Development of selective HDAC6 inhibitors with in vitro and in vivo anti-multiple myeloma activity

Article abstract of DOI:10.1016/j.bioorg.2021.105278

Histone deacetylase 6 (HDAC6) is a promising therapeutic target for the treatment of cancers, neurodegenerative diseases and autoimmune disorders. Herein a novel series of pyrrolo[2,3-d]pyrimidine-based HDAC inhibitors were designed, synthesized and biologically evaluated, among which compounds 7a, 12a1, and 16a1 exhibited potent inhibitory activities and selectivities against HDAC6. Notably, compared with the well-known HDAC6 inhibitor Tubastatin A, our pyrrolo[2,3-d]pyrimidine-based HDAC6 inhibitors showed superior in vitro antiproliferative activity against human multiple myeloma cell lines RPMI 8226, U266 and MM.1S, while maintaining the low cytotoxicity against human breast cancer cell line MDA-MB-231 and two normal cell lines. The HDAC6 selective inhibition of one representative compound 12a1 in RPMI 8226 cells was confirmed by western blot analysis. Although pyrrolo[2,3-d]pyrimidine is a privileged structure in many kinase inhibitors, compound 12a1 showed negligible inhibition against several kinases including JAK family members and Akt1, indicating its acceptable off-target profile. Besides, compound 12a1 exhibited desirable metabolic stability in mouse liver microsome. The in vivo anti-multiple myeloma potency of 12a1, alone and in combination with bortezomib, was demonstrated in a RPMI 8226 xenograft model.

Full text of DOI:10.1016/j.bioorg.2021.105278

Bioorganic Chemistry 116 (2021) 105278
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Development of selective HDAC6 inhibitors with in vitro and in vivo
anti-multiple myeloma activity
Shunda Lia, Chunlong Zhaoa, Guozhen Zhanga, Qifu Xua, Qian Liua, Wei Zhaoa,
,
*
C. James Choub, Yingjie Zhanga
a Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Cheeloo College of Medicine,
Shandong University, 44 West Wenhua Road, Ji’nan, Shandong 250012, PR China
b Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, Medical University of South Carolina, Charleston, South Carolina 29425,
United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
Histone deacetylase 6 (HDAC6) is a promising therapeutic target for the treatment of cancers, neurodegenerative
diseases and autoimmune disorders. Herein a novel series of pyrrolo[2,3-d]pyrimidine-based HDAC inhibitors
were designed, synthesized and biologically evaluated, among which compounds 7a, 12a1, and 16a1 exhibited
potent inhibitory activities and selectivities against HDAC6. Notably, compared with the well-known HDAC6
inhibitor Tubastatin A, our pyrrolo[2,3-d]pyrimidine-based HDAC6 inhibitors showed superior in vitro anti-
proliferative activity against human multiple myeloma cell lines RPMI 8226, U266 and MM.1S, while main-
taining the low cytotoxicity against human breast cancer cell line MDA-MB-231 and two normal cell lines. The
HDAC6 selective inhibition of one representative compound 12a1 in RPMI 8226 cells was confirmed by western
blot analysis. Although pyrrolo[2,3-d]pyrimidine is a privileged structure in many kinase inhibitors, compound
12a1 showed negligible inhibition against several kinases including JAK family members and Akt1, indicating its
acceptable off-target profile. Besides, compound 12a1 exhibited desirable metabolic stability in mouse liver
microsome. The in vivo anti-multiple myeloma potency of 12a1, alone and in combination with bortezomib, was
demonstrated in a RPMI 8226 xenograft model.
Histone deacetylase 6
Selective inhibitor
Multiple myeloma
Anticancer
Pyrrolo[2,3-d]pyrimidine
1. Introduction
potential anti-multiple myeloma target dependent on its important roles
in degradation of unfolded and misfolded ubiquitinated proteins [5]. To
Zinc2+-dependent histone deacetylases (HDACs), a family of hydro-
lases involved in epigenetic regulation and post-translational modifi-
cation by removing the acetyl groups from histones and non-histone
proteins, have been validated to be important anticancer targets based
on the successful launch of five inhibitors (vorinostat, belinostat, pan-
obinostat, romidepsin, and chidamide) [1,2]. Amongst, panobinostat
was approved in combination with bortezomib and dexamethasone for
the treatment of patients with multiple myeloma who have received at
least two prior treatment regimens, including bortezomib and an
immunomodulatory agent [3]. However, diarrhoea and cardiac toxic-
ities are two substantial toxicities of Panobinostat [4]. Considering that
panobinostat is a pan-HDAC inhibitor with little isoform selectivity, it
was presumed that isoform selective HDAC inhibitors could exhibit
improved tolerability.
be specific, HDAC6 contains a zinc finger ubiquitin-binding domain and
a dynein motor binding domain, through which polyubiquitinated
misfolded protein cargo is recruited by HDAC6 to dynein motors for
transport to aggresomes, then degraded by autophagy [6–8]. It has been
demonstrated that HDAC6 inhibition could induce accumulation of
polyubiquitinated proteins and inhibit the growth of multiple mye-
lomas, especially when combined with proteasome inhibitor bortezomib
[5,9]. More importantly, genetic ablation or pharmacological inhibition
of HDAC6 cause no lethality or toxicity typically associated with pan-
HDACs inhibition [10–13]. Currently, at least three HDAC6 inhibitors
(ACY-241, ACY-1215, and CS3003) have entered clinical trials for the
treatment of multiple myeloma [14]. The chemical structures and HDAC
inhibitory activities of ACY-241 and ACY-1215 are present in Fig. 1.
Pyrrolo[2,3-d]pyrimidine is a privileged structure in drug discovery,
especially in the design of protein kinase inhibitors. For example, many
Among the Zinc2+-dependent HDACs, HDAC6 was suggested to be a
* Corresponding author.
E-mail address: zhangyingjie@sdu.edu.cn (Y. Zhang).
https://doi.org/10.1016/j.bioorg.2021.105278
Received 17 June 2021; Received in revised form 11 August 2021; Accepted 16 August 2021
Available online 19 August 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

S. Li et al.
Bioorganic Chemistry 116 (2021) 105278
JAK inhibitors including the approved drugs ruxolitinib and tofacitinib
(Fig. 2) use the pyrrolo[2,3-d]pyrimidine group to form key hydrogen
bond interactions with the hinge region of JAK proteins [17]. Besides,
the clinical Akt inhibitor AZD5363 (Fig. 2) also contains the pyrrolo[2,3-
d]pyrimidine group [18]. Interestingly, two series of pyrrolo[2,3-d]py-
rimidine-based hydroxamates exemplified by compounds JMC-45 and
BMCL-13b respectively were developed as HDAC and JAK dual in-
hibitors [19,20], and one series of pyrrolo[2,3-d]pyrimidine-based
benzamides exemplified by compound EJMC-7 demonstrated potent
HDAC and tubulin polymerization dual inhibition [21] (Fig. 2). These
compounds indicated that pyrrolo[2,3-d]pyrimidine is also a privileged
structure in HDAC inhibitor design. In the present research, pyrrolo[2,3-
d]pyrimidine group was introduced to the terminal cap group of the
clinical HDAC6 inhibitors (ACY-241 and ACY-1215) and the HDAC6
inhibitor pharmacophore to design a novel series of pyrrolo[2,3-d]py-
rimidine-based hydroxamates. Structure-activity relationship (SAR)
study was focused on the linker part connecting the pyrrolo[2,3-d]py-
rimidine group and the hydroxamate group, leading to one selective
HDAC6 inhibitor 12a1 with remarkable in vitro and in vivo anti-
multiple myeloma potency.
converted into hydroxamic acids 5a5, 5b5, 5a6 and 5b6 by NH2OK in
dry methanol, respectively. In addition, the intermediate 3a and 3b
could react with NH2OTHP to afford 6a and 6b, respectively, which
were deprotected to get hydroxamic acids 7a and 7b under acidic
condition.
N-protection of the starting material 1 with tosyl chloride led to
compound 8, which reacted with various methyl aminobenzoates to
afford 9a0, 9a1, 9b0, and 9b1. The intermediates 9a1 and 9b1 could be
converted into hydroxamic acids 12a1 and 12b1 according to the
similar methods of synthesizing 7a and 7b from 2a and 2b. Besides,
methylation of the intermediates 9a0, 9a1, 9b0, and 9b1 by CH3I led to
13a0, 13a1, 13b0, and 13b1, respectively, which could also be con-
verted into the corresponding hydroxamic acids 16a0, 16a1, 16b0, and
16b1 using the similar methods of synthesizing 7a and 7b from 2a and
2b.
