Synthesis and bioactivity studies of covalent inhibitors derived from (-)-Chaetominine

Article abstract of DOI:10.1016/j.molstruc.2021.130694

We reported herein the synthesis and bioactivity studies of compounds derived from the natural product (-)-Chaetominine (6). The key feature of these compounds is the incorporation of electrophilic groups that are capable of forming covalent bonds with the cysteine or threonine residues of cellular proteins. The cell growth inhibition activities of these derivatives of 6 were tested in four cancer cell lines, i.e. a leukemia cell line K562, a multiple myeloma cell line MM1.S, an acute myeloid leukemia cell line MV4-11, and a colon cancer cell line RKO. The data show that cellular growth inhibition IC₅₀ values of 29, an acrylamide-containing molecule, are 9-17 folds more potent than that of 6, while other acrylamide-containing compounds are much less potent, suggesting 29 might have covalent interactions with cellular target proteins. Collectively, incorporation of an acrylamide moiety into 6 is a good strategy to improve its cell growth inhibition activity in cancer cell lines.

Full text of DOI:10.1016/j.molstruc.2021.130694

Journal Pre-proof
Synthesis and Bioactivity Studies of Covalent Inhibitors Derived from
(
-)-Chaetominine
Xian Zhou , Xuexin Feng , Dachi Wang , Deheng Chen ,
Gaoxing Wu , Ziqin Yan , Xilin Lyu , Huan Wang , Jin-Ming Yang ,
Yujun Zhao
PII:
S0022-2860(21)00827-9
DOI:
Reference:
https://doi.org/10.1016/j.molstruc.2021.130694
MOLSTR 130694
To appear in:
Journal of Molecular Structure
Received date:
Revised date:
Accepted date:
19 April 2021
6 May 2021
11 May 2021
Please cite this article as: Xian Zhou , Xuexin Feng , Dachi Wang , Deheng Chen , Gaoxing Wu ,
Ziqin Yan , Xilin Lyu , Huan Wang , Jin-Ming Yang , Yujun Zhao , Synthesis and Bioactivity Stud-
ies of Covalent Inhibitors Derived from (-)-Chaetominine, Journal of Molecular Structure (2021), doi:
https://doi.org/10.1016/j.molstruc.2021.130694
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Highlights
1
2
3
. More than 34 new analogues of Chaetominine have been synthesized;
. These new analogues feature versatile electrophilic warheads;
. Some analogues show improved antitumor activities in cancer cell lines
compared to Chaetominine

Synthesis and Bioactivity Studies of Covalent Inhibitors
Derived from (-)-Chaetominine
a,c,†
b,c,e,†
c
c,d
c,d
Xian Zhou,
Xuexin Feng,
Dachi Wang, Deheng Chen, Gaoxing Wu, Ziqin
c
c
c
b,e*
a,c,d,f,*
Yan, Xilin Lyu, Huan Wang, Jin-Ming Yang, Yujun Zhao
a) School of Chinese Materia Medica, Nanjing University of Chinese Medicine
b) School of Pharmacy, Yancheng Teachers University, Yancheng, Jiangsu 224007,
China
c) State Key Laboratory of Drug Research and Small-Molecule Drug Research
Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555
Zuchongzhi Rd., Shanghai, 201203, China
d) University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing,
1
00049, China
e) College of Chemical Engineering, Nanjing University of Technology, Nanjing
11816, China
2
f) School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001,
China
†
Equal Contributions
*
Corresponding authors, E-mail addresses: chemyjm@163.com for J.-M. Y.;
yjzhao@simm.ac.cn for Y. Z

Abstract
We reported herein the synthesis and bioactivity studies of compounds derived
from the natural product (-)-Chaetominine (6). The key feature of these compounds is
the incorporation of electrophilic groups that are capable of forming covalent bonds
with the cysteine or threonine residues of cellular proteins. The cell growth inhibition
activities of these derivatives of 6 were tested in four cancer cell lines, i.e. a leukemia
cell line K562, a multiple myeloma cell line MM1.S, an acute myeloid leukemia cell
line MV4-11, and a colon cancer cell line RKO. The data show that cellular growth
inhibition IC values of 29, an acrylamide-containing molecule, are 9-17 folds more
5
0
potent than that of 6, while other acrylamide-containing compounds are much less
potent, suggesting 29 might have covalent interactions with cellular target proteins.
Collectively, incorporation of an acrylamide moiety into 6 is a good strategy to
improve its cell growth inhibition activity in cancer cell lines.
Key words: Chaetominine, Cellular growth inhibition, Cancer cell line, Covalent
warhead

1
. Introduction
Nature products feature tremendously structural diversity and often overwhelming
three-dimensional complexity, yet in many cases they consist of reactive electrophilic
functional groups such as acrylamide, α,β-unsaturated ketone/ester, epoxide, β-lactam,
1
-6
and β-lactone, among others.
The natural products containing electrophilic
7
,8
9
functional groups are capable of forming covalent bonds with the cysteine, lysine,
1
0
6
threonine, and histidine residues of target proteins in cells, therefore manifesting
their multifaceted biological activities. These unique molecules have enlightened and
motivated many drug discovery campaigns, in which their synthetic analogues were
produced and their bioactivities were evaluated in vitro and in vivo.
Figure 1: representative examples of targeted covalent inhibitors (TCIs) derived from
natural products and (-)-Chaetominine
For example, E6201 (1, Figure 1), a MEK1 inhibitor was synthesized based on
natural product FR148083 and it was found that the MEK1 Cys207 residue and the
8
,11
conjugated enone moiety of E6201 formed C-S bond in vitro (PDB: 5HZE). E6201

1
2
(
1) showed inhibitory activity in several cell-based systems and is currently tested in
1
3
human for the treatment of metastases melanoma. In addition, the natural product
Wortmannin formed a C-N bond with the Lys-833 residue of PI3K through the
9
conjugated ester moiety, and therefore inhibited the PI3K kinases. PX-866 (2) was a
14
synthetic analogue of Wortmannin and had been advanced into phase II clinical
1
5-17
trials for treatment of several types of cancers.
CDDO (3) was a synthetic
1
8,19
analogue of oleanolic acid,
which inhibited Keap1/Nrf2 through covalently
7
modification of the Cyc151 residue of BTB, a subdomain of Keap1. 3 is currently
2
0
undergoing two phase III clinical trials for the treatment of kidney diseases. Both the
2
1
22
natural product 4 and the lipstatin-derived 5 consist of a β-lactone moiety. It has
been shown that 4 and 5 formed C-O bond with the Thr1 residue of 20S proteasome
10
23
subunit β5 and the Ser2308 residue of human fatty acid synthase (FAS)
respectively and therefore inhibited the corresponding target proteins. Currently, 4 is
undergoing late-stage clinical trials for the treatment of glioblastoma and
2
4
ependymoma, and 5 has been approved for the treatment of obesity. Evidently, the
so-called targeted covalent inhibitors (TCIs) derived from natural products are of
1
,3,25-31
compelling therapeutic potentials for the treatment of human diseases.
(-)-Chaetominine (6) is a natural product firstly isolated from an endophytic fungus
3
2
on the Adenophora axilliflora leaves and potently inhibited cell growth of the human
3
2,33
leukemia K562 and colon cancer SW1116 cell line.
6 features an interesting
tetracyclic ring system and to the best of our knowledge, covalent structural
modifications and medicinal chemistry studies of 6 are underexplored. As our ongoing

