Discovery of highly selective and potent p38 inhibitors based on a phthalazine scaffold

Article abstract of DOI:10.1021/jm8005417

Investigations into the structure-activity relationships (SAR) of a series of phthalazine-based inhibitors of p38 are described. These efforts originated from quinazoline 1 and through rational design led to the development of a series of orally bioavailable, potent, and selective inhibitors. Kinase selectivity was achieved by exploiting a collection of interactions with p38a including close contact to Ala157, occupation of the hydrophobic gatekeeper pocket, and a residue flip with Gy110. Substitutions on the phthalazine influenced the pharmacokinetic properties, of which compound 16 displayed the most desirable profile. Oral dosing (0.03 mg/kg) of 16 in rats 1 h prior to LPS challenge gave a >50% decrease in TNFα production.

Full text of DOI:10.1021/jm8005417

J. Med. Chem. 2008, 51, 6271–6279
6271
Discovery of Highly Selective and Potent p38 Inhibitors Based on a Phthalazine Scaffold#
Brad Herberich,*,† Guo-Qiang Cao,† Partha P. Chakrabarti,† James R. Falsey,† Liping Pettus,† Robert M. Rzasa,†
Anthony B. Reed,† Andreas Reichelt,† Kelvin Sham,† Maya Thaman,† Ryan P. Wurz,† Shimin Xu,† Dawei Zhang,† Faye Hsieh,‡
Matthew R. Lee,§ Rashid Syed,§ Vivian Li,§ David Grosfeld,§ Matthew H. Plant, Bradley Henkle, Lisa Sherman,
Scot Middleton, Lu Min Wong, and Andrew S. Tasker†
Departments of Medicinal Chemistry, Inflammation, Pharmacokinetics and Drug Metabolism, and Molecular Structure, Amgen, Inc.,
One Amgen Center DriVe, Thousand Oaks, California 91320-1799
ReceiVed May 9, 2008
Investigations into the structure-activity relationships (SAR) of a series of phthalazine-based inhibitors of
p38 are described. These efforts originated from quinazoline 1 and through rational design led to the
development of a series of orally bioavailable, potent, and selective inhibitors. Kinase selectivity was achieved
by exploiting a collection of interactions with p38R including close contact to Ala157, occupation of the
hydrophobic gatekeeper pocket, and a residue flip with Gly110. Substitutions on the phthalazine influenced
the pharmacokinetic properties, of which compound 16 displayed the most desirable profile. Oral dosing
(0.03 mg/kg) of 16 in rats 1 h prior to LPS challenge gave a >50% decrease in TNFR production.
Introduction
The excess production of the inflammatory cytokine tumor
necrosis factor-R (TNFRa) is associated with a number of
inflammatory diseases including rheumatoid arthritis and pso-
riasis.1 There are a number of anti-TNFR therapies that bind
and inhibit TNFR including etanercept (a soluble TNFR
receptor-immunoglobulin fusion protein), adalimumab (a fully
human monoclonal antibody), and infliximab (a mouse-human
chimeric TNFR antibody) for the treatment of inflammatory
diseases.1,2 Although these injectable biological drugs have made
a dramatic impact on patients’ lives, they have associated side
effects.3 The next generation of anti-inflammatory agents could
be orally available agents that decrease the production of TNFR.
The mitogen activated protein kinase (MAPK) p38 has been
shown to be a major signaling molecule in the production of
inflammatory cytokines, and its therapeutic potential has been
reviewed.4 Of the four isoforms, p38R appears to be the
dominant player.5 For more than a decade, extensive efforts have
Figure 1. Activity of quinazoline 1. Standard deviation is given when
been made to develop an orally available p38R inhibitor for
n > 2. The asterisk (/) designates an average of two values.
the treatment of inflammatory diseases,6 and in this report we
outline our progress toward this goal.
The initial quinazoline lead (1, Figure 1) demonstrated
nanomolar activity against p38R but suffered a lack of selectivity
against other kinases. An X-ray crystal structure of 1 bound to
p38R gave insights into a rational redesign to improve selectivity
(Figure 2). Quinazoline 1 binds to p38R, forming a classical
donor-acceptor pair interaction with Met109 on the linker
strand; the methyl group on the toluamide ring occupies the
hydrophobic “gatekeeper” pocket lined with Thr106, Val105,
#
Atomic coordinates and structure factors for cocrystal structures of
compounds 1 and 19 with p38R can be accessed using PDB codes 3DT1
and 3DS6, respectively.
* To whom correspondence should be addressed. Phone: 805-447-9963.
Figure 2. Cocrystal of quinazoline 1 and p38R.
Fax: 805-480-4532. E-mail: brad.herberich@amgen.com.
†
Department of Medicinal Chemistry.
Department of Pharmacokinetics and Drug Metabolism.
Department of Molecular Structure.
‡
and Leu104. The inhibitor accepts a hydrogen bond from the
backbone N-H of Asp168 located on the activation loop and
donates a hydrogen bond to the carboxylate side chain of Glu71
located on helix C. The morpholine side chain is solvent exposed
and does not significantly contribute to binding. As such,
compound 1 engages generic kinase architecture shared by the
§
Department of Inflammation.
a Abbreviations: TNFR, tumor necrosis factor R; MAPK, mitogen
activated protein kinase; PDGF, platelet-derived growth factor; JNK, c-Jun
N-terminal kinase; LPS, lipopolysaccharide; CL, clearance; F, bioavail-
ability; IL-8, interleukin 8.
10.1021/jm8005417 CCC: $40.75
2008 American Chemical Society
Published on Web 09/26/2008

6272 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Scheme 1. Synthesis of 1-Aminophthalazines 7-10a
Herberich et al.
a Reagents: (a) 4-(2-aminoethyl)morpholine, i-PrOH, 180 °C; (b) morpholine, K2CO3, CH3CN, 160 °C or (S)-3-methyl morpholine, Cs2CO3, CH3CN, 200
°C; (c) 6, Pd(PPh3)4, 2 M aqueous K2CO3, DME/EtOH, 90 °C; (d) (i) bis(pinacolato)diboron, Pd(dppf)Cl2, KOAc, dioxane, 80 °C, (ii) 11, Pd(PPh3)4, 1.5
M aqueous K2CO3, EtOH, 80 °C.
PDGF and Src family kinases, and this explains a lack of
selectivity against these proteins. Comparison of the ATP-
binding sites of p38R, Src and PDGF family kinases reveals a
number of residue differences. In PDGF, ATP forms a bidentate
interaction with the “hinge” or “linker stand” located between
the N- and C-terminal lobes of the kinase. It is supported from
below by Leu393 located on a ꢀ-sheet behind the linker strand.
This leucine is present in the vast majority of kinases with a
few exceptions; for example, this residue is methionine in cMet,
Akt, and insulin receptor kinase, a phenylalanine in the Raf
family, and valine in the JNK family. The p38 family is
somewhat unique in that this residue is an alanine (Ala157),
with ATP being supported by a leucine residue located on the
activation loop. This creates an open channel one could exploit
for selectivity. The substituent at the 2-postion of quinazoline
inhibitors such as 1 is incapable of accessing this space.
