A fast entry to the novel medicinally-important 9-anilinoacridine peptidyls by solid phase organic synthesis (SPOS)

Article abstract of DOI:10.1016/j.tetlet.2011.05.023

A new approach to the synthesis of novel medicinally-important mono- and bis-9-anilinoacridine (9-AnA) peptidyl derivatives is described. The method generates efficiently 9-AnAs with variable spacer lengths and charged, polar or hydrophobic residues at desired positions, which can increase binding affinity, conformational stability, intracellular transport and/or biological activity. The synthetic routes reported in this work are both general and applicable, and significantly expand the scope of potential 9-aminoacridine (9-AA) anticancer candidates.

Full text of DOI:10.1016/j.tetlet.2011.05.023

Tetrahedron Letters 52 (2011) 3640–3644
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A fast entry to the novel medicinally-important 9-anilinoacridine peptidyls
by solid phase organic synthesis (SPOS)
⇑
Tamara Brider, Yossi Gilad, Gary Gellerman
Department of Biological Chemistry, Ariel University Center of Samaria, PO Box 3, Ariel 40700, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
A new approach to the synthesis of novel medicinally-important mono- and bis-9-anilinoacridine (9-
AnA) peptidyl derivatives is described. The method generates efficiently 9-AnAs with variable spacer
lengths and charged, polar or hydrophobic residues at desired positions, which can increase binding affin-
ity, conformational stability, intracellular transport and/or biological activity. The synthetic routes
reported in this work are both general and applicable, and significantly expand the scope of potential
9-aminoacridine (9-AA) anticancer candidates.
Received 1 February 2011
Revised 11 April 2011
Accepted 6 May 2011
Available online 13 May 2011
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
9-Aminoacridine (9-AA)
9-Anilinoacridine (9-AnA)
Nucleophilic substitution
Peptidyl derivative
Solid phase organic synthesis
Many antitumour agents, including the anthracyclines, epipod-
ophyllotoxins and mitoxantrone target DNA topoisomerase II
arresting cell proliferation.1 Important 9-anilinoacridine (9AnA)
drugs (e.g., Amsacrine, AHMA) and their derivatives also play an
important role in medicine and are successful candidates for the
treatment of cancer, viral, and prion diseases.2a These compounds
have been classified as powerful DNA intercalators inhibiting
DNA replication by forming topoisomerase II–DNA complexes,
causing DNA strand breakdown and subsequent apoptosis. Evi-
dence has been presented showing that 9-aminoacridine based re-
agents are capable of targeting DNA without inducing DNA damage
and additional mutations in cellular genes. This phenomenon re-
duces side effects and the risk of developing secondary cancers,
thereby supporting the use of nongenotoxic DNA-binding small
molecules as anticancer therapies.2b The traditional solution syn-
theses of 9AnA analogs involve several steps and harsh reaction
conditions, requiring laborious purification of intermediates and fi-
nal compounds.3 Solid phase organic synthesis (SPOS) is a fast and
efficient technique that has become a productive approach in the
discovery of bioactive hits.
and major grooves are possible. Moreover, several approaches
based on dual amino-acridines (e.g., II, Fig. 1) have been reported
in the literature.5 These bisintercalators, including 9-aminoacri-
dine (9AA) moieties, exhibited both higher affinity6 and selectivity7
in binding compared to their mono counterparts. The improved
bioactivity of the covalent dimers of aminoacridine can be attrib-
uted to the increased local concentration of the active moiety. It
is noteworthy that all the methods mentioned above utilized pre-
formed 9-AA or 9AnA units.
We have previously demonstrated a new, highly-efficient, one-
pot derivatization of 9-AAs at the 9-amine position by simple SNAr
reactions yielding a series of substituted N(9)-anilinoacridines in
solution.8 However, purification of the products was problematic
in several of the syntheses. This encouraged us to develop a short
and efficient SPOS method for the rapid generation of new 9-AnA
core based compounds. In the context of exploring the rapid deriv-
atization of the 9-AnA scaffold we found that assembly of a 9-AnA
core on a solid support was a useful methodology, generating rel-
atively diverse and pure products without any purification step,
and, therefore, enhancing greatly their availability for examination
in biological systems.
