Design, synthesis, and biological evaluation of 2-substituted-2,3,4,9-tetrahydrospiro-β-carboline-3-carboxylic acid derivatives as first-in-class mast cell stabilizers

Article abstract of DOI:10.1002/ardp.201800019

Mast cell degranulation plays a momentous role in myriad diseases like asthma, eczema, allergic rhinitis, and conjunctivitis as well as anaphylactic shock; hence, there is an unmet need for developing new mast cells stabilizers. The reported mast cell sta

Full text of DOI:10.1002/ardp.201800019

Received: 24 January 2018
Revised: 19 March 2018
Accepted: 19 March 2018
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DOI: 10.1002/ardp.201800019
FULL PAPER
Design, synthesis, and biological evaluation of
2-substituted-2,3,4,9-tetrahydrospiro-β-carboline-3-carboxylic
acid derivatives as first-in-class mast cell stabilizers
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Jatinder Singh
Nirmal Singh
Ramanpreet Shah
Dhandeep Singh
Amteshwar S. Jaggi
Department of Pharmaceutical Sciences and
Drug Research, Punjabi University, Patiala,
India
Abstract
Mast cell degranulation plays a momentous role in myriad diseases like asthma, eczema,
allergic rhinitis, andconjunctivitis as well as anaphylactic shock; hence, thereis an unmet
need for developing new mast cells stabilizers. The reported mast cell stabilizers have a
heterocyclic moiety and an acidic group. Furthermore, the role of tryptophan in
suppression of mast cell activation is established. Hence, we prepared constrained
analogs of tryptophan, which are derivatives of 2,3,4,9-tetrahydrospiro-β-carboline-3-
carboxylic acid, and evaluated them for ex vivo inhibition of compound 48/80-induced
mast degranulation activity. By comparing IC50 (μM) values with that of the standard
drug sodium cromoglycate (IC50 = 0.489 0.003 μM), compounds with bulky
groups like heptyl (compound 9; IC50 = 0.389 0.015 μM) and octyl (compound 10;
IC50 = 0.354 0.023 μM) were found to be of similar potency as sodium cromoglycate.
Furthermore, the polar group-containing compounds like the chloropropyl (compound
16; IC50 = 0.382 0.083 μM) and benzoyl derivative (compound 14; IC50 = 00.469
0.032 μM) were also found to be of similar potency as sodium cromoglycate. This is a
seminal study of spiro-β-carboline mast cell stabilization having a wider scope in mast
cell research; yet, the mechanism of action remains elusive.
Correspondence
Dhandeep Singh, Department of
Pharmaceutical Sciences and Drug Research,
Punjabi University, Patiala 147002, Punjab,
India.
Email: ddd300@gmail.com
Funding information
University Grant Commission, Grant number:
41-1378/2012
K E Y W O R D S
β-carboline, compound 48/80, geminal and vicinal coupling, mast cell stabilizers, Pictet–
Spengler reaction
proliferation, fixation of blood born particles, etc.[1] Any type of
abnormal stimuli spur the mast cells (otherwise innocuous mast cells)
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1
INTRODUCTION
Mast cells are an integral part of the immune system and they
have pleiotropic physiological functions in our body: host defense,
response to stress, peripheral memory bank, aging, tissue growth and
to degranulate and release legions of inflammatory mediators like
histamine, cytokines, chemokines, and prostaglandins. These media-
tors are involved in myriad diseases like allergy, asthma, mastocytosis,
anaphylaxis, urticaria, and other miscellaneous mast cell diseases.[2,3]
The mast cell degranulation could occur in response to myriad stimuli
which could be from exogenous peptides (such as bacteria derived and
Jatinder Singh and Ramanpreet Shah contributed equally to the research work and to
drafting the manuscript.
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Arch Pharm Chem Life Sci. 2018;e1800019.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.201800019

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venom-derived peptides) and endogenous peptides (such as endo-
thelin).[2] Thus, preventing mast cells degranulation or mast cell
stabilization is a prudent approach to countervail the incendiary effects
of its mediators in mast cell-related disorders like allergic disorders,
anaphylactic shock, asthma, mastocytosis, urticaria, irritable bowel
syndrome, and atopic dermatitis.[2,3] Currently marketed mast cell
stabilizers such as azelastine, chromolyn, ketotifen, nedocromil,
olopatadine, and pemirolast are used to prevent or control certain
allergic disorders (Figure 1).
effects. The dichotomine C extracted from roots of Stellaria dichotoma
showed anti-allergic effects in RBL-2H3 cells.[10] Wen et al.[11]
reported that ML120B is a more selective and potent inhibitor of
IKKβ than PS-1142 and blocks LPS, TNFα, and IL-1β in human mast
cells. β-Carboline scaffolds have been well nourished by the nature,
since these compounds have prolific therapeutic potential in myriad
diseases: vertigo and hearing loss,[12] as antibiotics precursor,[13]
proliferative disorders/cancers,[14,15] neurodegenerative disorders,[16]
anxiolytic,[17] Alzheimer disease,[18] anti-platelet aggregation activ-
ity,[19] phosphodiesterase-5 inhibitory,[20] in ischemia/reperfusion
injury,[21] benzodiazepine agonistic activity,[22] anti-convulsant,[23]
anti-psychotic activity,[24] anti-viral.[25,26] Moreover, spiro-β-
carbolines have shown anti-malarial,[27] GHSR (growth hormone
secretagogue receptor) related disorders.[28]
The various natural compounds and medicines which are
implicated in other diseases also possess mast cell stabilization
activity.[4,5] Albeit, cromolyn was derived from structural modification
of furanochromone moiety of natural product khellin (extracted from
Ammi visnaga, an Eastern Mediterranean herb).[6,7] β-Carbolines
alkaloids contain parent nucleus of tricyclic pyrido[3,4-b]indole ring
system and they are ubiquitously found in plant kingdom (isolated as
diverse natural products), marine organisms, and insects.[8,9] More-
over, various β-carboline derivatives are reported to show anti-allergic
Cromolyn is the standard drug used as a mast cell stabilizer. The
mast cell stabilizers such as cromolyn, nedocromil, and pemirolast
(tetrazole is an isostere of carboxyl group) contain acidic moiety.
Therefore, these indicate the importance of acidic moiety, i.e., carboxyl
FIGURE 1 Currently marketed mast cell stabilizers

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group for mast cell stabilization activity. The setipiprant is a γ-carboline
derivative and it is useful in mast cell-related disorders like allergy,
asthma. Furthermore, there are key heterocyclic scaffolds and
pharmacophores like 4H-chromen-4-one (cromolyn), pyrido[1,2-a]-
pyrimidin-4-one (pemirolast), phthalazin-1-one (azelastine), pyrano
[3,2-g]quinolone (nedocromil), and cyclohepta[3,4-b]thiophen-4-one
(ketotifen) which are well tolerated for mast cell stabilization activity.
