Design, synthesis and biological evaluation of novel 1-phenyl phenanthridin-6(5H)-one derivatives as anti-tumor agents targeting TOPK

Article abstract of DOI:10.1016/j.ejmech.2018.11.007

T–lymphokine-activated killer cell–originated protein kinase (TOPK) is a serine-threonine mitogen-activated protein kinase that is highly expressed in many types of human cancer. Due to its important role in cancer progression, TOPK is becoming an attractive target in chemotherapeutic drug design. In this study, a series of 1-phenyl phenanthridin-6(5H)-one derivatives have been identified as a novel chemical class of TOPK inhibitors. Some of them displayed very potent anti-cancer activity with IC₅₀s less than 100 nM, superior than reference compound OTS964. The most potent compound, 9g suppressed the growth of cancer cells by apoptosis and specifically inhibited the activities of TOPK. Oral administration of 9g effectively suppressed tumor growth with TGI >79.7% in colorectal cancer xenograft models, demonstrating superior efficacy compared to OTS964. Pharmacokinetic studies reveal its good oral bioavailability. Our findings therefore show that 9g is a specific inhibitor of TOPK both in vitro and in vivo that may be further developed as a potential therapeutic agent against colorectal cancer.

Full text of DOI:10.1016/j.ejmech.2018.11.007

Accepted Manuscript
Design, synthesis and biological evaluation of novel 1-phenyl phenanthridin-6(5H)-
one derivatives as anti-tumor agents targeting TOPK
Quan-Fang Hu, Tian-Tao Gao, Yao-Jie Shi, Qian Lei, Zhi-Hao Liu, Qiang Feng, Zhen-
Jia Chen, Luo-Ting Yu
PII:
S0223-5234(18)30961-9
DOI:
https://doi.org/10.1016/j.ejmech.2018.11.007
EJMECH 10864
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 February 2018
Revised Date: 10 October 2018
Accepted Date: 4 November 2018
Please cite this article as: Q.-F. Hu, T.-T. Gao, Y.-J. Shi, Q. Lei, Z.-H. Liu, Q. Feng, Z.-J. Chen, L.-T.
Yu, Design, synthesis and biological evaluation of novel 1-phenyl phenanthridin-6(5H)-one derivatives
as anti-tumor agents targeting TOPK, European Journal of Medicinal Chemistry (2018), doi: https://
doi.org/10.1016/j.ejmech.2018.11.007.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design,
synthesis
and
biological
evaluation
of
novel
1-Phenyl
Phenanthridin-6(5H)-one derivatives as anti-tumor agents targeting TOPK
Quan-Fang Hu a,1, Tian-Tao Gao a,1, Yao-Jie Shi a, Qian Lei a, Zhi-Hao Liu a, Qiang Feng a,
Zhen-Jia Chen b, Luo-Ting Yu a,*
a 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
b Chengdu Chempartner Co., Ltd., 7th Floor, Building B3, Tianfu Life Science Park, No. 88, Keyuan South
Road, Hi-Tech Zone, Chengdu, China
Abstract
T–lymphokine-activated killer cell–originated protein kinase (TOPK) is a serine-threonine mitogen-activated
protein kinase that is highly expressed in many types of human cancer. Due to its important role in cancer
progression, TOPK is becoming an attractive target in chemotherapeutic drug design. In this study, a series of
1-phenyl phenanthridin-6(5H)-one derivatives have been identified as a novel chemical class of TOPK
inhibitors. Some of them displayed very potent anti-cancer activity with IC50s less than 100 nM, superior than
reference compound OTS964. The most potent compound, 9g suppressed the growth of cancer cells by
apoptosis and specifically inhibited the activities of TOPK. Oral administration of 9g effectively suppressed
tumor growth with TGI >79.7% in colorectal cancer xenograft models, demonstrating superior efficacy
compared to OTS964. Pharmacokinetic studies demonstrate its good oral bioavailability. Our findings
therefore show that 9g is a specific inhibitor of TOPK both in vitro and in vivo that may be further developed
as a potential therapeutic agent against colorectal cancer.
Keywords: TOPK inhibitor, 1-Phenyl Phenanthridin-6(5H)-one, Structure activity relationship, Colorectal
cancer, Pharmacokinetics
1. Introduction
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TOPK (T–lymphokine-activated killer cell–originated protein kinase), also known as PBK or
PDZ-binding kinase, is a member of the MAPKK protein family [1,2]. It is a serine-threonine
mitogen-activated protein kinase that is highly expressed in many types of human cancer, such as lung cancer,
breast cancer, colorectal cancer, lymphoma, leukemia, melanoma, cholangiocarcinoma and glioma [3-11].
However it is difficult to be detected in normal tissues except several fetal tissues and germ cells of the testis
[3,11]. TOPK could be a promising molecular target for drug development which is involved in many cellular
functions, including tumor development, cell growth, apoptosis, and inflammation [6,12-15]. Researchers
have found that bidirectional signals transduced by TOPK-ERK (extracellular regulated protein kinase)
interaction increases tumorigenesis of HCT116 colorectal cancer cells [6]. Also, studies have shown that
TOPK could phosphorylate histone H3 (H3) at Ser10 and serves as a molecular marker in breast cancer [3].
Increased levels of PBK/TOPK may contribute to tumor cell development and progression through
suppression of p53 function [13,16]. TOPK was also reported to increase cell migration by modulating a
PI3K/ PTEN/AKT-dependent signaling pathway [17].
Despite so many reports have indicated that TOPK plays crucial roles in tumorigenesis, the
three-dimensional structure of TOPK has not been reported. Only the crystal structure of an inactive dimer
was reported by C. Dong et al., which has two phospho-mimicking mutations T9E and T198E. The structural
data indicated that it exists in a conformational transition between dimers and monomers at different pH
conditions [18]. However the active state of TOPK is a monomer and does not form a dimer. The
unavailability of crystalline structure of TOPK makes the discovery of its inhibitors a little bit slow. There are
just several PBK/TOPK inhibitors reported in pre-clinical research (Fig.1). HI-TOPK-032 [10,19] effectively
suppresses colon cancer and glioma growth in vivo despite its inhibition in vitro is not so significant.
Ginsenoside Rh2 (GRh2) is the only reported natural product targeting TOPK which inhibits colon cancer cell
proliferation in vivo [20]. ADA-07 suppresses solar ultraviolet-induced skin carcinogenesis [21]. Ilaprazole
[22] and Pantoprazole [23] are two proton pump inhibitors that suppress colorectal cancer growth. OTS514
and OTS964 [24-28] have great suppression in lung cancer [24,26], acute myeloid leukemia [27] and ovarian
cancer [28]. Due to the toxicity of OTS514 with mild anemia and relatively severe leukocytopenia, whereas
the increasing of peripheral platelets in a dose-dependent manner, it was developed as liposomal formulation
which limited its application; On the contrary, OTS964 at some extend is better for oral administration,
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despite that hematopoietic toxicity is still a concern [24].
Fig. 1. Reported small molecular TOPK inhibitors
Although there have been significant advances in discovery of TOPK inhibitors as we discussed above, a
systematic structure−activity relationship (SAR) study to investigate TOPK potency has not been reported.
The potent TOPK inhibitor OTS964, which are promising therapeutic agents that may be applied to a wide
range of human malignancies, served as a useful template to explore new TOPK inhibitors.
Taking the preclinical inhibitor OTS964 as lead structures, here we attempted to design and synthesize a
novel series of anti-TOPK compounds and found candidates suitable for oral administration. Based on the
principle of bioisosteres, thiophene core in left side was substituted with phenyl core which proved to be an
effective pharmacophore for inhibitory activity, giving rise to the novel series of 1-phenyl
phenanthridin-6(5H)-one compounds. Preliminary binding study and biological evaluation had proved this
modified structure successfully maintained potent inhibitory activity. More than forty compounds were
prepared and afforded a systematic biological analysis. The preliminary structure-activity relationship (SAR)
was also summarized. And candidate was identified by high in vivo activity against colon cancer, as well as
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good oral bioavailability that may be further developed as potential therapeutic.
2. Results and discussion
2.1. Chemistry
We investigated the following four regions of the chemical series represented by OTS964 (Fig. 2): The
crucial thiophene core (green box), which we attempted to replace with phenyl core; the crucial aminoalkyl
group (light blue box), the alternative substituents of methyl (red box), the substitution on hydroxyl group
(dark blue box).
Fig. 2. Regions of OTS964 investigated for TOPK potency
The general synthesis of key intermediates boronic acids R1A~R3A are depicted in Scheme 1. Different
from the reference procedures [24], we synthesized the aryl boronic acids instead of corresponding pinacol
esters, which were easily purified by crystallization and gave all products as good off-white solids. R1A was
synthesized from the commercial available chiral amine R1-1 by three steps: protecting amino group with Boc,
converting to boronic ester via Suzuki-Miyaura reaction and then hydrolyzing under NaIO4 to yield R1A [29];
R2A was synthesized from R2-1 by bromination with DBDMH (1,3-dibromo-5,5-dimethylhydantoin) in
TFAA [24]; R3A was synthesized from corresponding nitrile R3-1, which was reduced to primary amine with
BH3•THF.
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Scheme 1. Synthesis of aryl boronic acid. i) Boc2O, DCM, rt; ii) 1) TFAA, DCM; 2) MeSO3H, DBDMH; iii) LiOH, H2O;
iv) BH3•THF, THF, rt; v) Bis(pinacolato)diboron, PdCl2(dppf), KOAc, dioxane, 90 °C, 18 h; vi) NaIO4, acetone/H2O,
40 °C.
The syntheses of another series of building blocks, 1-bromo-phenanthridin-6(5H)-ones are shown in
scheme 2. The bromo-benzoic acid was converted to bromo-benzoyl chloride, then reacted with aniline to give
amide, followed by protection amide with Boc [30, 31]. Then the protected amide was cyclized under catalytic
Pd(t-Bu3P)2 to
give
phenanthridin-6(5H)-one
through
C-H
direct
activation
[32-34].
1-bromo-phenanthridin-6(5H)-one was obtained by bromination with NBS in HOAc/DCM. DA6 and DD6
were converted to CA6 and CD6 in DMA by substitution of sodium methoxide. DA6 was converted to MA6
by substitution of aqueous dimethlyamine.
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Scheme 2. Synthesis of aryl bromide. i) 1) aryl acid in SOCl2/cat. DMF, reflux; 2) aryl amine/Et3N, DCM, rt; ii) DMAP,
o
Boc2O, DCM, rt; iii) Pd(t-Bu3P)2, KOAc, DMA, 120~130 C, argon; iv) NBS, HOAc/DCM, rt; v) MeONa, DMA, 50
~60 oC; vi) aq. dimethylamine, DMA, 110 oC.
The syntheses of final TOPK inhibitors are outlined in scheme 3. The above
1-bromo-phenanthridin-6(5H)-one was reacted with aryl boronic acid R1A~R3A under classical Suzuki
coupling condition to give 7. The weak base, NaHCO3 was best for the reaction which yielded lower
de-bromination by-product compared with other stronger bases. Inhibitors 8a~8t were obtained by direct
decarboxylation in HCl/MeOH. 9a~9t were obtained by de-methylation with BBr3 in DCM. They were further
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developed to 10a~10t by reductive amination. Compound 1, OTS514 and OTS964 were obtained by known
procedures [24,25].
Scheme 3. Synthesis of TOPK inhibitors. i) R1A~R3A, Pd(dppf)Cl2, dioxane/H2O, NaHCO3, 85 oC; ii) HCl/MeOH, rt;
iii) BBr3/DCM, rt; iv) paraformaldehyde, NaBH3CN, HOAc, MeOH, rt.
2.2. Structure -activity relationships
We synthesized compound 1 [25] which is an analog of OTS514/OTS964 and all of the prepared
compounds were first screened with a binding affinity assay against TOPK kinase [35,36] (Table 1). Inhibition
of cell proliferation was measured using the MTT assay with A549, HCT116 and HCT-15 cell lines which
observed high expression of TOPK [24]. Compound 1 has comparable binding effect with OTS514 although
less potent in cell-based assay. On the contrary, OTS964 is 30-fold lower than OTS514 in kinase activity but
almost as potent as the later at cell level. When exploring the SARs of OTS514 derivatives, firstly we
investigated the impact of replacing the thiophene rings with phenyl rings, which yielded nine 1-phenyl
phenanthridin-6(5H)-one inhibitors and the results are shown in table 1. SAR analysis revealed that most of
them were potent again TOPK. 9a is 48-fold more potent in the biochemical assay than 8a, and 9b is almost
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10-fold more potent than 8b. This confirms that the phenyl hydroxy function group is very important for
