Expedient synthesis and anticancer evaluation of dual-action 9-anilinoacridine methyl triazene chimeras

Article abstract of DOI:10.1111/cbdd.13776

The efficient synthesis of molecular hybrids including a DNA-intercalating 9-anilinoacridine (9-AnA) core and a methyl triazene DNA-methylating moiety is described. Nucleophilic aromatic substitution (S_NAr) and electrophilic aromatic substitution (EAS) reactions using readily accessible starting materials provide a quick entry to novel bifunctional anticancer molecules. The chimeras were evaluated for their anticancer activity. Chimera 7b presented the highest antitumor activity at low micromolar IC₅₀ values in antiproliferative assays performed with various cancer cell lines. In comparison, compound 7b outperformed DNA-intercalating drugs like amsacrine and AHMA. Mechanistic studies of chimera 7b suggest a dual mechanism of action: methylation of the DNA-repairing protein MGMT associated with the triazene structural portion and Topo II inhibition by intercalation of the acridine core.

Full text of DOI:10.1111/cbdd.13776

PROFESSOR GARY GELLERMEN (Orcid ID : 0000-0002-1336-8707)
Article type : Research Article
Expedient synthesis and anticancer evaluation of dual-action 9-anilinoacridine
methyl triazene chimeras
Dipak Walunj,1 Katarina Egarmina, 2,3,4 Helena Tuchinsky,4 Ofer Shpilberg,2,3 Oshrat Hershkovitz-
Rokah,2,3,4, Flavio Grynszpan, 1 and Gary Gellerman*1
Abstract
The efficient synthesis of molecular hybrids including a DNA intercalating 9-anilinoacridine (9-AnA)
core and a methyl triazene DNA methylating moiety is described. Nucleophilic aromatic substitution
(SNAr) and electrophilic aromatic substitution (EAS) reactions using readily accessible starting
materials provide a quick entry to novel bifunctional anticancer molecules. The chimeras were
evaluated for their anticancer activity. Chimera 7b presented the highest antitumor activity at low
micromolar IC50 values in antiproliferative assays performed with various cancer cell lines. In
comparison, compound 7b outperformed DNA intercalating drugs like Amsacrine and AHMA.
Mechanistic studies of chimera 7b suggest a dual mechanism of action: methylation of the DNA
repairing protein MGMT associated with the triazene structural portion and Topo II inhibition by
intercalation of the acridine core.
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Keywords: Anilinoacridine Triazene Molecular chimera SNAr EAS Anticancer
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differences between this version and the Version of Record. Please cite this article as doi:
10.1111/CBDD.13776
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Correspondence:
Gellerman G. Department of Chemical Sciences, Ariel University,
PO Box 3, Ariel 40700, Israel
garyg@ariel.ac.il
1
Department of Chemical Sciences, Ariel University,
PO Box 3, Ariel 40700, Israel
2
Institute of Hematology, Assuta Medical Centers,
Tel Aviv, Israel
3
Translational Research Lab, Assuta Medical Centers,
Tel Aviv, Israel
4
Department of Molecular Biology, Ariel University,
Ariel 40700, Israel
1. Introduction:
The discovery of small molecule multi-target drugs with combined pharmacological activities has
emerged as a widely used approach in medicinal chemistry (Lomberdino et al. 2004; Zhou et al.
2017). DNA intercalators, comprising a wide range of structurally diverse biologically active
compounds mostly with anticancer and antimalarial applications (Martínez et al. 2005; Almaqwashi et
al. 2016), have been proposed as advantageous components for successful dual-drug structural
hybrids - molecular chimeras.
During the last thirty years, the DNA intercalating 9-anilinoacridine (9-AnAs) has been frequently
used as structural core for hybrid drug candidates. This scaffold presents a planar structural fragment
with hydrogen bond donor and hydrogen bond acceptor groups that intercalates DNA and binds to
minor grooves stabilizing the DNA-drug complex (Gellerman et al. 2012), apparently reducing the
risk of secondary cancers (Godzieba et al. 2019). In addition, its DNA topoisomerase II (Topo II)
poisoning ability enhances the antiproliferative effects (Gamage et al. 1994; Auparakkitanon et al.
2000). One of the oldest drugs of this family is the antineoplastic Amsacrine that is still used in
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clinical treatment of acute lymphoblastic leukemia (Fig.1) (Nitiss et al. 2009; Horstmann et al. 2005).
Apparently, this drug suffers from fast deactivation by glutathione associated drug resistance. An
improved version of Amsacrine, (9-acridinylamino)-5-hydroxymethylaniline (AHMA) was designed
to avoid metabolic oxidation. Compared to Amsacrine, AHMA, has longer half-life in human plasma
accompanied by longer duration of its drug action (Rastogi et al. 2002). AHMA was also shown to
display better antitumor activity in vivo probably due to the substituent (NH2 and CH2OH, Fig. 1)
arrangement in the meta-positions of the aniline ring, which prevents undesired oxidation processes
(Chang et al. 2003; Afzal et al. 2016).
R2
R
N N
N
H
N
R1
HN
R3
R4
N
NH2
Dacarbazine (DTIC): R = Me
O
MTIC: R = H
N
O
N
N
Amsacrine: R2, R4, R1 = OMe,
R3 = NHSO2Me
N
N
N
AHMA:
R1, R3 = H, R2 = NH2,
R4 = CH2OH
NH2
Temozolomide (TMZ)
O
FIGURE 1 Structures of anticancer DNA intercalators, Topoisomerase II inhibitors and DNA
methylating agents.
DNA methylating agents like Dacarbazine (DTIC) and Temozolomide (TMZ), bearing a triazene
moiety (Fig. 1), are members of a different class of anticancer drugs applied to the treatment of
melanoma (DTIC) and glioblastoma (TMZ) (Su et al. 1999; Scarborough et al. 1996; Thomas et al.
2013). So far, DTIC and TMZ are the only triazene-based approved drugs currently in use in clinical
chemotherapy (Gerson et al. 2018).
The mechanism of DTIC and TMZ antitumor activity involves the methylation of nucleotides,
through in vivo generation of highly reactive diazomethane (the actual alkylating agent). Both TMZ
and DTIC are prodrugs of the active 5-(3-methyltriazen-1-yl) imidazole-4-carboximide (MTIC)
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intermediate (Meer et al. 1986; Jhang et al. 2012). Unlike DTIC, which requires metabolic activation
by cytochrome P450, otherwise remaining dormant, TMZ spontaneously converts to MTIC under
physiologic conditions (Kolar et al. 1980; Marchesi et al. 2007; Monteiro et al. 2013).
In general, the administration of drug combinations with differing mechanisms of action is a
favorable therapeutic treatment to augment the effectiveness of the treatment (Mort et al. 2014;
Sonpavde et al. 2016; Raja et al. 2013). Yet, combination chemotherapy by co-administration of two
or more drugs, does not appear to prevent the development of drug resistance in cancer cells and, in
addition, it is associated with increased toxicity (Szakacs et al. 2006; Pritchard et al. 2012; Shirota et
al. 2001). A useful approach to the design of unexplored promising anticancer candidates is the
structural combination of bioactive fragments of existing pharmaceutical agents with proven
effectiveness. This drug design strategy has led to the development of a sizable number anticancer
structural hybrids (chimeras) destined to preclinical and clinical development (Huang et al. 2014;
Naito et al. 2019; Oliveira et al. 2015). Thus and thus, we decided to design, synthesize and evaluate a
family of chimeras including 9-AnAs and methyl triazene moieties to investigate whether a single
molecule bearing two anticancer agents operating under two distinct mechanisms (Topo II inhibitors:
9-AnAs and DNA methylating agents: DTIC or TMZ) works better than each one of them alone.
In the past we developed the highly efficient one-pot derivatization of 9-anilonoacridines (9-AAs)
at the amino group by simple SNAr reaction using easily accessible haloaryl starting materials
(Gellerman et al. 2010; Gellerman et al. 2011). Such a ''reverse'' approach (Fig. 2) to synthesize 9-
AnAs complements the ''classical'' electrophilic aromatic substitution (EAS) approach to 9AnA
derivatives, in which 9-Cl acridine reacts with substituted anilines (Redko et al. 2012).