Recently the role of tryptophan in the suppression of mast cell
activation has been confirmed by Silwal et al.[29] Tetrahydro-β-
carboline is a constrained analog of tryptophan.[30,31] Furthermore,
dichotomine C, ML120B, and PS-1142 are derivatives of β-carboline
which possess potent anti-allergic effects. Walsh et al. demonstrated
the importance of rigid structures like biindeyl-1-ol and 1,2,3,4-
tetrahydro-1-naphthalenol are well tolerated for mast cell stabiliza-
tion.[32,33] The above all observations insinuate that spiro-tetrahydro-
β-carboline may have potential mast cell stabilizing activity (Figure 2).
Hence, it was thought worthwhile to synthesize spiro-tetrahydro-β-
carboline and test them for mast cell stabilizing activity ex vivo by
compound 48/80-induced rat peritoneal mast cells degranulation. This
is the first report of spiro-β-carboline derivatives possessing mast cell
stabilization activity. All structure of compounds were confirmed by
1H NMR, 13C NMR, HRMS, IR, and purity by HPLC.
concentrations. The results are expressed as half-maximal inhibitory
concentration (IC50) values presented in Table 1; the mean values of
experiments were performed in triplicate. As mast cell degranulation
results in release of myriad mediatiors. Out of these released mediators,
histamine is considered as crucial marker for mast cell degranulation and
it is determined by fluorescence method. In this method, to the mast cell
solution/suspension a specified amount of compound 48/80 (mast cell
degranulator) is added and kept for 20 min. This causes complete
degranulation resulting in histamine release. This released histamine is
complexed with OPT in NaOH solution for 2 min and then HCl is added
to stop this reaction and fluorescence is observed. This fluorescence is
maximum as no drug/compound is added and therefore called as 100%
degranulation. For testing of mast cell stabilizing activity of standard
drug/compounds, appropriate dilutions of these compounds were
mixed with mast cell solution and are incubated at 37°C for 15 min prior
to subjecting to degranulator addition (compound 48/80). Appropriate
observations for blanks, positive and negative controls were taken into
consideration. To negate false results of fluorescence appropriate
control values of various test solution were performed. In positive
control fluorescence of histamine with OPT was checked and maximum
fluorescence was observed. While, in negative control fluorescence due
to compounds/standard drug, compound 48/80, and OPT was checked
indicating no interaction of compound 48/80, standard drug, and
compounds with OPT. Spiro-β-carboline and all its derivatives
prevented the release of histamine caused by mast cell degranulation
in a dose-dependent manner as indicated by decreased histamine
fluorescence. The effect of alkyl chain at position-2 of spiro-β-carboline
was observed: longest alkyl chain compounds heptyl (compound 9;
IC50 = 0.389 0.015 μM) and octyl (compound 10; IC50 = 0.354
0.023 μM) derivatives were found to be of similar potency as that of
cromolyn (IC50 = 0.489 0.003 μM). Introduction of aromatic group like
benzene ring reduces the activity in comparison with heptyl and octyl
derivatives (compound 15; IC50 = 1.092 0.120 μM), while introduction
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2
RESULTS AND DISCUSSION
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2.1 Chemistry
Synthesis of compound 1 as shown in Scheme 1 is a Pictet–Spengler
reaction which involves the condensation of L-tryptophan with
cyclohexanone (ketone) in glacial acetic acid (dried with anhydrous
copper sulfate).[34,35] Synthesis of sulfonamide derivative compounds
11–13 is modified Schotten Baumann type reaction which involves
the reaction of compound 1 with alkyl/aryl sulfonyl chlorides in dry
pyridine.[36] Furthermore, similar reaction conditions were used to
synthesize benzoyl derivative (compound 14). The third type of
reaction is a nucleophilic substitution of alkyl halides which involves
the reaction of compound 1 with alkyl halides in presence of dry N,N-
dimethyl formamide (DMF) and dry potassium carbonate (K2CO3) to
yield compounds 2–10, 15, and 16.[37] Herein, we report an improved
method of using glacial acetic acid; glacial acetic acid serves as dual
purpose of a catalyst as well as solvent. Indeed, usage of dry glacial
acetic acid significantly reduced the reaction time and increased the
yield of the reaction. The pH adjustment is critical for this reaction to
get sufficiently pure precipitates.
of polar oxygen atom in benzoyl derivative (compound 14; IC50
=
00.469 0.032 μM) increases the activity. Introduction of sulfonyl
groups in side chain did not significantly affect the activity. Although,
introduction of bulkier group like benzenesulfonyl improved inhibitory
activity (compound 12; IC50 = 0.547 0.049 μM). There was significant
increase in activity with introduction of polar chloro group at propyl
chain (compound 16; IC50 = 0.382 0.083 μM). Furthermore, no per se
effect (compounds itself did not cause degranulation of mast cells) was
shown by any compound (at the highest concentration of 1 mg/mL). The
compounds at maximum concentration were tested for their per se
effect to check whether the compounds themselves can cause
degranulation of mast cell or not. So here mast cells were treated
with compounds at highest concentration and checked for release of
histamine as such. Indeed, this is further bolstered by Trypan blue
exclusion assay test (first to rule out cytotoxicity of compounds, second
to ensure that histamine release is not due to cytotoxicity) as mast cell
solution/suspension viability found to be 94%. Trypan blue also confirm
negative per se effect.
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2.2 Biological activity
All synthesized compounds were evaluated for their potential ex vivo
mast cell stabilization activity.[38] On treatment with standard cromolyn
prior to compound 48/80 reduced degranulation and hence reduces
amount of histamine released. A dose response curve was obtained by
using different concentrations of cromolyn and IC50 was calculated
accordingly. Similar to cromolyn all compounds were tested at different
Cromolyn is used as a standard drug to inhibit mast cell
degranulation induced by compound 48/80. Cromolyn stabilizes the

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FIGURE 2 Design strategy for mast cell stabilizers
mast cell membrane by either non-specific chloride channel inhibi-
tion,[39] or by inhibition of Ca2+ ions influx or via Annexin A or via
activation of GPR35.[40–43] Various evidences suggesting various
mechanisms of cromolyn, the mast cell stabilizing activity cannot be
attributed to a single mechanism. Despite this, cromolyn is extensively
used as a anti-allergic drug due to its profound activity. In this research
problem, our focus is on synthesis and evaluation by spiro-β-carboline
derivatives as mast cell stabilizers. Further experiments are required to
unravel the mechanism of mast cell stabilization.