Recently, several research groups have reported the solid phase
synthesis of novel DNA binding molecules by chemically modifying
known 9-anilinoacridine intercalating moieties with a variable
peptide appendage4 (e.g., I, Fig. 1). In such molecules the intercala-
tion domain will direct the peptide into a groove of the double heli-
cal secondary structure where sequence-specific contacts at minor
This Letter describes the facile solid phase synthesis of novel
mono- and bis-9-anilinoacridine (9-AnA) peptidyl derivatives
using Fmoc chemistry compatible with the protocol developed by
us.8 The synthetic strategy generates rapidly 9-AnAs with variable
spacer lengths and charged, polar or hydrophobic residues at
desired positions, which can lead to increased binding affinity,
conformational stability, intracellular transport, and biological
activity. The results described here should permit advanced
⇑
Corresponding author. Tel.: +972 3 937 1442; fax: +972 3 906 6634.
E-mail address: garyg@ariel.ac.il (G. Gellerman).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.05.023

T. Brider et al. / Tetrahedron Letters 52 (2011) 3640–3644
3641
O
H-AA
N
H
HN
N
L
NH
N
NH
N
R2
R1
R1
R2
II
O
N
H
AA NH2
I
L = polyamine linkers
R1, R2 = H, Cl, OMe, NO2,
CONHR3 (R3 = polyamine)
AA = amino acids
Figure 1. Examples of known potent 9-anilinoacridine peptides and polyamino bis-9-aminoacridines.
conjugation of 9-AnAs with biomolecules, such as peptides, anti-
bodies, and proteins, modulating their activity, bioavailability,
and applicability.
compatibility of carboxylated 9-AnA under standard peptide cou-
pling conditions. At this stage we decided to move to a solid phase
‘assembly’ approach toward 9-AnAs, enabling diverse derivatiza-
tion of the 9-AnA core.
Initially we decided to try coupling preformed 9-AnA 2b8 with
preloaded Fmoc-(
L
)Lys(Boc)-OH on Rink amide MBHA and Cl-Trt
Notably, the carboxylic acid group on 1b, released after cleavage
from the Cl-Trt acid-sensitive resin, can act as an anchor point for
conjugation chemistry. Cleavage of all compounds in this work was
carried out under strong acidic conditions [TFA/H2O/1,2-ethanedi-
thiol (EDT) (95:2.5:2.5)] due to the low solubility of the products in
most organic solvents. Previously, we had shown that the amine
(NH2) at C-9 of the 9-AA was nucleophilic enough to take part in
nucleophilic aromatic substitution (SNAr) yielding 9-anilinoacri-
dines.8 Most electrophilic haloaryl reagents involved in this reac-
tion bear two strongly electron-withdrawing (EW) groups, such
as CF3, CN, CO2H, and NO2 at ortho and para positions, favoring
resins as shown in Scheme 1. The purpose of this experiment
was to examine the straightforward coupling of a preformed
9AnA building block versus the 9AnA core assembly approach on
both resins. Thus, after applying the standard Fmoc chemistry pro-
tocol (PyBoP, NMM in DMF), 2b was successfully coupled to the
a-amino group of the preloaded (on Rink amide MBHA) lysine,
affording, after cleavage the corresponding Lys-9AnA peptide, 1a
in good yield and purity. When preloaded bifunctional Fmoc-
(L)Lys(Fmoc)-OH on Cl-Trt resin was used, the corresponding
bis-Lys(9AnA)2 1b was successfully obtained, confirming the
NO2
HO2C
NH2
NH
N
Fmoc-(L)Lys(Boc)-OH
2b
O
NH-(L)Lys(Boc)-H
NH2
b then d
HO
NO2
NH
b then c
(Rink amide)
N
H
O
N
1a (87%, 95% purity)
NO2
NH
HO2C
O
NO2
NH
HN
N
Fmoc-(L)Lys(Fmoc)-OH
2b
NH-(L)Lys-H
Cl
a then c
b then d
Conjugation
site
(Cl-Trt)
N
O
NO2
HO
N
H
O
NH
N
1b (84%, 89% purity)
Scheme 1. Synthesis of mono and bis-Lys-9-anilinoacridine derivatives 1a,b from 2b. Reaction conditions: (a) NMM, CH2Cl2, 40 min, rt; (b) PyBOP, NMM, DMF, 40 min, rt; (c)
20% piperidine/DMF (2 Â 20 min); (d) TFA/H2O/EDT (95:2.5:2.5).

