Design, synthesis, and evaluation of pyrrolo[2,1-f][1,2,4]triazine derivatives as novel hedgehog signaling pathway inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.12.055

A novel series of Hh signaling pathway inhibitors were designed by replacing the pyrimidine skeleton of our earlier reported lead compound 1 with pyrrolo[2,1-f][1,2,4]triazine scaffold. Starting from this new scaffold, SAR exploration was investigated based on structural modification on A-ring, C-ring and D-ring. And several much potent compounds were studies in vivo to profile their pharmacokinetic properties. Finally, optimization leads to the identification of compound 19a, a potent Hh signaling pathway inhibitor with superior potency in vitro and satisfactory pharmacokinetic properties in vivo.

Full text of DOI:10.1016/j.bmc.2013.12.055

Bioorganic & Medicinal Chemistry 22 (2014) 1429–1440
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and evaluation of pyrrolo[2,1-f][1,2,4]triazine
derivatives as novel hedgehog signaling pathway inhibitors
b
Minhang Xin a,b, , Liandi Zhang , Feng Tang b,c, Chongxing Tu b,c, Jun Wen b, Xinge Zhao b,c, Zhaoyu Liu b,
⇑
Lingfei Cheng b,c, Han Shen b
a Department of Medicinal Chemistry, School of Pharmacy, Health Science Center, Xi’an Jiaotong University, No. 76, Yanta West Road, Xi’an 710061, PR China
b Jiangsu Simcere Pharmaceutical Co. Ltd, Jiangsu Key Laboratory of Molecular Targeted Antitumor Drug Research, No 699-18, Xuan Wu District, Nanjing 210042, PR China
c Nanjing BioSciKin Co. Ltd, Small Molecule Drug Discovery Platform, No 699-18, Xuan Wu District, Nanjing 210042, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of Hh signaling pathway inhibitors were designed by replacing the pyrimidine skeleton of
our earlier reported lead compound 1 with pyrrolo[2,1-f][1,2,4]triazine scaffold. Starting from this new
scaffold, SAR exploration was investigated based on structural modification on A-ring, C-ring and D-ring.
And several much potent compounds were studies in vivo to profile their pharmacokinetic properties.
Finally, optimization leads to the identification of compound 19a, a potent Hh signaling pathway inhib-
itor with superior potency in vitro and satisfactory pharmacokinetic properties in vivo.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 15 October 2013
Revised 11 December 2013
Accepted 22 December 2013
Available online 3 January 2014
Keywords:
Hedgehog signaling pathway
Inhibitors
Pyrrolo[2,1-f][1,2,4]triazine
Novel
1. Introduction
overexpression were detected in many cancers such as pancreatic,
hepatocellular, esophageal, gastric, colorectal, lung, prostate, ovar-
The Hedgehog (Hh) pathway is a key signaling transduction
pathway which regulates many critical cellular processes including
cell proliferation, survival-signals management, embryological
development, tissue patterning, stem-cells maintenance.1 How-
ever, aberrant activation of the Hh signaling pathway has been
implicated in a variety of solid and hematologic tumors. Therefore,
Hh signaling pathway has emerged as an attractive molecular tar-
get and generated a great deal of interest in oncology community.
The Hh signaling cascade is triggered by binding of one of three
Hh ligands, sonic hedgehog (Shh), Indian hedgehog (Ihh), desert
hedgehog (Dhh), to their receptor patched (Ptch), which relieves
the suppressed GPCR-like receptor smoothened (Smo). Activated
Smo moves into the primary cilia to aid to dissociate Suppressor
of fused (Sufu) and Glioma-associated oncogene (Gli) complex,
resulting in activation of the Gli family of transcription factors.
Active Gli signaling then induces the expression of downstream
target genes and leads to cellular proliferation, differentiation.2
Despite Hh signaling pathway is usually inactive in adults, abnor-
mal activation of Hh signaling pathway has commonly occurred in
a number of cancer types. For examples, Strong evidences showed
that genetic mutations such as Ptch-mutation, Smo-mutation, and
Sufu-mutation were involved in basal cell carcinoma (BCC), medul-
loblastoma, and rhabdomyosarcoma. Additionally, Hh ligands
ian, melanoma, glioblastoma, leukemia, lymphoma, etc.3 Thus,
inhibition of Hh signaling pathway has great therapeutic benefit
for combating many tumor types.
The realization that abnormal activation of Hh signaling path-
way is so critically related to the development of a number of hu-
man cancer types promotes the research into the design and
synthesis of many Hh signaling pathway inhibitors and evaluation
of their therapeutic potentials. In recent years, many Hh signaling
pathway inhibitors as a novel class of antitumor drugs were
developed, and some of them were successfully advanced to clini-
cal trials including vismodegib (GDC-0449), sonidegib (NVP-
LDE225), LY-2940680, BMS-833923 (XL-139), saridegib (IPI-926),
PF-04449913, TAK-441 and NVP-LEQ506.4 Recently, vismodegib
has been approved by FDA in 2012 as a first-in-class Hh signaling
pathway inhibitor for treatment of metastatic BCC, meanwhile, it is
now ongoing in other solid and haematological tumors clinical tri-
als, such as medulloblastoma, multiple myeloma, leukemia, pan-
creatic, gastric, gastroesophageal, prostate, chondrosarcoma, and
small-cell lung cancers. Importantly, other clinical candidates are
also evaluated in many tumors clinics.5 Given the increasing inter-
est in the correlation between clinical therapeutic potentials of a
variety of tumors and the targeting Hh signaling pathway,
therefore, novel potent Hh signaling pathway inhibitors are still
emergently needed as probes to identify clinical profiles for Hh sig-
naling targeting strategy.
⇑
Corresponding author. Tel.: +86 25 85560000 3192.
E-mail address: xmhcpu@163.com (M. Xin).
0968-0896/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.12.055

1430
M. Xin et al. / Bioorg. Med. Chem. 22 (2014) 1429–1440
We have recently reported the discovery of a series of 4-(2-pyri-
and following chlorination using phosphoryl chloride afforded
the key building block 12. The readily available 12 were then trea-
ted with p-trifluoromethoxylphenylboronic acid using Pd-cata-
lyzed Suzuki coupling reaction to give 4-aryl-substituted
pyrrolo[2,1-f][1,2,4]triazine 13, and following Buchwald coupling
reaction of 13 with methyl 4-aminobenzoate under palladium ace-
tate catalysis afforded the intermediate 14, which was further
hydrolyzed to produce the free acid 15. The condensation of 15
with 2,6-dimethylaniline provided compound 16. And derivatives
19a–19d were obtained from intermediate 15 by three steps,
which were acylation with 3-amino-4-methylbenzyl alcohol to af-
ford 17, followed by chlorination with thionyl chloride to give 18,
and next by nucleophilic substitution with appropriate amines to
provide 19a–19d (Scheme 1).
The similar synthetic route was employed to prepare the com-
pounds 24a–24d using readily available 12 as starting material.
The intermediate 12 was reacted with 1-methylpyrazole-4-boronic
acid pinacol ester under palladium catalysis to afford the Suzuki
coupling product 20 in 88% yield. Coupling of 20 with 4-aminoben-
zoic acid under Buchwald conditions, followed by condensation,
chlorination, and substitution with corresponding amines, afforded
the compounds 24a–24d (Scheme 2).
Syntheses of A-ring analogues 31a–31c were accomplished
according to Scheme 3. For efficient and convenient preparation
of target compounds, the building block 29 was prepared following
a four-step synthetic route with excellent yield. Starting from com-
mercially available 4-nitrobenzoic acid 25, standard amide bond
coupling with 3-amino-4-methylbenzyl alcohol gave rise to 26.
Chlorination of 26 led to 27, which was subsequently substituted
by morpholine to afford 28. Then following catalytic hydrogenation,
the key building block 29 was obtained. At this point, the appropri-
ate condition of coupling reaction should be considered because of
the distinction of reactivity of arylboric substrates. Fortunately, 30a
was easily prepared in 56% yield by the above Suzuki coupling con-
dition, and the coupling of 12 with pyridine-3-boronic acid was also
achieved when using 1,10-bis(diphenylphosphino)ferrocene-palla-
dium(II)dichloride dichloromethane complex as Pd-catalyst to pro-
vide 30c in 22% yield. However, all our assays to perform Suzuki
coupling between 12 and pyridine-2-boronic reagents failed.9
Therefore, assembly of 2-pyridinyl function to 4-position of pyrrol-
o[2,1-f][1,2,4]triazine backbone was attempted by Stille coupling
reaction using 2-(tributylstannyl)pyridine instead.10 After the reac-
tion parameters such as temperature, reaction time, and solvent
were optimized, 30b was obtained in 30% yield. Accordingly, the
target compounds 31a–31c were achieved by treatment of 29 with
corresponding 30a, 30b, 30c under Buchwald coupling conditions,
respectively (Scheme 3).
midinylamino) benzamides as effective Hh signaling pathway
inhibitors. Preliminary structure–activity relationship (SAR) explo-
ration in this series pointed out that structural modification on
four regions (involving A-ring, B-ring, C-ring and D-ring) greatly
influenced the biological activities. In particular, it was found some
analogues substituted at C-5 and (or) C-6 of pyrimidine of B-ring
displayed significantly potent activity, such as 5-methyl (1),
5-ethyl (2), 6-methyl (3), 6-propyl (4), 5, 6-dimethyl (5) or other
substitutents.6 This founding seemed that the hydrophobic substit-
uents incorporated in both 5- and 6-positions exhibited consider-
able potential for affording enhanced activity. Separately to our
findings, a clinical Hh inhibitor BMS-833923 (also known as XL-
139) (6), investigated in Phase II, was recently disclosed to be
structurally featured by containing quinazoline core.7 Considering
the structural correlation between 5,6-disubstituted pyrimidine of
B-ring and quinazoline core, we speculated that the conceptual
cyclization of the position 5 and 6 of pyrimidine led to be capable
of obtaining a number of new fused-pyrimidine scaffolds as poten-
tial Hh signaling pathway inhibitors (Fig. 1).
As a continuation of our study toward the identification of
effective Hh signaling pathway inhibitors, we decided to take
advantage of the recently commonly-used pyrrolo[2,1-f][1,2,4]tri-
azine skeleton as a privileged scaffold for B-ring surrogate using
a combination of conceptual cyclization and bioisosterism strate-
gies and modify it on the A-ring, C-ring and D-ring based on the
previous SAR study, thereby designing a new series of pyrrol-
o[2,1-f][1,2,4]triazine analogues8 (Fig. 1). Furthermore, the phar-
macokinetic (PK) profiles of some potent compounds were also
investigated. Herein, in this report we disclose the synthesis and
Hh signaling pathway inhibition study of these analogues, and also
we report the PK evaluation in vivo.
2. Chemistry
As summarized in Table 1, the pyrrolo[2,1-f][1,2,4]triazine
derivatives (16, 19a–19d, 24a–24d, 31a–31c and 35a–35e) were
readily synthesized. The optimized synthetic routes were outlined
in Schemes 1–4.
To synthesize the pyrrolo[2,1-f][1,2,4]triazine derivatives, we
first prepared the key intermediate 2,4-dichloropyrrolo[2,1-
f][1,2,4]triazine 12, shown in Scheme 1.8 Treatment of 2-cyanopyr-
role 7 with chloramine under basic condition of sodium hydride
gave 1-amino-2-cyanopyrrole 8. And then the cyano group of 8
was hydrolyzed to amide 9, which was acylated with ethyl chloro-
formate to give the carbamate 10. Cyclization of 10 under basic
condition, the pyrrolo[2,1-f][1,2,4]triazine ring was constructed,
CF3
O
A
O
D
R1
5
R2
4
3
N
2
N
B
C
H
CF3
N
1
N
O
A
6
H
R5
cyclization
1
2
Ar
N
1
2
R =Me, R =H, IC50=1.3nM
our current design
5
series design
6
1
R =Et, R2=H, IC50=5.3nM
4
O
O
R3
D
3 R1=H, R2=Me, IC50=1.4nM
4 R1=H, R2=Pr, IC50=0.8nM
5 R1=Me, R2=Me, IC50=1.6nM
3
N
N
H
1
N
N
H
2
R4
B
N
C
N
Z2
N
N
H
Z1
N
H
O
pyrrolo[2,1-f][1,2,4] triazine series
H
N
N
N
H
N
N
H
6
(BMS-833923)
Figure 1. Our new compounds design.

