Novel cyclin-dependent kinase 9 (CDK9) inhibitor with suppression of cancer stemness activity against non-small-cell lung cancer

Article abstract of DOI:10.1016/j.ejmech.2019.07.038

A series of novel, highly potent, selective CDK9 inhibitors with cancer stem cells (CSCs) inhibition activity were designed and synthesized for non-small-cell lung cancer (NSCLC) therapy. Structure-activity relationship analysis based on enzymatic and cellular activities led to the discovery of a promising inhibitor 21e. 21e potently inhibited CDK9 with IC₅₀ value of 11 nM and suppressed the stemness properties of NSCLC effectively. It could decrease the stemness phenotypes of NSCLC cells, including tumor sphere formation, side-population and stemness markers abundance. 21e displayed good selectivity over the CDK family kinases and kinase profiling assay against 381 kinases. In addition, 21e inhibited cell proliferation, colony-formation, and cell cycle progression and induced apoptosis in NSCLC. In H1299 xenograft mouse model, a once-daily dose of compound 21e at 20 mg/kg significantly suppressed the tumor growth without obvious toxicity. Studies of mechanisms of action indicated that 21e efficiently inhibited CDK9 signaling pathway and stemness both in vitro and in vivo. Collectively, 21e as a novel CDK9 inhibitor with CSCs inhibition properties could be a promising agent for the treatment of NSCLC.

Full text of DOI:10.1016/j.ejmech.2019.07.038

Accepted Manuscript
Novel cyclin-dependent kinase 9 (CDK9) inhibitor with suppression of cancer
stemness activity against non-small-cell lung cancer
Xin Wang, Chenhua Yu, Cheng Wang, Yakun Ma, Tianqi Wang, Yao Li, Zhi Huang,
Manqian Zhou, Peiqing Sun, Jianyu Zheng, Shengyong Yang, Yan Fan, Rong Xiang
PII:
S0223-5234(19)30659-2
DOI:
https://doi.org/10.1016/j.ejmech.2019.07.038
EJMECH 11535
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 April 2019
Revised Date: 8 July 2019
Accepted Date: 10 July 2019
Please cite this article as: X. Wang, C. Yu, C. Wang, Y. Ma, T. Wang, Y. Li, Z. Huang, M. Zhou, P. Sun,
J. Zheng, S. Yang, Y. Fan, R. Xiang, Novel cyclin-dependent kinase 9 (CDK9) inhibitor with suppression
of cancer stemness activity against non-small-cell lung cancer, European Journal of Medicinal Chemistry
(2019), doi: https://doi.org/10.1016/j.ejmech.2019.07.038.
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ACCEPTED MANUSCRIPT
A selective, potent and oral CDK9 inhibitor, 21e (IC50 = 11 nM) with stemness
suppression properties of NSCLC was discovered. In H1299 xenograft mouse model,
2
1e at 20 mg/kg led to significant tumor regression without obvious toxicity.

ACCEPTED MANUSCRIPT
Novel Cyclin-dependent Kinase 9 (CDK9) Inhibitor
with Suppression of Cancer Stemness Activity against
Non-Small-Cell Lung Cancer
⊥
1
⊥
1
1
1
1
1
Xin Wang, Chenhua Yu, Cheng Wang, Yakun Ma, Tianqi Wang, Yao Li, Zhi
1
4
1, 5
6
7
Huang, Manqian Zhou, Peiqing Sun,
Jianyu Zheng, Shengyong Yang, Yan
1
,2
1, 2, 3
Fan* and Rong Xiang*
1
Department of Medicinal Chemistry, School of Medicine, Nankai University, 94 Weijin
Road, Tianjin 300071, China
2
2011 Project Collaborative Innovation Center for Biotherapy of Ministry of Education, 94
Weijin Road, Tianjin 300071, China
3
State Key Laboratory of Medicinal Chemical Biology, 94 Weijin Road, Tianjin 300071,
China
4
Department of Radiation Oncology, Tianjin Union Medical Center, Tianjin 300121, China.
5
Department of Cancer Biology, Wake Forest Comprehensive Cancer Center, Wake Forest
School of Medicine, Winston-Salem, NC, USA.
6
State Key Laboratory and Institute of Elemento-Organic Chemistry, Collaborative
Innovation Center of Chemical Science and Engineering, Nankai University, Tianjin 300071,
China.
1

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7
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan
University and Collaborative Innovation Center for Biotherapy, Chengdu 610041, China
*
Corresponding authors: Yan Fan* E-mail: yanfan@nankai.edu.cn; Rong Xiang*:
E-mail: rxiang@nankai.edu.cn.
⊥
Author Contributions: Xin Wang and Chenhua Yu contributed equally to this work.
2

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ABSTRACT
A series of novel, highly potent, selective CDK9 inhibitors with cancer stem cells
CSCs) inhibition activity were designed and synthesized for non-small-cell lung
(
cancer (NSCLC) therapy. Structure-activity relationship analysis based on enzymatic
and cellular activities led to the discovery of a promising inhibitor 21e. 21e potently
inhibited CDK9 with IC value of 11 nM and suppressed the stemness properties of
5
0
NSCLC effectively. It could decrease the stemness phenotypes of NSCLC cells,
including tumor sphere formation, side-population and stemness markers abundance.
2
1e displayed good selectivity over the CDK family kinases and kinase profiling
assay against 381 kinases. In addition, 21e inhibited cell proliferation,
colony-formation, and cell cycle progression and induced apoptosis in NSCLC. In
H1299 xenograft mouse model, a once-daily dose of compound 21e at 20 mg/kg
significantly suppressed the tumor growth without obvious toxicity. Studies of
mechanisms of action indicated that 21e efficiently inhibited CDK9 signaling
pathway and stemness both in vitro and in vivo. Collectively, 21e as a novel CDK9
inhibitor with CSCs inhibition properties could be a promising agent for the treatment
of NSCLC.
Keywords: CDK9, CSCs, Inhibitor, NSCLC
3

