Synthesis and biological evaluation of seliciclib derivatives as potent and selective CDK9 inhibitors for prostate cancer therapy

Article abstract of DOI:10.1007/s00706-020-02727-x

Seliciclib is a cyclin-dependent kinase (CDK) inhibitor that has been assayed in phase II clinical trials as an anticancer agent. This paper describes the synthesis of novel derivatives of seliciclib with improved potency, metabolic stability, aqueous solubility, and anti-proliferative activity. The new derivatives showed a novel CDKs selectivity profile. Replacement of ethyl alcohol at position 2 of purine with dimethylaminopropyl and fluorination of benzyl at position 6 of purine of seliciclib resulted in the formation of a derivative that potently and selectively inhibited CDK9 (26?nM vs. CDK9 and > 60-fold selectivity vs. CDK2/5/7). In comparison to seliciclib, this derivative shows lower metabolic clearance (25% lower in Cl_int), higher aqueous solubility and is more cytotoxic in androgen-independent prostate cancer cells. Graphic abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s00706-020-02727-x

Monatshefte für Chemie - Chemical Monthly (2021) 152:109–120
https://doi.org/10.1007/s00706-020-02727-x
ORIGINAL PAPER
Synthesis and biological evaluation of seliciclib derivatives as potent
and selective CDK9 inhibitors for prostate cancer therapy
Aisha A. Alsfouk1 · Hanan M. Alshibl2 · Najla A. Altwaijry1 · Bshra A. Alsfouk1 · Ebtehal S. Al‑Abdullah2
Received: 7 May 2020 / Accepted: 30 December 2020 / Published online: 13 January 2021
© Springer-Verlag GmbH Austria, part of Springer Nature 2021
Abstract
Seliciclib is a cyclin-dependent kinase (CDK) inhibitor that has been assayed in phase II clinical trials as an anticancer
agent. This paper describes the synthesis of novel derivatives of seliciclib with improved potency, metabolic stability, aque-
ous solubility, and anti-proliferative activity. The new derivatives showed a novel CDKs selectivity profle. Replacement of
ethyl alcohol at position 2 of purine with dimethylaminopropyl and fuorination of benzyl at position 6 of purine of selici-
clib resulted in the formation of a derivative that potently and selectively inhibited CDK9 (26 nM vs. CDK9 and >60-fold
selectivity vs. CDK2/5/7). In comparison to seliciclib, this derivative shows lower metabolic clearance (25% lower in Clint),
higher aqueous solubility and is more cytotoxic in androgen-independent prostate cancer cells.
Graphic abstract
Keywords Cyclin-dependent kinase · Purine · Metabolic stability · Roscovitine · CYC202
Introduction
3]. CDK9 is a member of the CDK family which dimerises
with cyclin T to form positive transcription elongation factor
b (p-TEFb), an elongation factor during RNA transcription
[4, 5]. CDK9 conducts its function by phosphorylating the
C-terminal domain (CTD) subunit of RNA polymerase II
[6].
Cyclin-dependent kinase (CDK) enzymes form heterodi-
mers with a specifc family of proteins called cyclins. These
CDK–cyclin complexes regulate the cell division cycle and
modulate gene transcription [1]. In cancer cells, the activ-
ity of CDK–cyclins is deregulated by various mechanisms
including hyperactivity of CDKs, overexpression of cyclins
and loss or inactivation of inhibitory proteins, e.g. p16 [2,
There is trong evidence that indicates that CDK9 is a
promising therapeutic opportunity in cancer through the
regulation of anti-apoptotic proteins that initiate cancer cell
immortality. Its pathogenic role has been established in a
number of malignancies including prostate cancer [6–14].
Prostate cancer is the most common cancer and the second
cause of death among men in the world [15, 16]. The current
treatment options are surgery, chemotherapy, radiotherapy,
and hormone deprivation with highly successful results.
However, 20% of prostate cancer patients develop castration-
resistance prostate cancer (CRPC), which is unresponsive to
* Bshra A. Alsfouk
baalsfouk@pnu.edu.sa
1
Department of Pharmaceutical Sciences, College
of Pharmacy, Princess Nourah Bint Abdulrahman University,
Riyadh, Saudi Arabia
2
Department of Pharmaceutical Chemistry, College
of Pharmacy, King Saud University, P.O. Box 2457,
Riyadh 11451, Saudi Arabia
Vol.:(0123456789)
1 3

110
A. A. Alsfouk et al.
conventional therapy and associated with poor prognosis.
Recently, CDK9 has been identifed as a key component in
CRPC through modulating the activity of androgen receptor
[10, 17–19]. This highlights the role of CDK9 in CRPC.
Since the identifcation of CDK9 as a valid therapeutic
target in cancer, a large number of small molecules have
been developed as CDK9 inhibitors. These inhibitors show
good anti-tumour activity and drug-likeness properties, sev-
eral of which have progressed into clinical trials as antican-
cer agents in various types of cancer [12, 14, 20–27]. The
frst generation evaluated in clinical trials is pan-CDK inhib-
itors including favopiridol, seliciclib, dinaciclib, SNS-032,
and RGB-286638, which can inhibit CDK9 as well as other
CDK isoforms and other kinases, as shown in Table 1. Fla-
vopiridol is the most studied CDK9 inhibitor and although
it is not a selective CDK9 inhibitor, its main mechanism of
action is believed to be through inhibition of CDK9 [28, 29].
Their clinical results show many adverse efects and events
[6, 23, 24, 30], which could be due to the lack of selectivity.
In 2017, researchers at Bayer identifed the frst highly selec-
tive CDK9 inhibitor, atuveciclib, which is currently under
evaluation in phase I clinical trials for the treatment of acute
leukemia [31].
Scheme 1
parent drug as a CDK2 inhibitor and, therefore, it is unlikely
to contribute to the observed anti-proliferative efcacy of
seliciclib [47].
The objective of the present study is to develop deriva-
tives of seliciclib with improved potency and selectivity
toward CDK9, as well as enhanced metabolic stability and
aqueous solubility. To achieve this, we designated and syn-
thesized derivatives with terminal amines to replace the
metabolically labile 2-hydroxyl of seliciclib. The majority
of the synthesized compounds contain ionisable amines at
physiological pHs and, therefore, can be formulated as salts.
Seliciclib (also known as CYC202 or roscovitine) is a
CDK inhibitor, mainly CDK2/E inhibitor with lower potency
versus CDK9/T, CDK7/H, CDK5/p53, and CDK4/D [32,
35]. It demonstrates a good selectivity profle against a panel
of other kinases [39]. Its evaluation in phase II clinical tri-
als did not show any adverse efects associated with its use,
but also did not show complete response to the patient’s
treatment [22, 40, 41]. Its anti-tumour efect is associated
with the inhibition of RNA polymerase II and Rb protein
phosphorylation, and reduction of the expression of cyclin
D1 protein [22, 30, 42]. Seliciclib has sub-optimum water
solubility but well-absorbed post-oral administration. It
shows high metabolic clearance, which was reported as the
major issue associated with its use in clinical studies [22,
34, 43, 44].
Results and discussion
Chemistry
In this work, seliciclib and its derivatives were synthe-
sized using the straightforward three-step procedure start-
ing from commercially available 2,6-dichloropurine. First,
replacement of the reactive chlorine at C6 of purine with
benzylamines. Second, alkylation of N9 of purine using
2-bromopropane. Third, substitution of the less reactive
2-chloropurine with various amines (Scheme 2).
In the frst step, 2,6-dichloropurine was treated with the
desired substituted benzylamines to replace the chlorine at
position C6 of purine. This is achieved by heating the reac-
tion mixture to 110 °C for 3 h using Et3N and n-butanol as
a solvent. The crude intermediates were recrystallized with
butanol and hexane to obtain 2-chloro-6-(substituted ben-
zylamino)purines 1a–1d in excellent yield (91–97%).