M. Xin et al. / Bioorg. Med. Chem. 22 (2014) 1429–1440
1431
Table 1
In vitro inhibition of Hh signaling pathway for pyrrolo[2,1-f][1,2,4]triazine derivatives
OCF3
R5
Ar
N
O
O
N
N
H
N
N
H
C
N
R4
N
N
H
N
N
H
19a-19d, 24a-24d, 31a-31c, 35a-35e
16
Compds
Ar
—
C ring
—
R4
—
R5
—
Gli-luc reporter IC50 (nM)
16
1.6
F3CO
F3CO
F3CO
F3CO
N
O
N
19a
19b
19c
19d
Me
Me
Me
Me
0.83
N
N
N
1.7
0.62
1.7
N
N
N
N
N
O
N
24a
24b
24c
24d
Me
Me
Me
Me
1.2
7.5
N
N
N
N
11.1
N
N
N
12.3
1.5
N
N
O
O
31a
31b
31c
Me
Me
Me
N
0.97
N
N
N
O
O
1.6
1.1
8.8
1.1
F3CO
35a
35b
35c
Me
H
N
N
N
F3CO
N
N
N
N
O
O
O
Me
N
35d
Me
0.73
F
N
N
O
35e
1
Me
—
1.0
1.3
N
—
—
—
Cl
O
Cl
N
N
H
GDC-0449
7.2
O
O
S
The similar synthetic routes employed to prepare the C-ring
analogues 35a–35e were described in Scheme 4. Beginning with
appropriately starting materials (13, 30b and 30c), the desired
Buchwald coupling products 32a–32e were achieved under Pd
catalyst, followed by condensation (33a–33e) and chlorination to
generate chlorides (34a–34e), subsequently nucleophilic substitu-
tion with morpholine to afford the target compounds 35a–35e
(Scheme 4).
and the vitro IC50 values were illustrated in Table 1. We started
by preparing and characterizing the pyrrolo[2,1-f][1,2,4]triazine
B-ring surrogate of the parent pyrimidine 1. Whereas the pyrrol-
o[2,1-f][1,2,4]triazine analogue 16 showed Hh inhibitory activity
with an IC50 of 1.6 nM, nearly comparable to parent lead 1
(1.3 nM) and more potent than GDC-0449 (7.2 nM). This result ap-
peared that pyrrolo[2,1-f][1,2,4]triazine scaffold for surrogate of
pyrimidine-ring exhibited considerable potential for developing
novel Hh signaling pathway inhibitors and prompted us to
investigate SAR in detail for the novel series of pyrrolo[2,1-
f][1,2,4]triazines.
3. Results and discussion
The Hh signaling pathway inhibitory activity of our series of
pyrrolo[2,1-f][1,2,4]triazine derivatives were initially assessed
using a luciferase reporter in NIH3T3 cell carrying a stably trans-
fected Gli-reporter construct (Gli-luciferase reporter cell lines)11
Our initial focus was on incorporating cyclic aliphatic amines
substitutions on C-5 of D-ring, which were considered to be helpful
for improving physicochemical properties based on the previous
SAR study. Therefore, some cyclic aliphatic amines were explored.

1432
M. Xin et al. / Bioorg. Med. Chem. 22 (2014) 1429–1440
OCF3
Cl
N
O
NH2
O
f
e
c
d
a
b
N
H
NH
N
H2N
N
O
N
OEt
N
N
H
N
H2N
N
78%
94%
56%
38%
54%
29%
CN
CN
Cl
N
H
11
O
H2N
O
12
N
9
8
7
10
N
OCF3
OCF3
OCF3
N
Cl
13
h
g
O
i
O
O
N
OH
N
N
OCH3 70%
32%
N
N
H
58%
N
N
N
N
N
N
N
N
H
H
H
j
15
14
16
61%
OCF3
OCF3
OCF3
O
O
O
k
l
R4
Cl
OH
N
N
N
H
N
N
N
N
N
H
97%
H
4
N
N
19a
19b
N
(59%, R =1-morpholinyl)
N
H
N
N
H
N
H
4
(42%, R =4-methylpiperazin-1-yl)
19c (38%, R4=1-piperidinyl)
19d (59%, R4=1-pyrrolidinyl)
18
17
Scheme 1. Reagents and conditions: (a) NaH, DMF, NH2Cl, rt, overnight; (b) KOH, H2O, rt, overnight; (c) pyridine, ClCO2Et, dioxane, 110 °C for 1 h; (d) EtOH, EtONa, reflux for
2 h; (e) POCl3, DIPEA, 120 °C for 48 h; (f) 4-trifluoromethoxyphenylboronic acid, Pd(PPh3)2Cl2, TEA, DMF, H2O, 80 °C for 4 h; (g) methyl 4-aminobenzoate, Pd(OAc)2, BINAP,
Cs2CO3, dioxane, 150 °C, microwave for 3 h; (h) NaOH, acetone/H2O, reflux for 5 h; (i) 2,6-dimethylaniline, HATU, TEA, DMF, rt for 16 h; (j) 3-amino-4-methylbenzyl alcohol,
HATU, DMF, TEA, 80 °C for 16 h; (k) SOCl2, CH2Cl2, rt for 5 h; (l) morpholine for 19a, 4-methylpiperazine for 19b, piperidine for 19c, pyrrolidine for 19d, CH3CN, K2CO3, reflux
for 6 h.
N
N
N
N
N
N
N
N
Cl
N
c
O
a
d
b
O
O
N
N
Cl
OH
N
88%
N
N
H
55%
91%
81%
N
OH
N
N
H
N
Cl
N
Cl
N
N
N
N
N
H
N
N
H
N
N
H
12
20
21
23
22
N
N
4
24a
24b
O
(82%, R=OCF3,R =1-morpholinyl)
4
e
R4
(89%, R=OCF3,R =4-methylpiperazin-1-yl)
N
N
H
24c (72%, R=OCF3,R4=1-piperidinyl)
24d (61%, R=OCF3,R4=1-pyrrolidinyl)
N
N
N
H
Scheme 2. Reagents and conditions: (a) 1-methyl-1H-pyrazole-4-boronic acid pinacol ester, Pd(PPh3)2Cl2, 2 N Na2CO3, dioxane, 70 °C for 16 h; (b) 4-aminobenzoic acid,
Pd(OAc)2, BINAP, Cs2CO3, dioxane, reflux for 16 h; (c) 3-amino-4-methylbenzyl alcohol, HATU, DMF, DIPEA, 80 °C for 16 h; (d) SOCl2, CH2Cl2, rt for 5 h; (e) morpholine for 24a,
4-methylpiperazine for 24b, piperidine for 24c, pyrrolidine for 24d, CH3CN, K2CO3, reflux for 6 h.
O
O
O
O
O
O
O
c
d
N
b
Cl
N
OH
a
OH
N
H
N
H
N
H
N
H
85%
100 %
100%
99%
O2N
H2N
O2N
O2N
O2N
29
28
27
25
26
O
N
B(OH)2
29
O
N
N
N
H
f, 28%
e, 56%
N
N
Cl
N
N
N
H
30a
31a
O
N
N
N
Cl
Sn(Bu)3
N
N
29
O
N
N
N
H
g, 30%
h, 55%
N
N
N
Cl
N
Cl
N
N
N
H
30b
12
31b
O
N
N
N
N
B(OH)2
29
O
N
j, 39%
N
N
i, 22%
N
H
N
Cl
N
N
N
H
30c
31c
Scheme 3. Reagents and conditions: (a) (1) SOCl2, reflux for 3 h; (2) 3-amino-4-methylbenzyl alcohol, THF, DIPEA, rt for 26 h; (b) SOCl2, CH2Cl2, rt for 2.5 h; (c) morpholine,
DMF, K2CO3, 85 °C for 2.5 h; (d) 10% Pd/C, H2, rt for 24 h; (e) phenylboronic acid, Pd(PPh3)2Cl2, DMF, TEA, H2O, 80 °C for 16 h; (f) intermediate 29, Pd(OAc)2, BINAP, Cs2CO3,
dioxane, 150 °C microwave for 3 h; (g) 2-(tributylstannyl)pyridine, Pd(PPh3)4, toluene, N2, reflux for 7 h; (h) 29, Pd(OAc)2, BINAP, Cs2CO3, dioxane, 150 °C microwave for 3 h;
(i) pyridine-3-boronic acid, Pd(dppf)Cl2ꢀCH2Cl2, DMF, TEA, H2O, 80 °C for 16 h; (j) 29, Pd(OAc)2, BINAP, Cs2CO3, dioxane, 150 °C microwave for 3 h.
It was found that the morpholine 19a (0.83 nM) and the piperidine
19c (0.62 nM) showed an approximate 2-fold more effective than
16, while N-methyl piperazine 19b (1.7 nM) and pyrrolidine 19d
(1.7 nM) displayed satisfactorily equipotent potency compared to
16. However, when trifluoromethoxyphenyl of A-ring was replaced
with 1-methylpyrazolyl, only compound 24a bearing the

M. Xin et al. / Bioorg. Med. Chem. 22 (2014) 1429–1440
1433
R
N
(A)
R
N
R
N
R
N
R
X
N
R5
X
N
R5
X
N
X
N
R5
O
X
N
O
b
O
O
a
c
d
O
OH
5
N
N
H
Cl
5
OH
N
H
N
H
N
N
N
N
N
N
N
N
N
N
H
33a
33b
N
H
Cl
N
H
N
N
H
5
13
(R=OCF3, X=C)
(65%, R=OCF3, X=C, R =CH3)
35a
35b
(78%, R=OCF3, X=C, R =CH3)
(76%, R=OCF3, X=C, R =H)
32a
(57%, R=OCF3, X=C)
32b=32a
32c
34a
34b
(100%, R=OCF3, X=C, R =CH3)
30b
(R=H, X=N)
5
5
(65%, R=OCF3, X=C, R =H)
5
(62%, R=OCF3, X=C, R =H)
33c (38%, R=H, X=N, R5=CH3)
35c (70%, R=H, X=N, R5=CH3)
(52%, R=H, X=N)
34c (98%, R=H, X=N, R5=CH3)
30b
from
e, 55%
(B)
N
N
N
N
O
N
O
O
N
O
O
Cl
h
g
47%
f
N
N
H
OH
OH
N
N
N
N
H
H
N
N
89%
41%
N
N
N
H
N
N
N
H
N
N
H
N
N
H
F
F
F
F
32d
34d
33d
35d
(C)
N
N
N
N
N
N
O
O
j
O
O
i
k
l
O
OH
N
N
N
Cl
OH
N
N
N
H
56%
N
N
H
92%
H
89%
73%
N
N
N
N
N
N
H
N
N
N
N
H
N
N
Cl
N
N
H
N
N
N
H
N
33e
35e
34e
30c
32e
Scheme 4. Reagents and conditions: (a) 5-amino-2-pyridinecarboxylic acid, Pd(OAc)2, BINAP, Cs2CO3, dioxane, reflux for 17 h; (b) 3-amino-4-methylbenzyl alcohol for 33a
and 33c, 3-aminobenzyl alcohol for 33b, HATU, DMF, DIPEA, 80 °C for 5 h; (c) SOCl2, CH2Cl2, rt for 5 h; (d) morpholine, DMF, K2CO3, 85 °C for 2.5 h; (e) 4-amino-3-
fluorobenzoic acid, Pd(OAc)2, BINAP, Cs2CO3, dioxane, reflux for 17 h; (f) phenylboronic acid, Pd(PPh3)2Cl2, DMF, TEA, H2O, 80 °C for 16 h; (f) 3-amino-4-methylbenzyl alcohol,
HATU, DMF, DIPEA, 80 °C for 5 h; (g) SOCl2, CH2Cl2, rt for 5 h; (h) morpholine, DMF, K2CO3, 85 °C for 2.5 h; (i) 6-amino-3-pyridinecarboxylic acid, Pd(OAc)2, BINAP, Cs2CO3,
dioxane, reflux for 17 h; (j) 3-amino-4-methylbenzyl alcohol, HATU, DMF, DIPEA, 80 °C for 5 h; (k) SOCl2, CH2Cl2, rt for 5 h; (l) morpholine, DMF, K2CO3, 85 °C for 2.5 h.
morpholine group gave encouraging potency with an IC50 = 1.2 nM.
