The novel benzopyran class of selective cyclooxygenase-2 inhibitors. Part III: The three microdose candidates

Article abstract of DOI:10.1016/j.bmcl.2010.07.059

In this manuscript, we report the discovery of the substituted 2-trifluoromethyl-2H-benzopyran-3-carboxylic acids as a novel series of potent and selective cyclooxygenase-2 (COX-2) inhibitors. We provide the structure-activity relationships, optimization of design, testing criteria, and human half-life data. The challenge of a surprisingly long half-life (t _1/2 = 360 h) of the first clinical candidate 1 and human t _1/2 had been difficult to predict based on allometric scaling for this class of highly ppb compounds. We used a microdose strategy which led to the discovery of clinical agents 18c-(S), 29b-(S), and 34b-(S) with human half-life of 57, 13, and 11 h.

Full text of DOI:10.1016/j.bmcl.2010.07.059

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7164–7168
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 III: The
three microdose candidates
*
Jane L. Wang , Karl Aston, David Limburg, Cindy Ludwig, Ann E. Hallinan, Francis Koszyk, Bruce Hamper,
David Brown, Matthew Graneto, John Talley, Timothy Maziasz, Jaime Masferrer, Jeffery Carter
Pfizer Global Research and Development, 700 Chesterfield PKWY West, Chesterfield, MO 63017, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
In this manuscript, we report the discovery of the substituted 2-trifluoromethyl-2H-benzopyran-3-car-
boxylic acids as a novel series of potent and selective cyclooxygenase-2 (COX-2) inhibitors. We provide
the structure–activity relationships, optimization of design, testing criteria, and human half-life data. The
challenge of a surprisingly long half-life (t1/2 = 360 h) of the first clinical candidate 1 and human t1/2 had
been difficult to predict based on allometric scaling for this class of highly ppb compounds. We used a
microdose strategy which led to the discovery of clinical agents 18c-(S), 29b-(S), and 34b-(S) with human
half-life of 57, 13, and 11 h.
Received 14 January 2010
Revised 12 July 2010
Accepted 14 July 2010
Available online 23 July 2010
Keywords:
Cyclooxygenase
Clinical candidate
Benzopyran
Ó 2010 Elsevier Ltd. All rights reserved.
Half-life
Plasma protein bound
Microdose
Microsomal
Metabolism
In Part I of this Letter we described the discovery of the substi-
tuted 2-trifluoromethyl-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)1 that had excellent potency and efficacy as an analge-
sic and anti-inflammatory agent.1 In Part II of this Letter, we de-
scribed the incorporation of metabolically labile moieties to the
first clinical candidate 1 to provide a path to shorten its half-life,
which led to discovery of second clinical candidate 2 (SC-75416)
(Fig. 1). Compound 2 (SC-75416) also had excellent potency and
efficacy as an analgesic and anti-inflammatory agent. It displayed
a much shorter human half-life (t1/2 = 34 h) compared to com-
pound 1 (t1/2 = 360 h), a half-life was appropriate for once-a-day
dosing.
During our ongoing chemistry program, our goal was to identify
potent and selective COX-2 inhibitors as backup candidates of
compound 2 (SC-75416) with good PK and metabolism. In both
Parts I and II of these Letters, we reported the results of exploration
of the 5-position and the 7-position of the benzopyran while keep-
ing the 6-choro substituent constant. We also described the explo-
ration of the 8-position of the benzopyran that extended into and
made contacts within the side-pocket binding region based on
Figure 1. 1 (SD-8381), a lead COX-2 inhibitor (see Part I of this Letter) and 2 (SC-
75416), a lead COX-2 inhibitor (see Part II of this Letter). (a) See Ref. 2c sec. 2.4 and
Ref. 3. (b) See Ref. 2c sec. 2.9. (c and d) See Ref. 2a–c sec. 2.10. (e) See Ref. 2a–c sec.
2.11.
celecoxib and compound 1 (SD-8381)’s X-ray structures. These
early modifications of substituents about the pyran nucleus
showed that the 2-trifluoromethyl, 3-carboxy, and 4-H substitutes
were key elements of the pharmacophore, and these compounds
* Corresponding author. Tel.: +1 636 527 5037; fax: +1 636 247 2086.