interaction with TOPK, presumably by the formation of hydrogen bonds. To our delight, 9a, 9b and 9c have
potency of less than 30 nM IC50 in the biochemical assay, and the potency of 9b , 9c was well translated into
a benefit of A549, HCT116 and HCT-15 cellular context, although 9a is much less active in cell-based assay.
When those compounds were methylated to give 10a, 10b, 10c, more than 5-fold binding potency reduced,
just similar to OTS964. The best substitution out of six R6 groups turned out to be R22. Encouragingly 9b was
almost 21-fold more potent than OTS514 at kinase level and has comparable cellular potency, which leaded us
to further optimization.
Table 1. Structure−activity relationship of substitutions to phenyl core
IC50 (nM) b
compound
R4
R5
R6
IC50 (nM) Kinase a
A549
HCT116
468 ± 11.03
18.89 ± 4.30
45 ± 1.22
HCT-15
256.9 ± 4.01
187.1 ± 2.31
73.08 ± 0.88
NT
Cpd 1
OTS514
OTS964
8a
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
R21
R22
R32
R21
R21
R31
R22
R22
R32
R23
R23
R33
23.00
10.69
353.7
142.5
2.94
360 ± 2.21
19.52 ± 0.23
20.41 ± 0.34
>1000
H
Me
H
>1000
9a
920 ± 6.52
250.3 ± 9.32
360 ± 2.09
78.7 ± 7.54
263.5 ± 4.90
894.3 ± 10.32
210.9 ± 2.78
>1000
>1000
>1000
10a
H
44.89
5.44
314.1 ± 1.05
357 ± 2.48
114.2 ± 7.56
299.3 ± 0.48
>1000
NT
8b
Me
H
277.7 ± 2.86
365 ± 4.09
599.5 ± 5.39
NT
9b
0.50
10b
8c
H
63.83
NT
Me
H
9c
25.1
231.2 ± 0.79
398.2 ± 6.34
NT
10c
H
143.9
949.9 ± 2.94
a [ATP] = 10 µM; Compounds were tested by 10 dose singlet assay; b Each compound was tested in triplicate assay,
IC50 values are presented as the mean ± SD; NT: not tested.
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Our next effort was to introduce multiple substituents of R1, R4 as well as R6 with the goal to identify
the best combination (Table 2, compounds 9d−10n). Small fluoro atom was substituted as R1 (9d, 9e) but it
was not well tolerated. Interestingly, when R1 was substituted by hydroxyl group, the kinase activity (9f, 9g
and 9h) was reduced with 60~660-fold (compared with 9a, 9b and 9c accordingly), but all the cytotoxic
activities were enhanced. The best cellular inhibition is 9g, its IC50 is as low as 5.4 nM to A549 and 7.8 nM to
HCT116, less than 10 nM with more than 30-fold potency positive shift from kinase level inhibition (IC50 0.33
µM). The high cellular potency encouraged us for its further optimization. When 9f, 9g and 9h were
methylated to their derivatives: 10f, 10g and 10h, the cellular inhibitions were slightly reduced just like our
results above (10a, 10b, 10c). After that, we tested 9i and 9j, chloro substitution compounds at R4. Both
compounds were almost as active as 9a/9b in the biochemical assay with IC50 less than 10 nM, but the
cytotoxic activities were not potent, presumably due to lower PKa value resulted from the chloro atom [37-
39]. Next we investigated the substitution of R4 with chloro atom and R1 with hydroxyl group, which
yielded three compounds 9k , 9l and 9m. However, all compounds just gave moderate enzyme and cytotoxic
activities, indicating the negative effect of combination hydroxyl group and chloro atom. Considering that
both hydroxyl group and halogen group are acidic substituents, we introduced a basic substitution group
dimethyl amine at the R1 position and obtained the compound 9n, 10n. But that substitution abolished the
potency completely. Comparing all those 20 compounds with different R6 substitutents, R22 still turned out to
be the best.
Table 2. Structure−activity relationship of further substituents R1, R4, R6
IC50 (nM) b
compound
R1
R4
R6
IC50 (nM) Kinase a
A549
>1000
HCT116
>1000
HCT-15
>1000
>1000
9d
9e
9f
F
F
Me R21
Me R22
Me R21
61.41
64.46
977.5
110 ± 4.70
438 ± 0.69
OH
82.52 ± 3.88 102.4 ± 1.06 96.69 ± 11.34
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10f
9g
OH
Me R31
4790
335.7
>10000
1505
128.5 ± 1.99 396.5 ± 1.39
5.405 ± 0.66 7.867 ± 5.42
162.4 ± 2.11
31.42 ± 0.46
75.97 ± 3.56
NT
OH
Me R22
Me R32
Me R23
Me R33
10g
9h
OH
113.3 ± 0.21
215 ± 0.56
OH
135.9 ± 2.45 44.73 ± 0.95
405.6 ± 0.39 141.2 ± 6.33
10h
9i
OH
ND
NT
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
R21
R31
R22
R32
R21
R31
R22
R32
R23
R33
2.71
26.13
4.59
65
600 ± 8.34
>1000
>1000
>1000
800.7 ± 7.80
>1000
308.9 ± 5.08
> 1000
NT
10i
9j
H
H
>1000
636 ± 1.26
>1000
10j
9k
H
>1000
OH
642.9
2440
321.3
2663
1266
5238
ND
554.1 ± 3.88 719.9 ± 9.53
515.6 ± 4.25 >1000
10k
9l
OH
NT
OH
OH
NT
102.7 ± 1.22 371.7 ± 3.05
554.1 ± 5.23 534.3 ± 2.60
437.4 ± 5.11 563.7 ± 4.31
10l
9m
10m
9n
NT
OH
NT
OH
NT
897.6 ± 7.60
>1000
>1000
>1000
>1000
Dimethyl amine
Dimethyl amine
Me R22
Me R32
NT
10n
ND
NT
>1000
a [ATP] = 10 µM; Compounds were tested by 10 dose singlet assay; b Each compound was tested in triplicate assay,
IC50 values are presented as the mean ± SD; NT: not tested; ND: not determined.
To explore the effect of alkyl substitution at phenyl core, we incorporated methyl substitution at different
position and the results are shown on table 3. However it seemed that those substitution groups had no
obvious positive effect on potency. When R1 was substituted by methyl, the potency of kinase activity was not
improved but the cytotoxic activity was much better (9r, IC50 against A549 is 25.8 nM, IC50 against HCT116
is 75.8 nM). When R2 was methyl (9o, 10o, 9p, 10p), the compounds turned out to be moderate active in
kinase assay (9o, 9p) but inactive in cell-based assay. When R3 was substituted by methyl, the potency of
kinase activity was improved to nanomole (9s, 9t) and had comparable inhibition with 9b in cell-based assay.
Generally methyl substitution of R1, R2, R3 obtained negligible potency optimization.
Here we summarize the structure−activity relationships of the investigated compounds as inhibitors of
the human TOPK: The crucial thiophene core was successfully substituted with phenyl core, the best
substituent of crucial aminoalkyl group is (R)-1-aminopropan-2-yl (R22), the alkyl substitution of phenyl core
is not necessary, the substitution on hydroxyl group completely abolish the potency.
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Table 3. Structure−activity relationship of methyl substitution at phenyl core
HO
R6
NH
R3
O
R1
R2
IC50 (nM) b
compound
R1
R2
R3
R6
IC50 (nM) Kinase a
A549
HCT116
242.2 ± 2.59
469 ± 4.68
75.85 ± 1.30
419.2 ± 4.77
571 ± 1.25
>1000
HCT-15
505.4 ± 3.08
397.3 ± 5.41
300.6 ± 0.61
337.3 ± 0.48
NT
9q
10q
9r
Me
Me
Me
Me
H
H
H
H
H
R21
R31
R22
R32
R21
R31
R22
R32
R21
R31
R22
R32
721.4
3013
187.3 ± 0.79
469.7 ± 0.94
25.86 ± 4.80
286.7 ± 3.59
>1000
H
H
586.1
>10000
894.3
6415
10r
9o
H
H
Me
Me
Me
Me
H
H
10o
9p
H
H
NT
>1000
H
H
NT
223.7
7055
>1000
962 ± 4.38
830 ± 5.90
384.8 ± 3.42
273.7 ± 0.95
156.1 ± 2.13
385.2 ± 1.49
10p
9s
H
H
NT
>1000
H
Me
Me
Me
Me
1.56
119.7 ± 2.78
316.9 ± 1.65
60.27 ± 3.42
466.2 ± 4.11
216.7 ± 2.06
467.3 ± 0.94
326.8 ± 2.09
501.3 ± 4.32
10s
9t
H
H
20.88
6.44
H
H
10t
H
H
19.06
a [ATP] = 10 µM; Compounds were tested by 10 dose singlet assay; b Each compound was tested in triplicate assay,
IC50 values are presented as the mean ± SD; NT: not tested.
2.3. Western blot analysis and colorectal cancer cell lines growth inhibition of 9g
Considering 9g has higher cell level activity (IC50<50 nM), we selected it for further western blot
analysis. Immunoblot were performed with the lysates of colorectal cancer cell lines SW620, HCT-15,
HCT116 and lung cancer cell line A549 after treated with 9g for 48 h (Fig. 3). The results undoubtedly
showed that TOPK was expressed in all four cell lines and 9g inhibited TOPK activity by directly binding
with TOPK. Treatment with 9g significantly decreased autophosphorylation of TOPK (Thr9), as well as
phosphorylation of histone H3 (Ser10) and ERK1/2 which is consistent with reported western blot results after
knockdown of TOPK [5,6,9]. It indicated that cell growth was significantly decreased in a manner of reducing
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TOPK expression. Next, we found that compound 9g inhibited the proliferation of three colorectal cells in a
time and concentration depended manner (Fig.4).
Fig. 3. 9g inhibited TOPK activity in vitro by directly binding with TOPK. Treatment with 9g (50 and 100 nM, 48 h)
reduced phosphorylation of TOPK together with its substrate, ERK1/2 and histone H3 (Ser10) in SW620, HCT-15,
HCT116 cells.
Fig.4. 9g inhibited the proliferation of SW620, HCT-15, HCT116 cells. Tumor cells were treated with 9g with different
concentrations after treatment for 24 h, 48 h and 72 h. MTT assays were performed to measure the proliferation of the
cells.
2.4. In vivo antitumor efficacy in human xenografts of 9g
HCT116 colon cancer is the most widely discussed xenograft model for TOPK efficacy evaluation in
vivo [19,20,22,23]. To compare the antitumor activity of 9g with reference compound OTS964, HCT116
colon cancer cells were engrafted subcutaneously into nude mice. 9g and OTS964 were orally administered at
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10 or 15 mg/kg once daily over a period of three weeks after the tumor size reached about 100 mm3. The
tumor size and body weight were measured every 3 days, and the percentage of tumor growth inhibition (TGI)
was calculated according to the formula [1 − (T − T0)/(C − C0)] × 100, where T and T0 are the mean tumor
volumes at day 21 and day 0 for the experimental group, and C and C0 are those for the vehicle control group.
Treatment of mice with 10 mg/kg of 9g significantly inhibited HCT116 tumor growth relative to the vehicle
group with TGI of 53.7%, better than OTS964 (TGI 49.4%). To our delight, when treated with 9g at 15 mg/kg,
TGI increased to 75.8%. In addition, mice seemed to tolerate treatment with 9g without adverse toxicity or
significant loss of body weight, similar to the vehicle group (Fig. 5).
Since 9g demonstrated superior efficacy compared to OTS964, we further evaluated its efficacy in
HCT-15 and SW620 colorectal cancer xenograft models. Oral administration of 9g at 10 and 15 mg/kg once
daily for 3 weeks resulted in final TGIs of 42.1% and 78.8% for HCT-15, 72.1% and 79.7% for SW620
respectively (Fig. 5a), with slightly body weight loss (Fig.5d). Those results indicated that the compound was
potent to substantially inhibit tumor growth at tolerated doses in mouse xenograft model, and supported
TOPK inhibition as a therapeutically relevant target.
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Fig. 5. In vivo efficacy of 9g in colorectal cancer xenograft models (n = 6). Nude mice bearing HCT116/HCT-15/SW620
were orally treated with vehicle control or 9g/OTS964 once daily for 3 weeks. (a) Effect of 9g/OTS964 on tumor growth
in mouse xenograft. (b) Tumor weight at day 21. (c) Tumor volume at day 21. (d) Corresponding body weight change.
2.5. Pharmacokinetic study of 9g
To support further pharmacological and toxicological study, a pharmacokinetic study of 9g was also
performed. 9g was administrated to rats via po and iv dose separately. Blood samples were collected at 0,
0.083, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h after dosing. The blood drug level was then determined with
LC-MS/MS. The mean 9g concentration in plasma versus time profile was presented in Fig. 6. The
pharmacokinetic parameters are listed in Table 4. 9g showed moderate systemic plasma clearance (2037.19 ±
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295.19 mL/h/kg), a high volume of distribution (20.25 ± 7.97 L/kg), and a long mean residence time via oral
administration (10.54 ± 0.29 h), suggesting the compound has good drug like characters. This together with a
good oral bioavailability (100.11 ± 24.39%) would predict further drug development of the small molecule as
anti-cancer agents.
Table 4. Pharmacokinetic properties of 9g in ratsa
Dose
Cmax
AUC(0-t)
Cl
Vss
t1/2
(h)
MRT(0-t)
(h)
F
Tmax (h)
(mg/kg)
(ng/mL)
(ng·h/mL)
(mL/h/kg)
(L/kg)
(%)
188.03 ±
45.43
0.08 ±
0.00
563.69 ±
131.54
2037.19 ±
295.19
20.25 ±
7.97
7.02 ±
2.74
3.24 ±
0.14
2 mg iv
250.83 ±
38.26
6.67 ±
1.15
4232.35 ±
1031.31b
10.54 ±
0.29
100.11 ±
24.39
15 mg po
a WinNonlin® software version 7.0 was used to estimate pharmacokinetic parameters; bAUC(0-24h)
.
300
250
200
150
9g PO 15 mg/kg
9g iv 2 mg/kg
100
50
0
0
5
10
15
20
25
30
time (h)
Fig. 6. Drug concentration time curve of 9g in plasma of SD rats after single oral/ intravenous administration. 9g was
administrated to rats (n = 3) iv 2mg/kg and po 15 mg/kg separately. Blood samples of 0.2 mL each were collected from
the jugular veins into heparized tubes at 0 h (before drug administration) and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h
after dosing. The blood drug concentration was then determined with LC-MS/MS.
3. Conclusion
We have demonstrated the development of series of novel 1-phenyl phenanthridin-6(5H)-one TOPK
15

