Design and synthesis of β-amino alcohol based β-lactam-isatin chimeras and preliminary analysis of in vitro activity against the protozoal pathogen Trichomonas vaginalis

Article abstract of DOI:10.1039/c3md00057e

We have described the synthesis of β-amino alcohol based β-lactam-isatin chimeras along with their in vitro evaluation against T. vaginalis. SAR studies showed dependence of activity on concentration as well as substituent on N-1 of β-lactam and C-5 of isatin with preference for a chloro-substituent for good activity.

Full text of DOI:10.1039/c3md00057e

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Design and synthesis of b-amino alcohol based
b-lactam–isatin chimeras and preliminary analysis of
in vitro activity against the protozoal pathogen
Trichomonas vaginalis†
Cite this: DOI: 10.1039/c3md00057e
Nisha,a Vishu Mehra,a Melissa Hopper,b Neal Patel,b Dominique Hall,b
b
b
a
*
Lisa A. Wrischnik, Kirkwood M. Land and Vipan Kumar
Received 19th February 2013
Accepted 9th April 2013
We have described the synthesis of b-amino alcohol based b-lactam–isatin chimeras along with their in vitro
evaluation against T. vaginalis. SAR studies showed dependence of activity on concentration as well as
substituent on N-1 of b-lactam and C-5 of isatin with preference for a chloro-substituent for good activity.
DOI: 10.1039/c3md00057e
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reported to exhibit a myriad of biological properties including
anti-HIV, anti-viral, anti-tumour, anti-fungal, anti-malarial and
Introduction
Trichomonas vaginalis is a protozoal parasite of the human
urogenital tract and the causative organism of trichomoniasis.1
The disease is a far more prevalent sexually transmitted disease
(STD) than the diseases caused by either Chlamydia trachomatis
or Neisseria gonorrhoea with approximately 5 million infections
in North America and 170 million infections worldwide, annu-
ally. Despite trichomoniasis being reported to have nearly as
high prevalence as chlamydia, gonorrhea, and syphilis combined,
it remains a neglected sexually transmitted disease, and is
regarded as a self-clearing ‘nuisance’.2,3 Metronidazole (MTZ) is
the current and only FDA approved drug for the treatment of
trichomoniasis and has been used for more than 40 years.4
There are ample reports regarding the development of resis-
tance to MTZ which in certain cases have shown to be tackled
with prolonged therapy, but occasionally the patients continue
to harbour the disease.5,6 Furthermore, it is now well estab-
lished that the complications related to trichomoniasis signif-
icantly increase the transmission and acquisition of the human
immunodeciency virus (HIV) by two fold, thus potentially
translating into a signicant number of global HIV infections.7,8
As evident, the development of novel and efficacious scaffolds
against trichomoniasis alone could signicantly minimize the
incidence of new HIV infections.
anti-oxidant activities. Cyclization of 1H-indole-2,3-dione to 4-
thiazoline,11 4-thiazolidinone,12 and pyridazinoindole13 was
efficient in increasing its antimicrobial efficacy. In particular,
SU-5416 (Semaxanib) and SU-11248 (simitinib) are the 2-oxoin-
dole based FDA approved drugs for the treatment of gastroin-
testinal stromal tumours and advanced renal cell carcinoma.
b-Lactam is considered as another privileged heterocyclic
scaffold with innumerable biological activities. b-Lactam anti-
biotics, including penicillins, cephalosporins and carbapenems,
have been extensively employed as chemotherapeutic agents for
treating bacterial infections and microbial diseases.14,15 These
structural units operate by forming a covalent adduct with
membrane bound bacterial transpeptidase, also known as
penicillin binding proteins (PBPs) involved in cell wall biosyn-
thesis.16,17 b-Lactams have also been shown to be potent inhibi-
tors of serine proteases, human leukocyte elastase and human
cytomegalovirus protease with some derivatives being efficient
against several types of cancer. The biological activity of the b-
lactam skeleton is generally believed to be associated with the
chemical reactivity of the four membered ring as well as the
nature of substituents especially at nitrogen of the 2-azetidinone.
