The novel benzopyran class of selective cyclooxygenase-2 inhibitors. Part 2: The second clinical candidate having a shorter and favorable human half-life

Article abstract of DOI:10.1016/j.bmcl.2010.07.054

In this Letter, we provide the structure-activity relationships, optimization of design, testing criteria, and human half-life data for a series of selective COX-2 inhibitors. During the course of our structure-based drug design efforts, we discovered two

Full text of DOI:10.1016/j.bmcl.2010.07.054

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7159–7163
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The novel benzopyran class of selective cyclooxygenase-2 inhibitors. Part 2: The
second clinical candidate having a shorter and favorable human half-life
*
Jane L. Wang , David Limburg, Matthew J. Graneto, John Springer, Joseph Rogier Bruce Hamper, Subo Liao,
*
Jennifer L. Pawlitz, Ravi G. Kurumbail, Timothy Maziasz, John J. Talley, James R. Kiefer , Jeffery Carter
Pfizer Global Research and Development, 700 Chesterfield PKWY West, Chesterfield, MO 63017, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
In this Letter, we provide the structure–activity relationships, optimization of design, testing criteria, and
human half-life data for a series of selective COX-2 inhibitors. During the course of our structure-based
drug design efforts, we discovered two distinct binding modes within the COX-2 active site for differently
substituted members of this class. The challenge of a undesirably long human half-life for the first clin-
ical candidate 1 t1/2 = 360 h was addressed by multiple strategies, leading to the discovery of 29b-(S)
(SC-75416) with t1/2 = 34 h.
Revised 12 July 2010
Accepted 14 July 2010
Available online 24 July 2010
Keywords:
Cyclooxygenase
COX-2 inhibitor
Clinical candidate
Benzopyran
Ó 2010 Elsevier Ltd. All rights reserved.
Half-life
Plasma protein bound
Microsomal
X-ray crystal structure
In part 1 of this investigation we described substituted 2-tri-
fluoromethyl-2H-benzopyran-3-carboxylic acids as a novel series
of potent and selective cyclooxygenase-2 (COX-2) inhibitors. We
discussed the discovery of the first clinical candidate 1 (SD-8381)
(Fig. 1) which had excellent potency and efficacy as an analgesic
and anti-inflammatory agent.1a
half-life of 1. We also incorporated polar groups to lower log P
and decrease plasma protein binding as a means of shortening
the in vivo half-life.
Despite its favorable half-life in rats (t1/2 = 10.1 h), dogs
(t1/2 = 20.4 h), and cyno (t1/2 = 14.2 h),1b its half-life in humans
was strikingly higher (t1/2 = 360 h). The route of clearance was
not consistent across species. The primary clearance pathway in
rat and dog was as parent (feces); whereas, in the cyno the primary
clearance was as the acyl-glucuronide phase-2 metabolite (urine).
In vitro human microsomal metabolism study of analog 1 (100%
remaining) showed no evidence of phase-1 metabolism. We rea-
soned that incorporation of a new clearance pathway, common
across multiple species, might provide a higher probability of iden-
tifying a compound with a more predictable human half-life.
Commonly, leads having too short a half life are re-engineered by
the iterative identification and blocking of metabolic sites with
oxidatively stable moieties. We reasoned that incorporation of
metabolically labile moieties might provide a path to shorten the
Figure 1. SD-8381, a lead COX-2 inhibitor. (see Part 1 of this Letter). (a) See Ref. 2
section 2.4 and note 3. (b) See Ref. 2 section 2.9. (c) and (d) See Ref. 2a–c section
2.10. (e) See Ref. 2a–c Section 2.11.
* Corresponding authors. Tel.: +1 636 527 5037; fax: +1 636 247 2086 (J.L.W.);
tel.: +1 636 207 1048 (J.R.K.).
E-mail addresses: jl47wang@yahoo.com (J.L. Wang), james.kiefer@att.net (J.R.
Figure 2. Compound 2, an analog of COX-2 inhibitor. (see Part 1 of this Letter). (a)
Kiefer).
