Click-chemistry approach to synthesis of functionalized isatin-ferrocenes and their biological evaluation against the human pathogen Trichomonas vaginalis

Article abstract of DOI:10.1016/j.jorganchem.2019.05.025

Copper-promoted azide-alkyne cycloadditions were attempted to synthesize a series of variedly functionalized 1H-1,2,3-triazole-linked isatin-ferrocene, ferrocenylmethoxy-isatin and isatin-ferrocenyl-chalcone conjugates. The synthesized scaffolds were assayed for their inhibitory activity against T. vaginalis as well as several common normal human flora bacteria. The observed inhibitory activities against T. vaginalis and undetectable inhibition of microflora bacteria suggest that these compounds may be specific against trichomonad protozoa and could serve as a new scaffold for synthesis of novel compounds against this important human pathogen.

Full text of DOI:10.1016/j.jorganchem.2019.05.025

Accepted Manuscript
Click-chemistry approach to functionalized isatin-ferrocenes and their biological
evaluation against the human pathogen Trichomonas vaginalis
Amandeep Singh, David Zhang, Christina C. Tam, Luisa W. Cheng, Kirkwood M.
Land, Vipan Kumar
PII:
S0022-328X(19)30217-7
DOI:
https://doi.org/10.1016/j.jorganchem.2019.05.025
JOM 20807
Reference:
To appear in: Journal of Organometallic Chemistry
Received Date: 19 April 2019
Revised Date: 24 May 2019
Accepted Date: 25 May 2019
Please cite this article as: A. Singh, D. Zhang, C.C. Tam, L.W. Cheng, K.M. Land, V. Kumar, Click-
chemistry approach to functionalized isatin-ferrocenes and their biological evaluation against the
human pathogen Trichomonas vaginalis, Journal of Organometallic Chemistry (2019), doi: https://
doi.org/10.1016/j.jorganchem.2019.05.025.
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ACCEPTED MANUSCRIPT
Copper-promoted azide-alkyne cycloadditions were attempted to synthesize a series of
variedly functionalized 1H-1,2,3-triazole-linked isatin-ferrocene, ferrocenylmethoxy-isatin
and isatin-ferrocenyl-chalcone conjugates. The synthesized scaffolds were assayed for their
inhibitory activity against T. vaginalis as well as several common normal human flora
bacteria. The observed inhibitory activities against T. vaginalis and undetectable inhibition of
microflora bacteria suggest that these compounds may be specific against trichomonad
protozoa and could serve as a new scaffold for synthesis of novel compounds against this
important human pathogen

