1-((S)-2-aminopropyl)-1H-indazol-6-ol: A potent peripherally acting 5-HT2 receptor agonist with ocular hypotensive activity

Article abstract of DOI:10.1021/jm050663x

Serotonin 5-HT₂ receptor agonists have been identified as a potential new class of agents for the treatment of ocular hypertension and glaucoma. The initially reported tryptamine analogues displayed either poor solution stability, potent centra

Full text of DOI:10.1021/jm050663x

318
J. Med. Chem. 2006, 49, 318-328
1-((S)-2-Aminopropyl)-1H-indazol-6-ol: A Potent Peripherally Acting 5-HT2 Receptor Agonist
with Ocular Hypotensive Activity
Jesse A. May,* Anura P. Dantanarayana, Paul W. Zinke, Marsha A. McLaughlin, and Najam A. Sharif
Ophthalmology DiscoVery Research, Alcon Research, Ltd., 6201 South Freeway, Fort Worth, Texas 76134
ReceiVed July 13, 2005
Serotonin 5-HT2 receptor agonists have been identified as a potential new class of agents for the treatment
of ocular hypertension and glaucoma. The initially reported tryptamine analogues displayed either poor
solution stability, potent central nervous system activity, or both of these undesirable characteristics and
were unacceptable for clinical evaluation. A series of 1-(2-aminopropyl)-1H-indazole analogues was
synthesized and evaluated for their suitability for consideration as clinical candidates. 1-((S)-2-Aminopropyl)-
1H-indazol-6-ol (9) was identified as a peripherally acting potent 5-HT2 receptor agonist (EC50 ) 42.7 nM,
Emax ) 89%) with high selectivity for the 5-HT2 receptors relative to other serotonergic receptor subtypes
and other families of receptors and has significantly greater solution stability than R-methyl-5-hydroxy-
tryptamine. Additionally, 9 potently lowers intraocular pressure in conscious ocular hypertensive monkeys
(-13 mmHg, 33%); this reduction appears to be through a local rather than a centrally mediated effect.
Compound 9 appears to be an excellent 5-HT2 receptor agonist for conducting further studies directed toward
a clinical proof-of-concept study for this class of ocular hypotensive agents.
Introduction
We have previously reported that 5-HT2 receptor agonists
can favorably reduce intraocular pressure (IOP) in a nonhuman
primate model of ocular hypertension.7-9 A reduction in IOP
was observed with a number of compounds that displayed potent
agonist activity at this receptor, including the prototypic selective
5-HT2 agonist (R)-2-(4-iodo-2,5-dimethoxyphenyl)-1-methyl-
ethylamine (R-DOI, 1), as well as the tryptamine analogues
5-hydroxy-R-methyltryptamine (2) and bufotenine. It was of
interest to identify other tryptamine analogues that would be
potent 5-HT2 agonists and lower IOP following topical ocular
administration but would not enter into the central nervous
system (CNS), thereby minimizing potential CNS-related side
effects. The favorable 5-HT2 affinity and functional activity
reported for isotryptamines10,11 suggested this template as a
potential lead for structural modification. The extremely poor
solution stability of 5-hydroxytryptamines, such as 2, coupled
with our interest in achieving stable solution formulations
suggested that we investigate tryptamine analogues that would
be expected to have greater chemical stability. The high electron
density of the pyrrole moiety of 5-HT contributes significantly
to the reactivity of this substituted indole. Benzimidazole
analogues would provide greater stability; however, a reported
analogue had only weak affinity for the 5-HT2 receptor.10 The
less nucleophilic indazole was of particular interest because of
its increased intrinsic chemical stability. We elected to pursue
the synthesis of a brief series of 1-(2-aminopropyl)-1H-indazol-
6-ol analogues and to assess their solution stability and their in
vitro 5-HT2 receptor profile as well as their propensity to enter
the brain, and, where warranted, to evaluate their efficacy as
ocular hypotensive agents in conscious ocular hypertensive
monkeys, a model that we have found to be highly predictive
of ocular hypotensive activity in humans.12 During the course
of this work, similar indazoles were disclosed in the patent
literature as potential CNS accessible anorectic agents.13-15
Diseases of aging are the most common causes of blindness
and low vision among members of all races and ethnicities. A
recent retrospective analysis of vision studies conducted since
1990 suggests different primary causes of blindness between
ethnicities; however, glaucoma was identified as one of the
major causes of blindness for each classification.1 Elevated
intraocular pressure (IOP) is considered to be a major risk factor
for the development of glaucoma and, if left untreated, can cause
damage to the optic nerve, leading to peripheral visual field
loss and blindness. Though effective therapies for the treatment
of ocular hypertension are available, such as prostaglandin FP
receptor agonists, â-adrenoceptor antagonists, and carbonic
anhydrase inhibitors, there are patients for whom the available
therapies are either not effective or are contraindicated due to
preexisting medical conditions. Thus, there is a continuing need
to identify new pharmacologic approaches to achieve a reduction
in IOP to provide alternative therapies for this blinding disease.
Serotonin (5-hydroxytryptamine, 5-HT) has been identified in
the aqueous humor of humans,2 and this has led to speculation
that 5-HT might be involved in the development of ocular
hypertension and the progression of glaucoma. The expression
of 5-HT2 receptor mRNA in human ciliary body,3 ciliary
muscle,4 and trabecular meshwork cells5 has been recently
established. Furthermore, functional 5-HT2 receptors are present
in the IOP-modulating ocular tissues such as the bovine ciliary
epithelium6 and in human ciliary muscle and trabecular mesh-
work cells.4 The presence of functionally active 5-HT2 receptors
in tissues known to be intimately involved in the control of
IOP suggests that serotonin may have an important role in the
regulation of IOP; however, what role the individual 5-HT2
receptor subtypes have with regard to IOP reduction has not
yet been determined. These findings lend encouragement and
support to the possibility that suitable 5-HT2 agonists could
provide a new therapeutic approach to controlling IOP.
Chemistry
The synthetic approach used to prepare 1-(2-aminoethyl)-1H-
indazol-6-ol (5-hydroxy-2-azaisotryptamine, 6) is outlined in
Scheme 1. Alkylation of 6-nitroindazole with 2-bromoethyl
* Corresponding author. Phone: 817-551-8150. Fax: 817-568-7661.
E-mail: jesse.may@alconlabs.com.
10.1021/jm050663x CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/01/2005

1-((S)-2-Aminopropyl)-1H-indazol-6-ol
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 319
Scheme 1a
a (a) Br(CH2)2OAc, DMF, K2CO3; (b) (i) K2CO3, MeOH; (ii) 10% Pd/C, EtOH, hydrogen; (c) H2SO4-H2O (1:1), NaNO2, 0 °C; (d) (i) C6H5CH2Br,
EtOH, K2CO3; (ii) CH2Cl2, Et3N, CH3SO2Cl; (iii) NaN3, DMF; (iv) 10% Pd/C, EtOH, HCO2NH4, hydrogen.
Scheme 2a
a (a) (S)-Propylene oxide, EtOH, NaOEt, rt; (b) MeSO2Cl, CH2Cl2, rt; (c) DMF, NaN3, 70 °C; (d) H2, 10% Pd/C, EtOH, HCO2NH4, rt; (e) NIS, THF, rt;
(f) CbzCl, NaHCO3, THF/H2O; (g) KOH/THF, HCHO/H2O; (h) Et3SiH, CH2Cl2, TFA, 60 °C; (i) MeI, DMF, K2CO3, rt; (j) 1-fluoropyridinium tosylate,
CH2Cl2, reflux; (k) NCS, THF, rt; (m) H2, 10% Pd/C, EtOAc/EtOH (5:1); (n) H2, 10% Pd/C, EtOH.
acetate followed by hydrolysis and reduction provided 2-(6-
amino-1H-indazol-1-yl)ethanol (4), which was converted to the
phenol (5) via the diazonium salt. Mesylation of 5 followed by
treatment with sodium azide and reduction provided 6. Substi-
tuted 1-((S)-2-aminopropyl)-1H-indazoles were prepared as
outlined in Scheme 2. Alkylation of 6-(benzyloxy)-1H-indazole
(7) with (R)-propylene oxide provided a mixture of the two
regioisomers, which were readily separated by column chro-
matography to provide the desired isomer 8, which was
converted to 9 via reduction of the azide intermediate. Protection
of the amino moiety of 9 with the carbobenzyloxy group
provided 11, a convenient intermediate for the preparation of a
series of 7-substituted analogues. Methylation of 11 with
iodomethane followed by deprotection gave the 6-methoxy
analogue 12. Reaction of 11 with aqueous formaldehyde
provided the 7-hydroxymethyl intermediate 13, which was
treated with triethylsilane followed by deprotection to give the
7-methyl analogue 14. Chlorination of 11 was readily ac-
complished with N-chlorosuccinimide, while treatment with
1-fluoropyridinium tosylate provided the fluoro intermediate.
Reductive cleavage of the protecting groups provided 15 and
16, respectively. Reaction of 9 with N-iodosuccinimide provided
17 directly.
