Synthesis and antiproliferative activity of new hybrids bearing neocryptolepine, acridine and α-aminophosphonate scaffolds

Article abstract of DOI:10.1007/s13738-019-01849-2

Synthesis of novel α-aminophosphonate hybrids 8a–f was accomplished by the reaction of 11-phenoxy-4-formylneocryptolepine 3, aminoalkylamino acridines 6a–f and diphenyl phosphite 7 in the presence of lithium perchlorate as Lewis acid catalyst in methanol.

Full text of DOI:10.1007/s13738-019-01849-2

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-019-01849-2
ORIGINAL PAPER
Synthesis and antiproliferative activity of new hybrids bearing
neocryptolepine, acridine and α‑aminophosphonate scaꢀolds
Abdullah A. S. Ahmed · Hanem M. Awad · Ibrahim El‑Tantawy El‑Sayed · Ahmed A. El Gokha1
1
2
1
Received: 18 October 2019 / Accepted: 27 December 2019
©
Iranian Chemical Society 2020
Abstract
Synthesis of novel α-aminophosphonate hybrids 8a–f was accomplished by the reaction of 11-phenoxy-4-formylneocryptole-
pine 3, aminoalkylamino acridines 6a–f and diphenyl phosphite 7 in the presence of lithium perchlorate as Lewis acid catalyst
in methanol. The starting aldehyde 3 was obtained by reaction of 11-chloroneocryptolpine 1 with 4-hydroxybenzaldehyde 2
in the presence of potassium carbonate in DMF, whereas 9-aminoalkylamino acridines 6a–f were synthesized by reaction of
9
-chloroacridine 4 with appropriate diamines 5a–f. The structure of all synthesized hybrids was conꢀrmed by spectroscopic
methods and showed spectra in consistence with the expected structures. The antiproliferative activity of all synthesized
hybrids was evaluated against HCT-116, MCF-7, HepG2 and A549 human cancer cell lines. The screened results proved
that most of the reported compounds are potent, especially hybrid 8a, which displayed the highest activity against MCF-7,
HepG2 and A549 cancer cell lines with IC 8.2, 23.1 and 19.4 µM, respectively. This activity is signiꢀcantly higher than
5
0
the standard drug doxorubicin. Moreover, hybrid 8f showed most potent activity against HCT-116 cancer cell line with IC
5
0
2
.4 µM higher than the reference drug doxorubicin (IC :10.90 µM).
50
Keywords Hybrid · Neocryptolpine · Acridine · α-Aminophosphonate · Antiproliferative activity
Introduction
resistance [16–18]. There is a rising need for development
of novel neocryptolepine analogues possessing potent antitu-
mor activities, but with reduced side eꢁects. A new approach
to reduce drug resistance is the synthesis of hybrid mol-
ecules with enhanced activities [19]. Hybridization of two
or three bioactive molecules often leads to increase activity
due to synergistic eꢁects. In light of the above-mentioned
considerations for anticancer agents with enhanced activity
and reduced side eꢁects, the objective of this study was to
synthesize potential anticancer compounds that are hybrids
of neocryptolepine, acridine and α-aminophosphonate scaf-
folds (Fig. 1). To examine this proposal, modiꢀcations were
made to the side chain at the 11-position of neocryptole-
pine skeleton with the installation of α-aminophosphonate
and acridine pharmacophores. The objective was to synthe-
size neocryptolepine hybrids based on the lead compound
reported in the literature, with the aim to fulꢀll the structure
activity relationships as well as to establish how these modi-
ꢀcations aꢁected anticancer activity.
Natural and synthetic neocryptolepine derivatives are a
series of heterocyclic compounds that are of considerable
interest for medicinal chemists and are widely used as anti-
malarial and antitumor agents [1–5]. Neocryptolepine is a
potential compound for developing new anticancer drugs
due to its planar structure that can bind to DNA by inter-
calation and inhibition of the DNA-topoisomerases I or II
that form the basis of its anticancer activity [6, 7]. Acridine
and α-aminophosphonate derivatives have gained attention
of medicinal chemists due to their wide range of biological
activities, which include antitumor, antiviral, antimicrobial,
antiparasitic and fungicidal eꢁects [8–15]. Unfortunately, the
use of these classes of biologically important compounds in
clinical use has been limited due to adverse eꢁect and drug
*
Ibrahim El-Tantawy El-Sayed
ibrahimtantawy@yahoo.co.uk
1
2
Department of Chemistry, Faculty of Science, Menouꢀa
University, Shebin El-Kom, Egypt
Department of Tanning Materials and Leather Technology,
National Research Centre, Dokki, Giza, Egypt
Vol.:(0
1
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3

Journal of the Iranian Chemical Society
potassium carbonate (1.04 g, 7.50 mmol) in DMF (5 mL).
The reaction mixture was reꢂuxed and monitored by TLC
until it ꢀnished, after 24 h. The mixture was cooled, then
poured into ice water, ꢀltered oꢁ and dried to yield yellow
solid of 11-phenoxy–4–formyl neocryptolepine 3.
