Design, Synthesis, Characterization, and In Vitro Evaluation of Isatin-Pomalidomide Hybrids for Cytotoxicity against Multiple Myeloma Cell Lines

Article abstract of DOI:10.1002/jhet.3365

Inspired by the concept of molecular hybridization, a series of new isatin-pomalidomide hybrids (9a–9g) were designed, synthesized, characterized, and evaluated for in vitro cytotoxic activity against U266B1 and RPMI 8226 multiple myeloma cell lines. Sandmeyer methodology and N-halomethylketo alkylation reaction are the two important reactions involved in the synthesis of isatin-pomalidomide hybrids (9a–9g). All the synthesized compounds (3a–3d, 4, 5, 6, and 9a–9g) were characterized by using IR, mass, ¹H-NMR, and ¹³C-NMR spectral techniques. The efficacy of all the synthesized compounds was tested against the aforementioned cell lines by employing MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) standard protocols while using pomalidomide as a standard. The test concentrations used in the MTT assay were 1, 10, 20, 30, and 40?μM, and the period of incubation was 24?h. All the synthesized compounds were found to have moderate to greater cytotoxic activity against the aforementioned cell lines. Among them, synthesized hybrids 9f (IC₅₀, U266B1?=?5.15?±?0.72?μM, RPMI8226?=?11.66?±?0.79?μM) and 9g (IC₅₀, U266B1?=?2.50?±?0.37?μM, RPMI8226?=?6.70?±?0.55?μM) displayed better cytotoxic activity against both the cell lines used in the present study.

Full text of DOI:10.1002/jhet.3365

Month 2018
Design, Synthesis, Characterization, and In Vitro Evaluation of
Isatin-Pomalidomide Hybrids for Cytotoxicity against Multiple Myeloma
Cell Lines
a
b
a
*
Shyam Panga, Naveen Kumar Podila, and Veeresham Ciddi
aUniversity College of Pharmaceutical Sciences, Kakatiya University, Vidyaranyapuri, Warangal,
Telangana 506009, India
bSynocule Research Lab, Navodaya Society, Banjara Hills, Hyderabad, Telangana 500034, India
*E-mail: ciddiveeresham@yahoo.co.in
Received February 9, 2018
DOI 10.1002/jhet.3365
Published online 00 Month 2018 in Wiley Online Library (wileyonlinelibrary.com).
Inspired by the concept of molecular hybridization, a series of new isatin-pomalidomide hybrids (9a–9g)
were designed, synthesized, characterized, and evaluated for in vitro cytotoxic activity against U266B1 and
RPMI 8226 multiple myeloma cell lines. Sandmeyer methodology and N-halomethylketo alkylation reaction
are the two important reactions involved in the synthesis of isatin-pomalidomide hybrids (9a–9g). All the
1
synthesized compounds (3a–3d, 4, 5, 6, and 9a–9g) were characterized by using IR, mass, H-NMR, and
13C-NMR spectral techniques. The efficacy of all the synthesized compounds was tested against the afore-
mentioned cell lines by employing MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide) standard protocols while using pomalidomide as a standard. The test concentrations used in the
MTT assay were 1, 10, 20, 30, and 40 μM, and the period of incubation was 24 h. All the synthesized com-
pounds were found to have moderate to greater cytotoxic activity against the aforementioned cell lines.
Among them, synthesized hybrids 9f (IC50, U266B1 = 5.15 0.72 μM, RPMI8226 = 11.66 0.79 μM)
and 9g (IC50, U266B1 = 2.50 0.37 μM, RPMI8226 = 6.70 0.55 μM) displayed better cytotoxic activity
against both the cell lines used in the present study.
J. Heterocyclic Chem., 00, 00 (2018).
INTRODUCTION
biological activities are presented and shown in Table 1
and Figure 1.
Design of new hybrid molecules by the covalent
conjugation of two or more bioactive fragments with
complementary or different mechanism of actions into a
single new hybrid moiety is called molecular
hybridization [1]. It is one of the simple and effective
ways of drug discovery with high incidence of success
rate, time saving, and economic as opposed to the
conventional process of drug discovery. Numerous
literature reports suggest that molecular hybrids are
known to possess additive or synergistic biological
activities unlike their parent moieties [2,3]. Some known
examples of such hybrid molecules with enhanced
The concept underlying this approach is minimizing the
dose of each individual drug to facilitate limited drug
exposure, reducing dose-dependent toxicity, and
decreasing the emergence of multidrug resistance [9].
Estramustine and nitric oxide-amino salicylic acid hybrids
are some of the best examples for the marketed and under
clinical trial products of molecular hybridization.