ACCEPTED MANUSCRIPT
Click-chemistry approach to functionalized isatin-ferrocenes and their
biological evaluation against the human pathogen Trichomonas vaginalis
Amandeep Singha, David Zhang b, Christina C. Tam c, Luisa W. Cheng c, Kirkwood M Land,b
Vipan Kumara*
a Department of Chemistry, Guru Nanak Dev University, Amritsar-143005, Punjab, India
b Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, USA
c Agricultural Research Service, United States Department of Agriculture, Albany, CA 94170, USA
Abstract: Copper-promoted azide-alkyne cycloadditions were attempted to synthesize a series of variedly functionalized
1H-1,2,3-triazole-linked isatin-ferrocene, ferrocenylmethoxy-isatin and isatin-ferrocenyl-chalcone conjugates. The
synthesized scaffolds were assayed for their inhibitory activity against T. vaginalis as well as several common normal
human flora bacteria. The observed inhibitory activities against T. vaginalis and undetectable inhibition of microflora
bacteria suggest that these compounds may be specific against trichomonad protozoa and could serve as a new scaffold for
synthesis of novel compounds against this important human pathogen.
Key
Words:
isatin-ferrocene
conjugates,
ferrocene
methoxy-isatin,
isatin-
ferrocenylchalcones, click chemistry, anti-trichomonad evaluation
Introduction
Trichomoniasis is a sexually transmitted disease caused by the protozoan parasite T. vaginalis
and responsible for 156 million cases world-wide in 2017.1 The standard treatment for
trichomoniasis involves 5-nitro-imidazole based drugs, including the most common
metronidazole (MTZ). There has been an increased incidence of clinical cases of
trichomoniasis resistant to metronidazole.2 These recalcitrant cases are often treated with
even higher dosages of the MTZ, which in turn may lead to higher levels of resistance and an
increase in patient side effects.3 Undoubtedly, new and alternative therapies are needed for
trichomoniasis.
Isatin is an indole chemical moiety containing both keto and lactam groups that has
been used extensively in the pharmacological and medicinal chemistry fields.4-5 Isatin
derivatives are known to possess anti-inflammatory, anti-fungal, anti-tubercular, anti-cancer,
anti-HIV, anti-viral, and anti-bacterial properties.6 These biological properties validated
isatin as a starting point for the design and preparation biological relevant chemical
libraries.7-8 SU11248 (Sutent), a 5-fluoro-3-substituted isatin derivative, was approved by the
FDA in 2006 for the treatment of advanced renal carcinoma and gastrointestinal stromal
tumors.9 Inhibitory activity of isatin-chromone against α-glucosidase has been reported by
Wang et al with the most potent compound showed IC50=3.8 µM.10 Isatin-Moxifloxacin
hybrids were evaluated against MTB H37Rv and MDR-TB strains of M. tuberculosis with the
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most potent compound demonstrating IC50=0.05 µM, two to seven times more active than
standard drugs moxifloxacin and rifampicin.11
In the past decade, the application of organometallic chemistry in the field of biology
and medicine has led to a new area called bioorganometallic chemistry.12 Ferrocene is the
most attractive among metallocenes because of its stability, biological activity, and non-
toxicity.13 Ferrocene is an ideal platform for the synthesis of new biologically active
compounds. Replacement of purely organic component with ferrocene has shown to improve
the biological potency of the target compounds, as demonstrated by ferrocenyl-conjugates of
commercial anti-estrogen tamoxifen and anti-androgen nilutamide.14 Ferroquine (FQ) is the
most exemplary contribution of ferrocene to the improvement of the anti-plasmodial potential
of chloroquine and is under clinical trials.15 In addition, the high lipophilicity of ferrocene
may allow interaction with or permeation of biological membranes, which is essential for
achieving their desired biological activities.16
Recent published studies from our laboratories describe the synthesis of isatin-based
scaffold viz. mono and bis-isatin, isatin-ferrocene, isatin ferrocenyl chalcones, and isatin
metronidazole conjugates along with their preliminary in vitro evaluation against T.
vaginalis.17 With an aim of discovering varied biologically relevant scaffolds, and extending
from previous studies, the present manuscript describes the synthesis of a series of
functionalized isatin-ferrocenes. These molecules were then evaluated for their growth
inhibition activities against T. vaginalis. The 1H-1,2,3-triazole core has drawn considerable
attention in the field of medicinal chemistry mainly due to its capability of forming hydrogen
bonds that eventually improves its solubility and interaction with biomolecular targets.18
Result and Discussion
The synthetic methodology for the synthesis of precursor C-5 substituted azido-hydroxy
isatin involved the initial base-promoted N-alkylation with methyl iodide or benzyl bromide
to yield 2. The treatment of 2 with trimethyl silyl iodide in dry DMF afforded spiro-isatin 3.
Epoxide ring opening of 3 with sodium azide led to the formation of 3-(azidomethyl)-3-
hydroxy-indolin-2-one 4. Cu-promoted click chemistry of 4 with ethynyl ferrocene with
subsequent purification via column chromatography using (5:95) methanol: chloroform
mixture as eluent gave the desired isatin-ferrocene conjugate 5 in good yields (Scheme 1).
Synthesis of isatin-ferrocenyl chalcones conjugates 7 involved the click reaction between 3-
(azidomethyl)-3-hydroxy-indolin-2-one 4 and ferrocenyl chalcone 6, synthesized via reported
protocols.18c The structure of conjugate 7 was characterized using spectral data and analytical
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1
evidences. For example, 7d exhibited a molecular ion peak at [M]+ 602.1662. Its H NMR