E-mail address: jl47wang@yahoo.com (J.L. Wang).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.059

J. L. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7164–7168
7165
exhibited very considerable in vivo potency. In addition, we dem-
onstrated that incorporation of metabolically labile moieties in this
series provided a method for reducing the half-life, a strategy we
continued to apply to achieve our goal in this work.
In Part I of this Letter, we described that the substituent at the
6-position was very important for COX potency both in vitro and
in vivo (air pouch model), so we maintained the 6-chloro as a cor-
nerstone in our initial SAR. We designed several compounds con-
taining alkyl, alkoxy, acetylene, and cyano, moieties to explore
the role of these substituents in COX-2 blockade. As described in
Scheme 1, salicylaldehydes 3a–g were reacted with commercially
available ethyl trifluorocrotonate 4 to produce the substituted
benzopyran ester (5a–g) in an efficient, one-step procedure4 in
K2CO3 and DMF. After hydrolysis of 5b–f, the desired acids 6b–f
were obtained. 6-Iodo 5g was coupled with TMS acetylene using
tetrakis(triphenylphospine)Pd(0) and CuI, followed by hydrolysis
to provide 6-ethynyl 7. Friedel–Crafts reaction of 5a using AlCl3
and acetyl chloride formed 6-acetyl 8, which was reduced by tri-
ethylsilane followed by hydrolysis to provide 8-ethyl 9. The 6-for-
myl 5d was reacted with hydroxylamine to form 5-aldoxime 10,
which was dehydrated by treatment with trifluoroacetic anhydride
to give 6-cyano 11. In Scheme 2, commercially available benzoyl
chloride 12 was reacted with 4-trifluoro-3-oxobutanoate and so-
dium hydride to form the 4-oxo benzopyran, which was reduced
by sodium borohydride to provide the 4-hydroxy chromene ester
13. Reaction of 13 with trifluoromenthanesulfonic anhydride pro-
duced the triflate 14. Subsequent treatment of 14 with tributyltin
hydride, followed by ester hydrolysis, provided the acid 15.
The 6-methyl 6c and 6-ethyl 9 demonstrated similar COX-2
inhibitory potency to 6-chloro 6b. The more polar 6-cyano analog
11 showed a twofold increase in potency and a 10-fold increase in
selectivity, but 6-formyl 6d exhibited a decreased potency. As
shown in Table 1, COX-2 inhibition by the electron-donating
Scheme 2. Reagents and conditions: (a) 4-trifluoro-3-oxobutanoate, NaH (60% oil
disp.), toluene, 105 °C, 24 h, 39% yield; (b) NaBH4, EtOH, THF, 0 °C, 1 h, 87% yield; (c)
2,6-di-tert-butylpyridine, (CF3SO2)2O, CH2Cl2, 48 h, 66% yield; (d) LiCl, tetra-
kis(PPh3)Pd(0), [CH3(CH2)3]3SnH, 50–65 °C, 3 h, 19% yield; (e) NaOH, THF, CH3OH,
H2O.
Table 1
In vitro activity of 6-substituted benzopyrans
a
Compd
R
Mod human IC50
(
lM)
hCOX-1/hCOX-2
COX-1
COX-2
6b
6c
6d
6e
6f
7
9
11
15
Cl
Me
29.2
224
500
17
100
94.2
0.12
115
4.39
0.32
0.25
0.73
0.029
100
0.073
0.33
91
896
685
773
1
1290
0.36
1045
107
CHO
OCF3
OCH3
CCH
Et
CN
CF3
0.11
0.041
IC50 curves were generated with each test concentration run in duplicate, each
curve was done n P 2. The high concentration was 500
lM.
a
see Ref. 2c sec. 2.4 and note 3.
6-methoxy substituted analog 6f was significantly diminished.
6-OCF3 6e, 6-ethynyl 7, and 6-CF3 15 analogs demonstrated
improved COX-2 inhibition. In the prophylactic rat air pouch
assay,2c the 6-ethynyl 7 provided 60% inhibition, whereas the
6-OCF3 6e and 6-CF3 15 provided complete inhibition, a difference
postulated to be due to different metabolic fates of these analogs.
The analog 6c suggests a similar metabolic trend, with the methyl
in the 6-position performing poorly (13%) in prophylactic rat air
pouch assay. However in the therapeutic rat air pouch assay,5
which was developed by modification of the prophylactic rat air
pouch assay to identify compounds that were metabolically labile,
the 6-ethyl analog 9 exhibited 90% inhibition of PGE2 production.
