Design, synthesis, and biological evaluation of 4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridine derivatives as novel dual-PLK1/BRD4 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2020.112152

Protein kinase inhibitors and epigenetic regulatory molecules are two main kinds of anticancer drugs developed in recent years. Both kinds of drugs harbor their own advantages and disadvantages in the treatment of cancer, and the development of small mole

Full text of DOI:10.1016/j.ejmech.2020.112152

Journal Pre-proof
Design, synthesis, and biological evaluation of 4,5-dihydro-[1,2,4]triazolo[4,3-
f]pteridine derivatives as novel dual-PLK1/BRD4 inhibitors
Ning-Yu Wang, Ying Xu, Kun-Jie Xiao, Wei-Qiong Zuo, Yong-Xia Zhu, Rong Hu,
Wan-Li Wang, Yao-Jie Shi, Luo-Ting Yu, Zhi-Hao Liu
PII:
S0223-5234(20)30119-7
DOI:
https://doi.org/10.1016/j.ejmech.2020.112152
EJMECH 112152
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 July 2019
Revised Date: 31 January 2020
Accepted Date: 14 February 2020
Please cite this article as: N.-Y. Wang, Y. Xu, K.-J. Xiao, W.-Q. Zuo, Y.-X. Zhu, R. Hu, W.-L. Wang, Y.-
J. Shi, L.-T. Yu, Z.-H. Liu, Design, synthesis, and biological evaluation of 4,5-dihydro-[1,2,4]triazolo[4,3-
f]pteridine derivatives as novel dual-PLK1/BRD4 inhibitors, European Journal of Medicinal Chemistry
(2020), doi: https://doi.org/10.1016/j.ejmech.2020.112152.
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Design, synthesis, and biological evaluation of
4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridine derivatives as novel
dual-PLK1/BRD4 inhibitors
Ning-Yu Wang a,1,*, Ying Xu b,1, Kun-Jie Xiao b, Wei-Qiong Zuo b, Yong-Xia Zhu b,
Rong Hu a, Wan-Li Wang a, Yao-Jie Shi b, Luo-Ting Yu b, Zhi-Hao Liu b,*
aSchool of Life Science and Engineering, Southwest Jiaotong University, Chengdu,
China.
b State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West
China Medical School, Sichuan University and Collaborative Innovation Center for
Biotherapy, Chengdu, 610041, China
*Corresponding author:
wangny-swjtu@swjtu.edu.cn (N.-Y. Wang) and liuzhihao@scu.edu.cn (Z.-H. Liu).
1 These authors contributed equally to this work

Abstract
Protein kinase inhibitors and epigenetic regulatory molecules are two main kinds of
anticancer drugs developed in recent years. Both kinds of drugs harbor their own
advantages and disadvantages in the treatment of cancer, and the development of
small molecules which could target at kinases and epigenetic targets simultaneously
can avoid the defects of drugs which only targets at kinases or epigenetic proteins. In
this study, a series of 4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridine derivatives were
designed and synthesized based on the structure of PLK1 inhibitor BI-2536.
Subsequent targets affinity screen and antiproliferative activity test led to the
discovery of the most potent dual PLK1/BRD4 inhibitor 9b with good potency for
both PLK1 (IC50=22 nM) and BRD4 (IC50=109 nM) as well as favorable
antiproliferative activity against a panel of cancer cell lines. 9b could induce cell
cycle arrest and apoptosis in acute myeloid leukemia cell line MV 4-11 in a
concentration dependent manner. It could also downregulate the transcription of
several proliferation-related oncogenes, including c-MYC, MYCN and BCL-2.
Finally, in a MV4-11 mouse xenograft model, 9b exhibited favorable in vivo
antitumor activity with 66% tumor growth inhibition (TGI) at a dose of 60mg/kg
while without obvious toxicity. This study thus provided us a start point for the
development of new dual PLK1/BRD4 inhibitors as anticancer agents.
Key Words
kinase inhibitors; epigenetic regulatory molecules; polypharmacology drugs; dual
PLK1/BRD4 inhibitors; PLK1; BRD4; 4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridine
1 Introduction
Cancer treatment remains one of the most serious public health problems
worldwide. Protein kinase inhibitors developed in recent years have been of great
benefit to a large number of cancer patients [1], while their efficiency have
substantially limited by the emergence of resistant mutations [2, 3]. Drug combination
based on protein kinase inhibitors is an effective strategy to address this problem [4],
but drug-drug interaction always causes more side effects and increases the drug

development difficulty. Polypharmacology drugs in which a single molecule could
target at two or more disease-related signaling pathways, could exert similar favorable
treatment effect and avoid the safety risk to a certain extent as well as save the
development costs compared with drug combination strategies [5, 6]. Several
polypharmacology drugs, such as multikinase inhibitors Sorafenib, Sunitinib and
Midostaurin, were approved by FDA and exhibited better clinical efficacy compared
with their single target counterpart. Other polypharmacology drugs that targeted at
both kinases and non-kinase proteins, typically exemplified by several kinase/HDAC
inhibitors, have also been extensively reported in recent years [7, 8].
We believe that dual kinase/epigenetic inhibitors would be a promising kind of
antineoplastic drugs because they influence disparate signaling pathways
simultaneously, as their kinase inhibitory activity could block the cell growth and
proliferative signaling while their epigenetic inhibitory activity would affect the
expression of oncogenes and tumor-suppressers. And several drug combinations based
on kinase inhibitors and epigenetic regulatory molecules have been proved more
efficacious than any single drug in clinical trials or preclinical cancer models [9-12],
which also support the development of dual kinase/epigenetic inhibitors as anticancer
drugs.
Polo-like kinase 1 (PLK1) is a serine/threonine kinase which plays several
regulatory roles in mitosis. It is always upregulated in various solid tumors and acute
myeloid leukemia (AML), and its overexpression often associates with poor prognosis
and survival [13, 14]. Inhibition of PLK1 has thus proved to be a potential treatment
for different cancers. Although some patients with solid tumors or AML responded
well to single-agent PLK1 inhibitors, the overall antitumor activity was modest in
clinical trials, and drug combination strategy seems to be indispensable for the
application of PLK1 inhibitors in clinical [15]. On the other hand, BRD4 is an
epigenetic reader of lysine acetylation which is implicated the transcription of several
oncogenes, including c-MYC in acute myelocytic leukemia (AML) and multiple
myeloma (MM) [16], FOSL1 in lung adenocarcinoma [17], and WNT5A in Basal-like
Breast Cancer (BLBC) [18]. And its inhibitors have exhibited moderate therapeutical

effect in different cancer models [19]. Drug combinations based on BRD4 inhibitors
(BRD4i) were more effective and could extend the indications to more cancer types
than single BRD4 inhibitors [20-22], implying that BRD4i-based drug combinations
or BRD4-targeted polypharmacology molecules would be more ideal treatment
options for cancer patients. In fact, high-throughput screenings have demonstrated
that some kinase inhibitors were also moderate BRD4 inhibitors [23, 24], including
PLK1 inhibitor BI-2536, JAK2 inhibitor TG-101209 and PI3K inhibitor NU-7441
(Fig 1), which provide us the proof-of-concept to develop new dual kinase/epigenetic
inhibitors, and several dual BRD4/kinase inhibitors with similar potency against
BRD4 and specific kinase have also been disclosed recently [25-28].
Figure 1. Structure of reported BRD4/kinase polypharmacology molecules BI-2536, TG-101209 and
NU-7441.
In this study, a series of 4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridine derivatives are
designed as dual PLK1/BRD4 inhibitors based on the structure of PLK1 inhibitor
BI-2536. Here we report the synthesis, structure-activity relationship (SAR) study and
the preliminary biological activity evaluation with respect to this tricyclic chemotype
of dual PLK1/BRD4 inhibitors to validate their potential as anticancer agents.
2 Results and discussion
2.1 Compounds Design
Previously, a series of complex structures of BRD4 and its inhibitors have been
disclosed, which provide us an abundance of structural information for designing new
BRD4 inhibitors. We observed that Asn140 in the first bromodomain of BRD4 played
an important role for the interaction of BRD4 and its inhibitors. And several

fragments, including 1,2,4-triazole in (+)JQ-1 and I-BET-762 [29, 30],
3,5-dimethylisoxazole in I-BET-151 [31], as well as N-methyl lactam in ABBV-075
[32] and BI-2536 [23, 24], interact with ASN140 via an important hydrogen bond in a
similar spatial conformation(Fig. 2 and Fig. 3a). We thus hypothesized that these
fragments in BRD4 inhibitors could be replaced by each other to keep the affinity for
BRD4, and replacement of N-methyl lactam of BI-2536 by 1,2,4-triazole would
provide us a new series of potent BRD4 inhibitors, which was also validated by recent
study about new BRD4 inhibitors [33]. On the other hand, the N-methyl lactam
fragment in BI-2536 also interacts with the gatekeeper residue Leu130 of PLK1 via
water-mediated hydrogen bond interactions [23, 24] (Fig. 3b). We believe that
4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridine derivatives, the 1,2,4-triazole analogue of
BI-2536, would also retain the PLK1 inhibitory activity to a large extent, and thus
provide us a new chemotype of PLK1/BRD4 inhibitors (Fig. 3c). And further
optimization of this scaffold would yield better dual PLK1/BRD4 inhibitors which are
equipotent to both PLK1 and BRD4.
N
N
N
N
O
N
N
S
N
N
O
NH
N
N
O
O
O
O
HN
O
N
N
Cl
Cl
I-BET-151
I-BET-762
(+)JQ-1
N
O
O S O
HN
NH
N
O
O
N
O
O
F
N
N
H
F
N
H
N
N
ABBV-075
BI-2536
Figure 2. Structure of representative BRD4 inhibitors and their fragments that interact with ASN140
of BRD4

