Carbamate derivatives of indolines as cholinesterase inhibitors and antioxidants for the treatment of Alzheimer's disease

Article abstract of DOI:10.1021/jm301411g

The cascade of events that occurs in Alzheimer's disease involving oxidative stress and the reduction in cholinergic transmission can be better addressed by multifunctional drugs than cholinesterase inhibitors alone. For this purpose, we prepared a large number of derivatives of indoline-3-propionic acids and esters. They showed scavenging activity against different radicals in solution and significant protection against cytotoxicity in cardiomyocytes and primary cultures of neuronal cells exposed to H₂O₂ species and serum deprivation at concentrations ranging from 1 nM to 10 μM depending on the compound. For most of the indoline-3-propionic acid derivatives, introduction of N-methyl-N-ethyl or N-methyl-N-(4-methoxyphenyl) carbamate moieties at positions 4, 6, or 7 conferred both acetyl (AChE) and butyryl (BuChE) cholinesterase inhibitory activities at similar concentrations to those that showed antioxidant activity. The most potent AChE inhibitors were 120 (3-(2-aminoethyl) indolin-4-yl ethyl(methyl)carbamate dihydrochloride) and 94 (3-(3-methoxy-3-oxopropyl)-4-(((4-methoxyphenyl)(methyl) carbamoyl)oxy)indolin- 1-ium hydrochloride) with IC₅₀s of 0.4 and 1.2 μM, respectively.

Full text of DOI:10.1021/jm301411g

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Article
Carbamate Derivatives of Indolines as Cholinesterase Inhibitors
and Antioxidants for the Treatment of Alzheimer’s Disease
Innesa Yanovsky, Efrat Finkin-Groner, Andrey Zaikin, Lena Lerman, Hila Shalom,
Shani Zeeli, Tehilla Weil, Isaac Ginsburg, Abraham Nudelman, and Marta Weinstock
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 14 Nov 2012
Downloaded from http://pubs.acs.org on November 16, 2012
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Carbamate Derivatives of Indolines as Cholinesterase Inhibitors and
Antioxidants for the Treatment of Alzheimer’s Disease
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Inessa Yanovsky, , Efrat FinkinꢀGroner, Andrey Zaikin, Lena Lerman, Hila
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Shalom, Shani Zeeli, Tehilla Weil, Isaac Ginsburg, Abraham Nudelman, and Marta
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Weinstock
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Department of Chemistry Bar Ilan University Ramat Gan, Israel. Institute of Drug
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Research Hebrew University Medical Center, Israel. Institute of Dental Research,
Hadassah School of Dental Medicine, The Hebrew University of Jerusalem, Jerusalem,
Israel.
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ABSTRACT
The cascade of events that occurs in Alzheimer’s disease involving oxidative stress and
the reduction in cholinergic transmission can be better addressed by multifunctional
drugs than cholinesterase inhibitors alone. For this purpose we prepared a large number
of derivatives of indolineꢀ3ꢀpropionic acids and esters. They showed scavenging activity
against different radicals in solution and significant protection against cytotoxicity in
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cardiomyocytes and primary cultures of neuronal cells exposed to H O species and
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serum deprivation at concentrations ranging from 1 nMꢀ10 ꢁM depending on the
compound. For most of the indolineꢀ3ꢀpropionic acid derivatives introduction of Nꢀ
methylꢀNꢀethyl or NꢀmethylꢀNꢀmethoxyphenyl carbamate moieties at positions 4, 6 or 7
conferred both acetyl (AChE) and butyryl (BuChE) cholinesterase inhibitory activities at
similar concentrations to those that showed antioxidant activity. The most potent AChE
inhibitors were 120 (3ꢀ(2ꢀaminoethyl) indolinꢀ4ꢀyl ethyl(methyl)carbamate
dihydrochloride), and 94 (3ꢀ(3ꢀmethoxyꢀ3ꢀoxopropyl)ꢀ4ꢀ(((4ꢀmethoxyphenyl)(methyl)
carbamoyl)oxy)indolinꢀ1ꢀium hydrochloride) with IC s of 0.4 and 1.2 ꢁM, respectively.
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INTRODUCTION
The sporadic form of Alzheimer’s disease (AD) is the most common cause of dementia in
the elderly. It is characterized by progressive neurodegeneration resulting in impaired
cognition and behavioral abnormalities. Cognitive dysfunction is associated with a
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reduction in cholinergic transmission in cortical and hippocampal neurons. This
observation led to the introduction of cholinesterase (ChE) inhibitors in an attempt to
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maintain cholinergic transmission. Of these, donepezil and galantamine are reversible,
selective inhibitors of acetylcholinesterase (AChE), while rivastigmine contains a
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carbamate moiety that binds covalently to the enzyme and remains attached to it until it is
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removed slowly by hydrolysis. Rivastigmine inhibits both AChE and (BuChE). As AD
progresses levels of AChE in the brain decline, but those of BuChE, found mainly in
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subcortical neurons and glial cells, remain unchanged or increase. Inhibition of BuChE
can increase levels of ACh in the brain and may also be able to impede the formation of
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abnormal beta amyloid, which can further impair neuronal activity. In addition, it was
shown that treatment with the dual AChE and BuChE inhibitor rivastigmine in women
with the wild type BuChE gene slowed progression of cognitive impairment and reduced
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white matter loss in the brain. Therefore, inhibitors of both AChE and BuChE may have
additional benefits in AD and also in dementias associated with subcortical pathology,
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such as that of Parkinson’s disease, Lewy body’s disease and also vascular dementia.
Oxidative stress is one of the earliest events in the pathogenesis of AD and may even
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occur before the onset of memory loss and the appearance of senile plaques and
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neurofibrillary tangles. Oxidative stress has also been shown to mediate tauꢀinduced
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neurodegeneration. The latter is more closely correlated with cognitive impairment than
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the number or size of βꢀamyloid plaques. Oxidative and nitrative stress results from the
formation of reactive oxygen (ROS) and reactive nitrogen (RNS) species, respectively.
ꢀ●
Nitric oxide (NO) released from activated microglia reacts with superoxide anion (O )
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to form peroxinitrite (ONOO ), which can damage proteins by causing nitration of
tyrosine residues. This can be detected in the brain with an antibody to 3ꢀnitrotyrosine in
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subjects with Mild Cognitive Impairment a prodromal phase of AD. Thus, drugs that
combine inhibition of AChE and BuChE with the ability to reduce oxidative and nitrative
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stress may be more efficacious than those that only inhibit ChE in preventing and/or
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treating AD.
The naturally occurring compound, indoleꢀ3ꢀpropionic acid (IPA) was shown to possess
radical scavenging properties and to protect primary neurons in culture against oxidative
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damage induced by beta amyloid. In previous research from our laboratory it was found
that introduction of an OH group into the 4 or 6ꢀpositions of the indole ring increased its
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antioxidant potential. Carbamoylation of the OH group with N,Nꢀdialkylꢀ or NꢀalkylꢀNꢀ
arylꢀcarbamoyl chlorides confers AChE and BuChE inhibitory activity on these
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molecules. Chyan et al. claimed that an electronꢀrich aromatic ring system in IPA is
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essential for antioxidant activity. Zaikin (2007) found that indolineꢀ3ꢀpropionic acid,
the reduced analogue of IPA, was more potent and effective than IPA as an antioxidant,
probably because it has an anilinic type of amino group that should enable it to undergo a
facile oxidation to the corresponding quinoidal type of compound (Fig. 1), whereas the
aromatic indole may be more resistant to oxidation. Thus, as the indolines are oxidized
they act as reducing agents more readily than the corresponding indoles.
The current study describes the preparation and in vitro activities of carbamate
derivatives of indolineꢀ3ꢀpropionic acid and esters as inhibitors of AChE and BuChE and
ROS and RNS radical scavengers. Their potential as protectants against cytotoxicity
induced in cardiomyocytes and neuronal cells by oxidative stress was also assessed. The
goal was to identify compounds that were equipotent in protecting cells against
cytotoxicity induced by ROS and RNS and inhibiting AChE and BuChE that could be
developed for the treatment of AD and other neurodegenerative diseases.
