Structure-based design, synthesis, and biological evaluation of novel inhibitors of human cyclophilin A

Article abstract of DOI:10.1021/jm050716a

Cyclophilin A is involved in many cellular processes, including protein folding and intracellular transports. Because cyclophilin A has been shown to interact with HIV-1 gag proteins and to enhance the viral infectivity, nonimmunosuppressive cyclophilin A ligands may represent a new class of therapeutic agents against HIV. Here, we report a virtual screening using structure- and pharmacophore-based design to identify original nonpeptidic cyclophilin ligands. Following a lead identification of compounds 1 [1-(3-benzyloxypyridin-2-yl)-3-(3-chlorophenyl)urea] and 2 [1-(3- benzyloxypyridin-2-yl)-3-(3-trifluoromethylphenyl)urea] (IC₅₀ = 0.3 μM), a series of molecules were synthesized from a diarylurea scaffold and evaluated for their in vitro ability to inhibit the cis-trans isomerase activity of cyclophilin A. Molecular modifications provided several more potent compounds, in particular analogues 4d and 4i with IC₅₀ of 14 and 20 nM, respectively. Then, we evaluated the effect of analogues 1 and 2 on HIV virion infectivity in both immortalized and primary cells. Both 1 and 2 reduced virion infectivity in the replication-defective one-round infection assay, but only 1 impaired wild-type HIV infection in human peripheral blood mononuclear cells.

Full text of DOI:10.1021/jm050716a

900
J. Med. Chem. 2006, 49, 900-910
Structure-Based Design, Synthesis, and Biological Evaluation of Novel Inhibitors of Human
Cyclophilin A
Jean-Franc¸ois Guichou,† Julien Viaud,† Cle´ment Mettling,‡ Guy Subra,† Yea-Lih Lin,‡ and Alain Chavanieu*,†
Centre de Biochimie Structurale, UMR 5048 CNRS, UMR 554 INSERM, UM1, Faculte´ de Pharmacie, BP14491,
15 AVenue Charles Flahault, 34093 Montpellier Cedex 5, France, and Institut de Ge´ne´tique Humaine,
Centre National de la Recherche Scientifique, Unite´ Propre de Recherche 1142, Montpellier Cedex 5, France
ReceiVed July 26, 2005
Cyclophilin A is involved in many cellular processes, including protein folding and intracellular transports.
Because cyclophilin A has been shown to interact with HIV-1 gag proteins and to enhance the viral infectivity,
nonimmunosuppressive cyclophilin A ligands may represent a new class of therapeutic agents against HIV.
Here, we report a virtual screening using structure- and pharmacophore-based design to identify original
nonpeptidic cyclophilin ligands. Following a lead identification of compounds 1 [1-(3-benzyloxypyridin-
2-yl)-3-(3-chlorophenyl)urea] and 2 [1-(3-benzyloxypyridin-2-yl)-3-(3-trifluoromethylphenyl)urea] (IC50
)
0.3 µM), a series of molecules were synthesized from a diarylurea scaffold and evaluated for their in vitro
ability to inhibit the cis-trans isomerase activity of cyclophilin A. Molecular modifications provided several
more potent compounds, in particular analogues 4d and 4i with IC50 of 14 and 20 nM, respectively. Then,
we evaluated the effect of analogues 1 and 2 on HIV virion infectivity in both immortalized and primary
cells. Both 1 and 2 reduced virion infectivity in the replication-defective one-round infection assay, but
only 1 impaired wild-type HIV infection in human peripheral blood mononuclear cells.
Introduction
neurotrophic effects in several animal models; however, the
mechanism remains to be elucidated.14,15 Interestingly, Wu et
al. identified a set of nonpeptidic cyclophilin ligands with
preliminary in vitro neuroprotective/neurotrophic activity.16 All
these studies suggest a potential therapeutic value for peptidic
and nonpeptidic cyclophilin ligands as novel anti-retroviral drugs
or in treating neurological disorders.
Herein, using a crystal structure of CsA in complex with
CypA (pdb code 1CWA), a virtual screening approach was
performed to identify novel inhibitors that can interfere with
the active site of cyclophilin. Following database docking,
commercially available compounds were selected and tested for
inhibitory activity on cyclophilin. Then, based on the most potent
new inhibitors, a set of nonpeptidic cyclophilin ligands was
synthesized and anti-HIV activity was evaluated.
The immunophilins are cytosolic enzymes that have been
observed abundantly and ubiquitously in a wide range of tissue
types and organisms.1,2 They are characterized by the ability to
catalyze the cis-trans isomerization of peptidylprolyl bonds3
(PPIases), which was identified as the rate-limiting step in
protein folding.4,5 Concurrently to their function in protein
maturation, immunophilins seems to play a key role in
fundamental cellular processes, as they either stabilize or
destabilize a protein-protein complex (immunosuppression,
calcium channels,6 steroid receptors,7 and tyrosine kinase
receptors8). Originally identified as the cellular targets of
immunosuppressant drugs, the immunophilins are represented
by two classes: FKBPs (FK506 binding proteins) and cyclo-
philins (Cyps) (reviewed in ref 9). Human cyclophilin A (CypA)
was first described as the binding partner of cyclosporin A10
(CsA), an immunosuppressive cyclic undecapeptide widely used
to prevent organ rejection after transplant operations. CsA binds
to the PPIase site on CypA, blocking its ability to direct cis-
trans isomerization. However, the immunosuppressive role of
CsA is not related to the inhibition of PPIase activity. Instead,
the high-affinity CsA/CypA complex binds to calcineurin and
inhibits its phosphatase activity, which in turn suppresses T cell
activation.11
Results and Discussion
Structure-Based Design and Virtual Screening. To identify
the inhibitory molecules of CypA, a structure-based approach
was used for selecting the initial compound library designed
for virtual screening. Several crystal structures of cyclosporin,
cyclosporin analogues, and various dipeptides, complexed with
CypA, are available in the Protein Data Bank. These structures
determined by X-ray crystallography or NMR were analyzed
to derive a pharmacophore model for potential cyclophilin
ligands. Cyclophilin A adopts an eight-stranded antiparallel
P-barrel structure in the CypA/CsA complex17-19 CsA and other
substrates bind a common relatively hydrophobic groove formed
by 11-13 residues via hydrogen bonds and polar and hydro-
phobic contacts. On the basis of X-ray crystal structures, the
most important residues of the active site were identified by
site-directed mutagenesis (Arg55, Phe60, Phe113, and His126).20
As shown on Figure 1, analysis of the contact surface of the
CypA/CsA complex (pdb code 1CWA) reveals that side chain
of the MeVal11 of CsA is buried in a left hydrophobic pocket.
By contrast, the side chain of the Abu2 of CsA lies in another
cavity, the Abu pocket. Localized between these two residues,
Recently, new potent applications for nonimmunosuppressive
analogues of CsA have been reported. CypA is implicated in
the formation of the viral capsid of HIV-1 through a binding
interaction with the HIV-1 Gag precursor polyprotein.12 Thus,
nonimmunosuppressant cyclosporin A analogue blocking the
PPIases activity was identified as potent inhibitors of HIV-1
replication.13 Also, both immunosuppressive and nonimmuno-
supressive agents have demonstrated potent neuroprotective and
* To whom correspondence should be addressed. Tel: +33 4 67 54 86
44. Fax: +33 4 67 52 96 23. E-mail: chat@cbs.cnrs.fr.
† Centre de Biochimie Structurale.
