6-(Methylamino)hexane-1,2,3,4,5-pentanol 4-(((1 S,2 S)-1-hydroxy-2,3- dihydro-1 H,1′ H -[2,2-biinden]-2-yl)methyl)benzoate (PH46A): A novel small molecule with efficacy in murine models of colitis

Article abstract of DOI:10.1021/jm300390f

The indane skeleton is found naturally and in several therapeutic molecules in medicinal chemistry. During our work on the anti-inflammatory activity of naturally occurring and synthetic indanes, we have synthesized a novel indane scaffold that has been optimized for both anti-inflammatory activity and bioavailability. We have evaluated our lead molecule, PH46A, in in vivo models of inflammatory bowel disease (IBD), an area of considerable unmet clinical need; current therapies are often unable to control the course of the disease. The compound significantly reduced histological damage and serum amyloid A (SAA) levels in IL-10^-/- colitis mice, was efficacious in the 5% dextran sulfate sodium (DSS) colitis model, and compared favorably with prednisolone in this model and supports its potential use to treat acute exacerbations of the disease. Further, the graded response to the compound may also lend itself to be used at a lower dose to maintain periods of remission.

Full text of DOI:10.1021/jm300390f

Article
pubs.acs.org/jmc
6-(Methylamino)hexane-1,2,3,4,5-pentanol 4-(((1S,2S)-1-Hydroxy-2,3-
dihydro-1H,1′H-[2,2-biinden]-2-yl)methyl)benzoate (PH46A): A Novel
Small Molecule With Efficacy In Murine Models Of Colitis
Neil Frankish* and Helen Sheridan*
Trinity College Dublin, Drug Discovery Group, School of Pharmacy and Pharmaceutical Sciences, College Green, Dublin 2, Ireland
ABSTRACT: The indane skeleton is found naturally and in several
therapeutic molecules in medicinal chemistry. During our work on the anti-
inflammatory activity of naturally occurring and synthetic indanes, we have
synthesized a novel indane scaffold that has been optimized for both anti-
inflammatory activity and bioavailability. We have evaluated our lead molecule,
PH46A, in in vivo models of inflammatory bowel disease (IBD), an area of
considerable unmet clinical need; current therapies are often unable to control
the course of the disease. The compound significantly reduced histological
damage and serum amyloid A (SAA) levels in IL-10−/− colitis mice, was
efficacious in the 5% dextran sulfate sodium (DSS) colitis model, and compared favorably with prednisolone in this model and
supports its potential use to treat acute exacerbations of the disease. Further, the graded response to the compound may also lend
itself to be used at a lower dose to maintain periods of remission.
INTRODUCTION
■
Inflammatory bowel disease (IBD), incorporating Crohn’s
disease (CD) and ulcerative colitis (UC), is a chronic disease
of the gastro-intestinal tract characterized by inflammation of the
large and/or small intestine, with symptoms of diarrhea,
abdominal pain, weight loss, and nausea. In extreme cases, it
may result in malnutrition, dehydration, and anemia, which may
be potentially fatal. In the U.S. alone, estimates point to 1.5
million persons being affected by the disease.1 Environmental
and the Dipterocarpaceae.14 The natural structures range from
and genetic factors are thought to influence the course of the
disease.2 While patients may experience no symptoms during
simple indanes and indanones with antibacterial activity, e.g., 1-
indanone (2) isolated from the cyanobacterium Nostoc,10 to the
periods of remission, this may be interrupted with “flare-ups”,
acute episodes of active inflammation. There is nothing in the
pterosin family of fern and fungal metabolites, e.g., Pterosin B
current pharmaceutical armamentarium to address the needs of a
substantial number of sufferers for an extended period, while
(3), which shows cytotoxicity toward the human leukemia HL-
60 cell line.11
many patients unable to find suitable treatment will often go on
The therapeutic potential of indane and indanone derived
molecules becomes more evident in the area of medicinal
to surgical intervention.3
IBD drug development has traditionally been very difficult as
chemistry where this scaffold is incorporated into an array of
the disease appears to be multifactorial in etiology and many
chemical structures that demonstrate significant therapeutic
targeted therapies have failed in the clinic.4,5 In this study, we
properties, e.g., the protease inhibitor Indinavir (4), in clinical
have assessed a lead molecule PH46A (1), representing a novel
use15,16 for the treatment of HIV-1, the indanone, Donepezil (5),
class of chemical compounds, in two mouse models of IBD: an
a potent acetylcholinesterase inhibitor, which is prescribed for
acute dextran sulfate sodium (DSS) model of murine colitis6 and
the treatment of Alzheimer’s disease,17 the propargalyl amine
the more chronic spontaneous model of IL-10−/− mice.7 We
show that 1 has the potential to have superior efficacy to standard
mainline therapy for induction of remission. Although these
models do not represent all aspects of IBD in man, they bring an
essential contribution to the development of much needed
therapeutic solutions.
The Indane scaffold is of key importance in the natural world.
Rasigiline, an irreversible inhibitor of monoamine oxidase used in
Parkinson’s disease (6),18 and the indanone Sulindac (7), a
nonsteroidal anti-inflammatory that has recently demonstrated
antiproliferative and apoptotic effects.19,20
The development of 1 originated in an investigation into the
traditional use of indanes, derived from Pteridophytes of the
It occurs in a range of biologically active natural products that
have been isolated from fungi,8,9 bacteria,10 Pteridophytes,11,12
and, recently, some higher plants including the Annonaceae13
Received: March 26, 2012
Published: June 4, 2012
© 2012 American Chemical Society
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The efficacy of 1 (3−30 mg/kg) was assessed in the 5% DSS
murine model of colitis, characterized by weight loss, shortening
of the colon, intestinal bleeding, and histological damage as well
as on the IL-10−/− murine model of colitis, a spontaneously
developing chronic colitis characterized by weight loss, raised
levels of serum amyloid A, and damage to the intestinal epithelial
layer without chemically induced tissue damage.
