Synthesis of novel class of n-alkyl-isatin-3-iminobenzoic acid derivatives and their biological activity in zebrafish embryos and human cancer cell lines

Article abstract of DOI:10.1248/bpb.b17-00674

Isatin (1H-indole-2,3-dione) and many of its derivatives are reported to have pharmacological properties. In this study, we report the synthesis and biological activity of a new class of N-alkyl-isatin-3-iminoben-zoic acid derivatives prepared via the condensation of N-alkyl isatin with 4-aminobenzoic acid by conventional, microwave, and ultrasonic methods. Microwave irradiation yielded the products in a shorter reaction time with higher yields and purities. The compounds were screened in zebrafish embryos, and also in three human cancer cell lines (MCF7, HepG2, and Jurkat) and one normal human cell line i.e., human foreskin cell line (HFF-1). Two compounds (3c, 3f) were found to be highly effective against hematopoiesis in live zebrafish embryo at 10μM concentration. The developmental stage-dependent treatment indicated that these compounds interfered with the differentiation of hemangioblasts to hematopoietic cells in zebrafish embryos. The comparative screening of semaxanib (SU5416) (a known isatin derivatives), to compounds synthesized in this study, revealed the contrasting effects of these two classes of isatin derivatives on zebrafish hematopoiesis. Most of the N-alkyl-isatin-3-iminobenzoic acid derivatives were toxic on cancer and non-cancer tested human cells lines, however, the compounds 3c and 3f specifically affected the cell viability of Jurkat cells (human hematological cell line) with least IC₅₀ values of 16.5 and 7.8μM. The structure–activity relationship (SAR) analysis indicated that the substitution pattern of the isatin at the 5-position was vital for activity. The in vivo and in vitro biological activities of these compounds suggested their potential use as pharmaceutical compounds for human leukemia treatment.

Full text of DOI:10.1248/bpb.b17-00674

Biological and Pharmaceutical Bulletin Advance Publication by J-STAGE
DOI:10.1248/bpb.b17-00674
Advance Publication
December 16, 2017
Biol. Pharm. Bull:
Regular article
Synthesis of novel class of N-alkyl-isatin-3-iminobenzoic acid derivatives
and their biological activity in zebrafish embryos and Human cancer cell
lines.
1
,*
2
1
Muhammad Farooq , Zainab M. Almarhoon , Nael Abu Taha,
1
1
2,3
Almohannad A. R. Baabbad , Mohammed A. Al-Wadaan , and Ayman El-Faham
1
Bioproducts Research Chair, College of Science, Department of Zoology, King Saud
University P.O. Box 2455, Riyadh 11451, Kingdom of Saudi Arabia.
Department of Chemistry, College of Science, King Saud University, P.O. Box 2455,
Riyadh 11451, Saudi Arabia;
2
3
Department of Chemistry, Faculty of Science, Alexandria University, P.O. Box 426,
Alexandria 21321, Egypt
*
Correspondence: fmuhammad@ksu.edu.sa
Ⓒ
2017 The Pharmaceutical Society of Japan

Isatin (1H-indole-2,3-dione) and many of its derivatives are reported to have
pharmacological properties. In this study, we report the synthesis and biological activity of a
new class of N-alkyl-isatin-3-iminobenzoic acid derivatives prepared via the condensation
of N-alkyl isatin with 4-aminobenzoic acid by conventional, microwave, and ultrasonic
methods. Microwave irradiation yielded the products in a shorter reaction time with higher
yields and purities. The compounds were screened in zebrafish embryos, and also in three
human cancer cell lines (MCF7, HepG2, and Jurkat) and one normal human cell line i.e
human foreskin cell line (HFF-1). Two compounds (3c and 3f) were found to be highly
effective against hematopoiesis in live zebrafish embryo at 10 µM concentration. The
developmental stage-dependent treatment indicated that these compounds interfered with the
differentiation of hemangioblasts to hematopoietic cells in zebrafish embryos. The
comparative screening of semaxanib (SU5416) (a known isatin derivatives), to compounds
synthesized in this study, revealed the contrasting effects of these two classes of isatin
derivatives on zebrafish hematopoiesis. Most of the N-alkyl-isatin-3-iminobenzoic acid
derivatives were toxic on cancer and non-cancer tested human cells lines, however, the
compounds 3c and 3f specifically affected the cell viability of Jurkat cells (human
hematological cell line) with least IC50 values of 16.5 and 7.8 µM. The Structure-activity
relationship (SAR) analysis indicated that the substitution pattern of the isatin at the 5-
position was vital for activity. The in vivo and in vitro biological activities of these
compounds suggested their potential use as pharmaceutical compounds for human leukemia
treatment.
Keywords: Isatin, N-alkyl-isatin-3-iminobenzoic acid, semaxanib (SU5416), suritinib,
zebrafish hematopoiesis, and leukemia
Biological and Pharmaceutical Bulletin Advance Publication

1
. Introduction
Leukemia, the cancer of the blood or bone marrow, is characterized by an abnormal
increase of immature white blood cells. The Middle East has the highest mortality rate from
leukemia in the world and, in a comparison of countries worldwide, Iraq ranked first, with a
1
)
mortality rate of 6.85 . Similarly, leukemia is the leading cancer among children in Saudi
Arabia and accounted for 38.8% of childhood cancers in 2013. Furthermore, leukemia
accounts for 5.9% of all newly diagnosed cancers in Saudi Arabia and was the third most
2
)
common cancer among males and the sixth most common cancer among females in 2013
.
The high mortality rate associated with leukemia in the Middle East is very alarming and has
necessitated to design and synthesis novel and effective chemotherapeutic agents. Isatin (1H-
indole-2,3-dione) and many isatin derivatives have been reported to possess diverse
3
-5)
biological activities . In a previous study, we reported the synthesis and anticancer activity
6
)
of isatin hydrazide-hydrazone derivatives with valproic acid (VPA) . These derivatives
have shown activity in cancer cell lines, but were particularly effective in the Jurkat cell line
(
human hematological cell line), compared with that in human liver cancer (HepG2) cells or
human embryonic kidney (HEK293) cells. Owing to the promising anticancer profile of isatin
hydrazide-hydrazone derivatives in a hematological cancer cell line, we have designed six
new structural derivatives of isatin-3-iminobenzoic acid and reporting the in vitro and in-vivo
biological activity. The newly synthesized compounds were first screened in zebrafish
embryos in order to evaluate the developmental toxicity and bioactivity (especially the anti-
hematopoietic profile) in an animal model. The results obtained from zebrafish embryos were
further verified in vitro by testing the cytotoxicity of these compounds in human
hematological cancer cell line (Jurakt) and two solid cancer lines i.e human liver cancer
(
HepG2) and human breast cancer (MCF7) cells. In order to assess the toxicity on normal
cells, the compounds were also tested in HFF-1 (ATCC® SCRC-1041) a cell line derived
from human foreskin cells.
2. Results
2
.1. Chemistry
In order to achieve the synthesis of the target compounds “3a–f” the N-alkylation of
2 3
isatin is usually prepared in the presence of K CO in DMF. This method is conducted for 48
7
)
h at room temperature , and has been modified using microwave irradiation (MW) or ultra-
Biological and Pharmaceutical Bulletin Advance Publication