‡ Centre National de la Recherche Scientifique.
10.1021/jm050716a CCC: $33.50 © 2006 American Chemical Society
Published on Web 01/10/2006

NoVel Inhibitors of Human Cyclophilin A
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 901
Figure 1. (A) The conformation of residues Abu2 and MeVal11 and the carbonyl oxygen of MeBmt1 of CsA in the CypA/CsA monomeric X-ray
crystal structure is shown (pdb code 1CWA). The left hydrophobic pocket of CypA is filled with the MeVa11 side chain and the Abu2 is positioned
at the beginning of the right pocket-Abu pocket. This image was generated and rendered with Pymol. The CypA surface was colored by atom
type. (B) Schematic diagram outlining the ideal properties of a CsA pharmacophore. Numbers represent distances in angstroms.
Figure 2. (A) Lead compounds 1 and 2 identified by virtual screening. (B) Putative binding mode of compounds 1 (upper, FlexX pose) and 2
(lower, Surflex pose) in the active site of human cyclophilin A. Possibilities of interaction with the surrounding amino acids include three hydrogen
bonds between the urea moiety and Gln63, Gly72, and Asn102 as well as several hydrophobic contacts.
the carbonyl oxygen of MeBmt1 is at hydrogen-bond distance
to the Nꢀ of Gln63. On the basis of information derived from
the cocrystal structure, three groups were chosen for the
development of the pharmacophore (Figure 1, panel B). For
two pharmacophoric points, a phenyl group (A, C) was selected
to mimic and potentially reinforce hydrophobic contacts of
MeVal11 and Abu2 with CypA. At first sight, the Abu pocket
of CypA appears to be a good location to design high-affinity
analogues with additional contacts. However, as illustrated by
Mikol et al.,21 three well-ordered water molecules that are parts
of the crystal structure could interfere during the binding. By
consequence, as a good compromise we decided to replace the
Abu side chain with an aromatic fragment not larger than a
phenyl group. Carbonyl oxygen (B) was kept for the central
pharmacophoric point. Then, the distances between carbonyl
oxygen and Câ of the two side chains were measured and added
to the pharmacophoric constraints. All residues that are within
10 Å from the Câ of MeVal11 in the CypA structure were
selected and used to define the binding site in our structure-
based computer screening.
Using the MDL ISIS/Base software of the ACD-3D (Avail-
able Chemicals Directory, which contains associated 3D data,
Release ACD 2004-2, 296 387 compounds in total), a 3D
database search was performed to identify compounds that map
all the pharmacophoric features. A subset of 3129 molecules
having molecular weights up to 650 Da was so defined. These
molecules were docked and evaluated by the FlexX program22
and then co-evaluated by using the X-score program.23 Mol-
ecules with FlexX energy scores from -25 to -40 or with
X-score energy scores from 5 to 7 were visually analyzed for
their associations with the left hydrophobic pocket of cyclophilin
and the capability to form hydrogen bond interactions through
their carbonyl oxygen. At the final stage, molecules that show
promising docking affinities in at least one of the scoring method
and a good binding mode in accordance with our pharmaco-
phoric definition were purchased. Taking into account the

902 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Guichou et al.
Table 1. Most Significant FlexX and Xscore Scores of Selected
and 2 were analyzed to suggest how the diarylurea scaffold may
be expanded and to further rationalize the structure-activity
relationship. In comparison with CsA, compounds 1 and 2 retain
two hydrogen bonds with critical residues Gln63 and Asn102
of CypA and present an additional bond with Gly72, one
nitrogen atom of the urea moiety is hydrogen bonded to the
carbonyl oxygen atom of Asn102, the carbonyl oxygen atom
of the urea function is hydrogen bonded to the Nꢀ atom of
Gln63, and the second nitrogen atom is bonded to the carbonyl
oxygen atom of Gly72. Furthermore, most of the hydrophobic
interactions between CypA and CsA were conserved within the
docking mode of compounds 1 and 2. Thus, Arg55, Phe60,
Ala101, Ala103, Phe113, Leu122, and His126 were found to
form hydrophobic contacts with molecule 1 and 2, whatever
the docking poses proposed by FlexX and Surflex. Interestingly,
as illustrated in Figure 2 (panel B), the 3-chlorophenyl and the
3-trifluoromethylphenyl group of 1 and 2, respectively, were
found to be directed toward a hydrophilic region of the Abu
pocket composed by residues Gly72-Thr73-Gly74, Thr107-
Asn108-Gly109-Ser110-Qln111, and Lys82. As described by
X-ray studies21 of several cyclophilin-cyclosporin complexes,
this region is normally occupied by three to four water
molecules, which form hydrogen bonds with hydrophilic
residues.
Compoundsa
IC50
IC50
compd (nM)
FlexX
Xscore compd (nM)
FlexX
Xscore
1
2
316 -26.03
303 -25.89
754 -23.57
nd
592 -25.98
60 amb
951 -22.90
71 amb
6.22
6.31
5.96
6.29
6.51
4d
4i
9c
11
12
15
14
20
-34.10
-24.75
6.27
5.64
4a
4s
4o
4c
4h
9b
7b
6b
4y
140 amb
60 amb
320 amb
nd not docked
-26.05
-27.59
5.64
16a nd
16b 4100 -18.66
17
18
19
6.49
5.38
5.92
528 -35.15
6.64
6.21
nd
nd
nd
-23.10
not docked
not docked
nd
nd
-33.00
not docked
a Compounds were docked/scored with FlexX and then rescored using
Xscore. For FlexX, more-negative scores are better. The Surflex score for
compounds 1 and 2 was 4.96 and 4.92, respectively. amb ) abnormal mode
of binding; nd ) not determined.
Scheme 1a
Chemistry and Structure-Activity Relationships. Accord-
ing to the docking study between the CypA structure and 1 or
2, the 3-benzyloxypyridine group was found to fulfill the left
pocket and the monosubstituted phenyl group positioned toward
the Abu pocket. Thus at a first step, we searched for substituents
that could afford additional contacts with the beginning or the
hydrophilic region of the Abu pocket, leaving the rest of the
molecule intact. Except for 4j, bisubstituted ureas 4a-z were
prepared by the condensation of 2-amino-3-benzyloxypyridine
(3) with aryl isocyanate in THF under reflux (Scheme 1) in
good to excellent yields. 4j was obtained by selective reaction
of the amino group of 3 with the isocyanate function of
4-(chlorosulfonyl)phenyl isocyanate at room temperature.25
Reduction26 of compounds 4b-d with SnCl2 in EtOH under
reflux afforded amines 5a-c. The amines 5a-c were acylated
with acetic anhydride to obtain the acetamide 6a-c or converted
to ureas 7a-c upon the action of potassium cyanate27 (Scheme
a Reagent: (a) R-NdCdO/THF.
availability of the compounds, we focused on a set of 31
chemicals for further characterization against the cis-trans
isomerase of cyclophilin A. Among them at 10 µM, five
compounds turned out to inhibit more than 80% of the isomerase
activity of cyclophilin A and four compounds were less potent
with a 40% inhibition. The IC50 values for the former five
compounds were experimentally determined as lying between
0.3 and 5 µM (data not shown). Compounds 1 [1-(3-benzyl-
oxypyridin-2-yl)-3-(3-chlorophenyl)urea from Menai] and 2
[1-(3-benzyloxypyridin-2-yl)-3-(3-trifluoromethylphenyl)urea from
Menai] were stronger inhibitors of the isomerase activity with
an IC50 of around 0.3 µM and 100% inhibition at 10 µM. As
shown in Figure 2 (panel A), 1 and 2 present a diarylurea
scaffold that is appropriate for rapid molecular optimization,
and thus, it was selected for further molecular modifications.