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inhibitors. This phenanthridinone scaffold exhibited more potent anti-TOPK activity compared to reference
compound OTS964. Based on the results of binding affinity and cell level screening, the preliminary SARs
were summarized. In the in vitro assays, 9b showed >700 fold stronger binding affinity at subnanomolar
level than OTS964 and 9g showed 2~6 fold superior activity against a panel of cancer cell lines at nanomolar
level. For the in vivo evaluation, compound 9g was shown to be effective in treating three mouse xenograft
models of colorectal cancer (>75% TGI) by oral administration without severe adverse events, superior than
OTS964 (49% TGI for HCT116) at the same dose (15 mg/kg, po). Its potency may be further developed and
may be applied to a wide range of human malignancies. In the pharmacokinetic study, compound 9g also
displayed favorable plasma stability and oral bioavailability. The in-vivo tumor suppression activity and
pharmacokinetic results of 9g suggest that the drug candidate has great clinical application potential.
In summary, the novel 1-phenyl phenanthridin-6(5H)-one derivatives exhibited potent TOPK inhibitory
activity both in vitro and in vivo, and showed excellent oral bioavailability. Developing inhibitors targeting
TOPK provides an exciting strategy for treatment of tumor.
4. Experimental
4.1. Chemistry
Unless otherwise noted, all materials were obtained from commercial suppliers and used without further
purification. The 1H and 13C NMR spectra were recorded on a Bruker Avance 400 spectrometer at 25 °C using
DMSO-d6, CD3OD or CDCl3 as the solvent. Chemical shifts (δ) are reported in ppm relative to Me4Si (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). High resolution mass 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). Visualization of TLC was accomplished with UV light and/or aqueous potassium permanganate or I2
in silica gel. Column chromatography was performed using silica gel 60 of 300−400 mesh (Qingdao Haiyang
Chemical, China).
16