Replacement with a phthalazine-based inhibitor with a substitu-
ent at the 1-position would enable a close contact to Ala157
and lead to increased selectivity. Additionally p38R contains a
glycine residue adjacent to the linker strand Met109. This
residue is present in only 40 kinases and is referenced for its
ability to “flip”, orienting the NH into the ATP binding pocket,
where in other proteins this would be a carbonyl.7 Unlike the
quinazoline ring system, the phthalazine scaffold contains a
nitrogen positioned so it could accept a hydrogen bond from
the N-H of Gly110 and would facilitate engagement of the
flipped glycine to potentially further improve selectivity.
pylamide tolyl fragment was appended as previously described
to furnish phthalazine ethers 15, 16, and 17. Separation of the
enantiomers, 16 and 17, was accomplished using supercritical
fluid chromatography on chiral stationary phase (see Experi-
mental Section). The 1-aryl substituted phthalazines (18 and
19) were accessed from 2 by sequential cross-coupling reactions.
Isoquinoline 20 was prepared from 4,7-dibromoisoquinoline10
by a selective Suzuki coupling of 6 followed by a palladium
mediated coupling of isopropanol (Scheme 3).
Results and Discussion
Replacement of the quinazoline ring with a phthalazine (1
versus 7) resulted in similar inhibitory activity for p38R (IC50
≈ 1 nM) but a large increase in selectivity against other kinases
(Table 1). The lower selectivity of 8 against cKit (440-fold)
could be increased to >1500-fold by introduction of a methyl
substituent (9). This effect was also seen in the 1-aryl series,
where 19 exhibited >2500-fold selectivity over cKit in contrast
to the unsubstituted congener 18. This increase of selectivity
was attributed to the methyl group being in proximity to Ala157
in p38R versus clashing with the more bulky Leu799 in cKit.
Removal of the methyl group on the toluamide ring (10) had a
negative impact on activity on all kinases. This is likely due to
the loss of an important hydrophobic interaction with the
gatekeeper pocket and the ability to more easily adopt a coplanar
relationship between the two rings. The ethers 15 and 16 showed
similar selectivity; expansion of the counter screening panel on
16 revealed modest activities against Lyn (IC50) 954 nM) and
PDGFR (IC50) 88 nM). The activity on the PDGF receptor
kinase was unexpected, since this protein, analogous to cKit
and Lyn, bears a leucine residue at the Ala157 position of p38R.
We can offer no structural rationale for the ability of PDGF
receptor kinase to bind compound 16, save possibly an increased
plasticity of this protein.
Chemistry
Scheme 1 outlines the synthetic routes used to access the
1-aminophthalazines (7-10) starting from 6-bromo-1-chlo-
rophthalazine (2).8 Nucleophilic displacement of the chloride
with an appropriate amine followed by a Suzuki coupling with
boronic ester 69 provided aminophthalazines 7-9. In situ
generation of the boronic ester of 4 followed by Suzuki coupling
with 11 afforded 10.
Phthalazine ethers were prepared in an analogous manner
(Scheme 2). Reaction of the sodium salt of i-PrOH or 1,1,1-
trifluoro-2-propanol with 2 afforded 12 and 13. The cyclopro-
A cocrystal structure of 19 bound to p38R (Figure 3) was
obtained; gratifyingly the phthalazine accepts hydrogen bonds
from the linker Met109 and the now flipped Gly110. As a
consequence of this rotation, the two N-terminal residues are
drawn in to engage the tolyl ring through van der Waals

p38 Inhibitors Based on a Phthalazine Scaffold
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6273
Scheme 2. Synthesis of 1-Ether and Arylphthalazines (15-19)a
a Reagents: (a) NaH, ROH; (b) 6, Pd(PPh3)4, 2 M aqueous Na2CO3, DME/EtOH, 90 °C; for 16 and 17: Pd (PPh3)4, 2 M aqueous Na2CO3, dioxane, 125
°C; (c) 6, Pd(PPh3)4, 2 M aqueous K2CO3, DME/EtOH, 70 °C; (d) ArB(OH)2, Pd(PPh3)4, 2 M aqueous K2CO3, DME/EtOH, 95 °C.
Scheme 3. Synthesis of Isoquinoline 20a
group delivered the enantiomers 16 and 17. The S-enantiomer
(16) maintained low single digit nanomolar potency, while 17
displayed a 2-fold decrease in potency. Both 18 and 19 were
very potent in the cellular (IC50 ≈ 1 nM) and whole blood (IC50
) 2-4 nM) assays.
Pharmacokinetics. In Sprague-Dawley rats, compound 7
displayed low bioavailability (1%) and exposure (39 ng ·h/mL),
while 8 exhibited a moderate plasma clearance (1.7 (L/h)/kg).
Studies in rat liver microsomes identified the morpholine ring
a Reagents: (a) (i) 6, Pd(PPh3)4, 1 M aqueous Na2CO3, DME, reflux;
(ii) i-PrOH, Pd(OAc)2, 2-di-tert-butylphosphino-1,1′-naphthyl, Cs2CO3,
as the major site of metabolism; incorporation of a methyl
substituent on the morpholine (9) somewhat ameliorated this
issue (CL) 0.8 (L/h)/kg). The phenyl series exemplified by
compound 19 also displayed less favorable pharmacokinetics
(CL ) 1.7 (L/h)/kg, t1/2 ) 1.4 h). Ether 16 demonstrated a lower
plasma clearance (0.35 (L/h)/kg) and consequently a longer half-
life (9.9 h) compared to its nonhalogenated congener 15 (CL
) 0.85 (L/h)/kg, t1/2 ) 1.8 h; Table 4).
In Vivo Studies. Compound 16 exhibited a dose-dependent
inhibition of the production of TNFR in rats stimulated by an
LPS challenge with an ED50 of <0.03 mg/kg (Figure 4). The
mean terminal plasma concentration at the ED50 was <3 nM,
which is consistent with its in vitro inhibitory effect in the
presence of whole blood (TNFR /IL-8 IC50 ) 2.7 nM, Table
3). The efficacy of 16 was also evaluated in a collagen induced
arthritis (CIA) disease study in Lewis rats (Figure 5). Compound
16 demonstrated an ED50 of 0.04 mg/kg, and the AUC (0-24
h) was estimated to be 123 nM ·h at the ED50.
toluene, 80 °C.
interactions. The methyl substituent forces the ring to adopt a
perpendicular conformer and engages Ala157 on the floor of
the ATP binding pocket. All other interactions are analogous
to those observed in the cocrystal of 1 and p38R (Figure 2).
To interrogate further the contribution of the glycine flip to
kinase selectivity, a matched pair of inhibitors that do not fully
engage Ala157 was prepared and evaluated. Phthalazine 15,
which is capable of inducing a conformational change in p38R
that would necessarily encounter a deleterious interaction with
the carbonyl group of non-glycine-containing enzymes, was
compared to isoquinoline 20, which is incapable of inducing a
conformational change but provides a C-H donor satisfying
both a nonflipped glycine and a non-glycine-containing enzyme.
We employed cKit as prototypical non-glycine-containing
enzyme (Table 2). While inhibitory activity against p38R for
15 and 20 is equivalent, the selectivity against cKit was
improved 10-fold for 15.
