Molecular Diversity (2023) 27:239–248

https://doi.org/10.1007/s11030-022-10423-7

ORIGINAL ARTICLE

Design, synthesis, and biological evaluation of 4‑(1H‑1,2,3‑triazol‑1‑yl)

benzamides as HSP90 inhibitors
Tingting He1 · Shulei Zhu1 · Wei Lu1

Received: 24 December 2021 / Accepted: 23 March 2022 / Published online: 16 April 2022
© The Author(s), under exclusive licence to Springer Nature Switzerland AG 2022

Abstract
Heat shock protein 90 (HSP90) is a promising anticancer drug target, which could be employed to construct HSP90 inhibitors-
based drug conjugates for selective tumor therapy. Herein, a series of 4-(1H-1,2,3-triazol-1-yl)benzamides were rationally
designed, synthesized as HSP90 inhibitors, and their structures were characterized by 1H NMR, 13C NMR, and HR-MS.
Preliminary HSP90 binding assay showed that compounds 6b, 6l, 6m, 6n, 6t, and 6u exhibited significant HSP90α bind-
ing affinity. Among these selected compounds, 6u displayed the most potent anti-proliferative activities and particularly
in Capan-1 cell line. Molecular modeling studies also confirmed possible mode of interaction between 6u and the binding
sites of HSP90 by hydrogen bond and hydrophobic interactions. Above all, these encouraging data indicated that 6u could
be used as a HSP90 inhibitor for further study and helped the recognition of the 4-(1H-1,2,3-triazol-1-yl)benzamide motif
as a new scaffold for HSP90 inhibitors.
Graphical abstract

Keywords Synthesis · HSP90 inhibitors · Anti-proliferative activities · Molecular docking

* Shulei Zhu Introduction

[email protected]
* Wei Lu Molecular chaperones are responsible for the assembly
[email protected] and correct folding of polypeptide chains, which prevent
1
Shanghai Engineering Research Center of Molecular the denaturation and inappropriate aggregation of proteins
Therapeutics and New Drug Development, School to maintain protein homeostasis [1]. Heat shock protein
of Chemistry and Molecular Engineering, East China Normal 90 (HSP90) is one of the most abundant and highly con-
University, 3663 North Zhongshan Road,
served molecular chaperones, which has received significant
Shanghai 200062, People’s Republic of China

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240 Molecular Diversity (2023) 27:239–248

attention as a therapeutic target for cancer treatment [2–4]. accumulates and is retained in tumors, resulting in a sus-
HSP90 comprises approximately 1%-3% of the cellular tained release of SN-38 [17–19]. We previously reported two
proteins and overexpresses up to tenfold under physiologic highly efficient tumor-targeting fluorescent probes NP-001
stress [5]. Presently, HSP90 has been identified as four iso- and Probe 2, which were integrated with HSP90 inhibitors
forms, including HSP90α, HSP90β, GRP94, and TRAP1. and 4-hydroxy-1,8-naphthalimide. NP-001 and Probe 2
It is essential for the cell viability, ATP-dependent folding, have shown a relatively longer retention time in tumor due
and assembling of its client proteins [6, 7]. To date, many to the binding to HSP90 [14, 20]. Based on these results,
HSP90’s clients, including PARP, Aurora A, PI3K, BRD4, an HSP90 inhibitor–SN-38 conjugate (18b) was also devel-
hTERT, and the estrogen/androgen receptors, have been oped, exhibiting superior antitumor activity and low toxicity
involved in tumorgenesis [8–11]. It has been shown that the in HCT116 and Capan-1 xenograft models [21]. Inspired by
natural products geldanamycin, radicicol, and their deriva- these results, further optimization and identification of novel
tives were known to be active in inhibition of HSP90, lead- HSP90 inhibitors, which could be employed in HSP90 drug
ing to degradation of client proteins by the ubiquitin–pro- conjugates, appear as an interesting challenge.
teasome pathway, which marked the beginning of the era Many researches indicated that core structures triazole
of HSP90 inhibitors [12, 13]. Recently, a large number [22–24] or benzamide [25–27] in HSP90 inhibitors would
of HSP90 inhibitors based on varied scaffolds have been contribute to improved efficacy and safety profile. These
reported. Most of them were reported to bind the N-terminal HSP90 inhibitors showed notable anticancer activities
ATP-binding pocket of HSP90, including the triazole scaf- in vitro and in vivo. Docking simulations suggested that the
fold and benzamide scaffold (Fig. 1) [14]. Unfortunately, triazoles and benzamide structure could penetrate the HSP90
none of them have been brought into market, because of ATP-binding site and form hydrogen bonds and hydrophobic
problematic off-target effects and related toxicities, such contacts with the amino acid residues. In this research, in
as ocular toxicities, excess hepatotoxicity, which compro- order to obtain more potent HSP90 inhibitors, a series of
mised their efficacy and led to resistance and metastasis [15]. 4-(1H-1,2,3-triazol-1-yl)benzamides (6a–6u) combined with
Therefore, it is urgent to find a new method to apply these triazole or benzamide structures were rationally designed
inhibitors. and synthesized (Scheme 1), of which their HSP90 bind-
Recently, many efforts have been focused on developing ing affinity and anti-proliferative activities against different
HSP90 inhibitors-based drug conjugates, which employed cancer cell lines were evaluated. Among them, compounds
HSP90 inhibitors as potential tumor-targeting ligands for 6u showed acceptable HSP90α binding affinity and potent
selective tumor chemotherapy. In these conjugates, HSP90 in vitro antitumor activity. Molecular modeling studies also
inhibitors were conjugated with fluorescent molecules or displayed possible mode of interaction between 6u and the
drugs through various linkers, which could improve the binding sites of HSP90.
delivery of the fluorescent molecules or drugs into tumor
cells via eHSP90-mediated endocytosis (Fig. 2) [14, 16–21]. Chemistry
Crowe, L. B. et al. has reported a cell impermeable far-red
fluorophore-tagged HSP90 inhibitor HS-131, which high- HSP90 inhibitors 6a–6u were synthesized using the route
lights the therapeutic relevance of selectively targeting summarized in Scheme 2. The phenyl hydrazine intermedi-
HSP90 internalization [16]. Tarveda Therapeutics also has ate (2) was prepared from commercially available 2,4-dif-
discovered a miniature drug conjugate PEN-866, which links luorobenzonitrile (1) via nucleophilic substitution with
a HSP90 inhibitor to SN-38 cytotoxic payload. PEN-866 hydrazine in methanol at reflux. Subsequently, intermediate

N
O N N
O CF3
N
HO N
N N N
N N
HO O O N
HO N O
N N NH2 O
N H
N OH OH N N HN H2 N
H2N O

triazole scaffold benzamide scaffold

Fig. 1  Typical HSP90 inhibitors with triazole scaffold and benzamide scaffold

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Molecular Diversity (2023) 27:239–248 241