Metabolic identifcation studies of seliciclib have shown
that 86% of the parent drug is readily metabolised by oxida-
tion and 60% of this metabolism is contributed to the forma-
tion of the carboxylate metabolite (Scheme 1) [45, 46]. The
carboxylate metabolite is seven times less active than the
Table 1 Selectivity profle of
Inhibitor
CDK9/T
CDK2/E
CDK5/p53
CDK7/H
CDK4/D
References
CDKs inhibitors in clinical
trials (IC50/nM)
Flavopiridol
Seliciclib
Dinaciclib
SNS-032
RGB-286638
Atuveciclib
11
950
4
4
1
282
100
1
48
3
–
160
1
340
5
1600
514
540
–
62
44
132
13,500
–
925
4
[32–34]
[32, 35]
[32, 36]
[32, 37]
[34, 38]
[31]
6
1000
>10,000
–
1 3

Synthesis and biological evaluation of seliciclib derivatives as potent and selective CDK9…
111
Scheme 2
In the second step, the 2-chloro-6-(substituted ben-
zylamino)purines 1a–1d were alkylated at position N9
of purine using 2-bromopropane and K2CO3 as a base in
DMSO at 20 °C for 16–48 h. The crude products were puri-
fed by recrystallization with isopropanol to yield 2-chloro-
6-(substituted benzylamino)-9-isopropylpurines 2a–2d in
good-to-excellent yield (87–95%).
changed in the new compounds as it showed the maximum
CDK activity in previous studies. Havlicek et al. showed
that N9-substitution by a hydrophobic residue is probably
important for a positive binding, and isopropyl appears to
be the most active group at position 9. Removal or change of
this side chain dramatically decreased the inhibitory activity
of the seliciclib and its derivatives [46, 48–53].
The frst two steps of the synthesis were switched (i.e.
alkylation of the 2,6-dicholorpurine) in an attempt to keep
the derivatization steps as the last two steps and minimize
the number of reactions required. This resulted in the forma-
tion of undesired N7 regioisomer in considerable amounts
(~40%), which requires column chromatography to separate
it from the desired N9 isomer. Therefore, it was decided to
replace the 6-chloro with benzylamine frst, then alkylate
the N9.
All new compounds were tested against CDK9/2. In addi-
tion, six of these compounds were selected to examine their
activity against CDK5/7. Also, the cytotoxic efect of all
compounds was assessed in PC3 cell line. The biological
results are illustrated in Tables 2 and 3.
The biological data indicate that the majority of the new
compounds showed better separation between CDK9 and
CDK2 activity in comparison to seliciclib, which is essen-
tially equipotent against the two isoforms. Several examples
show great selectivity (10–70-fold) for CDK9 over CDK2,
e.g. 3f, 3 g, 3j, 3l, and 3o, whereas other compounds exhib-
ited improved CDK2 selectivity over CDK9 but not exceed-
ing tenfold, e.g. 3d, 3h, and 3k.
In the final step, the 2-chloro-6-(substituted
benzylamino)-9-isopropylpurines 2a–2d were treated with
appropriate amines. The reaction temperature was 160 °C
for 8–24 h depending on the reactivity of the amines. The
final products were purified by recrystallization using
EtOAc:hexane (1:1). A further purifcation by column chro-
matography was required in some cases to obtain 2-amino-
6-(substituted benzylamino)-9-isopropylpurines 3a–3t. The
yield was varied from 34 to 94% depending on nucleophilic-
ity and size of the amine used.
Some of these compounds were assessed for their
CDK5/7 inhibitory activities as shown in Table 3. With the
exception of 3f and 3i activity against CDK7, all tested com-
pounds exhibit better selectivity for CDK5/7 than seliciclib.
In the PC3 cell line, most of the newly prepared compounds
show enhanced anti-proliferative activity in comparison to
seliciclib (Table 4).
Structure–activity relationship in biological assays
The impact of insertion of halogens to benzylamines was
examined. The results showed that 3,5-difuorobenzylamine
has unfavourable CDK9 activity and PC3 cytotoxicity in
comparison to 4-fuorobenzylamine, e.g. 3l vs. 3q, 3m vs.
3r, 3h vs. 3s, and 3o vs. 3t. The molecular docking study
revealed that the binding mode of the new compounds with
CDK9 shows a lysine residue (Lys35) close to m-fuoroben-
zylamine at position 6 of purine, which could account for
the lower activity of meta-fuorinated compounds (Fig. 1).
The impact of the replacement of a neutral hydroxyl
at position 2 of purine ring of seliciclib with positively
charged amines was investigated. Various amines with dif-
ferent steric hindrances and nucleophilicity were examined
The main objective of this work was to develop derivatives
of seliciclib with improved potency and selectivity toward
CDK9 isoform with potential utility in prostate cancer ther-
apy. In terms of chemical structure, the newly synthesized
compounds have two diferent substituents in comparison
to the standard compound, seliciclib: ionisable amino group
which replaces the 2-hydroxyl of seliciclib and the benzyl-
amines at position 6 substituted with halogens in various
positions. Previous studies of the purine scafold showed
that halogen substituents on the benzyl ring are associated
with high cytotoxic activity [48, 49]. Isopropyl has not been
1 3

112
A. A. Alsfouk et al.
Table 2 CDK9/2 biochemical
assay results of seliciclib and
its derivatives (data are means
(
SD) of three individual
experiments)
Comp.
Structure
R1 R2 R3
IC50 /nM
R4
CDK9
CDK2
CDK9
:2
ratio
Seliciclib
H
H
H
199.7( 12.9)
200.8( 7.9)
1:1
3a
3b
H
H
H
H
H
H
239.1( 14.0)
288.9( 12.8)
120( 6.9)
2:1
1:3
874.8(32.3)
3c
H
H
H
H
H
H
2785.7( 96.9)
608.38( 27.8)
1066.8( 47.5)
93.5( 5)
2.6:1
6.4:1
3d
3e
3f
H
H
H
H
Cl
Cl
H
H
H
498.2( 20.6)
150.7( 7.8)
388.5( 15.5)
2370.8 ( 84.5)
3673.7( 80.2)
6174( 180.1)
1:4.7
1:24
1:16
3g
3h
3i
H
H
Cl
Cl
H
H
508.8( 19.5)
304.7( 11.2)
330.6( 13.1)
238.5( 8.8)
1.5:1
1.3:1
3j
3k
3l
H
H
H
Cl
F
H
H
H
112.4( 6.1)
2288( 81.8)
26( 1.7)
1961.6( 67.9)
267.5( 16)
1:17
8.4:1
1:71
F
1852.6( 56.3)
3m
3n
H
H
F
F
H
H
835( 34)
2119.2( 64.8)
70.5( 4.6)
1:2.5
5.5:1
392.7( 15.8)
3o
3p
3q
H
F
F
F
H
F
F
131.1( 5.8)
288.7( 17.2)
444.4( 21.8)
17774.8( 61)
350.7( 12)
242.6( 8.4)
1:13
1:1.2
1.8:1
H
H
3r
F
H
F
1863.2( 69.5)
565.7( 34.1)
3.3:1
3s
3t
F
F
H
H
F
F
3578.6( 157.5)
443.2( 16.5)
778.8( 24.3)
1294.1( 44.2)
4.5:1
1:2.9
1 3

Synthesis and biological evaluation of seliciclib derivatives as potent and selective CDK9…
113
Table 3 CDK activity of
Comp
CDK9
CDK2
CDK5
CDK7
selected compounds expressed
as IC50/nM (data are means
3d
608.38 ( 27.8)
150.7 ( 7.8)
304.7 ( 11.2)
112.4 ( 6.1)
26 ( 1.7)
392.7 ( 15.8)
199.7 ( 12.9)
93.5 ( 5)
7093.4 ( 283.7)
1725.1 ( 50)
2843.2 ( 103.4)
932.8 ( 51.8)
2723.8 ( 103.7)
2213.6 ( 26.9)
507.8 ( 28.2)
5986.8 ( 226.9)
300 ( 25)
852.9 ( 25.8)
1813.9 ( 77.7)
1452.6 ( 51.8)
7774.7 ( 323.9)
1608 ( 56.4)
(
SD) of three individual
3f
3673.7 ( 80.2)
238.5 ( 8.8)
1961.6 ( 67.9)
1852.6 ( 56.3)
70.5 ( 4.6)
experiments)
3i
3j
3l
3n
Seliciclib
200.8 ( 7.9)
Table 4 PC3 cytotoxicity results of seliciclib and its derivatives (data
are means ( SD) of three individual experiments) and predicted top-
ological polar surface area (tPSA), log P (calculated using ChemBio-
Draw 15.0) and log D at pH 7.4 (calculated using ChemAxon)
3i vs. 3j and 3f, 3h vs. 3o and 3k, and 3s vs. 3t and 3p.
Figure 1C shows the binding mode of 3l and seliciclib with
CDK9 and CDK2. The terminal amino group of 3l and
hydroxyl of seliciclib interact with Asp167 and Asp145
residues in CDK9 and CDK2, respectively (distances are
2.038 and 2.277 Å, respectively). When the free rotat-
able amino and hydroxyl groups adopt a fipped pose, they
will interact with acidic residue (Asp109) in CDK9, which
will bind more tightly with the basic amino group of 3l
than the hydroxyl of seliciclib. This residue is replaced by
neutral Gln131 in CDK2, which shows no preference for
the amino group of 3l over hydroxyl group of seliciclib
and this could account for the better separation between
CDK9 and CDK2 observed with derivatives with a basic
amino group (in fip pose, the distances of cationic centre
of 3l and Asp109 of CDK9 is 3.067 Å and between 3 l and
Gln131 of CDK2 is 3.935 Å, respectively).