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CHEMISTRY
The syntheses of the various indoline derivatives are described in the Scheme 1, and the
synthetic intermediates and final products are listed in Table 1. The starting materials
were the commercially available hydroxy or benzyloxy substituted indoles 1ꢀ5 of general
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formula I. Initial carbamoylation of the phenolic OH with various R R NCOCl regents
gave the corresponding carbamates II. Vilsmeier–Haack formylation of the indolic
carbamates led to compounds III, which were subsequently converted into the α,βꢀ
unsaturated esters IV via a Knoevenagel condensation reaction. Selective reduction of the
sideꢀchain double bond of IV gave compounds V. In view of the poor aqueous solubility
of the latter and their low basicity, that prevented the formation of salts, the compounds
were further reduced to give the corresponding indolines VI, which were basic enough to
form water soluble salts readily VII. Some of the esters were hydrolyzed to their
corresponding acids, which in some cases were converted into the amides. To evaluate
the importance of the propionic acid or ester sideꢀchain linked at position 3 of the
indolines, several derivatives of formula VIII, prepared by reduction of the indoles II,
were converted into the corresponding waterꢀsoluble salts IX. To compare the activity of
the NH indolinic compounds to their corresponding NꢀR analogs, some of the compounds
were Nꢀmethylated with MeI, or alkylated with methyl acrylate to give compounds 113
and 114 belonging to category IX. To compare the biological activity of the indoleꢀ3ꢀ
propionic acid derivatives to that of compounds having a pattern of substitution
analogous to that of melatonin (without the acetamido group), compound 120 belonging
to category XIV was prepared from the nitro derivative 116, which underwent three
stages of reduction (Scheme 1). Compound 120 was isolated as the dihydrochloride salt.
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RESULTS AND DISCUSSION
Chemical and metabolic stability studies. The chemical stability in solution of two
carbamates 87 and 98 assessed by high performance liquid chromatography showed that
the mesylate salt of 87 is stable in aqueous solution of 1 mg/mL at room temperature
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(25°C) for at least 9 days. In the presence of human plasma at 37 °C, 40% of 87 was
hydrolyzed to 90 within 90 min, but the carbamate group remained intact. A concentrated
solution of the citrate salt of 98 (100 mg/mL) maintained at room temperature was
hydrolyzed to the acid 99 within a few days. Dilute solutions of 0.5 mg/mL were stable,
as solutions of 50 mg/mL or more if they were stored at ꢀ20 °C. In the presence of human
or mouse liver microsomes 87 or 98 were rapidly converted to their respective acids, 90
and 99. No other metabolic changes were detected such as the removal of the alkyl
radicals, Et and Me from the carbamate in contrast to what was shown for (6ꢀ(NꢀethylꢀNꢀ
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methylcarbamyloxy)ꢀN propargylꢀ1(R)ꢀaminoindan.
AChE and BuChE inhibitory activity. Three indole derivatives substituted with a
carbamate group (51, 53 and 69) that were tested for AChE inhibitory activity were found
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to be 2.5ꢀ20 times weaker than their indoline analogs (Table 2). They were also much
less active as antioxidants and therefore no further indoles were prepared or tested. We
have previously reported that N,Nꢀdimethyl carbamate derivatives of phenylethylamine
and Nꢀpropargylꢀaminoindan are more potent inhibitors of AChE than those in which one
alkyl is methyl and the second has a longer chain, such as ethyl, propyl or butyl, but their
duration of action is much shorter because of the more rapid hydrolysis of the
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carbamate.
When the second substituent is 4ꢀmethoxyphenyl, the AChE inhibitory
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activity is restored to that of the dimethyl analog but the rate of decarbamoylation of the
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enzyme is much slower, thereby increasing the duration of enzyme inhibition.
In preliminary studies we found that introduction of a carbamate into position 5 of the
indoline structure resulted in very weak AChE inhibitors with IC s >500 ꢁM because
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they readily undergo oxidation to quinoidal structures. Therefore in the current series,
the carbamate moiety was sited in positions 4 or 6 and in one compound 104, in position
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. Again, the N,Nꢀdimethylamino (111) and the NꢀmethylꢀNꢀ(4ꢀmethoxyphenyl) (94)
derivatives were the most potent AChE inhibitors. The greater AChE inhibitory activity
of 94 than that of carbamates with ethyl or butyl alky chains may be explained by the πꢀπ
interaction of the aryl substituent with phenylalanines 288 and 290 located at the bottom
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of the gorge adjacent to the active site of AChE. By contrast, compounds with Nꢀbutyl
or Nꢀmethoxyphenyl substituents 93, 94, 95, 96, 97, 100, 102 and 103) were much
weaker inhibitors of BuChE than their respective NꢀmethylꢀNꢀethyl analogs (87, 88, 89,
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phenylalanines of AChE are replaced by leucine and valine, thereby eliminating the πꢀπ
interaction. The gorge of BuChE is larger than that of AChE enabling the NꢀmethylꢀNꢀ
ethyl carbamates to interact more efficiently with the active site than with that of AChE.
However, this does not enable us to explain why compounds 93 and 100 were so much
weaker as BuChE inhibitors than their NꢀmethylꢀNꢀethyl analogs. This reduction in
BuChE inhibitory activity relative to that of the NꢀmethylꢀNꢀethyl analogs was not seen
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with the NꢀmethylꢀNꢀbutyl derivatives of aminoindan or phenylethylamine.
The propionic acid derivatives (90, 91, 95 and 99) were found to be weaker AChE
inhibitors than their respective methyl esters. Substitution of methyl ester by ethyl (89)
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decreased AChE inhibitory activity more than that of BuChE, while conversion of the
ester to an amide (92) caused a much larger reduction in BuChE inhibitory activity. With
the exception of compound 103, the Nꢀmethyl indolines were all weaker AChE and
BuChE inhibitors than their respective NꢀH analogs. On the other hand, substitution of
the propionic acid methyl ester side chain by a 2ꢀaminoethyl group resulted in the most
potent AChE inhibitor of this series (120). Its greater activity than that of the compounds
with an acidic or ester group in the same position may result from the interaction of the
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basic NH group with the peripheral anionic site situated at the entrance to the narrow
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gorge of the enzyme, thereby causing improved alignment of the molecule to the catalytic
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site situated near its base. The ability to interact with the peripheral anionic site could
confer an added advantage for treatment of AD as other AChE inhibitors that also bind to
the peripheral site the enzyme can prevent the aggregation of amyloidꢀbetaꢀpeptide into
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fibrils and plaques.
As previously reported for aminoindans and phenylethylamines, compounds with the
carbamate moiety in position 6 were 7ꢀ9 times less potent as AChE inhibitors than their
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counterparts in position 4, and 13ꢀ33 times less potent as BuChE inhibitors. The
reduction in AChE activity that was obtained by moving the carbamate from position 4ꢀ6
was 9ꢀfold (90 to 99), 35ꢀfold, (94 to 102), and 88ꢀfold (113 to 115). The 7ꢀsubstituted Nꢀ
methylꢀNꢀethyl carbamate (104) was also 8ꢀfold weaker as an AChE inhibitor than that in
position 4. The IC s for AChE and BuChE of 98 were also somewhat higher than that of
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ladostigil, (6ꢀ(Nꢀethyl,Nꢀmethylcarbamyloxy)ꢀNꢀpropargylꢀ1(R)ꢀaminoindan). The most
active BuChE inhibitor of all the compounds tested was 104 which also showed the
highest selectivity towards this enzyme (770ꢀfold). The lower AChE activity of 6ꢀ and 7ꢀ
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substituted carbamates may result from a change in the overall shape of the molecules
thereby making it more difficult for them to align correctly in the gorge leading to the
active site, as previously shown for derivatives of aminoindan. Such steric constraints are
less critical in the interaction with BuChE.
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Antioxidant activity. Two luminolꢀdependent chemiluminescenceꢀinducing cocktails
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were used to quantify the antiꢀoxidant scavenging abilities of indolines. Cocktail A
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generates a constant wave of light due to H O and Coꢀcatalyzed OH at a degree and rate
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that can be measured by chemiluminescence (Figure 2). Cocktail B generates O , NO
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and a constant wave of light due to ONOO . Although H O is not very reactive it may
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be converted to a number of ROS, including OH and HOO . Melatonin, a derivative of
IPA, which has a methoxy group in position 5 of the indole ring, was shown to scavenge
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ROS and RNS in solution.
In a study that also used a chemiluminometric method to
measure its antioxidant activity, the effective concentrations of melatonin (IC ) were 537
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M and 127 ꢁM for OH radical and peroxynitrite, respectively. In our system
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melatonin gave an IC for scavenging H O and OH generated by glucose oxidase (GO)
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of 95.8 ꢁM and for NO released by morpholino sydononimine (Sin1), the IC was 1.02
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mM. Many of carbamates tested were much more potent than melatonin, with IC s for
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scavenging of H O and OH , ranging from 70 nMꢀ24 ꢁM and for peroxinitrite formed
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by NO from 0.57ꢀ8.5 ꢁM (Table 3). Moreover, with the exception of 94, all the
compounds tested had antioxidant activity at concentrations at or below those that
inhibited AChE. While most of the compounds tested reduced luminescence induced by
the GO and Sin1 cocktails at similar concentrations, compounds 120 and 113 were about
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compounds showed similar antioxidant activity to that of the other carbamates when
tested against 2,2ꢀdiphenylꢀ1ꢀpicrylhydrazyl radical (DPPH). The apparently selective
effect against ROS generated by GO may have resulted from inhibition of the GO
enzyme thereby preventing their formation.
