3-Amino-7-phthalazinylbenzoisoxazoles as a novel class of potent, selective, and orally available inhibitors of p38α mitogen-activated protein kinase

Article abstract of DOI:10.1021/jm8005405

The p38 mitogen-activated protein kinase (MAPK) is a central signaling molecule in many proinflammatory pathways, regulating the cellular response to a multitude of external stimuli including heat, ultraviolet radiation, osmotic shock, and a variety of cy

Full text of DOI:10.1021/jm8005405

6280
J. Med. Chem. 2008, 51, 6280–6292
3-Amino-7-phthalazinylbenzoisoxazoles as a Novel Class of Potent, Selective, and Orally
Available Inhibitors of p38r Mitogen-Activated Protein Kinase†
Liping H. Pettus,*,‡ Shimin Xu,‡ Guo-Qiang Cao,‡ Partha P. Chakrabarti,‡ Robert M. Rzasa,‡ Kelvin Sham,‡ Ryan P. Wurz,‡
Dawei Zhang,‡ Scott Middleton,§ Bradley Henkle,§ Matthew H. Plant,§ Christiaan J. M. Saris,§ Lisa Sherman,§ Lu Min Wong,§
David A. Powers,# Yanyan Tudor,# Violeta Yu,# Matthew R. Lee, Rashid Syed, Faye Hsieh, and Andrew S. Tasker‡
Departments of Chemistry Research and DiscoVery, Inflammation, HTS Molecular Pharmacology, Molecular Structure, and Pharmacokinetics
and Drug Metabolism, Amgen Inc., One Amgen Center DriVe, Thousand Oaks, California 91320
ReceiVed May 9, 2008
The p38 mitogen-activated protein kinase (MAPK) is a central signaling molecule in many proinflammatory
pathways, regulating the cellular response to a multitude of external stimuli including heat, ultraviolet radiation,
osmotic shock, and a variety of cytokines especially interleukin-1ꢀ and tumor necrosis factor R. Thus,
inhibitors of this enzyme are postulated to have significant therapeutic potential for the treatment of rheumatoid
arthritis, inflammatory bowel disease, osteoporosis, and many other diseases where aberrant cytokine signaling
is the driver of disease. Herein, we describe a novel class of 3-amino-7-phthalazinylbenzoisoxazole-based
inhibitors. With relatively low molecular weight, these compounds are highly potent in enzyme and cell-
based assays, with minimal protein shift in 50% human whole blood. Compound 3c was efficacious (ED50
) 0.05 mg/kg) in the rat collagen induced arthritis (CIA) model.
Introduction
p38R mitogen-activated protein kinase (MAPKa) has been
shown to play a crucial role in regulating the biosynthesis of
proinflammatory cytokines including tumor necrosis factor R
(TNFR) and interleukin-1 ꢀ (IL-1ꢀ).1 Excessive production of
these two cytokines is implicated in many inflammatory
diseases,2 and their inhibition is a proven therapeutic strategy
in suppressing inflammation. Etanercept (a soluble TNFR
receptor), infliximab (a TNFR antibody), and adalimumab (a
TNFR antibody) are proven to be clinically effective in the
treatment of rheumatoid arthritis (RA), ankylosing spondylitis,
Crohn’s disease, and psoriasis by the blockade of TNFR
function.3 Therefore, the discovery and development of orally
active p38 MAP kinase inhibitors for the treatment of numerous
inflammatory diseases have been pursued by many pharmaceuti-
cal research groups.4
In our preceding communication, we reported the discovery
of 4-methyl-3-phthalazinylbenzamide 1 (Figure 1) as a potent
and selective p38 inhibitor.5 The crystal structure of p38R/
compound 1 complex reveals that the binding interaction of 1
and the p38R enzyme includes four H-bonding interactions: the
cyclopropylamide carbonyl oxygen and the backbone NH of
Asp168, the cyclopropylamide NH and the carboxylate of Glu71
located on the C-helix, the phthalazine ring nitrogen (N3) and
the linker NH of Met109, and a second ring nitrogen (N2)
intended to engage the NH of the flipped Gly110.6 The C1-o-
Figure 1. X-ray crystal structure of compound 1 bound in the ATP
binding site of unphosphorylated p38R. Bond distances are given in
angstroms. For the inhibitor, color-coding is as follows: C, green; N,
blue; O, red.
tolyl group, necessarily oriented 90° to the phthalazine ring,
has a hydrophobic interaction with Ala157 (rare as a floor
residue in the ATP binding site)7 that is drawn upward to meet
the inhibitor. Moreover, flipping Gly110 induces a conforma-
tional change in the backbone of the two residues Ala111 and
Asp 112, creating a narrow channel that is optimally occupied
by the C1-o-tolyl. In addition, the CH3-group in the benzamide
moiety occupies the Thr106 “gatekeeper pocket”. Finally the
cyclopropyl group is nested against Phe169 that resides DFG-
in.8 These strong interactions make for a highly potent (Ki )
0.4 nM) and selective p38 inhibitor.
To identify novel chemotypes with potentially improved
physical properties and PKDM profiles, we sought to replace
the benzamide moiety in compound 1 with an appropriate
bioisostere while still maintaining the good potency and
selectivity that compound 1 possesses (Figure 2). From the X-ray
structure, we recognized that the carbonyl oxygen utilized the
lone pair of electrons that were syn to the cyclopropylamine
moiety to engage the NH of Asp168. Conceptually, a benzamide
bioisostere that fused the carbonyl group to the benzene ring
would still have a lone pair of electrons in the right trajectory
for H-bonding with Asp168. Moreover, the cycle would need
to be small and lipophilic to be tolerated by the preceding
†
Atomic coordinates and structure factors for crystal structure of
compound 1 with p38R can be accessed using PDB code 3DS6.
* To whom correspondence should be addressed. Phone: 805-447-6964.
Fax: 805-480-1337. E-mail: lpettus@amgen.com.
‡
Department of Chemistry Research and Discovery.
Department of Inflammation.
Department of HTS Molecular Pharmacology.
Department of Molecular Structure.
§
#
Department of Pharmacokinetics and Drug Metabolism.
a Abbreviations: AUC, area under the curve; CIA, collagen-induced
arthritis; DFG, Asp-Phe-Gly sequence in ATP binding site; hWB, human
whole blood; IL, interleukin; MAPK, mitogen-activated protein kinase; RA,
rheumatoid arthritis; THP1, human acute monocytic leukemia cell line;
TNFR, tumor necrosis factor R; hERG, human ether-a-go-go related gene.
10.1021/jm8005405 CCC: $40.75
2008 American Chemical Society
Published on Web 09/26/2008

3-Amino-7-phthalazinylbenzoisoxazoles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6281
reduction, and subsequent oxidation to provide the aldehyde
24. Condensation of the aldehyde 24 with hydroxylamine
afforded oxime 25. Oxidation of 25 with N-chlorosuccimide
(NCS) gave the corresponding benzoyl chloride oxime, which
was treated with an amine to produce compounds 26a-c.
Heating of benzoxamidines 26a-c with 1,8-diazabicyclo[5.4.0]-
undec-7-ene (DBU) in THF in a microwave provided the desired
7-iodobenzoisoxazoles 27-29.12
As the need for greater quantities of 7-iodo-6-methylbenzo-
[d]isoxazol-3-amine 29 emerged, an alternative synthetic route
that required no microwave heating was developed. Thus, a one-
pot cyclization of ortho substituted benzonitriles to 3-amino-
1,2-benzoisoxazoles, as developed by Palermo,13 was employed
to convert benzonitrile 22 into 7-iodo-6-methylbenzo[d]isoxazol-
3-amine 30. Compound 30 was sequentially treated with
trifluoroacetic anhydride, alkylated with dimethyl sulfate, and
hydrolyzed in basic medium (NaOH) to produce 7-iodo-N,6-
dimethylbenzo[d]isoxazol-3-amine 29. The reaction of com-
pound 30 with acetyl chloride provided amide 31.
The synthesis of 3-amino-6-chloro-7-iodobenzoisoxazoles 36
and 37 is illustrated in Scheme 4. Benzyl alcohol 32 was
protected with a tert-butyldimethylsilyl (TBDMS) group. Lithia-
tion of compound 33 and quenching with iodine afforded
compound 34, from which the hydroxyl protecting group was
removed with tetrabutylammonium fluoride (TBAF) to give
benzyl alcohol 35. Compound 35 was converted to benzoisox-
azoles 36 and 37 via a five-step procedure similar to that
described in Scheme 3 for compounds 27-29.
Figure 2. Benzoisoxazole as a benzamide bioisostere.
Leu167. Upon replacement of the benzamide group present in
compound 1 with a benzoisoxazole, compound 2 was found to
inhibit p38R in the low nanomolar range. Herein, we report the
synthesis and biological evaluation of a unique class of 3-amino-
7-phthalazinylbenzoisoxazole derivatives 3 (Figure 2) as inhibi-
tors of p38 MAP kinase.5b Since several molecules in this series
were burdened with moderate to strong CYP3A4 inhibition, lead
optimization was focused on selecting compounds that were not
only potent but also free of CYP3A4 inhibition issues. Com-
pound 3c, in particular, showed good efficacy (ED50 ) 0.05
mg/kg) in the rat collagen induced arthritis (CIA) model.
Scheme 5 outlines the synthesis of phthalazinylindazole 40
and phthalazinylbenzoisothiazole 41. Heating of the benzonitrile
22 with hydrazine in refluxing THF provided the 7-iodo-6-
methyl-1H-indazol-3-amine 38. In a similar fashion, heating of
benzonitrile 22 with sulfur and ammonium hydroxide in
2-methoxyethanol gave the 3-aminobenzoisothiazole 39.14 Treat-
ment of compounds 38 and 39 with the (S)-1-(3-methylmor-
pholino)phthalazin-6-ylboronic acid 9 under Suzuki conditions
led to compounds 40 and 41, respectively.
Chemistry
The synthesis of compound 2 is shown in Scheme 1. The
known 1,6-dichlorophthalazine5b,9 (4) underwent selective Su-
zuki coupling at its C1-Cl site with o-tolylboronic acid to afford
the 6-chloro-1-o-tolylphthalazine 5, which was then converted
to the 1-o-tolylphthalazin-6-ylboronic acid 6 according to the
Miyaura protocol.10 Boronic acid 6 participated in a Suzuki
coupling reaction with the N-cyclopropyl-7-iodo-6-methylben-
zo[d]isoxazol-3-amine 27 (see Scheme 3) in the presence of
tetrakis(triphenylphosphine)palladium(0) to furnish compound
2.
Other phthalazinylbenzoisoxazole analogues were prepared
in a fashion similar to that described for compound 2, differing
only in the preparation of their precursors: the phthalazinylbo-
ronic acids 9-14, boronic ester 15, and the 7-iodobenzoisox-
azoles 27-31, 36, and 37 (Scheme 2). Amination of the known
6-bromo-1-chlorophthalazine5b,9 (7) afforded the 6-bromo-1-
aminophthalazines 8a-e, which were then transformed into
boronic acids 9-13 using Miyaura’s conditions followed by
acid hydrolysis. In a similar fashion, boronic acid 14 was derived
from 6-bromo-1-isopropoxyphthalazine 8f, which in turn was
obtained from the etherification of compound 7 with isopropanol
upon treatment with sodium hydride in THF. The iron-catalyzed
cross-coupling11 of the known 1-chloro-6-methoxyphthalazine
(16)5b,9 with isopropylmagnesium chloride followed by dem-
ethylation using BBr3 in dichloroethane afforded compound 18.
Triflation of this phenol and subsequent conversion of the triflate
19 to the boronic ester under Miyaura’s conditions furnished
1-isopropylphthalazin-6-ylboronic ester 15.
Results and Discussion
In Vitro SAR. Compounds were evaluated for their ability
to inhibit the phosphorylation of substrate activating transcription
factor 2 (ATF2) by recombinant human p38R and LPS-induced
TNFR production in THP1 cells. Compounds were further
evaluated against TNFR-induced IL-8 production in human
whole blood, an assay that was used as one of the major drivers
for compound selection. This assay was a more meaningful
measurement of inhibitor potency in a physiologically relevant
environment because of the presence of serum albumin and other
proteins.
Phthalazinylbenzoisoxazole inhibitors offered two major sites
for SAR modification (R1 and R3 in the generic structure 3,
Figure 2). To exemplify the influence induced by the R1 group,
R3 was maintained as the cyclopropyl group (Table 1).
Compound 2, (S)-3-methylmorpholino derivative 3a, piperazine
analogue 3f, and isopropylamino analogue 3g were nearly
equipotent in p38R enzyme, THP1 cell-based and whole blood
TNF/IL8 assays. Although all of the compounds in Table 1 had
no interference with CYP2D6 (IC50 > 25 µM), they were strong-
to-moderate inhibitors of CYP3A4 (IC50 ) 1.8-5.6 µM). Since
CYP inhibition has a possibility of causing serious drug-drug
interactions and liver toxicity in clinical use,15 the CYP3A4
inhibition was a concern for compounds made to date in this
series. Compound 2, the most hydrophobic (cLogP ) 5.0)
among the four compounds in Table 1, had the strongest
The synthesis of N-cyclopropyl-, N-ethyl-, and N-methyl-7-
iodo-6-methylbenzo[d]isoxazol-3-amine (27, 28, and 29) is
outlined in Scheme 3. Lithiation of the commercially available
2-fluoro-4-methylbenzonitrile 21 followed by iodination afforded
the 2-fluoro-3-iodo-4-methylbenzonitrile 22. Next, the nitrile
group was subjected to acid hydrolysis, borane-DMS complex

