Synthetic piperine amide analogs with antimycobacterial activity

Article abstract of DOI:10.1111/cbdd.13140

Piperine amide analogs are synthesized by replacement of the piperidine moiety with different types of cyclic amines, including adamantyl and monoterpene-derived fragments. The compounds are screened for activity against Mycobacterium tuberculosis H37Rv. The most potent compounds are the 1-adamantyl and the monoterpene-derived hybrids, which combine nanomolar antimycobacterial activity with low cytotoxicity against human cells. The presence of quaternary carbon atom as main structural requirement for anti-TB activity is pointed out by a QSAR study. The most promising compound is the (+)-isopinocampheylamine-derived amide which is characterized with selectivity index of 1387.8.

Full text of DOI:10.1111/cbdd.13140

DR GEORGI STAVRAKOV (Orcid ID : 0000-0002-8893-0346)
Article type : Research Article
Synthetic piperine amide analogues with antimycobacterial activity
Irena Philipovaa, Violeta Valchevab, Rositsa Mihaylovac, Mina Mateevac, Irini Doytchinovac, Georgi
Stavrakova,c,*
aInstitute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Acad.
Bonchev 9, Sofia 1113, Bulgaria
bInstitute of Microbiology, Bulgarian Academy of Sciences, Acad. Bonchev 26, Sofia 1113, Bulgaria
cFaculty of Pharmacy, Medical University of Sofia, str. Dunav 2, Sofia 1000, Bulgaria
*Corresponding author. Faculty of Pharmacy, str. Dunav 2, Sofia, BG-1000; tel +359 2 9236516 E-mail
address: gstavrakov@hotmail.com
Abstract
Piperine amide analogues are synthesized by replacement of the piperidine moiety with different
types of cyclic amines, including adamantyl and monoterpene derived fragments. The compounds
are screened for activity against Mycobacterium tuberculosis H37Rv. The most potent compounds
are the 1-adamantyl and the monoterpene derived hybrids, which combine nanomolar
antimycobacterial activity with low cytotoxicity against human cells. The presence of quaternary
carbon atom as main structural requirement for anti-TB activity is pointed out by a QSAR study. The
most promising compound is the (+)-isopinocampheylamine derived amide which is characterized
with selectivity index of 1387.8.
Keywords: piperine, terpene, camphane, tuberculosis
Introduction
Natural compounds have served the humanity as a valuable source of drugs for centuries. Their
diverse biological activities are very much ascribed to their structural complexity resulting in specific
interactions with complementary three dimensional molecular targets.1,2 However, due to some
well-known disadvantages of natural chemical components (i.e. low and often highly variable yields,
unfavourable physicochemical properties, pleiotropic biological effects, innate or induced drug-
resistance, etc.), research efforts nowadays are primarily focused on the development of synthetic
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analogues with improved pharmacological properties.3 Since the treatment of multidrug-resistant
tuberculosis (MDR-TB) is still problematic,4 the use of natural compounds as templates in drug
design remains a challenging task.5-7 A few natural products have been previously found to exhibit
potent antimycobacterial activity against isolates of MDR-TB, however they exhibit cytotoxic activity
towards normal cell lines.8,9
Monoterpenes and their derivatives play an important role in the synthesis of new biologically active
compounds including drugs.10 Monoterpenoid-based agent SQ 109 (Fig. 1), combining 2-
adamantylamine and geranylamine, is a novel anti-TB drug candidate with a unique multimodal
mechanism of action.11 Recently we have reported a series of studies focused on the synthesis of
anti-tuberculosis agents containing the bicyclo[2.2.1]heptane framework fragment.12-14 The latter is
combined with a number of pharmacophore groups resulting in improved physiological and
pharmacological properties. As evidenced, some of our monoterpene derivatives exhibit
pronounced in vitro antimycobacterial activity towards the chemosensitive MTB strain H37Rv and a
multi drug-resistant strain MDR 43 while showing low cytotoxicity against the human embryonal
kidney cell line 293T. Among them the product of condensation of 3-exo-aminoisoborneol with
cinnamic acid has proved as one of the leading structures (Fig. 1).12
Piperine (Fig. 1) is a naturally occurring alkaloid, isolated from black pepper (Piper nigrum L.), that is
well known for its wide spectra of pharmacological activities.15 Currently, piperine is used as a
promising scaffold for the development of prospective bioactive derivatives in drug discovery
programs.16 In addition, piperine analogues are reported to enhance the bioavailability of various
drugs through efflux pump inhibition (MDR – multidrug resistance protein or P-glycoprotein).17-20
Overexpression of P-gp in MDR-TB strains has been found to promote drug tolerance towards
numerous anti-TB drugs, i.e. rifampicin, isoniazid, moxifloxacin and bedaquiline.21,22 As expected,
recently published studies further reported that piperine synergistically affects the therapeutic
outcome of rifampicin treatment.23 Moreover, piperine analogs are found to exhibit antibacterial
activity,18,19 and piperine itself reveals protective efficacy against Mycobacterium tuberculosis.23
Inspired by the broad range of applications of piperine, the present study aims the synthesis,
evaluation of antimycobacterial activity and cytotoxicity of piperine amide analogues. The focus is
set on the replacement of the piperidine moiety with different types of cyclic amines, including
adamantyl and monoterpene derived fragments.
