Investigation of B,C-ring truncated deguelin derivatives as heat shock protein 90 (HSP90) inhibitors for use as anti-breast cancer agents

Article abstract of DOI:10.1016/j.bmc.2019.02.040

On the basis of deguelin, a series of the B,C-ring truncated surrogates with N-substituted amide linkers were investigated as HSP90 inhibitors. The structure activity relationship of the template was studied by incorporating various substitutions on the nitrogen of the amide linker and examining their HIF-1α inhibition. Among them, compound 57 showed potent HIF-1α inhibition and cytotoxicity in triple-negative breast cancer lines in a dose-dependent manner. Compound 57 downregulated expression and phosphorylation of major client proteins of HSP90 including AKT, ERK and STAT3, indicating that its antitumor activity was derived from the inhibition of HSP90 function. The molecular modeling of 57 demonstrated that 57 bound well to the C-terminal ATP-binding pocket in the open conformation of the hHSP90 homodimer with hydrogen bonding and pi-cation interactions. Overall, compound 57 is a potential antitumor agent for triple-negative breast cancer as a HSP90 C-terminal inhibitor.

Full text of DOI:10.1016/j.bmc.2019.02.040

Accepted Manuscript
Investigation of B,C-Ring Truncated Deguelin Derivatives as Heat Shock Pro-
tein 90 (HSP90) Inhibitors for Use as Anti-Breast Cancer Agents
Ho Shin Kim, Van-Hai Hoang, Mannkyu Hong, Kyung Chul Kim, Jihyae Ann,
Cong-Truong Nguyen, Ji Hae Seo, Hoon Choi, Jun Yong Kim, Kyu-Won Kim,
Woong Sub Byun, Sangkook Lee, Seungbeom Lee, Young-Ger Suh, Jie Chen,
Hyun-Ju Park, Tae-Min Cho, Ji Young Kim, Jae Hong Seo, Jeewoo Lee
PII:
S0968-0896(19)30117-8
https://doi.org/10.1016/j.bmc.2019.02.040
BMC 14776
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 January 2019
19 February 2019
19 February 2019
Please cite this article as: Kim, H.S., Hoang, V-H., Hong, M., Chul Kim, K., Ann, J., Nguyen, C-T., Seo, J.H., Choi,
H., Yong Kim, J., Kim, K-W., Sub Byun, W., Lee, S., Lee, S., Suh, Y-G., Chen, J., Park, H-J., Cho, T-M., Kim,
J.Y., Seo, J.H., Lee, J., Investigation of B,C-Ring Truncated Deguelin Derivatives as Heat Shock Protein 90 (HSP90)
Inhibitors for Use as Anti-Breast Cancer Agents, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/
10.1016/j.bmc.2019.02.040
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Investigation of B,C-Ring Truncated Deguelin Derivatives
as Heat Shock Protein 90 (HSP90) Inhibitors for Use as
Anti-Breast Cancer Agents
Ho Shin Kim a, Van-Hai Hoang a, Mannkyu Hong a, Kyung Chul Kim a, Jihyae Ann a, Cong-Truong
Nguyen a, Ji Hae Seo a, Hoon Choi a, Jun Yong Kim a, Kyu-Won Kim a, Woong Sub Byun a, Sangkook
Lee a, Seungbeom Lee a, Young-Ger Suh a,b, Jie Chen c, Hyun-Ju Park c, Tae-Min Cho d,e , Ji Young
Kim d, Jae Hong Seo d,e,* Jeewoo Lee a,*
a College of Pharmacy, Seoul National University, Seoul 08826, Korea
b College of Pharmacy, CHA University, Gyeonggi-do 11160, Korea
c School of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do 16419, South Korea
d Division of Medical Oncology, Department of Internal Medicine, Korea University College of
Medicine, Korea University, Seoul 152-703, Republic of Korea.
e Brain Korea 21 Program for Biomedical Science, Korea University College of Medicine, Korea
University, Seoul 152-703, Republic of Korea.
* Corresponding Authors
Jeewoo Lee, Ph.D.
Phone: 82-2-880-7846
Fax: 82-2-888-0649
E-mail: jeewoo@snu.ac.kr
Jae Hong Seo, Ph.D.
E-mail: cancer@korea.ac.kr
1

Key Words: Heat Shock Protein 90; HSP90; Hypoxia Inducible Factor-1; HIF-1; Antitumor;
Breast Cancer; Deguelin
Abstract
On the basis of deguelin, a series of the B,C-ring truncated surrogates with N-substituted
amide linkers were investigated as HSP90 inhibitors. The structure activity relationship of the
template was studied by incorporating various substitutions on the nitrogen of the amide linker and
examining their HIF-1α inhibition. Among them, compound 57 showed potent HIF-1α inhibition and
cytotoxicity in triple-negative breast cancer lines in a dose-dependent manner. Compound 57
downregulated expression and phosphorylation of major client proteins of HSP90 including AKT,
ERK and STAT3, indicating that its antitumor activity was derived from the inhibition of HSP90
function. The molecular modeling of 57 demonstrated that 57 bound well to the C-terminal ATP-
binding pocket in the open conformation of the hHSP90 homodimer with hydrogen bonding and pi-
cation interactions. Overall, compound 57 is a potential antitumor agent for triple-negative breast
cancer as a HSP90 C-terminal inhibitor.
2

1. Introduction
Heat shock protein 90 (HSP90) is a molecular chaperone involved in the folding, maturation,
and degradation of its client proteins, which are associated with hallmarks of cancer.1-3 Thus,
inhibition of HSP90 has attracted much attention as a new and effective cancer therapy, particularly
to overcome resistance to anticancer agents.4-6 Structurally, HSP90 is a homodimeric protein of ~90
kDa in which each monomer contains an N-terminal ATP-binding domain, a middle cochaperone and
client-binding domain, and a C-terminal dimerization domain. Most HSP90 inhibitors that have been
developed have been identified as the N-term inhibitors that bind to an ATP-binding domain in the
N-terminal.7,8 However, one of the main drawbacks in these inhibitors is the induction of a heat shock
response (HSR), which ultimately leads to an increase in HSP90 and anti-apoptotic proteins such as
HSP70 and HSP27. Therefore, the inhibition of another ATP-binding site in the C-terminal provides
an alternative strategy to overcome these side effects.9-12 Indeed, novobiocin, the first C-terminal
inhibitor discovered, has shown potency in several cancer cell lines as it degrades clients without
reported HSR.13 Hypoxia Inducible Factor-1 (HIF-1) is a transcription factor, one of the HSP90
client proteins, that regulates the cellular response for the survival of cells in hypoxia. Because this
regulation can induce angiogenesis, proliferation, metastasis and invasion of cancer cells,14,15 HIF-1α
inhibition through HSP90 modulation can inhibit the angiogenesis and proliferation of cancer cells
and reduce chemotherapy resistance.16,17
Triple-negative breast cancer (TNBC) is a highly heterogeneous and biologically aggressive
cancer which is associated with a poor clinical outcome due to the lack of established molecular
targets, such as estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth
factor receptor 2 (HER2).18 Elevated HSP90 levels are frequently found in TNBC patients and are
significantly correlated with a higher risk of recurrence and distant metastasis, leading to poor
prognoses.19 Emerging preclinical evidence highlights that HSP90 inhibition impairs cell
proliferation, cell invasion, motility and angiogenesis as well as cell propagation in solid cancers via
dysregulation of HSP90 client proteins, including EGFR, MEK, STAT, and AKT.20-23 Of particular
3

note, the growth of TNBC is tightly governed by the activation of these HSP90 clients24-26; thus,
targeting HSP90 may provide an effective therapeutic strategy for the treatment of TNBC.
Deguelin (1), a naturally occurring rotenoid, showed potent antitumor activity by inhibiting
ATP binding to the C-terminal of heat shock protein (HSP90), leading to the decomposition of HIF-
1.27 To discover a novel scaffold for a C-terminal HSP90 inhibitor based on deguelin, we have
investigated simplified deguelin analogues through a ring-truncation approach. Among them,
compounds 2 and 3, designed as B,C-ring truncated templates, exhibited excellent HIF-1α
suppression and potent cell growth inhibition in a human nonsmall-cell lung carcinoma cell line
(H1299) comparable to deguelin (Figure 1).28 Mechanistic studies revealed that these compounds
disrupted HSP90 function by binding to its C-terminal ATP-binding pocket and interfering with the
interaction with its cochaperones and client proteins, triggering their degradation.29
Figure 1. Deguelin and its B,C-ring-truncated surrogates
4