Results and Discussion
An assessment of the stability of indazole analogue 6 relative
to 5-HT showed it to have a dramatically improved solution
stability: the projected half-life (t1/2) for 6 was 3.7 years
compared to a projected t1/2 of 11 days for 5-HT (pH 7.4,
phosphate buffer), confirming our expectations. This observation
provided incentive to proceed with the preparation of analogues
bearing an R-methyl group, which was desirable for improved
metabolic stability. The R-methyl compounds 12-16 each
showed improved solution stability compared to 5-HT and 2
(Table 1). As anticipated, the ether 12 was the most stable of
the compounds with a projected t1/2 greater than 30 years. Not

320 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Table 1. Solution Stability in Phosphate Buffer at pH 7.4
May et al.
Activation of the 5-HT2B receptor has been implicated in
cardiac valvular hyperplasia and pulmonary hypertension, which
have been observed in some patients following chronic oral
therapy with compounds subsequently shown to be potent
5-HT2B receptor agonists.16-18 Most notable in this regard have
been the orally administered anorectic agent d-fenfluramine
(active metabolite, d-norfenfluramine) and the anti-Parkinsonism
ergolines pergolide and cabergoline, also potent 5-HT2B receptor
agonists.16,19-21 Hence, a compound devoid of agonist activity
at the 5-HT2B receptor would be desirable, although a compound
that does have significant 5-HT2B receptor agonist activity but
achieves only low plasma levels, especially when administered
topically to the eye, would not be anticipated to elicit the
aforementioned undesirable effects. The level of systemic
exposure following topical ocular dosing of 5-HT2 receptor
agonists remains to be determined.
In view of the overall profile for compound 9, the affinity of
this compound for a variety of other receptors was determined.
Only low to weak affinity or functional response was noted at
other 5-HT receptors and at the norepinephrine and 5-HT uptake
sites (Tables 3 and 4). Weak affinity at the R1A/1B receptors
and micromolar efficacy at R2A/B/C receptors was observed.
Compound 9 interacted only weakly with â-adrenergic receptors,
dopamine receptors, and other members of a panel of neu-
rotransmitter-related receptors, ion channels, enzymes, and
second messenger systems when tested at 1 nM, 100 nM, and
10 µM concentrations (see Supporting Information). The
maximum observed in any of these assays was 63% inhibition
of radioligand binding to a nonspecific opiate receptor and 51%
inhibition at a nonspecific dopaminergic receptor at a concentra-
tion of 10 µM. This concentration is significantly greater than
the nanomolar affinity and potency this compound exhibits for
the 5-HT2 receptors.
compd
t
1/2 (25 °C),a,b
y
compd
t1/2 (25 °C),a,b
y
5-HT
0.030 ( 0.000 (11 d)c
0.071 ( 0.001 (26 d)c
3.7 ( 0.1
12
14
15
16
30 ( 6
2
6
9
0.73 ( 0.00
1.4 ( 0.0
4.0 ( 0.1
4.6 ( 0.1
a Compound concentration 5 ppm, study temperature 75 °C, extrapolation
gave 25 °C value. b Mean of duplicates ( range. c Study conducted at 45
°C.
surprisingly, the phenol moiety significantly influences the
stability of these compounds, as is the case with 5-HT. The
stability of 9 and 16 was comparable to that of 6, while 14 and
15, substituted at C7 with methyl and fluoro, respectively,
showed a decreased stability relative to 9.
In vitro 5-HT2 receptor binding and functional response data
for the reported compounds are summarized in Table 2.
Compound 6 showed binding affinities comparable to those of
5-HT at the cloned human 5-HT2B and 5-HT2C receptors, but a
2-fold lower affinity at the 5-HT2A receptor; affinities for these
compounds at the rat 5-HT2A receptor were comparable.
Comparable functional potency was also observed for 6 and
5-HT at each of the rat 5-HT2 receptors. The R-S-methyl
analogue of 6, compound 9, has a modestly increased affinity
at the three cloned human 5-HT2 receptor subtypes, whereas
an R-methyl group at this position (compound 10) led to a 10-
fold decrease in affinity at the 5-HT2A and 5-HT2C receptors
and a 4-fold decrease at the 5-HT2B receptor compared to
compound 9. Methylation of the hydroxyl group of 9 (compound
12) did not alter the affinity at the 5-HT2A or 5-HT2B receptors
but did result in a 2-fold increase in affinity at the 5-HT2C
receptor, that is, 12 showed a 10-fold selectivity for 5-HT2C
relative to 5-HT2A. Substitution of methyl, chloro, or fluoro at
the C7 position of 9 did not result in dramatic changes in affinity
at any of the 5-HT2 receptors; however, a decrease in affinity
at each of the receptors was observed for the 7-iodo substituent
(compound 17) compared to the fluoro and chloro derivatives.
This same order of affinity was observed at the rat 5-HT2A
receptor, where 12 and 17 also displayed the lowest affinity of
the compounds tested. The functional response for compounds
at the rat receptors generally followed an order comparable to
that observed with affinity for the human receptors. That is,
compounds 6-16 were agonists that had comparable potencies
at the 5-HT2B and 5-HT2C receptors, which were modestly higher
than the potency observed at the 5-HT2A receptor (Table 2).
Thus, no substantial 5-HT2 receptor subtype selectivity was
observed for any of these compounds.
Low brain penetrability was of particular importance for
compounds with significant efficacy at the 5-HT2A receptor in
view of potential CNS side effects. Therefore, compounds were
evaluated for their ability to enter the CNS in two model
systems, a cell-based blood-brain barrier model and an in vivo
mouse behavioral model. A cell-based assay that has gained
considerable interest and utility for assessing the ability of
compounds to enter the brain uses Madin-Darby canine kidney
(MDCK) cells that have been transfected with the human
multiple drug resistance-1 (mdr1) gene that expresses a func-
tional P-glycoprotein (P-gp) transporter.22 This transporter has
been identified as an integral component of the blood-brain
barrier and has been demonstrated to play a major role in the
Table 2. in Vitro Binding and Functional Data for Compounds
5-HT2B
(rat fundus)
5-HT2A (rat)
EC50, nMc
5-HT2C (rat)
5-HT2 (cloned human) Ki, nMa
compd
IC50, nMb
Emax, %d
EC50, nMe
EC50, nMc
Emax, %d
2A
2B
2C
5-HTd
6
9 (S)
10 (R)
12
14
15
16
0.94 ( 0.07
0.77 ( 0.22
0.58 ( 0.21
2.08 ( 0.81
1.91 ( 0.82
0.74 ( 0.05
0.73 ( 0.36
0.54 ( 0.13
1.68 ( 0.47
0.21
57.9 ( 4.8
74.4 ( 8.6
42.7 ( 2.1
236 ( 34
54.0 ( 9.5
81.3 ( 8.4
58.1 ( 19.3
59.7 ( 13.8
204 ( 31
19.8
99
75
81
61
90
89
80
87
73
35
96
70
96
3.5 ( 0.4
8.6 ( 2.6
2.0 ( 0.5
85 ( 15
4.2 ( 0.6
8.9 ( 4.2
6.1 ( 1.9
5.9 ( 0.4
-
11.9 ( 3.3
4.2 ( 0.3
240 ( 30
2.7 ( 0.2
2.8 ( 0.3
4.5 ( 1.6
3.7 ( 0.8
87.3 ( 11
6.7 ( 2.0
7.3 ( 0.3
5.5 ( 1.3
4.9 ( 1.6
-
30.2 ( 2.3
1.8 ( 0.2
77.8 ( 6.3
3.1 ( 1.0
94
104
89
97
103
91
100
104
-
84
100
89
8.2 ( 1.6
20 ( 1.2
12 ( 3
105 ( 38
16 ( 1.7
1.8 ( 0.2
5.8 ( 0.8
12 ( 1.8
21 ( 2.9
0.65
13 ( 4.5
15 ( 3.6
8.1 ( 2.4
35 ( 11
7.5 ( 1.7
8.7 ( 4.4
1.7 ( 0.4
3.8 ( 1.0
6.0 ( 2.1
18
8.3 ( 2.6
9.6 ( 1.8
3.0 ( 0.4
34 ( 4.5
1.5 ( 0.2
3.6 ( 1.4
1.0 ( 0.3
1.2 ( 0.1
4.8 ( 1.0
4.0
17
1f
2f
3.5
9.53
2.0
58
462
47.5
12
15
4.6
13
52
7.8
7
42
8.3
18f
19f
103
a Radioligand [125I]DOI, (SEM. b Radioligand [125I]DOI, homogenized rat cerebral cortex, (SEM. c Intracellular calcium mobilization in rat vascular
smooth muscle cells (A7r5), (SEM. d Relative to maximal 5-HT-induced response. e (SEM. f Data are from ref 7, except for the 5-HT2B and 5-HT2C
results.

1-((S)-2-Aminopropyl)-1H-indazol-6-ol
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 321
Table 3. Binding of 9 to Serotonergic Receptors and R-Adrenergic
to basolateral permeability (PappA-B) was greater than 30 nm/s
in MDCK(MDR) cells. Conversely, a compound would be
expected to have a low propensity for entering the CNS if it
had a PappA-B value of less than 10 nm/s.