Based upon these data and our confidence in the therapeutic air
pouch model, we were gratified to identify efficacious compounds
possessing sites for metabolism such as 6-methyl (6c) and 6-ethyl
(9). Both 6c and 9 had previously failed to meet our potency crite-
rion, (100% inhibition at 2 mpk), for advancement as judged by
their performance in the prophylactic air pouch assay. Modifica-
tion of the testing scheme allowed a broader range of analogs to
be considered for advanced in vivo assessment. Phenols 16a–d
were converted to salicylaldehydes 17a–d, and further elaborated
to provide 18a–d as is shown in Scheme 3. In Scheme 4, salicylal-
dehyde 19 was treated with bromine to form bromo salicylalde-
hyde 20, which was converted to the chromene by our standard
procedure. Suzuki coupling with trimethylboroxine and hydrolysis
provided 6-Me-7-MeO analog 21. Stille coupling of 22 with tribu-
tyl(ethynyl)stannane, followed by hydrolysis gave acid 23, see
Scheme 1. Reagents and conditions: (a) K2CO3, DMF, 80–100 °C; (b) NaOH, THF,
CH3OH, H2O; (c) acetylene, tetrakis(PPh3)Pd(0), Cu(I)I, TEA, toluene; (d) AlCl3, acetyl
chloride, CH2Cl2, 0 °C to reflux, 6 days, 68% yield; (e) Et3Si, CH2Cl2, 89% yield; (f)
hydroxylamine HCl, CH3CO2Na, EtOH, H2O, 18 h, 55% yield; (g) (CF3CO)2O, dioxane,
25–85 °C, 16 h, 40% yield.

7166
J. L. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7164–7168
Scheme 7. Reagents and conditions: (a) NIS, DMF, 2 days, 97% yield; (b) 3, TEA,
DMF, 85 °C, 66 h, 90% yield; (c) trimethylboroxine, PdCl2(dppf)2 –CH2Cl2, Cs2CO3,
dioxane, 110 °C, 6 h, 83% yield; (d) TMS-CCH, CuI, Pd(PPh3)4, TEA, 4 days, 100%
yield; (e) TBAF, THF, 10 min, 73% yield; (f) CuI, PdCl2(dppf)2CH2Cl2, TEA, toluene,
propyne, À78 °C to 25 °C, 24 h, 89% yield; (g) 10%Pd/C, EtOH, 30 psi, 3 h, 100% yield;
(h) pinicolborane, PdCl2(dppf)2CH2Cl2, TEA, dioxane, 80 °C, 2 days, 59% yield; (i) 30%
H2O2, THF aq NaOH, 0 °C to 25 °C, 3.5 h, 91% yield; (j) CH3I, KI, K2CO3, DMF, 17 h,
100% yield; (k) NaOH, THF, CH3OH, H2O.
Scheme 3. Reagents and conditions: (a) CH3CH2MgBr, toluene, HMPA, p-formal-
dehyde, 90 °C, 3 h, ꢀ40% yield; (b) 3, K2CO3, DMF, 80–100 °C; (c) NaOH, THF,
CH3OH, H2O.
the COX-2 assay, but only the 6-Me-8-Me 18c exhibited very high
selectivity for COX-2 (Table 2). The 6-Me-8-OMe 21 exhibited
weak COX-2 inhibition, while the 6-ethyynl-8-Cl 23 and 6-Et-8-
Me 26 demonstrated good potency but poor selectivity for COX-
2. Analogs of 6-OCF3 29a–d demonstrated high COX-2 potency
and intermediate selectivity, except 29d, which displayed low
selectivity. Analogs 18c and 29a–c displayed good potency, selec-
tivity, and evidence of metabolism (data not shown) were sub-
jected to chiral resolution. The data (Table 3) indicated that the
S-isomers were more potent COX-2 inhibitors and possessed
in vivo efficacy in the therapeutic air pouch model.
As mentioned previously, compounds 30 (in Part I 5g) and 31
(in Part I 5h) (Fig. 2) were very potent and selective COX-2 inhib-
itors. To explore the utility of these tri-substituted COX-2 inhibi-
tors with reduced half-lives, we replaced two chloro groups with
methyl groups. Di-methyl substituted phenols 32a–c were con-
verted to salicylaldehydes 33a–c, followed by condensation with
ethyl trifluorocrotonate, chlorination, and ester hydrolysis to pro-
vide acids 34a–c, see Scheme 8.