While other compounds such as 24b with N-methyl piperazine
(7.5 nM), 24c with piperidine (11.1 nM), and 24d with pyrrolidine
(12.3 nM) exhibited significantly decreased activity compared to
16. These results indicated that the morpholine side chain was
favorable to the activity and thus morpholine at this position
was selected for subsequent investigation. On the other hand, the
biological data between 19a with 24a (IC50: 0.83 vs 1.2 nM)
showed that the A-ring trifluoromethoxyphenyl one was more po-
tent than 1-methylpyrazolyl one. The same cases were accounted
by compounds 19b and 24b (IC50: 1.7 vs 7.5 nM), 19c and 24c
(IC50: 0.62 vs 11.1 nM), as well as 19d and 24d (IC50: 1.7 vs
12.3 nM). This SAR trend was consistent with our previously re-
ported pyrimidine-core ones. We speculated A-ring aryl moiety
of the 6-membered ring to be preferred to 5-membered for main-
taining inhibitory activity, so it was reasonable to screen inhibitors
of 6-membered aryl ring in A-moiety. Thus, the representative
examples of 31a–31c were prepared. The Hh signaling inhibitory
activity revealed the 2-pyridinyl 31b (0.97 nM) provided a small
improvement in potency, while the activities of the phenyl 31a
(1.5 nM) and 3-pyridinyl 31b (1.6 nM) were similarly comparable,
in comparison to 16. Since the A-ring and D-ring were investigated,
we then turned our attention to C-ring. The selected examples
35a–35e were shown in Table 1. Compared 35a with 19a, it ap-
peared that replacement of phenyl with a more electron-deficient
pyridine was tolerated, and the same conclusion was also drawn by
the comparison 35c with 31b, as well as 35e with 31c. Addition-
ally, it appeared that introduction of electron-withdrawing fluoro
group at phenyl of C-ring (compound 35d), the potency was
slightly improved. However, an attempt to remove the methyl
group of D-ring which was presumed to be a metabolic point, led
to decrease inhibition (compound 35b). In a word, all the pyrrol-
o[2,1-f][1,2,4]triazine analogs showed good Hh signaling inhibitory
activity with IC50 varied from 0.62 to 12.3 nM. And the SAR study
showed 13 compounds displayed better inhibitory activities than
positive control GDC-0449, of which 8 ones exhibited superior
activities to our earlier reported compound 1.
selected for further investigating the PK properties in SD rats.
The results compared with GDC-0449 were outlined in Table 2.
After intravenous injection with 1 mg/kg in SD rats, 16 exhibited
moderate PK properties with an acceptable exposure
(AUC = 1427.65 h ng/mL) and low clearance (Cl = 579.86 mL/h/
kg), but long half-time (T1/2 = 15.15 h). Such a long elimination
phase half-life was likely due to its large volume of distribution
(Vz = 12587.00 mL/kg). While 19a bearing morpholine group
showed encouraging PK characteristics, the area-under-curve
(AUC) of 19a reached 2230.32 h ng/mL, and the clearance was
414.07 mL/h/kg. Furthermore, it was eliminated slowly with
approximately 5-fold longer time than GDC-0449 (T1/2 of 8.86 vs
1.64 h). Unfortunately, although 1-methylpyrazolyl 24a and 2-
pyrimidinyl 31b contained much hydrophilic group compared to
19a, they only displayed lower exposure, which were mainly due
to their high systemic clearance. However, similar to 19a, 35a
showed high exposure and low clearance, while its peak-plasma-
concentrations reached 4185.93 ng/mL. This means its easily
generating risk of toxicity. In a word, PK profiles suggested that
pyrrolo[2,1-f][1,2,4]triazine structure had encouraging PK proper-
ties, especially, compound 19a showing much comparable plasma
exposure and longer half-life. Therefore, based on the optimal
studies of combination of activity and PK properties, compounds
19a was considered to be the promising leading one, which exhib-
ited superior potency, satisfactory PK properties in iv adminis-
trated manner, compared to GDC-0449. And now 19a was
considered to be advanced into in vivo antitumor efficacy evalua-
tion of Hh signaling operative solid tumor models.
4. Conclusion
In summary, we have designed a novel series of Hh signaling
pathway inhibitors bearing pyrrolo[2,1-f][1,2,4]triazine framework
as the core scaffold to replace the pyrimidine skeleton of our earlier
reported lead compounds. Starting from the new scaffold, we have
done SAR exploration based on structural modification on A-ring,
C-ring and D-ring. Moreover, several much potent compounds
were investigated in vivo to profile their PK properties. Considering
the combination of Hh signaling pathway inhibitory activity
in vitro and PK profiles in vivo, 19a was identified to be the
To evaluate whether the novel pyrrolo[2,1-f][1,2,4]triazine
derivatives had an satisfactory druggability, of our most potent
inhibitors, several compounds 16, 19a, 24a, 31b and 35a were

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M. Xin et al. / Bioorg. Med. Chem. 22 (2014) 1429–1440
Table 2
Pharmacokinetic properties for represented compounds in SD ratsa
Compds
Cmax (ng/mL)
AUC (h ng/mL)
Vz (mL/kg)
Cl (mL/h/kg)
MRT(0 ꢁ t) (h)
T1/2 (h)
GDC-0449
16
19a
24a
31b
1249.82
2440.45
2720.52
1072.67
637.48
2455.65
1427.65
2230.32
790.59
764.22
1935.17
954.15
12587.00
5230.65
2188.02
2850.4
406.23
579.86
414.07
1267.67
1322.16
502.80
2.23
4.68
4.83
1.31
1.64
3.49
1.64
15.15
8.86
1.23
1.51
8.34
35a
4185.93
5973.95
a
Compound (including GDC-0449) was formulated using 5% DMA + 5% Tween-80, iv 1 mg/kg.
promising candidate compound, and further in vivo biological
evaluations are required to determine the antitumor efficacy.
5.1.4. Ethyl(2-carbamoyl-1H-pyrrol-1-yl)carbamate (10)
In a 25 mL reaction vial, pyridine (0.51 mL) was added to a solu-
tion of 9 (250 mg, 2 mmol) in dioxane (5 mL), and the mixture was
stirred for 15 min. Ethyl chloroformate (0.5 mL) was slowly added,
and the resulting mixture was stirred at reflux for 1 h. The reaction
mixture was concentrated in vacuo to give the crude product 10
(370 mg, 94%). MS (ESI) m/z: [M+H]+ = 198.
5. Experimental
5.1. Chemical synthesis
5.1.1. Materials
5.1.5. Pyrrolo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione (11)
In a 25 mL reaction vial, EtONa (182 mg, 2 mmol) was added to
a solution of 10 (250 mg, 1.3 mmol) in EtOH (10 mL). The reaction
mixture was stirred at reflux for 2 h. The reaction mixture was
cooled to room temperature and the precipitated solid was col-
lected by filtration and dried in vacuo to give 11 (169 mg, 56%)
as a white solid. MS (ESI) m/z: [MꢁH]ꢁ = 150. 1H NMR (400 M,
DMSO-d6) d 12.99 (br s, 1H, NH), 11.18 (br s, 1H, NH), 7.12 (m,
1H, ArH), 6.75 (m, 1H, ArH), 6.33 (m, 1H, ArH) ppm.
All chemicals were purchased from commercial sources and
used without further purification unless otherwise noted. Melting
points of compounds were measured on a Melt-Temp II apparatus
and uncorrected. 1H NMR spectra (400 MHz) and 13C NMR
(100 MHz) spectra were recorded on a Bruker BioSpin AG (Ultra-
shield Plus AV 400) spectrometer as deuterochloroform (CDCl3)
or dimethyl sulfoxide-d6 (DMSO-d6) solutions using tetramethyl-
silane (TMS) as an internal standard (d = 0) unless noted otherwise.
MS spectra was obtained on an Agilent technologies 6120 quadru-
pole LC/MS (ESI). High-resolution mass spectra (HRMS) were re-
corded on a Water Q-Tof micro mass spectrometer. The purity of
the compounds was verified by the HPLC study using a mixture
of solvent methanol/water or acetonitrile/water at the flow rate
of 2 mL/min and peak detection at 254 nm under UV. Microwave
reaction was operated on CEM microwave reactor. The solvents
(such as MeOH, EtOAc, EtOH, CH2Cl2 and others) were C.P. grade
purchased from Nanjing Chemical Co., Ltd and used without fur-
ther purification. Column chromatography (CC) was carried out
on silica gel (200–300 mesh, Qindao Ocean Chemical Company,
China). Thin-layer chromatography (TLC) analyses was carried
out on silica gel GF254 (Qindao Ocean Chemical Company, China).
Concentration and evaporation of the solvent after reaction or
extraction was carried out on rotary evaporator operated at re-
duced pressure.
5.1.6. 2,4-Dichloropyrrolo[2,1-f][1,2,4]triazine (12)
In a 25 mL reaction vial, a mixture of 11 (190 mg, 1.3 mmol) and
POCl3 (2 mL) and DIPEA (2 mL) was stirred at 120 °C for 24 h. After
cooling to room temperature, the reaction was poured slowly into
ice and stirred vigorously for 30 min, followed by extraction with
EtOAc. The organic layer was washed with water, and brine, dried
by anhydrous Na2SO4, and concentrated in vacuo. The residue was
purified by chromatography (petroleum ether/EtOAc, 8:1) to give
the title intermediate 12 (80 mg, 38%) as a yellow solid. MS (ESI)
m/z: [M+H]+ = 188. 1H NMR (400 M, DMSO-d6) d 7.88 (d, 1H,
J = 0.8 Hz, ArH), 7.07 (d, 1H, J = 3.6 Hz, ArH), 6.99 (dd, 1H,
J1 = 2.4 Hz, J2 = 4.8 Hz, ArH) ppm.
5.1.7. 2-Chloro-4-(4-(trifluoromethoxy)phenyl)pyrrolo[2,1-
f][1,2,4]triazine (13)
In a 50 mL reaction vial, Pd(PPh3)2Cl2 (30 mg, 0.042 mmol) and
NEt3 (121 mg, 1.2 mmol) were added to a solution of 12 (80 mg,
0.424 mmol) and 4-trifluoromethoxylphenylboronic acid (90 mg,
0.44 mmol) in DMF (28.5 mL) and H2O (0.5 mL). The mixture was
stirred at 80 °C for 4 h under N2 atmosphere. The reaction mixture
was cooled to room temperature and filtrated. The filtrate was di-
luted with H2O (100 mL) and then extracted with EtOAc, and the
organic layer was dried over anhydrous Na2SO4, after filtration,
the filtrate was evaporated and purified by chromatography
(petroleum ether/EtOAc, 20:1) to give the product 13 as a yellow
solid (97 mg, 78%). MS (ESI) m/z: [M+H]+ = 314. 1H NMR (400 M,
CDCl3) d 8.18 (d, 2H, J = 8.4 Hz, ArH), 7.92 (s, 1H, ArH), 7.42 (d,
2H, J = 8.4 Hz, ArH), 7.13 (d, 1H, J = 4.4 Hz, ArH), 7.03 (dd, 1H,
J1 = 2.4 Hz, J2 = 4.4 Hz, ArH) ppm.
5.1.2. 1-Amino-1H-pyrrole-2-carbonitrile (8)
In a 25 mL reaction vial, NaH (1370 mg, 60%) was slowly added
to a solution of 1H-pyrrole-2-carbonitrile (1800 mg, 19 mmol) in
DMF (10 mL), and the mixture was stirred for 1 h at room temper-
ature. The solution of NH2Cl (1950 mg, 38 mmol) in diethyl ether
(195 mL) was prepared according to literature,8 and the resulting
mixture was stirred overnight. The reaction mixture was filtrated
and the filtrate was evaporated, and then diluted with ice water
(50 mL) and extracted with EtOAc. After dried over anhydrous Na2-
SO4, the organic layer was concentrated and purified by chroma-
tography (petroleum ether/EtOAc, 8:1) to give the product 8 as
an oil (1122 mg, 54%). MS (ESI) m/z: [M+H]+ = 108. 1H NMR
(400 M, DMSO-d6) d 6.98 (m, 1H, ArH), 6.85 (m, 1H, ArH), 6.05
(m, 1H, ArH), 5.02 (s, 2H, NH2) ppm.
5.1.8. Methyl 4-((4-(4-(trifluoromethoxy)phenyl)pyrrolo[2,1-
f][1,2,4]triazin-2-yl)amino)benzoate (14)
5.1.3. 1-Amino-1H-pyrrole-2-carboxamide (9)
In a 50 mL reaction vial, 8 (1120 mg, 10 mmol) was added to a
solution of KOH (5870 mg, 100 mmol) in H2O (30 mL). The mixture
was stirred at stirred at room temperature. The mixture was ad-
justed to a pH = 7–8 with 2 M HCl in an ice bath. The precipitated
solid was collected by filtration and dried in vacuo to give 9
(380 mg, 29%) as a white solid. MS (ESI) m/z: [M+H]+ = 126.