Acridine
H
N
N
R1
NH
C
OPh
O
H P
4‑[(5‑Methyl‑5H indolo[2,3‑b]quinolin‑11‑yl)oxy]
benzaldehyde (3)
O
OPh
ꢀ
-Aminophosphonates
Yellow solid, Yield: (0.93 g, 70%), m.p: 108–110 °C, IR
−
1
(
KBr) cm υ: 2850 (CH), 1678 (C=O), 1635 (C=C, Ar)
N
N
1
1
1
570 (C=N). H-NMR (DMSO-d 400 MHz) δ: 9.64 (s,
6
CH3
H, CHO), 8.55–7.33 (m, 12H, CH ), 4.34 (s, 3H, CH ),
Ar
3
1
3
C-NMR(DMSO-d ,100 MHz)δ:192.11, 172.58,157.41,1
6
Neocryptolepine
4
1
7.78,154.20,152.37,138.83,131.26,127.52,126.53,124.16,
21.62,120.86, 119.37,117.37,116.02, 43.96. EIMS, m/z
Fig. 1 Structure of hybrids of neocryptolepine, acridine and
+
(
C H N O ) calcd, 352.39 [M] ; found, 352.12.
2
3
16
2
2
α-aminophosphonate scaꢁolds
Synthesis of aminoalkylamino acridine derivatives
6a‑f)
(
Materials and methods
Chemistry
To 9-chloroacridine 4 (0.12 g, 1 mmol) was added an excess
amount of appropriate amines 5a–f (3 mmol); then, the reac-
tion mixture was reꢂuxed for 1–2 h, until the complete con-
sumption of the starting materials occurred as monitored
by TLC. The mixture was cooled and poured into ice water,
General procedure
1
31
All H-NMR and P-NMR experiments were carried out
13
ꢀ
ltered oꢁ and dried to yield the desired product 6a–f.
with 500 MHz Jeol and 100 MHz Jeol for C-NMR at NMR
1
unit, Mansoura University. In addition, some of H-NMR
1
samples were measured with 400 MHz Varian at the main
chemical warfare laboratories, Ministry of Defense. Chemi-
cal shifts were reported in part per million (ppm) relative to
the respective solvent. The mass spectrometry experiments
were recorded on thermos scientiꢀc trace 1310 gas chroma-
tograph at Fungi National Centre, Al-Azhar University, and
IR spectroscopy was performed at Cairo University. Melt-
ing points (m.p) were recorded on Stuart scientiꢀc melting
point apparatus and are uncorrected. The anticancer activity
was executed at the National Research Center, Cairo, Egypt.
All reactions were followed by thin-layer chromatography
N ‑(acridin‑9‑yl)ethane‑1,2‑diamine (6b)
Yellow solid, yield: (0.15 g, 63%), m.p: 82–84 °C, IR
−
1
(
KBr) cm υ: 3402 (NH), 3340, 3286(NH ), 2823(C–H),
2
1
1
5
635 (C=C, Ar), 1558(C=N). H-NMR (DMSO d
6
00 MHz) δ: 8.40–7.18 (m, 8H, CH ), 3.97 (br.s, 2H,
Ar
1
3
CH ), 3.02 (m, 2H, CH ). C-NMR(DMSO-d ,100 MHz)
2
2
6
δ:153.36, 149.21, 131.61, 130.09, 126.59, 123.88, 121.65,
+
4
6.24, 40.52. EIMS, m/z (C H N ) calcd, 237.31 [M] ;
1
5
15
3
found, 237.18.
(
TLC) on silica gel F254 precoated plates (Merck). Solvents
1
N ‑(acridin‑9‑yl)propane‑1,3‑diamine (6c)
were used as received without further puriꢀcation. The start-
ing materials were either commercially available or synthe-
sized as 11-chloroneocryptolepine [1], 9-chloro acridine [9]
and 9-hydrazinylacridine [20] according to the literature
procedures.
Yellow solid, yield: (0.15 g, 60%), m.p: 88–90 °C, IR
−
1
(
KBr) cm υ: 3417 (NH, NH overlap), 1643 2924(C–H),
2
1
1
643(C=C, Ar), 1604(C=N). H-NMR (CDCl , 500 MHz),
3
δ ppm: 11.62(s, 1H, NH), 11.45 (s, 2H, NH ), 8.00–7.42 (m,
2
8
H, CH ), 3.64 (br.s, 2H, CH ), 2.42 (m, 2H, CH ), 1.61
Ar 2 2
Synthesis of 11‑phenoxy‑4‑formylneocryptolepine
1
3
(
m, 2H, CH ). C-NMR(DMSO-d ,100 MHz)δ:161.53,
2
6
(3)
1
42.30, 129.75, 127.82, 126.38, 120.81, 114.84, 41.32,
+
3
9.42, 31.54. EIMS, m/z (C H N ) calcd, 251.33 [M] ;
1
6
17
3
To 11-chloroneocryptolpine 1 (1 g, 3.75 mmol) were
added 4-hydroxybenzaldehyde 2 (0.92 g, 7.50 mmol) and
found 251.02.