34
interest in targeted covalent inhibitors, we incorporated a series of covalent
2
,26,35
warheads
into the tetracyclic ring system of 6, made a series of analogues, and
studied their bioactivities. Herein, we report the synthesis and bioactivity studies of
covalent-warhead-containing compounds derived from 6.
2
. Results and Discussion
Two design strategies were undertaken to modify structures of 6 as shown in Fig. 2.
Strategy 1 aims to modify the quinazolin-4(3H)-one moiety and strategy 2 aims to
modify the methyl group.
Figure 2: Modification strategies of 6
Previous studies have confirmed that the acrylamide and the propionate moieties of
3
6,37
38,39
BTK
and EGFR
inhibitors selectively formed covalent bonds with the cysteine
residues in the kinase active sites. In addition, chloroacetyl and vinyl sulfonyl groups
are electrophilic warheads that are capable of forming covalent bonds with active
3
5
cysteine residues . The oxirane-2-carboxamide moiety is also an electrophilic groups
40
in the design of a protease inhibitor , which forms a covalent bond with the active
site Thr residue. Therefore, the aforementioned electrophilic warheads were adopted
in our studies and incorporated into the core structure of 6.
First, the strategy 1 was explored and the quinazolin-4(3H)-one moiety was

replaced by electrophilic warheads as shown in Table 1. Compounds 7-11 were thus
synthesized and their cell growth inhibition activities were tested in leukemia cell line
3
2
K562, which was previously adopted as a model cell line to assay the activity of 6 .
In addition, a multiple myeloma cell line MM1.S, an acute myeloid leukemia cell line
MV4-11, and a colon cancer cell line RKO were also included to assay the cell
growth inhibition activities of 7-11. As shown in Table 1, 6 has an IC value of 98.62
5
0
μM in the K562 cell line, which is different from previous reported result (IC = 21
5
0
3
2
nM) using naturally isolated 6 . In order to examine this discrepancy, a FDA
approved drug Decitabine was included as a positive control, which inhibited K562
4
1,42
cell growth with IC value of 30 nM and is in line with previously reported data
.
5
0
The structure of in-house synthesized 6 was confirmed by X-ray crystallography
4
3
studies (SI, Table S1) . In addition, the isolated 6 from natural source has a specific
3
2
optical rotation value of -70 (c 0.48 in MeOH) , while the synthetic 6 has that of
4
4
45
-
49.7 (c 0.48 in MeOH) and -48 (c 0.45, MeOH) in literatures and of -51.3 (c 0.48
in MeOH) for our in-house data. The specific optical rotation value of natural 6 is
different from all synthetic ones and this inconsistency may partially explain the
discrepancy of their cellular activities.
In addition, 6 has IC values of 65.48, 79.23, and 169.53 μM in MM1.S, MV4-11,
5
0
and RKO cell lines, respectively. Generally, compounds 7-11 have similar cell growth
inhibition activities as that of 6. In particular, 8 was 3 folds more potent than 6 in
MM1.S cell line, whereas 11 was 5 folds and 3 folds more potent than 6 in K562 and
MM1.S cell lines, respectively. Compounds comprising highly electrophilic

acrylamide, chloroacetyl, and vinyl sulfonyl groups did not have strong toxicity but
comparable activity as 6, suggesting non-specific toxicity of these electrophilic
groups is minimal in these four cell lines.
Table 1: Cell growth inhibition activities of the derivatives of 6
a
IC (μM)
5
0
K562
MM.1S
MV4-11
79.23±1.06
64.47±5.97
RKO
6
(Chaetominine)
98.62±13.56 65.48±9.52
106.80±1.12 52.23±3.92
169.53±6.39
196.70±45.99
7
8
9
98.19±1.67
86.95±5.54
22.41±1.57 49.07±10.82 128.30±19.47
59.84±1.39
63.68±5.70
72.98±5.55
132.47±9.44
165.05±13.55
89.76±9.77
10
104.12±4.20 88.04±0.79
11
17.69±4.00
0.03±0.02
21.54±0.54 42.33±11.23
1.12±0.23 0.21±0.16
1
2 (Decitabine)
16.07±7.24
a
All the values are means of three independent experiments.
Next, the quinazolin-4(3H)-one moiety of 6 was degraded, while compounds
3-27 were synthesized and their cell growth inhibition activities were tested, the
1
results are summarized as shown in Table 2. In general, 15, 16, 17, 18, 20, 21, 24, 26,
and 27 had lower cell growth inhibition IC values compared to that of 6. Among
5
0
these, 15, 18, 21, 24, and 27 had para substitution pattern, while 17, 20, and 26 had
meta substitution pattern, indicating these two positions are optimal for improved
potency. The most potent compound is 18, featuring a chloroacetyl group at the para
position. However, the other two chloroacetyl-group-containing compounds also have

very low IC values, suggesting the activities may be non-specific.
5
0
Table 2: Cell growth inhibition activities of compounds derived from 6
a
IC (μM)
5
0
K562
MM.1S
54.09±1.77
66.97±4.64
61.30±4.21
13.98±0.941
3.89±0.12
MV4-11
73.75±1.88
58.61±14.01
9.79±2.80
RKO
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
106.17±3.42
67.88±2.08
30.15±7.94
22.22±1.365
7.04±0.59
116.80±3.71
107.83±7.22
88.67±3.41
36.04±3.649
18.02±1.38
14.59±0.93
101.68±7.53
93.94±2.43
90.09±2.33
96.11±7.43
97.56±5.85
94.07±8.31
66.48±5.52
83.87±1.10
83.82±0.94
20.28±4.347
3.39±0.13
4.92±0.23
4.80±0.67
4.40±0.36
60.23±25.97
17.69±5.02
24.46±0.51
93.52±5.04
55.34±13.83
40.87±7.09
29.50±1.89
39.02±2.33
36.07±2.34
17.68±3.90
34.47±2.09
24.14±2.62
82.57±10.32
56.83±10.62
47.25±4.27
26.09±0.75
22.80±0.92
21.23±1.00
29.40±24.16
25.13±1.48
16.04±0.98
77.23±2.43
66.30±2.33
24.77±5.29
41.72±21.24
17.75±1.09
15.36±2.48
a
All the values are means of three independent experiments.
The acrylamide moiety containing compound 15 has improved IC values in K562
5
0
and MM1.S cell line, while its close analogues 13 and 14 have equal IC values as
5
0
that of 6, suggesting the improved cellular activity of 15 requires optimal orientation

of the acrylamide moiety.
During our synthesis of 6, we found that its C=N bond was reduced to a C-N bond
in the presence of NaBH CN and 28 was obtained in good yield. Therefore, three
3
derivatives of 28 were made and tested in cell growth inhibition assays, the results are
summarized in Table 3.
Table 3: Cell growth inhibition activities of compounds derived from over-reduced 6
a
IC (μM)
5
0
K562
MM.1S
MV4-11
RKO
2
2
3
3
8
9
0
1
100.70±6.19 59.59±13.40 64.54±24.59 152.26±43.78
5.61±0.33
1.95±0.57
97.18±2.98
7.18±0.37
3.31±0.03
58.61±2.01
8.54±2.45
2.12±0.59
74.83±4.59
18.80±0.76
5.46±0.51
124.50±5.34
a
All the values are means of three independent experiments.
Interestingly, the cellular growth inhibition IC values of 29 are 5.61, 7.18, 8.54,
5
0
and 18.80 μM in K562, MM1.S, MV4-11, and RKO cell lines, respectively. These
numbers account for 17, 9, 9, and 9 folds improvement compared to that of 6. The
cellular growth inhibition IC values of 30 are 1.95, 3.31, 2.12, and 5.46 μM in K562,
5
0
MM1.S, MV4-11, and RKO cell lines, respectively, accounting for 50, 19, 37, and 31
folds improvement compared to that of 6. On the contrary, both 28 and 31 have
similar cell growth inhibition activities as that of 6. It is worthy of note that the
acrylamide-containing compounds 7, 13, 14, and 15 inhibit cell growth with much