In a 25 mL reaction vial, Pd(OAc)2 (14 mg, 0.064 mmol), BINAP
(80 mg, 0.128 mmol) and Cs2CO3 (208 mg, 0.64 mmol) were added
to a solution of 13 (100 mg, 0.32 mmol) and methyl 4-aminoben-
zoate (53 mg, 0.35 mmol) in dioxane (2 mL). The mixture was stir-
red at 150 °C for 3 h at automatic CEM microwave reactor. The

M. Xin et al. / Bioorg. Med. Chem. 22 (2014) 1429–1440
1435
reaction mixture was cooled to room temperature and filtrated.
The filtrate was evaporated and purified by chromatography
(petroleum ether/EtOAc, 40:1) to give the product 14 (80 mg,
58%) as a yellow product. MS (ESI) m/z: [M+H]+ = 429. 1H NMR
(400 M, DMSO-d6) d 10.04 (s, 1H, NH), 8.29 (d, 2H, J = 8.4 Hz,
ArH), 8.05 (s, 1H, ArH), 7.96 (m, 4H, ArH), 7.64 (d, 2H, J = 8.4 Hz,
ArH), 7.10 (d, 1H, J = 4.4 Hz, ArH), 6.93 (dd, 1H, J1 = 2.4 Hz,
J2 = 4.4 Hz, ArH), 3.83 (s, 3H, OCH3) ppm.
concentrated in vacuo. The residue was purified by chromatogra-
phy (petroleum ether/EtOAc, 2:1) to give the product 18 (80 mg,
97%) as a yellow solid. MS (ESI) m/z: [M+H]+ = 552. 1H NMR
(400 M, DMSO-d6) d 9.93 (s, 1H, CONH), 9.72 (s, 1H, NH), 8.32 (d,
2H, J = 8.4 Hz, ArH), 8.07 (s, 1H, ArH), 7.98 (m, 4H, ArH), 7.68 (d,
2H, J = 8.4 Hz, ArH), 7.33 (s, 1H, ArH), 7.23 (d, 1H, J = 7.6 Hz, ArH),
7.11 (d, 1H, J = 8.0 Hz, ArH), 7.08 (d, 1H, J = 8.0 Hz, ArH), 6.91 (m,
1H, ArH), 4.76 (s, 2H, CH2Cl), 2.23 (s, 3H, ArCH3) ppm.
5.1.9. 4-((4-(4-(Trifluoromethoxy)phenyl)pyrrolo[2,1-
f][1,2,4]triazin-2-yl)amino)benzoic acid (15)
5.1.13. N-(2-Methyl-5-(morpholinomethyl)phenyl)-4-((4-(4-
(trifluoromethoxy)phenyl)pyrrolo[2,1-f][1,2,4]triazin-2-
yl)amino)benzamide (19a)
In a 10 mL reaction vial, NaOH (20 mg, 0.5 mmol) and H2O
(1 mL) were added to a solution of 14 (80 mg, 0.19 mmol) in ace-
tone (5 mL). The mixture was stirred at reflux for 5 h. The reaction
mixture was cooled to room temperature, poured into ice water
(30 mL), and stirred vigorously. After acidification with 2 M HCl,
the precipitated solid was collected by filtration. The solid was
washed with ice water and dried in vacuo to give 15 (54 mg,
70%) as a yellow solid. MS (ESI) m/z: [M+H]+ = 415. 1H NMR
(400 M, DMSO-d6) d 12.35 (s, 1H, COOH), 9.78 (s, 1H, NH), 8.30
(d, 2H, J = 8.4 Hz, ArH), 8.07 (s, 1H, ArH), 7.98 (s, 4H, ArH), 7.65
(d, 2H, J = 8.4 Hz, ArH), 7.08 (d, 1H, J = 4.4 Hz, ArH), 6.95 (dd, 1H,
J1 = 2.4 Hz, J2 = 4.4 Hz, ArH) ppm.
In a 25 mL reaction vial, morpholine (20 mg, 0.23 mmol) and
K2CO3 (30 mg, 0.23 mmol) were added to a solution of 18 (40 mg,
0.073 mmol) in CH3CN (10 mL). The mixture was stirred at reflux
for 6 h. The reaction mixture was poured into ice water (50 mL)
and extracted with EtOAc. Then the organic layer was dried over
anhydrous Na2SO4, and concentrated in vacuo. The residue was
purified by chromatography (DCM/MeOH, 50:1) to give the inter-
mediate 19a (26 mg, 59%) as a yellow solid. mp 242–244 °C. HPLC:
95.9%. MS (ESI) m/z: [M+H]+ = 603.2. 1H NMR (400 M, DMSO-d6) d
9.92 (s, 1H, CONH), 9.68 (s, 1H, NH), 8.29 (d, 2H, J = 8.4 Hz, ArH),
8.05 (s, 1H, ArH), 7.96 (m, 4H, ArH), 7.65 (d, 2H, J = 8.0 Hz, ArH),
7.32 (s, 1H, ArH), 7.23 (d, 1H, J = 8.0 Hz, ArH), 7.08 (d, 2H,
J = 8.0 Hz, ArH), 6.90 (m, 1H, ArH), 3.57 (s, 4H, morpholine-H),
3.44 (s, 2H, ArCH2), 2.36 (s, 4H, morpholine-H), 2.23 (s, 3H, ArCH3)
ppm, 13C NMR (100 M, DMSO-d6) d 17.62, 53.10, 62.04, 66.16,
105.75, 113.34, 117.32, 118.17, 118.74, 120.52, 121.07, 126.38,
126.56, 126.95, 128.51, 130.01, 130.94, 132.16, 134.42, 135.51,
136.51, 143.45, 150.45, 152.75, 157.77, 164.83; HRMS(ESI) m/z
calcd for C32H30N6O3F3 [M+H]+ 603.2341, found 603.2331.
5.1.10. N-(2,6-Dimethylphenyl)-4-((4-(4-
(trifluoromethoxy)phenyl)pyrrolo[2,1-f][1,2,4]triazin-2-
yl)amino)benzamide (16)
In a 10 mL reaction vial, HATU (228 mg, 0.6 mmol) and TEA
(92 mg, 0.91 mmol) were added to a solution of 15 (100 mg,
0.24 mmol) and 2,6-dimethylaniline in DMF (5 mL). The mixture
was stirred at room temperature for 16 h under N2 atmosphere.
The reaction mixture was poured into ice water (30 mL) followed
by extraction with EtOAc. Then the organic layer was dried over
anhydrous Na2SO4, and concentrated in vacuo. The residue was
purified by chromatography (petroleum ether/EtOAc, 20:1) to give
the product 16 (40 mg, 32%) as a yellow solid. mp 253–255 °C.
HPLC: 99.0%. MS (ESI) m/z: [M+H]+ = 518.2. 1H NMR (400 M, CDCl3)
d 8.14 (d, 2H, J = 8.4 Hz, ArH), 7.97 (d, 2H, J = 8.4 Hz, ArH), 7.81 (d,
2H, J = 8.8 Hz, ArH), 7.42 (d, 2H, J = 8.0 Hz, ArH), 7.36 (s, 1H, ArH),
7.26 (s, 1H, CONH), 7.13 (s, 4H, ArH⁄3 + NH), 6.95 (d, 1H,
J = 4.4 Hz, ArH), 6.83 (s, 1H, ArH), 2.30 (s, 6H, 2⁄CH3) ppm, 13C
NMR (100 M, DMSO-d6) d 18.07, 105.73, 113.31, 117.41, 118.16,
118.74, 120.49, 121.06, 126.47, 126.52, 127.64, 128.38, 130.93,
134.42, 135.56, 135.72, 143.39, 150.43, 152.76, 157.77, 164.63;
HRMS(ESI) m/z calcd for C28H23N5O2F3 [M+H]+ 518.1811, found
518.1804.
5.1.14. N-(2-Methyl-5-((4-methylpiperazin-1-
yl)methyl)phenyl)-4-((4-(4-
(trifluoromethoxy)phenyl)pyrrolo[2,1-f][1,2,4]triazin-2-
yl)amino)benzamide (19b)
Similar procedure of 19a was employed to give compound 19b
(40 mg, 42%) as a yellow solid. mp 180–182 °C. HPLC: 95.1%. MS
(ESI) m/z: [M+H]+ = 616.3. 1H NMR (400 M, CDCl3) d 8.13 (d, 2H,
J = 8.4 Hz, ArH), 7.93 (d, 3H, J = 7.6 Hz, ArH), 7.82 (d, 2H,
J = 8.4 Hz, ArH), 7.65 (s, 1H, CONH), 7.43 (d, 2H, J = 8.0 Hz, ArH),
7.20 (d, 1H, J = 7.6 Hz, ArH), 7.10 (d, 1H, J = 4.0 Hz, ArH), 7.07 (s,
1H, NH), 6.96 (d, 1H, J = 4.8 Hz, ArH), 6.84 (m, 1H, ArH), 3.53 (s,
2H, ArCH2), 2.52 (s, 8H, piperazine-H), 2.24 (s, 3H, ArCH3), 2.23
(s, 3H, NCH3) ppm. 13C NMR (100 M, DMSO-d6) d 17.68, 45.40,
49.39, 52.63, 60.65, 105.76, 113.33, 117.30, 117.48, 118.17,
120.52, 121.08, 126.53, 128.53, 130.14, 130.94, 134.42, 136.62,
143.49, 150.45, 152.75, 157.78, 164.80; HRMS(ESI) m/z calcd for
5.1.11. N-(5-(Hydroxymethyl)-2-methylphenyl)-4-((4-(4-
(trifluoromethoxy)phenyl)pyrrolo[2,1-f][1,2,4]triazin-2-
yl)amino)benzamide (17)
C
33H33N7O2F3 [M+H]+ 616.2656, found 616.2648.
Similar to the preparation of 16, the title compound 17 (80 mg,
61%) was obtained as a yellow powder. MS (ESI) m/z: [M+H]+ = 534.
1H NMR (400 M, DMSO-d6) d 9.92 (s, 1H, CONH), 9.68 (s, 1H, NH),
8.29 (d, 2H, J = 8.4 Hz, ArH), 8.05 (s, 1H, ArH), 7.97 (m, 4H, ArH),
7.65 (d, 2H, J = 8.0 Hz, ArH), 7.33 (s, 1H, ArH), 7.23 (d, 1H,
J = 8.0 Hz, ArH), 7.11 (d, 1H, J = 8.0 Hz, ArH), 7.08 (d, 1H,
J = 7.6 Hz, ArH), 6.91 (m, 1H, ArH), 5.17 (m, 1H, OH), 4.49 (d, 2H,
J = 4.8 Hz, CH2OH), 2.22 (s, 3H, ArCH3) ppm.
5.1.15. N-(2-Methyl-5-(piperidin-1-ylmethyl)phenyl)-4-((4-(4-
(trifluoromethoxy)phenyl)pyrrolo[2,1-f][1,2,4]triazin-2-
yl)amino)benzamide (19c)
Similar procedure of 19a was employed to give compound 19c
(18 mg, 38%) as a yellow solid. mp 138–140 °C. HPLC: 95.7%. MS
(ESI) m/z: [M+H]+ = 601.3. 1H NMR (400 M, DMSO-d6) d 9.93 (s,
1H, CONH), 9.70 (s, 1H, NH), 8.30 (d, 2H, J = 8.0 Hz, ArH), 8.05
(s, 1H, ArH), 7.98 (m, 4H, ArH), 7.66 (d, 2H, J = 8.0 Hz, ArH), 7.32
(s, 1H, ArH), 7.24 (d, 1H, J = 7.6 Hz, ArH), 7.10 (d, 2H, J = 4.4 Hz,
ArH), 6.93 (m, 1H, ArH), 3.46 (s, 2H, ArCH2), 2.38 (s, 4H, piperi-
dine-H), 2.24 (s, 3H, ArCH3), 1.52 (s, 4H, piperidine-H), 1.23 (s,
2H, piperidine-H) ppm, 13C NMR (100 M, DMSO-d6) d 17.62,
23.78, 28.95, 53.65, 62.17, 105.75, 113.33, 117.31, 118.16, 120.52,
121.07, 126.44, 126.55, 127.01, 128.50, 129.97, 130.93, 132.18,
134.41, 136.40, 143.45, 150.45, 152.75, 157.77, 164.82; HRMS(ESI)
m/z calcd for C33H32N6O2F3 [M+H]+ 601.2547, found 601.2539.