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EWG
EDG
X
H2N
SNAr
EAS
b)
a)
Cl
NH2
N
N
9-aminoacridine
9-Chloroacridine
EDG - electron donating groups
EWG - electron withdrawing groups
X = halogen
FIGURE 2 (a) "Classical" – EAS and (b) "reverse" – SNAr approaches for the synthesis of
substituted 9-AnAs
Here, we report on the synthesis of chimeras built from 9-AnAs and methyl triazene structural
building blocks. Our synthetic strategy is based on our previous experience with the facile
derivatization of 9-AnA through aromatic substitution reactions. A total of nine chimeras were
prepared by this approach. Preliminary antiproliferative assays against H1299 (NSCLC) and WM-
266-4 (human metastatic melanoma) cancer cell lines helped us to identify a lead compound with
enhanced antitumor activity compared to the parent control drugs: Amsacrine and AHMA. Chimera
7b stands out among our synthesized hybrids, exhibiting remarkable cell growth inhibition with IC50 =
2.9 and 0.8 M against the above-mentioned cancer cell lines, respectively. Expectedly, the
chemostability of the synthesized chimeras varied according to the type of triazene used as substituent
on the aniline ring of 9-AnA. Here we demonstrate that chimera 7b can bestow its antiproliferative
effect by two independent mechanisms: DNA alkylation associated with methyl triazene structural
portion and Topo II inhibition affiliated with the 9-acridine core. The findings reported in this work
expand the scope of 9-anilinoacridine (9-AnA) based methyl triazene chimeras as efficient anticancer
candidates.
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2. Materials and methods
2.1 General information
Amsacrin, DTIC, and TMZ were purchased from Tzamal D-Chem Laboratories Ltd. Petah-Tikva,
Israel if not indicated otherwise. AHMA was synthesized according a reported literature procedure
(Su et al. 2006). All solvents were purchased from Bio-Lab Ltd. Jerusalem, Israel or Gas
Technologies Ltd. Kefar Saba, Israel. All reactions were performed in a round bottom flask and
monitored by TLC performed on aluminium plates (0.25 mm, E. Merck) precoated with silica gel
(Merck 60 F-254). Developed TLC plates were visualized under a short-wavelength UV lamp. Yields
refer to spectroscopically (1H, 13C NMR) homogeneous material obtained after column
1
13
chromatography performed on silica gel (Silica flash P60) supplied by Silicycle. H and C NMR
were recorded in CDCl3 and DMSO-d6 on a Bruker Avance III 400 MHz spectrometer. Chemical
shifts (δ) are quoted in ppm, relative to SiMe4 (δ = 0.0) as an internal standard. LC/MS analyses were
performed using an Agilent Technologies 1260 Infinity (LC) 6120 quadruple (MS), column Agilent
SB-C18, 1.8 mm, 2.1×50 mm, column temperature 50 °C, eluent water- acetonitrile (CH3CN) + 0.1%
formic acid. FT-IR spectra were recorded as KBr pellets on a Shimadzu FT-IR-8400S spectrometer.
High-resolution mass spectra (HRMS) were measured using an Agilent QTOF mass-spectrometer in
electrospray ionization (ESI) positive ion mode. Melting points were recorded on a standard melting
point apparatus.
2.2 Reagents and cell lines
All the cell lines were cultured in an RPMI medium supplemented with 2 mM glutamine, 10% fetal
bovine serum and penicillin streptomycin (100 IU/ml of each). The cell culture growth medium and
all additives were purchased from Biological Industries, Bet-Ha'emek, Israel. All cell cultures were
grown in an incubator at 37 °C in an environment containing 6% CO2. The cytotoxicity of the
materials was determined by measuring the mitochondrial enzyme activity, using a commercial XTT
assay kit (Biological Industries, Bet-Ha'emek, Israel). All samples contained DMSO at final
concentration below 0.05%. All cancer cell lines were kindly provided by Prof. Albert Pinhasov
(Ariel University, Israel).
2.3 Liquid chromatography
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Purification of the compounds was done via normal phase chromatography by using silica gel (Silica
flash @ P60) supplied by Merck. The column was kept at room temperature and eluted with either
petroleum ether, ethyl acetate or dichloromethane, neat or combined.
2.4 Liquid Chromatography Mass Spectrometry (LCMS)
Electron spray mass spectra (ESI-MS) were obtained using an Autoflex III smart-beam (MALDI,
Bruker), Q-TOF micro (Waters) or an LCQ FleetTM ion trap mass spectrometer (Finnigan/Thermo).
HPLC/LC-MS analyses were made using Agilent infinity 1260 connected to Agilent quadruple LC-
MS 6120 series equipped with ZORBAX SB-C18, 2.1 x 50 mm, 1.8 µm column. In all cases, the
eluent was A (0.1% formic acid (FA) in H2O) and B (0.1% FA in ACN) and the elution gradient
profile was: 100% A for first 3 min, followed by 5 min (from min 3 to min 8) during which it reached
100% B, followed by 5 min (from min 8 to min 13) of 100% B, followed by two min (from min 13 to
min 15) with a linear gradient back to 100% A, followed by 2 min (from min 15 to min 17) of 100%
A. The UV detector was set at 254 nm. The column temperature was kept at 50 °C. The flow rate was
of 0.3 ml/min. The MS fragmentor was tuned on 30 V or 70 V on positive or negative mode.
2.5 Chemostability studies
Stock solutions were prepared by dissolving 5 mg of the tested compound in 500 L of DMSO.
Aqueous stability was determined at pH 4.6 and 7.4. Aliquots (100 L) of stock solution were diluted
to a total of 2.5 mL with the desired buffer and then incubated at 37 °C. During the incubation period
250 L portions were drawn at different time intervals, filtered and analyzed by LC–MS.
2.6 Cytotoxicity test (XTT)
The cytotoxicity of the synthesized chimeras was determined by measuring the mitochondrial enzyme
activity, using a commercial XTT assay kit. All samples contained DMSO at final concentration
below 0.05%. Cells were cultured in micro wells at 5-10 × 104 cells/mL and incubated for 48 h and 72
h. After the first incubation period, the cultures were washed and then provided with fresh medium
containing different concentrations of the tested substances. At the end of the second incubation, XTT
reagent was added and the cells were re-incubated for additional 3 h. During that time, the
absorbencies in the wells were measured with a TECAN Infinite M200 ELISA reader at both 480 and
680 nm. The difference between these measurements was used for calculating the % growth inhibition
(GI) in test wells compared to two controls: cells that were exposed to the medium and solvent, and
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those which were exposed to a solvent-free medium. All the tests were done in tetra-plicate; each
experiment was conducted twice.
2.7 Western blot
HCT-116 cells (1 × 106 cells/well), were treated with DTIC, TMZ and Chimera 7b (100 µM) or
DMSO (Sigma Aldrich) for 48 h and were harvested using RIPA lysis buffer and protease inhibitor
cocktail (Sigma Aldrich). Equal amounts of protein were separated using 10% SDS- polyacrylamide
gel and blotted onto nitrocellulose membranes. Membranes were blocked with 5% non-fat dry milk in
Tris-buffered saline (TBS), containing 1% Tween20 for 2 h at room temperature, probed with the
appropriate primary antibody over night at 4 °C and then with the appropriate fluorescently labeled
secondary antibody (Li-Cor Biosciences). Membranes were scanned and analyzed using Licor-FC
Infrared Imaging System (Li-Cor Biosciences). Primary antibodies used: MGMT (Millipore) and
tubulin (Cell Signaling).
2.8 Human topoisomerase IIa kDNA decatenation assay
Topo II activity was measured by the ATP-dependent decatenation of kinetoplast DNA (kDNA). The
assay was performed according to manufacturer's (Inspiralis Limited, UK) protocol modified as
follows. The reaction mixture contained kDNA (200 ng), 3 units of Topo II and assay buffer with
ATP. AHMA and molecular chimera 7b were diluted in DMSO and added at the indicated
concentrations to the reaction mixture to a final volume of 15 ml. The same amount of DMSO was
added to control cultures without the drugs. The samples were incubated for 25 min at 37 °C and the
reaction was stopped by adding 1 ml of 10% SDS. Samples were electrophoresed in 1% agarose gel
in TAE buffer at 4 V/cm for 2 h. The gel was stained with ethidium bromide and photographed under
a UV trans-illuminator.
2.9 Chemistry
2.9.1 General procedure for the synthesis of the dimethyl triazene derivative of nitroaniline (1).
A suspension of nitroaniline (0.3 mole, 1 equiv.) in diluted hydrochloric acid in water having a ratio
of HCl:H2O (4:6) was diazotized at 0 °C with sodium nitrite (1.2 equiv.) in water and the resulting
solution was stirred for 45 min and to give a clear solution. The diazonium salt solution was treated
with 40% aqueous dimethylamine. At this stage, the triazene usually precipitated after reaching a pH
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between 7 to 8. The reaction mixture was quenched with a 1% solution of citric acid and extracted
with DCM. The organic layer was washed with brine, dried over Na2SO4, filtered and the solvents
were removed under reduced pressure to give a crude product which was then purified by silica gel
chromatography using EtOAc:petroleum ether (1:9), to give pure triazenes 1a-1i (Scheme 1) with 58
to94% yield.