greater IC values, i.e. much less potent. The significant difference between 29 and
5
0
its analogues 7, 13, 14, 15 suggests that the activity of 29 may be target-specific rather
than non-specific toxicity.
In addition to the strategy 1, additional modifications of 6 were performed on the
methyl group according to the strategy 2. Compounds 32-42 were synthesized and
tested in cell growth inhibition assay, the results are summarized in Table 4.
Table 4: Cell growth inhibition activities of compounds based on the strategy 2
a
IC (μM)
5
0
K562
MM.1S
MV4-11
RKO
3
2
123.06±7.48
84.15±7.55
70.89±11.26
138.4±16.85
3
3
3
3
3
3
3
4
4
4
3
4
5
6
7
8
9
0
1
2
163.35±102.47
29.18±3.89
103.63±6.74
102.67±6.16
14.85±8.88
94.79±9.83
38.02±3.13
96.97±11.15
100.08±11.39
45.77±14.03
87.64±7.94
13.49±0.86
88.43±4.96
82.37±3.26
51.53±21.79
83.56±15.76
35.69±13.00
90.45±13.31
86.10±1.54
88.23±25.43
64.42±10.26
24.63±5.76
70.35±15.45
70.72±11.76
61.30±5.92
159.4±4.40
43.84±3.85
131.4±8.05
163.8±34.38
85.23±13.78
92.42±11.98 177.77±33.32
43.16±11.17 245.55±22.27
95.70±21.36
148.60±8.63
86.89±13.18 143.75±13.51
61.02±18.98 116.31±22.27
a
All the values are means of three independent experiments.
In this series of compounds, a chloroacetyl group-containing compound 34 was

more potent than 6 in the cell growth inhibition assays, which is in line with the facts
that 16, 17, 18, and 30, all chloroacetyl-containing compounds, generally have higher
cell potency. 37 has no electrophilic warhead and it inhibited K562 growth with an
IC value of 14.85 μM, 4 folds more potent than that of 6. The other compounds have
5
0
similar cell growth inhibition activity as that of 6. Taken together, the modifications
according to the strategy 2 is less successful in term of improve cellular antitumor
potency in cancer cell lines.
3
. Chemistry
The syntheses of final compounds 7-11 have been summarized in Scheme 1. A
45,46
previously reported compound S4
was used as the starting material and
electrophilic warhead moieties were incorporated at the NH group, furnishing 7a-11a.
2
Removal of the TES group of 7a-11a yielded the final compounds 7-11 in moderate to
good yields.
Scheme 1: Syntheses of final compounds 7-11
Reaction Conditions: (a) Acryloyl chloride, Et N, THF, 2h; (b) Chloroacetyl chloride,
3
Et N, THF, 2h; (c) Oxirane-2-carboxylic acid , Isobutyl chloroformate, NMM, THF,
3
overnight; (d) 2-Butynoic acid, Isobutyl chloroformate, NMM,THF, overnight; (e)

(
2-Chloroethyl)sulphonyl chloride, Et N, THF, 2h; (f) HF, MeCN, overnight; (g) CsF,
3
DMSO, 2h.
The syntheses of 13-27 have been summarized in Scheme 2.
Scheme 2: Syntheses of final compounds 13-27
Reaction conditions: (a) Nitrobenzoyl chloride, Et N, THF, 2h; (b) Pd/C, H , THF,
3
2
overnight; (c) Acryloyl chloride, Et N, THF, 2h; (d) 2-Chloroacetyl chloride, Et N,
3
3
THF, 2h; (e) Oxirane-2-carboxylic acid, isobutyl chloroformate, NMM, THF,
overnight; (f) 2-Butynoic acid, isobutyl chloroformate, NMM, THF, overnight; (g)
(
2-Chloroethyl)sulphonyl chloride, Et N, THF, 2h; (h) HF, MeCN, overnight; (i) CsF,
3

DMSO, 2h.
4
5,46
In addition, S5-S7 were synthesized in good yields using S4
and three
nitrobenzoyl chlorides as the starting materials. Pd-catalyzed hydrogenation of the
NO groups of S5-S7 yielded aniline-containing compounds S8-S10. Subsequently,
-
2
S8-S10 were allowed to react with acryloyl chloride, 2-chloroacetyl chloride,
oxirane-2-carboxylic acid, 2-butynoic acid, or (2-chloroethyl)sulphonyl chloride
accordingly, affording 13a-27a. Finally, the TES groups of 13a-27a were removed
and the final compounds 13-27 were obtained in moderate to good yields.
The final compounds 28-31 were synthesized from a previously reported compound
47
S1 as outlined in Scheme 3.
Scheme 3: Syntheses of final compounds 28-31
Reaction conditions: (a) O , CH Cl , overnight; (b) NaBH CN, AcOH, MeOH, 5h;
2
2
2
3
then silica gel, NH ·H O:EtOH:Acetone:CH Cl =0.5:1:4:50, overnight. (c) Acryloyl
3
2
2
2
chloride, Et N, THF, 2h; (d) Chloroacetyl chloride, Et N, THF, 2h; (e) Propionyl
3
3
chloride, Et N, THF, 2 h.
3
4
8
Using a previously reported oxidation-reduction method, S1 was converted into 6,

while 28 was also obtained as a minor product, apparently through reduction of the
C=N bond of the quinazolin-4(3H)-one moiety. The structure and stereochemistry of 6
4
3
have been confirmed by X-ray crystallography analysis of its single crystals . Finally,
different electrophilic and the propionyl groups were incorporated onto 28 through the
corresponding condensation reactions with the NH group, providing final compounds
2
9-31.
The final compounds 37-42 were synthesized from a previously reported compound
4
9,50
S11
over 8 linear steps as shown in Scheme 4.
Scheme 4: Syntheses of final compounds 32-36
o
Reaction conditions: (a) Cl-COOiBu, NMM,THF, -30 C, rt, overnight; (b) Pd/C,
o
MeOH, rt, overnight; (c) (C H O) CH, p-TsOH, 30 C, overnight; (d) NCS, Et N,
2
5
3
3