5.1.12. N-(5-(Chloromethyl)-2-methylphenyl)-4-((4-(4-
(trifluoromethoxy)phenyl)pyrrolo[2,1-f][1,2,4]triazin-2-
yl)amino)benzamide (18)
In a 25 mL reaction vial, SOCl2 (0.5 mL) was added to a solution
of 17 (80 mg, 0.15 mmol) in CH2Cl2 (10 mL). The mixture was stir-
red at room temperature for 5 h. The reaction mixture was washed
with water and the organic layer dried over anhydrous Na2SO4, and

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M. Xin et al. / Bioorg. Med. Chem. 22 (2014) 1429–1440
5.1.16. N-(2-Methyl-5-(pyrrolidin-1-ylmethyl)phenyl)-4-((4-(4-
(trifluoromethoxy)phenyl)pyrrolo[2,1-f][1,2,4]triazin-2-
yl)amino)benzamide (19d)
NMR (400 M, DMSO-d6) d 9.73 (s, 1H, CONH), 9.69 (s, 1H, NH),
8.78 (s, 1H, ArH), 8.32 (s, 1H, ArH), 7.99 (m, 4H, ArH), 7.92 (s, 1H,
ArH), 7.48 (s, 1H, ArH), 7.31 (m, 3H, ArH), 6.85 (m, 1H, ArH), 4.78
(s, 2H, CH2Cl), 4.01 (s, 3H, ArCH3), 2.26 (s, 3H, ArCH3) ppm.
Similar procedure of 19a was employed to give compound 19d
(21 mg, 39%) as a yellow solid. mp 136–138 °C. HPLC: 95.1%. MS
(ESI) m/z: [M+H]+ = 587.2. 1H NMR (400 M, DMSO-d6) d 9.91 (s,
1H, CONH), 9.67 (s, 1H, NH), 8.29 (d, 2H, J = 8.4 Hz, ArH), 8.05
(s, 1H, ArH), 7.97 (m, 4H, ArH), 7.65 (d, 2H, J = 8.0 Hz, ArH), 7.31
(s, 1H, ArH), 7.22 (d, 1H, J = 7.6 Hz, ArH), 7.09 (d, 2H, J = 4.8 Hz,
ArH), 6.93 (m, 1H, ArH), 3.54 (s, 2H, ArCH2), 2.43 (s, 4H, pyrroli-
dine-H), 2.22 (s, 3H, ArCH3), 1.69 (s, 4H, pyrrolidine-H) ppm, 13C
NMR (100 M, DMSO-d6) d 17.61, 23.09, 53.41, 59.16, 105.74,
113.32, 117.31, 118.16, 120.51, 121.06, 126.48, 126.59, 128.49,
129.93, 130.93, 132.82, 134.41, 136.38, 137.22, 143.44, 150.45,
152.75, 157.76, 164.81; HRMS(ESI) m/z calcd for C32H30N6O2F3
[M+H]+ 587.2390, found 587.2382.
5.1.21. 4-((4-(1-Methyl-1H-pyrazol-4-yl)pyrrolo[2,1-
f][1,2,4]triazin-2-yl)amino)-N-(2-methyl-5-
(morpholinomethyl)phenyl)benzamide (24a)
Similar to the preparation of 19a, the title compound 24a
(41 mg, 82%) was obtained as a yellow powder. mp 158–160 °C.
HPLC: 95.4%. MS (ESI) m/z: [M+H]+ = 523.3. 1H NMR (400 M, CDCl3)
d 8.26 (s, 1H, CONH), 8.18 (s, 1H, NH), 7.97 (s, 1H, ArH), 7.92 (d, 2H,
J = 8.0 Hz, ArH), 7.81 (d, 2H, J = 8.0 Hz, ArH), 7.71 (s, 1H, ArH), 7.66
(s, 1H, ArH), 7.20 (d, 1H, J = 8.0 Hz, ArH), 7.11 (d, 1H,
J = 8.0 Hz,ArH), 7.01 (s, 1H, ArH), 6.97 (m, 1H, ArH), 6.80 (m, 1H,
ArH), 4.03 (s, 3H, ArCH3), 3.73 (m, 4H, morpholine-H), 3.52 (s,
2H, ArCH2), 2.48 (s, 4H, morpholine-H), 2.34 (s, 3H, ArCH3) ppm,
13C NMR (100 M, DMSO-d6) d 17.63, 40.06, 53.04, 61.97, 66.09,
104.52, 112.38, 117.17, 117.19, 118.13, 119.71, 126.23, 126.41,
126.99, 128.49, 130.02, 132.21, 132.94, 135.36, 136.48, 139.35,
143.74, 152.38, 152.80, 164.88; HRMS(ESI) m/z calcd for
5.1.17. 2-Chloro-4-(1-methyl-1H-pyrazol-4-yl)pyrrolo[2,1-
f][1,2,4]triazine (20)
In a 25 mL reaction vial, Pd(PPh3)2Cl2 (19 mg, 0.027 mmol) and
2 N Na2CO3 solution (0.27 mL, 0.54 mmol) were added to a solution
of 1-methyl-1H-pyrazole-4-boronic acid pinacol ester (55 mg,
0.27 mmol) and 12 (50 mg, 0.27 mmol) in dioxane (5 mL). The mix-
ture was stirred at 70 °C for 16 h under N2 atmosphere. The reac-
tion mixture was cooled to room temperature and filtrated. The
filtrate was diluted with H2O (100 mL) and then extracted with
EtOAc, and the organic layer was dried over anhydrous Na2SO4,
After filtration, the filtrate was evaporated and purified by chroma-
tography (petroleum ether/EtOAc, 5:1) to give the product 20 as a
yellow solid (55 mg, 88%). MS (ESI) m/z: [M+H]+ = 234. 1H NMR
(400 M, DMSO-d6) d 8.87 (s, 1H, ArH), 8.38 (s, 1H, ArH), 8.14 (s,
1H, ArH), 7.53 (d, 1H, J = 4.8 Hz, ArH), 7.10 (dd, 1H, J1 = 2.4 Hz,
J2 = 4.8 Hz, ArH), 3.98 (s, 3H, ArCH3) ppm.
C
29H31N8O2 [M+H]+ 523.2577, found 523.2570.
5.1.22. 4-((4-(1-Methyl-1H-pyrazol-4-yl)pyrrolo[2,1-
f][1,2,4]triazin-2-yl)amino)-N-(2-methyl-5-((4-
methylpiperazin-1-yl)methyl)phenyl)benzamide (24b)
Similar to the preparation of 19a, the title compound 24b
(40 mg, 89%) was obtained as a yellow powder. mp 142-144 °C.
HPLC: 98.9%. MS (ESI) m/z: [M+H]+ = 536.3. 1H NMR (400 M,
DMSO-d6) d 9.67 (d, 2H, CONH+NH), 8.77 (s, 1H, ArH), 8.32 (s,
1H, ArH), 7.97 (m, 4H, ArH), 7.92 (s, 1H, ArH), 7.29 (s, 1H, ArH),
7.26 (d, 1H, J = 4.8 Hz, ArH), 7.22 (d, 1H, J = 7.6 Hz, ArH), 7.08 (s,
1H, ArH), 6.84 (m, 1H, ArH), 4.02 (s, 3H, ArCH3), 3.43 (s, 2H, ArCH2),
2.38 (s, 8H, piperazine-H), 2.22 (s, 3H, ArCH3), 2.17 (s, 3H, NCH3)
ppm, 13C NMR (100 M, DMSO-d6) d 17.62, 40.07, 45.27, 52.07,
54.42, 56.02, 61.50, 104.52, 112.38, 117.17, 117.19, 118.13,
119.71, 126.24, 126.86, 128.49, 129.99, 132.10, 132.94, 135.80,
136.45, 139.35, 143.74, 152.39, 152.80, 164.88; HRMS(ESI) m/z
calcd for C30H34N9O [M+H]+ 536.2894, found 536.2886.
5.1.18. 4-((4-(1-Methyl-1H-pyrazol-4-yl)pyrrolo[2,1-
f][1,2,4]triazin-2-yl)amino)benzoic acid (21)
In a 25 mL reaction vial, Pd(OAc)2 (4 mg, 0.018 mmol), BINAP
(22 mg, 0.036 mmol) and Cs2CO3 (176 mg, 0.54 mmol) were added
to a solution of 20 (50 mg, 0.21 mmol) and 4-aminobenzoic acid
(53 mg, 0.35 mmol) in dioxane (5 mL). The mixture was stirred at
reflux for 16 h under N2 atmosphere. The reaction mixture was
cooled to room temperature and filtrated. The filtrate was poured
into water and adjusted to a pH=4ꢂ5 with 2 M HCl. The precipitated
solid was collected by filtration and dried in vacuo to give 21 (52 mg,
81%) as a yellow solid. MS (ESI) m/z: [M+H]+ = 335. 1H NMR (400 M,
DMSO-d6) d 12.50 (s, 1H, COOH), 9.74 (s, 1H, NH), 8.76 (s, 1H, ArH),
8.31 (s, 1H, ArH), 7.89 (s, 5H, ArH), 7.26 (d, 1H, J = 4.4 Hz, ArH), 6.84
(dd, 1H, J1 = 2.4 Hz, J2 = 4.8 Hz, ArH), 4.00 (s, 3H, ArCH3) ppm.
5.1.23. 4-((4-(1-Methyl-1H-pyrazol-4-yl)pyrrolo[2,1-
f][1,2,4]triazin-2-yl)amino)-N-(2-methyl-5-(piperidin-1-
ylmethyl)phenyl)benzamide (24c)
Similar to the preparation of 19a, the title compound 24c
(55 mg, 72%) was obtained as a yellow powder. mp 136–138 °C.
HPLC: 96.3%. MS (ESI) m/z: [M+H]+ = 521.3. 1H NMR (400 M,
DMSO-d6) d 9.76 (s, 1H, CONH), 9.71 (s, 1H, NH), 8.79 (s, 1H,
ArH), 8.32 (s, 1H, ArH), 8.02 (m, 4H, ArH), 7.93 (s, 1H, ArH), 7.39
(s, 1H, ArH), 7.28 (m, 2H, ArH), 7.19 (s, 1H, ArH), 6.85 (m, 1H,
ArH), 4.01(s, 3H, ArCH3), 3.39 (s, 2H, ArCH2), 3.06 (s, 4H, piperi-
dine-H), 2.25 (s, 3H, ArCH3), 1.58 (s, 4H, piperidine-H), 1.43 (s,
2H, piperidine-H) ppm, 13C NMR (100 M, DMSO-d6) d 17.70,
23.05, 24.31, 45.52, 52.96, 61.01, 104.54, 112.36, 117.15, 117.20,
118.11, 119.70, 126.16, 127.07, 127.75, 128.53, 130.14, 132.98,
136.60, 139.34, 143.79, 152.38, 152.81, 162.30, 164.87; HRMS(ESI)
m/z calcd for C30H33N8O [M+H]+ 521.2783, found 521.2777.
5.1.19. N-(5-(Hydroxymethyl)-2-methylphenyl)-4-((4-(1-
methyl-1H-pyrazol-4-yl)pyrrolo[2,1-f][1,2,4]triazin-2-
yl)amino)benzamide (22)
Similar procedure of 17 was employed to give compound 22
(200 mg, 55%) as a yellow solid. MS (ESI) m/z: [M+H]+ = 454. 1H
NMR (400 M, DMSO-d6) d 9.66 (s, 2H, CONH + NH), 8.76 (s, 1H,
ArH), 8.31 (s, 1H, ArH), 7.97 (m, 4H, ArH), 7.91 (s, 1H, ArH), 7.33
(s, 1H, ArH), 7.26 (d, 1H, J = 4.4 Hz, ArH), 7.22 (d, 1H, J = 7.6 Hz,
ArH), 7.11 (d, 1H, J = 7.6 Hz, ArH), 6.84 (m, 1H, ArH), 5.76 (m, 1H,
OH), 4.49 (d, 2H, J = 5.6 Hz, CH2OH), 4.00 (s, 3H, ArCH3), 2.23 (s,
3H, ArCH3) ppm.
5.1.24. 4-((4-(1-Methyl-1H-pyrazol-4-yl)pyrrolo[2,1-
f][1,2,4]triazin-2-yl)amino)-N-(2-methyl-5-(pyrrolidin-1-
ylmethyl)phenyl)benzamide (24d)
Similar to the preparation of 19a, the title compound 24d
(50 mg, 61%) was obtained as a yellow powder. mp 135–137 °C.