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0
Attachment of the carbamate to AChE or BuChE and consequent carbamoylation of the
enzyme releases the respective 4ꢀ or 6ꢀhydroxy indoline analogs (leaving groups). These
leaving groups, compounds 81, 84, and 110, were found to be as good as, or even better
than the parent carbamates at scavenging either ROS or RNS (Table 4). In general,
compounds with a carbamate or an OH group in position 4 were better scavengers of
ROS but not of RNS than those in position 6.
The antioxidant activity of the 4ꢀ and 6ꢀhydroxy indolines was also seen when ROS were
+3
generated in the presence of Fe and measured by a fluorescence method, or against the
+
5
+2
radical DPPH that does not depend on the presence of ROS, Se and Co . In general,
compounds with a carbamate or an OH group in position 4 were better scavengers of
ROS but not of RNS than those in position 6 (Table 4).
Protection against cytotoxicity induced by oxidative stress in cardiomyocytes. The H9c2
cardiomyocytes were chosen as a model in which to test the potential protective effect of
the compounds against cytotoxicity induced by ROS because they are more than 10 times
3
0, 31
more sensitive than neuronal cells to apoptosis induced by H O .
H9c2
2
2
cardiomyocyte cells derived from rat cardiac myoblasts were treated with 100 ꢁM H O
2
2
for 1 h, showed 30ꢀ50% cell mortality. Many of the compounds tested against H O
2
2
cytotoxicity caused a significant reduction in cell death at two concentrations ranges 1
pmꢀ100 nM and 10ꢀ100 ꢁM (Table 5 and Figure 3). At a concentration of 100 ꢁM none
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of the compounds showed any significant reduction of cell viability. Only two indole
derivatives were tested for antioxidant activity in cardiomyocytes. Of these, 67 was more
than 10,000 times weaker and 69 about 100 times weaker than their respective indoline
analogs (Table 5). With the exception of 91, the most potent indoline compounds that
significantly reduced apoptosis at concentrations of 10ꢀ100 pM were all carbamates and
methyl esters of propionic acid (87, 94, 98 and 102). The corresponding acid derivatives
of three of these carbamates (92, 96 and 99) were much less potent or inactive at
concentrations below 100 nM (Table 5). The exception was 91, which maximally reduced
cytotoxicity by 43ꢀ78% at concentrations of 1 pMꢀ1 nM and has a propionic acid group
and a methyl on the indoline nitrogen. The only other Nꢀmethyl derivatives tested (88 and
103), were both methyl indolineꢀ3ꢀpropionate and were much less active than 91. The
carbamates were also more potent than their respective leaving groups with an OH in
positions 4, 81, or 6, 84. This protective effect could have resulted from an action inside
the cell in which the compounds prevent the fall in the mitochondrial potential (∆Ψm)
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17
induced by oxidative stress as was shown by Zaikin, (2007), and was also reported for
3
2
ladostigil, a carbamate derivative of aminoindan.
Ladostigil also protected
cardiomyocites at a concentration range of 0.1ꢀ10 nM but to a somewhat lesser extent
than the Nꢀmethyl Nꢀethyl carbamate derivatives of indoline methyl esters 87 and 98. The
fall in ∆Ψm is thought to occur through the formation of pores in the mitochondria by
33
dimerized proꢀapoptotic proteins Bax or activated Bid, Bak, or Bad. Activation of these
proꢀapoptotic proteins is accompanied by the release of cytochrome C into the cytoplasm.
The more lipophilic carbamates and methyl indolineꢀ3ꢀpropionate derivatives are likely
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to penetrate the cell and mitochondrial membranes more readily than their respective OH
or acids derivatives.
As the concentration of the compounds increased above 100 nM their protective effect
declined but reappeared at 1 ꢁM, or more frequently at 10ꢀ100 ꢁM. The concentrations
constituting the second range of protective activity are compatible with those that
scavenged ROS and RNS. None of the compounds caused any measurable cytotoxicity in
cardiomyocytes at concentrations up to 100 ꢁM (Table 5).
0
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Protection against apoptosis induced by oxidative stress through serum deprivation in
chick neuronal cells. This experiment was performed in order to obtain evidence of a
protective effect of selected compounds against another insult that induces oxidative
stress in a primary neuronal cell culture. Serum deprivation induces apoptosis because of
34
a lack of necessary nutrients and trophic factors. Oxidative stress activates the intrinsic
(caspase 9ꢀdependent) apoptotic pathway causing the release of cytochrome C, and can
35
be prevented by drugs that scavenge or inactivate ROS. Four compounds comprising
two carbamates and their leaving groups (112, 110, 87 and 81) showed significant
protection at a concentration of 10 nM and also at those ranging from 1ꢀ10 ꢁM against
cytotoxicity induced in a primary culture of chick neuronal cells by serum deprivation
(Figure 4). This protective effect may also result from different actions of the drugs at
low and higher concentrations to reduce apoptosis.
■
CONCLUSION
We have described the synthesis and activity in vitro of indolines substituted at position 3
with propionic acids, their ester analogs or a 2ꢀaminoethyl group, and substituted on the
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aromatic ring with an NꢀmethylꢀNꢀethyl or NꢀmethylꢀNꢀbutyl, or NꢀmethylꢀNꢀ4ꢀ
methoxyphenyl carbamate moiety in positions 4, 6 or 7. These compounds were found to
act as inhibitors of AChE and BuChE and as antioxidants. Compounds with the
carbamate in position 4 are more potent AChE and BuChE inhibitors than those in
positions 6 or 7, and the methyl esters are more potent than their respective acids.
Compounds with an (NꢀmethylꢀNꢀethyl)carbamate are more potent inhibitors of BuChE
than of AChE and those with longer alkyl or aryl substituents. With the exception of 94
which has a (NꢀmethylꢀNꢀmethoxyphenyl)carbamate in position 4, all the compounds
tested, at concentrations similar to or lower than those inhibiting AChE, show antioxidant
10
11
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51
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60
+
5
activity against ROS and RNS generated by GO and Sin1 in the presence of Se and
+
2
Co . Carbamoylation of AChE releases the respective 4ꢀ or 6ꢀOH indoline analogs
(leaving groups). These leaving groups (compounds 81, 84, and 110) were as good as, or
better than the parent carbamates at scavenging either ROS and/or RNS. Many of the
compounds, particularly the methyl indolineꢀ3ꢀpropionate with a carbamate moiety are
able to reduce cytotoxicity induced by H O in cardiomyocytes at concentrations ranging
2
2
from 1 pmꢀ100 nM, and act by reducing the fall in the mitochondrial potential induced by
H O . At higher concentrations, compatible with those that scavenge ROS, almost all the
2
2
compounds also protect against cytotoxicity. Thus, several of these novel compounds
possess advantages over existing AChE inhibitors for the treatment of AD since they
interact at relevant concentrations with several important targets to reduce the
pathological changes occurring in this disease.
■
EXPERIMENTAL SECTION
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Chemistry. General remarks. 1HꢀNMR, 13CꢀNMR and 19FꢀNMR spectra were
obtained on Bruker Avanceꢀ200, AvanceꢀDPXꢀ300, AvanceꢀDMXꢀ600 and AvanceꢀIIIꢀ
7
00 spectrometers. Chemical shifts are expressed in ppm downfield from Me3Si (TMS)
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5
6
7
8
9
0
used as internal standard. The values are given in δ scale. The "t" is indicative of a
multiplet similar to a triplet with second order characteristics. Mass spectra (MS) were
obtained on a Varian Mat 731 spectrometer (CI+ = chemical ionization). HRMS were
obtained on an AutoSpec spectrometer (Water companyꢀUK) (CI+ CH4). Electron Spray
ionization (ESI) was obtained on a Micromass QꢀTOF Micro mass spectrometer
(
Micromass (Waters) UK). MALDI were obtained on Autoflex III in TOF/TOF mode
Bruker, Germany). Elemental analyses (CHNO) were obtained using a CHNSꢀO
(
analyzer using a Flash EA model (Italy). Progress of the reactions was monitored by TLC
on silica gel (Merck, Art. 5554). All the flash chromatographic procedures were carried
out on silica gel (Merck, Art. 9385). All moisture sensitive reactions were carried out in
flameꢀdried vessels. Melting points were determined on a FisherꢀJohns apparatus. The
nomenclature of the compounds was assigned according to ChemDraw Ultra version
11.0.1 and 12 (CambridgeSoft). The chemical shifts of compounds containing the
carbamate moiety are assigned to both rotamers. The 4ꢀ and 6ꢀhydroxyindoles 1 and 2,
and the 4ꢀ, 6ꢀ, and 7ꢀbenzyloxyindoles 3, 4 and 5 respectively, were commercial
products. Experimental procedures for compounds the biological activity of which was
assessed are listed below, and data and the synthetic intermediates, which were not
biologically tested, are found in the Supporting Information. The purity of the compounds
was determined by elemental analyses, confirming ≥95% purity.