Receptor Subtypes and Uptake Systemsa
receptor
Ki, nM
receptor
Ki, nM
5-HT1A
5-HT1B
5-HT1D
5-HT3
5-HT4
5-HT5A
5-HT6
141 ( 25b
>10000
7870
>10000
515
>10000
>10000
R1A
R1B
R2A
R2C
>30000
>30000
The MDCK(MDR) cell permeability data and distribution
coefficients for the 1-substituted-indazoles of the present study
and for two reference compounds, 1 and 5-methoxy-N,N-
dimethyl-tryptamine (18), are provided in Table 5. The passive
diffusion (PappPD) for a compound is the permeability in the
apical to basolateral direction (PappA-B) in the presence of a
P-gp inhibitor, cyclosporine A (CSA). The two centrally active
5-HT2 agonists, 1 and 18, which are known hallucinogenic
agents, both have high passive permeability in the MDCK-
(MDR) cells, 213 and 200 nm/s, respectively. These two
compounds substantiate the aforementioned guidelines for
estimating the CNS permeability with the MDCK(MDR) cell
assay. In contrast, the 1-substituted indazoles bearing a hydroxyl
group were less permeable than 1 or 18, having PappPD values
ranging from 2.8 to 20.3 nm/s and PappA-B values ranging from
6.4 to 24.9 nm/s. That is, all of the 6-hydroxyindazoles displayed
a passive diffusion of less than 50 nm/s, suggesting that these
compounds would have a low propensity for crossing the
blood-brain barrier by passive diffusion alone. A comparison
of the PappA-B values for these compounds suggests that 6, 9,
and 15, with values of 6.4, 7.6, and 11 nm/s, respectively, have
a low likelihood of entering the CNS; however, compounds 14,
16, and 17, with PappA-B values of 24.9, 16.5, and 12.2 nm/s,
respectively, would have an increased potential for entering the
CNS. Other studies will be needed to determine if they could
gain access to the brain, including assessment of other pathways.
The passive diffusion permeability of the 6-methoxy analogue
12, 187 nm/s, as well as the PappA-B value for this compound,
141 nm/s, was comparable to that of 1, suggesting that this
compound would readily enter the CNS.
2270 ( 210c
4700 ( 2100c
>10000
norepinephrine uptake
serotonin uptake
>10000
a Unless otherwise noted data are an average of duplicate determinations
performed at NovaScreen Biosciences, Corp. using their standardized
screening protocols. Inhibition constants (Ki) were determined using up to
seven concentrations of each compound. Typical interassay variation was
15-20%. Each value on the concentration plot was the mean of two
determinations. b Alcon data: IC50 value ( SEM, agonist radioligand [3H]-
8-OH-DPAT. c Alcon data: IC50 value ( SEM, agonist radioligand
[3H]clonidine.
Table 4. Functional Response of 9 and 10 in Selected Serotonergic and
Adrenergic Assays
EC50, nMa
assays
9
10
b
5-HT1A
1460 ( 162
5600 ( 1100
>10000
2890 ( 539
>10000
c
5-HT7
c
R2A
>10000
d
R2A
2600
1990
4710 ( 1480
5660 ( 1210
c
c
R2B
R2C
3050 ( 979
10000
a Mean ( SEM. b Inhibition of cAMP production; agonism, cloned
human receptor. c Adenylyl cyclase assay. d Tissue contraction assay per-
formed at MDS Pharma Services. Determination was made using eight
concentrations, and each value on the concentration plot was the mean of
two determinations. Typical interassay variation was 15-20%.
exclusion of a variety of xenobiotic compounds from the brain.23
A recent study profiled the permeability of a variety of currently
marketed CNS and non-CNS drugs in the MDCK(MDR) cell-
based assay.24 On the basis of the results of this study, it was
proposed that for access to the brain a compound should (1)
not be a substrate for P-gp, (2) have a bidirectional permeability
ratio less than 2.5 in MDCK(MDR) cells, and (3) have a passive
diffusion permeability (PappPD) greater that 150 nm/s. Con-
versely, for a compound to be excluded from the brain it should
(1) be a substrate for P-gp, (2) have a bidirectional permeability
ratio greater than 5, and (3) have a PappPD of less than 50 nm/s.
Another assessment of the utility of MDCK(MDR) cell perme-
ability for estimating the ability of a compound to enter the
CNS has recently been reported.25 This report compares the
MDCK(MDR) permeability of a set of compounds for which
the CNS permeability status was based on data obtained from
in vivo or in situ rat or mouse brain uptake experiments. On
the basis of the results of this study, it was proposed that a
compound is highly likely to enter the CNS if its apparent apical
None of the indazoles were substrates for the P-gp transporter.
Though the bidirectional permeability ratio [PappB-A/PappA-B
]
for 16 in the absence or presence of CSA suggests that P-gp is
not involved in the transport of this compound, the elevated
value of this ratio (i.e., >2) in the presence of CSA, suggests
that other transport systems may influence the permeability of
this particular compound.
Head-twitch induction in rodents, a stereotypical behavior
induced by stimulation of central 5-HT2A receptors, has been
used to assess the potential side effects of serotonergic
compounds. A good correlation has been observed between the
rodent head-twitch response for a compound and its ability to
invoke a hallucinogenic response in man.26-28 However, this
response has been shown to be modulated by agonist activity
at other receptors, such as 5-HT1A and 5-HT2C, so the results
of this assay must be viewed with some caution if other
Table 5. Permeability of Selected Compounds in MDCK(MDR) Cellsa
P
app, nm/s
PappPD, nm/sb
ratio
ratio
compd
DC7.4
A f B
B f A
[B f A/A f B]
A f B
B f A
[B f A/A f B]
6
9
0.254
0.693
2.08
1.28
0.394
1.09
3.00
2.72
3.30
6.4 ( 0.4
7.6 ( 1.7
141 ( 47
24.9 ( 0.0
11.1 ( 3.1
16.5 ( 1.0
12.2 ( 0.0
126 ( 5
6.0 ( 0.8
8.8 ( 1.5
195 ( 5
31.2 ( 1.6
16.6 ( 0.2
49.4 ( 5.7
34.6 ( 1.6
232 ( 52
144 ( 43
0.9
1.2
1.4
1.3
1.5
3.0
2.8
1.8
0.9
2.8 ( 0.8
7.0 ( 0.2
187 ( 17
20.3 ( 3.8
10.3 ( 1.5
13.6 ( 2.8
16.7 ( 4.8
213 ( 3
3.1 ( 0.1
5.8 ( 0.8
171 ( 21
16.6 ( 0.9
14.1 ( 4.7
32.5 ( 3.1
24.9 ( 2.6
191 ( 2
1.1
0.8
0.9
0.8
1.4
2.4
1.5
0.9
0.7
12
14
15
16
17
1
18
161 ( 13
200 ( 28
141 ( 17
a Papp values expressed as mean of duplicates ( range. b Permeability in the presence of a P-glycoprotein inhibitor, cyclosporin A.

322 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
May et al.
Table 7. IOP Response of 5-HT2 Agonists in the Lasered Monkey
Table 6. Effect of Compounds in Mouse Head-Twitch Response Assaya
b
b
ED5
ED5
postdose IOP reduction, %b,c
baseline IOP,
mmHgb
compd
(mg/kg)
compd
(mg/kg)
compd dose, µga
1 h
3 h
6 h
1
19
6
0.13
1.0
12
14
15
16
0.16
0.32
>30c
0.56
6
9
300
100
300
300
100
300
300
100
250
300
35.8(2.9)
35.9(3.2)
41.8(3.8)
38.4(2.2)
36.9(3.7)
37.2(3.1)
33.0(2.8)
31.9
8.3(4.7)
18.2d(3.5) 19.4d(4.9)
16.1d(3.7) 23.0d(4.1) 16.5d(4.3)
>30c
13.4e(2.7) 31.4d(3.2) 33.0d(3.1)
9
>30c
10
9.1(4.5)
13.7(7.4) 7.6(6.9)
13.6f(4.5) 15.1f(5.5) 5.0(5.4)
15.7d(3.2) 27.1d(5.0) 29.8d(4.3)
18.4f(5.4) 30.8d(4.6) 32.7d(5.3)
a Subcutaneous administration. b ED5 is the effective dose to produce
five head twitches in 10 min, value determined from dose response plot, n
) 5/dose (see Supporting Information for data). c Maximum response less
than 5 head twitches in 10 min for doses up to 30 mg/kg.
14
16
1g
11.0
21.6
12.0
25.3
35.2
25.4
34.4
33.4
26.1
2g
38.1
36.3
functional data are not available.29,30 Selected compounds from
the present study along with the well-known centrally active
standards 1 and 5-methoxy-R-methyltryptamine (19) were
evaluated for their ability to induce a head-twitch response in
mice following subcutaneous administration (Table 6). The
effective dose required to produce a mean of five head twitches
(ED5) during the 10-min scoring period was determined by
interpolation of the results from bracketing doses (see Supporting
Information); this value was used to provide a relative ranking
of tested compounds. The responses observed for 1 and 19 in
this assay, ED5 ) 0.13 and 1.0 mg/kg, respectively, were
consistent with the reported human hallucinogenic dose for these
two compounds, 2.3 and 2-4 mg, respectively.31,32 No perceiv-
able response was noted in this assay for 2 at doses ranging
from 3 to 30 mg/kg, which is consistent with the lack of
hallucinogenic activity noted for this compound. As anticipated
from the permeability data, 12 showed a high level of head-
twitch response, comparable to that of 1, with an ED5 of 0.16
mg/kg. Compounds 6, 9, and 15 did not induce the designated
five twitches for doses up to 30 mg/kg, so the ED5 is greater
than 30 mg/kg for these compounds, suggesting that they do
not readily enter the CNS, which is consistent with both the
PappPD and PappA-B permeability data discussed above. The
7-methyl (14) and 7-chloro (16) compounds had ED5 values of
0.32 and 0.56 mg/kg, respectively, which are considerably more
potent than anticipated from the MDCK(MDR) cell passive
diffusion permeability for these compounds (PappPD ) 20.3 and
13.6 nm/s, respectively). The head-twitch response observed for
14 and 16 are in better agreement with the CNS penetration
prediction on the basis of their PappA-B values (24.9 and 16.5
nm/s, respectively), which suggests that these compounds are
likely to enter the CNS. It appears, therefore, that for the
compounds of the present study the MDCK(MDR) cell PappA-B
permeability values are a better predictor of CNS permeability
than the PappPD values.