Compounds
PC3 cyto-
toxicity IC50/
µM
tPSA/Å2 log P Log D@
pH=7.4
Seliciclib (literature [54]) 30
84.6
2.75 2.3
Seliciclib (in house)
39.7 ( 1.3)
3a
3b
3c
3d
3e
3f
3g
3h
3i
22.6 ( 0.7) 58.8
41.4 ( 1.3) 67.6
14.1 ( 0.4) 67.6
20.6 ( 0.6) 64.8
3.01 2.6
2.57 0.35
2.46 1.0
2.85 2.8
2.63 1.1
3.57 3.2
3.13 0.45
3.02 1.6
3.41 3.5
3.19 1.6
3.17 2.7
2.73 0.5
2.62 1.1
3.01 3.0
2.79 3.0
3.33 2.8
2.89 0.6
2.78 1.3
3.17 3.0
2.95 1.3
33.4 ( 1)
67.6
40.8 ( 1.3) 58.8
50.2 ( 1.6) 67.6
67.8 ( 2.2) 67.6
8 ( 0.2)
10.7 ( 0.3) 67.6
8.4 ( 0.2)
32.5 ( 1)
58.5 ( 1.9) 67.6
21.6 ( 0.7) 64.8
53.3 ( 1.7) 67.6
18 ( 0.5)
10 ( 0.3)
249 ( 8.1)
149 ( 4.9)
64 ( 2.1)
64.8
Derivatives with a three-carbon distance of appendant
amine from purine core (dimethylpropylamine) showed
better activity than derivatives with two-carbon distanced
amine (dimethylethylamine) in terms of kinase activities and
cytotoxicity in PC3, e.g. 3q vs. 3r, 3l vs. 3m, 3g vs. 3h, and
3b vs. 3c. This could be due to the distance between the
terminal amines and Asp167 residue, the amine is closer
to Asp167 in propyl linker derivatives than in ethyl linker
derivatives (Fig. 1).
3j
3k
3l
58.8
67.6
3m
3n
3o
3p
3q
3r
3s
3t
58.8
67.6
67.6
64.8
67.6
The cytotoxic efects of seliciclib and the new compounds
were determined in androgen-independent prostate cancer
cells (PC3 cells) by MTT assay. In this assay, seliciclib
had an IC50 =39.7 µM (Table 4), which is comparable with
the reported IC50 of seliciclib in PC3 cells of 30 µM deter-
mined by Arisan et al. [54]. In other cell lines, the average
IC50 =16 µM of a 60 cell line panel (range from 8 to 30 µM)
[40, 55]. This low cellular activity of seliciclib could be due
to fast metabolic clearance and sub-optimum water solubil-
ity. Most of the new compounds in the present work show
improved cellular potency in comparison to seliciclib, with
a few compounds showing weak cellular cytotoxicity, e.g.
3g, 3o, and 3r. However, this weak cellular activity does not
correlate with in vitro CDK9 activity, which may be attrib-
uted to the physicochemical properties, e.g. lipophilicity and
chargeable amines, which could afect the permeability and
including cyclic versus acyclic, high versus low pKA, sec-
ondary vs. tertiary and hydrogen bond acceptor versus
hydrogen bond donor amines. The results indicate that
both hydroxyl and amine substituents are good for CDKs
activity and cytotoxicity, suggesting that both groups are
well tolerated and interact with the binding site. However,
amines give better separation between CDK2 and CDK9
activity than the hydroxyl group. Compounds with mor-
pholinyl substituents show favourable CDK2 activity and
cytotoxicity in comparison to compounds with piperazinyl
and methyl piperazinyl substituents, e.g. 3d vs. 3e and 3a,
1 3

114
A. A. Alsfouk et al.
A
B
C
2.039
2.277
Fig. 1 a Structures of 3l and seliciclib CDK9/2 inhibitory data
expressed IC50 values. b Proposed binding mode of 3l with CDK9-
ATP binding site (left) and schematic representation of 3l with CDK9
showing the key binding interactions (right). c Overlaid 3l (green)
and seliciclib (orange) with CDK9 (right) and CDK2 (left) showing
the key diference in amino acid residues between the two isoforms
that could account for selectivity (color fgure online)
account for the low cellular potency of these compounds
(Table 4).
major issue associated with the progression of seliciclib in
clinical trials. Protecting the terminal hydroxyl group against
oxidation can enhance the metabolic stability as reported by
Wilson et al. [43]. In this work, we replaced the metabolic
labile hydroxyl group on position 2 of purine with cyclic
amines, e.g. morpholine, piperazine, and methylpiperazine,
which are known to resist oxidative metabolism [56, 57]
or sterically hinder appendant tertiary amines, e.g. dimeth-
ylpropylamine and dimethylethylamine. Table 5 shows S9
Metabolic stability and water solubility
The major metabolic pathway of seliciclib is oxidation of
the terminal hydroxyl group to a carboxylic acid (Scheme 1),
which is rapidly formed during frst-pass metabolism [45,
46]. This high metabolic clearance has been reported as a
1 3

Synthesis and biological evaluation of seliciclib derivatives as potent and selective CDK9…
115
Table 5 S9 metabolic stability and kinetic aqueous solubility for
gel-precoated aluminium sheets (60 F254, Merck) with UV
visualization at 365 and 254 nm. A LC–MS purity check was
performed at King Abdullah International Medical Research
Center.
selected compounds
Compounds
t1/2/min
Clint mm3/min/im Aqueous
cells
solubility/
µM
2‑Chloro‑6‑(substituted benzylamino) purines 1a–1d Ben-
zylamine (6.35 mmol) and 1.2 cm3 triethylamine (8.6 mmol)
were added to a solution of 1 g 2,6-dichloropurine
(5.29 mmol) in 10 cm3 n-butanol. The reaction mixture was
kept at 110 °C for 3 h with continuous stirring. After cooling
to ambient temperature, the solid was fltered and washed
with cold n-butanol and hexane then dried.
3d
ND
ND
56
50
12
52
50
12
3f
64.3
62.5
69.3
ND
0.010784
0.011091
0.009998
ND
3j
3l
3q
Seliciclib
56.9
0.01218
ND not determined
N‑Benzyl‑2‑chloro‑9H‑purin‑6‑amine (1a) M.p.: 235–236 °C
metabolic stability results of selected examples (S9 assay
assesses the metabolic stability against both phase I and
phase II enzymes). All tested examples, with diferent types
of terminal amines, show lower metabolic clearance and
prolonged half-life than seliciclib.
(Ref. [58] 239–241 °C).
2‑Chloro‑N‑(4‑chlorobenzyl)‑9H‑purin‑6‑amine (1b,
1
C12H9Cl2N5) Yield 91%; m.p.: 249–250 °C; H NMR
(700 MHz, DMSO-d6): δ = 4.63 (br s, 2H), 7.38 (br
s, 4H), 8.16 (br s, 1H), 8.75 (br s, 1H), 13.12 (br s, 1H)
ppm; 13C NMR (176 MHz, DMSO-d6): δ=43.00, 118.35,
128.73, 129.70, 131.87, 138.85, 140.10, 151.09, 153.32,
155.17 ppm; HRMS (ESI): m/z calcd for C12H9Cl2N5
294.03078, found 294.03641.
Almost all tested compounds showed better aqueous solu-
bility than seliciclib. Furthermore, most of the synthesized
compounds contain ionisable amines at physiological pHs
and, therefore, can be formulated as salts.
Conclusion
2‑Chloro‑N‑(4‑fluorobenzyl)‑9H‑purin‑6‑amine (1c,
C12H9ClFN5) Yield 91%; m.p.: 239–241 °C; H NMR
1
This paper describes the synthesis and anti-CDK activ-
ity of novel derivatives of seliciclib. Most of the prepared
compounds demonstrated better separation between CDK9
and CDK2 inhibitory activity with enhanced metabolic
stability. Compound 3l is a potent and selective CDK9
inhibitor (IC50 for CDK9 = 26 nM, CDK2 = 1852.6 nM,
CDK5 = 2723.8 nM, and CDK7 = 1452.6 nM), show-
ing enhanced cytotoxicity against prostate cancer cells
(IC50 =32.5 µM), improved metabolic stability against phase
I and phase II metabolic pathways (S9 t1/2 = 60 min, 25%
increase in t1/2 comparing to seliciclib), and improved water
solubility (52 µM). Further mechanistic studies of this com-
pound in prostate cancer cells are currently ongoing.