Recent reports from our lab have shown the synthesis of 1H-
1,2,3-triazole tethered b-lactam–isatin hybrids along with their
in vitro evaluation against T. vaginalis.18 In continuation of our
interest in the synthesis of novel molecular frameworks with
biological potential,19 the present communication entails the
synthesis of b-amino alcohol based b-lactam–isatin conjugates
along with their preliminary in vitro evaluation studies against
T. vaginalis at 50 mM.
Isatin (1H-indole-2,3-dione) is considered as a structural
platform for chemical modication and is responsible for a
broad spectrum of biological properties in many synthetically
versatile molecules.9,10 The 1H-indole-2,3-dione moiety is
aDepartment of Chemistry, Guru Nanak Dev University, Amritsar-143005, Punjab,
India. E-mail: vipan_org@yahoo.com; Fax: +91-183-2258819-20; Tel: +91-183-
2258802 ext. 3320
bDepartment of Biological Sciences, University of the Pacic, Stockton, CA 95211, USA
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c3md00057e
The rationale behind using b-amino alcohol as a linker is
based on the diversity of this structural framework in nature as
well as in various pharmaceuticals.20 For example, compounds
with a hydroxyethylamine core have been reported to inhibit
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aspartic protease enzymes and are widely used as anti-HIV,21,22
antimalarial,23 anti-TB and anti-leishmanial activities.24
The compound 7a, for example, characterized as 1-[3-
hydroxy-2-(2-oxo-4-styryl-1-p-tolyl-azetidin-3-ylamino)-propyl]-
1H-indole-2,3-dione, was analysed for C29H27N3O4 and showed
a molecular ion peak at m/z 481.2 in its mass spectrum. The
salient features of its 1H NMR spectrum included a singlet at d
2.28 corresponding to methyl protons, another singlet d 3.74
corresponding to methylene protons, a doublet of doublet at d
5.01 corresponding to H1 ( J ¼ 5.4 and 5.8 Hz), another doublet
of doublet at d 5.61 corresponding to H2 ( J ¼ 5.7 and 7.5 Hz) and
a multiplet at d 5.76–5.92 corresponding to methine protons.
The cis-stereochemical relationship between H1 and H2 was
ascertained on the basis of the coupling constant J ¼ 5.7 Hz.
The appearance of characteristic signals at d 149.5, 163.9, 169.7
corresponding to three carbonyl (C]O) bonds along with the
required number of carbons in 13C NMR spectrum further
substantiated the assigned structure.
In vitro activity against Trichomonas vaginalis. The synthe-
sized scaffolds were evaluated for their anti-trichomonas activity
at 50 mM and the results are listed in Table 1. On comparing the
effect of substituents at N-1 of the b-lactam and C-5 of the isatin
ring, it has been observed that the scaffolds with the presence of
a chloro substituent at the N-aryl ring of the b-lactam ring as
well as the C-5 position of the isatin ring showed the best
growth inhibition among the test compounds as evident from
7b, 7e, 7h and 7k. The compounds 7a and 7c with an un-
substituted isatin ring and the presence of a p-tolyl and a
cyclohexyl group at N-1 of 2-azetidinone are exceptions exhib-
iting 95.26% and 93.56% growth inhibition, respectively. The
compounds 7e and 7h proved to be the most potent of the test
compounds exhibiting 97.63% and 97.09% growth inhibition
respectively at 50 mM. The active compounds from the prelim-
inary inhibition data were chosen in order to determine their
IC50 which is the minimum concentration required for 50%
growth inhibition and the results are tabulated in Table 2. As
evident from Table 2, although the synthesized conjugates are
not as active as the standard drug metronidazole, most of the
compounds showed potent activity against the G3 strain of T.
vaginalis. The compound 7h having a chloro-substituent on N-
aryl of the b-lactam ring as well as at the C-5 position of isatin
proved to be the most active chimeric scaffold among the test
series with an IC50 of 9.73 mM conrming the activity
enhancement role of chloro-substituents in the designed series
of novel scaffolds. The concentration–response curves for the
two most potent compounds viz. 7e and 7h are depicted in
Fig. 1. The most potent compound 7h has been further evalu-
ated for its cytotoxicity proles against HeLa cells and the
results of percent viability are compared with standard drugs
bleomycin and doxorubicin. Mammalian cell cytotoxicity was
comparable to bleomycin, with approximately 30% viability.
The low cytotoxicity of scaffold 7h further conrms the signi-
cance of this study towards the development of novel b-lactam–
isatin hybrids as new anti-trichomonas agents.
Result and discussion
Synthesis
The procedure for the synthesis of b-amino alcohol tethered
isatin–b-lactam hybrids was based on the well established acid
catalyzed epoxide ring opening of 1-oxiranylmethyl isatin 3 with
3-amino-2-azetidinone 6. The precursor 1-oxiranylmethyl
indole-2,3-dione 3 was prepared based on a combination of
literature methods25 involving an initial base assisted allylation
of substituted isatins to yield the corresponding N-allyl isatin 2
followed by its epoxidation using m-chloroperbenzoic acid
ꢀ
(mCPBA) in dry chloroform at 60 C (Scheme 1).
The diastereoselective synthesis of the second precursor viz.
3-amino-4-styryl-azetidine-2-one 6 was done according to our
recently reported procedure26 involving an initial Staudinger
reaction of 1-azadiene with azidoketene, generated in situ from
azidoacetic acid in the presence of p-toluene sulphonyl chloride
and triethylamine, to yield 5 with the subsequent reduction
using Zn/NH4Cl as depicted in Scheme 2.
The target compounds 7 were synthesized by reuxing
ꢀ
appropriate precursors 3 and 6 in dry toluene at 110 C in the
presence of catalytic amounts of p-toluene sulphonic acid
(Scheme 3). The purication of the reaction mixture, aer usual
work up, via column chromatography resulted in the isolation
of desired structural chimeras 7, the structures of which were
assigned on the basis of spectral data and analytical evidence.
Scheme 1 Synthesis of 1-oxiranylmethyl indole-2,3-dione.
Scheme 2 Synthesis of 3-amino-4-styryl-azetidine-2-one.
