 2.61 (mult, 1H), 2.49-2.30 (comp, 5H), 2.18
(mult, 1H), 1.96-1.72 (comp, 3H), 1.66-1.51 (comp, 2H), 1.22
(d, 3H, J ) 7.1 Hz), 1.05-1.15 (mult, 1H), 0.97 (d, 3H, J ) 6.4
Hz); 13C NMR (75 MHz) δ 216.3, 172.0, 171.8, 140.8, 137.2,
128.7, 128.3, 127.3, 66.4, 50.0, 42.6, 42.5, 40.4, 39.6, 38.3, 37.8,
32.9, 26.6, 26.4, 18.3, 14.1. Anal. (C22H29NO4) C, H, N.
4-[2-(3,5-Dim eth yl-2-oxocycloh exyl)-2-h yd r oxyeth yl]-
1-(4-cya n oben zyl)-2,6-p ip er id in ed ion e (7). In a typical
experiment 300 mg (1.07 mmol) of cycloheximide (1), 235 mg
(1.2 mmol) of 4-cyanobenzyl bromide, 10 mg (0.04 mmol) of
18-crown-6, and 200 mg (1.45 mmol) of K2CO3 in 3.0 mL of
acetone were stirred at 25 °C for 2 days. After filtration and
concentration in vacuo the crude product was purified by
column chromatography on silica gel (CH2Cl2/EtOAc (3:1)) to
yield 163 mg (38.4%) of compound 7 as a colorless, viscous oil:
1
MALDI-TOF-MS [M + H]+ ) 397.8 m/e; H NMR (300 MHz)
δ 7.67-7.35 (comp, 4H), 4.94 (s, 2H), 4.19 (d, 1H, J ) 10.9
Hz), 3.49 (s, 2H), 2.89 (d, 2H, J ) 12.8 Hz), 2.63 (m, 1H), 2.56-
2.32 (comp, 3H), 2.21 (mult, 1H), 1.99-1.73 (comp, 3H), 1.69-
1.52 (comp, 2H), 1.23 (d, 3H, J ) 7.1 Hz), 1.18-1.07 (mult,
1H), 0.98 (d, 3H, J ) 6.4 Hz); 13C NMR (75 MHz) δ 216.8,
172.1, 171.9, 138.6, 133.5, 132.4, 131.2, 129.2, 118.6, 112.6,
66.6, 50.1, 44.8, 42.0, 39.6, 38.4, 38.1, 31.5, 26.6, 26.0, 18.3,
14.1. Anal. (C23H28N2O4) C, H, N.
4-[2-(3,5-Dim eth yl-2-oxocycloh exyl)-2-h yd r oxyeth yl]-
2,6-p ip er id in ed ion e-1-(a cetic a cid a m id e) (8). To a solu-
tion of 500 mg (1.78 mmol) of cycloheximide (1) and 275 mg
(2.0 mmol) of bromoacetic acid amide in 5.0 mL of acetone were
added 10 mg (0.04 mmol) of 18-crown-6 and 300 mg (2.18
mmol) of K2CO3. The reaction mixture was stirred at 25 °C
for 3 days. After filtration and concentration in vacuo the crude
product was purified by column chromatography on silica gel
(CH2Cl2/EtOAc (3:1)) to yield 214 mg (35.5%) of compound 8
as a colorless, viscous oil: MALDI-TOF-MS [M + H]+ ) 339.9
4-[2-(3,5-Dim eth yl-2-oxocycloh exyl)-2-(oxyca r bon yl-1-
adam an tylam in o)eth yl]-2,6-piper idin edion e-1-(eth yl eth -
a n oa te) (4). To a solution of 300 mg (0.82 mmol) of 4-[2-(3,5-
dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]-2,6-piperidinedione-
1-(ethyl ethanoate) (8) in 5.0 mL of dry CH2Cl2 was added 145
mg (0.82 mmol) of 1-adamantyl isocyanate. The reaction
mixture was stirred at 25 °C for 3 h. After concentration in
vacuo the crude product was purified by column chromatog-
raphy on silica gel (CH2Cl2/EtOAc (5:1)) to give 394 mg (88.2%)
of compound 4 as a white solid: mp ) 73-74 °C; MALDI-TOF-
1
m/e; H NMR (500 MHz) δ 6.03 (mult, 2H), 4.43 (s, 2H), 4.16
(d, 1H, J ) 10.8 Hz), 2.95-2.80 (comp, 2H), 2.62 (comp, 2H),
2.54-2.41 (comp, 4H), 2.19 (mult, 1H), 1.93-1.83 (comp, 3H),
1.60 (td, 2H, J ) 13.3, 4.2 Hz), 1.33-1.26 (m, 1H), 1.22 (d,
3H, J ) 7.1 Hz), 0.97 (d, 3H, J ) 6.4 Hz); 13C NMR (75 MHz)
δ 216.3, 172.3, 172.1, 169.5, 66.5, 50.3, 42.6, 41.6, 40.8, 38.9,
38.0, 37.8, 33.4, 26.7, 26.4, 18.3, 14.2. Anal. (C17H26N2O5) C,
H, N.
