Synthesis, molecular modeling and biological evaluation of aza-proline and aza-pipecolic derivatives as FKBP12 ligands and their in vivo neuroprotective effects

Article abstract of DOI:10.1016/S0968-0896(03)00478-4

Nonimmunosuppressant ligands, exemplified by GPI 1046 (1), for the peptidyl-prolyl isomerase FKBP12 have been found to unexpectedly possess powerful neuroprotective and neuroregenerative effects in vitro and in vivo. We have extensively explored the thera

Full text of DOI:10.1016/S0968-0896(03)00478-4

Bioorganic & Medicinal Chemistry 11 (2003) 4815–4825
Synthesis, Molecular Modeling and Biological Evaluation of
Aza-Proline and Aza-Pipecolic Derivatives as FKBP12Ligands
and Their In Vivo Neuroprotective Effects
Douglas E. Wilkinson, Bert E. Thomas, IV, David C. Limburg, Agnes Holmes,
Hansjorg Sauer, Douglas T. Ross, Raj Soni, Yi Chen, Hong Guo, Pamela Howorth,
Heather Valentine, Dawn Spicer, Mike Fuller, Joseph P. Steiner, Gregory S. Hamilton
and Yong-Qian Wu*
Department of Research, Guilford Pharmaceuticals, Inc., Baltimore, MD 21224, USA
Received 19 April 2003; accepted 12 June 2003
Abstract—Nonimmunosuppressant ligands, exemplified by GPI 1046 (1), for the peptidyl-prolyl isomerase FKBP12 have been
found to unexpectedly possess powerful neuroprotective and neuroregenerative effects in vitro and in vivo. We have extensively
explored the therapeutic utility of FKBP12 ligands based on analogues of proline and pipecolic acid. As part of our ongoing
program to explore novel structural classes of FKBP12 ligands, we herein wish to report a new class of FKBP12 ligands
containing aza-proline and aza-pipecolic acid analogues. Details of the synthetic studies, together with biological activity will be
presented.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
regenerative properties in vitro and in vivo.6,7 The neuro-
trophic effects of these compounds are independent of
the immunosuppressive pathways because they lack the
effector domain of FK506.
FKBP12 is the cellular receptor for the immunosup-
pressant drug FK506. It belongs to a family of proteins
known as the immunophilins which possess peptidyl-
prolyl cis-trans isomerase activity (PPIase).1ꢀ3 PPIases
are also known to play critical roles in a variety of bio-
logical processes other than immunosuppression,
including protein trafficking and regulation of neurite
outgrowth.4 Recently it was discovered that FKBP12 is
10–40-fold more enriched in the brain than in the
immune tissues and that FK506 exhibits neurotrophic
properties in vitro and in vivo.5 FK506 possess two
distinct binding domains: a FKBP12-binding domain
and an effector domain, which mediates the interaction
of the immunophilin–drug complex with the secondary
protein target-calcineurin. Previously, we reported that
small molecule ligands for FKBP12, such as 1 (GPI-
1046, Fig. 1), possess neuroprotective and neuro-
Azapeptides are peptides where the asymmetric Ca car-
bon of an amino acid is replaced by nitrogen. They are
mimetics of natural peptides, however they are much
more resistant to protease cleavage. There are numerous
examples in the literature of azapeptides being utilized
as enzyme inhibitors or receptor antagonists, such as
protease papain inhibitors,8 elastase active-site titrants,9
and MHC II ligands.10
Analogues of proline and pipecolic acid have been
the focus of previous work exploring the SAR of
small molecules which mimic the FKBP-binding
domain of FK506.11ꢀ15 As part of our program to
explore novel classes of FKBP12 ligands that may
have enhanced metabolic stability and improved
DMPK profile, we herein wish to report the synthesis
and biological evaluation of a new class of FKBP12
ligands containing aza-proline and aza-pipecolic acid
analogues (Fig. 1).
*Corresponding author. Tel.: +1-410-631-6823; fax: +1-410-631-
6823; e-mail: wuy@guilfordpharm.com
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00478-4

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D. E. Wilkinson et al. / Bioorg. Med. Chem. 11 (2003) 4815–4825
Figure 1. FK506 and small molecule mimetics of the FKBP12 binding domain.
Chemistry
from bis-acylation products. Apparently, the nucleo-
philicity of the second nitrogen is significantly decreased
after the introduction of the first carbonyl group. Con-
sequently, the second acylation reactions require either
more electrophilic reagents (sulfonyl chloride, oxylate
chloride) or base promotion (KF on Celite), such as in
the case of 12f/13e.
The general synthetic route to aza-prolyl and aza-pipe-
colyl carbamate compounds is shown in Scheme 1. Di-
Boc hydrazine 2 was treated with sodium hydride, and
then reacted with dibromopropane and dibromobutane
to afford the five-membered ring pyzazolidine 3 and the
six-membered perhydropyridazine 4, respectively.
Deprotection of the Boc group using TFA led to 5/6, fol-
lowed by acylation with various imidazole-1-carboxylates
7 to provide the aza-prolyl and aza-pipecolyl esters.
Imidazole-1-carboxylates 7 were obtained easily by
treating carbonyl diimidazole with various alcohols.
Further acylation of 8/9 with methyl chlorooxoacetate
led to the oxamate intermediates 10/11. 10/11 were
treated with Grignard reagent 1,1-dimethylpropyl
magnesium bromide at ꢀ78ꢁC yielding the desired
diketoamide compounds 12a–d/13a–c (see Table 1).
Similarly, treatment of 8/9 with a-toluene sulfonyl
chloride and cyclohexyl isocyanate gave the respective
sulfonylamide 12e/13d and urea 12f/13e products. It is
worth noting that compounds 8/9 were obtained free
The general synthetic route to the aza-prolyl and aza-
pipecolyl amide compounds is shown in Scheme 2.
