Diastereoisomers of 2-benzyl-2, 3-dihydro-2-(1H-inden-2-yl)-1H-inden-1-ol: Potential anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmcl.2009.08.060

The synthesis and biological activity of the novel diastereoisomers of 2-benzyl-2,3-dihydro-2-(1H-inden-2-yl)-1H-inden-1-ol is reported. The 2,2-coupled indane dimers were synthesised by coupling of the silyl enol ether of 1-indanone with the dimethyl ket

Full text of DOI:10.1016/j.bmcl.2009.08.060

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5927–5930
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Bioorganic & Medicinal Chemistry Letters
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Diastereoisomers of 2-benzyl-2, 3-dihydro-2-(1H-inden-2-yl)-1H-inden-1-ol:
Potential anti-inflammatory agents
*
Helen Sheridan , John J. Walsh, Carina Cogan, Michael Jordan, Tom McCabe, Egle Passante, Neil H. Frankish
Trinity College Dublin, School of Pharmacy and Pharmaceutical Sciences, Dublin 2, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 July 2009
Revised 12 August 2009
Accepted 13 August 2009
Available online 21 August 2009
The synthesis and biological activity of the novel diastereoisomers of 2-benzyl-2,3-dihydro-2-(1H-inden-
2-yl)-1H-inden-1-ol is reported. The 2,2-coupled indane dimers were synthesised by coupling of the silyl
enol ether of 1-indanone with the dimethyl ketal of 2-indanone. The coupled product was directly alkyl-
ated to give the racemic ketone which was reduced to the diastereoisomeric alcohols. The alcohols were
separated and their relative stereochemistry was established by X-ray crystallography. These molecules
demonstrate significant anti-inflammatory activity in vivo and in vitro and may represent a new class of
anti-inflammatory agent.
Keywords:
Indane
Indane dimers
Mast cells
Ó 2009 Elsevier Ltd. All rights reserved.
Anti-inflammatory
RBL-2H3 cells
Introduction. Mast cells contain or may produce a wide range of
pro-inflammatory mediators and will degranulate following both
immunogenic (IgE) and non-immunogenic stimuli such as super-
oxides, complement, neuropeptides, and lipoproteins. In addition
to histamine, mast cells may also release leukotrienes, and prosta-
noids, many inflammatory cytokines and chemokines as well as
the protease enzymes, tryptase and chymase and many of these
mediators are key agents in inflammation.1 As a consequence of
their ‘sentinal’ locations and their panoply of pro-inflammatory
mediators, mast cells may play an active role in many inflamma-
tory diseases, such as allergy, asthma, arthritis, atherosclerosis,
pulmonary fibrosis, and parasitic diseases. Mast cells may also play
a vital role in host defense against pathogens as part of the innate
immune system since they express Toll-like receptors (TLR), and
respond to TLR ligands such as LPS and peptidoglycan.2,3
indane dimer 4 has shown significant inhibition of compound 48/
80-stimulated histamine release from rat peritoneal mast cells
together with moderate smooth muscle relaxation effects which
suggested that this class of molecule had potential in the treatment
of asthma and warranted further investigation.11 More recently we
have identified a second 1,2-coupled indane dimer 5 with signifi-
cantly increased mast cell stabilisation activity. However, we did
not succeed in our initial aim of combining bronchodilatory and
mast cell stabilisation activity in a single molecule.12
The indane skeleton features in a range of molecules that display
significant and diverse biological activity. The protease inhibitor
Indinavir 1 (Crixivan) is used as a component of Highly Active Retro-
viral Therapy (HART) to treat HIV infection and AIDS.4 Indacrinone 2
is a potentantidiuretic5 while indanocine 3 demonstrates significant
binding with microtubules.6 For some time this research group has
been working on a group of small biologically active indanes and
their dimers. We have established that a number of simple indanes
related to the pterosin group of natural products.7–9 and their 1,2-
coupled dimers10,11 demonstrate smooth muscle relaxant activity
and also inhibit histamine release from mast cells. The 1,2-coupled
We have subsequently synthesized and evaluated the activity of
related 2,2-coupled indane dimers. We now report on a pair of dia-
stereoisomers (6 and 7) which demonstrate significant mast cell
* Corresponding author. Tel.: +353 1 8962825/8.