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CONCLUSIONS
The derivatives of spiro-β-carboline were evaluated for mast cell
stabilization activity by compound 48/80 induced degranulation

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SCHEME 1 Synthesis of compound 1 by the Pictet–Spengler reaction
histamine release assay. It is the first report of mass cell stabilization
activity of spiro-β-carboline moiety. The 2-position of spiro-β-carboline
was explored by synthesizing alkyl, benzoyl, and sulfonyl derivatives.
The mast cell stabilization of heptyl (compound 9; IC50 = 0.389
0.015 μM), octyl (compound 10; IC50 = 0.354 0.023 μM), chloropropyl
(compound 16; IC50 = 0.382 0.083 μM), and benzoyl (compound 14;
IC50 = 00.469 0.032 μM) compounds were to be of similar potency as
that of standard drug sodium cromoglycate. This corroborated our initial
hypothesis of mast cell stabilization activity of spiro-β-carbolines.
This is a seminal study of spiro-β-carboline mast cell stabilization
having widerscopeinmast cell research, yet theirmechanismremainsto
be elusive. These four compounds could be taken as the lead for future
development of mast cell stabilizers.
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4
EXPERIMENTAL
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4.1 Chemistry
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4.1.1 General
Melting points reported are uncorrected. Synthetic procedures
employed were monitored for completion as well as purity of product

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TABLE 1 Mast cell stabilization activity of different compounds
(IC50 values)
kept overnight in a refrigerator. The precipitated product was collected
by filtration, washed thoroughly with water, and dried in vacuo. The
product was recrystallized from ethanol 95%.[34,35]
Compound
IC50 (μM)a
Cromolyn
0.489 0.003
1.479 0.133
3.328 0.299
1.620 0.486
1.548 0.268
1.324 0.253
0.875 0.166
1.588 0.095
1.319 0.369
0.389 0.015
0.354 0.023
1.389 0.375
0.547 0.049
1.113 0.022
0.469 0.032
1.092 0.120
0.382 0.083
1
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4.1.3 General procedure for preparation of
2
sulfonyl/acyl derivatives (11–14)
3
To 0.33 g (1.16 mmol) of compound 1 in 3 mL dry pyridine, 2.33
mmoles of methane sulfonyl chloride (0.265 g), benzene sulfonyl
chloride (0.410 g), tosyl chloride (0.442 g), and benzoyl chloride
(0.326 g) were added in small proportions and resultant mixture was
refluxed for 30 min. After completion of the reaction, the reaction
mixture was acidified with 2 M HCl and poured on crushed ice.[36] The
viscous products were quenched with chloroform, washed three to
four times with distilled water and dried with anhydrous sodium
sulfate. The products were purified using #230–400 mesh size silica to
give products 11–14, respectively. The products thus formed
contained two impurities. The first impurity (possibly the alkyl and
aryl sulfonic acids and benzoic acid) was eluted by column
chromatography using 99.50:0.5 petroleum ether/ethyl acetate. After
the removal of initial impurity, the polarity of solvent was changed to
96:04 petroleum ether/ethyl acetate to elute desired products and
leaving behind the second impurity back in the column.
4
5
6
7
8
9
10
11
12
13
14
15
16
an = 3.
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4.1.4 General procedure for alkylation reactions
by thin layer chromatography (TLC) employing 7 × 2.5 cm2 silica gel 60
F254 precoated TLC plates from Merck. Waters Micromass Q-tof
Micromass Spectrometer for HRMS. Nuclear magnetic resonance
spectra were recorded on Bruker Avance DPX-200 (400 MHz). In
1H NMR chemical shifts (δ) are reported in parts per million (ppm) using
tetramethylsilane (TMS) as the internal standard. Coupling constants
(J) are reported in Hz (Hertz). Multiplicities are reported using the
following abbreviations: br = broad, dist. = distorted, s = singlet,
d = doublet, t = triplet, q = quartet, m = multiplet, dd = double doublet,
sx = sextet, sp = septet. Mass spectra were recorded on a Bruker
Esquire 3000_00037. Waters HPLC system equipped with a pump, an
autosampler, a dual UV detector set at 280/320 nm, and an analytical
column (Waters, XTerra, RP18, 4.6 × 50 mm, 5 µm). The mobile phase
consisted of varying ratios of HPLC grade acetonitrile and methanol.
The flow rate was set at 1 mL/min. IR spectra were measured on a
Hitachi 270-30 infrared and Bruker Vector 22 spectrophotometers. IR
spectra were recorded as KBr pellets.
(2–10, 15, and 16)
To a solution of 0.33 g (1.16 mmoles) of compound 1 and 1.74 mmoles
of alkyl bromides/chlorides in 4 mL dry DMF, anhydrous potassium
carbonate (0.2 g) was added and refluxed for 18–20 h (alkyl bromides/
chlorides). The mixture was poured on crushed ice.[37] The viscous
products were quenched with chloroform, washed three to four times
with distilled water and dried with anhydrous sodium sulfate. The
products were purified using #230–400 mesh size silica to give
products 2–10, 15, and 16, respectively. The products were purified
using gradient elution starting from petroleum ether 100% to 90:10
petroleum ether/ethyl acetate mixture.
2′,3′,4′,9′-Tetrahydrospiro[cyclohexane-1,1′-pyrido[3,4-b]-
indole]-3′-carboxylic acid (1)
Yield: 87%; creamish white solid; mp: 220–224°C; 1H NMR (DMSO-d6,
400 MHz) δ: 10.78 (s, 1H, COOH), 7.37–7.36 (d, J = 7.68, 1H, Ar H),
7.31–7.29 (d, J = 7.96, 1H, Ar H), 7.04–7.00 (t, J = 7.46, 1H, Ar H),
6.96–6.92 (t, J = 7.24, 1H, Ar H), 3.78–3.69 [2H, s (broad), N─H
aromatic and N─H alicyclic]1, 3.05–3.00 (dd, J = 11.2, 4.36, 1H,
alicyclic H geminal and vicinal coupling), 2.72–2.65 (dd, J = 11.2, 4.36,
1H, alicyclic H geminal and vicinal coupling), 2.52–2.51 (t, 1H, alicyclic
H), 2.18–1.37 (m, 10H, spirocyclic H); 13C NMR (DMSO-d6, 100 MHz):
δ 173.29, 139.57, 135.83, 126.24, 120.65, 118.31, 117.49, 110.97,
105.36, 53.74, 52.18, 40.16, 39.95, 25.01, 24.85, 20.98, 20.69; IR
(cm−1) 3223, 3189, 3118, 2990, 2860, 2750, 2655, 2609, 2512, 2465,
1917, 1884, 1645, 1503, 1470, 1433, 1390, 1377, 1349, 1314, 1297,
1250, 1227, 1217, 1180, 1159, 1140, 1089, 1074, 1010, 960, 942,
The 1H and 13C NMR spectra, HPLC scans as well as the InChI
codes of the investigated compounds together with some biological
activity data are provided as Supporting Information.