In silico proling. The small library 7a–7l was proled in silico
for various physico-chemical and Absorption, Distribution,
Metabolism and Excretion (ADME) properties using the web-
based applications ChemAxon (http://www.chemaxon.com/)
and PreADMET (http://preadmet.bmdrc.org/). Basicity and
Scheme 3 Diastereoselective synthesis of b-lactam–isatin conjugates.
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Table 1 Inhibitory activity of the compound library against the G3 strain of T.
vaginalis tested at 50 and 100 mM
Table 1 (Contd. )
Average percentage
inhibition (50 mM) clog Pa,27
Average percentage
Compound Structure
Compound Structure
inhibition (50 mM) clog Pa,27
7g
59.80 ꢁ 5.19
6.08
7a
95.26 ꢁ 1.65
5.19
7h
97.09 ꢁ 1.61
6.43
7b
76.17 ꢁ 3.46
5.54
7i
61.84 ꢁ 10.56
71.91 ꢁ 0.81
77.19 ꢁ 3.56
5.68
5.51
5.86
7c
93.56 ꢁ 3.91
4.79
7j
7d
56.03 ꢁ 1.71
5.69
7k
7e
97.63 ꢁ 0.80
6.60
7l
64.63 ꢁ 1.69
5.10
7f
70.82 ꢁ 1.75
5.28
a
Calculated using Chem Draw Ultra 10.0.
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Table 2 IC50 determination of the synthesized hybrids
Table 4 In silico physicochemical parameters of isatin b-lactam conjugates
Compound
IC50 (mM) (G3) In silico physicochemical parametersa
No. of H No. of H
bond bond
acceptors donors log P log D5.0 log D7.4 violations
No. of
lipinski
7a
7b
7c
7e
7h
7k
19.20 ꢁ 1.08
24.71 ꢁ 1.12
18.51 ꢁ 1.18
14.62 ꢁ 1.12
09.73 ꢁ 1.13
27.45 ꢁ 1.16
0.72
Code Mw
7a
7b
7c
7d
7e
7f
7g
7h
7i
481.54
8
8
8
8
8
8
8
8
8
8
8
8
2
2
2
2
2
2
2
2
2
2
2
2
3.04 2.06
3.11 2.17
2.43 1.66
3.52 2.57
3.60 2.67
2.92 2.16
3.60 2.67
3.67 2.78
2.99 2.27
3.20 2.20
3.27 2.31
2.59 1.80
3.50
3.59
3.12
4.01
4.10
3.63
4.10
4.19
3.73
3.64
3.73
3.26
0
1
0
0
1
0
1
1
1
0
1
0
501.96
473.56
495.57
515.99
487.59
515.99
536.4
508.01
499.53
519.95
491.55
Metronidazolea
a
Current FDA approved drug for treatment of T. vaginalis infection.
7j
7k
7l
a
Calculator Plugins were used for structure property prediction and
Marvin 5.11.4, 2012, ChemAxon (http://
www.chemaxon.com).
calculation,
Fig. 1 Concentration–response curves of 7e and 7h.
accepted value of 5.0. All the derivatives comply with hydrogen
bond properties. The compounds that comply with the Lipinski
rule of ve are 7a, 7c, 7f and 7j while others show a singleviolation
viz. molecular weight. The results of this informative analysis
suggest that most of the compounds follow the computational
assessment and thus represent a pharmacologically active
framework that should be considered on progressing further
potential hits.
lipophilicity of the new compounds 7a–7l were predicted with
ChemAxon (Table 3), and caco-2, MDCK, BBB, HIA, plasma
protein binding and skin permeability data were predicted with
PreADMET (Table 4). The synthesized hybrids are predicted to
have lower blood–brain barrier permeation and thus are less
likely to cause neurotoxicity. The physicochemical properties of
the synthesized library of compounds 7a–7l were obtained using
chemaxon soware to assess their compliance with the Lipinski
rule of ve criteria (Table 4). The analysis of data revealed that (a)
all molecules have molecular weight in the range of 473–519
which is within the accepted limit of 500 (b) a log P lower than 5.0
indicates that the compounds are not very lipophilic and (c) a log
D at pH 5.0 and 7.4 (permeability) in the range of 1.66–2.78 and
3.12–4.19 respectively which lies well within the limits of the
Conclusions
In conclusion, the present manuscript describes the synthesis
of b-amino alcohol based b-lactam–isatin conjugates along with
their evaluation against T. vaginalis at 50 mM. The evaluation
data clearly revealed the dependence of activity on the substit-
uents at N-1 of b-lactam and C-5 of the isatin ring. Compound
Table 3 In silico ADME profiling of isatin b-lactam conjugates 7a–7l
Absorptiona
Distributiona
Human intestinal
absorption
HIA (percentage)
In vitro caco-2 cell
In vitro MDCK Cell
In vitro skin
In vitro plasma
protein binding
(percentage)
In vivo blood–brain
barrier penetration
(C.brain/C.blood)
permeability
permeability
permeability
Compound
(nm sꢂ1
)
(nm sꢂ1
)
(log Kp, cm hꢂ1
)
7a
7b
7c
7d
7e
7f
7g
7h
7i
96.10
96.54
95.54
96.22
96.65
95.64
96.65
97.02
96.07
96.11
96.55
95.54
22.79
22.91
22.51
23.24
23.40
22.94
23.40
23.80
23.06
23.62
23.23
23.24
0.04
0.04
0.05
0.04
0.04
0.04
0.04
0.04
0.04
0.04
0.04
0.04
ꢂ2.71
ꢂ2.85
ꢂ3.22
ꢂ2.63
ꢂ2.72
ꢂ3.18
ꢂ2.72
ꢂ2.77
ꢂ3.27
ꢂ2.98
ꢂ3.10
ꢂ3.56
91.70
96.53
81.78
90.24
92.01
82.56
92.33
98.46
86.43
90.89
96.64
82.71
0.05
0.03
0.03
0.04
0.03
0.04
0.03
0.03
0.04
0.03
0.02
0.03
7j
7k
7l
a
Predicted properties related to ADME using PreADMET using the web-based application PREDAMET (http://preadmet.bmdrc.org).