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4.1.1. Synthesis of (R)-(4-(1-((tert-butoxycarbonyl)amino)ethyl)phenyl)boronic acid (R1A)
Step1: (R)-tert-butyl (1-(4-bromophenyl)ethyl)carbamate (R1-2). (R)-1-(4-bromophenyl)ethanamine
(14.4 g, 72.0 mmol) was dissolved in 60 ml dichloromethane (DCM), a solution of Boc2O (17.3 g, 79.2 mmol,
1.1 eq) in 60 ml DCM was added dropwise at room temperature. The resulting solution was stirred at room
temperature for further 2 h after LCMS indicating completion. The mixture was concentrated and suspended
in hexane. The solid was collected and dried under vacuum to give 20.0 g white solid, yield: 92.6%.
Step2: (R)-tert-butyl (1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)ethyl)carbamate (R1).To
a suspension of product of step1 (20 g, 66.6 mmol) in dioxane (120 ml) was added Bis(pinacolato)diboron
(20.3 g, 80.0 mmol, 1.2 eq), KOAc ( 13.1 g, 133.2 mmol, 2 eq) and PdCl2(dppf) (1.95 g, 2.66 mmol, 0.04 eq).
The mixture was heated under nitrogen overnight, then filtered and concentrated. The residue was dissolved in
a mixture of hexane and ethylacetate (EA) (4:1) and filtered through silica gel pad, then concentrated and
directly used for hydrolysis. ESI-MS m/z 292.3 [M-56+H]+.
Step3: To the residue above was added acetone/H2O (200ml/100ml), NaIO4 (61.2 g, 286 mmol),
NH4OAc (14.7 g, 190.6 mmol). The suspension was stirred at 40 °C for 5 h, then filtered and the solid was
washed with acetone. The filtrate was concentrated and re-dissolved in DCM/MeOH (5/1), washed with water,
concentrated and suspended in hexane/EA (1:1), filtered to give 14.5 g R1A as off-white solid, yield: 82.1%.
1H NMR (400 MHz, DMSO-d6) δ 7.94 (s, 2H), 7.71 (d, J = 7.6 Hz, 2H), 7.35 (d, J = 7.2 Hz, 1H), 7.24 (d, J =
7.6 Hz, 2H), 4.67-4.51 (m, 1H), 1.36 (s, 9H), 1.29 (d, J = 6.8 Hz, 3H). ESI-MS m/z 210.2 [M-56+H]+.
4.1.2. Synthesis of (R)-(4-(1-((tert-butoxycarbonyl)amino)propan-2-yl)phenyl)boronic acid (R2A)
Step1: (R)-tert-butyl (2-(4-bromophenyl)propyl)carbamate (R2-3). A solution of trifluoro acetic
anhydride (23.0 mL, 164.4 mmol) in anhydrous DCM (150 mL) was cooled to 0 °C, and
(R)-2-phenylpropan-1-amine (20.2 g, 149.3 mmol) in anhydrous DCM (40 mL) was added dropwise. The
mixture was stirred at room temperature for 2 h. The flask was again cooled to 0 °C and methane sulfonic acid
(24.2 mL) was added, followed by 1,3-dibromo-5,5-dimethylhydantoin (21.3 g, 74.5 mmol) in one portion.
The mixture was stirred overnight, quenched with water (80 mL), and diluted with DCM (250 mL). The
17

ACCEPTED MANUSCRIPT
organic layers were separated and concentrated. The residue was diluted with water/EtOH (200ml/200ml),
LiOH•H2O (8 g, 190.5 mmol) was added and stirred at room temperature for 1 h. Then Boc2O (29.3 g, 134.4
mmol) was added and the mixture was stirred at room temperature overnight. The mixture was diluted with
H2O (200 ml) and extracted with DCM. DCM layer was concentrated to give yellow residue and the residue
was purified by column chromatography (silica, 0-30% ethyl acetate/heptane) to afford the desired product
(45.9 g, 98.0%) as yellow oil. ESI-MS m/z 260.1 [M-56+H]+.
Step2: Following procedure of step2 and step3 of R1A with above 45.9 g intermediate, R2A 20.8 g
(yield: 51%) was obtained as off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.71 (d, J = 8.0 Hz, 2H), 7.16
(d, J = 8.0 Hz, 2H), 3.08-3.01 (m, 2H), 2.87-2.82 (m, 1H), 1.35 (s, 9H), 1.14 (d, J = 6.8 Hz, 3H). ESI-MS m/z
224.1 [M-56+H]+.
4.1.3. Synthesis of (4-(1-(((tert-butoxycarbonyl)amino)methyl)cyclopropyl)phenyl)boronic acid (R3A)
Step1:
tert-butyl
((1-(4-bromophenyl)cyclopropyl)methyl)carbamate
(R3-3).
1-(4-bromophenyl)cyclopropanecarbonitrile (4.45 g, 20 mmol) was added to 1M Borane-THF complex ( 100
ml) and the mixture was stirred at room temperature overnight and then heated at 65 °C for 5 h. The mixture
was concentrated, diluted with MeOH, 1N HCl was added dropwise until PH<1. The mixture was heated at
65 °C while keeping the PH~1 by addition of 1N HCl intermittently, then cooled down and basified with solid
NaHCO3. The suspension was concentrated and re-dissolved in acetonitrile and filtered to give the acetonitrile
solution of free amine. Boc2O (4.1g, 18.7 mmol) in acetonitrile was added dropwise. The mixture was stirred
at room temperature for 3 h and then concentrated to give 6.1 g yellow oil, yield: 93.3%. ESI-MS m/z 272.0
[M-100+H]+.
Step2: Following procedure of step2 and step3 of R1A with above 6.1 g intermediate, R3A 3.9 g was
obtained as off-white solid, yield: 71.3%. 1H NMR (400 MHz, DMSO-d6) δ 7.93 (s, 2H), 7.69 (d, J = 7.6 Hz,
2H), 7.21 (d, J = 7.6 Hz, 2H), 6.82 (t, J = 5.6 Hz, 1H), 3.22 (d, J = 5.6 Hz, 2H), 1.34 (s, 9H), 0.85 (s, 2H),
0.71 (s, 2H). ESI-MS m/z 236.1 [M-56+H]+.
4.1.4. The representative procedure for the preparation of intermediate 5
18

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Step1: 2-bromo-N-(4-methoxy-2-methylphenyl)benzamide (BA3): 1 drop of DMF was added to a
suspension of 2-bromobenzoic acid (2 g, 9.95 mmol) in 5 ml SOCl2, the mixture was heated under reflux for 2
h, then concentrated and diluted with DCM 20 ml, concentrated again and re-diluted with DCM 20 ml and
concentrated. The residue was dissolved in 20 ml DCM, added dropwise to a solution of
4-methoxy-2-methylaniline (1.4 g, 9.95 mmol) and triethylamine (3.43 ml, 24.9 mmol) in 10 ml DCM, and
then stirred for 2 h. The mixture was concentrated to give brown solid, which was treated with EtOH/H2O
(10ml/20ml) to give off-white solid 2.5 g, yield: 78.4%. ESI-MS m/z 320.1 [M+H]+.
Step 2: tert-butyl (2-bromobenzoyl)(4-methoxy-2-methylphenyl)carbamate (BA4): BOC2O (2.0 g, 9.4
mmol) was added dropwise to a suspension of BA3 (2.5 g, 7.81 mmol) and 4-dimethylaminopyridine (DMAP)
(0.39 mmol, 47.7 mg, 0.05 eq) in 30 ml dry DCM, then stirred at room temperature overnight. It was
concentrated and treated with hexane/EA/H2O (20 ml/2 ml/20 ml) to precipitate the solid, the solid was
filtered and washed with hexane, dried under vacuum to give off-white solid 2.9 g, yield: 90%. ESI-MS m/z
366.1[M-56+H]+.
Step3: 2-methoxy-4-methylphenanthridin-6(5H)-one (BA5): A suspension o f BA3 (1.8 g, 4.28 mmol),
KOAc (1.68 g, 17.13 mmol) and Pd(t-Bu3P)2 (131 mg, 0.26 mmol) in dry N,N-Dimethylacetamide (DMA) (5
ml) was heated at 130~140 °C for 2 h under argon. The mixture was cooled down and quenched with cold
water, the precipitated solid was collected and washed with H2O, EtOH successively, then dried under
1
vacuum to yield BA4 as gray-white solid. Weight: 770 mg, yield: 75%. H NMR (400 MHz, DMSO-d6) δ
10.61 (s, 1H), 8.53 (d, J = 8.0 Hz, 1H), 8.32 (d, J = 8.0 Hz, 1H), 7.82 (t, J = 7.4 Hz, 1H), 7.72 (s, 1H), 7.62 (t,
J = 7.4 Hz, 1H), 7.00 (s, 1H), 3.84 (s, 3H), 2.43 (s, 3H). ESI-MS m/z 240.2 [M+H]+.
1
4.1.4.1. 7-fluoro-2-methoxy-4-methylphenanthridin-6(5H)-one (DA5). Yield (three steps): 35.6%. H NMR
(400 MHz, CDCl3) δ 8.51 (s, 1H), 8.04 (d, J = 8.4 Hz, 1H), 7.76-7.70 (m, 1H), 7.49 (d, J = 2.4 Hz, 1H),
7.28-7.23 (m, 1H), 6.99 (d, J = 2.0 Hz, 1H), 3.91 (s, 3H), 2.46 (s, 3H). ESI-MS m/z 258.3 [M+H]+.
1
4.1.4.2. 2-methoxy-4,8-dimethylphenanthridin-6(5H)-one (JA5). Yield (three steps): 43.9%. H NMR (400
MHz, CDCl3) δ 8.79 (s, 1H), 8.32 (d, J = 0.4 Hz, 1H), 8.12 (d, J = 8.4 Hz, 1H), 7.60 (dd, J = 8.0, 1.6 Hz, 1H),
7.51 (d, J = 2.8 Hz, 1H), 6.94 (d, J = 2.0 Hz, 1H), 3.91 (s, 3H), 2.53 (s, 3H), 2.48 (s, 3H). ESI-MS m/z 254.3
19