The isatin (1H-indole-2,3-dione) is a molecule with
immense and diverse biological profile, offering wide
array of applications in the field of medicinal chemistry
with ease of its synthesis. Some of the reported activities
of isatin in the literature are antibacterial [10], antifungal
© 2018 Wiley Periodicals, Inc.

S. Panga, N. K. Podila, and V. Ciddi
Vol 000
Table 1
Cytotoxic activities of the recently reported isatin-based hybrids.
S. no.
1.
Name of the hybrids
Cytotoxic activity against cancer cell lines
Ref.
[2]
Isatin-benzothiazole hybrids
MDA-MB231, MDA-MB468, and MCF7; also against two non-cancer
cell lines, 184B5 and MCF10A.
2.
3.
Isatin-thiazolidinone hybrids
Isatin-4-piperazinylquinoline hybrids
MDA-MB239 and MDA-MB435 (breast cancer cell lines variations)
[4]
[5]
MDA-MB468 (a PTEN defective, p53 positive, EGFR positive breast
adenocar-cinoma cell line) and MCF7 (p53+/ꢀ, invasive ductal breast carcinoma)
and two non-cancer breast epithelial cell lines (184B5 and MCF10A)
Human multiple myeloma MM1S, RPMI8226, U266 cells, and human
lung cancer A549 cells.
Liver HepG2, breast MCF-7, and colon HT-29 cancer cell lines, more
inhibition against HepG2 cell lines
HepG2 hepatocellular carcinoma, A549 lung cancer, and MCF-7 breast
cancer cell lines
4.
5.
6.
Curcumin-thalidomide hybrids
Isatin-quinazoline hybrids
Isatin-pyridine hybrids
[6]
[7]
[8]
Figure 1. Few examples for the recently reported isatin-based hybrids.
[11], antiviral [12], anti-HIV [13,14], anti-mycobacterial
[15], anticancer [16–20], anti-inflammatory [21],
anticonvulsant [22], anti-TB [23–25], antimalarial, and
anti-plasmodial [26–28] activities.
Gowda et al. [29] reported that isatin does possess anti-
multiple myeloma activity by inhibiting one of the key
enzymes of Bruton’s tyrosine kinase involved in the
pathogenesis of multiple myeloma. Thus, by combining
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Month 2018
Isatin-Pomalidomide Hybrids
these two pharmacophore units (isatin-pomalidomide) with
complimentary functions into single hybrid molecule, there
is a possibility of enhancing the efficacy of envisaged
hybrids.
essential for the elicitation of maximum anticancer
activity and claims that N-1 substitution on isatin favors
anticancer/cytotoxic activity [34,35]. Further, substitution
of electron-withdrawing groups at the 5th position of
N-alkyl-substituted isatin is associated with increased
biological activity [19,20].
Based on these observations, we designed the synthesis
of isatin-pomalidomide hybrids (9a–9g) by using
molecular hybridization technique [1]. According to this
approach, a series of new isatin-pomalidomide hybrid
molecules were engineered by the use of two different
bioactive drug fragments, pomalidomide and isatin (1H-
indole-2,3-dione), and the rationality behind the design of
isatin-pomalidomide hybrids (9a–9g) is illustrated in
Figure 3.
The focus of the present investigation is on the design,
synthesis, and characterization of series of novel
compounds obtained by hybridizing pomalidomide with
bioactive natural product scaffold, isatin. It also
involves the exploration of isatin-pomalidomide hybrids
potential as effective anticancer agents against RPMI
8226 and U266B1 multiple myeloma cell lines. Thus,
we here in report the unprecedented in vitro cytotoxic
activities of pomalidomide and its hybrids on multiple
myeloma cell lines.
Multiple myeloma is one of the hematological cancers
manifested by the proliferation of malignant monoclonal
B cells in the bone marrow. The other symptoms include
anemia, renal insufficiency, hypercalcemia, and
immunodeficiency [30]. The American Cancer Society’s
estimates for multiple myeloma in the USA are 30,770
diagnosed new cases (16,400 in men and 14,370 in
women) and 12,770 deaths are expected to occur (6,830
in men and 5,940 in women) in the year 2018 [31].
Thalidomide, lenalidomide, pomalidomide, bortezomib,
and its combination with dexamethasone are some of the
important drugs used in the treatment of multiple
myeloma [32]. Pomalidomide is
a
derivative of
thalidomide being approved in the USA and Europe for
the treatment of relapsed and refractory multiple myeloma
in adults. Pomalidomide is available in the market under
the brand name Pomalyst as capsule in the USA and as
Imnovid in Europe [32].
Pomalidomide is chemically described as 4-amino-
2-(2,6-dioxopiperodin-3-yl)-isoindoles-1,3-dione, and it
belongs to the class of phthalimide derivatives such as
thalidomide, lenalidomide, and pomalidomide whose
structures are shown in Figure 2.