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4.1.2 General procedure for preparation of
compound 1
To a suspension of L-tryptophan 1.0 g (4.90 mmol) in 15 mL glacial
acetic acid (dried with anhydrous copper sulfate), 0.50 mL (4.90 mmol)
of cyclohexanone was added. The resultant mixture was refluxed for
4 h, then cooled and adjusted the pH to 5 with ammonia solution and

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928, 848, 802, 759, 743, 687, 653, 596, 526, 496, 475, 458; HRMS:
285.1528 [ES+]; exact mass2 calcd. for C17H20N2O2 284.1524 [m/z];
HPLC purity 99.81% at 280 nm, tR = 4.519 min.
for C20H26N2O2 326.1994 [m/z]; HPLC purity 98.95% at 320 nm,
tR = 7.942 min.
2′-Isopropyl-2′,3′,4′,9′-tetrahydrospiro[cyclohexane-1,1′-
pyrido[3,4-b]indole]-3′-carboxylic acid (5)
2′-Methyl-2′,3′,4′,9′-tetrahydrospiro[cyclohexane-1,1′-
pyrido[3,4-b]indole]-3′-carboxylic acid (2)
Yield: 61%; yellowish oil; 1H NMR (DMSO-d6, 400 MHz): δ 7.82 (s, 1H,
COOH), 7.48–7.47 (d, J = 7.6, 1H, Ar H), 7.32–7.30 (d, J = 7.92, 1H, Ar
H), 7.24 (s, Ar N-H), 7.16–7.12 (t, J = 7.08, 1H, Ar H), 7.10–7.06 (t,
J = 7.26, 1H, Ar H), 5.18–5.11 [sp, J = 6.28, 1H, N-CH],4 3.82–3.76 (dd,
J = 11.4, 4.28, 1H, alicyclic H geminal and vicinal coupling), 3.09–3.04
(dd, J = 15.02, 4.32, 1H, alicyclic H geminal and vicinal coupling), 2.77–
2.71 (slightly distorted t, J = 13.04, 1H, alicyclic H), 1.96–1.51 (m, 10H,
spirocyclic H), 1.33–1.29 (d, J = 6.24, 6H, 2× N-CH3); 13C NMR
(DMSO-d6, 100 MHz): δ 173.33, 140.55, 135.73, 127.19, 121.63,
119.47, 118.14, 110.78, 106.88, 68.52, 52.70, 52.26, 38.58, 34.78,
26.16, 25.59, 21.89, 21.83, 21.48, 21.20; IR: (cm−1) 3329, 3145, 2963,
2849, 1708, 1631, 1567, 1498, 1437, 1253, 1176, 740, 718; HRMS:
327.1999 [ES+]; exact mass calcd. for C19H25N2O2 326.1994 [m/z];
HPLC purity 99.35% at 320 nm, tR = 7.153 min.
Yield: 81%; white solid; mp: 228°C; 1H NMR (DMSO-d6, 400 MHz): δ
7.81 (s, 1H, COOH), 7.51–7.49 (d, J = 7.56, 1H, Ar H), 7.31–7.29 (d,
J = 7.76, 1H, Ar H), 7.22 (s, Ar N-H), 7.16–7.07 (m, 2H, Ar H), 4.18–4.14
(dd, J = 11.6, 4.8, 1H, alicyclic H with geminal and vicinal coupling), 3.81
(s, 3H, N-CH3), 3.17–3.10 (slightly distorted t, J = 13.8, 1H), 2.85–2.80
(dd, J = 15.96, 4.84, 1H, alicyclic H with geminal and vicinal coupling),
1.83–1.45 (m, 10H, spirocyclic H); 13C NMR (DMSO-d6, 100 MHz): δ
174.03, 139.07, 135.74, 127.30, 121.50, 119.50, 118.24, 110.83,
106.23, 58.10, 57.14, 52.16, 37.87, 33.14, 31.05, 26.01, 21.43, 21.41,
18.90; IR (cm−1) 3410, 3280, 2940, 2849, 1719, 1642, 1565, 1410,
1178, 1253, 744, 719; HRMS: 299.1690 [ES+]; exact mass calcd. for
C18H22N2O2 298.1681 [m/z]; HPLC purity 98.02% at 280 nm,
tR = 6.163 min.
2′-Ethyl-2′,3′,4′,9′-tetrahydrospiro[cyclohexane-1,1′-
2′-Butyl-2′,3′,4′,9′-tetrahydrospiro[cyclohexane-1,1′-
pyrido[3,4-b]indole]-3′-carboxylic acid (3)
pyrido[3,4-b]indole]-3′-carboxylic acid (6)
Yield: 76%; creamish solid; mp: 238–240°C; 1H NMR (DMSO-d6,
400 MHz): δ 7.83 (s, 1H, COOH), 7.48–7.46 (d, J = 7.64, 1H, Ar H),
7.31–7.29 (d, J = 7.52, 1H, Ar H), 7.24 (s, Ar N-H), 7.16–7.12 (t, J = 7.1,
1H, Ar H), 7.11–7.07 (t, J = 7.2, 1H, Ar H), 4.31–4.25 [m, 2H, N-CH2],3
3.83–3.80 (dd, J = 11.02, 4.28, 1H, alicyclic H geminal and vicinal
coupling), 3.11–3.06 (dd, J = 15, 4.24, 1H, alicyclic H geminal and
vicinal coupling), 2.78–2.72 (slightly distorted t, J = 13.02, 1H, alicyclic
H), 1.96–1.50 (m, 10H, spirocyclic H), 1.36–1.33 (t, J = 7.08, 3H, N-
CH3); 13C NMR (DMSO-d6, 100 MHz): δ 174.03, 139.07, 135.74,
127.30, 121.50, 119.50, 118.24, 110.83, 106.23, 58.10, 57.14, 52.16,
Yield: 72%; creamish solid; mp: 236–238°C; 1H NMR (DMSO-d6,
400 MHz): δ 10.66 (s, 1H, COOH), 8.13 (s, 1H, Ar N-H), 7.35–7.33 (d,
J = 7.68, 1H, Ar H), 7.28–7.26 (d, J = 7.92, 1H, Ar H), 7.02–6.98 (t,
J = 7.46, 1H, Ar H), 6.94–6.90 (t, J = 7.2, 1H, Ar H), 4.21–4.11 (m, 2H,
N-CH2),3 3.74–3.70 (dd, J = 10.8, 3.92, 1H, alicyclic H geminal and
vicinal coupling), 2.96–2.92 (dd, J = 14.8, 4.16, 1H, alicyclic H geminal
and vicinal coupling), 2.65–2.59 (slightly distorted t, J = 12.86, 1H,
alicyclic H), 2.16–2.08 and 1.86–1.24 (m, 15H, 10H spirocyclic and 5H
N-C2H5), 0.98–0.89 (t, J = 7.32, 3H, N-CH3); 13C NMR (DMSO-d6,
100 MHz): δ 173.97, 140.69, 135.68, 127.18, 121.65, 119.49, 118.13,
110.77, 106.87, 66.59, 52.57, 52.26, 40.01, 38.97, 34.68, 27.57,
26.34, 25.64, 22.03, 21.46, 21.20, 12.81; IR: (cm−1) 3439, 3159, 2955,
2849, 1719, 1633, 1513, 1432, 1352, 1108, 731, 704; HRMS:
341.2158 [ES+]; exact mass calcd. for C21H28N2O2 340.2150 [m/z];
HPLC purity 100% at 320 nm, tR = 7.074 min.