The putative interaction mode of compounds 1 and 2 with
CypA was further docked and evaluated with a second docking
program, Surflex.24 As for FlexX, no water molecules were
present during the virtual screening calculations. Compounds
1 and 2 possessed similar FlexX, X-score, and Surflex energy
scores (∼26, ∼6, and ∼5, respectively) but were only top-ranked
by Surflex (Table 1). They appeared within the active site in a
comparable fashion with only small variations in distances to
active site residues with the benzyl group buried into the left
hydrophobic pocket and the phenyl ring positioned inside the
Abu pocket as shown in Figure 2, panel B (compound 1, FlexX
pose; compound 2, Surflex pose). The docked structures of 1
28
2). Hydrolysis of 4e-f with H2O2 under basic conditions
afforded amides 8a,b (Scheme 3). Saponification of ethyl ester
4g-i with NaOH gave carboxylic acids 9a-c and ethyl ester
4h was converted to methyl ester 10a upon the action of sodium
methanolate in MeOH (Scheme 4). Reactions of 4j with aqueous
NH3 or aqueous MeNH2 in THF gave the corresponding
sulfonamide 11 and 12 (Scheme 5). Reactions of 4k with
aqueous NH3 in THF gave a mixture of two compounds, 13
and 14, which were separated by chromatography on silica gel
(Scheme 6).
Then, we evaluated the influence of the 3-benzyl group.
Phenol derivative 15 was obtained through catalytic hydrogena-
Scheme 2a
a Reagents: (a) SnCl2/EtOH/reflux; (b) Ac2O/THF; (c) KOCN/H2O/AcOH/THF.

NoVel Inhibitors of Human Cyclophilin A
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 903
Scheme 3a
activity or no activity at all. For example, compound 9a with a
carboxylic acid function was completely inactive, whereas
compound 4g with an ethyl ester showed an IC50 of 880 nM.
These results suggest that ortho substitutions on the phenyl ring
might be unsuitable for compounds with an enhanced activity
and that additional contacts have to be found deeper inside the
Abu pocket.
Subsequently, we studied the effect of substituting the meta
position of the phenyl ring (Table 2). For meta-substituted
derivates, incorporation of an aliphatic chain (4m), an electron-
withdrawing group such as a bromide (4q), a nitrile (4e), or an
electron-donating group such as an acetamide moiety (6b) and,
to a lesser extent, an urea group (7b) resulted in a significant
decrease of the inhibition at 10 µM. Five other electron-
withdrawing groups were also tested. Among them, three
derivates (4h, 10a, 8a) showed less potent activity when
compared to the lead compound 1 with IC50 values from 361
to 951 nM. Interestingly, it is worthy of note that several
compounds reinforce the putative mode of binding and the
orientation of the phenyl ring toward a hydrophilic region of
the Abu pocket. First, comparison of compounds 4h, 10a, and
9b clearly indicates that hydrophobic contacts decrease the
efficiency of a molecule (IC50: ethyl ester 4h < methyl ester
10a < carboxylic acid 9b). Then, 4c and 9b, which present an
electron-withdrawing group that is able to form hydrogen bonds,
provide much better analogues, with an IC50 value of 60 and
71 nM, respectively.
a Reagents: (a) H2O2/Na2CO3/H2O/THF/DMF.
Scheme 4a
a Reagents: (a) NaOH/H2O/THF; (b) NaOMe/MeOH.
Scheme 5a
Various substitutions in the para position of the phenyl ring
were explored (Table 3). It was observed that aromatic (4y)
and electron-donating groups (6c, 7c) led to a sharp drop in
potency. Comparable to the SAR of meta-substituted derivates,
p-phenyl analogues with an electron-withdrawing group led to
various effects, from the most active compound in the series
(4d, IC50 ) 14 nM) to a compound with no activity at all (4f).
In this position, it is interesting to note that hydrogen-bonding
capability of substituents, preferably two double-bond oxygen
atoms, plays an important role in the enhanced activity of the
compound (4d, 4i, 11). On the another hand, di and trisubstituted
phenyl analogues give poor results and that is certainly due to
steric clashes with hydrophilic residues of the Abu pocket. Only
one difluorinated compound is active (4z, IC50 ) 458 nM),
which presents a hydrogen-bond-acceptor capability (Table 3).
The docking models revealed a good picture of the structure-
activity relationship for the p-phenyl substituted analogues.
Indeed, for instance, the nitrile group of the highly active
molecule 4d is localized near the coordinates normally occupied
by two water molecules in several structures of cyclophilin (e.g.,
W6 and W7 of pdb crystal structure 1CWA). In the same way
as these water molecules, the nitrile group of 4d could be
implicated in two hydrogen bonds with Thr107 and Gly109.
Moreover, the presence of an ethyl ester group in the para
position (4i) produced a very active compound, whereas the
ortho and meta positions (4g, 10a) conducted to a decrease in
activity. Again, this can be explain by docking models that show
a Reagents: (a) aq NH3/H2O/THF; (b) aq MeNH2/H2O/THF.
tion of 4a over Pd/C in EtOH. Urea 16a,b were prepared from
15; thus, treatment of a solution of 15 in dry THF with NaH,
followed by addition of alkylating agents at room temperature,
gave the corresponding urea 16a,b in good yields (Scheme 7).
At last, we prepared three compounds, 17-19, to confirm the
importance of the urea moiety. Thiourea 17 was prepared by
condensation of 3 with phenyl thioisocyanate in THF under
reflux. Reaction of 3 with benzoyl chloride and benzenesulfonyl
chloride gave the corresponding amide 18 and sulfonamide 19
in good yields (Scheme 8). All the chemical compounds were
characterized for their inhibitory potency of the cyclophilin
isomerase activity in a typical in vitro test.29
To improve the activity of meta-substituted compounds 1 and
2, our initial focus was to explore the Abu pocket accessed by
the phenyl ring. As indicated in Table 2, none of the ortho-
substituted derivates showed better activity than the two lead
compounds 1 or 2. Shifting the m-Cl group (1, IC50 ) 316 nM)
to the o-position (4o, IC50 ) 592 nM) resulted in reduction of
the IC50 value by a factor of 1.5-2, while removing the Cl group
(4a, IC50 ) 754 nM) reduces the IC50 value by a factor of 2.5.