HPLC: 95.7%. MS (ESI) m/z: [M+H]+ = 507.3. 1H NMR (400 M,
DMSO-d6) d 9.68 (d, 2H, CONH+NH), 8.77 (s, 1H, ArH), 8.31 (s,
1H, ArH), 8.00 (m, 4H, ArH), 7.92 (s, 1H, ArH), 7.34 (s, 1H, ArH),
7.26 (d, 1H, J = 4.0 Hz, ArH), 7.22 (d, 1H, J = 7.6 Hz, ArH), 7.11 (d,
5.1.20. N-(5-(Chloromethyl)-2-methylphenyl)-4-((4-(1-methyl-
1H-pyrazol-4-yl)pyrrolo[2,1-f][1,2,4]triazin-2-
yl)amino)benzamide (23)
Similar procedure of 18 was employed to give compound 23
(180 mg, 91%) as a yellow solid. MS (ESI) m/z: [M+H]+ = 472. 1H

M. Xin et al. / Bioorg. Med. Chem. 22 (2014) 1429–1440
1437
1H, J = 7.6 Hz, ArH), 6.84 (m, 1H, ArH), 4.04 (s, 3H, ArCH3), 3.61 (s,
2H, ArCH2), 3.34 (s, 4H, pyrrolidine-H), 2.23 (s, 3H, ArCH3), 1.72 (s,
4H, pyrrolidine-H) ppm, 13C NMR (100 M, DMSO-d6) d 17.66, 22.85,
45.45, 53.07, 58.06, 104.54, 112.39, 117.16, 118.11, 119.71, 126.16,
126.52, 127.25, 128.52, 130.21, 132.97, 136.59, 139.35, 143.78,
5.1.30. N-(2-Methyl-5-(morpholinomethyl)phenyl)-4-((4-
phenylpyrrolo[2,1-f][1,2,4]triazin-2-yl)amino)benzamide (31a)
In a 25 mL reaction vial, Pd(OAc)2 (7 mg, 0.032 mmol), BINAP
(40 mg, 0.64 mmol) and Cs2CO3 (104 mg, 0.32 mmol) were added
to a solution of 30a (50 mg, 0.16 mmol) and 29 (27 mg, 0.18 mmol)
in dioxane (5 mL). The mixture was stirred at 150 °C for 3 h at
automatic CEM microwave reactor. The reaction mixture was
cooled to room temperature and filtrated. The filtrate was evapo-
rated and purified by chromatography (petroleum ether/EtOAc,
2:1) to give the title compound 31a (30 mg, 28%) as a yellow solid.
mp 230–232 °C. HPLC: 95.1%. MS (ESI) m/z: [M+H]+ = 519.2. 1H
NMR (400 M, DMSO-d6) d 9.88 (s, 1H, CONH), 9.68 (s, 1H, NH),
8.15 (d, 2H, J = 6.8 Hz, ArH), 8.02 (s, 1H, ArH), 7.96 (s, 4H, ArH),
7.68 (m, 3H, ArH), 7.31 (s, 1H, ArH), 7.23 (d, 1H, J = 8.0 Hz, ArH),
7.10 (d, 1H, J = 7.2 Hz, ArH), 7.06 (d, 1H, J = 4.4 Hz, ArH), 6.89 (m,
1H, ArH), 3.58 (t, 4H, morpholine-H), 3.44 (s, 2H, ArCH2), 2.37 (t,
4H, morpholine-H), 2.23 (s, 3H, ArCH3) ppm, 13C NMR (100 M,
DMSO-d6) d 17.68, 53.10, 61.99, 66.15, 105.89, 110.82 114.03,
118.42, 120.75, 123.07, 123.94, 126.63, 126.97, 130.11, 131.14,
132.27, 135.62, 136.06, 136.41, 137.49, 148.08, 149.31, 151.81,
152.18, 154.95, 157.00, 163.53; HRMS(ESI) m/z calcd for
152.38, 152.79, 164.92; HRMS(ESI) m/z calcd for
C29H31N8O
[M+H]+ 507.2628, found 507.2621.
5.1.25. N-(5-(Hydroxymethyl)-2-methylphenyl)-4-
nitrobenzamide (26)
In a 25 mL reaction vial, the solution of 4-nitrobenzoic acid 25
(1000 mg, 5.98 mmol) in SOCl2 (5 mL) was stirred at reflux for
3 h. The reaction mixture was evaporated to dry to provide acyl
chloride, which was prepared into a solution in dry THF (1 mL).
Then to a solution of DIPEA (695 mg, 5.38 mmol) and 3-amino-4-
methylbenzyl alcohol (406 mg, 2.96 mmol) in THF (6 mL) at 0 °C,
the newly prepared solution of acyl chloride was slowly dropped.
The reaction mixture was warm to room temperature and stirred
for another 26 h. The mixture was diluted with water and ex-
tracted by EtOAc. After dried over anhydrous Na2SO4, the organic
layer was concentrated in vacuo and the residue was purified by
chromatography (petroleum ether/EtOAc, 10:1) to give the product
26 (800 mg, 99%) as a white solid. MS (ESI) m/z: [M+H]+ = 287. 1H
NMR (400 M, DMSO-d6) d 10.23 (s, 1H, CONH), 8.39 (d, 2H,
J = 8.0 Hz, ArH), 8.21 (d, 2H, J = 8.4 Hz, ArH), 7.30 (s, 1H, ArH),
7.25 (d, 1H, J = 8.0 Hz, ArH), 7.15 (d, 1H, J = 7.6 Hz, ArH), 5.21 (t,
1H, J = 5.2 Hz, OH), 4.49 (d, 2H, J = 4.0 Hz, CH2O), 2.22 (s, 3H, ArCH3)
ppm.
C
31H31N6O [M+H]+ 519.2464, found 519.2456.
5.1.31. 2-Chloro-4-(pyridin-2-yl)pyrrolo[2,1-f][1,2,4]triazine
(30b)
2-(Tributylstannyl)pyridine
(1000 mg,
2.72 mmol)
and
Pd(PPh3)4 (300 mg, 0.27 mmol) were added to a solution of 12
(500 g, 2.72 mmol) in toluene (100 mL). The mixture was stirred
at reflux for 7 h under N2 atmosphere. After cooled to room tem-
perature, the reaction mixture was quenched by adding water, fol-
lowed by extraction with EtOAc. Then the organic layer was dried
over anhydrous Na2SO4, and concentrated in vacuo. The residue
was purified by chromatography (petroleum ether/EtOAc, 8:1) to
give the 30b (190 mg, 30%) as a yellow solid. MS (ESI) m/z:
[M+H]+ = 231. 1H NMR (400 M, CDCl3) d 8.85 (d, 1H, J = 4.0 Hz,
ArH), 8.58 (d, 1H, J = 8.0 Hz, ArH), 7.96 (d, 1H, J = 4.0 Hz, ArH),
7.92 (m, 2H, ArH), 7.51 (m, 1H, ArH), 7.06 (m, 1H, ArH) ppm.
5.1.26. N-(5-(Chloromethyl)-2-methylphenyl)-4-
nitrobenzamide (27)
Similar to the preparation of 18, the title compound 27 (323 mg,
100%) was obtained as a white solid. MS (ESI) m/z: [M+H]+ = 305.
1H NMR (400 M, DMSO-d6) d 10.30 (s, 1H, NH), 8.41 (d, 2H,
J = 8.4 Hz, ArH), 8.23 (d, 2H, J = 8.4 Hz, ArH), 7.46 (s, 1H, ArH),
7.33 (m, 2H, ArH), 4.78 (s, 2H, CH2), 2.26 (s, 3H, CH3) ppm.
5.1.27. N-(2-Methyl-5-(morpholinomethyl)phenyl)-4-
nitrobenzamide (28)
5.1.32. N-(2-Methyl-5-(morpholinomethyl)phenyl)-4-((4-
(pyridin-2-yl)pyrrolo[2,1-f][1,2,4]triazin-2-yl)amino)benzamide
(31b)
Similar to the preparation of 19a, the title compound 28 (268 mg,
100%) was obtained as a white solid. MS (ESI) m/z: [M+H]+ = 356. 1H
NMR (400 M, DMSO-d6) d 8.36 (d, 2H, J = 8.4 Hz, ArH), 8.05 (d, 2H,
J = 8.4 Hz, ArH), 7.84 (s, 1H, NH), 7.66 (s, 1H, ArH), 7.22 (d, 1H,
J = 8.0 Hz, ArH), 7.16 (d, 1H, J = 7.6 Hz, ArH), 3.70 (t, 4H, CH2⁄2),
3.50 (s, 2H, CH2), 2.46 (t, 4H, CH2⁄2), 2.33 (s, 3H, CH3) ppm.
Similar to the preparation of 31a, the title compound 31b
(46 mg, 55%) was obtained as a yellow solid. mp 134–136 °C.
HPLC: 99.5%. MS (ESI) m/z: [M+H]+ = 520.2. 1H NMR (400 M,
CDCl3) d 8.86 (d, 1H, J = 4.0 Hz, ArH), 8.46 (d, 1H, J = 8.0 Hz,
ArH), 7.97 (s, 1H, CONH), 7.94 (m, 3H, ArH), 7.84 (s, 1H, ArH),
7.80 (d, 2H, J = 8.0 Hz, ArH), 7.70 (d, 1H, J = 4.0 Hz, ArH), 7.67 (s,
1H, NH), 7.51 (m, 1H, ArH), 7.21 (d, 1H, J = 8.0 Hz, ArH), 7.15 (s,
1H, ArH), 7.12 (d, 1H, J = 8.0 Hz, ArH), 6.88 (m, 1H, ArH), 3.74 (t,
4H, morpholine-H), 3.53 (s, 2H, ArCH2), 2.49 (s, 4H, morpholine-
H), 2.35 (s, 3H, ArCH3) ppm, 13C NMR (100 M, DMSO-d6) d
17.63, 53.03, 61.59, 66.08, 108.48, 113.43 117.27, 117.95,
120.07, 123.10, 126.19, 126.43, 126.51, 127.01, 128.54, 130.03,
132.24, 136.47, 137.29, 143.53, 149.52, 152.56, 153.64, 155.93,
164.85; HRMS(ESI) m/z calcd for C30H30N7O2 [M+H]+ 520.2467,
found 520.2461.
5.1.28. 4-Amino-N-(2-methyl-5-
(morpholinomethyl)phenyl)benzamide (29)
In a 25 mL reaction vial, a solution of compound 28 (256 mg,
0.72 mmol) in MeOH (10 mL), 10% Pd/C (20 mg) was added. The
suspension was stirred at room temperature and hydrogen was
bubbled for 24 h. The reaction mixture was filtered, and evapo-
rated. The residue was triturated with ether to give the title com-
pound (200 mg, 85%) as
a colorless powder. MS (ESI) m/z:
[M+H]+ = 326. 1H NMR (400 M, DMSO-d6) d 9.35 (s, 1H, NH), 7.71
(d, 2H, J = 8.4 Hz, ArH), 7.26 (s, 1H, ArH), 7.18 (d, 1H, J = 7.6 Hz,
ArH), 7.05 (d, 1H, J = 7.6 Hz, ArH), 6.60 (d, 2H, J = 8.4 Hz, ArH),
5.71 (s, 2H, NH2), 3.56 (t, 4H, CH2⁄2), 3.41 (s, 2H, CH2), 2.35 (s,
4H, CH2⁄2), 2.18 (s, 3H, CH3) ppm.
5.1.33. 2-Chloro-4-(pyridin-3-yl)pyrrolo[2,1-f][1,2,4]triazine
(30c)
.
Similar to the preparation of 13, while using Pd(dppf)Cl2 CH2Cl2
5.1.29. 2-Chloro-4-phenylpyrrolo[2,1-f][1,2,4]triazine (30a)
Similar to the preparation of 13, the title compound 30a (48 mg,
56%) was obtained as a yellow solid. MS (ESI) m/z: [M+H]+ = 230.
1H NMR (400 M, CDCl3) d 8.12 (d, 2H, J = 7.9 Hz, ArH), 7.89 (m,
1H, ArH), 7.62 (m, 3H, ArH), 7.15 (d, 1H, J = 4.7 Hz, ArH), 7.00 (m,
1H, ArH) ppm.
as catalyst and pyridine-3-boronic acid as a reactant, the title com-
pound 30c (258 mg, 22%) was obtained as a yellow solid. MS (ESI)
m/z: [M+H]+ = 231. 1H NMR (400 M, DMSO-d6) d 9.28 (d, 1H,
J = 1.6 Hz, ArH), 8.74(d, 1H, J = 3.6 Hz, ArH), 8.52 (d, 1H, J = 8.0 Hz,
ArH), 8.35 (s, 1H, ArH), 7.70 (m, 1H, ArH), 7.43 (d, 1H, J = 4.8 Hz,
ArH), 7.23 (m, 1H, ArH) ppm.