RESULTS
■
Chemistry. Compound 1 is synthesized via a key
intermediate 10 formed by the coupling of the silyl trimethyl
ether of 1-indanone (8) and the dimethyl ketal of 2-indanone (9)
with a catalytic amount of trimethylsilyltriflate over 3 Å
molecular sieves.28 Intermediate 10 was then alkylated with
methyl (4-bromomethyl) benzoate to yield the racemic keto
ester 11, as shown in Scheme 1.
This mixture of enantiomers was reduced with NaBH4 to yield
the diastereoisomeric esters 12 and 13, which were separated by
flash column chromatography using a TBME and EtOAc
gradient. The esters were hydrolyzed quantitatively to their
corresponding acids (14 and 15) by refluxing with 10% NaOH in
MeOH. Separation of 14 and 15 into their constituent
enantiomers 16, 17 and 18, 19, respectively (Scheme 2) was
achieved by chiral HPLC. The absolute stereochemistry of the 16
was established by single crystal X-ray analysis of its S-(−)-α-
methybenzylamine salt. Further, the absolute stereochemistry of
18 and 19 was established by conversion of the alcohols (16−19)
to their ketones and by correlation of their optical rotations.30
The acids 16 and 19 were converted into their N-methyl-(D)-
glucamine salts 1 and 20 by stirring the acids in MeOH with N-
methyl-D-glucamine.
Onychium family, in Taiwanese traditional medicine.21,22 In these
early studies, we established the smooth muscle relaxant and
mast cell stabilization effects of nature identical and synthetic
monomeric analogues.23−26 We subsequently synthesized and
investigated a novel series of indane dimers which were shown to
inhibit the release of histamine from rat peritoneal mast cells. The
impetus behind the research was to identify a novel molecule
which retained significant smooth muscle relaxant activity and
which possessed potent mast cell stabilizing activity, a molecule
which combined the effects of disodium cromoglycate with a
bronchodilator and which might find utility in the treatment of
asthma.27−29 However, while it appeared that the two activities
were mutually exclusive, we successfully identified a novel indane
scaffold with potential therapeutic value for the treatment of
inflammatory disease.28
PHARMACOLOGICAL EVALUATION
■
Effect of Enantiomers in DSS Colitis. Specific Pathogen-
Free female Balb/c mice, 6−8 weeks of age, given 5% DSS in
drinking water, were administered vehicle or 16−19 at 30 mg/kg
po as a suspension in carboxymethyl cellulose (0.5%)/2% Tween
80 daily for 7 days. Mice administered vehicle developed
progressive weight loss (Figure 1c) and increased disease activity
index (DAI) values following DSS treatment (Figure 1a,b).
There were no overt reactions following 7 days of daily oral (po
30 mg/kg) treatment with the compounds 16−19. The DAI
measures the extent of the disease in this model. 18 was without
activity on this variable, there not being any significant (P > 0.05)
difference in DAI at any time point (Figure 1a). At day 7, both 16
and 19 significantly (P < 0.01) reduced DAI by a considerable
margin, from 9.0 0.53 for vehicle controls to 2.5 0.71 for 16
More polar analogues of the active compounds previously
described28 were assessed in an inflammatory disease model, the
5% DSS model of murine colitis. Of these, 1 was chosen as the
lead candidate on the basis of its efficacy. The scaffold of 1
incorporates the indane skeleton of the original molecules and is
the N-glucamine salt of a single enantiomer of this novel drug.
and 3.2
0.73 for 19, there being no significant difference
between the two (Figure 1b). In comparison, 17 reduced DAI to
only 5.3 0.6. This was significantly (P > 0.05) less potent than
either 16 or 19. Further, while the DAI in 17-treated mice was
Scheme 1. Synthesis of Keto-ester 11 and Diastereoisomers 12 and 13 from Key Intermediate 10
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Scheme 2. Separation of Enantiomers of 12 and 13
Figure 1. Effect of vehicle (Veh), and 16−19 (30 mg kg) on disease activity index (DAI) (a), DAI on day 7 (b), bodyweight (c), and colon length (d).
Values are expressed as a mean SEM, n = 6. Asterisks indicate statistical significance at P < 0.01.
statistically (P < 0.01) less than vehicle controls at day 7, at day 6
there was no statistical (P > 0.05) difference between 17 and
vehicle (Figure 1a). In conclusion, of the four enantiomers, 16−
19, both 16 and 19 are highly active in this model at 30 mg/kg. 17
has minimal activity which is significantly (P < 0.01) less than 16
and 19, and 18 is almost devoid of activity in this DSS murine
colitis model. On colon length, all enantiomers increased colon
length compared to vehicle control in order of potency 16 > 19 >
17 > 18 (Figure 1d).
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Figure 2. Effect of vehicle (Veh) and 16, 19, 1, and 20 (10 mg kg) on disease activity index (DAI) (a), DAI on day 7 (b), body weight (c), and colon
length (d). Values are expressed as a mean SEM, n = 6. Asterisks indicate statistical significance at P < 0.01.
Effect of the Enantiomers 16 and 19 and Their N-
Glucamine Salts (1, 20) at 10 mg/kg in 5% DSS Colitis.