37.87, 33.14, 31.05, 26.01, 21.43, 21.41, 20.46, 18.90; IR: (cm−1
)
3316, 3046, 2975, 2832, 1709, 1631, 1568, 1490, 1461, 1345, 1246,
1148, 742, 718; HRMS: 313.1842 [ES+]; exact mass calcd. for
C19H24N2O2 312.1837 [m/z]; HPLC purity 99.78% at 320 nm,
tR = 7.199 min.
2′-Propyl-2′,3′,4′,9′-tetrahydrospiro[cyclohexane-1,1′-
2′-Pentyl-2′,3′,4′,9′-tetrahydrospiro[cyclohexane-1,1′-
pyrido[3,4-b]indole]-3′-carboxylic acid (4)
pyrido[3,4-b]indole]-3′-carboxylic acid (7)
Yield: 64%; creamish solid; mp: 238–240°C; 1H NMR (DMSO-d6,
400 MHz): δ 7.78 (s, 1H, COOH), 7.48–7.47 (d, J = 7.64, 1H, Ar H),
7.32–7.30 (d, J = 7.42, 1H, Ar H), 7.24 (s, Ar N-H), 7.16–7.12 (t, J = 6.44,
1H, Ar H), 7.11–7.07 (t, J = 6.72, 1H, Ar H), 4.24–4.13 [m, 2H, N-CH2],3
3.85–3.81 (dd, J = 11.04, 4.24, 1H, alicyclic H geminal and vicinal
coupling), 3.11–3.06 (dd, J = 15, 4.24, 1H, alicyclic H geminal and
vicinal coupling), 2.79–2.72 (slightly distorted t, J = 13.02, 1H, alicyclic
H), 1.99–1.49 (m, 12H, spirocyclic H and N-CH2), 1.02–0.98 (t, J = 7.94,
3H, N-CH3); 13C NMR (DMSO-d6, 100 MHz): δ 173.97, 140.69,
135.68, 127.18, 121.65, 119.49, 118.13, 110.77, 106.87, 66.59,
52.57, 52.26, 38.72, 34.76, 26.34, 25.64, 22.03, 21.46, 21.20, 10.44;
IR: (cm−1) 3327, 3126, 2975, 2852, 1706, 1621, 1567, 1498, 1467,
1252, 1142, 731, 708; HRMS: 327.2003 [ES+]; exact mass calcd.
Yield:78%;whitesolid;mp:238–242°C;1HNMR(DMSO-d6, 400 MHz):
δ 10.64 (s, 1H, COOH), 8.11 (s, 1H, Ar N-H), 7.35–7.33 (d, J = 7.68, 1H,
Ar H), 7.28–7.26 (d, J = 7.92, 1H, Ar H), 7.02–6.98 (t, J = 6.76, 1H, Ar H),
6.95–6.91 (t, J = 7.46, 1H, Ar H), 4.20–4.10 (m, 2H, N-CH2),3 3.76–3.72
(br dd, 1H, alicyclic H), 2.97–2.92 (dd, J = 14.8, 4.16, 1H, alicyclic H
geminal and vicinal coupling), 2.66–2.59 (slightly distorted t, J = 12.86,
1H, alicyclic H), 2.16–1.29 (m, 10H spirocyclic and 7H N-C3H7);
13C NMR (DMSO-d6, 100 MHz): δ 173.12, 141.26, 135.63, 126.44,
120.31, 118.10, 117.72, 110.77, 104.71, 64.19, 52.15, 51.81, 40.22,
39.80, 39.39, 33.68, 27.78, 25.94, 25.17, 21.78, 21.04, 20.71, 13.73; IR:
(cm−1) 3459, 3158, 2974, 2892, 1713, 1626, 1557, 1456, 1249, 1138,
732, 692; HRMS: 355.2314 [ES+]; exact mass calcd. for C22H30N2O2
354.2307 [m/z]; HPLC purity 99.64 % at 280 nm, tR = 3.865 min.

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2′-Hexyl-2′,3′,4′,9′-tetrahydrospiro[cyclohexane-1,1′-
2′-(Methylsulfonyl)-2′,3′,4′,9′-tetrahydrospiro[cyclohexane-
pyrido[3,4-b]indole]-3′-carboxylic acid (8)
1,1′-pyrido[3,4-b]indole]-3′-carboxylic acid (11)
Yield: 65%; yellow creamish semi-solid; 1H NMR (DMSO-d6,
400 MHz): δ 7.81 (s, 1H, COOH), 7.48–7.46 (d, J = 7.68, 1H, Ar H),
7.32–7.30 (d, J = 7.92, 1H, Ar H), 7.25 (s, 1H, Ar-NH), 7.16–7.13 (t,
J = 6.48, 1H, Ar H), 7.11–7.07 (t, J = 7.72, 1H, Ar H), 4.27–4.16 (m, 2H,
N-CH2),† 3.84–3.80 (distorted t, 1H, alicyclic H), 3.11–3.06 (dd, J = 15,
4.24, 1H, alicyclic H geminal and vicinal coupling), 2.79–2.73 (distorted
dd, 1H, alicyclic H), 1.97–1.50 (m, 10H, spirocyclic H), 1.44–1.25 (m,
8H, 4CH2), 0.93–0.88 (t, J = 7.0, 3H, CH3); 13C NMR (DMSO-d6,
100 MHz): δ 173.89, 140.58, 135.65, 127.12, 121.65, 119.47,
118.10, 110.77, 106.82, 65.22, 50.51, 55.63, 52.23, 38.64,
34.71, 31.42, 28.55, 26.24, 25.58, 22.15, 21.44, 21.16, 14.01;
IR: (cm−1) 3342, 3057, 2964, 2903, 1719, 1688, 1554, 1430,
1264, 1131, 728, 693; HRMS: 369.2458 [ES+]; exact mass calcd.
