Design, synthesis, and biological evaluation of novel phenol ether derivatives as non-covalent proteasome inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.10.056

A series of novel phenol ether derivatives were designed, synthesized, and evaluated as non-covalent proteasome inhibitors. Most compounds exhibited moderate to excellent proteasome inhibitory activity. In particular, compound 18x proved to be the most po

Full text of DOI:10.1016/j.ejmech.2018.10.056

Accepted Manuscript
Design, synthesis, and biological evaluation of novel phenol ether derivatives as non-
covalent proteasome inhibitors
Jianjun Yu, Lei Xu, Duidui Hong, Xiaotuan Zhang, Jieyu Liu, Daqiang Li, Jia Li, Yubo
Zhou, Tao Liu
PII:
S0223-5234(18)30927-9
DOI:
https://doi.org/10.1016/j.ejmech.2018.10.056
EJMECH 10842
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 July 2018
Revised Date: 22 October 2018
Accepted Date: 23 October 2018
Please cite this article as: J. Yu, L. Xu, D. Hong, X. Zhang, J. Liu, D. Li, J. Li, Y. Zhou, T. Liu,
Design, synthesis, and biological evaluation of novel phenol ether derivatives as non-covalent
proteasome inhibitors, European Journal of Medicinal Chemistry (2018), doi: https://doi.org/10.1016/
j.ejmech.2018.10.056.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis, and biological evaluation
of novel phenol ether derivatives as
non-covalent proteasome inhibitors
Jianjun Yua, 1, Lei Xud, b, c, 1, Duidui Honga, Xiaotuan Zhangb, c, Jieyu Liub, c, Daqiang
Lia, Jia Li b, c, Yubo Zhoub, c *, Tao Liu a, *
aZJU-ENS Joint Laboratory of Medicinal Chemistry, Zhejiang Province Key
Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences,
Zhejiang University, Hangzhou 310058, P. R. China
bNational Center for Drug Screening, State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai
201203, P. R. China
cUniversity of Chinese Academy of Sciences, Graduate School, No. 19A Yuquan Road,
Beijing 100049, P. R. China
dSchool of Life Science and Technology, ShanghaiTech University, Shanghai 201203,
P. R. China
*Corresponding authors: Tao Liu, E-mail: Lt601@zju.edu.cn; Yubo Zhou, E-mail:
ybzhou@simm.ac.cn
1Equal contribution
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Abstract
A series of novel phenol ether derivatives were designed, synthesized, and
evaluated as non-covalent proteasome inhibitors. Most compounds exhibited
moderate to excellent proteasome inhibitory activity. In particular, compound 18x
proved to be the most potent compound (chymotrypsin-like: IC50 = 49 nM), exhibiting
a 2-fold higher potency compared to the reported PI-1840. Besides, compound 18x
exhibited excellent metabolic stability and selective anti-proliferative activity against
solid cancer cell lines including HepG2 and HGC27, providing incentive for the
further development as a potential anticancer agent against solid cancers.
Key words: proteasome inhibitor, non-covalent, non-peptide, phenol ether, solid
cancers
Introduction
The ubiquitin-proteasome system (UPS) [1] represents the major pathway for
regulating the degradation of intracellular proteins [2] and is generally involved in
numerous physiologically important cellular processes, such as cell cycle progression
[3, 4], signal transduction [5], and immune response [6]. The 20S proteasome is a key
player in the pathway, consisting of four stacked heptameric rings: two outer α-rings
and two inner β-rings. Each β-ring contains the three proteolytic subunits β1c, β2c,
and β5c, which are responsible for caspase-like (C-L), trypsin-like (T-L), and
chymotrypsin-like (ChT-L) activity [7], respectively. These subunits can be replaced
by their interferon γ-inducible counterparts LMP2 (β1i), MECL-1 (β2i), and LMP7
(β5i), respectively, resulting in alternatively assembled immunoproteasomes. Because
of the crucial role of proteasome in a wide variety of cellular processes, the regulation
of its protease activity by inhibitors is of therapeutic interest [8].
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Figure 1. Proteasome inhibitors. (A) FDA-approved covalent proteasome inhibitor Bortezomib,
Carfilzomib, and Ixazomib. (B) Non-covalent proteasome inhibitors including the peptidic
inhibitor TMC-95A, compound 5, and non-peptidic inhibitor PI-1840.
FDA approval of the proteasome inhibitors Bortezomib, Carfilzomib, and Ixazomib
(1-3, Fig. 1A) has validated the proteasome as a promising target for cancer therapy.
All of the approved inhibitors carry an electrophilic warhead at the C-terminal end of
a peptidic backbone and are covalently bound to the catalytic Thr1 residues of the β
subunits [9]. However, the peptidic backbone and the electrophilic warhead of these
agents are believed to be the major cause of side effects, acquired drug resistance, and
unsatisfactory pharmacokinetic profiles [10-12]. Therefore, non-covalent inhibitors
have attracted significant attention in recent years. These inhibitors are devoid of any
electrophilic warhead and interact with the proteasome active site through a network
of interactions (hydrophobic, H-bonds, electrostatic, and van der Waals forces).
However, most of the non-covalent inhibitors are still equipped with a peptidic
backbone [13-18](e.g. compounds 4 and 5, Fig. 1B), are generally believed to be
unstable and less selective, with several potent non-peptide non-covalent inhibitors
having been published [19-21]. PI-1840 (6, Fig. 1B) represents one of the most potent
non-peptide non-covalent inhibitors with an IC50 value of 27 nM against CT-L [22].
Further studies indicated that PI-1840 suppresses the growth of human breast tumor
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xenografts in nude mice [23]. Nevertheless, according to a study reported by Boström,
J. et al. [24], 1,2,4-oxadiazole derivatives are more frequently recognized by
cytochrome P450 enzymes, leading to a higher metabolic turnover in human liver
microsomes (HLMs). To further verify this notion, we evaluated the metabolic
stability of PI-1840 in HLMs. The results indicated that PI-1840 displayed a high
metabolic susceptibility in HLMs, metabolized more than 50% within 30 min after
treatment with HLMs (Fig. 3). Considering this finding, our modification of PI-1840
prioritized replacing its 1, 2, 4-oxadiazole moiety with different isosteres containing
urea or amide to improve the metabolic stability of the compounds. After confirming
the optimal replacement of the 1,2,4-oxadiazole moiety, we designed and synthesized
a series of phenol ether derivatives to explore the SARs of ring A and the R group
residing on the phenyl ring (Fig. 2). The anti-proliferative activity against various
cancer cells of selected compounds were also evaluated in this manuscript.
Figure 2. Design of new non-covalent proteasome inhibitors based on PI-1840.
2. Results and discussion
2.1. Chemistry
The synthetic routes for the corresponding phenol ether derivatives are outlined in
Schemes 1 and 2. Substitution of tert-butyl (2-aminoethyl)carbamate 7 with
2-bromopropane in MeCN, afforded tert-butyl (2-(isopropylamino)ethyl)carbamate 8,
which was subsequently converted to compound 9 via condensation with chloroacetyl
chloride. Treatment of precursor 9 with 4-propylphenol in the presence of potassium
carbonate in refluxing acetonitrile, followed by removal of the Boc group yielded the
primary amine 11. Afterwards, the primary amine 11 reacted with pyridin-3-amine or
pyridin-3-ylmethanamine in the presence of triphosgene to produce urea 12a or 12b.
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In addition, the primary amine 11 was condensed with nicotinic acid using HOBt and
EDCI as condensation agents to provide amide 12c (Scheme 1).
Scheme 1. Synthetic route for target compounds 12a-12c. Reagents and conditions: (i)
2-bromopropane, TEA, MeCN, r.t., overnight; (ii) chloroacetyl chloride, TEA, THF, 0 °С - r.t., 30
min; (iii) 4-propylphenol, K2CO3, MeCN, reflux, 2 h; (iv) HCl-saturated ethyl acetate, 0 °С - r.t., 2
h, then NaHCO3 (aq.), 0 °С, 10min; (v) pyridin-3-amine or pyridin-3-ylmethanamine, triphosgene,
TEA, anhydrous DCM, 0 °С - r.t., 1 h; (vi) nicotinic acid, EDCI, HOBt, DIPEA, DCM, 0 °С - r.t.,
5 h.