(700 MHz, DMSO-d6): δ=4.63 (br s, 2H), 7.15 (br s, 2H),
7.40 (br s, 2H), 8.14 (br s, 1H), 8.74 (br s, 1H), 13.12 (br
s, 1H) ppm; 13C NMR (176 MHz, DMSO-d6): δ = 42.90,
115.49 (d, J =69.94 Hz), 118.35, 129.84, 136.02, 140.07,
151.05, 153.35, 155.17, 160.98, 162.36 ppm; HRMS (ESI):
m/z calcd for C12H9ClFN5 278.06033, found 278.06359.
2‑Chloro‑N‑(3,5‑difluorobenzyl)‑9H‑purin‑6‑amine (1d,
1
C12H8ClF2N5) Yield 97%; m.p.: 240–242 °C; H NMR
(700 MHz, DMSO-d6): δ = 4.68 (br s, 2H), 7.05–7.20 (m,
2H), 7.24–7.34 (m, 1H), 8.18 (br s, 1H), 8.74 (br s, 1H),
13.15 (br s, 1H) ppm; 13C NMR (176 MHz, DMSO-d6):
δ = 37.55, 115.57, 116.14, 117.15 (dd, J = 33.44, 8.8 Hz),
118.39, 128.60, 140.28, 151.17, 153.19, 155.06, 115.96,
157.32, 157.86, 159.21 ppm; HRMS (ESI): m/z calcd for
C12H8ClF2N5 296.05091, found 296.05723.
Experimental
Chemicals such as 2,6-dichloropurine, 2-bromopropane,
aliphatic and benzylamines were purchased from Sichuan
Benepure and Aldrich Chemical Companies. The melting
point measurements were carried out using the electrother-
mal melting point apparatus (UK). Bruker Ascend 700 NMR
spectrometer (Fällanden, Switzerland) was used to obtain
2‑Chloro‑6‑(substituted benzylamino)‑9‑isopropylpurines
2a–2d Dropwise addition of 0.85 cm3 2-bromopropane
(9 mmol) to a solution of 2-chloro-6-(substituted ben-
zylamino)purine 1 (3.6 mmol) and 1.5 g potassium car-
bonate (10.8 mmol) in 10 cm3 DMSO and the temperature
did not exceed 19 °C using isopropanol bath for 16–48 h.
After the reaction was completed, 25 cm3 of cold H2O was
added then the mixture was extracted with ethylacetate
(3×15 cm3). The organic layers were combined and washed
1
the NMR spectra at 700.17 MHz for H and 176.08 MHz
for 13C and the solvent used was DMSO-d6 in all samples.
TLC was performed for monitoring the reactions using silica
1 3

116
A. A. Alsfouk et al.
with brine (3×10 cm3) then dried over MgSO4 and evapo-
rated to dryness. In some cases, the product crystallized
using isopropanol.
and dried using MgSO4 then the organic solvent was evapo-
rated to get the dry product. The sticky residue was triturated
with hexane:EtOAc (1:1) to give the solid pure product. In
some cases, column chromatography was used to get pure
product.
N‑Benzyl‑2‑chloro‑9‑isopropyl‑9H‑purin‑6‑amine (2a,
1
C15H16ClN5) Yield 87%; m.p.: 177–179 °C; H NMR
(700 MHz, DMSO-d6): δ=1.49 (d, J=6.67 Hz, 6H), 4.62–
4.71 (m, 3H), 7.20–7.25 (m, 1H), 7.28–7.36 (m, 4H), 8.27
(s, 1H), 8.77 (br s, 1H) ppm; 13C NMR (176 MHz, DMSO-
d6): δ=22.54, 43.55, 47.26, 118.84, 127.31, 127.74, 128.76,
139.73, 139.89, 149.82, 153.21, 155.35 ppm; HRMS (ESI):
m/z calcd for C15H16ClN5 302.11670, found 302.12223.
N‑Benzyl‑9‑isopropyl‑2‑(4‑methylpiperazin‑1‑yl)‑9H‑pu‑
rin‑6‑amine (3a, C20H27N7) Yield 83%; m.p.: 138–140 °C;
1
LC–MS purity 99.9%; H NMR (700 MHz, DMSO-d6):
δ = 1.47 (d, J = 6.67 Hz, 6H), 2.18 (s, 3H), 2.30 (br s,
4H), 3.65 (br s, 4H), 4.51–4.68 (m, 3H), 7.17–7.23 (m,
1H), 7.29 (t, J = 7.53 Hz, 2H), 7.36 (d, J = 7.53 Hz, 2H),
7.85 (s, 1H), 7.97 (br s, 1H) ppm; 13C NMR (176 MHz,
DMSO-d6): δ = 22.55, 44.46, 46.17, 46.38, 54.97, 66.82,
114.13, 126.94, 127.90, 128.53, 136.27, 141.21, 150.95,
154.47, 158.69 ppm; HRMS (ESI): m/z calcd for C20H27N7
366.24007, found 366.2474.
2‑Chloro‑N‑(4‑chlorobenzyl)‑9‑isopropyl‑9H‑purin‑6‑amine
(2b, C15H15Cl2N5) Yield 95%; m.p.: 144–146 °C; 1H NMR
(700 MHz, DMSO-d6): δ=1.51 (d, J=6.45 Hz, 6H), 4.62 (d,
J=5.81 Hz, 2H), 4.68 (dt, J=13.44, 6.83 Hz, 1H), 7.37 (q,
J=8.60 Hz, 4H), 8.30 (s, 1H) 8.84 (br s, 1H) ppm; 13C NMR
(176 MHz, DMSO-d6): δ = 22.58, 42.96, 47.22, 118.92,
128.71, 129.64, 131.83, 138.85, 140.02, 149.90, 153.15,
155,29 ppm; HRMS (ESI): m/z calcd for C15H15Cl2N5
336.07773, found 336.08606.
N6‑Benzyl‑N2‑[3‑(dimethylamino)propyl]‑9‑isopro‑
pyl‑9H‑purine‑2,6‑diamine (3b, C20H29N7) Yield 44%;
1
LC–MS purity 93.6%; H NMR (700 MHz, DMSO-d6):
δ=1.47 (d, J=6.88 Hz, 6H), 1.62 (br s, 2H), 2.10 (s, 6H),
2.22 (t, J = 6.67 Hz, 2H), 3.23 (q, J = 6.60 Hz, 2H), 4.54
(dt, J = 13.34, 6.67 Hz, 1H), 4.62 (br s, 2H), 6.30 (br s,
1H), 7.18–7.23 (m, 1H), 7.28 (t, J=7.64 Hz, 2H), 7.35 (d,
J=7.31 Hz, 2H), 7.78 (s, 1H) ppm; 13C NMR (176 MHz,
DMSO-d6): δ = 22.54, 27.84, 43.19, 45.68, 46.04, 57.69,
79.64, 114.03, 126.89, 127.78, 128.52, 135.39, 141.27,
151.22, 154.89, 159.56 ppm; HRMS (ESI): m/z calcd for
C20H29N7 368.25572, found 368.26201.
2‑Chloro‑N‑(4‑fuorobenzyl)‑9‑isopropyl‑9H‑purin‑6‑amine
(2c, C15H15ClFN5) Yield 95%; m.p.: 152–154 °C; 1H NMR
(700 MHz, DMSO-d6): δ=1.50 (d, J=7.10 Hz, 6H), 4.62
(d, J=5.81 Hz, 2H), 4.68 (dt, J=13.55, 6.78 Hz, 1H), 7.15
(t, J=8.82 Hz, 2H), 7.39 (t, J=6.56 Hz, 2H), 8.30 (s, 1H),
8.82 (br s, 1H) ppm; 13C NMR (176 MHz, DMSO-d6):
δ = 22.58, 42.90, 47.21, 115.41 (d, J = 39.42 Hz), 118.90,
129.81 (d, J = 8.90 Hz), 135.99, 139.96, 149.87, 153.16,
155.27, 160.97, 162.35 ppm; HRMS (ESI): m/z calcd for
C15H15ClFN5 320.10728, found 320.11303.