Benzyl carbazate 14 was protected by a Boc group to
yield asymmetric di-protected hydrazine 15. Similarly,
the five-membered ring pyrazolidine 16 and the six-
membered perhydropyridazine 17 were obtained from
di-protected hydrazine when treated with sodium
hydride, followed by dibromopropane and dibromo-
butane in DMF. Mono deprotection of the Boc group
using TFA resulted in 18/19, which were then acylated by
methyl chlorooxoacetate yielding oxamate intermediates
20/21. The reaction of 20/21 with 1,1-dimethylpropyl-
magnesium bromide led to the formation of diketoa-
mides 22/23. The deprotection of the Cbz group under
Scheme 1. Conditions: (a) NaH, DMF; (b) TFA, CH2Cl2; (c/d) CH2Cl2 rt; (e) THF, 0 ꢁC, methyl oxalyl chloride; (f) CH2Cl2, a-toluene sulfonyl
chloride; (g) CH2Cl2, cyclohexylisocyanate, KF-Celite; (h) THF, ꢀ78 ꢁC, 1,1-dimethylpropylmagnesium bromide.

D. E. Wilkinson et al. / Bioorg. Med. Chem. 11 (2003) 4815–4825
Table 1. Structures and in vitro activity for azal-prolyl and aza-pipecolyl compounds
4817
No.
Structure
Formula
Anal.
IC50
12a
C19H26N2O4
C, H, N
15 mM
1.0 mM
3.8 mM
5.5 mM
3.2 mM
12b
12c
12d
12e
C20H28N2O4
C21H30N2O4
C19H27N3O4
C20H24N2SO4
C, H, N
C, H, N
C, H, N
C, H, N
12f
C20H29N3O3
C, H, N
>25 mM
>25 mM
12g
C19H26N2O3
C, H, N
12h
12i
12j
26
C20H28N2O3
C21H30N2O3
C22H32N2O3
C15H22N2O3
C20H25N3O3
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
9.0 mM
1.1 mM
>25 mM
>25 mM
5.5 mM
28
(continued)

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D. E. Wilkinson et al. / Bioorg. Med. Chem. 11 (2003) 4815–4825
Table 1 (continued)
No.
Structure
Formula
Anal.
IC50
12k
C20H29N3O3
C, H, N
750 nM
13a
13b
13c
C21H30N2O4
C22H32N2O4
C23H34N2O4
C, H, N
C, H, N
C, H, N
900 nM
1.9 mM
5.0 mM
13d
13e
C22H28N2S1O4
C, H, N
2.1 mM
C22H28N3O3
C, H, N
>25 mM
13f
13g
30
C23H34N2O3
C, H, N
C, H, N
5.0 mM
3.3 mM
120 nMa
C22H33N3O3
31
210 nMa
32
780 nMa
aRefs 14 and 15.

D. E. Wilkinson et al. / Bioorg. Med. Chem. 11 (2003) 4815–4825
4819
Scheme 2. Conditions: (a) (Boc)2O, Et3N; (b) NaH, DMF; (c) TFA, Ch2Cl2; (d) THF, 0 ꢁC, methyl oxalyl chloride; (e) THF, ꢀ78 ꢁC. 1,1-dime-
thylpropylmagnesium bromide; (f) Pd/C, H2; (g) RCOOH, Et3N, i-butylchloroformate; (h) 3-bromopyridine, Pd catalyst; (i) PtO2, H2.
hydrogenation conditions resulted in 24/25, which, in
turn, were coupled to various commercially available
phenylalkyl acids under mixed anhydride peptide cou-
pling conditions, affording final compounds 12g–j/13f
(see Table 1). For the synthesis of compounds 12k/13g,
no corresponding acids were commercially available.
Therefore, 24/25 were first converted to 26/27 after
coupling to acids containing a terminal alkyne. Then 26/
27 were subjected to a facile palladium catalyzed cou-
pling with 3-bromopyridine, resulting 28/29. The final
hydrogenation yielded the desired 12k/13g. Overall, the
reactions described above were smooth and of high
yield, and suitable to run in parallel synthesis.
compounds were evaluated for in vitro inhibitory activ-
ity against the PPIase activity of recombinant purified
human FKBP12, they were found to show significant
activity, though generally reduced, when compared to their
non-aza analogues, by at least an order of magnitude.
As the in vitro data shown in Table 1 indicate, the
compounds strongly preferred a ‘side arm’ aromatic
ring (e.g., 12b, 12i and 12k vs 26) and displayed an
optimal ‘side-arm’ chain length of five atoms from the
ring (12i vs 12g and 12j). Heterocycles such as a pyridine
ring can replace the phenyl ring (12k and 13g vs 12i and
13f). In addition, sulfonyl amides in both aza-prolyl and
aza-pipecolyl series were essentially as potent as their
diketoamides counterparts (12e and 13d vs 12b and
13b). These results were consistent with previous SAR
results in the regular prolyl and pipecolyl series (e.g., 30,
31 and 32).14,15 One noticeable difference was urea
compounds, such as 12f, which did not show appreci-
able activity up to 25 mM, while urea-based prolyl
ligands showed good activity4 (e.g., prolyl analogue of
12f having 1.8 mM activity).
Structure–Activity Relationship (SAR)
Aza-apeptides arise when asymmetric a-carbon of an
amino acid is replaced by nitrogen. It has been well
documented that constrained cyclic aza-prolyl and aza-
pipecolyl compounds adopt an asymmetric configur-
ation (induced chirality) at both nitrogens, although the
degree of pyramidalization at either nitrogen is not the
same as a tetrahedral carbon.16,17 It was expected that
the aza nitrogen in our inhibitors would need to be
pyramidalized in order to retain good affinity towards
FKBP12. It has already been shown that only the l-
stereoisomer of the prolyl/pipecolyl parent compounds
show significant inhibitory activity while d-stereo-
isomers have no inhibitory activity. When our aza
Molecular Modeling
There are many different effects that play a role in the
differential affinity of two ligands for the same target
protein. For example, differences in the enthalpic inter-
actions between the ligands and the target, entropy

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D. E. Wilkinson et al. / Bioorg. Med. Chem. 11 (2003) 4815–4825
effects and solvation effects can all play a role. The goal
of the modeling experiments was to identify the most
important effects in order to better understand the SAR
of the aza compounds.
those calculations what the energetic consequences of
the required pyramidalization of the aza nitrogen are. In
order to better determine the consequence of additional
pyramidalization of the aza nitrogen required for opti-
mal binding, we have quantum mechanically deter-
mined the structure of 1,2-diformylpyrazolidine at the
MP2/6-31G(d)//MP2/6-31G(d) level of theory.19 In this
structure the aza nitrogen is pyramidalized 35ꢁ from
planarity. In order to optimize the Ile56 NH interaction,
the aza nitrogen would need to pyramidalize ꢂ20ꢁ
more. We have approximated the energetic con-
sequences of this additional pyramidalization by freez-
ing the appropriate torision angle while optimizing the
rest of the molecule. At the MP2/6-31G(d)//MP2/6-
31G(d) level of theory the additional pyramidalization
of the aza nitrogen requires an additional 2.8 kcal/mol.