6282 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Pettus et al.
Scheme 1a
a Reagents and conditions: (a) o-tolylboronic acid, Pd(PPh3)4, 2 N Na2CO3, dioxane/EtOH, microwave, 78 °C, 53%; (b) (i) bis(pinacolato)diboron, Pd2(dba)3,
KOAc, P(Cy)3, dioxane, microwave, 120 °C; (ii) 2 N HCl, 38%; (c) compound 27, Pd(PPh3)4, 2 N Na2CO3, dioxane, microwave, 125 °C, 82%.
Scheme 2a
a Reagents and conditions: (a) HNR4R5, NMP, 165 °C; (b) isopropanol, NaH, THF; (c) (i) bis(pinacolato)diboron, Pd(dppf)Cl2, KOAc, dioxane, microwave,
120 °C; (ii) 2 N HCl; (d) Me2CHMgCl, 0.2 equiv of Fe(acac)3, room temp, 43%; (e) BBr3, DCE, 74%; (f) PhN(Tf)2, Et3N, CHCl3, 78%; (g)
bis(pinacolato)diboron, Pd(dppf)Cl2, KOAc, DMF, 80 °C, 40%; (h) Pd(PPh3)3, Na2CO3, dioxane, water, microwave, 120 °C.
CYP3A4 inhibition (IC50 ) 1.8 µM). However, a more general
correlation of the CYP3A4 affinity to the lipophilicity of the
inhibitor could not be made. The piperazine analogue 3f, with
the smallest cLogP of 2, still had an IC50 of 4.2 µM for
CYP3A4. The apparent cause of the CYP3A4 inhibition was
not clear to us. We thus undertook a structure-activity study
varying both termini of structure 3 (Figure 2) to identify
compounds potent on the target but devoid of CYP inhibitory
activity.
assay (Ki in the range of 0.3-0.7 nM). The decrease in cell
permeability on compound 3d may be attributed to the overall
increase in its polar surface area (PSA ) 90.3), as compared to
the ethyl analogue 3b (PSA ) 76.3). Compound 3d also
demonstrated the weakest potency in the whole blood TNFR/
IL-8 assay (IC50 ) 5.2 nM). From this study, we also noticed
that the variation of R3 group had a strong influence on the
CYP3A4 inhibitory activity. While the methyl analogue 3c and
the acetyl compound 3e did not present any potential issues
with CYP3A4 inhibition (IC50 > 25 µM), the ethyl analogue
3b and the cyclopropyl analogue 3a showed increased inhibition.
The effect of R2 and R3 groups on the p38R inhibitory activity
was studied while the R1 group was maintained as the (S)-3-
methylmorpholino group (Table 2). When R2 was a methyl,
small hydrophobic groups such as cyclopropyl, ethyl, methyl,
acetyl, and hydrogen were all well tolerated at the R3 site.
Compounds 3a-e proved to be equally potent in the enzyme
With replacement of the R2 methyl group with a chloride,
compounds 3h and 3i had comparable p38R enzyme inhibitory
activity to the methyl analogues 3a and 3c. Unfortunately,

3-Amino-7-phthalazinylbenzoisoxazoles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6283
Scheme 3a
40, the polar NH appears to be less tolerated by the aforemen-
tioned leucine, and for compound 41, the increased ring size of
the benzoisothiazole appears to create unfavorable steric interac-
tion with the leucine residue.
Pharmacokinetic (PK) studies carried out in Sprague-Dawley
rats on compounds 3c and 3j-n ultimately resulted in the
selection of 3c for further profiling. 3c displayed superior PK
properties evidenced by lower clearance and higher bioavail-
ability than analogs 3j-n. By intravenous (iv) administration
of 2 mg/kg, 3c showed a clearance of 1447 (mL/h)/kg, a
moderate volume of distribution of 2401 mL/kg, and an
elimination half-life of 2.8 h. Compound 3c was well-absorbed
(tmax ) 1.7 h, Cmax ) 1040 ng/mL) following oral administration
of 10 mg/kg [a solution dose in 1% Tween-80 in OralPlus (pH
2.2)], with an oral bioavailability (F) of 67%. Compound 3c
was 90% and 92% bound to rat and human plasma protein,
respectively (determined by the ultrafiltration methods). In rat
and human liver microsomes, 3c had a metabolic rate of 120
and 132 (µL/min)/(mg of protein), respectively.17
To evaluate kinase selectivity, compound 3c was tested for
inhibitory activity versus 32 kinases.18 With the exception of
p38ꢀ (Ki ) 1.8 nM), compound 3c (p38R, Ki ) 0.4 nM) showed
>1000-fold selectivity against all other kinases evaluated. As
commonly reported for p38R inhibitors,19 3c was selective
against p38γ and p38δ isoforms but not against p38ꢀ. In a
broader selectivity screen against a variety of G-protein-coupled
receptors and ion channels, 3c did not exhibit any off-target
interactions when tested at 10 µM concentration. Additional
profiling of 3c showed that it neither induced LDH release nor
caused human pregnane X receptor (hPXR) trans-activation. In
addition, it yielded an IC50 of 13 µM for blockade of the hERG
cardiac channel determined by manual electrophysiology. In
view of its promising pharmacokinetic and selectivity profiles,
3c was further evaluated in an animal disease model.
While an X-ray structure shows that benzamide 1 binds to
the ATP binding site in p38R protein (Figure 1), these data do
not exist for the benzoisoxazoles. Therefore, we undertook
classical Michaelis-Menten kinetic characterization of the
benzoisoxazoles,20 using 3c as a representative example. The
kinetic data of 3c, shown in Figures 4 and 5 and Table 5,
confirmed that 3c was an ATP competitive inhibitor of p38R
kinase. As for a competitive inhibitor, the Vmax does not vary
significantly while the ATP apparent Km shows the expected
linear relationship with concentration of 3c.
a Reagents and conditions: (a) LiTMP, I2, 48%; (b) H2SO4, dioxane, 92%;
(c) (i) BH3-SMe2, B(OMe)3, THF, 89%; (ii) MnO2, CH2Cl2, 89%; (d)
NH2OH, EtOH, 85%; (e) (i) NCS, DMF, 55 °C; (ii) R3NH2, THF; (f) DBU,
THF/NMP, 150 °C, 70 min, microwave; (g) acetohydroxamic acid, KOtBu,
DMF, 69%; (h) (i) (CF3CO)2O, CH2Cl2; (ii) K2CO3, Me2SO4; (iii) NaOH,
MeOH, 68% for three steps; (i) Cs2CO3, AcCl, CH2Cl2, 92%.
compounds 3h and 3i were also burdened with the issue of
moderate CYP3A4 inhibition (IC50 ) 4.5-6.0 µM).
The observation that compounds 3c and 3e were devoid of
CYP3A4 inhibition (IC50 > 25 µM) was encouraging. As shown
in Table 3, we focused our efforts on the modification of R1 to
determine if other substituents were well tolerated when R3 was
fixed as a methyl group. The (R)-3-methylmorpholine analogue
3j, isopropylamino compound 3k, and (S)-sec-butylamino
derivative 3l were equipotent to the (S)-3-methylmorpholine
analogue 3c in terms of p38R inhibition. The isopropyl ether
3m and the isopropyl analogue 3n, which were less polar than
the amino analogues, showed comparable THP1 cell based
potency. More significantly, however, was the observation that
all compounds in Table 3, regardless of their lipophilicity
(cLogP), had little CYP3A4 inhibition (IC50 > 25 µM). This
fact implied that the R1 substituents had little to no influence
on CYP inhibition when R3 was a methyl group. On the basis
of these findings, modification of R1 substituents appeared to
be a promising approach for improving PKDM profiles while
maintaining good potency on both p38R enzyme and cell based
assays.
In Vivo Pharmacology. The rat collagen induced arthritis
(CIA) model was chosen as the animal rheumatoid arthritis
disease model.21 Compound 3c was administered at 0.03, 0.1,
0.3, 1.0, and 3.0 mg/kg once a day to rats, beginning on day 10
through day 17 following immunization. As shown in Figure
6, 3c inhibited paw swelling in the CIA model in a dose
dependent manner with an ED50 of 0.05 mg/kg, corresponding
to an exposure of AUC (0-24 h) ) 22.4 nM ·h, with an
interpolated Cmax and C24h of 7.0 and 0.3 nM, respectively.
After optimization of R1 and R3 groups, an investigation of
possible replacement for the benzoisoxazole subunit was
undertaken. To facilitate this study, R1 was selected as (S)-3-
methylmorpholine and R3 was fixed as a hydrogen atom (for
synthetic simplicity). The benzoisoxazole ring oxygen was
replaced with either a sulfur atom or a NH group (Table 4).
Compared to the benzoisoxazole 3d, the indazole 40 and the
benzoisothiazole 41 showed a 50-fold and 7-fold potency loss
in p38R enzyme inhibition, respectively. These observations
supported our original hypothesis in that the cycle would need
to be small and lipophilic to be tolerated by the Leu167 residue
in the protein. Molecular modeling (FLAME)16 of compound
3c in the ATP binding site of unphosphorylated p38R (Figure
3) shows that the O-atom in the benzoisoxazole ring is in close
contact with the Leu167 residue in the protein. For the indazole
Conclusion
A structurally novel phthalazinylbenzoisoxazole series has
been discovered as p38 MAP kinase inhibitors. Structural
biology guided the design and modification for this series of
compounds. Through SAR studies, analogues with high affinity
to the p38 MAP kinase and no considerable CYP3A4 inhibition
were identified. As a representative example, optimized analogue
3c was potent and selective in vitro. Furthermore, with a