Conclusions
The compounds were evaluated for their inhibitory effect on
the production of TNFR in LPS-stimulated THP-1 cells and on
the production of IL-8 in TNF-stimulated human whole blood.
Phthalazine 7 was equipotent in the THP-1 cellular and 2-fold
more potent in the whole blood assays when compared to
quinazoline 1 (Table 3). Removal of the ethylene linker gave
8, which displayed a 35-fold increase in efficacy in the cellular
assay and a 5-fold increase in the whole blood assay versus 7.
The increased potency of 8 may be a result of the difference in
cellular permeability between 7 and 8. In the Caco-2 system, 7
exhibited lower permeability (Papp(A>B) ) 0.3 × 10-6 cm/s,
Papp(B>A) ) 31.0 × 10-6 cm/s) compared to 8 (Papp(A>B) )
10.5 × 10-6 cm/s, Papp(B>A) ) 39.8 × 10-6 cm/s). Compound
9 was equipotent to 8 in the cellular and whole blood assays.
The isopropyl ether analogue 15 exhibited low single digit
nanomolar activity in the cellular and whole blood assays.
Replacement of one of the methyl groups for a trifluoromethyl
The cocrystal of quinazoline 1 with p38R provided insights
to further exploit a number of features unique to this enzyme.
A phthalazine-based series (7-10, 15-19) was prepared and
shown to be potent and selective inhibitors of p38R. The X-ray
crystal structure of 19 bound to p38R illustrated that these
compounds engage an uncommon floor residue, Ala157, and
induce a conformational rotation of a glycine residue. Further
investigation led to 16, an agent with desirable pharmacokinetic
properties. In vivo studies revealed 16 exhibited potent inhibition
of TNF-R production (ED50 < 0.03 mg/kg) and low dose
efficacy in the CIA model (ED50 ) 0.04 mg/kg).
Experimental Section
Chemistry. All reactions were carried out under nitrogen or
argon atmosphere unless otherwise noted. All solvents were
purchased from Aldrich and used without further purification unless
otherwise noted. Reactions performed under microwave irradiation

6274 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Herberich et al.
Table 1. Selectivity Profile of Quinazoline 1 and Phthalazines 7-10 and 15-19a
a Standard deviations are given when n g 3. / designates an average of two values. ∧ indicates a value for n > 2.
Table 2. Inhibitory Activity of Phthalazine 15 and Isoquinoline 20
toward p38R and cKita
Figure 3. X-ray crystal structure of 19 bound to p38R.
were carried out on a Biotage Initiator Sixty Microwave Synthesizer.
Medium pressure chromatography was performed using a Combi-
Flash Companion (Teledyne ISCO) with RediSep normal-phase
silica gel (35-60 µM) columns and UV detection at 254 nm.
Preparative reverse-phase HPLC was performed on a Gilson 215
liquid handler equipped with a Phenomenex Synergi Max-RP
column (150 × 21.2 mm, 4.0 µm) with UV detection at 254 nm,
eluting with 10-100% CH3CN in water with 0.1% TFA for 12
min at 20 mL/min. Final analytical LCMS methods were performed
with an Aglient 1100 series instrument and UV detection at 254
nm, and a low resonance electrospray mode (ESI) with a binary
solvent system of 0.1% TFA in water (system A) and 0.1% TFA
in CH3CN (system B). Method LCMS-1 uses a Zorbax SB-C18
column (3.0 mm × 50 mm, 3.5 µm), 40 °C column temperature,
and 2.00 µL injection volume, eluting with the following gradients:
0.0-3.0 min, 10-95% B; 3.0-3.5 min, 95% B; 3.51 min, 10% B
a Standard deviations are given when n g 3. / designates an average of
two values.
at 1.5 mL/min. Method LCMS-2 uses a YMC ODS-AM C-18
column (100 mm × 21 mm, 5 µm), 40 °C column temperature, 3
µL injection volume, eluting with the following gradients: 0.0-0.5
min, 10% B; 0.5-7.0 min, 10-100% B; 7.0-9.5 min, 100% B;
9.5-10.0 min, 100-10% B at 0.5 mL/min. Method LCMS-3 uses
a Luna C18(2) column (100 mm × 4.6 mm, 5 µm), 40 °C column
temperature, 3 µL injection volume, eluting with the following

p38 Inhibitors Based on a Phthalazine Scaffold
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6275
Table 3. Cellular and Human Whole-Blood Activity of Quinazoline 1
and Phthalazines 7-9 and 15-19
gradients: 0.00-0.05 min, 0-20% B; 0.05-0.4 min, 20-80% B;
0.4-0.8 min, 80-100% B; 0.8-1.0 min, 100% B at 2.8 mL/min.
Method LCMS-5 uses a Phenomenex Synergi MAX-RP column
(2.0 mm × 50 mm, 4 µm), 40 °C column temperature, 3.00 µL
injection volume, eluting with the following gradients: 0.0-0.2 min,
0-10% B; 0.2-3.0 min, 10-100% B; 3.0-4.5 min, 100% B;
4.5-5.0 min, 100-10% B at 0.8 mL/min. Preparative chiral
supercritical fluid chromatography (SFC) was performed on a
Berger Multigram II SFC with a Chiralpak AD-H column (21 mm
× 250 mm, 5 µm) with liquid CO2 (system A) and MeOH (system
B): column temperature 40 °C, 60 mL/min flow rate, 100 bar outlet
pressure, isocratic elution 65:35 (A/B). Analytical chiral SFC was
performed on a Chiralpak AD-H (150 mm × 4.6 mm, 5 µm):
column temperature 40 °C, 4.0 mL/min flow rate, 100 bar outlet
pressure, isocratic elution 65:35 (A/B). 1H NMR spectra were
recorded on a Bruker AV-400 (400 MHz) or Bruker DPX-300 (300
MHz) spectrometer at ambient temperature. Elemental analyses
were performed by Atlantic Microlab, Inc. Norcross, GA, and were
within (0.4% of the theoretical values.
