Discovery of 4-ethoxy-7H-pyrrolo[2,3-d]pyrimidin-2-amines as potent, selective and orally bioavailable LRRK2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2018.03.045

Inhibition of LRRK2 kinase activity with small molecules has emerged as a potential novel therapeutic treatment for Parkinson's disease. Herein we disclose the discovery of a 4-ethoxy-7H-pyrrolo[2,3-d]pyrimidin-2-amine series as potent LRRK2 inhibitors id

Full text of DOI:10.1016/j.bmcl.2018.03.045

Accepted Manuscript
Discovery of 4-Ethoxy-7H-pyrrolo[2,3-d]pyrimidin-2-amines as Potent, Selec-
tive and Orally Bioavailable LRRK2 Inhibitors
Xiao Ding, Luigi Piero Stasi, Ming-Hsun Ho, Baowei Zhao, Hailong Wang, Kai
Long, Qiongfeng Xu, Yingxia Sang, Changhui Sun, Huan Hu, Haihua Yu,
Zehong Wan, Lizhen Wang, Colin Edge, Qian Liu, Yi Li, Kelly Dong, Xiaoming
Guan, F. David Tattersall, Alastair D. Reith, Feng Ren
PII:
S0960-894X(18)30238-5
https://doi.org/10.1016/j.bmcl.2018.03.045
BMCL 25699
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 January 2018
17 March 2018
17 March 2018
Please cite this article as: Ding, X., Stasi, L.P., Ho, M-H., Zhao, B., Wang, H., Long, K., Xu, Q., Sang, Y., Sun, C.,
Hu, H., Yu, H., Wan, Z., Wang, L., Edge, C., Liu, Q., Li, Y., Dong, K., Guan, X., Tattersall, F.D., Reith, A.D., Ren,
F., Discovery of 4-Ethoxy-7H-pyrrolo[2,3-d]pyrimidin-2-amines as Potent, Selective and Orally Bioavailable
LRRK2 Inhibitors, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.
2018.03.045
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Discovery of 4-Ethoxy-7H-pyrrolo[2,3-d]pyrimidin-
2-amines as Potent, Selective and Orally Bioavailable
LRRK2 Inhibitors
Leave this area blank for abstract info.
Xiao Dinga, Luigi Piero Stasia, Ming-Hsun Hob, Baowei Zhaoa, Hailong Wanga, Kai Longa, Qiongfeng Xua,
Yingxia Sanga, Changhui Suna, Huan Hua, Haihua Yua, Zehong Wana, Lizhen Wanga, Colin Edgec, Qian Liub,
Yi Lib, Kelly Dongb, Xiaoming Guana, F. David Tattersalla, Alastair D. Reithd, Feng Ren*, a

Bioorganic & Medicinal Chemistry Letters
Discovery of 4-Ethoxy-7H-pyrrolo[2,3-d]pyrimidin-2-amines as Potent, Selective
and Orally Bioavailable LRRK2 Inhibitors
Xiao Dinga, Luigi Piero Stasia, Ming-Hsun Hob, Baowei Zhaoa, Hailong Wanga, Kai Longa, Qiongfeng Xua, Yingxia
Sanga, Changhui Suna, Huan Hua, Haihua Yua, Zehong Wana, Lizhen Wanga, Colin Edgec, Qian Liub, Yi Lib, Kelly
Dongb, Xiaoming Guana, F. David Tattersalla, Alastair D. Reithd, Feng Ren*, a
aNeurodegeneration DPU, Neurosciences Therapeutic Area Unit, GSK Pharmaceuticals R&D, 898 Halei Road, Zhangjiang Hi-Tech Park, Pudong, Shanghai
201203, PR China.
bPlatform Technology Sciences, GSK Pharmaceuticals R&D, 898 Halei Road, Zhangjiang Hi-Tech Park, Pudong, Shanghai 201203, PR China.
cPlatform Technology Sciences, GSK Pharmaceuticals R&D, Medicines Research Centre, Gunnels Wood Road, Stevenage, Herts UK.
dNeurodegeneration DPU, Neurosciences Therapeutic Area Unit, GSK Pharmaceuticals R&D, Medicines Research Centre, Gunnels Wood Road, Stevenage,
Herts, UK.