o
CH Cl , 0 C-rt, overnight; (e) O , CH Cl , rt, overnight; (f) NaBH CN, AcOH,
2
2
2
2
2
3
o
MeOH,35 C, 5h; then silica gel, NH ·H O:EtOH:Acetone:CH Cl = 0.5 :1 :4 :50, 35
3
2
2
2
o
C, overnight; (g) CH Cl /TFA (v/v=4/1), rt, 2h; (h) Acryloyl chloride, Et N, THF, rt,
2
2
3
2
h; (i) 2-Chloroacetyl chloride, Et N, THF, rt, 2h; (j) Oxirane-2-carboxylic acid,
3
o
isobutyl chloroformate, NMM, THF, 0 C-rt, overnight; (k) 2-Butynoic acid, isobutyl
chloroformate, NMM, THF, rt, overnight.
The amide condensation of S11 and ε-Boc-L-lysine methyl ester provided S12,
which was reduced to aniline-containing S13. In the presence of p-TsOH and triethyl
4
6,48
orthoformate,
cyclization of S13 led to formation of the quinazolin-4(3H)-one
moiety of S14. In the presence of NCS, further cyclization of S14 led to formation
48
S15, which underwent oxidation-reduction cascade reactions and provided S16 as
the key intermediate. Subsequent intra-molecular cyclization of S16 was promoted by
4
8
the presence of wet silica gel, providing S17 in good yield. The Boc protecting
group of S17 was removed facilitated by treatment of TFA, furnishing 32. Finally, the
electrophilic warheads were installed using similar methods showing in Scheme 2 and
the desired products 33-36 were obtained accordingly.
As shown in Scheme 5, the final compounds 37-42 were synthesized from S11
and (2S)-2-amino-3-(Boc-amino)propionic acid methyl ester through practically same
synthetic sequences as shown in Scheme 4. The key difference is
(2S)-2-amino-3-(Boc-amino)propionic acid methyl ester was used instead of
ε-Boc-L-lysine methyl ester, which enables 37-42 with a shorter side chain compared
to that of 32-36.

Scheme 5: Syntheses of final compounds 37-42
o
Reaction conditions: (a) Cl-CO (i-Bu), NMM, THF, -30 C, rt, overnight; (b) Pd/C,
2
o
MeOH, rt, overnight; (c) (C H O) CH, p-TsOH, 30 C, overnight; (d) NCS, Et N,
2
5
3
3
o
CH Cl , 0 C-rt, overnight; (e) O , CH Cl , rt, overnight; (f) NaBH(OAc) , AcOH,
2
2
2
2
2
3
o
DCE, 35 C, overnight; (g) CH Cl /TFA (v/v= 4/1), rt, 2h; (h) Acryloyl chloride, Et N,
2
2
3
THF, rt, 2h; (i) 2-Chloroacetyl chloride, Et N, THF, rt, 2h; (j) Oxirane-2-carboxylic
3
o
acid, isobutyl chloroformate, NMM, THF, 0 C-rt, overnight; (k) 2-Butynoic acid,
o
isobutyl chloroformate, NMM, THF, 0 C-rt, overnight; (l) 2-Chloroethanesulfonyl
chloride, Et N, THF, rt, 2h.
3

4
. Conclusions
In summary, we have reported modifications of the natural product
(-)-Chaetominine (6) through two strategies. The key feature of these modifications is
incorporation of electrophilic groups that are capable of forming covalent bonds with
the cysteine or threonine residues of cellular proteins. The cell growth inhibition
activities of compounds derived from 6 were tested in four cancer cell lines, i.e. a
leukemia cell line K562, a multiple myeloma cell line MM1.S, an acute myeloid
leukemia cell line MV4-11, and a colon cancer cell line RKO. The data show that
cellular growth inhibition IC values of 29, an acrylamide-containing molecule, are
5
0
9
-17 folds more potent than that of 6, while other acrylamide-containing compounds
are much less potent, suggesting 29 might have covalent interactions with cellular
proteins. In addition, chloroacetyl-containing compounds are generally more potent
than 6 in cell growth inhibition assays, indicating chloroacetyl is over-reactive and
less selective. Collectively, the incorporation of an acrylamide moiety into 6 is a good
strategy to improve its antitumor activity in cancer cell lines. Further identification of
the cellular target proteins of 29 is underway and will be reported in due course.
5
. Experimental sections
5
.1 General Methods
All the final products were purified on a preparative HPLC (Waters 2545) with a C18
reverse phase column. The mobile phase used here was a gradient flow of solvent A (water,
.1% of TFA) and solvent B (CH CN, 0.1% of TFA) at a flow rate of 40 mL/min. The
0
3
analytical UPLC model was Waters Acquity H class (UV detection at 230 nm and 254 nm)

and the reverse phase column used was the Acquity UPLC® BEH (C18-1.7 µm, 2.1 × 50
mm). All final compounds were purified to ≥ 95% purity as determined by analytical UPLC
1
13
analysis. H and C NMR spectra were recorded with a Bruker Advance NMR 400
1
spectrometer and a Bruker Advance NMR 500 spectrometer. H NMR spectra were
calibrated with tetramethylsilane ( = 0 ppm), DMSO-d ( = 2.50 ppm), MeOH-d (
6
4
1
3
=
3.31 ppm), or CDCl ( = 7.26 ppm). C NMR spectra were calibrated with
3
DMSO-d ( = 39.5 ppm), MeOH-d ( = 49.0 ppm) or CDCl ( = 77.2 ppm).
6
4
3
Multiplicities are given as follows: s (singlet), d (doublet), t (triplet), q (quartet), dd
doublet of doublets), dt (doublet of triplets), and m (multiplets). High-resolution
(
mass spectral (HRMS) analysis was performed on UHPLC-QTOF (Agilent
Corporation) and Thermo DFS (Thermo Fisher Scientific).
a) Procedure for the synthesis of 7a (general Method A). S4 (49 mg, 0.13 mmol, 1.0
eq) and triethylamine (28 mg, 0.26 mmol, 2.0 eq) were dissolved in CH Cl (5 mL).
2
2
Acryloyl chloride (13 mg, 0.14 mmol, 1.1 eq) was added dropwise. The reaction was
stirred at room temperature for 2 h. The mixture was then diluted with water (20 mL)
and the aqueous layer was extracted with ethyl acetate (20 mL × 2). The combined
organic layers were dried over anhydrous sodium sulfate. The solvent was removed
on a rotary evaporator, and the remaining residues were purified by flash column
chromatography to provide 7a (39 mg, 71% yield). The syntheses of 13a, 14a, 15a,
2
9, 33, 38, and S25 were similar to that of 7a.
b) Procedure for the synthesis of 8a (general Method B). S4 (50 mg, 0.13 mmol, 1.0
eq) and triethylamine (28 mg, 0.26 mmol, 2.0 eq) were dissolved in CH Cl (5 mL).
2
2