Recently, we reported a series of fluorophenyl and pyridine surrogates of 2 and 3 with
modified linkers for HIF-1 inhibition.30 Among them, several compounds showed better HIF-1
inhibition than deguelin, 2 and 3. In particular, compound 4 exhibited promising anticancer and
antiangiogenic activities. The docking study of 4 indicated that its cytotoxicity was derived from HIF-
1α destabilization by binding to the C-terminal ATP-binding site of HSP90.30
To further optimize the B,C-ring-truncated templates of deguelin as HSP inhibitors, we
investigated its amide linker surrogates, represented in template I (Figure 1). In this study, we
demonstrated the structure-activity relationships of the template, in which the A-ring was substituted
with 3,4-dimethoxyphenyl, its fluorophenyl or pyridine surrogate, respectively, and the linker was
modified with N-substituted amide. The synthesized compounds were evaluated for their HIF-1α
inhibitions using western blot assay as the primary screening method. With selected potent inhibitors
in the series, we sought to exploit anticancer effect in breast cancer cell lines. In addition, we explored
the effect on major clients of HSP90 and performed molecular modeling to identify its mechanism
for HSP90 inhibition.
2. Results and discussion
2.1. Chemistry
The syntheses of N-substituted B,C-ring truncated deguelin derivatives were represented in
Scheme 1. For the synthesis of the N-alkylated amide compounds (12-74), commercially available
A-ring amines (5-7) were coupled with 2,2-dimethyl-5-methoxy-2H-chromene acid (8), which was
obtained by Pinnick oxidation of previously reported chromene aldehyde31 to form the amide
intermediates (9-11) under general EDC coupling conditions. Their amides were N-alkylated with the
corresponding halide to provide one part of final products directly or penultimate products, which
were deprotected, O-methylated or aminated to afford the rest of the final product.
5

For the synthesis of N-phenyl amide compounds (75-79), the A-ring amine (5) was
condensed with halobenzene through Buchwald-Hartwig amination to provide the corresponding
diphenylamines. The secondary amines were coupled with the acyl chloride of 8 in the presence of
K2CO3 to provide the desired final compounds (75-79).
o
Scheme 1. Reagents and conditions: (a) EDC·HCl, HOBt, NEt3, 1,4-dioxane, r.t to 50 C for 9-11; (b) RX,
NaH, DMF, 50-70 oC for 12-17, 24-26, 30-32, 42, 47, 63-74; (c) [TBS deprotection] TBAF, CH2Cl2, 0 oC for
18-20; [O-methylation] NaH, MeI, DMF, 0 oC for 21-23; [Boc deprotection] TFA, DCM, 0 oC for 33-35, 53;
[amination: Method A] (i) DPPA, DBU, DMF, 90 °C (ii) Lindlar’s catalyst, H2, MeOH, r.t for 27-29; [amination:
Method B] (i) DMP, CH2Cl2, r.t, (ii) NHR1R2, ZnCl2, NaBH4, CH2Cl2, MeOH, r.t for 36-41, 50-52, 54-62; [N-
acetylation] Ac2O, Pyridine, CH2Cl2, 0 oC for 44-46; [N-Boc protection] Boc2O, CH2Cl2, 0 oC for 47-48; (d)
Pd2(dba)3, BINAP, NaOtBu, p-R-halobenzene, 1,4-dioxane, reflux; (e) (i) 8, PPh3, Cl3CCN, CH2Cl2, reflux, (ii)
K2CO3, 1,4-dioxane, reflux for 75-79.
6

2.2. Structure-activity relationship for HIF-1 inhibition
As primary screening for HSP90 inhibitors, the compounds were evaluated for their HIF-1α
inhibition using western blot assay with human nonsmall cell lung carcinoma (H1299) cell lines.28
The H1299 cells were untreated or treated with the compounds at 100 nM for 72 h and subsequently
incubated under hypoxic conditions for 12 h. The resulting cell lysates were used for western blot
analysis with a monoclonal antibody against HIF-1. The results are summarized in Tables 1-3, and
deguelin (1) and 2, as references, were examined with 64% and 85.3% values, respectively, together
with the control (100%).
First, we explored N-alkyl amide linker analogues (Table 1). Unsubstituted amide linker
analogues with different A-regions (9-11) were found to be inactive for HIF-1 inhibition. N-Methyl
analogues (12-14) showed modestly improved inhibition. Whereas N-hydroxyethyl analogues (18-
20) were almost inactive, N-methoxyethyl and N-benzyloxyethyl analogues (21-26) exhibited good
inhibition. Surprisingly, the two N-aminoethyl analogues (27, 29) showed excellent HIF-1
inhibition, with values of 39.4 and 33.3, respectively, while 28 with the fluorophenyl A-ring was
inactive. N-Aminopropyl analogue, with a pyridine A-ring (35), also exhibited good inhibition.
Further modification in 27-29 and 33-35 was explored by incorporating methyl (36-38), dimethyl
(39-42), diethyl (43), acetyl (44-46) and t-butoxycarbonyl (47-48) groups into their amino group.
Most analogues with a pyridine A-ring displayed better inhibition than deguelin.
N-cycloalkylethyl analogues (49-62), including pyrrolidine, 1-piperidine, 4-piperidine,
morpholine, piperazine and N-methylpiperazine, were also examined. All showed good inhibition
that was similar to that of the corresponding N-dimethyl and N-diethyl analogues.
7

Table 1. HIF-1 inhibitory activities of the synthesized compounds
R/X
C-H
C-F
N
-H
147 (±30.0)
101 (±25.2)
88.4 (±19.5)
9
10
13
19
22
25
28
34
37
40
11
14
20
23
26
29
35
38
41
42
43
45
46
48
49
52
-Me
126 (±16.0)
133 (±38.1)
65.5 (±21.3)
74.4 (±20.2)
39.4 (±11.5)
100 (±28.1)
75.9 (±8.87)
71.0 (±15.7)
93.1 (±16.0)
124 (±16.9)
59.3 (±23.7)
65.4 (±22.4)
147 (±15.2)
68.5 (±27.5)
107 (±30.7)
53.3 (±14.3)
62.3 (±21.5)
87.9 (±10.5)
52.5 (±40.3)
57.2 (±10.3)
33.3 (±16.2)
45.3 (±10.1)
41.2 (±12.8)
37.9 (±14.0)
44.0 (±12.0)
64.4 (±14.7)
58.1 (±20.2)
76.6 (±11.3)
57.4 (±24.8)
66.0 (±26.4)
79.7 (±18.4)
12
18
21
24
27
33
36
39
127 (±10.1)
70.3 (±9.61)
83.7 (±18.9)
44
47
50
54.3 (±16.3)
51
8

55.2 (±11.5)
60.3 (±21.5)
59.4 (±10.2)
42.2 (±13.7)
53
56
59
62
117 (±11.1)
51.9 (±14.2)
61.9 (±19.3)
48.8 (±14.2)
54.9 (±19.4)
53.7 (±18.8)
54
57
60
55
58
61
Second, we examined N-benzyl and N-phenyl amide analogues (Table 2).
N-Benzyl analogues (63-65) displayed potent inhibition. Of these compounds, pyridine A-
ring surrogate (65) exhibited excellent inhibition to give a value of 38.6%. Its high potency prompted
us to investigate its SAR. The N-Benzyl group was modified with 3- or 4-substituted benzyl groups
(66-71) and isomeric pyridines (72-74). Among them, 4-fluorobenzyl (66), 4-chlorobenzyl (67) and
pyridin-4-yl (74) analogues exhibited good inhibition. The N-Phenyl analogue (75) also showed good
HIF-1 inhibition that was better than that of its benzyl surrogate (63). However, its substituted
analogues (76-79) displayed lower inhibition.
Table 2. HIF-1 inhibitory activities of the synthesized compounds
R/X
C-H
C-F
N
69.8 (±16.0)
60.8 (±51.3)
38.6 (±22.0)
63
64
65
66
67
63.1 (±14.5)
57.6 (±16.5)
9

77.0 (±16.1)
71.1 (±12.2)
85.7 (±11.4)
80.2 (±15.9)
97.0 (±14.6)
95.8 (±20.9)
55.2 (±15.2)
68
69
70
71
72
73
74
52.8 (±23.6)
70.2 (±20.0)
81.2 (±22.4)
61.9 (±17.8)
58.9 (±12.2)
75
76
77
78
79
Of the tested compounds, we selected 20 compounds having better HIF-1α inhibition than
deguelin and further evaluated their inhibition at a lower concentration (10 nM) to examine the
concentration-dependent inhibition (Table 3). Among them, 8 compounds (26, 27, 29, 41, 42, 45, 56,
57) displayed concentration-dependent inhibition and better inhibition than deguelin at 10 nM.
Table 3. HIF-1α inhibition of selected compounds
Code
Deguelin
14
WB (100 nM)
WB (10 nM)
94
64
62.3
57.2
39.4
90.4 (±19.5)
69.3 (±10.1)
62.1 (±9.2)
26
27
10