IC50 (nM)
compd
THP-1/TNFR
hWB TNFR/IL-8
1
7
8
9
15
16
17
18
19
24.7 ( 21.5
24.9 ( 18.9
0.7 ( 0.2
1.1 ( 0.6
0.4 ( 0.03
0.4 ( 0.2
0.9 ( 0.1
0.8 ( 0.3
0.4 ( 0.1
10.7 ( 4.8
4.3 ( 0.6
0.9 ( 0.3
1.2 ( 1.0
1.6 ( 0.9
2.7 ( 1.1
8.1 ( 2.7
3.5 ( 0.4
2.5 ( 0.8
Table 4. PK Profile in Sprague-Dawley Rats following Intravenous
(iv) and Oral (po) Dosea
iv (2.0 mg/kg in DMSO)
po (10 mg/kg)
AUC(0-∞) F
CL
Vdss
t1/2
compd
((L/h)/kg)
(L/kg)
(h)
(ng·h/mL)
(%)
N-Cyclopropyl-4-methyl-3-(2-((2-(4-morpholinyl)ethyl)amino)-
6-quinazolinyl)benzamide (1). A mixture of 6-bromo-N-(2-mor-
pholinoethyl)quinazolin-2-amine11 (200 mg, 0.68 mmol), 6 (225
mg, 0.75 mmol), Pd(PPh3)4 (78 mg, 0.068 mmol), and 2 M Na2CO3
(0.68 mL, 1.36 mmol) in 5 mL of 4/1 toluene/EtOH was heated at
80 °C for 3 h. After cooling, the reaction mixture was filtered
through a 0.45 µm filter. Saturated NaHCO3 (aq) was added, and
the mixture was extracted with EtOAc (3×). The combined organic
layers were dried over Na2SO4, filtered, and concentrated. The crude
product was purified by silica gel chromatography (0-5% MeOH
in CH2Cl2) to afford the title compound (170 mg, 58%) as an off-
white solid. 1H NMR (CDCl3, 400 MHz): δ 8.99 (s, 1H), 7.66-7.59
(m, 5H), 7.34 (d, J ) 8.53 Hz, 1H), 6.22 (s, 1H), 5.88 (s, 1H),
3.76-3.74 (m, 4H), 3.69-3.64 (m, 2H), 2.93-2.89 (m, 1H),
2.69-2.66 (m, 2H), 2.54 (br s, 4H), 2.32 (s, 3H), 0.90 (m, 2H),
0.64-0.60 (m, 2H). LCMS-1: m/z ) 432.2 [M + H]+, tR) 1.62
min (100%). HRMS ([M + H]+) calcd 432.239 40; found
432.234 27.
7
1.2
1.7
0.8
1.7
0.8
0.35
2.8
3.5
1.2
1.8
2.1
4.7
2.3
4.1
1.3
1.4
1.8
9.9
39c
1
22
57
40
92
72
8b
9
124c
7089d
2332c
10933d
21097d
19
15
16
a Values for are an average of three rats. b Dosed iv and po at 1 mg/kg.
c Vehicle ) 2% HPMC/1% Tween-80. d Vehicle ) 1% Tween-80 in
OraPlus, pH 2.2.
6-Bromo-N-(2-(4-morpholinyl)ethyl)-1-phthalazinamine (3).
A mixture of 28 (0.30 g, 1.23 mmol) and 4-(2-aminoethyl)mor-
pholine (0.32 mL, 2.46 mmol) in 1 mL of i-PrOH was heated in a
microwave at 180 °C for 5 min. The mixture was concentrated
and purified by silica gel chromatography (5% MeOH in CH2Cl2)
to furnish the title compound (320 mg, 77%) as a pale-yellow solid.
1H NMR (CDCl3, 400 MHz): δ 8.86 (s, 3 H), 7.97 (d, J ) 1.96
Hz, 1 H), 7.89 (dd, J ) 8.80, 1.96 Hz, 1 H), 7.67 (d, J ) 8.61 Hz,
1 H), 3.77-3.75 (m, 6 H), 2.79-2.75 (m, 2 H), 2.59-2.46 (m, 4
H). LCMS-2: m/z ) 337/339 [M + H]+, tR) 0.81 min (97.3%).
6-Bromo-1-(4-morpholinyl)phthalazine (4). A mixture of 2
(0.123 g, 0.5 mmol), morpholine (0.087 mL, 1.0 mmol), and K2CO3
(70.0 mg, 0.5 mmol) in 5 mL of CH3CN was heated in microwave
at 160 °C for 10 min. The mixture was concentrated and purified
by silica gel chromatography (5% MeOH in CH2Cl2) to give the
Figure 4. Effect of 16 on LPS-Induced TNFR in Lewis rats.
1
title compound (140 mg, 95%) as a pale-yellow solid. H NMR
(CDCl3, 400 MHz): δ 9.12 (s, 1H), 8.06 (s, 1 H), 7.91 (d, J ) 0.98
Hz, 2 H), 4.00-3.95 (m, 4H), 3.57-3.53 (m, 4 H). LCMS-3: m/z
) 294/296 [M + H]+, tR) 4.37 min (98.5%).
6-Bromo-1-((3S)-3-methyl-4-(morpholinyl)phthalazine (5). A
solution of (S)-3-methylmorpholine-PTSA salt (1.51 g, 5.5 mmol)
in MeOH (11 mL) was prepared using a sonicator. The solution
was treated with MP-carbonate (2.9 g), and the mixture was stirred
at room temperature for 3 h. It was then filtered through a sintered
glass frit, washed with CHCl3, and concentrated. The residue was
redissolved in CH3CN (6.9 mL) and combined with 2 (340 mg,
1.4 mmol) and Cs2CO3 (1.8 g, 5.5 mmol). This reaction mixture
was heated in the microwave for 30 min at 200 °C and then diluted
with 20 mL of EtOAc and washed three times with 20 mL of
saturated NaHCO3 (aqueous). The organic layer was dried over
Na2SO4, filtered, and concentrated. The crude material was purified
by silica gel chromatography (0-6% (2 M ammonia in MeOH) in
CH2Cl2) to afford the title compound (340 mg, 80% yield) as a
white solid. 1H NMR (CDCl3, 400 MHz): δ 9.14 (s, 1 H), 8.06 (d,
Figure 5. Efficacy of 16 in collagen-induced arthritis in Lewis rats.
gradients: 0.0-9.0 min, 5-95% B; 9.0-9.5 min, 95% B; 9.5-10.0
min, 5% B at 1.0 mL/min. Method LCMS-4 uses a Zorbax SB-
C18 column (4.6 mm × 30 mm, 1.8 µm), 45 °C column
temperature, 1 µL injection volume, eluting with the following

6276 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Herberich et al.
J ) 1.76 Hz, 1 H), 8.00-7.95 (m, 1 H), 7.94-7.88 (m, 1 H),
4.06-4.00 (m, 2 H), 4.00-3.88 (m, 2 H), 3.69-3.58 (m, 2 H),
3.41-3.33 (m, 1 H), 1.20 (d, J ) 6.26 Hz, 3 H). LCMS-2: m/z )
308, 310 [M + H]+, tR) 3.86 min (97.2%).
This reaction mixture was added to a mixture of 11 (111 mg, 388
µmol) and Pd(PPh3)4 (37 mg, 32 µmol) in EtOH (3.25 mL) and
1.5 M K2CO3 (aqueous, 0.86 mL, 1.29 mmol) and stirred for 2 h
at 80 °C. The reaction mixture was diluted with 75 mL of EtOAc
and washed three times with 50 mL of saturated NaHCO3. The
organic layer was dried over Na2SO4, filtered, and concentrated.
The crude material was purified by reverse-phase preparative HPLC.