Pomalidomide is found to be effective than its
precursors thalidomide and lenalidomide in the treatment
of multiple myeloma at an oral dose of 2–15 mg/day with
the inclusion of oxo and amino functional groups from
thalidomide and lenalidomide, respectively [33]. Despite
of much progress being made in the development of
novel anti-myeloma drugs, none of them could counter
effectively the emergence of more number of side effects,
drug resistance, and eventual relapse in the treatment of
multiple myeloma.
The present study attempts to address the
aforementioned problem by reducing the dose of
pomalidomide and replacing its part with the biologically
safe moiety and without getting compromised on the anti-
multiple myeloma activity against the multiple myeloma
cell lines.
RESULTS AND DISCUSSION
In the present study, isatin derivatives (3a, 3b, 3c, 3d, 4,
5, and 6) were synthesized via Sandmeyer methodology
initially, and later, these isatin derivatives were
conjugated with pomalidomide covalently through N-halo
methyl keto alkylation reaction as shown in Schemes 1
and 2. The physical data of all the synthesized
compounds are presented in the Experimental Methods
section. A total of 14 compounds have been synthesized,
out of which seven derivatives are unconjugated isatins
(3a, 3b, 3c, 3d, 4, 5, and 6) and the other seven
derivatives are pomalidomide conjugated isatin hybrids
(9a–9g).
Molecular hybridization is one of the simple and easiest
methods of the drug design to bring the best features from
their parent moieties to its corresponding progeny. The
main aim of the present study is to examine the role of
covalent conjugation and combination on the cytotoxic
Research investigations on isatin nucleus underscored
the importance of alkyl substitution with a carbon chain
length of 2–3 at N-1 position of isatin nucleus as it is
Figure 2. Chemical structures of thalidomide derivatives.
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S. Panga, N. K. Podila, and V. Ciddi
Vol 000
Figure 3. Rationale behind the design of isatin-pomalidomide hybrids (9a–9g).
Scheme 1. Synthesis of isatin and isatin derivatives (3a–3d, 4, 5, and 6) from p-aniline (1a–1d) and chloral hydrate.
activities of the isatin derivatives (3a, 3b, 3c, 3d, 4, 5, and
6) as well as their corresponding hybrids with
pomalidomide (9a–9g) against U266B1 and RPMI 8226
multiple myeloma cell lines. In order to highlight the role
of covalent conjugation and combination, their cytotoxic
activities have been evaluated separately as unconjugated
moieties (3a, 3b, 3c, 3d, 4, 5, and 6) and conjugated
moieties (9a–9g) against U266B1 and RPMI 8226
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Isatin-Pomalidomide Hybrids
Scheme 2. The synthetic pathway of isatin-pomalidomide hybrids (9a–9g).
multiple myeloma cell lines, which are presented in
Table 2. In addition to this, the hybrid (9g) with the best
IC50 value was selected, and its effect on the cytotoxicity
was evaluated as a mixture (1:1 molar ratio) so as to
understand the efficacy of the covalent conjugation over
the physical combination.
The IC50 values of unconjugated isatin derivatives (3a,
3b, 3c, 3d, 4, 5, and 6) and isatin-pomalidomide hybrids
(9a–9g) were found to be in the range of
35.82
2.16 μM to 2.50
0.37 μM (U266B1) and
Table 2
45.05 1.38 μM to 6.70 0.55 μM (RPMI 8226). This
shows that the synthesized derivatives are sensitive
towards U266B1 cells than RPMI 8226 cells. The
cytotoxic assay of covalently conjugated isatin-
pomalidomide hybrids (9a–9g) as well as unconjugated
isatin derivatives (3a–3d, 4, 5, and 6) indicated that isatin
nucleus conjugation and combination with pomalidomide
have enhanced the cytotoxic activity as observed from
their IC50 values in general. However, the extent of cell
growth inhibition varies from one conjugate to the other
conjugate when compared with pomalidomide. As per the
statistical analysis, it is understood that conjugation of
unsubstituted isatin (3a) and 5-substituted isatins (3b, 3c,
3d, 4, and 5) with pomalidomide have less effect on the
cytotoxicity against U266B1 and RPMI 8226 cell lines as
evident from its corresponding IC50 values of the
cytotoxic assay.
The IC50 data of the present study (from Figs. 4 and 5)
revealed the similarity in the cytotoxic activities of the 5-
substituted isatin-pomalidomide hybrids (9b, 9c, 9d, 9e,
and 9f) and pomalidomide. But, there is significant
difference (**P-value < 0.001, when compared with
pomalidomide) in the IC50 value of the 9g
hybrid/conjugate on both the cell lines U266B1
The half maximal effective concentration (IC50 in μM) of the isatin and its
derivatives (3a–3d, 4, 5, and 6) and isatin-pomalidomide hybrids (9a–9g)
against U266B1 and RPMI 8226 multiple myeloma cell lines.