The high permeability for 12 in the MDCK(MDR) cells and
the observed potency for producing head-twitches in mice
strongly suggested entry into the CNS. Topical ocular admin-
istration of 12 to rabbits in a routine preliminary acute toxicity
evaluation led to prominent hyperthermia with a compensatory
increase in breathing rate in the animals. This response is
consistent with previous observations for centrally acting 5-HT2A
receptor agonists,33 substantiating that 12 does enter the CNS
following topical ocular dosing in the rabbit. Compound 15 was
found to be unsuitable for further evaluation following the
observation of acute toxicity in the rabbit; this response included
a decrease in body temperature (hypothermia) and nonspecific
animal vocalizing, which appeared to be non-CNS related. The
low solubility of 17 precluded in vivo evaluations, even though
a 1% suspension of 17 did not elicit any untoward effects in an
acute rabbit toxicity study.
18g
a Phosphate buffer, pH 7.4. b (SEM. c The vehicle control group con-
ducted with each study did not exceed inherent model IOP variability of
(15%. d p < 0.001. e p < 0.01. f p < 0.05. g Data from ref 7.
Table 8. IOP Response Induced by 9 after Topical Ocular
Administration to the Normal Eyes of Conscious Cynomolgus Monkeys
postdose IOP reduction, %b,c
baseline IOP,
dose, µga
mmHgb
1 h
3 h
6 h
300, OSd
0, ODe
26.5(1.1)
39.5(3.5)
0.8(6.0)
2.4(3.2)
8.9(5.0)
8.6(3.8)
12.9(3.5)
9.1(3.7)
a Phosphate buffer, pH 7.4. b (SEM. c The vehicle control group con-
ducted with each study did not exceed inherent model IOP variability of
(15%. d Normal, normotensive left eye. e Lasered (hypertensive) right eye
contralateral to treatment.
model of ocular hypertension (Table 7). Following topical ocular
administration, compound 6 showed a maximum IOP reduction
(19%, -7.4 mmHg) comparable to that previously observed with
5-HT (18%).7 Unlike 5-HT, for which the IOP had returned to
the baseline level by 6 h, 6 maintained this modest IOP reduction
through the 6-h reading. Compound 6 appears to have a
moderately increased metabolic stability relative to 5-HT;
however, this has not been explored. Compounds 9, 14, and 16
all showed a very pronounced decrease in IOP of 30% or greater
(g-11 mmHg) at the 6-h reading in the ocular hypertensive
monkey after topical ocular administration. Although 9 was very
effective at lowering pressure after a 300 µg topical ocular dose,
after a 100 µg dose the response was reduced at both the 3-h
(23%, -8.6 mmHg) and 6-h (16%, -6.2 mmHg) readings. The
R enantiomer of 9, compound 10, showed no biologically
relevant reduction of IOP at either the 3- or 6-h postdose
readings. Topical ocular administration of 9 (300 µg) to the
normal (nonlasered normotensive) left eye of the monkeys did
not result in a pronounced reduction of IOP in the treated normal
eye or in the untreated (undosed) hypertensive contralateral eye
at the 6-h postdose reading (Table 8). Therefore, the IOP
reduction observed for 9 appears to be locally, rather than
centrally, mediated. No effect on the pupil was observed in the
treated eyes.
Conclusion
Compound 9 is a peripherally acting 5-HT2 receptor agonist
that exhibits high affinity, potency, and intrinsic efficacy at this
receptor and which has high selectivity for the 5-HT2 receptors
relative to other receptor families. Compound 9 has significantly
greater solution stability than 2. It potently lowers IOP in
conscious ocular hypertensive monkeys through a local rather
than a centrally mediated effect. Compound 9 appears to be an
excellent candidate for conducting a clinical proof-of-concept
study for this class of ocular hypotensive compounds.
Experimental Section
Compounds 6, 9, 10, 14, and 16, all of which showed an
acceptable profile in preliminary acute rabbit toxicity studies,
were evaluated in a conscious lasered cynomolgus monkey
Melting points were determined in open capillaries using a
Thomas-Hoover Uni-Melt Apparatus and are uncorrected. Organic
extracts were dried with magnesium sulfate. Chromatography refers

1-((S)-2-Aminopropyl)-1H-indazol-6-ol
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 323
to column chromatography conducted on 230-400 mesh silica gel
from E. Merck. Silica gel TLC plates were obtained from EM
Separation Technology. 1H and 13C NMR spectra were determined
on a Bruker AMX 200-MHz spectrometer or a Bruker DRX 600-
MHz spectrometer. Chemical shifts are reported in parts per million
(δ) relative to tetramethylsilane as internal standard. Mass spectra
were obtained on a Finnigan TSQ45 triple quadrupole mass
spectrometer, and HPLC-MS analyses were recorded on a Finnigan
LCQ Classic mass spectrometer. Optical rotations were determined
using a Jasco DIP-370 polarimeter. Elemental analyses were
performed by Atlantic Microlabs, Norcross, GA, and are within
(0.4% of the theoretical values. Evaporations were performed under
reduced pressure on a rotary evaporator at 40 °C unless otherwise
indicated.
2-(6-Nitro-1H-indazol-1-yl)ethyl Acetate (3). To a stirred
solution of 6-nitro-1H-indazole (6.0 g, 37 mmol) in DMF (100 mL)
was added potassium carbonate (15.4 g, 112 mmol) at room
temperature. After 30 min, 2-bromoethyl acetate (8.2 mL, 73 mmol)
was added and the solution heated at 70 °C for 18 h. The reaction
mixture was cooled to room temperature and diluted with a saturated
aqueous solution of ammonium chloride (10 mL), and this mixture
was extracted with ethyl acetate (3 × 65 mL). The extract was
washed with brine (10 mL), dried, and evaporated to a residue which
was purified by chromatography (silica, gradient from 30 to 50%
ethyl acetate in hexane) to give 3 as an oil (5.8 g, 63%): 1H NMR
(CDCl3) δ 8.49 (m, 1H), 8.23 (s, 1H), 8.05 and 8.01 (dd, J ) 2.0
and 10.0 Hz, 1H), 7.87 (d, J ) 10.0 Hz, 1H), 4.78 (t, J ) 5.0 Hz,
2H), 4.54 (t, J ) 5.0 Hz, 2H) 1.98 (s, 3H); MS (ES) m/z 250 (M
+ H+).
2-(6-Amino-1H-indazol-1-yl)ethanol (4). Potassium carbonate
(9.6 g, 70 mmol) was added to a solution of 3 (5.8 g, 23 mmol) in
methanol (20 mL) at room temperature and the mixture was stirred
for 18 h. The solvent was evaporated and the residue dissolved
with hydrochloric acid (2 N, 30 mL); this solution was extracted
with ethyl acetate (3 × 65 mL). The extract was washed with brine
(10 mL), dried, and evaporated to a residue [4.8 g, MS (ES) m/z
208 (M + H)+] which was dissolved in ethanol (50 mL). Palladium-
on-carbon (10%, 0.5 g) was added under a nitrogen atmosphere
and the suspension was stirred for 18 h under a hydrogen
atmosphere. The reaction mixture was passed through a filter aid
and the filtrate evaporated to a residue which was purified by
chromatography (silica, 5% methanol in dichloromethane) to give
4 as an oil (3.9 g, 96%): 1H NMR (DMSO-d6) δ 7.70 (s, 1H),
7.36 (d, J ) 9.2 Hz, 1H), 6.49 (dd, J ) 9.2 and 2.0 Hz, 1H), 5.28
(s, 2H), 4.84 (t, J ) 5.4 Hz, 1H), 4.18 (m, 2H), 3.78-3.69 (m,
2H); MS (ES) m/z 178 (M + H+).
1-(2-Hydroxyethyl)-1H-indazol-6-ol (5). A solution of 4 (3.90
g, 22 mmol) in H2SO4/H2O (1:1, 60 mL) was cooled to 0 °C and
a solution of NaNO2 (1.52 g, 22 mmol) in H2O (6 mL) was added
dropwise; this dark solution was stirred for 2 h and water (40 mL)
was added followed by heating at 110 °C for 2 h. The reaction
mixture was cooled to ambient temperature, carefully neutralized
with a saturated aqueous solution of NaHCO3 and extracted with
ethyl acetate (3 × 65 mL). The extract was washed with brine (10
mL), dried, and evaporated to give a residue which was purified
by chromatography (silica, gradient from 50% to 60% ethyl acetate
in hexane) to give an oil (2.5 g, 99%): 1H NMR (CDCl3) δ 9.60
(m, 1H), 7.84 (s, 1H), 7.50 (d, J ) 11.0 Hz, 1H), 6.82 (s, 1H),
6.66 (d, J ) 11.0 Hz, 1H), 4.86 (t, J ) 4.0 Hz, 1H), 4.30 (t, J )
4.0 Hz, 2H), 3.80 (t, J ) 4.0 Hz, 2H); MS (ES) m/z 179 (M +
H+).
