Design, synthesis and in vitro apoptotic mechanism of novel pyrrolopyrimidine derivatives

Article abstract of DOI:10.1016/j.bioorg.2018.10.060

In this work we described the synthesis and evaluation of cytotoxic and apoptotic activity of novel pyrrolopyrimidine derivatives against A549, PC3 and MCF-7 cells. Among the synthesized compounds, 6b, 8a, 9a and 7a, 8b displayed the significant cytotoxic

Full text of DOI:10.1016/j.bioorg.2018.10.060

Accepted Manuscript
Design, synthesis and in vitro apoptotic mechanism of novel pyrrolopyrimidine
derivatives
Zühal Kilic-Kurt, Filiz Bakar-Ates, Yeliz Aka, Ozgur Kutuk
PII:
S0045-2068(18)30603-5
DOI:
https://doi.org/10.1016/j.bioorg.2018.10.060
YBIOO 2594
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 June 2018
16 October 2018
29 October 2018
Please cite this article as: Z. Kilic-Kurt, F. Bakar-Ates, Y. Aka, O. Kutuk, Design, synthesis and in vitro apoptotic
mechanism of novel pyrrolopyrimidine derivatives, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/
j.bioorg.2018.10.060
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Design, synthesis and in vitro apoptotic mechanism of novel
pyrrolopyrimidine derivatives
Zühal Kilic-Kurta,*, Filiz Bakar-Atesb, Yeliz Akac and Ozgur Kutukc
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University,
Ankara, Turkey; bDepartment of Biochemistry, Faculty of Pharmacy, Ankara
University, Ankara, Turkey; cDepartment of Medical Genetics, School of Medicine,
Baskent University, Adana, TURKEY
*Zühal Kilic-Kurt, zkurt@ankara.edu.tr, Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, Ankara University, Ankara, Turkey
Abstract
In this work we described the synthesis and evaluation of cytotoxic and apoptotic activity of
novel pyrrolopyrimidine derivatives against A549, PC3 and MCF-7 cells. Among the
synthesized compounds, 6b, 8a, 9a and 7a, 8b displayed the significant cytotoxic activities against
A549 and PC3 cells with IC50 value of 0.35, 1.48, 1.56 and 1.04, 1.89 µM, respectively. It
wasfound that A549 cells were more sensitive to synthesized compounds than PC3 and MCF-7
cells. In order to evaluate the mechanism of cytotoxic activity in A549, compounds 6b, 8a and 9a
were selected for further studies. Annexin V binding assay and western blot analysis results revealed
that 6b, 8a and 9a induced apoptosis in A549 cells by intrinsic apoptotic pathway through the
activation pro-apoptotic proteins such as Bim, Bax, Bak, Puma and deactivation of anti-apoptotic
proteins including Bcl-2, Mcl-1 and Bcl-XL accompanied by the activation of caspase-3, caspase-9
and cleavage of PARP. Also, compounds 6b, 8a and 9a triggered apoptosis in HCT116 wt cells via
activation of caspase-3 and caspase-9, but not in HCT116 Bax/Bak KO cells, indicating resistance to
6b, 8a and 9a treatment.
Keywords: pyrrolopyrimidine derivatives; cytotoxicity; western blot analysis,
apoptosis mechanism

Introduction
Apoptosis, programmed cell death, plays essential role in the cellular development and
differentiation (homeostasis) [1]. Dysregulation of apoptosis lead to a variety of human
diseases, such as cancer, autoimmune disease and neurodegenerative disorders [2]. Two
types apoptotic pathways have been extensively described, including the extrinsic and
intrinsic pathway. These two process are highly regulated by Bcl-2 family of proteins
which are structurally and functionally classified as either anti-apoptotic (e.g. Bcl-2,
Bcl-xL, Bcl-w, Mcl-1) and pro-apoptotic (e.g. Bak, Bax, Bok) or BH-3 only pro-
apoptotic Bcl-2 proteins (Bad, Bim, Bmf, Bik, Hrk, Bid, Puma, Noxa) [3, 6].
Overexpression of anti-apoptotic proteins or the downregulation of pro-apoptotic
proteins is associated with resistance to cell death in cancer cells. Developing small
molecules that activate and induce apoptosis is promising strategy for the treatment of
cancer [7].
A number of promising new compounds with pyrrolo[2,3-d]pyrimidine scaffold have
been reported to possess anti-inflammatory [8], antimicrobials [9-10], and antiviral [11-
13] and anticancer [14-24] activities. Over the past years pyrrolo[2,3-d]pyrimidine-
based compounds have become one of the most extensively studied classes of
heterocycles in cancer drug discovery and development compounds. Well-known
anticancer agent pyrrolopyrimidine derivative Pemetrexed (Figure 1) is clinically
usedas thymidylate synthase (TS) inhibitors in malignant pleural mesothelioma and
first-line treatment in combination with cisplatin for the treatment of non-small cell lung
cancer (NSCLC) [14]. Toyocamycin [15] and Sangivamycin [16] (Figure 1), naturally
occurring nucleoside antibiotics, have been reported as inhibitors of protein kinase C
(PKC) and/or protein kinase A (PKA). Tofacitinib [17] and Ruxolitinib [18] (Figure 1)
bearing pyrrolopyrimidine scaffold are approved as small-molecule Janus-associated
kinase (JAK) inhibitors by FDA for the treatment of rheumatoid arthritis (RA) and
myelofibrosis, respectively. EC144 was found as a second generation inhibitor of
heatshock protein 90 (Hsp90) by Bimonte group with improved in vitro and in vivo
potency (IC50 = 1.1 nM) [19]. MLN4924 is a small-molecule inhibitor of NEDD8-
activating enzyme (NAE) with IC50 value of 4 nM and suppressed the growth of human
tumor xenografts in mice at well-tolerated concentrations [20-23]. Furthermore, it was
shownto inhibit tumor angiogenesis in in vivo models and to overcome resistance of
cancer cells to cell death. Compound I, 6-substituted pyrrolo[2,3-d]pyrimidine with a