1
3

Journal of the Iranian Chemical Society
1
2
N ‑(acridin‑9‑yl)‑N ‑(2‑aminoethyl)
ethane‑1,2‑diamine. (6d)
Diphenyl((2‑(acridin‑9‑yl)hydrazineyl)
(4‑((5‑methyl‑5H‑indolo[2,3‑b]quinolin‑11‑yl) oxy)
phenyl)methyl)phosphonate (8a)
Brown solid, yield: (0.15 g, 54%), m.p: 160–162 °C, IR
−
1
(
KBr) cm υ: 3363(NH, NH overlap), 2939 (C–H),
Brown solid, yield: (0.47 g, 72%), m.p: 162–164 °C, IR
2
1
−1
1
5
620 (C=C, Ar), 1566(C=N). H-NMR (CDCl ,
(KBr) cm υ: 3263 (NH), 2870 (C–H).1620 (C=C, Ar),
3
1
00 MHz) δ ppm: 7.80–7.29 (m, 8H, CH ), 4.24
1527(C=N), 1234 (P=O). H-NMR (DMSO-d , 400 MHz)
Ar
6
(
br.m, 2H, CH ), 3.90 (br.m, 4H, CH ), 2.92 (br.m, 2H,
δ: 12.07 (s, 1H, NH), 11.72 (s, 1H, NH), 8.22–7.08 (m,
2
2
1
3
CH ). C-NMR(DMSO-d ,100 MHz) δ:154.34, 148.23,
30H, CH ), 6.73 (d, 1H, CH–P, J = 8 MHz), 4.36 (s, 3H,
2
6
Ar
1
3
1
4
2
32.41, 130.16, 127.59, 121.88, 120.52, 52.37, 50.31, 43.91,
CH ). C-NMR (DMSO-d ,100 MHz) δ:177.85, 143.16,
3
6
+
0.71. EIMS, m/z (C H N ) calcd, 280.38 [M] ; found
141.55, 140.02, 135.30, 134.28, 132.92, 132.66, 131.14,
1
7
20
4
80.03.
130.17, 129.52, 127.99, 127.57, 126.78, 124.41, 124.01,
1
23.32, 121.75, 121.21, 118.07, 117,36, 166.55, 116.09,
1
3
31
N ‑(acridin‑9‑yl)‑N ‑(3‑aminopropyl)
propane‑1,3‑diamine (6e)
115.92, 67.60, 36.49. P-NMR (DMSO-d , 500 MHz) δ:
6
+
24.64. EIMS, m/z (C H N O P) calcd, 777.82 [M] found
48
36
5
4
7
79.30.
Brown solid, yield: (0.17 g, 51%), m.p: 148–150 °C, IR
−
1
(
(
KBr) cm υ: 3436 + 3286(NH, NH overlap), 2931
2
Diphenyl(((2‑(acridin‑9‑ylamino)ethyl)amino)
1
C–H).1627 (C=C, Ar), 1573(C=N). H-NMR (CDCl ,
3
(
4‑((5‑methyl‑5H‑indolo[2,3‑b]quinolin‑11yl)oxy)
5
2
3
00 MHz) δ: 11.97 (s, 1H, NH), 11.43 (s, 1H, NH), 11.09 (s,
phenyl) methyl) phosphonate (8b)
H, NH ), 8.01–7.32 (m, 8H, CH ), 4.22 (br.m, 2H, CH ),
2
Ar
2
1
3
.36 (br.m, 6H, CH ), 2.28 (br. m, 4H, CH ). C-NMR
2
2
Pale yellow solid, yield: (0.46 g, 68%), m.p: 160–162 °C,
(
DMSO-d ,100 MHz)δ:162.43, 141.34, 130.03, 129.82,
−1
6
IR (KBr) cm υ: 3410(NH), 2978 (C–H), 1620 (C=C, Ar),
1
25.38, 121.81, 115.74,46.35,41.62,39.44,31.96, 29.25.
1
1
527(C=N), 1257 (P=O). H- NMR (DMSO-d , 400 MHz)
6
+
EIMS, m/z (C H N ) calcd, 308.43 [M] ; found 308.25.
1
9
24
4
δ: 12.10(s, 1H, NH), 11.74(s, 1H, NH), 8.22–7.20 (m, 30H,
CH ), 6.73 (d, 1H, CH–P, J=8 MHz), 4.23 (m, 2H, CH ),
Ar
2
N‑(3‑(4‑(3‑aminopropyl)piperazin‑1‑yl)propyl)
acridin‑9‑amine (6f)
13
3
.97 (s, 3H, CH ), 3.82 (br.s, 2H, CH ). C-NMR (DMSO-
3
2
d ,100 MHz) δ:159.56, 156.08, 153.36, 151.47, 151.35,
6
1
1
1
3
49.20, 148.02, 139.07, 131.61, 130.08, 129.26, 128.81,
Yellow solid, yield: (0.19 g, 50%), m.p: 166–168 °C, IR
26.59, 125.68, 123.88, 122.98, 122.96, 122.82, 122.06,
−
1
(
(
KBr) cm υ: 3294(NH,NH overlap), 2931 (C–H).1635
2
21.64, 121.57, 119.38, 118.17, 113.76, 66.10, 48.77, 44.68,
1
C=C, Ar), 1596(C=N). H-NMR (CDCl , 500 MHz)
31
3
5.41. P-NMR (DMSO-d , 500 MHz) δ: 16.50. EIMS,
6
δ: 9.13 (br.s, 1H, NH), 8.27–7.34(m, 8H, CH ), 4.08
+
Ar
m/z (C H N O P) calcd, 805.87 [M] found 807.27.
5
0
40
5
4
(
br.s, 2H, CH ), 2.66 (br.s, 14H, CH ), 1.95 (br.s, 4H,
2
2
1
3
CH ). C-NMR(DMSO-d ,100 MHz) δ:164.31, 148.19,
2
6
Diphenyl(((3‑(acridin‑9‑ylamino)propyl)amino)
1
34.21, 131.36, 125.95, 120.82, 120.22, 58.26, 56.34, 51.83,
(
4‑((5‑methyl‑5H‑indolo[2,3‑b]quinolin‑11‑yl) oxy)
3
9.77, 27.11, 23.58. EIMS, m/z (C H N ) calcd, 377.54
2
3
31
5
+
phenyl)methyl)phosphonate (8c)
[
M] ; found 377.30.