H+)] which was dissolved in dichloromethane (10 mL) containing
triethylamine (1.0 mL, 7.4 mmol) and the mixture was cooled to 0
°C. This temperature was maintained while methanesulfonyl
chloride (0.58 mL, 7.4 mmol) was added. After 30 min, dichlo-
romethane (50 mL) was added to the reaction mixture followed by
water (50 mL). The organic layer was separated and the aqueous
layer was extracted with dichloromethane (2 × 50 mL). The
combined organic layers were washed with brine (30 mL), dried,
and evaporated to a residue that was dissolved in DMF (6 mL)
followed by the addition of sodium azide (0.96 g, 15 mmol). The
reaction mixture was heated at 60 °C for 7 h, poured into water,
and extracted with ether (3 × 50 mL). The combined extracts were
washed with brine, dried, and evaporated to a residue which was
purified by chromatography (silica, hexane to 10% ethyl acetate in
hexane) to give an oil [1.4 g, 71%; MS (ES) m/z 294 (M + H+)].
Ammonium formate (0.62 g, 9.8 mmol) was added to a solution of
this oil (0.72 g, 2.5 mmol) in ethanol (40 mL) under nitrogen and
at room temperature followed by the addition of palladium-on-
carbon (10%, 0.1 g). The mixture was stirred for 24 h at room
temperature and then filtered through a filter aid. The filtrate was
concentrated to a residue which was purified by chromatography
(silica, gradient from 5% to 20% methanol in dichloromethane) to
give a viscous oil, which was converted to a HCl salt that
crystallized from a mixture of methanol in ether to give 6 (0.23 g,
1
38%): mp 197-198 °C; H NMR (DMSO-d6) δ 8.33 (3H, brs),
8.05 (s, 1H), 7.54 (d, J ) 8.0 Hz, 1H), 6.90 (s, 1H), 6.76 (d, J )
8.0 Hz, 1H), 4.55 (t, J ) 8.0 Hz, 2H), 3.25 (m, 2H); MS (ES) m/z
178 (M + H+). Anal. (C9H11N3O·2HCl) C, H, N.
(R)-1-(6-Benzyloxyindazol-1-yl)propan-2-ol (8). To a stirred
solution of 1H-indazol-6-ol34 (2.5 g, 18 mmol) in ethanol (20 mL)
was added K2CO3 (4.1 g, 30 mmol) followed by benzyl bromide
(2.2 mL, 18 mmol); this mixture was heated at reflux temperature
for 6 h. The solvent was removed by evaporation, the resultant
residue was dissolved in 1 N HCl (80 mL), and this solution was
extracted with EtOAc (3 × 50 mL). The combined extracts were
washed with brine (30 mL), dried, and evaporated to an oil which
was purified by chromatography (silica, gradient from 30% to 50%
EtOAc in hexane) to give 7 as an oil (1.4 g, 35%): MS m/z 225
(M + H+). To a solution of 7 (0.96 g, 4.3 mmol) in ethanol (10
mL) at ambient temperature was added sodium ethoxide (2.5 mL,
6.4 mmol, 21% solution in ethanol). After 30 min, (R)-propylene
oxide (0.56 mL, 6.4 mmol) was added and the solution was stirred
for 18 h. The solution was diluted with a saturated aqueous
ammonium chloride solution (20 mL) and ethanol was evaporated
in vacuo. The aqueous solution was extracted with ethyl acetate (3
× 65 mL). The extract was washed with brine (30 mL), dried, and
evaporated to a residue which was purified by chromatography
(silica, 30% ethyl acetate in hexane) to give 8 as a solid (0.56 g,
1
47%): mp 95-97 °C; H NMR (DMSO-d6) δ 7.85 (s, 1H), 7.57
(d, J ) 8.0 Hz, 1H), 7.47-7.33 (m, 5H), 6.95 (dd, J ) 2.0 and 8.0
Hz, 2H), 4.35-4.07 (m, 3H), 1.25 (d, J ) 6.6 Hz, 3H); MS (ES)
m/z 283 (M + H+). Approximately 38% of the N2 substituted
regioisomer was also formed in this reaction.
1-((S)-2-Aminopropyl)-1H-indazol-6-ol Fumarate (9). To a
solution of 8 (1.2 g, 4.2 mmol) in dichloromethane (10 mL) was
added triethylamine (0.7 mL, 5 mmol) followed by methanesulfonyl
chloride (0.39 mL, 5 mmol) at 0 °C. After 30 min, a saturated
aqueous solution of ammonium chloride (20 mL) was added. The
organic layer was separated and the aqueous phase was extracted
with dichloromethane (3 × 50 mL). The organic phase was washed
with brine (30 mL), dried, and evaporated to a residue that was
dissolved in DMF (6 mL). Sodium azide (0.55 g, 8.4 mmol) was
added, and the reaction mixture was heated at 70 °C for 18 h, poured
into water (75 mL), and extracted with ethyl acetate (3 × 50 mL).
The extract was washed with brine, dried, and evaporated to a
residue which was purified by chromatography (silica, gradient from
hexane to 20% ethyl acetate in hexane) to give the azide (2.8 g).
To a solution of the azide (2.75 g, 8.95 mmol) in ethanol (10 mL)
was added palladium-on-carbon (10%, 0.5 g) followed by am-
monium formate (2.25 g, 35.8 mmol) under a nitrogen atmosphere
at room temperature. The suspension was stirred for 18 h at room
1-(2-Aminoethyl)-1H-indazol-6-ol Dihydrochloride (6). To a
solution of 5 (1.2 g, 6.7 mmol) in ethanol (20 mL) at ambient
temperature was added K2CO3 (1.0 g, 7.4 mmol) and benzyl
bromide (0.88 mL, 7.4 mmol); this dark solution was heated at
reflux temperature for 4 h. The solvent was removed by evaporation
and 2 N HCl (60 mL) was added to the residue. This mixture was
extracted with ethyl acetate (3 × 50 mL), and the combined extracts
were washed with brine (30 mL), dried, and evaporated to a residue.
The residue was purified by chromatography (silica, 40% ethyl
acetate in hexane) to give an oil [1.8 g, MS (ES) m/z 269 (M +

324 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
May et al.
temperature and the reaction mixture was filtered through a filter
aid. The filtrate was concentrated to a residue which was purified
by chromatography (silica, gradient from 5% to 20% methanol in
dichloromethane) to give a solid which was crystallized from
dichloromethane (0.91 g, 53%): mp 170-172 °C; 1H NMR
(DMSO-d6) δ 7.86 (s, 1H), 7.54 (d, J ) 8.6 Hz, 1H), 6.82 (d, J )
2.0 Hz), 6.67 (dd, J ) 2.0 and 8.6 Hz, 1H), 4.10-4.01 (m, 2H),
3.30 (m, 1H), 0.93 (d, J ) 6.6 Hz, 3H); MS (ES) m/z 191 (M+);
[R]D +3.82°, [R]405 +11.0° (c 0.44, CH3OH); %ee >97%, as
determined by HPLC (Chiralpak AS column, Chiral Technologies,
Inc.) for the trifluoroacetamide derivative relative to the racemate.
Anal. (C10H13N3O‚0.4H2O) C, H, N.
Treatment of the free base with fumaric acid provided the
fumarate salt: mp 178-180 °C; 1H NMR (DMSO-d6) δ 9.5 (br s,
2H), 7.93 (s, 1H), 7.55 (d, J ) 8.0 Hz, 1H), 6.91 (s, 1H), 6.72 (dd,
J ) 8.0 and 2.0 Hz, 1H), 6.49 (s, 1H), 4.47-4.26 (m, 2H), 3.65-
3.55 (m, 1H), 1.10 (d, J ) 8.0 Hz, 3H); [R]D +3.74° (c 0.508,
MeOH). Anal. (C10H13N3O‚1.2C4H4O4‚0.5H2O) C, H, N.
