Facile synthesis and antitumor activity of novel N(9) methylated AHMA analogs

Article abstract of DOI:10.1039/c2nj40567a

A facile synthesis of novel antitumor N(9)-methyl-3-(9-acridinylamino)-5- hydroxymethylaniline (AHMA) derivatives is described. Boc protection of aminobenzoic acids followed by LiAlH₄ reduction yielded novel methylaminobenzyl alcohol reactants. Their interaction with 9-chloroacridine provides N(9)-methylated AHMA derivatives for biological screening. A preliminary anti-proliferative assay against seven cancer cell lines identified compounds with low μM IC₅₀ values. The Royal Society of Chemistry and the Centre National de la Recherche Scientifique.

Full text of DOI:10.1039/c2nj40567a

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LETTER
Facile synthesis and antitumor activity of novel N(9) methylated
AHMA analogsw
Boris Redko,ab Amnon Albeckb and Gary Gellerman*a
Received (in Montpellier, France) 4th July 2012, Accepted 29th August 2012
DOI: 10.1039/c2nj40567a
A facile synthesis of novel antitumor N(9)-methyl-3-(9-acridinyl-
amino)-5-hydroxymethylaniline (AHMA) derivatives is described.
Boc protection of aminobenzoic acids followed by LiAlH4 reduction
yielded novel methylaminobenzyl alcohol reactants. Their inter-
action with 9-chloroacridine provides N(9)-methylated AHMA
derivatives for biological screening. A preliminary anti-proliferative
assay against seven cancer cell lines identified compounds with low
lM IC50 values.
half-life in human plasma.4,5 Consequently, the substituents
(NH2 and CH2OH) on the anilino ring of AHMA are located
in a meta-position to each other, thus preventing oxidation.
Therefore, AHMA had a longer half-life in human plasma
compared to m-AMSA.6
It was also noticed that 9-anilinoacridines undergo a rever-
sible amine exchange reaction at position nine under near-
physiological conditions in water.7,8 This thermodynamically
controlled reaction may have implications in understanding
the mode of action of 9-anilinoacridines in vivo and in the
future design of new drugs that will be based on this scaffold.9
These findings brought us to hypothesize that methyl substitu-
tion at amine nine of AHMA derivatives will influence such
exchange reactions or hydrolysis of the aniline moiety, therefore
shedding additional light on the SAR of AHMA analogs.
Surprisingly, N(9) methylated anilinoacridines are hardly
known in the literature, most probably due to poor commercial
availability of the corresponding N(Me) aromatic synthones.
Therefore furnishing the synthesis of suitable N(Me) arenes was
the first task we conducted. Here we report a facile synthesis of
novel antitumor N(9) methylated AHMA analogs for structure–
activity and bio-stability relationship studies. Preliminary anti-
cancer activity of these compounds is also reported.
The discovery of new compounds with antitumor activity has
become one of the most important goals in medicinal chemistry.
One interesting group of chemotherapeutic agents used in cancer
therapy comprises molecules that are DNA intercalators, from
natural products such as doxorubicin and actinomycin D to
synthetic drugs such as mitoxantrone and amsacrine.1
In previous studies on the development of potential 9-anilino-
acridine congeners, 3-(9-acridinylamino)-5-hydroxymethylaniline
(AHMA, 2, Fig. 1) exhibited both in vitro and in vivo potent
antitumor efficacy.2,3 AHMA displayed better antitumor efficacy
than the parent amsacrine (m-AMSA, 1) in mice bearing
mammary and lung carcinomas. Rational drug design for
AHMA derivatives was based on the study of the metabolic
pathway of m-AMSA (1), which was easily bio-oxidized to
form m-AMSA-quinonediimine (m-AQDI) and had a short
Initially, we prepared the required N(Me) arenes from the
corresponding aminoarenes, applying an improved two-step
synthesis described in the literature.10–12 It involves Boc protec-
tion of the aminobenzoic acids and subsequent reduction of both
the acid and the carbamate functional groups with LiAlH4 to
N-methyl benzyl alcohol. We suggest the following mechanism
Fig. 1 The structure of amsacrine, its oxidized metabolic product
m-AQDI, and AHMA.
a Department of Biological Chemistry, Ariel University Center of
Samaria, Ariel 40700, Israel. E-mail: garyg@ariel.ac.il;
Fax: +972 3 9066634; Tel: +972 3 9371442
b The Julius Spokojny Bioorganic Chemistry Laboratory,
Department of Chemistry, Bar-Ilan University, Ramat Gan 52900,
Israel. E-mail: amnon.albeck@biu.ac.il; Fax: +972 3 7384053;
Tel: +972 3 5318862
w Electronic supplementary information (ESI) available: 1H NMR,
13C NMR and HRMS data of all synthetic compounds. See DOI:
10.1039/c2nj40567a
Scheme 1 Proposed mechanism for reductive N-methylation of Boc-
protected anilines.
c
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for the conversion of BocNHAr to MeNHAr: N-Boc is first
reduced to formamide, followed by further reduction to aminal
and dehydration to form imine, which is finally reduced to
N(Me) (Scheme 1).