E-mail addresses: hsheridn@tcd.ie (H. Sheridan), nfrnkish@tcd.ie (N.H. Frankish).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.08.060

5928
H. Sheridan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5927–5930
stabilisation and anti-inflammatory effects in a range of in vivo and
in vitro screens. We feel that these diastereoisomers represents a
potential new class of indane compounds with potential to treat
a range of inflammatory conditions. The target 2,2-coupled indane
dimers were synthesised as shown in Scheme 1.13 The key inter-
mediate to this class of dimer 11, was prepared from 10 which
was synthesised by coupling of the silyl enol ether of 1-indanone
8 with the dimethyl ketal of 2-indanone 9. The coupled product
10 was alkylated to yield 11. This route has been successfully used
to synthesise a range of related dimers. Reduction of 11 yielded the
diastereoisomers 6 and 7. The relative stereochemistry of the pair
of diastereoisomers was established by single crystal X-ray analy-
sis of 6 as shown in Figure 1.14
Compounds 6 and 7 were evaluated for their smooth muscle
relaxant activity, their ability to inhibit histamine release from
rat peritoneal mast cells and to inhibit calcium ionophore
A23187-stimulated b-hexosaminidase release from RBL-2H3 cells;
these latter cells are a histamine-releasing cell line commonly used
to model mast cell degranulation.15 b-Hexosaminidase is released
in parallel with histamine and both histamine and b-hexosamini-
dase release from rat peritoneal mast cells and RBL-2H3 cells,
respectively, were used as markers for inhibition of release of
inflammatory mediators from the mast cell. The compounds were
also assessed for broad anti-inflammatory activity in vivo using the
arachidonic mouse ear swelling test.
Smooth muscle relaxant activity was measured by inhibition of
calcium contractures of potassium-depolarised isolated guinea-pig
ileum.16 Calcium (2.5 mM) induced a sustained contraction of gui-
nea-pig ileum which was inhibited by 48.1 2.3% by nifedipine
(1 Â 10À8 M). In contrast, 6 and 7 (1 Â 10À5 M) inhibited contrac-
tures by 18.9 2.16% and 36.37 5.7%, respectively (Table 1). The
difference between the two diastereoisomers was not statistically
different (P >0.05). It is unlikely that such a degree of smooth mus-
cle relaxation would be clinically useful.
Figure 1. X-ray crystal structure and relative stereochemistry of 6.
Table 1
Effect of nifedipine (1 Â 10À8 M), and compounds 6 and 7 (1 Â 10À5 M) on calcium-
induced (2.5 mM) contractions of potassium-depolarised guinea-pig ileum
Treatment
Mean
SEM
n
Nifedipine
48.1
2.3
6
6
6
7
18.88
36.37
2.16
5.667
12
as histamine release inhibitors in rat peritoneal mast cells.10 In
addition, we have used the histamine-releasing rat cell line RBL-
2H3 as a convenient alternative to rat peritoneal mast cells har-
vested by peritoneal lavage.