(E)-3,3-dimethyl-1-(2-nitrophenyl) triaz-1-ene (1a). The compound was purified by column
chromatography [ethyl acetate:petroleum ether (1:9), v/v]; yield: 363 mg (86%) Rf = 0.78, yellow
liquid; MS-ESI, (m/z) = 195 (M+1); 1H NMR (400 MHz, CDCl3 )  ppm): 7.64 (dd, J = 8.0, 1.34 Hz,
1H-a) 7.52 (dd, J = 8.2, 1.34 Hz, 1 H-b), 7.44 (, ddd, J = 8.3, 7.1, 1.5 Hz, 1 H-c), 7.16 (, ddd, J = 8.2,
7.1, 1.5 Hz, 1 H-d), 3.53 (s,3 H, CH3-e), 3.19 (s,3 H, CH3-f). 13C NMR (101 MHz, CDCl3)  (ppm):
144.9, 143.6, 132.1, 130.5, 124.6, 119.5, 36.1 (Me), 29.2 (Me).
(E)-3,3-dimethyl-1-(4-methyl-2-nitrophenyl) triaz-1-ene (1b). The compound was purified by
column chromatography [ethyl acetate:petroleum ether (1:9), v/v]; yield: 358 mg (87%), Rf = 0.73,
yellow liquid, MS-ESI, (m/z) = 209 (M+1, 100%), 1H NMR (400 MHz, CDCl3)  (ppm): 7.44 (d, J =
0.98 Hz, 1H -a ), 7.40 (d, J = 8.3 Hz, 1H -b), 7.25 (dd, J = 0.61, 1.96 Hz, 1H-c), 3.50 (s., 3H, CH3-d),
3.17 (s., 3H, CH3-e), 2.35 (s, 3H CH3-f); 13C NMR (101 MHz, CDCl3)  (ppm): 144.8, 141.5, 135.1,
133.1, 123.8, 119.5, 43.2, 36.2 (Me), 20.7 (Me).
(E)-1-(4-methoxy-2-nitrophenyl)-3,3-dimethyltriaz-1-ene (1c). The compound was purified by
column chromatography [ethyl acetate:petroleum ether (1:9), v/v]; yield: 0.364 g (91%), Rf = 0.66,
1
orange liquid, MS-ESI, (m/z) = 225 (M+1, 98%), H NMR (400 MHz, CDCl3)  ppm): 7.46 (d, J =
9.1 Hz, 1H-a), 7.17 (d, J = 2.8 Hz, 1H-b), 7.03 (dd, J = 2.8, 9.1 Hz, 1H-c), 3.82 (s, 3H, OCH3-d), 3.50
13
(s., 3H, CH3- e), 3.17 (s., 3H,CH3-f ); C NMR (101 MHz, CDCl3)  (ppm): 156.7, 145.2, 137.6,
120.7, 119.4, 107.8, 56.0 (OMe), 40.2 (Me), 36.3 (Me).
(E)-3,3-dimethyl-1-(3-nitrophenyl) triaz-1-ene (1d). The compound was purified by column
chromatography [ethyl acetate: petroleum ether (1:9), v/v]; yield: 347 mg (82%), Rf = 0.82, yellow
o
1
solid, MP:100-101 C, MS-ESI, (m/z) = 195 (M+1, 56%), 196 (M+2, 35%); H NMR (400 MHz,
CDCl3)  ppm 8.25 (t, J = 2.2 Hz, 1H-a), 7.94 (ddd, J = 0.98, 2.2, 8.1 Hz, 1H-b), 7.64 - 7.74 (m, 1H-
c), 7.44 (t, J = 8.1 Hz, 1H-d), 3.56 (s., 3H,CH3-e), 3.24 (s., 3H,CH3-f); 13C NMR (101 MHz, CDCl3) 
(ppm): 152.2, 149.1, 129.5, 127.0, 119.5, 114.9, 36.1 (Me), 32.5 (Me).
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(E)-1-(4-bromo-3-nitrophenyl)-3,3-dimethyltriaz-1-ene (1e). The compound was purified by
column chromatography [ethyl acetate:petroleum ether (1:9), v/v]; yield: 220 mg (58 %), Rf = 0.80,
o
1
light yellow solid, MP: 80-81 C, MS-ESI, (m/z) = 272.9 (M+1, 43%), 274.9 (M+3, 42%) H NMR
(400 MHz, CDCl3)  ppm): 8.24 (d, J = 2.7 Hz, 1H-a), 7.78 (dd, J = 2.7, 8.7 Hz, 1H-b), 7.69 (d, J =
8.7 Hz, 1H-c), 3.61 (s, 3H,CH3-d), 3.30 (s, 3H,CH3-e); 13C NMR (101 MHz, CDCl3)  (ppm): 149.2,
147.9, 133.7, 126.5, 119.8, 112.9, 43.6 (Me), 36.8 (Me).
Methyl (E)-4-(3,3-dimethyltriaz-1-en-1-yl)-3-nitrobenzoate (1f). The compound was purified by
column chromatography [ethyl acetate:petroleum ether (1:9), v/v]; yield: 293 mg (76%), Rf = 0.76,
o
1
yellow solid, MP: 96-97 C, MS-ESI, (m/z) = 253 (M+1, 68%), 254 (M+2, 29%). H NMR (400
MHz, CDCl3)  (ppm): 8.29 (d, J = 1.7 Hz, 1H-a), 8.08 (dd, J = 1.7, 8.6 Hz, 1H-b), 7.60 (d, J = 8.6
Hz, 1H-c), 3.92 (s, 3H,OCH3-d), 3.60 (s, 3H,CH3-e), 3.25 (s, 3H,CH3-f). 13C NMR (101 MHz, CDCl3)
 ppm): 165.4, 147.0, 137.2, 133.0, 126.3, 125.4, 119.3, 58.5, 43.8 (Me), 36.7(Me).
(E)-3,3-dimethyl-1-(4-nitrophenyl) triaz-1-ene (1g). The compound was purified by column
chromatography [ethyl acetate:petroleum ether (1:9), v/v]; yield: 375 mg (89 %), Rf = 0.84, yellow
o
1
solid, MP: 144-145 C, MS-ESI, (m/z) = 195(M+1, 66%), 196(M+2, 30%); H NMR (400 MHz,
CDCl3)  (ppm): 8.01 - 8.29 (dd, J = 9.2Hz, 2H-a), 7.41 - 7.55 (dd, J = 9.2Hz, 2H-b), 3.59 (s.,
3H,CH3-c), 3.27 (s., 3H,CH3-d). 13C NMR (101 MHz, CDCl3)  (ppm): 156.1, 144.7, 124.9, 120.7,
43.7 (Me), 36.4(Me).
(E)-1-(2-bromo-5-nitrophenyl)-3,3-dimethyltriaz-1-ene (1h). The compound was purified by
column chromatography [ethyl acetate:petroleum ether (1:9), v/v]; yield: 288 mg (74%), Rf = 0.80,
o
1
light yellow solid, MP: 112-113 C, MS-ESI, (m/z) = 272.9 (M+1, 48%), 274.9 (M+3, 47%). H
NMR (400 MHz, CDCl3)  ppm): 7.90 (d, J = 2.3 Hz, 1H-a), 7.63 (d, J = 8.7 Hz, 1H-b), 7.47 (dd, J =
2.3, 8.7 Hz, 1H-c), 3.57 (s.,3H,CH3-d), 3.24 (s., 3H,CH3-e); 13C NMR (101 MHz, CDCl3)  (ppm):
151.2, 150.8, 135.1, 125.5, 116.9, 109.0, 38.3 (Me), 32.1(Me).
(E)-1-(4-methoxy-3-nitrophenyl)-3,3-dimethyltriaz-1-ene (1i). The compound was purified by
column chromatography [ethyl acetate:petroleum ether (1:9), v/v]; yield: 364 mg (91 %), Rf = 0.68,
o
1
yellow solid, MP: 146-148 C, MS-ESI, (m/z) = 225 (M+1, 100%), H NMR (400 MHz, CDCl3) 
(ppm): 8.22 (d, J = 2.8 Hz, 1H-a), 8.03 (dd, J = 2.8, 9.05 Hz, 1H-b), 6.96 (d, J = 9.05 Hz, 1H-c), 3.99
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13
(s, 3H,OCH3-d), 3.57 (s., 3H,CH3-e), 3.29 (s., 3H,CH3-f); C NMR (101 MHz, CDCl3)  (ppm):
158.0, 141.9, 140.7, 121.7, 114.0, 111.1, 56.7, 43.5 (Me), 36.3(Me).
2.9.2 General Procedure for the SNAr synthesis of compounds 2a and 2b.
A solution of an aryl halide of triazene (1 equiv.) in dry DMF (6 ml) and Cs2CO3 (0.5 equiv.) was
added slowly to a stirred solution of 9-aminoacridine (1 equiv.), under positive nitrogen pressure at
o
room temperature. The reaction mixture heated to 90 C, and stirred for 12 h. The reaction was
monitored by TLC and LCMS. After reaching its completion, the reaction mixture was diluted with
water and extracted with EtOAc (DMF was removed carefully by successive water washings to the
organic layer). The organic layer was washed with brine, dried over Na2SO4 (anh.), filtered and
concentrated under reduced pressure to give a crude product which was then purified by silica gel
chromatography using EtOAc:CH2Cl2 (2:8), resulting in a pure compound, generally as a brown solid.
