Discovery of benzimidazole pyrrolidinyl amides as prolylcarboxypeptidase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.01.090

A series of benzimidazole pyrrolidinyl amides containing a piperidinyl group were discovered as novel prolylcarboxypeptidase (PrCP) inhibitors. Low-nanomolar IC₅₀'s were achieved for several analogs, of which compound 9b displayed modest ex vivo target engagement in eDIO mouse plasma. Compound 9b was also studied in vivo for its effect on weight loss and food intake in an eDIO mouse model and the results will be discussed.

Full text of DOI:10.1016/j.bmcl.2011.01.090

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1299–1305
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of benzimidazole pyrrolidinyl amides
as prolylcarboxypeptidase inhibitors
Hong C. Shen a, , Fa-Xiang Ding a, Changyou Zhou a, Yusheng Xiong a, Andreas Verras a, Renee M. Chabin b,
⇑
Suoyu Xu c, Xinchun Tong c, Dan Xie d, Michael E. Lassman d, Urmi R. Bhatt d, Margarita M. Garcia-Calvo d,
Wayne Geissler d, Zhu Shen d, Dunlu Chen d, Ranabir SinhaRoy d, Jeffery J. Hale a, James R. Tata a,
Shirly Pinto d, Dong-Ming Shen a, Steven L. Colletti a
a Department of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
b Department of Research Operations, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
c Department of Drug Metabolism, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
d Department of Metabolic Disorders, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of benzimidazole pyrrolidinyl amides containing a piperidinyl group were discovered as novel
prolylcarboxypeptidase (PrCP) inhibitors. Low-nanomolar IC50’s were achieved for several analogs, of
which compound 9b displayed modest ex vivo target engagement in eDIO mouse plasma. Compound
9b was also studied in vivo for its effect on weight loss and food intake in an eDIO mouse model and
the results will be discussed.
Received 8 January 2011
Revised 19 January 2011
Accepted 20 January 2011
Available online 26 January 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
PrCP
Prolylcarboxypeptidase
Inhibitors
Obesity
Benzimidazole pyrrolidinyl amides
Isolated from kidney extract,1 and later cloned from a human
kidney library,2 prolylcarboxypeptidase (PrCP) belongs to a class
of serine peptidases that cleaves the C-terminal amino acids linked
to proline in peptides such as angiotensin II (AngII), angiotensin III
(AngIII) and des-Arg9-bradykinin.3 PrCP is widely expressed in var-
ious organs including kidney, lung, adipose, liver, brain, heart, pan-
creas, etc.4 Within these organs, this enzyme is active in both
lysosomes and the membrane of human alveolar macrophages.5
The roles of PrCP to convert AngII to Ang 1-7 and AngIII to Ang
2-7 indicate a potential function of PrCP in both blood pressure
regulation and electrolyte balance.3 In addition, the human gene
for PrCP (11q14) may be related to essential hypertension.6
In the past several years, significant progress has been made in
understanding the potential therapeutic utility of PrCP inhibition.
In 2005, the Horvath group at Yale University suggested that PrCP
inhibition could lead to treatment of obesity, diabetes and related
conditions.7 This work is primarily based upon the PrCP À/À
mouse phenotype as well as rat pharmacology with a serine trap
PrCP inhibitor. It has also been reported that chromosomal region
D7Mit353, containing the PrCP gene, confers lean phenotype in
mice (50% less fat).8 In human genetic studies, a single cohort of
PrCP single nucleotide polymorphism (SNP) was found to be asso-
ciated with metabolic syndrome.9 Most recently, GlaxoSmithKline
also suggested the utility of PrCP inhibitors in treating obesity,
based on a human SNP analysis of PrCP in 937 obese individuals.
This study demonstrated a significant correlation between Prcp
gene permutation and high body mass index (BMI) values (>30).10
To explore the potential of PrCP inhibitors in treatment of obes-
ity,11 we began to investigate whether the inhibition of PrCP in-
deed led to mechanism-based weight loss in obesity models.
Furthermore, we attempted to understand the relevance of central
and/or peripheral PrCP inhibition to any observed efficacy. Herein,
we report our efforts in identifying a class of potent peripheral
PrCP inhibitors, and their pharmacodynamic (PD) effects in estab-
lished diet-induced obese (eDIO) wild type mice and eDIO/PrCP
knockout mice.