It was found that introduction of an aliphatic chain or an
aromatic ring resulted in a drastic loss of activity (4l, 4s).
Substitutions with more polar groups conduct to attenuated
Scheme 6a
a Reagent: (a) aq NH3/H2O/MeOH.

904 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Guichou et al.
Table 2. In Vitro Inhibitory Activities of Diarylurea Analogues Substituted on the Ortho Position (left) and Meta Position (right) of the Phenyl Ringb
a Number of determinations. b Data of the percent inhibition at 10 µM are obtained from triplicate measurements. IC50 data represent mean values of three
independent determinations and SD is typically lower than 8%. nd ) not determined.

NoVel Inhibitors of Human Cyclophilin A
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 905
Scheme 7a
experiments are under progress and will allow the design of
new analogues with single-digit nanomolar range activity.
Anti-HIV-1 Activity. The ability of compounds 1 and 2 to
interfere with the HIV-1 replication cycle was evaluated in a
one-round infection assay, where infectious but nonreplicative
virions harboring a luciferase reporter gene produced in 293T
cells were used to infect target HOS-CD4+-CCR5+ cells. When
10 µM of CsA or compounds 1 and 2 was added to both
producing and target cells, virions showed a dramatic decrease
of infectivity of respectively 96%, 97%, and 62% (Figure 3A).
This 96% decrease for CsA is slightly above the 85-88%
generally described.30,31 This could be due to different experi-
mental systems. Interestingly, as previously described for CsA,32
the decrease in infectivity is almost entirely due to CypA
inhibition in virions producing cells, since virions produced in
the presence of inhibitors showed a similar magnitude of
decrease in infectivity while the target cells are cultured in the
absence of inhibitors (Figure 3B). On the other hand, the
treatment of inhibitors in target cells only had a modest impact
on infectivity of control virions produced without inhibitors
treatment (Figure 3C). This effect has recently been shown to
be independent of the CypA-capside interaction; however, the
mechanism still needs to be defined.33,34
a Reagents: (a) Pd/C, H2/EtOH; (b) NaH/THF then R-X.
Scheme 8a
a Reagents: (a) Ph-NdCdS/THF; (b) Ph-COCl/THF; (c) Ph-SO2Cl/
THF.
additional van der Waals contacts with the Lys82 side chain
when the phenyl ring is para-substituted with an ethyl ester
group.
We next tested compounds 1 and 2 on the replication of the
pNL4.3 strain of HIV-1 in human peripheral blood mononuclear
cells (PBMC). We first tested that the compounds had no effect
on cell viability and proliferation. In contrast to CsA, which
interferes with T cell activation via the calcineurin pathway and
blocks potential cell proliferation,11,35 cells counts were com-
parable between different culture conditions with/without 1 or
2 inhibitors (data not shown). Interestingly, this result suggests
that compounds 1 and 2 could be nonpeptidic cyclophilin
inhibitors with nonimmunosuppressive activity. However, the
nonimmunosuppressive property of these diarylurea analogues
has to be confirmed with more accurate experiments such as
their incapacity to inhibit the activation of specific transcription
factors that regulate genes that are expressed early in T
lymphocyte activation.
As the block of PBMC growth per se is deleterious on HIV
production, the CsA inhibition of infectivity can be explained
by two different mechanisms: interaction with CypA during
virion production and inhibition of cell proliferation. We then
decided to compare the efficiency of the two compounds to that
of a potent anti-HIV drug without effect on cell proliferation,
AZT, a reverse transcriptase inhibitor. As the first viral inoculum
is produced without inhibitors, the chemical analogues will only
act on subsequent viral replication cycles. AZT (50 µM) was
then added 12 h after the initial infection in a control assay to
allow the first round of infection and inhibit further replicative
cycles. Compound 1 retained a dose dependent inhibitory effect
on replicative virions, 60% of the AZT effect at 10 µM, while
compound 2 had no effect at all on HIV-1 infectivity in these
primary cells (Figure 4). The later was already much less
efficient in the one-round infection assay, maybe due to poor
membrane permeability. It is interesting to note that compound
1 at 1 µM is more efficient as the number of replication cycle
increases (compare days 4 and 7 postinfection), which is
consistent with previous reports.36
We then investigated the influence of the benzyl group in
the inhibitory potency of our analogues. By virtual docking,
this benzyl group was found to sit in the hydrophobic left pocket
that is normally occupied by the side chain of MeVal11 of CsA.
Compounds 16a and 15 with either a bulky aromatic group or
no moiety present a sharp decrease in activity, whereas
compound 16b (IC50 ) 4.100 nM) with an aliphatic group was
less active than the parent compound 4a (IC50 ) 754 nM) (Table
4). Finally, to confirm the role of the urea moiety, which seems
implicated in several hydrogen bonds with the protein, we
replaced the central part of the molecule with a thiourea (17),
an amide (18), and a sulfonamide (19) and found these
compounds to be far less potent (19, 45% inhibition at 10 µM)
or even inactive. These results are in total agreement with the
binding model proposed for 1 and 2, which shows that the urea
moiety forms three hydrogen bonds with the protein.
Taken together, these results indicate that the pharmacophore
used in this study represented an efficient scaffold to mimic
critical interactions between a ligand and the human cyclophilin.
Five compounds over 31 commercially chemicals initially tested
were found to inhibit more than 80% of the cyclophilin activity.
On the basis of the reported SARs of our 47 original compounds,
we concluded that the two docking algorithms FlexX and Surflex
were able to generate correctly oriented docked poses for
molecules 1 and 2. However, as is usually the case with virtual
screening, it was not possible to differentiate between closely
related active and inactive analogues (Table 1). The correlation
between SARs and docking studies clearly reveals that inter-
molecular hydrogen bonding, between meta and para functional
groups of the phenyl ring and residues localized into the Abu
pocket of the cyclophilin, allowed an increase of the inhibitory
potency of our nonpeptidic analogues of CsA. The hydrophilic
part of the Abu pocket is normally stabilized via a hydrogen-
bonding network occurring through three well-ordered water
molecules.21 Here, we proposed that phenyl-substituted groups
with hydrogen-bonding capability either displace or replace at
least one of these water molecules. However, in absence of
X-ray analysis, the precise nature of residues implicated in the
hydrogen-bonding network between a compound and the
cyclophilin A remains to be defined. Indeed, X-ray structural
Conclusion
In summary, we employed a structure-based design to identify
compounds able to interfere with the active site of cyclophilin.
Five out of 31 selected compounds were found to induce an in
vitro inhibition of the isomerase activity of cyclophilin A. On

906 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Guichou et al.
Table 3. In Vitro Inhibitory Activities of Diarylurea Analogues Substituted on the Para Position of the Phenyl Ring (left) and with Multiple
Substitutions (right)b
a Number of determinations. b Data of the percent inhibition at 10 µM are obtained from triplicate measurements. IC50 data represent mean values of three
independent determinations and SD is typically lower than 8%. nd ) not determined.