Histamine release17 from rat peritoneal mast cells stimulated
by compound 48/80 was inhibited by disodium cromoglycate by
only approximately 11% (Table 2). However, 6 and 7 both inhibited
histamine release in a much more potent manner, by 84% and 93%
from 60.2 5.0% for 48/80-stimulated release to 9.3 1.3% and
4.1 0.4%, respectively, the difference between the two not being
Following our original observation that similar compounds
inhibited calcium contractions of smooth muscle, probably by inhi-
bition of calcium handling in excitation–contraction coupling in
smooth muscle cells6 we had extended our studies to encompass
a potential role in excitation–secretion coupling in secretory cells,
statistically different (P >0.05). Similarly, both 6 and 7 (10 lM)
inhibited calcium ionophore A23187-stimulated b-hexosamini-
dase release from RBL-2H3 cells,17–19 by approximately 45% and
40%, from a solvent (DMSO) control value of 35.2 4.8% to
19.2 3.2% and 21.0 2.8%, respectively (Fig. 2). In comparison,
quercetin (10 lM) inhibited enzyme release by approximately
70% to 11.0 1.0% of the total as shown in Figure 3. Furthermore,
such inhibition was dose-dependant; concentrations of both 6
(3–50
l
M) and 7 (3–50
l
M) gave a highly significant (P <0.0001)
M being 96% and
linear trend, with maximum inhibition at 50
l
75%, from a solvent (DMSO) control value of 80.5 9.4% to
20.1 0.2% and 3.2 0.4% for 6 and 7, respectively, though there
was no significant (P >0.05) difference between 6 and 7 at this
concentration.
These results suggest that both 6 and 7 are inhibitors of medi-
ator release from RPMCs and RBL-2H3 cells. Furthermore, in the
case of the latter, mediator release is dose-dependant. However,
Table 2
Histamine release from rat peritoneal mast cells stimulated by compound 48/80
(2
l
g mLÀ1) and the effect of DSCG (2 Â 10À5 M) and compounds 6 and 7 (2 Â 10À5 M)
Treatment
Mean
SEM
n
48/80 alone
DSCG
6
7
60.23
53.62
9.339
4.138
4.984
4.009
0.4251
1.316
15
15
5
5
Scheme 1. Synthetic route to compounds 6 and 7.

H. Sheridan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5927–5930
5929
Table 3
Effect of vehicle only, indomethacin (300
acid-induced (4 mg) mouse ear swelling
l
g), and 6 and 7 (300
l
g) on arachidonic
Treatment
Mean
SEM
n
Vehicle
Indomethacin
6
7
178.8
139.7
143.1
132.4
14.21
5.035
6.797
7.493
8
4
4
4
All treatments were administered topically in 100 lL acetone.
the positive control, indomethacin, suggesting that the compounds
may have anti-inflammatory activity in vivo, with possible clinical
potential. Further experiments should follow systemic administra-
tion of 6 and 7, to exclude the possibility of topical interaction be-
tween the compounds and arachidonic acid.
Conclusion. In conclusion, we report the synthesis and activity of
a pair of diastereomeric indane dimers that demonstrate inhibition
of histamine release from mast cells and demonstrate dose depen-
dent inhibition of b-hexosaminidase release from RBL-2H3 cells. In
addition these compounds demonstrate anti-inflammatory activity
in vivo in the arachidonic acid mouse ear model. In a preliminary
study,22 while we showed that IgE-stimulated histamine release
from RBL-2H3 cells was largely unaffected by the compounds, we
speculated that greater biological activity might reside in diastereo-
isomer 7, on the basis that the marginal effect did attain statistical
significance. However, we have been unable to confirm any such
difference in the activity of the two diastereoisomers in this study;
both 6 and 7 were equipotent in all systems studied. Furthermore,
we have shown significant anti-inflammatory activity in vivo and
these molecules are currently under investigation in other inflam-
matory disease models as potential novel therapeutic agents.
Figure 2. Effect on b-hexosaminidase release from RBL-2H3 cells stimulated by
calcium ionophore A23187 (5 g/mL) by quercitin (10 M), compounds 6 and 7
(10 M). The value obtained on incubation with the solvent control, DMSO (0.5%) is
shown for comparison. Values are expressed as a mean SEM, n as indicated.
l
l
l
References and notes
1. Metz, M.; Grimbaldeston, M. A.; Nakae, S.; Piliponsky, A. M.; Tsai, M.; Galli, S. J.
Immunol. Rev. 2007, 217, 304.
2. Puxeddu, I.; Piliponsky, A. M.; Bachelet, I.; Levi-Schaffer, F. Int. J. Biochem. Cell.
Biol. 2003, 35, 1601.