*Corresponding author. Tel.: +86 18616743377; fax: +86 021 61590730; e-mail address: 2648666968@qq.com (F. Ren).
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Inhibition of LRRK2 kinase activity with small molecules has emerged as a potential novel
therapeutic treatment for Parkinson’s disease. Herein we disclose the discovery of a 4-ethoxy-
7H-pyrrolo[2,3-d]pyrimidin-2-amine series as potent LRRK2 inhibitors identified through a
kinase-focused set screening. Optimization of the physicochemical properties and kinase
selectivity led to the discovery of compound 7, which exhibited potent in vitro inhibition of
LRRK2 kinase activity, good physicochemical properties and kinase selectivity across the
kinome. Moreover, compound 7 was able to penetrate into the CNS, and in vivo pharmacology
studies revealed significant inhibition of Ser935 phosphorylation in the brain of both rats (30
and 100 mg/kg) and mice (45 mg/kg) following oral administration.
Keywords:
pyrrolo[2,3-d]pyrimidine
LRRK2
Parkinson’s disease
kinase selectivity
CNS penetration.
2009 Elsevier Ltd. All rights reserved.
Parkinson’s disease (PD) is a slow, progressive and second
most common neurodegenerative disease which affects over 10
million people worldwide.1,2 Numerous treatments have been
developed including L-DOPA, dopamine agonists, MAO-B
inhibitors,1 but these compounds relieve the symptoms rather
than the progression of the disease. The development of disease-
modifying therapies therefore represents an unmet medical need.3
Through genome-wide association studies (GWAS), a number of
variants in genes such as those for α-synuclein, leucine-rich
repeat kinase 2 (LRRK2), Parkin and PTEN induced putative
kinase 1 (PINK1) have been reported to be associated with
increased risk of developing PD.4 Such variants in LRRK2
constitute the most common genetic cause of familial PD.5 In
particular, the most frequent G2019S mutation increases kinase
activity, suggesting that restoringLRRK2 kinase activity to
normal in the brain could be a potential therapeutic target for the
treatment of PD.6
including
2,4-diaminopyrimidine,9
indazole,10
pyrrolopyrimidine,11 indolinone12 and 3-cyanoquinoline13 cores.
Several representative compounds such as GNE-9605,9 PF-
06447475,11 and MLi-210 exhibited reasonable pharmacokinetic
(PK) profile with good central nervous system (CNS) penetration
and reasonable kinase selectivity. Many of these compounds
significantly reduced LRRK2 phosphorylation, (a surrogate
measure of the inhibition of LRRK2 kinase) both in vitro and in
vivo. Our group recently patented several novel scaffolds of
LRRK2 kinase inhibitors with high potency and good kinase
selectivity, including 5-substituent-N-arylbenzamide14 and 4-
substituent-7H-pyrrolo[2,3-d]pyrimidin-2-amine series15,16. The
benzamide compound we discovered, GSK2578215A,
demonstrated potent LRRK2 kinase inhibition and high
selectivity across the kinome17-18. This compound has been
widely used as a tool molecule for the exploration of the biology
of LRRK219,20. Herein, we describe the discovery of the 4-
substituted-7H-pyrrolo[2,3-d]pyrimidin-2-amine series as potent
LRRK2 kinase inhibitors and the lead optimization focusing on
improving the series’ physicochemical properties and kinase
In the past several years a number of small molecule LRRK2
inhibitors with distinct chemical structures have been disclosed7,8

selectivity. Extensive structure-activity relationship (SAR)
studies led to the discovery of a potent and selective LRRK2
inhibitor 7, which significantly inhibited Ser935 phosphorylation
in brain of both rats (30 and 100 mg/kg) and mice (45 mg/kg)
following oral administration.