NoVel Inhibitors of Human Cyclophilin A
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 907
Experimental Section
Table 4. In Vitro Inhibitory Activities of Compounds 15, 16a, and 16b
and Thiourea (17), Amide (18), and Sulfonamide (19) Analoguesb
General Methods. All reagents and solvents were purchased
from commercial sources and used without further purification.
Melting points were determined on a Kofler block and are
uncorrected. Mass spectra were acquired under fast atom bombard-
ment (FAB) conditions using a JEOL JMS DX300, and samples
were introduced on a matrix. Proton NMR spectra were collected
on a Bruker 400 MHz spectrometer using tetramethylsilane (TMS)
as internal standard, and chemical shift (δ) data are reported in
parts per million (ppm) relative to internal standard TMS. Thin-
layer chromatography (TLC) was performed using silica gel 60 F254
plates from Macherey-Nagel and visualized by UV light. Flash
chromatography was carried out using SDS silica gel 60 (35-70
mesh) with cyclohexane, ethyl acetate (EtOAc), and methanol
(MeOH) as eluents with chromatographic solvent proportion
expressed on a volume:volume basis. The reported chemical yields
were not optimized.
Synthesis of Ureas 4a-z: General Procedure (except for 4j).
2-Amino-3-benzyloxypyridine 3 (1 equiv) was dissolved in dry THF
(0.25 M). The aryl isocyanate (1.2 equiv) was added in one portion
and the reaction mixture was heated under reflux. After the reaction
was complete (TLC control), the reaction mixture was concentrated
and Et2O was added to precipitate the urea. The solid was filtrated,
washed with a small amount of Et2O, and dried under vacuum.
4-[3-(3-Benzyloxypyridin-2-yl)ureido]benzenesulfonyl Chlo-
ride (4j). 2-Amino-3-benzyloxypyridine 3 (200 mg, 1.0 mmol) was
dissolved in 4 mL of dry THF and this solution was cooled with
an ice bath. 4-Chlorosulfonylbenzene isocyanate (217 mg, 1.0
mmol) was added drop by drop. After the addition, the reaction
mixture was left for 4 h at room temperature and then concentrated
and Et2O was added to precipitate the urea. The solid was filtrated,
washed with a small amount of Et2O, and dried under vacuum to
give the urea 4j (180 mg, 43%) as a white solid.
Synthesis of 1-(2-Aminophenyl)-3-(3-benzyloxypyridin-2-yl)-
urea (5a). Compound 4b (1 equiv, 0.30 mmol) was dissolved in
absolute ethanol (0.008 M), and SnCl2 dihydrate (6 equiv, 1.80
mmol) was added in one portion. The reaction mixture was heated
under reflux for 4 h and the ethanol was evaporated. The residue
was taken up with EtOAc, and the organic phase was washed with
a solution of 1 M NaHCO3 and brine, dried over Na2SO4, filtrated,
and concentrated to afford amine 5a as a white solid (90%). This
product is unstable and it is used directly after isolation.
Synthesis of Amine 5b-c: General Procedure. Compounds
4c,d (1 equiv) were dissolved in absolute ethanol (0.008 M), and
SnCl2 dihydrate (6 equiv) was added in one portion. The reaction
mixture was heated under reflux for 4 h and the ethanol was
evaporated. The residue was taken up with EtOAc, and the organic
phase was washed with a solution of 1 M NaHCO3 and brine, dried
over Na2SO4, filtrated, and concentrated. The crude product was
chromatographed on a silica gel column (ethyl acetate/cyclohexane/
methanol 47.5/47.5/5). Evaporation and drying of the product
afforded amine 5b,c as a white solid.
a Number of determinations. b Data of the percent inhibition at 10 µM
are obtained from triplicate measurements. IC50 data represent mean values
of three independent determinations and SD is typically lower than 8%. nd
) not determined.
the basis of compounds 1 [1-(3-benzyloxypyridin-2-yl)-3-(3-
chlorophenyl)urea] and 2 [1-(3-benzyloxypyridin-2-yl)-3-(3-
trifluoromethylphenyl)urea], we developed a novel class of
potent diarylurea inhibitors and designed compounds with
enhanced activity (analogues 4d and 4i with IC50 of 14 and 20
nM, respectively). Then, we have shown that compounds 1 and
2 are potent inhibitors of the HIV-1 replication cycle on
immortalized cells or human peripheral blood mononuclear cells.
Therefore, these new nonpeptidic cyclophilin ligands might
serve as novel leads for further pharmacological investigations
as therapeutic agents against HIV-1.
Figure 3. Effect of various compounds on HIV-1 infectivity. Viral infectivity was determined by one-round infection as described in the Experimental
Section. Effects of CsA, 1, and 2 were expressed as the percentage of luciferase activity of the control. Inhibitors were added in both producing and
target cells (A), producing cells only (B) or target cells only (C). The results represent one of two independent experiments. Each experiment had
been performed in triplicate.

908 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Guichou et al.
Figure 4. Effect of different compounds on HIV-1 production in human PBMC. Viral production is monitored by p24 ELISA at days 4 (A) and
7 (B) postinfection. Increasing concentration of CsA (O), 1 (0), or 2 (4) inhibitors were added to the culture medium. Viral productions of control
(b) or 50 µM AZT (9) is shown with error bars.
Synthesis of Amide 6a-c: General Procedure. Compounds
5a-c (1 equiv) were dissolved in THF (0.03 M), and Ac2O (10
equiv) was added in one portion. The reaction mixture was left for
1 h at room temperature and the THF was evaporated. The crude
product was chromatographed on a silica gel column (ethyl acetate/
cyclohexane/methanol 45/45/10). Evaporation and drying of the
product afforded amide 6a-c as a white solid.
An aqueous solution of ammoniac (0.5 mL, 33%) was added drop
by drop. After 15 min, the reaction mixture was concentrated and
the residue was taken up with CH2Cl2. The organic phase was
washed with brine, dried over Na2SO4, filtrated, and concentrated.
The crude product was purified by flash chromatography (ethyl
acetate/cyclohexane/methanol 7/2/1) to afford the sulfonamide 11
(6 mg, 17%) as a white solid.
Synthesis of Urea 7a-c: General Procedure. Compounds
5a-c (1 equiv, 0.12 mmol) were dissolved in THF (1 mL) and
H2O (2 mL), and then a solution of KOCN (2 equiv, 0.24 mmol)
in 10% AcOH in H2O (2 mL) is added. The reaction mixture was
left for 2 h at room temperature and the solvent evaporated. The
crude product was chromatographed on a silica gel column (ethyl
acetate/cyclohexane/methanol 4/4/1). Evaporation and drying of the
product afforded urea 7a-c as a white solid.
Synthesis of Amide 8a-b: General Procedure. Compounds
4e,f (0.15 mmol) was dissolved in THF/EtOH/DMF 1/1/1 (3 mL),
and successively a solution of 3 N Na2CO3 (1 mL) and a 35%
solution of H2O2 (0.9 mL) were added. After 4 days at room
temperature, 4 mL of water was added and the reaction mixture
was extracted with EtOAc. The organic phase was washed with
brine, dried over Na2SO4, filtrated, and concentrated. The crude
product was triturated with Et2O to precipitate the amide. The solid
was filtrated, washed with a small amount of Et2O, and dried under
vacuum to give the amide 8a,b as a white solid.