3. Krishnaswamy, G.; Ajitawi, O.; Chi, D. S. Methods. Mol. Biol. 2006, 315, 13.
4. De Clercq, E. Int. J. Antimicrob. Agents 2009, 33, 307.
5. Abrams, W.; Irvin, J.; Tobert, J.; Ferguson, R.; Vlasses, P. Cardiovasc. Drug Rev.
2007, 3, 1.
6. Leoni, L.; Hamel, E.; Genini, D.; Shih, H.; Carrera, C. J.; Cottam, H. B.; Carson, D.
A. J. Natl. Cancer Inst. 2000, 92, 217.
7. Sheridan, H.; Lemon, S.; Frankish, N.; McCardle, P.; Higgins, T.; James, J.;
Bhandari, P. Eur. J. Med. Chem. 1990, 25, 603.
8. Farrell, R.; Kelleher, F.; Sheridan, H. J. Nat. Prod. 1996, 59, 446.
9. Sheridan, H.; Frankish, N.; Farrell, R. Eur. J. Med. Chem. 1999, 34, 953.
10. Sheridan, H.; Frankish, N.; Farrell, R. Planta Med. 1999, 65, 271.
11. Frankish, N.; Farrell, R.; Sheridan, H. J. Pharm. Pharmacol. 2004, 56, 1423.
12. Sheridan, H.; Walsh, J. J; Jordan, M.; Cogan, C.; Frankish, N. Eur. J. Med Chem.,
submitted for publication.
13. Compound 8: Triethylamine (12.7 mL, 90.8 mmol) followed by trime-
thylsilyltrifluoromethane sulfonate (TMS triflate) (15.0 mL, 83.3 mL), was
added slowly dropwise at 0 °C, to a stirred solution of 1-indanone (10.00 g,
75.7 mmol) in CH2Cl2 (150 mL) at 0 °C. After the addition, the solution was
allowed to warm to rt and stirred for 20 min before being cooled to 0 °C and
quenched by the addition of 5% aq. NaHCO3 (150 mL). The layers were
separated and the aqueous layer extracted with CH2Cl2 (2 Â 100 mL), the
combined organic extracts were washed with water and brine, dried over
MgSO4 and evaporated. The crude product was filtered through a short silica
pad and washed through with 100:0.5 hexane/ethyl acetate. The solvent was
again removed in vacuo with a bath temperature of not more than 40 °C, to
give 15.25 g (98%) of the product as a pale yellow oil. 1H NMR analysis was not
possible due to decomposition of the product over time in the NMR solvent.
mmax (neat): 3072, 3075, 2954, 2895, 1607, 1578, 1350, 1255 (Si–Me3), 1178,
1125 (Si–O), 1078, 884 (Si–O), 849 (Si–Me3), 755 (C–HAr). Compound 9:
Trimethyl orthoformate (20.7 mL, 189.2 mmol) and catalytic p-toluenesulfonic
Figure 3. The concentration effect relationship of compounds 6 and 7 (3–50
b-hexosaminidase release from RBL-2H3 cells stimulated by calcium ionophore
A23187 (5 g/mL). The value obtained on incubation with the solvent control,
DMSO (0.5%) and by quercetin (10 lM) is shown for comparison. Values are
expressed as a mean SEM, n = 3.
lM) on
l
we have previously shown in preliminary studies that both 6 and 7
were largely ineffective against IgE-mediated release of histamine
and b-hexosaminidase from RBL-2H3 cells.20 While the fact that
the compounds seem only to be effective against non-immuno-
genic stimuli may mitigate against an anti-allergic application of
these compounds, their mediator releasing inhibitory effects may
be applicable to inflammation in general.