thienoyl side chain, were reported as an inhibitor of KB and IGROV1 cell proliferation
and of catalytic activity of the target enzyme GARFTase both in vitro and in situ [24].
Compound II, 4,6-bis-anilino-1H-pyrrolo[2,3-d]pyrimidine derivative was discovered
as a nonselective inhibitor of the insulin-like growth factor 1 receptor (IGF-1R) [25].
Moreover, there are many pyrrolopyrimidine-based compounds showing anticancer
activity through different mechanims have been reported in the literatures [26-29].
Figure 1. Pyrrolopyrimidine derivatives having anticancer activities.
Urea derivatives have received great attention as potent anticancer agents. It has been
known that some urea-based compounds are used in treatment of various cancers
through a variety of mechanisms. Nitrosoureas, DNA alkylating agents, and
Nilutamide, androgen receptor agonist, are well-known compounds among the
anticancer agents [30]. Multikinase inhibitors bearing urea moiety such as Sorafenib
(Figure 2, VEGFR, PDGFR and Raf inhibitor) [31] and Regorafenib (Figure 2,
VEGFR1-3, Tie-2, PDGFR-β, FGFR, Kit, Ret, and Raf inhibitor) [32] have been
clinically approved for renal cell and metastatic colorectal cancer treatment,
respectively. Linifanib (Figure 2, VEGFR and PDGFR inhibitor) [33] is currently in
active clinical trials for non-small cell lung cancer, hepatocellular carcinoma, colorectal
cancer and acute myeloid leukemia treatment. In addition, several urea based
multikinase ihibitors (Figure 2), including Quizartinib [34], Tandutinib [35], BIRB-
796 [36], CP-547632 [37], compounds III-V [38-40] were reported in the literature.

Figure 2. Urea derivatives with anticancer activity.
Recently, several pyrrolopyrimidine derivatives which were substituted with
urea motif at 4-position of pyrrolopyrimidine ring were reported as anticancer agents
[29, 41]. Understanding that urea moiety and pyrrolopyrimidine scaffold are found in
compounds which are approved and in clinical trials for cancer treatment, and
pyrrolopyrimidine derivatives containing urea motif at 2-position have not been
explored as anticancer compounds in published reports encourage us to design novel
pyrrolo[2,3-d]pyrimidine derivatives with urea moiety at 2-position and to evaluate
their anticancer activity against breast (MCF-7), lung (A549), prostate (PC3) and colon
(HCT116) cancer cell lines.
Materials and methods
Chemistry
Synthesis of 2-amino-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (3)
The mixture of 2,4-diamino-6-hydroxypyrimidine (1, 5.14 g; 0.04 mol) and sodium
acetate (3.34 g; 0.04 mol) was dissolved in the mixture of DMF:H2O (5:1) at room
temperature. Then, chloroacetaldehyde (5.22 ml) was added and the mixture was stirred
for 48 h at room temperature. After completion of the reaction, solvent was evaporated

under vacuum and the brown solid was dissolved in water (25 mL). The resulting
precipitate was filtered. The filtrate was evaporated until one third of the solvent
volume was remained and the ethanol was added. The resulting precipitate was
collected and washed with little cold water and acetone to afford the compound 3 (5.28
g, 74%) as beige solid [42].
Synthesis of 4-chloro-7H-pyrrolo[2,3-d]pyrimidin-2-amine (4)
Compound 3 (2.95 g; 0.02 mol) was suspended in POCl3 (25 mL) and the mixture was
refluxed for 2 h. After cooling to room temperature, excess of POCl3 was
evaporatedunder vacuum and the residue was treated with ice water. The resulting
precipitate was filtered off and the pH of the filtrate was adjusted to 7 with aqueous
ammonia. Then, the filtrate was extracted with EtOAc (3x50mL). The organic phase
was dried over sodium sulfate and evaporated to afford compound 4 (0.33 g, 10%),
which was used for the next reaction without further purification [42].
Synthesis of 4-chloro-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-amine (5)
4-Chloro-7H-pyrrolo[2,3-d]pyrimidin-2-amine (4, 0.248 g; 1.47 mmol) was dissolved in
anhydrous DMF (3mL) and cooled to 0°C, NaH (35 mg; 1.47 mmol) was added into the
mixture and stirred for 1h. Then, MeI (0.1 ml; 1.62 mmol) was added dropwise to the
reaction mixture and stirring was continued overnight at room temperature. The reaction
was quenched with water and the solution was extracted with EtOAc (3x20 mL). The
organic phase was dried with anhydrous Na2SO4 and evaporated under vacuum. Crude
product was purified by column chromatography with 9:1 hexane/ethylacetate to afford
pure compound 5 (0.159 g; 38%), [43].
General procedure for the synthesis of compounds 6-10
4-Chloro-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-amine (5, 1 eq) was dissolved in
anhydrous dioxane under nitrogen atmosphere. Et3N (2.5 eq) and triphosgene (1 eq) in
anhydrous dioxane were added to the mixture dropwise at 0 °C and stirred for 5 h at the
room temperature. Then, corresponding anilines were added and the stirring was
continued overnight. At the end of the time, the solvent was evaporated and acetone was
added. Resulting precipitate was filtered and washed with cold water to afford desired
compounds 6-10 in moderate yield.