(E)-N-(4-(3,3-dimethyltriaz-1-en-1-yl)-2-nitrophenyl) acridin-9-amine (2a). The compound was
purified by column chromatography [EtOAc:CH2Cl2 (2:8) v/v]; yield: 97 mg (68%), Rf = 0.43, brown
o
1
solid, MP: 212-214 C; FT-IR (KBr, ν, cm−1): 2958, 1582,1501,1430,1380; H NMR (400 MHz,
DMSO-d6)  ppm): 7.99 (d, J = 2.3 Hz, 1H-a), 7.81 - 7.90 (dd, 2H-b), 7.50 - 7.63 (m, 4H-c), 7.37 -
7.42 (dd, 2H-d), 6.94 - 7.06 (m, 2H-e), 6.84 (d, J = 8.7 Hz, 1H-f), 3.49 (s,3H, CH3-g), 3.19 (s,
3H,CH3-f); 13C NMR (101 MHz, DMSO-d6)  (ppm): 151.5, 146.2, 144.3, 140.9, 139.9, 138.3, 131.9,
128.6, 121.5, 120.5, 117.5, 116.9, 115.7, 35.9 (Me), 33.7 (Me); HRMS-ESI(+) calculated for
C21H19N6O2, 387.1491; found 387.1856 (98%) [M+H]+
(E)-N-(2-(3,3-dimethyltriaz-1-en-1-yl)-4-nitrophenyl) acridin-9-amine (2b). the compound was
purified by column chromatography [EtOAc:CH2Cl2 (2:8) v/v]; yield: 88 mg, (62%), Rf = 0.48,
o
1
brown solid, MP: 204-206 C; FT-IR (KBr, ν, cm−1): 3010, 1576,1522,1410,1364; H NMR (400
MHz, DMSO-d6)  (ppm): 8.23 (dd, J = 1.4, 8.0 Hz, 1H-a), 7.92 - 8.12 (m, 2H-b), 7.78 - 7.92 (m, 2H-
c), 7.44 - 7.57 (m, 2H-d), 7.32 - 7.43 (m, 2H-e), 7.06 (d, J = 8.4 Hz, 1H-f), 6.96 (t, J = 7.4 Hz, 2H-g),
13
3.11 - 3.41 (s, 6H,2CH3-h); C NMR (101 MHz, DMSO-d6)  (ppm): 154.1, 152.8, 140.9, 137.7,
133.4, 131.7, 126.9, 126.0, 121.7, 117.9, 117.5, 116.3, 112.3, 31.1(Me), 29.8(Me) ; HRMS-ESI(+)
calculated for C21H19N6O2, 387.1491; found 387.1574 (96%) [M+H]+
2.9.3 General procedure for the EAS synthesis of dimethyltraizene 9-AnA derivatives 4a, 4b, 4c
and 4d.
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A catalytic amount of 10% Pd/C was added to a suspension of nitroaniline dimethyl triazene, (Scheme
1) (0.2 g, 1 equiv.) in ethanol (4 mL) under hydrogen atmosphere (1atm) and the whole reaction
mixture was stirred for 4 h room temperature. The reaction was monitored by TLC and LCMS. After
completion,the crude of the reaction was filtered through celite and the solvent was removed under
reduced pressure. After complete evaporation of the solvent, water was added and the product was
extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4 (anh.) and give a
crude product after solvent removal. The crude product was subsequently treated as follows: The
crude amine triazene (1 equiv.) was dissolved in absolute ethanol under a nitrogen atmosphere at 0 oC.
Then, a solution of 9-Cl-acridine (1 equiv.) and N,N-diisopropylethylamine (DIPEA) (1.5 equiv.) was
added. The reaction was stirred at room temperature for 12 h and monitored by TLC. After
completion, the reaction mixture was evaporated to dryness under reduced pressure. The residue was
dissolved in EtOAc and washed with distilled water. The aqueous layer was washed three times with
15 mL aliquots of EtOAc. The collected organic fractions were dried over Na2SO4 (anh.) and the
solvent was removed under reduced pressure to yield the crude product, which was then purified by
silica gel chromatography using EtOAc:CH2Cl2 (2:8 to 4:6) to afford the pure compound as a brown
solid.
(E)-N-(4-(3,3-dimethyltriaz-1-en-1-yl) phenyl) acridin-9-amine (4a). The compound was purified
by column chromatography [EtOAc:CH2Cl2 (5:5) v/v]; yield: 113 mg (64%), Rf = 0.64, brown solid,
o
1
MP: 234-236 C; FT-IR (KBr, ν, cm−1): 2932, 1488,1437,1395,1361; H NMR (400 MHz, CDCl3) 
(ppm): 8.03 (d, J = 8.56 Hz, 2H-a), 7.91 (s., 2H-b), 7.50 - 7.67 (m, 2H-c), 7.33 - 7.44 (dd, J = 8.8Hz,
2H-d), 7.24 (brs, 3H-e), 6.79 - 6.93 (dd, J = 8.8 Hz, 2H-f), 3.32 (s, 6H-2CH3-g); 13C NMR (101 MHz,
CDCl3)  (ppm): 146.2, 133.6, 130.7, 127.5, 125.0, 123.7, 122.1, 121.7, 119.7, 119.0, 116.5,
29.9(Me), 29.4 (Me); HRMS-ESI(+) calculated for C21H20N5, 342.1640; found 342.1712 (100%)
[M+H]+
(E)-N-(3-(3,3-dimethyltriaz-1-en-1-yl) phenyl) acridin-9-amine (4b). The compound was purified
by column chromatography [EtOAc:CH2Cl2 (5:5) v/v]; yield:132 mg (62%), Rf = 0.63, brown solid,
MP: 136-137 oC; FT-IR (KBr, ν, cm−1): 2945, 1470,1424,1400,1355; 1H NMR (400 MHz, CDCl3 ) 
(ppm): 8.02 (d, J = 8.6 Hz, 2H-a), 7.94 (d, J = 8.5 Hz, 2H-b), 7.57 (t, J = 7.5 Hz, 2H-c), 7.26 (s, 1H-
d), 7.15 - 7.23 (m, 3H-e), 7.09 - 7.15 (m, 1H-f), 7.07 (s, 1H-g), 6.61 - 6.72 (m, 1H-h), 3.29 (s,
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6H,2CH3-i); 13C NMR (101 MHz, CDCl3)  (ppm): 162.2, 147.2, 130.5, 129.7, 129.5, 126.8, 126.7,
124.7, 123.9, 120.2, 114.8, 114.4, 110.0, 29.7(Me), 29.6 (Me); HRMS-ESI(+) calculated for
C21H20N5, 342.1640; found 342.1712 (100%) [M+H]+
(E)-N-(4-bromo-3-(3,3-dimethyltriaz-1-en-1-yl) phenyl) acridin-9-amine (4c). The compound was
purified by column chromatography [EtOAc:CH2Cl2 (5:5) v/v]; yield: 96 mg (62%), Rf = 0.46, brown
o
1
solid, MP: 160-161 C; FT-IR (KBr, ν, cm−1): 2952, 1463,1502,1429,1348; H NMR (400 MHz,
DMSO-d6)  ppm): 8.23 (dd, J = 1.47, 8.0 Hz, 1H-a), 7.92 (br., s., 2H-b), 7.72 (ddd, J = 1.5, 6.9, 8.4
Hz, 1H-c), 7.38 - 7.62 (m, 5H-d), 7.25 (ddd, J = 1.0, 6.9, 8.0 Hz, 1H-e), 7.06 (s., 2H-f), 6.87 (d, J =
2.1 Hz, 1H-g), 6.51 (dd, J = 2.51, 8.4 Hz, 1H-h), 3.44 (br. s., 3H-CH3-i), 3.17 (brs, 3H,CH3-j) 13C
NMR (101 MHz, DMSO-d6)  (ppm): 148.5, 140.9, 133.6, 133.4, 131.7, 126.8, 126.7, 126.0, 121.0,
120.5, 117.3, 110.8, 107.9, 36.1 (Me), 31.3 (Me); HRMS-ESI(+) calculated for C21H19BrN5 Exact
Mass: 420.0746; found 419.0765, 421.0787 (47% : 53%) [M+H]+.