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1
. Introduction
Lung cancer is one of the most common malignant tumors characterized by high
incidence and mortality as well as poor prognosis.[1] More than 85% of lung cancers
are identified as non-small-cell lung cancer (NSCLC). Currently, conventional
chemotherapy is the systemic therapy and prognosis remains poor. Resistance to
chemotherapy, relapse in patients has always remained a challenging issue in NSCLC
treatment. This resistance is attributed to a small population of cancer cells known as
cancer stem cells (CSC).[2, 3] CSCs are with the key properties of self-renew and
potential differentiation, which may play a pivotal role in carcinogenesis, growth,
metastasis and drug resistance of NSCLC.[4-6] Traditional therapies, targeting rapidly
proliferating cell populations, may kill the bulk of the tumor, yet are inefficient at
targeting the CSCs. Elimination of CSCs is essential to improve the treatment of
cancer.
Compared with chemotherapy, targeted therapies have the advantage of
maximizing efficacy and reducing toxicity. Innovative adaptive study design
combining of targeted therapy with CSCs inhibition may be used to expedite effective
drug development. Preclinical studies have identified several potential targets, and
cyclin-dependent kinase 9 (CDK9) is one of the targets that have recently caused
increasing interest.[7-10] Inhibition of CDK9 selectively reduces many proteins
up-regulated in many cancers, leading to the inhibition of proliferation and the
promotion of apoptosis.[11-15] Recent studies have shown that blocking CDK9
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activity provides an appealing therapeutic approach to curb lung cancer
metastasis.[16]
CDK9 forms a functional complex with regulatory subunit cyclin T or cyclin K to
control transcription progression.[17] This CDK9/cyclin T complex named positive
transcription elongation factor b (P-TEFb) is a general transcription factor promoting
the RNA synthesis in genetic programmes for cell growth and differentiation.[10, 18]
As a component of the P-TEFb, CDK9 phosphorylates the C-terminal domain (CTD)
of the largest subunit of the most RNA polymerase II (pol II) to stimulate transcription
elongation of most protein coding genes.[19, 20] Phosphorylation of RNA pol II is
regarded as a marker in preclinical pharmacodynamic research, which is much more
involved in a variety of human pathological conditions, such as cancer.[8, 10] During
the past decade, attempts have been made toward the exploitation of CDK9 inhibitor
for anticancer potential. CDK9 shows high level of sequence conservation with other
CDK family members, thus the design of selective CDK9 inhibitor is challenging.
The first CDK9 inhibitor involved in clinical trial was flavopiridol with multiple CDK
inhibition. Flavopiridol inhibits CDKs that govern cell cycle progression and
phosphorylate the COOH-terminal domain of RNA polymerase II.[12, 21, 22] The
antitumor activity of flavopiridol in chronic lymphocytic leukemia appears to be the
CDK9-mediated downregulation of transcription of antiapoptotic proteins. While
there are also several pan-CDK inhibitors in clinical studies,[23-25] potent and
selective CDK9 inhibitors have only recently emerged.[26-28] These studies highlight
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the need for the development and validation of CDK9 small molecule inhibitors.
Simultaneous inhibition CDK9 and tumor stem cell could be a promising strategy
to enhance the antitumor activity. In an effort to discover CDK9 inhibitors with high
potency of CSCs inhibition against NSCLC, we recently performed a rational drug
design. In our study, we identified a series of novel compounds targeting CDK9
against human NSCLC. The compound 21e exhibits excellent inhibition against
CDK9 with IC50 value of 11 nM and inhibits stem cell properties of NSCLC
effectively. The main purpose of this study is to perform a comprehensive evaluation
to this compound, including in vitro and in vivo mechanism of action. Based on these
finding, compound 21e provides a potential treatment for the NSCLC.
2
2
. Results and discussion
.1. CDK9 inhibitors with CSCs inhibition activity design strategy.
On basis of our recent studies,[29, 30] combining the pyrrolo-[2,3-d]
pyrimidines-2-amine pharmacophore of ribociclib with other hydrophobic functional
group has been successful to enhance the CDK9 inhibition activity obviously. These
results support the view that the pyrrolo-[2,3-d] pyrimidines-2-amine scaffold might
be optimized to yield a useful CDK9 inhibitor. In order to achieve this goal, we have
performed docking studies to promote CDK9 specific inhibition. We docked
ribociclib in CDK9 and CDK6, as ribociclib is a CDK4/6 inhibitor applied in clinic
(Figure 1A and 1B). It shows that aminopyrimidine forms two key hydrogen bonds
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to the backbone residue VAL101 and CYS106 in the hinge region of CDK6 and
CDK9 respectively. By contrast, much bigger hydrophobic pocket formed obviously
by HIS108 and GLY112 adjacent to hinge region exits in CDK9. Meanwhile, the two
residues were substituted to residue GLN103 and THR107 at the same position in
CDK6 (Figure 1C). It means that the hydrophobic pocket in CDK9 could
accommodate more flexible and bigger fragment resulting in potent selectivity against
CDK6, which is consistent to our previous studies. Therefore, decision of the flexible
and bigger hydrophobic moiety appears to be particularly important in sustain the
scaffold of the aminopyrimidine to play multiple biological function.
Sulforaphane (SFN) is a well-known natural compound within the isothiocyanate
group and abrogates tumorigenesis and metastatic progression by targeting CSCs.
[
31-37] As reported, SFN potentiates the anticancer effects of gemcitabine,
doxorubicin, cisplatin, or 5-flurouracil on cancer cell line while also increasing
cytotoxicity of CSCs.[38-40] The combination of SFN with bafilomycin A1, as an
inhibitor of autophagy, enhances apoptotic effect in breast cancer.[41] Combination
therapies that SFN with other therapeutic agents favor a potential treatment modality
for cancer, which provide a promising strategy of drug design. [42, 43] Isothiocyanate
group with CSCs inhibition function as the pharmacophore of SFN molecule, was
chosen as the hydrophobic substituent to improve inhibitory and selectivity against
CDK9. [7] Thus, merged molecules, pyrrolo-[2,3-d] pyrimidines-2-amine from
ribociclib as CDK9 pharmacophore and isothiocyanate from SFN as targeting CSCs
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pharmacophore, were designed. The substitute group on the NH at the C2-position of
the pyrimidine was replaced by a tail substituent containing isothiocyanate, linker and
a substituted aromatic ring (Figure 1D). The diversity of the tail substituents was
performed to improve the inhibitory activity against CDK9 and CSCs.
2
.2. Chemistry.
Structural optimization was focused on the pyrimidine, Ar and linker regions
(Figure 1). The synthetic routes were carried out as shown in Scheme 1-3. The
synthesis of the final compounds 9a~9e and 10aa~10de were depicted in Scheme 1.
Briefly, the commercially obtained 1 or 2a~2d reacted with various
mono-Boc-diamine with different carbon lengths (3a~3e) to obtain the intermediates
4
a~4e and 5aa~5de with free aromatic amine, which then reacted with commercially
available 6 via Buchwald–Hartwig coupling catalyzed by palladium acetate to afford
the key intermediates 7a~7e and 8aa~8de. After deprotection by trifluoroacetic acid,
final compounds 9a~9e and 10aa~10de were readily prepared within carbon disulfide
and N,N’-dicylohexylcarbodiimide. In Scheme 2, aromatic diamine (11a~11c or 12)
condensed with Boc-NH-(CH ) -COOH (13a~13e) to afford intermediates 14a~14g
2
n
and 15. Then final compounds 20, 21a~21g and 22 were generated through three steps
using synthetic strategy in the similar manner of Scheme 1. As shown in Scheme 3,
p-nitroaniline analogs containing 2- or 3- substituent (23a~23c) were treated with Boc
anhydride, and then were reduced to give the corresponding primary amine (24a~24c).
After that, 24a~24c underwent Buchwald–Hartwig coupling reaction and deprotection
8