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7h having a chloro-substituent on N-aryl of the b-lactam ring as
well as at the C-5 position of isatin proved to be the most active
chimeric scaffold among the test series with an IC50 of 9.73 mM
along with low cytotoxicity. The physicochemical studies further
revealed that most of the compounds are compliant with the
Lipinski rules and the designed molecular framework can act as
therapeutic agents for the synthesis of new antiprotozoal drugs
targeting T. vaginalis.
General method for the preparation of b-lactam isatin
conjugates 7a–7l
To the stirred solution of 1-oxiranylmethyl isatin 3 (1 mmol) and
4-styryl-3-amino-2-azetidinone 6 (1 mmol) in dry toluene a
catalytic amount of p-toluene sulphonic acid was added and the
reaction mixture was reuxed at 110 ꢀC for 5 h. On completion
of the reaction, as monitored by TLC, water (15 mL) was added
to the reaction mixture and extracted with chloroform (2 ꢃ
50 mL). Combined organic layers were dried over anhydrous
sodium sulphate and concentrated under reduced pressure.
Purication of the reaction mixture thus obtained via column
chromatography using a hexane : ethylacetate (6 : 4) mixture as
eluent resulted in the isolation of the desired chimeras 7.
1-[3-Hydroxy-2-(2-oxo-4-styryl-1-p-tolyl-azetidin-3-ylamino)-
propyl]-1H-indole-2,3-dione (7a). White solid, yield 80%, m.p.
204–207 ꢀC, 1H NMR (300 MHz, CDCl3): d 2.28 (s, 3H, CH3), 3.74
(s, 2H, H5), 5.01 (dd, J ¼ 5.4 Hz, 5.8 Hz, 1H, H1); 5.16 (ddd, J ¼ 1.2
Hz, 10.2 Hz, 16.9 Hz, 2H, H7); 5.61 (dd, J ¼ 5.7 Hz, 7.5 Hz, 1H, H2);
5.76–5.92 (m, 1H, H6); 6.27 (dd, J ¼ 7.5 Hz, 16.1 Hz, 1H, H3); 6.49
(t, J ¼ 7.5 Hz, 1H, ArH); 6.60 (d, J ¼ 8.8 Hz, 1H, ArH); 6.75 (d, J ¼
16.0 Hz, 1H, H4); 6.85 (d, J ¼ 8.8 Hz, 1H, ArH); 7.08 (d, J ¼ 8.1 Hz,
2H, ArH); 7.21–7.37 (m, 9H, 8ArH + NH); 7.53 (s, 1H, OH,
exchangeable with D2O), 13C NMR (CDCl3, 75 MHz): 20.9, 45.2,
59.6, 60.1, 76.7, 111.9, 113.5, 114.8, 115.9, 117.2, 122.4, 126.7,
127.6, 128.4, 128.6, 129.5, 133.2, 134.1, 134.6, 134.9, 135.6, 136.1,
149.5, 163.9, 169.7; HRMS calculated for C29H27N3O4 [M]+
481.2002, found: 481.2008; analysis: calculated for C29H27N3O4:
C, 72.33; H, 5.65; N, 8.73, found: C, 72.41; H, 5.58; N, 8.69%.