1-(4-Chloro-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-3-(3-(trifluoromethyl)phenyl)
urea (6)
Yield 47%; mp: 216 °C; 1H NMR (400 MHz, DMSO-d6): δ= 3.84 (s, 3H, CH3), 6.54 (d,
1H, J= 3.2 Hz, H-5), 7.39 (d, 1H, J= 8 Hz), 7.51 (d, 1H, J= 4 Hz, H-6 ), 7.57 (t, 1H),
13
7.70 (d, 1H, J= 8.8 Hz), 8.15 (s, 1H), 10.28 (s, 1H, NH), 11.38 (s, 1H, NH); C NMR
(100 MHz, DMSO-d6): δ= 31.37, 98.66, 112.07, 114.99, 119.15, 122.68, 125.39,
129.61, 130.01, 130.75, 139.40, 150.62, 150.93, 151.18, 151.81. MS (ESI, 70 eV) m/z:
370.30 (M+H), 372.28 (M+H+2).
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-chloro-7-methyl-7H-pyrrolo[2,3-d]
pyrimidin-2-yl)urea (7)
Yield 41%; mp: 244 °C; 1H NMR (400 MHz, DMSO-d6): δ= 3.84 (s, 3H, CH3), 6.52 (d,
1H, J= 3.2 Hz, H-5), 7.52 (d, 1H, J= 3.6 Hz, H-6 ), 7.65 (d, 1H, J= 8.4 Hz), 7.72 (dd,
1H, Jo= 8.4 Hz, Jm= 2.4 Hz), 8.22 (d, 1H, Jm= 2.4 Hz), 10.38 (s, 1H, NH), 11.43 (s, 1H,
NH). 13C NMR (100 MHz, DMSO-d6): δ= 31.38, 98.64, 112.09, 117.53, 121.28, 123.40,
123.80, 126.74, 130.80, 132.04, 138.08, 150.46, 150.92, 151.12, 151.63. MS (ESI, 70
eV) m/z: 404.12 (M+), 406.62 (M+2).
1-(4-Chloro-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-3-(4-fluoro-3-(trifluoromethyl)
phenyl)urea (8)
1
Yield 75%; mp: 248-250 °C; H NMR (400 MHz, DMSO-d6): δ= 3.81 (s, 3H, CH3),
6.51 (d, 1H, J= 3.6 Hz, H-5), 7.43-7.49 (m, 2H), 7.72-7.74 (m, 1H), 8.12 (d, 1H, J= 4
13
Hz), 10.29 (s, 1H, NH), 11.31 (s, 1H, NH). C-NMR spectra could not be measured
since compound 8 is not enough soluble in DMSO-d6. MS (ESI, 70 eV) m/z: 388.20
(M+H), 389.77 (M+2).
1-(4-Chloro-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-3-(4-fluorophenyl)urea (9)
Yield 51%; mp: 260 °C; 1H NMR (400 MHz, DMSO-d6): δ= 3.75 (s, 3H, CH3), 6.75 (d,
1H, J= 3.6 Hz, H-5), 7.13 (t, 2H), 7.65 (d, 1H, J= 4.0 Hz, H-6), 7.80-7.96 (m, 2H),
13
10.27 (s, 1H, NH), 11.99 (s, 1H, NH). C NMR (100 MHz, DMSO-d6): δ= 30.40,
99.35, 110.75, 114.27, 115.49, 120.94, 121.6, 126.41, 130.05, 150.95, 151.60, 152.86.
MS (ESI, 70 eV) m/z: 320.15 (M+H), 322.26 (M+H+2).