The collected fractions were basified with saturated NaHCO3
(aqueous) and extracted with CHCl3. The organic phase was dried
over Na2SO4 and concentrated to give the title compound (22 mg,
N-Cyclopropyl-4-methyl-3-(1-((2-(4-morpholino)ethyl)amino)-
6-phthalazinyl)benzamide (7). A mixture of 3 (220 mg, 0.65
mmol) and 6 (197 mg, 0.65 mmol) in 20 mL of DME/EtOH (4/1)
was treated with 2 M K2CO3 (aqueous, 1 mL, 2 mmol) and
Pd(PPh3)4 (38 mg, 0.033 mmol). The mixture was stirred at 90 °C
for 1 h and then cooled to room temperature and diluted with 100
mL of CH2Cl2. The organic layer was washed with 20 mL of brine,
dried over anhydrous Na2SO4, filtered, and concentrated. The crude
material was purified by silica gel chromatography (1-10% MeOH
in CH2Cl2) to furnish the title compound (220 mg, 71%) as a pale-
yellow solid. 1H NMR (CDCl3, 400 MHz): δ 8.92 (s, 1 H),
7.89-7.84 (m, 1 H), 7.77-7.72 (m, 1 H), 7.74-7.66 (m, 3 H),
7.39-7.37 (m, 1 H), 6.51-6.41 (m, 1 H), 3.83-3.75 (m, 6 H),
2.99-2.88 (m, 1 H), 2.82-2.74 (m, 2 H), 2.58 (d, J ) 3.72 Hz, 4
H), 2.31 (s, 3 H), 0.93-0.83 (m, 2 H), 0.69-0.59 (m, 2 H). LCMS-
1: m/z ) 432 [M + H]+, tR) 1.18 min (98.3%). HRMS ([M +
H]+) calcd 432.239 40; found 432.235 68.
1
18%) as a white solid. H NMR (CDCl3, 400 MHz): δ 9.25 (s, 1
H), 8.20-8.01 (m, 3 H), 7.90 (d, J ) 6.06 Hz, 2 H), 7.76 (d, J )
7.63 Hz, 2 H), 6.31 (s, 1 H), 4.05-3.97 (m, 3 H), 3.65-3.55 (m,
3 H), 3.20-2.88 (m, 1 H), 0.98-0.86 (m, 1 H), 0.71-0.62 (m, 1
H). LCMS-1: m/z ) 375 [M + H]+, tR ) 1.25 min (99.4%). HRMS
([M + H]+) calcd 375.181 55; found 375.177 43.
6-Bromo-1-(1-methylethoxy)phthalazine (12). Sodium hydride
(60% in mineral oil, 493 mg, 12.32 mmol) was carefully added to
a suspension of 2 (600 mg, 2.46 mmol) in 5 mL of i-PrOH over 5
min at room temperature. The mixture was stirred at room
temperature for 1 h and then diluted with MeOH and adsorbed on
to silica gel. The crude product was purified by silica gel
chromatography (0-20% EtOAc in hexanes) to afford the title
compound (525 mg, 80%) as a yellow solid. 1H NMR (300 MHz,
CDCl3): δ 9.08 (s, 1 H), 8.10 (d, J ) 8.67 Hz, 1 H), 8.03 (d, J )
1.70 Hz, 1 H), 7.92 (dd, J ) 8.67, 1.88 Hz, 1 H), 5.76 (sept, J )
6.19 Hz, 1 H), 1.50 (d, J ) 6.22 Hz, 6 H). LCMS-2: m/z ) 267/
269 [M + H]+, tR) 5.12 min (98.3%).
N-Cyclopropyl-4-methyl-3-(1-(4-morpholino)-6-phthalazinyl)-
benzamide (8). 8 was prepared from 4 (130 mg, 0.44 mmol) in a
manner analogous to example 7. The crude material was purified
by silica gel chromatography (1-10% MeOH in CH2Cl2) to give
the title compound (150 mg, 90%) as a pale-yellow solid. 1H NMR
(CDCl3, 400 MHz): δ 9.21 (s, 1 H), 8.10 (d, J ) 8.41 Hz, 1 H),
7.83 (s, 1 H), 7.79 (dd, J ) 8.51, 1.47 Hz, 1 H), 7.70-7.67 (m, 2
H), 7.39 (d, J ) 8.02 Hz, 1 H), 4.04-3.99 (m, 4 H), 3.63-3.58
(m, 4 H), 2.94-2.90 (m, 1H), 2.32 (s, 3 H), 0.91-0.86 (m, 2 H),
0.65-0.61 (m, 2 H). MS m/z ) 389 [M + H]+. Anal. (C23H24N4O2)
C, H, N. HRMS ([M + H]+) calcd 389.239 40; found 389.188 84.
N-Cyclopropyl-4-methyl-3-(1-((3S)-3-methyl-4-morpholinyl)-
6-phthalazinyl)benzamide (9). A mixture of 6 (317 mg, 1.05
mmol), 5 (270 g, 876 µmol), Pd(PPh3)4 (101 mg, 88 µmol), and
1.5 M K2CO3 (aqueous, 1.75 mL, 2.63 mmol) in EtOH (9 mL)
was heated at 80 °C for 1 h. The reaction mixture was diluted with
100 mL of EtOAc and washed three times with 50 mL of saturated
NaHCO3 (aqueous). The organic layer was dried over Na2SO4 and
concentrated. The crude product was purified by reverse-phase
preparative HPLC, and the collected fractions were basified with
saturated NaHCO3 (aqueous). The aqueous phase was extracted with
CHCl3, and the organic layer was dried over anhydrous Na2SO4,
filtered, and concentrated to give the title compound (130 mg, 37%
N-Cyclopropyl-4-methyl-3-(1-(1-methylethoxy)-6-phthalazi-
nyl)benzamide (15). A solution of 12 (250 mg, 0.94 mmol), 7 (352
mg, 1.17 mmol), Pd(PPh3)4 (54 mg, 0.047 mmol), and 2 M Na2CO3
(aqueous, 1.40 mL, 2.81 mmol) in 5 mL of DME/EtOH (4/1) was
heated at 90 °C for 20 h. The cooled reaction was diluted with
CH2Cl2 and washed with saturated aqueous NaHCO3 solution, dried
over anhydrous MgSO4, filtered, and concentrated. The crude
product was purified by silica gel chromatography (1-5% MeOH
in CH2Cl2) to afford the title compound (240 mg, 71% yield) as an
1
off-white amorphous solid. H NMR (CDCl3, 400 MHz): δ 9.12
(s, 1 H), 8.26 (d, J ) 8.41 Hz, 1 H), 7.81-7.73 (m, 2 H), 7.73-7.66
(m, 2 H), 7.37 (d, J ) 7.82 Hz, 1 H), 6.38 (br s, 1 H), 5.85-5.73
(m, 1 H), 2.97-2.88 (m, 1 H), 2.29 (s, 3 H), 1.52 (d, J ) 6.06 Hz,
6 H), 0.88 (q, J ) 6.52 Hz, 2 H), and 0.67-0.60 (m, 2 H). MS m/z
) 362.2 [M + H]+. Anal. (C22H23N3O2) C, H, N. HRMS ([M +
H]+) calcd 362.186 30; found 362.190 46.
1
6-Bromo-1-(2,2,2-trifluoro-1-methylethoxy)phthalazine (13).
A solution of 1,1,1-trifluoropropan-2-ol (1.9 g, 17 mmol) in 10 mL
of THF and 3 mL of DMF at 0 °C was treated with NaH (60% in
mineral oil, 0.50 g, 12 mmol) and stirred for 10 min. Compound 2
(1.38 g, 5.7 mmol) was added as a solid in small portions. After
the reaction mixture was stirred at room temperature for 1 h, it
was poured into an ice-cold saturated NH4Cl solution and extracted
twice with EtOAc. The combined organic layers were dried over
Na2SO4 and concentrated. The residue was purified by silica gel
chromatography (20-70% EtOAc in hexanes) to give the title
compound (1.57 g, 86% yield) as an off-white amorphous solid.