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to produce 26a~26c. The condensation reaction between the key start material 13e
and amine analogs (including ribociclib, 26a~26c and 27) yielded intermediates 28,
2
9a~29c and 30. After removal of the N-Boc protecting group, the final compounds
1, 32a~32c, 33, 34 were prepared with carbon disulfide.
3
2
.3. Enzyme inhibitory activity and SAR.
The results of the enzymatic-inhibition assays are listed in Table 1-3 and S1 with
ribociclib as the positive control. At first, we introduced isothiocyanate to the para
position of the benzene to obtain compound 34 to explore the effect of isothiocyanate
group as the hydrophobic moiety on inhibition activity against CDK9. Compared with
ribociclib, the activity of 34 against CDK9 was improved significantly. Meanwhile,
the activity of 34 against CDK6 was decreased and CDK4 activity was still high.
Preliminary design of 34 supports our hypothesis, that hydrophobic substituent in the
tail might improve inhibitory activity against CDK9.
1
Further optimization was proceeding. When Ar is a phenyl with R = H,
compounds 9a~9e with linkers containing N-alkyl-sulfonamide and n ranging from 1
to 5, exhibit more potent CDK9 activity compared to ribociclib (Table 1). Due to the
decreasing activity against CDK4 and CDK6, Compound 9d and 9e (n = 4 or 5) were
selected to test the IC value to further identify CDK9 activity (Table 3, IC = 9
5
0
50
nM), more than 20 times than ribociclib. The preliminary results indicated that
extension of the linker might be helpful to promote the specificity of CDK9. When
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linkers were varied to N-alkyl-amide, the similar results on CDK9 were shown in
compound 10aa~10ae (Table 1), and no obvious difference on CDK4 and CDK6
except compound 10ae (n = 5). Then we changed back the phenyl to pyridine ring
(compound 10ba~10be) to determine the role of the pyridine ring of ribociclib for the
activity against CDK9. It turned out that pyridine had decreased the efficiency against
CDK9 (Table 1), compared to compounds 10aa~10ae. Modification of fluorine atom
to the phenyl ring resulted in analogues 10ca~10ce (Table 1), which retained the
potent potency against CDK9 (IC : 6 nM to 50 nM, Table 3), while decreased
5
0
inhibitory efficiency against CDK4 and CDK6. Hydroxyl at the same position
10da~10de, Table 1) displays high selective activity on CDK9. Unfortunately,
(
1
0da~10de lost about several times activity against CDK9 compared to the
1
compounds with same length linker, such as 10da (Table 3, IC : 68 nM, R = OH) vs
5
0
1
1
0ca (Table 3, IC : 10 nM, R = F), and especially 10dd and 10de show weak
50
1
activity to CDK9 as similar as ribociclib (IC : 197 nM). The substituent on the R (F
5
0
or OH) most likely contributes to the selectivity on CDK9, but F atom retains the high
inhibition activity on CDK9 (10ca~10ce). Through observed, compound 9d, 9e, 10ae,
1
0ce, 10dd, 10de (Ar = phenyl, n = 4 or 5) indicate more potent efficacy selective
activity on CDK9 than other same series compounds (n = 1~3). Therefore, it was
noteworthy that the selectivity on CDK9 might be also correlated with the length of
carbon linker and seven carbon atoms (n = 5) was optimal.
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Five compounds 21a~21e (Table 1) were thus made to further confirm the result
for the selectivity on CDK9. The results also showed the compound 21e (Table 3,
IC : 11 nM, n = 5) with 8-isothiocyanatooctanamide linker exhibited better
5
0
selectivity than compound 21a~21d (n = 1~4) and it is a potent and selective CDK9
inhibitor. As we designed, a more flexible and bigger group might be required at the
position of the linker resulting in potent selectivity against CDK9. Then the position
of the linker moving from 4- to 3- or 2- on the phenyl to obtain the compounds 21f or
2
1g (Table 2) based on the structure of the compound 21e. The enzymatic activity of
1f (Table 3, IC : 15 nM,) was no evident change compared with 21e, but all the
2
5
0
three kinases activity of 21g were lost, maybe because the bulky linker on the
-position of the phenyl was not tolerated in the binding site. Addition of the
2
methylene between the amide and phenyl group (22) decreased significantly the
selectivity on CDK9 (Table 2). Directly adding an 8-isothiocyanatooctanamide linker
to the ribociclib (31) maintained the similar activity in the comparison of ribociclib
and lost the selectivity on CDK9. In order to obtain additional SAR information,
fluorine atom or trifluoromethyl was introduced to the phenyl on the basis of
compound 21e to create the compound 32a~32c (Table 2). Compound 32a (Table 3,
IC : 25 nM) exhibited a similar inhibitory potency and selectivity to that of 10ce in
5
0
the enzymatic assays due to their similarity on structure with 10ce. While changing
the position of the fluorine atom to the 3-position, 32b, showed a decreased inhibitory
efficiency and selectivity against CDK9. Further replacement of trifluoromethyl (32c)
1
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compared to 32a, however, resulted in a significant decrease in activity on the three
kinases. Besides the linkers mentioned above, we made another compound that the Ar
in the linker was excluded (33), which lost its activity in the kinase-inhibitory assays.
In addition, we tried to change the core structure of pyrrole to triazole (20), only
showed moderate potency against CDK9 (Table 2). So we still focus on the core
structure of pyrimidine in this study. In all, we selected some compounds with high
potent activity against CDK9 as well as high selectivity for CDK9 to detect the cell
inhibition.
2
.4. Cellular Assay.
Since our goal here is to discover CDK9 inhibitors with high potency and
selectivity for NSCLC treatment, we screened compounds exhibiting better kinase
inhibitory to do the preliminary cell inhibition assays. Compounds except 10ba-10be,
1
0dd, 10de, 20, 21g, 31, 32b, 32c and 33 were chosen to test their cellular cytotoxic
activity (Table S2 and S3). NSCLC cell line A549 and H1299 inhibition profiling
assay with fixed concentrations of 10 µM and 1 µM was first carried out. We found
that compound 10ae and 21e had significant NSCLC cell inhibitory activity. We
further detected the selectivity of the two compounds against other cancer cells,
including breast cancer cell (MCF7), hepatocarcinoma cell (HepG2) and cervical
cancer cell (SiHa) (Table S4). The selectivity for NSCLC of 21e against other cancer
cell lines is higher than that of 10ae. Among these compounds, 21e, bearing carbon
atom at X position and hydrogen atom at R1 position with linker as
1
2