N O
N
N

O NH
N O N SO3H
H O
O H
H2 N O HO N
O N
N O
+ H O OH
- O N O
N OH
SO3
HS-131 NP-001

O
N
OH
HO
N N O HO
O N
N O
O O
N O
HO N O OH O N HN N
N
N OH O
HO O O
O
PEN866 18b

Fig. 2  Typical HSP90 probes and HSP90 conjugates

O NH2 Finally, derivatives 6a–6u carrying amide side chains linked

H to the phenyl ring were prepared from 5 through nucleo-
Benzamide core N
R philic replacements of the 2-fluoride group in a moderate
condition.
N
Triazole core N
N Results and discussion
O 6a-6u
The SAR for these compounds is reported in Table 1. To
validate the HSP90 binding affinity of these synthesized
Scheme 1  Structures of designed and synthesized compounds 6a–6u
inhibitors with different side chains, all of them were then
tested for their potential HSP90 inhibitory properties by
2 was oxidized in the presence of tert-butyl nitrite to obtain 3 fluorescence polarization (FP) assay. 17-AAG was selected
with a relative high yield, which was cyclized with 5,5-dime- as the positive control. As shown in Table 1, the binding
thyl-1,3-cyclohexadione in the presence of DBU in ethylene affinity values were illustrated and expressed as I­ C50 values.
glycol to afford compound 4. Next, the key intermediate 5 Our results showed that the positions of substitutions and the
was given through the hydration of the cyano group of 4. nature of the linker atoms indeed significantly affected the

Scheme 2  Reagents and conditions: (a) N

­ 2H4, MeOH, reflux, 3 h, 80%; (b) tert-butyl nitrite, MeCN, 0 °C, 3 h, 86%; (c) DBU, ­HOCH2CH2OH,
80 °C, 12 h, 90%; (d) H
­ 2O2, DMSO, EtOH, NaOH aq, reflux, 12 h, 65%; and (e) ­RNH2, ­K2CO3, DMF, rt, 3 h, 50–75%

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242 Molecular Diversity (2023) 27:239–248

Table 1  HSP90 binding affinity of 6a–6u and 17-AAG​ Table 2  Anti-proliferative activities of selected compounds
Compound Anti-proliferative activities ­IC50 (nM)a
A549 HCT116 MCF-7

6b 1111 ± 131 621 ± 82 472 ± 32

6l 553 ± 106 375 ± 59 220 ± 42
6m 472 ± 88 389 ± 47 192 ± 36
Compound R HSP90 ­IC50 (μM)a 6n 521 ± 112 289 ± 54 234 ± 65
6t 665 ± 89 335 ± 78 228 ± 68
6a Methyl 0.54 ± 0.06
6u 125 ± 35 92 ± 12 72 ± 9
6b n-Butyl 0.39 ± 0.02
17-AAG​ 255 ± 46 82 ± 21 8±3
6c n-Hexyl 1.62 ± 0.22
a
6d 2-Methoxyethyl 1.18 ± 0.11 IC50 values are indicated as the mean ± SD of at least three inde-
6e 2-(Methylsulfonyl)ethyl 1.17 ± 0.01 pendent experiments
6f 1,3-Dimethoxypropyl 1.55 ± 0.33
6g Cyclopropylmethyl 0.71 ± 0.21 potencies of these compounds supported this rationale. Par-
6h Cyclobutyl 0.67 ± 0.13 ticularly, compound 6u was shown with an ­IC50 value of
6i Cyclopentyl 0.77 ± 0.12 0.08 μM, which was comparable with that of 17-AAG.
6j Cyclohexyl 0.74 ± 0.09 Next, six selected compounds above were submitted to
6k Tetrahydrothiopyran-4-yl 0.69 ± 0.10 SRB assays in three different cancer cell lines, including A549
6l Tetrahydrofuran-3-yl 0.18 ± 0.09 (human lung cancer), HCT116 (human colon cancer), and
6m Tetrahydro-2H-pyran-4-yl 0.14 ± 0.01 MCF-7 (human breast cancer) to determine in vitro anticancer
6n 1,1-Dioxo-tetrahydrothiopyran-4-yl 0.29 ± 0.01 effects on cell growth. 17-AAG was used as a positive con-
6o Furan-2-methyl 0.55 ± 0.09 trol. Encouragingly, as shown in Table 2, all tested compounds
6p Thiophen-3-methyl 0.78 ± 0.12 exhibited appreciable anti-proliferative activities against the
6q Thiophen-2-methyl 0.79 ± 0.21 three cancer cells lines with ­IC50 at dozens of or hundreds of
6r 1-Methoxyphenyl 0.61 ± 0.19 nanomolar levels. Interestingly, MCF-7 responded more sen-
6s 1-Phenylethyl 0.55 ± 0.21 sitively than A549 and HCT116 to the exposure of all tested
6t 2-cis-Hydroxycyclopentyl 0.32 ± 0.03 compounds. Since HSP90 is higher expressed in MCF-7 cell
6u 4-cis-Hydroxycyclohexyl 0.08 ± 0.01 line than in A549 and HCT116 cell lines [28], we assumed
17-AAG​ / 0.08 ± 0.01 that HSP90 overexpression in MCF-7 mainly attributed to the
a
IC50 values are indicated as the mean ± SD of at least three inde-
anti-proliferative activities. Notably, the ­IC50 values of 6u were
pendent experiments.
observed to exhibit lower I­ C50s in A549, HCT116, and MCF-7
groups than those in 6b, 6l, 6m, 6n, 6t, which were consistent
binding affinities. Compounds 6a–c with alkyl chains dis- with previous HSP90 binding results, suggesting the antitumor
played ­IC50 values at hundreds of nanomolar level. Among treatment potential of 6u.
them, 6a with methyl group and 6c with hexyl group were Inspired by the previous results, the anti-proliferative
less potent than 6b with butyl group, indicating that proper capacities of 6u were further evaluated against another
chain length could affect the binding affinity with HSP90 seven cancer cell lines: HT-29, MDA-MB-231, LNCaP,
pocket. Open chain structures in compounds 6d, 6e, and 6f, Capan-1, A375, K562, and BGC823 and one normal cell
which were designed to improve flexibility and solubility, line: HL7702 cell. 17-AAG and NVP-AUY922 were used
showed reduced activity. Side chains with rings (6h–6j) as positive controls. As illustrated in Table 3, 6u exhibited
were also found less favored, because of having no hydrogen potent cytotoxic effect against all cancer cells, with ­IC50 at
bond acceptors and extra spacers. The favored side chains dozens of nanomolar levels. In detail, the ­IC50 values of 6u
were aminocyclic structures that contained oxygen or a weak were between 57 and 107 nM, which were consistent with
hydrogen bond acceptor such as the less stable alkene or sul- the previous HSP90 binding assay and preliminary anti-
fur (6l–6n). The aromatic side chains without H-bond accep- proliferative assay. In addition, the cytotoxic effects of 6u
tors (6o–6s) were shown not well tolerated. The main cause were observed to be weaker than those of NVP-AUY922 and
was due to the poor binding affinity with HSP90 pocket. stronger than those of 17-AAG against several cancer cell
The last members of compounds 6t–6u with hydrophobic lines, which is mainly due to the HSP90 binding affinities.
rings, coupled with hydrogen bond acceptors were designed, Notably, the I­ C50 values of 6u were both slightly lower than
in an effort to target HSP90 binding pocket. The improved those in 17-AAG and NVP-AUY922 groups against human
pancreatic cancer Capan-1, suggesting their potential as