,
IC50 μMa b
Entry
no.
Code
U266B1
RPMI8226
1
2
3
4
5
6
7
8
3a
3b
3c
3d
4
5
6
9a
35.82 2.16*** 45.05 1.38**
31.21 1.23*** 38.13 1.22**
25.84 1.72*** 35.41 1.03**
24.44 1.76*** 31.56 1.52**
22.42 1.24*** 33.61 0.94**
21.35 2.44*** 30.71 1.10**
15.37 2.01*** 24.09 0.99**
12.96 1.25*** 21.33 1.49**
9
9b
9c
9d
9e
9f
10.89 1.02*
9.73 1.17*
9.39 0.39*
8.23 0.78*
5.15 0.72*
2.50 0.37***
6.12 1.05**
18.57 1.48*
16.36 0.76*
15.68 0.57*
15.32 0.90*
11.66 0.79**
6.70 0.55***
11.57 0.42**
10
11
12
13
14
15
9g
PM + DBIS
(1:1)
16
Pomalidomide
(PM, Std)
7.49 0.84
15.54 0.92
IC50 was calculated from dose–response curves. Statistical analysis was
performed by applying two-way analysis of variance, nonparametric,
followed by Dunnet’s multiple comparisons test.
aSigmoidal dose–response curves (variable slope) were generated using
GraphPad Prism V. 6.00 (GraphPad Software Inc.).
bValues are the mean of triplicates of at least three independent
experiments.
*Not significant.
**P-value < 0.05.
***P-value < 0.001 when compared with pomalidomide.
(2.50
0.37 μM) and RPMI 8226 (6.70
0.55 μM)
when compared with pomalidomide IC50 value on both
the cell lines U266B1 (7.49 0.84 μM) and RPMI 8226
(15.54 0.92 μM), suggesting that 5,7 substitutions on
the isatin derivatives and its conjugation with
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S. Panga, N. K. Podila, and V. Ciddi
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Figure 4. A comparative view of the IC50 values of isatin derivatives (3a–3d, 4, 5, and 6) and its pomalidomide hybrids (9a–9g) against U266B1 multiple
myeloma cell line.
Figure 5. A comparative view of the IC50 values of isatin derivatives (3a–3d, 4, 5, and 6) and its pomalidomide hybrids (9a–9g) against RPMI8226 mul-
tiple myeloma cell line.
pomalidomide have increased the cytotoxic activity on
both the cell lines by significantly decreasing their
corresponding IC50 values.
In addition to this, combined effects of pomalidomide
(PM) and 5,7-di-bromoisatin (6) physical mixture (1:1)
were also tested against the cell line used in the current
study. The IC50 value of the physical mixture (PM and 6)
was compared with the IC50 value of its corresponding
covalent conjugate (9g). The results obtained from their
IC50 values demonstrated that covalent conjugation (9g)
has superior cytotoxicity [2.50
6.70 0.55 μM (RPMI8226)] than its physical mixture
(PM DBIS) [6.12 1.05 μM (U266B1);
11.57 0.42 μM (RPMI8226)].
As shown in Figures 4 and 5, the cytotoxic activity of
unconjugated isatin derivatives has been increased after
covalent conjugation with pomalidomide (9a–9g), which
is evident from their IC50 values, being decreased to half
of its value after covalent conjugation than its values
prior to conjugation. This confirms that the conjugation
0.37 μM (U266B1);
+
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Isatin-Pomalidomide Hybrids
of two pharmacophores with complementary functions
yields better activity than its original activity prior to
covalent conjugation.
The obtained results showed excellent consistency with
previously reported studies by Panga et al. [36], Modi et al.
[34], and Vine et al. [19], where substitutions at 5th and 7th
positions of isatin nucleus and conjugation at N-1 position
conferred better cytotoxicity. Similarly, in the present
study, the potent compounds 9g and 9f also have all the
stated characteristics as mentioned in the previous studies.
Preparation of 2-chloro-N-(1,3-dioxo-2-(2,6-dioxopiperidin-
3-yl)isoindolin-7-yl)-acetamide (8).
Pomalidomide (7)
(2.73 g, 0.01 moles) was dissolved in a mixture of 25 mL of
glacial acetic acid and 25 mL of saturated solution of
sodium acetate. To this, chloroacetyl chloride (1.5 mL, 0.013
moles) was added drop wise to avoid the vigorous reaction.
After half an hour, the product was filtered with suction,
washed several times with cold water, and then infrared
dried. The product was crystallized from methanol [34].
Procedure for the preparation of pomalidomide (7) and
5,7-di-bromoisatin (6) physical mixture (PM + DBIS) in
micromolar concentration. The test solutions (in μM) of
pomalidomide (7) and 5,7-di-bromoisatin (6) physical
mixture (PM + DBIS) were prepared in the ratio of 1:1
by using the following formula.