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8
-isothiocyanatooctanamide (Table 1), showed the high potency and selectivity for
NSCLC cell line against other cancer cells. Therefore, 21e was then carried out
further in in-depth in vitro and in vivo anti-NSCLC studies.
The anti-viability activities of 21e was tested against a panel of 14 tumor cell
lines from different origins including NSCLC, breast cancer, hepatocarcinoma,
cervical cancer, leukemia and lymphoma using CCK8 assay. Compound ribociclib
and SFN were used as positive controls. As denoted in Table 4, compound 21e
displayed exceptional potency against NSCLC cell lines, especial A549 and H1299
with IC values less than 0.5 µM. In the drug-resistant NSCLC cell line H1975,
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0
compound 21e also exhibited good inhibition potency with an IC value of 0.837 µM.
5
0
In HCC827 and PC-9 cell lines, compound 21e displayed higher inhibition potency
than two positive controls. In other cell lines, compound 21e exhibited weak or no
inhibition potency. These results demonstrated the good cellular selectivity of
compound 21e. Based on the high inhibitory activity, two typical NSCLC cell lines
A549 and H1299 were selected for further comparative studies.
2
.5. Kinase Selectivity Profile of Compound 21e.
To assess the kinase inhibitory activity and selectivity, the promising compound 21e
was investigated against a series of 381 kinases by the Eurofins kinases profiling at a
single concentration of 1 µM (Figure 2 and Table S5). Compound 21e showed poor
inhibition at the concentration against most of the kinases. A panel of CDKs family
1
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kinases is listed in Table 5. Interestingly, our compound 21e effectively inhibited the
activities of CDK9/cyclinT with IC value of 11 nM over other CDKs (such as IC
5
0
50
of CDK4/cyclinD at 148 nM and CDK6/cyclinD at 145 nM), indicating that
compound 21e is a potent CDK9 inhibitor. While CDK4/6 selective inhibitor
ribociclib was detected with IC values of 13 nM, 71 nM and 197 nM against CDK4,
5
0
CDK6, and CDK9 respectively. Based on the kinase-activity results, further IC50
values of selected kinases with high inhibitory rate over 95% at 1µM were examined
and also shown in Table 5. IC values of three human protein kinases were less than
5
0
5
0 nM, all of which are linked with malignant tumor and autoimmune diseases.
2
.6. Molecular Modeling of Compound 21e.
Predicted binding modes of compound 21e in CDK9 (PDB code: 4BCF) and CDK6
(PDB code: 4EZ5) were carried out by Discovery Studio 3.1. Hydrogen atoms were
added by Gold (version 5.0). The results of docking were created to image edited by
PyMOL.
The compound 21e is a potent and selective CDK9 inhibitor with IC value of 11
5
0
nM while ribociclib with CDK9 IC value of 197 nM. For comparison, the binding
5
0
mode of 21e was superimposed on that of ribociclib. As shown in Figure 3A,
compound 21e binds to the ATP-binding sites of CDK9 in similar orientation to that
of ribociclib. In both two binding models, each aminopyrimidine of two compounds
forms two key hydrogen bonds to the backbone CYS106 residue. Importantly, an
1
4