6284 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Pettus et al.
Scheme 4a
a Reagents and conditions: (a) TBDMSCl, imidazole, CH2Cl2, 84%; (b) LDA, I2, THF, 84%; (c) TBAF, THF, 97%; (d) (i) MnO2, CH2Cl2; (ii) NH2OH,
EtOH; (iii) NCS, DMF; (iv) R3NH2, THF; (v) DBU, THF/NMP, microwave, 165 °C.
Table 2. SAR: Influence of the R2 and R3 Substituentsa
Scheme 5a
THP1
hWB
CYP3A4
IC50
(µM)
p38R LPS/TNFR TNFR/IL-8
Ki (nM) IC50 (nM) IC50 (nM)
compd R2
R3
3a
3b
3c
3d
3e
3h
3i
Me cyclopropyl
Me Et
Me Me
0.7
0.3
0.4
0.3
0.6
0.2
0.4
0.6
0.4
0.4
3.6
0.7
1.3
3.6
1.3
2.0
3.3
5.2
2.0
3.3
5.6
10
>25
12
>25
6.0
4.5
a Reagents and conditions: (a) hydrazine hydrate, THF, reflux, 90%; (b)
compound 9, Pd(PPh3)4, Na2CO3, dioxane, water, microwave, 120 °C; (c)
S8, NH4OH, MeOCH2CH2OH, 130 °C, 31%.
Me
H
Me acetyl
Cl Me
Cl i-Pr
15
Table 1. SAR: Variation of the R1 Substituenta
a The p38R Ki and IC50 of cell based assays are mean values derived
from at least three independent dose-response curves. Variability around
the mean value was <50%. Midazolam was the substrate in CYP3A4
inhibition assay.
conducted with a Smith synthesizer from Personal Chemistry,
Uppsala, Sweden. Silica gel chromatography was performed using
either glass columns packed with silica gel (200-400 mesh, Aldrich
Chemical) or prepacked silica gel cartridges (Biotage and ISCO).
1H NMR spectra were obtained on a Bruker DRX 400 (400 MHz)
spectrometer and reported as ppm downfield from tetramethylsilane.
All final compounds were purified to >95% purity as determined
by LCMS analysis obtained on Agilent 1100; conditions are listed
in the Supporting Information. Low-resolution mass spectral (MS)
data were determined on a Perkin-Elmer SCIEX API 165 mass
spectrometer using ES ionization modes (positive or negative).
High-resolution mass spectral (MS) data were obtained on a Agilient
Technologies Mass Hunter ES-TOF Tunning Mix mass spectrometer.
1-o-Tolylphthalazin-6-ylboronic Acid (6). To a mixture of 1,6-
dichlorophthalazine (4) (1.03 g, 5.21 mmol), o-tolylboronic acid
(0.57 g, 4.17 mmol) and tetrakis(triphenylphosphine)palladium(0)
(301 mg, 0.26 mmol) in 1,4-dioxane (7.5 mL) in a 20 mL glass
tube was added 2.5 mL of EtOH, followed by 5 mL of 2 N Na2CO3.
The glass tube was sealed and heated in a Personal Chemistry
microwave at 78 °C for 45 min. The mixture was treated with 10
mL of 1 N NaOH and extracted with EtOAc (2 × 35 mL). The
combined EtOAc layers were dried and concentrated. Purification
on silica gel chromatography (eluted with 30-75% EtOAc in
hexanes) provided 6-chloro-1-o-tolylphthalazine (5) (543 mg, 53%
a The p38R Ki and IC50 of cell based assays are mean values derived
from at least three independent dose-response curves. Variability around
the mean value was <50%. Midazolam was the substrate in CYP3A4
inhibition assay. b Physicochemical properties were calculated using the
Daylight Toolkit,22 within Amgen’s proprietary software (ADAAPT).23
1
yield) as a brown amorphous solid. H NMR (DMSO-d6): δ 2.0
desirable pharmacokinetic profile, compound 3c exhibited
efficacy (ED50 of 0.05 mg/kg) in a rat collagen induced arthritis
model.
(s, 3H), 7.15-7.55 (m, 5H), 7.99 (d, J ) 9.0 Hz, 1H), 8.44 (s,
1H), 9.73 (s, 1H). MS (ESI, positive ion) m/z: 255 (M + 1).
In a sealed glass tube, a mixture of 6-chloro-1-o-tolylphthalazine
(127 mg, 0.50 mmol), bis(pinacolato)diboron (146 mg, 0.57 mmol),
potassium acetate (98 mg, 1.00 mmol), tris(dibenzylideneacetone)-
dipalladium(0) (18 mg, 0.02 mmol), and tricyclohexylphosphine
(11 mg, 0.04 mmol) in 2 mL of 1,4-dioxane was heated in a
Personal Chemistry microwave at 120 °C for 30 min. The reaction
mixture was diluted with 1 mL of dioxane, filtered through a pad
Experimental Section
Chemistry. All reagents and solvents were obtained from
commercial suppliers and used without further purification. All
reactions were carried out under an inert atmosphere of nitrogen
unless otherwise noted. All microwave-assisted reactions were

3-Amino-7-phthalazinylbenzoisoxazoles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6285
Table 3. Variation of R1-Phthalazinyl-N,6-dimethylbenzo[d]isoxazol-
3-aminea
Figure 3. Molecular modeling of compound 3c in the ATP binding
site of unphosphorylated p38R. Note that the O-atom of the benzoisox-
azole ring is in close contact with L167. Bond distances are given in
angstroms. For the inhibitor, color coding is as follows: C, gold; N,
blue; O, red.
a The p38R Ki and IC50 of cell based assays are mean values derived
from at least three independent dose-response curves. Variability around
the mean value was <50%. Midazolam was the substrate in CYP3A4
inhibition assay.
Figure 4. Michaelis-Menten plot for 3c.
Table 4. SAR: Replacement of the Benzoisoxazole Ringa
p38R THP1 LPS/TNFR hWB TNFR/IL8 CYP 3A4
compd
W
Ki (nM)
IC50 (nM)
IC50 (nM)
IC50 (µM)
3d
40
41
O
NH
S
0.3
16
2.0
3.6
120
29
5.2
153
50
12
Figure 5. Km/Vmax vs concentration of 3c (nM).
NTb
4.0
Table 5. Cconcentration of 3c, Vmax, and Apparent Km
a The p38R Ki and IC50 of cell based assays are mean values derived
from at least three independent dose-response curves. Variability around
the mean value was <50%. Midazolam was the substrate in CYP3A4
inhibition assay. b NT: not tested.
concn of
3c (nM)
Vmax (ratio/min)
Km (µM)
19
13
9
6
0
13.28
12.41
11.60
11.26
10.29
769.9
451.9
319.4
229.2
21.58
of Celite, and rinsed with 5 mL of EtOAc. The filtrate was
concentrated, and the dark residue was treated with 2 mL of 2 N
HCl. Hexanes (5 mL) were added, and the mixture was sonicated
for 5 min. The hexanes layer was discarded. The remaining acidic
layer was diluted with 1 mL of DMSO and loaded on a reverse
phase HPLC [using a gradient of 10-60% of (0.1% of trifluoro-
acetic acid in acetonitrile) in (0.1% of trifluoroacetic acid in water)].
The 1-o-tolylphthalazin-6-ylboronic acid (6) (50 mg, 38% yield)
was obtained as a white fluffy solid after drying the HPLC fractions
in a lyophylizer. 1H NMR (DMSO-d6): δ 2.01 (s, 3H), 7.19-7.55
(m, 5H), 8.30 (d, J ) 8.2 Hz, 1H), 8.65 (s, 1H), 9.76 (s, 1H). MS
(ESI, positive ion) m/z: 265 (M + 1).
N-Cyclopropyl-6-methyl-7-(1-o-tolylphthalazin-6-yl)benzo[d]-
isoxazol-3-amine (2). In a sealed glass tube, a mixture of
N-cyclopropyl-7-iodo-6-methylbenzo[d]isoxazol-3-amine (27) (52
mg, 0.16 mmol), 1-o-tolylphthalazin-6-ylboronic acid (6) (32 mg,
0.18 mmol), tetrakis(triphenylphosphine)palladium(0) (7.7 mg,
0.006 mmol) in 0.8 mL of 1,4-dioxane and aqueous solution of
Na2CO3 (2 N, 0.20 mL) was heated in a Personal Chemistry
microwave at 125 °C for 20 min. The mixture was treated with 1
mL of 1 N NaOH and extracted with EtOAc (3 × 5 mL). The
combined EtOAc layers were dried, concentrated, and purified on
a silica gel column (eluted with 2.5-15% MeOH in DCM) to
provide the title compound (55 mg, 82% yield) as a light-yellow
amorphous solid. 1H NMR (CDCl3): δ 0.87 (m, 2H), 0.72 (m, 2H),
2.19 (s, 3H), 2.42 (s, 3H), 2.88 (m, 1H), 4.75 (br s, 1H), 7.23 (m,
1H), 7.31-7.46 (m, 4H), 7.53 (d, J ) 8.0 Hz, 1H), 7.74 (d, J )
8.6 Hz, 1H), 7.88 (m, 1H), 8.13 (s, 1H), 9.59 (s, 1H). MS (ESI,
positive ion) m/z: 407 (M + 1). Anal. (C26H22N4O) C, H, N.
(S)-6-Bromo-1-(3-methylmorpholino)phthalazine (8a). In a
glass tube, to a solution of 6-bromo-1-chlorophthalazine (2.0 g,
8.2 mmol) and (S)-3-methylmorpholine (2.1 mL, 21 mmol) in 3

