Difluoroketones as inhibitors of matrix metalloprotease-13

Article abstract of DOI:10.1016/S0960-894X(00)00287-0

Substrate-like difluoroketones have been prepared as potential inhibitors of MMP-13. Weak inhibition was seen with the key target 2. This and the more potent activity of intermediate 7b illustrates that hydrated ketones can be used to inhibit MMP-13 and perhaps other members of this class of enzymes. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00287-0

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1581±1584
Di¯uoroketones as Inhibitors of Matrix Metalloprotease-13
Lawrence A. Reiter,* Gary J. Martinelli, Lisa A. Reeves and Peter G. Mitchell
P®zer Inc., Central Research Division, Eastern Point Road, Groton, CT 06340, USA
Received 15 December 1999; accepted 11 May 2000
AbstractÐSubstrate-like di¯uoroketones have been prepared as potential inhibitors of MMP-13. Weak inhibition was seen with the
key target 2. This and the more potent activity of intermediate 7b illustrates that hydrated ketones can be used to inhibit MMP-13
and perhaps other members of this class of enzymes. # 2000 Elsevier Science Ltd. All rights reserved.
The inhibition of matrix metalloproteases (MMP's) has
been e€ected with compounds containing a variety of zinc
binding groups including hydroxamic acids, carboxylic
acids, thiols and phosphinic acids.1 4 While potent
compounds containing each of these zinc ligands have
been described, each ligand class possesses properties
that have, in general, hampered their development into
therapeutic agents. As a result, we have continued to
examine other possible zinc ligands. In this regard, we
noted that a-¯uorinated ketones,5 binding in their
hydrated form, have been shown to be inhibitors of
metalloproteases such as carboxypeptidase A,6 8 angio-
tensin converting enzyme,6 metallo-b-lactamase,9 and
membrane-bound amino peptidase.10 Being transition
state mimics, a-¯uorinated ketones can readily bind in a
substrate-like fashion in pockets on both side of an
enzyme's catalytic residues. In this respect a-¯uorinated
ketones resemble phosphinic acid-based inhibitors. That
they are both monodentate ligands for the active site zinc
is another similarity observed in the available crystal
ligand nor the active site glutamate needs to be protonated
by bulk water in order to avoid an unfavorable charge-
charge interaction.1
The above aspects of a-¯uorinated ketone-based inhibitors
combined with our recent ®nding that phosphinate 1 is
a potent inhibitor of MMP-13 (IC50: 14 nM)11 led us to
prepare the di¯uoroketone analogue 2 as a probe to
examine the utility of this class of compounds for the inhi-
bition of MMP's. Di¯uoroketone 2 contains functionality
0
0
suitable for binding into the S2, S1 , and S3 pockets of
MMP-13.11 We chose to prepare 2 over the isomeric
a,a-di¯uoroketone 3 since the latter may be unstable
due to b-elimination of HF.16 18
structures;1,7,11,12 however,
a signi®cant di€erence
between them is their pKa's. Phosphinic acids will be
nearly completely deprotonated at physiological pH
(pKa's ꢀ3)13 and hydrated a-¯uorinated ketones will be
substantially uncharged (tri¯uoroacetone pKa=10.2).14
This di€erence may translate into better absorption for a
a-¯uorinated ketone-based inhibitor over a phosphinate-
based compound of otherwise similar structure. Orally
active a-¯uorinated ketone-based protease inhibitors
have been described.15 Owing to their not being depro-
tonated at physiological pH, a-¯uorinated ketone-based
inhibitors may also have an inherent potency advantage
over phosphinates in that upon binding neither the
In order to determine the best methods for approaching
the synthesis of 2, as well as to facilitate the interpretation
of NMR spectra through the elimination of diaster-
eomers, our initial target was 4, the derivative without
the phenethyl side chain. The successful0 synthetic route
parallels that of Hong18 in that the P1 carboxylate is
released through the oxidation of a terminal ole®n.