sonication (US). Microwave irradiation usually afforded the product in a shorter reaction time
with a higher yield (experimental section, Chart1).
Chart1. Synthesis of N-alkyl-isatin-3-iminobenzoic acid derivatives.
2.2. Biology
2
.2.1. N-Alkyl-isatin-3-iminobenzoic acid derivatives blocked the blood circulation in
zebrafish embryos
Zebrafish embryos were used as an in vivo animal model system to evaluate the
biological activity of these new synthesized N-Alkyl-isatin-3-iminobenzoic acid derivatives.
The embryos were soaked with serial dilutions (1–100 µM) of the compounds in embryo
water. Each compound showed dose-dependent toxicity against the embryos. As shown in
Table 1, compounds 3c, 3e, and 3f were more toxic to zebrafish embryos than were 3a, 3b,
and 3d. Compound 3c was the most toxic and 100% lethality was observed in exposed
embryos treated at ≥ 30 µM.
The sub-lethal concentrations (10–30 µM) of compounds 3c and 3f induced
teratogenicity and congenital malformations in exposed zebrafish embryos. The most
prominent phenotype observed after the exposure of these compounds to zebrafish embryos
was a lack of active circulation during exposure. Compound 3f was the most active and
hindered blood circulation at ≤ 10 µM. The embryos that remained exposed to compound 3f
Biological and Pharmaceutical Bulletin Advance Publication

were unable to restore normal circulation even up to 5 days post-fertilization. The
comparative anti-hematopoietic profiles of these compounds are presented in Table 1.
Table1: In vivo toxicity and hematopoietic defects induced by N-Alkyl-isatin-3-
iminobenzoic acid derivatives in live zebrafish embryos.
S.NO Com.ID Toxicity in zebrafish embryos
Minimum concentration at which
hematopoietic defects were observed in
zebrafish embryos (µM)
(
LC50 values) (µM)
1
2
3
4
5
6
3a
3b
3c
3d
3e
3f
Not active*
Not active*
28
Not active*
Not active*
20
Not active*
50
Not active*
Not active*
10
26
*
If a compound induce require phenotype/effects at ≥ 100 µM, was considered as not active.
All experiments were repeated three times with different batch of embryos every time and with at
least 35 embryos per treatment per concentration
In order to validate the hematopoietic defects in treated zebrafish embryos, the treated
embryos were stained with o-dianisidine to visualize the erythroid cells (red blood cells). As
shown in Figure 1, the zebrafish embryos treated with 10 µM compound 3f lacked positive o-
dianisidine staining in the yolk sac and in the posterior trunk, which confirmed that the lack
of circulation in treated embryos resulted from the absence of erythroid cells.
Biological and Pharmaceutical Bulletin Advance Publication

Figure 1. N-Alky-Isatin-3-iminobenzoic acid derivatives perturbed the blood formation process in
exposed zebrafish embryos. Representative images of zebrafish embryos at 48hpf processed for o-
dianisidine staining. The mock (1%V/V methanol) treated embryos had normal hematopoiesis as
indicated by positive O-dianisidine staining denoted by thin black arrow head, whereas the embryos
exposed to compound 3f (10 µM) were completely negative of-dianisidine staining.
2
.2.2. Compound 3c and 3f hampered the formation of blood cells in exposed zebrafish
embryo.
The zebrafish embryos were exposed to compound 3f at various developmental stages in
order to identify at which developmental stage the hematopoietic process was affected. First,
a batch of zebrafish embryos were exposed to compound 3f from the shield stage to 24 hpf
(
before the onset of hematopoiesis) up to 48 hpf and then processed for o-dianisidine staining.
Compound 3f was added only after 24 hpf to the second batch of embryos, which were
exposed for an additional 24 h before being processed for erythroid staining. The third batch
of embryos was exposed to compound 3f after 48 hpf (when the hematopoiesis process was
almost complete) and remained exposed for 24 h (until 72 hpf). As shown in Figure 3, the
zebrafish embryos that were exposed to compounds 3f from the shield stage to 24 hpf showed
positive o-dianisidine staining, whereas very weak staining was observed in embryos that
were exposed to 3f from 24 hpf to 48 hpf (Figure 2C). The zebrafish embryos that were
exposed after 48 hpf did not respond to the treatment and the blood cells formed normally
(
Figure 2D).
Biological and Pharmaceutical Bulletin Advance Publication

Figure 2. N-Alky-Isatin-3-iminobenzoic acid derivatives targeted the blood cells formation in
exposed zebrafish embryos. Representative images of zebrafish embryos treated at different
developmental stages as indicated and then processed for O- dianisidine staining to see the
formation of blood cells. A) Un treated or mock (1%V/V methanol) treated embryos had normal
hematopoiesis as indicated by positive O-dianisidine staining shown by white arrow head, B)
whereas the embryos which were exposed to compound 3f from shield stage until 48hpf did not
stain with O-dianisidine, C) A weaker staining was observed in those embryos which were exposed
compound 3f from 24-48hpf not before, D) The embryos which were exposed after 48hpf to
compound 3f did not respond at all to the treatment and the blood cells formed normally similar like
control normally shown by positive erythrocyte staining (white arrow)
8
)
Semaxanib (SU5416), which is an isatin derivative molecule was used as positive
9
)
control in this study. SU5416 is a VEGF receptor tyrosine kinase inhibitor
and
9
-11)
antiangiogenic activity of this compound has been reported previously
, however, the
hematopoietic activity of SU5614 in zebrafish embryos is largely unknown. In order to
determine the effects of SU5416 on zebrafish hematopoiesis, embryos were treated with 1–10
µM SU5416 from the shield stage and processed for o-dianisidine staining at 48 hpf. The
treated embryos had severe blood vessels formation defects upon treatment of SU5416 as
indicated by absence of intrasomatic blood vessels (isv) in Tg(Fli-1:EGFP) embryos at 48hpf
(
Figure 3 D), where the erythrocytes developed normally, as shown by the positive o-
dianisidine staining in SU5416 treated embryos (Figure 3, black arrow head in F). The o-
dianisidine staining was observed in posterior trunk in caudal vein, which has been shown in
small inset in Figure 3F.
Biological and Pharmaceutical Bulletin Advance Publication