1-(4-Chloro-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-3-(2,4-difluorophenyl)urea
(10)
Yield 68%; mp: 268 °C; 1H NMR (400 MHz, DMSO-d6): δ= 3.77 (s, 3H, CH3), 6.51 (d,
1H, J= 3.6 Hz, H-5), 7.03-7.08 (m, 1H), 7.32-7.38 (m, 1H), 7.48 (d, 1H, J= 4.0 Hz, H-
6), 8.25-8.32 (m, 1H), 10.39 (s, 1H, NH), 11.46 (s, 1H, NH). 13C NMR (100 MHz,
DMSO-d6): δ= 31.12, 98.77, 103.57, 103.80, 111.13, 112.08, 121.37, 123.55, 130.71,
150.44, 150.56, 150.96, 151.05, 151.89. MS (ESI, 70 eV) m/z: 338.40 (M+H).
General procedure for the synthesis of compounds 6-8a, 9a, 10a
Compounds 6-10 (1 eq) were dissolved in isopropanol and corresponding anilines (2 eq)
and 2-3 drops of conc. HCl was added and refluxed for 10-24 h. The reaction mixture
was cooled to room temperature and resulting precipitate was filtered and washed with
isopropanol. The crude product was purified by recrystallization from ethanol to give
pure compounds 6-8a, 9a, 10a.
1-(4-((3-Bromophenyl)amino)-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-3-(3-
(trifluoromethyl)phenyl)urea (6a)
1
Yield 36%; mp: 262-264 °C; H NMR (400 MHz, DMSO-d6): δ= 3.75 (s, 3H, CH3),
6.77 (d, 1H, J= 2.8 Hz, H-5), 7.16 (d, 1H, J= 3.6 Hz, H-6), 7.23-7.30 (m, 2H), 7.35 (d,
1H, J= 7.2 Hz), 7.44-7.52 (m, 2H), 7.99-8.0 (m, 3H), 10.08 (s, 2H, NH), 11.77 (s, 1H,
13
NH). C NMR (100 MHz, DMSO-d6): δ= 31.05, 99.37, 99.53, 114.86, 119.0, 120.33,
121.67, 122.57, 123.39, 125.80, 126.05, 129.39, 129.71, 129.89, 130.75, 139.44,
140.57, 149.17, 152.32. MS (ESI, 70 eV) m/z: 507.07 (M+2). Anal. calcd for
C21H16BrF3N6O·1.2 HCl: C 46.05, H 3.16, N 15.35, found: C 45.99, H 3.08, N 15.53.
1-(4-((3-Chloro-4-fluorophenyl)amino)-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-3-
(3-(trifluoromethyl)phenyl)urea (6b)
Yield 63%; mp: 258 °C; 1H NMR (400 MHz, DMSO-d6): δ= 3.78 (s, 3H, CH3), 6.77 (d,
1H, J= 3.2 Hz, H-5), 7.18 (d, 1H, J= 3.2 Hz, H-6), 7.34 (t, 1H), 7.38 (d, 1H, J= 7.2 Hz),
7.49-7.55 (m, 2H), 7.91-7.95 (m, 1H), 8.01 (s, 1H), 8.18 (dd, 1H, Jo= 6.4 Hz, Jm= 2.8
Hz), 9.92 (s, 1H, NH), 10.10 (s, 1H, NH), 11.90 (s, 1H, NH). 13C NMR (100 MHz,

DMSO-d6): δ= 30.08, 99.54, 99.95, 115.33, 117.07, 117.29, 119.49, 119.76, 119.95,
121.87, 122.80, 123.05, 126.04, 129.90, 130.21, 130.40, 137.10, 140.04, 149.67,
152.75, 153.35. MS (ESI, 70 eV) m/z: 479.30 (M+H), 481.27 (M+H+2). Anal. calcd for
C21H16ClF4N6O·1.5 H2O: C 49.86, H 3.58, N 16.61, found: C 49.94, H 3.23, N 16.76.
1-(4-((3-Bromophenyl)amino)-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-3-(4-chloro-
3-(trifluoromethyl)phenyl)urea (7a)
1
Yield 86%; mp: 265 °C.; H NMR (400 MHz, DMSO-d6): δ= 3.75 (s, 3H, CH3), 6.71
(d, 1H, J= 3.6 Hz, H-5), 7.14 (d, 1H, J= 3.6 Hz, H-6), 7.18 (d, 1H, J= 8.0 Hz), 7.24 (t,
1H), 7.54 (d, 1H, Jo= 8.8 Hz), 7.59 (d, 1H, J= 8.8 Hz), 7.99 (s, 1H), 8.02 (d, 1H, J= 8.4
Hz), 9.63 (s, 1H, NH), 9.97 (s, 1H, NH), 12.02 (s, 1H, NH). 13C NMR (100 MHz,
DMSO-d6): δ= 30.91, 99.15, 99.86, 117.50, 119.64, 121.51, 122.67, 123.03, 123.77,
125.12, 125.29, 126.53, 130.50, 131.86, 138.40, 141.41, 149.57, 152.01, 152.13,
153.26. MS (ESI, 70 eV) m/z: 540.73 (M+H), 542.15 (M+H+2). Anal. calcd for
C21H15BrClF3N6O·0.1 H2O: C 46.57, H 2.82, N 15.51, found: C 46.22, H 2.94, N 15.51.
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-((3-chloro-4-fluorophenyl)amino)-7-
methyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)urea (7b)
1
Yield 79%; mp: 276-278 °C, H NMR (400 MHz, DMSO-d6): δ= 3.74 (s, 3H, CH3),
6.68 (d, 1H, J= 3.6 Hz, H-5), 7.13 (d, 1H, J= 3.6 Hz, H-6), 7.29 (t, 1H), 7.57-7.63 (m,
2H), 7.86-7.90 (m, 1H), 7.96 (d, 1H, J= 2 Hz), 8.13 (d, 1H, Jo= 6.8 Hz, Jm= 2.8 Hz),
9.62 (s, 1H, NH), 10.02 (s, 1H, NH), 12.0 (s, 1H, NH). MS (ESI, 70 eV) m/z: 513.16
13
(M+), 515.69 (M+2). C NMR (100 MHz, DMSO-d6): δ= 30.93, 98.60, 99.56, 116.55,
117.49, 119.24, 120.87, 121.80, 123.08, 123.75, 125.38, 126.71, 131.88, 136.88,
138.32, 149.33, 151.64, 152.04, 152.17, 153.20, 154.04. Anal. calcd for C21H14Cl
2F4N6O·2H2O·2HCl: C 40.53, H 3.23, N 17.50, found: C 40.85, H 3.32, N 17.78.
1-(4-((3-Bromophenyl)amino)-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-3-(4-fluoro-
3-(trifluoromethyl)phenyl)urea (8a)