Scheme 4. Reagents and conditions: (a) bromine, HOAc, 0 °C, 2 h, 80% yield; (b) 3,
K2CO3, DMF, 85 °C,18 h, 63% yield; (c) trimethylboroxine, Pd(PPh3)4, K2CO3, DMF,
90 °C, 18 h, 79% yield; (d) NaOH, THF, CH3OH, H2O.
Scheme 5. 4-Acetyl phenol 24 was hydrogenated and treated with
MgCl2 and p-formaldehyde to form salicylaldehyde 25 which upon
further elaboration provided 26, see Scheme 6.
The favorable in vitro and in vivo activity of 6-chloro (6b), 6-tri-
fluoromethyl (6e), and 6-trifluoromethoxy (15) substituted ana-
logs suggested retaining these preferred 6-substituents along
with a metabolically labile group. Salicylaldehyde 27 was iodin-
ated with NIS, followed by formation of the chromene to provide
28, which were converted to analogs 29a–d, see Scheme 7. Analog
29a was obtained by coupling 28 with trimethylboroxine followed
by hydrolysis. The 8-ethyl 29b was prepared in four steps; by
coupling with TMS acetylene in the presence of CuI and Pd(0), fol-
lowed by removal the TMS group, hydrogenation, and saponifica-
tion. The 8-propyl 29c was prepared by coupling of 28 with
propyne using CuI and PdCl2(dppf)2CH2Cl2 as catalyst, followed
by hydrolysis. 8-Iodo 28 was converted to 8-boron ester by treating
with pinicol-diborane and PdCl2(dppf)2CH2Cl2, then converted to
the 8-OH ester, which was then alkylated and hydrolyzed to pro-
vide 29d.
The SAR indicated (Table 4) that tri-substituted chromene ana-
logs 34a–c were very potent and very selective COX-2 inhibitors.
Analogs 34b and 34c displayed evidence of metabolism (data not
shown) and were advanced to chiral resolution. The data (Table 5)
Table 2
In vitro activity of selected 6,8-disubstituted benzopyrans
The SAR indicated that the 6-Cl-8-Me 18a was a very potent but
not a highly selective COX-2 inhibitor. The analogs 6-Me-8-Cl 18b,
6-Me-8-Me 18c, and 6-OMe-8-Cl 18d displayed good potency in
Compd
R1, R2
Mod human IC50a
(lM)
hCOX-1/hCOX-2
COX-1
0.47
1.08
169b
25.6
COX-2
18a
18b
18c
18d
21
23
26
29a
29b
29c
29d
R1 = Cl, R2 = Me
R1 = Me, R2 = Cl
R1 = Me, R2 = Me
R1 = OMe, R2 = Cl
R1 = Me, R2 = OMe
R1 = CCH, R2 = Cl
R1 = Et, R2 = Me
R1 = OCF3, R2 = Me
R1 = OCF3, R2 = Et
R1 = OCF3, R2 = Pr
R1 = OCF3, R2 = iPr
0.084
0.19
5.6
5.7
704
107
2.9
5.4
0.5
9
<0.24b
0.24
Scheme 5. Reagents and conditions: (a) (CH3CH2)3SnCCH, tetrakis(PPh3)Pd(0),
toluene, reflux, 3 h, 76% yield; (b) NaOH, THF, CH3OH, H2O.
9.62
0.14
0.14
0.14
0.50
7
3.33
0.026
0.27
0.016
0.074
0.043
0.68
7
163
0.5
0.32
IC50 curves were generated with each test concentration run in duplicate, each
curve was done n P 2. The high concentration was 500
l
M.
a
See Ref. 2c sec. 2.4 and Ref. 3.
Scheme 6. Reagents and conditions: (a) 20% Pd(OH)2/C, HOAc, H2, 60 psi, 16 h, 93%
yield; (b) MgCl2, TEA, p-formaldehyde, ACN, reflux, 3 h, 53% yield; (c) 3, K2CO3, DMF,
80–100 °C, 78% yield; (d) NaOH, THF, CH3OH, H2O.
b
The traditional COX enzyme assay was run with longer reaction time compared
with the modified assay see Ref. 2c, sec. 2.4.