(E)-N-(3-(3,3-dimethyltriaz-1-en-1-yl)-4-methoxyphenyl) acridin-9-amine (4d). The compound
was purified by column chromatography [EtOAc:CH2Cl2 (5:5) v/v]; yield: 123 mg (74%,) Rf = 0.46 ,
brownish-black solid, MP: 246-247 oC, FT-IR (KBr, ν, cm−1): 3032, 1603, 1443, 1321; 1H NMR (400
MHz, DMSO-d6)  (ppm): 7.90 (br., s., 2H-a), 7.50 (br., s., 3H-b), 7.40 (br., s., 2H-c), 6.85 - 7.15 (m,
2H-d), 6.96 (d, J = 8.6Hz, 1H-e), 6.72 (br., s, 1H-f), 6.52 (dd, J = 2.57, 8.6Hz, 1H-g) , 3.79 (s, 3H-
13
OCH3-f), 3.10 - 3.40 (brs, 6H,2CH3- g); C NMR (101 MHz, DMSO-d6)  (ppm): 148.1, 140.8,
140.1, 138.3, 133.4, 131.0, 130.3, 125.8, 122.3, 120.5, 115.4, 114.5, 107.8, 56.3, 31.1(Me), 28.2
(Me); HRMS-ESI(+) calculated for C22H22N5O, 372.1746; found 372.1823 (100%) [M+H]+.
2.9.4 General procedure for the synthesis of the methyl triazene derivatives of nitroaniline (5).
A stirred suspension of a given nitroaniline (0.3 g, 1 equiv.) in 25 mL of hydrochloric acid diluted in
water in a ratio of HCl(c):H2O (4:6) was diazotized at 0oC with sodium nitrite (0.178 g, 1.2 equiv.) in
water. The reaction was stirred for 45 min to a clear solution. The diazonium salt solution was treated
with 40% aqueous methylamine, whereupon a triazene precipitate usually appeared (pH 7-8). The
reaction mixture was quickly extracted with CHCl3 (2 × 10 mL). The combined organic layers were
washed with NaHCO3 (4 × 10 mL), brine, dried over Na2SO4 (anh.) and the solvents were removed
under reduced pressure to afford a red-orange oil that was used without further purification. DIEA
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(1.130 mL) was added to a stirred solution of the above mentioned red-orange oil in CH2Cl2 (15 mL)
o
at 0 C followed by careful addition of ClCO2Me (0.187 mL, 1.1 equiv.) for 5a, 5c, and PhCOCl
(0.297 mL, 1.1 equiv.) was used for 5b. After stirring for 6 h, the reaction mixture was carefully
washed with dilute HCl several times until the aqueous layer reached a pH of 5. The organic layer
was washed with brine, dried over Na2SO4 (anh.) and the solvents removed under reduced pressure to
afford the crude product, which was purified by silica gel chromatography using EtOAc:petroleum
ether (2:8) to give pure products.
Methyl (E)-1-methyl-3-(2-nitrophenyl) triaz-2-ene-1-carboxylate (5a). The compound was
purified by column chromatography [ethyl acetate:petroleum ether (1:9), v/v]; yield: 393 mg (76 %),
Rf = 0.84, yellow liquid; MS-ESI, (m/z) = 239 (M+1, 26%), 261 (M+23, 62%); 1H NMR (400 MHz,
CDCl3)  ppm): 7.79 - 7.87 (m, 1H-a), 7.57 - 7.63 (m, 1H-b), 7.53 (dd, J = 1.28, 8.01 Hz, 1H-c), 7.40
- 7.46 (m, 1H-d), 3.98 (s, 3H-OCH3-e), 3.46 (s, 3H,CH3-f); 13C NMR (101 MHz, CDCl3)  ppm):
154.5, 145.9, 142.3, 133.2, 128.6, 124.3, 120.5, 54.5, 31.0 (Me) .
(E)-(1-methyl-3-(2-nitrophenyl) triaz-2-en-1-yl) (phenyl) methanone (5b). The compound was
purified by column chromatography [ethyl acetate:petroleum ether (1:9), v/v]; yield: .445 mg (72 %)
o
Rf = 0.84, yellow solid, MP: 136 - 137 C, MS-ESI, (m/z) = 284 (M+, 57%), 285 (M+1, 43%), 307
1
(M+23%); H NMR (400 MHz, CDCl3)  (ppm): 7.81 (dd, J = 1.34, 8.07 Hz, 1H-a), 7.65 - 7.71 (m,
2H-b), 7.38 - 7.55 (m, 5H-c), 7.24 (dd, J = 1.34, 8.07 Hz, 1H-d), 3.63 (s, 3H-CH3-e); 13C NMR (101
MHz, CDCl3)  ppm 171.9, 146.3, 141.7, 133.9, 132.9, 131.4, 129.9, 128.7, 127.9, 124.2, 119.9, 29.3
(Me).
Methyl (E)-1-methyl-3-(4-nitrophenyl) triaz-2-ene-1-carboxylate (5c). The compound was
purified by column chromatography [ethyl acetate:petroleum ether (1:9), v/v]; yield: 456 mg (88.2%
o
), Rf = 0.86, yellow solid, MP: 96 - 97 C, MS-ESI, (m/z) = 239 (M+1, 29%), 260.9 (M+23, 60%),
150 (M-88, 18%); 1H NMR (400 MHz, CDCl3) )  (ppm): 8.15 - 8.39 (d, J = 9.05 Hz, 2H), 7.67 - 7.80
(d, J = 9.05 Hz, 2H), 4.00 (s, 3H), 3.51 (s, 3H); 13C NMR (101 MHz, CDCl3)  (ppm): 154.4, 153.2,
147.3, 124.7, 122.7, 54.4, 30.8 (Me).
2.9.5 General procedure for the synthesis of methyl triazene derivatives of aniline (6).
A given derivative of nitro aniline 5 (0.2 g, 1 equiv.) was added to a suspension of methyl triazene
and catalytic amount of 10% Pd/C in ethanol (4 mL). The reaction was carried out under hydrogen
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(1atm) for 4 h at room temp. The reaction was monitored by TLC and LCMS. After completion, the
crude product was filtered through a Celite® bed and the solvent was removed under reduced pressure.
After complete evaporation of the solvent, water was added, and reaction mixture extracted with
EtOAc. The organic layer was washed with brine, dried over Na2SO4 (anh.) and concentrated under
reduced pressure to give a crude product purified by column chromatography.
Methyl (E)-3-(2-aminophenyl)-1-methyltriaz-2-ene-1-carboxylate (6a). The compound was
purified by column chromatography [ethyl acetate:petroleum ether (1:9), v/v]; yield: 150 mg (86%),
1
Rf = 0.54, yellow liquid; MS-ESI, (m/z) = 209 (M+1, 100%); H NMR (400 MHz, CDCl3)  ppm):
7.51 - 7.59 (m, 1H-a), 7.13 (ddd, J = 1.47, 7.15, 8.13 Hz, 1H-b), 6.69 - 6.80 (m, 2H-c), 5.03 (brs, 2H-
13
d), 3.97 (s, 3H-f), 3.46 (s, 3H-CH3-g); C NMR (101 MHz, CDCl3)  ppm): 155.1, 141.8, 133.3,
130.1, 124.7, 117.8, 116.9, 54. (CO2Me), 1, 29.7(Me).
(E)-(3-(2-aminophenyl)-1-methyltriaz-2-en-1-yl) (phenyl)methanone (6b). The compound was
purified by column chromatography [ethyl acetate:petroleum ether (1:9), v/v]; yield: 164 mg (92%),
1
Rf = 0.74, yellow liquid, MS-ESI, (m/z) = 255 (M+1, 95%); H NMR (400 MHz, CDCl3)  (ppm):
7.57 - 7.68 (m, 2H-a), 7.39 - 7.54 (m, 3H-b), 7.24 - 7.31 (m, 1H-c), 7.01 - 7.12 (m, 1H-d), 6.62 - 6.70
(m, 2H-e), 4.52 (brs, 2H-f), 3.61 (s, 3H-CH3-g); 13C NMR (101 MHz, CDCl3)  ppm): 172.0, 142.3,
134.9, 133.0, 130.7, 130.4, 129.3, 127.9, 122.8, 117.9, 116.7, 28.1(Me).
Methyl (E)-3-(4-aminophenyl)-1-methyltriaz-2-ene-1-carboxylate (6c). The compound was
purified by column chromatography [ethyl acetate:petroleum ether (1:9), v/v]; yield: 164 mg (94.2%),
o
1
Rf = 0.68, yellow solid, MP: 112 - 113 C, MS-ESI, (m/z) = 209 (M+1, 98%); H NMR (400 MHz,
CDCl3);  (ppm): 7.40 - 7.70 (d, J = 8.68 Hz, 2H-a), 6.51 - 6.72 (d, J = 8.93 Hz, 2H-b), 6.24 - 6.88
(m, 2H-c), 3.95 (s, 3H-d), 3.43 (s, 3H-CH3-e); 13C NMR (101 MHz, CDCl3) )  (ppm): 155.2, 147.5,
140.7, 123.7, 114.9, 53.8(CO2Me), 29.8(Me).