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M. Xin et al. / Bioorg. Med. Chem. 22 (2014) 1429–1440
5.1.34. N-(2-Methyl-5-(morpholinomethyl)phenyl)-4-((4-
(pyridin-3-yl)pyrrolo[2,1-f][1,2,4]triazin-2-
yl)amino)benzamide (31c)
122.61, 122.91, 124.80, 128.11, 130.03, 130.98, 134.27, 135.98,
138.37, 140.01, 141.71, 152.51, 157.96, 161.81; HRMS(ESI) m/z
calcd for C31H29N7O3F3 [M+H]+ 604.2291, found 604.2284.
Similar to the preparation of 31a, the title compound 31c
(40 mg, 39%) was obtained as a yellow solid. mp 220–222 °C. HPLC:
98.9%. MS (ESI) m/z: [M+H]+ = 520.2. 1H NMR (400 M, DMSO-d6) d
9.99 (s, 1H, CONH), 9.72 (s, 1H, NH), 9.30 (s, 1H, ArH), 8.87 (d,
1H, J = 4.0 Hz, ArH), 8.53 (d, 1H, J = 8.0 Hz, ArH), 8.09 (s, 1H, ArH),
7.98 (s, 4H, ArH), 7.71 (m, 1H, ArH), 7.32 (s, 1H, ArH), 7.24 (d,
1H, J = 8.0 Hz, ArH), 7.11 (s, 2H, ArH), 6.93(s, 1H, ArH), 3.58 (s,
4H, morpholine-H), 3.45 (s, 2H, ArCH2), 2.52 (s, 4H, morpholine-
H), 2.24 (s, 3H, ArCH3) ppm, 13C NMR (100 M, DMSO-d6) d 17.65,
53.10, 60.22, 62.76, 66.15, 105.73, 113.43 117.31, 118.25, 120.65,
123.89, 126.35, 126.93, 128.51, 130.00, 131.22, 132.15, 132.94,
135.51, 143.40, 149.15, 152.08, 152.72, 157.10, 164.75; HRMS(ESI)
m/z calcd for C30H29N7O2Na [M+ Na]+ 542.2287, found 542.2280.
5.1.39. N-(3-(Hydroxymethyl)phenyl)-5-((4-(4-
(trifluoromethoxy)phenyl)pyrrolo[2,1-f][1,2,4]triazin-2-
yl)amino)picolinamide (33b)
Similar to the preparation of 17, the title compound 33b
(163 mg, 65%) was obtained as a yellow solid. MS (ESI) m/z:
[M+H]+ = 521. 1H NMR (400 M, DMSO-d6) d 10.42 (s, 1H, CONH),
10.27 (s, 1H, NH), 9.16 (s, 1H, ArH), 8.50 (d, 1H, J = 8.0 Hz, ArH),
8.30 (d, 2H, J = 8.0 Hz, ArH), 8.15 (d, 1H, J = 8.0 Hz, ArH), 8.09 (s,
1H, ArH), 7.91 (s, 1H, ArH), 7.73 (d, 1H, J = 4.0 Hz, ArH), 7.66 (d,
2H, J = 8.0 Hz, ArH), 7.33 (t, 1H, J = 8.0 Hz, ArH), 7.14 (s, 1H, ArH),
7.08 (d, 1H, J = 8.0 Hz, ArH), 6.96 (s, 1H, ArH), 5.23 (s, 1H, OH),
4.51 (d, 2H, J = 4.0 Hz, CH2OH) ppm.
5.1.35. 5-((4-(4-(Trifluoromethoxy)phenyl)pyrrolo[2,1-
f][1,2,4]triazin-2-yl)amino)picolinic acid (32a)
5.1.40. N-(3-(Chloromethyl)phenyl)-5-((4-(4-
(trifluoromethoxy)phenyl)pyrrolo[2,1-f][1,2,4]triazin-2-
yl)amino)picolinamide (34b)
Similar to the preparation of 18, the title compound 34b
(106 mg, 62%) was obtained as a yellow solid. MS (ESI) m/z:
[M+H]+ = 539.
Similar to the preparation of 21, the title compound 32a
(415 mg, 57%) was obtained as a yellow solid. MS (ESI) m/z:
[M+H]+ = 416. 1H NMR (400 M, DMSO-d6) d 12.82 (br s, 1H, COOH),
10.25 (s, 1H, NH), 9.07 (s, 1H, ArH), 8.43 (d, 1H, J = 8.0 Hz, ArH),
8.29 (d, 2H, J = 8.0 Hz, ArH), 8.09 (s, 1H, ArH), 8.06 (d, 1H,
J = 8.0 Hz, ArH), 7.66 (d, 2H, J = 12.0 Hz, ArH), 7.13 (d, 1H,
J = 4.0 Hz, ArH), 6.95 (m, 1H, ArH) ppm.
5.1.41. N-(3-(Morpholinomethyl)phenyl)-5-((4-(4-
(trifluoromethoxy)phenyl)pyrrolo[2,1-f][1,2,4]triazin-2-
yl)amino)picolinamide (35b)
5.1.36. N-(5-(Hydroxymethyl)-2-methylphenyl)-5-((4-(4-
(trifluoromethoxy)phenyl)pyrrolo [2,1-f][1,2,4]triazin-2-
yl)amino)picolinamide (33a)
Similar to the preparation of 19a, the title compound 35b
(89 mg, 76%) was obtained as a yellow solid. mp 193–195 °C. HPLC:
97.3%. MS (ESI) m/z: [M+H]+ = 590.2. 1H NMR (400 M, DMSO-d6) d
10.46 (s, 1H, CONH), 10.28 (s, 1H, NH), 9.16 (d, 1H, J = 4.0 Hz,
ArH), 8.50 (d, 1H, J = 8.0 Hz, ArH), 8.30 (d, 2H, J = 8.0 Hz, ArH),
8.15 (d, 1H, J = 8.0 Hz, ArH), 8.09 (s, 1H, ArH), 7.91 (s, 1H, ArH),
7.79 (d, 1H, J = 8.0 Hz, ArH), 7.66 (d, 2H, J = 8.0 Hz, ArH), 7.33 (t,
1H, J = 8.0 Hz, ArH), 7.14 (d, 1H, J = 4.0 Hz, ArH), 7.06 (d, 1H,
J = 8.0 Hz, ArH), 6.96 (s, 1H, ArH), 3.60 (s, 4H, morpholine-H),
3.47(s, 2H, ArCH2), 2.38 (s, 4H, morpholine-H) ppm, 13C NMR
(100 M, DMSO-d6) d 53.14, 62.53, 66.17, 105.98, 113.63 118.24,
118.62, 118.72, 120.40, 120.64, 121.02, 121.28, 122.69, 124.20,
124.69, 128.41, 130.94, 134.25, 138.24, 138.33, 138.42, 139.89,
141.80, 150.50, 152.49, 157.58, 164.75; HRMS(ESI) m/z calcd for
Similar to the preparation of 17, the title compound 33a
(150 mg, 65%) was obtained as a yellow solid. MS (ESI) m/z:
[M+H]+ = 535. 1H NMR (400 M, DMSO-d6) d 10.28 (s, 1H, CONH),
10.07 (s, 1H, NH), 9.13 (d, 1H, J = 4.0 Hz, ArH), 8.52 (dd, 1H,
J1 = 4.0 Hz, J2 = 8.0 Hz, ArH), 8.30 (d, 2H, J = 8.0 Hz, ArH), 8.13 (m,
2H, ArH), 7.91 (s, 1H, ArH), 7.66 (d, 2H, J = 8.0 Hz, ArH), 7.23 (d,
1H, J = 8.0 Hz, ArH), 7.13 (d, 1H, J = 4.0 Hz, ArH), 7.06 (d, 1H,
J = 8.0 Hz, ArH), 6.96 (m, 1H, ArH), 5.20 (m, 1H, OH), 4.49 (t, 2H,
J = 4.0 Hz, CH2OH), 2.32 (s, 3H, ArCH3) ppm.
5.1.37. N-(5-(Chloromethyl)-2-methylphenyl)-5-((4-(4-
(trifluoromethoxy)phenyl)pyrrolo[2,1-f][1,2,4]triazin-2-
yl)amino)picolinamide (34a)
C
30H27N3O3F3 [M+H]+ 590.2136, found 590.2127.
Similar to the preparation of 18, the title compound 34a (127 mg,
100%) was obtained as a yellow solid. MS (ESI) m/z: [M+H]+ = 553.
1H NMR (400 M, DMSO-d6) d 10.29 (s, 1H, CONH), 10.09 (s, 1H,
NH), 9.15 (d, 1H, J = 4.0 Hz, ArH), 8.53 (dd, 1H, J1 = 4.0 Hz,
J2 = 8.0 Hz, ArH), 8.32 (d, 2H, J = 8.0 Hz, ArH), 8.15 (m, 2H, ArH),
7.93 (s, 1H, ArH), 7.67 (d, 2H, J = 8.0 Hz, ArH), 7.24 (d, 1H,
J = 8.0 Hz, ArH), 7.13 (d, 1H, J = 4.0 Hz, ArH), 7.06 (d, 1H, J = 8.0 Hz,
ArH), 6.96 (m, 1H, ArH), 4.72 (s, 2H, CH2Cl), 2.32 (s, 3H, ArCH3) ppm.
5.1.42. 5-((4-(Pyridin-2-yl)pyrrolo[2,1-f][1,2,4]triazin-2-
yl)amino)picolinic acid (32c)
Similar to the preparation of 21, the title compound 32c
(136 mg, 52%) was obtained as a yellow solid. MS (ESI) m/z:
[M+H]+ = 333. 1H NMR (400 M, DMSO-d6) d 10.18 (s, 1H, NH),
9.10 (d, 1H, ArH), 8.89 (s, 1H, ArH), 8.55 (m, 2H, ArH), 8.20 (m,
3H, ArH), 7.69 (d, 2H, J = 4.0 Hz, ArH), 6.94 (m, 1H, ArH) ppm.
5.1.43. N-(5-(Hydroxymethyl)-2-methylphenyl)-5-((4-(pyridin-
2-yl)pyrrolo[2,1-f][1,2,4]triazin-2-yl)amino)picolinamide (33c)
Similar to the preparation of 17, the title compound 33c (64 mg,
38%) was obtained as a yellow solid. MS (ESI) m/z: [M+H]+ = 452.
1H NMR (400 M, DMSO-d6) d 10.26 (s, 1H, CONH), 10.08 (s, 1H,
NH), 9.14 (d, 1H, J = 4.0 Hz, ArH), 8.90 (d, 1H, J = 4.0 Hz, ArH),
8.55 (m, 2H, ArH), 8.16 (m, 2H, ArH), 8.10 (m, 1H, ArH), 7.94 (s,
1H, ArH), 7.71 (m, 2H, ArH), 7.24 (d, 1H, J = 8.0 Hz, ArH), 7.06 (d,
1H, J = 8.0 Hz, ArH), 6.95 (m, 1H, ArH), 5.20 (t, 1H, OH), 4.48 (d,
2H, J = 4.0 Hz, CH2OH), 2.32 (s, 3H, ArCH3) ppm.
5.1.38. N-(2-Methyl-5-(morpholinomethyl)phenyl)-5-((4-(4-
(trifluoromethoxy)phenyl)pyrrolo[2,1-f][1,2,4]triazin-2-
yl)amino)picolinamide (35a)
Similar to the preparation of 19a, the title compound 35a
(108 mg, 78%) was obtained as a yellow solid. mp 255–257 °C.
HPLC: 96.8%. MS (ESI) m/z: [M+H]+ = 604.2. 1H NMR (400 M,
DMSO-d6) d 10.30 (s, 1H, CONH), 10.08 (s, 1H, NH), 9.12 (s, 1H,
ArH), 8.52 (d, 1H, J = 8.0 Hz, ArH), 8.30 (d, 2H, J = 4.0 Hz, ArH),
8.15 (d, 1H, J = 8.0 Hz, ArH), 8.12 (s, 1H, ArH), 7.93 (s, 1H, ArH),
7.66 (d, 2H, J = 4.0 Hz, ArH), 7.23 (d, 1H, J = 8.0 Hz, ArH), 7.14 (d,
1H, J = 4.0 Hz, ArH), 7.04 (d, 1H, J = 4.0 Hz, ArH), 6.95 (s, 1H, ArH),
3.59 (s, 4H, morpholine-H), 3.45(s, 2H, ArCH2), 2.37 (s, 4H, morpho-
line-H), 2.32 (s, 3H, ArCH3) ppm, 13C NMR (100 M, DMSO-d6) d
17.17, 53.10, 62.28, 66.16, 104.58, 113.70, 118.27, 120.78, 121.09,
5.1.44. N-(5-(Chloromethyl)-2-methylphenyl)-5-((4-(pyridin-2-
yl)pyrrolo[2,1-f][1,2,4]triazin-2-yl)amino)picolinamide (34c)
Similar to the preparation of 18, the title compound 34c (64 mg,
98%) was obtained as a yellow solid. MS (ESI) m/z: [M+H]+ = 470.