J. L. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7164–7168
7167
Table 3
Table 5
SAR of S and R isomers
SAR of S and R isomers of tri-substituted benzopyrans 33b and 33c
Compd
S isomer
R isomer
a
a
Mod hIC50
COX-1
(
l
M)
Air pouch
Inhibition
Mod hIC50
COX-1
(
l
M)
Air pouch
COX-2
COX-2
Inhibition
34b
34c
2.28
4.99
0.046
0.03
92%
60%
30.8
100
1.28
100
n.d.
n.d.
Compd R1, R2
S isomer
R isomer
IC50 curves were generated with each test concentration run in duplicate, each
a
a
curve was done n P 2. The high concentration was 500
lM.
Mod hIC50
M)
Air pouch
Mod hIC50
M)
Air pouch
a
See Ref. 2c sec. 2.4 and Ref. 3.
(
l
(l
COX-1 COX-2 Inhibition COX-1 COX-2 Inhibition
Table 6
18c
29a
29b
29c
R
1 = Me,
R2 = Me
1 = OCF3,
R2 = Me
1 = OCF3, 19.9
R2 = Et
1 = OCF3, 71.7
R2 = Pr
100
0.29
80%
n.d.
62.7
100
100
n.d.
100
0.86
1.7
n.d.
n.d.
n.d.
n.d
Pharmacology of selected S isomers
R
2.69 0.014 >68%
Compd
Air pouch
ED50
(mg/kg)
Edema
ED50
(mg/kg)
Hyperalgesia
ED50
Arthritis
ED50
Hmicre%
a
b
c
d
R
0.06
>72%
(mg/kg)
(mg/kg)
18c
29b
34b
2
1.87
0.41
8.6
3.8
8
20.7
5.6
7.6
10
0.8
3.2
41
99
15
R
0.042 >68%
IC50 curves were generated with each test concentration run in duplicate, each
a
b
c
See Ref. 2c sec. 2.9.
See Ref. 2c sec. 2.10
See Ref. 2c sec. 2.10
See Ref. 2c sec. 2.11.
curve was done n P 2. The high concentration was 500
l
M.
a
See Ref. 2c sec. 2.4 and notes 3.
d
e
Human microsomal assay, percent metabolized.
Table 7
PK of selected S isomers
Priority
Compd
Rat t1/2
Dog t1/2
Cyno t1/2
Projecteda
(h)
(h)
(h)
human t1/2 (h)
Ref. Part I
Ref. Part II
SD-8381
SC-75416
10
16
20
23
17
24
115
13
1
2
3
29b
18c
34b
2
8.7
4.7
7.7
19
23
12
28
5.4
5
67
169
a
Projected human t1/2 was generated by allometric scaling.
Figure 2. Examples of COX-2 inhibitors 30 and 31 (see Part 1 of this Letter). (a) See
Ref. 2c sec. 2.4 and Ref. 3.
indicated that the S-isomers were more potent COX-2 inhibitors and
displayed in vivo efficacy in the therapeutic air pouch model. These
data (Tables 3 and 5) demonstrated that analogs with small groups
attached to the 6,8-positions of benzopyran, containing the S-iso-
mers were more potent COX-2 inhibitors. This result was consistent
with our previous findings (see Parts I and II of this Letter).
Selected compounds from Tables 3 and 5 were advanced for
additional in vivo studies. All analogs in Table 6 demonstrated evi-
dence of in vitro metabolism and good rodent in vivo efficacy. In
vivo PK studies were conducted in multiple species with the objec-
tive of predicting human half-life by allometric scaling, see Table 7.
Although empirical, the approach has been widely used to extrap-
Scheme 8. Reagents and conditions: (a) MgCl2, ACN, TEA, p-formaldehyde, 90 °C,
6 h, ꢀ40% yield; (b) 3, K2CO3, DMF, 80–100 °C; (c) HOAc, Cl2(g), Zn dust; (d) NaOH,
THF, CH3OH, H2O.
Table 4
Exploration of 6-chloro di-substituted patterns of benzopyrans
R1, R2
Human IC50
(l
M)
hCOX-1/hCOX-2
a
Compd
COX-1
2.58
29.8
36.3
COX-2
34a
34b
34c
5-Me, 8-Me
5-Me, 7-Me
7-Me, 8-Me
0.21
0.13
0.12
12
229
302
IC50 curves were generated with each test concentration run in duplicate, each
curve was done n P 2. The high concentration was 500 M.
l
a
The traditional COX enzyme assay was run with a longer reaction time com-
Figure 3. Plasma concentration of compound
therapeutic dose in monkeys.