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General Procedures
36
Procedure A: Synthesis of carbamates. A carbamoyl chloride (15 mmol) was added to
a solution of a 4ꢀ, 5ꢀ or 6ꢀhydroxyindole; or to a 4ꢀ, 5ꢀ, 6ꢀ or 7ꢀhydroxyindoleꢀ3ꢀ
propanoic acid (or ester) (7.51 mmol) in dry CH Cl (50 mL) containing Et N (9.01
10
11
12
13
14
15
16
17
18
19
20
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22
23
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
2
3
mmol) and 4ꢀDMAP (10% mol). The mixture was stirred at room temperature for 24ꢀ72
h. The reaction was quenched by addition of water, and the mixture was extracted with
CH Cl . The organic layer was washed with 5% NaHCO and brine, dried over MgSO
2
2
3
4
and evaporated. The residual carbamates were purified either by chromatography, or by
extraction with 2N NaOH and CH Cl , to remove traces of unreacted hydroxyindoles and
2
2
carbamoyl chlorides, followed by elution of the organic phase through a plug of silica
gel, which was washed with EtOAc:hexane (1:1). The filtrate was evaporated and the
residue was crystallized from CH Cl and hexane.
2
2
37
Procedure B: Vilsmeier formylation. Phosphorus oxychloride (POCl ) (5.38 mmol)
3
was added dropwise with stirring to DMF (20 mmol) under N , the temperature being
2
o
kept at 10ꢀ20 C. A carbamate (4.9 mmol) in DMF (3 mL) was slowly added with
o
stirring, while maintaining the temperature of the mixture at 20ꢀ30 C. The mixture was
o
then stirred at 35 C for 45 min and was then cooled to room temperature. Cold water (10
mL) was added, followed by addition of a solution of 20% NaOH in water, until a
pH~13. Although the reaction was exothermic, the mixture was further heated to its
boiling point for 1 min, and was then stirred and allowed to cool to room temperature. In
some cases, the product, which solidified at this stage, was filtered and the crystals were
washed with water. In other cases, water was added followed by extraction with EtOAc.
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6
The organic phase was washed with water and brine, dried over MgSO and evaporated
4
to give the 3ꢀformylindoles.
38
Procedure C: Knoevenagel condensation. To a solution of malonic acid monomethyl
ester (2.18 mmol) in pyridine (8.09 mmol) containing a catalytic amount of piperidine (2ꢀ
3 drops) was added a 3ꢀformylindole (1.46 mmol). The mixture was refluxed for 5ꢀ6 h,
cooled to room temperature, diluted with EtOAc and acidified with 3N HCl to pH 7.0.
0
1
2
3
4
5
6
7
8
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3
4
5
6
7
8
9
0
The organic phase was separated, washed with brine, dried over MgSO and evaporated
4
and the product was isolated as the α,βꢀunsaturated ester.
39
Procedure D: Reduction of indoles. Method I: NaBH CN (4.23 mmol) was added
3
o
portion wise over 10 min at 0 C to a solution of an α,βꢀunsaturated ester or to an indole
(1.06 mmol) in AcOH (7.1 mL), and the mixture was stirred at room temperature from 1
h to overnight. Water (3 mL) was added and the solvent was removed under reduced
pressure. The residue was dissolved in EtOAc and washed with saturated NaHCO and
3
brine, dried over MgSO and evaporated. The residual indoline, was converted into the
4
hydrochloride salt upon dissolution in EtOAc followed by extraction with 2N HCl. The
aqueous phase was evaporated and the product was isolated as the salt. Alternatively, the
hydrochloride salts were obtained upon addition of HCl gas to a solution of the indolines
in dry ether.
4
0
Method II: To an ice cold stirred solution of an indole (1.64 mmol) in TFA (12.5 mL)
NaBH (3.30 mmol) was added and the mixture was stirred for 22ꢀ77 h at room
4
temperature. Water was then added followed by the addition of saturated NaHCO until a
3
pH = 7ꢀ8. The mixture was then extracted with EtOAc and brine, dried over Na SO and
2
4
concentrated and the residue was purified by chromatography.
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Procedure E: Removal of the benzyl group and reduction of α,βꢀunsaturated system.
4
1
Method I: To a solution of an indole substituted at position 3 with an α,βꢀunsaturated
ester/acid/amide or/and a phenolic compound protected with an Oꢀbenzyl group (6.72
mmol) in EtOH (50 mL) was added 10% Pd/C (0.16 g) and HCO NH (67 mmol). The
10
11
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60
2
4
mixture was refluxed for 1 h. The catalyst was removed by filtration through celite, the
filtrate was concentrated, and the product was isolated as the α,βꢀsaturated and/or the
debenzylated product.
Method II: To a solution of an α,βꢀunsaturated ester/acid/amide and/or a compound
protected with Oꢀbenzyl group or a compound containing a nitro group (1 eq) in EtOH,
was added 10% Pd/C (10% w/w). The mixture was stirred under H at a 1ꢀ3 atm pressure
2
for 4 hꢀ2 days at room temperature. The mixture was then filtered through celite and
concentrated.
42
Procedure F: NꢀMethylation of indoles. To a solution of methyl iodide (3 mmol) and
an indole (3 mmol) in CH Cl (50 mL) was added a solution of 50% NaOH (18 mmol) in
2
2
+
ꢀ
water (0.72 mL), followed by the addition of Bu N Br (9 mmol). The mixture was stirred
4
at room temperature for 16 h, extracted with CH Cl , washed with water and brine, dried
2
2
over MgSO , evaporated, and the product was isolated as the Nꢀmethylated indole.
4
4
3
Procedure G: Hydrolysis of methyl esters. Method I: To a solution of a methyl ester
(0.133 mmol) in MeOH (3.36 mL) a 2 N solution of NaOH (0.46 mL) was added, and the
obtained solution was stirred at room temperature overnight, evaporated and the residue
was dissolved in EtOAc to which 1N HCl was added until a pH = 1ꢀ2. The aqueous phase
was separated, extracted with EtOAc (x3), and the organic layers were combined, washed
with brine, dried over MgSO , filtered and evaporated to give the acid.
4
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Method II: To a solution of a methyl ester (0.1 mmol) in MeOH (0.1 mL) and H O (0.5
2
mmol), KOH (0.5 mmol) was added and the mixture was stirred at room temperature for
1
8 h. The solution was concentrated to remove the MeOH and to the residue EtOAc was
0
1
2
3
4
5
6
7
8
9
0
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0
1
2
3
4
5
6
7
8
9
0
added, and the mixture was extracted with 1N HCl. The organic layer was washed with
brine, dried over Na SO , filtered and evaporated to give the acid.
2
4
4
5
Method III: To an iceꢀcold solution of a methyl ester (0.65 mmol) in MeOH (15 mL)
and H O (2.5 mL), LiOH (6.5 mmol) was added. The obtained solution was refluxed for
2
3.5 h and was then diluted with EtOAc and extracted with 1N HCl. The organic layer was
separated, washed with brine, dried over Na SO , filtered and evaporated to give the acid.
2
4
Procedure H: Coupling with benzotriazoleꢀ1ꢀylꢀoxyꢀtrisꢀ(dimethylamino)ꢀphosphonium
4
6
hexafluorophosphate (BOP). To a solution of an indoleꢀ3ꢀpropionic acid (0.3 mmol) in
DMF (6 mL), under N , at room temperature was added Et N (0.3 mmol) and BOP (0.3
2
3
mmol). The mixture was stirred at room temperature for 45 min followed by addition of
NaNH (0.3 mmol). The mixture was stirred at room temperature overnight, diluted with
2
EtOAc and washed with water (x2), 1N HCl (x2), NaHCO3(aq) (x2), brine, dried over
MgSO , filtered and evaporated under reduced pressure.
4
Procedure I: Preparation of tartrate salts. To a stirred solution of an amine (0.04 mmol)
in EtOH/MeOH (1.5 mL) was added Lꢀ(+)ꢀtartaric acid (0.04 mmol). The mixture was
stirred for 30 min at rt, and evaporated and the product was isolated as the tartrate salt.