CONCLUSION
In the present study, molecular hybridization technique
was used to synthesize the isatin-pomalidomide hybrids
(9a–9g) with enhanced anti-multiple myeloma activity
than its parent compounds isatin derivatives (3a–3d, 4, 5,
and 6) and pomalidomide (PM). All the synthesized
derivatives (14) that consists of unconjugated isatin
derivatives and conjugated isatin derivatives with
pomalidomide have been evaluated for their cytotoxic
activity against U266B1 and RPMI 8226 multiple
myeloma cell lines by using MTT assay. It is observed
that among all the covalent hybrids of isatin and
pomalidomide, pomalidomide conjugation with 5,7-di-
bromoisatin derivative was found to be effective in
modifying the cytotoxic activity of the pomalidomide as
evident from its IC50 values. In addition to this, efforts
were laid to prove the superiority of covalent conjugation
over the physical combination by testing the most active
compounds against the aforementioned cell lines used in
the present study. The IC50 value obtained from this
study showed that the covalent conjugation of 5,7-di-
bromoisatin with pomalidomide has low IC50 value than
that of its physical mixture [5,7-di-bromoisatin and
pomalidomide (PM + DBIS)], concluding that covalent
conjugated compound (9g) was most effective against the
aforementioned two cell lines used in the present study.
Thus, it can be deduced from the aforementioned study
that the conjugation of isatin and pomalidomide was
proved to be beneficial. However, advanced studies such
as the stability of the hybrids in the systemic circulation
and their mode of action must be explored in the near
future to witness the real applications of molecular
hybridization in drug discovery and delivery.
M1 ꢁ V1 ¼ M2 ꢁ V2;
where M1
[weight
=
molarity of 5,7-di-bromoisatin (6)
=
mass/density; density
=
2.2 g/cm3];
V1 = volume of 5,7-di-bromoisatin (6); M2 = molarity of
pomalidomide (7); and V2 = volume of pomalidomide
(7) [weight = mass/density; density = 1.6 g/cm3].
The aforementioned equation is valid when the densities
of the compounds are equal. Hence, weights of the
compounds were divided by the densities of
pomalidomide (7) and 5,7-di-bromoisatin (6).
General
procedure
for
the
preparation
of
isatin-pomalidomide hybrids (9a–9g). Compounds 3a–3d,
4, 5, and 6 (1 g) were taken up in 30 mL anhydrous DMF
and ice cooled with stirring [34]. Solid K2CO3 (1 g, 0.0072
moles) was added in one portion, and the dark-colored
suspension was raised to room temperature and stirred for a
further 1 h. The appropriate amount of N-acylated
pomalidomide (8) (1 g, 0.0028 moles) and KI (0.5 g, 0.006
moles) was added, and the reaction mixture was stirred at
80°C for 2–24 h, until the reaction was over, which was
confirmed using TLC. The reaction mixture was poured
into six times its volume of water, and then diluted
hydrochloric acid was added until the pH value is between
three and four stirred for a further 10 min. The crude final
product was filtered with suction, washed several times
with cold water, and then infrared dried. The product was
crystallized from methanol. The yield, melting point, and
spectral data of each compound are given as follows.
1H-indole-2, 3-dione (3a).
Yield: 60%; M.P.: 193–
195°C; IR (KBr, t cmꢀ1): 3195 (NH of isatin NH), 1730
1
(C═O of isatin), 1696 (─NH─CO); H-NMR (300 MHz,
δ ppm, DMSO-d6): 6.95–8.03 (m, 4H, Ar─H), 10.20 (s,
1H, ─NH); CHN Anal. Calcd for C8H5NO2: C (65.31),
H (3.43), N (9.52). Found: C (65.11), H (3.20), N (9.71);
MS m/z (+ve):148.1 (M+).
EXPERIMENTAL METHODS
5-Chloro-1H-indole-2,3-dione (3b).
Yield: 68%; M.P.:
General procedure for the preparation of compounds (3a–
3d, 4, 5, and 6). Preparation of isatin and its derivatives
(3a–3d, 4, 5, and 6) was performed as per the already
reported procedures [19,20,37–39].
255–258°C; IR (KBr, t cmꢀ1): 3346 (NH of isatin), 1710
1
(C═O of isatin), 1683 (─NH─CO); H-NMR (300 MHz,
δ ppm, DMSO-d6): 7.50–7.80 (m, 3H, Ar─H), 8.94 (s,
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S. Panga, N. K. Podila, and V. Ciddi
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1H, ─NH); CHN Anal. Calcd for C8H4ClNO2: C (52.92),
H (2.22), N (7.71). Found: C (52.88), H (1.96), N (7.82);
MS m/z (+ve): 181.9 (M+), 183.9 (M + 2).