Pale yellow solid, yield: (0.35 g, 51%), m.p: 185–187 °C,
Synthesis of α‑aminophosphonate derivatives using
solid of 11‑phenoxy‑4‑formyl neocryptolepine
and aminoalkylamino acridine derivatives (8a‑f)
−
1
IR (KBr) cm υ: 3425(NH), 2985 (C–H), 1635(C=C,
1
Ar), 1597, 1527(C=N),1257(P=O). H-NMR(DMSO-
d , 400 MHz) δ: 12.08 (s, 1H, NH), 11.73 (s, 1H, NH),
6
8
4
2
.38–7.20 (m, 30H, CH ), 6.73 (d, 1H, CH–P, J=8 MHz),
To 11-phenoxy-4-formyl neocryptolepine 3 (0.3 g,
Ar
.17(m, 2H, CH ), 3.97(s, 3H, CH ), 3.40 (br.m, 2H, CH ),
0
.84 mmol) and aminoalkylamino acridine derivatives 6a–f
2
3
2
1
3
.45 (br.m, 2H, CH ). C-NMR (DMSO-d ,100 MHz)
(
0.84 mmol), diphenyl phosphite 7 (0.75 mL, 0.92 mmol),
2
6
δ:177.87, 172.61, 147.80, 141.76, 140.02, 134.27, 131.98,
lithium perchlorate (10 mol %) in methanol (5 mL) were
added and reꢂuxed for 48–72 h. TLC monitored the reaction
completion until all reactants are consumed. The desired
product ꢀltered oꢁ and crystallized by diethyl ether to yield
the corresponding products 8a–f in good yields.
1
1
1
31.93, 127.99, 127.71, 126.78, 125.43, 124.82, 124.26,
23.56, 122.41, 121.92, 121.74, 122.21, 120.87, 118.07,
3
1
15.92, 70.10, 46.78, 42.44, 35.41, 29.33. P-NMR
(
DMSO- d , 500 MHz) δ: 16.12. EIMS, m/z (C H N O P)
6
51 42
5
4
+
calcd, 819.90 [M] found 822.38.
1
3

Journal of the Iranian Chemical Society
Diphenyl(((2‑((2‑(acridin‑9‑ylamino)ethyl)amino)
ethyl)amino)(4‑((5‑methyl‑5H‑indolo[2,3‑b]
quinolin‑11‑yl)oxy)phenyl)methyl)phosphonate
135.57, 134.26, 131.99, 130.15, 126.76, 126.54, 125.53,
124.85, 123.55, 122.41, 121.91, 121.73, 121.20, 120.86,
118.06, 115.93, 70.09, 45.83, 44.69, 40.95, 42.69, 34.50,
31
(8d)
33.49. P-NMR (DMSO-d , 500 MHz) δ: -0.66. EIMS, m/z
6
+
(
C H N O P); calcd, 946.10 [M] found: 947.10.
58
56
7
4
Reddish brown solid, yield: (0.44 g, 61%), m.p: 180–182 °C,
−
1
IR (KBr) cm υ: 3433(NH), 2939(C–H), 1627 (C=C,
Biological activity
1
Ar), 1597, 1543(C=N), 1249 (P=O). H- NMR (DMSO -
d , 400 MHz) δ: 12.19 (s, 1H, NH), 11.80 (s, 2H, 2NH),
In vitro cytotoxic activity
6
8
4
3
1
1
1
1
.22–7.23 (m, 30H, CH ), 6.73(d, 1H, CH–P, J=8 MHz),
Ar
.31(s, 3H, CH ), 3.96 (br.s, 2H, CH ), 3.50 (br,s, 6H,
Cell culture of human colorectal carcinoma (HCT-116), hor-
mone-dependent human breast adenocarcinoma (MCF-7),
human hepatocellular carcinoma (HepG2) and human lung
carcinoma (A549) cell lines was purchased from the American
Type Culture Collection (Rockville, MD) and maintained in
DMEM which was supplemented with 10% heat-inactivated
FBS (fetal bovine serum), 100 U/ml penicillin and 100 U/mL
streptomycin. The cells were grown at 37 °C in a humidiꢀed
3
2
1
3
CH ). C-NMR (DMSO-d ,100 MHz) δ:177.81, 172.54,
2
6
47.81, 141.77, 138.41, 134.07, 133.04, 131.93, 130.11,
28.47, 128.05, 127.84, 126.71, 126.50, 125.40, 124.77,
24.48, 123.46, 122.34, 121.80, 121.65, 121.16, 119.27,
3
1
17.64, 115.92, 70.04, 50.03, 45.18, 35.52, 33.50. P-NMR
(
DMSO-d , 500 MHz) δ: 2.87. EIMS, m/z (C H N O P)
6
52 45
6
4
+
calcd, 848.94 [M] found 850.23.
atmosphere of 5% CO .