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[M+H]+.
1
4.1.4.3. 2-methoxy-4,7-dimethylphenanthridin-6(5H)-one (KA5). Yield (three steps): 42.7%. H NMR (400
MHz, CDCl3) δ 8.76 (s, 1H), 8.11 (d, J = 8.0 Hz, 1H), 7.62 (t, J = 7.8 Hz, 1H), 7.52 (d, J = 2.4 Hz, 1H), 7.36
(d, J = 7.6 Hz, 1H), 6.94 (d, J = 2.0 Hz, 1H), 3.90 (s, 3H), 2.98 (s, 3H), 2.47 (s, 3H). ESI-MS m/z 254.1
[M+H]+.
1
4.1.4.4. 2-methoxy-4,9-dimethylphenanthridin-6(5H)-one (LA5). Yield (three steps): 43.8%. H NMR (400
MHz, CDCl3) δ 9.00 (s, 1H), 8.40 (d, J = 8.0 Hz, 1H), 7.98 (s, 1H), 7.51 (d, J = 1.6 Hz, 1H), 7.41 (d, J = 7.6
Hz, 1H), 6.94 (d, J = 1.6 Hz, 1H), 3.91 (s, 3H), 2.57 (s, 3H), 2.49 (s, 3H). ESI-MS m/z 254.1 [M+H]+.
1
4.1.4.5. 4,7-difluoro-2-methoxyphenanthridin-6(5H)-one (DC5). Yield (three steps): 33.8%. H NMR (400
MHz, CDCl3) δ 8.60 (s, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.78-7.73 (m, 1H), 7.41 (d, J = 1.6 Hz, 1H), 7.33-7.28
(m, 1H), 6.93 (dd, J = 11.6, 2.8 Hz, 1H), 3.91 (s, 3H). ESI-MS m/z 262.3 [M+H]+.
1
4.1.4.6. 4-chloro-2-methoxyphenanthridin-6(5H)-one (BD5). Yield (three steps): 35.5%. H NMR (400 MHz,
CDCl3) δ 8.86 (s, 1H), 8.52 (d, J = 7.6 Hz, 1H), 8.19 (d, J = 8.0 Hz, 1H), 7.80 (t, J = 7.4 Hz, 1H), 7.64 (t, J =
7.4 Hz, 1H), 7.59 (d, J = 2.0 Hz, 1H), 7.16 (d, J = 2.4 Hz, 1H), 3.91 (s, 3H). ESI-MS m/z 260.1[M+H]+.
1
4.1.4.7. 4-chloro-7-fluoro-2-methoxyphenanthridin-6(5H)-one (DD5). Yield (three steps): 38.7%. H NMR
(400 MHz, CDCl3) δ 8.76 (s, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.78-7.73 (m, 1H), 7.56 (d, J = 2.4 Hz, 1H),
7.33-7.28 (m, 1H), 7.19 (d, J = 2.4 Hz, 1H), 3.91 (s, 3H). ESI-MS m/z 278.1 [M+H]+.
4.1.5. The representative procedure for the preparation of intermediate 6
1-bromo-2-methoxy-4-methylphenanthridin-6(5H)-one (BA6): BA5 (433 mg, 1.81 mmol) was added to a
mixture of DCM/HOAc (1.5 ml/1.5 ml) and stirred to give a solution, followed by addition of
N-bromosuccinimide (NBS) (322 mg, 1.81 mmol) by one portion. The mixture was stirred at room
temperature overnight. LCMS showed the reaction was not finished. Another portion of NBS (32 mg) was
added and then stirring was continued for 2 h. The reaction mixture was diluted with water, extracted with
DCM. DCM layer was separated and concentrated to give red-brown solid. The solid was suspended in
20

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EA/hexane (1:1) and heated under reflux for 30 min, cooled down and filtered, the solid was dried under
1
vacuum to give 460 mg yellow solid, yield: 79.9%. H NMR (400 MHz, CDCl3) δ 9.69 (d, J = 8.4 Hz, 1H),
8.66 (s, 1H), 8.56 (d, J = 8.0 Hz, 1H), 7.79 (t, J = 7.6 Hz, 1H), 7.64 (t, J = 7.6 Hz, 1H), 7.01 (s, 1H), 3.95 (s,
3H), 2.50 (s, 3H). ESI-MS m/z 320.0 [M+H]+.
1
4.1.5.1. 1-bromo-7-fluoro-2-methoxy-4-methylphenanthridin-6(5H)-one (DA6). Yield 88.5%. H NMR (400
MHz, CDCl3) δ 9.37 (d, J = 8.8 Hz, 1H), 8.36 (s, 1H), 7.76-7.71 (m, 1H), 7.34-7.29 (m, 1H), 7.03 (s, 1H),
3.96 (s, 3H), 2.47 (s, 3H). ESI-MS m/z 336.0 [M+H]+.
1
4.1.5.2. 1-bromo-2-methoxy-4,8-dimethylphenanthridin-6(5H)-one (JA6). Yield: 89.7%. H NMR (400 MHz,
CDCl3) δ 9.60 (d, J = 8.8 Hz, 1H), 8.72 (s, 1H), 8.37 (s, 1H), 7.61 (dd, J = 8.6, 1.8 Hz, 1H), 6.99 (s, 1H), 3.96
(s, 3H), 2.54 (s, 3H), 2.50 (s, 3H). ESI-MS m/z 334.1 [M+H]+.
4.1.5.3. 1-bromo-2-methoxy-4,7-dimethylphenanthridin-6(5H)-one (KA6). Yield: 96.2%. 1H NMR (400 MHz,
CDCl3) δ 9.27 (d, J = 8.0 Hz, 1H), 8.81 (s, 1H), 7.60 (t, J = 7.8 Hz, 1H), 7.40 (d, J = 7.6 Hz, 1H), 6.98 (s, 1H),
3.95 (s, 3H), 2.97 (s, 3H), 2.50 (s, 3H). ESI-MS m/z 332.0 [M+H]+.
4.1.5.4. 1-bromo-2-methoxy-4,9-dimethylphenanthridin-6(5H)-one (LA6). Yield: 93.4%. 1H NMR (400 MHz,
CDCl3) δ 9.52 (s, 1H), 8.57 (s, 1H), 8.45 (d, J = 8.0 Hz, 1H), 7.47 (d, J = 7.6 Hz, 1H), 7.01 (s, 1H), 3.96 (s,
3H), 2.59 (s, 3H), 2.49 (s, 3H). ESI-MS m/z 334.0 [M+H]+.
1
4.1.5.5. 1-bromo-4,7-difluoro-2-methoxyphenanthridin-6(5H)-one (DC6). Yield: 90.2%. H NMR (400 MHz,
CDCl3) δ 9.43 (d, J = 8.4 Hz, 1H), 7.74-7.68 (m, 1H), 7.32-7.27 (m, 1H), 6.97 (d, J = 11.2 Hz, 1H), 3.89 (s,
3H). ESI-MS m/z 340.1 [M+H]+.
1
4.1.5.6. 1-bromo-4-chloro-2-methoxyphenanthridin-6(5H)-one (BD6). Yield: 88.7%. H NMR (400 MHz,
CDCl3) δ 9.68 (d, J = 8.8 Hz, 1H), 8.90 (s, 1H), 8.57 (dd, J = 7.8, 1.4 Hz, 1H), 7.84-7.80 (m, 1H), 7.71-7.67
(m, 1H), 7.21 (s, 1H), 3.97 (s, 3H). ESI-MS m/z 338.0 [M+H]+.
1
4.1.5.7. 1-bromo-4-chloro-7-fluoro-2-methoxyphenanthridin-6(5H)-one (DD6) Yield: 91.7%. H NMR (400
MHz, CDCl3) δ 9.33 (d, J = 8.4 Hz, 1H), 8.76 (s, 1H), 7.77-7.71 (m, 1H), 7.37-7.32 (m, 1H), 7.19 (s, 1H),
21