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additional hydrogen bond exists between the isothiocyanate group of the compound
2
1e and HIS108 residue. This closer binding may explain the better binding affinity of
compound 21e to CDK9, about 15-fold more than ribociclib.
Since there is no 3D structure of CDK4-ligand complex reported at present, we
docked compound 21e into the CDK6 to attempt to account for the reason of the
selectivity of CDK9 among CDKs family. As shown in Figure 3B, 21e adopts a
similar binding mode within the CDK9 and CDK6 ATP binding sites and occupy the
similar positions. In both CDK9 and CDK6, 21e hydrogen-bonds with the kinase
hinge regions. The 2-aminopyrimidine formed two hydrogen bonds with the CYS106
residue in CDK9 (VAL101 in CDK6). At the back of the ATP binding site the
pyrrolo-[2,3-d]-pyrimidine group exploits the hydrophobic region close to the
gatekeeper residue PHE103 to form a favorable π-π interaction in CDK9. At the front
of the ATP binding pocket, the greater flexibility of the ATP-binding site of CDK9
enables the large flexible isothiocyanate of 21e, to be well accommodated by CDK9.
In contrast, the predicted binding mode of 21e bound to CDK6 shows that the CDK6
binding pocket is too crowded for 21e (Figure S1). Therefore, CDK9 has a more
flexible ATP binding pocket than CDK6. The differences in the ability of the CDKs to
readily adapt inhibitors offer an explanation for the high potency and selectivity of
2
2
1e toward CDK9.
.7. 21e Significantly Suppressed the Colony-formation Ability of NSCLC Cells.
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To complement the results from short-time treatments, long-term colony formation
assay was then performed to assess the cytoreductive activity of 21e. Colony
formation assay assesses the effect of a therapy on clonogenic survival which is one
of the most important parameter of therapy efficacy in oncology. As shown in Figure
4
A, 21e dose-dependently decreased the formation of colonies in all the two NSCLC
cell lines, while ribociclib and SFN reduced slightly.
2
.8. 21e Induced the Cell Cycle Progress at G2/M Phase of NSCLC Cells.
To understand how 21e inhibits cell proliferation, cell cycle analysis was performed.
The effect of 21e on cell cycle distribution was assessed in the A549 and H1299 cell
lines. The results indicated that 21e dose-dependently blocked the cell cycle at G2/M
phase compared to the vehicle-treated cells (Figure 4B). Of note, 21e at 1 µM was
able to increase the proportion of the cells in the G2/M phase drastically from
approximate 19.0% (DMSO) to 34.4%, and decreased the corresponding fraction of
the cells in the S phase from about 24.5% (DMSO) to 11.2% and basically unchanged
in the G0/G1 phase in H1299 (Figure 4B). Although the G2/M period was only
slightly increased in A549, it is consistent with the results of H1299. Therefore, our
results suggest that 21e inhibits cell proliferation by arresting cells in G2/M phase of
the cell cycle.
2
.9. 21e Induced Caspase-dependent Apoptotic Cell Death of NSCLC Cells.
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To determine whether 21e induces apoptosis cell death, we performed Annexin V-
FITV/PI staining. As shown in Figure 4C, 21e treatment for 48 h induced apoptosis
of A549 cells in a concentration-dependent manner. 21e at 0.25 to 1 µM increased the
apoptotic (Q2 late apoptotic + Q3 early apoptotic) cells by approximately 1.4-1.6-fold,
compared with the control. Similar results were observed in H1299, with about
2
.2-3.4-fold increase.
Bcl2-family members, including anti-apoptotic and pro-apoptotic proteins, are key
players in the regulation of apoptosis.[44] To determine how 21e induces apoptosis,
we next examined whether 21e could alter the expression of anti-apoptotic (Bcl2) and
pro-apoptotic (Bim) proteins in the cells. As shown in Figure 4D, 21e treatment with
different concentration for 24 h markedly downregulated the expression levels of Bcl2
and meanwhile upregulated the Bim level. Furthermore, we observed that 21e
increased the cleavage of caspase 3 (Figure 4D), indicating activation of caspase 3.
Therefore, our results indicated that 21e induced caspase-dependent apoptotic cell
death by upregulating the anti-apoptotic protein Bcl2 and downregulating the
pro-apoptotic protein Bim in NSCLC cells.
2
.10. 21e Suppressed the CDK9 Downstream Signaling Proteins of NSCLC cells.
CDK9 and cyclin T form the P-TEFb complex that was identified as an activity
controlling the early phase of transcription elongation via release of RNAPII from
inherent promoter-proximal pause sites.[45] CDK9 also phosphorylates the
1
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carboxyterminal heptarepeat (consensus sequence Y S P T S P S ) within CTD of the
1
2 3 4 5 6 7
largest subunit of RNAPII. RNAPII is recruited into the preinitiation complex with a
hypophosphorylated CTD, and the CTD is phosphorylated on Ser5-P during initiation
and then on Ser2-P during elongation.[46] The latter work is performed by
CDK9.[47] The level of phosphorylation at Ser2-P increases towards the 3’-ends of
genes, which correlates to binding of termination and RNA processing factors to the
CTD.[48] Then Ser2-P at promoter-proximal sites further helps to release RNAPII
from initiation and early elongation complexes.[49]
Thus, the effects of CDK9 inhibition by 21e in A549 and H1299 cells were
investigated. Firstly, we performed Western blot assay to evaluate the protein levels
of CDK9 inhibition (Figure 5). The abundance of CDK9 enzymes itself was not
affected by compound 21e. 21e slightly suppressed the expression of cyclin T1 in the
two cell lines. To evaluate whether the antiproliferative activity of compound 21e is
caused by inhibition of cellular CDK9, we selected inhibition of Rb phosphorylation
at the CDK-specific sites Ser 807/811 and Ser 780. The results showed that compound
2
1e effectively suppressed phosphorylation of Rb Ser 807/811 and Ser 780 at 0.25
µM, which confirmed that CDK9 is a target of 21e (Figure 5). Furthermore, one
established cellular target of CDK9 is Ser2 within the RNA polymerase CTD which
becomes phosphorylated (Ser2-P) during transcription elongation. After treated with
2
1e, both Ser2-P and Ser5-P were dramatically inhibited compared with DMSO
1
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treatment (Figure 5). Both analyses suggest that CDK9 is the main driver for Ser2-P
and cell proliferation inhibition.
2
.11. 21e Downregulated the Stem Cell Properties of NSCLC Cells.
There are mainly three methods for the identification of CSCs or CSC-like
properties: (1) identifying the side population (SP) in cancer cells, which enriches
CSC-like properties; (2) determining the growth properties of cells in serum-free
+
-
suspension culture;[50] (3) using of CSCs surface markers, such as CD44 CD24 ,
+
+ [51, 52]
CD133, CD44 /EpCAM .
Herein, in A549 and H1299 cells, we validated that
2
1e could decrease the sphere formation and the ratios of SP in a dose-dependent
manner. In sphere formation assay, 21e decreased the number of spheres formed by
A549 and H1299 cells, in comparison with the respective control, while SFN did not
present inhibition at the concentration of 1µM (Figure 6A). Consistent with these
observations, a side population that shows a higher efflux of DNA-binding dye
Hoechst 33342, as determined by flow cytometry, which is enriched with
hematopoietic stem cells (HSCs).[53] To some extent, the tumor cells with high side
population characteristics represents cancer stem cell.[54] We found that 21e reduced
the percentage of the side population in A549 and H1299 cells, as compared to the
cells treated with DMSO (Figure 6B). Furthermore, overexpression of Oct4
(
Octamer-binding transcription factor 4), SOX2 (SRY (sex determining region Y)-box
), Nanog, Klf4 (Kruppel-like factor 4), can induce somatic cells to acquire
pluripotency.[55] These proteins also serve as the CSC markers.[56, 57] We next
2
1
9

ACCEPTED MANUSCRIPT
investigated the effect of 21e on these CSC markers. Western blot analysis confirmed
that the expression level of stemness markers, including SOX2, Oct4, Nanog and
Klf4, was decreased in A549 and H1299 cells (Figure 6C). These results
demonstrated that 21e suppressed the stem cell-like properties of NSCLC cells.
2
.12. Pharmacokinetics of Compound 21e in Liver Microsomes of Multiple
Species.
The metabolic stability study of compound 21e was assessed using human liver
microsomes (HLM), dog liver microsomes (DLM), mouse liver microsomes (MLM),
rat liver microsomes (RLM, Table 6). The terminal half-lives (t ) were moderate in
1
/2
these four kinds of microsomes (ranging from 23.1 min to 40.8 min). In particularly,
compound 21e showed more stable in rat liver microsomes than others (Cl = 0.057).
int
The moderate pharmacokinetics in liver microsomes of compound 21e were
considered acceptable level for a drug development perspective.
2
.13. In Vivo Anti-tumor Activity and Mechanism of Action of 21e.
To evaluate the anti-tumor activity of 21e in vivo, H1299 xenograft model was used.
In this model, H1299 cells were injected subcutaneously into abdomen of each nude
mouse. The mice were randomized into five groups (5 mice per group) when the
3
tumors grew to a size of ~ 120mm . Next, the animals were orally given three doses
-1
-1
(
20, 40, and 80 mg Kg ) of 21e, one dose (80 mg Kg ) of ribociclib (positive control),
or vehicle (control) every day. As shown in Figure 7A, the growth of xenograft
2
0

ACCEPTED MANUSCRIPT
tumors was significantly inhibited by 21e in a dose-dependent manner. Tumor growth
inhibitions of 62.0%, 89.7%, 94.7% were observed in the H1299 model at doses of 20,
-1
4
0, and 80 mg Kg , respectively. In contrast, ribociclib (80 mg/kg) as the positive
control was less potent (59.5%) than 21e at the same dose. Of note, no obvious
toxicity was observed in all the treated groups (Figure 7B). In Figure 7C, an obvious
decrease in tumor size was also observed at the end of observation. The average
-1
tumor weight (Figure 7D) of the 21e-treated group (80 mg Kg ) was 0.18 g which
was less than that of ribociclib-treated group (0.49 g) and control group (2.17 g).
To elucidate the mechanism of anti-tumor action of 21e in vivo,
immunohistochemical analysis was conducted using H1299 tumor samples collected
from mice treated with vehicle, 21e and ribociclib, respectively. As shown in Figure
7
E, 21e remarkably inhibited the phosphorylation of Rb (Ser 780) and RNAPII CTD
(Ser 5), and led to almost unchanged CDK9 and cyclin T1 compared with the vehicle,
consistent with in vitro results. Moreover, 21e reduced the expression of stemness
marker Oct4. Taken together, our results indicated that 21e suppressed H1299
xenograft growth in mice by inhibiting CDK9 signaling and tumor CSCs, leading to
inhibition of cell proliferation and the stem cell properties.
3
. CONCLUSION
In this study, a series of new CDK9 inhibitors bearing isothiocyanate were
designed and synthesized. SAR study of these compounds was discussed based on
2
1