The crystal structure of (1R)-1,3-diphenyl-1-propyl (2S)-
1-(3,3-dimethyl-1,2-dioxopentyl)-2-piperdinecarboxylate
(SB3) bound to FKBP12 (PDB code: 1FKG) was used
for all experiments.18 SB3 was used as a template to
build the aza compounds into the active site of
FKBP12. Each compound was minimized in the active
site with FKBP12 held rigid.19
All of the aza compounds fit well into the active site of
FKBP12. The pharmacophore binding elements in the
active site for these types of ligands can best be descri-
bed as two hydrophobic pockets (one prolyl binding
pocket and a proximal spherical binding pocket) and
two hydrogen bond donors (Tyr82 side-chain OH and
Ile56 backbone NH). The aza compounds are predicted
to fill both hydrophobic pockets and bind the Tyr82 OH
(see Fig. 2) as well as their non-aza analogues. The
interaction with the Ile56 backbone NH is similar to the
non-aza analogues but requires further pyramidaliza-
tion of the aza nitrogen for the interaction to remain
optimal.
An additional consideration is the fact that there are
two pyramidalized conformation in which each of the
aza compounds exists. One conformation optimizes the
binding interactions to the target (productive) via
further pyramidalization; while the other one has the
wrong ‘stereochemistry’ (non-productive) and would
not result in significant binding to the target without
first interconverting to the productive conformation.
We believe that the energy required to further
pyramidalize the nitrogen in the aza compounds to
optimize the binding interactions, along with the con-
formational issue described above, primarily account
for the order of magnitude difference in the activity
between the aza and non-aza compounds.
In the non-aza analogues that have a tetrahedral carbon
in place of the aza nitrogen, the carbonyl interacts with
the Ile56 NH, and is perfectly located for this inter-
action. In order for the aza compounds to reproduce the
Ile56 NH interaction, the aza nitrogen would need to be
pyramidalized ꢂ55ꢁ from planarity. The crystal struc-
tures of the aza-tripeptides where the central amino acid
is replaced by an aza-proline have been solved. The aza
nitrogen is pyramidalized, on average, 40ꢁ,16,17 which if
extrapolated back to our aza compounds binding to
FKBP12, this would not result in an optimal interaction
between the carbonyl and the Ile56 NH. Since the for-
cefield used in these calculations is not explicitly para-
meterized for this type of molecule, it is unclear from
In Vivo Biology
The neuroprotective properties of the aza compounds
were evaluated in an in vivo model of neurodegenera-
tion. The nigrostriatal dopaminergic pathway in the
brain is particularly enriched in FKBP12. This pathway
is responsible for controlling motor movements. Par-
kinson’s disease is a serious neurodegenerative disorder
resulting from the degeneration of this motor pathway
and the subsequent decrease in dopaminergic transmis-
sion.20 N-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP) is a neurotoxin which selectively destroys
dopaminergic neurons.21 Lesioning of the nigrostriatal
pathway in animals with MPTP has been utilized
extensively as an animal model of Parkinson’s disease.
In a study utilizing a ‘protective’ or concurrent dosing
protocol, mice were treated with MPTP and the aza
compounds concurrently for 5 days.13 The aza com-
pounds were given for an additional 5 days. After 18
Table 2. In vivo data for representative examples
Compd
%Recovery
10 mg/kg, po
1
35.0+5.0
56.8+6.2
15.1+4.1
34.9+5.9
25.6+3.4
12k
13d
13f
28
Figure 2. The predicted binding mode of 12k complexed to FKBP12.
12k and FKBP12 residues Ile56 and Tyr82 are shown in capped sticks.
The surface of FKBP12 is shown in yellow.

D. E. Wilkinson et al. / Bioorg. Med. Chem. 11 (2003) 4815–4825
4821
Sigma-Aldrich Inc. (S)-Pipecolic acid was obtained as
the (R)-tartrate salt from Oxford Asymmetry. Analy-
tical thin-layer chromatography was carried out using
Merck DC-F254 precoated silica gel plates. Flash chro-
matography was performed using kieselgel 60 (230–400
mesh) silica gel. 1H NMR spectra were recorded on
either a Varian 300 MHz or a Bruker 400 MHz instru-
ment. Chemical shifts are reported in parts per million
(ppm). Mass spectral analyses were performed by
Oneida Research Services, Whitesboro, NY, USA. Ele-
mental analyses were determined by Atlantic Microlab,
Norcross, GA, USA and are withinꢃ0.4% of the
calculated values unless otherwise noted.
Figure 3. Dose–response curve for 13f.
General procedure for Scheme 1
days the animals were perfused and the brains were
fixed, cryoprotected and sectioned. Staining with an
antibody against tyrosine hydroxylase (TH) was used to
quantitate the survival of dopaminergic neurons.
tert-Butyl 2-[(tert-butyl)oxycarbonyl]perhydropyridazine-
carboxylate (4). A solution of di-Boc hydrazine (20 g,
84.4 mmol) in 150 DMF was added dropwise to a sus-
pended solution of 6.75 g (168.8 mmol) NaH in 75 mL
DMF under nitrogen. After the mixture was stirred for
30 min at room temperature, a solution of 1,4 dibromo-
butane (18.2 g, 84.4 mmol) in 25 mL DMF was added
dropwise. The reaction was allowed to stir overnight at
room temperature. The reaction was then concentrated
and partitioned between 200 mL CH2Cl2 and 200 mL
water. The aqueous layer was extracted with an addi-
tional 200 mL CH2Cl2. The combined organic layers
were dried over MgSO4, and filtered, and concentrated.