2 (SC-75416) in microdose and
pared with the modified assay see Ref. 2c, sec. 2.4 and note 3.

7168
J. L. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7164–7168
Figure 4. Utility of the microdose: plasma levels in man after oral doses of compound 2 (SC-75416).
29b-(S), and 34b-(S) as back up candidates for compound 2
Table 8
(SC-75416). 18c-(S), 29b-(S), and 34b-(S) showed human half-lives
of 57, 13, and 11 h and possess superior efficacy in in vivo animal
models. The microdose strategy permitted us to identify 29b-(S)
and 34b-(S) as candidates for advancement.
Human half-life of microdose candidates
Study
Compd
Human
Human
t1/2 (h)
Projecteda
human t1/2 (h)
hepta. CL int
Microdose
Microdose
Microdose
29b-(S)
18c-(S)
33b-(S)
67
23
10
13
57
11
5
67
169
Acknowledgments
a
Projected human t1/2 was generated by allometric scaling.
We thank Jim Gierse, Carol M. Kobldt, Yang Zhang, Ben S. Zwei-
fel for providing in vitro and in vivo data, Joe Collins, Pete Kleine,
Adam Libby, Katie Palmquist for scale up and chiral purification,
Mike Baratta, Rick Ridgewell, Alan Breau for in vitro metabolism
and PK study.
olate from animal to human.6 Historically human t1/2 had been dif-
ficult to predict based on allometric scaling for this class of highly
ppb compounds, since neither the microsomal data nor the hepa-
tocyte data simply predicted clearance. The data of 1 (SD-8381)
(pred. human t1/2 = 115 h, actual human t1/2 = 340 h) and 2 (SC-
75416) (pred. human t1/2 = 13 h, actual human t1/2 = 34 h) indi-
cated that allometric scaling in this series was largely ineffective.
We further sought to decreased risk in our development strat-
egy by developing and employing a screening microdose clinical
study.7 This method was thought to permit us to efficiently obtain
human pharmacokinetic data (sub-therapeutic dose) prior to selec-
tion of an agent for full clinical development. In the microdose clin-
ical trial, compounds were dosed at 1/100th the preclinical
efficacious dose, which streamlined safety profiling required (low
dose) and the amount of compound required. To determine if the
PK profile for this class was similar across species at both micro
and therapeutic dose levels, compound 2 (SC-75416) was dosed
at both micro and therapeutic dose levels in monkey and man.
The data demonstrated a similar PK profile in monkey and man
(Figs. 3 and 4). The PK profile was unchanged, suggesting that 2
had the same clearance mechanism at both micro and therapeutic
doses and in both monkey and man. Three backup candidates pre-
dicted to have an acceptable human t1/2 (Table 8) were advanced
for a clinical microdose study. Analogs 18c-(S), 29b-(S), and 34b-
(S) displayed human half-life of 57, 13, and 11 h.
References and notes
1. 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; (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; (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. The therapeutic rat air pouch assay was similar to prophylactic rat air pouch
assay: for prophylactic rat air pouch: compound was on board 1 h before dosing
carrangeenan in pouch and removing fluid and assay for PGE2 4 h after dosed the
compound. For therapeutic rat air pouch: compound was on board 1 h after
dosing carrangeenan in pouch and removing fluid and assay for PGE2 1 h after
dosed the compound.
In summary, we successfully applied a filter requiring Phase I
metabolism as a cornerstone in our compound design and advance-
ment criteria. We validated compounds in lower species (cyno) the
relative microdose to therapeutic dose by showing the same PK pro-
files (data not shown). We applied a microdose strategy to rapidly
obtain human PK parameters. We successfully discovered 18c-(S),
6. Caldwell, G. W.; Masucci, J. A.; Yan, Z.; Hageman, W. Eur. J. Drug Metab.
Pharmacokinet. 2004, 29, 133.
7. (a) European Agency for the Evaluation of Medicines for Human Use (EMEA),
Position Paper on the Nonclinical Safety Studies to Support Clinical Trials with a
Single Microdose, CPMP/SWP2599/0, Jan 28, 2003.; (b) Yamanea, N.; Tozuka, Z.;
Sugiyama, Y.; Tanimoto, T.; Yamazaki, A.; Kumagai, Y. J. Chromatogr. B 2007, 858,
118.