47
Procedure J: Preparation of nitro compounds. NH OAc (0.44 mmol), was added to a
4
stirred solution of an aldehyde (0.9 mmol) in MeNO (0.03 mmol) and the solution was
2
refluxed for 1ꢀ3 h, while being monitored by TLC (EtOAcꢀHex (1:2) until disappearance
of the starting aldehyde. The mixture was then diluted with EtOAc and extracted with
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water. The organic layer was washed with brine, dried over Na SO , filtered and
2
4
evaporated to give the nitro compound.
4
8
Procedure K: Reduction of an α,βꢀunsaturated nitro compounds with NaBH . To a
4
10
11
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13
14
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stirred mixture of a nitrostyrene (0.44 mmol), silica gel (1.3 g), isopropanol (1.8 mL) and
CHCl (9.6 mL) was added NaBH (1.8 mmol). The mixture was stirred at room
3
4
temperature for 30ꢀ90 min. The excess NaBH was decomposed with 1N HCl and the
4
mixture was filtered. The filtrate was washed with CH Cl and separated, the organic
2
2
phase was washed with brine, dried over Na SO , filtered and evaporated to give the
2
4
nitroethane.
Procedure L: NꢀAlkylation of an indole with methyl acrylate. To a stirred solution of an
indole, substituted at positions 4 or 6 with an NꢀethylꢀNꢀmethyl carbamate (0.46 mmol),
and methyl acrylate (0.69 mmol) in CH CN (2 mL) was added DBU (0.23 mmol). The
3
o
mixture was stirred at 50 C for 19 h, extracted with EtOAc, washed with 1N HCl, dried
over Na SO and evaporated.
2
4
3
ꢀ(2ꢀ(Methoxycarbonyl)ethyl)ꢀ1Hꢀindolꢀ4ꢀyl
ethyl(methyl)carbamate,
(51).
Synthesized from 37 or from 50 by Procedure E, was crystallized from EtOAcꢀhexane,
o
1
was isolated as a white solid in 92% yield, mp 122ꢀ124 C. HꢀNMR (300 MHz, acetoneꢀ
d6) ppm δ 10.16 (bs, 1H, Hꢀ1), 7.22 (d, J = 7.5 Hz, 1H, Hꢀ7), 7.09 (bs, 1H, Hꢀ2), 7.03 (t,
J = 7.5 Hz, 1H, Hꢀ6), 6.70 (d, J = 7.5 Hz, 1H, Hꢀ5), 3.67 (s, 3H, Hꢀ17), 3.37ꢀ3.56 (m, 2H,
Hꢀ11), 3.17+3.03 (s, 3H, Hꢀ13), 3.08 (t, J = 6.7 Hz, 2H, Hꢀ14), 2.65 (t, J = 6.7 Hz, 2H, Hꢀ
1
3
1
1
4
5), 1.22ꢀ1.31 (m, 3H, Hꢀ12); CꢀNMR (75 MHz, acetoneꢀd ) ppm δ 173.74, 155.24,
6
45.98, 139.89, 123.50, 123.34, 122.17, 113.81, 113.14, 112.93, 109.54, 51.47, 44.59,
+
+
4.42, 36.18, 36.05, 34.16, 34.09, 22.61, 13.56, 12.66; MS (CI ) m/z 305.149 (MH ,
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2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
.+
+
+
9
1
3
0.07), 304.139 (M , 81.83), 333.180 ([M+C H ] , 9.63), 273.120 ([MHꢀCH OH] ,
2
5
3
.
+
.+
+
8.86), 231.105 ([M ꢀC H O ], 8.38); HRMS calcd. for C H N O (M , DCI /CH )
3
5
2
16 20
2
4
4
+
+
04.1423 found 304.1385, for C H N O (MH , DCI /CH ) 305.1501 found 305.1488;
16
21
2
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Anal. Calcd. for C H N O (304.34 g/mol): C, 63.14; H, 6.62; N, 9.20. Found C,
16
20
2
4
63.299; H, 6.822; N, 8.966.
ꢀ(2ꢀ(Methoxycarbonyl)ethyl)ꢀ1ꢀmethylꢀ1Hꢀindolꢀ4ꢀyl
3
ethyl(methyl)carbamate,
42
(
53). Synthesized from 38 by Procedure E was isolated as a white solid (yield 96%), mp
o
1
>270 C, and was used as such without further purification; HꢀNMR (300 MHz,
acetoneꢀd ) ppm δ 7.18 (dd, J = 8, 1 Hz, 1H), 7.10 (t, J = 8 Hz, 1H), 6.98 (bs, 1H), 6.72
6
(bd, J = 7 Hz, 1H), 3.74 (s, 3H), 3.63 (s, 3H), 3.63ꢀ3.55 + 3.43ꢀ3.36 (m, 2H), 3.16 + 2.97
1
3
(
s, 3H), 3.04 ("t", J = 8 Hz, 2H), 2.66ꢀ2.61 (m, 2H), 1.28 + 1.17 (t, J = 7 Hz, 3H); Cꢀ
NMR (75 MHz, acetoneꢀd ) ppm δ 173.69, 155.19, 146.21, 146.05, 140.26, 127.99,
6
1
22.12, 113.18, 112.97, 107.57, 51.52, 44.63, 44.45, 36.23, 36.15, 34.12, 32.87, 22.46,
+
.+
1
3.60, 12.70; MS (CI+) m/z 319.164 (MH , 71.12), 318.160 (M , 58.45), 287.132 ([MHꢀ
+
+
+
CH OH] , 15.07), 245.114 (C H N O , 38.93), 86.053 (C H NO , 100); HRMS calcd.
3
14 17
2
2
4
8
+
+
for C H N O (MH ) 319.1658; found 319.1645.
1
7
23
2
4
3
ꢀ(2ꢀ(Methoxycarbonyl)ethyl)ꢀ1Hꢀindolꢀ6ꢀyl
butyl(methyl)carbamate,
(67).
Compound 67 prepared from 45 by Procedure E, and purified by chromatography eluted
1
with EtOAcꢀhexane (1:2), was isolated as a yellow oil in 52% yield. HꢀNMR (300 MHz,
acetoneꢀd ) ppm δ 9.98 (bs, 1H), 7.51 (d, J = 8.52 Hz, 1H), 7.14 (d, J = 1.70 Hz, 1H),
6
7.08 (m, 1H), 6.80 (dd, J = 8.52, 1.70 Hz, 1H), 3.61 (s, 3H), 3.30ꢀ3.50 (m, 2H),
3
.09+2.96 (s, 3H), 3.04 (t, J = 8.06 Hz, 2H), 2.68 (t, J = 8.06 Hz, 2H), 1.57ꢀ1.67 (m, 2H),
13
1
.29ꢀ1.42 (m, 2H), 0.89ꢀ0.99 (m, 3H); CꢀNMR (75 MHz, acetoneꢀd ) ppm δ 173.84,
6
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55.70, 148.37, 137.46, 125.65, 123.32, 118.99, 114.48, 105.40, 51.55, 49.40, 34.40,
+
+
3.67, 33.87, 29.32, 21.31, 20.51, 14.15; MS (CI ) m/z 332.171 (M , 81.29); HRMS
+
+
calcd. for C H N O (M , DCI /CH ) 332.1736 found 332.1707.
1
8
24
2
4
4
10
11
12
13
14
15
16
17
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19
20
21
22
23
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34
35
36
37
38
39
40
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42
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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Methyl 3ꢀ(6ꢀ((4ꢀmethoxyphenyl)(methyl)carbamoyloxy)ꢀ1Hꢀindolꢀ3ꢀyl)propanoate,
(69). Synthesized from 47 or from 60 by Procedure E, and purified by chromatography
eluted with EtOAc:hexane (1:1), was isolated as a white solid in 63% yield, mp 120ꢀ124
o
1
C. HꢀNMR (300 MHz, acetoneꢀd ) ppm δ 10.00 (bs, 1H), 7.50 (d, J = 8.4 Hz, 1H), 7.37
6
(dd, J = 6.7, 2.5 Hz, 2H), 7.12ꢀ7.13 (m, 2H), 6.96 (dd, J = 6.7, 2.5 Hz, 2H), 6.80 (bd, J =
8
.4 Hz, 1H), 3.80 (s, 3H), 3.60 (s, 3H), 3.34 (bs, 3H), 3.02 (t, J = 7.3 Hz, 2H), 2.67 (J =
1
3
7
.3 Hz, 2H); CꢀNMR (75 MHz, acetoneꢀd ) ppm δ 173.83, 158.92, 155.12, 148.23,
6
137.39, 128.29, 127.77, 125.74, 123.41, 119.03, 114.91, 114.67, 114.33, 105.30, 55.69,
+
+
+
5
1.55, 38.64, 35.31, 21.27; MS (CI ) m/z 382.151 (M , 56.01), 164.072 ([M ꢀ
+
+
+
C H NO ] , 93.42), 136.077 ([M ꢀC H NO ] , 99.97); HRMS calcd. for C H NO
12 12
3
13 12
4
9
10
2
+
+
+
+
(
[M ꢀC H NO ] , DCI /CH ) 164.0712 found 164.0724, for C H NO ([M ꢀ
1
2
12
3
4
8
10
+
+
C H NO ] , DCI /CH ) 136.0762 found 136.0773; Anal. calcd. for C H N O (MW
13
12
4
4
21 22
2
5
382.41 g/mol): C, 65.96; H, 5.80; N, 7.33; O, 20.92. Found C, 65.848; H, 5.968; N,
.959.