2
Diphenyl(((3‑((3‑(acridin‑9‑ylamino)propyl)amino)
propyl)amino)(4‑((5‑methyl‑5H‑indolo[2,3‑b]
quinolin‑11‑yl)oxy)phenyl)methyl)phosphonate
MTT cytotoxicity assay
(8e)
The cytotoxicity activities on HCT-116, MCF-7, HepG2
and A549 human cancer cell lines were estimated using the
3-[4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT) assay, which is based on the reduction of
the tetrazolium salt by mitochondrial dehydrogenases in via-
ble cells [21–23]. Cells were dispensed in a 96-well sterile
Reddish brown solid, yield: 0.35 g, 48%, m.p: 210–212 °C,
−
1
IR (KBr) cm υ: 3425(NH), 2985 (C–H), 1635 (C=C,
1
Ar), 1597, 1535(C=N), 1257 (P=O). H-NMR (DMSO-
d , 400 MHz) δ: 12.19 (s, 1H, NH), 11.80 (s, 2H, NH),
6
4
8
4
3
.47–7.32 (m, 30 H, CH ), 6.73 (d, 1H, CH–P, J=8 MHz),
microplate (5×10 cells/well) and incubated at 37 °C with
Ar
.27 (s, 3H, CH ), 3.97 (s, 2H, CH ), 3.49 (br.s, 2H, CH ),
series of diꢁerent concentrations, in DMSO, of each tested
compound or doxorubicin (positive control) for 48 h in a
serum-free medium prior to the MTT assay. After incuba-
tion, media were carefully removed; 40 µL of MTT (2.5 mg/
mL) was added to each well and then incubated for an addi-
tional 4 h. The purple formazan dye crystals were solubi-
lized by the addition of 200 µL of DMSO. The absorbance
was measured at 570 nm using a Spectra Max Paradigm
Multi-Mode microplate reader. The relative cell viability
was expressed as the mean percentage of viable cells com-
pared to the untreated control cells. All experiments were
conducted in triplicate and repeated on three diꢁerent days.
All the values were represented as mean ± SD. IC s were
3
2
2
.07(m, 4H, 2CH ), 1.72(m, 2H, CH ), 1.58 (m, 2H, CH ).
2
2
2
1
3
C-NMR (DMSO-d ,100 MHz) δ:177.96, 172.75, 156.46,
6
1
1
1
1
1
53.19, 149.96, 147.88, 141.80, 139.75, 138.49, 135.70,
34.39, 133.36, 132.13, 130.27, 128.70, 128.05, 126.60,
24.48, 122.90, 122.52, 121.99, 121.23, 118.15, 116.01,
13.29, 70.08, 52.00, 45.42, 42.69, 35.84, 33.74, 26.94,
3
1
8.20. P-NMR (DMSO-d , 500 MHz) δ: 2.31, 10.23.
6
+
EIMS, m/z (C H N O P) calcd, 877.00 [M] found
5
4
49
6
4
8
80.30.
Diphenyl(((3‑(4‑(3‑(acridin‑9‑ylamino)
propyl)piperazin‑1‑yl)propyl)amino)
5
0
(
4‑((5‑methyl‑5H‑indolo[2,3‑b]quinolin‑11‑yl)oxy)
determined by probit analysis by SPSS Inc. probit analysis
phenyl)methyl)phosphonate (8f)
(IBM Corp., Armonk, NY, USA).
Brown solid, yield: (0.37 g, 46%), mp: 150–152 °C, IR (KBr)
−
1
cm υ: 3425(NH), 2947 (C–H), 1635 (C=C, Ar), 1597,
Results and discussion
1
1
527(C=N), 1257 (P=O). H-NMR (DMSO-d , 400 MHz)
6
δ: 12.09 (s, 1H, NH), 11.74 (s, 1H, NH), 8.22–6.62 (m, 30 H,
Chemistry
CH ), 6.73(d, 1H, CH–P, J=8 MHz), 4.21 (m, 4H, 2CH ),
Ar
2
3
.97(s, 3H, CH ), 3.44 (m, 6H, CH ), 3.01 (m, 6H, CH ), 2.53
The starting 11-phenoxy-4-formyl neocryptolepine 3 was
synthesized in good yield through reaction of 11-chloro-
neocryptolpine 1 with 4-hydroxybenzaldehyde 2 in the
3
2
2
1
3
(
br.s, 2H, CH ), 2.40 (br.s, 2H, CH ). C-NMR (DMSO-
2
2
d ,100 MHz) δ:177.85, 172.25, 147.82, 141.75, 140.04,
6
1
3

Journal of the Iranian Chemical Society
presence of potassium carbonate in DMF under reꢂux for
substitution (S ) type A, followed by elimination of
NAr
2
4 h as illustrated in Scheme 1.
chlorine atom as HCl, aided by potassium carbonate as a
In the structure elucidation of 3, the IR spectrum
base catalyst as given in Scheme 2.