replaced with other hydrophilic amides such as morpholinol
amide. In addition, the 2,2,2-trifluoroethyl amine, which formed
hydrophobic interaction with the LRRK2 protein, could be
replaced with lipophilic alkoxyl groups. Compound 2 (Scheme
1) was thus quickly identified as a new hit with only two HBDs
and good potency both in HTRF (pIC50 = 8.1) and cellular assays
(pIC50 = 7.2). Not surprisingly, compound 2 showed much
improved membrane permeability (AMP = 510 nm/s) with a
reasonable TPSA of 92 Å. The FaSSIF solubility of compound 2
remained low (1.7 µg/mL) and required further optimization.
Both compounds 1 and 2 were progressed to kinase selectivity
assessment using standard radioactivity-based enzymatic assays
against a panel of 140 kinases,24 and at the concentration of 1
µM, they exhibited inhibitory activities of greater than 50% on
72 and 61 kinases, respectively, including inhibition of
MAP4K3, MLK1, JAK2, IRAK4, and SYK. Thus, a significant
improvement of the kinase selectivity profile was critical for
further progression of the series.
Scheme 1. Structures and profile of compounds 1 and 2
Our work was initiated from a kinase-focused set screening
(KCS) of GSK in house compounds by measuring the inhibition
of LRRKtide phosphorylation using a homogeneous time-
resolved fluorescence (HTRF) assay. Compound 1 (Scheme 1)
was identified as a hit with good LRRK2 inhibitory potency in
the HTRF assay (pIC50 = 7.9), and the activity was confirmed in
a SH-SY5Y cell assay where the effect on the phosphorylation of
LRRK2 Ser935 was measured. Compounds of similar
chemotypes have been widely reported as inhibitors of kinases
such as SYK21 and JAK22. Compound 1 was then docked in the
LRRK2 homology model,18 and was suggested to reside in the
ATP binding pocket of LRRK2 kinase (Figure 1). The scaffold
7H-pyrrolo[2,3-d]pyrimidin-2-amine formed three hydrogen-
bond interactions with the backbone of Glu1948 and Ala1950 in
the hinge region, and had close contact with the gatekeeper
residue Met1947. The 2,2,2-trifluoroethyl chain located inside
the pocket and pointed toward the residue His1998, while the
amide group pointed toward the solvent-exposed area.
Table 1. LRRK2 inhibition and kinase selectivity for
compounds 2‒13
Cell
HTRF
pIC50
Cmpd
R1
R2
H
KSb
a
pIC50
7.2
8.1
61/140
2
H
7.1
7.9
NDc
3
F
6.8
6.6
6.8
6.8
6.9
6.7
6.4
7.9
7.9
7.8
7.9
8.0
8.0
8.0
32/140
20/140
17/140
12/140
9/140
4
5
Cl
OCH3
6
OCH3
7
OCH2CH3
OCH2CH3
OCH(CH3)2
8
Figure 1. Predicted docking pose of compound 1 (purple) in the LRRK2
homology model (gray). Intermolecular hydrogen-bond interactions are
shown as blue dashed lines.
12/140
1/140
9
10
Further profiling of compound 1 revealed poor artificial
membrane permeability (AMP, 36 nm/s) and low solubility in
fasted state simulated intestinal fluids (FaSSIF, 8.6 µg/mL). We
attributed these poor physicochemical properties partly to its high
topological polar surface area (TPSA, 109 Å) and the presence of
five hydrogen-bond donors (HBDs) in the molecule. In addition,
high PSA and more HBDs have been reported to be detrimental
to CNS penetration properties (TPSA ≤ 90 Å and HBDs ≤ 2 were
recommended for CNS compounds).23 Initially, we focused on
reducing the molecules’ HBDs. Based on the modeling structure
(Figure 1), the two hydrogens on the 7H-pyrrolo[2,3-
d]pyrimidin-2-amine scaffold had to be maintained because they
involved in the bindings with the LRRK2 hinge region which are
pivotal for potency. Further investigation revealed that primary
amide group pointed towards the solvent region could be
OCH(CH3)2
OCHF2
6.2
6.8
5.5
7.9
>9.8
6.7
NDc
14/140
NDc
11
12
13
N(CH3)2
aThe average of at least two determinations.