6286 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Pettus et al.
1H), 7.91 (d, J ) 8.6 Hz, 1H), 8.02 (s, 1H), 8.09 (d, J ) 8.9 Hz,
1H), 9.07 (s, 1H). MS (ESI, positive ion) m/z: 268.0 (M + 1).
(S)-1-(3-Methylmorpholino)phthalazin-6-ylboronic Acid (9).
A mixture of (S)-6-bromo-1-(3-methylmorpholino)phthalazine 8a
(1.60 g, 5.2 mmol), bis(pinacolato)diboron (2.0 g, 7.8 mmol), and
potassium acetate (2.5 g, 26 mmol) in DMF (25 mL) was degassed
for 20 min. It was then treated with 1,1′-bis(diphenylphosphino)-
ferrocenepalladium dichloride (0.38 g, 0.52 mmol). The reaction
mixture was stirred at 85 °C for 18 h. After cooling to room
temperature, the reaction mixture was diluted with 50 mL of EtOAc
and filtered through a Celite pad. The organic solvent of the filtrate
was removed in vacuo. The remaining residue was treated with 50
mL of Et2O and 50 mL of 1 N aqueous HCl, stirred for 5 min, and
filtered through a Celite pad again. The filtrate was transferred to
a separatory funnel. The aqueous phase was separated, and the
organic phase was washed with 10 mL of 1 N aqueous HCl. The
combined aqueous phases were concentrated in vacuo. The residue
was dissolved in 2.5 mL of MeOH and 2.5 mL of DMSO, purified
via a reverse phase HPLC [using a gradient of 10-60% of (0.1%
of trifluoroacetic acid in acetonitrile) in (0.1% of trifluoroacetic
acid in water)]. The title compound (910 mg, 64% yield) was
obtained as a white fluffy solid after drying the HPLC fractions in
Figure 6. Effect of 3c on CIA in Lewis rats. Arthritis was induced by
intradermal injection of porcine type II collagen emulsified 1:1 in
incomplete Freunds adjuvant (IFA). Animals were assigned to treatment
groups at disease onset (study day 0), which occurred 10-12 days
following immunization. Compound 3c or vehicle (1% Tween-80 in
Oraplus, pH 2.2) was administered orally once a day for 7 days.
Dexamethasone was administered once daily, sc, for 7 days. Paw
diameter was measured daily from day 0 through day 7. Area under
the paw swelling curve (AUC) was calculated and used to determine
percent inhibition of inflammation compared with vehicle controls. Data
points represent mean ( STE (n ) 8 rats/group): (/) p e 0.05 vs vehicle
control.
1
a lyophylizer. H NMR (DMSO-d6): δ 1.25 (m, 3H), 3.60-3.80
(m, 4H), 3.98 (m, 2H), 4.21 (m, 1H), 8.22 (d, J ) 9.4 Hz, 1H),
8.41 (d, J ) 8.6 Hz, 1H), 8.61 (s, 1H), 8.68 (br, 2H), 9.50 (s, 1H).
MS (ESI, positive ion) m/z: 274 (M + 1).
A similar procedure was used to prepare boronic acids 10-14.
(S)-1-(4-Acetyl-2-methylpiperazin-1-yl)phthalazin-6-ylboro-
nic Acid (10). MS (ESI, positive ion) m/z: 315 (M + 1).
(R)-1-(3-Methylmorpholino)phthalazin-6-ylboronic Acid (11).
1H NMR (DMSO-d6): δ 1.25 (m, 3H), 3.60-3.80 (m, 4H), 3.98
(m, 2H), 4.21 (m, 1H), 8.22 (d, J ) 9.4 Hz, 1H), 8.41 (d, J ) 8.6
Hz, 1H), 8.61 (s, 1H), 8.68 (br, 2H), 9.50 (s, 1H). MS (ESI, positive
ion) m/z: 274 (M + 1).
mL of NMP was added triethylamine (1.4 mL, 9.9 mmol). The
glass tube was sealed, and the mixture was heated at 165 °C in an
oil bath for 1 h. After cooling to room temperature, the reaction
mixture was loaded onto a silica gel column. Elution with a gradient
of 1-5% of (2 M NH3 in MeOH) in DCM gave the title compound
1
(2.07 g, 82% yield) as a yellow solid. H NMR (CDCl3): δ 1.19
(d, J ) 6.2 Hz, 3H), 3.39 (m, 1H), 3.65 (m, 2H), 4.02 (m, 4H),
7.90-7.98 (m, 2H), 8.06 (d, J ) 1.8 Hz, 1H), 9.14 (s, 1H). MS
(ESI, positive ion) m/z: 310 (M + 1).
1-(Isopropylamino)phthalazin-6-ylboronic Acid (12). 1H NMR
(DMSO-d6): δ 1.36 (t, J ) 6.2 Hz, 6H), 4.31 (m, 1H), 8.42 (d, J
) 8.2 Hz, 1H), 8.51 (s, 1H), 8.67 (d, J ) 8.4 Hz, 1H), 8.75 (br,
2H), 9.01 (s, 1H), 9.21 (br, 1H). MS (ESI, positive ion) m/z: 232
(M + 1).
A similar procedure was used to prepare compounds 8b-e.
(S)-1-(4-(6-Bromophthalazin-1-yl)-3-methylpiperazin-1-yl)-
ethanone (8b). 1H NMR (CDCl3): δ 1.20 (m, 3H), 2.16 (s, 1.5H),
2.20 (s, 1.5H), 3.40-3.80 (m, 5H), 4.01-4.44 (m, 2H), 7.93 (m,
2H), 8.07 (s, 1H), 9.14 (s, 1H). MS (ESI, positive ion) m/z: 350
(M + 1).
1
(S)-1-(sec-Butylamino)phthalazin-6-ylboronic Acid (13). H
NMR (DMSO-d6): δ 0.94 (t, J ) 7.2 Hz, 3H), 1.32 (d, J ) 6.4 Hz,
3H), 1.64 (m, 1H), 1.76 (m, 1H), 4.21 (m, 1H), 8.41 (m, 1H), 8.48
(s, 1H), 8.68 (m, 3H), 8.98 (s, 1H), 9.18 (br, 1H). MS (ESI, positive
ion) m/z: 246 (M + 1).
(R)-6-Bromo-1-(3-methylmorpholino)phthalazine (8c). 1H NMR
(CDCl3): δ 1.19 (d, J ) 6.2 Hz, 3H), 3.39 (m, 1H), 3.65 (m, 2H),
4.02 (m, 4H), 7.90-7.98 (m, 2H), 8.06 (d, J ) 1.8 Hz, 1H), 9.14
(s, 1H). MS (ESI, positive ion) m/z: 310 (M + 1).
1-Isopropoxyphthalazin-6-ylboronic Acid (14). 1H NMR (DM-
SO-d6): δ 1.47 (d, J ) 6.2 Hz, 6H), 5.60 (m, 1H), 8.16 (d, J ) 8.3
Hz, 1H), 8.40 (d, J ) 8.0 Hz, 1H), 8.56 (s, 1H), 8.60 (br, 2H),
9.48 (s, 1H). MS (ESI, positive ion) m/z: 233 (M + 1).
6-Bromo-N-isopropylphthalazin-1-amine (8d). 1H NMR
(CDCl3): δ 1.38 (d, J ) 6.5 Hz, 6H), 4.61 (m, 1H), 4.87 (br, 1H),
7.63 (d, J ) 8.8 Hz, 1H), 7.85 (d, J ) 8.6 Hz, 1H), 7.95 (d, J )
1.5 Hz, 1H), 8.84 (s, 1H). MS (ESI, positive ion) m/z: 267 (M +
1).
1-Isopropyl-6-methoxyphthalazine (17). To a solution of
1-chloro-6-methoxyphthalazine (638 mg, 3.28 mmol) and iron(III)
acetylacetonate (58 mg) in tetrahydrofuran (32 mL) and 1-methyl-
2-pyrrolidinone (3.2 mL) was added isopropylmagnesium chloride
(2.46 mL of 2.0 M in ether, 5.92 mmol) via a syringe. The resulting
mixture was stirred for 10 min, then diluted with EtOAc and
carefully quenched with a few drops of 10% HCl. The mixture
was washed with saturated aqueous NaHCO3 solution. The organic
layer was dried over MgSO4 and concentrated in vacuo. Purification
on an ISCO 40 g column (20-70% EtOAc in hexanes) afforded
1-isopropyl-6-methoxyphthalazine (288 mg, 43% yield) as a golden
brown oil. 1H NMR (CDCl3): δ 1.53 (d, J ) 6.85 Hz, 6H),
3.77-3.91 (m, 1H), 3.99 (s, 3H), 7.17 (d, J ) 2.35 Hz, 1H), 7.48
(dd, J ) 9.10, 2.64 Hz, 1H), 8.08 (d, J ) 9.19 Hz, 1H), 9.33 (s,
1H). MS (ESI, positive ion) m/z: 203.2 (M + 1).
1-Isopropylphthalazin-6-ol (18). A solution of 1-isopropyl-6-
methoxyphthalazine (205 mg, 1.01 mmol) in 1,2-dichloroethane
(5.0 mL) was treated with boron tribromide (5.07 mL of 1.0 M
solution in dichloroethane, 5.07 mmol), and the resulting golden
brown mixture was stirred at 80 °C for 18 h. The cooled mixture
was diluted with DCM, and the reaction was quenched with
saturated aqueous NH4Cl solution. The layers were separated, and
the aqueous layer was basified to pH ∼7 with 5 N NaOH, resulting
(S)-6-Bromo-N-sec-butylphthalazin-1-amine (8e). 1H NMR
(methanol-d4) δ: 1.01 (t, J ) 7.28 Hz, 3H), 1.32 (d, J ) 6.53 Hz,
3H), 1.68 (sept, J ) 7.03 Hz, 1H), 1.78 (sept, J ) 7.03 Hz, 1H),
4.36 (sex., J ) 6.53 Hz, 1H), 7.99 (d, J ) 8.53 Hz, 1H), 8.14 (s,
1H), 8.21 (d, J ) 8.53 Hz, 1H), 8.76 (s, 1H). MS (ESI, positive
ion) m/z: 281 (M + 1).
6-Bromo-1-isopropoxyphthalazine (8f). To a solution of pro-
pan-2-ol (1.23 g, 20.53 mmol) in THF (5.0 mL) and DMF (1.5
mL) at 0 °C was added slowly sodium hydride (411 mg of 60%
wt, 10.26 mmol) under a nitrogen atmosphere. The mixture was
stirred at 0 °C for 10 min, and 6-bromo-1-chlorophthalazine (1.00
g, 4.10 mmol) was added as solid. After the reaction mixture was
stirred at room temperature for 4 h, it was cooled with an ice bath,
treated with saturated ammonium chloride (10 mL), and extracted
with EtOAc (2 × 50 mL). The combined organic phases were
washed with brine (10 mL), dried over Na2SO4, filtered, and
concentrated. The brown residue was purified by silica gel
chromatography (eluted with 30-70% EtOAc in hexanes) to give
967 mg of the title compound (88%) as an off-white amorphous
solid. 1H NMR (CDCl3): δ 1.50 (d, J ) 6.0 Hz, 6H), 5.76 (m,