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3.96 (s, 3H). ESI-MS m/z 356.0 [M+H]+.
4.1.5.8. 1-bromo-2,7-dimethoxy-4-methylphenanthridin-6(5H)-one (CA6). To a 150 ml two necked flask was
added D6 (5.4 g, 16.06 mmol), MeONa (4.3 g, 80.32 mmol), DMF (54 ml). The mixture was stirred at
50~60 °C for 1 h, cooled to room temperature and quenched with 200 ml water. The precipitated solid was
collected and washed with water and ethanol, dried under vacuum to give 4.5 g gray white solid. Yield: 80.5%.
1H NMR (400 MHz, CDCl3) δ 9.00 (d, J = 8.4 Hz, 1H), 8.18 (s, 1H), 7.69 (t, J = 8.4 Hz, 1H), 7.14 (d, J = 8.4
Hz, 1H), 6.99 (s, 1H), 4.05 (s, 3H), 3.95 (s, 3H), 2.44 (s, 3H). ESI-MS m/z 350.2 [M+H]+.
4.1.5.9. 1-bromo-4-chloro-2,7-dimethoxyphenanthridin-6(5H)-one (CD6). Yield: 88.4%. 1H NMR (400 MHz,
CDCl3) δ 8.98 (d, J = 8.4 Hz, 1H), 8.59 (s, 1H), 7.71 (t, J = 8.4 Hz, 1H), 7.18 (s, 1H), 7.17 (d, J = 7.2 Hz, 1H),
4.06 (s, 3H), 3.96 (s, 3H). ESI-MS m/z 370.0 [M+H]+.
4.1.5.10. 1-bromo-7-(dimethylamino)-2-methoxy-4-methylphenanthridin-6(5H)-one (MA6): DA6 (530 mg,
1.58 mmol), 33% dimethlyamine aqueous solution (3 ml), DMF (10 ml) were added to a 30 ml sealed tube
o
and the mixture was heated at 110 C overnight. It was cooled to room temperature and sold precipitated out.
The solid was collected and washed with water and EtOH, dried under vacuum to give 381 mg gray white
solid, yield: 66.9%. ESI-MS m/z 363.1 [M+H]+.
4.1.6. The representative procedure for the preparation of intermediate 7
(R)-tert-butyl (1-(4-(2-methoxy-4-methyl-6-oxo-5,6-dihydrophenanthridin-1-yl)phenyl)ethyl)carbamate
(7a): BA6 (210 mg, 0.66 mmol), R1A (228 mg, 0.86 mmol), NaHCO3 (166 mg, 1.98 mmol) and PdCl2(dppf)
(24 mg, 0.033 mmol) were added to 25 ml two necked flask equipped with a thermometer, followed by
DME/H2O (10 ml/0.5 ml). The mixture was heated at 85 °C overnight. Then it was concentrated, diluted with
water and extracted with DCM. The DCM layer was washed with water, concentrated and crystallized from
mixture of EA and hexane to give 160 mg desired target as grey solid, yield: 52.9%.
4.1.7. The representative procedure for the preparation of 8
22

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(R)-1-(4-(1-aminoethyl)phenyl)-2-methoxy-4-methylphenanthridin-6(5H)-one hydrochloride (8a): 7a
(160 mg) was stirred in 5 ml 4N HCl/dioxane overnight, concentrated and the residue was washed with DCM
1
and recrystallized from isopropanol to give 103 mg off-white solid, yield: 75%. H NMR (400 MHz,
DMSO-d6) δ 10.53 (s, 1H), 8.67 (s, 3H), 8.32 (d, J = 6.4 Hz, 1H), 7.64-7.60 (m, 2H), 7.50-7.40 (m, 1H),
7.29-7.23 (m, 4H), 6.87 (d, J = 8.8 Hz, 1H), 4.51-4.50 (m, 1H), 3.68 (s, 3H), 2.56 (s, 3H), 1.62 (d, J = 5.6 Hz,
3H). 13C NMR (100 MHz, DMSO-d6) δ 160.48, 151.54, 138.56, 138.50, 134.50, 131.13, 130.31, 129.70,
127.54, 127.36, 127.32, 127.18, 126.88, 124.84, 124.80, 117.10, 115.90, 56.33, 49.85, 20.88, 18.27. ESI-MS
m/z 359.2 [M+H]+. HRMS (ESI) Calcd for C23H23N2O2 [M+H]+ m/z 359.1754, found 359.1758.
4.1.7.1. (R)-1-(4-(1-aminopropan-2-yl)phenyl)-2-methoxy-4-methylphenanthridin-6(5H)-one hydrochloride
(8b). White solid, two step yield: 58.8%. 1H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 8.32-8.30 (m, 1H),
7.94 (s, 3H), 7.45-7.38 (m, 3H), 7.27 (s, 1H), 7.22-7.17 (m, 3H), 6.89 (d, J = 8.4 Hz, 1H), 3.68 (s, 3H),
3.16-3.06 (m, 3H), 2.51 (s, 3H), 1.35 (d, J = 6.0 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 160.50, 151.59,
142.05, 137.03, 134.63, 130.91, 130.43, 129.77, 127.71, 127.45, 127.33, 127.13, 126.97, 125.07, 124.66,
117.23, 115.93, 56.33, 44.79, 37.30, 19.49, 18.26. ESI-MS m/z 373.3 [M+H]+. HRMS (ESI) Calcd for
C24H25N2O2 [M+H]+ m/z 373.1911, found 373.1913.
4.1.7.2.
1-(4-(1-(aminomethyl)cyclopropyl)phenyl)-2-methoxy-4-methylphenanthridin-6(5H)-one
1
hydrochloride (8c). White solid, two step yield: 66%. H NMR (400 MHz, DMSO-d6) δ 10.48 (s, 1H),
8.32-8.29 (m, 1H), 8.09 (s, 3H), 7.46-7.42 (m, 3H), 7.27-7.17 (m, 4H), 6.94 (d, J = 8.4 Hz, 1H), 3.67 (s, 3H),
13
3.19 (s, 2H), 2.56 (s, 3H), 1.15-1.12 (m, 2H), 1.04-1.01 (m, 2H). C NMR (100 MHz, DMSO-d6) δ 160.50,
151.58, 140.17, 136.70, 134.58, 131.12, 130.27, 129.76, 128.38, 127.61, 127.27, 127.10, 126.91, 124.95,
124.66, 117.22, 115.86, 56.29, 46.66, 23.04, 18.24, 13.88. ESI-MS m/z 385.3 [M+H]+.
4.1.8. The representative procedure for the preparation of compound 9
(R)-1-(4-(1-aminoethyl)phenyl)-2-hydroxy-4-methylphenanthridin-6(5H)-one hydrochloride (9a). 8a
(136 mg) was suspended in 15% BBr3 in DCM (5ml) and stirred overnight. The mixture was concentrated and
treated with DCM 20 ml and concentrated, diluted with ice-water, neutralized with aq. ammonia until PH>9,
the precipitated solid was collected and dissolved with 3N HCl/MeOH, concentrated and crystallized with
23

ACCEPTED MANUSCRIPT
isopropanol to give 110 mg off-white solid, yield: 80%. 1H NMR (400 MHz, DMSO-d6) δ 10.43 (s, 1H), 9.27
( s, 1H), 8.64 (s, 3H), 8.31 (d, J = 8.0 Hz, 1H), 7.62-7.59 (m, 2H), 7.43 (t, J = 7.4 Hz, 1H), 7.29-7.27 (m, 2H),
7.24-7.20 (m, 1H), 7.07 (s, 1H), 6.90 (d, J = 8.4 Hz, 1H), 4.50 (t, J = 5.4 Hz, 1H), 2.47 (s, 3H), 1.62 (d, J = 6.8
Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 160.37, 149.79, 139.32, 137.99, 134.78, 130.84, 130.54, 128.70,
127.34, 127.29, 127.26, 126.88, 126.82, 124.53, 122.40, 119.38, 117.02, 49.85, 20.78, 18.03. ESI-MS: m/z
345.3 [M+H]+. HRMS (ESI) Calcd for C22H21N2O2 [M+H]+ m/z 345.1598, found 345.1558.
4.1.8.1. (R)-1-(4-(1-aminopropan-2-yl)phenyl)-2-hydroxy-4-methylphenanthridin-6(5H)-one hydrochloride
(9b). White solid, yield 51%. 1H NMR (400 MHz, DMSO-d6) δ 10.44 (s, 1H), 9.16 (s, 1H), 8.30 (d, J = 7.6 Hz,
1H), 8.13 (s, 3H), 7.44-7.38 (m, 3H), 7.22-7.18 (m, 3H), 7.06 (s, 1H), 6.99 (d, J = 8.4 Hz, 1H), 3.18-3.08 (m,
3H), 2.51(s, 3H), 1.36 (d, J = 6.8 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 160.38, 149.79, 141.75, 137.56,
134.88, 130.77, 130.56, 128.71, 127.69, 127.55, 127.40, 127.25, 126.82, 126.80, 124.35, 122.64, 119.38,
117.13, 44.87, 37.27, 19.48, 18.00. ESI-MS m/z 359.2 [M+H]+. HRMS (ESI) Calcd for C23H23N2O2 [M+H]+
m/z 359.1754, found 359.1758.
4.1.8.2.
1-(4-(1-(aminomethyl)cyclopropyl)phenyl)-2-hydroxy-4-methylphenanthridin-6(5H)-one
1
hydrochloride (9c). White solid, yield: 87%. H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 9.12 (s, 1H),
8.30 (dd, J = 8.0, 2.1 Hz, 1H), 8.04 (s, 3H), 7.46-7.40 (m, 3H), 7.27-7.19 (m, 3H), 7.05 (s, 1H), 7.04 (d, J =
8.4 Hz, 1H), 3.18 (d, J = 4.0 Hz, 2H), 2.47 (s, 3H), 1.13-1.10 (m, 2H), 1.06-1.00 (m, 2H). 13C NMR (100 MHz,
DMSO-d6) δ 160.40, 149.80, 139.79, 137.34, 134.84, 130.98, 130.43, 128.71, 128.46, 127.56, 127.23, 126.81,
126.75, 124.37, 122.53, 119.34, 117.13, 46.83, 25.46, 23.14, 17.99, 13.65. ESI-MS m/z 371.1 [M+H]+.
HRMS (ESI) Calcd for C24H23N2O2 [M+H]+ m/z 371.1754, found 371.1754.
4.1.8.3. (R)-1-(4-(1-aminoethyl)phenyl)-7-fluoro-2-hydroxy-4-methylphenanthridin-6(5H)-one (9d). Brown
solid, yield: 71.2%. 1H NMR (400 MHz, DMSO-d6) δ 10.32 (s, 1H), 9.28 (s, 1H), 8.48 (s, 3H), 7.57-7.54 (m,
2H), 7.30-7.26 (m, 2H), 7.19-7.16 (m, 2H), 7.05 (s, 1H), 6.87-6.85 (m, 1H), 4.49 (br s, 1H), 2.44 (s, 3H), 1.59
(d, J = 6.8 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 162.93, 160.33, 157.97, 157.93, 149.79, 139.05,
137.94, 137.70, 131.78, 131.67, 130.62, 129.25, 127.25, 127.20, 124.29, 123.83, 123.79, 122.30, 120.07,
116.30, 115.73, 115.70, 114.28, 114.07, 49.82, 20.72, 17.86. ESI-MS m/z 363.2 [M+H]+. HRMS (ESI) Calcd
for C22H20FN2O2 [M+H]+ m/z 363.1503, found 363.1498.
24