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Molecular Diversity (2023) 27:239–248 243

Table 3  Anti-proliferative activities of 6u

Compound Anti-proliferative activities ­IC50 (nM)a
6u 17-AAG​ NVP-AUY922

HT-29 57 ± 19 0.2 ± 0.02 19 ± 6

MDA-MB-231 92 ± 32 212 ± 68 35 ± 12
LNCaP 67 ± 23 78 ± 37 15 ± 13
Capan-1 107 ± 21 231 ± 28 630 ± 60
A375 82 ± 39 1134 ± 119 120 ± 50
K562 78 ± 21 132 ± 29 61 ± 13
BGC823 102 ± 31 67 ± 19 21 ± 9
HL7702 3382 ± 1313 3200 ± 890 1378 ± 1433
a
IC50 values are indicated as the mean ± SD of at least three inde-
pendent experiments

HSP90 inhibitors for pancreatic cancer treatment. The anti-

proliferative activities of 6u, 17-AAG, and NVP-AUY922
were also evaluated against the normal human liver HL7702
cell line, and all tested compounds showed lower cytotoxic
effects than those in cancer cell lines, indicating the cancer-
specific properties of these HSP90 inhibitors. Fig. 3  Binding modes of 6u to HSP90
Furthermore, to explore the mode of interactions between
HSP90 and 6u, molecular modeling studies were performed
with the N-terminal ATP-binding site of HSP90 (PDB code: hydrophobic interactions, further confirming the therapeu-
3D0B) [29] by means of AutoDock Vina program. Interac- tic potential of 6u. More importantly, these findings helped
tions with Asp93 are common to many HSP90 inhibitors and the recognition of the 4-(1H-1,2,3-triazol-1-yl)benzamides
can be reflected to be essential for interaction with HSP90. motif as a new scaffold for HSP90 inhibitors and also con-
As shown in Fig. 3, compound 6u formed important hydro- firmed the possibility of construction of HSP90 drug con-
gen bonding interaction at the benzamide site with Asp93. jugates for selective tumor therapy. Further optimization of
Besides, 6u also displayed hydrophobic interactions with the these compounds is ongoing.
amino acid residues Ile96, Met98, Leu103, Ala111, Val136,
Phe138, and Ala55. Molecular modeling studies of 6u were
consistent with the results of the binding affinity and the Experimental section
anti-proliferative activities against cancer cell lines, which
further confirmed the therapeutic potential of 6u. Instrumentation and materials

All reagents were obtained from commercial sources and

Conclusion dried prior to use unless noted otherwise. Anhydrous solu-
tions of reaction mixtures were transferred via an oven-
In summary, a series of 4-(1H-1,2,3-triazol-1-yl)benzamides dried syringe or cannula. All reactions were monitored by
have been rationally designed and synthesized as HSP90 thin-layer chromatography (TLC) on 25.4 mm × 76.2 mm
inhibitors. In vitro HSP90 binding affinity was tested first silica 394 gel plates GF-254 and UPLC-Mass on Waters
by FP assay and most of the compounds were found exhibit- ACQUITY UPLC H-Class. 1H NMR and 13C NMR spectra
ing notable HSP90 binding affinities with ­IC50 at nanomolar were recorded at 23 °C in ­CDCl3 and DMSO-d6 on a Bruker
level. Compounds 6b, 6l, 6m, 6n, 6t, and 6u exhibited sig- DRX-400 (400 MHz) using TMS as the internal standard.
nificant anti-proliferative activities against A549, HCT116, Chemical shifts were reported as δ (ppm) and signal split-
and MCF-7. Among them, 6u also exhibited potent cytotoxic ting patterns were described as singlet (s), doublet (d), triplet
effects against Capan-1 and much lower cytotoxic effects (t), quartet (q), quintet (quint), or multiplet (m), with cou-
against HL7702, suggesting their potential for pancreatic pling constants (J) in hertz. High-resolution mass spectra
cancer treatment. Furthermore, molecular docking stud- (HR-MS) were obtained on an electron spray injection (ESI)
ies also revealed that 6u could interact with N-terminal Thermo Fisher Scientific LTQ FTICR mass spectrometer.
ATP-binding site of HSP90 through hydrogen bond and Purity of all tested compounds was ≥ 95%, as estimated

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244 Molecular Diversity (2023) 27:239–248

by HPLC analysis performed on Agilent Diamonsil C18 4‑(6,6‑Dimethyl‑4‑oxo‑4,5,6,7‑tetrahydro‑1H‑benzo[d]