33.3
45.3
41.2
53.3
37.9
44.0
58.1
55.2
48.8
60.3
48.9
53.7
42.2
38.6
63.1
55.2
52.8
64.0 (±12.6)
111 (±21.9)
93.0 (±18.2)
97.9 (±14.1)
75.5 (±10.4)
65.6 (±18.5)
66.0 (±11.2)
97.2 (±18.2)
111 (±20.6)
72.9 (±14.8)
60.6 (±12.6)
81.2 (±23.8)
97.2 (±19.8)
83.4 (±14.1)
104 (±16.4)
108 (±12.5)
83.7 (±21.0)
29
35
38
40
41
42
45
53
55
56
57
61
62
65
66
74
75
2.3. Antitumor activity
To examine the antitumor activity of HSP90 inhibitors in breast cancer cell lines, we characterized
the cytotoxicities of the compounds in Table 3 in triple-negative breast cancer cell-lines (TNBC). Of
those compounds, only compound 57 showed a promising cytotoxic effect in MDA-MB-231
(invasive ductal carcinoma) and 4T1 (murine mammary carcinoma) at a concentration of 10 uM. To
further examine the effects of 57 on cell viability in TNBC, MDA-MB-231 and 4T1 cells were treated
with various concentrations of 57 (0.2, 0.5, 1, 2, 5, 10 or 20 µM) for 72 h. MTS assays revealed 57
(0.2-20 µM) significantly suppressed cell viability in both MDA-MB-231 and 4T1 cells in a time-
and does-dependent manner (Figure 2A). Following exposure to 57 (2-10 μM) for 72 h, MDA-MB-
231 cells exhibited significant morphological changes with concomitant cytoplasmic shrinkage
(Figure 2B).
11

Figure 2. The cytotoxic effect of 57 in triple-negative breast cancer cell-lines
(A) Effect of 57 on cell viability in MDA-MB-231 and 4T1 cells. Cells were treated with various
concentrations of 57 (0.2, 0.5, 1, 2, 5, 10 or 20 µM) for and 72 h. Cell viability was determined by
MTS assay. The results are presented as mean ± SEM of at least three independent experiments and
analyzed by ono-way ANOVA followed by Bonferroni’s post hoc test (* p<0.05, versus DMSO
control). (B) Changes in cellular morphology in MDA-MB-231 cells after 57 (2, 5 or 10 μM)
treatment for 72 h as seen through phase contrast microscopy.
2.4. Mechanism study
To identify the mechanism of compound 57 as a HSP90 inhibitor, we examined the influence
of 57 on the expression and activation of major clients of HSP90 including AKT (protein kinase B),
MEK (mitogen-activated protein kinase kinase) and STAT3 (signal transducer and activator of
transcription 3) in MDA-MB-231 cells in vitro. Treatment with 57 (5-10 μM, 72 h) resulted in a
significant downregulation of AKT, STAT3 and MEK protein content, concomitant with a marked
reduction of their phosphorylation (Figure 3A). The quantitative data further showed that 57
effectively suppressed activation of these HSP90 clients, as evidenced by a significant reduction in
12

the ratio of phospho-AKT/total-AKT, phospho-MEK/total-MEK and phospho-STAT3/total-STAT3
in MDA-MB-231 cells (Figure 3B). A mitogen-activated protein kinase (MAPK) signaling network
has been implicated in diverse aspects of cancer progression, and blockage of the MEK–ERK cascade
elicits antitumor activity in many cancer types.32 Exposure to 57 did not affect ERK protein content
but significantly hampered its phosphorylation and activity (Figure 3B), indicating that this response
is attributable to MEK inactivation by 57 challenge. The result revealed that 57 downregulated the
expression and phosphorylation of HSP90 client proteins, AKT, ERK and STAT3.
Figure 3. The influence of 57 on AKT/ERK/STAT3 expression and activation
(A) Immunoblot analyses of AKT, phospho-AKT (Ser473), STAT3, phospho-STAT3 (Tyr705), MEK,
phospho-MEK (Ser218/222), ERK and phospho-ERK (Thr202/Tyr204) protein expression in MDA-
MB-231 cells following 57 (2–10 µM, 72 h) challenge for 72 h. Actin was used as a loading control.
(B) Quantitative graphs represent (a) the ratio of total proteins (AKT, -STAT3, -MEK or -ERK)/actin,
(b) the ratio of phosphorylated proteins/actin, and (c) the ratio of phosphorylated-/total-proteins,
respectively. The results are presented as mean ± SEM of at least three independent experiments
analyzed by one-way ANOVA followed by Bonferroni's post hoc test (* p<0.05, versus DMSO
control).
13

2.5. Molecular Modeling
To explore the molecular basis of the interaction between 57 and HSP90, molecular docking
analysis was conducted using the Surflex-Dock program.33 Our previous hHSP90 homology model
structure29,30 was used as the receptor for docking. As expected, 57 binds into the C-terminal ATP-
binding cavity, anchoring at the cavity of dimerization interface of HSP90 homodimer with a “T-
shape” pose (Figure 4). The side-chain amine NH of Lys615(A) forms a hydrogen bond with the
methoxy group attached to the 2,2-dimethyl chromene ring, and it concurrently forms a π-cation
interaction with 3,4-dimethoxy phenyl ring. The side-chain OH of Ser677(A) forms bidentate
hydrogen bonds with 3,4-dimethoxy substituents on the phenyl ring. Upon examining the interaction
of 57 with chain B, the two nitrogen atoms in the piperazine ring serve as hydrogen bonding acceptors,
interacting with side-chain of Lys615(B) and Ser677(B). Our docking model suggests that compound
57 binds to the interface of the HSP90 homodimer, locking the open conformation of HSP90
homodimer, and therefore prohibiting N-terminal dimerization and the formation of the ATP binding
pocket.
Figure 4. Molecular docking model of 57 bound to the hHsp90 C-terminal domain
(A) Binding site for 57 in the dimerization interface of open state hHSP90. Chain A is rendered in
blue ribbon, and chain B is orange ribbon. The active site is shown as electrostatic property surface
map. Red, blue, and white colored regions correspond to negatively charged, positively charged, and
neutral areas, respectively. (B) Docked pose of 57 (carbon in green) in the hHSP90 C-terminal ATP
binding site. Key amino acid residues within the binding site are rendered in grey capped stick.
Hydrogen bonding interactions are depicted as yellow dashed lines and pi-cation interaction is
depicted as green dashed lines. A and B indicate chain names.
14

3. Conclusion
A series of the B,C-ring truncated surrogates of deguelin with N-substituted amide linkers
were investigated as HSP90 inhibitors by examining their HIF-1α inhibition as a primary screening
method. The structure-activity relationship of the template was investigated by alkylation, benylation
and phenylation on the nitrogen of the amide linker in the template. Among 68 compounds
synthesized, 8 compounds displayed better inhibition than deguelin at 100 and 10 nM with
concentration-dependent inhibition. The selected compounds were screened for antitumor activity in
triple-negative breast cancer cell-lines, and of those, compound 57 showed potent cytotoxicity in
MDA-MB-231 and 4T1 cells in a time- and does-dependent manner. The mechanistic study indicated
that 57 downregulated expression and phosphorylation of major client proteins of HSP90 including
AKT, ERK and STAT3, revealing that it induced antitumor activity by inhibiting the function of
HSP90. The docking study of 57 with the hHSP90 C-terminal ATP-binding site indicated that 57
binds with a T-shape pose to the C-terminal ATP-binding pocket in the open conformation of hHSP90
homodimer by hydrogen bonding and pi-cation interactions. Overall, compound 57 is a potential
antitumor agent for triple-negative breast cancer, and it acts as a HSP90 C-terminal inhibitor.
4. Experimental
4.1. Chemistry
4.1.1. General
All chemical reagents were commercially available. Melting points were determined on a Büchi
Melting Point B-540 apparatus and are uncorrected. Silica gel column chromatography was
performed on silica gel 60, 230-400 mesh, Merck. 1H NMR spectra were recorded on a a JEOL JNM-
LA 300 and a Bruker Avance 500 at 500 MHz. Chemical shifts are reported in ppm units with Me4Si
as a reference standard. Mass spectra were recorded on a VG Trio-2 GC-MS and 6460 Triple Quad
LC/MS.
15

4.1.2. General procedure
4.1.2.1. EDC coupling. To a solution of 8 (1.0 mmol) and appropriate aniline (5-6, 1.0 eq.) in 1,4-
dioxane, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC-HCl, 1.3 eq.), hydroxybenzotri-
azole (HOBt, 1.3 eq.) and triethylamine (1.3 eq.) were added and stirred at 50 °C for 15 h. After
completion, the reaction mixture was diluted with H2O and extracted with EtOAc. The combined
organic layers was washed with brine three times, dried over MgSO4 and concentrated in vacuo. The
residue was purified by silica gel column chromatography to obtain the desired compounds.
4.1.2.2. N-alkylation. To a solution of amide compound (9-11, 1.0 eq.) in DMF, sodium hydride
(NaH, 1.5 eq.) was added at 0 °C and stirred for 30 min. The appropriate alkylhalides (S1-S3, 2.0 eq.)
dissolved in DMF were added dropwise and stirred for 1 h. The reaction mixture was quenched by
dropwise addition of water at 0 °C and extracted with EtOAc, washed with brine several times, dried
over MgSO4, and concentrated in vacuo. The residue was purified by silica gel column
chromatography to obtain the desired compounds.
4.1.2.3. Silyl deprotection. To a solution of compound 15-17 (1.0 eq.) in DCM, TBAF 1 M in THF
(1.2 eq.) was added at 0 °C and stirred for 2 h at room temperature. The reaction mixture was
quenched with H2O and extracted with EtOAc. The organic layer was washed with brine, dried over
MgSO4, and concentrated in vacuo. The residue was purified by silica gel column chromatography
to obtain the desired compounds.
4.1.2.4. O-Methylation. An oven-dried round bottom flask was charged with compounds 18-20 (1.0
eq.), sodium hydride (1.2 eq.) and DMF. The mixture was stirred at 0 °C, and iodomethane (1.0 eq.)
was added dropwise. The mixture was warmed to room temperature for 1 h. The reaction was
quenched by the addition of water and extracted twice with EtOAc. The combined organic layer was
washed with brine, dried over MgSO4, filtered and concentrated in vacuo. The crude mixture was
purified by silica gel column chromatography to afford the desired product.
16