Given that both 16 and 19 show considerable activity in the 5%
DSS model at 30 mg/kg, we then re-examined their activity,
together with their N-glucamine salts (1, 20) at the lower dose of
10 mg/kg, given daily for 7 days as a suspension or solution in
carboxymethyl cellulose (0.5%)/2% Tween 80. No adjustment
was made in the dosages of the salts to compensate for their
increased molecular weight. Mice administered vehicle devel-
oped progressive weight loss (Figure 2c), and this was
significantly ameliorated by both 16 and 1 but not by 19 and
20 at 10 mg/kg. Both 19 and 20 at 10 mg/kg had no significant
(P >0.01) effect on DAI in the 5%-DSS murine colitis model
when compared to vehicle control (see Figure 2a,b). In contrast,
at day 7, both 16 and 1 at 10 mg/kg significantly (P < 0.01) and
potently reduced DAI from 9.3 0.51 (vehicle) to 2.1 0.7 and
3.3 0.52, respectively (Figure 2b). Both 16 and 1 significantly
increased colon length compared to vehicle control, whereas 19
and 20 had no significant effect (Figure 2d).
weight loss was reduced when DSS-mice were administered 1
(orally, per os (po)) daily, at all dose levels; at 30 mg/kg, 1
reduced weight loss to only 6.2% of non-DSS control mice. With
12.6% weight loss, prednisolone was only as effective as 10 mg/
kg 1 (12.8%) (Figure 3a). In addition, the DAI began to become
In conclusion, 16, together with its N-glucamine salt 1, is the
most potent of the four enantiomers by a considerable margin
and the only enantiomer to retain activity at this dose level. As a
consequence of the limited amount of material, much of the
comparison solubility and PK studies were performed on
compounds 19 and 20. Such studies showed that the
bioavailability of the acid, 19, was only 61%, whereas that of
the salt, 20, was 100%. Pharmacokinetic data on 1 has shown that
it is 100% bioavailable and >6000 μg mL aqueously soluble.31
Compound 1 was therefore chosen as the preferred compound.
Graded Response to 1 in Acute DSS Colitis and a
Comparison with Prednisolone. The values of a number of
disease indicators in mice treated with DSS were compared with
those in mice treated with the standard steroid therapy,
prednisolone (5 mg/kg), typically used for induction of
remission in patients suffering from active disease. BALB/c
mice, seven per group, in which an acute colitis had been induced
by administering 5% DSS in the drinking water for 7 days, were
assessed daily over this period. Body weight declined
progressively over the 7 days, by 18.55% at day 7, but this
Figure 3. Effect of vehicle (A), 1 (3, 10, 30 mg kg) (B, C, D),
prednisolone (5 mg kg) (E), and water only (non-DSS; N) on disease
activity index (DAI) (a), DAI on day 7 (b), body weight (c), and colon
length (d). Values are expressed as a mean SEM, n = 6−7. Asterisks
indicate statistical significance at P < 0.01.
elevated at day 3 and by day 4, and there was already apparent a
difference in treated and nontreated groups (Figure 3a). At day 7,
DAI was significantly and dose-dependently reduced by 1 to
24%, 54%, and 80% of the vehicle group for the 3, 10, and 30 mg/
kg doses, respectively, while prednisolone (5 mg/kg) reduced
DAI by 50% (Figure 3b). Another disease indicator measured
was colon length because treatment with DSS typically results in
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a shortening of the colon. By day 7, 1 reduced this shortening in a
dose-dependent manner, with 30 mg/kg 1 significantly (P <
0.01) more effective than prednisolone at 76% and 39%
inhibition, respectively (Figure 3d). Furthermore, colonic
MPO activity, representing the level of inflammatory neutrophil
cell infiltration into the gut wall, was increased by almost 8-fold
by the DSS treatment. By day 7, both 1 at 30 mg/kg and
prednisolone significantly reduced MPO by 63% and 54%,
respectively (Figure 4a). In addition, DSS-treatment resulted in
Figure 4. Effect of vehicle (A), 1 (3, 10, 30 mg kg) (B, C, D),
prednisolone (5 mg kg) (E), and water only (non-DSS; N) on colon
myeloperoxidase activity (MPO) (a), colon IL-6 levels (b), colon IL-1β
levels (c), and TNFα (4d). Values are expressed as a mean SEM, n =
6−7. Asterisks indicate statistical significance at P < 0.01.
raised levels of the colonic pro-inflammatory cytokines IL1b, IL6,
and TNFα, to 0.744 0.076 ng/mg, 1.057 0.1784 ng/mg, and
1.478
0.378 ng/mg, respectively. In each case, 1 caused a
Figure 5. Scoring of colons from DSS-treated mice administered (A), 1
(3, 10, 30 mg kg) (B, C, D), and prednisolone (5 mg kg) (E).
Representative H&E stained sections of distal colon of untreated
(normal) mice and animals treated with DSS and compounds as
indicated for 7 days. Colon of normal, non-DSS-treated, mouse
(normal). Colon of 5% DSS-treated mice injected with saline (A)
with 3, 10, and 30 mg kg 1 (B, C, D), or 5 mg kg prednisolone (E).
significant (P < 0.01) and dose-dependent reduction in these
cytokine levels. Prednisolone also reduced (p < 0.01) raised
cytokine levels; for each cytokine there was no significant
difference between the effect of prednisolone 5 mg/kg and 1 at
the higher dose at day 7 (Figure 4). The colons were scored for
histological damage. Treatment with 1 caused a marked and
dose-dependent decrease in such scores, with 1 (30 mg/kg)
reducing histological damage scores from 6.5 0.56 for vehicle
control to 1.6 0.24, i.e., by more than 75%. In comparison, the
effect of prednisolone on reducing histological damage scores, at
relative to mice treated with vehicle (score: 6.22 0.72) (Figure
6b). These data indicate that chronic treatment of IL-10−/− with
1 significantly ameliorates clinical indicators of colitis in this
model.
26% (4.8
significance (P > 0.05).