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T. Brider et al. / Tetrahedron Letters 52 (2011) 3640–3644
stabilized resonance structures.9 The NO2 group was found to be
the most effective, affording products in good yield and high pur-
ity. Following our preliminary biological results,10 in which com-
pound 2b exhibited remarkable anticancer activity in vitro, we
decided to investigate the influence of the positions of the NO2
and CO2H groups in halobenzene substrates 4a–d (Scheme 2) on
the effectiveness of SNAr reactions with 9-AA to form 9-AnAs on
a solid support, and on their structure–activity relationship (SAR).
Thus Rink amide11 and Cl-Trt12 preloaded fluoronitrocarboxy-
lic acids 5a–d and 5e–h, respectively, were submitted to SNAr
reactions with 9-AA (3 equiv) and excess Cs2CO3 in DMF at room
temperature for 24 h (Scheme 2). Unfortunately, only 4a yielded
the corresponding 9-AnAs 2a and known 2b in good yields and
purity, probably due to the bulkiness of the resin (for 5b,f) and
unfavorable resonance effects (for 5c,d,g,h with an EW group at
the meta position). Hence, we decided to distance the electro-
philic site from the resin by spacer hopping to eliminate the steric
problems. Thus, b-Ala preloaded on Rink amide was coupled suc-
cessfully to benzoic acids 4a–d under standard conditions to give
amides 6a–d which underwent further SNAr reactions with 9-AA.
In this case, the three starting reagents 4a, 4b, and 4c afforded
smoothly the corresponding b-Ala-9-AnAs 3a, 3b, and 3c. Warm-
ing the reaction mixtures up to 90 °C did not lead to better re-
sults. Apparently, steric problems can be overcome potentially
enabling the incorporation of 4a–c in the planned 9-AnA peptidyl
assemblies.
Amino acid prodrugs usually impart improved pharmacological
characteristics such as enhanced transport and higher resistance to
metabolism, exhibiting slower rates of enzymatic activation for
suspended release of a drug.13
Based on these facts we attempted the assembly of 9-AnA on
several representative amino acids as possible minor and major
groove contact spacers4 (and potential prodrug moieties) on Rink
amide for the synthesis of peptidyls 1a and 7a–c as shown in
Scheme 3. All the final compounds were prepared in good yields
and high purity regardless of the nature of the amino acid linkers.