4.1.2.5. Boc deprotection. To a solution of N-Boc protected compounds (30-32, 1.0 eq.) in DCM,
TFA (1.2 eq.) was added dropwise at 0 °C and stirred for 1 h at room temperature. Then, the reaction
mixture was quenched with saturated NaHCO3 solution and extracted with EtOAc. The organic layer
was washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was purified by
flash column chromatography on silica gel to afford the desired product.
4.1.2.6. Amination
Method A: To a solution of compound 18-20 (1.0 eq.) in DMF, 1,8-diazabicyclo(5.4.0)undec-7-ene
(DBU, 1.2 eq.) and diphenylphosphoryl azide (DPPA, 1.2 eq.) were added dropwise, and the reaction
mixture was stirred for 15 h at 80 °C. After completion, the mixture was cooled down to room
temperature, quenched with H2O and extracted with EtOAc. The combined organic layer was washed
with brine, dried over MgSO4, filtered and concentrated in vacuo. The crude mixture was purified by
silica gel column chromatography to afford the desired azide intermediate. To azide intermediates in
MeOH, Lindlar catalyst (0.1 eq.) was added and stirred for 1 h under H2 atmosphere at room
temperature. After completion, the mixture was filtered through a celite filter, and the filtrate was
concentrated in vacuo. The residue was purified by flash column chromatography on silica gel to
afford the desired product.
Method B: To a solution of compound 18-20 (1.0 eq.) in DCM, Dess−Martin periodinane (DMP, 1.0
eq.) was added and stirred for 1 h at room temperature. After completion, the reaction mixture was
neutralized with sodium bicarbonate solution (NaHCO3 sat.) and extracted with DCM. The combined
organic layer was washed with brine, dried over MgSO4, filtered and concentrated in vacuo. The
crude mixture was purified by silica gel column chromatography to afford the desired aldehyde
intermediate. To a solution of the aldehyde (1.0 eq.) in DCM, ZnCl2 (1.2 eq.) and NHR1R2 or its salt
(1.2 eq.) was added at 0 °C and stirred for 15 h at room temperature. Then, the reaction mixture was
concentrated and redissolved in MeOH. NaBH4 (2.0 eq.) was added to the reaction mixture at 0 °C
and stirred for 1 h at room temperature. After completion, the reaction mixture was quenched with
17

H2O and extracted with EtOAc. The combined organic layer was washed with brine, dried over
MgSO4, filtered and concentrated in vacuo. The crude mixture was purified by silica gel column
chromatography to afford the desired product.
4.1.2.7. N-Acetylation. To a solution of alkyl amine in DCM, pyridine (1.0 eq.) and acyl anhydride
(1.0 eq.) were added dropwise at 0 °C and stirred for 1 h at room temperature. After completion, the
reaction mixture was neutralized with saturated NaHCO3 solution and extracted with DCM. The
combined organic layer was washed with brine, dried over MgSO4, filtered and concentrated in vacuo.
The crude mixture was purified by silica gel column chromatography to afford the desired product.
4.1.2.8. N-tert-Butoxycarbonylation. To a solution of alkyl amine (1.0 eq.) in DCM, triethylamine
(2.0 eq.) and tert-butyloxycarbonyl anhydride (3 eq.) was added and stirred for 3 h at room
temperature. After completion, the reaction mixture was quenched with H2O and extracted with DCM.
The combined organic layer was washed with brine, dried over MgSO4, filtered and concentrated in
vacuo. The crude mixture was purified by silica gel column chromatography to afford the desired
product.
4.1.2.9. Buchwald reaction. To a solution of aromatic halide compound (1.0 eq.) in 1,4-dioxane,
appropriate aniline (1.0 eq.), Pd2(dba)3 (0.1 eq.), BINAP (0.1 eq.), NaOtBu (2.5 eq.) were added and
refluxed overnight. After completion, the mixture was filtered through a celite filter, and the filterate
was washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was purified by
flash column chromatography on silica gel to afford the desired secondary amine intermediate.
4.1.2.10. Acylation and coupling. To a solution of carboxylic acid (8, 1.0 eq.) in DCM, PPh3 (1.1
eq.), Cl3CCN (1.1 eq.) were added and refluxed for 2 h. After completion, the reaction mixture was
concentrated in vacuo to obtain 5-methoxy-2,2-dimethyl-2H-chromene-6-carbonyl chloride. The
appropriate amine intermediates (1.0 eq.) in 1,4-dioxane were added with K2CO3 (3.0 eq.) and
18

carbonyl chloride, and the solution was refluxed for 15 h. After completion, the reaction mixture was
quenched with H2O and extracted with DCM. The combined organic layer was washed with brine,
dried over MgSO4, filtered and concentrated in vacuo. The crude mixture was purified by silica gel
column chromatography to afford the desired product.
4.1.3. Chemical Spectra
4.1.3.1.
N-(3,4-Dimethoxyphenyl)-5-methoxy-2,2-dimethyl-2H-chromene-6-carboxamide (9).
Yield = 80%, white solid; 1H-NMR (CDCl3, 300 MHz) δ 9.60 (s, 1H), 7.94 (d, J = 8.6 Hz, 1H), 7.61
(d, J = 2.2 Hz, 1H), 6.93 (dd, J = 2.4, 8.6 Hz, 1H), 6.82 (d, J = 8.6 Hz, 1H), 6.70 (d, J = 9.0 Hz, 1H),
6.58 (d, J = 10.1 Hz, 1H), 5.71 (d, J = 10.1 Hz, 1H), 3.91 (s, 3H), 3.86 (s, 3H), 3.86 (s, 3H), 1.44 (s,
6H); HR-MS (FAB) Calcd for C21H24NO5 [M + H]+ 370.1654, found: 370.1651.
4.1.3.2.
N-(2-Fluoro-3,4-dimethoxyphenyl)-5-methoxy-2,2-dimethyl-2H-chromene-6-carbox-
amide (10). Yield = 65%, white solid, mp = 117-118 oC; 1H-NMR (CDCl3, 400 MHz) δ 10.05 (s, 1H),
8.11 (t, J = 8.7 Hz, 1H), 7.97 (d, J = 8.6 Hz, 1H), 6.68 (m, 2H), 6.58 (d, J = 10.0 Hz, 1H), 5.70 (d, J
= 10.0 Hz, 1H), 3.94 (s, 3H), 3.86 (s, 3H), 3.81 (s, 3H), 1.44 (s, 6H); HRMS (FAB) calcd for
C21H23FNO5 [M + H]+ 388.1560, found: 388.1559.
4.1.3.3.
N-(5,6-Dimethoxypyridin-2-yl)-5-methoxy-2,2-dimethyl-2H-chromene-6-carboxamide
(11). Yield = 55%, pale yellow solid, mp = 127-128 oC; 1H-NMR (CDCl3, 300 MHz) δ 9.99 (s, 1H),
7.96 (d, J = 8.6 Hz, 1H), 7.88 (d, J = 8.4 Hz, 1H), 7.13 (d, J = 8.4 Hz, 1H), 6.71 (d, J = 8.6 Hz, 1H),
6.61 (d, J = 10.1 Hz, 1H), 5.73 (d, J = 9.9 Hz, 1H), 3.99 (s, 3H), 3.91(s, 3H), 3.88 (s, 3H), 1.47 (s,
6H); HRMS (FAB) calcd for C20H23N2O5 [M + H]+ 371.1607, found: 371.1609.
4.1.3.4. N-(3,4-Dimethoxyphenyl)-5-methoxy-N,2,2-trimethyl-2H-chromene-6-carboxamide (12).
Yield = 85%, colorless oil; 1H NMR (CDCl3, 300 MHz) δ 6.60 - 6.47 (m, 6H), 5.57 (d, J = 9.7 Hz,
19