0.48) (Figure 5), did not achieve statistical
Treatment of IL-10 Deficient Mice with 1. IL-10−/−
BALB/c strain mice were treated from 6 to 8 weeks of age,
when colon inflammation is mild,7 with 1 (300 mg/kg/week) or
vehicle on a Mon/Wed/Fri dosing schedule over a period of 9
weeks. There was no significant differences in weight loss or overt
colitis−rectal bleeding or prolapse requiring humane killing of
mice between vehicle-treated (3/12 mice killed) and 1-treated
(1/12 mice killed) IL-10−/− mice. After 9 weeks, serum was
recovered from mice and SAA analyzed as a marker for the
severity of colitis. In IL-10−/− mice treated with 1, there were
significantly (P < 0.05) decreased SAA levels relative to vehicle-
treated mice (468.9 38.37 to 342.7 43.01 μg/mL) (Figure
6a). Histology sections of colons from IL-10−/− mice treated
with vehicle and 1 (Figure 6c,d) were scored for histological
damage. The extent of colon pathology was significantly reduced
(P < 0.05) in IL-10−/− mice receiving 1 (score: 4.18 0.48),
DISCUSSION
■
In this study, we evaluated the efficacy of the novel, anti-
inflammatory indane compound, 1, in two distinct murine colitis
models. Oral treatment of mice with 1 attenuated the severity of
colitis in an acute DSS model of colitis. Furthermore, chronic
administration of 1 to IL-10−/− mice ameliorated the
spontaneous development of colonic inflammation. As 1 has
efficacy in two distinct acute and chronic mouse models of IBD,
which are engendered by diverse mechanisms, the drug’s
therapeutic effect is independent of the model. These data
indicate that 1 has potential for the development of a drug
therapy in the treatment of IBD.
Traditionally, IBD patients were maintained on remission by
use of a 5-aminosalycilate. While its use in UC provides
considerable benefit, both in inducing remission in mild to
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CONCLUSIONS
■
Compound 1 has significant activity in the IL-10−/− model of
IBD and is highly efficacious in the acute DSS model. This
efficacy compares favorably with the potent steroid, predniso-
lone, and supports its potential use to treat acute exacerbations of
the disease. Further, the graded response to 1 may also lend itself
to be used at a lower dose to maintain periods of remission. This
small molecule represents a new class of compounds for the
treatment of IBD.
EXPERIMENTAL SECTION
■
Chemistry. Melting points were determined on a Me-Opta hot stage
and are uncorrected. Nuclear magnetic resonance spectra (NMR) were
recorded using a Bruker Avance 400 instrument. 1H NMR spectra were
recorded at 400.13 MHz, and 13C NMR spectra were recorded at 75.47
MHz. Chemical shifts are given in parts per million (ppm) and were
referenced to solvent signals. COSY, HSQC, and HBMC were
performed using Bruker microprograms. Spectra were analyzed using
MestRec Magnetic Resonance Companion version 4.4.1.0. High
resolution mass spectra were determined on a Micromass LCT
instrument operating in ES+ mode. HPLC was performed on a reverse
phase 250 mm × 4.6 mm Hypersil BDS C18, 5 μ, column using a Waters
600 chromatograph equipped with an auto sampler, column oven, and
dual wavelength detector. The flow rate was 1 mL/min (for ketone and
esters) and 0.6 mL/min (for acids and salts) with isochratic elution with
a mobile phase consisting of 70:30 CH3CN/0.1% aq acetic acid.
Detection: 210 nm. The column temperature was room temperature.
TLC was performed on commercially precoated plates (Merck,
Kieselgel 60F254). Flash column chromatography was carried out on
Merck Kieselgel 60 (200−400 mesh) (E. Merck, Art 9385, Darmstadt).
Visualization was by examination under visible light UV (254 nm) and
UV (365 nm) or by spraying with anisaldehyde/concd sulphuric acid
reagent followed by heating for 5−10 min at 110 °C. Test compounds 1
and 16−20 were >98% pure by HPLC.
Figure 6. Effect of 1 (300 mg kg week) in IL-10−/− mice. (a) Histology
scores of colon damage. (b) Serum amyloid A (SAA) levels.
Representative H&E stained sections of mouse colon of untreated IL-
10−/− mice. Values are expressed as mean SEM, n = 9 and 11 for
vehicle and 1 groups, respectively (c), showing breakdown of the
mucosal epithelium and crypt erosion with cell infiltration of mice
treated with 1 (d), showing substantially less epithelial damage and
limited cell infiltration.
moderate disease and in preventing relapse,32 its usefulness to
maintain remission in CD is questionable and no longer
recommended, leaving patients with mild CD few options for
maintenance therapy.1 In contrast, the mainstay of treatment of
active disease is a corticosteroid, used for limited periods to
return both UC and CD patients to remission. However,
systemic corticosteroids such as prednisolone are associated with
adverse effects, while nonsystemic budesonide, which has fewer
side-effects, does not maintain remission.33 Compound 1, with a
potentially more favorable toxicological profile than cortico-
steroids, could thus provide an alternative, at the least in cases of
steroid resistance, or in a steroid-sparing regimen. Indeed, our
toxicological and toxicokinetic studies have shown a maximum
tolerated dose (MTD) of >2000 mg/kg in rats and >500 mg/kg
in dogs.34 In addition, 1 could also help avoid serious adverse
effects3 of alternative treatments such as the immunosuppressive
drugs azathioprine and mercaptopurine as well as methotrexate
and cyclosporine. Further, effective alternatives such as the anti-
TNFα antibodies, infliximab, and adalimubab have, however, not
proven efficacious in all patients. There could therefore be an
opportunity to treat anti-TNFα nonresponders with 1 without a
generalized immunosuppression and subsequent development of
opportunistic infections.
In the acute DSS model of colitis, 1 can substantially reduce
disease severity, as ascertained by multiple parameters.
Furthermore, in the chronic spontaneous IL-10−/− mouse
model, 1 ameliorated development of colitis. In these two
models of colitis, disease is evoked by distinct processes that
range from maintenance of epithelial barrier and its integrity to
aberrant regulation of innate and adaptive immune homeostasis
in the gastro-intestinal tract. This infers that 1 suppresses colon
inflammation via a protective process, as yet unknown, that is
common to both models. Elucidation of the mechanism of action
of 1 in reducing inflammation in colitis will lead to new insights
into the development of IBD and opportunities to develop
further new therapeutic strategies.