The crude product was further purified by silica gel
chromatography to yield 20.2 g (82% yield) product.
The product was analyzed by GC/MSas pure com-
pound with M+ 286.
Table 2 presents the data from representative aza com-
pounds given to MPTP-treated animals in these experi-
ments as the percent recovery of striatal dopaminergic
innervation relative to MPTP-treated animals that
received vehicle. In animals treated with MPTP and
vehicle, a substantial loss of 60–70% of functional
dopaminergic terminals was observed as compared to
non-lesioned animals. Lesioned animals receiving test
compounds (10 mg/kg po) concurrently with MPTP
showed a striking preservation of TH-stained striatal
dopaminergic terminals, demonstrating the ability of
these compounds to block the degeneration of dopami-
nergic neurons produced by MPTP. Compounds such
as 12k and 13f produced a remarkable preservation of
striatal dopaminergic innervation up to 56.8 and 34.9%
of control levels, respectively. The dose-response curve
for 13f following oral administration in the protective
model is shown in Figure 3. It produced significant
effects at 10 mg/kg dose and produced maximum
preservation of about 45% at dose of 30 mg/kg.
Perhydropyridazine (6). 2.83 mL (36.7 mmol) TFA was
added dropwise to a solution of tert-butyl 2-[(tert-butyl)-
oxycarbonyl]perhydropyridazinecarboxylate 4 (1.5 g,
5.2 mmol) in 7 mL CH2Cl2, and the mixture was stirred
overnight. At this time, the reaction was completed and
5.85 mL (42 mmol) triethylamine was added to quench
the reaction. The reaction was concentrated and the
residue, which contained product, was used without
further purification.
Conclusions
4-Phenylbutyl perhydropyridazinecarboxylate (9b). A
solution containing 1,10-carbonyl diimidazole (0.893 g,
5.5 mmol) in 5 mL CH2Cl2 was added slowly to a solu-
tion of CH2Cl2 containing phenylbutyl alcohol (0.89
mL, 5.77 mmol). After stirring at room temperature for
1 h, this solution was then added slowly to the solution
containing perhydropyridazine 6 mentioned above. The
reaction was allowed to stir overnight. The crude mix-
ture was then concentrated and used without further
purification.
We have developed an efficient synthetic approach to
the synthesis of aza-prolyl and aza-pipecolyl com-
pounds. We believe these compounds are the first
reported examples of aza-proline and aza-pipecolic
derivatives as FKBP12 ligands. Further more, our pre-
liminary biological results suggest that some of these
compounds are potent neuroprotective agents with
potential therapeutic utility in treating degenerative dis-
orders of the nervous system, such as Parkinson’s disease.
Methyl 2-oxo-2-{2-[(4-phenylbutyl)oxycarbonyl]perhy-
dropyridazinyl} acetate (11b). A solution of CH2Cl2
containing previous crude product of 4-phenylbutyl
perhydropyridazinecarboxylate 9b from the last step
was cooled to 0 ꢁC, and a solution of methyl oxalyl
chloride (0.74 g, 5.77 mmol) in 5 mL CH2Cl2 was added
dropwise over 0.5 h. The resulting mixture was stirred at
0ꢁC for 4 h, and then warmed up to room temperature.
Experimental
General methods
All commercially available starting materials and sol-
vents were reagent grade. Anhydrous tetrahydrofuran
(THF) and diethyl ether were used as obtained from

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D. E. Wilkinson et al. / Bioorg. Med. Chem. 11 (2003) 4815–4825
The reaction mixture was diluted with 50 mL CH2Cl2
and washed with water. The organic layer was dried
over MgSO4, filtered, and concentrated. The crude pro-
duct was further purified by silica chromatography to
yield 1.8 g (62% overall yield for three steps) product.
1H NMR (CDCl3, 400 MHz): d 1.39 (m, 2H); 1.69 (m,
6H); 2.62(t, 2H, J=8); 2.83 (m, 1H); 3.1 0(m, 1H); 3.79
(s, 3H); 4.16 (m, 3H); 4.31 (m, 1H); 7.22 (m, 5H).
2-Phenylethyl 2-(3,3-dimethyl-2-oxopentanoyl)pyrazolidi-
1
necarboxylate (12a). Rf=0.5 (25% EtOAc/hexane). H
NMR (CDCl3, 400 MHz): d 0.83 (t, 3H, J=7.5 Hz);
1.18 (s, 6H); 1.63 (m, 2H); 1.99 (m, 2H); 2.97 (t, 2H,
J=7.1); 3.60 (br s, 4H); 4.35 (t, 2H, J=6.6 Hz); 7.19–
7.30 (m, 5H). Anal. calcd for C19H26N2O4: C, 65.88; H,
7.56; N, 8.09. Found: C, 65.82; H, 7.51; N, 8.02.
3-Phenylpropyl 2-(3,3-dimethyl-2-oxopentanoyl)pyrazoli-
dinecarboxylate (12b). Rf=0.4 (25% EtOAc/Hexane).
1H NMR (CDCl3, 400 MHz): d 0.86 (t, 3H, J=7.4 Hz);
1.22 (s, 6H); 1.66 (t, 2H, J=7.5 Hz); 2.00–2.12 (m, 4H);
2.72 (t, 2H, J=7.4 Hz); 3.60 (br s, 4H); 4.18 (t, 2H,
J=6.5); 7.18–7.31 (m, 5H). Anal. calcd for
C20H28N2O4: C, 66.64; H, 7.83; N, 7.77. Found: C,
66.73; H, 7.81; N, 7.72.
4-Phenylbutyl 2-(3,3-dimethyl-2-oxopentanoyl)perhydro-
pyridazine carboxylate (13b). A solution of methyl 2-
oxo-2-{2-[(4-phenylbutyl)oxycarbonyl]perhydropyrida-
zinyl} acetate 11b (1.2 g, 3.45 mmol) in 15 mL dry THF
was cooled to ꢀ78 ꢁC and treated with 5.2 mL of 1.0 M
solution of 1,1-dimethylpropylmagnesium chloride in
THF. After stirring the resulting homogeneous mixture
at ꢀ78 ꢁC for 4 h, the mixture was poured into saturated
ammonium chloride (20 mL) and extracted into ethyl
acetate. The organic layer was washed with water, dried
and concentrated. The crude material was purified by
silica gel column, eluting with 25% ethyl acetate in
hexane, to obtain 0.98 g product (73% yield). Rf=0.73
4-Phenylbutyl 2-(3,3-dimethyl-2-oxopentanoyl)pyrazoli-
dinecarboxylate (12c). Rf=0.6 (25% EtOAc/Hexane).