Yield: 53%; yellowish solid; mp: 226–230°C; 1H NMR (DMSO-d6,
400 MHz): δ 10.89 (s, 1H, COOH), 7.49 (d, J = 7.57, 1H, Ar H), 7.33 (d,
J = 7.86, 1H, Ar H), 7.09 (t, J = 7.68, 1H, Ar H), 6.97 (t, J = 7.16, 1H, Ar
H), 3.87 (s [broad], 4H, N-H, CH2), 2.9 (m, 1H, alicyclic H), 2.8 (dd,
J = 4.45, 11.72, 1H, alicyclic H), 2.57 (dd, J = 11.72, 4.45, 1H, alicyclic
H), 2.44–2.10 (m, 10H, spirocyclic H); 13C NMR (DMSO-d6, 100 MHz):
δ 174.21, 69.34, 68.49, 64.02, 58.79, 57.22, 52.18, 47.81, 40.32,
39.53, 33.18, 31.35, 26.51, 25.83, 24.68, 22.62, 21.16, 20.14;
IR: (cm−1) 3283, 2972, 1626, 1507, 1439, 1356, 1303, 1116,
1229, 748, 716; HRMS: 363.1302 [ES+]; exact mass calcd. for
C18H22N2O4S 362.1300 [m/z]; HPLC purity 95.08% at 320 nm,
tR = 7.081 min.
for
C
23H32N2O2 368.2463 [m/z]; HPLC purity 99.92% at
2′-(Phenylsulfonyl)-2′,3′,4′,9′-tetrahydrospiro[cyclohexane-
1,1′-pyrido[3,4-b]indole]-3′-carboxylic acid (12)
320 nm, tR = 7.347 min.
Yield: 48%; viscous brownish oil; 1H NMR (400 DMSO-d6, 400 MHz): δ
10.72 (s, 1H, COOH), 7.94–7.93 (m, 2H, phenyl H), 7.69–7.57 (m, 3H,
phenyl H), 7.50 (d, J = 7.08, 1H, Ar H), 7.33 (d, J = 7.23, 1H, Ar H), 7.30
(t, J = 7.29, 1H, Ar H), 7.26 (t, 6.92, 1H, Ar H), 3.40 (m, 1H, N-H), 3.22
(m, 1H, alicyclic H), 2.54 (dd, J = 11.38, 4.13, 1H, alicyclic H), 2.52 (dd,
J = 4.13, 11.38, 1H, alicyclic H), 1.09–0.7 (m, 10H, spirocyclic H);
13C NMR (DMSO-d6, 100 MHz): δ 174.28, 139.72, 138.16, 135.83,
132.89, 129.74, 129.11, 127.28, 126.75, 124.49, 120.65, 117.49,
110.97, 61.42, 59.47, 56.34, 55.02, 40.16, 39.95, 25.01, 24.85, 23.14,
22.97; IR: (cm−1) 3421, 3064, 2954, 2846, 1634, 1529, 1402, 1381,
1321, 1143, 1228, 743, 712; HRMS: 425.1461 [ES+]; exact mass calcd.
for C23H24N2O4S 424.1456 [m/z]; HPLC purity 98.06% at 320 nm,
tR = 7.609 min.
2′-Heptyl-2′,3′,4′,9′-tetrahydrospiro[cyclohexane-1,1′-
pyrido[3,4-b]indole]-3′-carboxylic acid (9)
Yield: 71%; creamish solid; mp: 254–256°C; 1H NMR (DMSO-d6,
400 MHz): δ 7.80 (s, 1H, COOH), 7.49–7.47 (d, J = 7.68, 1H, Ar H),
7.33–7.31 (d, J = 7.92, 1H, Ar H), 7.25 (s, 1H, Ar-NH), 7.17–7.13 (t,
J = 7.02, 1H, Ar H), 7.11–7.07 (t, J = 7.34, 1H, Ar H), 4.27–4.16 (m, 2H,
N-CH2), 3.85–3.81 (distorted t, 1H, alicyclic H), 7.11–7.06 (dd,
J = 15.04, 4.28, 1H, alicyclic
H geminal and vicinal coupling),
2.81–2.74 (distorted dd, 1H, alicyclic H), 1.98–1.25 (m, 20H,
spirocyclic H and 5 N-CH2), 0.91–0.88 (triplet, J = 6.92, 3H, CH3);
13C NMR (DMSO-d6, 100 MHz): δ 174.18, 141.52, 135.66, 127.10,
121.68, 119.50, 118.11, 110.79, 106.80, 65.25, 50.52, 55.61, 52.23,
38.63, 34.72, 31.72, 28.91, 26.18, 25.86, 25.55, 22.58, 21.44, 21.15,
14.09; IR: (cm−1) 3426, 3051, 2943, 1724, 1621, 1556, 1491, 1408,
1355, 1263, 1224, 1129, 731, 692; HRMS: 383.2617 [ES+]; exact mass
calcd. for C24H34N2O2 382.2620 [m/z]; HPLC purity 95.79% at
320 nm, tR = 7.683 min.
2′-(4″-Methylphenylsulfonyl)-2′,3′,4′,9′-
tetrahydrospiro[cyclohexane-1,1′-pyrido[3,4-b]indole]-3′-
carboxylic acid (13)
Yield: 52%; off-white solid; mp: 228–230°C; 1H NMR (DMSO-d6,
400 MHz): δ 10.75 (s, 1H, COOH), 7.60–7.58 (m, 2H, tolyl H), 7.42–
7.29 (m, 2H, tolyl H), 7.13 (d, J = 7.36, 1H, Ar H), 7.08 (d, J = 7.54, 1H, Ar
H), 7.01 (t, J = 7.61, 1H, Ar H), 6.93 (t, J = 7.04, 1H, Ar H), 3.31 (s, 1H,
N-H), 3.26 (m, 1H, alicyclic H), 3.08 (dd, J = 11.48, 4.39, 1H, alicyclic H),
3.01 (dd, J = 4.39, 11.48, 1H, alicyclic H), 1.01–0.8 (m, 13H, CH3 and
spirocyclic H); 13C NMR (DMSO-d6, 100 MHz): δ 174.62, 139.56,
138.15, 137.28, 135.64, 131.79, 129.16, 128.31, 126.47, 122.09,
120.78, 119.68, 117.26, 112.14, 106.82, 68.59, 67.12, 57.13, 56.19,
32.67, 26.71, 22.95, 21.34, 20.16; IR: (cm−1) 3265, 3056, 2948, 2847,
1625, 1508, 1421, 1345, 1301, 1151, 1224, 745, 718, 643; HRMS:
439.1607 [ES+]; exact mass calcd. for C24H26N2O4S 438.1613 [m/z];
HPLC purity 97.56% at 280 nm, tR = 5.110 min.