3-Amino-7-phthalazinylbenzoisoxazoles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6287
in the precipitation of a tan solid. Filtration, followed by drying
under high vacuum overnight, afforded 1-isopropylphthalazin-6-ol
(142 mg, 74% yield) as a tan solid. H NMR (DMSO-d6): δ 1.38
(d, J ) 6.85 Hz, 6H), 3.73-3.99 (m, 1H), 7.26 (d, J ) 2.15 Hz,
1H), 7.47 (dd, J ) 9.00, 2.35 Hz, 1H), 8.19 (d, J ) 8.80 Hz, 1H),
9.33 (s, 1H), 10.81 (br, 1H). MS (ESI, positive ion) m/z: 189.2 (M
+ 1).
it was poured onto 30 g of ice. The tan solid was filtered, washed
with water (2 × 5 mL) followed by EtOAc (2 × 30 mL), then
collected and dried to afford 2-fluoro-3-iodo-4-methylbenzoic acid
(3.3 g, LCMS >95% pure) as a tan crystalline solid. The filtrate
was transferred to a separatory funnel. The EtOAc layer was
separated, washed with brine (2 × 5 mL), dried, and concentrated
to afford additional 2-fluoro-3-iodo-4-methylbenzoic acid (1.6 g,
LCMS >95% pure) as a tan solid. H NMR (DMSO-d6): δ 2.48
(s, 3H), 7.27 (d, J ) 7.9 Hz, 1H), 7.75 (t, J ) 7.8 Hz, 1H), 12.01
(br, 1H). MS (ESI, positive ion) m/z: 280.9 (M + 1).
1
1
1-Isopropylphthalazin-6-yl Trifluoromethanesulfonate (19).
A mixture of 1-isopropylphthalazin-6-ol (208 mg, 1.11 mmol),
N-phenyltrifluoromethanesulfonimide (475 mg, 1.33 mmol), N,N-
dimethylpyridin-4-amine (13 mg), and triethylamine (0.23 mL, 1.66
mmol) was combined with chloroform (6.0 mL), and the mixture
was heated at 60 °C for 2 h, during which LCMS indicated the
reaction was complete. The cooled reaction mixture was diluted
with CH2Cl2 and washed with saturated aqueous NaHCO3 solution.
The organic layer was dried over MgSO4, filtered, and concentrated
in vacuo. Purification on an ISCO 12 g column (20-50% EtOAc
in hexanes) afforded 1-isopropylphthalazin-6-yl trifluoromethane-
2-Fluoro-3-iodo-4-methylbenzaldehyde (24). To a solution of
2-fluoro-3-iodo-4-methylbenzoic acid (2.0 g, 7.1 mmol) in anhy-
drous THF (10 mL) was added trimethyl borate (0.80 mL, 7.1
mmol) at room temperature. The resulting mixture was stirred at
room temperature for 15 min, then cooled with an ice bath and
treated with borane-dimethyl sulfide (6.4 mL of 2.0 M solution in
THF, 12.8 mmol) slowly. The reaction mixture was stirred at room
temperature for 4 h. MeOH (0.5 mL) was then added dropwise at
room temperature. After being stirred for 30 min, the reaction
mixture was concentrated in vacuo. The residue was diluted with
EtOAc and washed with saturated aqueous solution of sodium
bicarbonate followed by brine. The resulting organic solution was
then dried over magnesium sulfate and concentrated under reduced
pressure to give 2-fluoro-3-iodo-4-methylphenyl)methanol (1.69 g,
89% yield) as an off-white amorphous solid, which was used
1
sulfonate (277 mg, 78% yield) as a maize color solid. H NMR
(CDCl3): δ 1.56 (d, J ) 6.85 Hz, 6H), 3.84 - 3.96 (m, 1H), 7.79
(dd, J ) 9.10, 2.45 Hz, 1H), 7.87 (d, J ) 2.35 Hz, 1H), 8.32 (d, J
) 9.19 Hz, 1H), 9.47 (s, 1H). MS (ESI, positive ion) m/z: 321.1
(M + 1).
1-Isopropyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)phthalazine (15). Into a 50 mL round-bottomed flask under
argon were added 1-isopropylphthalazin-6-yl trifluoromethane-
sulfonate (269 mg, 0.84 mmol), 4,4,5,5-tetramethyl-2-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (320 mg,
1.26 mmol), dichloro[1,1′-bis(diphenylphosphino)ferrocene]palla-
dium(II) dichloromethane adduct (61 mg, 84 µmol), and potassium
acetate (412 mg, 4.2 mmol), followed by 1,4-dioxane (3.0 mL).
The mixture was heated at 80 °C for 2 h. The cooled reaction
mixture was diluted with EtOAc and filtered via a pad of Celite.
The filtrate was extracted with 10% HCl, and the organic layer
was discarded. The aqueous layer was neutralized with 1 N NaOH
to pH ∼7, resulting in the precipitation of the title compound (99
1
without further purification. H NMR (DMSO-d6): δ 7.34 (t, J )
7.6 Hz, 1H), 7.16 (8.0 Hz, 1H), 5.27 (t, J ) 5.7 Hz, 1H), 4.51 (d,
J ) 5.7 Hz, 2H), 2.40 (s, 3H).
A solution of the above-obtained (2-fluoro-3-iodo-4-methylphe-
nyl)methanol (1.69 g, 6.35 mmol) in 50 mL of DCM at room
temperature was treated with MnO2 (5.57 g, 63.5 mmol), and the
resulting mixture was stirred overnight. The mixture was filtered
through a pad of Celite and eluted with DCM. The filtrate was
concentrated. Purification was effected by flash chromatography
on silica gel (15-35% EtOAc in hexanes) to provide the title
compound (1.5 g, 89% yield) as an off-white solid. 1H NMR
(CDCl3): δ 2.56 (s, 3H), 7.19 (d, J ) 8.0 Hz, 1H), 7.74 (t, J ) 7.4
Hz, 1H), 10.29 (s, 1H). MS (ESI, positive ion) m/z: 264.9 (M +
1).
(E)-2-Fluoro-3-iodo-4-methylbenzaldehyde Oxime (25). A
solution of 2-fluoro-3-iodo-4-methylbenzaldehyde (1.26 g, 4.77
mmol) in EtOH (5 mL) at room temperature was treated with 5
mL of hydroxylamine (50% wt in water), and the reaction was
allowed to continue for 3 h, when the crude LCMS indicated no
remaining starting material. Volatiles were removed, and the residue
was treated with 5 mL of water and extracted with EtOAc (3 × 15
mL). The combined EtOAc layers were dried and concentrated.
Purification was effected by flash chromatography on silica gel
(10-50% EtOAc in hexanes) to provide the title compound (1.13
g, 85% yield) as an off-white crystalline solid. 1H NMR (CDCl3):
δ 2.49 (s, 3H), 7.06 (d, J ) 8.1 Hz, 1H), 7.42 (br, 1H), 7.63 (t, J
) 7.6 Hz, 1H), 8.34 (s, 1 H). MS (ESI, positive ion) m/z: 280.0
(M + 1).
N-Cyclopropyl-2-fluoro-N′-hydroxy-3-iodo-4-methylbenzami-
dine (26a). To a solution of 2-fluoro-3-iodo-4-methylbenzaldehyde
oxime (0.48 g, 1.7 mmol) in DMF (0.5 mL) was added N-
chlorosuccinimide (83 mg, 0.62 mmol), and the mixture was heated
at 55 °C for 5 min. The mixture was allowed to cool to <50 °C,
and additional N-chlorosuccinimide (166 mg, 1.24 mmol) was
added. Then the mixture was heated at 55 °C for 10 min. The
reaction mixture was allowed to cool to room temperature, treated
with 5 mL of water, and extracted with EtOAc (3 × 20 mL). The
combined EtOAc layers were washed with 5 mL of brine, dried,
and concentrated to afford the intermediate 2-fluoro-3-iodo-4-
methylbenzoyl chloride oxime as a light-yellow amorphous solid,
which was used without further purification. MS (ESI, positive ion)
m/z: 314.3 (M + 1).
1
mg, 40% yield) as a tan solid. H NMR (methanol-d4): δ 1.19 (s,
6H), 1.40 (s, 6H), 1.50 (d, J ) 6.6 Hz, 6H), 4.03 (m, 1H), 8.33 (m,
3H), 9.47 (s, 1H). MS (ESI, positive ion) m/z: 299.3 (M + 1).
2-Fluoro-3-iodo-4-methylbenzonitrile (22). A solution of 2,2,6,6-
tetramethyl-4-piperidine (TMP) (45 mL, 267 mmol) in THF (400
mL) was cooled below -80 °C. n-Butyllithium (2.5 M in hexane,
110 mL, 275 mmol) was added, slowly maintaining the temperature
of the mixture below -70 °C. After the addition was completed,
the reaction mixture was warmed to -50 °C and stirred at this
temperature for 30 min. The clear solution became turbid, indicating
salt formation. It was cooled to -80 °C again, and a solution of
2-fluoro-4-methylbenzonitrile (21) (32.4 g, 240 mmol) in THF (150
mL) was slowly added taking care that the temperature of the
reaction mixture remained below -70 °C. It was then warmed to
-50 °C and stirred for 30 min. The mixture was then cooled to
-70 °C, and a saturated solution of I2 (67 g, 264 mmol) in THF
was added slowly maintaining the temperature at -70 °C. After
complete addition, the mixture was warmed to ambient temperature.
It was added to a solution of Na2S2O3 (160 g in 1.5 L of water)
and stirred for an hour. The organic layer was separated, and the
aqueous layer was extracted with EtOAc. The organic layers were
combined, washed with brine, dried over Na2SO4, and filtered. The
volatiles were evaporated under reduced pressure. The crude product
was subjected to vacuum distillation. At about 60 °C, excess TMP
was removed. At about 100 °C, the starting compound 2-fluoro-
4-methylbenzonitrile and a small amount of 2-fluoro-3-iodo-4-
methylbenzonitrile were removed. Finally, at 115 °C, pure 2-fluoro-
3-iodo-4-methylbenzonitrile (22) was obtained (30 g, 48% yield)
as an off-white amorphous solid upon cooling to room temperature.
1H NMR (CDCl3): δ 2.56 (s, 3H), 7.15 (d, J ) 8.0 Hz, 1H), 7.49
(m, 1H). MS (ESI, positive ion) m/z: 261.9 (M + 1).
2-Fluoro-3-iodo-4-methylbenzoic Acid (23). A mixture of
2-fluoro-3-iodo-4-methylbenzonitrile (5.0 g, 19 mmol) in dioxane
(10 mL) and 60% H2SO4 (10 mL) was heated at 115 °C in an oil
bath for 18 h. After the mixture was cooled to room temperature,
Cyclopropylamine (0.60 mL, 8.6 mmol) was added dropwise to
an ice-cold solution of the above-obtained 2-fluoro-3-iodo-4-
methylbenzoyl chloride oxime in anhydrous THF (2 mL). The
reaction mixture was stirred at room temperature for 3 h, when