1H), 3.85 (s, 3H), 3.77 (s, 3H), 3.66 (s, 3H), 3.42 (s, 3H), 1.34 (s, 6H); HR-MS (ESI) m/z calcd for
C22H26NO5 [M + H]+ 384.1805, found 384.1807.
4.1.3.5.
N-(2-Fluoro-3,4-dimethoxyphenyl)-5-methoxy-N,2,2-trimethyl-2H-chromene-6-
carboxamide (13). Yield = 90%, pale yellow oil; 1H-NMR (CDCl3, 300 MHz) δ 6.92 (d, J = 8.3 Hz,
1H), 6.71 (t, J = 8.8 Hz, 1H), 6.40 (m, 3H), 5.57 (d, J = 10.1 Hz, 1H), 3.83 (s, 3H), 3.82 (s, 3H), 3.77
(s, 3H), 3.38 (s, 3H), 1.35 (s, 6H); HRMS (FAB) calcd for C22H25FNO5 [M + H]+ 402.1717, found:
402.1721.
4.1.3.6. N-(5,6-Dimethoxypyridin-2-yl)-5-methoxy-N,2,2-trimethyl-2H-chromene-6-carboxamide
(14). Yield = 90%, pale yellow oil; 1H-NMR (CDCl3, 300 MHz) δ 6.87 (m, 2H), 6.50 (m, 2H), 6.40
(d, J = 8.4 Hz, 1H,), 5.60 (d, J = 9.9 Hz, 1H), 3.88 (s, 3H), 3.86 (s, 3H), 3.80 (s, 3H), 3.47 (s, 3H),
1.39 (s, 6H); HRMS (FAB) calcd for C21H25N2O5 [M + H]+ 385.1763, found: 385.1768.
4.1.3.7. N-(3,4-Dimethoxyphenyl)-N-(2-hydroxyethyl)-5-methoxy-2,2-dimethyl-2H-chromene-6-
carboxamide (18). Yield = 70%, yellow oil; 1H-NMR (CDCl3, 300 MHz) δ 6.77 (d, J = 8.3 Hz, 1H),
6.68 (m, 3H), 6.44 (d, J = 9.9 Hz, 1H), 6.34 (d, J = 8.4 Hz, 1H), 5.59 (d, J = 9.9 Hz, 1H), 4.09 (t, J =
5.1 Hz, 2H), 3.88 (m, 5H), 3.79 (s, 3H), 3.71 (s, 3H), 3.27 (bs, 1H), 1.36 (s, 6H); HRMS (FAB) calcd
for C23H27NO6 [M]+ 413.1838, found: 413.1835.
4.1.3.8.
N-(2-Fluoro-3,4-dimethoxyphenyl)-N-(2-hydroxyethyl)-5-methoxy-2,2-dimethyl-2H-
chromene-6-carboxamide (19). Yield = 70%, pale yellow oil; 1H-NMR (CD3OD, 300 MHz) δ 6.95-
6.90 (m, 2H), 6.66-6.59 (m, 1H), 6.40 (d, J = 10.6 Hz, 1H), 6.37 (d, J = 8.6 Hz, 1H), 5.64 (d, J = 9.9
Hz, 1H), 4.20 (m, 2H), 3.79 (s, 3H), 3.76 (s, 3H), 3.70 (s, 3H), 3.69-3.63 (m, 2H), 1.31 (s, 6H); HRMS
(FAB) calcd for C23H27FNO6 [M + H]+ 432.1822, found: 432.1825.
20

4.1.3.9.
N-(5,6-Dimethoxypyridin-2-yl)-N-(2-hydroxyethyl)-5-methoxy-2,2-dimethyl-2H-
1
chromene-6-carboxamide (20). Yield = 65%, pale yellow oil; H-NMR (CDCl3, 400 MHz) δ 6.84
(m, 2H), 6.62 (m, 1H), 6.50 (d, J = 10.0 Hz, 1H), 6.37 (d, J = 8.3 Hz, 1H), 5.60 (d, J = 9.6 Hz, 1H),
4.09 (m, 2H), 3.90 (s, 3H), 3.83 (s, 3H), 3.79 (s, 3H), 1.38 (s, 6H); HRMS (FAB) calcd for
C22H26N2O6 [M]+ 414.1791, found 414.1792.
4.1.3.10. N-(3,4-Dimethoxyphenyl)-5-methoxy-N-(2-methoxyethyl)-2,2-dimethyl-2H-chromene-6-
carboxamide (21). Yield = 80%, pale yellow oil; 1H-NMR (CDCl3, 300 MHz) δ 6.77-6.61 (m, 4H),
6.44 (d, J = 9.9 Hz, 1H), 6.32(d, J = 8.4 Hz, 1H), 5.58 (d, J = 9.9 Hz, 1H), 4.04 (t, J = 5.7 Hz, 2H),
3.88 (s, 3H), 3.79 (s, 3H), 3.70 (s, 3H), 3.66 (t, J = 5.7 Hz, 2H), 3.39 (s, 3H), 1.35 (s, 6H); HRMS
(FAB) calcd for C24H30NO6 [M + H]+ 428.2073, found: 428.2077.
4.1.3.11.
N-(2-Fluoro-3,4-dimethoxyphenyl)-5-methoxy-N-(2-methoxyethyl)-2,2-dimethyl-2H-
1
chromene-6-carboxamide (22). Yield = 60%, pale yellow oil; H-NMR (CDCl3, 300 MHz) δ 6.90
(d, J = 8.7 Hz, 1H), 6.83 (t, J = 8.4 Hz, 1H), 6.71 (d, J = 9.9 Hz, 2H), 6.33 (d, J = 8.4 Hz, 1H), 5.53
(d, J = 10.2 Hz, 1H), 4.31-4.27 (m, 2H), 3.82 (s, 3H), 3.75 (s, 6H), 3.66-3.55 (m, 2H), 3.32 (s, 3H),
1.31 (s, 6H); HRMS (FAB) calcd for C24H29FNO6 [M + H]+ 446.1979, found: 446.1982.
4.1.3.12.
N-(5,6-Dimethoxypyridin-2-yl)-5-methoxy-N-(2-methoxyethyl)-2,2-dimethyl-2H-
chromene-6-carboxamide (23). Yield = 85%, pale yellow solid, mp = 60-61 oC; 1H-NMR (CDCl3,
400 MHz) δ 7.27 (m, 1H), 6.87 (m, 3H), 6.47 (d, J = 8.2 Hz, 1H), 5.59 (m, 1H), 4.19 (m, 2H), 3.88
(s, 3H), 3.86 (s, 3H), 3.82 (s, 3H), 3.68 (m, 2H), 3.45 (s, 3H), 1.44 (s, 6H); HRMS (FAB) calcd for
C23H29N2O6 [M + H]+ 429.2026, found 429.2018.
4.1.3.13.
N-(2-(Benzyloxy)ethyl)-N-(3,4-dimethoxyphenyl)-5-methoxy-2,2-dimethyl-2H-
chromene-6-carboxamide (24). Yield = 70%, pale yellow oil; 1H-NMR (CDCl3, 300 MHz) δ 7.35-
21

7.26 (m, 5H), 6.72 (m, 2H), 6.60 (m, 2H), 6.44 (d, J = 9.9 Hz, 1H), 6.31 (d, J = 8.4 Hz, 1H), 5.58 (d,
J = 9.9 Hz, 1H), 4.56 (s, 2H), 4.11 (m, 3H), 3.85 (s, 3H), 3.81 (t, J = 5.5 Hz, 2H), 3.77 (s, 3H), 3.56
+
(s, 3H), 1.35 (s, 6H); HRMS (FAB) calcd for C30H34NO6 [M + H]+ 504.2386, found: 504.2383.
4.1.3.14. N-(2-(Benzyloxy)ethyl)-N-(2-fluoro-3,4-dimethoxyphenyl)-5-methoxy-2,2-dimethyl-2H-
chromene-6-carboxamide (25). Yield = 70%, pale yellow oil; 1H-NMR (CDCl3, 300 MHz) δ 7.33-
7.26 (m, 5H), 6.90 (dd, J = 8.1, 0.9 Hz, 1H), 6.79 (t, J = 9.0 Hz, 1H), 6.42-6.33 (m, 3H), 5.54 (d, J =
9.9 Hz, 1H), 4.51-4.31 (m, 4H), 3.74 (s, 3H), 3.73 (s, 3H), 3.72 (s, 3H), 3.72-3.66 (m, 2H), 1.31 (s,
3H), 1.30 (s, 3H); HRMS (FAB) calcd for C30H33FNO6 [M + H]+ 522.2292, found: 522.2291.
4.1.3.15.
N-(2-(Benzyloxy)ethyl)-N-(5,6-dimethoxypyridin-2-yl)-5-methoxy-2,2-dimethyl-2H-
chromene-6-carboxamide (26). Yield = 82%, pale yellow solid, mp = 69-70 oC; 1H-NMR (CDCl3,
400 MHz) δ 7.39 (m, 1H), 7.32 (m, 5H), 6.83 (m, 2H), 6.50 (d, J = 10.0 Hz, 1H), 6.36 (d, J = 8.2 Hz,
1H), 5.59 (d, J = 10.4 Hz, 1H), 4.49 (m, 2H), 4.26 (m, 2H), 3.85 (s, 3H), 3.82 (s, 3H), 3.76 (s, 3H),
+
3.61 (m, 2H), 1.38 (s, 6H); HRMS (FAB) calcd for C29H33N2O6 [M + H]+ 505.2339, found 505.2334.
4.1.3.16. N-(2-Aminoethyl)-N-(3,4-dimethoxyphenyl)-5-methoxy-2,2-dimethyl-2H-chromene-6-
o
carboxamide (27). Yield = 82%, pale yellow solid, mp = 69-70 C; 1H-NMR (CDCl3, 400 MHz) δ
7.39 (m, 1H), 7.32 (m, 5H), 6.83 (m, 2H), 6.50 (d, J = 10.0 Hz, 1H), 6.36(d, J = 8.2 Hz, 1H), 5.59 (d,
J = 10.4 Hz, 1H), 4.49 (m, 2H), 4.26 (m, 2H), 3.85 (s, 3H), 3.82 (s, 3H), 3.76 (s, 3H), 3.61 (m, 2H),
+
1.38 (s, 6H); HRMS (FAB) calcd for C23H29N2O5 [M + H]+ 413.2339, found 413.2334.
4.1.3.17.
N-(2-Aminoethyl)-N-(2-fluoro-3,4-dimethoxyphenyl)-5-methoxy-2,2-dimethyl-2H-
chromene-6-carboxamide (28). Yield = 70%, pale yellow solid, mp = 105-106 oC; 1H-NMR (CDCl3,
300 MHz) δ 6.97 (d, J = 8.2 Hz, 1H), 6.88 (t, J = 8.8 Hz, 1H), 6.62 (dd, J = 9.0, 1.8 Hz, 1H), 6.40 (d,
J = 9.7 Hz, 1H), 6.87 (d, J = 7.9 Hz, 1H), 5.66 (d, J = 10.1 Hz, 1H), 4.06 (m, 2H), 3.78 (s, 3H), 3.76
22