The arachidonic acid mouse ear oedema model is useful to
screen potential anti-inflammatory compounds with an uncertain
mode of action, since it encompasses a wide range of potential tar-
gets.21 Application of arachidonic acid (4 mg) to a mouse ear gives
rise after 1 h to an increase in ear thickness of 78.8 14.2%. Prior
application (1 h) of indomethacin (300
lg) reduced ear swelling
by 50%, to 39.6 5.0%. In comparison, 6 and 7 (300
lg) reduced
acid (0.15 g, 1 mol %) were added to
a solution of 2-indanone (10.42 g,
ear swelling by approximately 45% and 59% to 43.0 7.5% and
32.4 6.8%, respectively (Table 3), the difference between the
two compounds, 6 and 7 not being statistically different (P
>0.05). The efficacy of both 6 and 7 was comparable to that of
78.8 mmol) in methanol (125 mL). The solution was allowed to stir at room
temperature for 2 h and then quenched by the addition of solid NaHCO3
(0.5 equiv). The methanol was evaporated in vacuo and the crude material
partitioned between water and ether (ca. 100 mL each). The layers were
separated and the aqueous layer extracted with ether (2 Â 100 mL). The

5930
H. Sheridan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5927–5930
combined organic layers were dried over Na2SO4 and concentrated in vacuo.
The crude product was purified by flash chromatography (98:2 hexane/ethyl
UK) when reaching 80% confluency and were plated at 1 Â 105 cells/cm2 in 24-
well plates for experiments.
acetate) dH (300 MHz, CDCl3): 3.14 (4H, s, 2 Â CH2), 3.29 (6H, s, 2 Â CH3), 7.12–
7.19 (4H, m, ArH). Compound 11: Benzyl bromide (1.9 mL, 16 mmol) was added
to a stirred solution of 10 (1.13 g, 4 mmol) in ether (34 mL) and tert-butanol
(6 mL). Potassium tert-butoxide (0.45 g, 4 mmol) in tert-butanol (20 mL) was
added drop wise and reaction was stirred for 3 h. The reaction was quenched
by the addition of satd NH4Cl and the layers separated. The reaction was
worked up and the crude product was purified by flash chromatography (9:2;
hexane/ethyl acetate). dH (300 MHz, CDCl3): 3.37 (2H, dd, J 13.8 Hz, CH2), [AB
system], 3.38–3.61 (4H, m, 2 Â CH2), 6.70 (1H, s, CH@C), 7.09–7.34 (10H, br m,
ArH), 7.36–7.39 (1H, m, ArH), 7.50–7.55 (1H, m, ArH), 7.91 (1H, d, J 7.7 Hz,
ArH). Compounds 6 and 7: Lithium tri-tert-butoxyaluminohydride (11.57 g,
45 mmol) was added in one portion to a stirred solution of the 11 (4.64 g,
14 mmol) in THF (115 mL). The solution was stirred at room temperature and
monitored by TLC (9:1 hexane/ethyl acetate). After work up the organic extract
was evaporated to yield a pale yellow oil (85%) containing of 6 and 7 (1:1). The
diastereoisomers were separated by flash chromatography using a n-hexane:
ethyl acetate gradient. Compound 6: dH (300 MHz, CDCl3) 2.51 (1H, d, J 5.3 Hz,
OH), 2.94 (2H, dd, J 13.4 Hz, CH2) [AB system ], 2.98 (2H, dd, J 15.6 Hz, CH2) [AB
system], 3.46 (2H, dd, J 22.5 Hz, CH2) [AB system], 5.17 (1H, d, J 5.1 Hz, CHOH),
6.44 (1H, s, CH@C), 6.82–6.85 (2H, m, ArH), 7.09–7.14 (4H, m, ArH), 7.22–7.26
(5H, m, ArH), 7.36–7.39 (2H, m, ArH). Compound 7: dH (300 MHz, CDCl3):3.00
(2H, dd, J 13.8 Hz, CH2) [AB, centered at 3.00], 3.08 (2H, dd, J 15.8 Hz, CH2) [AB
system], 3.41 (2H, dd, J 22.9 Hz, CH2) [AB system], 5.02 (1H, m, CHOH), 6.64
(1H, s, CH@C), 6.91–6.94 (2H, m, ArH), 7.10–7.31 (9H, br m, ArH), 7.35–7.40
(2H, m, ArH).