1
Yield 58%; mp: 262-264 °C, H NMR (400 MHz, DMSO-d6): δ= 3.73 (s, 3H, CH3),
6.77 (d, 1H, J= 2.8 Hz, H-5), 7.16 (d, 1H, J= 3.6 Hz, H-6), 7.22-7.29 (m, 2H), 7.41 (t,
1H), 7.52-7.56 (m, 1H), 7.90 (dd, 1H, Jo= 6.4 Hz, Jm= 2.8 Hz), 7.95-7.99 (m, 2H), 10.11
13
(s, 2H, NH), 11.76 (s, 1H, NH). MS (ESI, 70 eV) m/z: 523.21(M+), 525.05 (M+2). C
NMR (100 MHz, DMSO-d6): δ= 31.11, 99.41, 99.55, 116.77, 117.53, 117.74, 120.37,
121.76, 123.43, 125.03, 125.11, 125.86, 126.10, 130.82, 135.46, 140.61, 149.24,
151.15, 152.43, 152.90, 155.38. Anal. calcd for C21H15BrF4N6O·1.2HCl: C 44.58, H
2.88, N 14.86, found: C 44.48, H 2.97, N 14.97.
1-(4-((3-Chloro-4-fluorophenyl)amino)-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-3-
(4-fluoro-3-(trifluoromethyl)phenyl)urea (8b)
Yield 53%; mp: 279 °C, 1H NMR (400 MHz, DMSO-d6): δ= 3.74 (s, 3H, CH3), 6.75 (d,
1H, J= 3.2 Hz, H-5), 7.15 (d, 1H, J= 3.6 Hz, H-6), 7.31 (t, 1H), 7.42 (t, 1H), 7.59-7.63
(m, 1H), 7.86-7.89 (m, 2H), 8.14 (dd, 1H, Jo= 6.4 Hz, Jm= 2.8 Hz), 9.98 (s, 1H, NH),
10.10 (s, 1H, NH), 11.81 (s, 1H, NH). MS (ESI, 70 eV) m/z: 497.18 (M+H), 499.27
(M+H+2). 13C NMR (100 MHz, DMSO-d6): δ= 31.08, 99.19, 99.44, 116.63, 116.85,
117.51, 117.72, 119.32, 119.49, 121.49, 122.46, 125.03, 125.11, 125.65, 135.52,
136.48, 149.19, 152.37, 152.67, 152.88. Anal. calcd for C21H14ClF5N6O·1.2HCl: C
46.73, H 2.84, N 15.58, found: C 46.71, H 2.95, N 15.73.
1-(4-((4-Chlorophenyl)amino)-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-3-(4-
fluorophenyl)urea (9a)
Yield 85%; mp: 307 °C, 1H NMR (400 MHz, DMSO-d6): δ= 3.75 (s, 3H, CH3), 6.72 (d,
1H, J= 3.6 Hz, H-5), 7.09-7.13 (m, 3H), 7.31 (d, 2H, J= 8.8 Hz), 7.39-7.43 (m, 2H),
13
7.97 (d, 2H, J= 8.8 Hz), 9.62 (s, 1H, NH), 9.69 (s, 1H, NH), 11.75 (s, 1H, NH). C
NMR (100 MHz, DMSO-d6): δ= 31.10, 99.07, 99.71, 115.22, 115.44, 120.63, 120.71,
123.58, 125.73, 128.76, 134.73, 137.42, 148.69, 152.39, 156.60, 158.97. MS (ESI, 70
eV) m/z: 411.39 (M+H), 413.24 (M+H+2). Anal. calcd for C20H16ClFN6O·0.3H2O: C
57.71, H 4.01, N 20.19, found: C 57.87, H 4.12, N 19.95.
1-(4-((3-Bromophenyl)amino)-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-3-(2,4-
difluorophenyl) urea (10a)