showed a strong absorption band for the (C=O) of (CHO)
Moreover, the requested 9-aminoalkylamino acridines
6a–f were synthesized by reaction of 9-chloroacridine 4
with excess appropriate diamines 5a–f with diꢁerent car-
bon spacers to aꢁord aminoalkylamino acridines 6a–f in
good yields as depicted in Scheme 3. In the structure char-
acterization of 9-aminoalkylamino acridine derivatives 6a–f,
the IR analysis showed an absorption band ranging from υ:
−
1
group at υ: 1678 cm which conꢀrmed the presence of the
1
formyl (CHO) group. In addition, H-NMR spectrum for 3
showed a singlet with chemical shift at δ: 9.64 ppm, which
corresponds to the aldehydic proton. Further conꢀrma-
1
3
tion of present aldehydic group was observed in C-NMR
spectrum, which was showed a peak at 192.11 ppm for
the aldehydic carbon atom of 3. Furthermore, the EI mass
spectrum showed a molecular ion peak which corresponds
to 3. A proposed mechanism for formation of 3 involved
displacement of chlorine atom by the nucleophilic oxygen
of the hydroxy group of 2 throughout nucleophile aromatic
−
1
3417 to 3232 cm that represent the (NH and NH ) groups,
2
1
respectively. Moreover, H-NMR spectra showed character-
istic exchangeable peaks ranged from δ: 9.31 to 12.64 ppm,
which are characteristic to (NH and NH) groups. For the
2
mass spectroscopy, the presence of the expected molecular
Scheme 1 Synthesis of 11-phe-
noxy-4-formylneocryptolepine 3
Scheme 2 Suggested mechanism for formation of 3
1
3

Journal of the Iranian Chemical Society
Scheme 3 Synthesis of
aminoalkylamino acridine
derivatives
ion peaks of the target structures conꢀrms the formation of
the desired compounds. The plausible mechanism for the
showed the characteristic peak of (CH–P) ranging from 66.1
to 70.10 ppm which prove the installation of aminophos-
phonate moiety on the expense of the disappearance of the
formation of 6 is nucleophilic aromatic substitution (S
)
NAr
3
1
of the amine nitrogen to the chloride ion at position C-9 of
the acridine core through the formation of a resonance-stabi-
lized anion with a new C– N bond A as shown in Scheme 4.
Finally the hybrids of neocryptolepine, acridine and
α-aminophosphonate scaꢁolds 8a–f were synthesized by
installation of α-aminophosphonate moiety by three-com-
ponent one-pot reaction of 11-phenoxy-4-formylneocryp-
tolepine 3, 9-aminoalkayamino acridines 6a–f and diphenyl
phosphite 7 with heating in methanol and in the presence
aldehydic carbon at 192.11. Moreover, P-NMR conꢀrmed
the formation of α-aminophosphonate moiety with char-
acteristic (CH–P) ranging from − 0.66 to 24.64 ppm and
in consistence with reported range [24]. Furthermore, the
presence of (NH) was conꢀrmed by the presence of broad
singlet ranging from δ: 11.72 to 12.19 ppm. On the other
hand, the IR spectra of these compounds showed a charac-
teristic absorption bands for the NH group ranged from υ:
−
1
3433 to 3410 cm . In addition, the presence of absorption
−
1
of LiClO as Lewis acid catalyst as illustrated in Scheme 5.
band ranging from υ: 1257 to 1249 cm is characteristic
to the (P=O) group. Finally, the mass spectra of all synthe-
sized compounds showed molecular ion peaks, which are
in consistence with the expected molecular weight of the
desired compounds. A plausible mechanism for preparation
4
The newly synthesized hybrids 8 were character-
1
ized by H-NMR which revealed the presence of (CH–P)
proton with characteristic signals at δ: 6.72–6.74 ppm
appeared as doublet which confirms the installation of
1
3
α-aminophosphonate moiety, while C-NMR spectra
of α-aminophosphonates using LiClO as a catalyst is shown
4
Scheme 4 Suggested
mechanism for synthesis of
aminoalkyl amino acridine
derivatives
1
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Journal of the Iranian Chemical Society
Scheme 5 Synthesis of hybrids of neocryptolepine, acridine and α-aminophosphonates
in Scheme 6. First, the activation of carbonyl group of the
which had equipotent activities compared to doxorubicin
formyl group was achieved by Lewis acid catalyst, LiClO ,
(Fig. 3 and Table 1). In case of HepG2 human liver can-
cer, all compounds had signiꢀcantly more potent anticancer
eꢁect compared to that of the reference drug doxorubicin
(Fig. 4 and Table 1). In case of A549 cancer cells, twelve
compounds (8a, 1, 2, 6a, 6d, 6b, 8f, 3, 6c, 8c, 8b and 6e,
respectively) had signiꢀcantly more potent cytotoxic activi-
ties; two compounds (7 and 8f, respectively) had compara-
ble cytotoxic eꢁect; the rest of the compounds had slightly
less anticancer activities compared to that of doxorubicin
4
followed by condensation of the carbonyl group of 3 with
aminoalkylamino acridine derivatives 6 to aꢁord the inter-
mediate Schiꢁ base C. Then the nitrogen of Schiꢁ base was
activated by LiClO followed by the addition of phospho-
4
rus–hydrogen bond across the imine double bond to aꢁord
the target hybrids 8.
Biological evaluation
(
Fig. 5 and Table 1).
Cytotoxicity screening
From the above-mentioned results, one can conclude
that compounds 3, 7, 6a, 6c, 6e, 6f, 8a–c, 8f, 1 and 2 are
potent anticancer candidate drugs on all the four human
cancer types; compound 6d is specifically good antican-
cer candidate drug on human breast, liver and lung cancer
types rather than on human colon cancer type; compound
6b is potent anticancer candidate drug on human liver,
colon and lung cancer types but less active on breast
cancer type; compound 8d is potent anticancer candidate
drug on human liver, breast and colon cancer types but
not active on human lung cancer type; finally, compound
8e has better anticancer activity on human liver, colon
and breast cancer types than on human lung cancer type.
Sixteen compounds were examined in vitro for their activi-
ties against HCT-116, MCF-7, HepG2 and A549 human can-
cer cells using the MTT assay. The percentages of viable
cells were calculated and compared to those of the control.