Methods and Materials
1. Chemistry
Reagents were commercial grade and used without further purification. Thin layer
chromatography (TLC) was performed on aluminum sheets pre-coated with Merck Kieselgel 60 F254
0.25 mm (Merck). Flash column chromatography was carried out using Silica Gel 60 230-400 mesh
(Fluka). Commercially available solvents were used for reactions, TLC and column chromatography.
Melting points were determined in a capillary tube on SRS MPA100 OptiMelt (Sunnyvale, CA, USA)
automated melting point system (uncorrected). Optical rotation ((α)D20) were measured on JASCO P-
1
2010 polarimeter. The NMR spectra were recorded on a Bruker Avance II+ 600 (600.13 MHz for H
and 150.92 MHz for 13C NMR) spectrometer with TMS as internal standards for chemical shifts (δ,
1
ppm). H and 13C NMR data are reported as follows: chemical shift, multiplicity (s = singlet, d =
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doublet, t = triplet, q = quartet, br = broad, m = multiplet), coupling constants (Hz), integration,
1
13
identification. The assignment of the H and C NMR spectra was made on the basis of COSY and
HSQC experiments. LC–MS analyses were performed using a Q Exactive Plus Orbitrap Mass
spectrometer (Thermo Fisher Scientific, Bremen, Germany), equipped with an electrospray (ESI)
probe. The spectra were recorded on a positive mode using a MS Full Scan mode.
1.1. Procedure for the preparation of the (2E,4E)-piperic acid
Piperine (2.00g, 7 mmol) was added to 20% methanolic solution of potassium hydroxide (100 ml)
and was refluxed for 24 h. Reaction mixture was concentrated under reduced pressure. The residue
was dissolve in water and acidified with 6N HCl to pH< 1. The yellow precipitate was filtered and
washed with water and CH2Cl2. Solid product was recrystallized from methanol to give 1.406 g (92%)
piperic acid.
1.2. General Procedure for the preparation of the Amides (2a-i).
To a solution of (2E,4E)-piperinic acid (1 equiv) in tetrahydrofuran was added N-(3-
(dimethylamino)propyl)-N-ethylcarbodiimide (1.1 equiv) and 1-Hydroxybenzotriazole (1.1 equiv).
After half an hour the corresponding amine (1.1 equiv) was added to the mixture and stirring was
continued at room temperature until the starting material was completely consumed (TLC). The
mixture was quenched with water, extracted with CH2Cl2, washed with 2M HCl, sat. aq. NaHCO3 and
brine. The combined organic extracts were dried over Na2SO4 and the solvent was evaporated under
reduced pressure. The amides were purified by flash-column chromatography. Detailed analytical
1
13
data for piperine amide analogues 2b-i and copies of H and C NMR spectra of the most active
compounds are given in Supporting Information.
2. Antimycobacterial activity
The in vitro antimycobacterial activity of all compounds against reference strain Mycobacterium
tuberculosis H37Rv was evaluated using the resazurin microtiter assay (REMA) described by Martin
et al.24 Briefly, a 96-well microplate was used and in each well 100 μl suspension of Middlebrook 7H9
broth and tested compound (in DMSO) at different concentrations (5 μg/ml, 2 μg/ml, 1 μg/ml, 0.5
μg/ml, 0.25 μg/ml 0.125 μg/ml and 0.0625 μg/ml) were added. Additionally, in each well 100 μl of
the reference strain M. tuberculosis H37Rv (103-105 cells/ml) were added. Ethambutol (EMB) and
isoniazid (INH) were used as controls. After 7 days of incubation at 37 °C, bacterial growth was
measured by adding to each well the redox indicator – 0.1% of resasurin. The microplate was re-
incubated overnight at 37 °C; a change in color of the indicator resulting from a chemical
transformation of the reagent indicated bacterial growth in the presence of the tested compound at
different concentrations. A change from blue to pink indicated reduction of resazurin and therefore
bacterial growth. The minimum inhibitory concentration (MIC) was defined as the lowest drug
concentration that prevented this color change.