See Ref. 2c Section 2.4 and note 3.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.054

7160
J. L. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7159–7163
Scheme 1. Reagents and conditions: (a) K2CO3, DMF, 80–100 °C; (b) Et3N, DMSO; (c) (I) HOAc, Cl2, (II) Zn dust; (d) NaOH, THF, CH3OH, H2O.
Scheme 2. Reagents and conditions: (a) 3, K2CO3, DMF, 80–100 °C; (b) NaN3, DMSO, H2O, 85 °C, 15 h; (c) SnCl2–2H2O, MeOH; (d) 47% aq HI, KNO2, DMSO, 35 °C; (e) tetrakis
triphenylphosphine–palladium(0), K2CO3, B(Et)3, DMF, 110 °C, 5 h; (f) LiOH–H2O, THF, H2O, EtOH, 80 °C, 4 h; (g) 2-methylsulfonylethanol, NaH, DMF, 0 °C; (h) CH3CH2Br,
Cs2CO3, DMF; (i) HOPh or HSPh, K2CO3, DMF, 110 °C; (j) tetrakistriphenyl phosphine–palladium(0), RB(OH)2, DMA, 2.0 M aq Na2CO3, 95 °C, 16 h.
Table 1
SAR of 5-substituted benzopyran analogs
Table 2
SAR of 6,8-diCl-7-alkoxy analogs 23a–e
a
Compd
R
Mod human IC50
(lM)
c log P
COX-1
COX-2
a
7
12
13
15
17
18
19
20
Me
Et
7.44
66.7
>100
>100
39.8
48.9
68.4
0.37
0.12
28.8
1.48
>100
0.53
0.34
4.64
1.98
4.37
4.90
2.89
4.19
5.74
6.24
5.51
6.01
Compd
R
Mod human IC50
(
lM)
c log P
COX-1
COX-2
NH2
EtO
PhO
PhS
Ph
23a
23b
23c
23d
23e
–CH2c-pentyl
–CH2CH(CH2CH3)2
–C(CH3)3
–CH2CH(CH3)2
–CH2 c-hexyl
<0.14
<0.14
<0.14
0.002
3.79
<0.006
0.021
0.028
0.028
0.034
6.26
6.68
5.41
5.63
6.82
MePh
IC50 curves were generated with each test concentration run in duplicate, each
IC50 curves were generated with each test concentration run in duplicate, each
curve was done n P2. The high concentration was 500
lM.
curve was done n P2. The high concentration was 500
lM.
a
a
See Ref. 2c Section 2.4 and note 3.
See Ref. 2c Section 2.4 and note 3.

J. L. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7159–7163
7161
Scheme 3. Reagents and conditions: (a) 3, DMSO, Et3 N, heat; (b) polymer–PPh3, DEAD, THF; (c) Cl2, HOAc; (d) NaOH, EtOH, THF, H2O.
5-Amino derivative, 10, was prepared from 8 by conversion to 9,
followed by treatment with azide followed by reduction with
SnCl2. 5-I chromene 11 was obtained by treating 10 with KNO2
and HI. Palladium mediated coupling of 11 with triethylborane
and hydrolysis provided 12. 5-Amino acid 13 was obtained by
hydrolysis of ester 10. Treatment of 9 with 2-methylsulfonyl etha-
nol and sodium hydride produced phenol 14. Alkylation of 14 with
ethyl bromide followed by ester hydrolysis provided acid 15. Com-
pounds 16a and 16b were obtained by nucleophilic displacement
of 5-F chromene 9 with phenol, thiophenol. Analogs 16c and 16d
were prepared by Suzuki coupling of 11 with the appropriate boro-
nic acids. The esters (16a–d) were hydrolyzed to afford acids 17–
20. Table 1 showed while the 5,6-diCl acid 2 exhibited optimal po-
tency, all other groups at the 5-position (7, 12, 13, 15, 17–20) had
greatly decreased potency of hCOX-2.
Initial SAR exploration of the 5-position did not lead to a suffi-
ciently potent COX-2 inhibitor to warrant further consideration.