1
Yield 40%; mp: 308-309 °C, H NMR (400 MHz, DMSO-d6): δ= 3.76 (s, 3H, CH3),
6.78 (d, 1H, J= 3.6 Hz, H-5), 7.09 (t, 1H), 7.15 (d, 1H, J= 3.2 Hz, H-6), 7.18 (d, 1H, J=
8.0 Hz), 7.25 (t, 1H), 7.34-7.40 (m, 1H), 8.07 (t, 1H), 8.27 (d, 1H, J= 8.4 Hz), 8.33-8.38
(m, 1H), 9.54 (s, 1H, NH), 10.11 (s, 1H, NH), 11.96 (s, 1H, NH). MS (ESI, 70 eV)
13
m/z:473.24 (M+), 474.79 (M+H), 475.18 (M+2). C NMR (100 MHz, DMSO-d6): δ=
30.05, 99.24, 100.35, 111.50, 119.67, 121.86, 122.39, 125.29, 125.76, 131, 139.11,
142.08, 149.34, 152.47, 153.03, 154.09, 154.60, 155.88, 156.19, 159.28. Anal. calcd for
C20H15BrF2N6O·0.03H2O: C 50.69, H 3.20, N 17.73, found: C 50.59, H 3.24, N 17.68.
Biological activity
Cytotoxic activity determined by MTT assay
In vitro cytotoxic effects of the synthesized compounds were evaluated using
MTT assay. Briefly, a 180 μl of cultured cell suspension (5x104cell/ml)
wasseeded in 96 well plates and incubated for 24 hours. Following incubation,
various amounts of the synthesized compounds (0.1, 0.5, 2.5, 5, 10, 25, 50 and
100 μM) were added to the cells and incubated for 24 hours in a CO2 incubator.
The cells were than treated with 5 mg/ml MTT reagent for 2 hours and the viable
cell amount was determined by metabolic conversion of the soluble MTT dye.
The reduced crystals were dissolved in 180 μl of DMSO and the absorbance at
540 nm was measured in a spectrophotometer. The effect of compounds on cell
viability was calculated as a percentage of control cell growth obtained from
untreated cells. Imatinib was used as positive control. Each experiment
wasperformed in quadruplicate to assess the consistency of results. IC50 values
were calculated by using linear regression equations obtained from the
concentration vs viable cell amount % graphs.
Annexin V binding assay
The effects of synthesized compounds on apoptosis were analyzed using the
Muse Annexin V/Dead Cell (Merck Millipore) assay, according to the
manufacturer’s instructions. Briefly, the cells were seeded and incubated for 24
hours for attachment. The synthesized compounds were then applied with a final
concentration of IC50 values of each compound, which were determined from the
MTT measurements for 24 hours. The cells were then harvested and incubated

with Annexin V and 7-aminoactinomycin (7-AAD) dye, for 20 min, at room
temperature, in the dark. Four populations were differentiated by using Annexin
V and/or 7-AAD positivity on Muse Cell Analyzer (Merck, Millipore): non-
apoptotic live (7-AAD negative, apoptosis negative), non-apoptotic dead (7-AAD
positive, apoptosis negative), apoptotic live (7-AAD negative, apoptosis
positive), and apoptotic dead (7-AAD positive, apoptosis positive) cells.
Western blot analysis
Total cell lysates were separated on 10% SDS-PAGE gels. Following SDS-PAGE,
proteins were transferred onto PVDF membranes, blocked with 5% dried milk in PBS-
Tween20 and incubated with corresponding primary and secondary antibodies.
Immunoblots were developed by using Luminata Crescendo Western HRP substrate
(Millipore, MA, USA) and imaged with C-DiGit Blot Scanner (LI-COR Biosciences,
Bad Homburg, Germany). The following antibodies were used for immunoblotting:
BCL-2 (#2872, Cell Signaling), BCL-XL (#2762, Cell Signaling), MCL-1 (#5453, Cell
Signaling), BIM (#2819, Cell Signaling), NOXA (FL-54, Santa Cruz Biotechnology),
PUMA (#4967, Cell Signaling), BAX (#2774, Cell Signaling), BAK (#3814, Cell
Signaling), Cleaved Caspase-9 (Asp315) (#9505, Cell Signaling), Cleaved Caspase-3
(Asp175) (#9661, Cell Signaling), Cleaved PARP (Asp214) (#9541, Cell Signaling) and
Actin (#8457, Cell Signaling).
Cell culture
HCT-116 wild-type and HCT-116 BAX/BAK knockout cells grown in McCoy’s 5A
medium (ThermoFisher Scientific) supplemented with 10% heat-inactivated fetal
bovine serum (Sigma, St Louis, MO, USA), 100 IU/ml penicillin, and 100 mg/ml
streptomycin (Thermo Scientific) in a humidified incubator at 37°C and 5% CO2.
Caspase activation assays
The activity of caspase-3 and caspase-9 was determined by Apo Alert Caspase Assay
Plates (Clontech, Takara) as described by the manusfacturer. The release of
fluorochrome AMC was analyzed at 380ꢀnm excitation and 460ꢀnm emission using
Spectramax Gemini microplate fluorometer. Data shown are meanꢀ±ꢀSEM of three
independent experiments and expressed in arbitrary fluorescence units.

Statistical Analysis
The statistical significance for the assays were determined using GraphPad Prism
Software 6.0e version (Software MacKiev, Inc.). Data obtained from the cell culture
experiments were expressed as mean±SD and one-way ANOVA test was applied for
multiple comparisons. A p-value of less than 0.05 was considered as statistically
significant.
Result and discussion
Chemistry
The synthesis of the target pyrrolo[2,3-d]pyrimidine derivatives begins with reaction of
commercially available 2,4-diamino-6-hydroxypyrimidine with chloroacetaldehyde in
the mixture of DMF: H2O to obtain compound 3 [42]. Then, carbonyl group of
compound 3 was chlorinated by POCl3 in poor yield (10%) [42]. Alkylation of pyrrole-
nitrogen of compound 4 was afforded with MeI in the presence of NaH [43]. Converting
the exocyclic amino at 2-position of pyrrolopyrimidine ring to the corresponding urea
(6-10) using triphosgene and appropriate anilines was firstly performed by our group. .
Finally, target compounds (6-8a, 9a, 10a) were obtained via nucleophilic displacement
of 4-chloro moiety of urea intermediates (6-10) with corresponding anilines in the
presence of 2-3 drops of conc. HCl at reflux [44]. The structures of synthesized
1
13
compounds were confirmed by H NMR, C NMR, Mass-ESI and elemental analysis
data.

