2-Aryl-2,2-difluoroacetamide FKBP12 Ligands: Synthesis and X-ray Structural Studies

Article abstract of DOI:10.1021/ol0166909

2-Aryl-2,2-difluoroacetamido-proline and pipecolate esters are high affinity FKBP12 ligands whose rotamase inhibitory activity is comparable to that seen for the corresponding ketoamides. X-ray structural studies suggest that the fluorine atoms participat

Full text of DOI:10.1021/ol0166909

ORGANIC
LETTERS
2001
Vol. 3, No. 25
3987-3990
2-Aryl-2,2-difluoroacetamide FKBP12
Ligands: Synthesis and X-ray Structural
Studies
Gene M. Dubowchik,* Vivekananda M. Vrudhula, Bireshwar Dasgupta,
Jonathan Ditta, Ti Chen, Steven Sheriff,† Karin Sipman,† Mark Witmer,†
Jeffrey Tredup,† Dolatrai M. Vyas, Todd A. Verdoorn, Sagarika Bollini, and
Alexander Vinitsky
Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 5100,
Wallingford, Connecticut 06492-7660, and P.O. Box 4000,
Princeton, New Jersey 08543-4000
gene.dubowchik@bms.com
Received September 4, 2001
ABSTRACT
2-Aryl-2,2-difluoroacetamido-proline and pipecolate esters are high affinity FKBP12 ligands whose rotamase inhibitory activity is comparable
to that seen for the corresponding ketoamides. X-ray structural studies suggest that the fluorine atoms participate in discrete interactions with
the Phe36 phenyl ring and the Tyr26 hydroxyl group, with the latter resembling a moderate-to-weak hydrogen bond.
FK506 binding proteins (FKBPs) represent a growing family
of chaperones of the immunophilin class that perform
multiple tasks in all cell types.1 FKBPs facilitate the correct
folding of certain proteins by catalyzing cis-trans isomer-
ization around peptidyl-prolyl bonds (rotamase activity),
though this increasingly appears to be a minor role for these
proteins. Most well-known is the ability of FKBP12 to cause
immunosuppression by inhibition of calcineurin and FRAP
(also known as RAFT or mTOR) following binding of the
macrolides FK506 and rapamycin, respectively, that serve
as noncovalent bridging elements.2 However, FKBP12 is also
found in complex with intracellular calcium channels (RyR
and IP3R)3 as well as members of the TGFâ family of
receptors4 where it most likely modulates signaling.
Recently, FKBPs have been associated with recovery from
neuronal injury5 and implicated as targets for neuroregen-
eration and neuroprotection.6 The discovery that FK506
(Figure 1) possesses neurotrophic properties in vitro and in
vivo was quickly followed by reports of similar activity by
nonimmunosuppressive compounds such as V-10,3677 and
GPI-10468 that mimic only the FKBP12-binding portion of
FK506, though the effectiveness of the latter has been
(3) Gaburjakova, M.; Gaburjakova, J.; Reiken, S.; Huang, F.; Marx, S.
O.; Rosemblit, N.; Marks, A. R. J. Biol. Chem. 2001, 276, 16931.
(4) Huse, M.; Chen, Y.-G.; Massague, J.; Kuriyan, J. Cell 1999, 96, 425.
(5) Lyons, W. E.; Steiner, J. P.; Snyder, S. H.; Dawson, T. M. J. Neurosci.
1995, 15, 2985.
(6) (a) Hamilton, G. S.; Steiner, J. P. J. Med. Chem. 1998, 41, 5119. (b)
Gold, B. G. Mol. Neurobiol. 1997, 15, 285. (c) Gold, B. G.; Densmore, V.;
Shou, W.; Matzuk, M. M.; Gordon, H. S. J. Pharmacol. Exp. Ther. 1999,
289, 1202.
† P.O. Box 4000.
(1) Galat, A. Eur. J. Biochem. 1993, 216, 689.
(2) Schreiber, S. L. Science 1991, 251, 283.