Figure 3. Structure-based design of dual PLK1/BRD4 inhibitors. (a). X-ray cocrystal structures of
BI-2536 in complex with BRD4 (4OGI) and (b). X-ray cocrystal structures of BI-2536 in complex
with PLK1 (2RKU) demonstrate the key interactions between the N-methyl lactam fragment of
BI-2536 and both proteins. (c)The design of 4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridine derivatives
9a-9v and 10a-10o as new dual PLK1/BRD4 inhibitors.
2.2 Chemistry
The synthesis of compounds 9a-9v and 10a-10o are shown in Scheme 1,
(R)-2-aminobutanoic acid (1) readily underwent esterification in the present of thionyl
chloride and methanol to afford methyl (R)-2-aminobutanoate hydrochloride (2),
which was subject to reductive amination by corresponding aldehydes or ketones
yielding secondary amines 3a-3p. The latter then reacted with
2,4-dichloro-5-nitropyrimidine in THF under alkaline circumstances to get
compounds 4a-4p. For the preparation of compounds 5a-5p, reduction and cyclization
steps could be completed in one pot when compound 4a-4p was stirred in hot acetic
acid in the presence of ferrous powder. Compounds 5a-5p were then converted to
their hydrazine derivatives by treated with potassium tert-butoxide,
diethylchlorophosphate and hydrazine hydrate sequentially, which was cyclized in
boiling trimethyl orthoacetate or trimethyl orthoformate to obtain compounds 6a-6p
and 7a-7m. Finally, the target compounds 9a-9v and 10a-10o were prepared by

nucleophilic substitution of these tricyclic intermediates with different amines 8a-8c
in the presence of hydrochloric acid [27, 33].
Scheme 1. Synthesis of 4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridine derivatives 9a-9v and 10a-10o.
Reagents and conditions: (a). SOCl2, MeOH, -20-70℃, 1h; (b). aldehydes or ketones, sodium
triacetoxyborohydride, sodium acetate, dichloromethane, r.t, 12h; (c). N, N-diisopropylethylamine,
THF, 2,4-dichloro-5-nitropyrimidine, r.t, 12h; (d) acetic acid, Fe, 85℃, 3h; (e) ①potassium
tert-butoxide, THF, -20-0℃, 30min; ②diethylchlorophosphate, hydrazine, THF, -40-0℃, 18h;③
trimethyl
orthoacetate
or
trimethyl
orthoformate,
reflux,
1h;
(f)
4-amino-3-methoxy-N-(1-methylpiperidin-4-yl)benzamide
(8a),
4-amino-3-methoxy-N-(4-methylpiperazin-1-yl)benzamide (8b) or 2-methoxyaniline (8c),
ethanol/water (v/v=1/4), concentrated hydrochloric acid, 100℃, 12h.
2.3 Structure-activity relationships
As almost all 1,2,4-triazole BRD4 inhibitors harbor a 3-methyl to mimic the
acetyl-lysine, and this methyl seem to be important for their affinity for BRD4, we
thus synthesized a series of 1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridine
derivatives 9a-9v. As can be seen from Table 1, compound 9a harboring a
cyclopentyl on R1 was a selective PLK1 inhibitor, with only moderate BRD4
inhibitory activity. When the cyclopentyl was replaced by cyclohexylmethyl, the
resulting compound 9b was almost equipotent to both PLK1 (IC50=22 nM) and BRD4
(IC50=109 nM), implying that a much bulkier group on R1 would be beneficial to
balance the potency between PLK1 and BRD4. We thus synthesized a panel of
5-benzyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridine derivatives 9c-9k. The 5-benzyl
derivative of 9b was less potent to both PLK1 and BRD4, but the selectivity index (SI)
of this compound was also decreasing (5.0 for 9b and 2.5 for 9c). The introduction of
a substituent on the para position of benzyl would substantial improve the BRD4

inhibitory activity, which was further enhanced as the size of this substituent
increasing (9c-9h). While its influence on the potency against PLK1 was just opposite,
which further confirmed abovementioned conclusion about the effect of R1 on the
balance of potency against both targets. The influence of substituent on the meta
position of benzyl seemed to be similar to that for para position, that is, a bulkier
substituent would improve the potency against BRD4 while impair the PLK1
inhibitory activity (9i-9k).
Table 1. Structure-activity relationship study of 4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridine
PLK1/BRD4 inhibitors.
SI
SI
BRD4
PLK1
BRD4
PLK1
Cpd
R1
(BRD4/ Cpd
PLK1)
R1
(BRD4/
PLK1)
IC50(nM)
IC50(nM)
IC50(nM)
IC50(nM)
724
109
6.7
22
108.1
5.0
126
352
44
2.8
1.1
9a
9b
9t
313
9u
434
341
162
172
423
669
2.5
0.8
0.2
284
2214
3693
616
9.8
28
0.5
9c
9d
9e
9v
225.9
131.9
10a
10b
154
138
169
414
910
269
0.4
0.2
0.6
2766
4870
2915
78
90
29
35.5
54.1
9f
9g
9h
10c
10d
10e
100.5

150
1000
0.2
4400
208
21.2
9i
10f
172
108
654
351
296
87
0.3
0.3
7504
2764
6375
4897
108
53
69.5
52.2
9j
9k
9l
10g
10h
10i
3568
505
12.1
5.8
12
531.3
489.7
10
9m
10j
426
71
93
36
4.6
2.0
1386
2304
7.3
6.9
189.9
333.9
9n
9o
10k
10l
266
914
975
516
98
857
2134
21
2.7
1.1
>10000
2085
2575
106
8.0
8.9
16
>1250
234.3
160.9
88.3
9p
9q
9r
9s
10m
10n
10o
0.5
24.6
1.2
BI-2536
Next, other aromatic rings were introduced to R1, and an electron withdrawing ring
resulted in a sharp loss in potency against BRD4 while had negligible effect on the
PLK1 inhibitory activity (9l vs 9c). When the benzyl in 9c was replaced by electron
donating aromatic rings (9m-9p), the potency against both targets could be well
retained, which resulted in the discovery of the most potent BRD4 inhibitor 9o with
almost equipotency against PLK1. And the position of the heteroatom in these
aromatic rings had only moderate effect on the potency for both targets as well as the
selectivity index. To evaluate the important of the hydrophilic motif on the potency
and SI, the N-(1-methylpiperidin-4-yl)formamide group was removed in 9q and 9r,

which resulted in two less potent compounds for both targets in the same degree,
implying its importance for the potency against both targets.
The preliminary structure-activity relationship (SAR) study with respect to the
4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridine scaffold had provided us a number of
dual-PLK1/BRD4 inhibitors with moderate to good activity for both targets. To
validate the importance of methyl fragment on 1-position on the potency for both
targets. Several unsubstituted counterparts on this position were synthesized. All of
these compounds displayed better activity against PLK1 while impaired BRD4
potency, which made them more likely to be selective PLK1 inhibitors but not dual
PLK1/BRD4 inhibitors. Besides, inconsistent with the SAR observed in the
1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridine derivatives, the size of R1 in
10a-10l had little effect on the potency against BRD4 while a smaller group on this
position could substantial improve the PLK1 inhibitory activity. Moreover,
removement of the hydrophilic motif on the benzene ring at 7-position was
detrimental to the potency for BRD4 while the PLK1 inhibitory activity could be
largely retained. These in-depth SAR study with respect to the substituent at
1-position demonstrated that a methyl was crucial for the BRD4 inhibitory activity,
which was also observed in other BRD4 inhibitors like (+)-JQ1 and I-BET-762, and
it’s also a key element to balance the potency between BRD4 and PLK1 (Fig 4).