1-{2-[1-(4-Chloro-phenyl)-2-oxo-4-styryl-azetidin-3-ylamino]-
3-hydroxy-propyl}-1H-indole-2,3-dione (7b). White solid, yield
83%, m.p. 175–178 ꢀC, 1H NMR (300 MHz, CDCl3): d 3.76 (s, 2H,
H5), 4.98 (dd, J ¼ 5.4 Hz, 5.7 Hz, 1H, H1); 5.12 (ddd, J ¼ 1.2 Hz,
10.2 Hz, 16.8 Hz, 2H, H7); 5.65 (dd, J ¼ 5.5 Hz, 7.5 Hz, 1H, H2);
5.72–5.89 (m, 1H, H6); 6.25 (dd, J ¼ 7.4 Hz, 16.1 Hz, 1H, H3); 6.50
(t, J ¼ 7.9 Hz, 1H, ArH); 6.61 (d, J ¼ 8.8 Hz, 1H, ArH); 6.76 (d, J ¼
16.0 Hz, 1H, H4); 6.82 (d, J ¼ 8.8 Hz, 1H, ArH); 7.05 (d, J ¼ 8.3 Hz,
2H, ArH); 7.28–7.42 (m, 9H, 8ArH + NH); 7.59 (s, 1H, OH,
exchangeable with D2O), 13C NMR (CDCl3, 75 MHz): 45.3, 59.6,
60.2, 76.6, 112.0, 113.6, 114.8, 115.8, 117.3, 122.4, 126.7, 127.7,
128.5, 128.6, 129.6, 133.2, 134.2, 134.6, 134.9, 135.7, 136.1, 149.5,
164.0, 169.7; HRMS calculated for C28H24ClN3O4 [M]+ 501.1455,
found 501.1452; analysis: calculated for C28H24ClN3O4: C, 67.00;
H, 4.82; N, 8.37 found: C, 66.88; H, 4.91; N, 8.31%.
Materials and methods
Melting points were determined by open capillary using a Veego
Precision Digital Melting Point apparatus (MP-D) and are
1
uncorrected. H NMR spectra were recorded in deuterochloro-
form and DMSO-d6 with a Jeol 300 (300 MHz) spectrometer
using TMS as the internal standard. Chemical shi values are
expressed as parts per million downeld from TMS and J values
are in hertz. Splitting patterns are indicated as s: singlet, d:
doublet, t: triplet, m: multiplet, dd: doublet of a doublet, ddd:
doublet of a doublet of a doublet, and br: broad peak. 13C NMR
spectra were recorded on a Jeol 300 (75 MHz) spectrometer in
deuterochloroform and DMSO-d6 using TMS as the internal
standard. High resolution mass spectra were recorded on a
Bruker-micrOTOF-Q II spectrometer. Column chromatography
was performed on a silica gel (60–120 mesh).
Procedure for the synthesis of substituted N-allyl bromo
isatins 2
Isatin (1 mmol) was added to a stirred suspension of sodium
hydride (1.5 mmol) in dry DMF (10 mL), resulting in the
formation of purple coloured anions. The solution was stirred at
room temperature until the evolution of hydrogen ceased. To
this reaction mixture a solution of allyl bromide (1.1 mmol) in
DMF was added dropwise. The reaction mixture was heated to
60 ꢀC with constant stirring for about 6 hours. Aer the
completion of reaction, as evident from TLC, it was quenched by
the dropwise addition of water (20 mL) and subsequently
extracted with ethyl acetate (3 ꢃ 30 mL). The combined organic
layers were washed with brine solution, dried over anhydrous
Na2SO4 and concentrated under reduced pressure. Purication
of the reaction mixture via column chromatography using a
hexane : ethyl acetate (8 : 2) mixture furnished the desired N-
alkyl bromo derivatives in good yields.
1-[2-(1-Cyclohexyl-2-oxo-4-styryl-azetidin-3-ylamino)-3-hydroxy-
propyl]-1H-indole-2,3-dione (7c). White solid, yield 79%, m.p.
127–132 ꢀC, 1H NMR (300 MHz, CDCl3): d 1.56–1.64 (m, 10H,
cyclohexyl); 3.42–3.53(m, 1H, cyclohexyl); 3.64(s, 2H, H5); 4.57 (dd, J
¼ 4.8Hz,8.7Hz,1H,H1); 5.05 (ddd, J¼ 1.5 Hz, 10.2 Hz, 17.1 Hz, H7);
5.35 (dd, J¼ 4.8Hz,7.5Hz,1H,H2); 5.69–5.84 (m, 1H, H6); 6.15 (dd, J
¼ 7.5 Hz, 15.9 Hz, 1H, H3); 6.52 (d, J ¼ 9.0 Hz, 1H, ArH); 6.72 (d, J ¼
15.9 Hz, 1H, H4); 7.13–7.47 (m, 9H, 8ArH + NH), 13CNMR(CDCl3, 75
MHz):24.8, 25.2, 30.5, 32.0, 45.7, 52.4, 59.3, 59.9, 112.7, 113.0, 113.8,
114.0, 115.7, 120.0, 120.3, 124.6, 126.6, 128.3, 128.8, 134.8, 135.9,
146.4, 166.4, 168.7. HRMS calculated for C28H31N3O4 [M]+ 473.2315,
found 473.2319; analysis: calculated for C28H31N3O4: C, 71.01; H,
6.60; N, 8.87, found C, 71.13; H, 6.55; N, 8.82%.
Procedure for the synthesis of 1-oxiranylmethyl isatin 3
To a stirred solution of m-chloroperbenzoic acid (m-CPBA) (1.2
mmol) in dry CHCl3, the N-allyl bromo isatin 2 was added at 0 ꢀC
and the reaction mixture was reuxed at 60 ꢀC for 2 h. Aer the
completion of reaction as evident from TLC, the excess of m-
CPBA was quenched by washing with K2CO3 solution and the
organic layer was subsequently extracted with CHCl3 (3 ꢃ 30
mL). The combined organic layers were dried over anhydrous
Na2SO4 and concentrated under reduced pressure resulting in
the isolation of a crude solid which was recrystallized using a
9 : 1 hexane : chloroform mixture.