1H NMR (CDCl3, 300 MHz): δ 9.16 (s, 1 H), 8.10 (m, 2 H), 7.99
(m, 1 H), 6.16 (m, 1 H), 1.67 (d, J ) 6.5 Hz, 3 H). LCMS-5: m/z
) 321.0/323.0 [M + H]+, tR ) 3.22 min (100%).
N-Cyclopropyl-4-methyl-3-(1-((S)-2,2,2-trifluoro-1-methyl-
ethoxy)-6-phthalazinyl)benzamide (16) and N-Cyclopropyl-4-
methyl-3-(1-((R)-2,2,2-trifluoro-1-methylethoxy)-6-phthalazi-
nyl)benzamide (17). A mixture of 13 (3.0 g, 9.3 mmol), 6 (3.2 g,
11 mmol), Pd(PPh3)4 (0.54 g, 0.47 mmol) in 1,4-dioxane (15 mL),
and 2 M Na2CO3 (aqueous, 15 mL, 30.0 mmol) was heated at 125
°C in a microwave for 30 min. The reaction mixture was diluted
with 250 mL of EtOAc and washed with 10 mL of 1 N NaOH
(aqueous) followed by brine. The organic layer was dried over
MgSO4, filtered, and concentrated. The crude material was purified
by silica gel chromatography (45-85% EtOAc in hexanes) to give
N-cyclopropyl-4-methyl-3-(1-(1,1,1-trifluoropropan-2-yloxy)ph-
thalazin-6-yl)benzamide (3.34 g, 86% yield) as an off-white
yield) as a white solid. H NMR (CDCl3, 400 MHz): δ 8.12 (d, J
) 8.41 Hz, 1 H), 7.82-7.69 (m, 4 H), 7.37 (d, J ) 7.63 Hz, 1 H),
6.85 (s, 1 H), 4.13-3.85 (m, 4 H), 3.73-3.60 (m, 2 H), 3.40-3.27
(m, 1 H), 3.01-2.87 (m, 1 H), 2.31 (s, 3 H), 1.20 (d, J ) 6.46 Hz,
3 H), 0.90-0.78 (m, 2 H), 0.69-0.60 (m, 2 H). LCMS-3: m/z )
403 [M + H]+, tR) 5.23 min (100%). HRMS ([M + H]+) calcd
403.212 85; found 403.206 37.
N-Cyclopropyl-3-iodobenzamide (11). 3-Iodobenzoic acid (4.79
g, 19.3 mmol) was suspended in SOCl2 (19 mL) and stirred for
1 h at 70 °C. The reaction mixture was concentrated and
azeotropically dried with toluene. The residue was dissolved in
dioxane (19 mL), and N,N-diisopropylethylamine (13.5 mL, 77.3
mmol) and cyclopropylamine (6.77 mL, 96.6 mmol) were added
and stirred for 16 h at room temperature. The reaction mixture was
diluted with 75 mL of EtOAc, and the organic layer was partitioned
with saturated NaHCO3 (aqueous), washed twice with 50 mL of 3
N HCl (aqueous), dried over Na2SO4, filtered, and concentrated to
give the title compound (2.31 g, 42% yield) as an off-white solid.
1H NMR (CDCl3, 400 MHz): δ 7.77 (d, J ) 8.41 Hz, 2 H), 7.46
(d, J ) 8.41 Hz, 2 H), 6.22 (s, 1 H), 2.80-2.98 (m, 1 H), 0.77-0.99
(m, 2 H), 0.55-0.69 (m, 2 H). LCMS-1: m/z ) 288 [M + H]+,
tR) 1.77 min (99.4%).
N-Cyclopropyl-3-(1-(4-morpholinyl)-6-phthalazinyl)benza-
mide (10). A mixture of 4 (95 mg, 0.32 mmol), 1,1′-bis(diphe-
nylphosphino)ferrocenepalladium dichloride (24 mg, 0.032 mmol),
KOAc (95 mg, 0.97 mmol), and bis(pinacolato)diboron (90 mg,
0.36 mmol) in 1,4-dioxane (3 mL) was heated at 80 °C for 2.5 h.

p38 Inhibitors Based on a Phthalazine Scaffold
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6277
1
amorphous solid. H NMR (CDCl3, 300 MHz): δ 9.24 (s, 1 H),
with water. The layers were separated, and the aqueous layer was
extracted with CH2Cl2 (2×). The combined organic layers were
dried over anhydrous Na2SO4, filtered, and concentrated. The crude
material was purified by silica gel chromatography (1/1 to 6/1
EtOAc/hexane) to afford the title compound (50 mg, 0.14 mmol)
8.30 (d, J ) 8.2 Hz, 1 H), 7.87 (s, 1 H), 7.85 (s, 1 H), 7.69 (s, 1
H), 7.67 (s, 1 H), 7.40 (d, J ) 8.4 Hz, 1 H), 6.25 (br, 1 H), 6.20
(m, 1 H), 2.92 (m, 1 H), 2.31 (s, 3 H), 1.69 (d, J ) 6.5 Hz, 3 H),
0.87 (m, 2 H), 0.62 (m, 2 H). LCMS-5: m/z ) 416.1 [M + H]+, tR
) 3.08 min (100%).
1
as an off-white amorphous solid. H NMR (CDCl3, 400 MHz): δ
8.82 (s, 1 H), 8.24 (d, J ) 9.04 Hz, 1 H), 8.09 (s, 1 H), 7.74-7.66
(m, 2H), 7.34 (d, J ) 8.03 Hz, 1H), 6.68 (br s, 1H), 4.86-4.80
(m, 1H), 2.94-2.89 (m, 1H), 1.49 (d, J ) 6.02 Hz, 6H), 0.87 (m,
2H), 0.82 (m, 2H). LCMS-5: m/z ) 361[M + H]+, tR) 2.71 min
(100%). HRMS ([M + H]+) calcd 361.183 78; found 361.191 74.
p38 Kinase Reaction. The p38R kinase reaction was carried
out in a polypropylene 96-well black round-bottom assay plate in
a total volume of 30 µL of kinase reaction buffer (50 mM Tris, pH
7.5, 5 mM MgCl2, 0.1 mg/mL BSA, 100 µM Na3VO4, and 0.5
mM DTT). Recombinant activated human p38 enzyme (1 nM) was
mixed with 50 µM ATP and 100 nM GST-ATF2-Avitag, in the
presence or absence of inhibitor. The mixture was allowed to
incubate for 1 h at room temperature. The kinase reaction was
terminated, and phospho-ATF2 was revealed by addition of 30 µL
of HTRF detection buffer (100 mM HEPES, pH 7.5, 100 mM NaCl,
0.1% BSA, 0.05% Tween-20, and 10 mM EDTA) supplemented
with 0.1 nM Eu-anti-pTP and 4 nM SA-APC. After a 1 h incubation
at room temperature, the assay plate was read in a Discovery Plate
Reader (Perkin-Elmer). The wells were excited with coherent 320
nm light, and the ratio of delayed (50 ms after excitation) emissions
at 620 nm (native europium fluorescence) and 665 nm (europium
fluorescence transferred to allophycocyanin, an index of substrate
phosphorylation) was determined (Park, Y.-W.; Cummings, R. T.;
Wu, L.; Zheng, S.; Cameron, P. M.; Woods, A.; Zaller, D. M.;
Marcy, A. I.; Hermes, J. D. Homogeneous Proximity Tyrosine
Kinase Assays: Scintillation Proximity Assay versus Homogeneous
Time-Resolved Fluorescence. Anal. Biochem. 1999, 269, 94-104).