(250 mm × 4.6 mm). [1,2,3]triazol‑1‑yl)‑2‑fluorobenzamide (5) 1 M NaOH
(50 mL), ­H2O2 (30 mL), and DMSO (25 mL) were added
to a mixture of 4 (7.0 g, 24.6 mmol) in EtOH (70 mL). The
Synthesis routes of 6a–6u mixture was heated to reflux for 12 h. After completion of
the reaction, the mixture was cooled to room temperature
2‑Fluoro‑4‑hydrazineylbenzonitrile (2) and filtered to afford the crude product. The crude product
was then recrystallized with PE: EA (5:1) to give a yellow
Hydrazine (2.5 mL, 79.1 mmol) was added to a mixture solid 4.9 g. Yield: 65%. 1H NMR (400 MHz, ­CDCl3) δ 7.95
of 2,4-difluorobenzonitrile (1) (10.0 g, 71.9 mmol) in (dd, J = 7.5, 4.9 Hz, 1H), 7.46 (d, J = 12.3 Hz, 1H), 7.44 (dd,
methanol (100 mL). The mixture was heated to reflux for J = 7.5, 1.6 Hz, 1H), 7.30 (dd, J = 8.1, 1.5 Hz, 1H), 7.26 (d,
3 h. After completion of the reaction, the mixture was J = 12.4 Hz, 1H), 2.79 (s, 2H), 2.64 (s, 2H), 1.12 (s, 6H). 13C
cooled to room temperature and concentrated in vacuo NMR (100 MHz, C ­ DCl3) δ 192.71, 168.03, 160.43, 138.14,
to afford the crude product. The crude product was then 133.48, 131.94, 121.40, 117.71, 109.15, 49.37, 38.20, 35.36,
recrystallized with PE: EA (10:1) to give a pale yellow 28.21. LC − MS (ESI) m/z (M + H)+: 303.3.
solid 8.7 g. Yield: 80%. 1H NMR (400 MHz, DMSO-
d6) δ 8.05 (s, 1H), 7.48 (d, J = 8.7 Hz, 1H), 7.07 (s, 1H),
6.73 (d, J = 7.9 Hz, 1H), 4.39 (s, 2H). LC − MS (ESI) m/z General procedures of synthesis of 6a–6u
(M + H)+:152.2.
Substituted amino compounds (0.2 mmol) and K ­ 2CO 3
(50 mg, 0.34 mmol) were added to a mixture of 5 (50 mg,
4‑Azido‑2‑fluorobenzonitrile (3) 0.17 mmol) in DMF (3 mL). The mixture was stirred at room
temperature for 3 h. After completion of the reaction, the
Tert-butyl nitrite (7.9 mL, 66.2 mmol) was added slowly to mixture was diluted with H ­ 2O and extracted with EtOAc.
a mixture of 2 (5.0 g, 33.1 mmol) in MeCN (50 mL) at 0 The combined organic layers were then washed with satu-
℃ under N ­ 2. The mixture was stirred at 0 ℃ for 3 h. After rated brine, dried over anhydrous N
­ a2SO4, and concentrated
completion of the reaction, the mixture was diluted with in vacuo to afford the crude products. The crude products
EtOAc. The organic layer was then washed with saturated were then purified with column chromatography to give
­NaHCO3 and saturated brine, dried over anhydrous N­ a2SO4, 6a–6u. Yield: 50–75%.
and concentrated in vacuo to afford the crude product. The
crude product was then purified with column chromatog- 4‑(6,6‑Dimethyl‑4‑oxo‑4,5,6,7‑tetrahydro‑1H‑benzo[d]
raphy to give a yellow solid 4.6 g. Yield: 86%. 1H NMR [1,2,3]triazol‑1‑yl)‑2‑(methylamino)benzamide (6a) Yel-
(400 MHz, ­CDCl3) δ 7.57 (dd, J = 7.5, 5.0 Hz, 1H), 6.97 low solid. Yield: 75%. 1H NMR (400 MHz, DMSO-d6) δ
(dd, J = 7.5, 1.5 Hz, 1H), 6.89 (dd, J = 8.0, 1.5 Hz, 1H). 8.28 (d, J = 5.3 Hz, 1H), 8.01 (s, 1H), 7.82 (d, J = 8.3 Hz,
13
C NMR (100 MHz, C ­ DCl3) δ 164.05, 161.95, 143.73, 1H), 7.39 (s, 1H), 6.84 (d, J = 2.0 Hz, 1H), 6.79 (dd, J = 8.3,
133.98, 116.83, 114.13, 107.51, 97.42. LC − MS (ESI) m/z 2.0 Hz, 1H), 3.03 (s, 2H), 2.84 (d, J = 5.0 Hz, 2H), 1.24 (d,
(M + H)+: 163.1. J = 5.2 Hz, 3H), 1.05 (s, 6H). 13C NMR (100 MHz, DMSO-
d6) δ 189.73, 170.54, 151.44, 144.69, 141.03, 138.52,
4‑(6,6‑Dimethyl‑4‑oxo‑4,5,6,7‑tetrahydro‑1H‑benzo[d] 130.46, 114.70, 105.09, 35.49, 34.24, 29.25, 27.56. HR-MS
[1,2,3]triazol‑1‑yl)‑2‑fluorobenzonitrile (4) 5,5-Dimethyl- (m/z) (ESI), calcd for C ­ 16H20N5O16 [M + ­H]+: 314.3610;
1,3-cyclohexadione (3.9 g, 27.7 mmol) and DBU (426 mg, found: 314.3612. Melting point: 240–242 ℃.
2.8 mmol) were added to a mixture of 3 (4.5 g, 27.7 mmol)
in ethylene glycol (100 mL). The mixture was heated to 80 2‑(Butylamino)‑4‑(6,6‑dimethyl‑4‑oxo‑4,5,6,7‑tetrahy‑
℃ for 12 h. After completion of the reaction, the mixture dro‑1H‑benzo[d][1,2,3]triazol‑1‑yl)benzamide (6b) Yel-
was diluted with H ­ 2O and filtered to afford the crude prod- low solid. Yield: 55%. 1H NMR (400 MHz, DMSO-d6) δ
uct. The crude product was then purified with column chro- 7.74 (d, J = 8.3 Hz, 1H), 7.00 (s, 1H), 6.91 (d, J = 8.2 Hz,
matography to give a yellow solid 7.1 g. Yield: 90%. 1H 1H), 6.68 (s, 1H), 3.24 (dd, J = 13.1, 6.6 Hz, 2H), 3.05
NMR (400 MHz, ­CDCl3) δ 7.89 (dd, J = 7.5, 5.1 Hz, 1H), (d, J = 11.3 Hz, 2H), 2.31 (d, J = 58.1 Hz, 1H), 2.12 (d,
7.69 (dd, J = 7.5, 1.5 Hz, 1H), 7.35 (dd, J = 8.1, 1.5 Hz, 1H), J = 9.2 Hz, 1H), 1.62–1.52 (m, 2H), 1.37 (dd, J = 14.6,
2.89–2.68 (m, 2H), 2.64 (s, 2H), 1.12 (s, 6H). 13C NMR 7.2 Hz, 2H), 1.05 (s, 6H), 0.96–0.91 (m, 3H). 13C NMR
(100 MHz, ­CDCl3) δ 192.68, 160.81, 138.68, 137.75, (100 MHz, DMSO-d6) δ 190.19, 151.85, 145.39, 141.63,
133.36, 119.35, 114.54, 110.16, 99.48, 49.31, 38.12, 35.37, 140.43, 135.79, 117.76, 110.57, 106.19, 95.42, 52.35,
28.19. LC − MS (ESI) m/z (M + H)+: 285.3. 42.65, 35.98, 34.76, 30.68, 28.06, 20.04, 14.26. HR-MS

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Molecular Diversity (2023) 27:239–248 245

­ 19H26N5O2 [M + ­H]+: 356.4420;

(m/z) (ESI), calcd for C ­C20H28N5O4 [M + ­H]+: 402.2097; found: 402.2063. Melting
found: 356.4428. Melting point: 238–240 ℃. point: 240–242 ℃.