2′-Octyl-2′,3′,4′,9′-tetrahydrospiro[cyclohexane-1,1′-
pyrido[3,4-b]indole]-3′-carboxylic acid (10)
Yield: 67%; viscous light-brownish oil; mp: 254–256°C; 1H NMR
(DMSO-d6, 400 MHz): δ 7.76 (s, 1H, COOH), 7.48–7.46 (d, J = 7.72,
1H, Ar H), 7.33–7.31 (d, J = 7.88, 1H, Ar H), 7.25 (s, 1H, Ar N-H), 7.17–
7.13 (t, J = 7.06, 1H, Ar H), 7.11–7.07 (t, J = 7.54, 1H, Ar H), 4.27–4.16
(m, 2H, N-CH2),3 3.85–3.81 (slightly distorted t, J = 6.84, 1H, alicyclic
H), 3.11–3.06 (dd, 1H, J = 15.02, 4.28, alicyclic H geminal and vicinal
coupling), 2.80–2.74 (br dd, 1H, alicyclic H), 1.98–1.25 and 0.92–0.27
(m, 10H spirocyclic and 15H N-C7H15); 13C NMR (DMSO-d6,
100 MHz):
δ 173.14, 141.32, 135.61, 121.68, 119.54, 118.15,
117.72, 110.76, 104.73, 64.12, 52.27, 51.81, 40.22, 39.80, 34.80,
31.78, 29.21, 28.61, 27.78, 25.93, 22.65, 21.78, 21.46, 20.71, 14.08,
13.73; IR: (cm−1) 3450, 3200, 2963, 1730, 1620, 1559, 1452, 1353,
1246, 1228, 1131, 739, 695; HRMS: 397.2784 [ES+]; exact mass calcd.
for C25H36N2O2 396.2776 [m/z]; HPLC purity 99.96% at 320 nm,
tR = 7.125 min.
2′-Benzoyl-2′,3′,4′,9′-tetrahydrospiro[cyclohexane-1,1′-
pyrido[3,4-b]indole]-3′-carboxylic acid (14)
Yield: 58%; off-white solid; mp: 272°C; 1H NMR (DMSO-d6, 400 MHz):
δ 8.10–2.02 (d, J = 7.76, 1H, Ar H), 8.02–8.01 (d, J = 7.6, 1H, Ar H), 7.91
(s, 1H, COOH), 7.82–7.80 (d, J = 7.96, 1H, benzoyl Ar H), 7.60 (s, 1H, Ar

SINGH ET AL.
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N-H), 7.49–7.43 (m, 2H, benzoyl Ar H), 7.38–7.34 (t, J = 7.08, 1H, Ar
H), 7.22–7.18 (t, J = 7.4, 1H, Ar H), 6.99–6.97 (d, J = 8.04, 1H, benzoyl
Ar H), 2.96–2.90 (distorted t, 4H, spirocyclic H), 2.00–1.89 (distorted
qu, 6H, spirocyclic H); 13C NMR (DMSO-d6, 100 MHz): δ 173.18,
150.26, 141.34, 138.12, 133.47, 120.83, 126.18, 119.48, 118.36,
116.65, 110.86, 104.77, 64.12, 59.46, 50.85, 41.74, 35.01, 34.92,
32.03, 31.46, 30.34, 28.67, 25.93, 23.04, 21.19; IR: (cm−1) 3426, 3168,
2912, 2817, 1782, 1726, 1623, 1502, 1426, 1297, 932, 704; HRMS:
389.1761 [ES+]; exact mass calcd. for C24H24N2O3 388.1786 [m/z];
HPLC purity 100% at 320 nm, tR = 7.120 min.
of histamine from mast cells on degranulation. This histamine released
is quantitatively determined by a spectrofluorimetric method using
o-phthaldehyde.[38]
|
4.2.1 Materials
Histamine, o-phthaldehyde, cromolyn, compound 48/80, and Trypan
blue were procured from Sigma–Aldrich, USA.
|
4.2.2 Isolation of peritoneal mast cells
2′-Benzyl-2′,3′,4′,9′-tetrahydrospiro[cyclohexane-1,1′-
pyrido[3,4-b]indole]-3′-carboxylic acid (15)
Male Wistar rats were exsanguinated and injected intraperitoneally
with 15 mL of physiological salt solution (PSS) consisting of 137 mM
NaCl, 2.7 mM KCl, 1 mM MgCl2, 1.6 mM CaCl2, 10 mM HEPES, 0.05%
gelatin, and 0.41 mM NaH2PO4 in distilled water at pH 7.4. The
abdominal region was gently massaged for 1.5 min and then the
peritoneal exudate cells were collected in a polystyrene tube. The cell
suspensions were centrifuged at 265×g for 15 min at 4°C and washed
several times with the physiological suspension.[38]
Yield: 63%; off-white solid; mp: 260°C; 1H NMR (DMSO-d6, 400 MHz):
δ 10.69 (s, 1H, COOH), 7.43–7.37 (m, 7H, Ar H and benzyl H), 7.02–
6.98 (t, J = 7.92, 1H, Ar H), 6.94–6.90 (t, J = 7.24, 1H, Ar H), 5.23 (s, 1H,
Ar N-H), 3.87 (m, 1H, alicyclic H), 2.99–2.94 (dd, J = 11.52, 4.37, 1H,
alicyclic H), 2.93–2.65 (distorted dd, alicyclic H), 1.86–0.84 (m, 12H,
CH2 and spirocyclic H); 13C NMR (DMSO-d6, 100 MHz): δ 174.19,
143.82, 138.57, 135.26, 129.74, 128.71, 128.57, 128.39, 128.24,
128.16, 127.20, 121.85, 118.32, 116.17, 71.82, 63.78, 55.48, 52.31,
48.16, 32.49, 31.74, 28.76, 26.19, 21.49; IR: (cm−1) 3356, 3287, 3052,
2912, 2836, 1709, 1464, 1134, 741, 706; HRMS: 375.1988 [ES+];
exact mass calcd. for C24H26N2O2 374.1994 [m/z]; HPLC purity 98% at
320 nm, tR = 7.303 min.
|
4.2.3 Ex vivo mast cell stabilization assay
The 1000 μg/mL stock solution of each compound were made by
dissolving 10 mg of standard drug/compounds in 10 mL triple distilled
water and 100 μL DMSO was added for miscibility of compounds.
From this stock solution dilutions of each compound were made: 20,
40, 80, 160, 320, 640 μg/mL and distilled water added to make final
volume to 1 mL. Afterward, 1 mL mast cell solution was added to each
test tube and solution is incubated at 37°C for 15 min. Then 36 μg/mL
compound 48/80 (1 mL) was added to induce degranulation of mast
cells and kept for 20 min. After that 1 N sodium hydroxide (200 μL) and
1% ortho-phthaldehyde (50 μL) were added and kept for 2 min to
complete OPT complexing reaction with histamine in alkaline
conditions. Then this reaction was stopped by adding 200 μL of 2 N
HCl was added. At last readings of test tubes solutions were taken at
spectrofluorometer at 350 nm excitation and 450 nm emission
wavelength.[34] The 100% degranulation was assessed using mast
cell solution, compound 48/80, OPT, NaOH, and HCl (without the
addition of standard drug/compounds) as mentioned previously.