showed singlet at δ 3.10 of three protons corresponding to N-methyl substituent. The
presence of sharp singlet at δ 4.15 of 5H corresponds to the cyclopentadienyl ring of
ferrocene along with two singlets at δ 4.50 and at δ 4.61. The presence of absorption signals
at 175.0 and 188.1 ppm corresponding to the carbonyl carbon of isatin and chalcone
respectively, further confirmed the assigned structure.
Scheme1: Synthesis of 1H-1,2,3-triazole-linked Isatin-ferrocene conjugates 5a-h and 7a-h
The sequence of synthetic steps leading to ferrocene methoxy-isatin conjugates 11 is depicted
in Scheme 2. Ferrocene carboxaldehyde 8 on reaction with sodium borohydride yielded
ferrocene methanol 9 that was further treated with propargyl bromide in the presence of
sodium hydride as a base to result in O-propargylated ferrocene methanol 10. Cu-promoted
azide-alkyne cycloaddition reaction of 4 with 10 led to the isolation of crude solid, which was
purified via column chromatography using CHCl3: methanol (95:5) mixture as eluent, to
yield the desired conjugates 11 in good yields.
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Scheme 2: Synthesis of 1H-1,2,3-triazole-linked ferrocene methoxy-isatin conjugates 11a-h
The synthesized conjugates viz. 5a-h, 7a-h, 11a-h were evaluated for their growth inhibitory
activity against T. vaginalis as previously described.17a As evident from Table 1, the activity
of the synthesized was found to be depend upon the nature of the substituent at C-5 and N-1
position of the isatin ring as well as nature of linker used. A careful analysis of observed
inhibition data among isatin-ferrocene conjugates 5a-h revealed the presence of halogen
substituent (Cl, Br) for good activities as evident by 5b and 5c. The replacement of N-CH3
with N-benzyl improved the activities, in general, with conjugate 5g exhibiting approximately
94% growth inhibition.
Induction of ferrocenyl-chalcone moiety among the synthesized hybrids (7a-h) resulted in the
loss of activity especially in case of N-benzyl substituted scaffolds. The N-methylated
counterparts exhibited good inhibition with compound 7a (R=H, N-CH3) proving to be the
most potent in the series with growth inhibition of approximately 83%. Comparing the
observed activity profiles with ferrocene-methoxy isatin conjugates (11a-h) revealed an
interesting structure activity relationship with dependence on the substituent at N-1 of isatin
ring. As evident, the compounds (11a-d) with N-CH3 proved to be the ineffective in
inhibiting the growth. The replacement of N-CH3 to N-benzyl resulted in improvement in
activities with percentage inhibition in the range of 84.43-87.63 irrespective of the nature of
substituent at C-5 position of isatin ring.
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Table 1: Preliminary screening of isatin conjugates 5a-h, 7a-h, and 11a-h for growth
inhibition of the human pathogen T. vaginalis.
Entry
%
Entry
%
Entry
%
Inhibition
(+/-SE)
Inhibition
(+/- SE)
Inhibition (+/-
SE)
39.7 (1.4)
84.8 (1.5)
84.3 (2.2)
49.8 (1.2)
85.7 (1.6)
89.0 (1.2)
93.6 (2.2)
86.7 (2.1)
82.9 (2.1)
74.7 (2.2)
75.6 (1.1)
73.9 (1.8)
60.4 (2.1)
57.4 (2.2)
50.0 (1.8)
39.3 (1.2)
19.8 (1.1)
24.6 (2.1)
46.9 (0.8)
49.3 (0.4)
N.D
5a
5b
5c
5d
5e
5f
7a
7b
7c
7d
7e
7f
11a
11b
11c
11d
11e
11f
84.4 (1.3)
87.6 (2.3)
85.1 (1.1)
5g
5h
7g
7h
11g
11h
The most potent conjugates, identified from the percentage inhibition studies were evaluated
for their IC50 values (concentration that inhibited 50% parasite growth) and the results are
enlisted in Table 2. Conjugates 5f, 5g, 5h, 11f, and 11g showed IC50 values 28.9, 30.1, 28.3,
27.0, and 32.5 µM that were higher than the standard FDA-approved drug metronidazole.
Table 2: IC50 values of the most potent conjugates
Compounds
IC50 (µM)
52.5
51.6
50.4
28.9
30.1
28.3
55.2
27.0
32.5
59.1
0.7
5b
5c
5e
5f
5g
5h
7a
11f
11g
11h
MTZ
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In order to determine if the observed activities are specific to the parasite T. vaginalis
or non-specific because of their cytotoxicity, it was considered worthwhile to evaluate the
effect of these compounds on normal human flora since they occupy same niche as this
protozoal pathogen. Using the classic disc diffusion assay, we tested these compounds against
a panel of normal human flora consisting of the non-pathogenic strains, Escherichia coli K12
MG1655, Lactobacillus acidophilus, Lactobacillus rhamnosus LGG, and Lactobacillus
reuteri. No cytotoxic effects from any of these synthesized compounds were observed for any
of the normal flora.
Conclusion
A series of isatin-ferrocene conjugates linked with different functionalities were
synthesized and assayed for their inhibitory activities against T. vaginalis. As evident from
the biological data, the synthesized scaffolds are less active than standard drug
metronidazole. The most active conjugate of the series 11f with a benzyl substituent at the N-
1 position and chloro at C-5 position proved to be most active and non-cytotoxic with an IC50
value of 27 µM. This new scaffold can serve as a platform for the synthesis of new lead
compounds against this human pathogen.
Supplementary Material
Text and structures giving general experimental procedures, characterization data of (5a-h,
7a-h, 11a-h) compounds, and scanned (1H, 13C) NMR spectra for the compounds viz.. 5f, 7a,
7f, 11a, 11c, 11d.
Author Information
Corresponding Authors
*E-mail: vipan_org@yahoo.com and kland@pacific.edu
Abbreviations
SAR, Structure Activity Relationship.
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‹ Synthesis and anti trichomonad evaluation of isatin-ferrocene conjugates
‹ The presence of halogen at C-5 position proved to be essential for good activities
‹ The conjugate 11f displayed an IC50 value of 27 µM and was non-cytotoxic