1H NMR (CDCl3, 400 MHz): d 0.83 (t, 3H, J=7.5 Hz);
1.19 (s, 6H); 1.67 (t, 2H, J=7.5 Hz); 1.60–1.69 (m, 4H);
2.07 (t, 2H, J=7.4 Hz); 2.62 (t, 2H, J=6.4); 3.60 (br s,
4H); 4.13 (t, 2H, J=6.1 Hz); 7.28–7.15 (m, 5H). Anal.
calcd for C21H30N2O4: C, 67.35; H, 8.07; N, 7.48.
Found: C, 67.54; H, 8.31; N, 7.40.
1
(2:1 hexane: EtOAc). H NMR (CDCl3, 400 MHz): d
0.81 (t, 3H, J=7.1); 1.13 (s, 3H); 1.20 (s, 3H); 1.64 (m,
10H); 2.64 (m, 2H); 2.86 (m, 1H); 3.20 (m, 1H); 3.99 (m,
1H); 4.19 (m, 2H); 4.35 (m, 1H); 7.24 (m, 5H). Anal.
calcd for C22H32N2O4: C, 68.01; H, 8.30; N, 7.21.
Found: C, 68.10; H, 8.29; N, 7.15.
3-(3-Pyridyl)propyl 2-(3,3-dimethyl-2-oxopentanoyl)pyr-
1
azolidinecarboxylate (12d). Rf=0.1 (100% EtOAc). H
NMR (CDCl3, 400 MHz): d 0.85 (t, 3H, J=7.5 Hz);
1.21 (s, 6H); 1.67 (t, 2H, J=7.5 Hz); 2.00–2.13 (m, 4H);
2.72 (t, 2H, J=7.5 Hz); 3.62 (br, s, 4H); 4.19 (t, 2H,
J=6.4 Hz); 7.28 (br s, 1H), 7.54 (d, 1H, J=7.7 Hz); 8.48
(s, 2H). Anal. calcd for C19H27N3O4 0.35H2O: C, 62.06;
H, 7.59; N, 11.43. Found: C, 61.77; H, 7.53; N, 11.36.
4-Phenylbutyl 2-[benzylsulfonyl]perhydropyridazinecar-
boxylate (13d). A solution of a-toluene sulfonyl chlor-
ide (1.12 g, 5.77 mmol) in CH2Cl2 was added to a
CH2Cl2 solution containing 4-phenylbutyl perhy-
dropyridazinecarboxylate 9b (1.37 g, 5.2 mmol) and
triethylamine (0.83 mL, 6 mmol). The reaction was stir-
red overnight at room temperature under nitrogen, and
then diluted to 50 mL CH2Cl2. The organic layer was
washed with water, dried, and concentrated. The crude
material was purified by silica gel column to yield 1.4 g
(64%) final product as clear oil. Rf=0.60 (2:1 hexane:
.
3-Phenylpropyl 2-[benzylsulfonyl]pyrazolidinecarboxylate
1
(12e). Rf=0.5 (40% EtOAc/hexane). H NMR (CDCl3,
400 MHz): d 2.01–2.17 (m, 4H); 2.72 (t, 2H, J=7.8 Hz);
3.68 (br s, 4H); 4.23 (t, 2H, J=6.6 Hz); 4.51 (s, 2H);
7.17–7.50 (m, 10H). Anal. calcd for C20H24N2SO4: C,
61.83; H, 6.23; N, 7.21; S, 8.25. Found: C, 61.63; H,
6.21; N, 7.05; S, 8.07.
1
EtOAc). H NMR (CDCl3, 400 MHz): d 1.68 (m, 8H);
2.67 (m, 2H); 2.90 (m, 1H); 3.38 (m, 2H); 4.22 (m, 5H);
7.32 (m, 10H). Anal. calcd for C22H28N2S1O4 C, 63.44;
H, 6.78; N, 6.73, S, 7.70. Found: C, 63.86; H, 6.83; N,
6.41, S, 7.58.
3-Phenylpropyl 2-(N-cyclohexylcarbamoyl)pyrazolidine-
carboxylate (12f). Rf=0.5 (60% EtOAc/Hexane). 1H
NMR (CDCl3, 400 MHz): d 1.09–2.00 (m, 15H); 2.69 (t,
2H, J=7.8 Hz); 3.70 (br s, 4H); 4.18 (t, 2H, J=6.4 Hz);
5.46 (d, 1H, J=8.2 Hz); 7.16–7.30 (m, 5H). Anal. calcd
for C20H29N3O3: C, 66.83; H, 8.13; N, 11.69. Found: C,
66.73; H, 8.28; N, 11.59
4-Phenylbutyl 2-(N-cyclohexylcarbamoyl)perhydropyri-
dazinecarboxylate (13e). Cyclohexylisocyanate (0.38 g,
3.0 mmol) was added to a CH2Cl2 solution containing
4-phenylbutyl perhydropyridazinecarboxylate 9b (0.72
g, 2.75 mmol) and 50 wt.% KF-celite (348 mg, 3 mmol).
The reaction was stirred overnight at room temperature
under nitrogen atmosphere, and then diluted to 50 mL
CH2Cl2. The organic layer was washed with water,
dried, and concentrated. The crude material was pur-
ified by silica gel column to yield 0.95 g (89%) final
3-Phenylpropyl 2-(3,3-dimethyl-2-oxopentanoyl)perhy-
dropyridazine carboxylate (13a). Rf=0.73 (2:1 hexane/
1
EtOAc). H NMR (CDCl3, 400 MHz): d 0.82 (t, 3H,
J=7.4 Hz); 1.16 (s, 3H); 1.22 (s, 3H); 1.67 (m, 6H); 2.00
(m, 2H); 2.69 (t, 2H, J=7.9 Hz); 2.86 (m, 1H); 3.23
(m, 1H); 4.00 (m, 1H); 4.20 (m, 2H); 4.37 (m, 1H); 7.23
(m, 5H). Anal. calcd for C21H30N2O4: C, 67.35; H, 8.07;
N, 7.48. Found: C, 67.51; H, 8.11; N, 7.39.