(7) Costantini, L. C.; Isacson, O. Exp. Neurol. 2000, 164, 60.
(8) Ross, D. T.; Guo, H.; Howorth, P.; Chen, Y.; Hamilton, G. S.; Steiner,
J. P. Neurosci. Lett. 2001, 297, 113.
10.1021/ol0166909 CCC: $20.00 © 2001 American Chemical Society
Published on Web 11/17/2001

the corresponding ketoacid 2 was carried out with selenium
dioxide in pyridine.13 Initially, we converted 2 to the methyl
ester (not shown) using methanolic HCl for the subsequent
diethylaminosulfur trifluoride (DAST) reaction, followed by
ester hydrolysis to give 4. Although this sequence gave 4 in
good overall yield, we found it more efficient to treat 2
directly with DAST, giving a mixture of diethylamide 3
(50%) and acid 4 (15%) that could be subjected to basic
hydrolysis followed by acidification to give 4 cleanly.
Smooth conversion to the acid chloride 5 using oxalyl
chloride and catalytic DMF was carried out as needed prior
to amide formation.
Prolyl and pipecolylamine esters 8 (Scheme 2) were
prepared as previously described,14 with various secondary
Scheme 2. Synthesis of Aryl Difluoroamides 9a-oa
Figure 1. FKBP12 ligands.
questioned.9 Although no obvious mechanism for this activity
is apparent, several potential pathways have been proposed
including destabilization of steroid receptor complexes
containing the chaperones FKBP52 and hsp906c and modula-
tion of intracellular calcium bursts by binding to FKBP12.10
Recently, an intriguing report suggests that neuronal inhibi-
tory factor (NIF), a protein that prevents growth of injured
neurons, requires correct folding around two peptidyl-prolyl
bondssa process in which an immunophilin could be
utilizedsbefore it is active.11 Compounds that have been
reported to be high affinity FKBP12 ligands are pipecolyl
or prolyl ketoamides, where X-ray crystal structures some-
times indicate a hydrogen-bonding interaction between the
ketone carbonyl oxygen and the Tyr26 hydroxyl group.12 In
our search for novel FKBP12-binding compounds, we found
that replacement of the ketone carbonyl with a gem-
difluoromethylene group gives FKBP12 ligands that show
activity that is comparable to and sometimes much better
than that of the corresponding keto-compounds.
a (a) EDC, DMAP; (b) 4 M HCl/dioxane; (c) DIEA.
alcohols 7 generated from Grignard additions. The synthesis
of 1-phenyl-7-(3-pyridyl)-heptan-4-ol 7o (Scheme 3) was
carried out in a different manner than previously reported,12
via alkylation of 3-picoline.15 Amide couplings proceeded
smoothly to give the final products 9, which were purified
by flash chromatography on silica gel.16
For purposes of comparison, several ketoamides and
methyleneamides were synthesized. Compounds 10a and 10b
(Table 1) were prepared as previously described by carbo-
The gem-difluoro compounds were prepared as follows.
Oxidation of 3,4,5-trimethoxyacetophenone 1 (Scheme 1) to
Scheme 3. Synthesis of 1-Phenyl-7-(3-pyridyl)-heptan-4-ola
Scheme 1. Synthesis of Acid Chloride 5a
a (a) TBDMSCl, Et3N, cat. DMAP, ether; (b) 3-picoline, LDA,
THF; (c) TBAF, THF; (d) (1) oxalyl chloride, DMSO; (2) Et3N,
CH2Cl2; (e) THF, 0 °C to rt.
a (a) SeO2, pyridine; (b) DAST, CH2Cl2; (c) NaOH; (d) oxalyl
chloride, DMF (cat.), CH2Cl2.