2.3. In vitro HDACs inhibitory activity
Considering the clinical HDAC6 inhibitors ACY-125 and ACY-241
not only inhibit HDAC6, but also showed considerable inhibition
against HDAC1, HDAC2, HDAC3 and HDAC8 (Fig. 1) [15,16], all our
target compounds were firstly evaluated by determining their inhibitory
2. Results and discussion
rates against HDAC2, HDAC6 and HDAC8 at 0.5 μM. It has been showed
2.1. Compound design
that most target compounds exhibited over 50% HDAC6 inhibition at
0.5 M, indicating their potent HDAC6 inhibitory activities (Table 1).
μ
Compounds 5a5, 5a6, 5b5, 5b6 were designed by substituting pyr-
rolo[2,3-d]pyrimidine group for the terminal two phenyl groups in ACY-
1215 and ACY-241. Note that the pyrimidine group in ACY-1215 and
ACY-241 was replaced with phenyl group to keep the number of
hydrogen bond acceptor below ten, complying with the Lip-
inski’s rule of five (Fig. 3).
Compounds with aliphatic linkers (5a5, 5a6, 5b5, 5b6) also possessed
considerable HDAC2 inhibitory potencies, while N-hydroxybenzamide-
based compounds, such as 7a, 12a1 and 16a1 showed better HDAC6
selectivity. For N-hydroxybenzamide-based compounds, generally, para-
substituted N-hydroxybenzamides were more potent HDAC6 inhibitors
than their meta- substituted analogs (7a vs. 7b, 12a1 vs. 12b1, 16a1 vs.
16b1), which was in line with the HDAC6 inhibitor pharmacophore
summarized in Fig. 4. The methyl group in the N-hydroxybenzamides
showed no definitive effects on HDAC6 inhibition. To further confirm
and profile their HDAC6 inhibitory activities and selectivities, the IC50
values of seven selected compounds against HDAC2 and HDAC6 were
determined, with the pan-HDAC inhibitor vorinostat (SAHA) and
HDAC6 selective inhibitor Tubastatin A as positive controls. The results
confirmed that compounds with aliphatic linkers (5a5, 5a6, 5b5, 5b6)
showed no dramatic HDAC6 selectivity over HDAC2, which was similar
to SAHA. Note that compound 5a5 was even more potent against
Moreover, based on the structures of reported HDAC6 inhibitors
including ours [12,22–26], we can summarize a general HDAC6 inhib-
itor pharmacophore, where the common N-hydroxybenzamide group is
connected with the terminal bulky aromatic cap group via one or two
carbon/nitrogen atoms (Fig. 4). Introduction of the bulky aromatic
pyrrolo[2,3-d]pyrimidine to the terminal cap of HDAC6 inhibitor
pharmacophore resulted in the target compounds 7a, 7b, 12a1, 12b1,
16a0, 16b0, 16a1, 16b1 (Fig. 4).
2.2. Chemistry
HDAC2 (IC50 = 0.015 μM) than HDAC6 (IC50 = 0.139 μM). Satisfyingly,
The procedures to synthesize all target compounds were outlined in
Scheme 1. The commercially available C4-chloro-7H-pyrrolo[2,3-d]py-
rimidine (1) reacted with various methyl aminobenzoates to afford 2a
and 2b, which were hydrolyzed to obtain the key intermediates 3a and
3b. Then compound 3a and 3b were condensed with various methyl
aminoalkanoates to produce 4a5, 4b5, 4a6 and 4b6, which were
N-hydroxybenzamide-based compounds 7a, 12a1, and 16a1 showed
about 13-, 53- and 24-fold selectivity against HDAC6 over HDAC2,
respectively. It is worth noting that the highest HDAC6-selective N-
hydroxybenzamide 12a1 was also the most potent one, which possessed
superior HDAC6 inhibitory activity (IC50 = 0.010
μ
M) than both SAHA
(IC50 = 0.082 M) and Tubastatin A (IC50 = 0.085
μ
μM).
Fig. 1. The structures and HDAC inhibitory activities of two clinical HDAC6 inhibitors, ACY-1215 [15] and ACY-241 [16].
2

S. Li et al.
Bioorganic Chemistry 116 (2021) 105278
Fig. 2. The structures of representative compounds containing the pyrrolo[2,3-d]pyrimidine scaffold, which is shown in red.
Fig. 3. The design strategy of target compounds 5a5, 5a6, 5b5, 5b6.
Fig. 4. The structures of reported HDAC6 inhibitors, and the design strategy of target compounds 7a, 7b, 12a1, 12b1, 16a0, 16b0, 16a1, 16b1.
2.4. In vitro anti-proliferative activity
multiple myeloma cell lines more effectively than Tubastatin A, mean-
while maintained the negligible effects on MDA-MB-231, indicating the
selective cytotoxicity to multiple myeloma of HDAC6 selective in-
hibitors. Of particular note was the HDAC6 selective inhibitor 12a1,
whose anti-multiple myeloma potency was comparable to the pan-
HDAC inhibitor 5b6. More importantly, compound 12a1 showed satis-
Due to their potent HDAC6 inhibitory potencies, compounds 5b6,
7a, 12a1, and 16a1 were progressed to in vitro antiproliferative assay
against three human multiple myeloma cell lines (RPMI 8226, U266 and
MM.1S) and one breast cancer cell line MDA-MB-231. The HDAC6 se-
lective inhibitor Tubastatin A was used as the positive control. Accord-
ing to the results in Table 2, compound 5b6 exhibited greater anti-
multiple myeloma potency than the other compounds, however this
compound was also more cytotoxic to breast cancer cell line MDA-MB-
231, which could be attributed to its more potent inhibition against
HDAC2 (Table 1) and other class I HDAC isoforms. Note that compounds
7a, 12a1, and 16a1 could attenuate the growth of all three human
factory safety profile in normal cells, with IC50 values being over 50 μM
against the human embryonic kidney cell line HEK293 and the human
normal hepatic cell line LO2. Among the three tested multiple myeloma
cell lines, RPMI8226 showed the most sensitivity to our inhibitors.
3

S. Li et al.
Bioorganic Chemistry 116 (2021) 105278
Scheme 1. Reagents and conditions: (a) various methyl aminobenzoate, isopropanol, conc HCl, reflux; (b) MeOH, 2.5 N NaOH, reflux; (c) various methyl amino-
alkanoates, TBTU, TEA, anhydrous DMF; (d) NH2OH⋅HCl, KOH, anhydrous CH3OH; (e) NH2OTHP, EDCI, HOBt, TEA, DMF; (f) HCl, anhydrous EtOAc; (g) TsCl, TEA,
DMAP, DCM; (h) Cs2CO3, CH3I, DMF.
2.5. Western blot analysis
nanomolar IC50 values against JAK1 and Akt1, respectively. However, at
the concentration of 0.5 μM, compound 12a1 showed inhibitory rates
Western blot analysis was performed to evaluate the intracellular
HDACs inhibitory effects of compound 12a1 in RPMI 8226 cells. The
HDAC6 selective inhibitor Tubastatin A and pan-HDAC inhibitor Pan-
obinostat were used as positive controls. From the results in Fig. 5, it was
found that, similar to Tubastatin A, compound 12a1 could dose-
dependently increase the intracellular levels of the HDAC6 substrate
far less than 50% against all five tested kinases, indicating a desirable
off-target profile.
2.7. In vitro mouse liver microsome stability
The mouse liver microsome assay was carried out to preliminarily
evaluate the metabolic stability of 12a1. The reduced nicotinamide
adenine dinucleotide phosphate (NADPH) was used as cofactor. The
results in Table 4 showed that the remaining of 12a1 after 60 min in-
cubation was 83%, indicating a good metabolic stability. In contrast, the
acetyl-α-tubulin without influencing the class I HDACs substrates acetyl-
histone H3 and acetyl-histone H4. In contrast, the pan-HDAC inhibitor
Panobinostat could increase the intracellular levels of both acetyl-
α
-tubulin and acetyl-histones. These results confirmed the HDAC6
inhibitory activity and selectivity of 12a1.
t
1/2 of the approved hydroxamate-based HDAC inhibitor SAHA in mouse
liver microsome was only 28 min [27].
2.6. In vitro kinases inhibitory activity
2.8. In vivo antitumor activity
As discussed in the introduction part, pyrrolo[2,3-d]pyrimidine is
widely used as hinge binding motif in many kinase inhibitors, such as
the JAK inhibitors ruxolitinib, tofacitinib and the Akt inhibitor AZD5363
(Fig. 2). To preliminarily investigate the off-target effects of our pyrrolo
[2,3-d]pyrimidine-based HDAC inhibitors, compound 12a1 was tested
in kinase inhibitory assay against all four JAK family members (JAK1,
JAK2, JAK3 and TYK2) and one representative Akt family member
(Akt1). Our results in Table 3 revealed that the approved JAK inhibitor
ruxolitinib and the clinical Akt inhibitor AZD5363 exhibited single-digit
Based on the favorable in vitro properties of 12a1, a RPMI 8226
xenograft model in nude mice was established to evaluate its in vivo
antitumor potency with the approved proteasome inhibitor bortezomib
as reference compound. Previous research demonstrated that combina-
tion of HDAC6 inhibitors and bortezomib could exhibit enhanced anti-
multiple myeloma activity [5,9,24], therefore the in vivo antitumor ef-
fect of 12a1 combined with bortezomib was also evaluated. After 19
consecutive days of treatment, all treatment groups showed significant
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S. Li et al.
Bioorganic Chemistry 116 (2021) 105278
Table 1
Table 2
HDAC2, HDAC6 and HDAC8 inhibitory activities of all target compounds.