*Corresponding author. Tel.: +1-860-441-3681: fax: +1-860-441-3803;
e-mail: lawrence_a_reiter@groton.p®zer.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00287-0

1582
L. A. Reiter et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1581±1584
Accordingly, the a-ketoester 519 was treated with
DAST20 and the resultant a,a-di¯uoroester converted in
three standard steps to the Weinreb amide 6 (see
Scheme 1). This was treated with 3-butenylmagnesium
bromide giving the desired ketone 7a. In an earlier
unsuccessful route, we found that production of a d,d-
di¯uoro-g-keto acid led to the formation of a stable
cyclic derivative which we could not induce to react in
its open-chain carboxylate form.21 Thus, we did not
wish to release the terminal carboxylic acid until after
the ketone was appropriately protected. Since protec-
tion of this highly electron-de®cient ketone as a ketal
was not expected to be facile,22,23 we reduced 7a to the
alcohol 8a and protected this as its acetate. Ruthenium
catalyzed cleavage of the ole®n24 gave the expected
carboxylic acid 9a. Coupling of the carboxylate with
N-methyl-tert-leucine and deacetylation gave the penul-
timate alcohol 10a, oxidation of which with Dess±Martin
reagent25 yielded the desired a,a-di¯uoroketone 4.
NMR spectroscopy was used to demonstrate that the
target compounds hydrated in the presence of water and
did not exist as hydroxy lactams.29 Thus, the 19F NMR
of 7a in dry DMSO-d6 shows a singlet at 105.1 ppm
(Table 1). Dilution with D2O results in the appearance
of another signal in the 19F NMR spectrum at 108.8
ppm. This shift is consistent with that expected for
hydration of a ¯uoroketone.30,31 The formation of the
1
hydrate was further con®rmed through the 13C and H
NMR spectra. In dry DMSO the carbonyl carbon C(2)
was observed at 198.6 ppm, the ¯uorine substituted
carbon C(1) at 115.4 ppm, and the protons H(3) on the
carbon adjacent to the ketone at 2.80 ppm. In the pre-
sence of D2O additional signals appeared at 94.1 ppm
for C(2), at 120.7 ppm for C(1), and at 1.52 ppm for the
methylene group, all of which are consistent with hydra-
tion of the ketone. The percent hydration observed after
1
24 h was determined by integration of the 19F and H
NMR signals and was on the order of 27%. The NMR
spectra of 4 under the same conditions revealed corre-
sponding shifts in the signals for the ¯uorine, carbon and
hydrogen atoms. Hydration after 24 h was determined to
be 42%. That similar shifts were observed for 7a and 4
indicates that hydroxy lactam formation by cyclization
of the secondary amide of 4 onto the ketone moiety was
not occurring to any signi®cant degree. This point was
reinforced by the 19F NMR of the tertiary amide 1132
(see Table 2 for structure) which, like 7a, is incapable of
forming a cyclic structure and which shows a comparable
shift between the 19F resonances for the ketone and the
In order to prepare 2, a Grignard reagent corresponding
to 3-butenylmagnesium bromide but containing a 2-
phenethyl side chain was required. This was prepared in
two steps: a SN20 reaction of 1,4-dibromo-2-butene with
phenethylmagnesium chloride26 in the presence of
CuCNꢁ 2LiCl27 followed by Grignard formation using
Rieke magnesium.28 This Grignard reagent and 6 yielded
the expected ketone 7b. As before, reduction gave the
alcohol 8b which was acetylated, oxidized, coupled to
N-methyl-tert-leucine and deacetylated yielding alcohol
10b. Oxidation gave the target compound 2.
1
hydrate. Since 2 was a mixture of diastereomers the H
and 13C NMR spectra were too complex to be inter-
pretable at the same level of detail; however, the 19F
NMR showed parallel shifts of the diastereotopic ¯uorine
atoms for both diastereomers. While the percent hydration
of 2 was only 4% after 24 h,33 in a higher concentration
Scheme 1. Synthesis of di¯uoroketones 2 and 4. Reaction conditions. (i) Neat, rt, 18 h; (ii) 10% aq ethanol, rt 18 h; (iii) cat. DMF, CH2Cl2, rt, 2 h;
(iv) DIEA, CH2Cl2, rt, 18 h; (v) THF, 0ꢂC to rt, 18 h; (vi) ethanol, rt, 1 h; (vii) pyridine, rt, 60 h; (viii) CCl4/CH3CN/H2O, rt, 2 h; (ix) BOP, CH2Cl2, rt, 18 h;
(x) MeOH, rt, 2 h; (xi) CH2Cl2, rt, 2 h. All compounds were characterized by 1H and 19F NMR and MS and purity assesed by reverse-phase HPLC.