ACCEPTED MANUSCRIPT
enzymatic and cellular activities. In cellular assays, compound 21e exhibited
considerable anti-viability potencies against human NSCLC cell lines and good
selectivity against other cancer cells. It could significantly decrease the number of
colonies, induced NSCLC cell apoptosis, and arrested the cell cycle in the G2 phase.
Meanwhile, 21e significantly decreased cancer stem-like cells fraction, tumor sphere
formation and stemness markers expression of NSCLC cells. In H1299 xenograft
mouse models, 21e displayed potent anti-tumor activities. Immunohistochemistry of
tumor tissues showed that 21e efficiently inhibited CDK9 signaling and CSCs marker.
This work offer a new potential approach based on single target CDK9 to inhibit stem
cell for treatment of NSCLC.
4
4
. EXPERIMENTAL SECTION
.1. General Methods for Chemistry.
The commercially obtained chemicals were used directly without further
purification. Solvents were purified and distilled following the standard procedures.
All the reactions were monitored by thin-layer chromatography (TLC). The NMR
1
spectra were taken on a Bruker AV-400 MHz spectrometer (400 MHz for H and 101
1
3
MHz for C) and chemical shifts were expressed in ppm downfield using
tetramethylsilane as the internal standard. High-resolution mass spectra (HRMS) were
performed on a VG ZAB-HS mass spectrometer under eluectron spray ionization
(ESI). All the derivatives for testing bioactivity were purified to >95% purity which
2
2

ACCEPTED MANUSCRIPT
was determined by HPLC analysis on a Shimadzu Prominence-i LC-2030C 3D
system (column, InertSustain C18, 4.6 mm × 250 mm, 5 µM; mobile phase, gradient
elution of methanol/H O (90:10); low rate, 1.0 mL/min; UV wavelength, 190−800
2
nm; temperapture, 40 °C; injection volume, 10 µL). Melting point were tested on the
Mettler Toledo MP50 Melting Point System that can present results with a decimal
precision.
4
.1.1.
7
-Cyclopentyl-2-((4-(N-(3-isothiocyanatopropyl)sulfamoyl)phenyl)amino)-N,N-dimeth
yl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (9a). To a stirred solution of 7a (382
mg, 0.65mmol) in DCM, trifluoracetic acid (3 mL) was added dropwise in ice-water
bath, then stirred 1 h at RT. After removing solvent in vacuo, the residue was
dissolved in n-butanol (10 mL) and washed with saturated Na CO aquous solution
2
3
(10 mL). The organic layer was concentrated under reduced pressure to use directly
without purification. After dissolving the resulting light-yellow oil in THF (5 mL),
carbon disulphide (800 µL, 20 equiv), DCC (148 mg, 0.72 mmol) were added in
successively, stirred overnight at RT. The solvent was concentrated in vacuo and crude
product was purified by silica gel column chromatography to give title compound 9a
in 57% yield as light-yellow solid; mp 176.3 °C. IR (film): 3234.3, 3151.2, 3122.5,
3
1
032.9, 2995.9, 2950.2, 2873.1, 2194.4, 2163.2, 2067.2, 1604.7, 1588.8, 1567.7,
546.2, 1527.4, 1483.7, 1428.6, 1401.7, 1353.8, 1328.1, 1244.5, 1176.4, 1155.8,
-1 1
1
138.5, 1091.3, 1027.4 cm . H NMR (400 MHz, CDCl ) δ 8.68 (s, 1H, 4-H), 8.06 (s,
3
2
3

ACCEPTED MANUSCRIPT
1
H, NH), 7.89 (d, J = 8.8 Hz, 2H, 3', 5'-H), 7.80 (d, J = 8.8 Hz, 2H, 2', 6'-H), 6.47 (s,
H, 5-H), 5.44 (t, J = 6.3 Hz, 1H, NH), 4.79 (m, 1H, CH), 3.62 (t, J = 6.3 Hz, 2H,
1
CH ), 3.15 (s, 6H, 2CH ), 3.09 (q, J = 6.4 Hz, 2H, CH ), 2.61 – 2.52 (m, 2H, CH ),
2
3
2
2
2
.11 – 2.00 (m, 4H, 2CH ), 1.88 (p, J = 6.5 Hz, 2H, CH ), 1.71 (q, J = 6.1 Hz, 2H,
2 2
13
CH ); C NMR (101 MHz, CDCl ) δ 163.9 (1C, Ar-C), 154.4 (1C, C=O), 151.7 (1C,
2
3
Ar-C), 151.5 (1C, Ar-C), 144.6 (1C, Ar-C), 133.0 (1C, Ar-C), 131.9 (1C, Ar-C), 131.0
1C, S=C=N), 128.5 (2C, Ar-C), 117.8 (2C, Ar-C), 113.3 (1C, Ar-C), 101.0 (1C, Ar-C),
(
5
8.2 (1C, CH), 42.3 (1C, CH ), 40.1 (1C, CH ), 39.5 (2C, 2CH ), 35.3 (1C, CH ),
2 2 3 2
+
3 2
3
0.3 (2C, 2CH ), 24.8 (2C, 2CH ). HRMS (ESI, m/z) calcd for C H N O S [M +
2
2
24 30
7
+
H] , 528.1846, found 528.1851. HPLC purity 97% (t = 3.68 min).
R
4
.1.2.
7
-Cyclopentyl-2-((4-(N-(4-isothiocyanatobutyl)sulfamoyl)phenyl)amino)-N,N-dimethy
l-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (9b). Compound 9b was prepared
using 7b instead of 7a in the similar manner of compound 9a to afford the title
product as light-yellow solid in 65% yield; mp 185.9 °C. IR (film): 3354.2, 3260.1,
3
1
1
108.5, 2948.4, 2867.7, 2180.7, 2095.4, 2065.7, 1908.1, 1732.9, 1626.3, 1605.9,
587.2, 1563.6, 1541.9, 1521.2, 1485.8, 1428.1, 1396.5, 1358.9, 1333.3, 1290.0,
-1 1
248.4, 1186.4, 1158.6, 1135.8, 1090.7, 1074.3, 1025.1 cm . H NMR (400 MHz,
CDCl ) δ 8.67 (s, 1H, 4-H), 8.06 (s, 1H, NH), 7.89 (d, J = 8.5 Hz, 2H, 3', 5'-H), 7.80
3
(d, J = 8.5 Hz, 2H, 2', 6'-H), 6.47 (s, 1H, 5-H), 5.28 (t, J = 6.3 Hz, 1H, NH), 4.78 (m,
1
H, CH), 3.49 (t, J = 6.3 Hz, 2H, CH ), 3.15 (s, 6H, 2CH ), 3.00 (q, J = 6.5 Hz, 2H),
2
3
2
4