bKinase selectivity (KS) was measured using standard radioactivity-based
enzymatic assays against a panel of 140 kinases (Dundee profiling) at 1 µM:
data are shown as numbers of kinases displaying >50% activity.
cNot determined.

Table 2. LRRK2 potency, developability, and kinase selectivity for compounds 7, 14‒22
Cell
pIC50
HTRF
pIC50
hClib
(mL/min/g)
Pgp/BCRPc PRd/
PP(nm/s) e
Solubilityf
(μg/mL)
Cmpd
R3
ClogDg
PFIh
KSi
a
6.8
6.9
7.9
7.9
1.1
0.8
3.1/319
3.8/56
127.3
25.5
4.5
4.1
7.5
7.1
12/140
14/140
7
14
7
8.1
7.8
1.4
0.8
1.8/406
2.2/294
153.7
78.8
5.0
5.1
8.0
8.1
15/140
17/140
15
16
7.1
6.7
6.5
7.2
8.2
1.5
1.1
NDj
4.4
5.7
5.7
8.7
8.7
NDj
17
18
1.5/276
39.7
12/140
6.8
7.2
7.2
8.2
1.1
NDj
6.2
4.3
3.0
7.3
6.3
NDj
NDj
19
20
<0.777
5.0/84
620.7
7.1
6.8
8.5
8.1
3.2
2.5
2.3/294
309.4
300.1
4.9
4.9
7.9
7.9
NDj
21
22
4.0/270
17/140
c
aThe average of at least two determinations. bIn vitro human liver microsomal clearance. MDCKII-MDR1 transduced with BacMam2-BCRP cell line.
f
dPermeability ratio, A→B (apical to basolateral) with GF120918/A→B without GF120918. ePassive Permeability, A→B with GF120918. FaSSIF
i
Solubility was measured after 4 h incubation. gMeasured ChromLogD7.4. hPFI = Chrom log DpH7.4 + # Ar. Kinase selectivity, standard radioactivity-based
j
enzymatic assays against a panel of 140 kinases (Dundee profiling) at 1 uM, quoted as numbers of kinase displaying >50% activity. Not determined.

The poor kinase selectivity of compounds 1 and 2 was
proposed to be due to the binding in the ATP pocket, which is
highly conserved across the human kinome. Unique structure
motifs or functional groups to enable more selective specific
interaction(s) of compounds with LRRK2 kinase over other
protein kinases might provide improved selectivity. Our
homology model showed that among the 21 residues which
formed the ATP binding pocket of LRRK2, five resided in close
contact with the docked compound 1 and 2, i.e. Met1947,
Leu1949, His1998, Leu2001, and Ala2016 (Figure 1 and Figure
S1). Multiple sequence alignments of these specific bind binding
site residues were performed across the human kinome of
LRRK2 over other kinases.25,26 Among these, Leu1949 was
identified as the most distinct residue for LRRK2 and 60% of its
equivalent residue in other human kinases was determined to be
either Tyr or Phe, which were larger in size than Leu. Leu1949
resided in close contact of the R2 group of the series (Table 1),
and we thus started to explore SAR on R2 aiming for specific
interaction(s) with LRRK2 over other kinases for improved
kinase selectivity.