2-(5-Chloro-2,3-dioxo-2,3-dihydro-indol-1-yl)-N-[2-(2,6-
dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl]-
acetamide (9b). Yield: 50%; M.P.: 205–207°C; IR (KBr,
t cmꢀ1): 3300 (NH of ─NH─CO), 1720 (C═O of isatin),
5-Fluoro-1H-indole-2,3-dione (3c).
Yield: 56%; M.P.:
1
224–227°C; IR (KBr, t cmꢀ1): 3338 (NH of isatin), 1740
(C═O of isatin), 1672 (─NH─CO), 1227 (C–F); 1H-
NMR (300 MHz, δ ppm, DMSO-d6): 6.95–7.70 (m, 3H,
Ar─H), 11.00 (s, 1H, ─NH); CHN Anal. Calcd for
C8H4FNO2: C (58.19), H (2.44), N (8.48). Found: C
(58.10), H (2.38), N (8.69); MS m/z (+ve): 166.1 (M+).
1680 (─NH─CO), 560 (─C─Cl); H-NMR (300 MHz,
d ppm, DMSO-d6): 2.10–2.35 (m, 4H, 2 × CH2), 3.92 (s,
2H, H2C─CO), 4.00–4.20 (t, 1H, ─CH), 7.50–8.13 (m,
6H, Ar─H), 9.80 (s, 1H, ─NH), 10.2 (bs, 1H,
─CONHCO─); 13C-NMR (300 MHz,
d
ppm,
DMSO-d6): 21.2, 29.8, 54.0, 54.8, 119.0, 123.0, 123.4,
124.2, 125.0, 130.0, 130.5, 132.1, 132.6, 135.0, 140.3,
146.0, 160.4, 168.0, 168.4, 169.2, 173.0, 183.8; CHN
Anal. Calcd for C23H15ClN4O7: C (55.83), H (3.06), N
(11.32). Found: C (55.76), H (3.02), N (11.25); MS m/z
5-Iodo-1H-indole-2,3-dione (3d).
Yield: 40%; M.P.:
276–280°C; IR (KBr, t cmꢀ1): 3216 (NH of ─NH─CO),
1744 (C═O of isatin), 1658 (─NH─CO); 1H-NMR
(300 MHz, δ ppm, DMSO-d6): 7.60–8.70 (m, 3H,
Ar─H), 8.86 (s, 1H, ─NH); CHN Anal. Calcd for
C8H4INO2: C (35.19), H (1.48), N (5.13). Found: C
(35.12), H (1.41), N (5.20); MS m/z (+ve): 273.9 (M+).
(+ve): 461.2 (M+), 462.2 (M + 2).
N-[2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-
isoindol-4-yl]-2-(5-fluoro-2,3-dioxo-2,3-dihydro-indol-1-yl)-
acetamide (9c). Yield: 52%; M.P.: 281–283°C; IR (KBr, t
5-Nitro-1H-indole-2,3-dione (4).
Yield: 50%; M.P.:
cmꢀ1): 3300 (NH of ─NHCO), 1715 (C═O of isatin),
251°C; IR (KBr, t cmꢀ1): 3238 (NH of ─NH─CO),
1730 (C═O of isatin), 1663 (─NH─CO), 1500–1480 and
1350–1318 (─NO2); 1H-NMR (300 MHz, d ppm,
DMSO-d6): 7.98–8.72 (m, 3H, Ar─H), 10.01 (s, 1H,
─NH); CHN Anal. Calcd for C8H4N2O4: C (50.10), H
(2.10), N (14.58). Found: C (49.99), H (1.86), N (14.92);
MS m/z (+ve): 193.02 (M+).
1
1668 (─NH─CO), 1260 (─C─F); H-NMR (300 MHz,
δ ppm, DMSO-d6): 2.05–2.32 (m, 4H, 2 × CH2), 3.90 (s,
2H, H2C─CO), 4.38–4.42 (t, 1H, ─CH), 7.46–8.08 (m,
6H, Ar─H), 9.40 (s, 1H, ─NH), 9.98 (bs, 1H,
─CO─NH─CO─); 13C-NMR (300 MHz,
δ ppm,
DMSO-d6): 20.9, 29.0, 53.3, 54.0, 115.0, 119.3, 121.5,
123.0, 124.0, 125.1, 131.9, 132.6, 140.4, 144.0, 159.0,
160.3, 168.0, 168.4, 169.5, 172.4, 183.2; CHN Anal.
Calcd for C23H15FN4O7: C (57.75), H (3.16), N (11.71).
Found: C (53.03), H (3.11), N (6.32); MS m/z (+ve):
5-Bromo-1H-indole-2,3-dione (5).