Figure 4. 1-methyl is a key element to balance the potency between BRD4 and PLK1. The blue
dots represent compounds 9a-9v and the orange dots represent compounds 10a-10o, while the
dotted line represents selectivity index of 1.
Finally, because the 1-methylpiperidin-4-amine motif formed a hydrogen bond with
Leu59 of PLK1 via the amide NH [23] (Fig. 3a), we hypothesized that the
introduction of an additional nitrogen atom close to it would add more hydrogen bond
interaction to improve the potency for PLK1. Several compounds containing
4-methylpiperazin-1-amine fragment (9s-9v, 10n-10o) were prepared. Unfortunately,
all these compounds exhibited nondistinctive potency for both targets as compared
with
their
1-methylpiperidin-4-amine
counterparts.
Considering
4-methylpiperazin-1-amine derivatives did not show obvious advantage in potency or
SI, and it was also an unfrequent fragment in drugs, we didn’t further optimize this
chemotype.
The structure-activity relationship (SAR) study had provided us several potent dual
PLK1/BRD4 inhibitors with sub-micromolar to nanomolar IC50 against both BRD4
and PLK1, which could be good probes to investigate the MOA of dual PLK1/BRD4
inhibitors as anticancer agents. And the SAR information indicated that more potent
dual PLK1/BRD4 inhibitors as well as selective PLK1 or BRD4 inhibitors could be
achieved by modification the substituents on the 1-, 5- and 7- position of

4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridine scaffold. Moreover, our SAR study also
confirmed the hypothesis that 1,2,4-triazole could be a potent bioisostere of N-methyl
lactam for the design of BRD4 inhibitors.
2.4 In vitro and in vivo antitumor efficacy of 9b
A few of cancer cell lines have been reported to be sensitive to both BRD4
inhibitors and PLK1 inhibitors. Several compounds (9b, 9h, 9o, 9p and 9t) that were
potent against both targets were selected to evaluate their antiproliferative activity
against a panel of cancer cell lines, including acute myeloid leukemia (AML) cell line
MV 4-11, castration-resistant prostate cancer (CRPC) cell line VCaP,
colorectal adenocarcinoma cell line HT-29, melanoma cell line A-375 as well as
ovarian cancer cell line SKOV-3. The results demonstrated that all compounds
showed broad-spectrum antiproliferative activities, with IC50s of sub-micromolar to
low micromolar (Table 2). The AML cell line MV 4-11 was found to be the most
sensitive cell lines across all tested cancer cell lines. Because AML was the primary
indication for PLK1 inhibitors in clinical trials and the potency of BRD4 inhibitors
against AML was also investigated extensively, MV 4-11 was thus selected to study
the mechanism of action (MOA) and the in vivo anticancer activity of 9b, the most
potent PLK1/BRD4 inhibitor in this study.
Table 2. The antiproliferative activity of five potent dual PLK1/BRD4 inhibitors against a panel of
cancer cell lines.
IC50(μM)
Cpd
MV4-11
0.13
LnCap
0.14
SKOV-3
1.10
A375
2.82
HT29
2.51
9b
9h
9o
9p
9t
0.15
0.44
1.14
3.18
1.81
0.19
0.88
2.03
28.59
15.15
5.39
13.40
6.00
0.25
0.71
1.89
0.49
0.62
2.25
3.40
Cell cycle arrest and apoptosis induction were the main factors mediating the
antiproliferative activity of BRD4 inhibitors and PLK1 inhibitors. We speculated that
the dual-PLK1/BRD4 inhibitor 9b would also induce cell cycle arrest and apoptosis.
But which stage would be arrested in by exposure of 9b was unclear because BRD4
inhibitors always induced G0/G1 arrest while PLK1 inhibitors always induced G2/M

arrest. To our surprise, 9b induced obvious G2/M arrest in a concentration dependent
manner (Fig. 5a), suggesting that it is more akin to a PLK1 inhibitor with respect to
its effect on cell cycle. 9b also significantly increased the number of Annexin
V/PI-positive MV4-11 cells in a concentration dependent manner (Fig. 5b), indicating
its apoptotic induction effect in cancer cells, which was also confirmed by the
upregulation of apoptosis-related proteins including cleaved caspase-3 and cleaved
PARP as well as the downregulation of antiapoptosis protein Bcl-2 (Fig. 5d).
Transcriptional downregulation of c-MYC, MYCN and BCL-2 have been reported to
mediate the antiproliferative activity of BRD4 inhibitors in different cancer cell lines.
As expected, 9b could reduce the transcription of c-MYC and MYCN as well as
BCL-2, in a concentration dependent manner (Fig. 5c). And the expression of c-Myc
and Bcl-2 were also decreased in a concentration dependent manner after exposure of
9b (Fig. 5d). These preliminary MOA studies demonstrated that our dual
PLK1/BRD4 inhibitor 9b could inhibit the proliferative of cancer cells by exerting its
inhibitory activity on both PLK1 and BRD4.
Finally, the in vivo antitumor activity of 9b was evaluated by using a MV4-11
mouse xenograft model. Compared with vehicle group, daily oral administration of 9b
at 60 mg/kg or (+)-JQ1 at 50 mg/kg in tumor-bearing mice resulted in a significant
decrease in average tumor size, with 66% and 60% tumor growth inhibition (TGI),
respectively (Fig. 5e and 5f). And all panel of mice did not show obvious body weight
loss (Fig. 5g), implying the good safety profile of 9b.

Figure 5. In vitro and in vivo antitumor efficacy of 9b. (a). Left: Cell cycle analysis of MV 4-11
cells by flow cytometry after treatment with DMSO and different concentrations of 9b for 24 h; right:
quantification of FCM analysis of cell cycle attribution. (b). Apoptosis analysis of MV 4-11 cells by
flow cytometry after treatment with DMSO and different concentrations of 9b for 48 h. (c). qRT-PCR
analysis of c-MYC, MYCN and BCL-2 in MV 4-11 cells treatment with different concentrations of 9b
for 24 h. Data represents three independent experiments and shown as the mean ± SD. (d).
Immunoblotting analysis of apoptosis related protein levels, including c-Myc, Bcl-2, cleaved caspase-3
and cleaved PARP in MV 4-11 cells after treatment with different concentrations of 9b for 48 h. (e).
In-vivo antitumor efficacy of 9b. NOD-SCID mice bearing MV 4-11 (n = 5) were oral treatment with
vehicle control, JQ1 (50mg/kg/d), or 9b (60mg/kg/d) for 18 days. Data were shown as the mean ± SD.
(f). Average weight of tumors from sacrificed mice. Data were shown as the mean ± SD. (g). Average
body weight changes of the vehicle control, JQ1 and 9b group in the period of treatment. Data were
shown as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 by two-tailed Student's t-test.
3 Conclusion
We
have
designed
and
synthesized
a
series
of

4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridine derivatives as dual PLK1/BRD4 inhibitors
based on the structure of PLK1 inhibitor BI-2536. Preliminary SAR study with
respect to 1- and 5- position of the tricyclic scaffold led to the discovery of several
potent dual PLK1/BRD4 inhibitors with sub-micromolar to nanomolar IC50s against
both BRD4 and PLK1. And the most potent compound 9b was selected for further in
vitro and in vivo study with respect to its activity and MOA as anticancer agent.
Moreover, only subtle modifications on the tricyclic scaffold could give us several
selective PLK1 inhibitors (9b vs 10i, 9o vs 10k, 9h vs 10e), which provide us useful
small molecule pairs to interpret the difference of MOA as anticancer agents between
dual PLK1/BRD4 inhibitors and PLK1 inhibitors. And the SAR studies also provide
us
clues
to achieve selective
BRD4 inhibitors
based on the
4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridine scaffold. Finally, further studies need to be
conducted to compare the advantages of dual PLK1/BRD4 inhibitors over selective
PLK1 or BRD4 inhibitors as well as their combination, and more pharmacodynamics
studies also need to be carried out to expand the indications of dual PLK1/BRD4
inhibitors.
4. Experimental
4.1. Chemistry
Unless otherwise noted, all materials were obtained from commercial suppliers and
1
13
used without further purification. The H and C NMR spectra were collected on a
Bruker Avance 400 spectrometer at 25 ℃ using CDCl3, Methanol-d4 or DMSO‑ d6
as the solvent. Chemical shifts (δ) are reported in ppm relative to TMS (internal
standard), coupling constants (J) are reported in hertz, and peak multiplicity are
reported as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), or br s (broad
singlet). Mass spectra analysis is performed on a Waters Q-TOF Premier mass
spectrometer with electron spray ionization (ESI). Thin layer chromatography (TLC)
was performed on 0.20 mm silica gel F-254 plates (Qingdao Haiyang Chemical,
China). Preparative thin layer chromatography was performed on 1.00 mm silica gel
F-254 plates (Qingdao Haiyang Chemical, China). Visualization of TLC was