2
-Chloroacetyl chloride (18 mg, 0.15 mmol, 1.1 eq) was added dropwise. The
reaction was stirred at room temperature for 2 h. The mixture was then diluted with
water (20 mL) and the aqueous layer was extracted with ethyl acetate (20 mL×2). The
combined organic layers were dried over anhydrous sodium sulfate. The solvent was
removed on a rotary evaporator and the remaining residues were purified by flash
column chromatography to provide 8a (40 mg, 67% yield). The syntheses of 16a, 17a,
1
8a, 30, 34, and 39 were similar to that of 8a.
c) Procedure for the synthesis of 9a (general Method C). N-Methyl morpholine (26
mg, 0.24 mmol, 2.0 eq) and isobutyl chlorocarbonate (41 mg, 0.3 mmol, 2.5 eq) were
sequentially added to a solution of oxirane-2-carboxylic acid (26 mg, 0.24 mmol, 2.0
o
o
eq) in anhydrous THF (5 mL) at 0 C. After being stirred at 0 C for 1 h, the
suspension was slowly added to a solution of S4 (46 mg, 0.12 mmol, 1.0 eq) and
o
N-methyl morpholine (26 mg, 0.24 mmol, 2.0 eq) in anhydrous THF (5 mL) at 0 C.
The reaction was stirred at room temperature for 12 h. The mixture was then diluted
with water (20 mL) and the aqueous layer was extracted with ethyl acetate (20 mL×2).
The combined organic layers were dried over anhydrous sodium sulfate. The solvent
was removed on a rotary evaporator and the remaining residues were purified by flash
column chromatography to provide 9a (37 mg, 69% yield). The syntheses of 19a, 20a,
2
1a, 35, and 40 were similar to that of 9a.
d) Procedure for the synthesis of 10a (general Method D). N-Methyl morpholine (30
mg, 0.52 mmol, 2.0 eq) and isobutyl chlorocarbonate (35 mg, 0.24 mmol, 0.9 eq)
were successively added to a solution of but-2-ynoic acid (22 mg, 0.26 mmol, 1.0 eq)

o
o
in anhydrous THF (5 mL) at 0 C. After being stirred at 0 C for 1 h, the suspension
was slowly added to a solution of S4 (100 mg, 0.26 mmol, 1.0 eq) and N-methyl
o
morpholine (22 mg, 0.26 mmol, 1.0 eq) in anhydrous THF (5 mL) at 0 C. The
reaction was stirred at room temperature for 12 h. The mixture was then diluted with
water (20 mL) and the aqueous layer was extracted with ethyl acetate (20 mL×2). The
combined organic layers were dried over anhydrous sodium sulfate. The solvent was
removed on a rotary evaporator and the remaining residues were purified by flash
column chromatography to provide 10a (46 mg, 40% yield). The syntheses of 22a,
2
3a, 24a, 36, and 41 were similar to that of 10a.
e) Procedure for the synthesis of 11a (general Method E). S4 (35 mg, 0.09 mmol, 1.0
eq) was dissolved in CH Cl (5 mL). 2-Chloroethane-1-sulfonyl chloride (1.8 mg,
2
2
0
.11 mmol, 1.2 eq) and triethylamine (14 mg, 0.14 mmol, 1.5 eq) were added
sequentially. The reaction was stirred at room temperature for 12 h. The mixture was
then diluted with water (20 mL) and the aqueous layer was extracted with ethyl
acetate (20 mL×2). The combined organic layers were dried over anhydrous sodium
sulfate. The solvent was removed on a rotary evaporator and the remaining residues
were purified by flash column chromatography to provide 11a (18 mg, 42% yield).
The syntheses of 25a, 26a, 27a, and 42 were similar to that of 11a.
f) Procedure for deprotection of triethylsilyl using hydrofluoric acid (general Method
F). 7a (60 mg, 0.14 mmol, 1.0 eq) was dissolved in acetonitrile (5 mL). Hydrofluoric
acid (49% in water, 0.29 mL, 5.0 eq) was added and the reaction was stirred at room
temperature for 12 h. The solvent was removed on a rotary evaporator and the

remaining residues were purified by reverse phase HPLC to provide 7 (23 mg, 53%
yield). The syntheses of 8, 9, 10, 13, 14, 16, 17, 19, 20, 22, 23, and 25 were similar to
that of 7.
g) Procedure for deprotection of triethylsilyl using cesium fluoride (general Method
G). 11a (18 mg, 0.04 mmol, 1.0 eq) was dissolved in DMSO (2 mL). Cesium fluoride
(9 mg, 0.06 mmol, 1.5 eq) was added and the reaction was stirred at room temperature
for 2 h. The water (3 mL) was added and the solution was purified by reverse phase
HPLC to provide 11 (7.8 mg, 57% yield). The syntheses of 15, 18, 21, 24, 26, and 27
were similar to that of 11.
5
.2 Synthesis of Final Compounds
1
1
(
2S,2a S,4R,5aS)-5a-hydroxy-2-methyl-4-(4-oxoquinazolin-3(4H)-yl)-2a ,4,5,5a-te
trahydro-3H-2a,9b-diazacyclopenta[jk]fluorene-1,3(2H)-dione (1, (-)-Chaetomi
nine) (6). S1 (1.6g, 3.8 mmol) was dissolved in CH Cl (10 mL). The reaction
-
2
2
system was degassed, filled with oxygen and stirred at room temperature for 12 h. The
solvent was removed on a rotary evaporator and the remaining residues were purified
by flash column chromatography. S2 was isolated in 55% yield (910 mg). Then S2
was dissolved in methanol (10 mL), followed by acetic acid (0.6mL) and sodium
o
cyanoborohydride (264 mg, 4.2 mmol). The reaction was stirred at 35 C for 5 h, and
quenched by saturated sodium bicarbonate solution (20 mL). The mixture was
extracted with ethyl acetate (20 mL×2). The combined organic layers were dried over
anhydrous sodium sulfate. The solvent was removed on a rotary evaporator. The
remaining residues were dissolved in the mixed solvent (10 mL, NH -H O : EtOH :
3
2

Acetone : CH Cl = 0.5:1:4:50) followed by addition of silica gel (2.7 g). The reaction
2
2
o
was stirred at 35 C for 12 h. The solvent was removed on a rotary evaporator and the
remaining residues were purified by flash column chromatography. 6 was obtained as
1
the major product in 36%, while 28 was isolated in 18% yield (151 mg). H NMR
(
400 MHz, DMSO-d ): δ 8.29 (s, 1H), 8.18 (d, J = 7.9 Hz, 1H), 7.90-7.82 (m, 1H),
6
7
7
.69 (d, J = 8.1 Hz, 1H), 7.58 (t, J = 7.6 Hz, 1H), 7.50 (t, J = 7.9 Hz, 2H), 7.43 (t, J =
.7 Hz, 1H), 7.25 (t, J = 7.4 Hz, 1H), 6.69 (s, 1H), 5.91 (s, 1H), 5.60 (s, 1H), 4.61 (q,
J = 6.9 Hz, 1H), 2.93 (t, J = 12.8 Hz, 1H), 2.58-2.50 (m, 1H), 1.60 (d, J = 7.0 Hz, 3H).
1
3
C NMR (125 MHz, DMSO-d ): δ 172.1, 160.0, 147.4, 138.7, 136.7, 134.7, 129.9,
6
1
27.3, 127.2, 126.4, 125.5, 124.9, 114.5, 82.5, 76.4, 59.6, 38.2, 14.0. HRMS (ESI)
+
20 ºC
D
calculated for C H N O [M+H] = 403.1401, obtained: 403.1401. [푎]
= - 51.3
2
2
19
4
4
o
(c = 0.48, MeOH).
1
(
2S,2a S,4R,5aS)-5a-Hydroxy-2-methyl-4-(4-oxo-1,4-dihydroquinazolin-3(2H)-yl)
1
-
2a ,4,5,5a-tetrahydro-3H-2a,9b-diazacyclopenta[jk]fluorene-1,3(2H)-dione (28)
1
H NMR (400 MHz, DMSO-d ): δ 7.80 (dd, J = 7.9, 1.6 Hz, 1H), 7.57 (d, J = 7.9 Hz,
6
1
7
H), 7.50 (dd, J = 7.7, 1.2 Hz, 1H), 7.42 (td, J = 7.8, 1.3 Hz, 1H), 7.33 (ddd, J = 8.5,
.3, 1.6 Hz, 1H), 7.26 (td, J = 7.6, 1.1 Hz, 1H), 6.88-6.76 (m, 2H), 5.52 (s, 1H), 4.65
(d, J = 9.4 Hz, 1H), 4.56 (q, J = 6.9 Hz, 1H), 2.63-2.44 (m, 2H), 1.70 (d, J = 7.0 Hz,
1
3
3
1
1
4
H). C NMR (125 MHz, DMSO-d ): δ 172.4, 166.3, 163.4, 149.3, 138.8, 137.1,
6
33.1, 129.7, 128.2, 125.4, 124.9, 117.6, 115.8, 114.7, 114.5, 82.3, 76.4, 59.2, 37.2,
-
4.2. HRMS (ESI) calculated for C H N O [M-H] = 403.1412, obtained:
2
2
19
4
4
03.1403. [푎]2
0 ºC
= - 32.0 (c = 0.7, MeOH).
o
D