Inspired by a potent PrCP inhibitor reported by Merck col-
leagues earlier (1 in Fig. 1),12 we attempted to remove both the
a-aminoisobutyl amide group and the b-methyl group in order to
eliminate chiral centers and simplify the structures. Furthermore,
the introduction of a cyclic secondary amine could replace the in-
ner phenyl group of the biphenyl moiety of analog 1, thereby
⇑
Corresponding author. Tel.: +1 732 594 1755; fax: +1 732 594 9473.
E-mail address: hong_shen@merck.com (H.C. Shen).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.090

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H. C. Shen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1299–1305
R2
NH2
H
N
O
N
O
O
R1
Cl
Cl
N
N
Cl
Cl
N
N
N
H
N
H
2
1
R2
N
R2
N
O
+
O
OtBu
R1
R1
H
OH
3
5
6
+
H
N
Cl
Cl
N
N
H
4
Figure 1. Design and synthesis of PrCP inhibitors.
increasing the polarity and potentially reducing the serum shift of
these compounds. This simplified design of analogs also allowed
for parallel synthesis and thus rapid establishment of structure–
activity relationship (SAR). As shown in Figure 1, the synthesis of
the analogs described as general structure 2 involved the amide
coupling of b-amino acid 3 and benzimidazole pyrrolidine 4. The
requisite b-amino acid 3 could then be derived from the 1,4-addi-
tion of amine 5 to t-butyl acrylate 6.
Besides the human PrCP inhibition assay, a subset of com-
pounds were also tested in the mouse PrCP inhibition assay and/
or the AngIII assay which was conducted in the whole mouse plas-
ma. The AngIII assay enabled the assessment of plasma serum shift
of compounds. In this assay, the mouse plasma was first treated
with protease inhibitors to suppress the cleavage of AngIII by
non-PrCP proteases. A PrCP inhibitor was then added to study
the cleavage of AngIII in comparison with the vehicle, in which this
conversion was primarily promoted by PrCP.
Aided by the human PrCP enzyme inhibition assay, the optimal
ring size of the cyclic secondary amine was investigated first
(Table 1, 7a–7d). 4-Phenyl piperidinyl analog 7a and 4-phenyl
homopiperidinyl analog 7b had similar IC50’s for PrCP, but com-
pound 7a is structurally simpler by having one less chiral center
compared with 7b. Compound 7a was also more potent than lower
homologs 7c and 7d. In addition, piperazine analog 7e provided
similar activity. However, despite the good enzyme inhibition,
these compounds appeared to have an unexpectedly large serum
shift, which was reflected by the mouse AngIII assay data. It was
then envisioned that further replacement of the terminal phenyl
group with polar heteroaryls may reduce the observed serum
shifts. Thus, several analogs containing five- or six-membered
heteroaryls (7f–r) were prepared. Indeed, polar pyrazolepiperidine
analog 7f, and 4-pyridylpiperidine 7o led to enhanced activity in
mouse whole serum (IC50 = 290 nM and 430 nM, respectively).
Due to the reduced serum shift, reasonable PrCP inhibitory
activity of pyrazole 7f and oxadiazole 7n, their derivatives were
further explored using readily available building blocks. The SAR
regarding the attached aryl or heteroaryl to either of these two
heteroaryls is summarized in Tables 2 and 3. Several compounds
(8a, 8c, 8f, 8g, 9a, 9b, 9f) in both the oxadiazole (Table 2) and
the pyrazole (Table 3) series exhibited excellent PrCP inhibitory
activity in the absence of mouse serum. In addition, polar terminal
groups such as pyridyl (8c, 9b, 9c) or phenyl sulfone (8g) analogs
demonstrated excellent PrCP inhibitory activity in mouse plasma
(IC50 <40 nM). Unfortunately, modification of piperidine by adding
a carbonyl group (8i) or introducing a geminal-dimethyl group (8j)
did not offer significant advantages regarding PrCP inhibition, ser-
um shift reduction, or PK profiles (Table 4) with respect to analog
8a.