Preparation of (1H-inden-3-yloxy)trimethylsilane (8), 2,2-dime-
thoxy-2,3-dihydro-1H-indene (9), and 2,3-dihydro-2-(2,3-dihydro-2-
methoxy-1H-inden-2-yl)inden-1-one (10) are outlined in Sheridan et al
2009.28
Methyl 4-((2,3-Dihydro-2-(1H-inden-2-yl)-1-oxo-1H-inden-2-yl)-
methyl)benzoate (11). To a stirred solution of 10 (1.00 g, 4 mmol)
in tert-butanol (5 mL) and diethyl ether (30 mL) was added methyl (4-
bromomethyl)benzoate (1.41 g, 6 mmol). To this was added a solution
of potassium tert-butoxide in tert-butanol (30 mL), slowly, dropwise.
With each drop, the mixture turned a yellow color and then reverted to
its original gray color. The mixture was stirred for further 3 h until the
TLC (hexane/EtOAc, 4:1) showed no more starting material. The
reaction was quenched by the addition of satd NH4Cl. The layers were
separated and the aqueous layer extracted with diethyl ether (2 × 120
mL). The combined organic extracts were washed with water and brine,
dried over MgSO4, and evaporated in vacuo. The solid product
precipitated from the crude on removal of most of the solvent. This was
filtered off and washed with cold diethyl ether to give compound 11
(0.98 g, 62%) as a cream solid; mp 143−147 °C. 1H NMR (400 MHz,
CDCl3) δH (ppm) 7.87 (d, J = 7.52 Hz, 2H), 7.74 (d, J = 7.68 Hz, 1H),
7.57 (t, J = 7.48 Hz, 1H), 7.40 (d, J = 7.56 Hz, 2H), 7.36 (t, J = 7.48 Hz,
1H), 7.30−7.14 (m, 5H), 6.74 (s, 1H), 3.88 (s, 3H), 3.61−3.31 (m, 6H).
13C NMR (100 MHz, CDCl3) δC (ppm) 205.2, 166.5, 151.8,
148.3,143.7, 142.7, 142.5, 134.7, 2 × 129.6, 2 × 129.0, 128.2, 128.0,
127.3, 126.0, 125.7, 124.3, 124.2, 123.1, 120.3, 56.7, 51.6, 41.8, 38.4,
37.2. HRMS (ESI) m/z calculated for C27H22O3 (M + Na)+, 471.1474;
found, 417.1461.
Methyl 4-((2,3-Dihydro-1-hydroxy-2-(1H-inden-2-yl)-1H-inden-2-
yl)methyl)benzoate (12) and (13). To a stirred solution of 11 (2.00 g,
5.07 mmol) in MeOH (20 mL) and tetrahydrofuran (20 mL) was added
NaBH4 (0.29 g, 7.61 mmol), slowly (10 min), portionwise at 0 °C. The
temperature of the reaction was maintained between 0 and 5 °C
throughout the reaction. The mixture was stirred for further 3 h until the
TLC (hexane/EtOAc/DCM, 5:1:1) showed no more starting material.
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On completion, the reaction was quenched by the addition of water (30
mL) and MeOH was removed from the reaction mixture under vacuum.
The resulting residue was extracted between water (30 mL) and diethyl
ether (3 × 25 mL). The combined organic extracts were washed with
water and brine, dried over MgSO4, and evaporated in vacuo. The
residue was purified by flash column chromatography on silica gel
(eluent: hexane/EtOAc/DCM, 5:1:1). All homogeneous fractions were
collected and the solvent was evaporated to afford compounds 12 (1.07
g, 54%) and 13 (0.97 g, 49%) as white amorphous solids.
2 × 129.9, 2 × 129.3, 128.2, 128.1, 126.6, 126.5, 126.0, 124.5, 123.9,
123.5, 123.1, 120.2, 82.4, 55.5, 39.5, 38.2, 38.1. HRMS (ESI) m/z
calculated for C26H22O3 (M − H)+, 381.1485; found, 381.1494.
Separation of (15) to Yield (18) and (19). Enantiomers were
separated by preparative Chiral HPLC: column 250 mm × 76 mm
CHIRALCEL OJ 20 μm, detection UV 203 nm, flow rate 500 mL/min.
Mobile phase methanol, Rt (18) 13.6 min; Rt (19) 21.4 min.
4-(((1R,2S)-2,3-Dihydro-1-hydroxy-2-(1H-inden-2-yl)-1H-inden-2-
yl)methyl)benzoic Acid (18). Melting point 136−140 °C. Optical
rotation [α]D20 = −39.3° (MeOH, 0.66%, 20 °C). 1H NMR (400 MHz,
CDCl3) δH (ppm) 7.93 (d, J = 8.08 Hz, 2H), 7.26−7.47 (m, 7H), 7.19 (t,
J = 7.34 Hz, 1H), 7.05 (d, J = 8.08 Hz, 2H), 6.70 (s, 1H), 5.08 (s, 1H),
2.90−3.59 (m, 6H). 13C NMR (100 MHz, CDCl3) δc (ppm) 171.2,
149.8, 144.1, 143.8, 143.0, 142.7, 141.0, 130.4, 2 × 129.8, 2 × 129.4,
128.5, 126.9, 126.7, 126.0, 124.7, 124.5, 124.1, 123.2, 120.3, 81.6, 55.9,
43.1, 39.9, 37.9. HRMS (ESI) m/z calculated for C26H22O3 (M − H)+,
381.1485; found, 381.1494.
4-(((1S,2R)-2,3-Dihydro-1-hydroxy-2-(1H-inden-2-yl)-1H-inden-2-
yl)methyl)benzoic Acid (19). Melting point 195−196 °C. Optical
rotation [α]D20 = +32.1° (MeOH, 1.18%, 20 °C). 1H NMR (400 MHz,
CDCl3) δH (ppm) 7.93 (d, J = 8.12 Hz, 2H), 7.26−7.47 (m, 7H), 7.19 (t,
J = 7.34 Hz, 1H), 7.05 (d, J = 8.12 Hz, 2H), 6.70 (s, 1H), 5.08 (s, 1H),
2.94−3.59 (m, 6H). 13C NMR (100 MHz, CDCl3) δc (ppm) 171.2,
149.8, 144.1, 143.8, 143.0, 142.7, 141.0, 130.4, 2 × 129.8, 2 × 129.4,
128.5, 126.9, 126.7, 126.0, 124.7, 124.5, 124.1, 123.2, 120.3, 81.6, 55.9,
43.1, 39.9, 37.9. HRMS (ESI) m/z calculated for C26H22O3 (M − H)+,
381.1485; found, 381.1488.