6288 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Pettus et al.
LCMS indicated the reaction to be completed. The volatiles were
removed under reduced pressure. Purification was effected by flash
chromatography on silica gel (25-60% EtOAc in hexanes) to
provide the title compound (505 mg, 89% yield) as an amorphous
2,2,2-trifluoro-N-(7-iodo-6-methylbenzo[d]isoxazol-3-yl)aceta-
mide as an off-white solid, which was used prior to further
purification. MS (ESI, positive ion) m/z: 370.9 (M + 1).
To the above-obtained 2,2,2-trifluoro-N-(7-iodo-6-methylben-
zo[d]isoxazol-3-yl)acetamide in acetone (5.0 mL) at room temper-
ature were added dimethyl sulfate (766 mg, 6.07 mmol) and
potassium carbonate (1.40 g, 10.12 mmol). The mixture was heated
at 50 °C in an oil bath for 5 h, then cooled to room temperature,
filtered through a pad of Celite, and rinsed with additional acetone
(2 × 5 mL). The filtrate was concentrated to dryness to provide
2,2,2-trifluoro-N-(7-iodo-6-methylbenzo[d]isoxazol-3-yl)-N-methy-
lacetamide, which was used prior to further purification. MS (ESI,
positive ion) m/z: 385.0 (M + 1).
1
off-white solid. H NMR (CDCl3): δ 7.23 (d, J ) 7.3 Hz, 1H),
7.08 (d, J ) 7.9 Hz, 1H), 5.60 (br, 1H), 2.51 (s, 3H), 2.43 (m,
1H), 0.40 (m, 4H). MS (ESI, positive ion) m/z: 335 (M + 1).
N-Cyclopropyl-7-iodo-6-methylbenzo[d]isoxazol-3-amine (27).
1,8-Diazabicyclo [5.4.0]-7-undecene (0.25 mL, 1.65 mmol) was
added to N-cyclopropyl-2-fluoro-N′-hydroxy-3-iodo-4-methylben-
zamidine (26a) (500 mg, 1.5 mmol) in anhydrous THF (2.0 mL)
and NMP (0.5 mL), then heated in a microwave at 150 °C for 70
min. Volatiles were evaporated under reduced pressure. Purification
was effected by flash chromatography on silica gel (25-75% EtOAc
in hexanes) to provide the title compound (338 mg, 72% yield) as
At room temperature, the above-obtained 2,2,2-trifluoro-N-(7-
iodo-6-methylbenzo[d]isoxazol-3-yl)-N-methylacetamide was dis-
solved in 10 mL of MeOH and treated with 10 mL of 1 N NaOH.
After the mixture was stirred at room temperature for 2 h, the
solvents were removed under reduced pressure. The residue was
treated with saturated ammonium chloride solution (20 mL) and
extracted with EtOAc (3 × 50 mL). The combined organic layers
were dried over Na2SO4 and concentrated. Purification was effected
by flash chromatography on silica gel (25-65% EtOAc in hexanes)
to provide 7-iodo-N,6-dimethylbenzo[d]isoxazol-3-amine (794 mg,
1
an off-white crystalline solid. H NMR (CDCl3): δ 0.70 (m, 2H),
0.86 (m, 2H), 2.57 (s, 3H), 2.83 (m, 1H), 4.65 (s, 1H), 7.08 (d, J
) 7.9 Hz, 1H), 7.39 (d, J ) 7.8 Hz, 1H). MS (ESI, positive ion)
m/z: 315.0 (M + 1).
A similar procedure was used to prepare compounds 28 and 29.
N-Ethyl-7-iodo-6-methylbenzo[d]isoxazol-3-amine (28). 1H
NMR (CDCl3): δ 1.34 (t, J ) 7.3 Hz, 3H), 2.57 (s, 3H), 3.46 (m,
2H), 4.10 (br, 1H), 7.08 (d, J ) 8.0 Hz, 1H), 7.29 (d, J ) 7.9 Hz,
1H). MS (ESI, positive ion) m/z: 303 (M + 1).
1
68% yield) as an off-white crystalline solid. H NMR (CDCl3): δ
7-Iodo-N,6-dimethylbenzo[d]isoxazol-3-amine (29). 1H NMR
(CDCl3): δ 2.57 (s, 3H), 3.09 (s, 3H), 4.12 (br, 1H), 7.07 (d, J )
7.9 Hz, 1H), 7.29 (d, J ) 7.9 Hz, 1H). MS (ESI, positive ion) m/z:
289 (M + 1).
2.57 (s, 3H), 3.09 (s, 3H), 4.12 (br, 1H), 7.07 (d, J ) 7.9 Hz, 1H),
7.29 (d, J ) 7.9 Hz, 1H). MS (ESI, positive ion) m/z: 289.0 (M +
1).
(4-Chloro-2-fluorobenzyloxy)(tert-butyl)dimethylsilane (33).
To a solution of (4-chloro-2-fluorophenyl)methanol (4.60 g, 28.6
mmol) in DCM (100 mL) was added imidazole (1.95 g, 28.6 mmol)
and tert-butylchlorodimethylsilane (4.32 g, 28.6 mmol) at room
temperature. The reaction mixture was stirred for 12 h at room
temperature. The white precipitate was removed by filtration, and
the filtrate was washed with 10% HCl. The separated aqueous phase
was extracted with DCM (2 × 40 mL). The combined organic
phases were washed with saturated aqueous solution of sodium
bicarbonate followed by brine. The resulting organic solution was
dried over magnesium sulfate and concentrated under reduced
pressure. Purification was effected by flash chromatography on silica
gel (eluted with hexanes) to give the title compound (6.64 g, 84.3%
yield) as a colorless oil. 1H NMR (CDCl3): δ 0.00 (s, 6H), 0.81 (s,
9H), 4.64 (s, 2H), 6.91 (dd, J ) 9.9, 1.8 Hz, 1H), 7.01 (d, J ) 8.3
Hz, 1H), 7.31 (t, J ) 8.1 Hz, 1H).
7-Iodo-6-methylbenzo[d]isoxazol-3-amine (30). To a 250 mL
three-necked round-bottomed flask equipped with a mechanical
stirrer and under nitrogen containing anhydrous DMF (100 mL)
was added acetohydroxamic acid (4.65 g, 62 mmol). After a clear
solution was obtained, potassium tert-butoxide (6.94 g, 62 mmol)
was added to the mixture. The cloudy white mixture was allowed
to stir for 30 min. 2-Fluoro-3-iodo-4-methylbenzonitrile 22 (8.0 g,
31 mmol) was added, and the reaction mixture was allowed to stir
at room temperature for 6 h. Additional reagents of acetohydroxamic
acid (1.16 g, 15.5 mmol) and potassium tert-butoxide (1.74 g, 15.5
mmol) were added to the reaction mixture, and the stirring was
continued for 12 h. The reaction mixture was distilled under reduced
pressure to remove most of the DMF, and the remaining residue
was partitioned between EtOAc (250 mL) and saturated ammonium
chloride solution (50 mL). The water layer was washed with EtOAc
(2 × 100 mL). The combined organic layers were washed with
brine (50 mL), dried over MgSO4, and evaporated under reduced
pressure. Purification was effected by flash chromatography on silica
gel (20-70% EtOAc in hexanes) to provide the title compound
(5.8 g, 69% yield) as an off-white crystalline solid. 1H NMR
(CDCl3): δ 2.59 (s, 3H), 4.24 (br, 2H), 7.13 (d, J ) 8.1 Hz, 1H),
7.35 (d, J ) 8.1 Hz, 1H). MS (ESI, positive ion) m/z: 275.0 (M +
1).
N-(7-Iodo-6-methylbenzo[d]isoxazol-3-yl)acetamide (31). To
a mixture of 7-iodo-6-methylbenzo[d]isoxazol-3-amine (0.30 g, 1.09
mmol) and cesium carbonate (0.43 g, 1.31 mmol) in DCM (10.0
mL) was added acetyl chloride (95 mg, 1.20 mmol). After the
mixture was stirred at room temperature for 20 min, it was treated
with 5.0 mL of water and stirred for 10 min. The resulting mixture
was extracted with EtOAc (3 × 30 mL). The combined organic
phases were washed with saturated aqueous solution of sodium
bicarbonate followed by brine. The resulting organic solution was
dried over magnesium sulfate and concentrated under reduced
pressure to give the title compound (0.32 g, 92% yield) as an off-
white amorphous solid. 1H NMR (CDCl3): δ 2.02 (s, 3H), 2.46 (s,
3H), 7.13 (d, J ) 8.0 Hz, 1H), 7.36 (d, J ) 8.0 Hz, 1H), 8.00 (br,
1H). MS (ESI, positive ion) m/z: 317.0 (M + 1).
(4-Chloro-2-fluoro-3-iodobenzyloxy)(tert-butyl)dimethylsi-
lane (34). To a solution of diisopropylamine (3.09 mL, 21.8 mmol)
in THF (60 mL) under N2 at 0 °C was slowly added n-butyllithium
(8.72 mL of 2.5 M solution in hexanes, 21.8 mmol) over 5 min.
After being stirred at 0 °C for 20 min, the reaction mixture was
cooled to -78 °C, and a solution of silyl ether 33 (5.00 g, 18.2
mmol) in 15 mL of THF was added slowly over 5 min. After the
mixture was stirred for 2 h at -78 °C, a solution of iodine (5.54 g,
21.8 mmol) in 25 mL of THF was added slowly over 10 min. The
reaction mixture was allowed to stir for 20 min at -78 °C and
warmed to room temperature in 20 min. The reaction was quenched
with saturated sodium thiosulfate solution and extracted with Et2O
(2 × 60 mL). The combined organic phases were washed with
saturated sodium bicarbonate and brine. The resulting organic
solution was then dried over magnesium sulfate and concentrated
under reduced pressure. Purification was effected by flash chro-
matography on silica gel (5-10% DCM in hexanes) to give the
1
title compound (6.11 g, 84% yield) as a colorless oil. H NMR
(CDCl3): δ 0.01 (s, 6H), 0.80 (s, 9H), 4.65 (s, 2H), 7.17 (d, J )
7.1 Hz, 1H), 7.30 (t, J ) 7.5 Hz, 1H).
(4-Chloro-2-fluoro-3-iodophenyl)methanol (35). To a solution
of (4-chloro-2-fluoro-3-iodobenzyloxy)(tert-butyl)dimethylsilane 34
(6.64 g, 16.6 mmol) in THF (60 mL) was added tetrabutylammo-
nium fluoride (19.9 mL of 1.0 M solution in THF, 19.9 mmol).
The reaction solution was stirred at room temperature for 1 h. The
volatiles were removed via vacuum. The brown residue was diluted
with 150 mL of Et2O and washed with saturated aqueous solution
of sodium bicarbonate and brine. The resulting organic solution
Alternative Procedure for the Synthesis of 7-Iodo-N,6-
dimethylbenzo[d]isoxazol-3-amine (29). At room temperature,
trifluoroacetic anhydride (0.63 mL, 4.45 mmol) was added to a
solution of 7-iodo-6-methylbenzo[d]isoxazol-3-amine 30 (1.11 g,
4.05 mmol) in 10 mL of DCM. After the mixture was stirred for
6 h, the volatiles were removed under reduced pressure to provide