1
product as clear oil. Rf=0.28 (2:1 hexane:EtOAc). H
NMR (CDCl3, 400 MHz): d 1.10 (m, 3H); 1.33 (m, 3H);
1.69 (m, 10H); 1.88 (m, 2H); 2.62 (m, 2H); 2.72 (m, 1H);
2.87 (m, 1H); 3.60 (m, 1H); 4.13 (m, 3H); 4.38 (m, 1H);
5.11 (d, 1H, J=8.3 Hz); 7.23 (m, 5H). Anal. calcd for
5-Phenylpentyl 2-(3,3-dimethyl-2-oxopentanoyl)perhydro-
pyridazine-carboxylate (13c). Rf=0.74 (2:1 hexane/
.
C22H28N3O3 0.14H2O: C, 67.75; H, 8.60; N, 10.77.
Found: C, 67.75; H, 8.45; N, 10.90.
1
EtOAc). H NMR (CDCl3, 400 MHz): d 0.82 (t, 3H,

D. E. Wilkinson et al. / Bioorg. Med. Chem. 11 (2003) 4815–4825
4823
J=7.4); 1.14 (s, 3H); 1.21 (s, 3H); 1.38 (m, 2H); 1.65 (m,
10H); 2.62 (t, 2H, J=7.6 Hz); 2.83 (m, 1H); 3.20 (m,
1H); 3.98 (m, 1H); 4.15 (m, 2H); 4.33 (m, 1H); 7.23 (m,
5H). Anal. calcd for C23H34N2O4: C, 68.63; H, 8.51; N,
6.96. Found: C, 68.70; H, 8.47; N, 7.08.
water, dried and concentrated. The crude material was
purified by silica gel column, eluting with 10% ethyl
acetate in hexane, to obtain 7.0 g product (69% yield) as
1
clear oil. H NMR (CDCl3, 400 MHz): d 0.76 (t, 3H,
J=7.0 Hz); 1.06 (s, 6H); 1.69 (m, 6H); 2.80 (m, 1H);
3.15 (m, 1H), 4.03 (m, 1H); 4.13 (m, 1H), 5.18 (m, 2H),
7.36 (m, 5H).
(tert-Butoxy)-N-[benzylamino]formamide (15). A solu-
tion of benzyl carbazate (25 g, 150.4 mmol), Boc anhy-
dride (42.7 g, 195.5 mmol), triethylamine (19.8 g, 195.5
mmol), DMAP (0.9 g, 7.5 mmol) in 650 mL CH2Cl2 was
stirred for 24 h. The mixture was concentrated and
purified by silica gel column, eluting with 20% ethyl
acetate in hexane, to yield 36 g (90%) product. 1H
NMR (CDCl3, 400 MHz): d 1.40 (m, 9H); 5.25 (m, 2H);
7.36 (m, 5H).
3,3-Dimethyl-1-perhydropyridazinylpentane-1,2-dione (25).
1 g 10% Pd/C was added to a solution of 3,3-dimethyl-
1-[2-benzylperhydropyridazinyl] pentane-1,2-dione (7.0
g, 20.2 mmol) in 70 mL EtOH. The mixture was under
hydrogenation at room pressure (1 atm) overnight. The
product was obtained as white solid after filtering Pd
catalyst and concentration (3.8 g, 89%). 1H NMR
(CDCl3, 400 MHz): d 0.88 (t, 3H, J=7.0 Hz), 1.19 (s,
6H), 1.65 (m, 4H), 1.79 (m, 2H), 2.85 (m, 2H), 3.42 (m,
1H), 3.56 (m, 1H).
tert-Butyl 2-benzylperhydropyridazinecarboxylate (17).
A solution of (tert-butoxy)-N-[benzylamino]formamide
15 (35 g, 131 mmol) in 300 mL DMF was added drop-
wise to a suspended solution of 6.3 g (262 mmol) NaH
in 130 mL DMF under nitrogen. After the mixture was
stirred for 30 min at room temperature, a solution of 1,4
dibromobutane (28.4 g, 131 mmol) in 50 mL DMF was
added dropwise. The reaction was allowed to stir over-
night at room temperature. The reaction was then con-
centrated, followed by partition between 200 mL
CH2Cl2 and 200 mL water. The aqueous layer was
extracted with additional 200 mL CH2Cl2. The com-
bined organic layers were dried over MgSO4, and fil-
tered and concentrated. The crude product was further
purified by silica chromatography to yield 13.5 g (32%
3,3-Dimethyl-1-[2-(6-phenylhexanoyl)perhydropyridazinyl]-
pentane-1,2-dione (13f). To a solution of 5-phenylvalaric
acid (0.2 g, 1.1 mmol) in 3 mL CH2Cl2 was added tri-
ethylamine (0.15 mL, 1.1 mmol), followed by isobutyl
chloroformate (0.15 g, 1.1 mmol) at 0ꢁC. After stirring
for 5 min, a solution of 3,3-dimethyl-1-perhydropyr-
idazinylpentane-1,2-dione 25 (0.212 g, 1 mmol) in 1 mL
CH2Cl2 was added. The reaction was gradually warmed
up to room temperature. The crude material was subject
to silica gel purification to yield final product as clear oil
(0.20 g, 55%). Rf=0.58 (33% EtOAc/hexane). 1H
NMR (CDCl3, 400 MHz): d 0.89 (t, 3H, J=7.5 Hz), 1.24
(s, 6H), 1.37 (m, 2H), 1.68 (m, 6H), 1.74 (m, 4H), 2.23
(m, 2H), 2.62 (t, 2H, J=7.60 Hz), 2.80 (m, 2H), 4.53 (m,
2H), 7.21 (m, 5H). Anal. calcd for C23H34N2O3: C, 71.47;
H, 8.87; N, 7.25. Found: C, 71.54; H, 8.80; N, 7.32.
1
yield) product. H NMR (CDCl3, 400 MHz): d 1.46 (m,
9H); 1.64 (m, 4H); 2.88 (m, 2H); 4.20 (m, 2H); 5.16 (m,
2H); 7.31 (m, 5H).