Based on the desirable characteristics of 1 (SD-8381), we intro-
duced polar moieties or metabolizable groups at the 7-position of
1 to examine their effect on activity. Compounds 23a–e were pre-
pared from the salicylaldehyde 21 to form the chromene ester, fol-
lowed by a Mitsunobu reaction to provide 7-alkoxy esters 22a–e,
which upon chlorination and hydrolysis provided 7-alkoxy acids
23a–e (Scheme 3). Table 2 shows that these analogs were very po-
tent COX-2 inhibitors, but not as selective as compound 1. We also
observed a departure from the basic SAR trends previously seen in
the benzopyran series when lengthy 7-substitutions were made
(data not shown). To explore this divergence in SAR, we deter-
mined the crystal structure of bound (R,S) 23d at 2.2 Å resolution
(see Supplementary data for methods and data statistics). Only
the (R-isomer) was bound in the active site. It was observed that
23d-(R) had inverted and that the binding mode of the compound
was rotated 180° (Fig. 3a and Note 5). This result was further con-
firmed by obtaining the crystal structure of the resolved 23d-(R)
and failing to obtain crystals with 23d-(S). In this new binding
mode, the R-isomer bound much like typical NSAIDs and had
formed a 2.5 Å hydrogen bond to Tyr355 (numbering based on
ovine COX-1) and a 3.0 Å ion pair with Arg120.6 The trifluoro-
methyl group formed van der Waals interactions with Leu351
and Phe367, similar to celecoxib,1a though the connection angle
to the core differs. The 7-alkoxy tail extended into the top of the
active site, sweeping out approximately the same space as the tri-
fluoromethyl group of the S-isomer binding orientation and of the
4-methyl aryl ring of celecoxib. In this orientation, the 5-position
rather than the 8-position of the compound pointed towards the
side pocket of the active site. By contrast, the 8-position, which
could tolerate large substituents in the S-enantiomer binding mode
was flanked by a hydrophobic patch of residues including Val345
and Leu531 with limited space for elaboration. Since the binding
mode and stereochemical preference changed with the addition
of long 7-alkoxy substitutions, an entirely new SAR pattern gov-
erns the binding of R-enantiomers in this sub-series. Intriguingly,
COX-2 selectivity was retained in the R-isomer binding mode in
spite of changing every protein–inhibitor interaction and the anec-
dotal observation that NSAIDs formed ion pairs to Arg120 tend to
lack COX isoform selectivity.
Figure 3. Crystal structures of inhibitors (a) 23d-(R) and (b) 29b-(S) bound at the
COX-2 active site [composed with PyMol]. Note that the orientation of the
compounds differs by approximately 180° and that the chirality of the stereo
center inverts between the two structures.
As discussed in part 1 of this Letter, 6-substitution was required
for potency. Based on the biological data of compound 2 (Fig. 2)
(analog 5a in part 1 of this Letter), we modified the 5-position of
the benzopyran by introducing alkyl and aryl groups to further
optimize the activity while maintaining the chloro at the 6-posi-
tion (Scheme 2). The 5-Me-6-Cl acid 7 was prepared via a three
step-reaction;4 first formation of the 5-Me chromene 5, followed
by chlorination (6) and then hydrolysis as described in Scheme 1.

7162
J. L. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7159–7163
Scheme 4. Reagents and conditions: (a) K2CO3, Et3N, DMF, 60–90 °C, 84% yield; (b) Cl2 (g), AcOH, Zn dust, 80% yield; (c) NBS, (BzO)2, CCl4, reflux, 85% yield; (d) HNR1R2, K2CO3,
DMF, 0 °C; (e) NaOH, THF, CH3OH, H2O, two steps 18–57% yield.
Table 3
Table 4
Analogs with polar groups at the 7-position decreased hCOX-2 inhibition
Analogs with metabolized groups at the 7-position displayed hCOX-2 inhibition
R1/R2
Mod human IC50
(lM)
c log P
a
Compd
COX-1
COX-2
26a
26b
26c
26d
26e
–N(CH3)(CH2CH3)
–N(CH3)[CH(CH3)2]
2,5-diMe pyrrolidine
–N[CH(CH3)2]2
100
100
100
100
100
21.3
100
96
100
62.2
1.96
2.27
3.11
3.11
3.66
2,6-diMe piperidine
a
Compd
R
IC50 Mod.
h COX-2
Micros.