In vitro anti-proliferative activity of selected compounds.
a
Compounds
Structures
Inhibitory rates at 0.5
μ
M (IC50
)
Cpd
IC50
(μ
M)a
HDAC2
HDAC6
HDAC8
23%
Multiple myeloma
Breast
cancer
MDA-
Normal cell
HEK293
5a5
99%
89%
RPMI8226
U266
MM.1S
LO2
(0.015
(0.139
MB-231
μ
M)
μM)
5b6
7a
0.46 ±
0.12
1.60 ±
0.31
1.74 ±
0.27
23.3 ±
3.8
NDb
NDb
> 50
NDb
>50
NDb
NDb
5a6
5b5
96%
100%
18%
38%
2.58 ±
0.16
6.92 ±
1.14
5.97 ±
1.20
> 50
(0.095
(0.022
μ
M)
μM)
12a1
16a1
0.62 ±
0.08
1.69 ±
0.26
2.12 ±
0.39
> 50
> 50
> 50
>
50
2.17 ±
0.33
4.04 ±
1.13
5.25 ±
0.77
NDb
98%
100%
(0.091
(0.038
Tubastatin
A
9.45 ±
1.62
18.6 ±
4.01
26.7 ±
4.63
>
μ
M)
μM)
50
a
5b6
7a
100%
99%
31%
57%
Assay were performed in replicate (n ≥ 3), values are shown as mean ± SD.
b
(0.045
(0.018
Not determined.
μ
M)
μM)
32%
99%
(1.24
(0.090
μ
M)
μM)
7b
6%
45%
43%
12a1
12b1
46%
99%
58%
49%
(0.53
(0.010
μ
M)
μM)
5%
64%
Fig. 5. RPMI 8226 cells were treated with different concentrations of com-
pounds or DMSO for 6 h. The levels of acetyl-α-tubulin (Ac-tub), total tubulin
(tub), acetyl-histone H3 (Ac-HH3), acetyl-histone H4 (Ac-HH4) and total his-
tone H3 (HH3) were determined by immunoblotting.
16a0
13%
25%
57%
Table 3
Kinases inhibitory activities of compounds 12a1, ruxolitinib and AZD5363.
16a1
16b0
40%
100%
50%
59%
a
(0.70
(0.029
Inhibitory rates at 0.5 μM and IC50
μ
M)
μM)
JAK1
JAK2
JAK3
TYK2
Akt1
4%
63%
12a1
22%
19%
NDb
NDb
37%
NDb
NDb
12%
NDb
NDb
5%
ruxolitinib
IC50 = 5.5 nM
NDb
AZD5363
NDb
IC50 = 8.4 nM
a
Assay were performed in replicate (n ≥ 2).
b
16b1
5%
48%
49%
Not determined.
Table 4
SAHA
92%
98%
NDb
NDb
In vitro mouse liver microsome stability of compound 12a1.
(0.18
(0.082
Incubation time
μ
M)
μM)
Tubastatin
A
7%
99%
0 min
100%
5 min
95%
15 min
98%
30 min
94%
45 min
93%
60 min
83%
(>5
μ
M)
(0.085
Remaininga
a
μ
M)
Assay were performed in replicate (n ≥ 2).
a
Assays were performed in replicate (n ≥ 2), the SD values of IC50s are < 20%
inhibition compared with 12a1 or bortezomib alone, which could be at
least partially attributed to the simultaneous inhibition of HDAC6 and
proteasome. Mice body weights were monitored regularly to evaluate
the toxicity of different treatment. As shown in Fig. 7, bortezomib or
12a1 treatment alone showed no obvious effects on mice body weights.
Although mice treated by 12a1 and bortezomib combination lost
weights slightly during treatment, they recovered gradually before the
of the means.
b
Not determined.
tumor growth inhibition (TGI) and relative increment ratio (T/C) rela-
tive to the control group, which were presented in Table 5. The tumor
growth curve was showed in Fig. 6. Remarkably, the combination
treatment group presented significantly more potent tumor growth
5

S. Li et al.
Bioorganic Chemistry 116 (2021) 105278
G619, respectively. The phenyl group of 12a1 was sandwiched between
Table 5
In vivo antitumor efficacy of 12a1, bortezomib and their combination in a RPMI
F680 and F620, forming π-π stacking interactions. The pyrrolo[2,3-d]
8226 xenograft mode.
Treatment groups
pyrimidine group was solvent-exposed, interacting with S568 and M682
around the entrance to the active site via two hydrogen bonds. Although
12a1 could also fit into the active site of HDAC2, with the phenyl group
T/C (%)a
TGI (%)a
12a1 (50 mg/kg/d, ip)
70
65
41
29
34
61
forming π-π stacking interaction with F155 (Fig. 8B), its hydroxamate
bortezomib (0.5 mg/kg/d, iv)
12a1 (50 mg/kg/d, ip) + bortezomib (0.5 mg/kg/d, iv)
group only displayed monodentate chelation with the catalytic Zn2+
.
Besides, only two hydrogen bonds between 12a1 and HDAC2 were
observed. Therefore, the proposed bidentate hydroxamate-Zn2+ binding
mode and more hydrogen bonds between 12a1 and HDAC6 rationalized
its considerable HDAC6 inhibitory activity and selectivity.
a
Compared with the control group, all treated groups showed statistically
significant (P < 0.05) T/C and TGI by Student’s two-tailed t test.
3. Conclusion
To discover novel HDAC6 inhibitors as anti-multiple myeloma
agents, a novel series pyrrolo[2,3-d]pyrimidine-based hydroxamates
were designed, synthesized and evaluated. Structure-activity relation-
ship (SAR) studies have been focused on the linker part between the
hydroxamate group and the pyrrolo[2,3-d]pyrimidine group, leading to
compound 12a1 with not only potent HDAC6 inhibitory activity but also
considerable selectivity over HDAC2. More importantly, compound
12a1 exhibited remarkable in vitro and in vivo anti-multiple myeloma
potency and no significant toxicity, which could be further developed as
antitumor lead compound.
4. Experimental section
4.1. Chemistry
Unless specified otherwise, all starting materials, reagents and sol-
vents were commercially available. 1H NMR and 13C NMR spectra were
obtained using a Bruker DRX spectrometer at 400 and 100 MHz
respectively. Chemical shifts were reported in parts per million (ppm).
Multiplicity of 1H NMR signals was reported as singlet (s), doublet (d),
triplet (t), quartert (q) and multiplet (m). ESI-MS data was recorded on
an API 4000 spectrometer. High resolution mass spectra were conducted
by Shandong Analysis and Test Center in Ji’nan. Melting points were
determined on an electrothermal melting point apparatus and were
uncorrected.
Fig. 6. Growth curve of implanted RPMI 8226 xenograft in nude mice. Data are
expressed as the mean ± SD. * p < 0.05 by Student’s two-tailed t test.
4.1.1. Methyl 4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)benzoate (2a)
To a solution of 1 (0.50 g, 3.25 mmol) and methyl 4-aminobenzoate
(0.54 g, 3.58 mmol) in isopropanol was added 2 drops of conc. HCl, and
the mixture was heated to reflux with stirring for 10 h. The solvent was
evaporated under vacuum. The crude product was purified by silica gel
column chromatography (MeOH/CH2Cl2, 1/50 to 1/20) to afford com-
pound 2a (0.50 g, 57% yield), white solid. 1H NMR (400 MHz,
DMSO‑d6) δ 12.65 (s, 1H), 11.05 (s, 1H), 8.49 (s, 1H), 8.03 (d, J = 8.5
Hz, 2H), 7.95 (d, J = 8.5 Hz, 2H), 7.45 (t, J = 2.8 Hz, 1H), 7.07 (dd, J =
3.5, 1.7 Hz, 1H), 3.87 (s, 3H). ESI-MS, m/z = 269.13 [M+H]+.
Compound 2b was prepared from compound 1 in a similar manner as
described for compound 2a
4.1.2. Methyl 3-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)benzoate (2b)
Pink solid. 71% yield. 1H NMR (400 MHz, DMSO‑d6) δ 12.66 (s, 1H),
11.11 (s, 1H), 8.42 (s, 1H), 8.27 (s, 1H), 8.06 (d, J = 7.6 Hz, 1H), 7.83 (d,
J = 7.4 Hz, 1H), 7.61 (t, J = 7.7 Hz, 1H), 7.42 (s, 1H), 6.99 (s, 1H), 3.87
(s, 3H). ESI-MS, m/z = 269.12 [M+H]+.
Fig. 7. Body weight curve of nude mice. Data are expressed as the mean ± SD.
end of treatment, suggesting the acceptable toxicity of drug
combination.