1-((S)-2-Aminopropyl)-7-methyl-1H-indazol-6-ol (14). To a
stirred solution of 11 (2.2 g, 6.8 mmol) in THF (30 mL) was added
KOH (3.3%, 5 mL) and aqueous formaldehyde (37%, 1.6 mL);
this solution was heated at 55 °C for 2 h followed by stirring for
18 h at ambient temperature. A saturated ammonium chloride
solution (30 mL) was added to the reaction mixture followed by
extraction with EtOAc (3 × 50 mL). The extract was washed with
brine (30 mL), dried, and evaporated to a residue which was purified
by chromatography (silica, 50% EtOAc in hexane) to give 13 as
an oil (2.2 g, 91%) that was used directly in the next reaction: MS
(ES) m/z 356 (M + H+). Triethylsilane (0.62 mL, 4.0 mmol) was
added to a solution of 13 (0.24 g, 0.67 mmol) in dichloromethane
(5 mL) followed by trifluoroacetic acid (0.62 mL, 8.0 mmol) and
the mixture was heated for 5 h at 60 °C. The reaction mixture was
cooled to ambient temperature and a saturated aqueous solution of
sodium bicarbonate (30 mL) was added followed by extraction with
ethyl acetate (3 × 50 mL). The extract was washed with brine (30
mL), dried, and evaporated to give a residue (0.19 g, 82%) that
was used without further purification [MS (ES) m/z 339 (M+)]. To
a solution of the residue (0.18 mg, 0.53 mmol) in EtOH (10 mL)
was added palladium-on-carbon (10%, 30 mg) and the mixture was
stirred under a hydrogen atmosphere for 20 h. The solution was
passed through a filter aid and the filtrate was evaporated to a
residue that was purified by chromatography (silica, 10% methanol
in dichloromethane) to give a solid (0.10 g, 92%), which was
recrystallized from ethyl acetate: mp 156-157 °C; 1H NMR
(DMSO-d6) δ 7.90 (s, 1H), 7.37 (d, J ) 6.8 Hz, 1H), 6.80 (d, J )
6.8 Hz, 1H), 6.47 (s, 2H), 4.56 (m, 2H), 3.58 (m, 1H), 2.45 (s,
3H), 1.04 (d, J ) 6.0 Hz, 3H); 13C NMR (DMSO-d6) δ 167.6,
154.1, 140.5, 135.0, 133.6, 118.5, 118.4, 112.5, 102.9, 53.9, 47.3,
17.1, 10.3; MS (ES) m/z 206 (M + H+); [R]D +20.0°, [R]405 +44.6°
(c 1.10, MeOH). Anal. (C11H15N3O) C, H, N.
1-((R)-2-Aminopropyl)-1H-indazol-6-ol Fumarate (10). The
R enantiomer was prepared in an identical manner as that described
above for the preparation of 9 but using (S)-propylene oxide: mp
1
170-171 °C; H NMR (DMSO-d6) δ 7.86 (s, 1H), 7.54 (d, J )
8.6 Hz, 1H), 6.82 (d, J ) 2.0 Hz, 1H), 6.67 (dd, J ) 2.0 and 8.6
Hz, 1H), 4.10-4.01 (m, 2H), 3.30 (m, 1H), 0.93 (d, J ) 6.6 Hz,
3H); [R]D -5.8°, [R]405 -12.0° (c 0.45, MeOH); MS (ES) m/z 191
(M+). Anal. (C10H13N3O‚0.21H2O) C, H, N. Treatment of the free
base with fumaric acid provided the fumarate salt: mp 177-179
°C; 1H NMR (DMSO-d6) δ 8.2 (br s, 2H), 7.91 (s, 1H), 7.54 (d, J
) 8.4 Hz, 1H), 6.89 (s, 1H), 6.71 and 6.70 (dd, J ) 8.4 and 1.8
Hz, 1H), 4.34-4.23 (m, 2H), 3.50 (m, 1H), 1.04 (s, J ) 6.6 Hz,
3H); [R]D -5.86° (c 0.53, MeOH). Anal. (C10H13N3O‚0.8C4H4O4)
C, H, N.
1-((S)-2-Aminopropyl)-7-fluoro-1H-indazol-6-ol Hydrochlo-
ride (15). To a solution of 11 (1.9 g, 5.8 mmol) in dichloromethane
(10 mL) was added 1-fluoropyridinium triflate (2.8 g, 12 mmol)
and the mixture was heated at reflux temperature for 24 h. A
saturated aqueous solution of ammonium chloride (20 mL) was
added and the mixture was extracted with ethyl acetate (3 × 50
mL). The extract were washed with brine, dried, and evaporated
to a residue which was purified by chromatography (silica, 60%
ethyl acetate in hexane) to give an oil (0.7 g, 35%): MS (ES) m/z
344 (M + H+). Palladium-on-carbon (10%, 0.01 g) was added to
a solution of this oil (0.6 g, 1.7 mmol) in ethanol (10 mL) and the
mixture was stirred at ambient temperature under an atmosphere
of hydrogen for 18 h. The reaction mixture was passed through a
filter aid and the filtrate concentrated to give a solid (0.35 g), which
was dissolved in methanol (20 mL). To this solution was added a
solution of 1 N hydrochloric acid in ethanol (1 mL) and the mixture
was evaporated to give a gum, which solidified from a mixture of
[(S)-2-(6-Hydroxyindazol-1-yl)-1-methylethyl]carbamic Acid
Benzyl Ester (11). 1-((S)-2-Aminopropyl)-1H-indazol-6-ol (9) (2.1
g, 11 mmol) was suspended in THF (20 mL), and saturated aqueous
sodium bicarbonate (10 mL) and benzyl chloroformate (1.5 mL,
11 mmol) were added. The mixture was stirred at ambient
temperature until all of the starting amine dissolved (ca. 45 min).
Saturated aqueous sodium bicarbonate (150 mL) was added and
the reaction mixture extracted with ethyl acetate (3 × 150 mL).
The extract was dried, filtered, and evaporated to give 11 as a tan
foam (2.6 g, 78%): LC/MS (+APCI) m/z 326 (M + H+); 1H NMR
(DMSO-d6) δ 9.61 (s, 1H), 7.84 (s, 1H), 7.52 (d, J ) 9.6 Hz, 1H),
7.32 (m, 6H), 6.80 (s, 1H), 6.70 (d, J ) 9.6 Hz, 1H), 4.97 (s, 2H),
4.25 (m, 3H), 1.01 (d, J ) 7.8 Hz, 3H). This material was used in
subsequent reactions without further purification.
(S)-2-(6-Methoxyindazol-1-yl)-1-methylethylamine Fumarate
(12). To a solution of 11 (0.28 g, 0.86 mmol) in DMF (10 mL) at
room temperature was added potassium carbonate (0.14 g, 1.0
mmol) followed by iodomethane (0.23 mL, 3.7 mmol). After 5 h,
a saturated aqueous solution of ammonium chloride (30 mL) was
added and the mixture was extracted with ethyl acetate (3 × 65
mL). The combined extracts were washed with brine (10 mL), dried,
and evaporated to give a residue which was purified by chroma-
tography (silica, 30% ethyl acetate in hexane) to give the methyl
ether (0.24 g, 82%); MS (ES) m/z 340 (M + H+). To a solution of
the methyl ether in ethanol (25 mL) at ambient temperature was
added palladium-on-carbon (10%, 0.10 g) under a nitrogen atmo-
sphere; this suspension was stirred for 20 h under an atmosphere
of hydrogen. The reaction mixture was passed through a filter aid
and the filtrate concentrated to a residue which was purified by
chromatography (silica, gradient from 5% to 10% methanol in
dichloromethane) to give a syrup (0.17 g, 97%) that was converted
to the fumarate salt. Crystallization from a mixture of methanol
1
methanol and ethyl acetate (0.25 g, 58%): mp 240 °C; H NMR
(DMSO-d6) δ 10.13 (s, 1H), 8.22 (br s, 3H), 8.06 (s, 1H), 7.39 (d,
J ) 8.8 Hz, 1H), 6.90 (d, J ) 8.8 Hz, 1H), 4.64-4.52 (m, 2H),
3.71 (m, 1H), 1.16 (d, J ) 6.8 Hz, 3H); LC/MS m/z 210 [M +
H+]; [R]D +10.2°, [R]405 +30.2° (c 0.965, MeOH). Anal. (C10H12-
FN3O‚HCl‚0.2H2O) C, H, N.
1-((S)-2-Aminopropyl)-7-chloro-1H-indazol-6-ol Hydrochlo-
ride (16). A solution of 11 (0.92 g, 2.8 mmol) in THF (20 mL)
was cooled to 0 °C and N-chlorosuccinimide (0.51 g, 3.8 mmol)
added. The solution was stirred for 2 h, poured into a saturated
aqueous solution of sodium bicarbonate (20 mL), and extracted
with EtOAc (2 × 25 mL). The extract was dried and evaporated to
an oil, which was purified by chromatography (silica, 40% ethyl
acetate in hexane) to give an oil (0.48 g, 47%) [LC/MS m/z 360
(M + H+)]. To a solution of this oil (0.30 g, 0.84 mmol) in a
mixture of ethyl acetate (20 mL) and ethanol (4 mL) was added
palladium-on-carbon (10%, 0.05 g) and the mixture was stirred
under a hydrogen atmosphere for 15 h. The solution was passed
through a filter aid and the filtrate evaporated to a residue which
was purified by chromatography (reverse phase: MeCN, H2O, 0.1%
TFA as a gradient from 100% to 0%) to furnish 16 as the
trifluoroacetate salt. Dissolution of this salt in 0.1 N HCl (5 mL),
1
and ether gave a colorless solid (0.11 g): mp 150-152 °C; H
NMR (DMSO-d6) δ 9.65 (br s, 2H), 7.98 (s, 1H), 8.28 (s, 1H),
7.63 (d, J ) 8.8 Hz, 1H), 7.24 (s, 1H), 6.77 (d, J ) 8.8 Hz, 1H),
6.48 (s, 2H), 4.54-4.43 (m, 2H), 3.85 (s, 3H), 3.61 (m, 1H), 1.11
(d, J ) 6.8 Hz, 3H); MS (ES) m/z 206 (M + H+); [R]D +2.3°,
[R]405 +6.9° (c 0.52, MeOH). Anal. (C11H15N3O‚1.2C4H4O4) C,
H, N.