The phenol ether derivatives 18a-18z, 18aa-18ae, and 19 were prepared as shown
in Scheme 2. Ethyl 2-chloroacetate 13a, benzyl 2-chloroacetate 13b, or ethyl
3-bromopropanoate 13c was first allowed to react with propan-2-amine in the
presence of triethylamine in MeCN at room temperature to produce intermediate
14a-14c, followed by condensation with chloroacetyl chloride to obtain compounds
15a-15c. Reaction of 15a-15c with various phenols furnished intermediates 16a-16l,
which were hydrolyzed or debenzylated to afford the corresponding carboxylic acids
17a-17l. Finally, the carboxylic acids 17a-17l were condensed with the corresponding
amines to produce the target compounds 18a-18z and 18aa-18ae. In this step, we
adopted two different condensation methods (vi and vii, Scheme 2), due to the
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different nucleophilicities of the amines. Compound 19 was synthesized from
compound 18ae via reduction with sodium borohydride in the presence of CaCl2.
Most of final compounds were obtained in isomer mixture, we have carried out
1
variable temperature H NMR experiments to confirm the existence of atropisomers.
As the temperature was raised for 18x, the two sets of peaks observed in the 1H NMR
1
spectrum at 20 °С coalesced at 100 °С. The H NMR spectra of 18x at different
temperature were provided in supplemental material.
Scheme 2. Synthetic routes for the target compounds 18a-18z, 18aa-18ae, and 19. Reagents and
conditions: (i) propan-2-amine, TEA, MeCN, r.t., overnight; (ii) chloroacetyl chloride, TEA, THF,
0 °С - r.t., 30 min; (iii) K2CO3, MeCN, reflux, 2 h; (iv) 1N NaOH (aq), 0 °С - r.t., 2 h, then 1N
HCl (aq.), 0 °С, PH =2~3; (v) H2, 10% Pd/C, MeOH, r.t., 2 h; (vi) HOBt, EDCI, DIPEA, DCM,
0 °С - r.t., 5 h; (vii) isobutyl chloroformate, 4-methylmorpholine, anhydrous THF, –10 °С - r.t., 6
h; (viii) NaBH4, CaCl2, MeOH, 0 °С - r.t., 30 min.
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2.2. Biological evaluation
2.2.1. Proteasome inhibitory activity and metabolic stability in liver microsomes
All synthesized target compounds were evaluated for their 20S proteasome
chymotrypsin-like inhibitory activity in vitro. In this study, PI-1840 was employed as
positive control. The results are summarized in Tables 1-3. Most compounds
exhibited moderate to excellent proteasome inhibitory activity.
Table 1.
20S proteasome chymotrypsin-like inhibitory activity of target compounds (12a-12c and
18a-18d).
Inhibitory rate at
Compd.
L
10 ꢀM (%)a
13.80 ± 6.64
12a
12b
12c
18a
18b
18c
38.68 ± 1.76
30.79 ± 7.57
15.50 ± 3.20
12.66 ± 5.11
1.16 ± 6.53
58.18 ± 0.02
80.20 ± 2.10
18d
-
PI-1840
a The inhibitory rate is shwon as an average of three independent determinations.
First, the modification of PI-1840 prioritized replacing its 1, 2, 4-oxadiazole moiety
with different isosteres containing urea or amide to afford compounds 12a-12c and
18a-18d. As illustrated in Table 1, only compound 18d showed 58.18% inhibition at
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10 ꢀM, all other compounds demonstrated disappointing results (<50% inhibition at
10 ꢀM). For 18d, replacing the 1,2,4-oxadiazole moiety of PI-1840 with an amide led
to a decrease in inhibitory potency to some extent, but the following stability test of
compound 18d and PI-1840 in HLMs indicated that compound 18d was more stable
than PI-1840 in HLMs (Fig. 3). Encouraged by this finding, we identified compound
18d as a lead compound for further modification.
Human liver microsomes
18d
PI-1840
100
50
0
0
10
20
30
Time (min)
Figure 3. Metabolic stability of compound 18d and PI-1840 in human liver microsomes.
Table 2.
20S proteasome chymotrypsin-like inhibitory activity of target compounds (18d-18u).
Compd.
18d
A
IC50 (nM)a
1682 ± 42
836 ± 137
100 ± 10
280 ± 18
395 ± 30
89 ± 25
18e
18f
18g
18h
18i
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>10000
18j
2000 ± 340
952 ± 180
4726 ± 1251
1197 ± 231
922 ± 386
18k
18l
18m
18n
18o
230 ± 72
18p
361 ± 23
18q
18r
18s
18t
3863 ± 1131
2011 ± 323
5907 ± 1648
1111 ± 257
18u
-
92 ± 5
PI-1840
a The IC50 values are shown as an average of three independent determinations.
In order to determine the SARs around ring A present in 18d, we replaced ring A
with various heteroaryl rings (compounds 18e-18i and compounds 18r and 18s),
substituted aryl rings (compounds 18j-18q), and fused rings (compounds 18t and 18u).
Firstly, compared with 18d, 18e, and 18f, we found that the position of the nitrogen
atom in the pyridine ring was critical to potency. Replacing the m-pyridyl moiety in
18d with an o-pyridyl (as in 18e) and a p-pyridyl (as in 18f) moiety increased the
potency 2-fold and 16-fold, respectively. Furthermore, replacing the m-pyridyl moiety
in 18d with pyrimidine (as in 18g), pyrazine ( as in 18h), or pyridazine (as in 18i) also
led to potency increase, and the compound 18i containing 4-substituted pyridazine
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exhibited a comparable potency to PI-1840. Meanwhile, in order to explore the most
potent compound, various substituted aryl rings were also introduced to ring A.
Similarly to the pyridine ring, the most suitable position of the methoxy substituent in
the phenyl ring was determined to be in para-position (18l), but the CT-L inhibitory
activity was found to be lost when the methoxy substituent was moved into
ortho-position (18j, IC50 > 10 ꢀM) or meta-position (18k, IC50 = 2 ꢀM). Moreover,
substituting the 4-methoxyphenyl in 18l by 3,4-dimethoxyphenyl (as in 18m) led to a
5-fold loss of potency. Therefore, we introduced different substituents in the
para-position on the phenyl ring to afford the compounds 18n, 18o, 18p, and 18q with
IC50 values of 1197, 922, 230, and 361 nM, respectively. However, all compounds in
which the pyridazine rings were replaced by substituted aryl rings exhibited a loss of
CT-L inhibitory activity compared to 18i. In addition, replacing ring A with
five-membered heteroaryl rings (compounds 18r, 18s) and fused rings (compounds
18t, 18u) was also found to be of no benefit to CT-L inhibitory potency. According to
above-mentioned modification of the ring A, we identified the 4-substituted
pyridazine ring as optimal group.
Table 3.
20S proteasome chymotrypsin-like inhibitory activity of target compounds (18v-18z, 18aa-18ae,
and 19).
Compd.
18v
R
IC50 (nM)a
481 ± 131
243 ± 57
89 ± 25
18w
18i
49 ± 12
18x
118 ± 38
64 ± 13
18y
18z
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579 ± 242
18aa
18ab
18ac
18ad
259 ± 34
952 ± 180
405 ± 100
1561 ± 13
18ae
968 ± 179
92 ± 5
19
-
PI-1840
a The IC50 values are shown as an average of three independent determinations.
In the next modification, the influence of the R group in para-position of the
phenyl ring was assessed, with a series of compounds (18v-18z, 18aa-18ae and 19)
being synthesized. The length of the alkyl group in the para position of the phenyl
ring was shown to be important, increasing the length of the R group from methyl
(18v) to ethyl (18w), propyl (18i), and butyl (18x) resulted in progressive
improvement of inhibitory activity from 481 to 243, 89, and 49 nM, respectively.