Mouse pharmacokinetic (PK) profiles of compounds 8a–9b
(Table 4) were characterized by high clearance, low oral bioavail-
ability and low oral exposure. To ascertain whether compound
9b was a pgp (p-glycoprotein) substrate, a PK study in CF1 wild
type and pgp deficient mice was conducted. At a 0.5 mpk (mg/
kg) dose in CF1 wild type mice, the brain/plasma ratio was 0.20
(10 nM/54 nM) at half an hour post dosing, and 0.88 (4.0 nM/
4.6 nM) at 4 h post dosing. In contrast, in mdr (multidrug
resistance) 1aÀ/À mice with deficient pgp transporter, the brain/
plasma compound ratio was 1.4 (70 nM/51 nM) at half an hour
post dosing, and 13 (54 nM/4 nM) at 4 h post dosing. Since 9b
was a pgp substrate in vivo, it was utilized as a tool compound
to evaluate whether peripheral PrCP inhibition could lead to
weight loss in eDIO mice.
To measure the ex vivo target engagement of 9b in plasma, the
wild type eDIO mice were dosed with 9b subcutaneously (sc) using
continuous infusion pumps in three different groups at 10, 30, and
60 mg/kg (mpk), respectively, for seven days. On the seventh day,
compound 9b exhibited a similar target engagement in plasma at
all three different doses (B, C, D in Fig. 2). Furthermore, at the
60 mpk dose, the maximum efficacy was not achieved. The highest
inhibition observed was 55% for group D versus 80% for group D+,
in which 2
inhibition. The similar plasma levels of 9b in all three groups
(0.09–0.16 M) explained why there was a lack of optimal target
lM of added compound 9b provided additional PrCP
l
engagement despite higher doses. The lack of improvement of drug
exposure in spite of increasing dosage was most likely due to the
observed compound precipitation during the subcutaneous
infusion, particularly at the 30 and 60 mpk doses. Therefore, in a
subsequent PD study, 12 mpk was selected as the dose level.

H. C. Shen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1299–1305
1301
Table 1
In vitro SAR on human and mouse PrCP inhibition and mouse AngIII assaysa
O
Cl
N
Compound No.
PrCP IC50 (h, m)b, nM
Mouse AngIII IC50, nM
N
N
H
Cl
7a
7b
10, —
25, —
>10000
—
N
N
7c
93% at 5
lM, —
—
N
7d
7e
141, —
16, —
—
N
>10000
N
N
N
HN
7f
7g
7h
18, —
290
—
N
N
34, —
N
N
HN
N
N
N
22, 107
—
HN
7i
7j
129, —
155, —
—
N
N
N
N
N
N
N
7k
7l
42, —
71, —
30, —
N
N
N
N
N
N
N
N
N
7m
2900
N
O
N
N
7n
7o
7p
64, —
21, —
93, —
—
N
430
—
N
N
N
N
N
N
N
N
7q
7r
44, —
24, —
—
N
N
8000
N
a
Values are based on one or two experiments, each in triplicate.
h = human, m = mouse.
b
In a seven-day pharmacodynamic (PD) study, food intake and
weight and food intake effects of compound 9b were not due to
PrCP inhibition. At the end of the study, drug levels of all treated
mice were obtained. It should be noted that only small differences
were observed for the average drug levels of compound 9b in the
body weight of the wild type and PrCP knockout eDIO mice13 were
measured daily during the continuous sc infusion of compound 9b
(12 mpk q.d.) and vehicle, respectively (Fig. 3). Despite the obser-
vation of a decrease in both food intake and body weight in the
wild type group treated with compound 9b (turquoise curve) with
respect to the vehicle group (blue curve), the same magnitude of
body weight and food intake effects were also manifested in the
PrCP knockout group (lavender and red curves for vehicle and trea-
ted groups, respectively). This observation suggested that the body
PrCP wild type and in knockout groups (0.21 0.10
type vs 0.29 0.14 M in PrCP knockout mice, n = 8). Comparing
the drug level (0.21 0.10 M) with the vide supra target engage-
M), it is reason-
lM in wild
l
l
ment study (ꢀ50% for drug exposure of 0.09–0.16
l
able to assume that ꢀ50% of plasma target engagement was
achieved in this PD study. In light of this result and an earlier

1302
H. C. Shen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1299–1305
Table 2
In vitro SAR on human and mouse PrCP inhibition and mouse AngIII assaysa
O
Cl
N
Compound No.