N-Methyl-(D)-glucamide Salt of 4-{[(1S,2S)-2,3-Dihydro-1-hy-
droxy-2-(1H-inden-2-yl)-1H-inden-2-yl]methyl}benzoic Acid (1). To
the MeOH (HPLC grade, 10 mL) suspension of the acid 16 (1.08 g, 2.8
mmol) at 55 °C was added N-methyl-D-glucamine (0.55 g, 2.8 mmol, 1
equiv) portionwise. This mixture was stirred at 55 °C until a clear
solution was obtained. The solution was then hot-filtered into a clean
flask and allowed to cool. Isopropyl alcohol (20 mL) was then added to
precipitate the salt. The salt was then filtered and washed with
acetonitrile (20 mL), sucked dry, and then dried in a vacuum oven at 40
°C. The title salt (1.61 g, 98%) was obtained as a white solid; mp 165−
167 °C. Optical rotation: [α]D20 = −88.6° (MeOH, 70 mg/10 mL, 20
°C). 1H NMR (400 MHz, DMSO-d6) δH (ppm) 7.62 (d, J = 7.96 Hz,
2H), 7.39−7.33 (m, 2H), 7.25−7.18 (m, 4H), 7.15 (t, J = 7.34 Hz, 1H),
7.06 (t, J = 7.26 Hz, 1H), 6.81 (d, J = 8.04 Hz, 2H), 6.39 (s, 1H), 5.81
(broad s, 1H), 5.03 (s, 1H), 3.84−3.80 (m, 1H), 3.64 (broad d, J = 4.92
Hz, 1H), 3.59−3.35 (m, 6H), 3.14 (d, J = 13.56 Hz, 1H), 3.00−2.76 (m,
4H), 2.64 (d, J = 13.60 Hz, 1H), 2.42 (apparent s, 3H). 13C NMR (150
MHz, DMSO-d6) δc (ppm) 169.5, 154.2, 145.1, 144.4, 142.8, 141.7,
140.3, 133.6, 2 × 129.2, 2 × 128.5, 127.5, 127.0, 126.2, 126.0, 124.4,
124.2, 123.7, 123.4, 120.1, 81.0, 71.2, 70.5, 70.3, 69.3, 63.5, 55.7, 51.8,
39.4, 38.2, 37.9, 33.9. HRMS (ESI) m/z calculated for C33H39O8N (M −
H)+, 576.2592; found, 576.2612.
Compound (12). Melting point 141−143 °C. 1H NMR (400 MHz,
CDCl3) δH (ppm) 6.99 (d, J = 7.72 Hz, 2H), 7.46 (d, J = 7.04 Hz, 2H),
7.20−7.31 (m, 6H), 6.97 (d, J = 7.80 Hz, 2H), 6.50 (s, 1H), 5.29 (d, J =
24.16 Hz, 1H), 3.91 (s, 3H), 3.60 (d, J = 22.68 Hz, 1H), 3.48 (d, J =
22.88 Hz, 1H), 3.28 (d, J = 13.24 Hz, 1H), 3.06 (d, J = 15.64 Hz, 1H),
3.51 (d, J = 16.00 Hz, 1H), 2.86 (d, J = 13.28 Hz, 1H). 13C NMR (100
MHz, CDCl3) δC (ppm) 166.9, 152.3, 144.1, 143.9, 143.4, 142.4, 140.0,
2 × 129.8, 2 × 128.6, 128.1, 128.0, 127.5, 126.6, 126.0, 124.4, 123.9,
123.6, 123.2, 120.2, 82.4, 55.5, 51.6, 39.6, 38.2, 38.0. HRMS (ESI) m/z
calculated for C27H24O3 (M + Na)+, 419.1606; found, 419.1618.
Compound (13). Melting point 161−163 °C. 1H NMR (400 MHz,
CDCl3) δH (ppm) 7.87 (d, J = 8.12 Hz, 2H), 7.45 (d, J = 7.04 Hz, 1H),
7.42 (d, J = 7.40 Hz, 1H), 7.35−7.26 (m, 5H), 7.19 (d, J = 7.40 Hz, 1H),
7.02 (d, J = 8.16 Hz, 2H), 6.69 (s, 1H), 5.07 (d, J = 7.92 Hz, 1H), 3.90 (s,
3H), 3.55 (d, J = 22.68 Hz, 1H), 3.37 (d, J = 22.76 Hz, 1H), 3.27 (d, J =
15.88 Hz, 1H), 3.26 (d, J = 13.48 Hz, 1H), 2.95 (d, J = 15.76 Hz, 1H),
2.93 (d, J = 13.60 Hz, 1H). 13C NMR (100 MHz, CDCl3) δC (ppm)
166.6, 149.9, 143.8, 143.1, 143.0, 142.7, 141.0, 130.3, 2 × 129.7, 2 ×
128.8, 128.4, 127.8, 126.7, 126.0, 124.6, 124.5, 124.1, 123.1, 120.3, 81.6,
55.9, 51.6, 43.0, 39.9, 37.9. HRMS (ESI) m/z calculated for C27H24O3
(M + Na)+, 419.1605; found, 417.1618.
Hydrolysis of Methyl Ester (12) or (13). The methyl ester was placed
in a round-bottomed flask and 10% aq NaOH (1 mL) was added to it,
followed by sufficient MeOH to form a solution (6 mL). The resulting
mixture was stirred under reflux conditions, and the reaction was
monitored by TLC (hexane/EtOAc, 4:1). After 24 h, no further ester
was seen. The mixture was cooled, and satd aq NH4Cl (10 mL) was
added (solution at pH = 12). The mixture was acidified with 2 M aq HCl
until pH = 2. The product was extracted from the cloudy solution into
EtOAc (3 × 20 mL). The combined extracts were dried over MgSO4 and
evaporated in vacuo to give target compound (quantitative). The crude
material could be isolated in very pure form by slurry in a solvent mixture
of hexane/MTBE (v/v, 80:20). The yield from this step was generally
>95% of 14 or 15 as cream solids.