1
MS [M + H]+ ) 545.9 m/e; H NMR (300 MHz) δ 5.07 (mult,
1H), 4.49 (s, 2H), 4.16 (q, 2H, J ) 7.1 Hz), 3.00 (dd, 1H, J )
17.2, 3.0 Hz), 2.82-2.37 (comp, 6H), 2.27 (mult, 1H), 2.16
(mult, 1H), 2.07 (br, 3H), 1.92 (br, 8H), 1.73-1.55 (comp, 10H),
1.31-1.20 (comp, 6H), 0.98 (d, 3H, J ) 6.4 Hz); 13C NMR (75
MHz) δ 212.5, 171.7, 171.5, 167.9, 61.4, 50.8, 49.8, 42.9, 41.8,
40.8, 40.6, 38.9, 38.6, 37.9, 36.5, 36.3, 29.4, 26.8, 26.6, 18.2,
14.2, 14.1. Anal. (C30H44N2O7) C, H, N.
4-[2-(3,5-Dim eth yl-2-oxocycloh exyl)-2-h yd r oxyeth yl]-
2,6-p ip er id in ed ion e-1-(4-eth yl bu ta n oa te) (9). To a solu-
tion of 500 mg (1.78 mmol) of cycloheximide (1) and 390 mg
(2.0 mmol) of 4-bromobutanoic acid ethyl ester in 10 mL of
dimethylformamide was added 414 mg (3.0 mmol) of K2CO3.
This reaction mixture was stirred at 25 °C for 3 days.
Subsequently, the solvent and other volatile organics were
removed under low pressure. The remaining residue was
dissolved in a mixture of 7.0 mL of CHCl3 and 3.0 mL of
EtOAc. After filtration and concentration in vacuo the crude
product was purified by column chromatography on silica gel
(CHCl3/EtOAc (2:1)) to yield 197 mg (27.8%) of compound 9
as a colorless, viscous oil: MALDI-TOF-MS [M + H]+ ) 397.8
4-[2-(3,5-Dim eth yl-2-oxocycloh exyl)-2-h yd r oxyeth yl]-
1-m eth yl-2,6-p ip er id in ed ion e (5). To a solution of 300 mg
(1.07 mmol) of cycloheximide (1) and 170 mg (1.2 mmol) of
CH3I in 3.0 mL of acetone were added 10 mg (0.04 mmol) of
18-crown-6 and 200 mg (1.45 mmol) of K2CO3. The reaction
mixture was stirred at 25 °C for 48 h. After filtration and
concentration in vacuo the crude product was purified by
column chromatography on silica gel (CH2Cl2/EtOAc (3:1)) to
give 147 mg (46.5%) of compound 5 as a colorless, viscous oil:
1
m/e; H NMR (300 MHz) δ 4.20 (d, 1H, J ) 10.8 Hz), 3.91 (t,
1
MALDI-TOF-MS [M + H]+ ) 296.5 m/e; H NMR (300 MHz)
2H, J ) 6.7 Hz), 3.68 (s, 1H), 3.45-3.23 (comp, 3H), 3.10-
1.50 (comp, 16H), 1.33-0.95 (comp, 10H). Anal. (C21H33NO6)
C, H, N.
δ 4.12 (d, 1H, 10.7 Hz), 3.03 (s, 3H), 2.87-2.68 (comp, 3H),
2.55 (mult, 1H), 2.42 (mult, 1H), 2.37-2.21 (comp, 3H), 2.12
(mult, 1H), 1.91-1.72 (comp, 3H), 1.61-1.44 (comp, 2H), 1.19-
1.07 (comp, 4H), 0.90 (d, 3H, J ) 6.4 Hz); 13C NMR (75 MHz)
δ 216.0, 172.3, 172.1, 66.2, 50.0, 42.4, 40.3, 39.2, 38.0, 33.0,
26.5, 26.4, 26.0, 18.1, 14.0. Anal. (C16H25NO4) C, H, N.