electronic properties was well tolerated with good potency in
both cell and HTRF assays, including acyclic (14), cyclic
(15‒18), spiro- (19 and 20), and fused- (21 and 22) amides. The
observation was consistent with the predicted binding mode that
the amide group pointed towards the opened solvent-exposed
area of LRRK2 kinase, enabling the tolerance of substitutions in
this region. All the tested compounds demonstrated good
metabolic stability in human liver microsomes with intrinsic
clearance less than 3 mL/min/g except for compounds 21 (3.2
mL/min/g). None of them was a significant Pgp/BCRP efflux
transporter substrate when measured in a MDCKII cell line
double-transduced with human Pgp and BCRP transporters, in
combination with the high passive permeability (> 100 nm/s
except for compounds 14 and 20), indicating the potential good
CNS penetration property of these compounds. Relatively lower
passive permeability (< 100 nm/s) was observed for compounds
14 and 20 which might be due to an additional HBD for 14 and
higher basicity for 20. Moreover, most of compounds showed
good FaSSIF solubility (>100 g/mL) except for compounds 14
and 1619. Several compounds were further screened in a panel
of 140 kinases and they all showed comparable kinase selectivity
as compound 7.
As illustrated in Table 1, compounds with various substituents
on R2 position were synthesized, and their potency and kinase
selectivity were determined. Most of the compounds
demonstrated comparable or slightly compromised LRRK2
inhibitory potency compared to compound 2, and significant
improvement of kinase selectivity was achieved. For instance,
introducing halo groups such as fluoro (4) or chloro (5) on the R2
position resulted in slightly decreased cellular potency and much
improved kinase selectivity, hitting only 32 and 20 kinases out of
the 140-kinase panel with over 50% inhibition at 1 µM
concentration, respectively. Side-by-side comparisons among
compounds 46 and 8 revealed that increasing the sizes at the R2
position resulted in the improvement of kinase selectivity
(OCH2CH3 > OCH3  Cl > F). This observation was consistent
with our homology model and the literature reports of similar
structural scaffolds, that alkoxy groups with appropriate sizes
were favored for the R2 binding pocket of LRRK2 over other
kinases.27,28 Further increasing the steric hindrance of the R2
substitution (10) led to significant reduction of LRRK2 potency
especially in the cell-based assay, even though the kinase
selectivity was excellent (only 1 out of 140 kinases was inhibited
with > 50% at 1 µM of compound 10). The homology model also
suggested that the R2 group was projected proximity to Leu1949
of the LRRK2 binding pocket and therefore steric bulky
substituents such as isopropoxy (10 and 11) and dimethylamino
(13) were not well tolerated. Considering the potential de-
methylation metabolism risk of the N-methyl piperazine
analogues, the morpholine derivatives (compounds 7, 9, and 11)
were favored. Among them, compound 7 was selected for further
progression due to its lower molecular weight than compound 9
and higher potency than compound 11. Further profiling of
Table 3. PK profile of compounds 7 and 22 in rats
DNAUC0~t
in bloodc
DNAUC0~t
in brain
Fu% in
braind
Cmpd
Kp (br/bl)
7a
1034
487
252
246
0.24
0.51
1.0
22b
<0.5
a10 mg/kg, oral dosing. b2 mg/kg, oral dosing. cDNAUC = dose
d
normalized area under the curve, (ng·h/mL(g))/(mg/kg). Free fraction
in rat brain.
With the balanced profile of potency, permeability, solubility,
and metabolic stability, compounds 7 and 22 were progressed to
in vivo evaluation for their CNS penetration and pharmacokinetic
properties after dosing by oral gavage at 10 mg/kg and 2 mg/kg,
respectively (compound 5 demonstrated very similar profile as 7
but with slightly less favored kinase selectivity, in combination
with their high structure similarity, compound 5 was de-selected
for further progression). As shown in Table 3, both compounds 7
and 22 exhibited good CNS exposure in rats with brain to blood
ratios of 0.24 and 0.51, respectively, as measured by total drug
concentrations. However, compound 22 was observed lower
unbound fraction in rat brain (<0.5%) in comparison to 7 (1.0 %).