However, an excessive length of the R group such as pentyl (as in 18z) was
demonstrated to be detrimental to the inhibitory activity. Meanwhile, compared
with compounds 18x and 18y, we could find that a linear alkyl substituent was
better to potency than a branched alkyl substituent of the phenyl ring. Indeed, an
isobutyl substituent present in compound 18y resulted in a 2-fold loss of CT-L
inhibitory activity compared to a butyl substituent present in 18x. Moreover, that
the R group prefers hydrophobic group to hydrophilic group with a propyl
substituent (as in 18i) being 10-fold more potent than a hydroxyethyl substituent (as
in 19). However, replacing the alkyl substituent with an alkoxy substituent was
shown to be detrimental to the CT-L inhibitory activity (18w vs 18aa, 18i vs 18ab).
Furthermore, we also attempted to introduce various other groups to the phenyl ring
such as trifluoromethyl (18ac), bromide (18ad), and 2-methoxy-2-oxoethyl (18ae),
but all of these compounds demonstrated unsatisfactory CT-L inhibitory activity.
Overall, we identified compound 18x as the most potent compound exhibiting an
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almost 2-fold higher potency than PI-1840. Furthermore, subsequent assays of the
metabolic stability of 18x and PI-1840 in human liver microsomes showed that
compound 18x was more stable than PI-1840 (Fig. 4).
Human liver micosomes
100
18x
PI-1840
50
0
0
10
20
30
Time (min)
Figure 4. Metabolic stability of 18x and PI-1840 in human liver microsomes.
Table 4.
Inhibition for β1c, β2c, β5c and β5i of 18p, 18x, PI-1840 and carfilzomib
IC50(nM)
Compd.
C-L (β1c)
>10000
T-L(β2c)
>10000
CT-L(β5c)
230 ± 72
49 ± 12
92 ± 5
LMP7(β5i)
>10000
>10000
>10000
27 ± 7
18p
18x
>10000
>10000
PI-1840
Carfilzomib
>10000
>10000
1452 ± 400
825 ± 305
5 ± 0
a The IC50 values are shown as an average of three independent determinations.
We have also assessed the subunits selective inhibitory activity of 18p and 18x.
The experimental results demonstrated that 18p and 18x exhibited the same
selective inhibitory activity as PI-1840 with only targeting β5c (Table 4), but
carfilzomib can inhibit several subunits. These results indicated that our
compounds was β5c selective proteasome inhibitors which were different from
approved carfilzomib.
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2.2.2. Anti-proliferation activity against cancer cell lines
Table 5.
Anti-proliferation activity of compound 18p and 18x against different cancer cell lines
Anti-proliferative activity (IC50, ꢀM)a
Compd.
HepG2
HGC27
RPMI 8226
>20
MM1S
>20
MV-4-11
>20
18p
18x
2.87 ± 0.51
3.91 ± 0.95
2.95 ± 0.77
2.47 ± 0.12
4.62 ± 1.44
3.35 ± 1.09
>20
>20
>20
>20
>20
>20
PI-1840
a The IC50 values are shown as an average of three independent determinations.
We selected the two potent compounds 18p and 18x to further assess their
anti-proliferative activity in vitro against various cancer cell lines including HepG2,
HGC27, RPMI 8226, MM1S, and MV-4-11. Interestingly, we found that the two
selected compounds exhibited more potent anti-proliferation activity against solid
cancer cell lines compared to blood cancer cell lines. Compounds 18p and 18x
inhibited the viability of the two tested solid cancer cell lines HepG2, HGC27, with
IC50 values ranging from 2 ꢀM to 5 ꢀM. However, both compounds were shown to
only weakly inhibit the viability of the three blood cancer cell lines RPMI 8226,
MM1S, and MV-4-11, with IC50 values of more than 20 ꢀM. Several studies[25, 26]
have shown that higher immunoproteasome expression over constitutive
proteasome in B-cell malignancies, suggesting the importance of the
immunoproteasome in hematologic malignancies[27, 28]. As described in table 4,
18p and 18x shown selectivity for β5c inhibitory activity with no β5i inhibition,
this may be the reason for a lack of efficacy in blood cancer cells versus solid
cancer cells. Therefore, other than approved inhibitors that may only be applied to
blood cancers, these compounds could be applied to solid cancers.
2.3. Binding mode analysis
To elucidate the binding modes of this class of compounds within the β5 site of
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proteasome, molecular docking calculations were performed. First, the ligand in the
crystal structure of 3MG6 (PDB ID) was extracted and re-docked into the binding
pocket [29-31]. The root mean square deviation (RMSD) between the docked pose
and the original conformation of the ligand was calculated, and a RMSD = 0.3 Å
suggests that the Glide docking with the SP scoring can successfully recognize the
near-native conformations. Then, the binding mode of compound 18x within the
proteasome was predicted by the Glide docking. As depicted in Fig. 5, the
4-butylphenyl moiety of compound 18x appears to provide a well fit for the important
hydrophobic S3 pocket (Fig. 5B), while four critical hydrogen bonds were also
observed (Fig. 5A and Fig. 5C). As discussed in SARs, the para-position of A ring was
important to inhibitory activity, we found that p-nitrogen atom of pyridazine ring
formed a hydrogen bond with Arg91. Particularly, two other hydrogen bonds were
formed between the ligand and Ala50N and Asp114Oγ via a bridge water molecule.
Besides, carbonyl group (near to phenyl ring) was predicted to form the last hydrogen
bond with Ala49N. The docking studies may allow the rational design of more potent
proteasome inhibitors. While a hydrogen bond acceptor group or atom was needed in
the para-position of A ring in this class and the R group prefer to be hydrophobic
group to bind to S3 pocket.
Figure 5. Binding modes of compound 18x within the β5 site of proteasome. A: Binding modes
of compound 18x within the β5 site of proteasome displayed as Connolly surface. B: Close-up
view of the S3 pocket to illustrate occupancy by the 4-butylphenyl moiety of compound 18x in
the β5 active site. C: Binding modes of compound 18x within the β5 site of proteasome
represented as a ribbon model. H-bonds are shown as dashed yellow lines
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3. Conclusions
In this study, we found that the reported PI-1840 displayed a high metabolic
susceptibility in HLMs due to the presence of a 1, 2, 4-oxadiazole moiety. In order
to explore novel proteasome inhibitors with higher metabolic stability, we
rationally designed and synthesized a series of phenol ether derivatives based on
the structural features of PI-1840. Most of these compounds exhibited moderate to
excellent proteasome inhibitory activity. Compound 18x in particular was found to
be the most potent compound with being 2-fold more potent than PI-1840. In
addition, compound 18x was also found to be more stable than PI-1840 in HLMs.
Moreover, further test results confirmed that compounds 18p and 18x exhibited
selective anti-proliferative activity against solid cancer cell lines, including HepG2
and HGC27. Docking studies were also carried out to predict the binding modes of
this class of compounds within proteasome. Taken in concert, we identified a new
class of non-covalent proteasome inhibitors with higher metabolic stability and
potent activity, providing incentive for the further development of such compounds
as potential anticancer agents in the treatment of solid cancers.
4. Experimental section
13
1H and C NMR spectra were recorded on Brüker 400/500 MHz spectrometer
(Brüker Bioscience, Billerica, MA, USA) with CDCl3 or DMSO-d6 as solvent
(Instead of 18x, all NMR experiments performed at 20 °С). Chemical shifts (d)
were reported in parts per million (ppm) relative to internal TMS, and coupling
constants (J) were reported in Hertz (Hz). Splitting patterns were designated as
singlet (s), broad singlet (brs), doublet (d), double doublet (dd), triplet (t), quartet (q)
and multiplet (m). Melting points were determined using a Buchi B-540 capillary
melting point apparatus and are uncorrected. Electrospray ionization mass
spectroscopy (ESI-MS) spectra were obtained with a Shimadzu LCMS-2020 mass
spectrometer with mobile phases as methanol and water containing 0.1% formic
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acid. High resolution mass spectra (HRMS) were measured on an Agilent 6224
TOF LC/MS spectrometer using ESI-TOF (electrospray ionization-time of flight).
