Novel compounds with potent CDK9 inhibitory activity for the treatment of myeloma

Article abstract of DOI:10.1016/j.bmcl.2018.01.002

Cyclin-dependent kinases (CDKs) and Polo-like kinases (PLKs) play key role in the regulation of the cell cycle. The aim of our study was originally the further development of our recently discovered polo-like kinase 1 (PLK1) inhibitors. A series of new 2,4-disubstituted pyrimidine derivatives were synthesized around the original hit, but their PLK1 inhibitory activity was very poor. However the novel compounds showed nanomolar CDK9 inhibitory activity and very good antiproliferative effect on multiple myeloma cell lines (RPMI-8226).

Full text of DOI:10.1016/j.bmcl.2018.01.002

Accepted Manuscript
Novel compounds with potent CDK9 inhibitory activity for the treatment of
myeloma
Zsófia Czudor, Mária Balogh, Péter Bánhegyi, Sándor Boros, Nóra Breza, Judit
Dobos, Márk Fábián, Zoltán Horváth, Eszter Illyés, Csaba Szántai-Kis, Péter
Markó, Anna Sipos, Bálint Szokol, László Őrfi
PII:
S0960-894X(18)30002-7
https://doi.org/10.1016/j.bmcl.2018.01.002
BMCL 25528
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 July 2017
14 December 2017
1 January 2018
Please cite this article as: Czudor, Z., Balogh, M., Bánhegyi, P., Boros, S., Breza, N., Dobos, J., Fábián, M.,
Horváth, Z., Illyés, E., Szántai-Kis, C., Markó, P., Sipos, A., Szokol, B., Őrfi, L., Novel compounds with potent
CDK9 inhibitory activity for the treatment of myeloma, Bioorganic & Medicinal Chemistry Letters (2018), doi:
https://doi.org/10.1016/j.bmcl.2018.01.002
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Novel compounds with potent CDK9
inhibitory activity for the treatment of
myeloma
Leave this area blank for abstract info.
OMe
N
H
N
F
H
R1
R2
R3
R2
R3
R1
N
N
F
N
S
Cl
N
Cl
N
O
O
H
N
R8
R7
S
54 CDK9 IC50: 0.4 nM
N
Cl
N
N
R4
R4
N
N
R5
OMe
R6
H
N
N
N
N
N
66 RPMI-8226 IC50: 1.34 uM

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Novel compounds with potent CDK9 inhibitory activity for the treatment of
myeloma
Zsófia Czudora, Mária Baloghb, Péter Bánhegyib, Sándor Borosb, Nóra Brezab, Judit Dobosb, Márk Fábiána,
Zoltán Horváthb, Eszter Illyésb, Csaba Szántai-Kisb, Péter Markób, Anna Siposb, Bálint Szokolb, László
Őrfia, b∗
a Department of Pharmaceutical Chemistry, Semmelweis University, Hőgyes Endre u. 9. 1092, Budapest, Hungary
b Vichem Chemie Research Ltd., Herman Ottó u. 15., 1022, Budapest, Hungary
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Cyclin-dependent kinases (CDKs) and Polo-like kinases (PLKs) play key role in the regulation
of the cell cycle. The aim of our study was originally the further development of our recently
discovered polo-like kinase 1 (PLK1) inhibitors. A series of new 2,4-disubstituted pyrimidine
derivatives were synthesized around the original hit, but their PLK1 inhibitory activity was very
poor. However the novel compounds showed nanomolar CDK9 inhibitory activity and very
good antiproliferative effect on multiple myeloma cell lines (RPMI-8226).
Keywords:
2017 Elsevier Ltd. All rights reserved
.