(%) rem.
human/rat
Air pouch
(10 mpk)b
(%)
IC50 curves were generated with each test concentration run in duplicate, each
curve was done n P2. The high concentration was 500
(lM)
l
M.
a
See Ref. 2c Section 2.4 and note 3.
29a
29b
29c
29d
29e
32a
32b
33
–CH(CH3)2
–C(CH3)3
–CH2CH2CH3
–CH2CH(CH3)2
–(CH2)2C(CH3)2
–SCH2CH(CH3)2
–N(CH3)CH2CH(CH3)2
–OCH2CH(CH2CH3)2
0.30
0.005
0.15
0.020
0.094
0.019
0.070
0.046
n.d.
51/n.d.
n.d.
100
49
62
51
30
52
42
0.71/0.26
0.49/0.44
0.86/0.86
0.75/0.38
0.54/0.51
0.76/0.73
Keeping the key 6-chloro substituent and removing the 8-
chloro to reduce the c log P, we explored the 7-position extensively
by both parallel and conventional chemical synthesis. First, we ex-
plored the 7-position by introduction of polar moieties such as
methyl amino groups to lower the c log P ranging from 2 to 3.7.
Methyl salicylaldehyde 24 was converted to the chromene and
then chlorinated to form the ester 25 via similar steps described
at Scheme 1. After bromination of 7-methyl 25, we replaced the
bromo with various amines, then hydrolyzed the ester to provide
acids 26a–e (Scheme 4). Unfortunately, none of these amine ana-
logs maintained COX inhibition (Table 3).
We focused on analogs without basic moieties, but having rea-
sonable lipophilicity and bearing the metabolizable groups to
reach our goal in lowering half-life. Compounds were designed
and prepared according to Scheme 5. Starting with phenol 27,
the salicylaldehyde was formed, which was then converted to
the chromene ester 28.4 Acids 29a–e were obtained first via
9-BBN coupling with 7-iodo chromene 28, followed by chlorination
and hydrolysis. Analogs 32a–b were prepared by nucleophilic dis-
placement of 7-fluoro chromene 30 with nucleophilic thiol or
amine to form the 7 substituted analogs 31a–b, followed by hydro-
lysis as described in Scheme 6. Alkoxy analog 33 was prepared
IC50 curves were generated with each test concentration run in duplicate, each
curve was done n P2. The high concentration was 500
lM.
a
See Ref. 2c Section 2.4 and note 3.
See Refs. 2a–c Section 2.9.
b
according to Scheme 3 except less chlorine gas was used compared
when preparing di-chloro analogs. The SAR in Table 4 revealed that
compound 29a, with a smaller group at the 7-position, was a less
potent COX-2 inhibitor. However, analogs 29b–e, 32a–b, and 33,
with bulky or longer groups at the 7-position, displayed potent
COX-2 inhibition.
We determined the 2.8 Å resolution crystal structure of the
most potent enantiomer of 29b and observed the S-enantiomer
bound at the COX-2 active site. No co-crystals were obtained from
the opposite enantiomer. The orientation of 29b-(S) matches that
of the previously reported S-isomer structure (1)1a and preserves
many of the same contacts between the protein and inhibitor.
Scheme 5. Reagents and conditions: (a) MgCl2, ACN, p-formaldehyde, TEA, 10–72 °C, 2 h, 79% yield; (b) K2CO3, DMF, 80–100 °C, 71% yield; (c) R-9BBN, THF, Pd(dppf)ClÁCH2Cl2,
K3PO4(aq), 60 °C, 4 h, 56–76% yield; (d)HOAc, Cl2, Zn dust, 100% yield; (e) NaOH, THF, CH3OH, H2O, 91% yield.