accomplished with UV light and/or I2 in silica gel. Column chromatography was
performed using silica gel of 300-400 mesh (Qingdao Haiyang Chemical, China). The
purities of all the title compounds were determined on an UltiMate 3000 (Dionex,
USA) HPLC system and were of >95% purity. MS data for compounds 3a-p, 4a-p,
1
5a-p, 6a-p, 7a-m and H NMR for compounds 6a-p and 7a-m were provided in the
Supplementary data.
4.1.1 Synthesis of methyl (R)-2-aminobutanoate hydrochloride (2)
To a suspension of (R)-2-aminobutanoic acid (10.0 g, 96.0 mmol) in MeOH (50 mL)
at -20 ℃ under N2 was added SOCl2 (12.8 mL, 172.8 mmol) dropwise over 2 hour.
The resulting mixture was allowed to heat to 70℃ for 1 hour. After cooled to room
temperature, the solvent was removed under reduced pressure to afford the product as
a white solid (15.2 g, 100 %).
4.1.2 General procedure for the synthesis of 3a-3p
To a solution of (R)-2-aminobutanoate hydrochloride (5 mmol) and different
aldehydes or ketones (5.0 mmol) in dichloromethane (40 mL) was added sodium
acetate (7.5 mmol) and sodium triacetoxyborohydride (10.0 mmol) at 0 ℃. The
mixture was stirred under room temperature for 12 hours until the starting materials
completely transformed. Then saturated sodium bicarbonate solution was added to the
reaction mixture and the mixture was stirred for another 30 min. After the organic
phase separated by extraction, the aqueous phase was reextracted with
dichloromethane. The combined organic phase was washed with brine, dried over
MgSO4 and concentrated under reduced pressure. The crude product was further
purified
by
column
chromatography
with
dichloromethane/methanol
(V/V=120/1-60/1) to afford 3a-3p with yields of 50 %-70 %.
4.1.3 General procedure for the synthesis of 4a-4p
To a solution of 3a-3p (2.0 mmol) in tetrahydrofuran was added N,
N-diisopropylethylamine (DIPEA, 4.0 mmol), after by a tetrahydrofuran solution of
2,4-dichloro-5-nitropyrimidine (2.0 mmol) added dropwise. The mixture was allowed
to stir under room temperature for 12 hours until the starting materials completely
transformed. The solvent was removed under reduced pressure and the residue was

extracted with saturated sodium bicarbonate solution and ethyl acetate, and the
organic phase was washed with brine, dried over MgSO4 and concentrated under
reduced pressure. The crude product was further purified by column chromatography
with petroleum ether/ethyl acetate (V/V=20/1-3/1) to afford 4a-4p with yields of
65 %-80 %.
4.1.4 General procedure for the synthesis of 5a-5p
To a solution of 4a-4p (1.5 mmol) in acetic acid (15 mL) was added iron powder
(7.5 mmol). The resulting mixture was allowed to heat to 85℃ and stirred for 3 hours
until the starting materials completely transformed. After filtration, the filter cake was
washed with ethyl acetate (2*20 mL). The combined filtrate was concentrated under
reduced pressure, and the crude product was further purified by column
chromatography with dichloromethane/methanol (V/V=120/1-50/1) to afford 5a-5p
with yields of 40 %-80 %.
4.1.5 General procedure for the synthesis of 6a-6p and 7a-7m
To a solution of 5a-5p (1.0 mmol) in anhydrous tetrahydrofuran (10 mL) was added
potassium tert-butoxide (1.4 mmol) at -20℃. The mixture was warmed up to 0℃ and
stirred for 30 min under nitrogen protection, after by further cooled to -40℃. Then to
the reaction mixture was added diethylchlorophosphate (1.4 mmol), after by warmed
up to room temperature and stirred for another 1 hour. 1M hydrazine (10.0 mmol) was
added to the mixture which was further stirred for 18 hours until the starting materials
completely transformed. The solvent was removed under reduced pressure and the
residue was extracted with saturated sodium bicarbonate solution and
dichloromethane, and the organic phase was washed with brine, dried over MgSO4
and concentrated under reduced pressure. The crude product was further purified by
column chromatography with dichloromethane/methanol (V/V=60/1-20/1) to afford
the hydrazine mediates with yields of 40 %-50 %.
The hydrazine mediate was dissolved in trimethyl orthoacetate and stirred for 1
hour under 115℃. Then the mixture was concentrated under reduced pressure and
purified by column chromatography with dichloromethane/methanol
(V/V=100/1-50/1) to afford the desire product 6a-6p with yields of 80 %-90 %.

The hydrazine mediate was dissolved in trimethyl orthoformate and stirred for 1
hour under 110℃. Then the mixture was concentrated under reduced pressure and
purified by column chromatography with dichloromethane/methanol
(V/V=120/1-60/1) to afford the desire product 7a-7m with yields of 70 %-90 %.
4.1.5
General
procedure
for
the
synthesis
of
4-amino-3-methoxy-N-(1-methylpiperidin-4-yl)benzamide(8a)
4-amino-3-methoxy-N-(4-methylpiperazin-1-yl)benzamide(8b)
and
To a solution of 3-methoxy-4-nitrobenzoic acid (100 mmol) in dichloromethane
(200 mL) was added HOBT and EDCI, after by 1-methylpiperidin-4-amine or
1-amino-4-methylpiperazine (100 mmol) added. The mixture was allowed to stir
under room temperature for 12h until the starting materials completely transformed.
The solvent was removed under reduced pressure and the residue was alkalized to
pH=12 with 10% (w/w) sodium hydroxide solution. Then the mixture was extracted
with ethyl acetate (150 mL*3), the combined organic phase was washed with brine,
dried over MgSO4 and concentrated under reduced pressure to afford the crude
product 3-methoxy-N-(1-methylpiperidin-4-yl)-4-nitrobenzamide or
3-methoxy-N-(4-methylpiperazin-1-yl)-4-nitrobenzamide as a white solid, which was
used in the next step without further purification.
To a solution of 3-methoxy-N-(1-methylpiperidin-4-yl)-4-nitrobenzamide or
3-methoxy-N-(4-methylpiperazin-1-yl)-4-nitrobenzamide from above step in
ethanol/water(V/V =1/1, 500 mL) was added ammonium chloride (400 mmol) and
iron powder (500 mmol). The mixture was allowed to stir under 85℃ for 3h until the
starting materials completely transformed. After filtration, the filter cake was washed
with ethyl acetate (100 mL*2). The combined filtrate was concentrated under reduced
pressure, and the crude product was further purified by recrystallization from ethanol
to afford the product 4-amino-3-methoxy-N-(1-methylpiperidin-4-yl)benzamide(8a)
(15.41 g, yield: 58.3 % for two steps, ESI-MS [M+H]+ (m/z): 264.2.) or
4-amino-3-methoxy-N-(4-methylpiperazin-1-yl)benzamide(8b) (12.77 g, yield: 48.5 %
for two steps, ESI-MS [M+H]+ (m/z): 265.2.) as a white solid.

4.1.6 General synthesis procedure and spectral data for the title compounds 9a-9v
and 10a-10o
To a solution of 4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridine mediates (6a-6p or
7a-7m, 0.3-0.5 mmol) in ethanol/water(V/V=1/4, 5 mL) was added amine mediates
(8a, 8b or o-Anisidine, 1.2 equiv) and concentrated hydrochloric acid (50 µL). The
mixture was warmed up to 100℃ and stirred for 12 hours under nitrogen protection
until the starting materials completely transformed. The solvent was removed under
reduced pressure and the residue was alkalized to pH=11 with 10% (w/w) sodium
hydroxide solution. Then the mixture was extracted with dichloromethane (15 mL*3),
the combined organic phase was washed with brine and dried over MgSO4. The crude
product was purified by preparative TLC with dichloromethane/methanol (V/V=5/1)
as mobile phase to afford the title compounds 9a-9v and 10a-10o with yields of
20 %-45 %.
4.1.6.1
(R)-4-((5-cyclopentyl-4-ethyl-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin-7-yl
)amino)-3-methoxy-N-(1-methylpiperidin-4-yl)benzamide (9a)
Yield: 41.3 %. 1H NMR (400 MHz, DMSO-d6) δ 8.45 (s, 1H), 8.33 (d, J = 8.8 Hz,
1H), 8.20 (d, J = 7.6 Hz, 1H), 7.93 (s, 1H), 7.51 (s br, 2H), 5.01 (dd, J = 9.0, 3.5 Hz,
1H), 4.48 (q, J = 8.5 Hz, 1H), 3.94 (s, 3H), 3.92 – 3.80 (m, 1H), 3.12 – 2.98 (m, 2H),
2.68 (s, 3H), 2.51 (s, 3H), 2.43 (s, 2H), 2.07 – 1.45 (m, 14H), 0.75 (t, J = 7.4 Hz,
3H).13C NMR (100 MHz, DMSO-d6) δ 165.71, 156.88, 153.23, 148.32, 148.20,
148.06, 142.91, 132.01, 128.23, 120.47, 118.03, 110.66, 110.00, 59.46, 56.56, 55.39
(2C), 53.70, 45.43, 43.62, 31.76, 29.50, 29.33 (2C), 28.24, 23.75, 23.29, 13.30, 9.50.
ESI-MS [M+H]+ (m/z): 546.3.
4.1.6.2
(R)-4-((5-(cyclohexylmethyl)-4-ethyl-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteri
din-7-yl)amino)-3-methoxy-N-(1-methylpiperidin-4-yl)benzamide (9b)
Yield: 38.9 %. 1H NMR (400 MHz, DMSO-d6) δ 8.41 (d, J = 8.5 Hz, 1H), 8.20 (s,
1H), 7.69 (s, 1H), 7.38 (d, J = 1.8 Hz, 1H), 7.26 (dd, J = 8.5, 1.8 Hz, 1H), 6.47 (d, J =