Separation of (14) to Yield (16) and (17). Enantiomers were
separated by preparative Chiral HPLC: column 250 mm × 76 mm
CHIRALPAK AZ 20 μm, detection UV 203 nm, flow rate 250 mL/min.
Mobile phase 100:0.1, methanol/acetic acid. Rt (16) 6.65 min; Rt (17)
8.48 min.
4-(((1S,2S)-2,3-Dihydro-1-hydroxy-2-(1H-inden-2-yl)-1H-inden-2-
yl)methyl)benzoic Acid (16). Melting point 184−185 °C. Optical
20
1
N-Methyl-(D)-glucamine Salt of 4-(((1S,2R)-2,3-Dihydro-1-hy-
droxy-2-(1H-inden-2-yl)-1H-inden-2-yl)methyl)benzoic Acid (20).
To the MeOH (HPLC grade, 1 mL) solution of the acid 19 (0.2 g,
0.52 mmol, 1 equiv) at 55 °C was added a MeOH (2 mL) suspension of
N-methyl-D-glucamine (0.102 g, 0.52 mol, 1 equiv). This mixture was
stirred at room temperature overnight. Since no filterable material was
obtained, the solvent was evaporated completely under reduced
pressure and the resulting solid was slurried in EtOAc. The salt was
filtered, washed with EtOAc, and then dried in vacuum oven at 40 °C.
The title salt (0.222 g, 74%) was obtained as a pale-yellow solid; mp 70−
rotation [α]D = −162.5° (MeOH, 40 mg/10 mL, 20 °C). H NMR
(400 MHz, CDCl3) δH (ppm) 7.90 (d, J = 8.04 Hz, 2H), 7.44 (d, J = 7.08
Hz, 2H), 7.25−7.34 (m, 5H), 7.18 (dt, J = 7.03, 1.75 Hz, 1H), 7.00 (d, J
= 8.08 Hz, 2H), 6.49 (s, 1H), 5.27 (s, 1H), 3.58 (d, J = 22.64 Hz, 1H),
3.46 (d, J = 22.64 Hz, 1H), 3.29 (d, J = 13.32 Hz, 1H), 3.07 (d, J = 15.64
Hz, 1H), 3.02 (d, J = 15.60 Hz, 1H), 2.87 (d, J = 13.52 Hz, 1H). 13C
NMR (100 MHz, CDCl3) δc (ppm) 171.0, 152.0, 144.7, 144.0, 143.2,
142.3, 140.0, 2 × 129.9, 2 × 129.3, 128.2, 128.1, 126.6, 126.5, 126.0,
124.5, 123.9, 123.5, 123.1, 120.2, 82.4, 55.5, 39.5, 38.2, 38.1. HRMS
(ESI) m/z calculated for C26H22O3 (M − H)+, 381.1485; found,
381.1501.
20
80 °C (decomp/hygroscopic). Optical rotation: [α]D = +63.3°
1
(MeOH, 32 mg/10 mL, 20 °C). H NMR (400 MHz, DMSO-d6) δH
4-(((1R,2R)-2,3-Dihydro-1-hydroxy-2-(1H-inden-2-yl)-1H-inden-2-
(ppm): 7.69 (t, J = 7.92 Hz, 2H), 7.37 (d, J = 7.32 Hz, 1H), 7.33−7.20
(m, 5H), 7.15 (t, J = 7.46 Hz, 1H), 7.06 (t, J = 7.28 Hz, 1H), 6.91 (d, J =
8.00 Hz, 2H), 6.38 (s, 1H), 5.45 (broad s, 1H), 4.97 (s, 1H), 3.89−3.87
(m, 1H), 3.67 (broad d, J = 4.80 Hz, 1H), 3.61−3.38 (m, 6H), 3.17 (t, J =
14.84 Hz, 1H), 2.98−2.80 (m, 4H), 2.47 (apparent s, 3H). 13C NMR
(100 MHz, DMSO-d6) δC (ppm) 170.2, 152.7, 144.9, 144.7, 143.5,
141.4, 140.6, 135.1, 2 × 129.3, 2 × 128.6, 128.3, 127.7, 126.3, 125.9,
124.5, 124.4, 123.5, 123.3, 120.0, 80.9, 71.3, 70.5, 70.3, 68.9, 63.5, 55.9,
yl)methyl)benzoic Acid (17). Melting point 195−196 °C. Optical
20
1
rotation [α]D = +127.5° (MeOH, 40 mg/10 mL, 20 °C). H NMR
(400 MHz, CDCl3) δH (ppm) 7.90 (d, J = 7.32 Hz, 2H), 7.44 (d, J = 6.96
Hz, 2H), 7.25−7.31 (m, 5H), 7.18 (t, J = 6.90 Hz, 1H), 7.00 (d, J = 7.56
Hz, 2H), 6.49 (s, 1H), 5.27 (s, 1H), 3.58 (d, J = 22.60 Hz, 1H), 3.46 (d, J
= 22.52 Hz, 1H), 3.29 (d, J = 13.28 Hz, 1H), 3.07 (d, J = 16.00 Hz, 1H),
3.02 (d, J = 15.80 Hz, 1H), 2.87 (d, J = 13.32 Hz, 1H). 13C NMR (100
MHz, CDCl3) δc (ppm) 171.2, 152.0, 144.7, 144.0, 143.2, 142.3, 140.0,
5503
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Journal of Medicinal Chemistry
Article
51.5, 42.4, 39.6, 38.2, 33.4. HRMS (ESI) m/z calculated for C33H39O8N
(M − H)+, 576.2592; found, 576.2616.