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1-[3-Hyroxy-2-(2-oxo-4-styryl-1-p-tolyl-azetidin-3-ylamino)-
5-Chloro-1-{2-[1-(4-chloro-phenyl)-2-oxo-4-styryl-azetidin-3-yl-
propyl]-5-methyl-1H-indole-2,3-dione (7d). Pale yellow, yield amino]-3-hydroxy-propyl}-1H-indole-2,3-dione (7h). White
80%, m.p. 196–198 ꢀC, 1H NMR (300 MHz, CDCl3): d 2.07 (s, 3H, solid, yield 76%, m.p.173–180 ꢀC, 1H NMR (300 MHz, CDCl3): d
CH3), 2.30 (s, 3H, CH3); 3.73 (s, 2H, H5), 5.04 (dd, J ¼ 5.1 Hz, 5.7 3.75 (s, 2H, H5); 5.04 (dd, J ¼ 4.8 Hz, 5.6 Hz, 1H, H1); 5.16 (ddd,
Hz, 1H, H1); 5.14 (ddd, J ¼ 1.5 Hz, 10.2 Hz, 17.1 Hz, 2H, H7); 5.63 J ¼ 1.2 Hz, 10.8 Hz, 17.1 Hz, H7); 5.56 (dd, J ¼ 4.8 Hz, 7.8 Hz, 1H,
(dd, J ¼ 5.1 Hz, 7.8 Hz, 1H, H2); 5.79–5.88 (m, 1H, H6); 6.24 (dd, J H2); 5.72–5.81 (m, 1H, H6); 6.25 (dd, J ¼ 7.5 Hz, 16.1 Hz, 1H, H3);
¼ 6.9 Hz, 16.2 Hz, 1H, H3); 6.53 (d, J ¼ 8.9 Hz, 1H, ArH); 6.72–6.77 6.51 (d, J ¼ 8.9 Hz, 1H, ArH); 6.74 (d, J ¼ 16.0 Hz, 1H, H4); 7.15–
(m, 2H, H4 + 1ArH), 7.04–7.38 (m, 12H, 10ArH + NH + OH, 7.35 (m, 13H, 11ArH + NH + OH, exchangeable with D2O), 13C
exchangeable with D2O); 13C NMR (CDCl3, 75 MHz): 20.1, 20.9, NMR (CDCl3, 75 MHz): 45.4, 59.7, 59.9, 76.7, 112.2, 113.8, 114.8,
45.5, 59.7, 59.8, 76.7, 112.2, 113.7, 115.8, 117.2, 122.3, 124.0, 115.8, 117.3, 122.3, 124.1, 126.6, 127.8, 128.2, 128.5, 129.6,
126.7, 127.7, 128.4, 128.6, 129.6, 134.2, 134.2, 134.9, 134.9, 135.6, 134.3, 134.8, 134.8, 135.6, 135.8, 147.4, 163.7, 169.8; HRMS
135.9, 147.4, 163.7, 169.7; HRMS calculated for C30H29N3O4 [M]+ calculated for C28H23Cl2N3O4 [M]+ 535.1066, found 535.1061;
495.2158, found 495.2152; analysis: calculated for C30H29N3O4: analysis: calculated for C28H23Cl2N3O4: C, 62.69; H, 4.32; N,
C, 72.71, H, 5.90; N, 8.48, found: C, 72.65, H, 5.86, N, 8.55%.
7.83, found: C, 62.77; H, 4.26; N, 7.88%.
1-{2-[1-(4-Chloro-phenyl)-2-oxo-4-styryl-azetidin-3-ylamino]-
5-Chloro-1-[2-(1-cyclohexyl-2-oxo-4-styryl-azetidin-3-ylamino)-
3-hydroxy-propyl}-5-methyl-1H-indole-2,3-dione
(7e).