Scheme 1. Synthesis of target pyrrolo[2,3-d]pyrimidine derivatives. Reagent and conditions: a)
NaOAc, DMF:H2O (5:1), 48h, rt. b) POCl3, 110 °C, 2h c) CH3-I, NaH, 0°C →rt. d) triphosgene,
Et3N, anhydrous dioxane, 0 °C →rt, 5h, then appropriate aniline, rt. e) isopropanol:DMF (1:1),
three drops conc. HCl, appropriate aniline, 10-24h, reflux.
Evaluation of Biological activity
In vitro cytotoxic activity
Target compounds (6-8a-b; 9a, 10a) have been evaluated for in vitro cytotoxic activity
in various human cancer cell lines, including PC3 (prostat), A549 (lung) and MCF-7
(breast), using MTT assay. The results are summarized in Table 1. Imatinib was used
as positive control. Majority of the synthesized compounds showed more
cytotoxic activity than imatinib. Among the tested carcinoma cell lines, A549 cells
seemed to be more sensitive in response to the growth inhibition with IC50 values
between 0.35 and 5.68 µM. Synthesized compounds were found to be moderately active
against MCF-7 cell line (23.29-71.90 µM). Compound 6b, 8a and 9a exhibited the
remarkably cytotoxic activity against A549 with IC50 value of 0.35, 1.48 and 1.56 µM,
respectively. Compound 7a, bearing 4-chloro-3-(trifluoromethyl)phenyl)urea moiety
was more active than corresponding 6a, 8a and 10a on PC3 cells (IC50= 1.04 µM)
suggesting that bulky urea moiety enhance the activity. In addition, 7a was less
cytotoxic against A549 (IC50= 5.68 µM) and MCF-7 (IC50= 24.85 µM) cells. Among
the compounds substituted with 3-chloro-4-fluorophenylamino, 8b bearing 3-
(trifluoromethyl)phenylurea moiety, substituted at 4-position with an electron
withdrawing fluoro atom showed better cytotoxic activity against PC3 (IC50= 1.89 µM)
and A549 (IC50= 5.17 µM) cell lines than MCF-7 (IC50= 26.19 µM).
Table 1. In vitro cytotoxicity of synthesized compounds 6-8a-b; 9a, 10a against A549,
PC3 and MCF-7 cells (IC50 in μM).
IC50 (µM)
Compd
R1
R2
R3
R4
R5
A549
PC3
MCF-7

6a
6b
-H
-H
-Cl
-Cl
-F
-CF3
-CF3
-CF3
-CF3
-CF3
-CF3
-H
-H
-H
-H
-H
-H
-H
-H
-F
-Br
-Cl
-Br
-Cl
-Br
-Cl
-H
-H
-F
5.26
0.35
5.68
5.36
1.48
5.17
1.56
4.09
30.65
32.30
35.69
1.04
23.29
23.84
24.85
39.01
43.24
26.19
24.85
7a
-H
-F
7b
27.56
44.63
1.89
8a
-H
-F
8b
-F
9a
-F
-Cl
-H
31.01
12.46
39.71
10a
Imatinib
-F
-H
-Br
71.90
3.01
The cells were treated with different concentrations of compounds ranged between 0.1 to 100
μM and the cytotoxicity was determined by MTT assay. IC50 values of experiments conducted
in triplicates.
Determination of apoptosis
The apoptotic efficiency of the most active compounds including 6b, 8a and 9a were
determined by Annexin V binding assay in A549 cells. The obtained results are shown
in Figure 3. Flow cytometry analysis revealed that A549 cells treated with compounds
6b, 8a and 9a showed significant increase in the percentage of late apoptotic cells by
2.8-, 2.7- and 2.5-folds compared to control, respectively. These results clearly
demonstrated that the compounds 6b, 8a and 9a had strong cytotoxic activity against
A549 cell line through induction of apoptosis.

Figure 3. The apoptotic effects of compound 6b, 8a and 9a on A549 cells. The cells were
treated with IC50 concentrations of compounds for 24 hours and the cell population % were
determined by Muse cell analyzer (Merck Millipore). The apoptotic cell population were
determined by the Annexin V positivity and dead cells were determined by the nuclear dye 7-
aminoactinomycin D (7-AAD) positivity. The cell analyzer determined four different group of
population via cytofluorometric seperation as follows: non-apoptotic live (7-AAD negative,
apoptosis negative), non-apoptotic dead (7-AAD positive, apoptosis negative), apoptotic live (7-
AAD negative, apoptosis positive), and apoptotic dead (7-AAD positive, apoptosis positive)
cells. The figure represents cytofluorimetric dot plots of three independent experiments
performed for annexin V detection and mean±SD of late apoptotic population %. Statistical
analysis was performed with one-way ANOVA for three independent data and the differences
are identified as * from control, (p<0.01).
Effects on the level of Bcl-2 family members
We tested whether treatment with 6b, 8a and 9a altered BCL-2 protein family members
in A549 cells. As shown in Figure 4, treatment with 6b, 8a and 9a led to decreased
levels of BCL-XL and MCL1, although only 9a treatment reduced BCL-2 levels. We
detected increased levels of BIM with 8a and 9a treatment as well as increased BAX
expression upon treatment with all three molecules. 6b and 8a treatment resulted in
increased BAK levels, which was more prominent following 8a exposure. NOXA was
not altered in response to 8a and 9a treatment, 6b treatment led to decreased NOXA
levels. In addition, we observed increased PUMA expression following treatment with