Melting points were determined using a Buchi B-540 capillary melting point
apparatus and are uncorrected. HPLC analysis was performed using an Agilent
1100 Series system with a COSMOSIL 5C18-MS-II (4.6 mm I.D. × 250 mm)
column and detected at 254 nm wavelength. The details of the HPLC methods can
be found in supplementary data. Reagents and solvents were purchased from
common commercial suppliers and were used without further purification unless
stated otherwise. Column chromatography was performed using silica gel (200-300
mesh). All yields are unoptimized and generally represent.
4.1. Chemistry
4.1.1. tert-Butyl (2-(isopropylamino)ethyl)carbamate 8
To a solution of tert-butyl (2-aminoethyl) carbamate 7 (123 mg, 1.0 mmol) and
TEA (202 mg, 2.0 mol) in 5 mL MeCN was added 2-bromopropane (160 mg, 1.0
mmol) at 0 °С. Then the reaction mixture was warmed to room temperature and
stirred overnight. The MeCN was evaporated, and the residue was dissolved in
EtOAc (15 mL), washed with water (10 mL × 3) and brine (10 mL × 3). The
organic layer was dried over Na2SO4, concentrated under vacuum and purified by
SiO2 chromatography (ethyl acetate: petroleum ether=1:1) to afford the compound
8 as a colourless liquid (144 mg, 70%).1H NMR (500 MHz, DMSO-d6) δ 6.88 –
6.55 (m, 1H), 3.44 – 3.13 (m, 1H), 3.02 – 2.88 (m, 2H), 2.72 – 2.63 (m, 1H), 2.53 –
2.48 (m, 2H), 1.38 (s, 9H), 0.95 (d, J = 6.5 Hz, 6H). ESI-MS: m/z = 203.1 [M+H] +.
4.1.2. tert-Butyl (2-(2-chloro-N-isopropylacetamido)ethyl)carbamate 9
To a solution of 8 (130 mg, 0.64 mmol) and triethylamine (129 mg, 1.28 mmol)
in anhydrous THF (5 mL) under a nitrogen atmosphere at 0 °С was added
chloroacetyl chloride (108 mg, 0.96 mmol) dropwise. After stirring at room
temperature for 30 min, 10 mL H2O was added to the reaction mixture at 0 °С. The
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THF was evaporated concentrated and the crude residue was extracted with ethyl
acetate (10 mL × 3). The organic layer was washed with saturated aqueous Na2CO3
(10 mL × 3) and brine (10 mL × 3), dried over anhydrous Na2SO4, evaporated and
purified by SiO2 chromatography to afford the compound 9 as a colourless liquid
1
(158 mg, 89%). The H NMR showed 3:1 ratio of atropisomers. 1H NMR (500
MHz, CDCl3) δ 5.23 (s, 1H), 4.06 (s, 2H [4.14 minor isomer]), 4.02 – 3.94 (m,
1H [4.46 minor isomer]), 3.40 – 3.12 (m,4H), 1.51 – 1.29 (m, 9H), 1.21 (d, J = 6.5
Hz, 6H [ 1.15 minor isomer]). ESI-MS: m/z = 279.1 [M+H] +.
4.1.3. tert-Butyl (2-(N-isopropyl-2-(4-propylphenoxy)acetamido)ethyl)carbamate
10
To a solution of 9 (158 mg, 0.57 mmol) in anhydrous 5 mL MeCN was added
K2CO3 (236 mg, 1.71 mmol) at 0°С, followed by 4-propylphenol (86 mg, 0.70
mmol). The reaction mixture was heated at reflux under nitrogen atmosphere for 2h.
After cooling to room temperature, the reaction mixture was concentrated and
dissolved with ethyl acetate (10 mL), washed with water (10 mL × 3) and brine (10
mL × 3). The organic layer was dried over Na2SO4, concentrated under vacuum and
purified by SiO2 chromatography (ethyl acetate: petroleum ether=1:5) to afford the
compound 10 as a colourless liquid (198 mg, 92%). 1H NMR (500 MHz, CDCl3) δ
7.32 – 7.23 (m, 2H), 7.15 – 7.13 (m, 2H), 6.67 (s, 1H), 4.49 (s, 2H), 3.40 – 3.38 (m,
2H), 3.15 – 3.02 (m, 1H), 2.89 (t, J = 6.0 Hz, 2H), 2.50 (t, J = 6.5 Hz, 2H), 1.60 –
1.50 (m, 2H), 1.44 (s, 9H), 1.15 (d, J = 6.5 Hz, 6H), 0.92 (t, J = 7.5 Hz, 3H).
ESI-MS: m/z = 379.2 [M+H] +.
4.1.4. N-(2-Aminoethyl)-N-isopropyl-2-(4-propylphenoxy)acetamide 11
To a solution of 10 (378 mg, 1.00 mmol) in 2 mL ethyl acetate was added
saturated hydrogen chloride in ethyl acetate (4 mL) at 0°С. After stirring at room
temperature for 2 h, the reaction mixture was concentrated and dissolved with ethyl
acetate (15 mL), with 15 mL saturated aqueous Na2CO3 was added at 0°С. The
mixture was kept at 0°С and stirred for 10min. Then the organic layer was washed
17

ACCEPTED MANUSCRIPT
with brine (10 mL × 3), dried over Na2SO4, concentrated under vacuum and
purified by SiO2 chromatography to afford the compound 11 as pale yellow oil
1
(256 mg, 92%). H NMR (500 MHz, DMSO-d6) δ 8.37 (t, J = 5.6 Hz, 2H), 7.14 –
7.05 (m, 2H), 6.93 – 6.86 (m, 2H), 4.46 (s, 2H), 3.43 – 3.36 (m, 2H), 3.14 – 3.01
(m, 1H), 2.85 (t, J = 6.0 Hz, 2H), 2.48 (t, J = 6.5 Hz, 2H), 1.59 – 1.49 (m, 2H), 1.14
(d, J = 6.5 Hz, 6H), 0.87 (t, J = 7.5 Hz, 3H). ESI-MS: m/z = 279.2 [M+H] +.
4.1.5. N-Isopropyl-2-(4-propylphenoxy)-N-(2-(3-(pyridin-3-yl)ureido)ethyl)
acetamide 12a
Pyridin-3-amine (94 mg, 1.00 mmol) and triethylamine (303 mg, 3.00 mmol)
dissolved in 3 mL anhydrous DCM , and the mixture was added to a solution of
triphosgene (95 mg, 0.32 mmol) in anhydrous DCM (5 mL) under a nitrogen
atmosphere at 0 °С. After stirring at room temperature for 1 h, compound 11 (278
mg, 1.00 mol) dissolving in 1 mL anhydrous DCM was added to the reaction
mixture at 0 °С. The reaction mixture was warmed to room temperature and stirred
for 1 h. Then the reaction mixture was concentrated and dissolved with ethyl
acetate (10 mL), washed with water (10 mL × 3) and brine (10 mL × 3), dried over
anhydrous Na2SO4, and then evaporated. The residues purified by SiO2
chromatography (DCM: MeOH=12:1) to afford the compound 12a as a pale yellow
solid (230 mg, 58%). Mp: 113.1-114.3 °С. HPLC purity = 99.99%, HPLC tR =
1
14.84 min (Method A). H NMR (500 MHz, CDCl3) δ 8.92 (d, J = 2.5 Hz, 1H),
8.78 (s, 1H), 8.24 (d, J = 4.5 Hz, 1H), 8.20 – 8.11 (m, 1H), 7.32 – 7.23 (m, 1H),
7.21 (dd, J = 8.5, 4.5 Hz, 1H), 7.15-7.08 (m, 2H), 6.88-6.77 (m, 2H), 4.75 – 4.59
(m, 1H), 4.55 (s, 2H), 3.53 – 3.42 (m, 2H), 3.39 – 3.30 (m, 2H), 2.54 (t, J = 7.5 Hz,
13
2H), 1.66 – 1.56 (m, 2H), 1.15 (d, J = 6.5 Hz, 6H), 0.92 (t, J = 7.5 Hz, 3H); C
NMR (125 MHz, CDCl3) δ 170.87, 155.29, 155.12, 143.28, 141.57, 137.32, 136.68,
129.71, 126.78, 123.29, 114.39, 67.23, 45.34, 40.52, 39.43, 37.09, 24.65, 20.86,
+
13.72. HRMS (ESI) m/z: calcd for C22H31N4O3 [M + H] , 399.2391; found
399.2421.