Figure 3. The tyrosine kinase inhibitor Semaxanib (SU5416) did not interfere with the
hematopoietic process in exposed zebrafish embryos. The representative micrograph of zebrafish
embryos from Tg(fli 1:EGFP) either mock treated (left panel) or treated with 1µM of SU5416 from
shield until 48hpf (right panel). The control embryos had normal blood vessel formation as shown by
white arrow (C), and also blood cells formed normally (E), the embryos treated with SU5416 had
malformed blood vessels (D), whereas the blood cells formed normally as indicated by black arrow in
F. The inset in F shows the same embryo at higher magnification showing the pooling of blood cells
in caudal vein area.
2
.2.3 N-alkyl-isatin-3-iminobenzoic acid derivatives did not interfere with endothelial
cells (blood vessels) formation in zebrafish embryos
1
2)
The embryos from transgenic zebrafish Tg(fli1:EGFP)y¹ were treated with serial dilutions
of N-alkyl-isatin-3-iminobenzoic acid derivatives in order to evaluate their effects on blood
vessel formation. As shown in Figure 4, compound 3f did not hinder angiogenic blood vessel
formation in treated embryos. Similarly, no alkyl-isatin-3-iminobenzoic acid derivatives were
effective in blocking the angiogenic blood vessel formation in treated embryos at all the
tested concentrations.
Biological and Pharmaceutical Bulletin Advance Publication

Figure 4 N-alkyl-isatin-3-iminobenzoic acid derivatives could not temper the blood vessels
formation in zebrafish embryos. The representative micrograph of zebrafish embryos from
Tg(fli1:EGFP)y¹ line, control (A,C) or treated with compound 3f (B,D) at or before shield stage and
photographed at 48hpf. All the blood vessels (green fluorescents) including angiogenic blood vessels
(
intrasomatic blood vessels, isv) as indicated by white arrow formed normally in control embryos.
Zebrafish embryo treated with 3f (10µM) had no circulation and cardiac edema can be seen in bright
field images (B) but the blood vessels formed normal in the treated embryos as indicated by white
arrow showing normal structure of intersomatic blood vessels (isv).
2
.2.4. N-Alkyl-isatin-3-iminobenzoic acid derivatives were cytotoxic in human leukemia
cell line.
The newly synthesized N-alkyl-isatin-3-iminobenzoic acid derivatives were screened
via an in vitro colorimetric MTT cell viability assay in three human cancer cell lines, human
breast cancer (MCF7), human liver cancer (HepG2), and human leukemia (Jurkat) to assess
their cytotoxicity in cancer cells and also in normal cells human foreskin cell line (HFF-1)
The known anticancer drugs 5-fluorouracil (5-FU) and SU5416 were used as positive control
to assess the comparative biological activity of N-alkyl-isatin-3-iminobenzoic acid
derivatives with known anticancer molecules. As shown in table 2 and Figures 5-7, most of
the compounds affected cell viability of cancer cell lines and showed weaker to moderate
level of cytoxicity in normal human foreskin cells (table 2 and figure 8), however, the
cytotoxicity of compounds 3c and 3f was much stronger in hematological cell line (Jurkat)
with IC50 values (16.5 and 7.8 µM respectively) as compared with their activity in human
liver cancer (HepG2) and human breast cancer (MCF7) cells. The cytotoxicity of compound
Biological and Pharmaceutical Bulletin Advance Publication

3
c in MC7 and HepG2 was 24.14 and 37µM, while the IC50 values of compounds 3f were
4.61 and 66 µM in MCF7 and HepG2 respectively. The cytoxicity of these two compounds
3
was very much weak in normal human foreskin cells. The compound 3c was not active (with
IC50 values more than 200µM), while the IC 50 values for compound 3f was 91.53 µM (table
2
) in normal human foreskin cells (HFF-1). In comparison with compounds 3c and 3f, the
cytotoxicity of SU5416 was very weak in tested cancer cell lines. The IC values of SU5416
5
0
in HepG2 and Jurkat were 112 and 90 µM, whereas in MCF7 the IC50 value of SU5416 was
calculated as 41.39 µM (Table 2). The next level of cytotoxicity was found in compounds 3a
and 3e. The IC50 values for compounds 3a and 3e were 14.3 and 24.3 µM respectively in
Jurkat cells. While compound 3a was not active in MCF7 and HepG2 cells and the IC50
values for compound 3e were 23.59 and 41.00 µM in MCF7 and HepG2 cells respectively. In
order to assess the cytotoxicity of these compounds in normal (non-cancers) cells, the human
foreskin cell HFF-1 were treated and IC50 values were calculated. As shown in table 2 and
figure 8, the IC50 values of HFF-1 cells indicated that most of these compounds required
very high concentration to suppress 50% survival of the normal cells as compared with their
activity in cancer cells. The compound 3c which ranked second active compound in Jurkat
cells with IC50 values of 16.5 µM was non active in human normal foreskin cells line (table
2
), whereas compound 3f (the most active compound against Jurkat cells) needed 91.53µM to
inhibit the 50% survival of human normal foreskin cells.
Biological and Pharmaceutical Bulletin Advance Publication

Table 2- The cytotoxicity of N-Alkyl-isatin-3-iminobenzoic acid derivatives in human
cancer and normal cell lines.
Cytotoxicity of N-Alkyl-isatin-3-iminobenzoic acid derivatives
in human cell lines
IC50 values µM*
Compounds ID
MCF7
NA**
66
HepG2
NA
91
Jurkat
14.3
61.4
HFF-1
3
a
61.29
NA
3
b
3
c
25.14
NA
23.59
34.61
NA
37
NA
41
66
38
16.5
28.03
24.3
7.8
39.26
89.63
NA
3
d
81.66
22.8
3
e
3
-FU
f
91.53
37.87
31.86
5
SU-5416
41.39
NA
*
IC50 values were calculated based on five different concentrations of the compounds and an
average three biological repeats using the Origin Pro version 8.5.
*
* In case of IC50 values more than 100µM the compounds were scored as not active (NA)
Biological and Pharmaceutical Bulletin Advance Publication