Synthesis of Benzoic Acid 9a-c: General Procedure. Com-
pounds 4g-i (1 equiv) were dissolved in THF/MeOH (1/1) (0.02
M) and a solution of 1 M NaOH (10 equiv) was added drop by
drop. The reaction mixture was left overnight at room temperature.
The reaction mixture was neutralized with a solution of 5 M HCl,
and solvents were evaporated. The residue was taken up with 40
mL of water and this solution was saturated with NaCl. The aqueous
phase was extracted with EtOAc (3 × 50 mL). The organic phases
were combined, dried over Na2SO4, filtered, and concentrated to
afford the benzoic acid 9a-c as a white solid.
Synthesis of 3-[3-(3-Benzyloxypyridin-2-yl)ureido]benzoic
Acid Methyl Ester (10a). Compound 4h (1 equiv, 0.26 mmol)
was dissolved in dry MeOH/THF (5/1) (0.02 M). A solution of 1.3
M sodium methanolate (10 equiv, 2.6 mmol) in dry methanol was
added drop by drop. The reaction mixture was left for 1 h at room
temperature and neutralized with a solution of 1 M HCl, and the
solvents were evaporated. The residue was taken up with EtOAc,
and the organic phase was washed with a solution of saturated
NaHCO3 and brine, dried over Na2SO4, filtrated, and concentrated
to give the methyl ester 10a as a white solid.
Synthesis of 4-[3-(3-Benzyloxypyridin-2-yl)ureido]-N-methyl-
benzenesulfonamide (12). Compound 4j (41.7 mg, 0.1 mmol) was
dissolved in 2 mL of dry THF, and this solution was cooled with
an ice bath. An aqueous solution of methylamine (0.5 mL, 40%)
was added drop by drop. After 15 min, the reaction mixture was
concentrated and the residue was taken up with CH2Cl2. The organic
phase was washed with brine, dried over Na2SO4, filtrated, and
concentrated. The crude product was purified by flash chromatog-
raphy (ethyl acetate/cyclohexane/methanol 7/2/1) to afford the
sulfonamide 12 (18 mg, 43%) as a white solid.
Synthesis of 5-[3-(3-Benzyloxypyridin-2-yl)ureido]isophthal-
amic Acid Methyl Ester (13) and 5-[3-(3-Benzyloxypyridin-2-
yl)ureido]isophthalamide (14). Compound 4k (100 mg, 0.23
mmol) was dissolved in 40 mL of MeOH. This solution was cooled
with an ice bath and ammoniac was bubbled in for 15 min. The
reaction mixture was left for 5 days at room temperature (each day
ammoniac was bubbled in for 15 min with an ice bath). The reaction
mixture was concentrated and the residue was taken up with EtOAc.
The organic phase was washed with brine, dried over Na2SO4,
filtrated, and concentrated. The crude products were purified by
flash chromatography (ethyl acetate/cyclohexane/methanol 7/2/1)
to afford 13 (4 mg, 4%) and 14 (20 mg, 20%).
Synthesis of 1-(3-Hydroxypyridin-2-yl)-3-phenylurea (15).
Compound 4a (1.0 g, 3.13 mmol) was dissolved in 70 mL of dry
MeOH and 5 mL of dry THF. This solution was degassed with
nitrogen and Pd/C (120 mg) was added. The resulting solution was
submitted to a pressure of hydrogen (60 mbar) for 1 h. The reaction
mixture was filtered (Celite) and the filtrate was concentrated. The
residue was triturated with Et2O, filtrated, and washed with a small
amount of Et2O to afford 15 as a white solid (458 mg, 64%).
Synthesis of Ether 16a,b: General Procedure. Compound 15
(115 mg, 0.5 mmol) was slowly added to a suspension of NaH in
4 mL of dry DMSO. After 30 min at room temperature, the reaction
mixture was cooled with an ice bath and the alkylating agent (1.2
equiv, 0.6 mmol) was added drop by drop. The temperature of the
resulting solution rose to room temperature. After 3 h, water was
added and the reaction mixture was extracted with ethyl acetate.
The organic phase was washed with brine, dried over Na2SO4,
filtrated, and concentrated. The crude product was purified by flash
chromatography (ethyl acetate/cyclohexane 3/7) to afford the ether
as a white solid.
Synthesis of 4-[3-(3-Benzyloxypyridin-2-yl)ureido]benzene-
sulfonamide (11). Compound 4j (41.7 mg, 0.1 mmol) was dissolved
in 2 mL of dry THF and this solution was cooled with an ice bath.

NoVel Inhibitors of Human Cyclophilin A
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 909
Synthesis of 1-(3-Benzyloxypyridin-2-yl)-3-phenylthiourea
(17). 2-Amino-3-benzyloxypyridine 3 (200 mg, 1.0 mmol) was
dissolved in 4 mL of dry THF. The phenyl isothiocyanate (135
mg, 1,2 mmol) was added in one portion and the reaction mixture
was heated under reflux for 72 h. The reaction mixture was
concentrated and the crude product was purified by flash chroma-
tography (ethyl acetate/cyclohexane 2/8) to afford the thiourea 17
(242 mg, 73%).
Synthesis of N-(3-Benzyloxypyridin-2-yl)benzamide (18). Ben-
zoyl chloride (116 µL, 1 mmol) was dissolved in 10 mL of dry
THF, and then a solution of 2-amino-3-benzyloxypyridine 3 (1.0
g, 5 mmol) in 10 mL of dry THF was added drop by drop. After
the addition, the reaction mixture was left for 4 h at room
temperature and then concentrated. The crude product was purified
by flash chromatography (ethyl acetate/cyclohexane 4/6) to afford
the amide 18 (241 mg, 81%) as a white solid.
Synthesis of N-(3-Benzyloxypyridin-2-yl)benzenesulfonamide
(19). 2-Amino-3-benzyloxypyridine 3 (100 mg; 0.5 mmol) was
dissolved in 10 mL of dry THF. Then benzene sulfonyl chloride
(89 mg, 0.5 mmol) is added in one portion and the reaction mixture
was heated under reflux for 40 h. The reaction mixture was
concentrated, the residue was taken up with CH2Cl2, and the organic
phase was washed with water and brine, dried over Na2SO4,
filtrated, and concentrated. The crude product was purified by flash
chromatography (EtOAc/cyclohexane 15/85) to afford the sulfon-
amide 19 (50 mg; 30%).
Ability of Compounds To Interfere with the HIV-1 Replica-
tion Cycle. Cells. HOS-CD4+-CCR5+ cells (AIDS Reagent
Program) and SV40 T antigen-transformed human embryonic
kidney 293T cells were grown in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal calf serum (FCS)
and antibiotics.