19. b-Hexosaminidase assay: RBL-2H3 cells (1 Â 105 cells/cm2) were seeded and
they were let to adhere for at least 3 h. Then the supernatant was removed and
replaced with fresh medium containing calcium ionophore at 5
compounds at the desired concentration (0.3–50 M). Plates were incubated
for 30 min at 5% CO2 and 37 °C. The b-hexosaminidase activity was assayed
according to a previously published method, [2]. Briefly, 30 L of supernatant
were transferred into a 96-well plate. Fifty microliters of substrate solution
were added (1.3 mg/mL of p-nitrophenyl N-acetyl- -glucosaminide (Sigma) in
citrate buffer, pH 4.5) and the plate was incubated for 1 h at 37 °C. The reaction
was stopped by adding 80 L of NaOH (0.5 M) to each well and the formed p-
lg/mL and test
l
l
D
l
nitrophenolate was measured spectrophotometrically in a 96-well plate reader
(FLUOstar OPTIMA, BMG Labtech, Aylesbury, UK) at a wavelength of 405 nm.
The absorption was converted into the percentage of total cellular b-
hexosaminidase activity by comparison with the absorption produced by a
Triton X-100 (Sigma) lysate of the same cells according to the following
*
equation:% total enzyme activity = [(secreted À spontaneous)/total content] 100.
Incubations were performed in triplicate. The percentage of the vehicle was
always maintained at 0.5% of the volume of the supernatant.
20. Passante, E.; Carsten, E.; Sheridan, H.; Frankish, N. FASEB J. 2007, 21, A442.
21. Mouse ear oedema: The mouse ear oedema model was performed using Balb/C
mice (25–35 g), of either sex. The left ear was treated by the topical application
(10
compound (300
dexamethasone (100
topical application of arachidonic acid (10
l
L) of solvent (acetone 100%). To the right ear was applied 10
g/ear in acetone), indomethacin (300 g/ear in acetone) or
g/ear in acetone). After 1 h, oedema was induced by the
L of 400 mg/mL in acetone). The
lL test
l
l
l
l
14. Sheridan, H.; Walsh, J. J.; McCabe, T.; Passante, E.; Cogan, C.; Jordan, M.;
Frankish, N., X-ray crystal data deposit. CCDC deposition number: 733630, 2009.
15. Passante, E.; Carsten, E.; Sheridan, H.; Frankish, N. Inflamm. Res. 2009, 58, 611.
16. Smooth muscle relaxant activity: Measurement of smooth muscle relaxant
activity was performed as previously described.6
17. Histamine release: Measurement of inhibition of inhibition of histamine release
from rat peritoneal mast cells was carried out as decribed previously.9
18. RBL-2H3 Cell culture; RBL-2H3 cells were purchased from LCG Promochem
width of each ear was measured, both before and 60 min after the induction of
oedema, using a micrometer screw gauge. Ear oedema was calculated by
comparing the left and right ear width after induction of oedema and
expressed as percentage normal. Values are expressed as a mean SEM and
statistical comparisons between groups was performed by one way Anova,
followed by Dunnet’s Multiple Comparison test as a post-test. Data were
displayed and statistical analysis was performed using Prism
4 software.
Animals were sacrificed according to guidelines laid down by the working
party report (Laboratory Animals (1996) 30, 293–316, Laboratory Animals
(1997) 31, 1–32), on Directive 86/609/EEC (No. L 358, ISSN 0378-6978), which
is endorsed by the Bioresources Ethical Review Committee of the University.
22. Passante, E.; Ehrhardt, C.; Sheridan, H.; Frankish, N. Inflamm. Res. 2008, 57, 15.
(Teddington, UK) and were cultured in
a-MEM (GIBCO, Paisley, UK)
supplemented with 10% foetal bovine serum (FBS), (Biosera, Ringmer, UK)
and 100 U/mL penicillin–streptomycin (GIBCO) at 5% CO2 and 37 °C. Cells were
subcultured using either trypsin–EDTA (GIBCO) or AccutaseÒ (Bioquote, York,