L. A. Reiter et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1581±1584
Table 1. Selected NMR data for di¯uoroketones 2, 4, 7a, and 11
1583
Compound
Solventa
19Fb,c
13C(1)c
13C(2)c
1H(3)c
7a
Dry DMSO
105.1
s
108.8
(28%)d
103.8 s
115.4 t
(J=254)
120.7 t
(J=252)
115.3 t
(J=252)
120.5 t
(J=253)
Ð
198.6 t
(J=30.3)
94.1 t
(J=30.3)
198.7 t
(J=31.8)
93.7 t
2.80
m
1.52
(26%)b
2.89 m
Wet DMSO
Dry DMSO
Wet DMSO
Dry DMSO
Wet DMSO
Dry DMSO
Wet DMSO
4
108.8
(42%)d
103.6/ 104.3
2d (J=251)
108.7/ 109.0
(23%)d
103.5/ 104.4
2d (J=252)
108.90/ 108.93
(4%)d
1.69
(39%)b
Ð
(J=29.2)
Ð
11
2
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
103.9 s
108.8 s
aDry DMSO: sample dissolved in 1.0 mL DMSO-d6 (from an ampoule) in an oven dried NMR tube; wet DMSO: above sample diluted with 0.10 mL
D2O.
bppm relative to CFCl3.
cs, singlet, d, doublet, t, triplet, m, multiplet.
dPercentage of hydrate present 24 h after dilution of the sample.
of water (0.20 mL D2O in 1.0 mL DMSO-d6) and after
48 h, 14% of the material existed in its hydrated form.
Table 2. MMP-13 Activity of di¯uoroketones and intermediates
Evaluation of the target ketones 2 and 4 as well as some
of the intermediates revealed them to be, in general,
weak inhibitors of MMP-13 (Table 2).34 The key target 2
has an IC50 of 5 mM, more than 100-fold higher than
phosphinate 1. Despite this relatively weak inhibition,
the di¯uoroketone moiety does appear to be critical to
the observed inhibition. Thus, 10b, the alcohol precursor
of 2, has an IC50 greater than 30 mM. Even more telling
in this regard is the comparison of 7b and 8b. Ketone 7b
is the most potent compound in the series having a sub-
mM IC50 while the corresponding alcohol is nearly 100-
fold less active. Although the hydrated di¯uoroketone
moiety may be acting as a zinc ligand as desired, a
comparison of the di€erences in activity between 2, 4,
and 11 suggests that the compounds are not binding to
the enzyme in the expected substrate-like manner. In
other series0of substrate-like MMP inhibitors, removing
the key P1 side chain leads to a large reduction in
binding anity.1 This appears to be re¯ected in the dif-
ference between 7a and 7b; however, in the case of the
Compound
X
R
Z
MMP-13 IC50
(mM) (n)
7a
4
7b
8b
10b
2
ˆO
ˆO
ˆO
HÐ
HÐ
PhCH2CH2
-CHˆCH2
-COTlea
-CHˆCH2
-CHˆCH2
-COTlea
-COTlea
>>3 0
12.0+4.2 (2)
0.29+0.1 (2)
27
-OH,-H PhCH2CH2
-OH,-H PhCH2CH2
>3 0
ˆO
ˆO
ˆO
PhCH2CH2
PhCH2CH2 -CON(CH2)5
PhCH2CH2 -H
5.1+3.8 (3)
6.1+1.5 (2)
>30
11
12
aCOTle:-N-carbonyl-N0-methyl-tert-leucine amide.
be small enough to escape a negative steric interaction
with the enzyme upon binding. In contrast, the larger
amide functionalities of 2 and 11 may drive these mole-
cules into a favorable overall conformation but be too
large to allow as ecient binding to the enzyme. Ketone
12, which lacks a substituent b to the ketone, may simply
not adopt a conformation favorable for binding.