ACCEPTED MANUSCRIPT
4.1.8.4.
(R)-1-(4-(1-aminopropan-2-yl)phenyl)-7-fluoro-2-hydroxy-4-methylphenanthridin-6(5H)-one
1
hydrochloride (9e). Brown solid, yield: 78.3%. H NMR (400 MHz, DMSO-d6) δ 10.29 (s, 1H), 9.22 (br s,
1H), 8.10 (s, 3H), 7.38-7.35 (m, 2H), 7.21-7.11 (m, 4H), 7.07 (s, 1H), 6.89 (d, J = 7.6 Hz, 1H), 3.17-3.03 (m,
3H), 2.44 (s, 3H), 1.34 (d, J = 6.4 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 162.88, 160.29, 157.95, 149.81,
141.69, 137.81, 137.30, 131.72, 131.63, 130.64, 129.24, 127.55, 124.06, 123.94, 122.58, 120.12, 116.40,
115.70, 115.66, 114.21, 113.99, 44.86, 37.23, 19.46, 17.79. ESI-MS m/z 377.3 [M+H]+. HRMS (ESI) Calcd
for C23H22FN2O2 [M+H]+ m/z 377.1660, found 377.1658.
4.1.8.5. (R)-1-(4-(1-aminoethyl)phenyl)-2,7-dihydroxy-4-methylphenanthridin-6(5H)-one hydrochloride (9f).
Brown solid, yield: 77%. 1H NMR (400 MHz, DMSO-d6) δ 13.86 (s, 1H), 10.93 (s, 1H), 9.35 (s, 1H), 8.44 (s,
3H), 7.57-7.54 (m, 2H), 7.28-7.25 (m, 2H), 7.09-7.7.05 (m, 2H), 6.77 (d, J = 8.0 Hz, 1H), 6.43 (d, J = 8.4 Hz,
1H), 4.52-4.49 (m, 1H), 2.50 (s, 3H), 1.59 (d, J = 6.8 Hz). 13C NMR (100 MHz, DMSO-d6) δ 165.09, 161.51,
150.65, 139.19, 137.93, 135.98, 133.19, 130.48, 127.68, 127.22, 127.15, 125.29, 122.61, 119.71, 117.86,
117.82, 113.48, 110.72, 49.83, 20.74, 18.09. ESI-MS m/z 361.3 [M+H]+. HRMS (ESI) Calcd for C22H21N2O3
[M+H]+ m/z 361.1547, found 361.1548.
4.1.8.6.
(R)-1-(4-(1-aminopropan-2-yl)phenyl)-2,7-dihydroxy-4-methylphenanthridin-6(5H)-one
1
hydrochloride (9g). Off-white solid, yield: 81.4%. H NMR (400 MHz, CD3OD) δ 7.51 (dd, J = 8.0, 1.6 Hz,
1H), 7.41 (dd, J = 7.8, 1.8 Hz, 1H), 7.32 (dd, J = 8.0, 1.2 Hz, 1H), 7.26 (dd, J = 8.0, 1.6 Hz, 1H), 7.07-7.03 (m,
2H), 6.78 (d, J = 8.0 Hz, 1H), 6.69 (d, J = 8.4 Hz, 1H), 3.33-3.19 (m, 3H), 2.53 (s, 3H), 1.49 (d, J = 6.0 Hz,
3H). 13C NMR (400 MHz, DMSO-d6) δ 165.09, 161.45, 150.62, 141.68, 137.43, 136.06, 133.16, 130.50,
127.71, 127.56, 127.46, 125.12, 122.87, 119.70, 117.97, 117.93, 113.42, 110.72, 44.84, 37.28, 19.46, 18.05.
ESI-MS m/z 375.2 [M+H]+. HRMS (ESI) Calcd for C23H23N2O3 [M+H]+ m/z 375.1703, found 375.1707.
4.1.8.7.
1-(4-(1-(aminomethyl)cyclopropyl)phenyl)-2,7-dihydroxy-4-methylphenanthridin-6(5H)-one
hydrochloride (9h). White solid, yield: 71.4%. 1H NMR (400 MHz, DMSO-d6) δ 13.84 (s, 1H), 10.89 (s, 1H),
9.26 (s, 1H), 8.01 (s, 3H), 7.43 (d, J = 8.0 Hz, 2H), 7.17 (d, J = 8.0 Hz, 2H), 7.11 (t, J = 8.2 Hz, 1H), 7.08 (s,
1H), 6.76 (d, J = 8.4 Hz, 1H), 6.49 (d, J = 8.4 Hz, 1H), 3.17 (d, J = 5.2 Hz, 2H), 2.48 (s, 3H), 1.12-1.09 (m,
13
2H), 1.02-0.99 (m, 2H). C NMR (100 MHz, DMSO-d6) δ 165.10, 161.42, 150.68, 139.77, 137.20, 136.01,
133.38, 130.38, 128.32, 127.68, 125.11, 122.76, 119.69, 118.10, 117.95, 113.39, 110.66, 46.80, 23.12, 18.06,
25

ACCEPTED MANUSCRIPT
13.66. ESI-MS m/z 387.1 [M+H]+. HRMS (ESI) Calcd for C24H23N2O3 [M+H]+ m/z 387.1703, found
387.1705.
4.1.8.8.
(R)-1-(4-(1-aminoethyl)phenyl)-4-chloro-2-hydroxyphenanthridin-6(5H)-one
hydrochloride(9i).
1
Brown solid, yield: 55.7%. H NMR (400 MHz, CD3OD) δ 8.42 (d, J = 8.0 Hz, 1H), 7.65-7.62 (m, 2H),
7.52-7.48 (m, 1H), 7.43-7.40 (m, 2H), 7.29 (s, 1H), 7.27-7.20 (m, 2H), 4.62 (q, J = 6.8 Hz, 1H), 1.77 (d, J =
7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 159.96, 150.41, 138.51, 138.45, 138.24, 133.96, 131.34,
130.29, 127.82, 127.54, 127.47, 127.41, 127.01, 126.50, 124.53, 118.77, 117.37, 49.77, 20.74. ESI-MS m/z
365.1 [M+H]+. HRMS (ESI) Calcd for C21H18ClN2O2 [M+H]+ m/z 365.1051, found 365.1053.
4.1.8.9. (R)-1-(4-(1-aminopropan-2-yl)phenyl)-4-chloro-2-hydroxyphenanthridin-6(5H)-one hydrochloride
1
(9j). Brown solid, yield: 72.7%. H NMR (400 MHz, DMSO-d6) δ 10.43 (s, 1H), 9.72 (s, 1H), 8.32 (dd, J =
7.6, 1.2 Hz, 1H), 7.99 (s, 3H), 7.48 (t, J = 7.6 Hz, 1H), 7.41 (dd, J = 5.2, 2.4 Hz, 1H), 7.33 (s, 1H), 7.26-7.21
(m, 3H), 6.96 (d, J = 8.4 Hz, 1H), 3.15-3.07 (m, 3H), 1.36 (d, J = 6.0 Hz, 3H). 13C NMR (100 MHz, DMSO-d6)
δ 159.97, 150.43, 142.26, 136.53, 134.06, 131.26, 130.30, 127.84, 127.76, 127.55, 127.46, 127.00, 126.52,
124.80, 118.90, 118.58, 117.37, 44.78, 37.31, 19.51. ESI-MS m/z 379.1 [M+H]+. HRMS (ESI) Calcd for
C22H20ClN2O2 m/z [M+H]+ 379.1208, found 379.1208.
4.1.8.10. (R)-1-(4-(1-aminoethyl)phenyl)-4-chloro-2,7-dihydroxyphenanthridin-6(5H)-one hydrochloride (9k).
White solid, yield: 68.9%. 1H NMR (400 MHz, DMSO-d6) δ 13.63 (s, 1H), 11.04 (s, 1H), 10.00 (s, 1H), 8.55
(s, 3H), 7.61-7.59 (m, 2H), 7.39 (s, 1H), 7.30-7.27 (m, 2H), 7.13 (t, J = 8.2 Hz, 1H), 6.85 (d, J = 8.0 Hz, 1H),
13
6.42 (d, J = 8.4 Hz, 1H), 4.52-4.49 (m, 1H), 1.60 (d, J = 6.8 Hz, 3H). C NMR (100 MHz, DMSO-d6) δ
165.05, 161.54, 151.20, 138.42, 138.18, 135.11, 133.66, 130.25, 127.40, 127.35, 125.62, 124.70, 119.55,
119.45, 118.10, 117.76, 114.60, 110.84, 49.79, 20.72. ESI-MS m/z 381.1[M+H]+. HRMS (ESI) Calcd for
C21H18ClN2O3 [M+H]+ m/z 381.1000, found 381.1001.
4.1.8.11.
(R)-1-(4-(1-aminopropan-2-yl)phenyl)-4-chloro-2,7-dihydroxyphenanthridin-6(5H)-one
hydrochloride (9l). White solid, yield: 87.7%. 1H NMR (400 MHz, DMSO-d6) δ 13.61 (s, 1H), 11.01 (s, 1H),
9.84 (s, 1H), 7.98 (s, 3H), 7.41-7.39 (m, 2H), 7.36 (s, 1H), 7.23-7.18 (m, 2H), 7.12 (t, J = 8.2 Hz, 1H), 6.83 (d,
13
J = 8.0 Hz, 1H), 6.43 (d, J = 8.4 Hz, 1H), 3.13-3.06 (m, 3H), 1.35 (d, J = 6.0 Hz, 3H). C NMR (100 MHz,
26