LPS-Induced TNF-r Production in THP-1 Cells. THP-1 cells
were resuspended in fresh THP-1 media (RPMI 1640, 10% heat-
inactivated FBS, 1× PGS, 1× NEAA, plus 30 µM ꢀME) at a
concentration of 1.5 × 106 cells/mL. An amount of 100 µL of cells
per well was plated in a flat-bottom polystyrene 96-well tissue
culture plate. Then 2 µg/mL of bacterial LPS (Sigma) was prepared
in THP-1 media and transferred to the first 11 columns of a 96-
well polypropylene plate. Column 12 contained only THP-1 media
for the LO control. Compounds were dissolved in 100% DMSO
and serially diluted 3-fold in a polypropylene 96-well microtiter
plate (drug plate). Columns 6 and 12 were reserved as controls
(HI controls and LO controls, respectively) and contained only
DMSO. One microliter of inhibitor compound from the drug plate
followed by 10 µL of LPS was transferred to the cell plate. The
treated cells were induced to synthesize and secrete TNFR in a 37
°C humidified incubator with 5% CO2 for 3 h. TNFR production
was determined by transferring 50 µL of conditioned media to a
96-well small spot TNFR plate (MSD, Meso Scale Discovery)
containing 100 µL of 2× Read buffer P supplemented with an anti-
TNFR polyclonal Ab labeled with ruthenium (MSD-Sulfo-TAG-
NHS ester). After an overnight incubation at room temperature with
shaking, the reaction was read on the Sector Imager 6000 (MSD).
A low voltage was applied to the ruthenylated TNFR immune
complexes, which in the presence of TPA (the active component
in the ECL reaction buffer, Read buffer P) resulted in a cyclical
redox reaction generating light at 620 nm. The amount of secreted
TNFR in the presence of compound compared with that in the
presence of DMSO vehicle alone (HI control) was calculated using
the following formula: % control (POC) ) (compd - average LO)/
(average HI - average LO) × 100.
The racemic material was dissolved in DME and subjected to
preparative chiral SFC to provide 1.46 g of N-cyclopropyl-4-methyl-
3-(1-((R)-1,1,1-trifluoropropan-2-yloxy)phthalazin-6-yl)benza-
mide (17) as a white solid. Analytical chiral SFC: tR ) 1.51 min.
Anal. (C22H20F3N3O2) C, H, N. HRMS ([M + H]+) calcd
416.158 04; found 41.161 12. Also obtained was 1.50 g of N-cy-
clopropyl-4-methyl-3-(1-((S)-1,1,1-trifluoropropan-2-yloxy)phthalazin-
6-yl)benzamide (16) as an off-white solid. Analytical chiral SFC:
tR ) 2.45 min. Anal. (C22H20F3N3O2) C, H, N. HRMS ([M + H]+)
calcd 416.158 04; found 416.150 93.
3-(1-Chloro-6-phthalazinyl)-N-cyclopropyl-4-methylbenza-
mide (14). A mixture of 2 (500 mg, 2.05 mmol), 6 (617 mg, 2.05
mmol), Pd(PPh3)4 (118 mg, 0.10 mmol), and 2 M K2CO3 (aqueous,
3.07 mL, 6.15 mmol) in 16 mL of DME/EtOH (4/1) was stirred at
70 °C for 1 h and then concentrated. The crude product was purified
by silica gel chromatography (5% (2 M ammonia in MeOH) in
CH2Cl2) to afford the title compound (650 mg, 94%) as a yellow
1
solid. H NMR (CDCl3, 400 MHz): δ 9.46 (s, 1 H), 8.36 (d, J )
8.41 Hz, 1 H), 7.98 (dd, J ) 8.41, 1.56 Hz, 2.30 (s, 3H), 0.91-0.86
(m, 2 H), 0.64-0.61 (m, 2 H). LCMS-5: m/z ) 338[M + H]+, tR
) 2.91 min (95.8%).
N-Cyclopropyl-4-methyl-3-(1-phenyl-6-phthalazinyl)benza-
mide (18). A mixture of 14 (98 mg, 0.29 mmol), phenylboronic
acid (35 mg, 0.29 mmol), and Pd(PPh3)4 (17 mg, 0.015 mmol) in
2 mL of DME/EtOH (4/1) was treated with 2 M K2CO3 (aqueous,
0.44 mL, 0.87 mmol) and then stirred at 95 °C for 30 min. The
mixture was cooled down to room temperature and concentrated.
The crude product was purified by silica gel chromatography (2-6%
(2 M ammonia in MeOH) in CH2Cl2) to give the title compound
(60 mg, 55%) as a pale-yellow solid. 1H NMR (CDCl3, 400 MHz):
δ 9.45 (s, 1H), 8.06 (d, J ) 8.53 Hz, 1H), 7.86 (s, 1H), 7.79-7.69
(m, 3H), 7.67-7.61 (m, 2H), 7.57-7.49 (m, 3H), 7.32 (d, J )
8.03 Hz, 1H), 6.33 (2, 1H), 2.89-2.79 (m, 1H), 2.26 (s, 3H),
0.85-0.75 (m, 2H), 0.59-0.52 (m, 2H). LCMS-1: m/z ) 380.2
[M + H]+, tR) 1.65 (98.8%). HRMS ([M + H]+) calcd 380.175 74;
found 380.169 05.
N-Cyclopropyl-4-methyl-3-(1-(2-methylphenyl)-6-phthalazi-
nyl)benzamide (19). 19 was prepared from 14 (130 mg, 0.44 mmol)
and o-tolylboronic acid (64 mg, 0.47 mmol) in a manner analogous
to example 18. The crude material was purified by silica gel
chromatography (2-6% (2 M ammonia in MeOH) in CH2Cl2) to
give the title compound (130 mg, 70%) as a pale-yellow solid. 1H
NMR (CDCl3, 400 MHz): δ 9.57 (s, 1 H), 7.95 (s, 1 H), 7.80-7.76
(m, 1 H), 7.72-7.68 (m, 3 H), 7.46-7.44 (m, 1 H), 7.42-7.36
(m, 4 H), 6.35 (s, 1 H), 2.94-2.90 (m, 1 H), 2.33 (s, 3 H), 2.16 (s,
3 H), 0.88-0.85 (m, 2 H), 0.64-0.60 (m, 2 H). LCMS-1: m/z )
394 [M + H]+, tR) 1.77 min (99.3%). HRMS ([M + H]+) calcd
394.191 39; found 394.191 62.