6
Methyl 3ꢀ(4ꢀhydroxyindolinꢀ3ꢀyl)propanoate hydrochloride, (81). Upon addition of
HCl gas to a solution of 73 in ether, compound 81 was obtained as a hygroscopic white
1
solid in quantitative yield. HꢀNMR (300 MHz, CD OD) ppm δ 7.27 (t, J = 9.6 Hz, 1H),
3
6
.89 (t, J = 9.6 Hz, 1H), 3.87 (ABq, J = 14.5, 4.8 Hz, 1H), 3.72ꢀ3.69 (m, 1H), 3.67 (s,
H), 3.60 (dd, J = 14.5, 4.8 Hz, 1H), 2.48 (t, J = 9.6 Hz, 2H), 2.28ꢀ2.24 (m, 1H), 1.97ꢀ
3
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13
1
1
3
.92 (m, 1H); CꢀNMR (75 MHz, CD OD) ppm δ 175.27, 156.69, 138.97, 131.38,
3
31.28, 131.17, 125.24, 117.06, 110.46, 52.17, 51.88, 40.87, 32.39, 28.99.
ꢀ(4ꢀHydroxyindolinꢀ3ꢀyl)propanoic acid hydrochloride, (82). Compound 82 was
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
isolated as a yellow oil in quantitative yield from 73 upon addition of 3N HCl to a
1
solution of 73 in MeOH. HꢀNMR (300 MHz, CD OD) ppm δ 7.27 (t, J = 8.2 Hz, 1H),
3
6
.92 (bt, J = 8.2 Hz, 2H), 3.89 (bt, J = 8.1 Hz, 1H), 3.71ꢀ3.61 (m, 2H), 2.44 (t, J = 8.1 Hz,
1
3
2
H), 2.35ꢀ2.20 (m, 1H), 1.97ꢀ1.87 (m, 1H); CꢀNMR (75 MHz, CD OD) ppm δ 173.72,
3
154.58, 137.98, 131.32, 125.59, 117.49, 110.91, 51.68, 40.84, 32.49, 28.98.
Methyl 3ꢀ(4ꢀhydroxyꢀ1ꢀmethylindolinꢀ3ꢀyl)propanoate hydrochloride,
(83).
Synthesized from 34 by Procedure D, Method I. The crude product consisted of a mixture
of the methyl ester and the acid. Thus, it was dissolved in MeOH and few drops of
concentrated HCl were added. The mixture was stirred over night. The solution was
1
evaporated and the ester was isolated as an oil (yield 69%); HꢀNMR (300 MHz,
CD OD) ppm δ 7.34 (t, J = 8 Hz 1H), 7.05 (d, J = 8 Hz, 1H), 6.94 (d, J = 8 Hz, 1H),
3
3
7
1
.97ꢀ3.91 (bm, 1H), 3.84ꢀ3.81 + 3.60 (m, 2H), 3.67 (s, 3H), 3.26 (s, 3H), 2.51 ("t", J =
13
.5 Hz, 2H), 2.40ꢀ2.29 + 2.00ꢀ1.88 (m, 2H); CꢀNMR (75 MHz, CD OD) ppm δ 175.11,
3
56.62, 143.14, 131.74, 125.04, 118.05, 109.90, 62.76, 52.18, 43.31, 39.79, 32.48, 28.68.
Methyl 3ꢀ(6ꢀhydroxyindolinꢀ3ꢀyl)propanoate hydrochloride, (84). Synthesized from
2 or from indoline 74 by Procedure D, was stirred at room temperature for 2 h. The
4
residue was crystallized from MeOH/Et O to provide the 84 as a green hygroscopic solid
2
1
in 68% yield. HꢀNMR (300 MHz, CD OD) ppm δ 7.29 (bd, J = 8.4 Hz, 1H), 6.92 (dd, J
3
=
8.4, 1.8 Hz, 1H), 6.91 (s, 1H), 3.96 (ABq, J = 0.8 Hz, 1H), 3.66 (s, 3H), 3.51ꢀ3.53 (m,
2
H), 2.45 (t, J = 7.7 Hz, 2H), 2.13 (td, J = 14.0, 7.4 Hz, 1H), 1.89 (J = 14.0, 7.4 Hz, 1H);
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CꢀNMR (75 MHz, CD OD) ppm δ 174.93, 159.60, 137.60, 129.79, 127.13, 118.46,
3
+
+
1
07.34, 52.48, 52.25, 41.53, 32.31, 30.28; MS (CI ) m/z 222.110 (MH , 100), 221.103
+
+
+
+
(M , 46.37), 190.087 ([MH ꢀCH OH], 26.79), 134.076 ([M ꢀC H O ] , 42.54); HRMS
3 4 7 2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
+
calcd. for C H NO (MH , DCI /CH ) 222.1130 found 222.1104.
1
2
16
3
4
3ꢀ(6ꢀHydroxyindolinꢀ3ꢀyl) propanoic acid hydrochloride, (85). Synthesized from 62 or
1
from 74 by Procedure D, was isolated as a brown hygroscopic oil in 23% yield. HꢀNMR
(
300 MHz, CD OD) ppm δ 7.31 (d, J = 8.0 Hz, 1H), 6.88ꢀ6.97 (m, 2H), 3.97 (m, 1H),
3
13
3.46ꢀ3.68 (m, 2H), 2.45 (t, J = 8.0 Hz, 2H), 2.01ꢀ2.18 (m, 1H), 1.77ꢀ1.95 (m, 1H); Cꢀ
NMR (75 MHz, CD OD) ppm δ 173.62, 158.39, 136.34, 128.40, 125.75, 117.14, 116.88,
3
ꢀ
.+
1
05.89, 51.50, 51.24, 40.26, 39.04, 30.80, 28.94, 28.58; MS (CI ) m/z 207.090 (M ,
ꢀ
.+
ꢀ
69.86), 148.033 (C H NO , 34.36); HRMS calcd. for C H NO (M , DCI /CH )
9
10
11 13
3
4
2
07.0895 found 207.0904.
ꢀ(6ꢀHydroxyꢀ1ꢀmethylindolinꢀ3ꢀyl)propanoic acid) hydrochloride, (86). Synthesized
3
from 64 by Procedure D, was stirred at room temperature for 2 h, was isolated as a
1
colorless hygroscopic oil in 63% yield. HꢀNMR (300 MHz, CD OD) ppm δ 7.33 (bs,
3
1H), 6.98 (m, 1H), 4.08 (t, J = 10.8 Hz, 1H), 3.67 (t, J = 10.8 Hz, 1H), 3.55 (m, 1H), 3.25
1
3
(
s, 3H), 2.48 (t, J = 7.2 Hz, 2H), 2.15ꢀ2.26 (m, 1H), 1.80ꢀ1.94 (m, 1H); CꢀNMR (75
MHz, CD OD) ppm δ 176.35, 160.09, 142.74, 129.32, 127.16, 106.33, 63.71, 43.18,
3
+
.+
+
40.45, 32.35+32.24, 29.91+29.80; MS (CI ) m/z 221.105 M , 71.83), 222.112 (MH ,
+
+
7
0.38); HRMS calcd. for C H NO (MH
,
DCI /CH ) 222.1130 found 222.1123, for
12
16
3
4
.+
+
C H NO (M , DCI /CH ) 221.1052 found 221.1046.
12 15
3
4
3
ꢀ(2ꢀ(Methoxycarbonyl)ethyl)indolinꢀ4ꢀyl ethyl(methyl)carbamate hydrochloride,
(
87). Synthesized from 37 or 51 or from 75 by Procedure D, was stirred at room
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5
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5
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temperature over night, was isolated as a yellow oil in 40% yield. HꢀNMR (300 MHz,
CD CN) ppm δ 7.16ꢀ7.48 (m, 3H), 3.70ꢀ3.86 (m, 1H), 3.60 (s, 3H), 3.46ꢀ3.62 (m, 3H),
3
3
.35+3.37 (m, 1H), 3.06+2.95 (s, 3H), 2.36ꢀ2.40 (m, 2H), 2.05ꢀ2.11 (m, 1H), 1.80ꢀ1.85
0
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9
0
1
2
3
4
5
6
7
8
9
0
13
(
m, 1H), 1.15ꢀ1.36 (m, 3H); CꢀNMR (75 MHz, CD CN) ppm δ 173.79, 153.98, 149.63,
3
138.13, 131.93, 130.78, 124.69, 125.29, 118.24, 117.68, 52.14, 50.45, 44.92, 44.78,
+
+
4
2
0.52, 34.64, 34.35, 31.73, 28.51, 13.45, 12.61; MS (CI ) m/z 335.204 ([M+C H ] ,
2 5
+
+
8.34), 307.170 (MH , 100), 306.164 (M, 46.78), 275.143 ([MH ꢀCH OH], 27.57);
3
+
+
HRMS calcd. for C H N O (MH , DCI /CH ) 307.1658 found 307.1702.