4‑(6,6‑dimethyl‑4‑oxo‑4,5,6,7‑tetrahydro‑1H‑benzo[d] 2‑((cyclopropylmethyl)amino)‑4‑(6,6‑dime ‑
[1,2,3]triazol‑1‑yl)‑2‑(hexylamino)benzamide (6c) Yellow thyl‑4‑oxo‑4,5,6,7‑tetrahydro‑1H‑benzo[d][1,2,3]tria‑
solid. Yield: 50%. 1H NMR (400 MHz, C ­ DCl3) δ 8.00 (d, zol‑1‑yl)benzamide (6g) Yellow solid. Yield: 68%. 1H NMR
J = 7.5 Hz, 1H), 7.71 (d, J = 6.0 Hz, 1H), 7.69–7.59 (m, (400 MHz, ­CDCl3) δ 7.99–7.94 (m, 2H), 7.69–7.56 (m, 2H),
2H), 7.41 (d, J = 1.4 Hz, 1H), 7.17 (dd, J = 7.5, 1.5 Hz, 7.40 (d, J = 1.5 Hz, 1H), 7.21 (dd, J = 7.5, 1.5 Hz, 1H), 3.27
1H), 3.29 (td, J = 7.1, 6.0 Hz, 2H), 2.69 (d, J = 21.4 Hz, (dd, J = 6.9, 6.0 Hz, 2H), 2.68 (d, J = 34.8 Hz, 4H), 1.76–
4H), 1.67 (p, J = 7.1 Hz, 2H), 1.46–1.25 (m, 6H), 1.08 (s, 1.71 (m, 1H), 1.67–1.63 (m, 2H), 1.17–1.13 (m, 2H), 1.09
6H), 0.95–0.77 (m, 3H). 13C NMR (100 MHz, ­CDCl3) δ (s, 6H). 13C NMR (100 MHz, ­CDCl3) δ 193.18, 168.88,
193.54, 169.04, 169.02, 137.69, 133.88, 113.34, 105.35, 147.12, 137.53, 134.22, 131.08, 115.59, 113.96, 105.90,
49.35, 44.33, 38.18, 35.36, 31.09, 29.07, 28.12, 27.36, 49.35, 47.87, 38.22, 35.36, 27.96, 16.48, 4.47. HR-MS
22.18, 14.11. HR-MS (m/z) (ESI), calcd for ­C21H29N5O2 (m/z) (ESI), calcd for ­ C19H24N5O2 [M + ­H]+: 354.1885;
[M + ­H]+: 384.4960; found: 384.4962. Melting point: found: 354.1889. Melting point: 217–219 ℃.
245–247 ℃.
2‑(cyclobutylamino)‑4‑(6,6‑dimethyl‑4‑oxo‑4,5,6,7‑tetrahy‑
4‑(6,6‑dimethyl‑4‑oxo‑4,5,6,7‑tetrahydro‑1H‑benzo[d] dro‑1H‑benzo[d][1,2,3]triazol‑1‑yl)benzamide(6h) Yellow
[1,2,3]triazol‑1‑yl)‑2‑((2‑methoxyethyl)amino)benzamide solid. Yield: 64%. 1H NMR (400 MHz, DMSO-d6) δ 8.55
(6d) Yellow solid. Yield: 58%. 1H NMR (400 MHz, C ­ DCl3) (d, J = 6.0 Hz, 1H), 8.04 (s, 1H), 7.84 (d, J = 8.4 Hz, 1H),
δ 8.34–8.12 (m, 1H), 7.96 (d, J = 7.5 Hz, 1H), 7.61 (s, 2H), 7.41 (s, 1H), 6.81 (d, J = 8.4 Hz, 1H), 6.74 (s, 1H), 3.97
7.39 (d, J = 1.6 Hz, 1H), 7.21 (dd, J = 7.5, 1.5 Hz, 1H), (q, J = 7.7, 7.1 Hz, 1H), 3.01 (s, 2H), 2.41 (d, J = 7.6 Hz,
3.78–3.57 (m, 4H), 3.36 (s, 3H), 2.69 (d, J = 34.3 Hz, 4H), 2H), 1.82 (td, J = 18.1, 8.4 Hz, 4H), 1.36–1.13 (m, 3H), 1.05
1.10 (s, 6H). 13C NMR (100 MHz, ­CDCl3) δ 193.18, 168.86, (s, 6H). 13C NMR (100 MHz, DMSO-d6) δ 189.72, 170.53,
147.28, 137.53, 134.22, 131.09, 115.25, 113.96, 105.37, 149.18, 144.68, 141.03, 138.42, 130.66, 114.47, 51.87,
70.86, 58.74, 49.17, 44.72, 38.33, 35.36, 27.96. HR-MS 46.92, 35.48, 34.29, 30.24, 27.55, 14.97. HR-MS (m/z)
(m/z) (ESI), calcd for ­ C18H24N5O3 [M + ­H]+: 358.4140; (ESI), calcd for ­C19H24N5O2 [M + ­H]+: 354.1882; found:
found: 358.4138. Melting point: 225–227 ℃. 354.1884. Melting point: 225–227 ℃.