0
amide-containing analogues 2 and 4, removal of the P1
side chain leads to only a ꢀ2-fold decrease in activity.
0
0
Truncation of the P2 ±P3 region also typically leads to
substantial potency reduction.1 Such a potency change
is not observed in the present case as 2 and 11 are
essentially equi-active. The binding di€erences observed
for 2, 7b, 11 and 1235 may derive from an interplay
between the conformational and steric e€ects conferred by
the substituent b to the ketone. Thus, the vinyl group of 7b
may drive the molecule into a favorable conformation and
The apparent failure of 2 and the other di¯uoroketones
to bind in the expected manner could be due to a number
of factors. For example, examination of 2 `bound' into a
model of MMP-13 in a substrate-like orientation reveals
a potential negative dipole interaction between one of
the ¯uorine atoms and the carbonyl of Ala-186, which
are closer than a typical O-F van der Waals contact.36
Another relevant factor may be the bond angle for the

1584
L. A. Reiter et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1581±1584
C±CF2±C group in 2 which is likely to be more obtuse37
as compared to the corresponding C±CH2±C of 1, a
change which will substantially alter the position of the
terminal P2 phenyl ring.
16. Schirlin, D.; Baltzer, S.; Altenburger, J. M.; Tarnus, C.;
Remy, J. M. Tetrahedron 1996, 52, 305.
17. Damon, D. B.; Hoover, D. J. J. Am. Chem. Soc. 1990,
112, 6439.
18. Hong, W.; Dong, L.; Cai, Z.; Titmas, R. Tetrahedron Lett.
1992, 33, 741.
In conclusion, we have described the synthesis of a series
of di¯uoroketones that were designed to be inhibitors
of MMP-13. While the key target 2 was only a weak
inhibitor of the enzyme, our hypothesis regarding the
use of di¯uoroketones as inhibitors of MMP's is validated
by the activity observed, especially the sub-mM activity
of 7b. Although the binding orientation of the active
compounds does not appear to be as predicted, our
results suggest that the potency may be improved by
varying both of the groups b to the ketone. This and an
assessment of any stereochemical bias imparted by these
groups may lead to a better understanding of the binding
mode of these compounds.
19. Cavallini, G.; Massarani, E.; Nardi, D.; Mauri, L.; Bar-
zaghi, F.; Mantegazza, P. J. Am. Chem. Soc. 1959, 81, 2564.
20. Middleton, W. J.; Bingham, E. M. J. Org. Chem. 1980, 45,
2883.
21. Contrasting with our experience, Gelb (ref 6) reported
successful carbodiimide-induced couplings of a ¯uorinated g-
ketoacid with amines. Others have reported related ¯uorinated
g-ketoacids which exist to some degree in cyclic forms, but
have not reported subsequent reactions on the terminal car-
boxylic acid (refs 18 and 39).
22. Guthrie, J. P. Can. J. Chem. 1975, 53, 898.
23. Deprotecting an electron de®cient ketal was also not
expected to be readily accomplished (ref 40). While a-¯uori-
nated ketones have been protected as semicarbazides or
hydrazones (e.g. ref 41 and 42), we did not investigate these
since the formation of cyclic intermediates remains possible.
24. Carlsen, P. H. J.; Katsuki, T.; Martin, V. S.; Sharpless, K.
B. J. Org. Chem. 1981, 46, 3936.
Acknowledgements
We thank Drs. Kim F. McClure and Julian Blagg for
helpful discussions during the preparation of this manu-
script and Ethan J. Stam for technical assistance.
25. Lindermann, R. J.; Graves, D. M. J. Org. Chem. 1989, 54,
661.
26. Mesnard, D.; Miginiac, L. C. R. Acad. Sci., Ser. C. 1973,
277, 567.
27. Yanagisawa, A.; Noritake, Y.; Nomura, N.; Yamamoto,
H. Synlett 1991, 251. Yanagisawa, A.; Nomura, N.; Yama-
moto, H. Tetrahedron 1994, 50, 6017.
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lier work (ref 11). Since Km>>[S], IC50's ꢃ Ki's. Pre-incuba-
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of substrate did not lead to a signi®cant change in its IC50
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