N6‑Benzyl‑N2‑[2‑(dimethylamino)ethyl]‑9‑isopropyl‑9H‑pu‑
rine‑2,6‑diamine (3c, C119H27N7) Yield 34%; m.p.: 87–89 °C;
LC–MS purity 96%; H NMR (700 MHz, DMSO-d6):
δ = 1.47 (d, J = 6.67 Hz, 6H), 2.16 (br s, 6H), 2.40 (br s,
2H), 3.30–3.33 (m, 2H), 4.54 (dt, J=13.50, 6.70 Hz, 1H),
4.63 (br s, 2H), 6.10 (br s, 1H), 7.18–7.22 (m, 1H), 7.29 (t,
J = 7.53 Hz, 2H), 7.34 (d, J = 7.74 Hz, 2H), 7.80 (s, 1H)
ppm; 13C NMR (176 MHz, DMSO-d6): δ = 22.52, 33.41,
43.11, 45.59, 46.12, 58.81, 114.15, 126.88, 127.69, 128.53,
135.54, 141.22, 151.20, 154.94, 159.38 ppm; HRMS (ESI):
m/z calcd for C19H27N7 354.24007, found 354.24650.
2‑Chloro‑N‑(3,5‑difluorobenzyl)‑9‑isopropyl‑9H‑pu‑
rin‑6‑amine (2d, C15H14ClF2N5) Yield 92%; m.p.: 182–
1
184 °C; H NMR (700 MHz, DMSO-d6): δ = 1.51 (d,
J = 6.45 Hz, 6H), 4.62–4.75 (m, 3H), 7.11–7.20 (m, 2H),
7.22–7.29 (m, 1H), 8.33 (s, 1H), 8.83 (br s, 1 H) ppm;
13C NMR (176 MHz, DMSO-d6): δ=22.57, 37.49, 47.24,
115.61 (dd, J=33.44, 8.8 Hz), 116.18, 117.14 (dd, J=33.44,
7.04 Hz), 119.03, 128.55, 140.19, 150.01, 153.02, 155.26,
155.91, 157.27, 157.86, 159.21 ppm; HRMS (ESI): m/z
calcd for C15H14ClF2N5 338.09786, found 338.10403.
N‑Benzyl‑9‑isopropyl‑2‑morpholino‑9H‑purin‑6‑amine (3d,
C19H24N6O) Yield 78%; m.p.: 158–159 °C; LC–MS purity
96.7%; 1H NMR (700 MHz, DMSO-d6): δ=(d, J=6.88 Hz,
6H), 3.61 (s, 8H), 4.53–4.67 (m, 3H), 7.18–7.22 (m,
1H), 7.29 (t, J = 7.64 Hz, 2H), 7.36 (d, J = 7.53 Hz, 2H),
7.88 (s, 1H), 8.03 (br s, 1H) ppm; 13C NMR (176 MHz,
DMSO-d6): δ=22.54, 43.50, 45.19, 46.24, 66.52, 114.38,
126.95, 127.89, 128.55, 136.46, 141.17, 150.84, 154.51,
158.74 ppm; HRMS (ESI): m/z calcd for C19H24N6O
353.20844, found 353.21753.
Amination of 2‑chloro‑6‑(substituted benzylamino)‑9‑iso‑
propylpurines (synthesis of 3a–3t) 2-Chloro-6-(substituted
benzylamino)-9-isopropylpurine 2 (3.3 mmol) and appro-
priate amine (49.5 mmol) were stirred at 160 °C for 8–24 h
depending on amine’s reactivity. After the reaction was com-
pleted, the mixture was allowed to cool and treated with 7
cm3 cold water for an hour. After that, it was extracted with
ethyl acetate (3×5 cm3). The organic layers were combined
1 3

Synthesis and biological evaluation of seliciclib derivatives as potent and selective CDK9…
117
N‑Benzyl‑9‑isopropyl‑2‑(piperazin‑1‑yl)‑9H‑purin‑6‑amine
(3e, C19H25N7) Yield 72%; m.p.: 116–118 °C; LC–MS
N‑(4‑Chlorobenzyl)‑9‑isopropyl‑2‑(piperazin‑1‑yl)‑9H‑pu‑
rin‑6‑amine (3j, C19H24ClN7) Yield 69%; LC–MS purity
1
1
purity 98.8%; H NMR (700 MHz, DMSO-d6): δ = 1.47
94.2%; H NMR (700 MHz, DMSO-d6): δ = 1.46 (d,
(d, J = 6.67 Hz, 6H), 2.68 (br s, 4H), 3.54–3.62 (m, 4H),
4.51–4.68 (m, 3H), 7.17–7.22 (m, 1H), 7.28 (t, J=7.53 Hz,
2H), 7.36 (d, J=7.53 Hz, 2H), 7.84 (s, 1H), 7.94 (br s, 1H)
ppm; 13C NMR (176 MHz, DMSO-d6): δ = 22.54, 43.50,
45.79, 46.00, 46.15, 114.03, 126.92, 127.90, 128.52, 136.17,
141.27, 151.01, 154.45, 158.85 ppm; HRMS (ESI): m/z
calcd for C19H25N7 352.22441, found 352.23135.
J = 6.67 Hz, 6H), 2.68 (br s, 4H), 3.57 (br s, 4H), 4.56
(dt, J = 13.18, 6.64 Hz, 3H), 7.32–7.39 (m, 4H), 7.85 (s,
1H), 7.99 (br s, 1H) ppm; 13C NMR (176 MHz, DMSO-
d6): δ = 22.52, 42.92, 45.67, 45.89, 46.18, 113.99,
128.47, 129.68, 131.41, 136.30, 140.32, 151.04, 154.32,
158.79 ppm; HRMS (ESI): m/z calcd for C19H24ClN7
386.18545, found 386.19246.
N‑(4‑Chlorobenzyl)‑9‑isopropyl‑2‑(4‑methylpipera‑
zin‑1‑yl)‑9H‑purin‑6‑amine (3f, C20H26ClN7) Yield 56%;
m.p.: 97–99 °C; LC–MS purity 95.9%; 1H NMR (700 MHz,
DMSO-d6): δ = 1.47 (d, J = 6.67 Hz, 6H), 2.18 (s, 3H),
2.29 (br s, 4H), 3.63 (br s, 4H), 4.49–4.66 (m, 3H), 7.36
(q, J = 8.53 Hz, 4H), 7.86 (s, 1H), 8.02 (br s, 1H) ppm;
13C NMR (176 MHz, DMSO-d6): δ=22.54, 42.94, 44.46,
46.17, 46.38, 54.96, 114.13, 128.48, 129.69, 131.42, 136.38,
140.30, 150.94, 154.36, 158.66 ppm; HRMS (ESI): m/z
calcd for C20H26ClN7 400.20116, found 400.20871.
N‑(4‑Fluorobenzyl)‑9‑isopropyl‑2‑(4‑methylpipera‑
zin‑1‑yl)‑9H‑purin‑6‑amine (3k, C20H26 FN7) Yield 88%; m.p.:
1
107–109 °C; LC–MS purity 99.4%; H NMR (700 MHz,
DMSO-d6): δ = 1.47 (d, J = 6.88 Hz, 6H), 2.19 (s, 3H),
2.27–2.36 (m, 4H), 3.65 (br s, 4H), 4.57 (dt, J = 13.50,
6.70 Hz, 3H), 7.11 (t, J=8.93 Hz, 2H), 7.39 (dd, J=8.39,
5.81 Hz, 2H), 7.86 (s, 1H), 7.99 (br s, 1H) ppm; 13C NMR
(176 MHz, DMSO-d6): δ = 22.54, 42.83, 44.47, 46.17,
46.39, 54.98, 114.13, 115.16 (d, J = 55.95 Hz), 129.79
(d, J = 7.63 Hz), 136.33, 137.41, 150.96, 154.39, 158.69,
160.79, 162.16 ppm; HRMS (ESI): m/z calcd for C20H26 FN7
384.23065, found 384.23702.
N6‑(4‑Chlorobenzyl)‑N2‑[3‑(dimethylamino)propyl]‑9‑isopro‑
pyl‑9H‑purine‑2,6‑diamine (3g, C20H28ClN7) Yield 81%; LC–
MS purity 96.7%; 1H NMR (700 MHz, DMSO-d6): δ=1.46
(d, J=6.88 Hz, 6H), 1.60 (br s, 2H), 2.09 (s, 6H), 2.20 (br s,
2H), 3.15–3.26 (m, 2H), 4.42–4.57 (m, 2H), 4.59 (br s, 1H),
6.31 (br s, 1H), 7.35 (q, J=8.53 Hz, 4H), 7.79 (s, 1H) ppm;
13C NMR (176 MHz, DMSO-d6): δ=22.53, 27.84, 42.60,
45.66, 46.05, 57.6, 79.64, 114.01, 128.46, 129.59, 131.38,
135.39, 140.33, 151.25, 154.79, 159.53 ppm; HRMS (ESI):
m/z calcd for C20H28ClN7 402.21675, found 402.22476.