One-Round Infection Assay. To produce replication-defective
HIV virions, 293T cells were cotransfected with the pNL4.3-
env-Luc+ (AIDS Reagent Program) plasmid and with the pCMV-
Ad8-Env plasmid encoding the envelope of HIV-1 prototype Ad8
with increasing amounts of CsA analogues. HOS-CD4+-CCR5+
cells were plated in a 24-well plate at a density of 5 × 104 cells/
well. They were exposed to 30 ng of p24 equivalents of Ad8-
pseudotyped defective virions for 18 h, washed, and cultured for
72 h. Luciferase activity was measured by luminometry, using the
Luciferase Assay System (Promega).
Cell Infection. Peripheral blood mononuclear cells were isolated
by Ficoll-Hypaque density gradient centrifugation and stimulated
for 48 h with phytohemagglutinin (5 µg/mL, Difco Laboratories)
and 100 U/mL of interleukin-2 (Boehringer-Mannheim). Cells (1.5
× 105) were then incubated for 1 h with or without the inhibitory
compounds and exposed to 40 ng of p24 equivalent of the pNL4.3
HIV-1 strain for 24 h in 96-wells plates, washed extensively, and
cultured at 2 × 106 cells/mL in 0.5 mL RPM-I1640 (GIBCO)
supplemented with 10% fetal calf serum, antibiotics, glutamine,
and with or without compounds. Half of the medium was changed
with fresh medium every 3 days, and infection was monitored by
p24 ELISA (Beckmann Coulter).
spectrophotometer (Varian), and the rate constants were determined
from the absorbance versus time data files.
Virtual Screening. The structure-based design of inhibitors was
performed using the crystal structure of the CypA/CsA complex
(pdb code 1CWA). A total of 3129 molecules with specific
pharmacophoric constraints and molecular weights up to 650 Da
was retrieved from the ACD (Available Chemicals Directory,
Release ACD 2004-2, 296 387 compounds in total). To take into
account the interatomic distances of the pharmacophore, the ACD-
3D database that includes the 3D atomic coordinates was used. This
set of molecules retrieved in a multi-SDF file were converted to
3D structures with CORINA (Molecular Networks GmbH) and then
docked and evaluated by the FlexX program.23 Standard parameters
of the FlexX 1.13.1 program were used for iterative growing and
subsequent scoring of FlexX poses. Active-site atoms were defined
as a sphere of 10 Å from the center point defined by Phe113. A
receptor description file was automatically defined from the pdb
coordinates of the hydrogen-free protein/active site coordinates.
Molecules with a score higher than -25 and docked further than
10 Å of the Phe113 side chain of CypA were eliminated by using
the LEAD3D program developed by Douguet et al.37 All poses were
rescored with X-Score23 to rerank them and to give a more accurate
estimation of the binding free energies of the molecules. Inspection
of the hit lists and their docked structures fitted into the complex
were visualized with the program XmMol.38 Visual inspection of
the suggested binding modes of FlexX, according to the scoring
values of FlexX and X-Score, was used to select a set of compounds
to be ordered and tested. In a second set of experiments, each
molecule found to be active against the isomerase activity of
cyclophilin A was docked and evaluated with another docking
program called Surflex.24 This new docking study between the
CypA structure and, for example, compounds 1 or 2 was realized
to define more accurately the putative binding mode of the
compounds. It has to be noticed that for virtual screening calcula-
tions performed with FlexX and Surflex, no water molecules were
added during the binding-site or the protomol generation, respec-
tively.
Acknowledgment. The authors thank Dr. Dominique Dou-
guet (for software development of LEA3D), Dr. Yannick Bessin
(for protein purification), Mr. Jean-Luc Pons (for computing
assistance), Dr. Andre Padilla (for NMR assistance), and Prof.
Manfred Mutter (for the gift of Cyclosporin A). We are grateful
to the AIDS Research and Reference Reagent Program for the
pNL4.3-env-Luc+ plasmid and the HOS-CD4+-CCR5+ cells.
We are also grateful to Mr. Terry Page for his critical reading
of the manuscript. This work was sponsored by the CNRS and
INSERM.
Supporting Information Available: Table providing results
from purity analyses by HPLC for key target compounds and
chemical shifts of synthesized compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
Inhibition of Isomerase Activity of Cyclophilin A. The cis-
trans isomerization of an alanine-proline bond in a model substrate,
N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, was monitored on a
spectrophotometer (Varian) in a chymotrypsin-coupled assay. The
inhibition of this reaction induced by different concentrations of
inhibitor was determined, and the data were analyzed as a change
in first-order rate constant as a function of inhibitor concentration,
which yields the Ki value (GraphPad software). The following were
added to a cuvette: 840 µL of ice cold assay buffer (40 mM
HEPES, pH 7.9, 100 mM NaCl), 70 µL of Cyp A (0.36 µM in 40
mM HEPES, pH 7.9, 100 mM NaCl), 70 µL of chymotrypsin (50
mg/mL in 1 mM HCl), and 10 µL of test compound, at various
concentrations, in dimethyl sulfoxide. The reaction was initiated
by the addition of 10 µL of substrate (N-succinyl-Ala-Ala-Pro-Phe-
p-nitroanilide, 2 mg/mL in 500 mM LiCl in trifluoroethanol). The
absorbance at 390 nm versus time was monitored for 90 s using a
References
(1) Harding, M. W.; Handschumacher, R. E.; Speicher, D. W. Isolation
and amino acid sequence of cyclophilin. J. Biol. Chem. 1986, 261,
8547-8555.
(2) Hunter, T. Prolyl isomerases and nuclear function. Cell 1998, 92,
141-143.
(3) Galat, A. Peptidylproline cis-trans-isomerases: Immunophilins. Eur.
J. Biochem. 1993, 216, 689-707.
(4) Schreiber, S. L. Chemistry and biology of the immunophilins and
their immunosuppressive ligands. Science 1991, 251, 283-287.
(5) Gothel, S. F.; Marahiel, M. A. Peptidyl-prolyl cis-trans isomerases,
a superfamily of ubiquitous folding catalysts. Cell. Mol. Life Sci.
1999, 55, 423-436.
(6) Marks, A. R. Cellular functions of immunophilins. Physiol. ReV. 1996,
76, 631-649.
(7) Pratt, W. B.; Toft, D. O. Steroid receptor interactions with heat shock
protein and immunophilin chaperones. Endocr. ReV. 1997, 18, 306-
360.

910 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Guichou et al.
(8) Chen, Y. G.; Liu, F.; Massague, J. Mechanism of TGFbeta receptor
inhibition by FKBP12. EMBO J. 1997, 16, 3866-3876.
(9) Galat, A. Peptidylprolyl cis/trans isomerases (immunophilins): Bio-
logical diversitystargetssfunctions. Curr. Top. Med. Chem. 2003,
3, 1315-1347.
(25) Leban, J.; Pegoraro, S.; Dormeyer, M.; Lanzer, M.; Aschenbrenner,
A.; Kramer, B. Sulfonyl-phenyl-ureido benzamidines: A novel
structural class of potent antimalarial agents. Bioorg. Med. Chem.
Lett. 2004, 14, 1979-1982.