ACCEPTED MANUSCRIPT
DMSO-d6) δ 165.03, 161.45, 151.22, 142.20, 136.44, 135.17, 133.61, 130.24, 127.70, 127.64, 125.58, 124.97,
119.63, 119.24, 118.21, 117.74, 114.50, 110.80, 44.80, 37.27, 19.48. ESI-MS m/z 395.0 [M+H]+. HRMS (ESI)
Calcd for C22H20ClN2O3 [M+H]+ m/z 395.1157, found 395.1157.
4.1.8.12.
1-(4-(1-(aminomethyl)cyclopropyl)phenyl)-4-chloro-2,7-dihydroxyphenanthridin-6(5H)-one
hydrochloride (9m). White solid, yield: 79.4%. 1H NMR (400 MHz, DMSO-d6) δ 13.61 (s, 1H), 11.00 (s, 1H),
9.93 (s, 1H), 8.02 (s, 3H), 7.45 (d, J = 8.4 Hz, 2H), 7.39 (s, 1H), 7.20 (d, J = 8.4 Hz, 2H), 7.15 (d, J = 8.4 Hz,
1H), 6.83 (d, J = 8.0 Hz, 1H), 6.49 (d, J = 8.4 Hz, 1H), 3.18 (s, 2H), 1.12-1.10 (m, 2H), 1.03-1.00 ( m, 2H).
13C NMR (100 MHz, DMSO-d6) δ 165.04, 161.43, 151.30, 140.28, 136.22, 135.14, 133.85, 130.11, 128.44,
125.55, 124.87, 119.62, 119.24, 118.39, 117.73, 114.48, 110.75, 46.74, 23.11, 13.74. ESI-MS m/z 407.1
[M+H]+. HRMS (ESI) Calcd for C23H20ClN2O3 [M+H]+ m/z 407.1157, found 407.1164.
4.1.8.13.
(R)-1-(4-(1-aminopropan-2-yl)phenyl)-7-(dimethylamino)-2-hydroxy-4-methylphenanthridin-6(5H)-one
1
dihydrochloride (9n). Light yellow solid, yield: 33.5%. H NMR (400 MHz, DMSO-d6) δ 14.83 (br s, 1H),
11.56 (br s, 1H), 9.60 (s, 1H), 8.18 (s, 3H), 8.08 (s, 1H), 7.50-7.38 (m, 3H), 7.22-7.16 (m, 4H), 3.31 (s, 6H),
3.20-3.05 (m, 3H), 2.54 (s, 3H), 1.35 (d, J = 6.4 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 162.42, 151.37,
144.86, 142.17, 136.76, 136.51, 132.31, 130.48, 130.42, 127.95, 127.74, 127.46, 125.45, 122.58, 120.96,
120.36, 117.25, 115.61, 45.94, 44.84, 37.16, 19.49, 18.13. ESI-MS m/z 402.2 [M+H]+. HRMS (ESI) Calcd for
C25H28N3O2 [M+H]+ m/z 402.2176, found 402.2180.
4.1.8.14. (R)-1-(4-(1-aminoethyl)phenyl)-2-hydroxy-4,8-dimethylphenanthridin-6(5H)-one hydrochloride (9o).
Off-white solid, yield: 79.4%. 1H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 9.22 (s, 1H), 8.60 (s, 3H), 8.12
(d, J = 0.8 Hz, 1H), 7.61-7.58 (m, 2H), 7.28-7.24 (m, 2H), 7.04-7.01 (m, 2H), 6.84 (d, J = 8.4 Hz, 1H),
13
4.51-4.48 (m, 1H), 2.46 (s, 3H), 2.34 (s, 3H), 1.62 (d, J = 6.8 Hz, 3H). C NMR (100 MHz, DMSO-d6) δ
160.34, 149.72, 139.39, 137.97, 136.46, 132.31, 132.00, 130.54, 130.51, 128.33, 127.36, 127.28, 127.20,
127.08, 126.76, 124.40, 122.16, 118.90, 117.12, 49.86, 20.82, 20.51, 18.01. ESI-MS m/z 359.1 [M+H]+.
HRMS (ESI) Calcd for C23H23N2O2 [M+H]+ m/z 359.1754, found 359.1761.
4.1.8.15.
(R)-1-(4-(1-aminopropan-2-yl)phenyl)-2-hydroxy-4,8-dimethylphenanthridin-6(5H)-one
27

ACCEPTED MANUSCRIPT
1
hydrochloride (9p). Off-white solid, yield: 72.6%. H NMR (400 MHz, DMSO-d6) δ 10.35 (s, 1H), 9.04 (s,
1H), 8.11(s, 1H), 8.06 (s, 3H), 7.38 (d, J = 8.0 Hz, 2H), 7.19 (t, J = 6.0 Hz, 2H), 7.02-7.08 (m, 2H), 6.85 (d, J
13
= 8.4 Hz, 1H), 3.16-3.07 (m, 3H), 2.45 (s, 3H), 2.34 (s, 3H), 1.36 (d, J = 6.4 Hz, 3H). C NMR (100 MHz,
DMSO-d6) δ 160.35, 149.73, 141.74, 137.62, 136.43, 132.40, 131.94, 130.54, 128.33, 127.67, 127.51, 127.37,
127.01, 126.73, 124.21, 122.41, 118.89, 117.22, 44.87, 37.25, 20.50, 19.48, 17.99. ESI-MS m/z 373.3 [M+H]+.
HRMS (ESI) Calcd for C24H25N2O2 [M+H]+ m/z 373.1911, found 373.1875.
4.1.8.16. (R)-1-(4-(1-aminoethyl)phenyl)-2-hydroxy-4,7-dimethylphenanthridin-6(5H)-one hydrochloride (9q).
Off-white solid, yield: 77.2%. 1H NMR (400 MHz, DMSO-d6) δ 10.08 (s, 1H), 9.14 (s, 1H), 8.45 (s, 3H), 7.52
(t, J = 7.2 Hz, 2H), 7.26 (t, J = 6.8 Hz, 2H), 7.18-7.16 (m, 1H), 7.02-6.97 (m, 3H), 4.48-4.46 (m, 1H), 2.82 (s,
3H), 2.43 (s, 3H), 1.58 (d, J = 6.8 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 161.63, 149.51, 140.31, 139.42,
137.60, 136.30, 130.78, 130.73, 130.19, 129.54, 129.00, 127.12, 126.91, 126.32, 125.18, 123.71, 122.08,
119.31, 117.45, 49.81, 23.96, 20.73, 17.68. ESI-MS 359.2 m/z [M+H]+. HRMS (ESI) Calcd for C23H23N2O2
[M+H]+ m/z 359.1754, found 359.1755.
4.1.8.17.
(R)-1-(4-(1-aminopropan-2-yl)phenyl)-2-hydroxy-4,7-dimethylphenanthridin-6(5H)-one
1
hydrochloride (9r). Off-white solid, yield: 79.1%. H NMR (400 MHz, DMSO-d6) δ 10.05 (s, 1H), 9.04 (s,
1H), 8.02 (s, 3H), 7.33 (d, J = 8.4 Hz, 2H), 7.19-7.15 (m, 3H), 7.02-6.98 (m, 3H), 3.11-3.07 (m, 1H), 2.81 (s,
3H), 2.43 (s, 3H), 1.34 (d, J = 6.4 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 161.66, 149.54, 141.44, 140.18,
137.65, 136.41, 130.75, 130.14, 129.51, 128.96, 127.44, 127.26, 126.42, 125.13, 123.46, 122.36, 119.31,
117.56, 44.88, 37.20, 23.95, 19.42, 17.66. ESI-MS m/z 373.1[M+H]+. HRMS (ESI) Calcd for C24H25N2O2
[M+H]+ m/z 373.1911, found 373.1913.
4.1.8.18. (R)-1-(4-(1-aminoethyl)phenyl)-2-hydroxy-4,9-dimethylphenanthridin-6(5H)-one (9s). Brown solid,
yield: 77.9%. 1H NMR (400 MHz, DMSO-d6) δ 10.31 (s, 1H), 9.21 (s, 1H), 8.57 (s, 3H), 8.19 (d, J = 8.0 Hz,
1H), 7.66 (d, J = 8.0 Hz, 1H), 7.54 (d, J = 8.0 Hz, 1H), 7.30 (d, J = 8.0 Hz, 1H), 7.25 (d, J = 8.0 Hz, 2H), 7.04
(s, 1H), 6.68 (s, 1H), 4.52 (d, J = 6.0 Hz, 1H), 2.46 (s, 3H), 1.95 (s, 3H), 1.61 (d, J = 6.8 Hz, 3H). 13C NMR
(100 MHz, DMSO-d6) δ 160.34, 149.65, 140.36, 139.52, 138.26, 134.69, 130.60, 130.38, 128.85, 127.95,
127.82, 127.31, 126.46, 124.48, 124.44, 122.46, 119.26, 116.95, 49.92, 21.55, 21.33, 18.03. ESI-MS m/z
359.1[M+H]+. HRMS (ESI) Calcd for C23H23N2O2 [M+H]+ m/z 359.1754, found 359.1757.
28