ACCEPTED MANUSCRIPT
2
.57 (dd, J = 13.0, 7.4 Hz, 2H, CH ), 2.05 (dd, J = 19.4, 8.0 Hz, 4H, 2CH ), 1.71 (d, J
2
2
1
3
=
8.3 Hz, 4H, 2CH ), 1.65 – 1.57 (m, 2H, CH ); C NMR (101 MHz, CDCl ) δ 163.9
2 2 3
(1C, Ar-C), 154.5 (1C, C=O), 151.7 (1C, Ar-C), 151.5 (1C, Ar-C), 144.6 (1C, Ar-C),
1
33.0 (1C, Ar-C), 132.1 (1C, S=C=N), 131.2 (1C, Ar-C), 128.4 (2C, Ar-C), 117.8 (2C,
Ar-C), 113.3 (1C, Ar-C), 101.0 (1C, Ar-C), 58.2 (1C, CH), 44.7 (1C, CH ), 42.4 (1C,
2
CH ), 39.5 (2C, 2CH ), 30.3 (2C, 2CH ), 27.1 (1C, CH ), 26.8 (1C, CH ), 24.8 (2C,
2
3
2
2
2
+
+
2
CH ). HRMS (ESI, m/z) calcd for C H N O S [M + H] , 542.2003, found
2 25 32 7 3 2
5
42.2007. HPLC purity 99% (t = 3.79 min).
R
4
.1.3.
7
-Cyclopentyl-2-((4-(N-(5-isothiocyanatopentyl)sulfamoyl)phenyl)amino)-N,N-dimeth
yl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (9c). Compound 9c was prepared
using 7c instead of 7a in the similar manner of compound 9a to afford the title
product as light-yellow solid in 63% yield; mp 77.7 °C. IR (film): 3270.8, 3182.4,
3
109.0, 2941.6, 2866.6, 2178.3, 2099.6, 2000.1, 1624.1, 1606.4, 1587.1, 1563.3,
-1
1
519.9, 1486.8, 1427.4, 1398.2, 1355.7, 1322.5, 1248.0, 1138.6, 1092.0, 1024.2 cm .
1
H NMR (400 MHz, CDCl ) δ 8.67 (s, 1H, 4-H), 8.04 (s, 1H, NH), 7.89 (d, J = 8.7 Hz,
3
2
H, 3', 5'-H), 7.81 (d, J = 8.7 Hz, 2H, 2', 6'-H), 6.47 (s, 1H, 5-H), 5.12 (t, J = 6.2 Hz,
H, NH), 4.77 (m, 1H, CH), 3.45 (t, J = 6.5 Hz, 2H, CH ), 3.15 (s, 6H, 2CH ), 2.97 (q,
1
2
3
J = 6.7 Hz, 2H, CH ), 2.63 – 2.52 (m, 2H, CH ), 2.11 – 2.00 (m, 4H, 2CH ), 1.70 (d, J
2
2
2
=
6.3 Hz, 2H, CH ), 1.66 – 1.59 (m, 2H, CH ), 1.51 (q, J = 7.2 Hz, 2H, CH ), 1.40 (dq,
2 2 2
1
3
J = 8.9, 3.6 Hz, 2H, CH ); C NMR (101 MHz, CDCl ) δ 163.9 (1C, Ar-C), 154.5
2
3
2
5

ACCEPTED MANUSCRIPT
(1C, C=O), 151.7 (1C, Ar-C), 151.5 (1C, Ar-C), 144.5 (1C, Ar-C), 133.0 (1C, Ar-C),
1
31.4 (1C, Ar-C), 130.0 (1C, S=C=N), 128.4 (2C, Ar-C), 117.7 (2C, Ar-C), 113.3 (1C,
Ar-C), 101.0 (1C, Ar-C), 58.2 (1C, CH), 45.0 (1C, CH ), 42.9 (1C, CH ), 39.5 (2C,
2
2
2
CH ), 30.3 (2C, 2CH ), 29.5 (1C, CH ), 29.0 (1C, CH ), 24.8 (2C, 2CH ), 23.7 (1C,
3 2 2 2 2
+
3 2
+
CH ). HRMS (ESI, m/z) calcd for C H N O S [M + H] , 556.2159, found
2
26 34
7
5
56.2155. HPLC purity 98% (t = 3.93 min).
R
4
.1.4.
7
-Cyclopentyl-2-((4-(N-(6-isothiocyanatohexyl)sulfamoyl)phenyl)amino)-N,N-dimethy
l-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (9d). Compound 9d was prepared
using 7d instead of 7a in the similar manner of compound 9a to afford the title
product as light-yellow solid in 82% yield; mp 169.5 °C. IR (film): 3627.0, 3486.6,
3
1
234.0, 3170.1, 3081.1, 3033.1, 2995.6, 2936.7, 2860.3, 2177.2, 2092.5, 1607.3,
591.7, 1569.3, 1556.1, 1530.7, 1484.4, 1427.4, 1402.1, 1356.5, 1328.3, 1244.0,
-1 1
1
158.5, 1138.9, 1093.1, 1028.6 cm . H NMR (400 MHz, CDCl ) δ 8.67 (s, 1H, 4-H),
3
8
6
2
.00 (s, 1H, NH), 7.89 (d, J = 8.7 Hz, 2H, 3', 5'-H), 7.80 (d, J = 8.6 Hz, 2H, 2', 6'-H),
.46 (s, 1H, 5-H), 5.02 (t, J = 6.3 Hz, 1H, NH), 4.79 (m, 1H, CH), 3.45 (t, J = 6.5 Hz,
H, CH ), 3.15 (s, 6H, 2CH ), 2.95 (q, J = 6.7 Hz, 2H, CH ), 2.62 – 2.52 (m, 2H,
2
3
2
CH ), 2.13 – 2.01 (m, 4H, 2CH ), 1.71 (p, J = 6.6, 6.1 Hz, 2H, CH ), 1.62 (t, J = 7.0
2
2
2
13
Hz, 2H, CH ), 1.52 – 1.45 (m, 2H, CH ), 1.32 (dt, J = 9.3, 5.5 Hz, 4H, 2CH ); C
2
2
2
NMR (101 MHz, CDCl ) δ 164.0 (1C, Ar-C), 154.5 (1C, C=O), 151.8 (1C, Ar-C),
3
1
51.5 (1C, Ar-C), 144.4 (1C, Ar-C), 133.0 (1C, Ar-C), 131.5 (1C, Ar-C), 129.9 (1C,
2
6