1
1
N-((2S,2a S,4R,5aS)-5a-Hydroxy-2-methyl-1,3-dioxo-1,2,2a ,4,5,5a-hexahydro-3
H-2a,9b-diazacyclopenta[jk]fluoren-4-yl)acrylamide (7) Method F using 7a (60 mg,
1
0
1
1
5
1
.14 mmol): 23 mg, 53% yield. H NMR (400 MHz, DMSO-d ): δ 8.39 (d, J = 8.5 Hz,
6
H), 7.50-7.43 (m, 2H), 7.39 (t, J = 7.7 Hz, 1H), 7.22 (t, J = 7.5 Hz, 1H), 6.34 (dd, J =
7.1, 10.2 Hz, 1H), 6.15 (dd, J = 17.1, 2.2 Hz, 1H), 5.65 (dd, J = 10.1, 2.2 Hz, 1H),
.46 (s, 1H), 4.85-4.69 (m, 1H), 4.56 (q, J = 6.9 Hz, 1H), 2.42 (dd, J = 12.9, 3.3 Hz,
1
3
H), 1.93 (t, J = 12.8 Hz, 1H), 1.54 (d, J = 6.9 Hz, 3H). C NMR (125 MHz,
MeOD-d ): δ 174.2, 169.8, 168.0, 140.5, 137.5, 131.8, 131.1, 127.4, 126.9, 125.6,
4
-
1
3
2
16.2, 84.4, 77.9, 61.0, 40.7, 15.1. HRMS (ESI) calculated for C H N O [M-H] =
1
7
16
3
4
26.1146, obtained: 326.1141.[푎] 2D 0 ºC = - 9.4 (c = 0.6, MeOH).
o
1
-Chloro-N-((2S,4R,5aS)-5a-hydroxy-2-methyl-1,3-dioxo-1,2,2a ,4,5,5a-hexahydr
o-3H-2a,9b-diazacyclopenta[jk]fluoren-4-yl)acetamide (8). Method F using 8a (40
1
mg, 0.09 mmol): 18 mg, 61% yield. H NMR (400 MHz, MeOD-d ): δ 7.55 (d, J =
4
7
5
2
.9 Hz, 1H), 7.47 (d, J = 7.5 Hz, 1H), 7.41 (t, J = 7.7 Hz, 1H), 7.26 (t, J = 7.6 Hz, 1H),
.50 (s, 1H), 4.85 (dd, J = 12.7, 3.4 Hz, 1H), 4.56 (q, J = 6.9 Hz, 1H), 4.17 (s, 2H),
1
3
.59 (dd, J = 13.1, 3.4 Hz, 1H), 2.07 (t, J = 12.8 Hz, 1H), 1.68 (d, J = 6.9 Hz, 3H). C
NMR (125 MHz, MeOD-d ): δ 174.1, 169.5, 169.2, 140.5, 137.5, 131.1, 126.9, 125.6,
4
3
5
1
16.2, 84.4, 77.8, 61.0, 43.2, 40.3, 15.1. HRMS (ESI) calculated for C H ClN O4
16 15 3
-
20 ºC
D
o
[
M-H] = 348.0757, obtained: 348.0748. [푎]
= - 7.4 (c = 1.0, MeOH).
1
N-((2S,4R,5aS)-5a-Hydroxy-2-methyl-1,3-dioxo-1,2,2a ,4,5,5a-hexahydro-3H-2a,9
b-diazacyclopenta[jk]fluoren-4-yl)oxirane-2-carboxamide (9). Method F using 9a
1
(
37 mg, 0.09 mmol): 10 mg, 35% yield. H NMR (400 MHz, MeOD-d ): δ 7.55 (d, J
4

=
7.9 Hz, 1H), 7.47 (d, J = 7.6 Hz, 1H), 7.41 (t, J = 7.7 Hz, 1H), 7.25 (t, J = 7.6 Hz,
H), 5.49 (s, 1H), 4.88-4.81 (m, 1H), 4.54 (q, J = 6.9 Hz, 1H), 3.48 (dd, J = 4.5, 2.5
Hz, 1H), 3.01 (dd, J = 6.1, 4.4 Hz, 1H), 2.96 (dd, J = 6.1, 2.5 Hz, 1H), 2.53 (dd, J =
1
1
3
1
3.1, 3.4 Hz, 1H), 2.13-2.00 (m, 1H), 1.67 (d, J = 6.9 Hz, 3H). C NMR (125 MHz,
MeOD-d ): δ 174.1, 171.7, 169.5, 140.5, 137.5, 131.1, 126.9, 125.6, 116.2, 84.4, 77.9,
4
-
6
3
1.0, 49.9, 47.5, 40.3, 15.0. HRMS (ESI) calculated for C H N O [M-H] =
1
7
16
3
5
42.1095, obtained: 342.1088. [푎] 2D 0 ºC = - 24.9 (c = 0.3, MeOH).
o
N-((2S,4R,5aS)-5a-Hydroxy-2-methyl-1,3-dioxo-1,2,2a1,4,5,5a-hexahydro-3H-2a,
9
b-diazacyclopenta[jk]fluoren-4-yl)but-2-ynamide (10). Method F using 10a (46
1
mg, 0.10 mmol): 23 mg, 67% yield. H NMR (400 MHz, MeOD-d ): δ 7.55 (d, J = 7.9
4
Hz, 1H), 7.47 (d, J = 7.6 Hz, 1H), 7.41 (t, J = 7.8 Hz, 1H), 7.25 (t, J = 7.6 Hz, 1H),
5
1
3
1
.48 (s, 1H), 4.84 (dd, J = 12.7, 3.8 Hz, 1H), 4.54 (q, J = 6.9 Hz, 1H), 2.52 (dd, J =
3.1, 3.4 Hz, 1H), 2.06 (dd, J = 12.4, 8.6 Hz, 1H), 2.01 (s, 3H), 1.67 (d, J = 6.9 Hz,
1
3
H). C NMR (125 MHz, MeOD-d ): δ 174.1, 171.2, 169.2, 140.5, 137.4, 131.1,
4
26.9, 125.6, 116.1, 85.7, 84.3, 77.8, 75.1, 61.0, 40.4, 15.1, 3.1. HRMS (ESI)
-
20 ºC
D
o
calculated for C H N O [M-H] = 338.1146, obtained: 338.1138. [푎]
= + 1.3
1
8
16
3
4
(c = 0.5, MeOH).
1
N-((2S,2a S,4R,5aS)-5a-hydroxy-2-methyl-1,3-dioxo-1,2,2a1,4,5,5a-hexahydro-3H
-2a,9b-diazacyclopenta[jk]fluoren-4-yl)ethenesulfonamide (11). Method G using
1
1
1a (49 mg, 0.13 mmol): 39 mg, 71% yield. H NMR (500 MHz, MeOD-d ): δ 7.52 (d,
4
J = 7.9 Hz, 1H), 7.47-7.44 (d, J = 7.5 Hz, 1H), 7.39 (t, J = 7.8 Hz, 1H), 7.23 (t, J = 7.5
Hz, 1H), 6.78 (dd, J = 16.6, 9.9 Hz, 1H), 6.22 (d, J = 16.6 Hz, 1H), 5.96 (d, J = 9.9 Hz,