PrCP IC50 (h, m)b, nM
Mouse AngIII IC50, nM
N
N
H
Cl
O
N
N
N
N
N
8a
8b
8c
8d
12, 3.5
18, —
173
155
29
N
O
N
N
O
N
N
N
N
N
1.7, 0.53
13, —
N
O
N
N
435
N
O
N
N
N
N
N
8e
8f
19, —
3.7, —
745
—
N
O
N
N
N
O
N
N
N
8g
8h
2.3, 0.41
17, —
10
MeO2S
HO2C
O
N
N
N
241
O
O
N
N
8i
8j
8.5, —
15, 19
425
113
N
MeO2S
O
N
N
N
N
a
Values are based on one or two experiments, each in triplicate.
h = human, m = mouse.
b
observation that a PrCP inhibitor caused mechanism-based effi-
cacy,12 higher plasma target engagement is most likely required
for PrCP-dependant weight loss. Thus, it would be more desirable
to test compounds with good oral exposure and PrCP inhibitory
activity via oral dosing to avoid compound precipitation and other
potential side effects caused by continuous infusion.
As shown in Scheme 1, the synthesis of analog 9b commenced
with carboxylic acid 10.14 A Weinreb amide method provided
methyl ketone intermediate 12. This was followed by a Claisen
condensation to yield diketone 14. The subsequent condensation
with methylhydrazine afforded two pyrazole regioisomers. Com-
pound 15, the major regioisomer, was obtained by supercritical
fluid chromatography using a chiral OJ column. Deprotection of
the piperidine followed by a 1,4-addition to acrylate generated
intermediate 17, which then underwent ester cleavage and an
ensuing amide formation using benzimidazole-substituted pyrrol-
idine 412 to generate analog 9b.
In conclusion, benzimidazole pyrrolidinyl amides containing a
piperidinyl group have been shown to be a class of potent PrCP
inhibitors in vitro. A representative compound (9b) displayed

H. C. Shen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1299–1305
1303
Table 3
In vitro SAR on human and mouse PrCP inhibition and mouse AngIII assaysa
O
Cl
N
Compound No.
PrCP IC50 (h, m)b, nM
Mouse AngIII IC50, nM
N
N
H
Cl
N
N
N
N
N
N
N
N
9a
4.7, —
—
N
9b
9c
9d
1.4, 0.8
32, —
6.7
39
N
N
N
N
N
HN
27, —
485
N
F
N
N
9e
9f
25, —
7, —
—
—
N
N
N
N
O
O
a
Values are based on one or two experiments, each in triplicate.
h = human, m = mouse.
b
Table 4
Mouse PK profiles of selected PrCP inhibitorsa
5
3
1
wt/veh
wt/39b
ko/veh
ko/39b
Compds F% Cl (mL/min/
Vdss (L/
kg)
T1/2
(h)
AUCNpo
mg)
(l
M h kg/
kg)
8a
8c
8g
8i
11
8
14
3
56
38
54
115
60
4.8
5.7
7.7
3
1.7
4.9
2.2
1.3
3.1
4.1
0.11
0.07
0.07
0.007
0.06
0.02
8j
3
12
9b
4
65
9.4
a
Formulations: 1 mg/mL ethanol/PEG/water (20:40:40). IV dose: 1 mg/kg (n = 2).
PO dose: 2 mg/kg (n = 3). Blood concentration was determined by LC/MS/MS fol-
lowing protein precipitation with acetonitrile.
Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7
-1
2
A: WT Vehicle
B: WT 10 mpk
C: WT 30 mpk
D: WT 60 mpk
100
80
60
40
20
0
0
-2
-4
-6
-8
Day 0
Day 1 Day 2
Day 3 Day 4
Day 5
Day 6 Day 7
µ
9b
M
A+: A + 2
µ
B+: B + 2 M 9b
µ
C+: C + 2 M 9b
µ
D+: D + 2 M 9b
A
B
C
D
A+ B+ C+ D+
wt/veh
wt/93b
ko/veh
ko/39b
-20
Figure 2. Target engagement of compound 9b in wild type eDIO mouse (7th day, sc
continuous pump infusion, normalized to vehicle).