Figure 5. Dose response curves (MTT-test in Jurkat cells)
The line graph showing the effect of compounds on Jurkat cells line. The inhibition in cancer cell line
was measured by MTT assay, and represented as % of control (untreated) cells. The average values
of at least five replicates are presented
.
Figure 6. Dose response curves (MTT-test in HepG2 cells)
The line graph showing the effect of compounds on HepG2 cells line. The inhibition in cancer cell line
was measured by MTT assay, and represented as % of control (untreated) cells. The average values
of at least five replicates are presented.
Biological and Pharmaceutical Bulletin Advance Publication

Figure 7. Dose response curves (MTT-test in MCF7 cells)
The line graph showing the effect of compounds on MCF7 cell line. The inhibition in cancer cell line
was measured by MTT assay, and represented as % of control (untreated) cells. The average values
of at least five replicates are presented.
Biological and Pharmaceutical Bulletin Advance Publication

Figure 8. Dose response curves (MTT-test in HFF-1 cells)
The line graph showing the effect of compounds on HFF-1 cells line. The inhibition in cancer cell line
was measured by MTT assay, and represented as % of control (untreated) cells. The average values
of at least five replicates are presented.
Biological and Pharmaceutical Bulletin Advance Publication

3
. Discussion
3
.1. Chemistry
The N-alkyl-isatin-3-iminobenzoic acid derivatives (3a–f) were prepared via the
condensation of N-alkyl isatin with 4-aminobenzoic acid in absolute ethanol and several
drops of glacial acetic acid by conventional, microwave, and ultrasonic sound methods
7
)
(
Chart1) . Microwave irradiation yielded the products in a shorter reaction time with higher
yields and purities, as indicated by spectral data.
1
As a prototype, the H NMR spectrum of 3c (Figure 9) measured in CDCl
3
showed six
resonances, located at δ 5.02 (s, integrated to two protons), δ 6.55 (d, integrated to one
proton), δ 6.71–6.78 (m, integrated to three protons), δ 7.10 (d, integrated to two protons), δ
7
.24–7.37 (m, integrated to five protons), and δ 8.21 (d, integrated to two protons), which
were assigned to CH protons, proton of H-4, protons of H-5, H-6, H-7, protons of H-2'', H-6'',
protons of C (H-2', H-3', H-4', H-5', and H-6', and protons of H-3'' and H-5'', respectively.
The C NMR spectrum of 3c exhibited a signal up-field at δ 42.83 ppm, which was assigned
to the aliphatic CH group. The three most down-field signals at δ 161.63, 169.84, and 178.80
2
6
H
5
1
3
2
ppm were attributed to C=O, C=N, and COOH respectively, whereas the aromatic carbons
appeared at δ 119.33–153.92 ppm.
Figure 9: The structure of compound 3c
Biological and Pharmaceutical Bulletin Advance Publication

3
.2. Biology
Isatin (1H-indole-2,3-dione) has been reported to have diverse biological activities.
Many of its derivatives have been synthesized and have been reported to possess novel
1
3-15)
pharmacological effects
. In this study, we have reported the synthesis and biological
activity of six N-alkyl-isatin-3-iminobenzoic acid derivatives.
Zebrafish embryos are an excellent animal model for the study of hematopoiesis,
because the zebrafish embryos do not need an active circulation for up to 6–7 days post
fertilization; the oxygen requirements are met from the dissolved oxygen in water and the
nutrients from the yolk. The primary in vivo screening of N-alkyl-isatin-3-iminobenzoic acid
derivatives in zebrafish embryos revealed that they have affected the hematopoiesis as 100%
of treated embryos did not have active circulation. Two compounds (3c and 3f) were very
effective and interfered with hematopoiesis in zebrafish embryos with IC50 values in the
micromolar range (20 and 10 µM, respectively).
The molecular events between developmental hematopoiesis and pathological leukemia
1
6-18)
are correlated
. Zebrafish has served as a suitable model to study both of these
1
9-21)
processes
. Similar to mammals, zebrafish have primitive and definitive hematopoiesis.
Primitive hematopoiesis begins during the gastrula segmentation (6–18 hpf) and active
circulation can be seen at approximately 24 hpf. The next round of hematopoiesis, definitive
2
2, 23)
hematopoiesis, starts at the pharyngula stage from 30 hpf and completes at 48 hpf
. The
primitive blood cells in zebrafish embryos originate from the intermediate cell mass, which
21)
contains both vascular and hematopoietic precursors . In this study, the hematopoiesis
defects were observed in zebrafish embryos when embryos were exposed to N-Alky-Isatin-3-
iminobenzoic acid derivatives (especially 3c and 3f) during the gastrula segmentation stage
(
6–18 hpf). In order to determine whether these compounds interfered with blood cell
formation process (effecting the morphogenesis of blood cells) or destroyed the blood cells,
after blood cells have been formed (cytotoxic towards blood cells) a time-dependent
experiment was conducted in which zebrafish embryos were exposed to compound 3f either
before or after the onset of primary and definitive hematopoiesis. As shown in Figure 3, the
time-dependent treatment of zebrafish embryos revealed that N-Alky-Isatin-3-iminobenzoic
acid derivatives turned out to be most effective inhibitor of the hematopoietic process in
zebrafish embryos. The o-dianisidine positive cells (erythrocytes) were not observed in
zebrafish embryos when they were exposed to compound 3f before the onset of
hematopoiesis, i.e. before the gastrula segmentation stage (6–18 hpf), whereas o-dianisidine
Biological and Pharmaceutical Bulletin Advance Publication