Pale 3-hydroxy-propyl]-1H-indole-2,3-dione (7i). White solid, yield
1
1
yellow, yield 83%, m.p. 204–207 ꢀC, H NMR (300 MHz, CDCl3): 84%, m.p. 170–172 C, H NMR (300 MHz, CDCl3): d 1.54–1.62
d 2.29 (s, 3H, CH3); 3.74 (s, 2H, H5); 5.01 (dd, J ¼ 4.8 Hz, 5.7 Hz, (m, 10H, cyclohexyl); 3.44–3.55 (m, 1H, cyclohexyl); 3.66 (s, 2H,
1H, H1); 5.16 (ddd, J ¼ 1.2 Hz, 10.8 Hz, 17.1 Hz); 5.59 (dd, J ¼ 4.8 H5); 4.56 (dd, J ¼ 4.8 Hz, 8.7 Hz, 1H, H1); 5.08 (ddd, J ¼ 1.5 Hz,
Hz, 7.5 Hz, 1H, H2); 5.72–5.81 (m, 1H, H6); 6.23 (dd, J ¼ 7.5 Hz, 10.2 Hz, 17.1 Hz, H7); 5.38 (dd, J ¼ 4.8 Hz, 7.5 Hz, 1H, H2); 5.67–
16.1 Hz, 1H, H3); 6.51 (d, J ¼ 8.8 Hz, 1H, ArH); 6.74 (d, J ¼ 16.0 5.81 (m, 1H, H6); 6.16 (dd, J ¼ 7.5 Hz, 15.9 Hz, 1H, H3); 6.49 (d, J
Hz, 1H, H4); 7.15–7.39 (m, 13H, 11ArH + NH + OH, exchangeable ¼ 9.0 Hz, 1H, ArH); 6.71 (d, J ¼ 15.9 Hz, 1H, H4); 7.15 (dd, J ¼ 2.2
with D2O), 13C NMR (CDCl3, 75 MHz): 20.9, 45.4, 59.4, 59.8, 76.8, Hz, 8.8 Hz, 1H, ArH); 7.21–7.45 (m, 7H, 6ArH + NH), 13C NMR
112.3, 113.7, 115.9, 117.2, 122.3, 124.1, 126.7, 127.8, 128.4, (CDCl3, 75 MHz): 24.8, 25.1, 30.4, 31.8, 45.6, 52.6, 59.3, 59.8,
128.7, 129.7, 134.2, 134.3, 134.9, 134.9, 135.5, 135.8, 147.4, 112.7, 112.8, 113.9, 114.1, 115.8, 120.1, 120.3, 124.6, 126.7,
163.8, 169.7; HRMS calculated for C29H26ClN3O4 [M]+ 515.1612, 128.3, 128.7, 134.7, 135.9, 146.4, 166.5, 166.9; HRMS calculated
found 515.1618; analysis: calculated for C29H26ClN3O4: C, 67.50; for C28H30ClN3O4 [M]+ 507.1925, found 507.1921; analysis:
ꢀ
H, 5.08; N, 8.14, found C, 67.43; H, 5.17; N, 8.23%.
calculated for C28H30ClN3O4: C, 66.20; H, 5.95; N, 8.27, found:
1-[2-(1-Cyclohexyl-2-oxo-4-styryl-azetidin-3-ylamino)-3-hydroxy- C, 66.28; H, 5.89; N, 8.34%.
propyl]-5-methyl-1H-indole-2,3-dione (7f). White solid, yield
5-Fluoro-1-[3-hydroxy-2-(2-oxo-4-styryl-1-p-tolyl-azetidin-3-yl-
1
ꢀ
81%, m.p. 140–145 C, H NMR (300 MHz, CDCl3): d 1.55–1.64 amino)-propyl]-1H-indole-2,3-dione (7j). White solid, yield 77%,
(m, 10H, cyclohexyl); 2.30 (s, 1H, CH3); 3.46–3.58 (m, 1H, cyclo- m.p. 186–188 ꢀC, 1H NMR (300 MHz, CDCl3): d 2.30 (s, 3H, CH3);
hexyl); 3.68 (s, 2H, H5); 4.54 (dd, J ¼ 4.8 Hz, 8.7 Hz, 1H, H1); 5.10 3.74 (s, 2H, H5); 5.00 (dd, J ¼ 4.8 Hz, 5.7 Hz, 1H, H1); 5.18 (ddd, J
(ddd, J ¼ 1.5 Hz, 10.2 Hz, 17.1 Hz, H7); 5.35 (dd, J ¼ 4.8 Hz, 7.5 Hz, ¼ 1.2 Hz, 10.5 Hz, 17.1 Hz, H7); 5.59 (dd, J ¼ 4.9 Hz, 7.8 Hz, 1H,
1H, H2); 5.62–5.78 (m, 1H, H6); 6.15 (dd, J ¼ 7.5 Hz, 15.9 Hz, 1H, H2); 5.74–5.83 (m, 1H, H6); 6.27 (dd, J ¼ 7.5 Hz, 16.2 Hz, 1H, H3);
H3); 6.50 (d, J ¼ 9.0 Hz, 1H, ArH); 6.70 (d, J ¼ 15.9 Hz, 1H, H4); 7.14 6.56 (d, J ¼ 8.9 Hz, 1H, ArH); 6.77 (d, J ¼ 16.1 Hz, 1H, H4); 7.18–
(dd, J ¼ 2.2 Hz, 8.8 Hz, 1H, ArH); 7.22–7.50 (m, 8H, 6ArH + NH + 7.40 (m, 13H, 11ArH + NH + OH, exchangeable with D2O), 13C
OH, exchangeable with D2O), 13C NMR (CDCl3, 75 MHz): 20.9, NMR (CDCl3, 75 MHz): 20.9, 45.2, 59.7, 60.0, 76.8, 111.9, 113.5,
24.9, 25.0, 30.4, 31.9, 45.6, 52.5, 59.2, 59.7, 112.8, 113.1, 113.9, 114.9, 115.8, 117.3, 122.5, 126.6, 127.5, 128.4, 128.7, 129.5,
114.1, 115.8, 120.1, 120.4, 124.7, 126.7, 128.2, 128.8, 134.7, 135.8, 133.3, 134.1, 134.5, 134.9, 135.6, 136.2, 149.6, 163.9, 169.8;
146.3, 166.4, 168.8; HRMS calculated for C29H33N3O4 [M]+ HRMS calculated for
C
29H26FN3O4 [M]+ 499.1907, found
487.2471, found 487.2476; analysis: calculated for C29H33N3O4: 499.1903; analysis: calculated for C29H26FN3O4: C, 69.73; H,
C, 71.44; H, 6.82; N, 8.62, found: C, 71.53; H, 6.75; N, 8.72%.