(s, 3H), 3.71 (s, 3H), 2.85 (t, J = 6.8 Hz, 2H), 1.31 (s, 6H); HRMS (FAB) calcd for C23H28FN2O5 [M
+ H]+ 431.1982, found: 431.1979.
4.1.3.18.
N-(2-Aminoethyl)-N-(5,6-dimethoxypyridin-2-yl)-5-methoxy-2,2-dimethyl-2H-
chromene-6-carboxamide (29). Yield = 70%, pale yellow solid, mp = 105-106 oC; 1H-NMR (CDCl3,
300 MHz) δ 6.97 (d, J = 8.2 Hz, 1H), 6.88 (t, J = 8.8 Hz, 1H), 6.62 (dd, J = 9.0, 1.8 Hz, 1H), 6.40 (d,
J = 9.7 Hz, 1H), 6.87 (d, J = 7.9 Hz, 1H), 5.66 (d, J = 10.1 Hz, 1H), 4.06 (m, 2H), 3.78 (s, 3H), 3.76
(s, 3H), 3.71 (s, 3H), 2.85 (t, J = 6.8 Hz, 2H), 1.31 (s, 6H); HRMS (FAB) calcd for C22H28N3O5 [M
+ H]+ 414.1982, found: 414.1979.
4.1.3.19. N-(3-Aminopropyl)-N-(3,4-dimethoxyphenyl)-5-methoxy-2,2-dimethyl-2H-chromene-6-
carboxamide (33). Yield = 70%, pale yellow oil; 1H-NMR (CD3OD, 300 MHz) δ 6.80-6.69 (m, 4H),
6.46 (d, J = 9.9 Hz, 1H), 6.32 (d, J = 8.4 Hz, 1H), 5.71 (d, J = 10.1 Hz, 1H), 4.03 (t, J = 6.6 Hz, 2H),
3.85 (s, 3H), 3.72 (s, 3H), 3.67 (s, 3H), 3.10 (t, J = 7.3 Hz, 2H), 1.97-1.92 (m, 2H), 1.33 (s, 6H);
HRMS (FAB) calcd for C24H31N2O5 [M + H]+ 427.2233, found: 427.2221.
4.1.3.20.
N-(3-Aminopropyl)-N-(2-fluoro-3,4-dimethoxyphenyl)-5-methoxy-2,2-dimethyl-2H-
chromene-6-carboxamide (34). Yield = 70%, pale yellow oil; 1H-NMR (CD3OD, 300 MHz) δ 6.92-
6.87 (m, 2H), 6.67 (d, J = 9.0 Hz, 1H), 6.41 (d, J = 9.9 Hz, 1H), 6.36 (d, J = 8.6 Hz, 1H), 5.68 (d, J
= 9.9 Hz, 1H), 3.91 (m, 2H), 3.80 (s, 3H), 3.77 (s, 3H), 3.70 (s, 3H), 3.12-3.07 (m, 2H), 1.93 (m, 2H),
1.32 (s, 6H); HRMS (FAB) calcd for C24H30FN2O5 [M + H]+ 445.2139, found: 445.2130.
4.1.3.21.
N-(3-Aminopropyl)-N-(5,6-dimethoxypyridin-2-yl)-5-methoxy-2,2-dimethyl-2H-
1
chromene-6-carboxamide (35). Yield = 45%, yellow oil; H-NMR (CDCl3, 300 MHz) δ 6.78 (m,
2H), 6.46 (d, J = 10.0 Hz, 1H), 6.41 (d, J = 8.0 Hz, 1H), 6.33 (d, J = 8.4 Hz, 1H), 5.61 (d, J = 10.0
23

Hz, 1H), 4.16 (m, 2H), 3.87 (s, 3H), 3.83 (s, 3H), 3.77 (s, 3H), 3.16 (m, 2H), 2.00 (m, 4H), 1.37 (s,
6H); HRMS (FAB) calcd for C23H30N3O5 [M + H]+ 428.2185, found 428.2185.
4.1.3.22.
N-(3,4-Dimethoxyphenyl)-5-methoxy-2,2-dimethyl-N-(2-(methylamino)ethyl)-2H-
chromene-6-carboxamide (36). Yield = 45%, pale yellow oil; 1H-NMR (CD3OD, 300 MHz) δ 6.88
(d, J = 8.4 Hz, 1H), 6.78-6.70 (m, 3H), 6.44 (d, J = 10.1 Hz, 1H), 6.34 (d, J = 8.4 Hz, 1H), 5.68(d, J
= 10.1 Hz, 1H), 4.07 (t, J = 6.6 Hz, 2H), 3.83 (s, 3H), 3.72 (s, 3H), 3.67 (s, 3H), 2.94 (t, J = 6.6 Hz,
2H), 2.51 (s, 3H), 1.32 (s, 6H); HRMS (FAB) calcd for C24H31N2O5 [M + H]+ 427.2233, found:
427.2247.
4.1.3.23. N-(2-Fluoro-3,4-dimethoxyphenyl)-5-methoxy-2,2-dimethyl-N-(2-(methylamino)-ethyl)-
2H-chromene-6-carboxamide (37). Yield = 70%, pale yellow solid, mp = 84-85 oC; 1H-NMR (CDCl3,
300 MHz) δ 6.99 (d, J = 7.7 Hz, 1H), 6.87 (t, J = 8.8 Hz, 1H), 6.63 (dd, J = 9.0, 1.8 Hz, 1H), 6.40 (d,
J = 9.9 Hz, 1H), 6.38 (d, J = 8.1 Hz, 1H), 5.67 (d, J = 9.9 Hz, 1H), 4.08-3.87 (m, 2H), 3.78 (s, 3H),
3.76 (s, 3H), 3.72 (s, 3H), 3.03 (t, 2H, J = 6.6 Hz), 2.60 (s, 3H), 1.31 (s, 6H); HRMS (FAB) calcd for
C24H30FN2O5 [M + H]+ 445.2139, found: 445.2146.
4.1.3.24. N-(5,6-Dimethoxypyridin-2-yl)-5-methoxy-2,2-dimethyl-N-(2-(methylamino)ethyl)-2H-
chromene-6-carboxamide (38). Yield = 80%, pale yellow solid, mp = 88-89 oC; 1H-NMR (CD3OD
300 MHz) δ 7.03 (d, J = 7.9 Hz, 1H), 6.91 (d, J = 8.2 Hz, 1H), 6.59 (m, 1H), 6.47 (d, J = 9.9 Hz, 1H),
6.38 (d, J = 8.2 Hz, 1H), 5.69 (d, J = 9.9 Hz, 1H), 4.10 (t, J = 6.6 Hz, 2H), 3.84 (s, 3H), 3.81 (s, 3H),
3.74 (s, 3H), 2.88 (t, J = 6.6 Hz, 2H), 2.43 (s, 3H), 1.35 (s, 6H); HRMS (FAB) calcd for C23H30N3O5
[M + H]+ 428.2185, found: 428.2186.
4.1.3.25.
N-(3,4-Dimethoxyphenyl)-N-(2-(dimethylamino)ethyl)-5-methoxy-2,2-dimethyl-2H-
chromene-6-carboxamide (39). Yield = 45%, pale yellow oil; 1H-NMR (CD3OD, 300 MHz) δ 6.84
24