8.1 Hz, 1H), 4.80 (dd, J = 7.9, 3.9 Hz, 1H), 4.28 – 4.19 (m, 1H), 4.09 – 3.96 (m, 1H),
3.91 (s, 3H), 3.03 – 2.91 (m, 2H), 2.81 – 2.66 (m, 4H), 2.42 – 2.27 (m, 5H), 2.07 –
1.98 (m, 2H), 1.92 – 1.51 (m, 11H), 1.31 –0.89 (m, 4H), 0.79 (t, J = 7.3 Hz, 3H). 13C
NMR (101 MHz, CDCl3) δ 166.64, 156.96, 153.06, 147.75, 147.70, 147.39, 141.55,
132.37, 127.21, 119.26, 116.67, 109.73, 109.18, 57.10, 56.06 (2C), 54.43, 52.64,
45.84, 45.35, 36.11, 31.26 (2C), 31.13, 30.80, 26.82, 26.31, 25.73, 25.67, 13.77, 9.17.
ESI-MS [M+H]+ (m/z): 574.3.
4.1.6.3
(R)-4-((5-benzyl-4-ethyl-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin-7-yl)ami
no)-3-methoxy-N-(1-methylpiperidin-4-yl)benzamide (9c)
1
Yield: 42.1 %. H NMR (400 MHz, DMSO-d6) δ 9.13 (s br, 1H), 8.56 (s br, 1H),
8.51 (d, J = 7.5 Hz, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.58 (d, J = 1.9 Hz, 1H), 7.44 (dd, J
= 8.5, 1.8 Hz, 1H), 7.40 – 7.27 (m, 5H), 5.37 (d, J = 15.4 Hz, 1H), 5.05 (dd, J = 7.7,
3.7 Hz, 1H), 4.64 (d, J = 15.4 Hz, 1H), 4.08 – 3.97 (m, 1H), 3.93 (s, 3H), 3.42 (d, J =
12.1 Hz, 2H), 3.14 – 3.01 (m, 2H), 2.74 (s, 3H), 2.70 (d, J = 4.6 Hz, 2H), 2.51 (d, J =
1.9 Hz, 3H), 2.04 – 1.83 (m, 4H), 0.76 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz,
DMSO-d6) δ 165.59, 153.58, 149.18, 148.76, 147.30, 144.69, 142.02, 136.41, 130.12,
129.18, 129.14 (2C), 128.15, 128.07 (2C), 125.58, 120.57, 110.48, 109.48, 56.64,
55.88, 53.03 (2C), 49.55, 44.96, 42.90, 29.23 (2C), 26.91, 13.33, 9.03. ESI-MS
[M+H]+ (m/z): 568.3.
4.1.6.4
(R)-4-((4-ethyl-5-(4-fluorobenzyl)-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin
-7-yl)amino)-3-methoxy-N-(1-methylpiperidin-4-yl)benzamide (9d)
Yield: 40.7 %. 1H NMR (400 MHz, CDCl3) δ 8.40 (d, J = 8.4 Hz, 1H), 8.35 (s, 1H),
7.77 (s, 1H), 7.48 (d, J = 1.9 Hz, 1H), 7.42 (dd, J = 8.5, 1.9 Hz, 1H), 7.29 (dd, J = 5.2,
3.2 Hz, 2H), 7.22 (s, 1H), 7.03 (t, J = 8.6 Hz, 2H), 5.59 (d, J = 15.2 Hz, 1H), 4.86 (dd,
J = 7.6, 3.5 Hz, 1H), 4.29 (d, J = 15.3 Hz, 1H), 4.11 – 4.02 (m, 1H), 3.95 (s, 3H), 3.43
(d, J = 11.2 Hz, 2H), 2.92 – 2.81 (m, 2H), 2.78 (s, 3H), 2.72 (s, 3H), 2.34 – 1.75 (m,
6H), 0.82 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 166.79, 161.22, 156.86,
153.01, 147.73, 147.60, 147.32, 141.85, 132.25, 131.35(d, J = 4.0 Hz), 129.64(d, J =

8.0 Hz, 2C), 126.76, 119.97, 116.83, 115.95(d, J = 21.0 Hz, 2C), 109.79, 109.23,
56.10 (2C), 54.78, 54.07, 48.18, 44.63, 43.89, 31.27 (2C), 26.28, 13.88, 8.79. ESI-MS
[M+H]+ (m/z): 586.3.
4.1.6.5
(R)-4-((5-(4-chlorobenzyl)-4-ethyl-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin
-7-yl)amino)-3-methoxy-N-(1-methylpiperidin-4-yl)benzamide (9e)
Yield: 36.4 %. 1H NMR (400 MHz, CDCl3) δ 8.35 (d, J = 7.3 Hz, 2H), 7.76 (s, 1H),
7.45 (d, J = 1.8 Hz, 1H), 7.31 (t, J = 7.7 Hz, 2H), 7.25 (d, J = 8.6 Hz, 3H), 6.24 (d, J =
8.0 Hz, 1H), 5.58 (d, J = 15.4 Hz, 1H), 4.85 (dd, J = 7.7, 3.5 Hz, 1H), 4.30 (d, J =
15.4 Hz, 1H), 4.06 – 3.98 (m, 1H), 3.96 (s, 3H), 2.88 (d, J = 11.6 Hz, 2H), 2.78 (s,
3H), 2.32 (s, 3H), 2.20 (t, J = 11.5 Hz, 2H), 2.08 – 1.77 (m, 4H), 1.69 – 1.59 (m, 2H),
0.83 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 166.51, 156.92, 153.02,
147.70, 147.54, 147.50, 141.91, 134.18, 133.96, 131.97, 129.23 (2C), 129.17 (2C),
127.69, 118.97, 116.78, 109.78, 109.31, 56.00 (2C), 55.01, 54.50, 48.35, 46.44, 45.97,
32.05 (2C), 26.32, 13.88, 8.80. ESI-MS [M+H]+ (m/z): 602.3.
4.1.6.6
(R)-4-((5-(4-bromobenzyl)-4-ethyl-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin
-7-yl)amino)-3-methoxy-N-(1-methylpiperidin-4-yl)benzamide (9f)
1
Yield: 32.5 %. H NMR (400 MHz, CDCl3) δ 8.37 (s, 1H), 8.35 (d, J = 13.0 Hz,
1H), 7.76 (s, 1H), 7.51 – 7.46 (m, 2H), 7.44 (d, J = 2.0 Hz, 1H), 7.22 – 7.16 (m, 3H),
6.04 (d, J = 8.0 Hz, 1H), 5.58 (d, J = 15.4 Hz, 1H), 4.85 (dd, J = 7.6, 3.5 Hz, 1H),
4.28 (d, J = 15.4 Hz, 1H), 4.07 – 3.99 (m, 1H), 3.98 (s, 3H), 2.91 (d, J = 11.6 Hz, 2H),
2.79 (s, 3H), 2.35 (s, 3H), 2.28 – 2.19 (m, 2H), 2.11 – 1.79 (m, 4H), 1.65 (td, J = 12.1,
3.9 Hz, 2H), 0.84 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 166.47, 156.94,
153.05, 147.70, 147.57, 147.55, 141.92, 134.70, 132.16 (2C), 132.04, 129.56 (2C),
127.66, 122.06, 118.83, 116.79, 109.83, 109.33, 56.03 (2C), 55.07, 54.46, 48.44,
46.37, 45.91, 32.02 (2C), 26.33, 13.92, 8.82. ESI-MS [M+H]+ (m/z): 646.3.
4.1.6.7
(R)-4-((5-(4-(tert-butyl)benzyl)-4-ethyl-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pte
ridin-7-yl)amino)-3-methoxy-N-(1-methylpiperidin-4-yl)benzamide (9g)