Cyclin Dependent Kinase inhibitor
2,4-disubstituted pyrimidine
Multiple myeloma
Polo-like Kinase
———
∗ Corresponding author. Tel.: +3614591500/53030; fax: +3612170891; e-mail: orfi.laszlo@pharma.semmelweis-univ.hu

The improper operation of the cell cycle may result in the
appearance of cancer cells; therefore, overexpression or
amplification of cell cycle regulator kinases (CDKs, PLKs,
CHKs, Aurora kinases) play pivotal role in the formation and
progression of different tumors. Cyclin-dependent kinases
(CDKs) and Polo-like kinases (PLKs), the focus of our
research, play an essential role in the regulation of the cell
cycle.1, 2
F
O
N
H
N
H
N
N
S
O
O
O
N
Example 18
Figure 1. Best hit of the patented PLK1 inhibitor series (PLK1 IC50
0.040 µM).20
:
In addition to the regulation of this process, transcription is
significantly affected by CDKs.1 These protein kinases belong
to the serine/threonine kinases and their activity depends on
cyclin which is a regular subunit.3 These kinases are activated
by the phosphorylation of a threonine residue.4 CDKs can be
divided into subfamilies based on sequence similarity. One of
them is the cell cycle related sub-group (CDK1-CDK4 and
CDK6), the other is the transcriptional regulator sub-group
(CDK3, CDK7, CDK8, CDK9, CDK10) and there are several
CDKs with non-cell cycle function (CDK11-CDK20). CDK9
is involved in the regulation of transcription.1, 5, 6 Through the
hexamethylene bisacetamide-inducible protein 1 (HEXIM1),
CDK9 can be associated with the development of HIV.7
Furthermore, inhibition of CDK9 might result in the
destruction of cancer cells. This is due to CDK9 being
responsible for the synthesis of Mcl-1 and XIAP antiapoptotic
proteins that maintain necessary conditions for cancer cells to
survive8, 9, e.g. in chronic lymphocytic leukemia or in breast
cancer.10, 11 Thus inhibitors of CDK9 could be utilized either in
the treatment of HIV or cancer.
To keep at the sulfisoxazole moiety, several intermediates
of 1-20 were reacted with sulfisoxazole, yielding compounds
21, 22, 25, 26, 29, 30, 32, 33, 34, 37 (Table 1). These
derivatives were found to be less potent inhibitors of PLK1
kinase than 4,6-pyrimidine analogs of the PLK1 Vichem20,
with the best molecule (cpd. 26) reaching 75% inhibitory
activity (10 µM) in PLK1 biochemical assay (data shown in
Table 1).
Table 1. Compounds tested in PLK1 biochemical assay
Cpd
R1
R2
R3
R4
PLK1 inhibition (%)a
21
22
25
26
29
30
32
33
34
37
MeO
MeO
MeO
MeO
MeO
EtO
EtO
iPrO
H
H
F
H
H
H
H
H
F
56
35
37
75
34
11
5
F
F
H
F
MeO
F
H
H
H
H
H
F
H
Cl
F
H
H
13
65
55
PLKs are also classified as serine/threonine kinases and this
kinase family has an important function in the cell
proliferation.2, 12 Based on the structural homology, four groups
are generally distinguished: PLK1, PLK2 (or Snk), PLK3 (or
Fnk) and PLK4 (or Sak). Each of these enzymes regulate the
M-phase of mitosis in cell proliferation.13,14 The
overexpression or amplification of PLK1 can also lead to
malignant processes. Some studies show correlation between
several gastrointestinal cancer types and overexpressed PLK1.
Inter alia it might be the cause of esophageal squamous cell
carcinoma or colon cancer.15, 16 In addition, this enzyme may
affect the development of acute myeloid leukemia (AML).17
High prevalence of these types of cancer has been observed in
OECD countries, fatal outcomes of the disease can be
measured in tens of thousands.18
H
iPrO
F
F
H
H
aCompound concentration was 10 µM
Because these molecules did not prove to be particularly
efficient PLK1 inhibitors, but the compounds were novel, we
sought to identify other kinase target. CDK9 enzyme was
chosen due to the fact that several potent 4,6-pyrimidine-based
CDK9 inhibitors are well known already, for instance, the
structure shown in Figure 2.21 We hypothesized that in this
case it could be meaningful to investigate the role of the
positions of the nitrogen atoms in the pyrimidine ring to
explore the structure–activity relationship.