2.9. Molecular docking study
4.1.3. 4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)benzoic acid (3a)
To a solution of the compound 2a (0.22 g, 0.82 mmol) in MeOH (20
mL) was added 2.5 N NaOH. The mixture was heated to reflux with
stirring for 2 h. Once the reaction appeared complete by TLC analysis,
MeOH was evaporated from the reaction mixture, the resultant solution
The proposed binding modes of 12a1 in HDAC6 and HDAC2 were
¨
investigated using Maestro 11.5 module in the Schrodinger 2018-1. As
shown in Fig. 8A, compound 12a1 could fit well in the active site of
HDAC6, with the hydroxamate group coordinating the catalytic Zn2+
bidentately and forming three hydrogen bonds with Y782, H610 and
◦
was cooled to 0 C and acidified to pH 2 with 1 N HCl. The resultant
precipitated product was collected by vacuum filtration and washed
6

S. Li et al.
Bioorganic Chemistry 116 (2021) 105278
Fig. 8. The proposed binding modes of compound 12a1 (green) in the active sites of HDAC6 (A, bluewhite surface and sticks, PDB: 5EDU) and HDAC2 (B, gray
surface and sticks, PDB: 3MAX). The Zn2+ coordination and hydrogen bonds are shown by black dashed lines.
with water to afford compound 3a (0.16 g, 76%), white solid. 1H NMR
(400 MHz, DMSO‑d6) δ 12.62 (s, 1H), 10.99 (s, 1H), 8.46 (s, 1H), 8.01
(d, J = 8.4 Hz, 2H), 7.89 (d, J = 8.4 Hz, 2H), 7.45 (t, J = 2.8 Hz, 1H),
7.07 – 6.98 (m, 1H). ESI-MS, m/z = 253.06 [Mꢀ H]-.
4.1.7. Methyl 6-(3-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)benzamido)
hexanoate (4b5)
White solid. 75% yield. 1H NMR (400 MHz, Methanol‑d4) δ 11.84 (s,
1H), 9.52 (s, 1H), 8.35 (s, 1H), 8.29 (t, J = 5.7 Hz, 1H), 8.02 (d, J = 8.8
Hz, 2H), 7.84 (d, J = 8.5 Hz, 2H), 7.29 (t, J = 2.8 Hz, 1H), 6.85 (dd, J =
3.6, 1.8 Hz, 1H), 3.59 (s, 3H), 3.25 (q, J = 6.7 Hz, 2H), 2.31 (t, J = 7.4
Hz, 2H), 1.54 (q, J = 7.4 Hz, 4H), 1.32 (p, J = 3.4 Hz, 4H).
Compounds 3b was prepared from compound 2b in a similar manner
as described for compound 3a.
4.1.4. 3-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)benzoic acid (3b)
1
White solid. 53% yield. H NMR (400 MHz, DMSO‑d6) δ 12.76 (s,
4.1.8. Methyl 7-(3-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)benzamido)
1H), 11.27 (s, 1H), 8.42 (s, 1H), 8.21 (d, J = 2.0 Hz, 1H), 7.97 (dd, J =
7.9, 2.2 Hz, 1H), 7.87 (d, J = 7.7 Hz, 1H), 7.62 (t, J = 7.9 Hz, 1H), 7.45
(t, J = 2.9 Hz, 1H), 6.98 (s, 1H). ESI-MS, m/z = 253.04 [Mꢀ H]-.
heptanoate (4b6)
1
White solid. 69% yield. H NMR (400 MHz, DMSO‑d6) δ 11.78 (s,
1H), 9.47 (s, 1H), 8.41 (t, J = 5.7 Hz, 1H), 8.30 (s, 1H), 8.24–8.18 (m,
2H), 7.49–7.37 (m, 2H), 7.25 (t, J = 2.8 Hz, 1H), 6.83 (dd, J = 3.5, 1.9
Hz, 1H), 3.58 (s, 3H), 3.26 (q, J = 6.7 Hz, 2H), 2.31 (t, J = 7.4 Hz, 2H),
1.53 (h, J = 7.0 Hz, 4H), 1.35–1.28 (m, 4H).
4.1.5. Methyl 6-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)benzamido)
hexanoate (4a5)
To a solution of 3a (0.25 g, 0.98 mmol) in anhydrous DMF (20 mL) in
ice bath was added 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluro-
nium tetrafluoroborate (TBTU, 0.38 g, 1.18 mmol), followed by Et3N
(0.15 g, 1.47 mmol). 30 min later, methyl 6-aminocaproate hydro-
chloride (0.21 g, 1.08 mmol) and additional Et3N (0.15 g, 1.47 mmol)
were added. 6 h later, the solution was diluted by water and extracted
with ethyl acetate. The combined organic extract was washed with
saturated NaHCO3 and brine, dried over Na2SO4 overnight, and the
solvent was evaporated under vacuum. The crude product was purified
by silica gel column chromatography (MeOH/CH2Cl2, 1/50 to 1/20) to
afford compound 4a5 (0.25 g, 67% yield), yellow solid. 1H NMR (400
MHz, DMSO‑d6) δ 11.83 (s, 1H), 9.52 (s, 1H), 8.34 (s, 1H), 8.29 (t, J =
5.6 Hz, 1H), 8.00 (d, J = 8.7 Hz, 2H), 7.83 (d, J = 8.7 Hz, 2H), 7.30–7.26
(m, 1H), 6.84 (dd, J = 3.3, 1.8 Hz, 1H), 3.58 (s, 3H), 3.24 (q, J = 6.7 Hz,
2H), 2.32 (t, J = 7.4 Hz, 2H), 1.55 (dp, J = 15.1, 7.3 Hz, 4H), 1.32 (p, J
= 7.5, 6.9 Hz, 2H).
4.1.9. 4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-(6-
(hydroxyamino)-6-oxohexy-l)benzamide (5a5)
KOH (28.55 g, 509 mmol) and NH2OH⋅HCl (23.84 g, 343 mmol)
were dissolved, respectively, in 70 and 120 mL of MeOH to get solution
A and solution B. Then solution A was added dropwise to solution B.
After filtering the precipitate (KCl), a NH2OK solution was obtained.
Compound 4a5 (0.20 g, 0.52 mmol) was dissolved in 30 mL of NH2OK
solution and stirred for 2 h. After the reaction was complete, it was
evaporated under vacuum. The residue was acidified by addition of 1 N
HCl to pH 5–6. The resulting precipitate was collected by filtration and
dried to get the crude product, which was purified by recrystallization to
get compound 5a5, white solid (0.20 g, 83% yield). Mp: 210–212 ◦C. 1H
NMR (400 MHz, DMSO‑d6) δ 11.84 (s, 1H), 10.36 (s, 1H), 9.54 (s, 1H),
8.68 (s, 1H), 8.34 (s, 1H), 8.31 (t, J = 5.7 Hz, 1H), 8.02 (d, J = 8.5 Hz,
2H), 7.84 (d, J = 8.5 Hz, 2H), 7.28 (t, J = 2.5 Hz, 1H), 6.86 (d, J = 3.4
Hz, 1H), 3.25 (q, J = 6.7 Hz, 2H), 1.97 (t, J = 7.4 Hz, 2H), 1.53 (dd, J =
10.6, 5.0 Hz, 4H), 1.30 (q, J = 7.9 Hz, 2H). 13C NMR (101 MHz,
DMSO‑d6) δ 169.57, 166.25, 153.63, 151.49, 151.10, 143.58, 128.24,
128.10, 123.06, 119.28, 104.54, 99.28, 32.73, 29.50, 26.63, 25.43.
HRMS (AP-ESI) m/z calcd for C19H22N6O3 [M+H]+ 383.1826, found
383.1829.
Compounds 4a6, 4b5 and 4b6 were prepared from compounds 3a,
3b in a similar manner as described for 4a5, respectively.
4.1.6. Methyl 7-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)benzamido)
heptanoate (4a6)
Yellow solid. 70% yield. 1H NMR (400 MHz, Methanol‑d4) δ 11.84 (s,
1H), 9.52 (s, 1H), 8.35 (s, 1H), 8.29 (t, J = 5.7 Hz, 1H), 8.02 (d, J = 8.8
Hz, 2H), 7.84 (d, J = 8.5 Hz, 2H), 7.29 (t, J = 2.8 Hz, 1H), 6.85 (dd, J =
3.6, 1.8 Hz, 1H), 3.59 (s, 3H), 3.25 (q, J = 6.7 Hz, 2H), 2.31 (t, J = 7.4
Hz, 2H), 1.54 (q, J = 7.4 Hz, 4H), 1.32 (p, J = 3.4 Hz, 4H).
Compounds 5a6, 5b5 and 5b6 were prepared from compounds 4a6,
4b5 and 4b6 in a similar manner as described for 5a5, respectively.
7

S. Li et al.
Bioorganic Chemistry 116 (2021) 105278
4.1.10. 4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-(7-
at room temperature was added dropwise 1.0 mL of 4 N HCl in ethyl
acetate. Completion of the reaction was confirmed by TLC. The resultant
precipitated product was collected by vacuum filtration, affording
compound 7a (0.028 g, 37% yield), White solid. Mp: >240 ◦C. 1H NMR
(400 MHz, DMSO‑d6) δ 12.87 (s, 1H), 11.48 (s, 1H), 8.44 (s, 1H), 7.89
(d, J = 8.4 Hz, 2H), 7.74 (d, J = 8.3 Hz, 2H), 7.48 (t, J = 2.9 Hz, 1H),
7.05 (s, 1H). 13C NMR (101 MHz, DMSO‑d6) δ 163.87, 151.10, 144.00,
139.39, 130.57, 128.57, 125.43, 123.97, 103.67, 102.95. HRMS (AP-
ESI) m/z calcd for C13H11N5O2 [M+H]+ 270.0986, found 270.0989.