3988
Org. Lett., Vol. 3, No. 25, 2001

presence of an additional hydrogen bond, albeit most likely
a weaker one, between the ketone carbonyl oxygen and the
Tyr26 hydroxyl group is suggested in some but not all
ketoamide ligands. The trimethoxyphenyl ring (or the tert-
alkyl group in related compounds14a) interacts with a lipo-
philic wall of the pocket that includes the Ile90 and Ile97
side chains. In the case of unbranched esters such as GPI-
1046, the arylalkyl side chain runs out of the primary site
through a shallow hydrophobic groove where the aryl ring
resides in a promiscuous secondary binding site. For
compounds such as V-10,367, the additional branch of the
side chain tends to make self-binding interactions with the
trimethoxyphenyl ring as well as fill the void between that
ring and the Phe46 phenyl on the opposite wall of the active
site.12,14a
Inhibition of the rotamase activity of FKBP12 was
measured as described previously,18 except that the assay
was run at 10 °C instead of 0 °C to avoid water condensation
on the walls of the cuvette. Table 1 shows FKBP12 rotamase
inhibition data for a simple series of prolyl amide esters.
Both difluoroamides, 9a and 9b, show activity that is
comparable to or somewhat better than that of the corre-
sponding ketoamides, 10a and 10b. It is important to note
that the methyleneamides, 11a and 11b, showed no detectable
inhibitory activity at 10 µM,19 suggesting that the fluorine
atoms are not maintaining ligand binding energy simply by
occupying steric space but are most likely participating in
specific interactions in the FKBP12 active site.
Table 1. FKBP12 Rotamase Inhibition Data for 9a-11b
compd
X
Y
Ki, µM
9a
F2
O
H2
F2
O
N
N
N
CH
CH
CH
0.872
4.00
nia
1.30
2.20
nia
10a
11a
9b
10b
11b
H2
a ni: no detectable inhibition at 10 µM.
diimide coupling of the intermediates 8 and 2 shown in
Scheme 1.14a Methyleneamides 11a and 11b were similarly
made by the coupling of 8 with commercially available 3,4,5-
trimethoxyphenylacetic acid. Several attempts to prepare 2,2-
difluoromethylene carboxylates of 2-tert-alkyl-containing
acids or esters analogous to 2 were unsuccessful, probably
as a result of steric constraints against difluorination.
Because the compounds of interest contain benzylic
halogens that are in a para-relationship to an electron-
donating methoxy group, it was important to ensure their
hydrolytic stability. In this regard, a representative example
(9o) was found to be unchanged over 27 h in 9:1 phosphate-
buffered saline/DMSO containing bovine serum albumin at
pH 7.4 and in fetal calf serum, both at room temperature.
X-ray and NMR structural data shows that, for compounds
that mimic the FKBP12-binding portion of FK506 such as
V-10,36712 and GPI-1046,17 the pipecolate or proline ring
lies at the bottom of the largely hydrophobic binding pocket,
whose floor includes a Trp59 indole ring. Important hydrogen-
bonding interactions exist between the amide carbonyl
oxygen and the Tyr82 hydroxyl group and between the ester
carbonyl oxygen and the Ile56 backbone amide NH. The
The FKBP12 inhibitory activities of a series of branched
ester prolyl and pipecolyl difluoroamides are shown in Table
2. The activities of corresponding pipecolyl and proline esters
are comparable. The exceptions are compounds 9o (Ki )
19 nM) and 9n (Ki ) 104 nM), and 9h (Ki ) 40 nM) and
Table 2. FKBP12 Rotamase Inhibition Data for 9c-o
(9) (a) Harper, S.; Bilsland, J.; Young, L.; Bristow, L.; Boyce, S.; Mason,
G.; Rigby, M.; Hewson, L.; Smith, D.; O’Donnell, R.; O’Connor, D.; Hill,
R. G.; Evans, D.; Swain, C.; Williams, B.; Hefti, F. Neuroscience 1999,
88, 257. (b) Becker, D. B.; Jensen, J. N.; Myckatyn, T. M.; Doolabh, V.
B.; Hunter, D. A.; Mackinnon, S. E. J. Reconstr. Microsurg. 2000, 16, 613.