positive cells were present in zebrafish embryos when they were exposed after 48 hpf. The
formation of blood vessels and blood cells are interconnected developmental processes. The
hematopoietic cells (the cells that will give rise to different blood cell lineage) and
endothelial cells (the cells that will give rise to blood vessels) share a common progenitor,
2
4)
which is called the hemangioblast . In this study, it is quite clear that N-alkyl-isatin-3-
iminobenzoic acid derivatives have affected the differentiation of hemangioblasts to
hematopoietic cells, but not the endothelial cells. Undeveloped or malformed blood vessels
could also be a reason for ineffective circulation, and hence, in order to address this point, the
development of blood vessels was also checked in N-alkyl-isatin-3-iminobenzoic acid
derivative-treated zebrafish embryos. The embryos from Tg(fli1:EGFP)y¹ had normal
vasculature, which were exposed to N-alkyl-isatin-3-iminobenzoic acid derivatives (Figure 4),
which also proved the derivatives targeted only hematopoietic cells and not the endothelial
cells.
The hematopoietic activity of the N-alkyl-isatin-3-iminobenzoic acid derivatives
synthesized in this study was compared with the known isatin derivative semaxinib (SU5416)
Flk-1/KDR inhibitor, which is also a specific inhibitor of tyrosine kinase and interferes with
angiogenesis activity by obstructing the vascular endothelial growth factor VEGF to its
9
, 10)
receptor VEGFR2
. However, the effect of SU5416 on hematopoiesis in zebrafish
embryos is largely unknown. As shown in Figure 4, zebrafish embryos treated with 1 µM
SU-5416 had undeveloped/malformed vasculature, but were positive for o-dianisidine
staining. As shown in Figure 4, the zebrafish embryos treated with SU5416 had positive o-
dianisidine staining localized more in the anterior trunk, mainly in the posterior cardinal vein
(
PCV) and the caudal vein (CV). SU5416 is a very strong antiangiogenic compound, which
induces the severe malformation of blood vessels in treated zebrafish embryos, even at ≤ 1
µM. The positive o-dianisidine-stained cells in SU5416 embryos indicated that the blood
cells had formed normally in these embryos. However, the blood cells pooled and clogged in
the caudal and posterior cardinal veins and these cells failed to circulate back to the heart
owing to the absence of active blood vessels and, typically, active circulation is not seen in
these embryos. Previously, o-dianisidine staining of SU5416-treated zebrafish embryos has
not been used to assess the status of erythrocytes. Hence, this study also reported that
SU5416 did not affect hematopoiesis in zebrafish embryos. Interestingly, contrasting activity
of these two classes of isatin derivatives, i.e., semaxinib (SU5416) and N-alkyl-isatin-3-
iminobenzoic acid derivatives has been observed for zebrafish hematopoiesis. As observed in
Biological and Pharmaceutical Bulletin Advance Publication

this study, semaxinib (SU5416), which is an isatin derivative, targeted the differentiation of
hemangioblasts towards endothelial cells and had no effect on hematopoiesis; whereas
compound 3f, which is an N-alkyl-isatin-3-iminobenzoic acid derivative, targeted the
differentiation of hemangioblasts towards hematopoiesis, but had no effect on endothelial
cells.
Newly synthesized N-alkyl-isatin-3-iminobenzoic acid derivatives showed very specific
activity that interfered with hematopoiesis in live zebrafish embryos. In order to know
whether these compounds could also affect the viability of hematological cancer cells, the
anti-proliferative profile of these compounds were tested in Jurkat cells, which is a human
leukemia cell line. The cytotoxicity of these compounds have also been checked in two solid
cancer cell lines i.e. human liver cancer (HepG2) and human breast cancer (MCF7) cells and
normal cell line derived from human foreskin (HFF-1). The cell viability assays have shown
that most of these compounds showed promising cytotoxicity in tested cancer cells as
compared to known anti-cancer drug 5-FU, however, the compounds 3c and 3f showed
specific cytotoxicity towards Jurkat (leukemia cells) at very low IC50 values (16.5 and 7.8
µM, respectively). Compounds 3c and 3f were more effective than the known anticancer drug
5-FU. The IC50 value of 5-FU was 39.26 µM in Jurkat cells. Moreover, the compounds 3c
and 3f were more cytotoxic in MCF7, HepG2, and Jurkat cells compared with SU5416. The
biological activity of compounds 5-FU in MCF7, HepG2 and Jurkat cells which has been
6, 25-29)
observed in this study is in accordance with other published reports
The compounds 3c and 3f showed very weak toxicity in human foreskin HFF-1 cells, the
IC50 values of compound 3c in HFF-1 cells was more than 100 µM and hence designated as
not active while the IC50 value for compound 3f in HFF-1 cells was 91.53 µM. The strong
cytotoxicity of compounds 3c and 3f in human hematological cancer cells (Jurkat) and
comparatively weaker level of toxicity in normal human foreskin cells (HFF-1) warrants, the
further studies of these two molecule for the designing and treatment of anticancer drugs to
treat human leukemia.
In this study, the parent compound (isatin itself) did not alter the hematopoiesis in
zebrafish embryos nor cell viability in Jurkat cells within the tested range of concentrations.
However, the cytotoxicity of isatin derivatives in leukemia cell lines have been reported in
3
0, 31)
other studies.
. However, to the best of our knowledge, we have not encountered any
Biological and Pharmaceutical Bulletin Advance Publication

study that reports the action of isatin or any of its derivatives as an anti-hematopoietic
molecule in live animals such as zebrafish. Hence, the compounds which are synthesized in
this study are unique because they have been shown as effective anti-hematopoietic
molecules in vivo (in live zebrafish embryos) and as an effective antileukemia molecule in
vitro (in Jurkat cells). The specificity of these compounds towards hematopoiesis in zebrafish
embryos indicated that they are highly specific inhibitors of molecular events, such as
transcription factors and critical pathways, which activate blood cell formation in zebrafish
and could be applied to treat leukemia in humans.
3.. Structure activity relationships (SAR)
In this study, we have reported the synthesis of six N-alkyl-isatin-3-iminobenzoic acid
derivatives (the target products). These compounds were designed based on the reported
biological activities of isatin itself (I), semaxanib (SU5416) II, and sunitinib III, (previously
known as SU11248), Beside FDA approved anti-cancer drug for the treatment of renal cell
carcinoma and gastric cancer, the Sunitinib is also in phase I/II clinical trial for the treatment
3
2)
33-35)
of acute myeloid leukemia via inhibiting the FLT3 gene
. Finally, other derivatives IV
1
5, 36)
[
13, 24], which have been reported with strong anticancer activity (Figure 10).
Zebrafish embryos were used as an in vivo animal model system to evaluate the
biological activity of these compounds. The isatin derivatives were then screened in vitro to
assess their cytotoxicity by a colorimetric MTT cell viability assay. Three human cancer cell
lines, liver carcinoma cells (HepG2), human breast carcinoma (MCF7), and a human acute T
cell leukemia cell line (Jurkat) were treated with a serial dilution of the target products. The
known anticancer drugs 5-FU and SU5416 were used as positive control in this study. The
results from the zebrafish and the human cancer cell lines suggested that the isatin derivatives
were markedly influenced by the different substituents on the isatin ring at position 5 and at
the N-terminal of the isatin moiety.
The zebrafish embryos did not develop blood vessels when treated with 1 µM SU-5416,
but positive o-dianisidine staining indicated that this tyrosine kinase inhibitor did not affect
the blood cells formation in zebrafish embryos. In contrast, compounds 3c and 3f specifically
inhibited blood cell formation, but did not affect the blood vessel formation in zebrafish
embryos, whereas the other compounds did not affect the zebrafish embryos. This indicated
that the fluorine at position 5 conferred greater activity than chlorine and bromine atoms, but,
with respect to hematopoietic targeting, the benzyl group was more active than the methyl
Biological and Pharmaceutical Bulletin Advance Publication