(d, J = 8.4 Hz, 1H), 6.75 (m, 3H), 6.44 (d, J = 9.9 Hz, 1H), 6.33 (d, J = 8.0 Hz, 1H), 5.67 (d, J = 9.9
Hz, 1H), 4.02 (m, 2H), 3.83 (s, 3H), 3.73 (s, 3H), 3.67 (s, 3H), 2.62 (m, 2H), 2.33 (s, 6H), 1.32 (s,
6H); HRMS (FAB) calcd for C25H33N2O5 [M + H]+ 441.2389, found: 441.2380.
4.1.3.26.
N-(2-(Dimethylamino)ethyl)-N-(2-fluoro-3,4-dimethoxyphenyl)-5-methoxy-2,2-dime-
thyl-2H-chromene-6-carboxamide (40). Yield = 60%, pale yellow oil; 1H-NMR (CD3OD, 300 MHz)
δ 6.94 (d, J = 8.4 Hz, 1H), 6.89 (t, J = 8.4 Hz, 1H), 6.62 (m, 1H), 6.40 (d, J = 8.6 Hz, 1H), 6.37 (d, J
= 7.9 Hz, 1H), 5.65 (d, J = 10.1 Hz, 1H), 4.11 (m, 2H), 3.78 (s, 3H), 3.76 (s, 3H), 3.70 (s, 3H), 2.65
(t, J = 6.8 Hz, 2H), 2.35 (s, 6H), 1.31 (s, 6H); HRMS (FAB) calcd for C25H32FN2O5 [M + H]+
459.2295, found: 459.2292.
4.1.3.27. N-(5,6-Dimethoxypyridin-2-yl)-N-(2-(dimethylamino)ethyl)-5-methoxy-2,2-dimethyl-2H-
1
chromene-6-carboxamide (41). Yield = 65%, pale yellow oil; H-NMR (CDCl3, 400 MHz) δ 7.24
(m, 1H), 6.84 (m, 2H), 6.47 (m, 1H), 6.38 (d, J = 8.3 Hz, 1H), 5.60 (m, 1H), 4.18 (m, 2H), 3.91 (s,
3H), 3.85 (s, 3H), 3.79 (s, 3H), 2.89 (m, 2H), 2.45 (s, 6H), 1.43 (s, 6H); HRMS (FAB) calcd for
C24H32N3O5 [M + H]+ 442.2342, found 442.2350.
4.1.3.28. N-(5,6-Dimethoxypyridin-2-yl)-N-(3-(dimethylamino)propyl)-5-methoxy-2,2-dimethyl-
1
2H-chromene-6-carboxamide (42). Yield = 50%, pale yellow oil; H-NMR (CD3OD, 300 MHz) δ
7.04 (d, J = 7.9 Hz, 1H), 6.85 (d, J = 8.2 Hz, 1H), 6.59 (m, 1H), 6.48 (d, J = 9.9 Hz, 1H), 6.36(d, J =
8.4 Hz, 1H), 5.70 (d, J = 10.1 Hz, 1H), 4.02 (m, 2H), 3.83 (s, 3H), 3.82 (s, 3H), 3.74 (s, 3H), 2.71 (m,
2H), 2.44 (s, 6H), 2.03-1.89 (m, 2H), 1.35 (s, 6H); HRMS (FAB) calcd for C25H34N3O5 [M + H]+
456.2498, found: 456.2495.
4.1.3.29. N-(2-(Diethylamino)ethyl)-N-(5,6-dimethoxypyridin-2-yl)-5-methoxy-2,2-dimethyl-2H-
1
chromene-6-carboxamide (43). Yield = 45%, yellow oil; H-NMR (CDCl3, 300 MHz) δ 6.97 (m,
25

2H), 6.49-6.38 (m, 3H), 5.70 (d, J = 10.1 Hz, 1H), 4.09 (m, 2H), 3.85 (s, 3H), 3.82 (s, 3H), 3.74 (s,
3H), 2.90 (m, 2H), 2.68 (m, 4H), 1.35 (s, 6H), 1.09-1.00 (m, 6H); HRMS (FAB) calcd for C26H36N3O5
[M + H]+ 470.2655, found: 470.2649.
4.1.3.30. N-(2-Acetamidoethyl)-N-(3,4-dimethoxyphenyl)-5-methoxy-2,2-dimethyl-2H-chromene-
6-carboxamide (44). Yield = 80%, white solid, mp = 79-80 oC; 1H-NMR (CDCl3, 300 MHz) δ 6.70-
6.62 (m, 4H), 6.44 (d, J = 9.9 Hz, 1H), 6.32 (d, J = 8.2 Hz, 1H), 5.61 (d, J = 9.9 Hz, 1H), 4.05 (t, J =
5.3 Hz, 2H), 3.88 (s, 3H), 3.79 (s. 3H), 3.72 (s, 3H), 3.52 (m, 2H), 2.03 (s, 3H), 1.36 (s, 6H); HRMS
(FAB) calcd for C25H31N2O6 [M + H]+ 455.2182, found: 455.2184.
4.1.3.31.
N-(2-Acetamidoethyl)-N-(5,6-dimethoxypyridin-2-yl)-5-methoxy-2,2-dimethyl-2H-
chromene-6-carboxamide (45). Yield = 45%, yellow oil; 1H-NMR (CDCl3, 300 MHz) δ 6.81 (t, J =
8.6 Hz, 2H), 6.70 (m, 1H), 6.48 (d, J = 9.5 Hz, 1H), 6.43 (m, 1H), 6.37 (d, J = 8.4 Hz, 1H), 5.62 (d,
J = 9.9 Hz, 1H), 4.15 (m, 2H), 3.92 (s, 3H), 3.85 (s, 3H), 3.78 (s, 3H), 3.53 (m, 2H), 1.99 (s, 3H),
1.38 (s, 6H); HRMS (FAB) calcd for C24H30N3O6 [M + H]+ 456.2135, found 456.2135.
4.1.3.32.
N-(3-Acetamidopropyl)-N-(5,6-dimethoxypyridin-2-yl)-5-methoxy-2,2-dimethyl-2H-
chromene-6-carboxamide (46). Yield = 80%, pale yellow solid, mp = 45-46 oC; 1H-NMR (CDCl3,
300 MHz) δ 6.99 (m, 1H), 6.79 (d, J = 8.4 Hz, 1H), 6.51 (d, J = 10.1 Hz, 1H), 6.44 (m, 1H), 6.35 (d,
J = 8.3 Hz, 1H), 5.63 (d, J = 9.9 Hz, 1H), 4.11 (m, 2H), 3.89 (s, 3H), 3.87 (s, 3H), 3.79 (s, 3H), 3.89
(m, 2H), 2.05 (s, 3H), 1.72 (m, 2H), 1.38 (s, 6H); HRMS (FAB) calcd for C25H32N3O6 [M + H]+
470.2291, found: 470.2291.
4.1.3.33.
tert-Butyl
(2-(N-(3,4-dimethoxyphenyl)-5-methoxy-2,2-dimethyl-2H-chromene-6-
o
1
carboxamido)ethyl)carbamate (47). Yield = 80% , white solid, mp = 70-71 C; H-NMR (CDCl3,
300 MHz) δ 6.73-6.63 (m, 4H), 6.45 (d, J = 9.7 Hz, 1H), 6.31 (d, J = 8.4 Hz, 1H), 5.59 (d, J = 10.1
26

Hz, 1H), 5.22 (s, 1H), 4.00 (t, J = 5.7 Hz, 2H), 3.89 (s, 3H), 3.78 (s, 3H), 3.70 (s, 3H), 3.41 (m, 2H),
1.44 (s, 9H), 1.36 (s, 6H); HRMS (FAB) calcd for C28H36N2O7 [M]+ 512.2523, found: 512.2528.
4.1.3.34. tert-Butyl (2-(N-(5,6-dimethoxypyridin-2-yl)-5-methoxy-2,2-dimethyl-2H-chromene-6-
carboxamido)ethyl)carbamate (48). Yield = 90% yield, pale yellow solid, mp = 64-65 oC; 1H-NMR
(CDCl3, 400 MHz) δ 8.09 (s, 1H), 7.74 (m, 1H), 7.32 (m, 1H), 7.14 (m, 1H), 6.75 (m, 1H), 6.46 (d, J
= 8.2 Hz, 1H), 5.67 (m, 1H), 4.34 (m, 2H), 3.91 (s, 3H), 3.88 (s, 3H), 3.83 (s, 3H), 3.35 (m, 2H), 1.45
(s, 6H), 1.26 (s, 9H); HRMS (FAB) calcd for C27H36N3O7 [M + H]+ 514.2553, found 514.2548.
4.1.3.35. N-(5,6-Dimethoxypyridin-2-yl)-5-methoxy-2,2-dimethyl-N-(2-(pyrrolidin-1-yl)ethyl)-2H-
1
chromene-6-carboxamide (49). Yield = 65%, pale yellow oil; H-NMR (CDCl3, 400 MHz) δ 6.85
(d, J = 8.4 Hz, 1H), 6.81 (d, J = 8.4 Hz, 1H), 6.45 (m, 2H), 6.40 (d, J = 8.3 Hz, 1H), 5.62 (d, J = 10.0
Hz, 1H), 4.46 (t, J = 7.2 Hz, 2H), 3.87 (s, 3H), 3.83 (s, 3H), 3.81 (s, 3H), 3.43 (t, J = 7.6 Hz, 2H),
2.14 (m, 4H), 2.00 (m, 2 H), 1.93 (m, 2H), 1.38 (s, 6H); HRMS (FAB) calcd for C26H34N3O5 [M +
H]+ 468.2498, found 468.2488.
4.1.3.36.
N-(3,4-Dimethoxyphenyl)-5-methoxy-2,2-dimethyl-N-(2-(piperidin-1-yl)ethyl)-2H-
1
chromene-6-carboxamide (50). Yield = 70%, yellow oil; H-NMR (CDCl3, 300 MHz) δ 6.81-6.60
(m, 4H), 6.47 (d, J = 9.9 Hz, 1H), 6.32 (d, J = 8.3 Hz, 1H), 5.58 (d, J = 9.9 Hz, 1H), 4.03 (t, J = 6.8
Hz, 2H), 3.88 (s, 3H), 3.79 (s, 3H), 3.68 (s, 3H), 2.58 (t, J = 6.8 Hz, 2H), 2.49 (m, 4H), 1.60 (m, 4H),
1.45 (m, 2H), 1.35 (s, 6H); HRMS (FAB) calcd for C28H37N2O5 [M + H]+ 481.2702, found: 481.2710.
4.1.3.37. N-(2-Fluoro-3,4-dimethoxyphenyl)-5-methoxy-2,2-dimethyl-N-(2-(piperidin-1-yl)ethyl)-
1
2H-chromene-6-carboxamide (51). Yield = 50%, pale yellow oil; H-NMR (CD3OD, 300 MHz) δ
6.94-6.87 (m, 2H), 6.67-6.60 (m, 1H), 6.40 (d, J = 9.8 Hz, 1H), 6.36 (d, J = 8.2 Hz, 1H), 5.65 (d, J =
10.1 Hz, 1H), 4.22-4.10 (m, 2H), 3.79 (s, 3H), 3.76 (s, 3H), 3.70 (s, 3H), 2.65-2.51 (m, 6H), 1.63-
27