Yield: 42%; M.P.:
247–250°C; IR (KBr, t cmꢀ1): 3260 (NH of ─NH─CO),
1728 (C═O of isatin), 1695 (─NH─CO), 678 (C─Br);
1H-NMR (300 MHz, δ ppm, DMSO-d6): 6.97–7.80 (m,
3H, Ar─H), 10.00 (s, 1H, ─NH); CHN Anal. Calcd for
C8H4BrNO2: C (42.51), H (1.78), N (6.20). Found: C
(42.30), H (1.70), N (6.50); MS m/z (+ve): 225.9 (M+),
479.2 (M+), 479.1 (M + 1).
N-[2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-
isoindol-4-yl]-2-(5-iodo-2,3-dioxo-2,3-dihydro-indol-1-yl)-
acetamide (9d). Yield: 36%; M.P.: 248–250°C; IR (KBr, t
227.9 (M + 2).
cmꢀ1): 3400 (OH of ─NH─CO), 1730 (C═O of isatin),
5,7-Di-bromo-1H-indole-2,3-dione (6). Yield: 36.2%; M.
1
P.: 252–255°C; IR (KBr, t cmꢀ1): 3386 (NH of
1660 (─NH─CO), 580 (─C─I); H-NMR (300 MHz, δ
ppm, DMSO-d6): 1.98–2.29 (m, 4H, 2 × CH2), 3.86 (s,
2H, H2C─CO), 3.98–4.01 (t, 1H, ─CH2), 7.58–8.23 (m,
6H, Ar─H), 9.90 (s, 1H, ─NH), 10.50 (bs, 1H,
─NH─CO), 1725 (C═O of isatin), 1698 (─NH─CO);
1H-NMR (300 MHz, δ ppm, DMSO-d6): 7.80 (s, 1H,
Ar─H), 7.90 (s, 1H, Ar─H), 9.81 (s, 1H, ─NH); CHN
Anal. Calcd for C8H3Br2NO2: C (31.51), H (0.99), N
(4.59). Found: C (31.42), H (0.90), N (4.73); MS m/z
(+ve): 304.8 (M+), 302.8 (M ꢀ 2), 306.8 (M + 2).
─CO─NH─CO─); 13C-NMR (300 MHz,
δ ppm,
DMSO-d6): 21.0, 29.6, 52.8, 54.2, 89.6, 119.0, 123.2,
124.0, 124.4, 125.2, 132.2, 133.0, 138.0, 140.5, 144.0,
147.0, 160.4, 168.0, 168.4, 169.0, 172.3, 184.3; CHN
Anal. Calcd for C23H15IN4O7: C (47.12), H (2.58), N
(9.50). Found: C (47.18), H (2.40), N (9.23); MS m/z
N-(1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)isoindolin-7-yl)-2-
(2,3-dioxoindolin-1-yl)-acetamide (9a). Yield: 52%; M.P.:
220–222°C; IR (KBr, t cmꢀ1): 3250 (NH of ─NH─CO),
1695 (C═O of isatin), 1666 (─NH─CO); 1H-NMR
(300 MHz, δ ppm, DMSO-d6): 2.00–2.30 (m, 4H,
2 × CH2), 3.83 (s, 2H, H2C─CO), 4.49–4.53 (t, 1H,
─CH), 7.20–8.15 (m, 7H, Ar─H), 9.92 (s, 1H, ─NH),
10.0 (bs, 1H, ─CONHCO─); 13C-NMR (300 MHz, δ
ppm, DMSO-d6): 21.0, 29.3, 53.2, 54.1, 117.4, 122.0,
123.0, 124.0, 124.4, 125.0, 130.0, 132.4, 132.3, 134.8,
140.4, 148.1, 160.4, 168.0, 168.2, 169.0, 172.4, 184.0;
CHN Anal. Calcd for C23H16N4O7: C (60.00), H (3.50),
N (12.17). Found: C (59.60), H (3.48), N (12.10); MS m/
z (+ve): 461.04 (M+), 462.1 (M + 1).
(+ve): 587.0 (M+), 588.0 (M + 1).