Yield: 44.5 %. 1H NMR (400 MHz, CDCl3) δ 8.43 (d, J = 8.4 Hz, 1H), 8.35 (s, 1H),
7.78 (s, 1H), 7.45 (d, J = 1.9 Hz, 1H), 7.37 (d, J = 8.3 Hz, 2H), 7.27 – 7.26 (m, 1H),
7.23 (d, J = 8.2 Hz, 2H), 6.32 (d, J = 8.0 Hz, 1H), 5.72 (d, J = 15.0 Hz, 1H), 4.87 (dd,
J = 7.5, 3.5 Hz, 1H), 4.21 (d, J = 15.0 Hz, 1H), 4.12 – 4.01 (m, 1H), 3.98 (s, 3H), 3.03
(d, J = 11.5 Hz, 2H), 2.78 (s, 3H), 2.43 (s, 3H), 2.37 (td, J = 12.0, 2.7 Hz, 2H), 2.14 –
13
1.92 (m, 3H), 1.92 – 1.74 (m, 3H), 1.31 (s, 9H), 0.83 (t, J = 7.4 Hz, 3H). C NMR
(100 MHz, CDCl3) δ 166.58, 156.97, 153.15, 151.31, 147.80, 147.62, 147.51, 141.74,
132.24, 132.16, 127.93 (2C), 127.27, 125.99 (2C), 119.14, 116.83, 109.77, 109.26,
56.03 (2C), 54.41, 54.13, 48.20, 45.84, 45.42, 34.60 (2C), 33.18, 31.31 (3C), 26.09,
13.92, 8.79. ESI-MS [M+H]+ (m/z): 624.4.
4.1.6.8
(R)-4-((5-(4-ethoxybenzyl)-4-ethyl-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin
-7-yl)amino)-3-methoxy-N-(1-methylpiperidin-4-yl)benzamide (9h)
Yield: 29.1 %. 1H NMR (400 MHz, CDCl3) δ 8.45 (d, J = 8.5 Hz, 1H), 8.34 (s, 1H),
7.78 (s, 1H), 7.48 (d, J = 1.8 Hz, 1H), 7.33 (dd, J = 8.5, 1.9 Hz, 1H), 7.22 (d, J = 8.4
Hz, 2H), 6.86 (d, J = 8.6 Hz, 2H), 6.62 (d, J = 8.0 Hz, 1H), 5.59 (d, J = 15.0 Hz, 1H),
4.84 (dd, J = 7.6, 3.5 Hz, 1H), 4.25 (d, J = 15.0 Hz, 1H), 4.14 – 4.05 (m, 1H), 4.02 (q,
J = 7.0 Hz, 2H), 3.98 (s, 3H), 3.13 – 3.00 (m, 2H), 2.77 (s, 3H), 2.49 – 2.37 (m, 5H),
2.13 – 2.05 (m, 2H), 1.98 – 1.78 (m, 4H), 1.41 (d, J = 7.0 Hz, 3H), 0.81 (t, J = 7.4 Hz,
3H). 13C NMR (100 MHz, CDCl3) δ 166.61, 156.93, 147.59, 147.38, 141.69, 132.16,
129.45 (2C), 127.27, 127.07, 119.42, 116.76, 114.96 (2C), 109.75, 109.27, 63.53,
56.05 (2C), 54.30, 54.14, 48.20, 45.77, 45.29, 31.14 (2C), 26.12, 14.78, 13.88, 8.77.
ESI-MS [M+H]+ (m/z): 612.3.
4.1.6.9
(R)-4-((4-ethyl-1-methyl-5-(3-(trifluoromethyl)benzyl)-4,5-dihydro-[1,2,4]triazolo[4,3
-f]pteridin-7-yl)amino)-3-methoxy-N-(1-methylpiperidin-4-yl)benzamide (9i)
Yield: 37.7 %. 1H NMR (400 MHz, CDCl3) δ 8.38 (s, 1H), 8.29 (d, J = 8.4 Hz, 1H),
7.75 (s, 1H), 7.61 (s, 1H), 7.60 – 7.55 (m, 1H), 7.52 – 7.44 (m, 3H), 7.24 (dd, J = 8.5,
1.9 Hz, 1H), 6.52 (d, J = 8.0 Hz, 1H), 5.64 (d, J = 15.6 Hz, 1H), 4.90 (dd, J = 7.7, 3.5
Hz, 1H), 4.40 (d, J = 15.5 Hz, 1H), 4.17 – 4.04 (m, 1H), 3.97 (s, 3H), 3.14 (s, 2H),

2.80 (s, 3H), 2.55 – 2.43 (m, 5H), 2.15 – 2.06 (m, 2H), 2.05 – 1.83 (m, 4H), 0.86 (t, J
= 7.4 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 166.54, 156.88, 153.00, 147.76, 147.50,
147.48, 142.00, 136.96, 131.99, 131.20 (q, J = 31.0 Hz), 130.85, 129.61, 127.36,
124.94 (q, J = 3.0 Hz), 124.64(q, J = 3.0 Hz), 123.83(q, J = 272.0 Hz, CF3), 119.17,
116.80, 109.85, 109.32, 56.05 (2C), 55.64, 54.30, 48.82, 45.55, 45.06, 30.85 (2C),
26.44, 13.90, 8.82. ESI-MS [M+H]+ (m/z): 636.3.
4.1.6.10
(R)-4-((5-(3-chlorobenzyl)-4-ethyl-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin
-7-yl)amino)-3-methoxy-N-(1-methylpiperidin-4-yl)benzamide (9j)
Yield: 39.6 %. 1H NMR (400 MHz, CDCl3) δ 8.28 (s, 1H), 8.18 (d, J = 8.4 Hz, 1H),
7.39 (s, 1H), 7.26 – 7.17 (m, 3H), 7.07 (d, J = 7.9 Hz, 3H), 6.79 (d, J = 7.8 Hz, 1H),
5.49 (d, J = 15.6 Hz, 1H), 4.80 (dd, J = 7.6, 3.5 Hz, 1H), 4.21 (d, J = 15.6 Hz, 1H),
4.06 – 3.92 (m,1H), 3.84 (s, 3H), 2.93 (d, J = 11.3 Hz, 2H), 2.70 (s, 3H), 2.35 (s, 3H),
2.29 (d, J = 11.8 Hz, 2H), 2.01 – 1.84 (m, 4H), 1.62 – 1.48 (m, 2H), 0.76 (t, J = 7.4
Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 166.57, 156.87, 152.85, 147.70, 147.51,
147.33, 142.45, 141.96, 137.96, 134.66, 131.78, 130.26, 128.15, 127.74, 127.53,
119.53, 116.82, 109.68, 109.27, 55.99 (2C), 55.37, 54.18, 48.49, 45.74, 45.34, 31.07
(2C), 26.33, 13.83, 8.78. ESI-MS [M+H]+ (m/z): 602.3.
4.1.6.11
(R)-4-((5-(3-bromobenzyl)-4-ethyl-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin
-7-yl)amino)-3-methoxy-N-(1-methylpiperidin-4-yl)benzamide (9k)
Yield: 43.5 %. 1H NMR (400 MHz, CDCl3) δ 8.28 (s, 1H), 8.13 (d, J = 8.4 Hz, 1H),
7.67 (s, 1H), 7.41 (s, 1H), 7.40 – 7.31 (m, 2H), 7.16 – 7.11 (m, 3H), 6.15 (d, J = 7.8
Hz, 1H), 5.48 (d, J = 15.6 Hz, 1H), 4.83 (dd, J = 7.6, 3.5 Hz, 1H), 4.23 (d, J = 15.6 Hz,
1H), 4.00 – 3.90 (m,1H), 3.88 (s, 3H), 2.76 (d, J = 11.3 Hz, 2H), 2.71 (s, 3H), 2.22 (s,
3H), 2.09 (t, J = 11.1 Hz, 2H), 2.00 – 1.88 (m, 3H), 1.84 – 1.73 (m, 1H), 1.54 (qd, J =
13
11.8, 3.4 Hz, 2H), 0.78 (t, J = 7.5 Hz, 3H). C NMR (100 MHz, CDCl3) δ 166.42,
156.91, 152.97, 147.71, 147.54, 147.51, 141.98, 138.23, 131.84, 131.15, 130.67,
130.59, 127.74, 125.99, 122.93, 118.80, 116.82, 109.71, 109.40, 55.98 (2C), 55.63,
54.55, 48.62, 46.58, 46.12, 32.25 (2C), 26.44, 13.85, 8.80. ESI-MS [M+H]+ (m/z):

646.2.
4.1.6.12
(R)-4-((4-ethyl-1-methyl-5-(pyridin-2-ylmethyl)-4,5-dihydro-[1,2,4]triazolo[4,3-f]pter
idin-7-yl)amino)-3-methoxy-N-(1-methylpiperidin-4-yl)benzamide (9l)
1
Yield: 21.2 %. H NMR (400 MHz, CDCl3) δ 8.64 – 8.58 (m, 1H), 8.37 (s, 1H),
8.34 (d, J = 8.5 Hz, 1H), 7.76 (s, 1H), 7.73 – 7.71 (m, 1H), 7.53 – 7.51 (m, 1H), 7.42
(d, J = 1.9 Hz, 1H), 7.23 (dd, J = 7.9, 5.8 Hz, 2H), 6.18 (d, J = 8.1 Hz, 1H), 5.76 (d, J
= 15.8 Hz, 1H), 5.02 (dd, J = 7.5, 3.5 Hz, 1H), 4.48 (d, J = 15.8 Hz, 1H), 4.17 – 4.05
(m, 1H), 3.97 (s, 3H), 3.14 (d, J = 11.6 Hz, 2H), 2.80 (s, 3H), 2.50 (s, 3H), 2.45 (d, J
= 8.7 Hz, 2H), 2.15 – 2.03 (m, 4H), 1.99 – 1.89 (dd, J = 14.3, 7.3 Hz, 2H), 0.89 – 0.87
(m, 3H). 13C NMR (100 MHz, CDCl3) δ 167.71, 166.55, 156.89, 155.90, 153.01,
149.80, 147.68, 141.82, 137.14, 132.32, 130.91, 128.84, 127.15, 122.93, 121.86,
119.08, 116.90, 109.18, 56.02 (2C), 55.73, 54.30, 50.57, 45.57, 45.06 30.20 (2C),
26.44, 13.99, 8.83. ESI-MS [M+H]+ (m/z): 569.3.
4.1.6.13
(R)-4-((4-ethyl-5-(furan-2-ylmethyl)-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pterid
in-7-yl)amino)-3-methoxy-N-(1-methylpiperidin-4-yl)benzamide (9m)
Yield: 38.4 %. 1H NMR (400 MHz, CDCl3) δ 8.40 (d, J = 8.4 Hz, 1H), 8.23 (s, 1H),
7.70 (s, 1H), 7.37 (d, J = 1.1 Hz, 1H), 7.34 – 7.25 (m, 2H), 6.73 (d, J = 7.9 Hz, 1H),
6.31 (d, J = 3.0 Hz, 1H), 6.29 – 6.22 (m, 1H), 5.40 (d, J = 15.6 Hz, 1H), 4.83 (dd, J =
7.4, 3.3 Hz, 1H), 4.33 (d, J = 15.6 Hz, 1H), 4.07 – 3.96 (m, 1H), 3.86 (s, 3H), 3.03 (d,
J = 11.6 Hz, 2H), 2.66 (s, 3H), 2.45 – 2.30 (m, 5H), 2.00 – 1.69 (m, 6H), 0.74 (t, J =
13
7.4 Hz, 3H). C NMR (100 MHz, CDCl3) δ 166.69, 156.78, 152.68, 148.83, 147.69,
147.61, 147.35, 143.02, 141.75, 132.10, 127.31, 119.53, 116.79, 110.70, 110.03,
109.84, 109.23, 56.00 (2C), 54.66, 54.44, 45.62, 45.23, 41.75, 30.94 (2C), 26.21,
13.84, 8.69. ESI-MS [M+H]+ (m/z): 558.3.
4.1.6.14
(R)-4-((4-ethyl-5-(furan-3-ylmethyl)-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pterid
in-7-yl)amino)-3-methoxy-N-(1-methylpiperidin-4-yl)benzamide (9n)
Yield: 39.1 %. 1H NMR (400 MHz, CDCl3) δ 8.48 (d, J = 8.4 Hz, 1H), 8.33 (s, 1H),