2.9.6 General procedure for the synthesis of methyl triazene 9-AnA derivatives (7).
Sodium hydride (1.2 equiv.) was added in small portions during aperiod of 15 min to the stirred
solution of a given amine (6) (0.1 g, 1 equiv.), in dry DMF (6 mL) at 0 oC. Then, 9-chloroacridine was
o
slowly added to the reaction mixture under positive nitrogen pressure. After stirring at 0 C for 45
min the reaction mixture was kept at room temperature for 11 h. The reaction was monitored by TLC
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and LCMS. After completion, the crude product was diluted with a saturated solution of NH4Cl and
extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4 (anh.), filtered
and the solvent removed under reduced pressure to give a product, which was later purified by
column chromatography.
Methyl (E)-3-(2-(acridin-9-ylamino) phenyl)-1-methyltriaz-2-ene-1-carboxylate (7a). The
compound was purified by column chromatography [EtOAc:CH2Cl2 (5:5) v/v]; yield: 136 mg, (74
o
%,) Rf = 0.53, brown solid, MP: 132 - 133 C; FT-IR (KBr, ν, cm−1): 3005, 1703, 1657, 1420, 1374;
1H NMR (400 MHz, CDCl3)  (ppm): 8.23 (br. s., 2H-a), 8.12 (d, J = 8.68 Hz, 2H-b), 7.70 - 7.82 (m,
3H-c), 7.44 (t, J = 7.40 Hz, 2H-d), 6.92 - 7.05 (m, 2H-e), 6.54 (d, J = 8.07 Hz, 1H-f), 3.83 (s, 3H-g),
13
3.57 (s, 3H-CH3-h); C NMR (101 MHz, CDCl3)  (ppm): 154.8, 134.4, 131.0, 130.7, 130.6, 129.8,
129.6, 125.3, 124.2, 124.0, 122.9, 121.8, 120.1, 116.2, 54.3 (CO2Me), 29.8 (Me); HRMS-ESI (+)
calculated for C22H20N5O2, 386.1539; found 386.1636 (100%) [M+H]+.
(E)-(3-(2-(acridin-9-ylamino) phenyl)-1-methyltriaz-2-en-1-yl)(phenyl) methanone (7b). The
compound was purified by column chromatography [EtOAc:CH2Cl2 (5:5) v/v]; yield: 114 mg (67 %,)
o
1
Rf = 0.57, brown solid, MP: 228 - 229 C; FT-IR (KBr, ν, cm−1): 2990, 1660, 1531, 1433, 1302; H
NMR (400 MHz, CDCl3)  (ppm): 8.21 (d, J = 10.64 Hz, 1H-a), 8.09 - 8.17 (m, 2H-b), 7.87 (d, J =
8.68 Hz, 1H-c), 7.75 (t, J = 7.34 Hz, 1H-d), 7.56 - 7.63 (m, 2H-e), 7.43 - 7.56 (m, 4H-f), 7.34 - 7.41
(m, 2H-g), 7.06 - 7.16 (m, 1H-h), 6.96 - 7.05 (m, 2H-i), 6.80 - 6.96 (m, 2H-k), 3.66 (s, 3H-CH3-l); 13C
NMR (101 MHz, CDCl3)  (ppm): 172.1 (C=O), 147.2, 147.0, 146.3, 140.7, 134.5, 134.4, 131.4,
130.8, 130.6, 130.1, 129.6, 129.2, 128.4, 127.9, 125.1, 124.4, 117.8; 29.8 (Me); HRMS-ESI(+)
calculated for C27H22N5O, 432.1746; found 432.1815 (100%) [M+H]+.
Methyl (E)-3-(4-(acridin-9-ylamino) phenyl)-1-methyltriaz-2-ene-1-carboxylate (7c). The
compound was purified by column chromatography [EtOAc:CH2Cl2 (5:5) v/v]; yield: 144 mg, (78
o
%,) Rf = 0.54, brown solid, MP: 138 - 139 C; FT-IR (KBr, ν, cm−1): 2897, 1690, 1608, 1510, 1409;
1H NMR (400 MHz, CDCl3)  (ppm): 7.92 - 8.16 (d, J = 8.44 Hz, 2H-a), 7.43 - 7.64 (m, 5H-b), 7.21
13
(brs, 3H-c), 6.78 - 6.94 (d, J = 8.68 Hz, 2H-d), 3.96 (s, 3H-e), 3.46 (s, 3H-CH3-f); C NMR (101
MHz, CDCl3) )  (ppm): 155.1, 143.3, 139.3, 130.9, 126.0, 125.8, 124.5, 123.9, 123.7, 119.9, 119.1,
118.0, 54.0 (CO2Me), 31.4 (Me); HRMS-ESI (+) calculated for C22H20N5O2, 386.1539; found
386.1636 (100%) [M+H]+.
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3. Results and discussion
3.1 Synthesis of dimethyl triazene nitroaryls 1a - 1i.
To demonstrate our synthetic approach, we initially synthesized dimethyl triazene nitroaryl fragments
1a - 1i mimicking the dacarbazine bioactive core structure (Scheme 1). These building blocks are
decorated with representative electron withdrawing (EWG) and electron donating (EDG) groups
designed to examine their impact on the course of the acridine derivatization reaction.
R
R
NH2
N
N
a,b
N
NO2
NO2
1 a-i
N
N
N
N
N
N
N
O2N
N
N
N
N
O2N
NO2
, 86%
1d
, 82%
1g
,89%
1a
N
N
O2N
Br
N
N
N
O2N
N
N
NO2
, 87%
Br
1b
1e
, 58%
1h
, 74%
N
N
N
N
O2N
N
N
N
N
N
O
NO2
, 91%
O
O
NO2
, 76%
1c
O
1f
1i
, 91%
Reagents and conditions: a) NaNO2/HCl, 0 oC, 45 min; b) aq. 40% sol. dimethylamine.
SCHEME 1 Synthesis of the starting dimethyl triazene nitroaryls 1a-1i
The synthesis of theses derivatives involves two consecutive steps. Diazotization of a starting
nitroaniline by sodium nitrite in diluted HCl at 0 °C followed by the condensation of the
corresponding diazonium salt with 40% aqueous dimethyl amine, without isolation of the
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intermediate. This process led us to the desired dimethyl triazene derivatives 1a - 1i of the
corresponding nitroaryls with H, Me, OMe, CO2Me and Br substituents at the phenyl ring.
Initially, we followed a literature procedure where neutralization of diazotation reaction mixture using
sodium carbonate is followed by the addition 1.2 equiv. of 1 M solution of dimethyl amine in THF
(White et al.. 1973; Diana et al. 2011). Unfortunately, the products were obtained in low yields below
20% probably due to the decomposition of the diazonium salts during the neutralization process. We
modified the original procedure by skipping the neutralization step and directly adding excess 40%
dimethyl amine (aq. sol.) reaching pH 8. This procedure afforded the corresponding triazene
compounds 1a - 1i in moderate to high yields (58 - 91%) (Scheme 1).
3.2 Synthesis of dimethyl triazene anilinoacridine chimeras 2a - 2b by SNAr.
Previously, we reported on the SNAr reaction of electrophilic haloaryls bearing one or two strongly
electron-withdrawing groups to obtain antiproliferative 9-AnA derivatives (also dubbed: ''reverse''
approach) (Gellerman et al. 2010; Gellerman et al. 2011). Here, we employed a similar synthetic
strategy to obtain two new molecular chimeras. Treatment of arylbromides 1e and 1h with 9-AnA in
the presence of 1 equiv. of Cs2CO3 in DMF at 90 °C for 12 h afforded the dimethyl triazene
derivatives of 9-AnA 2a and 2b in good yields (68% and 62%, respectively, Scheme 2).
NO2
N
N
N
N
N
N
N
N
N
Br
O2N
HN
N
N
NO2
1e
N
NO2
a
1h
NH2
Br
a
HN
N
N
2a
, 68%
2b
, 62%
Reagents and conditions: a) Cs2CO3, DMF, 90 °C, 12 h.
SCHEME 2 Synthesis of dimethyltraizene 9-AnA derivatives 2a, 2b by SNAr reaction
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Chimeras 2a and 2b are unlikely to be obtained using the ''classical'' EAS approach (Fig. 2) due to
low reactivity of the putative amine group on the required anilines substituted with strong electron
withdrawing nitro groups.