N-[2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-
isoindol-4-yl]-2-(5-nitro-2,3-dioxo-2,3-dihydro-indol-1-yl)-
acetamide (9e). Yield: 40%; M.P.: 236–238°C; IR (KBr, t
cmꢀ1): 3360 (NH of ─NH─CO), 1720 (C═O of isatin),
1640 (─NH─CO), 1348 (─C─NO2); 1H-NMR
(300 MHz, δ ppm, DMSO-d6): 2.11–2.39 (m, 4H,
2 × CH2), 3.94 (s, 2H, H2C─CO), 4.35–4.38 (t, 1H,
─CH), 7.70–8.75 (m, 6H, Ar─H), 9.80 (s, 1H,
─NH─CO), 10.03 (bs, 1H, ─CO─NH─CO─); 13C-
NMR (300 MHz, δ ppm, DMSO-d6): 21.2, 29.5, 53.0,
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2018
Isatin-Pomalidomide Hybrids
54.5, 119.0, 122.8, 123.0, 124.4, 124.9, 125.3, 132.2,
132.6, 140.7, 144.8, 154.3, 160.0, 168.3, 168.8, 169.3,
173.0, 184.6; CHN Anal. Calcd for C23H15N5O9: C
(54.66), H (2.99), N (13.86). Found: C (54.40), H (3.01),
inactivated fetal bovine serum (Himedia, Mumbai, India)
in 25-cm2 tissue culture flasks. All cells were incubated at
37°C in a humidified atmosphere of 95% air and 5% CO2.
MTT assay for cytotoxicity evaluation of the test
compounds. To detect growth inhibition by the series
N (13.78); MS m/z (+ve): 506.1 (M+), 507.1 (M + 1).
2-(5-Bromo-2,3-dioxo-2,3-dihydro-indol-1-yl)-N-[2-(2,6-
dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl]-
acetamide (9f). Yield: 50%; M.P.: 210–212°C; IR (KBr,
of compounds, cells were seeded onto 96-well plates at
a density of 5 × 103 per well before compound
treatment. Cytotoxicity of the series of compounds in a
variety of cell lines was determined using the
MTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide assay after incubation of cells with these
compounds at 1-, 10-, 20-, and 40-μM concentrations for
48 h. IC50 (the concentration of 50% inhibition of cell
growth) was determined from the dose–response curves [40].
t cmꢀ1): 3300 (─NH─CO), 1710 (C═O of isatin), 1683
1
(C═O of ─NH─CO), 650 (C─Br); H-NMR (300 MHz,
δ ppm, DMSO-d6): 1.80–2.32 (m, 4H, 2 × CH2), 3.84 (s,
2H, H2C─CO), 4.61–4.64 (t, 1H, ─CH2), 7.20–8.19 (m,
6H, Ar─H), 8.90 (s, 1H, ─NH─CO), 10.20 (s, 1H,
─CO─NH─CO─); 13C-NMR (300 MHz,
δ ppm,
DMSO-d6): 22, 31, 54, 55, 120, 123.1, 123.3, 123.8,
131.9, 132.0, 141.4, 141.8, 144.8, 160.0, 168.1, 169.1,
169.9, 171.8, 184.8; CHN Anal. Calcd for
C23H15BrN4O7: C (51.22), H (2.80), N (10.39). Found: C
(50.98), H (2.73), N (10.40); MS m/z (+ve): 539.0
Statistical analysis. Graphs were plotted by taking Log
concentration on X-axis and % inhibition on Y-axis. The
IC50 values were obtained by nonlinear regression using
GraphPad Prism 6.0. IC50 measurements for each
compound were performed three times. All the values
(M + 1), 541.0 (M + 2).
were expressed as mean
SD. Two-way analysis of
2-(5,7-Di-bromo-2,3-dioxo-2,3-dihydro-indol-1-yl)-N-[2-(2,6-
dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl]-
acetamide (9g). Yield: 38%; M.P.: 201–202°C; IR (KBr, t
variance was performed to validate the data obtained
from cytotoxic assay. All the values were found to be
statistically significant at P < 0.05.
cmꢀ1): 3460 (NH of ─NH─CO), 1717 (C═O of isatin),
1
1666 (─NH─CO), 648 (─C─Br); H-NMR (300 MHz,
δ ppm, DMSO-d6): 2.11–2.39 (m, 4H, 2 × CH2), 3.94 (s,
2H, H2C─CO), 4.35–4.38 (t, 1H, ─CH), 7.70–8.75 (m,
6H, Ar─H), 9.80 (s, 1H, ─NH─CO), 10.03 (bs, 1H,
Acknowledgments. One of the authors Mr. Panga Shyam wishes
to thank UGC, New Delhi, India, for providing Basic Scientific
Research (BSR) Fellowship to carry out doctoral research work
at Kakatiya University, Warangal, Telangana, India.
─CO─NH─CO─); 13C-NMR (300 MHz,
δ ppm,
DMSO-d6): 19.2, 21.8, 51.5, 51.8, 121.5, 122.0, 122.6,
124.0, 124.4, 134.8, 141.2, 141.9, 146.7, 148.0, 160.0,
161.8, 168.2, 169.4, 171.9, 183.4; CHN Anal. Calcd for
C23H14Br2N4O7: C (44.69), H (2.28), N (9.06). Found: C
(44.60), H (2.12), N (9.20); MS m/z (+ve): 617.9
(M + 1), 615.9 (M ꢀ 2), 619.9 (M + 2).
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet