Discovery and synthesis of novel 4-aminopyrrolopyrimidine Tie-2 kinase inhibitors for the treatment of solid tumors

Article abstract of DOI:10.1016/j.bmcl.2013.03.012

The synthesis and biological evaluation of novel Tie-2 kinase inhibitors are presented. Based on the pyrrolopyrimidine chemotype, several new series are described, including the benzimidazole series by linking a benzimidazole to the C5-position of the 4-a

Full text of DOI:10.1016/j.bmcl.2013.03.012

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3059–3063
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and synthesis of novel 4-aminopyrrolopyrimidine Tie-2
kinase inhibitors for the treatment of solid tumors
Joel T. Arcari, Jean S. Beebe, Martin A. Berliner, Vincent Bernardo, Merin Boehm, Gary V. Borzillo,
Tracey Clark, Bruce D. Cohen, Richard D. Connell, Heather N. Frost, Deborah A. Gordon
William M. Hungerford, Shefali M. Kakar, Aaron Kanter, Nandell F. Keene, Elizabeth A. Knauth
⇑
Susan D. LaGreca, Yong Lu, Louis Martinez-Alsina, Matthew A. Marx, Joel Morris, Nandini C. Patel ,
Doug Savage, Cathy I. Soderstrom, Carl Thompson, George Tkalcevic, Norma J. Tom, Felix F. Vajdos,
⇑
James J. Valentine, Patrick W. Vincent, Matthew D. Wessel, Jinshan M. Chen
Worldwide Research and Development, Pfizer Inc., Eastern Point Road, Groton, CT 06340, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and biological evaluation of novel Tie-2 kinase inhibitors are presented. Based on the
pyrrolopyrimidine chemotype, several new series are described, including the benzimidazole series by
linking a benzimidazole to the C5-position of the 4-amino-pyrrolopyrimidine core and the ketophenyl
series synthesized by incorporating a ketophenyl group to the C5-position. Medicinal chemistry efforts
led to potent Tie-2 inhibitors. Compound 15, a ketophenyl pyrrolopyrimidine urea analog with improved
physicochemical properties, demonstrated favorable in vitro attributes as well as dose responsive and
robust oral tumor growth inhibition in animal models.
Received 13 December 2012
Revised 25 February 2013
Accepted 4 March 2013
Available online 21 March 2013
Keywords:
Tie-2
Kinase inhibitor
Pyrrolopyrimidine
Angiogenesis
Cancer
Ó 2013 Elsevier Ltd. All rights reserved.
Solid tumor growth and invasion depend on an adequate blood
supply, and angiogenesis remains an area of high importance for
anticancer drug discovery.1 Tie-2 is a receptor tyrosine kinase
(RTK) that is essential for the formation of the embryonic vascula-
ture and is strongly implicated in tumor angiogenesis.2 Whereas
VEGF and other RTKs deliver the primary proliferation signals to
the endothelium, Tie-2 signals largely regulate vessel integrity,
and play roles in the dynamic modeling of vessels required for
angiogenesis.3 Tie-2 and its various angiopoeitin ligands (Ang-1,
Ang-2 and Ang-4) can either promote vessel integrity and decrease
permeability (via Ang-1 stimulation of Tie-2),3 or can drive vessel
destabilization and permeabilization as a prelude to sprouting
and angiogenesis (Ang-2 inhibition of Tie-2).4 It has been sug-
gested that alteration of Tie-2 activity in either direction: constitu-
tive activation leading to an overly stable vasculature, or inhibition
leading to protracted destabilization, could result in impediments
to both tumor angiogenesis and tumor growth.5
O
S
HN
O
O
O
S
O
H
N
O
S
O
Ar
HN
Cl
NH2
O
NH2
NH2
N
N
N
N
NH
NH2
N
N
N
N
5
N
N
4
N
7
N
N
N
N
1
3
2a
2b
N
N
Figure 1. Tie-2 active pyrrolopyrimidines.
esis. The desired inhibitors would need to have adequate ADMET
attributes for safe, oral cancer treatment. Prior to our effort, pyrrol-
opyrimidines had been reported to bind to the Tie-2 kinase domain.
However, specific details were not available with regard to Tie-2
kinase/cellular potency, selectivity, SAR, ADMET and in vivo attri-
butes. For example, compound 1 (Fig. 1), with a chlorine substituted
phenyl linked directly to the 5-position of the 4-amino-7-cyclopen-
tyl-pyrrolopyrimidine core and an aryl sulfonamide attached to the
Our objective was to develop potent novel Tie-2 inhibitors with
good selectivity against other kinases closely involved in angiogen-
⇑
Corresponding authors. Tel.: +1 617 395 0707.
E-mail addresses: michael.j.chen@pfizer.com (J.M. Chen), nandini.c.patel@
pfizer.com (N.C. Patel).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.012

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J. T. Arcari et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3059–3063
para-position of the phenyl linker, was reported to bind to the
catalytic domain of human Tie-2 in a co-crystal structure,6 no fur-
ther information was available in the literature. Pyrrolopyrimidines
have also been reported to inhibit Lck7 and Src8 with weak Tie-2
activity. For example, compound 2a had an IC50 of 15 nM against
Lck and 250 nM against Tie-2 at 1 mM ATP.7a Furthermore, com-
pound 2b was reported to inhibit Tie-2 with an IC50 of 137 nM,
whereas its IC50s against Lck, Src and kdr were greater than
3000 nM.7b In recent years there have been several reports on
non-pyrrolopyrimidine based Tie-2 inhibitors,9 but few with the
kinase selectivity attributes of our interest at the time. Herein we
report the discovery and synthesis of novel pyrrolopyrimidines as
potent Tie-2 inhibitors with good selectivity against other key angi-
ogenesis kinases, adequate ADMET properties, and robust tumor
growth inhibition in xenograft models.
We designed and synthesized a number of analogs with new
linkers and eventually identified a few active pyrrolopyrimidine
analogs with a benzimidazole linker and arylsulfonamide head
groups (3, Table 1). For example, compound 4, with a 2,6-di-
fluoro-phenylsulfonamide head group, had Tie-2 kinase and cellu-
lar IC50s of 568 and 518 nM, respectively.10 The corresponding urea
analog compound 5 was then prepared which proved to be more
potent in the Tie-2 kinase and cellular assays, with IC50s of 241
and 112 nM, respectively. Subsequently, we designed and synthe-
sized the ketophenyl linker and synthesized the corresponding
meta-sulfonamide 6 and meta-urea 7. Similar to the benzimidazole
analogs, the urea analog 7 was more potent than the corresponding
sulfonamide analog 6. This trend was more pronounced in the cel-
lular assay, with an IC50 of 67 nM for compound 7 and about four-
fold reduced activity (253 nM) of the corresponding sulfonamide
analog 6. Alternative substitution positions on both ketophenyl
and benzimidazole linkers resulted in analogs of weaker activity.
For example, the para-substituted ketophenyl analogs 8 and 9 were
less active than the corresponding meta-analogs 6 and 7, respec-
tively. Furthermore, extending the head functional group into car-
bamates, carboxamides and amines (G = CO(O), CO, CH2) also led to
diminished potency in general. One of the hypotheses employed in
the design at this stage was to minimize the rotational freedom of
the bond connecting the linker to the pyrrolopyrimidine core. For
example, a benzimidazole linker may bring in potential rigidity
via an intramolecular hydrogen-bond between a pyrimidine NH
and imidazole ring N; as could a pyrimidine NH with the carbonyl
O in the ketophenyl analogs. Compound 10, where the carbonyl
group is reduced to the methylene, was significantly less active
(>10,000 nM). This appeared to suggest a critical role of the H-bond
acceptor in this region of the molecule and was consistent with the
initial hypothesis.
These early analogs appeared to have some selectivity against
the kinases we intended to avoid the most. For example, com-
pound 7 had kinase IC50 values of 3100 and >5000 nM against
KDR and PDGFR, respectively. Some of them exhibited significant
inhibition against Trk, a family of receptor tyrosine kinases origi-
nally identified as modulators of the maintenance and survival of
neuronal cells.11 In the last two decades, evidence has suggested
that Trk kinases (TrkA and TrkB) play key roles in malignant trans-
formation, chemotaxis, metastasis, and survival signaling in hu-
man tumors. As a result, Trk kinases have attracted significant
attention as drug discovery targets for cancer and pain therapy.12
For example, TrkB has been found to be over expressed in some
small cell lung cancer and prostate cancers. Thus a dual Tie-2/Trk
inhibitor might be hypothesized to provide additional efficacy
against these tumors.
Compounds 4 through 7 had MW around 500, clogP of 4–5, and
TPSA of 115–140 (Table 2), all approaching the upper limits for or-
ally available drugs.13 These molecules appeared to have reason-
able permeability, but could be potential substrates for P-gp
mediated efflux. For example, compound
7 had a value of
7.7 Â 10À6 cm/s for the apical to basal measurement in the Caco-
2 cell line, with a high Caco-2 BA/AB ratio of 3.5. In addition, rat li-
ver microsomal stability of these compounds was poor, with unfa-
vorably high rat liver microsomal extraction ratio (RLM Er >0.60).
Furthermore, solubility of these analogs was typically very low
(<5 lg/mL).
Enzyme kinetics studies carried out on some of these analogs
suggested that they were reversible and ATP competitive Tie-2 ki-
nase inhibitors. The ketoureas appeared to have a much slower off-
rate than the ketosulfonamide, which could potentially translate to
advantages in pharmacological effects.14 Our initial lead optimiza-
tion efforts mostly focused on improving Tie-2 potency (thereby
selectivity), addressing metabolic stability, P-gp mediated efflux
and solubility. Our medicinal chemistry strategy was to fully ex-
plore the SAR of head aryl group for optimal lipophilic binding
interaction in this region, as well as to reduce size and lipophilicity
via significant modification of the cyclopentyl tail group.
Table 1
Impact of linker on kinase and cellular potency
F
F
NH2
N
G
L
N
N
The team subsequently synthesized diverse analogs of the
benzimidazole and the meta-ketophenyl series including sulfona-
mides, ureas, and a smaller number of other analogs (carbamates,
carboxamides, and amines). All analogs synthesized at this stage
maintained the cyclopentyl tail group for SAR continuity. Out of
this effort a total of 350 analogs were made, and their potency dis-
tribution versus chemotype is plotted in Figure 2. The most potent
analogs were ketophenyl ureas.
L
G
Compound
IC50 (nM) kinase/cell
H
N
SO2
CONH
4
5
568/518
241/122
N
N
H
O
SO2
CONH
6
7
423/253
361/67
H
N
O
SO2
CONH
8
9
745/769
345/254
Table 2
Molecular properties of initial leads
Compound
MW
clogP
TPSA
RLM Er
Caco-2 BA/AB
N
H
4
5
6
7
509
488
497
476
4.69
4.48
4.11
4.05
140
127
128
115
0.69
0.90
ND
ND
3.2
4.7
3.5
H
N
SO2
10
>10,000/ND
0.83

J. T. Arcari et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3059–3063
3061
lipophilicity and MW of these analogs. A series of new tail groups
of various lipophilicities and sizes were designed and the penulti-
mate amine intermediates were synthesized with these tail groups
installed. Each of these templates were utilized to synthesize a set
of 20 ureas and 20 sulfonamides. The selection of isocyanate and
sulfonyl chloride building blocks for this effort was directed by
SAR results of the cyclopentyl ketophenyl series. The average
RLM Er and cellular IC50s of these subsets are listed in Table 4.
Several observations were made from these data: (1) smaller
groups (R = H, Me, i-Pr) were preferred over the larger groups
(THP, cyclopentyl, methoxyethyl) for better microsomal stability
in general; (2) the methoxyethyl tail was the least stable, in spite
of a favorable clogP and moderate size, probably due to metabolic
demethylation; (3) larger groups (cyclopentyl, THP, i-Pr) were fa-
vored in terms of Tie-2 potency. Overall, i-Pr analogs imparted
moderately improved microsomal stability (RLM Er 0.56) while
Figure 2. Chemotype versus Tie-2 IC50. bzs, benzimidazole sulfonamides; bzu,
benzimidazole ureas; ks, meta-ketophenyl sulfonamides; m, amides, carbamates,
amines.
maintaining potency equivalent to cyclopenyl group (0.29 lM).
From these efforts ketophenyl urea compound 15 emerged and
the attributes of 15 are listed inFigure 3. Compound 15 had low sin-
gle digit nM Tie-2 cellular IC50 and equally potent TrkA cellular
activity (TrkA kinase/cell IC50 of 0.4 and 4 nM, respectively). Its
MW of 448, clogP of 4.7, and TPSA of 115 were all within the bound-
aries of oral drugs. These appeared to have a positive impact on the
microsomal stability, with both rat and human liver microsomal
extraction ratios close to 0.5. Compound 15 also had improved sol-
Four of the more potent cyclopentyl ketophenyl ureas, com-
pounds 11, 12, 13, and 14, are listed in Table 3. All had lipophilic
substituents (Cl or Me) at the meta- or para-position of the termi-
nal phenyl group. The cellular IC50s of these were in the low single
digit nanomolar to picomolar range (4.6, 1.2, 0.89, and 0.69 nM,
respectively).15 The most potent compounds in the cell assays
exhibited a hydrogen bond acceptor at the 2-position of the termi-
nal phenyl ring (F in compound 13, O in compound 14), which
might hydrogen bond to the adjacent urea NH thus leading to
potentially favorable conformation and/or permeability. As the
MW increased and clogP remained high for these analogs, RLM
Er and Caco-2 BA/AB ratio were still problematically high.16
Medicinal chemistry efforts were then focused on the cyclopen-
tyl tail group of the ketophenyl ureas. Correlation between RLM Er
and clogP of these analogs was poor in general suggesting that
multiple metabolic pathways were potentially involved. One
objective was to modify the cyclopentyl group to reduce overall
ubility (35
l
g/mL) and permeability (Caco-2 BA: 9.5 Â 10À6 cm/s).
With a Caco-2 BA/AB ratio of 1.5, this compound was less likely a
P-gp mediated efflux substrate. Inhibition of cytochrome P450 iso-
zymes by compound 15 was low (IC50 >3 lM). Compound 15 had
modest kinase selectivity15 against the key oncology targets we in-
tended to avoid. For example, its IC50s against KDR, FGFR, and EGFR
were >10,000 nM and PDGFR at 1400 nM. Compound 15 was nega-
tive in the Biolums AMES assays, but positive in the in vitro micro-
nucleus assay. It also inhibited the hERG potassium ion channel
with an iKr IC20 of 1000 nM.
Table 3
Cyclopentyl meta-ketophenyl ureas
Ar
HN
O
HN
O
H2N
N
N
N
Ar
Compound
IC50 kinase/cell
45/4.6
MW
475
clogP
RLM Er
0.7
Caco-2 BA/AB
17
11
5.3
Cl
Cl
Cl
12
13
ND/1.2
12/0.89
509
472
5.5
4.8
ND
0.8
1.9
9.2
F
Me
Me
O
14
8.8/0.69
484
5
0.69
9.4

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J. T. Arcari et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3059–3063
Table 4
Tail groups, average RLM Er and cellular potency
Ar
G
NH
O
H2N
N
N
R
N
G = SO2, C(O)NH
H
Me
Figure 5. C6 tumor growth inhibition of compound 15.
O
In tumor growth inhibition (TGI) studies, compound 15 exhib-
ited robust efficacy in several xenograft models. For example, in
the C6 (uninfected parental C6 cells lacking human Tie-2) rat glio-
blastoma model, compound 15 was dosed orally once daily (po qd)
at 10 or 33 mg/kg through the 10 day study (Fig. 5). At 10 mg/kg po
qd, ꢀ40% TGI was achieved whereas 33 mg/kg po qd resulted in
ꢀ60% TGI. For the bid dose, 10 mg/kg furnished ꢀ70% TGI and
33 mg/kg resulted in a slightly higher TGI. The observation that
bid dosing promotes minimal enhancement of tumor inhibition
at the 33 mg/kg dose relative to 10 mg/kg dose is consistent with
our observations that Tie-2 inhibitors may block new blood vessel
formation and tumor growth, but do not generally induce tumor
shrinkage when administered as single agents. Thus, the tumor
inhibitions observed for the bid doses may reflect near-maximal
activity in this model. No significant loss of body weight was ob-
served in any animal during these studies.
The synthesis of compound 20, the penultimate intermediate to
compound 15, began with alkylation of compound 16 (Scheme 1).18
The alkylation product compound 17, was brominated to furnish
compound 18. Compound 18 was subjected to halogen-metal ex-
change and the resulting species was trapped with 3-nitro-benzoyl
chloride to afford compound 19. Amination of 19 followed with
nitro reduction furnished the penultimate amine intermediate 20.
The final derivatization step, usually formation of sulfonamide
from sulfonyl chloride and urea from isocyanates using the penul-
timate intermediates, was typically carried out in parallel format.
Final analogs were prepared in 15–25 mg quantity all purified via
reversed phase semi preparative HPLC. We instituted an intuitive
OMe
kinase/cell
MW/ cLogP/ TPSA
40 / 3.7 nM
449/ 4.7 / 115
0.52 / 0.46
Cl
RLM/ HLM Er
HN
Caco-2BA& BA/AB 9.5x10-6 cm/s& 1.5
HN
O
Cypsinhibition IC50
solubility
KDR, FGFR, EGFR >10,000 nM
PDGFR
Biolums AMES
IVmicronucleus
>3 µM
35µg / mL
O
NH2
N
N
15
1400 nM
negative
positive
N
iKr IC
1000 nM
20
Figure 3. Attributes of compound 15.
Compound 15 inhibited Tie-2 phosphorylation in an ex vivo
model using a cloned C6 rat glioblastoma cell line over expressing
human Tie-2 via a retrovirus construct.17 This inhibition was dose
responsive and plasma concentration dependent (Fig. 4). In PK
studies, 0.164 mg/kg IV dose in fed male Sprague–Dawley rats led
to the observation of a clearance (Cl) of 38 mL/min/kg (54% Q), a
steady state volume of distribution (VDss) of 0.9 L/kg and a T1/2 of
0.4 h. Oral suspension dose in ultrafasted rats at 4.2 mg/kg led to
a systemic Cmax of 1715 ng/mL, Tmax of 0.75 h, hepatic extraction
(Eh) of 27 mL/min/kg (38%) and oral bioavailability (F%) of 100%.
Cl
Cl
Br
Cl
a
N
N
b
N
N
N
N
N
N
H
N
16
17
18
O2N
H2N
c
d, e
O
O
f
15
Cl
Cl
N
N
N
N
N
19
N
20
Scheme 1. Synthesis of compound 15. Reagents and conditions: (a) i-PrI, Cs2CO3,
DMF, rt, 5 h, 94%; (b) NBS, DCM, rt, 12 h, 90%; (c) n-BuLi, ether, À78 °C, 0.5 h then
Weinreb amide, 73%; (d) dioxane, NH4OH (1:1), 45 °C, 90%; (e) Fe, NH4Cl, dioxane/
EtOH/H2O, 100 °C, 80–100% (2 steps); (f) p-Cl-Ph-NCO, 80 °C, 68%.
Figure 4. Inhibition of Tie-2 phosphorylation versus plasma concentration.

J. T. Arcari et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3059–3063
3063
Figure 6. Analytical LCMS—semipreparative HPLC purification paradigm.
Med. Chem. Lett. 2009, 19, 424; (d) Hodous, B.; Geuns-Myer, S.; Hughes, P.;
Albrecht, B.; Bellon, S.; Bready, J.; Caenepeel, S.; Cee, V.; Chaffee, S.; Coxon, A.;
Emery, M.; Fretland, J.; Gallant, P.; Gu, Y.; Hoffman, D.; Johnson, R.; Kendall, R.;
Kim, J.; Long, A.; Morrison, M.; Olivieri, P.; Patel, V.; Polverino, A.; Rose, P.;
Tempest, P.; Wang, L.; Whittington, D.; Zhao, H. J. Med. Chem. 2007, 50, 611; (e)
Hasegawa, M.; Nishigaki, N.; Washio, Y.; Kano, K.; Harris, P.; Sato, H.; Mori, I.;
West, R.; Shibahara, M.; Toyoda, H.; Wang, L.; Nolte, R.; Veal, J.; Cheung, M. J.
Med. Chem. 2007, 50, 4453. and also see references cited therein.
paradigm to effectively leverage the analytical LCMS to customize
gradient windows for each HPLC purification run for maximal
resolution (Fig. 6).19 For example, if the desired product retention
time t on the analytical LCMS corresponded to x% B on the gradient
chart, we configured our systems such that the desired product
usually eluted off the column during the semipreparative purifica-
tion within a gradient window of 5 to x% B. This paradigm was de-
ployed routinely and handle the bulk of our samples. For example,
dozens of analogs were made with N5-cyclohexyl tail on which
further substitutions resulted in cis- and trans-isomers (as in com-
pound 2). These isomeric mixtures were routinely separated using
this paradigm.
10. Details of the Tie-2 kinase and cellular assays are disclosed in Ref. 18. (a) Briefly
the kinase ELISA assay used a GST-Tie-2 kinase fusion protein and poly-Glu-Tyr
coated 96-well plates. Reactions were performed in the presence of 100
lM
ATP and phosphorylated products were detected with horseradish
a
peroxidase-conjugated monoclonal antibody to phosphotyrosine (HRP-PY20).
For compound reversibility studies the enzyme was pre-incubated with
compound for 15 min before the addition of ATP. Enzyme kinetics was
determined by carrying out the above procedure using titrated ATP dilutions.
In summary, we have established several novel pyrrolopyrimi-
dine series of Tie-2 inhibitors. The ketophenyl urea analogs are
reversible and ATP competitive Tie-2 inhibitors with potent activ-
ity in whole-cell assays. Medicinal chemistry efforts culminated in
the identification of compound 15, which demonstrated exquisite
potency against Tie-2/Trk, and robust dose-responsive oral
efficacy. Subsequent efforts to refine attributes of pharmacokinet-
ics, hERG/iKr, and other parameters resulted in a compound ad-
vanced into human clinical trial studies.20
(b) The cellular assay used NIH/3T3 fibroblasts stably transfected with
a
chimeric receptor composed of the extracellular domain of human EGFR and
intracellular domain of human Tie-2. The cells were incubated with
compounds for 60 min and then stimulated with EGF before being fixed to
the plate. Phosphotyrosine was detected using an Eu-labeled antibody and
DELFIA detection reagents. For washout experiments the compound containing
media was removed and replaced with fresh media. At indicated time points
the cells were stimulated and the assay carried out as described above.
11. Huang, E.; Reichardt, L. Annu. Rev. Biochem. 2003, 72, 609.
12. Wang, T.; Yu, D.; Lamb, M. Expert Opin. Ther. Patents 2009, 19, 305.
13. (a) Lipinski, C. A. J. Pharmacol. Toxicol. Methods 2000, 44, 235; (b) Vebber, D. F.;
Johnson, S. R.; Cheng, H.-Y.; Smith, B. R.; Ward, K. W.; Kopple, K. D. J. Med.
Chem. 2002, 45, 2615.
14. Copeland, R.; Pompliano, D.; Meek, T. Nat. Rev. Drug Disc. 2006, 5, 730.
15. The observed disconnect between the kinase and cellular IC50s is believed to be
in part due to the artifacts of kinase constructs and screens. Cellular IC50s may
be a more accurate measure of selectivity. This will be discussed in greater
details in Ref. 20.
Acknowledgments
We would like to thank Drs. Mark Flanagan and Jeremy Starr for
critical reading of this manuscript.
16. Didziapetris, R.; Japertas, P.; Avdeef, A.; Petrauskas, A. J. Drug Target. 2003, 11, 391.
17. Ex vivo ELISA: 5 Â 106 C6 rat glioma cells stably expressing human Tie-2 were
injected with 50% matrigel s.c. into the right flank of each mouse. Treatment
was initiated when tumors in all mice in each experiment ranged in size from
250 to 350 mm3. Tumors and plasma were harvested at indicated time points
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following
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dropwise to
a solution of 5-bromo-4-chloro-7-isopropyl-7H-pyrrolo[2,3-
d]pyrimidine (15.0 g, 54.64 mmol) in ether (1.2 L) at À78 °C. After 1 h
a
solution of N-methoxy-N-methyl-3-nitrobenzamide (15 g, 71.03 mmol) in
ether (100 mL) was added by drop wise addition. After 1 h the reaction was
quenched with saturated aqueous NH4Cl and warmed to room temperature.
The layers were separated and the aqueous layer was extracted with EtOAc
(3 Â 200 mL). The organic layers were combined and washed with water (one
time) and brine (one time), then dried over Na2SO4 and concentrated.
Purification by flash column chromatography (hexanes/ethyl acetate 75:25)
afforded the desired product as a white solid (15.21 g, 73%). LRMS: m/z 345.2
[M+H], C16H13ClN4O3 calculated 344. 1H NMR (400 MHz, DMSO-d6, d): 1.45 (d,
J = 6.6, 6H), 5.12–5.05 (m, 1H), 7.84 (t, J = 7.89, 1 H), 8.26 (d, J = 7.5, 1H), 8.48–
8.53 (m, 3H), 8.78 (s, 1H). Additional experimental details on the synthesis of
these analogs can be found in the various published patent applications: Acari,
J.; Chen, J.; LaGreca, S.; Marx, M. A.; Wessel, M. WO 2004056830.; (b) Wessel,
M. D.; Chen, J.; Marx, M. A.; Lagreca, S. D. WO 2005047289 A1.; (c) Marx, M. A.;
La Greca, S. D.; Chen, J.; Wessel, M. D.; Arcari, J.T. WO 2005116035 A1.
19. (a) Chen, J. Presented at the Cambridge Healthtech Institute’s 2nd Annual High-
throughput Organic Synthesis & Purification: San Diego, CA, 2007.; (b) Chen, J.
Abstract of Papers, 39th Midwest Regional Meeting of the American Chemical
Society, Manhattan, KS, 2004; Abstract MID04-263.
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20. Optimization of compound 15 into a human clinical trial phase I compound is
the subject of a future communication.