Similarly, bis-9-AnAs were prepared from several di-Fmoc pro-
tected Lys analogs. A large variety of commercially available
L
and D diamino aliphatic acids can be used to synthesize rapidly
bis-intercalators with variable distances between the two 9-AnAs
that sandwich two base pairs, and thread their side chains through
the helix to form hydrogen bonding contacts with the O6/N7atoms
of guanine in the major or minor grooves.14 It has been shown that
bis(9-aminoacridine-4-carboxamides) bis-intercalate via a thread-
ing mode and that their complexes dissociate many orders of mag-
nitude more slowly than simple intercalators.15
Moving forward, representative diFmoc protected
L-Lys, L-Orn
and -DAB were loaded on resins using standard methods. After
L
Fmoc release (Scheme 3) compounds 4a and 4b were coupled with
the intermediate preloaded amino acids 9, and the products sub-
mitted to SNAr reactions with a large excess of 9-AA (4 equiv)
and Cs2CO3 in DMF for 24 h at rt. Upon completion of the
NO2
HO2C
F
O
NO2
X
4a, 2-nitro-4-carboxy
4b, 2-carboxy-4-nitro
NO2
O
NH
4c, 2-carboxy-5-nitro
c then d
4d, 3-carboxy-2-nitro
X
X
F
a for Cl-Tr
b for Rink
Amide
N
5a-d
(X=NH)
X = NH2 (Rink amide)
X = Cl (Cl-Trt)
5e-h (X=O)
2a X=NH2, 96% (91% purity)
2b X=OH, 81% (92% purity)
O
O
NO2
NH
H2N
N
H
N
NO2
O
3a, 89% (94% purity)
O
O
4a-d
c then d
NH2
N
H
N
N
O
O
NO2
b
H
H
F
NH2
N
H
(Rink amide)
6a-d
NH
N
3b 4-NO2, 82% (86% purity)
3c 5-NO2, 87% (93% purity)
Scheme 2. Synthesis of nitro-carboxy and carboxamide-9-anilinoacridine derivatives 2a and 3a–c by SNAr reactions on solid phase. Reaction conditions: (a) NMM, CH2Cl2,
40 min, rt; (b) PyBOP, NMM, DMF, 40 min, rt; (c) 20% piperidine/DMF (2 Â 20 min); (d) 9-AA, Cs2CO3, DMF, 24 h, rt; (e) TFA/H2O/EDT (95:2.5:2.5).

T. Brider et al. / Tetrahedron Letters 52 (2011) 3640–3644
3643
O
NO2
NH
(1) Fmoc(L)Gln(Trt)-OH or
AA
N
H
H2N
Fmoc(L)Ser(tBu)-OH or
Fmoc(L)Lys(Boc)-OH or
Fmoc(L)Arg(Pbf)-OH,
b
4a,
(1)
b
NH-AA(PG)-H
NH2
(2) d
(3) e
Rink Amide
(2) c
N
1a AA = Lys (95%, 92% purity)
7a AA = Gln (84%, 91% purity)
7b AA = Ser (93%, 93% purity)
7c AA = Arg (86%, 89% purity)
O
NO2
m
p
o
O
X C CH
N
H
Cl
NH
Fmoc-(L)Lys(Fmoc)-OH or
Fmoc-(L)Orn(Fmoc)-OH or
Fmoc-(L)DAB(Fmoc)-OH
(Cl-Trt)
(CH2)n
NH
(1) 4a or 4b, b
X-(L)AA(H)-H
(X = O, NH)
or
(2) d
(3) e
Cl-Trt: (1) amino acid, a; (2) c.
Rink Amide: (1) amino acid, b;
(2) c.
N
9
NO2
NH
N
NH2
O
(Rink amide)
1b X = OH,
8a X = NH2,
8b X = NH2,
n = 4 (88%, 91% purity)
n = 4 (92%, 93% purity)
n = 3 (79%, 86% purity)
n = 2 (85%, 90% purity)
m-NO2, p-9-AA,
m-NO2, p-9-AA,
p-NO2, o-9-AA,
m-NO2, p-9-AA,
8c X = NH ,
2
Scheme 3. Synthesis of mono and bis peptidyl-9-anilinoacridine derivatives 1a, 7a–c, 1b, 8a–c by SNAr reactions on solid phase. Reaction conditions: (a) NMM, CH2Cl2,
40 min, rt; (b) PyBOP, NMM, DMF, 40 min, rt; (c) 20% piperidine/DMF (2 Â 20 min); (d) 9-AA (4 equiv), Cs2CO3, DMF, 24 h, rt; (e) TFA/H2O/EDT (95:2.5:2.5).
Howell, L. A.; Howman, A.; O’Connell, M. A.; Searcey, M. Bioorg. Med. Chem. Lett.
2009, 19, 5880–5883; (d) Elueze, E. I.; Croft, S. L.; Warhurst, D. C. J. Antimicrob.