3.3 Synthesis of dimethyltriazene 9-AnA chimeras 4a - 4d by EAS.
In order to further extend the scope of the library of 9-AnA dimethyl triazene hybrids, we employed
the “classical” electrophilic aromatic substitution (EAS) approach, where 9- chloroacridine reacts
with a nucleophilic aniline (Blaziak et al. 2016). The reduction of the nitro group in nitroaryl triazenes
1a - 1i was planned to provide the required anilino triazene nucleophiles. However, during the
reduction of 1a - 1i, we noticed that the formation of anilino triazene derivatives from nitroaryls we
observed that only the reduction of meta- and para- nitro triazenes led to the desired products. In
contrast to the reduction of ortho-nitro triazenes resulted in a different product (Scheme 3). Based on
1
experimental and spectral data (reaction conditions, H, 13C NMR, and MS see supportive
information), we concluded that this product is the corresponding benzotriazole 3 (Scheme 3), which
forms in 100% conversion from nitro triazenes 1a, b, c, and f regardless of other substituents in the
ring, namely electron-donating or withdrawing groups.
N
N
N
N
a
N
N
R
R
NO2
1a,b,c,f
NH2
N
N
N
R
H
3a
3b
3c
3d
R = OMe (86%)
R = Me
R = H
(82%)
(81%)
R = CO2Me (72%)
SCHEME 3 First attempt towards the synthesis of o-dimethyltriazene aniline building blocks
This observation has been previously documented by reports describing the triazene conversion to
benzotriazole via an intramolecular cyclization (Kumar et al. 2011; Zhou et al. 2011). A possible
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mechanism for the benzotriazole formation following the reduction of corresponding ortho-
nitrotriazenes is depicted in Scheme 4. Initially, the ortho-nitrotriazene compound is reduced to an
ortho-aminotriazene in the presence of Pd/C under a hydrogen atmosphere (1 atm) (A). Then, the
amine lone pair of electrons resonates through the phenyl ring to form zwitterion B, which rapidly
rearomatizes via intramolecular cyclization to intermediate C followed by proton transfer to the 2-
triazole amine D. Finally, the formation of the aromatic benzotriazole 3 provides the driving force for
the elimination of dimethyl amine.
N
N
N
N
N
N
N
N
N
N
Reduction
N
N
NH2
NH2
R
R
NO2
R
NH2
R
A
B
C
H
H
N
N
N
N
N N
N
N
H
R
H
SCHEME
4
Proposed
reaction
3
D
mechanism for the formation of benzotriazole 3
We decided not to pursue the ortho derivatives by this approach and move forward towards the
meta and para 9-AnA dimethyl triazenes 4a, b, c, and d. To that end, triazene nitroaryls 1d, g, h, and
i were reduced using hydrogen and 10% Pd/C in EtOH. Filtration of the catalyst followed by EAS
reaction with 9-Cl acridine finally led to the desired products with good yields (62-74% after two
steps without isolation of intermediates, Scheme 5).
R
N
N
N
N
SCHE
ME 5
Synthe
sis of
dimeth
yltriaz
N
HN
N
N
HN
a,b
a,b
1d,h,i
1g
N
4b
4c
4d
, R = H, 68% (after two steps)
R = Br, 62% (after two steps)
R = OMe, 74% (after two steps)
4a
64%
Reagents and conditions: a) 10% Pd/C, H2 (1 atm), EtOH, r.t., 3h; b) 9-Cl acridine, DIPEA, EtOH ,r.t.,12h.
ene 9-AnA derivatives 4a - 4d by EAS
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3.4 Synthesis of protected monomethyl triazene chimeras 7a - 7c by EAS.
Temozolomide (TMZ) is an anticancer drug, which contains an embedded monomethyl triazene
structural tether. The therapeutic activation of TMZ involves the spontaneous decomposition of a
cyclic urea core, forming the unstable MTIC (Fig. 1), which under physiologic conditions releases
diazomethane (a very reactive DNA methylating agent). Therefore, we presumed that the protection
of mono methyl amine intermediates using carbamate or amide biodegradable groups (Beckerab et al.
2018) could result in molecular candidates better fit to release DNA methylating agents, similarly but
with slower kinetics than TMZ (Fang et al. 2012). In addition, we realized that the N-protection of
monomethyl triazene in the corresponding ortho-nitroaryls could help to overcome the formation of
the un-reactive benzotriazole upon reduction of NO2 to NH2 as mentioned above. The ortho- and
para-monomethyl triazene nitroaryls 5a, b, and c bearing methyl carbamate and benzamide
protections, respectively (Scheme 6), were prepared and subjected to catalytic hydrogenation (H2,
10% Pd/C) directly leading to the protected ortho triazene anilines 6a and 6b and the para triazene
aniline 6c ready to be coupled with 9-Cl acridine. Finally, 6a - c were reacted with 9-Cl acridine using
NaH in DMF. Following this approach we were able to generate the desired 9-AnA monomethyl
triazene chimeras 7a - c in 74%, 67% and 78% yield, respectively.
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N
N
NH2
N
N
N
R
N
b
R
a
NH2
NO2
NO2
6a
b
c
o-nitro, R = CO2Me (86%)
o-nitro, R = COPh (92%)
p-nitro, R = CO2Me (94%)
5a
b
c
o-nitro, R = CO2Me (76%)
o-nitro, R = COPhe (72%)
p-nitro, R = CO2Me (83%)
N
N
N
N
O
R
N
N
O
c
HN
HN
6a,b,c
or
N
N
7a
b
R = CO2Me (74%)
R = COPh (67%)
7c
(78%)
Reagents and conditions: a) i. NaNO2/HCl, 0 °C, 30 min., ii. aq.methylamine, iii. ClCO2Me,
5a, 5c 5b
DIPEA, DCM, 0 °C to r.t, 3h for
; PhCOCl, DIPEA, DCM, 0 °C to r.t, 3h for . b) 10%
Pd/C, H2 (1 atm), EtOH, r.t., 3 h; c) 9-Cl acridine, NaH, DMF, 0 °C to r.t, 12 h.
SCHEME 6 Synthesis of protected monomethyl triazene 9-AnA chimeras 7a - 7c by EAS
3.5 Chemostability of triazene chimeras in acetate and PBS buffers.
Chemostability studies of the three chimeras 7a, 7b and 7c as well as the dimethyl triazene 9-
AnAs 2a – b and 4a - d hybrids were performed at two pH values: 4.6 and 7.4. The results
were compared to the chemostabilities of TMZ and DTIC under the same conditions. The two
pHs were chosen to mimic the relatively acidic milieu present in some common cancer
intracellular fluids (pH 4.6) and the slightly basic physiological environment (pH 7.4),
respectively. For this study, samples of all compounds were incubated at 37 oC. Aliquots were
taken at selected time intervals and were analyzed by LC-MS (Fig. 3). In PBS buffer (pH 7.4,
Fig. 3A) the dimethyl triazene 9-AnAs 2a – b and 4a - d exhibited similar stability (t1/2 = 10 -
15 h) as DTIC (t1/2 = 10.5 h), while monomethyl triazene counterparts 7a - c (t1/2 = 4.8 - 10.6 h)
and TMZ (t1/2 = 5.5 h) decomposed faster. However, at the acidic pH (Fig. 3B) a reversed
stability pattern, revealed that the protected monomethyl triazenes 7a - c are more stable than
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the dimethyl triazenes 2 and 4. Our results are consistent with the reported stability of the
control drugs TMZ and DTIC in various hydrolytic media (Mirzaei et al. 2015). The results of
these experiments also indicate very little difference in stability between methyl carbamate and
benzamide degradable groups in 7a, 7b and 7c. From the data we can conclude that the
stability of both series of chimeras is in a comparable range as that established by TMZ and
DTIC and therefore the herein described chimeras appear to be promising candidates to display
potent anticancer activities.
FIGURE 3 Chemostability of dimethyl triazene chimeras 2a, b, 4a - 4d, and monomethyl chimeras
7a - 7c in PBS (pH 7.4) and acetate (pH 4.6) buffers
3.6 Cell cytotoxicity of triazene chimeras
A preliminary anticancer study to estimate the potential antitumor activity of all compounds was
performed. The chimeras were subjected to a cytotoxicity assay against three cancer cell lines H1299
(NSCLC), WM266.4 (human metastatic melanoma) and HCT116 (colorectal carcinoma). HCT116
cell line expresses elevated levels of MGMT, the enzyme O(6)-methylguanine-DNA
methyltransferase that repairs alkylating damage, such as that induced by temozolomide (see
supporting info, Fig. S1) (Yusein-Myashkova et al. 2016; Zheng et al. 2008; van Nifterick et al.