O
Due to the significant biological role of CDK9 and PLK1
they are promising therapeutic targets therefore numerous
inhibitors have been developed against these kinases.
Dinaciclib is one of the most known CDK9 inhibitors in Phase
II Clinical trials, . Dinaciclib was used as standard in our
biological evaluations (CDK9 IC50: 4 nM). It can be used
against triple negative breast cancer.11, 19 Volasertib, a potent
PLK inhibitor used against AML, is in Phase II clinical
trialalso.17
H
N
O
P
N
N
O
Example 52a
Figure 2. Example of a potential CDK9 inhibitor (G. Nemeth et al.
CDK9 IC50: 197 nM).21
Our 2,4-pyrimidine derivatives (21, 22, 25, 26, 29, 30, 32,
33, 34, and 37) were tested in biochemical assay and it was
concluded that these novel molecules are effective CDK9
enzyme inhibitors; therefore, further analogs were prepared
according to the scheme shown in Figure 3-4 (biological data
is listed in Table 2-3) and the structure-activity relationship
(SAR) was examined.
Inhibitors of the kinases mentioned above had already been
investigated by our research group demonstrating very
favorable behavior in terms of inhibition potency of these
enzymes. 20, 21
Example 18, shown in Figure 1, has exceptional PLK1
enzyme inhibition potency (IC50: 0.040 µM).20 In the current
study our goal was to explore the relationship between the
position of nitrogen atoms on this pyrimidine ring and potency
was of particular interest. Novel compounds with 2,4-
pyirimidine core structures were synthesized, the synthesis
scheme of the associated intermediates is shown in Figure 3-4.
Altogether fifty-one compounds with 2,4-pyrimidine core
structure were prepared. Their structure and inhibition activity
of CDK9 enzyme are shown in Table 2-3.

R2
R3
R1
N
Cl
Cl
Table 2. The synthesized 2,4-pyrimidines (cpd 21-57) with inhibition
a
activity of CDK9 enzyme.
Cl
N
N
R4
N
CDK9
Cpd
R1
R2
R3
R4
Ar
1-20
IC50 (nM)a
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
H
F
H
H
F
H
H
H
F
3,4-dimethyl-isoxazole
3,4-dimethyl-isoxazole
3,4-dimethyl-isoxazole
3,4-dimethyl-isoxazole
3,4-dimethyl-isoxazole
3,4-dimethyl-isoxazole
3,4-dimethyl-isoxazole
3,4-dimethyl-isoxazole
3,4-dimethyl-isoxazole
3,4-dimethyl-isoxazole
3,4-dimethyl-isoxazole
3,4-dimethyl-isoxazole
3,4-dimethyl-isoxazole
3,4-dimethyl-isoxazole
3,4-dimethyl-isoxazole
3,4-dimethyl-isoxazole
3,4-dimethyl-isoxazole
3,4-dimethyl-isoxazole
3,4-dimethyl-isoxazole
3,4-dimethyl-isoxazole
4,5-dimethyl-isoxazole
4,5-dimethyl-isoxazole
benzo[d]isoxazole
3
2
c
b
2
R
R1
H
H
F
9
1
R
R2
R3
H
N
H
F
1
N
H
N
8
R
3
N
R
H
N
4
H
F
8
R
N
4
R
S
Ar
N
5
R
R7
H
Cl
H
F
1
O
O
R6
21-57
H
H
H
H
H
H
H
iPrO
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
Cl
H
H
F
12
25
9
58-69
Figure 3. Synthesis of novel 2,4-pyrimidine derivatives. a: substituted
phenyl boronic acid, Pd(PPh3)4, Na2CO3, H2O, DME, inert atmosphere, 4
h, 120 °C, b: corresponding aniline23, hydrogen chloride solution 4.0 M in
dioxane, tert-butanol, 5 h, 130 °C, microwave, c: corresponding aniline24-
26, hydrogen chloride solution 4.0 M in dioxane, tert-butanol, 5 h, 130 °C,
microwave.