Methyl
2-oxo-2-[2-benzylperhydropyridazinyl]acetate
(21). 20% TFA in CH2Cl2 was cooled to 0ꢁC and
added dropwise to a solution of tert-butyl 2-benzylper-
hydropyridazinecarboxylate 17 (13.34 g, 41.7 mmol) in
10 mL CH2Cl2. The mixture was stirred overnight. At
this time, the mixture was cooled to 0 ꢁC and 12.66 mL
(125 mmol) triethylamine was added, followed by addi-
tion dropwise of methyl oxalyl chloride (5.62, 45.9
mmol) in 5 mL CH2Cl2. The mixture was allowed to stir
2 h at 0 ꢁC and warm up to room temperature over-
night. The reaction was diluted with addition of CH2Cl2
and washed with water. The organic layer was dried
over MgSO4, filtered, and concentrated. The crude pro-
duct was further purified by silica chromatography to
1-(2-Hex-5-ynoylperhydropyridazinyl)-3,3-dimethylpen-
tane-1,2-dione (27). To a solution of 5-hexynoic acid
(0.467 g, 4 mmol) in 10 mL CH2Cl2 was added triethyl-
amine (0.56 mL, 4 mmol), followed by isobutyl chloro-
formate (0.53 mL, 4 mmol) at 0 ꢁC. After stirring for 5
min, a solution of 3,3-dimethyl-1-perhydropyridazinyl
pentane-1,2-dione 25 (0.424 g, 2 mmol) in 1 mL CH2Cl2
was added. The reaction was gradually warmed up to
room temperature. The crude material was subject to
silica gel purification to yield final product as clear oil
1
(0.385 g, 63%). H NMR (CDCl3, 400 MHz): d 0.91 (t,
3H, J=7.0 Hz); 1.26 (s, 6H); 1.76 (m, 8H); 2.28 (m, 2H);
2.50 (m, 2H); 2.88 (m, 2H); 3.60 (m, 1H); 4.50 (m, 2H).
1
yield 9.2 g (72.4% yield) product as clear oil. H NMR
(CDCl3, 400 MHz): d 1.72 (m, 4H); 2.85(m, 1H);
3.12(m, 1H); 3.67 (s, 3H); 4.15 (m, 1H); 4.35 (m, 1H);
5.20 (m, 2H); 7.35 (m, 5H).
3,3-Dimethyl-1-[2-(6-(3-pyridyl)hex-5-ynoyl)perhydropyr-
idazinyl] pentane-1,2-dione (29). To a solution of 1-(2-
hex-5-ynoylperhydropyridazinyl)-3,3-dimethylpentane-
1,2-dione 27 (0.384 g, 1.25 mmol) in 10 mL CH2Cl2
under nitrogen was added 3-iodopyridine (0.283 g, 1.38
mmol), (Ph3P)2PdCl2 (0.044 g, 0.06 mmol), CuI (0.0024
g, 0.013 mmol) and triethylamine (0.3 mL, 2 mmol).
The reaction mixture was stirred 30 min at room tem-
perature and then refluxed overnight. The mixture was
concentrated and purified by silica gel column, eluting
with 30% ethyl acetate in hexane, to yield product as
light yellow oil (0.31 g, 65%). 1H NMR (CDCl3,
400 MHz): d 0.84 (t, 3H, J=7.4 Hz); 1.21 (s, 6H); 1.70
3,3 - Dimethyl - 1 - [2- benzylperhydropyridazinyl]pentane -
1,2-dione (23). A solution of methyl 2-oxo-2-[2-benzyl-
perhydropyridazinyl]acetate 21 (9.0 g, 29.4 mmol) in 30
mL dry THF was cooled to ꢀ78 ꢁC and treated with 35
mL of 1.0 M solution of 1,1-dimethylpropylmagnesium
chloride in THF. After stirring the resulting homo-
geneous mixture at for 5 h, the mixture was poured into
saturated ammonium chloride (150 mL) and extracted
into ethyl acetate. The organic layer was washed with

4824
D. E. Wilkinson et al. / Bioorg. Med. Chem. 11 (2003) 4815–4825
(m, 6H); 1.96 (m, 2H), 2.52 (m, 3H); 2.90 (m, 2H); 3.60
(m, 1H); 4.42 (m, 2H); 7.20 (m, 1H); 7.66 (m, 1H);
8.49(m, 1H); 8.62 (m, 1H).
(CDCl3, 400 MHz): d 0.87 (t, 3H, J=7.5 Hz); 1.22 (s,
3H); 1.26 (s, 3H); 1.63 (m, 2H); 2.1 (m, 2H); 2.73 (m,
4H); 3.20–3.85 (m, 4H); 7.19 (m, 1H); 7.66 (m, 1H); 8.5
(m, 2H). Anal. calcd for C20H25N3O3: C, 67.67; H, 6.91;
N, 10.63. Found: C, 64.58; H, 7.09; N, 10.82.
3,3-Dimethyl-1-[2-(6-(3-pyridyl)hexanoyl)perhydropyri-
dazinyl] pentane-1,2-dione (13g). 0.1 g PtO2 was added
to a solution of 3,3-dimethyl-1-[2-(6-(3-pyridyl)hex-5
ynoyl)perhydropyridazinyl] pentane-1,2-dione 35 (0.3 g,
0.8 mmol) in 20 mL dry MeOH. The mixture was under
hydrogenation at room pressure (1 atm) overnight. The
product was obtained as clear oil after filtering the cat-
alyst, concentrating and purifying on a silica gel (0.125
g, 41%). Rf=0.18 (EtOAc). 1H NMR (CDCl3,
400 MHz): d 0.89 (t, 3H, J=7.4 Hz); 1.24 (s, 6H); 1.38
(m, 2H); 1.66 (m, 10H); 2.14 (m, 2H); 2.63 (m, 2H); 2.82
(m, 2H); 4.60 (m, 2H); 7.23 (m, 4H). Anal. calcd for
C22H33N3O3: C, 68.19; H, 8.58; N, 10.84. Found: C,
68.40; H, 8.52; N, 10.62.