18

ACCEPTED MANUSCRIPT
4.1.6. N-Isopropyl-2-(4-propylphenoxy)-N-(2-(3-(pyridin-3-ylmethyl)ureido)ethyl)
acetamide 12b
This compound was prepared from pyridin-3-ylmethanamine (108mg, 1.00
mmol), triethylamine (303 mg, 3.00 mmol) and triphosgene (95 mg, 0.32 mmol) in
a similar manner as described for compound 12a. The product was obtained as pale
yellow oil (260 mg, 63%). HPLC purity = 96.32%, HPLC tR = 12.96 min (Method
1
A). H NMR (500 MHz, CDCl3) δ 8.57 (d, J = 1.5 Hz, 1H), 8.43 (dd, J = 5.0, 1.5
Hz, 1H), 7.72 – 7.66 (m, 1H), 7.38 (s, 1H), 7.19 (dd, J = 7.5, 5.0 Hz, 1H), 7.09 (d, J
= 8.5 Hz, 2H), 6.81 – 6.75 (m, 3H), 4.51 – 4.38 (m, 5H), 3.41 – 3.31 (m, 2H), 3.27
– 3.18 (m, 2H), 2.51 (t, J = 7.5, 2H), 1.63 – 1.51 (m, 2H), 1.08 (d, J = 6.5 Hz, 6H),
13
0.90 (t, J = 7.5 Hz, 3H); C NMR (125 MHz, CDCl3) δ 170.30, 157.96, 155.20,
149.11, 148.10, 136.53, 135.94, 135.62, 129.65, 123.42, 114.40, 67.24, 45.58,
42.29, 40.36, 39.62, 37.09, 24.66, 20.92, 13.72. HRMS (ESI) m/z: calcd for
C23H33N4O3 [M + H] +, 413.2547; found 413.2540.
4.1.7. N-(2-(N-Isopropyl-2-(4-propylphenoxy)acetamido)ethyl)nicotinamide 12c
To a solution of nicotinic acid (128 mg, 1.00 mmol) in DCM (5 mL) were added
HOBt (162 mg, 1.20 mmol) and EDCI (228 mg, 1.50 mmol) at 0 °С. The reaction
mixture was stirred for 30 min. Then compound 11 (278 mg, 1.00 mmol) and
diisopropylethylamine (0.5 mL, 3.00 mmol) were added. After stirring at room
temperature for another 5 h, the resulting mixture was washed with aqueous
NaHCO3 solution (10 mL × 3), brine (10 mL × 3) and dried over Na2SO4. The
organic layer was evaporated in vacuo and the crude product was purified by SiO2
chromatography (ethyl acetate: petroleum ether: TEA=1:1:3%) to afford the
compound 12c as a white solid (341 mg, 89%). Mp: 54.5-56.8 °С. HPLC purity =
1
99.10%, HPLC tR = 11.30 min (Method A). H NMR (500 MHz, CDCl3) δ 8.67 –
8.62 (m, 1H), 8.59 (s, 1H), 7.64 (d, J = 7.5 Hz, 1H), 7.44 (s, 1H), 7.37 – 7.27 (m,
1H), 7.11 – 7.04 (m, 2H), 6.82 (d, J = 7.5 Hz, 2H), 4.45 (s, 2H), 3.98 – 3.85 (m,
1H), 3.69 – 3.50 (m, 4H), 2.51 (t, J = 7.5 Hz, 2H), 1.64 – 1.51 (m, 2H), 1.31 – 1.05
(m, 6H), 0.93 – 0.85 (m, 3H); 13C NMR (125 MHz, CDCl3) δ 169.96, 169.11,
19

ACCEPTED MANUSCRIPT
155.37, 150.54, 147.05, 136.31, 134.12, 132.71, 129.58, 123.51, 114.47, 67.43,
50.84, 39.76, 39.00, 37.10, 24.66, 21.17, 13.73. HRMS (ESI) m/z: calcd for
C22H30N3O3 [M + H] +, 384.2282; found 384.2291.
4.1.8. Ethyl isopropylglycinate 14a
To a solution of propan-2-amine (4.72 g, 80.00 mmol) and TEA (2.00 g, 20.00
mol) in 50 mL MeCN was added ethyl 2-chloroacetate (2.44 g, 20.00 mmol) at
0 °С. Then the reaction mixture was warmed to room temperature and stirred
overnight. The MeCN was evaporated, and the residue was dissolved in EtOAc (50
mL), washed with water (50 mL × 3) and brine (50 mL × 3). The organic layer was
dried over Na2SO4, concentrated under vacuum and the crude product was purified
SiO2 chromatography (ethyl acetate: petroleum ether =1:4) to afford the compound
14a as a colourless liquid (2.10 g, 72%).1H NMR (400 MHz, DMSO-d6) δ 4.12 –
4.02 (m, 2H), 3.42 – 3.19 (m, 3H), 2.78 – 2.65 (m, 1H), 1.19 (t, J = 7.2 Hz, 3H),
0.94 (d, J = 6.4 Hz, 6H). ESI-MS: m/z = 146.1 [M+H] +.
4.1.9. Benzyl isopropylglycinate 14b
This compound was prepared from propan-2-amine (4.72 g, 80.00 mmol),
triethylamine (2.00 g, 20.00 mmol) and benzyl 2-chloroacetate 13b (4.16 g, 20.00
mmol) in a similar manner as described for compound 14a. The product was
1
obtained as a colourless liquid (3.22 g, 78%). H NMR (400 MHz, DMSO-d6) δ
7.40 – 7.28 (m, 5H), 5.12 (s, 2H), 3.37 (s, 2H), 2.72 (hept, J = 6.2 Hz, 1H), 1.85 (s,
1H), 0.94 (d, J = 6.2 Hz, 6H). ESI-MS: m/z = 208.1 [M+H] +.
4.1.10. Ethyl 3-(isopropylamino)propanoate 14c
This compound was prepared from propan-2-amine (4.72 g, 80.00 mmol),
triethylamine (2.00 g, 20.00 mmol) and ethyl 3-bromopropanoate 7c (3.60 g, 20.00
mmol) in a similar manner as described for compound 14a. The product was
obtained as a colourless liquid (2.20, 69%).1H NMR (400 MHz, DMSO-d6) δ 4.04
(q, J = 7.2 Hz, 2H), 3.17 (brs, 1H), 2.77 – 2.58 (m, 3H), 2.38 (t, J = 6.8 Hz, 2H),
20

ACCEPTED MANUSCRIPT
1.17 (t, J = 7.2 Hz, 3H), 0.94 (d, J = 6.4 Hz, 6H). ESI-MS: m/z = 160.1 [M+H] +.
4.1.11. Ethyl N-(2-chloroacetyl)-N-isopropylglycinate 15a
This compound was prepared from 14a (0.93 g, 6.40 mmol), triethylamine (1.29
g, 12.80 mmol) and chloroacetyl chloride (1.08 g, 9.60 mmol) in a similar manner
as described for compound 9. The product was obtained as a colourless liquid (1.2
1
g, 90%). The H NMR showed 5:2 ratio of atropisomers. 1H NMR (500 MHz,
CDCl3) δ 4.25 – 4.15 (m, 3H, [4.84-4.76 minor isomer]), 4.14 (s, 2H, [4.02 minor
isomer]), 3.90 (s, 2H, [3.97 minor isomer]), 1.31 – 1.24 (m, 3H), 1.23 (d, J = 6.5 Hz,
6H, [1.09-1.08 minor isomer]). ESI-MS: m/z = 222.1 [M+H] +.