MeO MeO
EtO
EtO
EtO
iPrO
H
F
H
Cl
F
8
2
H
H
F
17
49
458
13
6
H
F
MeS
Me
H
H
H
F
F
F
O
F
F
11
21
55
50
4
NH2
H
N
F
O
N
F
MeO
F
a
b
O
N
Cl
O
N
+
Cl
H
H
H
H
H
H
H
F
O
N
O
Cl
Cl
H
F
70
2
MeO
MeO
MeO
MeO
MeO
MeO
EtO
EtO
MeO
MeO
MeO
MeO
MeO
MeO
O
F
F
O
3
H
N
H
N
N
c
N
d
F
5
O
N
O
N
H
F
thiazole
33
122
2
OH
71
Cl
thiazole
72
H
F
thiazole
O
F
H
N
H
H
H
H
H
F
thiazole
210
378
12
6
N
Cl
H
F
thiazole
O
N
2-methyl-[1,3,4]thiadiazole
2-methyl-[1,3,4]thiadiazole
2-methyl-[1,3,4]thiadiazole
2-methyl-[1,3,4]thiadiazole
2-methyl-[1,3,4]thiadiazole
2-methyl-[1,3,4]thiadiazole
2-methyl-[1,3,4]thiadiazole
2-methyl-[1,3,4]thiadiazole
2-methyl-[1,3,4]thiadiazole
HN
73
O
N
H
H
F
9
H
F
2
Figure 4. Synthesis of novel 2,4-pyrimidine derivatives. a: cesium
H
F
10
0.4
14
2
carbonate, palladium acetate, BINAP, dioxane, 16 h, 90 °C, b: NaOH,
THF, EtOH, H2O, 4 h, 50 °C, c: thionyl chloride, 3 h, RT, d: 5-Amino-3,4-
dimethylisoxazole, pyridine, 4 h, reflux.
H
F
MeO MeO
H
H
H
H
H
H
EtO
F
Twenty intermediates (cpd 1-20) were coupled with
sulfisoxazole, yielding compounds 21-40. Several of these
structures (cpds 21-26, 29-31, 35, 36) had an IC50 value below
10 nM as shown in Table 2. Seven of these compounds had a
methoxy group in R1-position and were hydrogen- or fluorine-
substituted in positions R2, R3 and R4. It was found that the
number of fluorine atoms and their position on the ring had no
significant effect on the IC50 value. Compound 29 had a
methoxy substituent in both positions R1 and R2 demonstrating
the same potency as the former seven inhibitors.
After analysis of the effect of the R1-methoxy group, R1-
ethoxy and R1-isopropoxy analogs were synthesized. Two of
the most potent compounds (22 and 24) were tested via
replacement of the methoxy group to ethoxy and isopropoxy
groups. In position R1 ethoxy substitution (30 and 31)
influenced inhibition inconsiderably; on the other hand,
substitution by isopropoxy moiety (33 and 34) reduced IC50
significantly.
EtO
Cl
5
aDinaciclib as reference compound, IC50: 4 nM
Table 3. The synthesized 2,4-pyrimidines (cpd 58-69) with inhibition
activity of CDK9 enzyme.
CDK9
Cpd
R1
R2
R3
R4
R5
R6
R7
R8
IC50 (nM)a
1576
58
59
60
61
62
63
64
65
66
67
68
69
MeO
MeO
MeO
MeO
EtO
H
F
H
H
H
F
H
H
F
F
F
H
H
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
H
*
*
> 12500
> 12500
> 12500
> 12500
> 12500
> 12500
> 12500
3
H
F
F
H
*
H
H
H
H
H
H
H
F
F
H
*
F
H
H
H
H
H
H
H
H
F
H
*
EtO
Cl
F
F
H
*
iPrO
F
F
H
*
F
F
H
*
MeO
MeO
MeO
MeO
H
F
H
H
H
H
**
**
**
***
H
H
H
H
H
4
H
H
2
F
H
H
> 12500
aDinaciclib as reference compound, IC50: 4 nM
*carboxamide
**1H-benzimidazole-1-yl-methyl
***3,5-dimethyl-isoxazole

The effect of the introduction of other halogens on the
benzyl ring on the IC50 values was also investigated.