(26) Mathis, C. A.; Wang, Y.; Holt, D.; Huang, G.-H.; Debnath, M. L.;
Klunk, W. E. Synthesis and Evaluation of 11C-Labeled 6-Substituted
2-Arylbenzothiazoles as Amyloid Imaging Agents. J. Med. Chem.
2003, 46, 2740-2754.
(27) Laudien, R.; Mitzner, R. Phenylureas. Part 1. Mechanism of the basic
hydrolysis of phenylureas. J. Chem. Soc., Perkin Trans. 2 2001, 11,
2226-2229.
(28) Kabalka, G. W.; Deshpande, S. M.; Wadgaonkar, P. P.; Chatla, N.
The transformation of nitriles into amides using sodium percarbonate.
Synth. Commun. 1990, 20 (10), 1445-1451.
(29) Kofron, J. L.; Kuzmic, P.; Kishore, V.; Colon-Bonilla, E.; Rich, D.
H. Determination of kinetic constants for peptidyl prolyl cis-trans
isomerases by an improved spectrophotometric assay. Biochemistry
1991, 30, 6127-6134.
(30) Saphire, A. C.; Bobardt, M. D.; Gallay, P. A. Cyclophilin a plays
distinct roles in human immunodeficiency virus type 1 entry and
postentry events, as revealed by spinoculation. J. Virol. 2002, 76,
4671-4677.
(31) Thali, M.; Bukovsky, A.; Kondo, E.; Rosenwirth, B.; Walsh, C. T.;
et al. Functional association of cyclophilin A with HIV-1 virions.
Nature 1994, 372, 363-365.
(32) Braaten, D.; Franke, E. K.; Luban, J. Cyclophilin A is required for
the replication of group M human immunodeficiency virus type 1
(HIV-1) and simian immunodeficiency virus SIV(CPZ)GAB but not
group O HIV-1 or other primate immunodeficiency viruses. J. Virol.
1996, 70, 4220-4227.
(33) Saphire, A. C.; Bobardt, M. D.; Gallay, P. A. trans-Complementation
rescue of cyclophilin A-deficient viruses reveals that the requirement
for cyclophilin A in human immunodeficiency virus type 1 replication
is independent of its isomerase activity. J. Virol. 2002, 76, 2255-
2262.
(34) Sokolskaja, E.; Sayah, D. M.; Luban, J. Target cell cyclophilin A
modulates human immunodeficiency virus type 1 infectivity. J. Virol.
2004, 78, 12800-12808.
(35) Clipstone, N. A.; Crabtree, G. R. Identification of calcineurin as a
key signaling enzyme in T-lymphocyte activation. Nature 1992, 357,
695-697.
(36) Towers, G. J.; Hatziioannou, T.; Cowan, S.; Goff, S. P.; Luban, J.;
et al. Cyclophilin A modulates the sensitivity of HIV-1 to host
restriction factors. Nat. Med. 2003, 9, 1138-1143.
(37) Douguet, D.; Munier-Lehmann, H.; Labesse, G.; Pochet, S.
LEA3D: A computer-aided ligand design for structure-based drug
design. J. Med. Chem. 2005, 48, 2457-68.
(38) Tuffery, P. XmMol: An ×11 and motif program for macro-
molecular visualization and modeling. J. Mol. Graph. 1995, 13, 67-
72, 62.
(10) Fischer, G.; Wittmann-Liebold, B.; Lang, K.; Kiefhaber, T.; Schmid,
F. X. Cyclophilin and peptidyl-prolyl cis-trans isomerase are
probably identical proteins. Nature 1989, 337, 476-478.
(11) Liu, J.; Farmer, J. D., Jr.; Lane, W. S.; Friedman, J.; Weissman, I.;
et al. Calcineurin is a common target of cyclophilin-cyclosporin A
and FKBP-FK506 complexes. Cell 1991, 66, 807-815.
(12) Luban, J. Absconding with the chaperone: Essential cyclophilin-
Gag interaction in HIV-1 virions. Cell 1996, 87, 1157-1159.
(13) Mlynar, E.; Bevec, D.; Billich, A.; Rosenwirth, B.; Steinkasserer,
A. The nonimmunosuppressive cyclosporin A analogue SDZ NIM
811 inhibits cyclophilin A incorporation into virions and virus
replication in human immunodeficiency virus type 1-infected primary
and growth-arrested T cells. J. Gen. Virol. 1997, 78, 825-835.
(14) Hansson, M. J.; Mattiasson, G.; Mansson, R.; Karlsson, J.; Keep,
M. F.; et al. The nonimmunosuppressive cyclosporin analogues
NIM811 and UNIL025 display nanomolar potencies on permeability
transition in brain-derived mitochondria. J. Bioenerg. Biomembr.
2004, 36, 407-413.
(15) Sosa, I.; Reyes, O.; Kuffler, D. P. Immunosuppressants: Neuropro-
tection and promoting neurological recovery following peripheral
nerve and spinal cord lesions. Exp. Neurol. 2005, 31, 31.
(16) Wu, Y. Q.; Belyakov, S.; Choi, C.; Limburg, D.; Thomas, I. B.; et
al. Synthesis and biological evaluation of nonpeptidic cyclophilin
ligands. J. Med. Chem. 2003, 46, 1112-1115.
(17) Kallen, J.; Spitzfaden, C.; Zurini, M. G.; Wider, G.; Widmer, H.; et
al. Structure of human cyclophilin and its binding site for cyclosporin
A determined by X-ray crystallography and NMR spectroscopy.
Nature 1991, 353, 276-279.
(18) Mikol, V.; Kallen, J.; Pflugl, G.; Walkinshaw, M. D. X-ray structure
of a monomeric cyclophilin A-cyclosporin A crystal complex at 2.1
A resolution. J. Mol. Biol. 1993, 234, 1119-1130.
(19) Zhao, Y.; Ke, H. Mechanistic implication of crystal structures of the
cyclophilin-dipeptide complexes. Biochemistry 1996, 35, 7362-
7368.
(20) Zydowsky, L. D.; Etzkorn, F. A.; Chang, H. Y.; Ferguson, S. B.;
Stolz, L. A.; et al. Active site mutants of human cyclophilin A
separate peptidyl-prolyl isomerase activity from cyclosporin A
binding and calcineurin inhibition. Protein Sci. 1992, 1, 1092-1099.
(21) Mikol, V.; Papageorgiou, C.; Borer, X. The role of water molecules
in the structure-based design of (5-hydroxynorvaline)-2-cyclo-
sporin: Synthesis, biological activity, and crystallographic analysis
with cyclophilin A. J Med. Chem. 1995, 38, 3361-3367.
(22) Rarey, M.; Wefing, S.; Lengauer, T. Placement of medium-sized
molecular fragments into active sites of proteins. J. Comput.-Aided
Mol. Des. 1996, 10, 41-54.
(23) Wang, R.; Lai, L.; Wang, S. Further development and validation of
empirical scoring functions for structure-based binding affinity
prediction. J. Comput. Aided Mol. Des. 2002, 16, 11-26.
(24) Jain, A. N. Surflex: Fully automatic flexible molecular docking using
a molecular similarity-based search engine. J. Med. Chem. 46, 499-
511. 2003.
JM050716A