(10) Sauer, H.; Francis, J. M.; Jiang, H.; Hamilton, G. S.; Steiner, J. P.
Brain Res. 1999, 842, 109.
(11) Hasler, D. W.; Jensen, L. T.; Zerbe, O.; Winge, D. R.; Vasak, M.
Biochemistry 2000, 39, 14567.
(12) Armistead, D. M.; Badia, M. C.; Deininger, D. D.; Duffy, J. P.;
Saunders: J. O.; Tung, R. D.; Thomson, J. A.; Decenzo, M. T.; Futer, O.;
Livingston, D. J.; Murcko, M. A.; Yamashita, M. M.; Navia, M. A. Acta
Crystallogr., Sect. D 1995, 51, 522.
compd
y
n
m
Ki, µM
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
1
2
1
2
1
2
1
2
2
1
2
1
2
2
2
2
2
2
2
2
2
3
3
3
3
3
0
0
1
1
2
2
3
3
0
2
2
3
3
0.084
0.174
0.642
1.00
0.170
0.040
0.120
0.090
0.059
0.060
0.050
0.104
0.019
(13) Taylor, M. J.; Hoffman, T. Z.; Yli-Kauhaluoma, J. T.; Lerner, R.
A.; Janda, K. D. J. Am. Chem. Soc. 1998, 120, 12783.
(14) (a) Holt, D. A.; Luengo, J. I.; Yamashita, D. S.; Oh, H. J.; Konialian,
A. L.; Yen, H. K.; Rozamus, L. W.; Brandt, M.; Bossard, M. J.; Levy, M.
A.; Eggleston, D. S.; Liang, J.; Schultz, L. W.; Stout, T. J.; Clardy, J. J.
Am. Chem. Soc. 1993, 115, 9925. (b) Dubowchik, G. M.; Ditta, J.; Herbst,
J. J.; Bollini, S.; Vinitsky, A. Bioorg. Med. Chem. Lett. 2000, 10, 559.
(15) Davies-Coleman, M. T.; Faulkner, D. J.; Dubowchik, G. M.; Roth,
G. P.; Polson, C.; Fairchild, C. J. Org. Chem. 1993, 58, 5925.
9m
9n
9o
Org. Lett., Vol. 3, No. 25, 2001
3989

9g (Ki ) 170 nM), where there are >4-fold differences
favoring the larger ring. In general, activities are in the range
reported by others for analogous ketoamide-containing
compounds.20
Potential interactions between the gem-difluoromethylene
group of these ligands and FKBP12 active-site residues are
apparent from X-ray structural data of compound 9o in
complex with FKBP12 (Figure 2). One fluorine atom is
Figure 3. Overlay of X-ray structures of FKBP12 active site with
bound difluoroamide 9o (orange) and SB3 (pink), showing distances
between the Tyr26 hydroxyl oxygen and potential hydrogen bond
accepting atoms.
The distances as well as the relative orientations of these
groups are maintained in seven X-ray structures of related
compounds (data not shown) and are notable in that they
resemble favorable F-H interactions calculated for fluoro-
benzene/benzene and seen for fluoroaromatic inhibitors of
carbonic anhydrase.23 In addition, a series of gem-difluoro-
phosphonic acid inhibitors of protein tyrosine phosphatases
has also been shown to be high affinity ligands where the
corresponding methylene compounds are inactive.24 Here too,
interactions with both a hydrogen bond donor (an amide NH)
and a side chain phenyl ring are implicated.
The other prominent difference seen in the structure of
9o in comparison with that published for V-10,36712 is a
narrower tilt angle for the trimethoxyphenyl ring due to the
sp3 hybridization of the difluoromethylene carbon. This has
the effect of tilting the ring in the direction of orthogonality
in relation to the floor of the binding pocket. In future
publications we will show how the aryl-2,2-difluroacetamide
moiety has conferred high-affinity FKBP12 binding activity
on non-ester-containing chemotypes where the corresponding
ketoamides show dramatically lower affinity.