N2‑[3‑(Dimethylamino)propyl]‑N6‑(4‑fuorobenzyl)‑9‑isopro‑
pyl‑9H‑purine‑2,6‑diamine (3l, C20H28FN7) Yield 85%; LC–
MS purity 98.7%; 1H NMR (700 MHz, DMSO-d6): δ=1.46
(d, J=6.67 Hz, 6H), 1.62 (br s, 2H), 2.10 (s, 6H), 2.21 (br s,
2H), 3.23 (q, J=6.53 Hz, 2H), 4.53 (dt, J=13.34, 6.67 Hz,
2H), 4.59 (br s, 1H), 6.31 (br s, 1H), 7.10 (t, J = 8.82 Hz,
2H), 7.39 (dd, J = 8.28, 5.92 Hz, 2H), 7.79 (s, 1H) ppm;
13C NMR (176 MHz, DMSO-d6): δ=22.53, 27.84, 37.44,
42.48, 45.87, 46.04, 57.69, 114.01, 115.20 (d, J=29.25 Hz),
129.69 (d, J = 7.63 Hz), 135.44, 137.43, 151.10, 154.80,
159.55, 160.77, 162.14 ppm; HRMS (ESI): m/z calcd for
C20H28FN7 386.2463, found 386.25355.
N6‑(4‑Chlorobenzyl)‑N2‑[2‑(dimethylamino)ethyl]‑9‑isopro‑
pyl‑9H‑purine‑2,6‑diamine (3h, C19H26ClN7) Yield 80%;
m.p.: 99–101 °C; LC–MS purity 98.3%; 1H NMR (700 MHz,
DMSO-d6): δ = 1.47 (d, J = 6.67 Hz, 6H), 2.12 (br s, 6H),
2.34 (br s, 2H), 3.29 (q, J = 6.02 Hz, 2H), 4.48–4.57 (m,
1H), 4.59 (br s, 2H) 6.09 (br s, 1H), 7.35 (s, 4H), 7.80 (s,
1H) ppm; 13C NMR (176 MHz, DMSO-d6): δ=22.51, 37.42,
42.55, 45.71, 46.14, 58.89, 114.14, 128.48, 129.51, 131.38,
135.62, 140.30, 151.21, 154.82, 159.36 ppm; HRMS (ESI):
m/z calcd for C19H26ClN7 388.20110, found 388.20881.
N2‑[2‑(Dimethylamino)ethyl]‑N6‑(4‑fluorobenzyl)‑9‑iso‑
propyl‑9H‑purine‑2,6‑diamine (3m, C19H26FN7) Yield
1
42%; m.p.: 95–97 °C; LC–MS purity 97.9%; H NMR
(700 MHz, DMSO-d6): δ=1.47 (d, J=6.67 Hz, 6H), 2.13
(br s, 6H), 2.35 (br s, 2H), 3.31 (q, J =6.45 Hz, 2H), 4.54
(dt, J=13.34, 6.67 Hz, 1H), 4.59 (br s, 2H), 6.09 (br s, 1H),
7.11 (t, J=8.82 Hz, 2H), 7.35–7.40 (m, 2H), 7.79 (s, 1H),
7.84 (br s, 1H) ppm; 13C NMR (176 MHz, DMSO-d6):
δ=22.51, 40.48., 42.48, 45.72, 46.13, 58.91, 114.14, 115.22
(d, J=40.69 Hz), 129.61 (d, J=8.90 Hz), 135.57, 137.39,
151.27, 154.84, 159.38, 160.77, 162.15 ppm; HRMS (ESI):
m/z calcd for C19H26FN7 372.23065, found 372.23750.
N‑(4‑Chlorobenzyl)‑9‑isopropyl‑2‑morpholino‑9H‑pu‑
rin‑6‑amine (3i, C19H23ClN6O) Yield 79%; m.p.: 142–144 °C;
1
LC–MS purity 95.8%; H NMR (700 MHz, DMSO-d6):
δ = 1.47 (d, J = 6.67 Hz, 6H), 3.60 (br s, 8H), 4.50–4.66
(m, 3H), 7.36 (q, J = 8.60 Hz, 4H), 7.88 (s, 1H), 8.08 (br
s, 1H) ppm; 13C NMR (176 MHz, DMSO-d6): δ = 22.53,
42.90, 45.16, 46.26, 86.51, 114.37, 128.50, 129.70, 131.44,
136.56, 140.24, 150.90, 154.41, 158.70 ppm; HRMS (ESI):
m/z calcd for C19H23ClN6O 387.1646, found 387.17714.
N‑(4‑Fluorobenzyl)‑9‑isopropyl‑2‑morpholino‑9H‑pu‑
rin‑6‑amine (3n, C19H23 FN6O) Yield 79%; m.p.: 149–151 °C;
1
LC–MS purity 98.5%; H NMR (700 MHz, DMSO-d6):
1 3

118
A. A. Alsfouk et al.
δ = 1.47 (d, J = 6.67 Hz, 6H), 3.61 (s, 8H), 4.58 (quin,
J = 6.67 Hz, 3H), 7.11 (t, J = 8.82 Hz, 2H), 7.39 (dd,
J = 8.17, 5.81 Hz, 2H), 7.88 (s, 1H), 8.05 (br s, 1H) ppm;
13C NMR (176 MHz, DMSO-d6): δ=22.54, 42.80, 45.18,
46.25, 66.52, 114.37, 115.26 (d, J=48.32 Hz), 129.79 (d,
J = 26.70 Hz), 136.52, 137.35, 150.87, 154.40, 158.72,
160.79, 162.17 ppm; HRMS (ESI): m/z calcd for C19H23
FN6O 371.19901, found 371.20758.
2H), 3.22–3.32 (m, 2H), 4.54 (dt, J = 13.39, 6.75 Hz,
1H), 4.65 (br s, 2H), 6.14 (br s, 1H), 7.11 (dd, J = 7.42,
3.98 Hz, 2H), 7.22 (td, J=9.41, 3.98 Hz, 1H), 7.83 (s, 1 H)
ppm; 13C NMR (176 MHz, DMSO-d6): δ = 22.50, 36.93,
45.63, 46.18, 58.84, 79.63, 114.13, 115.03 (dd, J=31.68,
7.04 Hz), 115.65, 116.90 (dd, J= 33.44, 8.8 Hz), 130.19,
135.67, 151.33, 154.75, 155.75, 157.10, 157.92, 159.27,
159.32 ppm; HRMS (ESI): m/z calcd for C19H25F2N7
390.22123, found 290.22781.
N‑(4‑Fluorobenzyl)‑9‑isopropyl‑2‑(piperazin‑1‑yl)‑9H‑pu‑
rin‑6‑amine (3o, C19H24FN7) Yield 94%; LC–MS purity
N‑(3,5‑Difluorobenzyl)‑9‑isopropyl‑2‑morpholino‑9H‑pu‑
rin‑6‑amine (3s, C19H22F2N6O) Yield 93%; m.p.: 141–143 °C;
1
95.3%; H NMR (700 MHz, DMSO-d6): δ = 1.47 (d,
1
J = 6.67 Hz, 6H), 2.64–2.74 (m, 4H), 3.54–3.63 (m, 4H),
4.49–4.63 (m, 3H), 7.11 (t, J = 8.82 Hz, 2H), 7.39 (dd,
J = 8.17, 5.81 Hz, 2H), 7.84 (s, 1H), 7.96 (br s, 1H) ppm;
13C NMR (176 MHz, DMSO-d6): δ=22.54, 42.79, 45.78,
45.99, 46.15, 114.02, 115.24 (d, J = 36.88 Hz), 129.77
(d, J = 7.63 Hz),136.23, 137.44, 151.04, 154.34, 158.83,
160.77, 162.15 ppm; HRMS (ESI): m/z calcd for C19H24FN7
370.2150, found 370.2269.
LC–MS purity 98.8%; H NMR (700 MHz, DMSO-d6):
δ = 1.47 (d, J = 6.88 Hz, 6H), 3.59 (s, 8H), 4.55–4.72 (m,
3H), 7.07–7.13 (m, 1H), 7.14–7.19 (m, 1H), 7.22 (td,
J=9.20, 4.41 Hz, 1H), 7.91 (s, 1H), 8.08 (br s, 1H) ppm; 13C
NMR (176 MHz, DMSO-d6): δ=22.52, 36.94, 45.08, 46.30,
66.46, 114.38, 115.18 (dd, J=33.44, 8.8 Hz), 116.07, 116.93
(dd, J = 33.44, 7.04 Hz), 129.99, 136.79, 151.02, 154.31,
155.31, 155.82, 157.18, 157.88, 159.24 ppm; HRMS (ESI):
m/z calcd for C19H22F2N6O 389.1859, found 389.19707.