Scheme 6. Reagents: (a) HSR or HNR1R2, K2CO3, DMF, 80–100 °C, 48 h, 80–90% yield; (b) NaOH, THF, CH3OH, H2O, 90% yield.

J. L. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7159–7163
7163
Table 5
and
inhibition
played excellent in vitro potency, selectivity and efficacy in the
air pouch (100% inhibition at 2 mpk). The PK profile of 29b-(S)
met all of our design criteria and 29b-(S) was advanced to further
in vivo studies. Both acute and chronic in vivo studies demon-
strated that 29b-(S) was superior to NSAIDs and other COX-2 selec-
tive inhibitors (Table 6).2c
S
R enantiomers of 7-substituted chromenes displayed different hCOX-2
In summary, we have discovered a new series of potent, orally
bioavailable, selective COX-2 inhibitors structurally very distinct
from the diaryl heterocycle class. In vivo they are among the most
potent and anti-inflammatory and analgesic COX-2 inhibitors yet
described. Additionally, compounds from these series possess high
water solubility as their corresponding carboxylate salts providing
the possibility of alternative formulation and dosing strategies. We
successfully applied a filter requiring phase-1 metabolism as a cor-
nerstone in our selection criteria, which resulted in the discovery
29b-(S) (SC-75416). 29b-(S) exhibited a human half-life of 34 h,
appropriate for once-a-day dosing and demonstrated very analge-
isc efficacy in a clinical phase II trial of post surgical dental pain.2c
In part 2 of this series, we described application of phase-1 metab-
olism as a cornerstone in our selection criteria. Part 3 of this series
will detail our strategy for compound selection using a microdos-
ing clinical protocol.
Compd
R
S-isomer
R-isomer
a
a
Mod h IC50
(l
M) Mod h IC50
(
lM)
COX-1 COX-2
COX-1
COX-2
29b
29c
29d
29e
32a
32b
–C(CH3)3
1.02 0.062
1.95 0.14
136
5.15
–CH2CH2CH3
–CH2CH(CH3)2
–(CH2)2C(CH3)2
–SCH2CH(CH3)2
0.44 0.005
0.35 0.007
1.63 0.068
3.66 0.008
100
41.6
22.6
0.91
1.13
0.15
0.87
–N(CH3)CH2CH(CH3)2 100
15.8
0.085
IC50 curves were generated with each test concentration run in duplicate, each
curve was done n P2. The high concentration was 500
lM.
a
See Ref. 2c Section 2.4 and note 3.
Acknowledgments
Table 6
Pharmacology of 29b-(S) (SC-75416)
The authors thank J. Gierse, C. M. Kobldt, Y. Zhang, B. S. Zweifel
for providing in vitro and in vivo data, J. Collins, P. Kleine, A. Libby,
K. Palmquist for scale up and chiral purification, M. Baratta, R.
Ridgewell, A. Breau for in vitro metabolism and PK study, J. L.
Pierce for protein expression, J. K. Gierse for protein purification,
T. A. Stults and H. E. Narepekha for crystallization, and W. C. Stal-
lings for helpful discussions.
AirPouch
ED50
Edema
ED50
Hyperalgesia
ED50
Arthritis
ED50
hCOX-2
IC50
a
b
c
d
e
Supplementary data
0.43 mg/kg
2.7 mg/kg
4 mg/kg
0.08 mg/kg
0.0625 lM
a
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.07.054.
See Ref. 2c Section 2.9.
See Ref. 2c Section 2.10.
See Ref. 2c Section 2.11.
b,c
d
e
See Ref. 2c Section 2.4 and note 3.
References and notes
1. (a) Wang, J. L.; Carter, J.; Kiefer, J. R.; Kurumbail, R. G.; Pawlitz, J. L.; Brown, D.;
Hartmann, S. J.; Graneto, M. J.; Seibert, K. S.; Talley, J. J. Bioorg. Med. Chem. Lett.,
submitted for publication; (b) The Pfizer Institutional Animal Care and Use
Committee reviewed and approved the animal use in these studies. The animal
care and use program is fully accredited by the Association for Assessment and
Accreditation of Laboratory Animal Care, International.