3-Amino-7-phthalazinylbenzoisoxazoles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6289
was dried over magnesium sulfate and concentrated under reduced
pressure. Purification was effected by flash chromatography on silica
gel (eluted with DCM) to give the title compound (4.60 g, 97%
yield) as a colorless oil. H NMR (CDCl3) δ 1.90 (t, J ) 5.7 Hz,
1H), 4.66 (d, J ) 5.7 Hz, 2H), 7.16-7.32 (m, 2H). MS (ESI,
positive ion) m/z: 289.0 (M + 1).
N,6-Dimethyl-7-(1-((S)-3-methylmorpholino)phthalazin-6-yl)-
benzo[d]isoxazol-3-amine (3c). 1H NMR (CDCl3): δ 1.25 (d, J )
6.4 Hz, 3H), 2.41 (s, 3H), 3.10 (d, J ) 5.2 Hz, 3H), 3.43 (m, 1H),
3.70 (m, 2H), 3.91-4.15 (m, 4H), 4.50 (m, 1H), 7.21 (d, J ) 8.1
Hz, 1H), 7.43 (d, J ) 8.0 Hz, 1H), 7.92 (d, J ) 8.4 Hz, 1H), 7.99
(s, 1H), 8.20 (d, J ) 8.4 Hz, 1H), 9.23 (s, 1H). MS (ESI, positive
ion) m/z: 390 (M + 1). Anal. (C22H23N5O2) C, H, N.
6-Methyl-7-(1-((S)-3-methylmorpholino)phthalazin-6-yl)ben-
zo[d]isoxazol-3-amine (3d). 1H NMR (CDCl3): δ 1.24 (d, J ) 6.4
Hz, 3H), 2.43 (s, 3H), 3.43 (m, 1H), 3.71 (m, 2H), 3.91-4.15 (m,
5H), 4.42 (s, 1H), 7.27 (d, J ) 8.3 Hz, 1H), 7.49 (d, J ) 8.3 Hz,
1H), 7.91 (d, J ) 8.4 Hz, 1H), 7.98 (s, 1H), 8.22 (d, J ) 8.4 Hz,
1H), 9.25 (s, 1H). MS (ESI, positive ion) m/z: 376 (M + 1).
1
6-Chloro-7-iodo-N-methylbenzo[d]isoxazol-3-amine (36). Fol-
lowing the procedure described for the synthesis of compound 27,
using (4-chloro-2-fluoro-3-iodophenyl)methanol (35), the title
compound was obtained (43% overall yield for five steps) as an
1
off-white solid. H NMR (CDCl3): δ 7.34 (d, J ) 8.4 Hz, 1H),
7.28 (d, J ) 8.4 Hz,1H), 4.16 (br, 1H), 3.08 (d, J ) 4.7 Hz, 3H).
MS (ESI, positive ion) m/z: 309 (M + 1).
6-Chloro-7-iodo-N-isopropylbenzo[d]isoxazol-3-amine (37).
Following the procedure described for the synthesis of compound
27, using (4-chloro-2-fluoro-3-iodophenyl)methanol (35), the title
compound was obtained (41% overall yield for five steps) as an
off-white solid. H NMR (CDCl3): δ 7.34 (d, J ) 8.4 Hz, 1H),
7.28 (d, J ) 8.4 Hz, 1H), 4.01 (br, 1H), 3.97 (m, 1H), 1.34 (d, J
) 6.2 Hz, 6H). MS (ESI, positive ion) m/z: 338 (M + 1).
7-Iodo-6-methyl-1H-indazol-3-amine (38). A solution of 2-fluoro-
3-iodo-4-methylbenzonitrile (200 mg, 0.77 mmol) in hydrazine
hydrate (0.48 mL, 1.53 mmol) in 0.5 mL of THF was heated at 80
°C in an oil bath for 16 h. The mixture was cooled to room
temperature and partitioned between EtOAc and water. The EtOAc
layer was separated, dried, and concentrated. Purification was
effected by flash chromatography on silica gel (50-80% EtOAc
in hexanes) to provide the title compound (189 mg, 90% yield) as
an off-white amorphous solid. 1H NMR (CDCl3): δ 8.80 (br, 1H),
7.41 (d, J ) 8.0 Hz, 1H), 6.96 (d, J ) 8.0 Hz, 1H), 4.18 (br, 2H),
2.55 (s, 3H). MS (ESI, positive ion) m/z: 274.0 (M + 1).
N-(6-Methyl-7-(1-((S)-3-methylmorpholino)phthalazin-6-yl)-
benzo[d]isoxazol-3-yl)acetamide (3e). H NMR (CDCl3) δ 1.25
1
(d, J ) 6.4 Hz, 3H), 2.03 (s, 3H), 2.43 (s, 3H), 3.45 (m, 1H), 3.71
(m, 2H), 3.91-4.13 (m, 4H), 4.42 (s, 1H), 7.27 (d, J ) 8.3 Hz,
1H), 7.49 (d, J ) 8.3 Hz, 1H), 7.91 (d, J ) 8.4 Hz, 1H), 7.98 (s,
1H), 8.22 (m, 1H), 9.25 (s, 1H). MS (ESI, positive ion) m/z: 418
(M + 1). HRMS (EI) m/z calcd for C23H24N5O3 [M + H]+:
418.1879; found, 418.1897.
1
1-((S)-4-(6-(3-(Cyclopropylamino)-6-methylbenzo[d]isoxazol-
1
7-yl)phthalazin-1-yl)-3-methylpiperazin-1-yl)ethanone (3f). H
NMR (CDCl3) δ 0.73 (m, 2H), 0.88 (m, 2H), 1.24 (m, 3H), 2.19
(s, 3H), 2.43 (s, 3H), 2.86 (m, 1H), 3.54-3.86 (m, 4H), 4.13 (m,
3H), 4.75 (d, J ) 3.7 Hz, 1H), 7.22 (m, 1H), 7.53 (m, 1H), 8.01
(m, 2H), 8.17 (m, 1H), 9.24 (s, 1H). MS (ESI, positive ion) m/z:
457.0 (M + 1).
6-(3-(Cyclopropylamino)-6-methylbenzo[d]isoxazol-7-yl)-N,N-
1
dimethylphthalazin-1-amine (3g). H NMR (CDCl3) δ 0.72 (m,
2H), 0.86 (m, 2H), 1.41 (d, J ) 6.4 Hz, 6H), 2.39 (s, 3H), 2.86 (m,
1H), 4.71 (m, 1H), 4.74 (br, 1H), 4.95 (br, 1H), 7.20 (d, J ) 8.2
Hz, 1H), 7.50 (d, J ) 8.0 Hz, 1H), 7.85 (m, 3H), 8.94 (s, 1H). MS
(ESI, positive ion) m/z: 373.8 (M + 1).
7-Iodo-6-methylbenzo[d]isothiazol-3-amine (39). A mixture of
2-fluoro-3-iodo-4-methylbenzonitrile (130 mg, 0.50 mmol), sulfur
(16 mg, 0.50 mmol), and ammonium hydroxide (28-30%, 0.25
mL) in 2-methoxyethanol (1.5 mL) was heated in an oil bath at
135 °C for 12 h. Volatiles were removed under reduced pressure.
Purification was effected by flash chromatography on silica gel
(25-50% EtOAc in hexanes) to provide the title compound (45
6-Chloro-N-cyclopropyl-7-(1-((S)-3-methylmorpholino)ph-
1
thalazin-6-yl)benzo[d]isoxazol-3-amine (3h). H NMR (CDCl3)
δ 9.21-9.30 (s, 1H), 8.22 (d, J ) 8.6 Hz, 1H), 8.14 (s, 1H), 8.03
(d, J ) 8.4 Hz, 1H), 7.51 (d, J ) 8.4 Hz, 1H), 7.41 (d, J ) 8.4 Hz,
1H), 4.63 (s, 1H), 3.92-4.16 (m, 4H), 3.64-3.74 (m, 2H), 3.44
(m, 1H), 3.11 (d, J ) 4.7 Hz, 3H), 1.25 (d, J ) 6.3 Hz, 3H). MS
(ESI, positive ion) m/z: 410.2 (M + 1). HRMS (EI) m/z calcd for
C21H21ClN5O2 [M + H]+: 410.1384; found, 410.1399.
6-Chloro-N-isopropyl-7-(1-((S)-3-methylmorpholino)phthalazin-
6-yl)benzo[d]isoxazol-3-amine (3i). 1H NMR (CDCl3): δ 9.25 (s,
1H), 8.21 (d, J ) 8.6 Hz, 1H), 8.14 (s, 1H), 8.03 (dd, J ) 8.6, 1.6
Hz, 1H), 7.39-7.56 (m, 3H), 4.33 (d, J ) 7.4 Hz, 1H), 3.93-4.14
(m, 4H), 3.66-3.75 (m, 2H), 3.42 (m, 1H), 1.36 (d, J ) 2.0 Hz,
6H), 1.24 (d, J ) 4.0 Hz, 3H). MS (ESI, positive ion) m/z: 438.3
(M + 1). HRMS (EI) m/z calcd for C23H25ClN5O2 [M + H]+:
438.1691; found, 438.1705.
1
mg, 31% yield) as a yellow crystalline solid. H NMR (DMSO-
d6): δ 7.65 (d, J ) 8.1 Hz, 1H), 7.19 (d, J ) 8.0 Hz, 1H), 3.35 (br,
2H), 2.51 (s, 3H). MS (ESI, positive ion) m/z: 291.0 (M + 1).
N-Cyclopropyl-6-methyl-7-(1-((S)-3-methylmorpholino)ph-
thalazin-6-yl)benzo[d]isoxazol-3-amine (3a). A mixture of (S)-
1-(3-methylmorpholino)phthalazin-6-ylboronic acid 9 (0.11 g, 0.41
mmol), N-cyclopropyl-7-iodo-6-methylbenzo[d]isoxazol-3-amine 27
(0.10 g, 0.32 mmol), tetrakis(triphenylphosphine)palladium(0) (18
mg, 0.016 mmol) in 2.0 mL of 1,4-dioxane, and sodium carbonate
(0.32 mL of 2 N solution) in a sealed glass tube was heated in a
Personal Chemistry microwave at 130 °C for 20 min. The mixture
was diluted with EtOAc (30 mL) and washed with 1 N NaOH (5
mL) followed by brine. The organic phase was dried over
magnesium sulfate and concentrated under reduced pressure.
Purification was effected by flash chromatography on silica gel
(eluted with 80-100% EtOAc in hexanes) to give the title
N,6-Dimethyl-7-(1-((R)-3-methylmorpholino)phthalazin-6-yl)-
benzo[d]isoxazol-3-amine (3j). 1H NMR (CDCl3): δ 1.25 (d, J )
6.4 Hz, 3H), 2.41 (s, 3H), 3.10 (d, J ) 5.2 Hz, 3H), 3.43 (m, 1H),
3.70 (m, 2H), 3.91-4.15 (m, 4H), 4.50 (m, 1H), 7.21 (d, J ) 8.1
Hz, 1H), 7.43 (d, J ) 8.0 Hz, 1H), 7.92 (d, J ) 8.4 Hz, 1H), 7.99
(s, 1H), 8.20 (d, J ) 8.4 Hz, 1H), 9.23 (s, 1H). MS (ESI, positive
ion) m/z: 390 (M + 1).
N-Isopropyl-6-(6-methyl-3-(methylamino)benzo[d]isoxazol-7-
yl)phthalazin-1-amine (3k). 1H NMR (CDCl3): δ 1.41 (d, J )
6.3 Hz, 6H), 2.38 (s, 3H), 3.11 (d, J ) 5.3 Hz, 3H), 4.27 (br, 1H),
4.70 (m, 1H), 4.95 (br, 1H), 7.21 (d, J ) 8.1 Hz, 1H), 7.41 (d, J
) 8.0 Hz, 1H), 7.86 (m, 3H), 8.94 (s, 1H). MS (ESI, positive ion)
m/z: 347.8 (M + 1).
N-(S)-sec-Butyl-6-(6-methyl-3-(methylamino)benzo[d]isoxazol-
7-yl)phthalazin-1-amine (3l). 1H NMR (CDCl3): δ 1.05 (m, 3H),
1.38 (m, 3H), 1.60-1.80 (m, 2H), 2.43 (s, 3H), 3.22 (s, 3H), 4.28
(br, 1H), 4.58 (m, 1H), 4.96 (br, 1H), 7.20 (d, J ) 7.6 Hz, 1H),
7.28 (m, 1H), 7.41 (d, J ) 7.9 Hz, 1H), 7.86 (m, 2H), 8.93 (s, 1H).
MS (ESI, positive ion) m/z: 362 (M + 1).
1
compound (59 mg, 45% yield) as a yellow amorphous solid. H
NMR (CDCl3): δ 0.79-0.91 (m, 3H), 1.16-1.31 (m, 4H), 2.41 (s,
3H), 2.81 (m, 1H), 3.43 (m, 1H), 3.60 (m, 2H), 3.91-4.15 (m,
4H), 5.03 (s, 1H), 7.21 (d, J ) 8.0 Hz, 1H), 7.56 (d, J ) 8.0 Hz,
1H), 7.91 (d, J ) 8.4 Hz, 1H), 8.00 (s, 1H), 8.20 (d, J ) 8.4 Hz,
1H), 9.23 (s, 1 H). MS (ESI, positive ion) m/z: 416 (M + 1). Anal.
(C24H25N5O2) C, H, N.
Similar procedures were used to prepare compounds 3b-n, 40,
and 41.
N-Ethyl-6-methyl-7-(1-((S)-3-methylmorpholino)phthalazin-
6-yl)benzo[d]isoxazol-3-amine (3b). 1H NMR (CDCl3): δ 1.27 (d,
J ) 6.6 Hz, 3H), 1.36 (t, J ) 7.0 Hz, 3H), 2.42 (s, 3H), 3.48 (m,
3H), 3.70 (m, 2H), 3.91-4.15 (m, 5H), 7.23 (d, J ) 8.2 Hz, 1H),
7.43 (d, J ) 8.0 Hz, 1H), 7.92 (d, J ) 8.4 Hz, 1H), 8.01 (s, 1H),
8.20 (d, J ) 8.4 Hz, 1H), 9.28 (s, 1H). MS (ESI, positive ion) m/z:
404 (M + 1). Anal. (C23H25N5O2) C, H, N.