Substitution by chlorine decreased the potency by an order of
magnitude (cpds 27, 28, 32).
affects CDK9 enzyme inhibition potency. Analogs of cpds 21,
22 and 24 were synthesized and as Table 3 shows, each of the
three compounds (66-68) showed a remarkably decreased IC50
value.
Compound 36, a sulfisoxazole-linked inhibitor bearing a
methyl group in position R1 (R2-F, R3-H and R4-H), showed
low nanomolar IC50 value; however, replacement of the
methoxy, ethoxy or methyl substituent of position R1 with
other small-size groups such as methylthio groups or halogens
decreased CDK9 enzyme inhibition (cpds 35 and 37-40).
Surprisingly, compound 38 showed far lower potency
compared to cpds 25 or 26, whereas the substituents on the
ring were the same differing only in their order. This fact
supports the importance of R1-methoxy, -ethoxy or –methyl
group.
Building on the SAR above, our goal was to understand the
role of the sulfonamide linker region; therefore, cpds 69 and 73
were synthesized. The structure of cpd 69 lacks the
sulfonamide or acid amide group between isoxazole and the
benzene ring. This molecule was not capable to inhibit the
activation of CDK9 enzyme. Cpd 73 is the analog of cpd 22,
differing in the carboxyl motif only which was changed from
sulfonamide to amide group. Comparison of the inhibition
potency of these two structures shows that the sulfonamide
bearing compound had IC50 value two magnitudes lower, cpd
73 showing an IC50 value of 135 nM. The synthesis of cpd 73
was carried out through intermediate cpd 71 via chlorination
by thionyl chloride followed by acylation in pyrimidine. Other
coupling methods were ineffective. This intermediate
compound was also measured and surprisingly it was found to
be an equipotent inhibitor to some sulfisoxazole-coupled
derivatives, with an IC50 value of 66 nM.
The inhibitors discussed so far (cpds 21-40) were coupled
with 3,4-dimethylisoxazole via the sulfonamide linker region.
Our aim was to investigate how the structure of the isoxazole
ring influences CDK9 enzyme inhibition; therefore, 4,5-
dimethylisoxazole analogs of two effective inhibitors, cpds 21
and 22, were prepared. The IC50 value of these compounds (41
and 42) was found to be practically the same as their 3,4-
isoxazole counterparts, thus it can be presumed that the
potency of inhibition is independent of the isomerism.
Due to the therapeutic importance of CDK2 and CDK7 27-29
,
the selectivity of our most potent 2,4-pyrimidine CDK9
inhibitors were tested on these cyclin dependent kinases (Table
4). Additional measurement was also carried out for PLK1
inhibition (IC50 values are included in Table 4). Experimental
data suggests that our derivatives inhibit CDK9 selectively.
In an effort to better explore the SAR of CDK9 enzyme
inhibition, the isoxazole moiety was replaced with
benzisoxazole (cpd 43), and it was concluded that the presence
of this bicycle on our molecule caused basically no difference
compared to its isoxazole analog, cpd 42.
Table 4. Cyclin dependent kinase and PLK1 inhibition activity.
Cpd
CDK2 inhibition (%)a CDK7 inhibition (%)a
PLK1 IC50 (µM)
The next step in our research was the replacement of
isoxazole ring with other five-membered heterocycles, such as
thiazole (cpds 44-48) and 2-methyl-[1,3,4]thiadiazole (cpd 49-
57). In case of molecules containing thiazole, CDK9 enzyme
inhibition was less potent (by 1 - 2 orders of magnitude) than
their isoxazole-containing analogs. There was an exception:
compound 46 (R1-methoxy, R2-H, R3-H, R4-F substituted
similarly to cpd 24), demonstrating an IC50 value of 2 nM.