M. Xin et al. / Bioorg. Med. Chem. 22 (2014) 1429–1440
1439
5.1.45. N-(2-Methyl-5-(morpholinomethyl)phenyl)-5-((4-
(pyridin-2-yl)pyrrolo[2,1-f][1,2,4]triazin-2-
yl)amino)picolinamide (35c)
ppm, 13C NMR (100 M, DMSO-d6) d 17.64, 53.12, 62.01, 66.18,
108.58, 113.66, 114.52, 114.73, 118.18, 119.93, 121.52, 123.22,
124.07, 126.61, 127.00, 128.69, 128.75, 130.07, 131.07, 131.18,
132.31, 135.63, 136.15, 137.29, 149.49, 151.44, 152.63, 153.64,
153.88, 155.79, 163.69; HRMS(ESI) m/z calcd for C30H29N7O2F
[M+H]+ 538.2374, found 538.2367.
Similar to the preparation of 19a, the title compound 35c
(35 mg, 70%) was obtained as a yellow solid. mp 222–224 °C. HPLC:
95.5%. MS (ESI) m/z: [M+H]+ = 521.2. 1H NMR (400 M, CDCl3) d 9.95
(s, 1H, CONH), 8.87 (d, 1H, J = 4.0 Hz, ArH), 8.84 (d, 1H, J = 4.0 Hz,
ArH), 8.43 (m, 2H, ArH), 8.33 (d, 1H, J = 8.0 Hz, ArH), 8.27 (s, 1H,
NH), 7.95 (m, 1H, ArH), 7.84 (s, 1H, ArH), 7.74 (m, 1H, ArH), 7.51
(m, 1H, ArH), 7.21 (d, 1H, J = 4.0 Hz, ArH), 7.18 (d, 1H, J = 4.0 Hz,
ArH), 7.08 (d, 1H, J = 8.0 Hz, ArH), 6.90 (m, 1H, ArH), 3.74 (t, 4H,
morpholine-H), 3.54 (s, 2H, ArCH2), 2.50 (s, 4H, morpholine-H),
2.43 (s, 3H, ArCH3) ppm, 13C NMR (100 M, DMSO-d6) d 17.19,
53.13, 62.32, 66.18, 108.76, 113.75, 118.06, 120.30, 122.59,
122.81, 123.13, 124.71, 125.08, 126.23, 127.98, 130.00, 135.85,
136.02, 137.29, 138.32, 140.07, 141.66, 149.55, 152.31, 152.52,
156.05, 161.77; HRMS(ESI) m/z calcd for C29H28N8O2Na [M+Na]+
543.2240, found 543.2233.
5.1.50. 6-((4-(Pyridin-3-yl)pyrrolo[2,1-f][1,2,4]triazin-2-
yl)amino)nicotinic acid (32e)
Similar to the preparation of 20, the title compound 32e
(175 mg, 56%) was obtained as a yellow solid. MS (ESI) m/z:
[M+H]+ = 333. 1H NMR (400 M, DMSO-d6) d 10.47 (s, 1H, NH),
9.32 (s, 1H, ArH), 8.88 (m, 2H, ArH), 8.55 (d, 1H, J = 8.0 Hz, ArH),
8.30 (d, 2H, J = 8.0 Hz, ArH), 8.11 (s, 1H, ArH), 7.70 (t, 1H,
J = 4.0 Hz, ArH), 7.21 (d, 1H, J = 4.0 Hz, ArH), 6.99 (s, 1H, ArH) ppm.
5.1.51. N-(2-Methyl-5-(morpholinomethyl)phenyl)-6-((4-
(pyridin-3-yl)pyrrolo[2,1-f][1,2,4]triazin-2-
yl)amino)nicotinamide (35e)
5.1.46. 3-Fluoro-4-((4-(pyridin-2-yl)pyrrolo[2,1-f][1,2,4]triazin-
2-yl)amino)benzoic acid (32d)
Similar to the preparation of 19a, the title compound 35e
(120 mg, 89%) was obtained as a yellow solid. mp 248–250 °C.
HPLC: 95.4%. MS (ESI) m/z: [M+H]+ = 521.2. 1H NMR (400 M,
DMSO-d6) d 10.32 (s, 1H, CONH), 9.93 (s, 1H, NH), 9.35 (s, 1H,
ArH), 8.94 (d, 2H, J = 8.0 Hz, ArH), 8.56 (s, 1H, ArH), 8.33 (s, 2H,
ArH), 8.14 (s, 1H, ArH), 7.70 (s, 1H, ArH), 7.33 (m, 4H, ArH), 7.00
(s, 1H, ArH), 3.59(t, 4H, morpholine-H), 3.46(s, 2H, ArCH2), 2.38
(s, 4H, morpholine-H), 2.25 (s, 3H, ArCH3) ppm, 13C NMR (100 M,
DMSO-d6) d 17.68, 53.10, 61.99, 66.15, 105.89, 110.82 114.03,
118.42, 120.75, 123.07, 123.94, 126.63, 126.97, 130.11, 131.14,
132.27, 135.62, 136.06, 136.41, 137.49, 148.08, 149.31, 151.81,
152.18, 154.95, 157.00, 163.53; HRMS(ESI) m/z calcd for C29H28N8-
O2Na [M+Na]+ 543.2240, found 543.2233.
Similar to the preparation of 20, the title compound 32d
(168 mg, 55%) was obtained as a yellow solid. MS (ESI) m/z:
[M+H]+ = 350. 1H NMR (400 M, DMSO-d6) d 9.25 (s, 1H, COOH),
8.88 (s, 1H, NH), 8.55 (d, 1H, J = 8.0 Hz, ArH), 8.29 (t, 1H,
J = 8.0 Hz, ArH), 8.10 (t, 1H, J = 8.0 Hz, ArH), 7.99 (s, 1H, ArH),
7.82 (d, 1H, J = 8.0 Hz, ArH), 7.68 (m, 3H, ArH), 6.92 (s, 1H, ArH)
ppm.
5.1.47. 3-Fluoro-N-(5-(hydroxymethyl)-2-methylphenyl)-4-((4-
(pyridin-2-yl)pyrrolo [2,1-f][1,2,4]triazin-2-
yl)amino)benzamide (33d)
Similar to the preparation of 17, the title compound 33d
(200 mg, 89%) was obtained as a yellow solid. MS (ESI) m/z:
[M+H]+ = 469. 1H NMR (400 M, DMSO-d6) d 9.87 (s, 1H, CONH),
9.22 (s, 1H, NH), 8.89 (d, 1H, J = 4.0 Hz, ArH), 8.56 (d, 1H,
J = 8.0 Hz, ArH), 8.27 (t, 1H, J = 8.0 Hz, ArH), 8.11 (t, 1H, J = 8.0 Hz,
ArH), 7.98 (s, 1H, ArH), 7.90 (d, 2H, J = 12.0 Hz, ArH), 7.71 (m, 2H,
ArH), 7.31 (s, 1H, ArH), 7.24 (d, 1H, J = 8.0 Hz, ArH), 7.13 (d, 1H,
J = 8.0 Hz, ArH), 6.92 (m, 1H, ArH), 5.21 (t, 1H, J = 4.0 Hz, OH),
4.50 (d, 2H, J = 8.0 Hz, CH2OH), 2.23 (s, 3H, ArCH3) ppm.
5.2. In vitro Gli-luciferase reporter assay
Compounds were evaluated using a luciferase reporter assay in
NIH3T3 cells carrying a stably transfected Gli-reporter construct
(Gli-luc reporter cell line). NIH3T3/Gli-luc cells were treated with
DMEM + 10% FBS + 1 lg/mL Puromycin. The cells were seeded onto
96-well plates at 2 ꢃ 104 cells/well and cultured in the condition of
5% CO2 and 37 °C overnight. After incubation, all test compounds
(including GDC-0449, as one test compound for internal standard
control) diluted in a serial 8ꢃsolution (0.05–300 nM) containing
5.1.48. N-(5-(Chloromethyl)-2-methylphenyl)-3-fluoro-4-((4-
(pyridin-2-yl)pyrrolo[2,1-f][1,2,4]triazin-2-yl)amino)benzamide
(34d)
0.5% FBS and 0.7 lg/mL Sonic Hedgehog agonist were added to
each well (n = 4 wells per concentration). The cells were incubated
for an additional 48 h. To determine the assay window, cells were
incubated in media containing 0.1% DMSO with or without Sonic
Hedgehog (0% or 100% inhibition control) respectively. Cells were
then harvested and lysed in reporter lysis buffer, and luciferase
activities were measured using a Dual-LuciferaseÒ Reporter Assay
System (Promega E1910). The activity of the Gli reporter was de-
fined as the ratio of Firefly/Renilla luciferase activities.
Similar to the preparation of 18, the title compound 34d
(100 mg, 47%) was obtained as a yellow solid. MS (ESI) m/z:
[M+H]+ = 487. 1H NMR (400 M, CDCl3) d 8.86 (d, 1H, J = 4.0 Hz,
ArH), 8.76 (t, 1H, J = 4.0 Hz, ArH), 8.50 (d, 1H, J = 8.0 Hz, ArH),
8.09 (s, 1H, CONH), 7.95 (t, 1H, J = 4.0 Hz, ArH), 7.81 (s, 1H, NH),
7.76 (m, 4H, ArH), 7.51 (m, 1H, ArH), 7.36 (d, 1H, J = 8.0 Hz, ArH),
7.25 (d, 1H, J = 8.0 Hz, ArH), 7.18 (d, 1H, J = 8.0 Hz, ArH), 6.90 (m,
1H, ArH), 4.61 (s, 2H, CH2Cl), 2.33 (s, 3H, ArCH3) ppm.
5.3. Pharmacokinetic studies in SD rats
5.1.49. 3-Fluoro-N-(2-methyl-5-(morpholinomethyl)phenyl)-4-
((4-(pyridin-2-yl)pyrrolo[2,1-f][1,2,4]triazin-2-
yl)amino)benzamide (35d)
Test compounds were administered to SD rats at a dose of 1 mg/
kg for iv administration. After administration, blood samples col-
lected for PK analyses (n = 3) were centrifuged at 4000 rpm for
5 min at 4 °C. Plasma samples were analyzed after protein precip-
itation with acetonitrile acidified with1% formic acid. LC/MS/MS
analysis of test compounds was performed under optimized condi-
tions to obtain the best sensitivity and selectivity of the analyte in
selected reaction monitoring mode (SRM) containing an internal
standard. Plasma concentration-time data were measured by a
noncompartmental approach using the software WinNonlin Enter-
prise, version 5.2 (Pharsight Co.,Mountain View, CA).
Similar to the preparation of 19a, the title compound 35d
(41 mg, 41%) was obtained as a yellow solid. mp 166–168 °C. HPLC:
95.0%. MS (ESI) m/z: [M+H]+ = 538.2. 1H NMR (400 M, CDCl3) d 8.86
(d, 1H, J = 4.0 Hz, ArH), 8.76 (t, 1H, J = 8.0 Hz, ArH), 8.50 (d, 1H,
J = 8.0 Hz, ArH), 7.95 (m, 2H, ArH), 7.82 (s, 1H, CONH), 7.73 (m,
3H, ArH), 7.65 (s, 1H, NH), 7.51 (m, 1H, ArH), 7.36 (d, 1H,
J = 4.0 Hz, ArH), 7.21 (d, 1H, J = 8.0 Hz, ArH), 7.12 (d, 1H,
J = 8.0 Hz, ArH), 6.90 (m, 1H, ArH), 3.72(s, 4H, morpholine-H),
3.51(s, 2H, ArCH2), 2.47 (s, 4H, morpholine-H), 2.35 (s, 3H, ArCH3)

1440
M. Xin et al. / Bioorg. Med. Chem. 22 (2014) 1429–1440
Stanley, M. S.; Stibbard, J. H.; Sutherlin, D. P.; Ubhayaker, S.; Wang, S.; Wong, S.;
Acknowledgments
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This work was supported by the National Major Science and
Technology Project of China (Innovation and Development of
New Drugs, No. 2011ZX09401-008).
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