N‑(3,5‑Difluorobenzyl)‑9‑isopropyl‑2‑(4‑methylpipera‑
zin‑1‑yl)‑9H‑purin‑6‑amine (3p, C20H25F2N7) Yield 84%;
m.p.: 103–105 °C; LC–MS purity 99.1%; 1H NMR
(700 MHz, DMSO-d6): δ=1.47 (d, J=6.67 Hz, 6H), 2.17
(s, 3H), 2.27 (br s, 4H), 3.62 (br s, 4H), 4.54–4.70 (m, 3H),
7.06–7.14 (m, 1H), 7.14–7.19 (m, 1H), 7.22 (td, J = 9.20,
4.41 Hz, 1H), 7.89 (s, 1H), 8.03 (br s, 1H) ppm; 13C NMR
(176 MHz, DMSO-d6): δ = 22.53, 36.98, 44.38, 46.22,
46.36, 54.91, 114.14, 115.14 (dd, J=33.44, 8.8 Hz), 115.96,
116.91(dd, J = 35.20, 8.8 Hz), 130.13, 136.59, 151.09,
154.27, 155.83, 157.20, 157.88, 158.58, 159.23 ppm;
HRMS (ESI): m/z calcd for C20H25 F2N7 402.22123, found
402.22991.
N‑(3,5‑Difuorobenzyl)‑9‑isopropyl‑2‑(piperazin‑1‑yl)‑9H‑pu‑
rin‑6‑amine (3t, C19H23F21N7) Yield 92%; m.p.: 167–169 °C;
LC–MS purity 98.5%; H NMR (700 MHz, DMSO-d6):
δ = 1.47 (d, J = 6.67 Hz, 6H), 2.66 (br s, 4H), 3.55 (br s,
4H), 4.57–4.61 (m, 3H), 7.07–7.13 (m, 1H), 7.14–7.19 (m,
1H), 7.22 (td, J=9.14, 4.52 Hz, 1H), 7.87 (s, 1H), 8.00 (br
s, 1H) ppm; 13C NMR (176 MHz, DMSO-d6): δ = 22.52,
36.97, 45.71, 45.94, 46.20, 114.03, 115.12 (dd, J=33.44,
8.8 Hz), 116.07, 116.90 (dd, J = 33.44, 8.8 Hz), 130.21,
136.49, 151.20, 154.26, 155.83, 157.19, 157.86, 158.74,
159.23 ppm; HRMS (ESI): m/z calcd for C19H23F2N7
388.20558, found 388.21513.
N6‑(3,5‑Difuorobenzyl)‑N2‑[3‑(dimethylamino)propyl]‑9‑iso‑
propyl‑9H‑purine‑2,6‑diamine (3q, C20H27F2N7) Yield 83%;
Kinase inhibition assays
1
LC–MS purity 91.3%; H NMR (700 MHz, DMSO-d6):
δ=1.47 (d, J=6.67 Hz, 6H), 1.58 (br s, 2H), 2.08 (s, 6H),
2.18 (br s, 2H), 3.14–3.25 (m, 2H), 4.54 (dt, J = 13.28,
6.59 Hz, 1H), 4.65 (br s, 2H), 6.36 (br s, 1H), 7.06–7.15
(m, 2H), 7.22 (td, J=9.14, 4.52 Hz, 1H), 7.82 (s, 1H) ppm;
13C NMR (176 MHz, DMSO-d6): δ=22.51, 27.79, 36.89,
40.43, 45.62, 46.10, 57.63, 114.04, 114.55 (dd, J=33.44,
10.56 Hz), 115.80, 116.87 (dd, J=33.44, 8.8 Hz), 130.14,
135.73, 151.38, 154.76, 155.78, 157.14, 157.91, 159.26,
159.48 ppm; HRMS (ESI): m/z calcd for C20H27F2N7
404.23688, found 404.24336.
The CDK2 assay kit was purchased from Promega Cor-
poration ( Madison, WI, USA) and the anti-CDK2 efect
was measured following the manufacturer’s instructions
(CDK2/CyclinA2 Kinase Assay Catalogue# V2971). The
remaining kinase assay kits (CDK5, 7, and 9) crystallized
from BPS Biosciences (San Diego, CA, USA) and the anti-
CDK efect was measured following the manufacturer’s
instructions (CDK5 Kinase Assay Kit Catalogue# 79,600,
CDK7 Kinase Assay Kit Catalogue# 79,603, CDK9
Kinase Assay Kit Catalogue# 79,628). The kinase inhibi-
tory activity was expressed as IC50 for three individual
experiments. In all kinase assays, seliciclib was used as
positive control and DMSO was used as negative control.
N6‑(3,5‑Difuorobenzyl)‑N2‑[2‑(dimethylamino)ethyl]‑9‑iso‑
propyl‑9H‑purine‑2,6‑diamine (3r, C19H25F2N7) Yield 79%;
1
LC–MS purity 97.1%; H NMR (700 MHz, DMSO-d6):
δ = 1.47 (d, J = 6.67 Hz, 6H), 2.11 (br s, 6H), 2.32 (br s,
1 3

Synthesis and biological evaluation of seliciclib derivatives as potent and selective CDK9…
119
2. Shapiro GI (2006) J Clin Oncol 24:1770
MTT cytotoxicity assay
3. Shapiro GI, Edwards CD, Rollins BJ (2000) Cell Biochem Bio-
phys 33:189
PC3 cells were treated with compounds in a concentration
range from 0.1 to 10 µM for 48 h, 10% v/v reconstituted
MTT was added and incubated for 3 h before reading the
plates on Wallac Victor2 1420 multilabel counter in fuores-
cence mode at a wavelength of 460/590 nm (ex/em). Cyto-
toxicity was expressed as IC50 for three individual experi-
ments. Seliciclib was used as positive control and DMSO
was used as negative control in this assay.
4. Yik JHN, Chen R, Nishimura R, Jennings JL, Link AJ, Zhou Q
(2003) Mol Cell 12:971
5. Baumli S, Lolli G, Lowe ED, Troiani S, Rusconi L, Bullock AN,
Debreczeni JÉ, Knapp S, Johnson LN (2008) EMBO J 27:1907
6. Morales F, Giordano A (2016) Cell Cycle 15:519
7. Wang J, Dean DC, Hornicek FJ, Shi H, Duan Z (2019) FASEB
J 33:5990
8. Ma H, Seebacher NA, Hornicek FJ, Duan Z (2019) EBioMedi-
cine 39:182
9. Kretz AL, Schaum M, Richter J, Kitzig EF, Engler CC, Leithäu-
ser F, Henne-Bruns D, Knippschild U, Lemke J (2017). Tumor
Biol. https://doi.org/10.1177/1010428317694304
10. Franco LC, Morales F, Bofo S, Giordano A (2018) J Cell Bio-
chem 119:1273
S9 metabolic stability assay
The tested inhibitors (5 µM) were incubated with Tris-bufer
(0.1 M), mouse liver S9, NADPH, PAPS, and UDPGA at
37 °C for 1 h. Then the protein in the samples was partici-
pated, centrifuged, and fltered. The disappearance of the
parent compounds was analysed using LC–MS/MS.
11. Bofo S, Damato A, Alfano L, Giordano A (2018) J Exp Clin
Cancer Res 37:36
12. McInnes C (2008) Drug Discov Today 13:875
13. Jacobs A (1997) Understanding organic reaction mechanisms.
Cambridge University Press, Cambridge
14. Krystof V, Baumli S, Furst R (2012) Curr Pharm Des 18:2883
15. Siegel RL, Miller KD, Jemal A (2018) CA Cancer J Clin 68:7
16. Mellado B, Codony J, Ribal MJ, Visa L, Gascón P (2009) Clin
Transl Oncol 11:5
Kinetic solubility assay
17. Rahaman MH, Kumarasiri M, Mekonnen LB, Yu M, Diab S,
Albrecht H, Milne RW, Wang S (2016) Endocr Relat Cancer
23:211
The tested compounds (30 µM) in DMSO were diluted with
aqueous bufer and turbidimetry was measured as end point
at 450 nm absorbance.