4.1.12. Benzyl N-(2-chloroacetyl)-N-isopropylglycinate 15b
This compound was prepared from 14b (1.32 g, 6.40 mmol), triethylamine (1.29
g, 12.80 mmol) and chloroacetyl chloride (1.08 g, 9.60 mmol) in a similar manner
as described for compound 9. The product was obtained as a colourless liquid (1.68
1
g, 93%). The H NMR showed 3:1 ratio of atropisomers.1H NMR (500 MHz,
CDCl3) δ 7.41 – 7.30 (m, 5H), 5.17 (s, 2H [5.20 minor isomer]), 4.20 – 4.11 (m, 3H
[4.80 minor isomer], [ 4.07 minor isomer]), 3.99 – 3.94 (m, 2H), 1.22 (d, J = 6.5 Hz,
6H [ 1.06 minor isomer]). ESI-MS: m/z = 284.1 [M+H] +.
4.1.13. Ethyl 3-(2-chloro-N-isopropylacetamido)propanoate 15c
This compound was prepared from 14c (1.02 g, 6.40 mmol), triethylamine (1.29
g, 12.80 mmol) and chloroacetyl chloride (1.08 g, 9.60 mmol) in a similar manner
as described for compound 9. The product was obtained as a colourless liquid (1.43
1
g, 95%). The H NMR showed 3:1 ratio of atropisomers.1H NMR (500 MHz,
CDCl3) δ 4.17 – 4.07 (m, 3H), 4.05 (s, 2H), 3.46 (t, J =7.5 Hz, 2H), 2.60 (t, J =7.5
Hz, 2H), 1.30 – 1.12 (m, 9H). ESI-MS: m/z = 236.1 [M+H] +.
4.1.14. Ethyl N-isopropyl-N-(2-(p-tolyloxy)acetyl)glycinate 16a
This compound was prepared from 15a (180 mg, 0.81 mmol), p-cresol (105 mg,
21

ACCEPTED MANUSCRIPT
0.98 mmol) and K2CO3 (449 mg, 3.26 mmol) in a similar manner as described for
1
compound 10. The product was obtained as colourless oil (187 mg, 79%). The H
NMR showed 2:1 ratio of atropisomers.1H NMR (400 MHz, CDCl3) δ 7.12 – 7.02
(m, 2H), 6.90 – 6.75 (m, 2H), 4.73 (s, 2H, [5.62 minor isomer]), 4.34 – 4.22 (m, 1H,
[4.82 minor isomer]), 4.21 – 4.13 (m, 2H), 3.92 (s, 2H, [4.07 minor isomer]), 2.28
(s, 3H), 1.29 – 1.22 (m, 3H), 1.20 (d, J = 6.6 Hz, 6H, [1.09 minor isomer]). ESI-MS:
m/z = 294.1 [M+H] +.
4.1.15. Ethyl N-(2-(4-ethylphenoxy)acetyl)-N-isopropylglycinate 16b
This compound was prepared from 15a (180 mg, 0.81 mmol), 4-ethylphenol
(119 mg, 0.98 mmol) and K2CO3 (449 mg, 3.26 mmol) in a similar manner as
described for compound 10. The product was obtained as colourless oil (225 mg,
90%). The 1H NMR showed 2:1 ratio of atropisomers.1H NMR (400 MHz, CDCl3)
δ 7.13 – 7.04 (m, 2H), 6.92 – 6.80 (m, 2H), 4.73 (s, 2H, [4.62 minor isomer]), 4.33
– 4.22 (m, 1H, [4.81 minor isomer]), 4.20 – 4.12 (m, 2H), 3.92 (s, 2H, [4.07 minor
isomer]), 2.58 (q, J = 7.6 Hz, 2H), 1.33 – 1.15 (m, 12H, [1.09 minor isomer]).
ESI-MS: m/z = 308.2 [M+H] +.
4.1.16. Ethyl N-isopropyl-N-(2-(4-propylphenoxy)acetyl)glycinate 16c
This compound was prepared from 15a (180 mg, 0.81 mmol), 4-propylphenol
(133 mg, 0.98 mmol) and K2CO3 (449 mg, 3.26 mmol) in a similar manner as
described for compound 10. The product was obtained as colourless oil (228 mg,
87%). The 1H NMR showed 2:1 ratio of atropisomers. 1H NMR (500 MHz, CDCl3)
δ 7.13 – 7.09 (m, 2H), 6.91 – 6.88 (m, 2H, [6.87-6.85 minor isomer] ), 4.76 (s, 2H,
[4.65 minor isomer]), 4.36 – 4.26 (m, 1H, [4.89-4.80 minor isomer]), 4.20 (m, 2H),
3.95 (s, 2H, [4.10 minor isomer]), 2.69 – 2.43 (m, 2H), 1.69 – 1.57 (m, 2H), 1.26 (t,
J = 7.0 Hz, 3H), 1.23 (d, J = 6.5 Hz, 6H, [1.12 minor isomer]), 0.96-0.93 (m, 3H).
ESI-MS: m/z = 322.2 [M+H] +.
4.1.17. Ethyl N-(2-(4-butylphenoxy)acetyl)-N-isopropylglycinate 16d
22

ACCEPTED MANUSCRIPT
This compound was prepared from 15a (150 mg, 0.68 mmol), 4-butylphenol
(122 mg, 0.82 mmol) and K2CO3 (375 mg, 2.72 mmol) in a similar manner as
described for compound 10. The product was obtained as colourless oil (160 mg,
71%). The 1H NMR showed 2:1 ratio of atropisomers.1H NMR (500 MHz, CDCl3)
δ 7.11 – 7.05 (m, 2H), 6.89 – 6.85 (m, 2H, [6.84 minor isomer]), 4.73 (s, 2H, [4.62
minor isomer]), 4.32 – 4.24 (m, 1H, [4.82 minor isomer]), 4.17 – 4.13 (m, 2H),
3.92 (s, 2H, [3.91 minor isomer]), 2.58 – 2.49 (m, 2H), 1.61 – 1.49 (m, 2H), 1.37 –
1.24 (m, 5H), 1.21 (d, J = 6.5 Hz, 6H, [1.1 minor isomer]), 0.94 – 0.88 (m, 3H).
ESI-MS: m/z = 336.2 [M+H] +.
4.1.18. Ethyl N-isopropyl-N-(2-(4-isobutylphenoxy)acetyl)glycinate 16e
This compound was prepared from 15a (150 mg, 0.68 mmol), 4-isobutylphenol
(122 mg, 0.82 mmol) and K2CO3 (375 mg, 2.72 mmol) in a similar manner as
described for compound 10. The product was obtained as colourless oil (157 mg,
69%). The 1H NMR showed 2:1 ratio of atropisomers.1H NMR (500 MHz, CDCl3)
δ 7.08 – 7.02 (m, 2H), 6.89 – 6.85 (m, 2H, [1.10 minor isomer]), 4.74 (s, 2H, [4.63
minor isomer]), 4.33 – 4.25 (m, 1H, [4.82 minor isomer]), 4.18 – 4.15 (m, 2H),
3.93 (s, 2H, [4.01 minor isomer]), 2.43 – 2.37 (m, 2H), 1.86 – 1.76 (m, 1H), 1.29 –
1.25 (m, 3H), 1.21 (d, J = 6.5 Hz, 6H, [1.10 minor isomer]), 0.90 – 0.85 (m, 6H).
ESI-MS: m/z = 336.2 [M+H] +.
4.1.19. Ethyl N-isopropyl-N-(2-(4-pentylphenoxy)acetyl)glycinate 16f
This compound was prepared from 15a (150 mg, 0.68 mmol), 4-pentylphenol
(134 mg, 0.82 mmol) and K2CO3 (375 mg, 2.72 mmol) in a similar manner as
described for compound 10. The product was obtained as colourless oil (167 mg,
70%). The 1H NMR showed 2:1 ratio of atropisomers. 1H NMR (500 MHz, CDCl3)
δ 7.11 – 7.06 (m, 2H), 6.89 – 6.85 (m, 2H, [6.84 minor isomer]), 4.73 (s, 2H, [4.62
minor isomer]), 4.32 – 4.24 (m, 1H, [4.82 minor isomer]), 4.19 – 4.13 (m, 2H),
3.93 (s, 2H, [4.08 minor isomer]), 2.57 – 2.48 (m, 2H), 1.61 – 1.52 (m, 2H), 1.33 –
1.26 (m, 7H), 1.21 (d, J = 6.5 Hz, 6H, [1.12 minor isomer]), 0.92 – 0.86 (m, 3H).