Blanks of each compound at each concentration were taken using
OPT, NaOH, and HCl in order to assess any false fluorescence of
compounds themselves or their interaction with OPT. All blanks were
subtracted to get accurate readings.
2′-(2-Chloroethyl)-2′,3′,4′,9′-tetrahydrospiro[cyclohexane-1,1′-
pyrido[3,4-b]indole]-3′-carboxylic acid (16)
Yield: 84%; dark brownish oil; 1H NMR (DMSO-d6, 400 MHz): δ 7.76 (s,
1H, COOH), 7.48–7.45 (distorted d, 1H, Ar H), 7.32–7.30 (d, J = 7.8, 1H,
Ar H), 7.25 (s, Ar N-H), 7.17–7.13 (t, J = 7.44, 1H, Ar H), 7.13–7.07 (t,
J = 7.8, 1H, Ar H), 4.40–4.35 [m, 2H, N-CH2],3 3.87–3.80 and 3.69–3.65
(distorted qu, 4H, N-CH2 and CH2Cl), 3.54–3.50 (t, J = 6.52, 1H, alicyclic
H), 3.11–3.05(distorteddd, 1H, alicyclicH), 2.80–2.73(distorteddd, 1H,
alicyclic H), 2.29–2.26 (t, J = 6.28, 1H, spirocyclic C-H), 2.22–2.16 (qu,
J = 6.32, 1H, spirocyclic C-H), 1.96–1.51 and 1.32–1.25 (m, 10H,
spriocyclic H); 13C NMR (DMSO-d6, 100 MHz): δ 172.91, 140.58,
135.69, 127.11, 121.74, 119.56, 118.11, 110.77, 106.69, 61.76, 52.67,
52.23, 52.13, 41.15, 38.69, 34.83, 26.29, 25.60, 22.03, 21.45, 21.19; IR:
(cm−1) 3418, 3017, 2957, 1716, 1620, 1565, 1437, 1162, 671, 864, 762,
708; HRMS: 362.1610 [M+2]; exact mass calcd. for C20H25ClN2O2
360.1604 [m/z]; HPLC purity 99.35% at 320 nm, tR = 7.303 min.
|
4.2 Biological activity
|
4.2.4 Trypan blue exclusion test
The experimental protocol was approved by Institutional Animal Ethics
Committee (IAEC) and care of animals were taken as per guidelines of
Committee for the Purpose of Control and Supervision of Experiment
on Animals (CPCSEA), Ministry of Environment and Forest Govern-
ment of India (Reg. No. 107/1999/CPCSEA).
Trypan blue exclusion was carried out using established method.[44,45]
ACKNOWLEDGMENTS
Mast cell stabilization of a compound is indicated by prevention of
degranulation induced by compound 48/80 on isolated rat peritoneal
mast cells. The extent of mast cell degranulation is measured by release
We would like to thank University Grant Commission, New Delhi
for providing research grant (F. No. 41-1378/2012) for mast cell
stabilizers research. The author sought to sincerely thank S. Avtar

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SINGH ET AL.
|
Singh for his erudition toward NMR (SAIF, Panjab University,
Chandigarh, India). We also like to thank Department of Chemistry
and Biochemistry, Brigham Young University (BYU), Provo, Utah,
United States for their help. We would also like to thank Prof. K. S.
Batra, Lotika Bajaj (PhD Pharmaceutics, Mercer University, USA),
Ankur Jalan and Dhruv Shah (PhD Organic Chemistry; PhD Analytical
Chemistry, BYU) for their valuable suggestions and timely help.
Notre Dame De La Paix, Universite Libre Bruxelles, assignee. Beta
carboline derivatives useful in the treatment of proliferative
disorders. United States patent US 9,168,247. 2015 Oct 27.
[15] X. F. Zhang, R. Q. Sun, Y. F. Jia, Q. Chen, R. F. Tu, K. K. Li, X. D. Zhang,
R. L. Du, R. H. Cao, Sci. Rep. 2016, 6, 33204.
[16] P. Melnyk, P. Carato, S. Burlet, E. Nguyen, P. Verwaerde, N.
Sergeant, C. Estrella, inventors; Alzprotect, Universite De Lille 2
Droit Et Sante, Inserm (Institut National De La Sante Et De La
Recherche Medicale), assignee. Carboline compounds usable in the
treatment of neurodegenerative diseases. United States patent US
9,714,245. 2017 Jul 25.
CONFLICT OF INTEREST
[17] D. N. Stephens, H. H. Schneider, W. Kehr, J. S. Andrews, K. J. Rettig, L.
Turski, R. Schmiechen, J. D. Turner, L. H. Jensen, E. N. Petersen, J.
Pharmacol. Exp. Ther. 1990, 253(1), 334.
The authors do not have a conflict of interest of any kind.
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Heilmann, J. Lehmann, T. Winckler, J. Med. Chem. 2010, 53(9), 3611.
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ENDNOTES
1 Disappeared in D2O exchange.
2 http://www.sisweb.com/referenc/tools/exactmass.htm.
3 Triplet get distorted due to geminal coupling of N-CH2 with carbonyl bond
of COOH.
4 Septet not get distorted to multiplet, because only one H present at N─H
[20] N. S. Ahmed, B. D. Gary, H. N. Tinsley, G. A. Piazza, S. Laufer, A. H.
Abadi, Arch. Pharm. Chem. Life Sci. 2011, 11, 149.
[21] W. Bi, Y. Bi, P. Xue, Y. Zhang, X. Gao, Z. Wang, M. Li, M. Baudy-Floc’h,
N. Ngerebara, K. M. Gibson, L. Bi, Eur. J. Med. Chem. 2011, 46, 1453.
[22] J. Timonty, P. Skolnick, J. M. Cook, J. Med. Chem. 1987, 30, 750.
[23] S. A. Pogosyan, N. P. Gregoryan, R. G. Paronikyan, J. Pharm. Chem.
2007, 41, 17.
and it is not involved in geminal coupling with carbonyl bond of COOH.
[24] M. Abou-Gharbia, U. R. Patel, J. A. Moyer, E. A. Muth, J. Med. Chem.
1987, 30, 100.
ORCID
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Dhandeep Singh
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How to cite this article: Singh J, Shah R, Singh D, Jaggi AS,
Singh N. Design, synthesis, and biological evaluation of
2-substituted-2,3,4,9-tetrahydrospiro-β-carboline-3-
carboxylic acid derivatives as first-in-class mast cell
stabilizers. Arch Pharm Chem Life Sci. 2018;1–11.
https://doi.org/10.1002/ardp.201800019
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