1
6
23
2
4
4
3
ꢀ(2ꢀ(Methoxycarbonyl)ethyl)ꢀ1ꢀmethylindolinꢀ4ꢀyl
ethyl(methyl)carbamate
hydrochloride, (88). Compound 88 was prepared from 53 by Procedure D, Method I.
The crude product consisted of a mixture of the methyl ester and the acid. 43% of the
crude was dissolved in MeOH and 6 drops of 3N HCl were added. The mixture was
stirred over three nights. The solution was evaporated and 88 was isolated as an oil (yield
1
6
2%); HꢀNMR (300 MHz, D O) ppm δ 7.57 (t, J = 8 Hz, 1H), 7.47 (d, J = 8 Hz, 1H),
2
7
.28 (d, J = 8 Hz, 1H), 4.08ꢀ4.01 (m, 1H), 3.87ꢀ3.77 (m, 2H), 3.67 (s, 3H), 3.58ꢀ3.51 +
3.42ꢀ3.35 (m, 2H), 3.30 (s, 3H), 3.13 + 2.99 (s, 3H), 2.49 (t, J = 7.5 Hz, 2H), 2.22ꢀ2.13 +
13
1
.99ꢀ1.86 (m, 2H), 1.25 + 1.17 (t, J = 7 Hz, 3H); CꢀNMR (75 MHz, D O) ppm δ
2
175.78, 154.94, 147.98, 142.23, 131.02, 129.95, 124.65, 124.50, 116.07, 61.30, 52.40,
4.53, 44.41, 42.81, 38.53, 34.081, 30.99, 26.88, 12.41, 11.68; MS (CI+) m/z 321.181
4
+
.+
+
(
MH , 100), 320.175 (M , 69.90), 86.070 (C H NO , 58.93); HRMS calcd. for
4 8
+
+
C H N O (MH ) 321.1814; found 321.1813.
17
25
2
4
Ethyl 3ꢀ(4ꢀ(ethyl(methyl)carbamoyloxy)indolinium citrate, (89). Synthesized from 76
1
by Procedure I, was isolated as a yellow oil in quantitative yield. HꢀNMR (300 MHz,
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CD OD) ppm δ 7.05 (t, J = 7.9 Hz, 1H), 6.59 (d, J = 7.9 Hz, 1H), 6.47 (bd, J = 7.9 Hz,
3
1H), 4.10 (q, J = 7.6 Hz, 2H), 3.66ꢀ3.59 (m, 1H), 3.57ꢀ3.48 (m, 1H), 3.43ꢀ3.36 (m, 2H),
3.30ꢀ3.25 (m, 1H), 2.98+3.11 (s, 3H), 2.85 (ABq, J = 14.0 Hz, 4H), 2.34 (t, J = 7.9 Hz,
2H), 2.10ꢀ1.99 (m, 1H), 1.87ꢀ1.75 (m, 1H), 1.23 (t, J = 6.7 Hz, 3H), 1.33ꢀ1.15 (m, 3H);
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
3
CꢀNMR (75 MHz, CD OD) ppm δ 176.84, 174.97, 173.48, 153.31, 149.68, 141.50,
3
1
4
30.04, 125.78, 114.82, 114.60, 110.21, 109.48, 74.13, 61.46, 52.89, 52.79, 45.24, 45.15,
+
3.84, 41.19, 34.54, 34.38, 32.35, 29.31, 29.23, 14.54, 13.52, 12.69; MS (CI ) m/z
.
+
+
.+
320.178 (M , 67.08), 321.178 (MH , 38.14); HRMS calcd. for C H N O (M ,
1
7
24
2
4
+
DCI /CH ) 320.1736 found 320.1775.
4
3
ꢀ(3ꢀOxobutyl)indolinꢀ4ꢀyl ethyl(methyl)carbamate hydrochloride, (90). Synthesized
from 52 by Procedure D, was stirred at room temperature over night, was isolated as an
1
offꢀwhite hygroscopic crystals in 56% yield. HꢀNMR (300 MHz, CD CN) ppm δ 7.17ꢀ
3
7
3
1
.46 (m, 3H), 3.82 ("t", J = 9.90 Hz, 1H), 3.62ꢀ3.63 (m, 1H), 3.58 ("t", J = 9.9 Hz 1H),
.38+2.99 (bs, 5H), 2.35 ("t", J = 7.4 Hz, 2H), 2.02ꢀ2.09 (m, 1H), 1.73ꢀ1.85 (m, 1H),
13
.15+1.34 (m, 3H); CꢀNMR (75 MHz, CD CN) ppm δ 175.37, 154.36, 149.38, 137.70,
3
131.99, 130.96, 124.93, 118.33, 50.97, 44.96, 39.46, 34.59, 31.84, 28.34, 13.30, 12.73;
+
+
.+
+
MS (CI ) m/z 293.150 (MH , 100), 292.144 (M , 76.31), 275.138 ([MHꢀH O] , 56.34),
2
.
+
.+
+
2
19.115 ([M ꢀC H O ], 13.73); HRMS calcd. for C H N O (M , DCI /CH )
3 5 2 15 20 2 4 4
+
+
292.1423; found 292.1438, for C H N O (MH , DCI /CH ) 293.1501 found 293.1497,
1
5
21
2
4
4
+
+
for C H N O ([MHꢀH O] , DCI /CH ) 275.1396 found 275.1384.
1
5
19
2
3
2
4
3
ꢀ(4ꢀ((Ethyl(methyl)carbamoyl)oxy)ꢀ1ꢀmethylindolinꢀ3ꢀyl)propanoic
acid
hydrochloride, (91). Compound 77 was dissolved in CH Cl and stirred with 3N HCl at
2
2
room temperature for 2 d. The aqueous phase was evaporated to provide 91 as a colorless
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1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
oil in quantitative yield. HꢀNMR (300 MHz, CD OD) ppm δ 7.59ꢀ7.50 (m, 2H), 7.31 (d,
3
J = 7.5 Hz, 1H), 4.04ꢀ3.97 (m, 1H), 3.92ꢀ3.85 (m, 1H), 3.79ꢀ3.77 (bs, 1H), 3.56+3.42 (q,
J = 7.2 Hz, 2H), 3.40 (s, 3H), 3.15+3.01 (s, 3H), 2.44 (t, J = 7.2 Hz, 2H), 2.30ꢀ2.20 (m,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
1
H), 1.92ꢀ1.84 (m, 1H), 1.28+1.20 (t, J = 7.2 Hz, 3H); CꢀNMR (175 MHz, CD OD)
3
ppm δ 174.58, 154.87, 149.91, 149.87, 143.40, 143.34, 131.81, 131.63, 131.54, 131.53,
1
3
25.91, 125.74, 116.83, 62.35, 62.24, 45.51, 45.30, 43.35, 40.20, 34.72, 34.49, 32.98,
+
.+
+
1.91, 28.52, 28.45, 13.50, 12.61; MS (CI ) m/s 306.159 (M , 57.80), 307.167 (MH ,
+
+
100); HRMS calcd. for C H N O (MH DCI /CH ) 307.1658 found 307.1668.
16
23
2
4
4
3
ꢀ(3ꢀAminoꢀ3ꢀoxopropyl)indolinꢀ4ꢀyl ethyl(methyl)carbamate hydrochloride, (92).