4‑(6,6‑dimethyl‑4‑oxo‑4,5,6,7‑tetrahydro‑1H‑benzo[d] 2‑(cyclopentylamino)‑4‑(6,6‑dimethyl‑4‑oxo‑4,5,6,7‑tet‑
[1,2,3]triazol‑1‑yl)‑2‑((2‑(methylsulfonyl)ethyl)amino)ben‑ rahydro‑1H‑benzo[d][1,2,3]triazol‑1‑yl)benzamide (6i) Yel-
zamide (6e) Yellow solid. Yield: 58%. 1H NMR (400 MHz, low solid. Yield: 63%. 1H NMR (400 MHz, DMSO-d6) δ
­CDCl3) δ 8.27 (t, J = 7.3 Hz, 1H), 8.00 (d, J = 7.5 Hz, 1H), 8.50 (d, J = 6.5 Hz, 1H), 8.02 (s, 1H), 7.83 (d, J = 8.4 Hz,
7.67 (s, 2H), 7.41 (d, J = 1.4 Hz, 1H), 7.16 (dd, J = 7.5, 1H), 7.36 (s, 1H), 6.88 (d, J = 2.1 Hz, 1H), 6.78 (dd, J = 8.3,
1.5 Hz, 1H), 3.70 (q, J = 7.2 Hz, 2H), 3.28 (t, J = 7.1 Hz, 2.1 Hz, 1H), 3.85 (q, J = 6.2 Hz, 1H), 3.02 (s, 2H), 2.01 (dq,
2H), 2.94 (s, 3H), 2.81–2.67 (m, 2H), 2.67 (s, 2H), 1.08 (s, J = 12.6, 6.4 Hz, 2H), 1.64 (ddd, J = 25.2, 10.6, 5.2 Hz, 4H),
6H). 13C NMR (100 MHz, ­CDCl3) δ 193.54, 169.02, 145.29, 1.45 (dq, J = 12.0, 5.9 Hz, 2H), 1.24 (d, J = 6.0 Hz, 1H),
137.75, 137.20, 134.67, 131.09, 115.89, 112.78, 105.45, 1.05 (s, 6H). 13C NMR (100 MHz, DMSO-d6) δ 189.72,
54.60, 48.90, 41.21, 39.43, 38.15, 35.34, 28.19. HR-MS 170.67, 150.06, 144.71, 141.01, 138.39, 130.67, 114.36,
(m/z) (ESI), calcd for ­C18H24N5O4S [M + ­H]+: 406.1506; 108.11, 106.14, 52.96, 51.88, 35.47, 34.30, 32.68, 27.56,
found: 406.1504. Melting point: 228–229 ℃. 23.53. HR-MS (m/z) (ESI), calcd for ­C20H26N5O2 [M + ­H]+:
368.2042; found: 368.2048. Melting point: 228–229 ℃.
2‑((1,3‑dimethoxypropan‑2‑yl)amino)‑4‑(6,6‑dime ‑
thyl‑4‑oxo‑4,5,6,7‑tetrahydro‑1H‑benzo[d][1,2,3]tria‑ 2‑(cyclohexylamino)‑4‑(6,6‑dimethyl‑4‑oxo‑4,5,6,7‑tetrahy‑
zol‑1‑yl)benzamide (6f) Yellow solid. Yield: 65%. 1H NMR dro‑1H‑benzo[d][1,2,3]triazol‑1‑yl)benzamide (6j) Yellow
(400 MHz, ­CDCl3) δ 7.96 (d, J = 7.5 Hz, 1H), 7.65 (s, 2H), solid. Yield: 52%. 1H NMR (400 MHz, DMSO-d6) δ 8.00
7.51 (d, J = 1.6 Hz, 1H), 7.31 (d, J = 11.7 Hz, 1H), 7.15 (dd, (d, J = 7.5 Hz, 1H), 7.71–7.59 (m, 2H), 7.51–7.43 (m, 2H),
J = 7.5, 1.5 Hz, 1H), 3.84 (dp, J = 11.7, 7.0 Hz, 1H), 3.44 7.14 (dd, J = 7.4, 1.5 Hz, 1H), 3.43 (dp, J = 11.0, 7.0 Hz,
(dd, J = 12.4, 7.0 Hz, 4H), 3.22 (s, 6H), 2.82–2.68 (m, 2H), 1H), 2.70 (d, J = 23.4 Hz, 4H), 1.62 (q, J = 7.2 Hz, 4H),
2.67 (d, J = 2.9 Hz, 2H), 1.08 (s, 6H). 13C NMR (100 MHz, 1.53–1.47 (m, 3H), 1.42–1.35 (m, 3H), 1.08 (s, 6H). 13C
­CDCl3) δ 193.95, 169.04, 145.59, 138.19, 137.20, 134.67, NMR (100 MHz, DMSO-d6) δ 193.54, 169.02, 145.42,
130.73, 116.39, 112.78, 107.17, 70.72, 58.07, 54.97, 137.69, 133.88, 130.74, 116.35, 113.34, 107.36, 52.45,
48.84, 38.42, 35.36, 28.18. HR-MS (m/z) (ESI), calcd for 49.35, 38.36, 35.36, 33.39, 28.18, 26.04, 25.03. HR-MS

13
246 Molecular Diversity (2023) 27:239–248

­ 21H28N5O2 [M + ­H]+: 382.2198;