1-((S)-2-Aminopropyl)-1H-indazol-6-ol
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 325
followed by concentration (repeated twice) afforded the hydro-
chloride salt (0.081 g, 21%): mp 159-161 °C; H NMR (CD3-
In Vitro Binding Assays. Serotonin Human 5-HT1A Receptor
Binding. The procedure was previously described.7 In brief, the
binding of [3H]-8-OH-DPAT (0.25 nM final) to Chinese hamster
ovary cell membranes expressing the recombinant human 5HT1A
receptor was performed in 50 mM Tris-HCl buffer (pH 7.4) in a
total volume of 0.5 mL for 1 h at 27 °C. Unlabeled 8-OH-DPAT
(10 µM final) was used to define the nonspecific binding. The assays
were terminated by rapid vacuum filtration and the samples counted
on a scintillation counter. The data were analyzed using a nonlinear,
iterative curve-fitting computer program.38-40
Serotonin Rat 5-HT2A Receptor Binding. The procedure was
previously described.7 In brief, the relative affinities of compounds
at the 5-HT2A receptor were determined by measuring their ability
to compete for the binding of the agonist radioligand [125I]DOI to
rat brain 5-HT2A receptors. Aliquots of postmortem rat cerebral
cortex homogenates (400 µL) dispersed in 50 mM Tris-HCl buffer
(pH 7.4) were incubated with [125I]DOI (80 pM final) in the absence
or presence of methiothepin (10 µM final) to define total and
nonspecific binding, respectively, in a total volume of 0.5 mL. The
assay mixture was incubated for 1 h at 23 °C in polypropylene
tubes, and the assays were terminated by rapid vacuum filtration
over Whatman GF/B glass fiber filters previously soaked in 0.3%
polyethyleneimine using ice-cold buffer. The samples were counted
on a â-scintillation counter and the data analyzed using a nonlinear,
iterative curve-fitting computer program.38-40
Serotonin Cloned Human 5-HT2 Receptors Binding. Binding
affinity of compounds at the cloned human 5-HT2A, 5-HT2B, and
5-HT2C receptors expressed in Chinese hamster ovary cells using
the agonist [125I]DOI (0.2 nM, 15 min at 37 °C) as the radioligand
for each receptor was determined and reported as Ki values. These
studies were conducted, and the data analyzed, at Cerep (Poitiers,
France) using a standard radioligand binding techniques as described
above.
Determination of Binding at Other Receptors and Cellular
Elements. Binding assays for 5-HT1B, 5-HT1D, 5-HT3, 5-HT4, and
5-HT6 serotonergic receptors and R1A, R1B and R2B adrenergic
receptors and other receptor, enzyme, ion channel profiling were
conducted at NovaScreen Biosciences (Hanover, MD) using their
standard documented screening protocols.
1
OD) δ 8.02 (s, 1H), 7.56 (d, J ) 8.4 Hz, 1H), 6.90 (d, J ) 8.4 Hz,
1H), 5.02-4.90 (m, 2H), 3.90 (m, J ) 5.2 Hz, 1H), 1.34 (d, J )
6.8 Hz, 3H); LC/MS m/z 226 (M + H+); [R]D +20.0°, [R]405 +50.0°
(c 0.51, MeOH). Anal. (C10H12ClN3O‚2HCl‚0.5H2O) C, H, N.
1-((S)-2-Aminopropyl)-7-iodo-1H-indazol-6-ol (17). To a solu-
tion of 9 (1.5 g, 7.6 mmol) in THF (10 mL) at ambient temperature
was added N-iodosuccinimide (1.9 g, 8.4 mmol). After 1 h, the
solution was purified by chromatography (silica, 10% methanol in
dichloromethane) to yield a solid (0.56 g, 44%): mp 132-134 °C;
1H NMR (DMSO-d6) δ 7.91 (s, 1H), 7.55 (d, J ) 8.4 Hz, 1H),
6.82 (d, J ) 8.4 Hz, 1H), 4.67-4.53 (m, 2H), 3.37 (m, 1H), 0.96
(d, J ) 6.6 Hz, 3H); 13C NMR (DMSO-d6) δ 156.5, 140.5, 132.7,
121.7, 119.6, 111.2, 61.3, 56.4, 48.6, 20.2; MS (ES) m/z 318 (M
+ H+); [R]D +22.0°, [R]405 +192.0° (c 0.54, MeOH). Anal. (C10H12-
IN3O‚0.1H2O) C, H, N.
Determination of Compound Stability. The aqueous stability
of compounds was determined in pH 7.4, 0.025 M sodium
phosphate buffer. Each compound was dissolved [5 µg/mL (0.0005%)
and/or 1%] in buffer, and the solutions were heated at either 75 °C
for up to 4 weeks or at 45 °C for up to 12 weeks. Water for injection
was used for buffer preparation. For pH adjustment, 0.6 N HCl
and 1.0 N NaOH stored in glass containers were used. An HPLC
method was developed for the analysis of stability samples of each
compound. The stability results (percent degradation) were used
for calculation of the predicted half-life of a compound at 25 °C.
This prediction was based on the time required for a 10% loss of
compound (T90) and the fact that the rate of degradation for a first-
order reaction decreases 50% for every 10 °C drop in temperature.
Determination of Distribution Coefficients. Partitioning of
compounds between n-octanol and aqueous buffer was determined
at pH 7.4 using 0.1 M phosphate buffer. The initial concentration
(C1) of compound in buffer and the buffer concentration following
extraction with n-octanol (C2) were determined by RP-HPLC
analysis against concentration standards for the specific compound.
The distribution coefficient (DC) of a compound at a given pH
was calculated using the equation DCpH ) (C1 - C2)/C2.
In Vitro Permeability and P-Glycoprotein Efflux. Permeability
and transport studies were conducted and the data analyzed at
Absorption Systems, Exton, PA, using methods previously de-
scribed.35,36 Briefly, MDCK(MDR) monolayers were grown to
confluence on collagen-coated, microporous, polycarbonate mem-
branes in 12-well Costar Transwell plates. To ensure monolayer
integrity, the transepithelial electrical resistance (TEER) was
measured. Only cell monolayers with TEER values >1900 Ω‚cm2
were used. The permeability assay buffer was Hank’s balanced salt
solution containing 10 mM HEPES and 15 mM glucose at a pH of
7.0-7.2. Permeability through a cell-free (blank) membrane
determined nonspecific binding and free diffusion of the test article
through the device. Solution concentrations of the test articles were
10 µM in assay buffer. At each time point, 1 and 2 h, a 200-µL
aliquot was taken from the receiver chamber and replaced with
fresh assay buffer. Cells were dosed on the apical side [apical-to-
basolateral, absorptive transport, (A-B)] or basolateral side [ba-
solateral-to-apical, secretory transport, (B-A)] and incubated at 37
°C with 5% CO2 at 90% relative humidity. Each determination was
performed in duplicate. Lucifer Yellow permeability was measured
for each monolayer after the experiment to ensure that the cell
monolayer integrity and viability was not compromised by the test
article. Postexperiment Lucifer Yellow permeability in monolayers
was 0.24-0.75 nm/s.
Adrenergic Cloned Human r2A and r2C Receptor Binding.
Membranes from Sf9 cells expressing the cloned human R2A and
R
2C receptor (Biosignal, Montreal, Canada) were diluted to 32 and
48 µg/mL protein, respectively, in 75 mM Tris-HCl containing 12.5
mM MgCl2 and 2 mM EDTA (pH 7.4). The membranes were
resuspended using a Branson Sonifier 450 (Branson Ultrasonics
Corp., Danbury, CT) (<20 s). Drug dilutions were made in 1:1
DMSO:ethanol (v/v) using a Biomek 2000 robot (Beckman
Instruments, Fullerton, CA). The diluted compounds (25 µL),
followed by a volume of 200 µL of receptor preparation and finally
25 µL of [3H]clonidine (28 nM final concentration), were added
by the Biomek 2000 robot to a 96-well plate. The incubations (60
min at 23 °C) were terminated by rapid vacuum filtration on a
Tomtec Harvester 96 (TomTech, Hamden, CT) using Whatman
GF/C glass fiber filters that were previously soaked in 0.3%
polyethyleneimine. The filters were washed with ice-cold 50 mM
Tris-HCl, pH 7.4. The samples were counted on a TopCount
scintillation counter (Packard Instruments, Meriden, CT).
In Vitro Functional Assays. Serotonin Human 5-HT1A Recep-
tor Activity: Inhibition of cAMP Production in Cultured Cells.
The procedure was previously described.7 Briefly, Chinese hamster
ovary (CHO) cells expressing the cloned human 5-HT1A receptor
were preincubated with the phosphodiesterase inhibitor 3-isobutyl-
1-methylxanthine (1 mM final) for 20 min at 23 °C, followed by
the addition of the test compounds, and the incubation continued
for another 20 min. The adenylyl cyclase activator forskolin (10
µM) was added and the incubation terminated after 10 min using
ice-cold 0.1 M acetic acid. The measurement of cAMP was
performed using an enzyme immunoassay as previously described.41
The inhibition of forskolin-induced cAMP production by the test
compounds was analyzed using a nonlinear, iterative curve-fitting
computer program.38-40
To determine the transport of compounds in the absence of
functional P-gp activity, the above experimental conditions were
used but in the presence of the P-gp inhibitor cyclosporin A
(CSA).37 Cells were preincubated for 30 min with the inhibitor (10
µM) and then washed. During the permeation determination period,
CSA (10 µM) was present on both sides of the membrane. The
PappA-B determined in the presence of CSA was taken as an estimate
of the permeability attributed to passive diffusion for the compound
(PappPD).