In conclusion, we have found that 2-aryl-2,2-difluoro-
acetamide proline and pipecolate esters are high affinity
FKBP12 ligands whose rotamase inhibitory activity is
comparable to that of the corresponding ketoamides. X-ray
structural studies suggest that the fluorine atoms participate
in discrete interactions with the Phe36 phenyl ring and the
Tyr26 hydroxyl group, with the latter resembling a moderate-
to-weak hydrogen bond.
Supporting Information Available: Experimental pro-
cedures for the preparation of compound 9o, characterization
data for compounds 9a-o, and details of X-ray crystal-
lographic studies. This material is available free of charge
via the Internet at http://pubs.acs.org.
Figure 2. X-ray structure of FKBP12 active site with bound
difluoroamide 9o showing close contacts between the fluorine atoms
and active-site residues. PDB Accession number: 1J4R.
within moderate hydrogen bonding distance (3.2 ( 0.2 Å)
of the same Tyr26 hydroxyl group that forms a hydrogen
bond with the ketone oxygen of V-10,367 (3.4 Å),12 making
what appears to be at least an electrostatic interaction if not
a true hydrogen bond. This is shown more clearly in Figure
3 where 9o is overlayed with SB3,14a a compound whose
ketone carbonyl oxygen appears to make only a weak
electrostatic interaction with the Tyr26 hydroxyl. Although
the idea of organic fluorine as a hydrogen bond acceptor is
controversial, partly because of the apparent lack of a good
theoretical basis,21 there are cases where the likelihood
appears to be strong.22 The second fluorine atom is within
van der Waals contact distance of CE1 and CD1 of the Phe36
side chain phenyl ring (3.0 and 3.3 ( 0.2 Å, respectively).
(16) All new compounds gave satisfactory analytical data. For 9o: MS
1
625.1 (MH)+; H NMR δ 8.38-8.34 (m, 2H), 7.75-7.00 (m, 7H), 6.76-
6.63 (m & s, 2H), 5.25-4.10 (4xm, 2H), 3.80-3.50 (m, 10H), 3.00-2.85
(m, 1H), 2.52 (m, 2H), 2.25-1.00 (4xm, 16H). Anal. Calcd for
C35H42N2O6F2: C, 67.29; H, 6.78; N, 4.48; F, 6.08. Found: C, 67.40; H,
6.85; N, 4.23; F, 5.86.
(17) Sich, C.; Cowley, D. J.; Guenet, C.; Merly, J. P.; Tuefel, M.; Saudek,
V. Eur. J. Biochem. 2000, 267, 5342.
(18) Kofron, J. L.; Kuzmic, P.; Kishore, V.; Colon-Bonilla, E.; Rich, D.
H. Biochemistry 1991, 30, 6127.
(19) The lack of FKBP12 inhibitory activity of related compounds in
which the ketone carbonyl has been replaced by methylene has been
demonstrated previously: Wang, G. T.; Lane, B.; Fesik, S. W.; Petros, A.;
Luly, J.; Krafft, G. A. Bioorg. Med. Chem. Lett. 1994, 4, 1161.
(20) Hamilton, G. S.; Steiner, J. P. Curr. Pharm. Des. 1997, 3, 405.
(21) Dunitz, J. D.; Taylor, R. Chem. Eur. J. 1997, 3, 89.
(22) Barbarich, T. J.; Rithner, C. D.; Miller, S. M.; Anderson, O. P.;
Strauss, S. H. J. Am. Chem. Soc. 1999, 121, 4280 and leading references
therein.
OL0166909
(23) DerHovanessian, A.; Doyon, J. B.; Jain, A.; Rablen, P. R.; Sapse,
A.-M. Org. Lett. 1999, 1, 1359.
(24) Groves, M. R.; Yao, Z. J.; Roller, P. P.; Burke, T. R.; Barford, D.
Biochemistry 1998, 37, 17773. Associated PDB assession number: 1BZC.
3990
Org. Lett., Vol. 3, No. 25, 2001