Activities of these compounds against the four cancer cell
lines were compared to the activity of doxorubicin as refer-
ence drug as well. All compounds suppressed the four cancer
human cells in a dose-dependent manner (Figs. 2, 3, 4, 5).
In case of HCT-116 human colorectal carcinoma cells, both
Fig. 2 and Table 1 show that all compounds had signiꢀcantly
more potent cytotoxic activities except for two compounds
(
6e and 6d, respectively) which had slightly less activities
compared to doxorubicin.
In vitro antiproliferative activity
In case of MCF-7 human breast cancer cells, all com-
pounds had signiꢀcantly more potent cytotoxic activities
except for three compounds (6e, 2 and 6b, respectively)
The results of the cytotoxic activity in vitro were expressed
as the IC –concentration of the compound (in μM) that
5
0
1
3

Journal of the Iranian Chemical Society
Scheme 6 A plausible mechanism for synthesis of hybrids 8
3.125 µM
6.25 µM
12.5 µM
25 µM
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
Fig. 2 Dose-dependent cytotoxic activities of sixteen compounds on HCT-116 cancer cells according to the MTT assay
inhibits proliferation of the cells by 50% as compared to
the untreated control cells. The inhibition concentration (IC)
values were separately calculated for each experiment, and
the mean values ± SD were recorded from at least 3 inde-
pendent experiments. The results on the antiproliferative
activity of α-aminophosphonate derivatives are summa-
rized in Table 1, along with the data of the anticancer drug,
doxorubicin. The cytotoxicity test of the combined struc-
ture of neocryptolepine, acridine and α-aminophosphonate
scaꢁolds as a chemically modiꢀed hybrids indicated a syn-
ergetic eꢁect on anticancer activity. It is worth to note that
the anticancer activity of the modiꢀed analogues is found
to be higher than the reference drug doxorubicin as well
as the starting 11-phenoxy-4-formyl neocryptolepine 3.
Based on the antiproliferative activities shown in Table 1,
the most potent compounds with very strong and selective
anticancer activity among all tested derivatives are 8a–f with
IC : 5.8, 7.3, 7.0, 4.7, 5.7 and 2.4 μM against colorectal
5
0
1
3

Journal of the Iranian Chemical Society
Table 1 IC50 of the compounds against the four cancer types accord-
33.4 μM against human lung carcinoma cell line (A549).
ing to the MTT assay
It is worth to note that the hybrid structures 8a, 8d, 8e and
8
f showed better antiproliferative activity against colorectal
Compound code IC50 (µM)± SD
carcinoma (HCT-116) comparing with each starting scaf-
fold independently as well as reference drug doxorubicin.
The same result and trend were observed with the hybrids
8a, 8b and 8f when evaluated with human breast carcinoma
cell line (MCF7), while in case of human lung carcinoma
cell line (A549) only hybrids 8a and 8f exhibited synergistic
activity than each scaꢁold alone.
HCT-116 MCF-7
HepG2
A549
1
2
3
6
6
6
6
6
6
7
8
8
8
8
8
8
5.2± 1.3 13.1± 3.2 25.2± 2.7 19.6± 2.9
5.7± 1.2 26.6± 3.5 28.1± 3.5 20.6± 3.1
6.2± 1.5 20.6± 2.9 21.6± 2.7 21.5± 2.3
4.5± 1.3 13.9± 2.1 22.2± 2.3 20.7± 2.2
7.6± 1.8 27.5± 3.1 22.2± 2.2 20.8± 2.1
9.3± 1.7 25.2± 2.7 21.9± 2.1 23.5± 2.5
12.5± 2.1 18.6± 2.1 21.4± 2.3 20.7± 2.2
11.6± 1.9 26.2± 2.5 24.7± 2.5 26.0± 2.7
10.0± 1.9 19.6± 2.1 23.6± 2.5 28.0± 3.1
8.5± 1.9 18.0± 2.2 27.2± 3.7 27.7± 3.2
a
b
c
d
e
f
Study of structure activity relationships (SARs)
The study of structure activity relationship was investigated
in order to correlate between the structure and the pharma-
cological activity in view of searching for lead compound for
further optimization. The variety of all synthesized hybrids
speciꢀed in the presence of carbon spacer incorporated
with neocryptolepine, acridine and α-aminophosphonate
scaꢁolds.
a
b
c
d
e
f
5.8± 1.4
8.2± 1.7 23.1± 2.4 19.4± 2.3
7.3± 1.6 15.9± 2.1 24.6± 2.7 25.6± 3.1
7.0± 1.7 18.3± 2.1 27.8± 2.9 25.2± 3.2
4.7± 1.3 18.4± 2.1 28.0± 3.1 33.4± 3.7
5.7± 1.5 18.8± 2.3 26.8± 2.9 29.0± 3.5
2.4± 0.4 16.0± 2.1 27.1± 3.1 20.9± 2.9
10.9± 2.3 26.5± 3.7 32.8± 3.6 27.9± 4.1
Doxorubicin
It is noticeable from above-mentioned results that hybrid
8
f containing piperazine spacer between two amino groups
gave the highest antiproliferative activity against HCT-116
cell line, revealing the impact of this pharmacophore group
on enhancement of antitumor activity of 8f [25], while the
linkage of four- and six-carbon spacers included internal NH
group illustrated in hybrids 8d and 8e, respectively, showed
high anticancer activity but less than 8f containing pipera-
zine spacer. Moreover, hybrids 8a–c containing hydrazinyl,
two- and three-carbon spacers, respectively, showed high
antitumor activity but less than 8f containing piperazine
spacer.
carcinoma (HCT-116), respectively, while they exhibited the
same strong activity against breast carcinoma (MCF-7) with
IC : 8.2, 15.9, 18.3, 18.4, 18.8 and 16.0 μM, respectively.