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2010). The cells were exposed to increasing doses of the compounds, and after 48 h or 72 h of
incubation, metabolic activities of the cells were measured using the XTT assay. Antiproliferative
tests of compounds 2, 4 and 7 identified them as bioactive chimeras against all cancer cell lines in the
micromolar range. These compounds exhibited increased cytotoxic effects compared to Amsacrine
and AHMA (Table 1). In general, protected monomethyl triazenes 7 were more active that dimethyl
triazene derivatives 2 and 4, probably because they do not require activation by cytochrome P450 of
the dimethyltriazene tether, which occurs only in vivo. Among triazenes 7, compound 7b is of special
interest. This chimera exhibited the highest anticancer activity against H1299 and WM266.4 cancer
cell lines (over ten times more active than Amsacrine and AHMA) with IC50 values of 2.9 M and 0.8
M after 72 h of incubation, respectively. HCT116 cell line was, in general, the most resistant to
anticancer treatment. Hybrid 7b also showed the highest cytotoxic effect on this cell line compared to
all other tested candidates and control drugs. Such elevated activity could be the result of a dual
action mechanism only forged ahead by a chimera, acting both as a DNA intercalating and alkylating
agent. Additional testing to support this idea was performed, see below. The fact that 7b, as well as all
other tested compounds, is less effective on HTC116 can be attributed to its MGMT associated DNA
repairing activity. Notably, TMZ was not active in all in vitro experiments at the tested concentrations
(IC50 > 100) (Le Calve et al. 2010). The results pinpoint 7b as a potent anticancer chimeric candidate
to be considered for further development.
TABLE 1 IC50 (half maximal inhibitory concentration) values (M) for 2a - b, 4a - d, 7a - c, AHMA
and Amsacrine in H1299, WM266.4 and HTC116 cell lines for 48 h and 72 h incubation. IC50 values
were calculated using three-parameter non-linear regression. IC50 values marked with * were
calculated using four-parameter non-linear regression. N/A means IC50 values and shifts could not be
reliably calculated
Comp.
2a
2b
4a
4b
4c
4d
7a
7b
7c
Amsacrine AHMA TMZ
Cell line
27.7
28.6
22.2
31.4*
27.6
24.6
5.3
25.2
25.8
36.9*
H1299-48h
N/A
>100
(+0.12) (+0.95) (+0.18) (+0.41) (+0.09)
(+0.84) (+0.23) (+1.02)
(+2.31)
(+0.77)
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17.1
18.2
12.3
21.3
20.5
33.2
14.4
2.9
5.6
16.5
17.7
H1299-72h
>100
>100
>100
>100
>100
(+0.26) (+0.18) (+0.35) (+0.46) (+1.89) (+1.17) (+0.76) (+0.43) (+0.62)
(+0.03)
(+0.80)
35.1
32.3
46.8
28.6
29.9
13.5
27.7
3.6
13.5
20.2
24.3
WM264.4-48h
WM264.4-72h
HCT116-48h
HCT116-72h
(+0.33) (+1.52) (+0.74) (+0.68) (+0.73) (+0.86) (+0.12) (+0.48) (+0.54)
(+1.46)
(+2.30)
28.3
19.8
35.1
18.5
17.0
9.4
0.8
12.8
11.5
15.8
N/A
(+0.49) (+0.78) (+0.81) (+1.37) (+0.11)
(+1.19) (+0.37) (+0.94)
(+0.98)
(+1.23)
48.4
57.1
45.3
30.8
46.0
36.8
41.2
N/A
N/A
N/A
51.3
N/A
48.0
N/A
46.9
(+3.44) (+2.32)
(+1.97)
(+3.56) (+3.69)
(+0.92)
(+1.73)
41.1
38.7
37.1
15.4
37.5
31.9
32.2
(+2.35) (+3.63) (+4.49) (+2.92) (+1.28) (+2.76) (+1.68) (+1.05)
(+1.33)
(+2.41)
3.7 Western blot analysis of 7b
Based on the preliminary antiproliferative studies we decided to take a closer look at hybrid 7b to
confirm its anticancer activity and assess its mode of action. Western blot analysis to define O(6)-
Methylguanine DNA methyltransferase (MGMT) protein levels in the HTC116 cell line was
performed in the presence of 7b. MGMT is a DNA repairing enzyme that removes the mutagenic
alkyl adducts introduced at the O(6) position of guanine and the O(4) position of thymine introduced
by various exogenous and endogenous agents (Kaina et al. 2007). Unlike other DNA repairing
pathways, human MGMT works as a single protein to remove the guanine O(6) bound alkyl groups
by the nucleophilic action of the thiol group on cysteine145 located in its active site (Hegi et al.
2008). Since the amount of MGMT determines repair level of DNA alkylated adducts, the MGMT
expression level provides important information about cancer susceptibility and the chances of
success of potential drugs based on alkylating mechanisms of action (Christmann et al. 2011).
Temozolomide (TMZ) and Dacarbazine (DTIC) belong to a class of drugs known as alkylating
agents. The efficacy of treatment with these agents may be limited by inherent or developed
resistance, particularly, but not exclusively, due to the expression of MGMT in a significant
proportion of tumors, specifically in glioblastoma (Fan et al. 2013). In order to study the effect of
chimera 7b on the level of MGMT we treated HCT116 cells with 100 μM of TMZ, DTIC and chimera
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7b for 48 h. Lysates from compound-treated cells were subjected to Western blot analysis for MGMT
protein detection. Compared with the two controls, TMZ and DTIC, we found that chimera 7b was
more effective in reducing the expression of MGMT by 50% (Fig. 4). This study indicates that
chimera 7b promotes DNA methylation with certainty. In order to demonstrate that 7b may rely on a
dual action mechanism to exert its enhanced antitumor activity we also investigated its ability to
inhibit the enzymatic activity of Topo II.
FIGURE 4 HCT-116 cells were treated with DTIC, TMZ and Chimera 7b in concentration of 100
µM for 48 h. Protein samples were prepared and resolved by SDS-PAGE. MGMT and tubulin
(loading control) antibodies were used to detect protein levels. The graph represent the quantification
of the protein level as evaluated by image studio software. Values represent a mean of at least 3
experiments. *P-value < 0.05. DTIC - Dacarbazine, TMZ - Temozolomide
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3.8 Chimera 7b inhibits enzymatic activity of Topo II
AHMA is an anticancer agent that reversibly binds at the minor groove of DNA and stabilizes the
Topo II DNA complex interfering with its enzymatic activity (Delgado et al. 2018). Chimera 7b is a
structural combination of AHMA and a triazene moiety. The modification of the AHMA scaffold may
affect its original DNA-intercalation and DNA-Topo II stabilization effectiveness. The Topo II
inhibitory activities of AHMA and chimera 7b were evaluated employing the kDNA decatenation
enzymatic assay. In this assay, catenated DNA (kDNA) remains at the top of the electrophoretic gel,
whereas the smaller decatenated DNA, minicircles, produced from enzymatic activity of Topo II runs
to the bottom of the gel. The inhibitory activity of the two compounds were assessed at 25 M and
100 M. At the higher concentration both compounds showed no development of minicircles,
corroborating that AHMA and 7b strongly inhibit Topo II. At the lower concentration, AHMA was
not capable to fully repress the formation of minicircles while 7b showed no visible signs of them
(Fig. 5). The results of the kDNA decatenation enzymatic assay indicates that chimera 7b is a stronger
inhibitor of Topo II than AHMA.
FIGURE 5 Topo II enzymatic inhibition by 7b and AHMA. Chimera 7b or AHMA were added to a
cell-free lysate containing Topo II and DNA. After incubation the products were separated by agarose
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electrophoresis. The upper bands are catenated DNA (due to enzyme inhibition) while the lower
bands (minicircles) are decatenated DNA
4. Conclusions
In summary, we developed molecular chimeras toward dual-action anticancer agents. These chimeras
consist of a DNA intercalating core tethered to a methyl triazene DNA methylating moiety. An
efficient preparation method of aromatic triazene synthetic fragments, followed by their EAS
coupling with 9-chloroacridine or SNAr with 9-aminoacridine, afforded novel molecular chimeras in
good yields. Chemostability studies indicated that the chimeras are stable enough to perform as
anticancer agents. At physiological pH dimethyl triazenes are more stable than the monomethyl
counterparts protected by amide or carbamate degradable groups. In acidic media, the dimethyl
triazene derivatives were found to be the less stable bunch. An in vitro assay revealed benzamide
monomethyl triazene 7b, which efficiently inhibits growth of MGMT deficient H1299 and WM266.4
cancer cell lines after 72 h incubation with IC50 values of 2.9 M and 0.8 M, correspondingly, as a
promising dual-action candidate to further studies. Positive results from MGMT western blot and
Topo II inhibition studies indicate that chimera 7b may exert its antiproliferative effect either by DNA
alkylation or via DNA intercalation. The two inhibitory mechanisms may work independently or,
most likely, in a coexistent synchronized fashion. Chimera 7b was identified as a potent dual-action
anticancer candidate for preclinical studies.
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Conflict of interest -The author declares no conflict of interests.
Acknowledgements
The authors thank Mrs. Cherna Moskowitz for generous stipend to D.W. The authors also thank the Authority
for Research & Development of the Ariel University for financial support. F.G. is incumbent of the Cosman
endowment for organic chemistry research.
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ORCID
Gary Gellerman http://orcid.org/0000-0002-213368707
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