The procedure works smoothly, yielding N(Me) arenes
12–20 in good yields (Table 1). Structure determination of
12–20 was confirmed by 2D NMR experiments (see ESIw).
Notably, several building blocks underwent unexpected
benzylic OH elimination at ortho and para positions to the
N(Me). For example, in compound 7, only the meta positioned
carboxyl group was reduced to the corresponding alcohol,
Scheme 2 Proposed mechanism for reductive deoxygenation of
N(Me) methyleneoxy anilines.
Table 1 Reaction data for reductive N-methylation of aminoanilines
by Boc protection and LiAlH4 reduction
while the para CO2H was further reduced to produce the methyl
substitution, yielding compound 16. Such a phenomenon was also
observed in 9, 10 and 11, yielding dehydrated products 18, 19 and
20, respectively, as opposed to aminobenzoic acids 3 and 4, bearing
carboxylic groups at meta positions to the amine group. Based on
the above results we assume that such dehydration reactions occur
through 1,4-elimination (for ortho) and similar 1,6-elimination (for
para) of the intermediate aluminum complex, followed by further
reduction. Such a mechanism could not be realized in the meta
hydroxymethyl anilines, preserving it from reductive dehydration
(Scheme 2). The extra elimination–reduction reactions and the
formation of a reactive amino quinone methide intermediate may
explain the somewhat lower yields in the reduction reactions
involving the ortho and para amino carboxylic acids.
Yield (%)
(for the two steps)
Entry Substrate
Product
1
87
2
86
Next, we proceeded to the synthesis of the N(9)Me AHMA
derivatives from the corresponding N-methylated arenes. The
other component, 9-chloroacridine, was prepared by micro-
wave assisted synthesis.13 Finally, the reaction of aminoarenes
12–16 with 9-chloroacridine under mild basic conditions
(NMM) in a chloroform–ethanol mixture at room tempera-
ture afforded new N(9)-Me AHMA derivatives in good yields
(Scheme 3). Unfortunately, aminoarenes 17–20 did not afford
the corresponding 9-anilinoacridines. Only the starting materials
were recovered under various reaction conditions. This might be
3
4
93a
91
5
75a
6
7
77
62a
8
9
70a
63a
a
After chromatography.
Scheme 3 Synthesis of AHMA analogs.
c
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Table 2 Cell growth inhibition assay of the synthetic AHMA derivatives against a non-tumorigenic breast epithelial cell line and seven cancer cell linesa
AHMA derivatives
MFA-10A
H 1299
MCF7 MDR
MCF7 MITO
OVCAR8
NCI ADR
MDA-MD-131
HT29
AHMA
21
22
23
24
11.2
23.7
NA
30.1
NA
NA
5.3
4.5
NA
NA
0.7
5.8
1.8
NA
NA
NA
NA
NA
2.9
NA
NA
NA
NA
1.5
1.5
2.5
5.5
4.1
2.3
2.7
0.6
0.9
0.9
1.9
1.2
0.7
1.7
1.6
NA
8.8
NA
1.2
1.79
NA
NA
NA
NA
25
3.1
a
Cell growth inhibition assay after 48 h incubation. Values reported are IC50 (mM) (n = 3, all SE were between 0.02 and 0.06 mM). Boldfaced
values mark combinations that exhibit a significantly higher cytotoxic effect than that of AHMA. Cell lines are as follows: MDA-MD-131 (renal
cancer), OVCAR8 (ovarian cancer), NCI-ADR (ovarian cancer associated with multidrug resistance (MDR)), MCF-7mito (mitoxantrone selected
breast cancer associated with MDR), HT29 (colon carcinoma), H1299 (lung carcinoma), and MFA-10A (noncancerous epithelial cells). NA – IC50
values above 40 mM; no effect was observed with the vehicle (DMSO) alone.
due to steric hindrance caused by the presence of a methyl
group in an ortho position to the aromatic N(Me).
methanol (33 mL). The solution was stirred vigorously, and
then t-butoxycarbonyl anhydride (28.8 g, 132 mmol) was
added in portions. The mixture was stirred at room tempera-
ture for 24 h. The solution was concentrated, and the residue
was partitioned between ethyl acetate and 1 M potassium
hydrogen sulfate. The organic layer was separated, washed
with water, and dried over magnesium sulfate before the
removal of ethyl acetate.