Figure 3. Compound 9b-induced food intake and body weight loss of wild type
eDIO mouse and PrCP knock/out eDIO mouse over seven days (n = 8 for all four
groups; 12 mpk, sc continuous pump infusion; veh = vehicle, ko = PrCP knock out,
wt = wild type; top: daily food intake (g/mouse), bottom: cumulative body weight
gain (g/mouse)).
excellent human PrCP inhibitory activity (IC50 = 1 nM) and mouse
whole serum PrCP inhibitory activity (IC50 = 7 nM). It was found
that this compound was a pgp substrate. Due to its suboptimal oral

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H. C. Shen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1299–1305
O
O
O
a
c
b
N
O
HO
N
O
N
N
Boc
Boc
N
Boc
O
11
12
10
N
13
O
O
N
N
N
d
e
N
N
NBoc
N
NH
NBoc
N
16
15
14
N
N
N
f
N
g
N
Ot-Bu
OH
N
Ot-Bu
N
O
O
O
18
6
17
Cl
Cl
h
N
NH
N
N
N
Cl
Cl
N
HN
H
N
N
N
O
9b
4
Scheme 1. Reagents and conditions: (a) MeNHOMe, EDC, Et3N, CH2Cl2, rt, 16 h, 77%; (b) MeMgBr, THF, À78 °C to 0 °C, 1 h, 96%; (c) 13, NaH, THF, 0 °C to rt, 16 h, 75%; (d)
MeNHNH2, EtOH, reflux, 2 h, chiral OJ column, 57%; (e) trifluoroacetic acid, CH2Cl2, rt, 1 h, 100%; (f) 6, Et3N, CH3CN, 85 °C, 16 h, 92%; (g) trifluoroacetic acid, CH2Cl2, rt, 1 h,
100%; (h) 4, EDC, HOBt, Et3N, CH2Cl2, rt, 5 h, 52%.
14. To the mixture of 10 (47.0 g, 205 mmol) and N,O-dimethylhydroxylamine
PK, 9b was dosed at 12 mpk per day sc for seven days, and signif-
icantly reduced the body weight of the eDIO mice. However, analog
hydrochloride (20.8 g, 213 mmol) in CH2Cl2 (1 L) was added EDC (44.6 g,
232 mmol) in one portion followed by Et3N (32.4 mL, 232 mmol) dropwise at
9b also showed similar efficacy in PrCP knockout eDIO mice, sug-
gesting that compound 9b-induced weight loss in wild type eDIO
mice was due to certain off-target effect. This result indicates that
higher than 50% peripheral and/or certain level of central target
engagement may be required to achieve efficacy.12 The discovery
of other structurally diverse and orally bioavailable PrCP inhibitors
acting at peripheral and/or central tissues may better define the
role of PrCP in regulating food intake and body weight. The results
will be reported in due course.
rt. The resulting solution was stirred at rt for 16 h. The solution was washed
with brine (800 mL Â 4) and a saturated NaHCO3 solution (500 mL), and then
dried over Na2SO4. After evaporating the solvent, the residue was purified on
silica gel using 60%EtOAc/hexane to give 11 (43.2 g, 77%) as an oil.
To the solution of 11 (43.2, 158 mmol) in THF (1.3 L) at À78 °C was added
methylmagnesium bromide (106 mL, 3 M, 317 mmol) dropwise. The solution
was warmed to 0 °C for 1 h before it was quenched with
a saturated
ammonium chloride solution. To this mixture was added EtOAc (1000 mL)
and the organic phase was washed with brine (500 mL Â 2), dried over Na2SO4
to give 12 (34.8 g, 96%) as an oil.
To the solution of 12 (7.99 g, 35.2 mmol) and 13 (5.78 g, 42.2 mmol) in THF
(72 mL) was added NaH (1.69 g, 42.2 mmol, 60%) at 0 °C and the resulting
mixture was stirred at rt for 16 h. The reaction mixture was cooled to À78 °C
before it was quenched by methanol (7 mL) dropwise followed by the addition
of saturated ammonium chloride solution (200 mL) at 0 °C. The resulting
mixture was then extracted with EtOAc (200 mL Â 3). The combined organic
phase was washed with brine (500 mL), and dried over Na2SO4. After removing
the volatile components, the residue was purified on silica gel eluting with 20–
100% EtOAc/hexane to give 14 (8.75 g, 75%) as an oil.