326 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
May et al.
Serotonin 5-HT2A Receptor Functional Activity: [Ca2+
]
subcultured with 0.05% trypsin/0.53 mM EDTA in 48-well plates
with confluence being reached in approximately 3 days. Growth
medium was replaced with fresh medium 24 h before assay of
confluent cells.
The neuroblastoma × glioma rat-mouse hybrid NH108-15 cells
expressing endogenous R2C receptors were grown in DMEM with
high glucose supplemented with 10% (v/v) heat-inactivated fetal
bovine serum in a humidified atmosphere of 5% CO2/95% air. Cells
were subcultured with 0.05% trypsin/0.53 mM EDTA in poly-D-
lysine-coated 48-well plates with confluence being reached in
approximately 3 days. Growth medium was replaced with fresh
medium 24 h before assay of confluent cells in order to avoid the
nutrient exhaustion and acidic pH.
i
Mobilization Assay. The procedure was previously described.7
Briefly, the receptor-mediated mobilization of intracellular calcium
([Ca2+]i) was studied with the fluorescence imaging plate reader
(FLIPR) using rat vascular smooth muscle cells (A7r5, expressing
native 5-HT2 receptors) in 96-well culture plates.42 An aliquot (25
µL) of the test compound was added to the Ca2+-sensitive dye-
loaded cells, and the fluorescence data were collected in real time
at 1.0-s intervals for the first 60 s and at 6.0-s intervals for an
additional 120 s. Responses were measured as peak fluorescence
intensity minus basal and, where appropriate, were expressed as a
percentage of a maximum 5-HT-induced response (Emax). The
concentration-response data were analyzed using a nonlinear,
iterative curve-fitting computer program.42
Cyclic AMP Functional Assays. Confluent cultures of HT29,
OK, and NG108 cells were washed twice with 0.5 mL of 15 mM
HEPES-buffered DMEM (DMEM/F12) and then incubated with
0.5 mL of DMEM/F12 containing 1.0 mM 3-isobutyl-1-methylx-
anthine (IBMX) and 0.8 mM ascorbate at room temperature for 20
min. At the end of this period, the appropriate serially diluted test
agents in ethanol were added, such that the final ethanol concentra-
tion was 1%. Cells were incubated for a further 10 min. Then the
appropriate concentration of forskolin (for HT29, cells 4 µM; OK
cells, 10 µM; and NG108 cells, 1 µM) was added, and the cells
were incubated an additional 10 min. At the end of the incubation
period, the media was aspirated and 150 µL of 0.1 M acetic acid
pH 3.5 was added for termination of cAMP synthesis and activation
of cell lysis. The plates were incubated at 4 °C for 20 min. Then
220 µL of 0.1 M sodium acetate pH 11.5-12 was added to
neutralize the samples prior to analysis of cAMP levels using an
enzyme immunosorbant assay kit as previously described.41 Con-
centration-response curves were constructed and the data analyzed
as described above to obtain the potency values of the compounds.
In Vivo Assays. Mouse Head-Twitch Studies. These studies
were conducted at Calvert Laboratories, Inc., Olyphant, PA, using
male Sprague-Dawley mice that were 4-10 weeks old and
weighing 20-40 g, under conditions previously reported.46,47
Briefly, the desired dose of the test compound, as a solution of the
free base or a suitable salt, was administered to each mouse by
subcutaneous injection (saline, pH 6.5-7.5, bolus of 20 mL/kg),
followed by placing the mouse in an observation area. The head-
twitch response was scored at 2 min intervals during the 10-20
min postdose period. Five mice were used for each concentration
of the compound evaluated. Mean scores ((SEM) for each 2 min
interval as well as the total mean score ((SEM) over the 10-min
period were recorded. The effective dose required to produce a mean
of five twitches during the total scoring period (ED5) was calculated
by interpolation of the results from bracketing doses. The primary
reference compound was R-DOI, which was determined to have
an ED5 of 0.11 mg/ kg in this assay.
Acute Ocular Hypotensive Response in Monkeys. Compounds
were evaluated for their ability to lower intraocular pressure in
conscious cynomolgus monkeys with laser-induced ocular hyper-
tension in the right eyes.7 Briefly, IOP was determined with an
applanation tonometer after light corneal anesthesia with 0.1%
proparacaine; eyes were rinsed with saline after each measurement.
After a baseline IOP measurement, the test compound was instilled
in one 30-µL aliquot to the right eyes only of eight or nine
cynomolgus monkeys. Vehicle was instilled in the right eyes of
five or six additional animals. Subsequent IOP measurements were
taken at 1, 3, and 6 h. The significance of the IOP response was
evaluated using Student’s t-test to compare difference from baseline
for each time point. A compound is considered efficacious in this
model of ocular hypertension if there is a decrease from baseline
IOP in the lasered eye of at least 20% following topical administra-
tion.
Serotonin 5-HT2B Receptor Functional Activity: Rat Isolated
Stomach Fundus. This assay was conducted by MDS Pharma
Services, Bothell, WA, using methods previously described.43 In
brief, longitudinal stomach fundus strips dissected from adult Wistar
rats were mounted in 25-mL organ baths containing oxygenated
Krebs buffer (pH 7.4) maintained at 37 °C. After a 45-min
equilibration period, 10-µL aliquots of test agents were added to
the organ bath (10 mL volume), and isometric tension was recorded
via an FT03 transducer. Cumulative contractile dose-response
curves were constructed for test agonists. R-Methyl-5-HT was used
as a standard reference agonist. Dose-response data were analyzed
as described above to obtain the potency values (EC50) of test
agents.
Serotonin 5-HT2C Receptor Functional Activity: [Ca2+
]
i
Mobilization Assay. These assays were performed as for the
5-HT2A receptor above, except that SR3T3 cells expressing the
recombinant rat 5-HT2C receptor were utilized.
Serotonin 5-HT7 Receptor Functional Activity. SV-40 im-
mortalized human corneal epithelial cells (CEPI-17-CL4 cell) were
cultured in KGM (keratinocyte growth medium) with 0.15 mM
CaCl2. Amphotericin B and gentamicin were replaced by penicillin
(100 units/mL) and streptomycin (100 µg/mL).44 Media and other
supplements were products of Bio-Whittaker (Walkersville, MD).
Agonist-dependent cyclic AMP formation was measured by a
method described previously.45 In brief, compounds of interest were
diluted in ethanol such that the final ethanol concentration was 1%.
Upon reaching confluence, the cells were rinsed twice with 0.5
mL of DMEM/F-12. The cells were incubated for 20 min in the
presence or absence of 5-HT receptor antagonists with DMEM/F-
12 containing 0.8 mM ascorbate and 1.0 mM of the phosphodi-
esterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, Sigma, St.
Louis, MO) at room temperature (∼22 °C). Serotonin receptor
agonists were added at the end of this period, and the reaction was
allowed to proceed for 15 min. 5-Carboxamidotryptamine (5-CT;
100 nM) was used as a positive control. After aspiration of the
reaction medium, ice-cold 0.1 M acetic acid (150 µL, pH 3.5) was
added for the termination of cAMP synthesis and cell lysis. Finally,
ice-cold 0.1 M sodium acetate (225 µL, pH 11.5-12.0) was added
to neutralize the samples prior to analysis by the EIA.45 Cyclic
AMP production was measured using an EIA kit purchased from
Amersham Pharmacia Biotech (Piscataway, NJ). This assay was
conducted according to the package insert in an automated manner
using the Biomek 2000 robot (Beckman Instruments, Fullerton,
CA).45
Adrenergic r2A, r2B, r2C Receptor Functional Assays. Cell
Culture. HT29 human clonic adenocarcinoma cells expressing
endogenous R2A receptors were grown in McCoy’s 5A medium
modified supplemented with 10% (v/v) heat-inactivated fetal bovine
serum in a humidified atmosphere of 5% CO2/95% air. Cells were
subcultured with 0.05% trypsin/0.53 mM EDTA in 48-well plates
with confluence being reached in approximately 4 days. Growth
medium was replaced with fresh medium 24 h before assay of
confluent cells in order to avoid nutrient exhaustion.
Opposum kidney (OK) proximal tubule kidney cells expressing
endogenous R2B receptors were grown in DMEM with high glucose
supplemented with 10% (v/v) heat-inactivated fetal bovine serum
in a humidified atmosphere of 5% CO2/95% air. Cells were
Acknowledgment. We thank Gary Williams, Parvaneh
Katoli, Colene Drace, Shouxi Xu, Daniel Scott, Tony Wallace,
Lanaya Wood, and Drs. Curtis Kelly and Julie Crider for their
expert technical assistance; Dr. John Liao of our Physical

1-((S)-2-Aminopropyl)-1H-indazol-6-ol
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Supporting Information Available: Mouse head-twitch re-
sponse data, additional ligand binding data for 9, and elemental
analysis data. This material is available free of charge via the
Internet at http://pubs.acs.org.
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