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3. Cytotoxicity
The human embryonal kidney cell line 293T cells were obtained from the German Collection of
Microorganisms and Cell Cultures. Cells were kept in controlled environment e RPMI-1640 medium,
supplemented with 10% heat-inactivated fetal calf serum and 2mM L-glutamine, at 37 ºC in a
‘Heraeus’ incubator with 5% CO2 humidified atmosphere.
The cytotoxicity of the newly synthesized compounds was assessed using the MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]-dye reduction assay as described by
Mossman with some modifications.25,26 In brief, exponentially growing cells were seeded in 96-well
microplates (100 µl/well) at a density of 3.5 105 cell/ml and allowed to grow for 24 h prior the
exposure to the studied compounds. Stock solutions of the tested compounds were freshly prepared
in DMSO and thereafter were subset to serial dilutions with growth medium in order to obtain the
desired final concentrations. At the final dilutions the solvent concentration never exceeded 0.5%.
Cells were exposed to the tested agents for 72 h, whereby for each concentration a set of at least 8
separate wells was used. After the exposure period MTT solution (10 mg/ml in phosphate-buffered
saline) aliquots (100 µl/well) were added to each well. The plates were further incubated for 4 h at
37 ºC and the MTT-formazan crystals formed were dissolved through addition of 110 ml of 5%
HCOOH in 2-propanol. The MTT-formazan absorption of the samples was measured by a multimode
microplate reader DTX 880 (Beckman Coulter) at 580 nm. Cell survival fractions were calculated as
percentage of the untreated control. The experimental data were fitted to sigmoidal concentration-
response curves and the corresponding IC50 values (concentrations causing 50% reduction of cellular
survival vs. the untreated control) via non-linear regression (GraphPad Prizm software for PC).
4. Quantitative structure-antimycobacterial activity relationships (QSAR)
The QSAR study was performed by MDL QSAR version 2.2 (MDL Information Systems, Inc., San
Leandro, USA). The chemical structure of the studied compounds was described by 178 molecular
descriptors including molecular connectivity indices, shape indices, electrotopological state indices,
molecular properties and 3D molecular descriptors. Genetic algorithm was used to select the most
relevant to the anti-TB activity descriptors.27 The final model was derived by multiple linear
regression and assessed by r2, SEE, F-ratio, p-value and q2.
Results and Discussion
Chemistry
A set of piperine amide analogues 2a-i was synthesized by condensation of selected amines 1a-i with
piperic acid (Scheme 1). The latter was prepared quantitatively by alkaline hydrolysis of piperine by
refluxing in 20% methanolic solution of potassium hydroxide for 24 h. The amide formation was
optimized for cyclohexylamine, and the optimal results were achieved when using N-(3-
dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole
hydrate (HOBT) as activating agents and THF as solvent. The proper choice of solvent was crucial due
to the low solubility of piperic acid.
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The synthesis of 1-E,E-piperinoyl-cyclohexylamine 2a has already been reported via initial activation
of the piperic acid with methane sulfonyl chloride, followed by reaction with the amine.28 However,
the classical peptide synthetic methodology was preferred due to mild conditions and easy product
isolation. The same protocol was applied for the preparation of all other piperine amide analogues
2b-i. The products were obtained in very good yields and excellent purity after flash column
1
chromatography (Table 1). All compounds were identified by H NMR, 13C NMR and HRMS data. The
spectral analyses were in accordance with the assigned structures (Supporting Information).
The choice of the cyclic amines was directed by one main factor: proof of concept for the role of the
terpene fragment for the antimycobacterial activity. Thus, the cyclohexylamine 1a was chosen as a
reference being a simple cyclic amine. The adamantylamines 1b and 1c targeted resemblance with
SQ 109. A non-terpene bicyclo[2.2.1]heptane framework was introduced by exo-2-aminonorbornane
1d. The monoterpenes were represented by the (+)-camphor-derived isobornylamine 1e,
bornylamine 1f, and 3-exo-aminoisoborneol 1g.29-31 Finally, (–)- and (+)-isopinocampheylamines, 1h
and 1i, were selected as an extension of the terpene series.
Antimycobacterial activity
The synthesized compounds were evaluated for their in vitro activity against Mycobacterium
tuberculosis H37Rv using the resazurin microtiter assay (REMA).32 Ethambutol and isoniazid were
used as reference antituberculous agents. The N-cyclohexyl and N-norbornyl based amides, 2a and
2d, revealed activities exceeding the one of ethambutol and comparable with isoniazid. The same
was relevant for the adamantyl derivative 2c, while 2b reached MIC of 0.36 M. All (+)-camphor
derived amides displayed prominent MICs, outlining the 3-exo-aminoisoborneol derived amide 2g
with MIC of 0.17 M. Interesting were the results exhibited by (–)- and (+)-isopinocampheyl amides
2h and 2i, in which the (+)-enantiomer revealed much higher activity with MIC of 0.18 M (Table 1).