group at position 1 in the isatin moiety. The combination between fluorine and benzyl group
in this series of compounds exerted a great effect on the zebrafish embryos.
The MTT cell viability assay on three cancer cell lines showed that all the tested
compounds were cytotoxic in cancer cells, and cytotoxicity was better than known anticancer
drug 5FU and SU-5416 against Jurkat cells. The best results were obtained again with 3c, and
3f whereas, 3b had the lowest efficacy; the IC50 values for 3a, 3b, 3c, 3d, 3e, and 3f were
14.3, 61.4, 16.5, 28.0, 24.3, and 7.8 µM, respectively, compared with 39.3 and 89.6 µM for
5FU and SU-5416, respectively in Jurkat cells. Similarly, 3c and 3f were more effective on
MCF7 (IC50 25.14 and 31.6 µM, respectively) as compared with 5FU (301 µM) and SU-5416
41.3 µM). The IC50 values for 3c and 3f were 37 and 66 µM, respectively, in HepG2
(
compared with 5FU (38 µM) and SU-5416 (112 µM); 3f was similar to 5FU, but more
effective than SU-5416.
This rough SAR analysis (Figure 10) corroborated that the substitution pattern of the
isatin at the 5-position and isatin-3-iminobenzoic acid derivatives here tested were vital for
activity. The most potent compounds identified were those exhibiting polar substituents
(
electronegative atoms) at position 5 of the isatin moiety, namely compound 3f, whereas
compounds with less polar groups, namely 3a and 3b were less active. This suggested that
electrostatic interaction (e.g., dipolar or hydrogen bond) might be critical for activity. In
contrast, the N-methyl derivative (3b) showed lower activity than the N-benzyl derivative
(
3c), so it could be assumed that the lipophilicity of the isatin moiety at the 1-position
provided a positive contribution to the activity.
Biological and Pharmaceutical Bulletin Advance Publication

Figure 10: The structure activity relationship between compound 3f, semaxinib, Sunitinib,
and others imino-isatin.
We have not come across to any report about effect of Sunitinib in zebrafish embryos,
however, the anti- cancer activity of Sunitinib in Jurkat cells has been reported. The
cytotoxicity activity compound 3f in Jurkat cells is in accordance to thee published reports.
3
7)
38)
The IC50 values of sunitinib has been reported as 6 µM
and 2.3 µM
in Jurkat cells,
whereas the IC50 values of compound 3f was 7.8 µM in Jurkat cells in this study. The
structural comparison between compound 3f and sunitinib revealed that the compound 3f is
very much structurally related to sunitinib, as both of the compounds share fluorine as their
moiety in isatin ring (Figure 10) which might be center for the functional activity of these two
compounds. Further, we have tested the toxicity of newly synthesized isatin derivatives in
Biological and Pharmaceutical Bulletin Advance Publication

live zebrafish embryos. This assay has an advantage over other in vitro based studies that the
in vivo toxicity of the compound would indicate whether the compound would be suitable to
be further tested in clinical trials. The Phase III clinical trials of semaxinib for the treatment
of advanced colorectal cancer were prematurely ended in February 2002 due to discouraging
39, 40)
results
. The fate of SU-5416 clearly indicates that in vivo animal studies in relevant
animal models are very much necessary to assess the potential toxicity of the compounds and
must be done prior to clinical trials. The zebrafish assays in this study clearly indicates that
compound 3f induced hematopoietic defects at lower concentration without inducing gross
teratogenicity or toxicity (table 1). The hematopoietic defects were noticed ≤ 10µM, whereas
it needed more than 26µM to induce the lethality or toxicity in zebrafish embryos. The
promising bioactivity of compound 3f in Jurkat cells and zebrafish embryos warrants the
design and synthesizes of similar molecules and also to check the molecular targets for
example FLT3 in leukemia cells with these compounds, which is currently in progress in our
lab. The promising activity means, that the compound 3f has the therapeutic potential to be
developed as drug to treat leukemia after exploring the mechanism of action in leukemia cells.
4
. Materials and Methods
4.1. Materials
All solvents used were of HPLC reagent grade. The melting points were determined
with Mel-Temp apparatus and were uncorrected. Fourier transform infrared spectroscopy
FTIR) spectra were recorded on a Nicolet 560 sepectrometer. Nuclear magnetic resonance
(
1
13
spectra ( H NMR and C NMR) were recorded on a JEOL 400 MHz spectrometer with
chemical shift values reported in ppm (δ units) relative to an internal standard. Microwave
irradiation was conducted using a multimode reactor (Synthos 3000, Anton Paar GmbH;
1
1
400W maximum magnetron). Reactions were performed in teflon vessels with a capacity of
0 mL. The target temperature was set and fixed during the irradiation. The settings and
readings for power (W) and pressure were obtained from the instrument (J. P. Selecta, Spain;
0 Hz, 770 W). The elemental analyses were performed on a Perkin-Elmer 2400 elemental
6
analyzer and the obtained values were within ± 0.3% of the theoretical values. The follow-up
of the reactions and purity assessment of the compounds were achieved by thin layer
chromatography (TLC) on silica gel-protected aluminum sheets (GF254, Merck) and the
spots were detected by exposure to a UV-lamp at λ=254 nm for a few seconds. A Perkin
Elmer FT-IR Spectrometer (Spectrum 1000) was used to record infrared (FTIR) spectra of
Biological and Pharmaceutical Bulletin Advance Publication