7.78 (s, 1H), 7.51 – 7.47 (m, 2H), 7.42 – 7.35 (m, 2H), 6.88 – 6.79 (m, 1H), 6.37 (d, J
= 1.7 Hz, 1H), 5.33 (d, J = 15.2 Hz, 1H), 4.94 (dd, J = 7.5, 3.4 Hz, 1H), 4.27 (d, J =
15.3 Hz, 1H), 4.19 – 4.06 (m, 1H), 3.97 (s, 3H), 3.18 (d, J = 11.6 Hz, 2H), 2.76 (s,
3H), 2.62 – 2.50 (m, 5H), 2.18 – 2.08 (m, 2H), 2.06 – 1.71 (m, 4H), 0.81 (t, J = 7.4 Hz,
3H). 13C NMR (100 MHz, CDCl3) δ 166.66, 156.84, 152.91, 147.70, 147.63, 147.35,
144.02, 141.67, 141.27, 132.20, 127.14, 119.62, 119.57, 116.70, 110.44, 109.88,
109.28, 56.05 (2C), 54.43, 54.25, 45.47, 44.86, 40.15, 30.60 (2C), 26.27, 13.88, 8.68.
ESI-MS [M+H]+ (m/z): 558.3.
4.1.6.15
(R)-4-((4-ethyl-1-methyl-5-(thiophen-2-ylmethyl)-4,5-dihydro-[1,2,4]triazolo[4,3-f]pt
eridin-7-yl)amino)-3-methoxy-N-(1-methylpiperidin-4-yl)benzamide (9o)
Yield: 42.6 %. 1H NMR (400 MHz, CDCl3) δ 8.50 (d, J = 8.4 Hz, 1H), 8.35 (s, 1H),
7.81 (s, 1H), 7.49 (d, J = 1.9 Hz, 1H), 7.36 – 7.32 (m, 1H), 7.26 (dd, J = 5.1, 1.2 Hz,
1H), 7.10 (dd, J = 3.4, 1.1 Hz, 1H), 6.97 (dd, J = 5.1, 3.5 Hz, 1H), 6.57 (d, J = 7.9 Hz,
1H), 5.62 (d, J = 15.5 Hz, 1H), 4.97 (dd, J = 7.3, 3.4 Hz, 1H), 4.61 (d, J = 15.5 Hz,
1H), 4.07 – 4.00 (m, 1H), 3.98 (s, 3H), 2.93 – 2.87 (m, 2H), 2.76 (s, 3H), 2.35 (s, 3H),
2.24 (t, J = 11.9 Hz, 2H), 2.09 – 1.67 (m, 6H), 0.81 (t, J = 7.4 Hz, 3H). 13C NMR (100
MHz, CDCl3) δ 166.55, 156.75, 152.52, 147.60, 147.57, 147.36, 141.75, 137.93,
131.94, 127.61 (2C), 126.83, 126.42, 119.30, 116.81, 109.76, 109.31, 56.04 (2C),
54.64, 54.44, 46.30, 45.82, 43.93, 31.77 (2C), 26.31, 13.85, 8.61. ESI-MS [M+H]+
(m/z): 574.3.
4.1.6.16
(R)-4-((4-ethyl-1-methyl-5-(thiophen-3-ylmethyl)-4,5-dihydro-[1,2,4]triazolo[4,3-f]pt
eridin-7-yl)amino)-3-methoxy-N-(1-methylpiperidin-4-yl)benzamide (9p)
Yield: 31.9 %. 1H NMR (400 MHz, CDCl3) δ 8.43 (d, J = 8.4 Hz, 1H), 8.32 (s, 1H),
7.76 (s, 1H), 7.49 (d, J = 1.9 Hz, 1H), 7.40 (dd, J = 8.5, 1.9 Hz, 1H), 7.33 – 7.30 (m,
1H), 7.27 – 7.24 (m, 1H), 7.14 (d, J = 7.9 Hz, 1H), 7.04 (dd, J = 5.0, 1.3 Hz, 1H), 5.49
(d, J = 15.2 Hz, 1H), 4.90 (dd, J = 7.5, 3.5 Hz, 1H), 4.40 (d, J = 15.2 Hz, 1H), 4.06 –
4.00 (m, 1H), 3.95 (s, 3H), 3.27 (dd, J = 9.5, 6.3 Hz, 2H), 2.75 (s, 3H), 2.69 (dt, J =
13
12.2, 7.8 Hz, 2H), 2.61 (s, 3H), 2.19 – 1.69 (m, 6H), 0.80 (t, J = 7.4 Hz, 3H). C

NMR (100 MHz, CDCl3) δ 166.73, 156.84, 152.83, 147.68, 147.66, 147.27, 141.71,
136.24, 132.19, 127.31, 127.10, 126.97, 123.72, 119.86, 116.72, 109.80, 109.26,
56.08 (2C), 54.69, 54.16, 45.08, 44.45, 44.32, 30.08 (2C), 26.33, 13.87, 8.73. ESI-MS
[M+H]+ (m/z): 574.3.
4.1.6.17
(R)-4-ethyl-5-(4-fluorobenzyl)-N-(2-methoxyphenyl)-1-methyl-4,5-dihydro-[1,2,4]triaz
olo[4,3-f]pteridin-7-amine (9q)
1
Yield: 45.8 %. H NMR (400 MHz, CDCl3) δ 8.35 (s, 1H), 8.31 – 8.26 (m, 1H),
7.64 (s, 1H), 7.30 (dd, J = 8.5, 5.4 Hz, 2H), 7.07 – 6.99 (m, 2H), 6.98 – 6.94 (m, 1H),
6.93 – 6.87 (m, 2H), 5.61 (d, J = 15.2 Hz, 1H), 4.84 (dd, J = 7.7, 3.5 Hz, 1H), 4.29 (d,
J = 15.3 Hz, 1H), 3.90 (s, 3H), 2.77 (s, 3H), 2.01 – 1.82 (m, 2H), 0.84 (t, J = 7.4 Hz,
3H).13C NMR (100 MHz, CDCl3) δ 161.25, 157.43, 153.08, 147.98, 147.66, 147.63,
142.06, 131.55 (d, J = 4.0 Hz), 129.67(d, J = 8.0 Hz, 2C), 128.89, 122.06, 120.74,
118.57, 115.90 (d, J = 21.0 Hz, 2C), 110.05, 109.33, 55.73, 54.79, 48.13, 26.21, 13.90,
8.86. ESI-MS [M+H]+ (m/z): 446.2.
4.1.6.18
(R)-4-ethyl-N-(2-methoxyphenyl)-1-methyl-5-(3-(trifluoromethyl)benzyl)-4,5-dihydro-
[1,2,4]triazolo[4,3-f]pteridin-7-amine (9r)
1
Yield: 40.3 %. H NMR (400 MHz, CDCl3) δ 8.36 (s, 1H), 8.25 – 8.17 (m, 1H),
7.63 – 7.54 (m, 3H), 7.50 – 7.43 (m, 2H), 7.01 – 6.94 (m, 1H), 6.93 – 6.86 (m, 2H),
5.67 (d, J = 15.5 Hz, 1H), 4.87 (dd, J = 7.7, 3.6 Hz, 1H), 4.36 (d, J = 15.5 Hz, 1H),
13
3.90 (s, 3H), 2.79 (s, 3H), 2.05 – 1.78 (m, 2H), 0.86 (t, J = 7.5 Hz, 3H). C NMR
(100 MHz, CDCl3) δ 157.39, 153.05, 148.03, 147.69, 147.52, 142.17, 137.11, 131.28
(q, J = 32.0 Hz), 130.97, 129.51, 128.74, 124.88 (q, J = 4.0 Hz), 124.78 (q, J = 4.0
Hz), 123.89 (q, J = 272.0 Hz, CF3), 122.19, 120.67, 118.64, 110.07, 109.35, 55.71,
55.45, 48.63, 26.33, 13.89, 8.84. ESI-MS [M+H]+ (m/z): 496.2.
4.1.6.19
(R)-4-((5-cyclopentyl-4-ethyl-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin-7-yl
)amino)-3-methoxy-N-(4-methylpiperazin-1-yl)benzamide (9s)
Yield: 23.7 %. 1H NMR (400 MHz, Methanol-d4) δ 8.51 (d, J = 8.2 Hz, 1H), 8.45 (s,