1.55 (m, 4H), 1.48-1.43 (m, 2H), 1.31(s, 6H); HRMS (FAB) calcd for C28H36FN2O5 [M + H]+
499.2608, found: 499.2598.
4.1.3.38. N-(5,6-Dimethoxypyridin-2-yl)-5-methoxy-2,2-dimethyl-N-(2-(piperidin-1-yl)ethyl)-2H-
1
chromene-6-carboxamide (52). Yield = 65%, pale yellow oil; H-NMR (CDCl3, 400 MHz) δ 7.27
(m, 1H), 6.85 (m, 2H), 6.49 (d, J = 10.0 Hz, 1H), 6.38 (d, J = 8.3 Hz, 1H), 5.60 (d, J = 9.6 Hz, 1H),
4.13 (m, 2H), 3.91 (s, 3H), 3.87 (s, 3H), 3.75 (s, 3H), 2.72 (m, 2H), 2.52 (m, 4H), 1.58 (m, 4H), 1.54
(m, 2H), 1.38 (s, 6H); HRMS (FAB) calcd for C27H36N3O5 [M + H]+ 482.2655, found 482.2648.
4.1.3.39. N-(5,6-Dimethoxypyridin-2-yl)-5-methoxy-2,2-dimethyl-N-(2-(piperidin-4-yl)ethyl)-2H-
chromene-6-carboxamide (53). Yield = 45%, pale yellow solid, mp = 46-47 oC; 1H-NMR (CDCl3,
300 MHz) δ 7.03 (d, J = 7.1 Hz, 1H), 6.82 (d, J = 7.1 Hz, 1H), 6.55-6.35 (m, 3H), 5.69 (d, J = 10.1
Hz, 1H), 3.99 (m, 2H), 3.83 (s, 3H), 3.81 (s, 3H), 3.74 (s, 3H), 3.04 (m, 2H), 2.63-2.56 (m, 2H), 1.74
(m, 2H), 1.51 (m, 3H), 1.35 (s, 6H), 1.18 (m, 2H); HRMS (FAB) calcd for C27H36N3O5 [M + H]+
482.2655, found: 482.2664.
4.1.3.40.
N-(3,4-Dimethoxyphenyl)-5-methoxy-2,2-dimethyl-N-(2-morpholinoethyl)-2H-
chromene-6-carboxamide (54). Yield = 45%, pale yellow oil; 1H-NMR (CD3OD, 300 MHz) δ 6.82-
6.76 (m, 4H), 6.46 (d, J = 9.7 Hz, 1H), 6.32 (d, J = 8.3 Hz, 1H), 5.68 (d, J = 10.1 Hz, 1H), 4.04 (t, J
= 6.8 Hz, 2H), 3.85 (s, 3H), 3.72 (s, 3H), 3.68 (m. 4H), 3.59 (s, 3H), 2.57 (t, J = 6.6 Hz, 2H), 2.50 (m,
4H), 1.32 (s, 6H); HRMS (FAB) calcd for C27H35N2O6 [M + H]+ 483.2495, found: 483.2500.
4.1.3.41. N-(2-Fluoro-3,4-dimethoxyphenyl)-5-methoxy-2,2-dimethyl-N-(2-morpholinoethyl)-2H-
o
1
chromene-6-carboxamide (55). Yield = 50%, pale yellow oil, mp = 55-59 C; H-NMR (CD3OD,
300 MHz) δ 6.95-6.90 (m, 2H), 6.66-6.60 (m, 1H), 6.41 (d, J = 10.1 Hz, 1H), 6.37 (d, J = 8.4 Hz,
1H), 5.66 (d, J = 9.9 Hz, 1H), 4.25-4.15 (m, 2H), 3.80 (s, 3H), 3.76 (s, 3H), 3.70 (s, 3H), 3.65 (m,
28

4H), 2.60-2.40 (m, 6H), 1.31 (s, 6H); HRMS (FAB) calcd for C27H34FN2O6 [M + H]+ 501.2401, found:
501.2408.
4.1.3.42.
N-(5,6-Dimethoxypyridin-2-yl)-5-methoxy-2,2-dimethyl-N-(2-morpholinoethyl)-2H-
chromene-6-carboxamide (56). Yield = 65%, pale yellow oil, mp = 45-46 oC; 1H-NMR (CDCl3, 400
MHz) δ 7.27 (m, 1H), 6.84 (m, 2H), 6.49 (d, J = 10.0 Hz, 1H), 6.38 (d, J = 8.3 Hz, 1H), 5.61 (d, J =
10.0 Hz, 1H), 4.12 (m, 2H), 3.91 (s, 3H), 3.87 (s, 3H), 3.75 (s, 3H), 3.67 (m, 2H), 2.59 (m, 4H), 1.65
(m, 4H), 1.38 (s, 6H); HRMS (FAB) calcd for C26H34N3O6 [M + H]+ 484.2448, found 484.2442.
4.1.3.43.
N-(3,4-Dimethoxyphenyl)-5-methoxy-2,2-dimethyl-N-(2-(piperazin-1-yl)ethyl)-2H-
chromene-6-carboxamide (57). Yield = 50%, pale yellow solid, mp = 90-91 oC; 1H-NMR (CD3OD,
300 MHz) δ 6.82-6.76 (m, 4H), 6.45 (d, J = 10.1 Hz, 1H), 6.32 (d, J = 8.3 Hz, 1H), 5.67 (d, J = 9.9
Hz, 1H), 4.10-4.04 (m, 2H), 3.85-3.64 (m, 4H), 3.84 (s, 3H), 3.72 (s, 3H), 3.66 (s, 3H), 2.60-2.40 (m,
6H), 1.32 (s, 6H); HRMS (FAB) calcd for C27H36N3O5 [M + H]+ 482.2655, found: 482.2655.
4.1.3.44. N-(2-Fluoro-3,4-dimethoxyphenyl)-5-methoxy-2,2-dimethyl-N-(2-(piperazin-1-yl)ethyl)-
o
1
2H-chromene-6-carboxamide (58). Yield = 50%, pale yellow solid, mp = 88-89 C; H-NMR
(CD3OD, 300 MHz) δ 6.94-6.89 (m, 2H), 6.64-6.61 (m, 1H), 6.40 (d, J = 10.4 Hz, 1H), 6.37 (d, J =
9.5 Hz, 1H), 5.65 (d, J = 9.9 Hz, 1H), 4.18-4.11 (m, 2H), 3.80-3.68 (m, 4H), 3.79 (s, 3H), 3.76 (s,
3H), 3.70 (s, 3H), 2.60-2.22 (m, 6H), 1.31 (s, 6H); HRMS (FAB) calcd for C27H35FN3O5 [M + H]+
500.2561, found: 500.2561.
4.1.3.45. N-(5,6-Dimethoxypyridin-2-yl)-5-methoxy-2,2-dimethyl-N-(2-(piperazin-1-yl)ethyl)-2H-
chromene-6-carboxamide (59). Yield = 60%, pale yellow solid, mp = 53-54 oC; 1H-NMR (CD3OD,
300 MHz) δ 7.03 (d, J = 7.9 Hz, 1H), 6.87 (d, J = 8.0 Hz, 1H), 6.56 (m, 1H), 6.50 (d, J = 9.9 Hz, 1H),
6.37 (d, J = 8.2 Hz, 1H), 5.69 (d, J = 9.9 Hz, 1H), 4.11 (m, 2H), 3.83 (s, 6H), 3.74 (s, 3H), 2.81 (m,
29