2. (a) Zhang, Y.; Shaffer, A.; Portanova, J.; Seibert, K.; Isakson, P. C. J. Pharmacol. Exp.
Ther. 1997, 283, 1069–1075; (b) Portanova, J. P.; Zhang, Y.; Anderson, G. D.;
Hauser, S. D.; Masferrer, J. L.; Seibert, K.; Gregory, S. A.; Isakson, P. C. J. Exp. Med.
1996, 184, 883–891; (c) Gierse, J.; Nickols, M.; Leahy, K.; Warner, J.; Zhang, Y.;
Cortes-Burgos, L.; Carter, J.; Seibert, K.; Masferrer, J. Eur. J. Pharmacol. 2008, 588,
93.
3. The modified enzyme assays (Ref. 4) was created to identify inhibitors that
exhibit slow ‘off-rate’ kinetics due to tight binding to their target enzyme, the
traditional enzyme assay (Ref. 2c) may not be optimal for characterization of
compounds from alternative classes that exhibit different kinetics for inhibition.
Further, such assays may provide substantially different IC50 values with subtle
changes in assay protocol.
4. Aston, K. W.; Brown, D. L.; Carter, J. S.; Deprow, A. M.; Fletcher, T. R.; Hallinan, E.
A.; Hamper, B. C.; Huff, R. M.; Kiefer, J. R., Jr.; Koszyk, F.; Kramer, S. W.; Liao, S.;
Limburg, D.; Springer, J. R.; Tsymbalov, S.; Wang, L. J.; Xing, L.; Yu, Y. WO
04087687 A1, 2004.
5. X-ray structure coordinates for all complexes have been deposited into the
Protein Data Bank with accession codes 3NTG (23d-(R)) and 3MQE (29b-(S)).
Data for the crystal structures described herein were collected at IMCA-CAT (17-
ID) beamline at the Advanced Photon Source and was supported by the
companies of the Industrial Macromolecular Crystallography Association
through a contract with the Center for Advanced Radiation Sources at the
University of Chicago.
6. (a) Picot, D.; Loll, P. J.; Garavito, R. M. Nature 1994, 367, 243; (b) Kurumbail, R. G.;
Stevens, A. M.; Gierse, J. K.; McDonald, J. J.; Stegeman, R. A.; Pak, J. Y.; Gildehaus,
D.; Miyashiro, J. M.; Penning, T. D.; Seibert, K.; Isakson, P. C.; Stallings, W. C.
Nature 1996, 384, 644.
However, to accommodate the bulky 7-t-butyl substituent, the en-
tire membrane binding helix cluster moved ꢀ0.7 Å away from the
active site, and the side chain of Tyr355 moved 1.6 Å. The detailed
interaction of the COX enzymes with the membrane are not
known, but it is interesting that such a large concerted motion is
required for binding of this compound and that its in vitro and
in vivo potency appear unimpaired.
Based on these data in Table 4, we evaluated the 6-Cl-7-subiti-
tuted analogs in an in vitro human liver microsomal assay. Com-
pounds containing alkyl, alkoxy, and alkyl-amino moieties
showed greater than 15% phase-1 oxidative or reductive metabo-
lism in vitro. A group of compounds displaying good in vivo po-
tency in the rat air pouch model (>50% inhibition of PGE2) were
resolved by chiral chromatography (Table 5).
The benzopyran series described here represents two distinct
chemical series with respect to the COX-2 binding mode, preferred
chirality, and resulting SAR pattern. Potent and selective COX-2
inhibitors can be made from both the S- and R-isomers, depending
on the length of the 7-substituent. When the analogs contain short
(up to two non-hydrogen atoms) groups at the 7-position, the S-
isomer 29b-(S) dominates. Analogs (29c–e and 32a–b) with a long-
er chain at the 7-position (three or more non-hydrogen atoms)
bind preferentially as the R-isomer. Retention of affinity and selec-
tivity while completely altering the binding mode and chirality is
both novel and unexpected. Compounds 29b-(S) (SC-75416) dis-