Given the superior free unbound fraction, in combination with its
good oral exposure especially in brain, compound 7 was
progressed to an in vivo pharmacodynamic (PD) study.
compound
7 revealed good metabolic stability in liver
microsomes (Cli = 1.1 mL/min/g)29 and good solubility in
FaSSIF (127.3 µg/mL). Kinase selectivity of 7 was further
evaluated using the HotSpot assay platform of over 340 other
kinases and it was confirmed a highly selective LRRK2 inhibitor
with only three kinases (ALK, IRR and TSSK1) exhibiting an
ambit score of <10 in the KINOMEscan profile30.
We then focused our SAR on the exploration of the amide
group (R3) aiming to further improve the overall developability
profile (Table 2). Property forecast index (PFI),31 determined
from calculated or measured ChromlogD (pH = 7.4), was
reported as a key indicator for compounds’ developability
properties, thus we designed our compounds with PFIs < 9. A
variety of different amide groups with diversified steric and

Fu% in blood/brain (rat)
Fu% in blood/brain (mouse)
Fu% in serum (human)
AMP (nm/s)
3.2/1.0
3.1/1.0
5.9
480
CYP inhibition pIC50 (3A4)
hPXR pEC50
3.8
<4.3
4.9
OATP1B1 pIC50
hERG binding pIC50
<4.2
Having demonstrated good in vivo pharmacology in rodents,
compound 7 was further evaluated for its developability profile
(Table 4). The unbound fractions of 7 in blood and brain were
determined to be 3.2% and 1.0%, respectively, in rat, in good
agreement with the values in mouse (3.1% and 1.0%,
respectively). In addition, a good unbound fraction property of 7
was also observed in human serum wherein a 5.9% unbound
fraction was observed. The high passive permeability of the
compound was also confirmed in the artificial membrane
permeability assay (480 nm/s). Compound 7 demonstrated no
meaningful inhibition on human CYP3A4 (pIC50 = 3.8),
OATP1B1 (pIC50 < 4.9), and PXR (pIC50 < 4.3), suggesting low
concern for drug-drug interactions. Further, the hERG binding
assay was conducted to evaluate the preliminary cardiac safety,
and the compound was determined to have a low risk of QT
interval prolongation (hERG pIC50 < 4.2).
Figure 2. (a) Pharmacodynamic analysis for compound 7 in
Han Wistar rats. (b) Pharmacodynamic analysis for compound
7 in C57/BL6 mice. Pharmacodynamic study of compound 7
from brain, spleen, lung and kidney following oral gavage at
the indicated doses. Tissues were collected, and endogenous
LRRK2 was resolved in SDS-PAGE followed by western blot
with an antibody directed against LRRK2 phospho-Ser935 or
total LRRK2.
The synthesis of compound 7 was shown in Scheme 1.
Treatment of 2,4-dichloro-7H-pyrrolo-[2,3-d]pyrimidine with
sodium ethoxide in ethanol gave intermediate 23, which then
reacted with 4-methylbenzene-1-sulfonyl chloride in the presence
of sodium hydride in DMF under room temperature to afford
compound 24. Amide coupling reaction between 3-methoxy-4-
nitrobenzoic acid and morphine proceeded smoothly in the
presence of EDC and HOBT to provide intermediate 7a, which
was subjected to the hydrogenation reaction to give aniline
intermediate 7b. The final product 7 was obtained through
Buchwald coupling reaction between 24 and 7b using Pd2(dba)3
as the catalyst and Xphos as the ligand.