Compounds 7b was prepared from compound 6b in a similar manner
as described for compound 7a.
(hydroxyamino)-7-oxohept-yl)benzamide (5a6)
White solid. 83% yield. Mp: 198–200 ◦C. 1H NMR (400 MHz,
DMSO‑d6) δ 11.88 (s, 1H), 10.37 (s, 1H), 9.64 (s, 1H), 8.67 (s, 1H), 8.33
(d, J = 8.8 Hz, 2H), 8.01 (d, J = 8.5 Hz, 2H), 7.84 (d, J = 8.5 Hz, 2H),
7.29 (t, J = 2.8 Hz, 1H), 6.88 (dd, J = 3.5, 1.7 Hz, 1H), 3.25 (q, J = 6.7
Hz, 2H), 1.95 (t, J = 7.3 Hz, 2H), 1.51 (q, J = 7.0 Hz, 4H), 1.29 (tq, J =
13.7, 7.8, 7.1 Hz, 4H). 13C NMR (101 MHz, DMSO‑d6) δ 169.62, 166.22,
153.50, 151.17, 150.68, 143.36, 128.34, 128.26, 123.13, 119.54,
104.50, 99.61, 32.72, 29.64, 28.85, 26.74, 25.59. HRMS (AP-ESI) m/z
calcd for C20H24N6O3 [M+H]+ 397.1983, found 397.1987.
4.1.11. 3-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-(6-
4.1.16. 3-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-
(hydroxyamino)-6-oxohexy-l)benzamide (5b5)
hydroxybenzamide (7b)
White solid. 69% yield. Mp: 208–210 ◦C. 1H NMR (400 MHz,
DMSO‑d6) δ 11.79 (s, 1H), 10.35 (s, 1H), 9.49 (s, 1H), 8.67 (s, 1H), 8.42
(t, J = 5.7 Hz, 1H), 8.30 (s, 1H), 8.21 (d, J = 8.1 Hz, 2H), 7.44 (dd, J =
18.5, 7.7 Hz, 2H), 7.25 (t, J = 2.8 Hz, 1H), 6.83 (d, J = 3.4 Hz, 1H), 3.26
(q, J = 6.7 Hz, 2H), 1.97 (t, J = 7.3 Hz, 2H), 1.53 (t, J = 7.6 Hz, 4H),
1.35–1.26 (m, 2H). 13C NMR (101 MHz, DMSO‑d6) δ 169.59, 166.81,
153.88, 151.33, 151.10, 140.95, 135.80, 128.78, 123.13, 122.82,
120.80, 119.94, 104.22, 99.31, 32.74, 29.39, 26.62, 25.42. HRMS (AP-
ESI) m/z calcd fo C19H22N6O3 [M+H]+ 383.1826, found 383.1834.
Off-white solid. 62% yield. Mp: 210–212 ◦C. 1H NMR (400 MHz,
DMSO‑d6) δ 12.84 (s, 1H), 11.45 (s, 1H), 8.41 (s, 1H), 8.00 (t, J = 1.9 Hz,
1H), 7.81 (dd, J = 7.9, 2.1 Hz, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.58 (t, J =
7.8 Hz, 1H), 7.47 (t, J = 2.9 Hz, 1H), 6.99 (s, 1H). 13C NMR (101 MHz,
DMSO‑d6) δ 163.95, 144.40, 136.94, 134.58, 130.05, 127.23, 125.27,
125.05, 123.22, 103.23, 102.52. HRMS (AP-ESI) m/z calcd for
C
13H11N5O2 [M+H]+ 270.0986, found 270.0987.
4.1.17. 4-Chloro-7-tosyl-7H-cyclopenta[d]pyrimidine (8)
To a mixture of compound 1 (1.0 g, 6.5 mmol) in 50 mL of CH2Cl2
were added TsCl (1.3 g, 6.9 mmol), Et3N (1.30 g, 1.3 mmol), and DMAP
(0.03 g, 0.2 mmol). The reaction mixture was stirred for 5 h at room
temperature. When the reaction was complete, the solution was poured
into 100 mL CH2Cl2, then washed with water, 1 N citric acid for 2–3
times, dried over anhydrous MgSO4, filtered and concentrated to give
compound 8 (1.81 g, 90.5% yield) as an off-white solid. 1H NMR (400
MHz, DMSO‑d6) δ 8.82 (s, 1H), 8.13 (d, J = 4.0 Hz, 1H), 8.08–8.03 (m,
2H), 7.48 (d, J = 8.1 Hz, 2H), 6.97 (d, J = 4.0 Hz, 1H), 2.37 (s, 3H).
4.1.12. 3-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-(7-
(hydroxyamino)-7-oxohept-yl)benzamide (5b6)
White solid. 76% yield. Mp: 186–188 ◦C. 1H NMR (400 MHz,
DMSO‑d6) δ 12.16 (s, 1H), 10.36 (s, 1H), 10.12 (s, 1H), 8.48 (t, J = 5.7
Hz, 1H), 8.33 (s, 1H), 8.15 (s, 1H), 8.06 (d, J = 8.1 Hz, 1H), 7.60 (d, J =
7.7 Hz, 1H), 7.48 (t, J = 7.9 Hz, 1H), 7.34 (t, J = 2.8 Hz, 1H), 6.85 (d, J
= 3.4 Hz, 1H), 3.26 (q, J = 6.7 Hz, 2H), 1.95 (t, J = 7.4 Hz, 2H), 1.51 (h,
J = 7.5 Hz, 4H), 1.35–1.22 (m, 4H). 13C NMR (101 MHz, DMSO‑d6) δ
169.60, 166.50, 152.88, 149.88, 148.81, 139.52, 136.07, 129.18,
124.55, 123.67, 122.41, 121.22, 103.85, 100.35, 32.72, 29.49, 28.84,
26.72, 25.59. HRMS (AP-ESI) m/z calcd for C20H24N6O3 [M+H]+
397.1983, found 397.1991.
4.1.18. Methyl 4-((7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)
benzoate (9a0)
To a solution of 8 (0.61 g, 2.00 mmol) and methyl 4-aminobenzoate
(0.34 g, 2.20 mmol) in isopropanol was added 2 drops of conc.HCl, and
the mixture was heated to reflux with stirring for 2 h. Once the reaction
appeared complete by TLC analysis, The reaction solution was cooled to
room temperature, filtered, washed with isopropanol for 1–2 times, and
dried in vacuum to afford compound 9a0 (0.76 g, 89% yield), yellow
solid. 1H NMR (400 MHz, DMSO‑d6) δ 9.97 (s, 1H), 8.51 (s, 1H), 8.02 (d,
J = 8.5 Hz, 4H), 7.96 (d, J = 8.8 Hz, 2H), 7.78 (d, J = 4.0 Hz, 1H), 7.46
(d, J = 8.1 Hz, 2H), 7.18 (d, J = 4.0 Hz, 1H), 3.83 (s, 3H), 2.36 (s, 3H).
ESI-MS, m/z = 421.13 [Mꢀ H]-.
4.1.13. 4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-((tetrahydro-2H-
pyran-2-yl)o-xy)benzamide (6a)
To a solution of 3a (0.45 g, 1.77 mmol) in anhydrous DMF (50 mL) in
ice bath, was added EDCI (0.41 g, 2.12 mmol), HOBT (0.29 g, 2.12
mmol), followed by Et3N (0.42 g, 4.24 mmol). 30 min later, O-2-tetra-
hydro-2H-pyranhydroxylamine (0.25 g, 2.12 mmol) was added. 12 h
later, the solution was diluted by water and extracted with ethyl acetate.
The combined organic extract was washed with saturated NaHCO3 and
brine, dried over Na2SO4 overnight, and the solvent was evaporated
under vacuum to get compound 6a (0.12 g, 20% yield), white solid. 1H
NMR (400 MHz, DMSO‑d6) δ 11.85 (s, 1H), 11.51 (s, 1H), 9.57 (s, 1H),
8.35 (s, 1H), 8.03 (d, J = 8.6 Hz, 2H), 7.77 (d, J = 8.7 Hz, 2H), 7.29 (t, J
= 2.9 Hz, 1H), 6.85 (dd, J = 3.5, 1.9 Hz, 1H), 5.00 (t, J = 3.0 Hz, 1H),
4.06–4.00 (m, 1H), 3.55–3.50 (m, 1H), 1.78–1.67 (m, 3H), 1.60–1.52
(m, 3H). ESI-MS, m/z = 352.15 [Mꢀ H]-.
Compounds 9a1, 9b0 and 9b1 were prepared from compound 8 in a
similar manner as described for compound 9a0, respectively.