4-[2-(3,5-Dim eth yl-2-oxocycloh exyl)-2-h yd r oxyeth yl]-
2,6-p ip er id in ed ion e-1-(eth yl eth a n oa te) (10). To a solution
of 5.0 g (17.8 mmol) of cycloheximide (1) and 3.0 mL (27.0
mmol) of bromoacetic acid ethyl ester in 35 mL of acetone were
added 100 mg (0.38 mmol) of 18-crown-6 and 3.0 g (21.6 mmol)
of K2CO3. The reaction mixture was stirred at 25 °C for 3 days.
After the potassium salts were filtered off, the filtrate was
concentrated in vacuo. The oil was purified by column chro-
matography on silica gel (CHCl3/EtOAc (2:1)). Recrystallization
from EtOAc afforded 4.3 g (65.7%) of compound 10 as white
crystals: mp ) 100-101 °C; [R]D) +8.7° (pyridine); MALDI-
TOF-MS [M + H]+ ) 368.8 m/e; 1H NMR (300 MHz) δ 4.50 (s,
4-[2-(3,5-Dim eth yl-2-oxocycloh exyl)-2-h yd r oxyeth yl]-
1-ben zyl-2,6-p ip er id in ed ion e (6). In a typical experiment
300 mg (1.07 mmol) of cycloheximide (1), 205 mg (1.2 mmol)
of 4-benzyl bromide, 10 mg (0.04 mmol) of 18-crown-6, and 200
mg (1.45 mmol) of K2CO3 in 3.0 mL of acetone were stirred at
25 °C for 2 days. After filtration and concentration in vacuo
the crude product was purified by column chromatography on
silica gel (CH2Cl2/EtOAc (3:1)) to yield 186 mg (46.8%) of

3620 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Christner et al.
2H), 4.27-4.13 (comp, 3H), 2.97-2.81 (comp, 3H), 2.63 (mult,
1H), 2.55-2.43 (comp, 4H), 2.21 (mult, 1H), 2.02-1.78 (comp,
3H), 1.69-1.55 (comp, 2H), 1.33-1.22 (comp, 7H), 0.99 (d, 3H,
J ) 6.4 Hz); 13C NMR (75 MHz) δ 216.5, 171.7, 171.6, 167.9,
66.6, 61.4, 50.1, 42.6, 40.5, 39.1, 37.7, 37.6, 33.0, 26.7, 26.4,
18.3, 14.1, 14.0. Anal. (C19H29NO6) C, H, N.
Australia), and cycloheximide-N-(ethyl ethanoate) (10; n ) 7)
were applied directly to the anastomoses in dosages of 1 and
30 mg/kg in 5% DMSO, respectively. The reconstructed nerve
was covered with a tube of BActerial SYnthesized Cellulose
(BASIC)39 serving as a drug depot. Corresponding dosages of
the dipeptide Ala-Ala-OH (n ) 6) were used as a placebo. All
animals showed a complete paralysis of the concerned leg after
neurotomy. Functional recovery was studied by weekly as-
sessment of the walking behavior during 8 and 10 weeks,
respectively. Following criteria were applied: usage and
movement coordination of the operated leg, foot and toe
posture, toe movement. The situation was quantified blindly
using a point system: +1, positive; 0, inconspicuous; -1,
negative. Obtained data were evaluated by calculation of the
percentage of clinical signs of functional recovery relative to
the placebo; 8 and 10 weeks after neurotomy the animals were
again anesthetized and the EDL from both hind legs were
prepared and removed. The muscle weights were determined
in relation to protein concentration, and the percentages of
weight differences of EDL muscles of the left neurotomized
hind limbs in comparison to the corresponding right unlesioned
legs were calculated relative to the placebo.
In Vitr o Assa ys. PPIases were kindly provided by Dr. J .-
U. Rahfeld (rhFKBP12, E. coli FKBP26, FKBP22 from Pho-
tobacterium sp., rhPin1, E. coli parvulin), Dr. B. Schmidt (L.
pneumophila FKBP25 (Mip)), S. Hottenrott (E. coli SlyD), Dr.
T. Zarnt (rabbit FKBP52), and Dr. G. Stoller (E. coli trigger
factor; Halle, Germany). Human Cyp18 was produced recom-
binantly in E. coli.4c All substrates were synthesized by Dr.