The solution of 14 (9.82 g, 29.5 mmol) and methyl hydrazine (1.90 mL,
35.5 mmol) in ethanol (250 mL) was heated at reflux for 2 h. After removing
the volatile, the residue was purified on silica gel using EtOAc as the eluent to
give a mixture of two regioisomers, which was further purified on chiral HPLC
(OJ column, SFC method) using 25% MeOH/CO2 to give pyrazole 15 (5.73 g,
57%) as a solid.
To the solution of 15 (7.0 g, 20.4 mmol) in CH2Cl2 (120 mL) was added
trifluoroacetic acid (60 mL) and the resulting solution was stirred at rt for 1 h.
After removing the volatile components, the residue was dissolved in CH2Cl2
(300 mL), and washed with NaOH (500 mL, 2 N). The aqueous phase was
extracted with CH2Cl2 (300 mL Â 3). The combined organic phase was dried
over Na2SO4 and concentrated to give 16 (5.15 g) as an oil.
The solution of 16 (5.15 g, 21.3 mmol), Et3N (3.55 mL, 25.5 mmol), and 6
(9.25 mL, 63.8 mmol) in acetonitrile (200 mL) was heated at reflux for 16 h. The
volatile components were evaporated and the residue was purified on silica gel
using 2–10% MeOH/CH2Cl2 to give 17 (7.24 g, 92% in two steps) as an oil.
To the solution of 17 (4.38 g, 11.8 mmol) in CH2Cl2 (10 mL) was added
trifluoroacetic acid (32 mL) and the resulting solution was stirred at rt for 2 h.
After removing the volatile components, the residue was dissolved in
acetonitrile/water (2/1, 500 mL) and lyophilized to give 18 (7.37 g, 95%) as a
TFA salt.
References and notes
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Hum. Genet. 2003, 114, 87.
10. Chissoe, S. US20080108080.
11. Palmiter, R. D. J. Clin. Invest. 2009, 119, 2130.
12. Zhou, C.; Garcia-Calvo, M.; Pinto, S.; Lombardo, M.; Feng, Z.; Bender, K.; Pryor,
K. D.; Bhatt, U. R.; Chabin, R. M.; Geissler, W. M.; Shen, Z.; Tong, X.; Zhang, X.;
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13. Male C57BL/6NT wild-type and PrCP À/À mice (N6 C57BL/6NT background)
were purchased from Taconic Farm, Inc.

H. C. Shen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1299–1305
1305
To the solution of 18 (5.03, 7.66 mmol), 4 (3.78 g, 11.5 mmol), HOBt (1.17 g,
7.66 mmol), and Et3N (8.54 mL, 61.3 mmol) in CH2Cl2 (100 mL) was added EDC
(2.94 g, 15.3 mmol), and the resulting solution was stirred at rt for 4 h. The
reaction solution was then diluted with CH2Cl2 (600 mL), washed with
saturated NaHCO3 (500 mL Â 3), and dried over Na2SO4. After evaporating
the volatile, the residue was purified on silica gel using 5–20% MeOH/CH2Cl2
(0.25% Et3N) to give 9b (2.22 g, 52%) as a white foam. 1H NMR (chloroform-d,
500 MHz): d 8.70 (d, J = 6.2 Hz, 2H), 7.82 (s, 1H), 7.54 (s, 1H), 7.36 (d, J = 6.0 Hz,
2H), 6.24 (s, 1H), 5.44–5.42 (m, 1H), 3.93 (s, 3H), 3.74–3.64 (m, 1H), 3.61–3.56
(m, 1H), 3.17–3.09 (m, 2H), 3.01–2.94 (m, 1H), 2.88–2.75 (m, 4H), 2.64–2.61
(m, 1H), 2.42–2.23 (m, 4H), 2.17–2.12 (m, 1H), 2.12–2.05 (m, 2H), 1.85–1.85
(m, 2H). Exact mass calcd for C28H31Cl2N7O: 551.2 (100%); 553.2 (64%); found
[M+H+]: 552.1 (100%); 554.0 (66%).