1
H), 5.42 (s, 1H), 4.50 (q, J = 7.0 Hz, 1H), 4.23 (dd, J = 12.5, 3.4 Hz, 1H), 2.66 (dd, J
1
3
=
13.2, 3.4 Hz, 1H), 1.97 (t, J = 12.9 Hz, 1H), 1.65 (d, J = 7.0 Hz, 3H). C NMR
(
125 MHz, MeOD-d ): δ 174.0, 169.4, 140.5, 138.3, 137.4, 131.1, 127.0, 126.2, 125.6,
4
1
16.1, 84.2, 77.9, 61.0, 52.8, 42.7, 15.1. HRMS (ESI) calculated for C H N O S
16 18 3 5
+
20 ºC
D
o
[
M+H] = 364.0962, obtained: 364.0959. [푎]
= + 15.3 (c = 0.4, MeOH).
1
1
2
-Acrylamido-N-((2S,2a S,4R,5aS)-5a-hydroxy-2-methyl-1,3-dioxo-1,2,2a ,4,5,5a-
hexahydro-3H-2a,9b-diazacyclopenta[jk]fluoren-4-yl)benzamide (13). Method F
1
using 13a (23 mg, 0.04 mmol): 8 mg, 44% yield. H NMR (500 MHz, Methanol-d ):
4
δ 8.50 (d, J = 8.3 Hz, 1H), 7.73 (dd, J = 7.8, 1.6 Hz, 1H), 7.60-7.37 (m, 4H),
7
5
1
3
1
5
4
3
.29-7.20 (m, 2H), 6.51 (dd, J = 17.0, 10.2 Hz, 1H), 6.38 (dd, J = 17.1, 1.5 Hz, 1H),
.83 (dd, J = 10.2, 1.5 Hz, 1H), 5.08 (dd, J = 12.8, 3.4 Hz, 1H), 4.61 (q, J = 6.9 Hz,
H), 2.63 (dd, J = 13.2, 3.4 Hz, 1H), 2.19 (t, J = 12.9 Hz, 1H), 1.72 (d, J = 7.0 Hz,
1
3
H). C NMR (125 MHz, Methanol-d ): δ 172.6, 169.5, 168.7, 164.7, 139.1, 137.6,
4
36.1, 131.8, 131.5, 127.6, 126.5, 125.6, 124.3, 123.5, 121.2, 114.8, 83.1, 76.6, 60.1,
+
9.7, 48.3, 38.8, 13.7, 13.1. HRMS (ESI) calculated for C H N O [M+H] =
2
4
23
4
5
47.1663, obtained: 447.1666. [푎] 2D 0 ºC = - 4.0°(c = 0.3, MeOH).
1
1
-Acrylamido-N-((2S,2a S,4R,5aS)-5a-hydroxy-2-methyl-1,3-dioxo-1,2,2a ,4,5,5a-
hexahydro-3H-2a,9b-diazacyclopenta[jk]fluoren-4-yl)benzamide (14). Method F
1
using 14a (13 mg, 0.02 mmol): 7 mg, 68% yield. H NMR (500 MHz, Methanol-d ):
4
δ 8.15 (t, J = 2.0 Hz, 1H), 7.86 (dd, J = 8.1, 2.1 Hz, 1H), 7.64 (dt, J = 7.7, 1.3 Hz, 1H),
7
.57 (d, J = 7.9 Hz, 1H), 7.52-7.39 (m, 3H), 7.27 (td, J = 7.5, 1.0 Hz, 1H), 6.52-6.37
(m, 2H), 5.82 (dd, J = 9.7, 2.2 Hz, 1H), 5.55 (s, 1H), 5.08 (dd, J = 12.7, 3.4 Hz, 1H),

4
1
1
1
.58 (q, J = 6.9 Hz, 1H), 2.65 (dd, J = 13.1, 3.4 Hz, 1H), 2.19 (t, J = 12.9 Hz, 1H),
1
3
.71 (d, J = 7.0 Hz, 3H). C NMR (125 MHz, Methanol-d ): δ 172.8, 168.6, 168.4,
4
64.8, 139.1, 138.7, 136.2, 134.9, 130.9, 129.7, 128.7, 126.8, 125.6, 124.3, 123.1,
22.7, 119.1, 114.8, 83.0, 76.6, 59.6, 48.4, 39.3, 13.7. HRMS (ESI) calculated for
+
20 ºC
D
C H N O [M+H] = 447.1663, obtained: 447.1667. [푎]
= - 5.3°(c = 0.3,
24
23
4
5
MeOH).
1
1
4
-Acrylamido-N-((2S,2a S,4R,5aS)-5a-hydroxy-2-methyl-1,3-dioxo-1,2,2a ,4,5,5a-
hexahydro-3H-2a,9b-diazacyclopenta[jk]fluoren-4-yl)benzamide (15). Method G
1
using 15a (26 mg, 0.0 5mmol): 3.5 mg, 16% yield. H NMR (500 MHz, Methanol-d ):
4
δ 7.88 (d, J = 8.5 Hz, 2H), 7.79-7.74 (m, 2H), 7.55 (d, J = 7.9 Hz, 1H), 7.47 (d, J =
7
1
.5 Hz, 1H), 7.40 (td, J = 7.7, 1.3 Hz, 1H), 7.24 (td, J = 7.5, 1.1 Hz, 1H), 6.45 (dd, J =
6.9, 9.6 Hz, 1H), 6.39 (dd, J = 16.9, 2.2 Hz, 1H), 5.80 (dd, J = 9.6, 2.3 Hz, 1H), 5.53
(s, 1H), 5.09-5.03 (m, 1H), 4.56 (q, J = 6.9 Hz, 1H), 2.61 (dd, J = 13.1, 3.4 Hz, 1H),
1
3
2
.17 (t, J = 12.9, 1.7 Hz, 1H), 1.69 (d, J = 7.0 Hz, 3H). C NMR (125 MHz,
DMSO-d ): δ 172.9, 168.0, 165.6, 163.4, 141.7, 139.0, 136.8, 131.6, 129.7, 128.8,
6
1
28.3, 127.5, 125.4, 124.8, 118.5, 114.4, 82.4, 76.3, 58.8, 47.7, 14.6. HRMS (ESI)
-
20 ºC
D
o
calculated for C H N O [M-H] = 445.1517, obtained: 445.1515. [푎]
= + 5.2
2
4
21
4
5
(c = 0.25, DMSO).
1
2
-(2-Chloroacetamido)-N-((2S,2a S,4R,5aS)-5a-hydroxy-2-methyl-1,3-dioxo-1,2,2
1
a ,4,5,5a-hexahydro-3H-2a,9b-diazacyclopenta[jk]fluoren-4-yl)benzamide (16).
Method F using 16a (23 mg, 0.04 mmol): 4.3 mg, 23% yield. 1H NMR (500 MHz,
Methanol-d ): δ 8.44 (dd, J = 8.4, 1.1 Hz, 1H), 7.77 (dd, J = 7.9, 1.5 Hz, 1H),
4