22
24
26
31
42
46
52
54
66
68
43
44
83
14
0
3
1
2
0
0
0
2
7
0
0
>12.5
12.35
5.91
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
46
29
50
9
Inhibition data of 44-48 derivatives (Table 2) reveals that
the introduction of the thiazole functional group does not result
in more potent inhibitors, in contrary to coupling with
thiadiazole ring. Amongst the latter our most potent CDK9
enzyme inhibitor is cpd 54, with an IC50 value measured in
subnanomolar region. This inhibitor has similar structure to the
isoxazole bearing compound 26 (IC50: 1 nM); both of them are
having the same substituents on the benzyl ring (R1-methoxy,
R2-H, R3-F, R4-F). The other thiadiazole analogs were also
proved to be potent enzyme inhibitors. This replacement of the
original heterocycle provided compounds that were equipotent
inhibitors with their isoxazole analogs, with the exception of
cpd 54 resulting in an IC50 value of 0.4 nM.
44
aCompound concentration was 1 µM.
Based on previous reports 30-32, our novel 2,4-pyrimidine
derivatives were tested in multiple myeloma cell lines (RPMI-
8226). Two of our CDK9 inhibitors showed considerable
proliferation inhibitory effect: cpds 66 and 68. As shown in
Table 3, both of these molecules are substituted with a
benzimidazole ring. The IC50 values are as follows: cpd 66:
1.34 µM, cpd 68: 1.78 µM; in case of other derivatives, which
are not shown in Table 5, they were over 30 µM (Table 5).
Table 5. Multiple myeloma inhibition activity.
Compounds 58-65 are examples of analogs where the five-
membered heteroaromatic ring was omitted; instead, functional
groups containing heteroatoms were substituted on the ring
beside the linker nitrogen (the sulfonamide linker region was
also omitted whose role was also investigated, see the end of
this section). This modification resulted in remarkable decrease
in CDK9 enzyme inhibition.
Cpd
RPMI-8226 IC50 (µM)
Cpd
RPMI-8226IC50 (µM)
21
24
41
43
44
29.15
26.58
12.32
15.51
24.68
54
58
60
66
68
11.66
21.52
20.46
1.34
1.78
Based on our previous research, 4,6-pyrimidine analogues
of compounds 66-68 (benzimidazole substitution through
methylene connection) are potent proliferation inhibitors on
many cell lines. We were particularly interested in how this
methylene-benzimidazole structure on the 2,4-pyrimidine core
In summary, in this paper we disclosed the synthesis and
biological evaluation of novel 2,4-pyrimidine derivatives
which inhibit CDK9 enzyme in low nanomolar range.

14. Swallow, C.J.; et al. Sak/Plk4 and mitotic fidelity. Oncogene,
2005, 24, 306.
15. Zhao, C.; et al. Overexpression of Plk1 promotes malignant
progress in human esophageal squamous cell carcinoma. J
Cancer Res Clin Oncol, 2010, 136, 9.
Introducing the sulfisoxazole motif markedly improved
efficacy; nevertheless, the most effective proved to be cpd 66
on RPMI-8226 cell line bearing 1H-benzimidazole moiety.
16. Takahashi, T.; et al. Polo-like kinase 1 (PLK1) is overexpressed
in primary colorectal cancers. Cancer Sci, 2003, 94, 148.
17. Saygin, C.; et al. Emerging therapies for acute myeloid
leukemia. J Hematol Oncol, 2017, 10, 1.
18. http://data.euro.who.int/hfamdb/ (october 2017)
19. Sonawane, Y.A.; et al. Cyclin dependent kinase 9 inhibitors for
cancer therapy. J Med Chem, 2016, 59, 8667.
Supplementary Material
Supplementary data (detailed synthetic methods, analytical
data, biological protocols) associated with this article can be
found in the online version.
20. Greff, Z.; et al., US2015/0368209 (A1); 2015.
21. Németh, G.; et al. Synthesis and evaluation of phosphorus
containing, Sspecific CDK9/CycT1 inhibitors. J Med Chem,
2014, 57, 3939.
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a potent
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4. Brown, N.R.; et al. The structural basis for specificity of
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Nat Cell Biol, 1999; 1, 438.
5. Malumbres, M.; et al. Mammalian cyclin-dependent kinases.
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6. Peyressatre, M.; et al. Targeting Cyclin-Dependent Kinases in
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