M. Schutkowski (Halle, Germany). PPIase activities were
measured according to Fischer et al.1 using R-chymotrypsin
(Merck, Darmstadt Germany; 410 µg/mL) as isomer-specific
protease and the peptide Suc-Ala-Phe-Pro-Phe-pNA (24.0 µM)
as a substrate. In the case of the E. coli SlyD and hPin1, the
protease trypsin (Boehringer/Mannheim, Germany; 210 µg/
mL) and the peptides Suc-Ala-Phe-Pro-Arg-pNA (34.0 µM) and
Ac-Ala-Ala-Ser(P)-Pro-Arg-pNA (26.0 µM), respectively, were
applied instead. In general, the test was performed by observ-
ing the released 4-nitroaniline at 390 nm with a Hewlett-
Packard 8452A diode array UV/vis spectrophotometer at 10
°C. For inhibition experiments, the enzymes were incubated
with the effectors (0.01-200 µM) in 1200 µL of 35 mM HEPES
(pH 7.8) for at least 5 min. Effectors were freshly diluted from
10-20 mM stock solutions in DMSO. The amount of organic
solvent was kept constant within each experiment, usually
below 0.1% (v/v). The reactions were started by addition of 2-3
µL of a peptide stock solution (10 mg/mL in DMSO). Under
the condition [S0] , Km, recorded progression curves can be
described by first-order kinetics. From the observed rate
constant kobs a first-order rate constant kenz for enzymatic
catalysis of cis to trans isomerization can be calculated as kobs
) k0 + kenz, where k0 represents the first-order rate constant
for the uncatalyzed cis/ trans isomerization. Inhibitor con-
stants (IC50, Ki) were determined by analyzing the concentra-
tion dependence of each inhibitor on PPIase activity. Apparent
Ki values were obtained by fitting the data by nonlinear
regression to a mass action ratio-based equation for competi-
tive binding inhibition. The Michaelis-Menten kinetics for the
inhibition of hFKBP12 by cycloheximide (1) was determined
according to the method of Kofron et al.38 For the assay a 100
µg/mL solution of the substrate Suc-Ala-Leu-Pro-Phe-pNA
(final concentration 0.02-1.0 mM) in 0.5 M LiCl/trifluoro-
ethanol was used, thereby ensuring a proportion of peptide
molecules in cis conformation of ∼40%. Data were analyzed
by means of the software programs SigmaPlot Scientific
Graphing System version 4.0 (J andel Corp., Chicago, IL) and
TREND (Martin-Luther-University, Halle-Wittenberg, Ger-
many).
The eukaryotic translation assay was performed using a
rabbit reticulocyte type I translation kit (Boehringer/Man-
nheim, Germany) according to the manufacturer’s instructions
with the following changes: R-factor mRNA, generously
provided by Dr. S. Panzner (Halle, Germany), was chosen as
a substrate. To inhibit degradation of sample mRNA, Rnasin
ribonuclease inhibitor (40 U/µL; Promega, Heidelberg, Ger-
many) was added to the reaction mixtures containing reticu-
locyte lysate, 1 mM amino acid mixture without Met, 10 mCi/
mL [35S]Met, 100 µg/mL RNA, and 100-0.08 µM cycloheximide
(1) or cycloheximide-N-(ethyl ethanoate) (10) diluted in nu-
clease-free H2O, respectively.
In Vivo Stu d ies. For the sciatic nerve neurotomy experi-
ment,30 4-month-old female rats (HAN:Wist) were anesthetized
by intramuscular application of 16 mg/kg RompunTS (Bayer,
Leverkusen, Germany) and 125 mg/kg Ketavet (Pharmacia &
Upjohn GmbH, Erlangen, Germany). The sciatic nerve of the
left hind leg was exposed; the three fascicles were separated
and then dissected. Immediately after dissection the nerve was
microsurgically reconstructed by three interfascicular anas-
tomoses with Ethilon 10/0 (Ethicon, Hamburg, Germany).
FK506 (n ) 4), kindly provided by Dr. A. Lawen (Clayton,
Ack n ow led gm en t. This work was supported by the
Fonds der Chemischen Industrie. The authors thank Dr.
M. Dahse and Dr. A. Ha¨rtl for performance of cytotoxic
assays and also Dr. M. Zerlin for stimulating discussions
and assistance in drug screening experiments. We
express our thanks to Dr. S. Panzner for a generous gift
of S. cerevisiae R-factor mRNA and U. Udhard for
providing the BASYC.
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