6290 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
7-(1-Isopropoxyphthalazin-6-yl)-N,6-dimethylbenzo[d]isoxazol-
Pettus et al.
LPS-Induced TNFr Production in THP-1 Cells. THP1 cells
were resuspended in fresh THP1 media (RPMI 1640, 10% heat-
inactivated FBS, 1× PGS, 1× NEAA, plus 30 µM ꢀME) at a
concentration of 1.5 × 106 cells/mL. One hundred microliters of
cells per well were plated in a flat bottom polystyrene 96-well tissue
culture plate. Then 200 µg/mL of bacterial LPS (Sigma) was
prepared in THP1 media and transferred to the first 11 columns of
a 96-well polypropylene plate. Column 12 contained only THP1
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 control and LO control, respectively) and contained
only DMSO. An amount of 1 µL 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 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.
1
3-amine (3m). H NMR (methanol-d4) δ: 1.56 (d, J ) 6.02 Hz,
6H), 2.40 (s, 3H), 2.98 (s, 3H), 5.72 (t, J ) 6.02 Hz, 1H), 7.30 (d,
J ) 8.53 Hz, 1H), 7.67 (d, J ) 8.03 Hz, 1H), 8.03 (d, J ) 8.03
Hz, 1H), 8.14 (s, 1H), 8.38 (d, J ) 8.53 Hz, 1H), 9.25 (s, 1H). MS
(ESI, positive ion) m/z: 348.8 (M + 1). Anal. (C20H20N4O2) C,
H, N.
7-(1-Isopropylphthalazin-6-yl)-N,6-dimethylbenzo[d]isoxazol-
1
3-amine (3n). H NMR (CDCl3): δ 1.59 (d, J ) 6.85 Hz, 6H),
2.41 (s, 3H), 3.11 (d, J ) 5.09 Hz, 3H), 3.89 - 4.04 (m, 1H), 4.27
(br, 1H), 7.23 (d, J ) 8.22 Hz, 1H), 7.43 (d, J ) 8.02 Hz, 1H),
7.98 (d, J ) 8.61 Hz, 1H), 8.03 (s, 1H), 8.27 (d, J ) 8.41 Hz, 1H),
9.43 (s, 1H). MS (ESI, positive ion) m/z: 333.2 (M + 1). HRMS
(EI) m/z calcd for C20H21N4O [M + H]+: 333.1715; found,
333.1729.
6-Methyl-7-(1-((S)-3-methylmorpholino)phthalazin-6-yl)-1H-
1
indazol-3-amine (40). H NMR (CDCl3): δ 1.25 (d, J ) 6.3 Hz,
3H), 2.33 (s, 3H), 3.41 (m, 2H), 3.49 (s, 1H), 3.70 (m, 3H),
3.90-4.11 (m, 4H), 7.08 (d, J ) 8.3 Hz, 1H), 7.51 (d, J ) 8.2 Hz,
1H), 7.87 (m, 1H), 7.94 (s, 1H), 8.21 (d, J ) 8.5 Hz, 1H), 9.21 (s,
1H). MS (ESI, positive ion) m/z: 374.9 (M + 1).
6-Methyl-7-(1-((S)-3-methylmorpholino)phthalazin-6-yl)ben-
1
zo[d]isothiazol-3-amine (41). H NMR (CDCl3): δ 1.27 (d, J )
6.3 Hz, 3H), 2.37 (s, 3H), 3.43 (m, 1H), 3.70 (m, 2H), 3.90-4.13
(m, 4H), 4.93 (br, 2H), 7.41 (d, J ) 8.3 Hz, 1H), 7.66 (d, J ) 8.0
Hz, 1H), 7.88 (d, J ) 8.6 Hz, 1H), 7.96 (s, 1H), 8.23 (d, J ) 8.5
Hz, 1H), 9.25 (s, 1H). MS (ESI, positive ion) m/z: 392.2 (M + 1).
Anal. (C21H21N5OS) C, H, N.
Biological Methods. p38r MAP Kinase in Vitro Assay. The
p38R kinase reaction was carried out in a polypropylene 96-well
black round-bottomed 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 1 h of 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 fluo-
rescence transferred to allophycocyanin (an index of substrate
phosphorylation)) was determined (reference, Park, Y. W.; Cum-
mings, R. T.; Wu, L. Homogeneous Proximity Tyrosine Kinase
Assays: Scintillation Proximity Assay versus Homogeneous Time-
Resolved Fluorescence. Anal. Biochem. 1999, 269, 94-104). The
proportion of substrate phosphorylated in the kinase reaction in
the presence of compound compared with that phosphorylated 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. Data (consisting of POC
and inhibitor concentration in µM) were fitted to a four-parameter
equation y ) A + [(B - A)/(1 + (x/C)D)], where A is the minimum
y (POC) value, B is the maximum y (POC), C is the x (compound
concentration) at the point of inflection, and D is the slope factor,
using a Levenburg-Marquardt nonlinear regression algorithm. The
inhibition constant (Ki) of the inhibitor was estimated from the IC50
(compound concentration at the point of inflection, C) using the
Cheng-Prusoff equation: Ki ) IC50 /(1 + S/Km), where S is the
ATP substrate concentration and Km is the Michaelis constant for
ATP as determined experimentally.
TNF-Challenged IL-8 Production in Human Whole Blood
Cells. 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 the blood and incubated
for 1 h at 37 °C with 5% CO2. TNFR (Amgen) with a final
concentration of 1 nM was then added to the blood and incubated
overnight (16-18 h) at 37 °C with 5% CO2. Plasma was harvested,
and cytokines (IL8) were measured by MSD (Meso Scale Discov-
ery) 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).
CYP3A4 IC50 in Human Liver Microsomes. Midazolam 1′-
hydroxylation was used to monitor the CYP3A4 activity. Inhibitor
(0.41-100 µM) was co-incubated with midazolam (5 µM) in the
presence of human liver microsomes (0.1 mg/mL) and NADPH (1
mM) at 37 °C for 5 min. Incubation containing no inhibitor was
used as solvent control. At the end of the incubation, the reaction
was terminated by the addition of 0.05% formic acid in acetonitrile.
The concentration of 1′-hydroxymidazolam was determined by
LCMS analysis. The inhibition of CYP3A4 activity was assessed
by comparing the amount of 1′-hydroxymidazolam formed in
presence of varying concentrations of inhibitor to the amount of
1′-hydroxymidazolam formed in the solvent control. The IC50 transit
values were calculated by Excel-fit, version 4.1.1. In each study, a
CYP3A4 potent and specific inhibitor, ketoconzaole (0.037 µM),
was used as positive control.
Acknowledgment. We thank Dr. Randy Jensen for providing
2D-NOESY NMR analyses.
Supporting Information Available: X-ray data of cocrystal of
1 in p38R protein and HPLC purity (at two different conditions)
or combustion analysis data for each biological testing compound.
This material is available free of charge via the Internet at http://
pubs.acs.org.
ATP Competitive Assay. The method for this experiment was
the same as for the p38R enzyme assay except that the ATP
concentration was varied in the range of 1.3 µM to 5 mM. For
kinetic studies, the concentrations of the inhibitor (3c) were 0
(DMSO solution), 6, 9, 13, and 19 nM.

3-Amino-7-phthalazinylbenzoisoxazoles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6291
of Phthalazine, Aza- and Diaza-phthalazine Compounds as Protein
Kinase, Especially p38 Kinase, Inhibitors for Treating Inflammation
and Related Conditions. PCT Int. Appl. WO 2006094187, 2006.
(6) For other examples of Met-109 and Gly-110 “peptide flip”, see the
following: (a) Fitzgerald, C. E.; Patel, S. B.; Becker, J. W.; Cameron,
P. M.; Zaller, D.; Pikounis, V. B.; O’Keefe, S. J.; Scapin, G. Structural
Basis for p38R MAP Kinase Quinazolinone and Pyridol-Pyrimidine
Inhibitor Specificity. Nat. Struct. Biol. 2003, 10 (9), 764–769. (b)
Bemis, G. W.; Salituro, F. G.; Duffy, J. P.; Harrington, E. M.
Preparation of Pyrido[1,2-c]pyrimidin-3-ones or 1,2-Dihydro-py-
rido[1,2-c]pyrimidin-3-ones as Inhibitors of p38. U.S. Patent 6147080,
2000. (c) A review on this subject can be found in the following: Lee,
M. R.; Dominguez, C. MAP Kinase p38 Inhibitors: Clinical Results
and an Intimate Look at Their Interactions with p38R Protein. Curr.
Med. Chem. 2005, 12 (25), 2979–2994.
(7) Alanine is rare as a floor residue in the ATP binding pocket. Only 7
out of the 518 kinases have alanine as the floor residue: Manning, G.;
Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam, S. The Protein
Kinase Complement of the Human Genome. Science 2002, 298, 1912–
1916.
(8) For references comparing DFG-in and DFG-out binding, see the
following: (a) Tokarski, J. S.; Newitt, J. A.; Chang, C. Y. J.; Cheng,
J. D.; Wittekind, M.; Kiefer, S. E.; Kish, K.; Lee, F. Y. F.; Borzillerri,
R.; Lombardo, L. J.; Xie, D.; Zhang, Y.; Klei, H. E. The Structure of
Dasatinib (BMS-354825) Bound to Activated ABL Kinase Domain
Elucidates Its Inhibitory Activity against Imatinib-Resistant ABL
Mutants. Cancer Res. 2006, 66 (11), 5790–5797. (b) Nagar, B.;
Bornmann, W. G.; Pellicena, P.; Schindler, T.; Veach, D. R.; Miller,
W. T.; Clarkson, B.; Kuriyan, J. Crystal Structures of the Kinase
Domain of c-Abl in Complex with the Small Molecule Inhibitors
PD173955 and Imatinib (STI-571). Cancer Res. 2002, 62 (15), 4236–
4243.
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