Preliminary anti-proliferative tests of the synthetic compounds
identified low micromolar leads against MDA-MD-131 (renal
cancer), OVCAR8 (ovarian cancer), NCI-ADR (ovarian cancer
associated with MDR phenomena), MCF-7mito (mitoxantrone
selected breast cancer associated with MDR phenomena), HT29
(colon carcinoma) and almost insensitive to chemotherapy
H1299 (lung carcinoma). Some promising compounds exhibited
enhanced cytotoxic effect as compared with AHMA, while
showing very moderate cytotoxic effect on the non-tumorigenic
breast epithelial MFA10A cell line (Table 2). Interestingly,
significant differences in the sensitivity to the tested compounds
were observed between these cell lines. Of special interest is
compound 24, exhibiting the best activity against the H1299 cell
line (almost an order of magnitude better than AHMA and the
other tested compounds), and against the ovarian cancer
OVCAR8 cell line (against which AHMA exhibits no activity).
In addition, compound 24 exhibited no cytotoxicity against the
non-malignant MFA-10A cell line. These results are very
promising since the H1299 lung carcinoma cell line is very
resistant to other chemotherapies, and ovarian cancer is one of
the deadliest cancers in women, with a 5 year survival rate of
only 47%.14 Thus, compound 24 is an attractive target for
further development of anticancer drugs.
Lithium aluminum hydride (4.9 g, 129 mmol) was sus-
pended in THF (200 mL) at 0 1C. The protected aniline
(32 mmol) was added slowly in small portions. The reaction
mixture was heated at reflux overnight and then cooled
to room temperature. Saturated K2CO3 was added slowly.
Filtration and evaporation of the solvent gave a colorless oil.
Compounds that did not precipitate were purified by flash
column chromatography on silica gel 60 (20% EtOAc in
petroleum ether) to yield pure products.
Data for 12 (2.36 g, 87% yield): 1H NMR (300 MHz,
CDCl3): 6.02 (d, 2H, J = 1.5 Hz), 5.81 (br s, 1H), 4.53
(s, 2H), 2.82 (s, 6H); 13C NMR (75 MHz, CDCl3): 151.0,
143.4, 101.3, 95.9, 66.1, 31.1; HRMS (CI, m/z) calcd for
C9H14N2O (MH+) 166.1106, found 166.116.
General procedure for the synthesis of 21–25
A solution of 9-chloroacridine (3.12 g, 14.6 mmol) in CHCl3
(5 mL) was added dropwise to a mixture of the corre-
sponding aniline (14.6 mmol) and 4-methylmorpholine
(3.21 mL, 29.2 mmol) in EtOH (20 mL) at an ice bath
temperature. After stirring for 1 h, the temperature was
raised to room temperature and the mixture was stirred
further for 24 h. The precipitated orange product was collected
by filtration, washed with EtOH and dried. Compounds that
did not precipitate were purified by flash column chromato-
graphy on silica gel 60 (5% MeOH in CHCl3) to yield pure
products.
Data for 21 (2 g, 89% yield): 1H NMR (400 MHz, DMSO-d6):
8.18 (d, 2H, J = 8.7 Hz), 7.81 (dd, 2H, J = 7.3 Hz), 7.63
(d, 2H, J = 8.7 Hz), 7.49 (dd, 2H, J = 7.3 Hz), 6.35 (s, 1H),
5.91 (s, 1H), 5.70 (br s, 1H), 4.67 (m, 1H), 3.71 (br s, 1H), 3.60
(d, 2H, J = 4.8 Hz), 2.83 (d, 3H, J = 4.8 Hz), 2.48 (d, 3H, J =
4.5 Hz); 13C NMR (75 MHz, DMSO-d6): 158.9, 158.4, 155.8,
147.4, 146.7, 142.0, 141.1, 127.7, 127.3, 126.4, 121.5, 103.3,
96.6, 61.0, 32.7, 29.9; HRMS (CI, m/z) calcd for C22H21N3O
(MH+) 343.1685, found 343.1690.
In conclusion, we introduce a useful method for the efficient
synthesis of medicinally important N(9)Me AHMA derivatives.
Efficient two-step preparation of N(Me) aromatic synthones,
followed by simple coupling with 9-chloroacridine, afforded
novel AHMA analogs that can provide valuable information
on the bio-mechanistic issues associated with N(9) substitution.
The new compounds were tested as anti-proliferative agents
against seven cancer cell lines. Some of these compounds
exhibited significant anti-tumor activity. In three cases, the
activity was an order of magnitude or more better than that of
AHMA. Detailed biological tests and mechanistic studies are
in progress and will be published in due course.
Experimental
General procedure for the synthesis of 12–20 via Boc protection
and reduction
To a solution of a substituted aniline (33 mmol) in methanol
(30 mL) was added a 10% solution of triethylamine in
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Abbreviations
AHMA, 3-(9-acridinylamino)-5-hydroxymethylaniline; m-AMSA,
amsacrine; m-AQDI, m-AMSA-quinonediimine; Boc, benzyloxy-
carbonyl; MDR, multidrug resistance; NMM, N-methyl-
morpholine; SAR, structure–activity relationship.
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Acknowledgements
The authors acknowledge Dr Stella Aronov from Ariel University
Center for elaborating the biological tests.
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