5
0
On the other hand, the same trend is seen against the hepa-
tocellular carcinoma (HepG2) with IC : 23.1, 24.6, 27.8,
5
0
2
8
1
8.0, 26.8 and 27.1 μM, respectively. Moreover, compounds
a–c, 8e and 8f exhibited a very potent activity with IC :
5
0
9.4, 25.6, 25.2, 29.0 and 20.9 μM against (A549), respec-
tively. Based on the above-mentioned results most of the
compounds showed anticancer activity signiꢀcantly higher
than the reference drug doxorubicin (cf. table 1). However,
compound 8d is the only one that showed antiproliferative
activity less than the reference drug doxorubicin with IC50
It also reported that hybrids 8a, 8d, 8e and 8f have syner-
gistic activity comparing with their single scaꢁolds, 11-phe-
noxy-4-formylneocryptolepine 3, diphenyl phosphite 7 and
substituted acridines at position 9 with hydrazinyl, four- and
3
.125 µM
6.25 µM
12.5 µM
25 µM
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
Fig. 3 Dose-dependent cytotoxic activities of sixteen compounds on MCF-7 cancer cells according to the MTT assay
1
3

Journal of the Iranian Chemical Society
3
.125 µM
6.25 µM
12.5 µM
25 µM
5
4
3
2
1
0
0
0
0
0
0
Fig. 4 Dose-dependent cytotoxic activities of sixteen compounds on HepG2 cancer cells according to the MTT assay
3
.125 µM
6.25 µM
6
5
4
3
2
1
0
0
0
0
0
0
0
Fig. 5 Dose-dependent cytotoxic activities of sixteen compounds on A549 cancer cells according to the MTT assay
six-carbon spacers including internal NH group in addition
to piperazine spacer 6a, 6d, 6e and 6f, respectively. On the
other hand, hybrid 8a containing hydrazinyl linkage showed
the highest antiproliferative activity against MCF-7 com-
paring with other hybrids containing two- or three-carbon
spacer, four- or six-carbon spacer with internal amino group
and piperazine spacer. The synergistic activity was reported
in hybrids containing hydrazinyl, two-carbon spacers and
piperazine spacer, revealing the importance of the pres-
ence of these linkages among the three scaꢁolds. In addi-
tion, hybrid 8a containing hydrazinyl linkage gave highest
antitumor activity against HepG2 followed by hybrids two-
carbon spacer, six-carbon spacer with internal amino group,
piperazine spacer three-carbon spacer and four spacer with
internal amino group, respectively. Unfortunately, there is no
synergistic eꢁect reported for synthesized hybrids. Finally,
for A549 cell line, it is illustrated that 8a containing hydrazi-
nyl linkage has the best antitumor activity comparing with
piperazine spacer, three-carbon spacers, two-carbon spac-
ers, six-carbon spacers with internal amino group except 8d
containing four carbon spacers with internal amino group,
showing lower activity than hybrids and reference drug.
Moreover, hybrids containing hydrazinyl and piperazine
spacer 8a and 8f also have synergistic eꢁect comparing with
11-phenoxy-4-formylneocryptolepine 3, diphenyl phosphite
7 and substituted acridines at position C-9 with hydrazinyl,
piperazine spacer 6a and 6f scaꢁolds.
In conclusion, our initial goal to make hybrids of neocryp-
tolepine, acridine and α-aminophosphonate scaꢁolds was
achieved. This led to have a small series of modiꢀed struc-
ture with synergistic antiproliferative activity against HCT-
116, MCF-7, HepG2 and A549 human cancer cell lines.
Conclusion
Novel strong antiproliferative hybrids 8a–f were success-
fully synthesized based on neocryptolepine scaꢁold through
three-component one-pot reaction of 3, with 6a–f and 7 in
the presence of lithium perchlorate as Lewis acid catalyst
and were elucidated by spectroscopic methods. All syn-
thesized hybrids were screened against HCT-116, MCF-7,
1
3

Journal of the Iranian Chemical Society
HepG2 and A549 human cancer cell lines and revealed that
most of hybrids showed strong antiproliferative activity
against reference drug doxorubicin with synergistic eꢁect.
12. M.A. Hamed, A.A. El Gokha, A.F.A. Ahmed, A. El Sayed, M.
Sabry, R. Tarabee, A.El S. Abdel Megeed, I.E.T. El Sayed, Int. J.
Pharm. Sci. Rev. Res. 34, 205 (2015)
1
3. P. Sreelakshmi, R.N. Maheshwara, S. Santhisudha, G. Mohan,
N. Saichaithanya, M.S. Shaik, K. Peddanna, C. Nagaraju, S.R.
Cirandur, Med. Chem. Res. 28, 528 (2019)
Funding We would like to acknowledge ꢀnancial support by Menouꢀa
University throughout the Project Number Ib-C2013.
14. A.B. Danne, S.V. Akolkar, T.R. Deshmukh, M.M. Siddiqui, B.B.
Shingate, J. Iran. Chem. Soc. 16, 953 (2019)
1
1
5. H.A.L. El-Boraey, A.A. El-Gokha, I.E.T. El Sayed, Med. Chem.
Res. 24, 2142 (2015)
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