all three molecules. Likewise, 6b, 8a and 9a induced activation of caspase-3, caspase-9
and PARP cleavage, indicating the activation of apoptosis in A549 cells.
Figure 4. A549 cells were treated with 6b, 8a and 9a for 24 h. BCL-2, BCL-XL, MCL1, BIM,
NOXA, PUMA, BAX, BAK, active caspase-9, active caspase-3, cleaved PARP levels were
detected by using immunoblot analysis. Actin was probed as loading control.
Apoptosis effect on HCT116 wt and HCT116 BAX/BAK KO cells
To further confirm the involvement of intrinsic apoptotic pathway in 6b, 8a and 9a-
induced cell death, we took advantage of HCT116 wt and HCT116 BAX/BAK KO
cells. We initially verified the absence of BAX and BAK in HCT116 BAX/BAK KO by
immunoblot analysis (Figure 5A). As demonstrated in Figure 5B, 6b, 8a and 9a induced
apoptosis in HCT116 wt cells although HCT116 BAX/BAK KO cells were significantly
resistant to 6b, 8a and 9a treatment. Correspondingly, 6b, 8a and 9a treatment triggered
activation of caspase-3 and caspase-9 in HCT116 wt cells but not in HCT116
BAX/BAK KO cells (Figure 5C).

Figure 5A) Expression of BAX and BAK in HCT116 wt and HCT116 BAX/BAK KO cells
were analyzed by using immunoblot analysis. Actin was probed as loading control. B) HCT116
wt and HCT116 BAX/BAK KO cells were treated with 6b, 8a and 9a for 48 h. Cell death was
evaluated by Annexin V/PI staining and flow cytometry. Columns, mean % Annexin V/PI
positive cells from three independent experiments; bars, SE;**P<0.01. C) HCT116 wt and
HCT116 BAX/BAK KO cells were treated with 6b, 8a and 9a for 48 h. Activation of caspase-3
and caspase-9 was evaluated by fluorometric caspase activation assays. Columns, mean relative
fluorescence units from three independent experiments; bars, SE; *P<0.05, **P<0.01.
Conclusions
In this work, we have designed and synthesized novel pyrrolo[2,3-d]pyrimidine
derivatives and analyzed their cytotoxic activities against three cancer cell lines (MCF-
7, A549 and PC3). Most of the synthesized compounds showed more cytotoxicity
against A549 cell than other tested cell lines. Compounds 6b, 8a and 9a displayed the
highest activity against A549 with IC50 value of 0.35, 1.48 and 1.56 µM, respectively
and they induced intrinsic apoptosis in A549 cells confirmed by western blot analysis of
pro-apoptotic and anti-apoptotic protein levels. In order to verify the intrinsic apoptotic
induction of compounds 6b, 8a and 9a, apoptotic effects of them on HCT116 wt and

HCT116 BAX/BAK KO cells were evaluated. Although HCT116 wt cells treated with
all of three compounds showed significant increase in the percent of annexin V-FTIC-
positive apoptotic cells, HCT116 BAX/BAK KO cells were found significantly resistant
to compounds 6b, 8a and 9a. These results were consistent with the levels of caspase-3
and caspase-9 activation in HCT116 wt and HCT116 BAX/BAK KO cells.
The obtained potent cytotoxic effects through activation of the mitochondrial apoptotic
pathway encourage further modifications of the compounds 6b, 8a and 9a and to
develop more potent anticancer agents. Also, further investigation to reveal mechanism
of action of these compounds will be conducted.
Acknowledgements
This work was supported by The Scientific and Technological Research Council of
Turkey Grant [SBAG-214S573]. We thank Prof. Hakan Goker and Assoc. Prof.
Mehmet Alp from Central Instrumentation Laboratory of Faculty of Pharmacy, Ankara
University for NMR and elemental analyses.
Disclosure statement
The authors declare no conflict of interest, financial or otherwise.
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Highlights




A series of pyrrolopyrimidine derivatives were synthesized and evaluated for
anticancer activity against A549, PC3 and MCF-7 cells.
Compounds 6b, 8a, 9a and 7a, 8b displayed the significant cytotoxic activities against A549
and PC3 cells with IC50 values ranging from 0.35 to 1.89 µM.
Annexin V binding assay and western blot analysis results indicated that 6b, 8a and 9a
induced apoptosis in A549 cells by intrinsic apoptotic pathway.
Also, compounds 6b, 8a and 9a induced apoptosis in HCT116 wt cells. Bax/Bak KO cells
were found resistance to 6b, 8a and 9a treatment.