(m/z) (ESI), calcd for C 149.70, 145.27, 141.58, 139.62, 139.09, 131.39, 130.48,
found: 382.2200. Melting point: 245–246 ℃. 116.07, 115.35, 113.63, 112.38, 109.51, 52.37, 49.31,
36.01, 34.67, 29.93, 28.22. HR-MS (m/z) (ESI), calcd for
4‑(6,6‑dimethyl‑4‑oxo‑4,5,6,7‑tetrahydro‑1H‑benzo[d] ­C20H26N5O4S [M + ­H]+: 432.1661; found: 432.1669. Melt-
[1,2,3]triazol‑1‑yl)‑2‑((tetrahydro‑2H‑thiopyran‑4‑yl)amino) ing point: 260–261 ℃.
benzamide (6k) Yellow solid. Yield: 58%. 1H NMR
(400 MHz, DMSO-d6) δ 8.01 (d, J = 7.5 Hz, 1H), 7.73–7.56 4‑(6,6‑dimethyl‑4‑oxo‑4,5,6,7‑tetrahydro‑1H‑benzo[d]
(m, 2H), 7.55–7.46 (m, 2H), 7.15 (dd, J = 7.5, 1.5 Hz, 1H), [1,2,3]triazol‑1‑yl)‑2‑((furan‑2‑ylmethyl)amino)benza‑
3.53 (dp, J = 10.6, 7.0 Hz, 1H), 2.80–2.67 (m, 2H), 2.67 mide (6o) Yellow solid. Yield: 55%. 1H NMR (400 MHz,
(s, 2H), 2.64 (td, J = 7.1, 4.1 Hz, 4H), 1.86 (dq, J = 12.4, DMSO-d6) δ 8.77 (t, J = 5.9 Hz, 1H), 8.06 (s, 1H), 7.85 (d,
7.1 Hz, 2H), 1.66 (dq, J = 12.3, 7.0 Hz, 2H), 1.08 (s, 6H). J = 8.4 Hz, 1H), 7.61 (s, 1H), 7.45 (s, 1H), 6.98 (d, J = 2.0 Hz,
13
C NMR (100 MHz, DMSO-d6) δ 193.54, 169.02, 145.42, 1H), 6.86 (dd, J = 8.4, 2.0 Hz, 1H), 6.42 (dd, J = 3.3, 1.8 Hz,
138.00, 137.20, 134.22, 130.73, 116.21, 112.78, 107.38, 1H), 6.36 (d, J = 3.2 Hz, 1H), 4.48 (d, J = 5.8 Hz, 2H),
51.71, 48.90, 38.36, 35.36, 33.48, 28.29, 28.12. HR-MS 2.91 (s, 2H), 1.36–1.18 (m, 3H), 1.04 (s, 6H). 13C NMR
(m/z) (ESI), calcd for ­C20H26N5O2S [M + ­H]+: 400.1763; (100 MHz, DMSO-d6) δ 189.69, 170.51, 152.10, 150.00,
found: 400.1770. Melting point: 258–260 ℃. 144.64, 142.46, 138.30, 115.06, 110.41, 109.22, 107.23,
105.92, 51.89, 34.23, 27.60. HR-MS (m/z) (ESI), calcd for
4‑(6,6‑dimethyl‑4‑oxo‑4,5,6,7‑tetrahydro‑1H‑benzo[d] ­C20H22N5O3 [M + ­H]+: 380.1678; found: 380.1680. Melting
[1,2,3]triazol‑1‑yl)‑2‑((tetrahydrofuran‑3‑yl)amino)benza‑ point: 255–257 ℃.
mide (6l) Yellow solid. Yield: 67%. 1H NMR (400 MHz,
DMSO-d6) δ 8.59 (d, J = 6.6 Hz, 1H), 8.00 (d, J = 25.1 Hz, 4‑(6,6‑dimethyl‑4‑oxo‑4,5,6,7‑tetrahydro‑1H‑benzo[d]
1H), 7.84 (d, J = 8.4 Hz, 1H), 7.41 (d, J = 20.8 Hz, 1H), 6.89 [1,2,3]triazol‑1‑yl)‑2‑((thiophen‑3‑ylmethyl)amino)benza‑
(s, 1H), 6.82 (d, J = 8.4 Hz, 1H), 5.78 (s, 2H), 4.38–4.02 mide (6p) Yellow solid. Yield: 59%. 1H NMR (400 MHz,
(m, 1H), 3.04 (s, 2H), 2.84 (dd, J = 15.9, 7.5 Hz, 2H), 2.20 DMSO-d6) δ 8.01 (d, J = 7.5 Hz, 1H), 7.65 (s, 2H), 7.41 (d,
(d, J = 16.7 Hz, 2H), 1.24 (s, 3H), 1.06 (s, 6H). 13C NMR J = 1.4 Hz, 1H), 7.37 (dd, J = 7.5, 2.9 Hz, 1H), 7.18–7.08 (m,
(100 MHz, DMSO-d6) δ 189.72, 170.60, 149.74, 144.73, 2H), 6.96 (dd, J = 7.5, 1.5 Hz, 1H), 6.77 (t, J = 8.1 Hz, 1H),
141.02, 138.39, 130.76, 128.95, 114.65, 108.43, 105.94, 4.50 (d, J = 8.1 Hz, 2H), 2.83–2.56 (m, 4H), 1.08 (s, 6H).
13
51.90, 50.67, 35.48, 34.28. HR-MS (m/z) (ESI), calcd for C NMR (100 MHz, DMSO-d6) δ 193.54, 169.04, 146.67,
­C19H24N5O3 [M + ­H]+: 370.1834; found: 370.1838. Melting 137.92, 137.20, 134.22, 131.09, 126.89, 126.30, 122.17,
point: 250–252 ℃. 115.94, 112.78, 106.24, 48.90, 45.41, 38.36, 35.36, 28.18.
HR-MS (m/z) (ESI), calcd for ­ C20H22N5O2S [M + ­H]+:
4‑(6,6‑dimethyl‑4‑oxo‑4,5,6,7‑tetrahydro‑1H‑benzo[d] 396.1450; found: 396.1456. Melting point: 241–242 ℃.
[1,2,3]triazol‑1‑yl)‑2‑((tetrahydro‑2H‑pyran‑4‑yl)amino)
benzamide (6m) Yellow solid. Yield: 55%. 1H NMR 4‑(6,6‑dimethyl‑4‑oxo‑4,5,6,7‑tetrahydro‑1H‑benzo[d]
(400 MHz, DMSO-d6) δ 8.53 (d, J = 7.4 Hz, 1H), 8.04 (s, [1,2,3]triazol‑1‑yl)‑2‑((thiophen‑2‑ylmethyl)amino)benza‑
1H), 7.84 (d, J = 8.5 Hz, 1H), 7.40 (s, 1H), 6.99 (s, 1H), mide (6q) Yellow solid. Yield: 58%. 1H NMR (400 MHz,
6.79 (d, J = 8.4 Hz, 1H), 3.85 (d, J = 11.3 Hz, 2H), 3.48 (t, DMSO-d6) δ 7.99 (d, J = 7.5 Hz, 1H), 7.65 (s, 2H), 7.44
J = 11.1 Hz, 2H), 3.00 (s, 1H), 1.96 (d, J = 13.2 Hz, 2H), (dd, J = 6.8, 2.2 Hz, 1H), 7.41 (d, J = 1.4 Hz, 1H), 7.16 (dd,
1.43 (t, J = 11.8 Hz, 2H), 1.24 (s, 3H), 1.05 (s, 3H), 0.85 J = 7.5, 1.5 Hz, 1H), 7.06 (t, J = 7.2 Hz, 1H), 7.01–6.95 (m,
(s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 190.23, 171.10, 2H), 4.76 (d, J = 7.3 Hz, 2H), 2.91–2.41 (m, 4H), 1.08 (s,
151.02, 145.20, 141.49, 138.97, 131.09, 114.97, 108.73, 6H). 13C NMR (100 MHz, DMSO-d6) δ 193.54, 169.02,
106.15 (s), 52.40, 47.28, 35.99, 34.72, 28.06, 10.81. HR-MS 146.29, 141.73, 137.77, 137.20, 134.22, 131.09, 127.22,
(m/z) (ESI), calcd for C ­ 20H26N5O3 [M + ­H]+: 384.1991; 126.17, 125.13, 115.82, 112.78, 106.19, 48.90, 43.75, 38.27,
found: 384.1995. Melting point: 228–230 ℃. 35.36, 28.12. HR-MS (m/z) (ESI), calcd for ­C20H22N5O2S
[M + ­H]+: 396.1450; found: 396.1456. Melting point: 250–
4‑(6,6‑dimethyl‑4‑oxo‑4,5,6,7‑tetrahydro‑1H‑benzo[d] 252 ℃.
[1,2,3]triazol‑1‑yl)‑2‑((1,1‑dioxidotetrahydro‑2H‑thi‑
opyran‑4‑yl)amino)benzamide (6n) Yellow solid. Yield: 4‑(6,6‑dimethyl‑4‑oxo‑4,5,6,7‑tetrahydro‑1H‑benzo[d]
69%. 1H NMR (400 MHz, DMSO-d6) δ 8.68 (d, J = 7.1 Hz, [1,2,3]triazol‑1‑yl)‑2‑((2‑methoxyphenyl)amino)benza‑
1H), 8.18 (s, 1H), 8.00–7.45 (m, 1H), 7.29 (d, J = 6.6 Hz, mide (6r) Yellow solid. Yield: 57%. 1H NMR (400 MHz,
1H), 7.15–6.73 (m, 1H), 3.94 (s, 1H), 3.41 (s, 8H), 3.10 (d, DMSO-d6) δ 8.87 (s, 1H), 7.98 (d, J = 7.5 Hz, 1H), 7.86
J = 10.4 Hz, 2H), 2.35 (s, 1H), 1.98 (s, 1H), 1.11 (s, 6H). (d, J = 1.6 Hz, 1H), 7.66 (s, 2H), 7.31 (dd, J = 7.5, 1.5 Hz,
13
C NMR (100 MHz, DMSO-d6) δ 190.19, 171.22, 162.20, 1H), 7.24–7.12 (m, 1H), 7.04–7.00 (m, 2H), 6.99–6.95 (m,