the prepared compounds as KBr pellets. The instrument is available at King Saud University,
College of Science, Chemistry Department.
4
.2. General procedure for synthesis of 1-substituted-5-substituted-indoline-2,3-diones, 2b–f.
4
.2.1. Conventional method (A)
Benzyl chloride (4.1 mmol) was added dropwise to a mixture of isatin derivatives 1 (3.4
mmol) and potassium carbonate (5.1 mmol) in DMF (10 mL) at 0 °C and the reaction was
stirred at room temperature for 48 h. The precipitate was poured into an ice-water mixture.
The resulting solid was filtered, washed with water, dried, and re-crystallized from ethanol.
4
.2.2. Microwave method (B)
A mixture of isatin 1 (5 mmol) and potassium carbonate (8 mmol) in DMF (10 mL) was
stirred for 10 min at room temperature. Alkyl halides (6 mmol; benzyl bromide for
preparation of 2c, 2d, 2e and 2f and CH I for the preparation of 2b) was added dropwise to
3
the reaction mixture and then the reaction was microwave irradiated using a multimode
reactor (Synthos 3000, Anton Paar GmbH, Graz, Austria) with 1,400 W maximum magnetron.
The vessels were heated for 5 min at 80 °C and held at the same temperature for a further 5
min (400 W). A fan was used to provide cooling (5 min). The final product was dried and
recrystallized from ethanol. All the spectral data obtained for the products were in good
agreement with the reported data.
4
.2.3. Ultrasonic method (C)
Benzyl chloride (4.1 mmol) was added dropwise to a mixture of isatin derivatives 1 (3.4
mmol) and potassium carbonate (5.1 mmol) in DMF (10 mL) at 0 °C and the reaction was
subjected to ultrasound for 4 h. The precipitate was poured into an ice-water mixture. The
resulting solid was filtered, washed with water, dried, and then recrystallized from ethanol.
4
.2.4. 1-Methylisatin (2b)
The product was obtained as orange red crystals in the following yields: 90 % (A),
4
1, 42)
1
9
2 % (B), and 87 % (C); mp 126–127 °C [Lit.
mp 126–127 °C; 73 % yield]. H NMR
3
(CDCl ): δ = 3.36 (s, 3H, CH3), 6.78 (d, J = 7.8 Hz, 1H, Ar), 7.12 (t, J = 7.4 Hz, 1H, Ar),
7
.47 (t, J = 8.1 Hz, 1H, Ar), 7.61 (d, J = 8.1 Hz, 1H, Ar).
Biological and Pharmaceutical Bulletin Advance Publication

4
.2.5. 1-Benzylisatin (2c)
The product was obtained as orange red crystals in the following yields: 83% (A); 88 %
4
1, 42)
1
(
B); 82% (C); mp 134–136 °C. [Lit.
. mp 134–136 °C; 88 % yield]. H NMR (CDCl
3
): δ
=
4.93 (s, 2H, C H CH ), 6.77 (d, J = 7.7 Hz, 1H, Ar), 7.08 (t, J =7.4 Hz, 1H, Ar), 7.33 (s, 5H,
6 5 2
Ar), 7.47 (t, J = 8.1, 1H), 7.61 (d, J = 8.1, 1H, Ar).
4
.2.6. 1-Benzyl-5-bromoisatin (2d)
The product was obtained as red orange crystals in the following yields: 98 % (A); 92 %
4
2)
1
(
B); 94% (C); mp 149–151 °C [Lit. mp 152–153 °C; 95 % yield]. H NMR (CDCl
3
): δ =
4
8
.91 (s, 2H, C H CH ), 6.69 (d, J = 8.1 Hz, 1H, Ar), 7.24-7.32 (m, 5 H, Ar), 7.45 (dd, 1H, J =
6 5 2
.1 Hz, 2.2 Hz, Ar), 7.56 (d, 1H, J = 2.2 Hz, Ar).
.2.7. 1-Benzyl-5-chloroisatin (2e)
4
The product was obtained as orange crystals in the following yields: 98% (A); 92% (B);
1
9
4 % (C); mp 136 °C [Lit. [26] mp 134 °C; 65 % yield]. H NMR (CDCl
3
): δ 4.94 (s, 2H,
C
6
H
5
2
CH ), 6.69 (d, J = 8.1 Hz, 1H, Ar), 7.2–7.34 (m, 5 H, Ar), 7.45 (dd, 1H, J = 8.1 Hz, 2.3
Hz, Ar), 7.56 (d, 1H, J = 2.3 Hz, Ar).
4
.2.8. 1-Benzyl-5-fluoroisatin (2f)
The product was obtained as red powder, mp: 134 °C; yields: 88 % (A); 89 % (B); 90
1
(
C) %; H NMR (CDCl
3
): δ 4.92 (2H, s, C
6
H
5
CH
2
), 6.72 (1H, dd, J = 8.79, 3.0 Hz), 7.18 (1H,
1
3
dd, J = 8.79, 3.0 Hz), 7.21 (1H, d, J =3.0 Hz) 7.28–7.38 (5H, m); C-NMR (CDCl
10.9, 111.3, 123.2, 123.5, 126.1, 127.0, 127.8, 132.9, 145.35. Anal. calc. for C H FNO
2
3
): δ 42.9,
1
1
5
10
(255.25): C, 70.58; H, 3.95; N, 5.49. Found: C, 70.77; H, 4.05; N, 5.22.
4
.3. General procedure for synthesis of 4-(1-substituted-5-substituted-2-oxoindolin-3-
ylideneamino] benzoic acid (3a–f)
4
.3.1. Conventional method (A)
A mixture of isatin derivatives (2a–f) (0.01 M) and 4-amino-benzoic acid (0.01 M) in
absolute ethanol (20 mL) was refluxed for 0.5 h in the presence of 2–3 drops of glacial acetic
Biological and Pharmaceutical Bulletin Advance Publication

acid. After cooling, the mixture was filtered and the crude solid was recrystallized from
ethanol.
4
.3.2. Microwave method (B)
A mixture of isatin derivatives (2a–f) (0.01 M) and 4-amino-benzoic acid (0.01 M) in
absolute ethanol (2 mL) was irradiated in the presence of two drops of glacial acetic acid in
MW for 20 min at 600 W.
4
.3.3. 4-((2-oxoindolin-3-ylidene)amino)benzoic acid (3a)
The product was obtained as an off-white solid, mp: 286–289 °C; yield: 99% (A); 98% (B);
4
3)
-1
[
Lit ; mp 288–290 °C; yield 90%). IR (KBr, cm ): 3500–2500 (br, OH, NH), 1725 (C=O,
isatin ring), 1686 (C=O, acid), 1616 (C=N).
4
.3.4. 4-((1-Methyl-2-oxoindolin-3-ylidene)amino)benzoic acid (3b)
The product was obtained as yellow powder, mp: 186–188 °C; yield %: 99(A); 98 (B).
-1
1
IR (KBr, cm ): 3422 (OH), 1702, 1659 (C=O), 1602. (C=N); H-NMR (CDCl
H, CH )), 6.55 (1H, d, J = 7.35 Hz), 6.71–6.78 (3H, m), 7.10 (2H, d, J = 8.7), 8.21 (2H, d, J
3
8.7 Hz); C-NMR (CDCl ): δ 30.2, 114.3, 121.7, 125.2, 126.2, 126.7, 127.7, 133.4, 133.6,
3
): δ 3.36 (s,
3
3
1
3
=
1
4
46.1, 161.6 , 169.8 (C=N), 178.8 (COOH). Anal. calc. for C16
.32; N, 9.99. Found: C, 68.88; H, 4.43; N, 10.09.
12 2 3
H N O (280.08): C, 68.56; H,
4
.3.5. 4-(1-Benzyl-2-oxoindolin-3-ylideneamino)benzoic acid (3c)
The product was obtained as yellow powder, mp: 176–178 °C; yield %: 99(A); 98 (B).
-1
1
IR (KBr, cm ): 3422 (OH), 1702, 1659 (C=O), 1602 (C=N); H-NMR (CDCl
3
): δ 5.02 (2H, s,
C H CH ), 6.55 (1H, d, J = 7.35 Hz), 6.71-6.78 (3H, m), 7.10 (2H, d, J = 8.7 Hz), 7.24-7.37
6
5
2
1
3
(
5H, m, C
6
H
5
CH
16.4, 121.7, 125.2, 126.2 , 126.7, 127.7, 130.8, 133.4, 133.6, 146.1, 153.9, 161.6 (CO),
69.8 (C=N), 178.8 (COOH). Anal. calc. for C22 (356.38): C, 74.15; H, 4.53; N, 7.86.
2 3
), 8.21 (2H, d, J = 8.7 Hz); C NMR (CDCl ): δ 42.83, 109.33, 114.29,
1
1
16 2 3
H N O
Found: C, 74.34; H, 4.64; N, 7.99.
4
.3.6. 4-(1-Benzyl-5-bromo-2-oxoindolin-3-ylideneamino)benzoic acid (3d)
The product was obtained as yellow powder, mp: 260–262 °C; yield: 54% (A); 60 %
-1
1
(
3
B). IR (KBr, cm ): 3415 (OH), 1741, 1678 (C=O), 1602 (C=N); H-NMR (CDCl ): δ 5.00
Biological and Pharmaceutical Bulletin Advance Publication