4.1.19. Methyl 3-(((7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)
methyl)benzoate (9a1)
Yellow solid. 74% yield. 1H NMR (400 MHz, DMSO‑d6) δ 8.52 (t, J =
6.1 Hz, 1H), 8.22 (s, 1H), 8.00–7.94 (m, 2H), 7.89 (d, J = 8.1 Hz, 2H),
7.59 (d, J = 3.9 Hz, 1H), 7.42 (dd, J = 8.2, 4.6 Hz, 4H), 6.91 (d, J = 4.0
Hz, 1H), 4.75 (d, J = 6.0 Hz, 2H), 3.81 (s, 3H), 2.33 (s, 3H). ESI-MS, m/z
= 437.09 [M+H]+.
Compound 6b was prepared from compound 3b in a similar manner
as described for compound 6a.
4.1.14. 3-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-((tetrahydro-2H-
pyran-2-yl)o-xy)benzamide (6b)
4.1.20. Methyl 3-((7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)
benzoate (9b0)
1
White solid. 31% yield. H NMR (400 MHz, DMSO‑d6) δ 11.79 (s,
1H), 11.65 (s, 1H), 9.53 (s, 1H), 8.29 (s, 1H), 8.22–8.18 (m, 2H), 7.43 (t,
J = 7.8 Hz, 1H), 7.37 (d, J = 7.6 Hz, 1H), 7.25 (t, J = 2.9 Hz, 1H),
6.85–6.78 (m, 1H), 5.02 (t, J = 3.2 Hz, 1H), 4.09–4.00 (m, 2H), 1.73 (s,
3H), 1.60–1.53 (m, 3H). ESI-MS, m/z = 352.12 [Mꢀ H]-.
Yellow solid. 70% yield. 1H NMR (400 MHz, DMSO‑d6) δ 10.08 (s,
1H), 8.45 (s, 1H), 8.38 (d, J = 1.9 Hz, 1H), 8.25–8.22 (m, 1H), 8.01 (d, J
= 8.2 Hz, 2H), 7.75 (d, J = 4.0 Hz, 1H), 7.66 (dt, J = 7.8, 1.3 Hz, 1H),
7.51 (t, J = 8.0 Hz, 1H), 7.46 (d, J = 8.1 Hz, 2H), 7.23 (d, J = 4.0 Hz,
1H), 3.87 (s, 3H), 2.36 (s, 3H). ESI-MS, m/z = 423.12 [M+H]+.
4.1.15. 4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-
hydroxybenzamide (7a)
To a stirred solution of 6a (0.1 g, 0.28 mmol) in 4 mL of ethyl acetate
8

S. Li et al.
Bioorganic Chemistry 116 (2021) 105278
4.1.21. Methyl 3-(((7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)
methyl)benzoate (9b1)
calcd for C14H13N5O2 [M+H]+ 284.1142, found 284.1147.
Yellow soild. 69% yield. 1H NMR (400 MHz, DMSO‑d6) δ 8.57 (t, J =
6.0 Hz, 1H), 8.23 (s, 1H), 7.97 (d, J = 8.3 Hz, 2H), 7.92 (d, J = 1.7 Hz,
1H), 7.82 (dt, J = 7.8, 1.4 Hz, 1H), 7.63–7.57 (m, 2H), 7.47 (d, J = 7.7
Hz, 1H), 7.43 (d, J = 8.0 Hz, 2H), 6.93 (d, J = 4.0 Hz, 1H), 4.73 (d, J =
6.0 Hz, 2H), 3.82 (s, 3H), 2.35 (s, 3H). ESI-MS, m/z = 437.16 [M+H]+.
Compounds 10a1, 10b1 were prepared from 9a1, 9b1 in a similar
manner as described for compound 3a, respectively.
4.1.28. Methyl 4-(methyl(7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)
amino)benzoate (13a0)
To a solution of compound 9a0 (0.30 g, 0.71 mmol) in DMF (10 mL)
was added cesium carbonate (0.46 g, 1.42 mmol). The reaction mixture
was stirred for 30 min under nitrogen. Iodomethane (0.78 g, 5.48 mmol)
was added, and the reaction mixture was stirred at room temperature for
2 h. The reaction mixture was poured into ice cold water and stirred for
30 min. The resulting precipitate was filtered , washed with water, to
afford the desired compound 13a0, yellow solid (0.23 g, 74%). 1H NMR
(400 MHz, DMSO‑d6) δ 8.46 (s, 1H), 8.08–8.03 (m, 2H), 7.96 (d, J = 8.1
Hz, 2H), 7.53 – 7.50 (m, 2H), 7.46–7.42 (m, 3H), 5.00 (d, J = 4.1 Hz,
1H), 3.89 (s, 3H), 3.53 (s, 3H), 2.36 (s, 3H). ESI-MS, m/z = 437.10
[M+H]+.
4.1.22. 4-(((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)methyl)benzoic acid
(10a1)
1
White solid, 82% yield. H NMR (400 MHz, DMSO‑d6) δ 11.54 (s,
1H), 8.09 (t, J = 6.7 Hz, 1H), 8.06 (s, 1H), 7.88 (d, J = 7.8 Hz, 2H), 7.44
(d, J = 7.9 Hz, 2H), 7.10 (s, 1H), 6.58 (s, 1H), 4.76 (d, J = 6.0 Hz, 2H).
ESI-MS, m/z = 267.08 [Mꢀ H]-.
Compounds 13a1, 13b0 and 13b1 were prepared from compounds
12a1, 12b0 and 12b1 in a similar manner as described for compound
13a0, respectively.
4.1.23. 3-(((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)methyl)benzoic acid
(10b1)
1
White soild. 69% yield. H NMR (400 MHz, DMSO‑d6) δ 12.94 (s,
4.1.29. Methyl 4-((methyl(7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)
1H), 11.60 (s, 1H), 8.14–8.09 (m, 2H), 7.93 (d, J = 1.7 Hz, 1H), 7.81 (dt,
J = 7.7, 1.4 Hz, 1H), 7.63–7.57 (m, 1H), 7.45 (t, J = 7.7 Hz, 1H),
7.13–7.08 (m, 1H), 6.59 (dd, J = 3.4, 1.8 Hz, 1H), 4.77 (d, J = 6.1 Hz,
2H). ESI-MS, m/z = 267.06 [Mꢀ H]-.
amino)methyl) benzoate (13a1)
1
Yellow solid. 13% yield. H NMR (400 MHz, DMSO‑d6) δ 8.26 (s,
1H), 8.02–7.98 (m, 2H), 7.90 (d, J = 8.1 Hz, 2H), 7.61 (d, J = 4.2 Hz,
1H), 7.44 (d, J = 8.2 Hz, 2H), 7.35 (d, J = 8.1 Hz, 2H), 6.83 (s, 1H), 5.04
(s, 2H), 3.82 (s, 3H), 3.32 (s, 3H), 2.36 (s, 3H). ESI-MS, m/z = 451.08
[M+H]+.
Compounds 11a1, 11b1 were prepared from 10a1, 10b1 in a similar
manner as described for compound 6a.
4.1.24. 4-(((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)methyl)-N-
((tetrahydro-2H-pyra-n-2-yl)oxy)benzamide (11a1).
4.1.30. Methyl 3-(methyl(7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)
amino)benzoate (13b0)
1
Yellow solid, 37% yield. 1H NMR (400 MHz, DMSO‑d6) δ 11.58 (s,
1H,), 11.55 (s, 1H), 8.07 (s, 1H,), 8.03 (t, J = 6.1 Hz, 1H), 7.71 (d, J =
8.1 Hz, 2H), 7.41 (d, J = 8.1 Hz, 2H), 7.11–7.08 (m, 1H), 6.61–6.54 (m,
1H), 4.98 (t, J = 3.1 Hz, 1H), 4.75 (d, J = 6.0 Hz, 2H), 4.08–4.00 (m,
1H), 3.53–3.48 (m, 1H), 1.73–1.68 (m, 3H), 1.57–1.50 (m, 3H). ESI-MS,
m/z = 368.10 [M+H]+.
Yellow solid. 64% yield. H NMR (400 MHz, DMSO‑d6) δ 8.43 (s,
1H), 8.05–8.03 (m, 1H), 7.95 (d, J = 8.2 Hz, 2H), 7.91 (d, J = 2.1 Hz,
1H), 7.70–7.68 (m, 2H), 7.43 (d, J = 8.1 Hz, 2H), 7.40 (d, J = 4.1 Hz,
1H), 4.81 (d, J = 4.1 Hz, 1H), 3.85 (s, 3H), 3.51 (s, 3H), 2.36 (s, 3H). ESI-
MS, m/z = 437.08 [M+H]+.
4.1.31. Methyl 3-((methyl(7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)
amino)methyl) benzoate (13b1)
4.1.25. 3-(((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)methyl)-N-
((tetrahydro-2H-pyra-n-2-yl)oxy)benzamide (11b1)
Off-white solid. 62% yield. 1H NMR (400 MHz, DMSO‑d6) δ 8.28 (s,
1H), 8.00 (d, J = 8.3 Hz, 2H), 7.87–7.82 (m, 2H), 7.62 (d, J = 4.1 Hz,
1H), 7.52–7.47 (m, 2H), 7.47–7.41 (m, 2H), 6.85 (s, 1H), 5.03 (s, 2H),
3.81 (s, 3H), 3.31 (s, 3H), 2.36 (s, 3H). ESI-MS, m/z = 451.12 [M+H]+.