Chemother. 1996, 37, 511–518; (e) Wainwright, M. J. Antimicrob. Chemother.
solid-phase synthesis, the resin-bound bis-9-AnAs were cleaved by
treatment with TFA/EDT/H2O (95:2.5:2.5) to give 1b (prepared ear-
lier from preformed 2b, see Scheme 1) and 8a–c in good yields and
purity, confirming that Rink amide MBHA and Cl-Trt resins were
suitable for this synthetic route. The products were characterized
by MS and NMR spectrometry (see Supplementary data). The pur-
ity of the synthesized compounds was determined by HPLC
(MeCN/0.1% TFA in H2O).
In conclusion, we have developed a new method for the effi-
cient synthesis of mono- and bis-9-anilinoacridine (9-AnA) pepti-
dyl derivatives via SNAr reactions on solid phase. The resulting
9-AnA peptidyls were obtained in good yields rapidly affording no-
vel potential DNA intercalators with variable spacer lengths and
charged, polar or hydrophobic residues at desired positions. All
the products were obtained from commercially available reagents.
The in vitro anticancer activity of the synthesized compounds is
under evaluation.
2001, 47, 1–13.
2. (a) Doh-ura, K.; Iwaki, T.; Caughey, B. J. Virol. 2000, 74, 4894–4897; (b) Gurova,
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9. Solomons, G. T. W.; Fryhle, G. B.; Organic Chemistry, 8th ed., Chapter 15,
‘‘Reactions of Aromatic Compounds’’, Wiley, 2007.
10. Unpublished results.
11. General procedure for the solid phase synthesis on Rink amide MBHA resin: The
procedure for the synthesis on Rink amide MBHA resin was identical to the
synthesis on Cl-Trt resin except for the loading: Loading: The Fmoc protecting
group from rink amide was removed by reaction with 20% piperidine in NMP
(2 Â 15 min, 5 mL each) and subsequent washing (2 Â CH2Cl2, 2 Â DMF, 5 mL
each). Next, a preactivated solution of protected amino acid (0.78 mmol acid,
0.78 mmol, PyBOP, 2.34 mmol DIEA in 4.5 mL of DMF) was added to the resin
and shaken for 2 h. The resin was washed with 2 Â DMF and 2 Â CH2Cl2 (3 mL
each). Cleavage was performed as for Cl-Trt resin. Data for 7c: yellowish
powder (0.083 g, 86% yield), IR (KBr): 3200–2800 (br, s), 1650 (C@O), 1630,
Acknowledgment
The authors thank Dr. Alexandra Massarwa for the HRMS mea-
surements of all new compounds.
Supplementary data
1240 cmÀ1
;
HRMS (CI, m/z) calcd for C26H27N8O4 (MH+) 515.207, found
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2011.05.023.
515.276; 1H NMR (300 MHz, DMSO-d6): 8.80 (m,1H), 8.63 (m,1H), 8.92–8.88
(m, 2H), 8.50–8.35 (m, 4H), 7.8 (d, J = 8 Hz, 2H), 7.60–7.45 (m, 4H), 7.14–6.95
(m, 3H), 4.21 (m, 1H), 3.26–3.02 (m, 4H), 1.55 (m, 2H); 13C NMR (75 MHz,
CDCl3): 168.2, 164.2, 160.1, 158.4, 153.3, 145.5, 143.1, 133.7, 131.0, 128.7,
128.6, 127.0, 124.1, 123.6, 121.9, 121.3, 57.8, 44.3, 25.0, 21.7.