randomly alphabetically labeled. When mice were killed, a 1 cm piece of
the proximal colon was removed from IL-10-mice. All tissues were fixed
in 10% formaldehyde−saline, and sections were prepared and stained
with hematoxylin and eosin, as described.36 All histology scoring was
performed in a blinded fashion independently by two observers. Colon
sections from mice were graded using a histological index ranging from 0
to 4, based on the degree of epithelial layer erosion, goblet cell depletion,
and inflammatory cell infiltrate37 (0 = normal, 1 = minimal evidence of
inflammatory infiltrate, 2 = significant evidence of inflammatory
infiltrate (cryptitis, crypt abscesses), 3 = significant evidence of
inflammatory infiltrate with goblet cell depletion, 4 = significant
evidence of inflammatory infiltrate with erosion of the mucosa). The
maximum possible score was 4. Serum amyloid A (SAA) was measured
by sandwich ELISA using a kit from Life Diagnostics Inc. (PA, USA).
SAA ELISAs were performed following the manufacturer’s instructions.
All data are means and SEM from 11 1-treated mice and nine vehicle
mice, and statistical analysis was made by Student’s t test.
Biological Assays. Animals and Experimental Design. Specific
Pathogen-Free female BALB/c strain mice, 6−8 weeks of age, were
obtained from a commercial supplier (Harlan, UK). Mice were fed
irradiated diet and housed in individually ventilated cages (Tecniplast,
UK) under positive pressure. IL-10−/− mice, on a BALB/c strain
background, were initially purchased from Jackson Laboratories (USA)
and then bred under specific pathogen-free (SPF) conditions at Trinity
College Dublin. In mouse studies, experimental groups were randomly
alphabetically labeled. Throughout experiments, all data recording and
analysis was performed in a blind manner.
All animal experiments were performed in compliance with Irish
Department of Health and Children regulations and were approved by
the Trinity College BioResources ethical review board.
Compounds. DSS (35−50000 kDa) was purchased from (ICN
Biomedical Inc., USA). Prednisolone was obtained from Sigma Aldrich.
All compounds were prepared for oral gavage (0.1 mL po per 10 g body
weight) as an aqueous solution in distilled water. Distilled water was
used as a vehicle control.
AUTHOR INFORMATION
■
Corresponding Author
Dextran Sulfate Sodium (DSS) Model. A 5% DSS solution was
prepared in drinking (tap) water, with fresh DSS solution provided every
second day. The mice were checked each day for morbidity and the
weight of individual mice recorded. Induction of colitis was determined
by weight loss, fecal blood, stool consistency, and, upon autopsy, length
of colon and histology. Approximately 1 cm of distal colon was removed
for histology. The rest of the colon was recovered, snap-frozen, and
stored at −20 °C for immunological analysis. To quantify the severity of
colitis, a disease activity index (DAI) was determined based on previous
studies of DSS-induced colitis.6 DAI was calculated for individual mice
on each day based on weight loss, occult blood, and stool consistency. A
score was given for each parameter, with the sum of the scores used as
the DAI (Weight loss 0% > 9%: 0−4. Stool consistency: 0, 2, 4 as normal,
loose stool, and diarrhea. Stool blood: 0, 2, 4 as none, visible, and gross
bleeding. Maximum score is 12).
Sections of distal colon were fixed in 10% formaldehyde−saline.
Tissue was paraffin-embedded and 5 μm serial sections cut. Slides were
stained with hematoxylin and eosin. Histolological grading was based on
a scoring system (Severity of cell infiltration: 0−3 for none, slight,
moderate, and severe. Extent of injury: 0−3 for none, mucosal, mucosal
and submucosal, transmural. Crypt damage: 0−4 for none, basal 1/3
damaged, basal 2/3 damaged, only surface epithelium intact, loss of
entire crypt, and epithelium.) modified from previous studies on DSS-
induced colitis and histology scoring was performed in a blinded fashion
independently by two observers. The combined score from each feature
graded was calculated for individual mice with the maximum score being
10. After removal of approximately 1 cm of colon for histology, the
remainder of the colon was snap-frozen and stored. Individual colon
tissue samples were thawed, and gut contents were removed, chopped
finely, and homogenized. The protein concentration of the supernatant
was determined. Colon levels of myeloperoxidase (MPO) were
determined using a standard method.35 Cytokines in colon supernatants
were analyzed using conventional sandwich ELISAs.35 Coating
antibodies, standards, and detecting antibodies were obtained from
BDPharMingen or R&D Systems. Levels of cytokines and MPO are
expressed relative to colon protein.
*For N.H.F.: phone, +353-1-8962825; fax, 353-1-89628210; E-
mail, nfrnkish@tcd.ie. For H.S.: phone, +353-1-8962828; fax,
353-1-89628210; E-mail, hsheridn@tcd.ie.
Author Contributions
The authors contributed equally to this work.
Notes
The authors declare the following competing financial
interest(s): N Frankish and H Sheridan are co-founders of
Trino Therapeutics Ltd.
ACKNOWLEDGMENTS
■
This work was supported by The Wellcome Trust, grant
reference no. 067033/Z/02/A, Enterprise Ireland, and Trino
Therapeutics Ltd.
ABBREVIATIONS USED
■
Aq, aqueous; DCM, dichloromethane; EtOAc, ethyl acetate;
NH4Cl, ammonium chloride; MgSO4, magnesium sulfate;
MeOH, methanol; NaBH4, sodium borohydride; Rt, retention
time; CD, Crohn’s disease; DSS, dextran sulfate sodium; DAI,
disease activity index; IBD, inflammatory bowel diseases; IFNγ,
interferon gamma; IL-10, interleukin-10; LPS, lipopolysacchar-
ide; MPO, myeloperoxidase; NF-kB, nuclear factor-kappaB;
NRs, nuclear receptors; SAA, serum amyloid A; UC, ulcerative
colitis; SPF, specific pathogen-free; MPO, myeloperoxidase
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