Compounds 14a0, 14a1, 14b0 and 14b1 were prepared from com-
pounds 13a0, 13a1, 13b0 and 13b1 in a similar manner as described for
compound 3a, respectively.
Yellow solid. 40% yield. 1H NMR (400 MHz, DMSO‑d6) δ 11.65 (s,
1H, NH), 11.54 (s, 1H, NH), 8.08 (s, 1H), 8.01 (t, J = 6.1 Hz, 1H), 7.77
(d, J = 1.7 Hz), 7.62 (dt, J = 7.7, 1.4 Hz), 7.53–7.49 (m, 1H), 7.40 (t, J =
7.6 Hz, 1H), 7.11–7.08 (m, 1H), 6.58 (dd, J = 3.5, 1.9 Hz, 1H), 4.98 (t, J
= 3.1 Hz, 1H), 4.75 (d, J = 6.0 Hz, 2H), 4.09–4.02 (m, 1H), 4.01 (d, J =
7.1 Hz, 1H), 3.51 (d, J = 11.5 Hz, 1H), 1.70 (m, 3H), 1.58–1.51 (m, 3H).
ESI-MS, m/z = 366.12 [Mꢀ H]-.
Compounds 12a1, 12b1 were prepared from compounds 11a1,
11b1 in a similar manner as described for 7a, respectively.
4.1.32. 4-(Methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)benzoic acid
(14a0)
Yellow solid. 65% yield. 1H NMR (400 MHz, DMSO‑d6) δ 11.71 (s,
1H), 8.34 (s, 1H), 8.02–7.98 (m, 2H), 7.47–7.41 (m, 2H), 6.98 (dd, J =
3.5, 2.4 Hz, 1H), 4.91 (dd, J = 3.5, 1.9 Hz, 1H), 3.58 (s, 3H). ESI-MS, m/
z = 267.04 [Mꢀ H]-.
4.1.26. 4-(((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)methyl)-N-
hydroxybenzamide (12a1)
Yellow solid. 37% yield. Mp: 222–228 ◦C.1H NMR (400 MHz,
DMSO‑d6) δ 12.82 (s, 1H), 11.26 (s, 1H), 10.41 (s, 1H), 8.32 (s, 1H), 7.77
(d, J = 8.0 Hz, 2H), 7.53 (d, J = 8.0 Hz, 2H), 7.43 (t, J = 2.9 Hz, 1H),
7.12 (s, 1H), 4.96 (d, J = 6.3 Hz, 2H). 13C NMR (101 MHz, DMSO‑d6) δ
164.23, 150.48, 142.81, 140.45, 132.47, 127.96, 127.62, 124.91,
102.70, 102.26, 44.88. HRMS (AP-ESI) m/z calcd for C14H13N5O2
[M+H]+ 284.1142, found 284.1145.
4.1.33. 4-((Methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)methyl)
benzoic acid (14a1)
Yellow solid. 66% yield. 1H NMR (400 MHz, DMSO‑d6) δ 11.69 (s,
1H), 8.13 (s, 1H), 7.90 (d, J = 8.0 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H), 7.12
(t, J = 2.9 Hz, 1H), 6.50 (d, J = 3.5 Hz, 1H), 5.08 (s, 2H), 3.35 (s, 3H).
ESI-MS, m/z = 281.08 [Mꢀ H]-.
4.1.27. 3-(((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)methyl)-N-
hydroxybenzamide (12b1)
4.1.34. 3-(Methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)benzoic acid
(14b0)
Yellow solid. 51% yield. Mp: 156–158 ◦C. 1H NMR (400 MHz,
DMSO‑d6) δ 12.80 (s, 1H), 11.30 (s, 1H), 10.21 (s, 1H), 8.36 (s, 1H), 7.83
(s, 1H), 7.70 (d, J = 7.7 Hz, 1H), 7.59 (d, J = 7.7 Hz, 1H), 7.49–7.42 (m,
2H), 7.06 (s, 1H), 4.92 (d, J = 6.0 Hz, 2H). 13C NMR (101 MHz,
DMSO‑d6) δ 164.34, 151.27, 143.05, 137.62, 133.56, 130.77, 129.11,
126.67, 126.40, 124.83, 102.55, 102.22, 45.08. HRMS (AP-ESI) m/z
Yellow solid. 65% yield. 1H NMR (400 MHz, DMSO‑d6) δ 13.17 (s,
1H), 11.66 (s, 1H), 8.30 (s, 1H), 7.96 (dt, J = 6.6, 1.9 Hz, 1H), 7.84 (d, J
= 2.0 Hz, 1H), 7.66–7.60 (m, 2H), 6.91 (t, J = 3.0 Hz, 1H), 4.67 (dd, J =
3.6, 1.9 Hz, 1H), 3.55 (s, 3H). ESI-MS, m/z = 267.06 [Mꢀ H]-.
9

S. Li et al.
Bioorganic Chemistry 116 (2021) 105278
4.1.35. 3-((Methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)methyl)
benzoic acid (14b1)
4.1.42. N-hydroxy-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)
benzamide (16b0)
Yellow solid. 70% yield. 1H NMR (400 MHz, DMSO‑d6) δ 11.70 (s,
1H), 8.14 (s, 1H), 7.83 (t, J = 3.5 Hz, 2H), 7.51–7.42 (m, 2H), 7.13 (t, J
= 2.9 Hz, 1H), 6.51 (d, J = 2.7 Hz, 1H), 5.07 (s, 2H), 3.33 (s, 3H).
Compounds 15a0, 15a1, 15b0 and 15b1 were prepared from com-
pounds 14a0, 14a1, 14b0 and 14b1 in a similar manner as described for
compound 6a, respectively.
White solid. 60% yield. Mp: 230–232 ◦C. 1H NMR (400 MHz,
DMSO‑d6) δ 12.85 (s, 1H), 11.49 (s, 1H), 8.50 (s, 1H), 8.00–7.96 (m,
2H), 7.76–7.66 (m, 2H), 7.19 (t, J = 2.9 Hz, 1H), 4.75 (d, J = 3.4 Hz,
1H), 3.73 (s, 3H). 13C NMR (101 MHz, DMSO‑d6) δ 163.14, 152.45,
146.87, 143.99, 143.82, 135.18, 130.86, 130.76, 127.81, 126.86,
124.53, 103.20, 102.58, 41.78. HRMS (AP-ESI) m/z calcd for
C
14H13N5O2 [M+H]+ 284.1142, found 284.1147.
4.1.36. 4-(Methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-
((tetrahydro-2H-pyran-2-yl)ox-y)benzamide (15a0)
4.1.43. N-hydroxy-3-((methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)
methyl)benza-mide (16b1)
Yellow solid. 61% yield. 1H NMR (400 MHz, DMSO‑d6) δ 11.74 (s),
11.69 (s, 1H, NH), 8.32 (s), 7.89–7.83 (m), 7.48–7.42 (m), 6.96 (dd, J =
3.5, 2.4 Hz, 1H), 5.03 (t, J = 2.9 Hz), 4.85 (dd, J = 3.5, 1.9 Hz),
4.15–4.02 (m), 3.56 (s, 3H), 3.55–3.51 (m, 1H,), 1.74 (s, 3H,), 1.62–1.51
(m, 3H). ESI-MS, m/z = 366.15 [Mꢀ H]-.
White solid. 58% yield. Mp: 134–136 ◦C. 1H NMR (400 MHz,
DMSO‑d6) δ 12.84 (s, 1H), 11.28 (s, 1H), 8.42 (s, 1H), 7.73–7.67 (m,
1H), 7.67 (s, 1H), 7.47 (d, J = 6.3 Hz, 2H), 7.41 (t, J = 3.0 Hz, 1H), 6.77
(s, 1H), 5.19 (s, 2H), 3.48 (s, 3H). 13C NMR (101 MHz, DMSO‑d6) δ
164.16, 150.05, 143.96, 136.64, 133.74, 130.16, 129.39, 126.63,
125.57, 124.34, 104.67, 101.95, 60.78, 32.78. HRMS (AP-ESI) m/z calcd
for C15H15N5O2 [M+H]+ 298.1299, found 298.1309.
4.1.37. 4-((Methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)methyl)-N-
((tetrahydro-2H-pyra-n-2-yl)oxy)benzamide (15a1)
Yellow solid. 60% yield. 1H NMR (400 MHz, DMSO‑d6) δ 11.68 (s,
1H), 11.59 (s, 1H), 8.13 (s, 1H), 7.71 (d, J = 8.0 Hz, 2H), 7.33 (d, J = 8.0
Hz, 2H), 7.12 (t, J = 3.0 Hz, 1H), 6.49 (s, 1H), 5.05 (s, 1H), 4.98 (d, J =
3.3 Hz, 1H), 4.06–4.00 (m, 1H), 3.53–3.48 (m, 1H), 1.71 (m, 3H),
1.56–1.51 (m, 3H). ESI-MS, m/z = 382.06 [M+H]+.