13
Molecular Diversity (2023) 27:239–248 247

1H), 3.83 (s, 3H), 2.81–2.67 (m, 2H), 2.67 (s, 2H), 1.08 (s, compounds and FITC-labeled geldanamycin were added to
6H). 13C NMR (100 MHz, DMSO-d6) δ 193.95, 168.95, each reaction well. Recombinant HSP90 protein was diluted
148.53, 143.41, 138.33, 136.61, 134.32, 133.71, 130.76, with the reaction buffer (20 mM 4-(2-hydroxyethyl)-1-piper-
123.99, 118.96, 112.58, 110.06, 56.01, 48.84, 38.42, 35.36, azineethanesulfonic acid pH 7.3, 50 mM KCl, 5 mM M ­ gCl2,
­ 22H24N5O3 [M + ­H]+:
28.13. HR-MS (m/z) (ESI), calcd for C 20 mM ­Na2MoO4, 0.01% Triton X-100, 100 μg/mL bovine
406.1834; found: 406.1836. Melting point: 255–257 ℃. serum albumin, and 2 mM dithiothreitol) and added to each
well to get the final concentration of 22.2 nM. The enzyme
(R)‑4‑(6,6‑dimethyl‑4‑oxo‑4,5,6,7‑tetrahydro‑1H‑benzo[d] reaction was initiated as soon as the purified HSP90 was
[1,2,3]triazol‑1‑yl)‑2‑((1‑phenylethyl)amino)benzamide added. After incubation for 60 min at 4 °C, the fluorescent
(6s) Yellow solid. Yield:52%. 1H NMR (400 MHz, polarization of the samples was determined by a microtiter-
DMSO-d6) δ 8.01 (d, J = 7.5 Hz, 1H), 7.71–7.59 (m, 2H), plate reader (Envision, PerkinElmer).
7.53 (d, J = 1.5 Hz, 1H), 7.40 (tt, J = 1.6, 0.9 Hz, 1H),
7.39 (dt, J = 1.7, 0.9 Hz, 1H), 7.32–7.30 (m, 1H), 7.29 (d,
Cytotoxicity assay
J = 0.7 Hz, 1H), 7.28–7.27 (m, 1H), 7.26–7.23 (m, 1H),
7.15 (dd, J = 7.5, 1.5 Hz, 1H), 4.56 (dd, J = 9.5, 7.9, 6.8,
Cells were seeded in 96-well plates at proper density in
5.9 Hz, 1H), 2.80–2.68 (m, 2H), 2.67 (d, J = 4.4 Hz, 2H),
growth media. After 24 h, a range of concentrations of
1.55 (d, J = 6.8 Hz, 3H), 1.08 (d, J = 25.1 Hz, 6H). 13C NMR
the test compounds were added and the plates were incu-
(100 MHz, DMSO-d6) δ 193.95, 169.06, 145.82, 136.61,
bated at 37 °C for 72 h. Cell proliferation was determined
134.32, 130.74, 128.42, 127.54, 127.14, 117.38, 112.81,
by SRB assay. ­IC50 values were calculated by concentra-
107.28, 56.61, 48.84, 38.36, 35.36, 28.19, 22.15. HR-MS
tion–response curve fitting using a SoftMax probased four-
(m/z) (ESI), calcd for C ­ 23H26N5O2 [M + ­H]+: 404.2042;
parameter method.
found: 404.2042. Melting point: 250–252 ℃.

4‑(6,6‑dimethyl‑4‑oxo‑4,5,6,7‑tetrahydro‑1H‑benzo[d] Molecular docking

[1,2,3]triazol‑1‑yl)‑2‑(((1R,2R)‑2‑hydroxycyclopentyl)
amino)benzamide (6t) Yellow solid. Yield:59%. 1H NMR The molecular structure 6u was drawn by ChemDraw Ultra
(400 MHz, DMSO-d6) δ 8.49 (s, 1H), 8.03 (s, 1H), 7.83 (d, 14.0 and then saved as mol2 file. The crystal structure of
J = 8.2 Hz, 1H), 7.37 (s, 1H), 7.02 (s, 1H), 6.80 (d, J = 7.9 Hz, HSP90 alpha (PDB: 3D0B) was obtained from the PDB
1H), 3.88 (s, 1H), 3.54 (s, 1H), 3.03 (s, 2H), 2.17 (s, 1H), database (https://w
​ ww.r​ csb.o​ rg/s​ truct​ ure/3​ D0B) [27], which
1.99 (s, 1H), 1.89–1.60 (m, 4H), 1.55 (s, 1H), 1.41 (s, 1H), was applied as the target for molecular docking by PISA
1.05 (d, J = 4.8 Hz, 6H). 13C NMR (100 MHz, DMSO-d6) software. The docking models were analyzed by the Auto-
δ 189.73, 170.66, 150.25, 144.67, 141.02, 138.39, 114.36, Dock Vina program. Pymol software was used to show the
108.40, 106.39, 76.40, 51.91, 35.47, 32.88, 30.59, 27.67, docking models.
21.17. HR-MS (m/z) (ESI), calcd for C ­ 20H26N5O3 [M + ­H]+:
384.1991; found: 384.1994. Melting point: 260–261 ℃. Supplementary Information The online version contains supplemen-
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 11030-0​ 22-1​ 0423-7.

4‑(6,6‑dimethyl‑4‑oxo‑4,5,6,7‑tetrahydro‑1H‑benzo[d] Acknowledgements This research was funded by the Fundamental

[1,2,3]triazol‑1‑yl)‑2‑(((1r,4r)‑4‑hydroxycyclohexyl)amino) Research Funds for the Central Universities and the National Natural
benzamide (6u) Yellow solid. Yield:55%. 1H NMR Science Foundation of China, Grant Numbers 22077034 and 82104000.
(400 MHz, DMSO-d6) δ 8.40 (s, 1H), 8.00 (s, 1H), 7.80 (s,
1H), 7.34 (s, 1H), 6.91 (s, 1H), 6.76 (s, 1H), 4.57 (s, 1H), 3.36
(s, 2H), 3.01 (s, 2H), 1.99 (s, 2H), 1.81 (s, 2H), 1.31 (s, 2H),
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