23

ACCEPTED MANUSCRIPT
ESI-MS: m/z = 350.2 [M+H] +.
4.1.20. Ethyl N-isopropyl-N-(2-(4-methoxyphenoxy)acetyl)glycinate 16g
This compound was prepared from 15a (180 mg, 0.81 mmol), 4-methoxyphenol
(121 mg, 0.98 mmol) and K2CO3 (449 mg, 3.26 mmol) in a similar manner as
described for compound 10. The product was obtained as colourless oil (205 mg,
82%). The 1H NMR showed 2:1 ratio of atropisomers. 1H NMR (500 MHz, CDCl3)
δ 6.93 – 6.85 (m, 2H), 6.85 – 6.81 (m, 2H), 4.71 (s, 2H, [4.60 minor isomer]), 4.34
– 4.23 (m, 1H, [4.83 minor isomer]), 4.21 – 4.15 (m, 2H), 3.92 (s, 2H, [4.07 minor
isomer]), 3.77 – 3.75 (m, 3H), 1.28 – 1.22 (m, 3H), 1.21 (d, J = 6.5 Hz, 6H, [1.10
minor isomer]). ESI-MS: m/z = 310.2 [M+H] +.
4.1.21. Ethyl N-(2-(4-ethoxyphenoxy)acetyl)-N-isopropylglycinate 16h
This compound was prepared from 15a (180 mg, 0.81 mmol), 4-ethoxyphenol
(135 mg, 0.98 mmol) and K2CO3 (449 mg, 3.26 mmol) in a similar manner as
described for compound 10. The product was obtained as colourless oil (190 mg,
73%). The 1H NMR showed 2:1 ratio of atropisomers. 1H NMR (500 MHz, CDCl3)
δ 6.91 – 6.84 (m, 2H), 6.84 – 6.78 (m, 2H), 4.70 (s, 2H, [4.59 minor isomer]), 4.33
– 4.23 (m, 1H, [4.82 minor isomer]), 4.20 – 4.14 (m, 2H), 4.00 – 3.94 (m, 2H), 3.92
(s, 2H, [4.07 minor isomer]), 1.41 – 1.35 (m, 3H), 1.28 – 1.22 (m, 3H), 1.20 (d, J =
6.5 Hz, 6H, [1.09 minor isomer]).ESI-MS: m/z = 324.2 [M+H]+.
4.1.22. Ethyl N-isopropyl-N-(2-(4-(trifluoromethyl)phenoxy)acetyl)glycinate 16i
This compound was prepared from 15a (180 mg, 0.81 mmol),
4-(trifluoromethyl)phenol (158 mg, 0.98 mmol) and K2CO3 (449 mg, 3.26 mmol)
in a similar manner as described for compound 10. The product was obtained as
1
1
colourless oil (260 mg, 93%). The H NMR showed 2:1 ratio of atropisomers. H
NMR (500 MHz, CDCl3) δ 7.57 – 7.51 (m, 2H), 7.05 – 6.99 (m, 2H), 4.87 – 4.79
(m, 2H, [4.71 minor isomer]), 4.26 – 4.14 (m, 3H), 3.94 (s, 2H, [4.02 minor
isomer]), 1.31 – 1.18 (m, 9H, [1.11 minor isomer]). ESI-MS: m/z = 348.1 [M+H]+ .
24

ACCEPTED MANUSCRIPT
4.1.23. Ethyl N-(2-(4-bromophenoxy)acetyl)-N-isopropylglycinate 16j
This compound was prepared from 15a (180 mg, 0.81 mmol), 4-bromophenol
(158 mg, 0.98 mmol) and K2CO3 (449 mg, 3.26 mmol) in a similar manner as
described for compound 10. The product was obtained as colourless oil (262 mg,
91%). The 1H NMR showed 2:1 ratio of atropisomers. 1H NMR (400 MHz, CDCl3)
δ 7.46 – 7.33 (m, 2H), 6.88 – 6.79 (m, 2H), 4.74 (s, 2H, [4.63 minor isomer]), 4.27
– 4.12 (m, 3H, [4.81 minor isomer]), 3.92 (s, 2H, [4.02 minor isomer]), 1.31 – 1.23
(m, 3H), 1.21 (d, J = 6.7 Hz, 6H, [1.09 minor isomer]). ESI-MS: m/z = 358.0
[M+H] +.
4.1.24. Benzyl N - isopropyl- N - (2 - (4- (2 – methoxy - 2- oxoethyl) phenoxy)
acetyl)glycinate 16k
This compound was prepared from 15b (1.00 g, 3.50 mmol), methyl
2-(4-hydroxyphenyl)acetate (0.71 g, 4.20 mmol) and K2CO3 (2.42 g, 17.5 mmol) in
a similar manner as described for compound 10. The product was obtained as
1
colourless oil (0.98 g, 68%). The H NMR showed 2:1 ratio of atropisomers. 1H
NMR (400 MHz, CDCl3) δ 7.39 – 7.28 (m, 5H), 7.22 – 7.13 (m, 2H), 6.93 – 6.88
(m, 2H, [6.83 minor isomer]), 5.15 (s, 2H, [5.16 minor isomer]), 4.74 (s, 2H, [4.60
minor isomer]), 4.29 – 4.19 (m, 1H), 3.98 (s, 2H, [ 4.24 minor isomer]), 3.67 (s,
3H), 3.55 (s, 2H), 1.19 (d, J = 6.6 Hz, 6H, [1.07 minor isomer]). ESI-MS: m/z =
414.2 [M+H] +.
4.1.25. Ethyl 3-(N-isopropyl-2-(4-propylphenoxy)acetamido)propanoate 16l
This compound was prepared from 15c (588 mg, 2.50 mmol), 4-propylphenol
(374 mg, 2.75 mmol) and K2CO3 (1.04 g, 7.5 mmol) in a similar manner as
1
described for compound 10. The product was obtained as colourless oil. H NMR
(400 MHz, CDCl3) δ 7.07 (d, J = 8.4 Hz, 2H), 6.84 (d, J = 8.4 Hz, 2H), 4.64 (s, 2H),
4.25 – 4.04 (m, 3H), 3.49 (t, J = 8.0 Hz, 2H), 2.67 – 2.57 (m, 2H), 2.53 (t, J = 8.0
Hz, 2H), 1.60 – 1.45 (m, 2H), 1.26 – 1.13 (m, 9H), 0.90 (t, J = 7.2 Hz, 3H).
25

ACCEPTED MANUSCRIPT
ESI-MS: m/z = 336.2 [M+H] +.
4.1.26. General procedure for the synthesis of carboxylic acids 17a-17j and 17l
To a solution of compounds 16l-16j and 16l (3.00 mmol) in 6 mL EtOH/THF
(VEtOH:VTHF=1:1) at 0 °С was added 6 mL 1N NaOH (aq). The mixture was stirred
at room temperature for 1 h and EtOH and THF were evaporated in vacuo. The
residue was acidified to pH=2~3 with 1 N HCl and extracted with ethyl acetate (10
mL × 3). The combined organic layers were washed with brine, dried over Na2SO4,
filtered and concentrated under reduced pressure. The crude product 17a-17j and
17l were put into next step without further purification.
4.1.27. N-Isopropyl-N-(2-(4-(2-methoxy-2-oxoethyl)phenoxy)acetyl)glycine 17k
Compound 16k (413 mg, 1.00 mmol) was dissolved in methanol (5 mL) in the
presence of 10% palladium on carbon (10 mol%), and the reaction mixturewas
stirred under an atmosphere of H2 for 2 h. The Pd/C was removed via filtration
through celite, and the solvent was evaporated to obtain compound 17k in
quantitative yield. The crude product was used directly in the next step.
4.1.28.