N-Cyclopropyl-4-methyl-3-(4-(1-methylethoxy)-7-isoquinoli-
nyl)benzamide (20). A solution of 4,7-dibromoisoquinoline10 (322
mg, 1.12 mmol), 6 (338 mg, 1.12 mmol), Pd(PPh3)4 (65 mg, 0.056
mmol), and 1 M sodium carbonate (aqueous, 2.24 mL, 2.24 mmol)
in 7 mL of DME was heated to reflux and stirred for 16 h. After
the mixture was cooled to room temperature, water was added and
the mixture was extracted with EtOAc (3×). The combined organic
layers were dried over anhydrous Na2SO4, filtered, and concentrated.
The crude material was purified by silica gel chromatography
(1-4% MeOH in CH2Cl2) to afford 3-(1-bromo-6-phthalazinyl)-
N-cyclopropyl-4-methylbenzamide (114 mg, 0.30 mmol).
A solution of 3-(1-bromo-6-phthalazinyl)-N-cyclopropyl-4-me-
thylbenzamide (220 mg, 0.58 mmol), i-PrOH (0.22 mL, 2.90 mmol),
Cs2CO3 (560 mg, 1.70 mmol), Pd(OAc)2 (16 mg, 0.069 mmol),
and 2-di-tert-butylphosphino-1,1′-naphthyl (34 mg, 0.087 mmol)
in 3 mL of toluene was stirred at 80 °C for 20 h. After cooling to
room temperature, the mixture was diluted with CH2Cl2 and washed
TNFr Induced IL-8 Production in Human Whole Blood.
Whole blood was drawn from healthy, nonmedicated volunteers
into sodium heparin tubes. An amount of 100 µL of blood was
then plated into 96-well tissue culture plates (BD). Ten point
compound titrations were added to blood and incubated for 1 h at
37 °C, 5% CO2. TNFR (Amgen) with a final concentration of 1
nM was then added to blood and incubated overnight (16-18 h)
at 37 °C, 5% CO2. Plasma was harvested, and cytokines were

6278 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Herberich et al.
measured by MSD (Meso Scale Discovery) ECL based antibody
sandwich assay. All reagents were prepared in RPMI 1640, 10%
v/v human serum AB (Gemini Bio-Products), 1× Pen/Strep/Glu.
Final concentration of human whole blood was 50%. Data were
analyzed using XLfit/Activity Base software package (IDBS).
Pharmacokinetic Studies. Male Sprague-Dawley rats were
administered the compound intravenously as a solution in DMSO
(1 or 2 mg/kg dose) or orally as a suspension in 2% hydroxypro-
pylmethylcellulose with 1% Tween-80 or in 1% Tween-80 in
OraPlus, pH 2.2 (1 or 10 mg/kg dose). Samples were taken at
various times after dosing and analyzed for parent compound by
LCMS.
to determine percent inhibition of inflammation compared with
vehicle controls. Data points represent the mean ( STE (n ) 8
rats/group).
X-ray Crystallography. The protein construct expression and
purification were based on the work described previously with some
modification (Wilson, K. P.; Fitzgibbon, M. J.; Caron, P. R.;
Griffith, J. P.; Chen, W.; McCaffrey, P. G.; Chambers, S. P.; Su,
M. S. J. Biol. Chem. 1996, 271, 27696-27700). Briefly, full length
p38R was expressed as a fusion protein with an amino terminal
poly-histidine tag and a thrombin cleavage site. The protein was
expressed in E. coli, and the cells were lysed by sonication. After
centrifugation, the p38R protein was captured with Talon resin
(Clontech). The resin was extensively washed, and thrombin was
added to the slurry to cleave the poly-histidine tag. The protein
was further purified on a HiTrap Blue column followed by a HiTrap
Q column (GE HealthcareLife Sciences). For both resins the protein
was eluted with a 0-1 M NaCl gradient in a 20 mM Tris-HCl, pH
7.5, 5 mM dithiothreitol, 10% glyerol buffer. The protein was
dialized against the final storage buffer of 50 mM HEPES at pH
7.5, 100 mM NaCl, 10 mM dithiothreitol, and 5% glycerol.
For crystallization trials, the protein was concentrated to 15 mg/
mL and a 10-fold molar excess of either compound 1 or 19 was
added. A wide range of crystallization conditions were tested using
the vapor diffusion method. Crystals of p38R with 1 were obtained
with 1.6 M ammonium sulfate and 100 mM HEPES at pH 7.5 at
room temperature. Crystals of p38R with 19 were obtained with
31% PEG 4000, 100 mM Na citrate, pH 5.6, 200 mM ammonium
acetate, and 10 mM dithiothreitol at room temperature.
Caco-2 Cell Permeability Studies. Human colon carcinoma cell
line (Caco-2) was obtained from the American Type Culture
Collection (ATCC, Manassas, VA). Cells were cultured in Dul-
becco’s modified Eagle medium (DMEM) (Invitrogen Corp.,
Carlsbad, CA) supplemented with a final concentration of 1% MEM
nonessential amino acids, 1 mM L-glucose, 2% penicillin-strepto-
mycin-glutamine, and 10% fetal bovine serum (Invitrogen Corp.,
Carlsbad, CA). Supplemented medium was filtered through a 0.2
µm filter prior to use. Cells were seeded at 1.4 × 105 cells/cm2
(passages 36-44, day 0) onto 24-well BIOCOAT HTS Fibrillar
Collagen MultiwellTM insert plates (BD Biosciences, Billerica,
MA) and were cultured for 20 days in a CO2 (5%) incubator
maintained at 37 °C (model BBD 6220, Kendro Laboratory
Products, Newtown, CT). Cell culture media were replaced every
2-3 days throughout the course of the culture. Prior to the transport
experiment, culture medium was aspirated from both apical and
basolateral wells and Caco-2 cells were rinsed twice with warmed
(37 °C) Hank’s balanced salt solution supplemented with 10 mM
Hepes at pH 7.4 (HHBSS, Invitrogen, Grand Island, NY). HHBSS
was removed from wells prior to dosing with test drugs at 10 µM
in HHBSS in either apical or basolateral chambers. Receiver
chambers were supplemented with 2% bovine serum albumin (BSA)
in order to ensure the optimal recovery of transported agents. All
apical and basolateral chambers contained 250 and 900 µL of
HHBSS, respectively, during all transport experiments. Transport
studies were incubated for 1 h at 37 °C on a shaking platform. At
the end of the incubation period, 150 µL samples were collected
from receiver reservoirs and analyzed by LCMS/MS on an API4000
(Applied Biosystems, Foster City, CA) triple quadrupole mass
spectrometer interfaced with turbo IonSpray operating in positive
mode using Analyst 1.3.1 software. The apparent permeability
coefficient (Papp) of all tested agents was estimated from the slope
of a plot of cumulative amount of the agent versus time based on
the following equation:
The structures were solved by molecular replacement using epmr,
refined using CNX (Accelrys), and manual rebuilding was done
using the graphics program Quanta (Accelrys). The structure of 1
and p38R was refined to 2.8 Å with an R-factor of 0.214 and free
R-factor of 0.279. The structure of 19 and p38R was refined to 2.9
Å with an R-factor of 0.228 and free R-factor of 0.319.
Supporting Information Available: Table of elemental analysis
results of compounds 8 and 15-17, HPLC analyses of compounds
1, 7, 9, 10, and 18-20, SFC analysis of 16 and 17, and crystal
X-ray structure data for 1 and 19. This material is available free of
charge via the Internet at http://pubs.acs.org.
References
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