Table 1. Structures, antimycobacterial activity and cytotoxicity of piperine amide analogues 2a-i.
b
Compound
Structure,
yield
MTB MICa
Cytotoxicity IC50
SIc IC50/MIC
(M)
(M)
2a
1.67
0.36
1.42
1.60
ND
209.3 ± 15.4
ND
ND
2b
2c
2d
581.4
ND
ND
ND
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2e
2f
0.35
0.35
0.17
198.9 ± 16.4
146.5 ± 13.1
33.1 ± 2.3
568.3
418.6
194.7
2g
2h
2i
1.14
ND
ND
0.18
249.8 ± 19.5
1387.8
EMB.2HCld
Isoniazidd
7.22
1.82
aAnti-mycobacterial activity towards reference strain of Mycobacterium tuberculosis H37Rv; bIn vitro
c
cytotoxicity towards human embryonal kidney cell line 293T, ND – Not Determined; Selectivity
d
index; EMB.2HCl – ethambutol dihydrochloride (reference compound) and Isoniazid (reference
compound).
Cytotoxicity
In order to examine the selectivity of the antiproliferative effects, the cytotoxicity of the most active
compounds was assessed against the human embryonal kidney cell line 293T, after 72 h exposure.
Evident from the IC50 values, the compounds were generally of low cytotoxicity against the human
cells. The tested compounds induced 50% inhibition of cellular viability at concentrations exceeding
100 M; with the only exception of 2g, which exerted moderate cytotoxicity with the IC50 value of
33.1 M. Thus, the compounds combine low cytotoxicities with high antimycobacterial activities,
best represented by their selectivity indexes (SI > 194). The results are extremely in favour of (+)-
isopinocampheyl amide 2i with SI 1387.8 (Table 1).
Quantitative structure-antimycobacterial activity relationships (QSAR)
Quantitative structure-antimycobacterial activity relationship (QSAR) model was derived to guide
further lead optimization. The most relevant descriptors for the antimycobacterial activity of the
tested compounds were selected by genetic algorithm. The final model is derived by linear
regression and is given below:
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pMIC = 0.295(±0.156) SssssC_acnt + 0.393(±0.496) ABSQ + 4.425
n = 9 r2 = 0.644 SEE = 0.288 F2,6 = 5.425 p-value = 0.045 q2 = 0.504,
where pMIC is the negative logarithm of MIC, descriptor SssssC_acnt accounts for all quaternary
carbon atoms in the molecule, ABSQ shows the sum of absolute values of the charges on each atom
of molecule, r2 is the explained variance, SEE – standard error of estimate, q2 – cross-validated
correlation coefficient.
The descriptor SssssC_acnt takes values 0, 1 or 2 for the tested compounds. The positive coefficient
means that compounds with more quaternary carbon atoms have higher antimycobacterial activity.
Indeed, the most active compounds 2f, 2e and 2g have 2 quaternary carbons. The descriptor ABSQ
ranges from 3.574 to 4.521. The most active compound 2g has the highest value of ABSQ because of
the extra OH-group. Obviously, this group is very important for the anti-TB activity as it was found
before.12
Conclusion
In conclusion, a series of nine piperine derived amide analogues was synthesized and screened for
antimycobacterial activity against Mycobacterium tuberculosis H37Rv. All compounds displayed
activities better than ethambutol and comparable or exceeding the one of isoniazid. The
monoterpene derived hybrids revealed prominent activities. The most active were the piperine
amides derived from 3-exo-aminoisoborneol and (+)-isopinocampheylamine with MICs of 0.17 and
0.18 M respectively. The most potent compounds proved to exert low level of cytotoxic activity
against a human embryonal kidney cell line 293T. The (+)-isopinocampheyl amide outlined with
selectivity index of 1387.8. The QSAR study pointed out the presence of quaternary carbon atom as
main structural requirement for anti-TB activity. This additionally supported the role of the terpene
compact lipophilic moiety for the anti-mycobacterial activity.
Acknowledgment
We acknowledge the Medical Science Council and National Science Fund for financial supports
(Grants 8-S/2015 and DN03-9/2016). Authors are thankful to Prof. P. Nedyalkov for the HRMS
analysis and to Prof. G. Momekov for the fruitful discussions.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
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Figure legends
Fig. 1. Structures of SQ 109, 3-exo-Aminoisoborneol derivative, and Piperine.
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Scheme 1. Synthesis of piperine amide analogues.
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