ACCEPTED MANUSCRIPT
S=C=N), 128.4 (2C, Ar-C), 117.7 (2C, Ar-C), 113.3 (1C, Ar-C), 101.0 (1C, Ar-C),
5
8.2 (1C, CH), 45.0 (1C, CH ), 43.1 (2C, 2CH ), 34.0 (1C, CH ), 30.3 (2C, 2CH ),
2 3 2 2
2
9.8 (1C, CH ), 29.5 (1C, CH ), 26.2 (1C, CH ), 25.9 (1C, CH ), 24.8 (2C, 2CH ).
2
2
2
2
2
+
+
HRMS (ESI, m/z) calcd for C H N O S [M + H] , 570.2316, found 570.2314.
27
36
7
3 2
HPLC purity 99% (t = 4.11 min).
R
4
.1.5.
7
-Cyclopentyl-2-((4-(N-(7-isothiocyanatoheptyl)sulfamoyl)phenyl)amino)-N,N-dimeth
yl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (9e). Compound 9e was prepared
using 7e instead of 7a in the similar manner of compound 9a to afford the title
product as light-yellow solid in 89% yield; mp 186.1 °C. IR (film): 3378.7, 3136.3,
3
1
1
085.7, 2941.2, 2908.7, 2862.0, 2176.7, 2139.2, 2114.1, 1735.7, 1625.1, 1603.6,
583.4, 1561.5, 1546.4, 1506.9, 1477.8, 1462.6, 1420.3, 1400.5, 1344.8, 1312.1,
-1
1
271.1, 1247.3, 1181.5, 1151.0, 1138.6, 1112.0, 1091.6, 1079.2, 1059.3 cm . H
NMR (400 MHz, CDCl ) δ 8.68 (s, 1H, 4-H), 7.95 (s, 1H, NH), 7.89 (d, J = 8.6 Hz,
3
2
H, 3', 5'-H), 7.81 (d, J = 8.5 Hz, 2H, 2', 6'-H), 6.46 (s, 1H, 5-H), 4.90 (t, J = 6.2 Hz,
H, NH), 4.77 (m, 1H, CH), 3.44 (t, J = 6.6 Hz, 2H, CH ), 3.15 (s, 6H, 2CH ), 2.95 (q,
1
2
3
J = 6.8 Hz, 2H, CH ), 2.58 (qd, J = 9.1, 6.3, 4.0 Hz, 2H, CH ), 2.12 – 2.03 (m, 4H,
2
2
2
CH ), 1.71 (q, J = 6.1 Hz, 2H, CH ), 1.63 (d, J = 7.2 Hz, 2H, CH ), 1.47 (t, J = 7.1
2 2 2
1
3
Hz, 2H, CH ), 1.35 – 1.24 (m, 6H, 3CH ); C NMR (101 MHz, CDCl ) δ 163.9 (1C,
2
2
3
Ar-C), 154.6 (1C, C=O), 151.8 (1C, Ar-C), 151.5 (1C, Ar-C), 144.4 (1C, Ar-C), 133.0
1C, Ar-C), 131.6 (1C, Ar-C), 129.8 (1C, S=C=N), 128.5 (2C, Ar-C), 117.7 (2C,
(
2
7

ACCEPTED MANUSCRIPT
Ar-C), 113.3 (1C, Ar-C), 101.0 (1C, Ar-C), 58.2 (1C, CH), 45.1 (1C, CH ), 43.2 (1C,
2
CH ), 39.5 (2C, 2CH ), 30.3 (2C, 2CH ), 29.9 (1C, CH ), 29.6 (1C, CH ), 28.4 (1C,
2
3
2
2
2
CH ), 26.5 (1C, CH ), 26.4 (1C, CH ), 24.8 (2C, 2CH ). HRMS (ESI, m/z) calcd for
2
2
2
2
+
+
C H N O S [M + H] , 584.2472, found 584.2467. HPLC purity 98% (t = 4.41
28
38
7
3
2
R
min).
4
.1.6.
7
-Cyclopentyl-2-((4-((3-isothiocyanatopropyl)carbamoyl)phenyl)amino)-N,N-dimethy
l-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (10aa). Compound 10aa was prepared
using 8aa instead of 7a in the similar manner of compound 9a to afford the title
product as white solid in 33% yield; mp 205.6 °C. IR (film): 3355.2, 3303.7, 13180.6,
3
1
1
107.8, 13068.2, 6042.1, 3015.4, 2950.4, 2922.6, 12871.7, 2177.7, 2108.5, 2082.7,
731.0, 1623.8, 1611.5, 589.6, 1549.2, 1519.7, 1502.8, 1483.8, 1428.2, 1403.6,
-1
1
337.0, 1301.8, 1285.0, 1246.1, 1186.8, 1159.1, 1139.5, 1045.2, 1024.6 cm . H
NMR (400 MHz, DMSO-d ) δ 9.85 (s, 1H, NH), 8.79 (s, 1H, 4-H), 8.41 (t, J = 5.6 Hz,
6
1
H, NH), 7.92 (d, J = 8.4 Hz, 2H, 3', 5'-H), 7.82 (d, J = 8.5 Hz, 2H, 2', 6'-H), 6.61 (s,
1
H, 5-H), 4.76 (p, J = 8.9 Hz, 1H, CH), 3.74 (t, J = 6.4 Hz, 2H, CH ), 3.35 (q, J = 6.3
2
Hz, 2H, CH ), 3.06 (s, 6H, 2CH ), 2.49 – 2.43 (m, 2H, CH ), 2.10 – 1.98 (m, 4H,
2
3
2
1
3
2
CH ), 1.89 (p, J = 6.6 Hz, 2H, CH ), 1.69 (q, J = 6.0 Hz, 2H, CH ); C NMR (101
2 2 2
MHz, DMSO-d6) δ 166.0 (1C, C=O), 162.8 (1C, Ar-C), 155.0 (1C, C=O), 152.1 (1C,
Ar-C), 151.0 (1C, Ar-C), 143.7 (1C, Ar-C), 132.1 (1C, Ar-C), 127.9 (2C, Ar-C), 127.2
(1C, S=C=N), 126.1 (1C, Ar-C), 116.8 (2C, Ar-C), 112.0 (1C, Ar-C), 100.6 (1C, Ar-C),
2
8