3-(N-Isopropyl-2-(4-propylphenoxy)acetamido)-N-(pyridin-3-yl)propanamide 18a
This compound was prepared from 17l (921 mg, 3.00 mmol), pyridin-3-amine
(282 mg, 3.00 mmol), HOBt (486 mg, 3.60 mmol), EDCI (864 mg, 4.50 mmol) and
DIPEA (1.5 mL, 9.00 mmol) in a similar manner as described for compound 12c.
The product was obtained as pale yellow oil (402mg, 43%). HPLC purity = 97.73%,
HPLC tR = 12.17 min (Method A). The 1H NMR showed 7:1 ratio of atropisomers.
1H NMR (500 MHz, CDCl3) δ 9.62 (s, 1H), 8.68 (s, 1H), 8.29 (s, 1H), 8.12 (d, J =
8.5 Hz, 1H), 7.24 – 7.15 (m, 1H), 7.03 (d, J = 8.5 Hz, 2H, [6.98 minor isomer]),
6.77 (d, J = 8.5 Hz, 2H), 4.69 (s, 2H), 4.21 – 3.98 (m, 1H), 3.60 (t, J = 7.0 Hz, 2H,
[3.66 minor isomer]), 2.66 (t, J = 7.0 Hz, 2H), 2.48 (t, J = 7.5 Hz, 2H), 1.63 – 1.51
(m, 2H), 1.25 (d, J = 6.5 Hz, 6H, [1.17 minor isomer]), 0.90 (t, J = 7.5 Hz, 3H).13C
NMR (125 MHz, CDCl3) δ 170.31, 168.48, 155.86, 144.69, 141.40, 136.07[135.48
26

ACCEPTED MANUSCRIPT
minor isomer], 129.49, 126.88, 123.52, 114.25, 67.46, 48.71, 38.01, 37.59, 37.10,
24.67, 21.15[20.16 minor isomer], 13.77. HRMS (ESI) m/z: calcd for C22H30N3O3
[M + H] +, 384.2282; found 384.2289.
4.1.29.
3-(N-Isopropyl-2-(4-propylphenoxy)acetamido)-N-(pyridin-3-ylmethyl)propanami
de 18b
This compound was prepared from 17l (307 mg, 1.00 mmol),
pyridin-3-ylmethanamine (108 mg, 1.00 mmol), HOBt (162 mg, 1.20 mmol), EDCI
(188 mg, 1.50 mmol) and DIPEA (0.5 mL, 3.00 mmol) in a similar manner as
described for compound 12c. The product was obtained as pale yellow oil (325mg,
1
82%) . HPLC purity = 98.03%, HPLC tR = 10.86 min (Method A). The H NMR
showed 7:1 ratio of atropisomers. 1H NMR (500 MHz, CDCl3) δ 8.49 (s, 1H), 8.44
(s, 1H), 7.58 (d, J = 7.5 Hz, 1H), 7.36 (t, J = 5.5 Hz, 1H), 7.20 (t, J = 4.5 Hz, 1H),
7.04 (d, J = 8.0 Hz, 2H,[7.01 minor isomer]), 6.78 (d, J = 8.0 Hz, 2H), 4.59 (s, 2H,
[4.63 minor isomer]), 4.34 (d, J = 6.0 Hz, 2H), 4.11 – 4.03 (m, 1H), 3.49 (t, J = 7.5
Hz, 2H,[3.58 minor isomer]), 2.55 – 2.41 (m, 4H), 1.62 – 1.49 (m, 2H), 1.19 (d, J =
6.5 Hz, 6H, [1.14 minor isomer]), 0.89 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz,
CDCl3) δ 171.41[170.40 minor isomer], 168.09, 155.95, 149.10, 148.47, 135.97,
135.62, 134.35, 129.46, 123.54, 114.33[114.48 minor isomer], 67.66, 48.58[47.51
minor isomer], 40.85[39.91 minor isomer], 38.17, 37.09[37.49 minor isomer],
36.41, 24.68, 21.10[20.13 minor isomer], 13.76. HRMS (ESI) m/z: calcd for
C23H31N3O3 [M + H] +, 398.2438; found 398.2440.
4.1.30.
N-Isopropyl-N-(2-oxo-2-((pyridin-3-ylmethyl)amino)ethyl)-2-(4-propylphenoxy)ac
etamide 18c
This compound was prepared from 17c (293 mg, 1.00 mmol),
pyridin-3-ylmethanamine (108 mg, 1.00 mmol), HOBt (162 mg, 1.20 mmol), EDCI
(188 mg, 1.50 mmol) and DIPEA (0.5 mL, 3.00 mmol) in a similar manner as
27

ACCEPTED MANUSCRIPT
described for compound 12c. The product was obtained as pale yellow oil (302mg,
1
79%). HPLC purity = 99.99%, HPLC tR = 11.22 min (Method A). The H NMR
showed 7:1 ratio of atropisomers. 1H NMR (500 MHz, CDCl3) δ 8.44 (dd, J = 5.0,
1.0 Hz, 1H), 8.40 (s, 1H), 7.52 (d, J = 7.5 Hz, 1H), 7.21 – 7.14 (m, 1H), 7.09 – 6.98
(m, 3H), 6.81 (d, J = 8.5 Hz, 2H, [6.71 minor isomer]), 4.71 (s, 2H, [4.56 minor
isomer]), 4.29 (d, J = 6.1 Hz, 2H, [4.34 minor isomer]), 4.27 – 4.18 (m, 1H), 3.94 (s,
2H, [3.96 minor isomer]), 2.47 (t, J = 7.5 Hz, 2H), 1.60 – 1.49 (m, 2H), 1.19 (d, J =
13
7.0 Hz, 6H, [1.04 minor isomer]), 0.88 (t, J = 7.5 Hz, 3H). C NMR (125 MHz,
CDCl3) δ 169.88, 169.29 [168.98 minor isomer], 155.67, 148.86 [149.30 minor
isomer], 148.59, 136.18 [136.07 minor isomer], 135.27 [135.83 minor isomer],
133.97 [133.73 minor isomer], 129.57, 123.51, 114.27 [114.36 minor isomer],
67.44 [68.18 minor isomer], 49.09 [46.50 minor isomer], 45.28 [45.90 minor
isomer], 40.71 [41.04 minor isomer], 37.07, 24.65, 20.94 [19.50 minor isomer],
+
13.76. HRMS (ESI) m/z: calcd for C22H30N3O3 [M + H] , 384.2282; found
384.2299.
4.1.31. N-Isopropyl-N-(2-oxo-2-(pyridin-3-ylamino)ethyl)-2-(4-propylphenoxy)
acetamide 18d
This compound was prepared from 17c (293 mg, 1.00 mmol), pyridin-3-amine
(94 mg, 1.00 mmol), HOBt (162 mg, 1.20 mmol), EDCI (188 mg, 1.50 mmol) and
DIPEA (0.5 mL, 3.00 mmol) in a similar manner as described for compound 12c.
The product was obtained as pale yellow oil (147mg, 40%). HPLC purity = 99.55%,
HPLC tR = 12.10 min (Method A). The 1H NMR showed 10:1 ratio of atropisomers.
1H NMR (500 MHz, CDCl3) δ 9.05 (s, 1H), 8.51 (d, J = 2.5 Hz, 1H, [8.48 minor
isomer]), 8.30 (d, J = 4.0 Hz, 1H), 7.94 (d, J = 8.5 Hz, 1H, [8.03 minor isomer]),
7.20 (dd, J = 8.5, 4.5 Hz, 1H), 7.08 (d, J = 9.0 Hz, 2H, [7.01 minor isomer]), 6.87
(d, J = 8.5 Hz, 2H, [6.77 minor isomer]), 4.80 (s, 2H, [4.69 minor isomer]), 4.37 –
4.26 (m, 1H), 4.08 (s, 2H, [4.15 minor isomer]), 2.50 (t, J = 7.5 Hz, 2H), 1.62 –
1.51 (m, 2H), 1.30 (d, J = 6.5 Hz, 6H, [1.17 minor isomer]), 0.90 (t, J = 7.5 Hz, 3H).
13C NMR (125 MHz, CDCl3) δ 169.65[169.23 minor isomer], 168.31, 155.74,
28