18. Gordon V, Bhadel S, Wunderlich W, Zhang JA, Ficarro SB,
Mollah SA, Shabanowitz J, Hunt DF, Xenarios I, Hahn WC,
Conaway M, Carey MF, Gioeli D (2010) Mol Endocrinol
24:2267
Molecular docking
19. Pawar A, Gollavilli PN, Wang S, Asangani IA (2018) Cell Rep
22:2236
20. Senderowicz AM (1999) Invest New Drugs 3:313
21. Kumar SK, Fruth B, Roy V, Erlichman C, Stewart AK (2015)
Blood 125:443
The three-dimensional crystal structure of the proteins
(PDB: 3LQ5, 2A4L) was obtained from RCSB Protein Data
Bank. The programs utilized for performing the molecular
docking were Discovery Studio, ChemBio3D Ultra 14.0
(PerkinElmer informatics), and PyRx (the Scripps Research
Institute, La Jolla, CA, USA). The preparation of protein was
conducted using Discovery Studio, which involved remov-
ing water and sulfate molecules as well as the co-crystal-
lized compound, and it was saved as a PDB fle. Then, the
AutoDock Tools in PyRx were used to introduce the polar
hydrogens and it was saved as a PDBQT fle. The ligands
(compound 3 l and seliciclib) were prepared using Chem-
Bio3D Ultra 14.0 and saved as PDB fles. Lastly, Grid box
and AutoDock Vina in PyRx software were used to perform
the docking. The minimum energy pose of 3 l was compared
with that of seliciclib.
22. Benson C, White J, De BJ, Donnell AO, Raynaud F, Cruick-
shank C, Mcgrath H, Walton M, Workman P, Kaye S, Cassidy
J, Judson I, Twelves C (2007) Br J Cancer 96:29
23. Tong W, Chen R, Plunkett W, Siegel D, Sinha R, Harvey RD,
Badros AZ, Popplewell L, Coutre S, Fox JA, Mahadocon K,
Chen T, Kegley P, Hoch U, Wierda WG (2010) J Clin Oncol
28:3015
24. Van Der BDAJ, Burger H, De BP, Lamers CHJ, Naus N, Loferer
H, Wiemer EAC, Mathijssen RHJ, DeJonge MJA (2014) Clin
Cancer Res 20:4776
25. Le Tourneau C, Faivre S, Laurence V, Delbaldo C, Vera K, Girre
V, Chiao J, Armour S, Frame S, Green SR, Gianella-Borradori A,
Diéras V, Raymond E (2010) Eur J Cancer 46:3243
26. Walsby E, Pratt G, Shao H, Abbas AY, Fischer PM, Bradshaw TD,
Brennan P, Fegan C, Wang S, Pepper C (2014) Oncotarget 5:375
27. Zhai S, Senderowicz AM, Sausville EA, Figg WD (2002) Ann
Pharmacother 36:905
28. Chao SH, Price DH (2001) J Biol Chem 276:31793
29. Blagosklonny MV (2004) Cell Cycle 12:1537
30. Cersosimo RJ (2012) Ann Pharmacother 46:1518
31. Lücking U, Scholz A, Lienau P, Siemeister G, Kosemund D,
Bohlmann R, Briem H, Terebesi I, Meyer K, Prelle K, Denner K,
Bömer U, Schäfer M, Eis K, Valencia R, Ince S, von Nussbaum F,
Mumberg D, Ziegelbauer K, Klebl B, Choidas A, Nussbaumer P,
Baumann M, Schultz-Fademrecht C, Rühter G, Eickhof J, Brands
M (2017) ChemMedChem 12:1776
Acknowledgements This research was funded by the Deanship of
Scientifc Research at Princess Nourah bint Abdulrahman University,
through the Research Funding Program (Grant No#FRP-1440-8).
References
32. Sonawane YA, Taylor MA, Napoleon JV, Rana S, Contreras JI,
Natarajan A (2016) J Med Chem 59:8667
1. Malumbres M, Barbacid M (2009) Nat Rev Cancer 9:153
1 3

120
A. A. Alsfouk et al.
33. Byth KF, Thomas A, Hughes G, Forder C, McGregor A, Geh C,
Oakes S, Green C, Walker M, Newcombe N, Green S, Growcott
J, Barker A, Wilkinson RW (2009) Mol Cancer Ther 8:1856
34. Mariaule G, Belmont P (2014) Molecules 19:14366
35. Heathcote DA, Patel H, Kroll SH, Hazel P, Periyasamy M, Alikian
M, Kanneganti SK, Jogalekar AS, Scheiper B, Barbazanges M,
Blum A (2010) J Med Chem 53:8508
46. McClue SJ, Stuart I (2008) Drug Metab Dispos 36:561
47. Oxane K, Noßke E, Maurer M, Węsierska-Gądek J (2011) J Exp
Ther Oncol 9:1
48. Zatloukal M, Jorda R, Gucký T, Řezníčková E, Voller J, Pospíšil
T, Malínková V, Adamcová H, Kryštof V, Strnad M (2013) Eur J
Med Chem 61:61
49. Imbach P, Capraro H-G, Furet P, Mett H, Meyer T, Zimmermann
J (1999) Bioorg Med Chem Lett 9:91
36. Parry D, Guzi T, Shanahan F, Davis N, Prabhavalkar D, Wiswell
D, Seghezzi W, Paruch K, Dwyer MP, Doll R, Nomeir A, Windsor
W, Fischmann T, Wang Y, Oft M, Chen T, Kirschmeier P, Lees
EM (2010) Mol Cancer Ther 9:2344
50. Veselý J, Havliček L, Strnad M, Blow JJ, Arianna Donella-Deana
Lorenzo P, Letham DS, Kato J, Detivaud L, Leclerc S, Meijer L
(1994) Eur J Biochem 224:771
37. Conroy A, Stockett DE, Walker D, Arkin MR, Hoch U, Fox JA,
Hawtin RE (2009) Cancer Chemother Pharmacol 64:723
38. Cirstea D, Hideshima T, Santo L, Eda H, Mishima Y, Nemani
N, Hu Y, Mimura N, Cottini F, Gorgun G, Ohguchi H, Suzuki R,
Loferer H, Munshi NC, Kenneth C, Raje N, Hospital MG, Lipper
J, Myeloma M (2014) Leukemia 27:2366
51. Havlıcek L, Hanus J, Vesely J, LeClerc S, Meijer L, Shaw G,
Strnad M (1997) J Med Chem 40:408
52. Chang Y, Gray NS, Rosania GR, Sutherlin DP, Kwon S, Norman
TC, Sarohia R, Leost M, Meijer L, Schultz PG (1999) Chem Biol
6:361
53. Schow SR, Mackman RL, Blum CL, Brooks E, Horsma AG, Joly
A, Kerwar SS, Lee G, Shifman D, Nelson MG, Wang X, Wick
MM, Zhang X, Lum RT (1997) Bioorg Med Chem Lett 7:2697
54. Arisan E, Obakan P, Coker-Gurkan A, Calcabrini A, Agostinelli
E, Unsal N (2014) Curr Pharm Des 20:180
39. Knockaert M, Reinhardt J, Lozach O, Schmitt S, Baratte B, Koken
M, Coburn SP, Tang L, Jiang T, Liang D, Dierick J, Pinna LA,
Meggio F, Totzke F, Lerman AS, Carnero A, Wan Y, Gray N,
Meijer L (2005) J Biol Chem 280:31208
40. McClue SJ, Blake D, Clarke R, Cowan A, Cummings L, Fischer
PM, MacKenzie M, Melville J, Stewart K, Wang S, Zhelev N
(2002) Int J Cancer 102:463
55. Meijer L, Borgne A, Mulner O, Chong JPJ, Blow JJ, Inagaki N,
Inagaki M, Delcros JG, Moulinoux JP (1997) Eur J Biochem
243:527
41. Yeo W, Goh B, Le TC, Green SR, Chiao JH, Siu LL (2009) J Clin
Oncol 27:6026
56. Jean DJS, Fotsch C (2012) J Med Chem 55:6002
57. Wu Y-J (2012) Prog Heterocycl Chem 24:1
58. Steklov MY, Tararov VI, Romanov GA, Mikhailov SN (2011)
Nucleosides. Nucleotides Nucleic Acids 30:503
42. Whittaker SR, Walton MI, Garrett MD, Workman P (2004) Cancer
Res 64:262
43. Wilson SC, Atrash B, Barlow C, Eccles S, Fischer PM, Hayes A,
Kelland L, Jackson W, Jarman M, Mirza A, Moreno J, Nutley BP,
Raynaud FI, Sheldrake P, Walton M, Westwood R, Whittaker S,
Workman P, Mcdonald E (2011) Bioorg Med Chem 19:6949
44. Aldoss IT, Tashi T, Ganti AK (2009) Expert Opin Investig Drugs
18:1957
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
45. Nutley BP, Raynaud FI, Wilson SC, Fischer PM, Hayes A, God-
dard PM, Mcclue SJ, Jarman M, Lane DP, Workman P (2005) Mol
Cancer Ther 4:125
1 3