4.2. In vitro HDACs inhibition assay
HDACs inhibitory activity was determined by fluorescence assay as
previously described [28]. Briefly, 10 μL of enzyme solution (HDAC2,
HDAC6 or HDAC8) was mixed with different concentrations of tested
compound (50
L). The mixture was incubated at 37 ◦C for 5 min, fol-
lowed by adding 40
4.1.38. 3-(Methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-
((tetrahydro-2H-pyran-2-yl)ox-y)benzamide (15b0)
μ
μ
L fluorogenic substrate (Boc-Lys(acetyl)-AMC for
Yellow solid. 60% yield. 1H NMR (400 MHz, DMSO‑d6) δ 11.74 (s,
1H), 11.66 (s, 1H), 8.30 (s, 1H), 7.82 (dt, J = 7.4, 1.5 Hz, 1H,), 7.77 (d, J
= 2.1 Hz, 1H), 7.63–7.53 (m, 2H), 6.91 (t, J = 2.9 Hz, 1H), 4.99 (s, 1H),
4.67 (dd, J = 3.5, 1.9 Hz, 1H), 4.08–4.00 (m, 1H), 3.55 (s, 3H),
3.52–3.48 (m, 1H), 1.71 (brs, 3H), 1.60–1.50 (m, 3H). ESI-MS, m/z =
368.07 [M+H]+.
HDAC2 HDAC6, Boc-Lys(trifluoroacetyl)-AMC for HDAC8). After incu-
bation at 37 ◦C for 30 min, the mixture was quenched by addition of 100
μ
L of developer containing trypsin and Trichostatin A. Over another 20
min of incubation at 37 ◦C, fluorescence intensity was measured using a
microplate reader at excitation and emission wavelengths of 390 and
460 nm, respectively. The inhibitory rates were calculated from the
fluorescence intensity readout of tested wells relative to those of control
wells, and the IC50 values were calculated using nonlinear regression
with normalized dose–response fit in Prism GraphPad software.
4.1.39. 3-((Methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)methyl)-N-
((tetrahydro-2H-pyra-n-2-yl)oxy)benzamide (15b1).
Yellow solid. 65% yield. 1H NMR (400 MHz, DMSO‑d6) δ 11.65 (s,
1H), 11.54 (s, 1H), 8.08 (s, 1H), 8.01 (t, J = 6.1 Hz, 1H), 7.77 (d, J = 1.7
Hz, 1H), 7.62 (dt, J = 7.7, 1.4 Hz, 1H), 7.53–7.49 (m, 1H), 7.40 (t, J =
7.6 Hz, 1H), 7.11–7.08 (m, 1H), 6.58 (dd, J = 3.5, 1.9 Hz, 1H), 4.98 (t, J
= 3.1 Hz, 1H), 4.75 (d, J = 6.0 Hz, 2H), 4.09–4.02 (m, 1H), 4.01 (d, J =
7.1 Hz, 1H), 3.51 (d, J = 11.5 Hz, 1H), 1.70 (s, 3H), 1.58–1.51 (m, 3H).
ESI-MS, m/z = 366.12 [Mꢀ H]-.
4.3. In vitro anti-proliferation assay
All cell lines were maintained in RPMI1640 medium containing 10%
◦
FBS at 37 C in a 5% CO2 humidified incubator. Anti-proliferative ac-
tivity was determined by the MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-
diphenyl-2 h-tetrazolium bromide) method. Briefly, cells were passaged
the day before dosing into a 96-well plate, allowed to grow for 12 h, and
then treated with different concentrations of compound for 48 h. A 0.5%
MTT solution was added to each well. After incubation for another 4 h,
formazan formed from MTT was extracted by adding 200 uL of DMSO.
Absorbance was then determined using an ELISA reader at 570 nm, and
the IC50 values were calculated using nonlinear regression in Prism
GraphPad software.
Compounds 16a0, 16a1, 16b0 and 16b1 were prepared from com-
pounds 15a0, 15a1, 15b0 and 15b1 in a similar manner as described for
compound 7a, respectively.
4.1.40. N-hydroxy-4-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)
benzamide (16a0)
White solid. 66% yield. Mp: 228–230 ◦C. 1H NMR (400 MHz,
DMSO‑d6) δ 12.86 (s, 1H), 11.44 (s, 1H), 8.51 (s, 1H), 7.98 (d, J = 8.2
Hz, 2H), 7.65 (d, J = 8.3 Hz, 2H), 7.21 (t, J = 3.0 Hz, 1H), 4.86 (s, 1H),
3.72 (s, 3H). 13C NMR (101 MHz, DMSO‑d6) δ 166.93, 163.43, 145.93,
143.91, 133.63, 131.64, 129.30, 128.04, 124.63, 103.31, 102.62, 41.79.
HRMS (AP-ESI) m/z calcd for C14H13N5O2 [M+H]+ 284.1142, found
284.1147.
4.4. Western blot analysis
RPMI 8226 cells were treated with compounds or DMSO for 6 h.
Then the cells were washed twice with cold PBS and lysed in ice-cold
RIPA buffer. Lysates were cleared by centrifugation. Protein concen-
trations were determined using the BCA assay. Equal amounts of cell
extracts were then resolved by SDS-PAGE, transferred to nitrocellulose
4.1.41. N-hydroxy-4-((methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)
methyl)benza-mide (16a1)
membranes and probed with acetyl α-tubulin antibody, total-α-tubulin
White solid. 56% yield. Mp: 160–162 ◦C. 1H NMR (400 MHz,
DMSO‑d6) δ 12.89 (s, 1H), 11.32 (s, 1H), 8.40 (s, 1H), 7.77 (d, J = 8.1
Hz, 2H), 7.39 (d, J = 7.6 Hz, 3H), 6.72 (s, 1H), 5.21 (s, 2H), 3.49 (s, 3H).
13C NMR (101 MHz, DMSO‑d6) δ 146.74, 132.29, 128.12, 127.76,
127.23, 123.26, 103.58, 102.12, 101.41, 61.84, 54.29, 28.36, 25.19,
18.76. HRMS (AP-ESI) m/z calcd for C15H15N5O2 [M+H]+ 298.1299,
found 298.1307.
antibody, acetyl histone H3 antibody, acetyl histone H4 antibody and
total histone H3 antibody, respectively. Blots were imaged using an
enhanced chemiluminescence system.
4.5. In vitro kinases inhibition assay
JAK1, JAK2, JAK3, TYK2 and Akt1 inhibitory activities were tested
10

S. Li et al.
Bioorganic Chemistry 116 (2021) 105278
in HUAWEI PHARMA (Ji’nan, China) using Kinase-GloTM Luminescent
Kinase Assay, which measures kinase activity by quantitating the
amount of ATP remaining in solution following a kinase reaction. In
brief, the tested compounds, kinases, substrate, and ATP were diluted in
kinase buffer to the indicated concentrations according to the instruc-
tion, and incubated at 30 ◦C for 40 min, then the Kinase-Glo reagent was
added. Over an additional 15 min of incubation, the luminescence was
recorded on a microplate reader (SpectraMax M5) to calculate the
inhibitory rate. The IC50 values were calculated using nonlinear
regression with normalized dose–response fit in Prism GraphPad
software.
Molecular docking was carried out via Ligand Docking module with
Standard-Precision (SP) mode. Other parameters were set as default
values. PyMOL (http://www.pymol.org/) was used to visualize the
docked poses and generate the figures.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
4.6. In vitro mouse liver microsome stability assay
This work was supported by Natural Science Foundation of Shan-
dong Province (Grant No. ZR2018QH007), Key Research and Develop-
ment Program of Shandong Province (2017CXGC1401), Young Scholars
Program of Shandong University (YSPSDU, 2016WLJH33).
Mouse liver microsome (0.5 mg/mL) containing compound 12a1
(1.0 μ
M) was incubated with NADPH (1.0 mM) at 37 ◦C. At the specific
time points, samples were added to acetonitrile to terminate the reac-
tion, then subjected to vortex mixing for 5 min and stored in a freezer at
ꢀ 80 ◦C. Before analysis, the samples were centrifuged at 4000 rpm for
15 min. The remaining of 12a1 in the supernatants were analyzed by LC-
MS/MS.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105278.
4.7. In vivo antitumor experiment in RPMI 8226 xenograft model
References
All experiments involving laboratory animals were performed with
the approval of local ethics committee for animal experimentation. In
vivo human tumor xenograft models were established according to
previously described method with minor modification [24]. In brief, 1 ×
107 RPMI 8226 cells were inoculated subcutaneously in the right flank of
male athymic nude mice (BALB/c-nu, 5–6 weeks old, Beijing HFK
Bioscience Co., Ltd.). When the tumors reached an average volume of
100 mm3, the mice were randomized into four groups (n = 5) and
treated with vehicle (PBS with 5% DMSO, ip), compound 12a1 (50 mg/
kg/d, ip), bortezomib (0.5 mg/kg/d, iv) or 12a1 (50 mg/kg/d, ip) plus
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end of treatment to reveal the antitumor effects in tumor weight and
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