(
2H, s, C
8.43 Hz), 7.22-7.36 (6H, m, Ar), 8.23 (2H, d, J = 8.43 Hz); C-NMR (CDCl
16.1, 126.1, 127.8, 130.8, 139.4, 153.9, 161.4 (CO), 169.6 (C=N), 178.9 (COOH). Anal.
calc. for C22 (434.03): C, 60.71; H, 3.47; N, 6.44. Found: C, 60.94; H, 3.65; N,
6 5 2
H CH ), 6.55 (1H, d, J = 2.19 Hz), 6.69 (1H, dd, J = 8.43, 2.19 Hz), 7.09 (2H, d, J
1
3
=
3
): δ 43.0,
1
2 3
H15BrN O
6
.67.
4
.3.7. 4-(1-Benzyl-5-chloro-2-oxoindolin-3-ylideneamino)benzoic acid (3e)
The product was obtained as yellow powder, mp: 242–244 °C; yield %: 54(A); 60 (B).
-
1
1
IR (KBr, cm ): 3415 (OH), 1741, 1678 (C=O), 1602 (C=N); H-NMR (CDCl
CH ), 6.55 (1H, d, J = 2.19 Hz), 6.69 (1H, dd, J = 8.43, 2.19 Hz), 7.09 (2H, d, J = 8.43
Hz,), 7.22–7.36 (6H, m, Ar), 8.23 (2H, d, J = 8.43 Hz); C NMR (CDCl
26.1, 127.8, 130.8, 139.4, 153.9, 161.6 (CO), 169.8 (C=N), 178.8 (COOH). Anal. calc. for
(390.08): C, 67.61; H, 3.87; N, 7.17. Found: C, 67.87; H, 4.06; N, 7.33.
3
): δ 5.00 (2H, s,
C
6
H
5
2
1
3
3
): δ 42.99, 116.1,
1
22 2 3
C H15ClN O
4
.3.8. 4-(1-Benzyl-5-fluoro-2-oxoindolin-3-ylideneamino)benzoic acid (3f)
The product was obtained as yellow powder, mp: 216–218 °C; yield %: 80 (A); 89
-
1
1
(
(
B). IR (KBr, cm ): 3415 (OH), 1744, 1678 (C=O), 1603 (C=N); H-NMR (CDCl
3
): δ 5.01
2H, s, C CH ), 6.29 (1H, dd, J = 8.07, 4.0 Hz), 6.70 (1H, dd, J = 8.79, 4.0 Hz), 6.98 (1H,
6
H
5
2
td, J = 8.79 Hz), 7.09 (2H, d, J = 8.43 Hz), 7.29-7.37 (5H, m, Ar), 8.23 (2H, d, J = 8.43
1
3
Hz); C NMR (CDCl
3
): δ 41.7, 115.9, 125.9, 126.69, 127.6, 130.8, 142.1 . Anal. calc. for
22 2 3
C H15FN O
(374.37): C, 70.58; H, 4.04; N, 7.48. Found: C, 70.84; H, 4.33; N, 7.77.
4
.3. Animal biology
The adult zebrafish were raised and maintained following the guidelines described in the
4
4)
zebrafish book
.
4.4. Animal treatment
The wild type (AB Tubingen) and transgenic Tg (fli-1:EGFP) zebrafish embryos were
4
5)
obtained by natural pairwise mating and treated as described previously . Three biological
replicate trials were conducted for each experiment.
SU-5416 (1,3-Dihydro-3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylene]-2H-indol-2-one) was
purchased from Sigma Aldrich, Cat # S8442 Sigma lot # 052M4611V
Biological and Pharmaceutical Bulletin Advance Publication

4.5. O-Dianisidine staining for erythroid cells
The presence of erythroid cells were evaluated by o-dianisidine staining, as previously
4
6)
described
.
4
.6. Cell culture and cell viability assay
N-Alkyl-isatin-3-iminobenzoic acid derivatives were screened in human liver cancer
HepG2), breast cancer (MCF7), and leukemia (Jurkat) cell lines to assess the in vitro
(
6
)
cytotoxicity activity by MTT assay as previously described .
.7. Statistics
Origin (Version 6.1052; Origin Lab Corp Northampton, MA 01060, USA) was used for
4
statistical analysis. The data were considered statistically significant for values of P < 0.05.
Acknowledgments: The authors extend their sincere appreciation to Vice Deanship of
Research Chairs, King Saud University, for supporting this study.
Author Contributions: Muhammad Farooq designed the study conducted the experiments in
zebrafish embryos, performed the in vitro studies in cancer cell lines and statistical analysis
of the data, made the tables and figures, and wrote the manuscript. Ayman El-Faham and
Zainab M. Almarhoon synthesized the compounds, designed the studies, and wrote the
chemical-related sections of the manuscript. Nael Abu Taha performed the in vitro studies in
the cancer cell lines. Mohammad A. M. Wadaan conceived the study, participated in its
design, and drafted the manuscript. All of the authors read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
Biological and Pharmaceutical Bulletin Advance Publication

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