References and notes
12. General procedure for the solid phase synthesis on Cl-Trt resin: To 2-chlorotrityl
resin (0.2 g, 0.28 mmol loading) in a jacketed fritted peptide vessel was added a
solution of protected amino acid (0.26 mmol) in dry DMF (3.5 mL), and after
1. (a) Baguley, B. C.; Leteurtre, F.; Riou, J. F.; Finlay, G. J.; Pommier, Y. Eur. J. Cancer
1997, 33, 272–279; (b) Denny, W. A. Curr. Med. Chem. 2002, 9, 1655–1665; (c)

3644
T. Brider et al. / Tetrahedron Letters 52 (2011) 3640–3644
addition of diisopropylethylamine (DIEA, 185 mL, 1.04 mmol) the reaction
to give after the usual work-up (fast purification using the solid-phase
extraction pack RP-18, first washed with H2O and then extracted with MeCN,
5 mL each). Data for 1b: yellowish powder (0.141 g, 88% yield). IR (KBr): 3500–
mixture was shaken for 1.5 h. After completion of the loading, dry MeOH
(1.5 mL) was poured into the vessel and shaking was continued for an
additional 20 min. The solvent was removed by filtration and the following
washings were performed sequentially: 2 Â CH2Cl2/MeOH/DIEA (17:2:1),
2 Â CH2Cl2, 2 Â DMF, 2 Â CH2Cl2, 2 Â CH2Cl2/DMF (1:1) (3 mL each). The
Fmoc protecting group was removed by reaction with 20% piperidine in NMP
(2 Â 15 min, 5 mL each) and subsequent washing (2 Â CH2Cl2, 2 Â DMF, 5 mL
each). Next, a preactivated solution of fluoronitrobenzoic acid (0.78 mmol acid,
0.78 mmol PyBOP, 2.34 mmol DIEA in 4.5 mL of DMF) was added to the resin
and shaken for 2 h. The resin was washed with 2 Â DMF and 2 Â CH2Cl2 (3 mL
each) and the aromatic substitution using 9-AA (4 equiv) with 0.5 g of Cs2CO3
in 3 mL of DMF was performed for 24 h. After washing with 2 Â H2O, 2 Â DMF,
2 Â MeOH, 2 Â CH2Cl2 and 2 Â DMF (3 mL each) the resin was transferred to a
vial for cleavage. Cleavage: a cold solution of 2.5% H2O/2.5% triisopropylsilane
in 95% TFA (2 mL) was added. After shaking for 1.5 h, the solution was collected
and the resin washed with cold TFA (2 Â 1 mL each). After combining the TFA
solutions, the solvent was evaporated first under N2 stream and then in vacuo
3080 (br, s), 1700 (C@O), 1650, 1180 cmÀ1
,
HRMS (CI, m/z) calcd for
C
46H37N8O8 (MH+) 829.273, found 829.285; 1H NMR (300 MHz, DMSO-d6):
8.83–8.65 (m,4H), 8.50–8.35 (m, 4H), 8.18–8.02 (m, 2H), 7.83–7.79 (m, 2H),
7.67–7.55 (m, 3H), 7.54–7.46 (m, 3H), 7.20–6.85 (m, 6H), 4.40 (m, 1H), 3.25–
3.07 (m, 4H), 1.89–1.80 (m, 2H), 1.58-1.40 (m, 4H). 13C NMR (75 MHz, CDCl3):
178.0, 165.1, 163.2, 161.9, 160.1, 159.0, 158.4, 156.3, 153.2, 144.6, 144.1, 143.1,
136.8, 133.3, 133.0, 132.9, 130.0, 129.5, 127.6, 126.4, 125.3, 124.2, 120.6, 120.2,
119.5, 118.5, 115.4, 56.6, 50.1, 33.6, 30.2, 22.3.
13. (a) Deshmukh, M.; Chao, P.; Kutscher, H. L.; Sinko, P. J. J. Med. Chem. 2010, 53,
1038–1047; (b) Landowski, C. P.; Song, X.; Amidon, G. L. Pharm. Res. 2005, 22,
1510–1518.
14. Adams, A.; Guss, J. M.; Collyer, C. A.; Wakelin, L. P. Biochemistry 1999, 38, 9221–
9931.
15. Wakelin, L. P.; Bu, X.; Eleftheriou, A.; Stewart, B. W. J. Med. Chem. 2003, 46,
5790–5798.