5.25; N, 8.41, found: C, 69.81; H, 5.32; N, 8.52%.
5-Chloro-1-[3-hydroxy-2-(2-oxo-4-styryl-1-p-tolyl-azetidine-3-yl-
1-{2-[1-(4-Chloro-phenyl)-2-oxo-4-styryl-azetidin-3-ylamino]-
amino)-propyl]-1H-indole-2,3-dione (7g). White solid, yield 75%, 3-hydroxy-propyl}-5-uoro-1H-indole-2,3-dione (7k). White
m.p. 188–194 ꢀC, 1H NMR (300 MHz, CDCl3):d2.30(s, 3H, CH3); 3.72 solid, yield 75%, m.p. 187–189 ꢀC, 1H NMR (300 MHz, CDCl3): d
(s, 2H, H5); 5.02 (dd, J¼ 4.8 Hz, 5.7 Hz, 1H, H1); 5.14 (ddd, J¼ 1.2 Hz, 3.73 (s, 2H, H5); 5.03 (dd, J ¼ 4.8 Hz, 5.7 Hz, 1H, H1); 5.15 (ddd, J
10.8 Hz, 17.1 Hz, H7); 5.57 (dd, J ¼ 4.9 Hz, 7.8 Hz, 1H, H2); 5.76–5.85 ¼ 1.2 Hz, 10.6 Hz, 17.1 Hz, H7); 5.55 (dd, J ¼ 4.8 Hz, 7.5 Hz, 1H,
(m, 1H, H6); 6.21 (dd, J ¼ 7.5 Hz, 16.5 Hz, 1H, H3); 6.53 (d, J ¼ 8.9 Hz, H2); 5.76–5.85 (m, 1H, H6); 6.22 (dd,cyclohexyl); 3.44–3.54 (m,
1H, ArH); 6.76 (d, J ¼ 16.3 Hz, 1H, H4); 7.10–7.38 (m, 13H, 11ArH + 1H, cyclohexyl); 3.66 (s, 2H, H5); 4.57 (dd, J ¼ 4.8 Hz, 8.7 Hz, 1H,
NH + OH, exchangeable with D2O), 13C NMR (CDCl3, 75 MHz): 20.9, H1); 5.09 (ddd, J ¼ 1.2 Hz, 10.2 Hz, 15.9 Hz, 2H, H7); 5.39 (dd, J ¼
45.2, 59.8, 60.2, 76.7, 111.9, 113.8, 114.4, 115.8, 117.3, 122.4, 124.0, 4.9 Hz, 7.5 Hz, 1H, H2); 5.71–5.83 (m, 1H, H6); 6.16 (dd, J ¼ 9.0
126.4, 127.8, 128.3, 128.7, 129.6, 134.3, 134.9, 135.0, 135.6, 136.1, Hz, 15.9 Hz, 1H, H3); 6.50 (dd, J ¼ 4.8 Hz, 9.3 Hz, 1H, ArH); 6.69
147.5, 163.7, 169.8; HRMS calculated for C29H26ClN3O4 [M]+ (d, J ¼ 15.9 Hz, 1H, H4); 6.84 (dd, J ¼ 3.0 Hz, 9.0 Hz, 1H, ArH);
515.1612, found 515.1617; analysis: calculated for C29H26ClN3O4: C, 7.20–7.41 (m, 8H, 6ArH + NH + OH, exchangeable with D2O), 13
C
67.50; H, 5.08; N, 8.14, found: C, 67.59; H, 5.15; N, 8.07%.
NMR (CDCl3, 75 MHz): 24.9, 25.1, 30.5, 31.9, 45.6, 52.4, 59.2,
Med. Chem. Commun.
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59.8, 112.8, 112.9, 113.8, 114.1, 115.9, 120.0, 120.3, 124.6, 126.7, 13 V. S. Velezheva, P. J. Brennan, V. Y. Marshakov, D. V. Gusev,
128.2, 128.6, 134.7, 135.8, 146.3, 166.3, 168.8; HRMS calculated
for C28H30FN3O4 [M]+ 491.2220, found 491.2227; analysis:
calculated for C28H30FN3O4: C, 68.42; H, 6.15; N, 8.55, found: C,
68.52; H, 6.23; N, 8.47%.
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Acknowledgements
Financial Assistance from University Grants Commission (UGC)
under Rajiv Gandhi Fellowship for SC/ST students (Nisha) is
gratefully acknowledged. Financial assistance from Board of
Research in Nuclear Sciences under DAE Research Award for
Young Scientist Scheme (VK) is gratefully acknowledged.
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