3,3-Dimethyl-1-[2-(5-(3-pyridyl)pentanoyl)pyrazolidinyl]-
pentane-1,2-dione (12k). Rf=0.3 (EtOAc). 1H NMR
(CDCl3, 400 MHz): d 0.87 (t, 3H, J=7.5 Hz); 1.22 (s,
3H); 1.26 (s, 3H); 1.37 (m, 2H); 1.65 (m, 6H); 2.1 (m,
2H); 2.30 (m, 1H); 2.62 (m, 3H); 3.20–3.85 (m, 4H); 7.19
(m, 1H); 7.66 (m, 1H); 8.5 (m, 2H). Anal. calcd for
C20H29N3O3: C, 65.74; H, 8.06; N, 11.09. Found: C,
65.98; H, 8.20; N, 10.89.
Molecular Modeling
Most of the molecular modeling was performed using
SYBYL modeling.22 The crystal structure of (1R)-1,3-
3,3-Dimethyl-N-[2-(3-phenylpropanoyl)tetrahydro-1H-1-
pyrazolyl]-1,2-pentane-dione (12g). Rf=0.60 (2:1 hex-
ane/EtOAc). H NMR (CDCl3, 300 MHz): d 0.80–0.85
(t, 3H); 1.11–1.15 (m, 8H); 1.58–2.02 (m, 6H): 2.50–2.95
(m, 4H); 7.17–7.28 (m, 5H). Anal. calcd for
C19H26N2O3: C, 69.06; H, 7.93; N, 8.48. Found: C,
68.98; H, 7.90; N, 8.41.
diphenyl-1-propyl
pentyl)-2-piperidinecarboxylate
(2S)-1-(3,3-dimethyl-1,2- dioxo-
(SB3) bound to
1
FKBP12 (PDB code: 1FKG) was used for all experi-
ments.18 SB3 was used as a template to build the aza
compounds into the active site of FKBP12. Each com-
pound was minimized in the active site with the struc-
ture of the enzyme held rigid. The Tripos forcefield23
with Gasteiger–Huckel atomic charges24 were used in all
˚
3,3 - Dimethyl - 1 - [2- (4 - phenylbutanoyl)pyrazolidinyl]-
pentane-1,2-dione (12h). Rf=0.5 (Hexane:EtOAc 1:1).
1H NMR (CDCl3, 400 MHz): d 0.87 (t, 3H, J=7.5 Hz);
1.22 (s, 3H); 1.26 (s, 3H); 1.64 (m, 2H); 1.92–2.07 (m,
5H), 2.20 (m, 1H), 2.63 (m, 2H); 3.25 (m, 2H); 3.80 (m,
2H); 7.27 (m 5H, aromatic). Anal. calcd for
C20H28N2O3: C, 69.05; H, 8.27; N, 8.06. Found: C,
69.02; H, 8.22; N, 8.05.
calculations. A non-bonded cutoff of 25 A was utilized
with a distance dependent dielectric. The ab initio quan-
tum mechanical calculations were performed using the
program GAMESS.25 Molecules were optimized at the
MP2 level of theory using the 6-31G(d) basis set.26ꢀ29
Biology
3,3-Dimethyl-N-[2-(5-phenylpentanoyl)tetrahydro-1H-1-
pyrazolyl]-1,2-pentane-dione (12i). Rf=0.25 (2:1 hexane/
EtOAc). H NMR (CDCl3, 300 MHz): d 0.81–0.83 (m,
3H); 1.14 (s, 6H); 1.21 (m, 2H); 1.55–1.62 (m, 8H): 2.02
(m, 2H); 2.61 (m, 4H); 7.14–7.28 (m, 5H). Anal. calcd
for C21H30N2O3: C, 70.36; H, 8.44; N, 7.81. Found: C,
70.10; H, 8.41; N, 7.77.
Inhibition of the rotamase activity of FKBP-12 by test
compounds was assayed as described by Kofron,12
using the peptide N-succinyl Ala-Leu-Pro-Phe p-nitro-
anilide (Bachem) as substrate.
1
MPTP lesioning of dopaminergic neurons in mice was
used as an animal model of Parkinson’s disease as pre-
viously described.13 Four-week-old male CD1 white
mice (20–25 g) were dosed ip with 30 mg/kg of MPTP in
saline vehicle for 5 days. Test compounds suspended in
Cremaphor or vehicle, were administered by daily oral
dosing occurred 30 min prior to each MPTP injection
and on each of 5 subsequent days. All experimental
mice and matching MPTP/vehicle and vehicle/vehicle
control groups were sacrificed 18 days after their first
MPTP injection. The striata were dissected and homo-
genized. Immunostaining was performed on saggital
and coronal brain sections using anti-tyrosine hydox-
ylase Ig to quantitate survival and recovery of dopami-
nergic neurons.
3, 3-Dimethyl-1-[2-(6-phenylhexanoyl)pyrazolidinyl]pen-
tane-1,2-dione (12j). Rf=0.5 (hexane/EtOAc 1:1). 1H
NMR (CDCl3, 400 MHz): d 0.87 (t, 3H, J=7.5 Hz);
1.22 (s, 3H); 1.26 (s, 3H); 1.35 (m, 2H); 1.59 (m, 6H);
2.07 (m, 2H), 2.20 (m, 1H), 2.60 (m, 3H); 3.25 (m, 2H);
3.70 (m, 2H); 7.26 (m 5H, aromatic). Anal. calcd for
C22H32N2O3: C, 70.65; H, 8.70; N, 7.36. Found: C,
70.94; H, 8.66; N, 7.52.
3,3-Dimethyl-1-(2-pent-4-ynoylpyrazolidinyl)pentane-1,2-
dione (26). Rf=0.45 (EtOAc). 1H NMR (CDCl3,
400 MHz): d 0.87 (t, J=7.5 Hz); 0.90 (m, 2H); 1.22 (s,
3H); 1.26 (s, 3H); 1.64 (m, 2H); 2.03–2.20 (m, 3H), 2.52
(m, 2H), 2.63 (m, 1H); 3.69 (m, 3H). Anal. calcd for
C15H22N2O3 : C, 64.55; H, 7.98; N, 9.98. Found: C,
64.73; H, 7.97; N, 10.06.
References and Notes
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1
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