7
5
1
.63-7.47 (m, 3H), 7.43 (td, J = 7.7, 1.3 Hz, 1H), 7.29-7.20 (m, 2H), 5.57 (s, 1H),
.08 (dd, J = 12.7, 3.3 Hz, 1H), 4.60 (d, J = 6.9 Hz, 2H), 4.32 (s, 2H), 2.64 (dd, J =
13
3.1, 3.4 Hz, 1H), 2.20 (t, J = 12.9 Hz, 1H), 1.72 (d, J = 7.0 Hz, 3H). C NMR (126
MHz, MeOD-d ): δ 172.7, 169.2, 168.6, 139.1, 131.7, 129.8, 127.7, 125.6, 124.3,
4
1
23.8, 121.2, 114.8, 83.1, 76.6, 59.7, 42.7, 38.9, 13.7, HRMS (ESI) calculated for
35
+
20 ºC
D
C H ClN O [M+H] = 469.1273, obtained: 469.1272. [푎]
= - 4.0°(c = 0.15,
23
22
4
5
MeOH).
-(2-Chloroacetamido)-N-((2S,2a S,4R,5aS)-5a-hydroxy-2-methyl-1,3-dioxo-1,2,2
1
3
a1,4,5,5a-hexahydro-3H-2a,9b-diazacyclopenta[jk]fluoren-4-yl)benzamide (17).
1
Method F using 17a (27 mg, 0.05 mmol): 8.1 mg, 37% yield. H NMR (500 MHz,
Methanol-d ): δ 8.10 (t, J = 1.9 Hz, 1H), 7.81 (dd, J = 8.1, 2.2 Hz, 1H), 7.69-7.64 (m,
4
1
H), 7.57 (d, J = 8.0 Hz, 1H), 7.52-7.40 (m, 3H), 7.27 (td, J = 7.6, 1.1 Hz, 1H), 5.55
(
(
s, 1H), 5.08 (dd, J = 12.6, 3.4 Hz, 1H), 4.58 (q, J = 6.9 Hz, 1H), 4.23 (s, 2H), 2.64
13
dd, J = 13.1, 3.4 Hz, 1H), 2.19 (t, J = 12.9 Hz, 1H), 1.71 (d, J = 7.0 Hz, 3H). C
NMR (126 MHz, Acetone-d ): δ 171.1, 166.9, 166.6, 164.6, 137.4, 136.5, 134.5,
6
1
4
4
4
33.2, 128.0, 127.1, 123.9, 122.6, 121.4, 117.5, 113.1, 81.3, 74.9, 58.0, 46.7, 45.4,
35
22
0.9, 37.5, 27.6, 12.0. HRMS (ESI) calculated for C H ClN O [M+H]+ =
2
3
4
5
69.1273, obtained: 469.1277. [푎] 2D 0 ºC =-11.2 ° (c = 0.3, MeOH).
1
-(2-Chloroacetamido)-N-((2S,2a S,4R,5aS)-5a-hydroxy-2-methyl-1,3-dioxo-1,2,2
1
a ,4,5,5a-hexahydro-3H-2a,9b-diazacyclopenta[jk]fluoren-4-yl)benzamide (18).
1
Method G using 18a (28 mg, 0.06 mmol): 14 mg, 50 % yield. H NMR (500 MHz,
Methanol-d ): δ 7.87 (d, J = 8.7 Hz, 2H), 7.71 (d, J = 8.7 Hz, 2H), 7.54 (d, J = 7.9 Hz,
4

1
1
H), 7.46 (d, J = 7.5 Hz, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.24 (t, J = 7.5 Hz, 1H), 5.52 (s,
H), 5.12-4.98 (m, 1H), 4.55 (q, J = 6.9 Hz, 1H), 4.20 (s, 2H), 2.60 (dd, J = 13.1, 3.4
1
3
Hz, 1H), 2.16 (t, J = 12.9 Hz, 1H), 1.68 (d, J = 6.9 Hz, 3H). C NMR (125 MHz,
Methanol-d ): δ 174.1, 170.1, 169.3, 167.6, 142.7, 140.5, 137.6, 131.1, 130.9, 129.5,
4
1
26.9, 125.7, 120.5, 116.2, 84.4, 78.0, 61.0, 49.7, 44.1, 40.7, 15.1. HRMS (ESI)
35
22
+
20 ºC
D
calculated for C H ClN O [M+H] = 469.1273, obtained: 469.1277. [푎]
= -
2
3
4
5
o
0
.6 (c = 1.0, MeOH).
1
N-(2-(((2S,2a S,4R,5aS)-5a-Hydroxy-2-methyl-1,3-dioxo-1,2,2a1,4,5,5a-hexahydr
o-3H-2a,9b-diazacyclopenta[jk]fluoren-4-yl)carbamoyl)phenyl)oxirane-2-carbox
1
amide (19). Method F using 19a (12 mg, 0.02 mmol): 3.8mg, 41% yield. H NMR
(
500 MHz, Methanol-d ): δ 8.46 (d, J = 8.4 Hz, 1H), 7.78 (dd, J = 7.9, 1.5 Hz, 1H),
4
7
1
.62-7.48 (m, 3H), 7.47-7.39 (m, 1H), 7.29-7.20 (m, 2H), 5.56 (s, 1H), 5.03 (dd, J =
2.7, 3.4 Hz, 1H), 4.60 (q, J = 7.0 Hz, 1H), 3.58-3.54 (m, 1H), 3.09 (dd, J = 5.8, 4.4
Hz, 1H), 3.00-2.95 (m, 1H), 2.67-2.58 (m, 1H), 2.23 (t, J = 12.9 Hz, 1H), 1.71 (d, J =
1
3
7
1
3
4
.0, 3H). C NMR (125 MHz, Methanol-d ): δ 172.7, 168.5, 139.1, 137.4, 136.2,
4
31.9, 129.8, 127.7, 125.6, 124.3, 123.6, 121.2, 114.8, 83.0, 76.6, 59.7, 49.3, 46.5,
+
8.9, 13.7. HRMS (ESI) calculated for C H N O [M+H] = 463.1612, obtained:
2
4
23
4
6
63.1603. [푎]2
0 ºC
= - 5.9°(c = 0.3, MeOH).
D
1
1
N-(3-(((2S,2a S,4R,5aS)-5a-hydroxy-2-methyl-1,3-dioxo-1,2,2a ,4,5,5a-hexahydro
-3H-2a,9b-diazacyclopenta[jk]fluoren-4-yl)carbamoyl)phenyl)oxirane-2-carboxa
1
mide (20). Method F using 20a (21 mg, 0.04 mmol): 4.5 mg, 27% yield. H NMR
(
500 MHz, Methanol-d ): δ 8.10 (d, J = 2.0 Hz, 1H), 7.83 (dd, J = 8.2, 2.1 Hz, 1H),
4