Prior to the in vivo PD evaluation, we firstly confirmed the in
vitro pharmacology of compound 7 on endogenous LRRK2 in
human lymphoblastoid cells derived from a healthy subject
(AHE) and a Parkinson’s disease patient with homozygous
LRRK2[G2019S] mutation (ANK)32. A concentration-dependent
inhibition of LRRK2 Ser935 phosphorylation was observed with
measured pIC50 values of 7.5 in AHE and 7.8 in ANK. The in
vivo pharmacology of 7 was then evaluated by measuring
inhibition of LRRK2 Ser935 phosphorylation in brain, lung,
spleen, and kidney following oral administration to rats at 10
mg/kg, 30 mg/kg and 100 mg/kg (Figure 2a). Compound 7
demonstrated dose dependent reductions on phosphorylation of
LRRK2 Ser935 in brain, with around 20%, 50%, and 75%
reductions at 10 mg/kg, 30 mg/kg and 100 mg/kg oral doses,
respectively. As expected, the peripheral PD effects were greater
with almost complete inhibition of Ser935 phosphorylation in all
peripheral tissues (lung, spleen, and kidney) at all doses. A
single-dose (45 mg/kg) time-course in vivo pharmacology
experiment for compound 7 in mice was also conducted (Figure
2b). In the study, the maximal reductions of Ser935
phosphorylation were observed at 1 h after oral administration,
with around 50% reduction in brain and 80% reduction in both
kidney and lung.
In summary, we discovered a series of 7H-pyrrolo[2,3-
d]pyrimidin-2-amine derivatives as potent LRRK2 kinase
inhibitors through kinase-focused set screening (KCS). Further
optimization of the kinase selectivity and physicochemical
properties led to the discovery of compound 7 with high in vitro
inhibition of LRRK2 kinase activity and good selectivity over
hundreds of other kinases. The compound proved to be CNS
penetrant, and in vivo pharmacology experiments revealed
significant inhibition of Ser935 phosphorylation of LRRK2 in
brain of both rats (30 mg/kg and 100 mg/kg) and mice (45
mg/kg) following oral administration. In addition, compound 7
demonstrated good developability profile which enabled it to be a
good tool for the exploration of LRRK2 biology both in vitro and
in vivo. Further optimization of this series towards a clinical
candidate will be reported in due course.
Table 4. Developability profile of compound 7
compound
7

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7b
e
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Scheme 1. Synthesis of compound 7.
Reagents and
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condition: (a) EtONa, EtOH, 90 oC, overnight; (b) TsCl, NaH,
DMF, 23 oC, 1 h; (c) morphine, EDC, HOBT, Et3N, DCM, 23
oC, overnight; (d) Pd/C, H2, MeOH, 23 C, overnight; (e) 24,
o
Pd2(dba)3, Xphos, K2CO3, 2-butanol, 125 °C, overnight.
19. Steger, M.; Tonelli, F.; Ito, G.; Davies, P.; Trost, M.; Vetter, M.;
Wachter, S.; Lorentzen, E.; Duddy, G.; Wilson, S.; Baptista, M. A.;
Fiske, B. K.; Fell, M. J.; Morrow, J. A.; Reith, A. D.; Alessi, D. R.;
Mann, M. elife 2016, 5.
Acknowledgements
We thank Mr. Liang Huang for HRMS analysis, and Mr. Lijun
Ge, Mr. Yan Chen, and Mr. Yong Zhang for their in vivo works
for compound testing.
20. Ito, G.; Katsemonova, K.; Tonelli, F.; Lis, P.; Baptista, M. A.; Shpiro,
N.; Duddy, G.; Wilson, S.; Ho, P. W.; Ho, S. L.; Reith, A. D.; Alessi, D.
R. Biochem. J. 2016, 473, 2671.
Supplementary Material
21. Ancliff, R. A.; Atkinson, F. L.; Barker, M. D.; Box, P. C.; Daniel, C.;
Gore, P. M.; Guntrip, S. B.; Hasegawa, M.; Inglis, G. G. A.; Kano, K.;
Miyazaki, Y.; Patel, V. K.; Ritchie, T. J.; Swanson, S.; Walker, A. L.;
Wellaway, C. R.; Woodrow. WO2007042299. 2007.
Supplementary data associated with this article can be found in
the online version.
22. J. Salas Solana, J.; Almansa Rosales, C.; Soliva, R.; Fontes Ustrell, M.;
Vendrell Escobar, M. WO2008119792. 2008.
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