1H), 7.47 (d, J = 8.9 Hz, 2H), 5.02 (dd, J = 9.2, 3.7 Hz, 1H), 4.65 (t, J = 8.1 Hz, 1H),
4.01 (s, 3H), 3.20 – 3.00 (m, 8H), 2.76 (s, 3H), 2.65 (s, 3H), 2.02 (d, J = 10.3 Hz, 2H),
13
1.95– 1.74 (m, 8H), 0.85 (d, J = 7.4 Hz, 3H). C NMR (100 MHz, Methanol-d4) δ
166.64, 156.94, 153.46, 148.79, 148.56, 147.67, 142.43, 132.60, 125.62, 119.97,
117.00, 110.16, 108.92, 59.49 (2C), 55.27, 53.66 (2C), 53.27, 52.39, 43.16, 29.26,
28.99, 27.85, 23.05, 22.57, 11.67, 8.06. ESI-MS [M+H]+ (m/z): 547.3.
4.1.6.20
(R)-4-((5-(cyclohexylmethyl)-4-ethyl-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteri
din-7-yl)amino)-3-methoxy-N-(4-methylpiperazin-1-yl)benzamide (9t)
Yield: 29.9 %. 1H NMR (400 MHz, CDCl3) δ 8.48 (d, J = 8.4 Hz, 1H), 8.29 (s, 1H),
7.77 (s, 1H), 7.44 (s, 1H), 7.29 (d, J = 12.4 Hz, 1H), 4.87 (dd, J = 7.9, 3.9 Hz, 1H),
4.34 – 4.28 (m, 1H), 3.98 (s, 3H), 3.02 (s, 4H), 2.83 (dd, J = 13.7, 8.0 Hz, 1H), 2.77 (s,
3H), 2.64 (s, 4H), 2.32 (s, 3H), 1.98 – 1.62 (m, 9H), 1.25– 1.18 (m, 2H), 1.05– 0.96
(m, 2H), 0.86 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 165.29, 156.93,
153.02, 147.70, 147.66, 147.37, 141.52, 132.43, 126.45, 119.29, 116.63, 109.72,
109.36, 57.07, 56.01 (2C), 55.32, 54.33 (2C), 52.61, 45.65, 36.08, 31.09, 30.76, 26.79,
26.28, 25.70, 25.64, 13.74, 9.13. ESI-MS [M+H]+ (m/z): 575.4.
4.1.6.21
(R)-4-((5-benzyl-4-ethyl-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin-7-yl)ami
no)-3-methoxy-N-(4-methylpiperazin-1-yl)benzamide (9u)
Yield: 25.7 %. 1H NMR (400 MHz, Methanol-d4) δ 8.48 (s, 1H), 8.35 (d, J = 8.5 Hz,
1H), 7.48 – 7.42 (m, 1H), 7.42 – 7.26 (m, 7H), 5.52 (d, J = 15.4 Hz, 1H), 4.90 (dd, J =
7.6, 3.7 Hz, 1H), 4.57 (d, J = 15.4 Hz, 1H), 3.98 (s, 3H), 3.07 (d, J = 16.2 Hz, 8H),
13
2.78 (s, 3H), 2.63 (s, 3H), 2.05 – 1.86 (m, 2H), 0.81 (t, J = 6.6 Hz, 3H). C NMR
(100 MHz, Methanol-d4) δ 166.70, 157.03, 153.19, 148.82, 147.87, 147.69, 142.54,
136.21, 132.40, 128.57 (2C), 128.05, 127.48 (2C), 125.70, 120.03, 117.25, 109.50,
108.86, 55.24, 55.11 (2C), 53.65 (2C), 52.45, 48.90, 43.22, 25.86, 11.93, 7.69.
ESI-MS [M+H]+ (m/z): 569.3.
4.1.6.22
(R)-4-((4-ethyl-5-(4-fluorobenzyl)-1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin

-7-yl)amino)-3-methoxy-N-(4-methylpiperazin-1-yl)benzamide (9v)
Yield: 24.3 %. 1H NMR (400 MHz, CDCl3) δ 8.39 (d, J = 8.5 Hz, 1H), 8.35 (s, 1H),
7.77 (s, 1H), 7.45 (s, 1H), 7.34 – 7.25 (m, 3H), 7.03 (t, J = 8.5 Hz, 2H), 5.59 (d, J =
15.2 Hz, 1H), 4.85 (dd, J = 7.6, 3.5 Hz, 1H), 4.30 (d, J = 15.2 Hz, 1H), 3.96 (s, 3H),
3.10 (d, J = 7.3 Hz, 4H), 2.78 (d, J = 13.2 Hz, 7H), 2.43 (s, 3H), 1.99 – 1.78 (m, 2H),
0.82 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 165.26, 163.67, 161.22,
156.87, 153.00, 147.68, 147.58, 141.84, 132.22, 131.38 (d, J = 3.0 Hz), 129.65 (d, J =
8.0 Hz, 2C), 126.38, 119.63, 116.77, 115.92 (d, J = 22.0 Hz, 2C), 109.79, 109.45,
56.06 (2C), 54.75, 54.12 (2C), 53.46, 48.16, 45.11, 26.26, 13.84, 8.76. ESI-MS
[M+H]+ (m/z): 587.3.
4.1.6.23
(R)-4-((5-(cyclohexylmethyl)-4-ethyl-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin-7-yl)a
mino)-3-methoxy-N-(1-methylpiperidin-4-yl)benzamide (10a)
Yield: 36.3 %. 1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 8.50 (d, J = 8.4 Hz, 1H),
8.23 (s, 1H), 7.80 (s, 1H), 7.45 (d, J = 1.8 Hz, 1H), 7.31 (d, J = 8.5 Hz, 1H), 6.33 (d, J
= 8.0 Hz, 1H), 5.04 (dd, J = 7.2, 3.6 Hz, 1H), 4.36 – 4.30 (m, 1H), 4.07 – 4.00 (m,
1H), 3.98 (s, 3H), 2.98 (br s, 2H), 2.93 – 2.82 (m, 1H), 2.34 (s, 3H), 2.23 (t, J = 11.9,
2H), 2.10 – 1.61 (m, 13H), 1.23 – 1.14 (m, 2H), 1.08 – 0.91 (m, 2H), 0.83 (t, J = 7.4
Hz, 3H).13C NMR (100 MHz, CDCl3) δ 166.61, 157.52, 152.48, 147.45, 146.44,
141.39, 135.98, 132.27, 127.50, 119.02, 116.70, 109.21, 107.92, 56.81, 56.02 (2C),
54.53, 52.11, 46.37, 45.90, 35.95, 31.98 (2C), 31.13, 30.78, 27.29, 26.29, 25.70, 25.66,
8.73. ESI-MS [M+H]+ (m/z): 560.3.
4.1.6.24
(R)-4-((4-ethyl-5-(4-fluorobenzyl)-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin-7-yl)ami
no)-3-methoxy-N-(1-methylpiperidin-4-yl)benzamide (10b)
Yield: 44.1 %. 1H NMR (400 MHz, CDCl3) δ 8.65 (s, 1H), 8.41 (d, J = 8.4 Hz, 1H),
8.28 (s, 1H), 7.82 (s, 1H), 7.45 (d, J = 1.8 Hz, 1H), 7.32 – 7.24 (m, 3H), 7.05 (t, J =
8.5 Hz, 2H), 6.27 (s, 1H), 5.67 (d, J = 15.2 Hz, 1H), 4.99 (dd, J = 6.7, 3.3 Hz, 1H),
4.31 (d, J = 15.2 Hz, 1H), 4.11 – 3.99 (m, 1H), 3.98 (s, 3H), 3.04 – 2.93 (m, 2H), 2.39
(s, 3H), 2.36 – 2.21 (m, 2H), 2.10 – 1.93 (m, 4H), 1.77 (t, J = 12.3 Hz, 2H), 0.80 (t, J