Synthesized from 55 by Procedure D, was stirred at room temperature over night, was
1
isolated as a colorless oil in 38% yield. HꢀNMR (300 MHz, CD OD) ppm δ 7.24ꢀ7.60
3
(
m, 3H), 3.94ꢀ4.22 (m, 1H), 3.70ꢀ3.78 (m, 2H), 3.40ꢀ3.56 (m, 2H), 3.14+3.00 (s, 3H),
1
3
2
.43 ("t", J = 7.9 Hz, 2H), 2.27ꢀ2.34 (m, 1H), 1.85ꢀ1.91 (m, 1H), 1.22ꢀ1.46 (m, 3H); Cꢀ
NMR (75 MHz, CD OD) ppm δ 178.65, 154.65, 150.02, 138.26, 132.50, 132.14, 131.70,
3
1
31.41, 125.27, 126.02, 117.93, 51.37, 51.16, 45.45, 45.30, 40.59, 34.74, 34.56, 33.07,
+
+
32.95, 29.67, 29.39, 13.59, 12.66; MS (CI ) m/z 362.239 ([M+C H ] , 49.69), 334.217
2
11
+
+
+
+
(
M+C H ] , 99.97), 320.20 ([M+C H ] , 79.22), 292.165 (MH , 23.11), 275.136 ([MH ꢀ
3 7 2 5
+
+
NH ], 23.50), 86.01 (C H NO , 88.33); HRMS calcd. for C H N O ([M+C H ] ,
3
4
8
17 26
3
3
2
5
+
+
+
DCI /CH ) 320.1974 found 320.2004, for C H N O (MH , DCI /CH ) 292.1661 found
4
15 22
3
3
4
2
92.1655.
ꢀ(2ꢀ(Methoxycarbonyl)ethyl)ꢀ1ꢀmethylindolinꢀ4ꢀyl
3
butyl(methy)lcarbamate
hydrochloride, (93). Synthesized from 56 by Procedure D, Method I, was dissolved in
MeOH and 6 drops of 3N HCl were added. The mixture was stirred for 72 h. The
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obtained solution was evaporated and the product was isolated as an oil (33% yield); Hꢀ
NMR (300 MHz, CD OD) ppm δ 7.42 (bs, 1H), 7.31ꢀ7.30 (bm, 1H), 7.14ꢀ7.09 (m, 1H),
3
3
.82 (bm, 1H), 3.76ꢀ3.70 (bm, 2H), 3.67 (s, 3H), 3.51 + 3.38 (bm, 2H), 3.21 (s, 3H), 3.15
3.01 (s, 3H), 2.48ꢀ2.34 (bt, J = 7 Hz, 2H), 2.20 + 1.88 (bs, 2H), 1.70ꢀ1.57 (m, 2H),
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
1
3
1
.45ꢀ1.33 (m, 2H), 1.02ꢀ0.94 (m, 3H); CꢀNMR (75 MHz, CD OD) ppm δ 173.34,
3
1
54.01, 148.38, 145.10, 144.98, 130.00, 129.90, 129.80, 121.56, 121.16, 112.92, 112.71,
60.99, 60.87, 50.85, 48.93, 40.40, 38.74, 33.93, 33.660, 30.67, 30.60, 29.89, 29.12, 27.34,
+
.+
2
7.22, 19.60, 19.50, 12.81; MS (CI+) m/z 349.212 (MH , 70.32), 348.205 (M , 44.07),
+
+
+
1
14.091 (C H NO , 100); HRMS calcd. for C H N O (MH ) 349.2127; found
6 12 19 29 2 4
349.2124.
3
1
ꢀ(3ꢀMethoxyꢀ3ꢀoxopropyl)ꢀ4ꢀ(((4ꢀmethoxyphenyl)(methyl)carbamoyl)oxy) indolinꢀ
ꢀium hydrochloride, (94). Compound 94 was isolated as a hygroscopic yellow solid in
1
quantitative yield upon addition of HCl gas to a solution of 78 in ether. HꢀNMR (300
MHz, CD OD) ppm δ 7.47 (bt, J = 7.3 Hz, 1H), 7.36 (d, J = 9.3 Hz, 2H), 7.37ꢀ7.32 (m,
3
2H), 6.97 (d, J = 9.3 Hz, 2H), 3.93ꢀ3.81 (m, 1H), 3.81 (s, 3H) 3.67 (s, 3H), 3.62ꢀ3.54 (m,
1
3
2
H), 3.33 (bs, 3H), 2.27 (bt, J = 5.5 Hz, 2H), 1.89 (bs, 1H), 1.66 (bs, 1H); CꢀNMR (75
MHz, CDCl ) ppm δ 174.60, 160.37, 154.37, 149.99, 138.37, 136.48, 131.84, 131.35,
3
128.91, 124.88, 117.54, 115.58, 55.96, 52.24, 51.35, 41.21, 39.19, 31.96, 28.69; MS
+
.+
+
+
(
CI ) m/z 384 (M , 10.22), 385 (MH , 22.77), 386 (MH , 6.92).
2
3
ꢀ(4ꢀ((4ꢀMethoxyphenyl)(methyl)carbamoyl)indolinꢀ3ꢀyl)propanoic
acid
hydrochloride (95). Compound 94 was dissolved in 3N HCl and stirred over two nights
1
and then evaporated. The HCl salt was isolated in 93% yield; HꢀNMR (300 MHz, D O)
2
ppm δ 7.49ꢀ7.25 (m, 5H), 6.99 (d, J = 8.5 Hz, 2H), 3.95ꢀ3.86 (m, 1H), 3.78 (s, 3H), 3.63
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1
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
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5
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5
5
5
6
13
+
3.57 (m, 2H), 3.43 + 3.27 (bs, 3H), 2.20 (m, 2H), 1.74 + 1.61 (m, 2H); CꢀNMR (50
MHz, D O) ppm δ 178.90, 160.31, 151.64, 149.83, 138.81, 137.15, 132.66, 132.38,
2
1
29.77, 125.93, 119.27, 116.87, 57.70, 54.31, 41.40, 40.36, 32.93, 29.11.
ꢀ(2ꢀ(Methoxycarbonyl)ethyl)ꢀ1ꢀmethylindolinꢀ4ꢀyl 4ꢀmethoxyphenyl(methyl)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
carbamate hydrochloride, (96). Compound 96 prepared from 58, was dissolved in
MeOH and a few drops of concentrated HCl were added. The mixture was stirred over
night, and the obtained solution was evaporated. The product was isolated as an oil (yield
1
9
3
1
1
9%). HꢀNMR (300 MHz, CD CN) ppm δ 7.45ꢀ7.27 (m, 5H), 6.95 (d, J = 9 Hz, 2H),
3
.79 (s, 3H), 3.73 + 3.45 (bs, 3H), 3.65 (s, 3H), 3.27 + 3.07 (s, 3H), 2.24 (bs, 2H), 1.87 +
13
.71 (bs, 2H); CꢀNMR (75 MHz, CD CN) ppm δ 173.75, 157.76, 153.54, 149.38,
3
43.11, 136.34, 131.13, 128.88, 124.83, 116.30, 115.25, 61.26, 56.12, 52.21, 42.57,
39.58, 39.19, 31.94, 28.19.
ꢀ(4ꢀ((4ꢀMethoxyphenyl)(methyl)carbamoyl)ꢀ1ꢀmethylindolinꢀ3ꢀyl)propanoic acid
3
hydrochloride, (97). Synthesized from 59 by Procedure D, Method I, was isolated in
1
65% yield. HꢀNMR (300 MHz, D O) ppm δ 7.43ꢀ7.31 (m, 2H), 7.25 (d, J = 9 Hz, 2H),
2
7
.16 (d, J = 8 Hz, 1H), 6.86 (d, J = 9 Hz, 2H), 3.89 + 3.82 (m, 1H), 3.66 (bs, 4H), 3.47
(bs, 1H), 3.31 + 3.21 (bs, 3H), 3.16 (bs, 3H), 2.17ꢀ2.12 (m, 2H), 1.69 + 1.51 (bm, 2H);
13
CꢀNMR (50 MHz, D O) ppm δ 178.56, 160.25, 156.16, 149.61, 143.72, 137.08,
2
1
3
3
32.67, 132.78, 131.67, 129.68, 126.39, 118.20, 116.80, 63.14, 57.65, 45.10, 40.39,
3.03, 28.86 (Cꢀ18).
ꢀ(2ꢀ(Methoxycarbonyl)ethyl)ꢀ1ꢀindolineꢀ6ꢀyl
ethyl(methyl)carbamate
hydrochloride, (98). Synthesized form 99 or 79. A solution of compound 99 in MeOH
containing a few drops of 1N HCl was stirred over night, the solvent was then evaporated
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o
and the product was isolated as brown crystals in 43% yield, mp 100ꢀ105 C.
Alternatively, the HCl salt was obtained by bubbling HCl gas into a solution of indoline
1
7
9 in ether or CH Cl . HꢀNMR (300 MHz, CD OD) ppm δ 7.53 (bd, J = 6.4 Hz, 1H),
2
2
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7
.35 (bs, 1H), 7.18 (bd, J = 6.4 Hz, 1H), 4.05 (m, 1H), 3.68 (s, 3H), 3.56ꢀ3.64 (m, 1H),
3.35ꢀ3.52 (m, 2H), 3.35ꢀ3.41 (m, 1H), 3.11+2.99 (s, 3H), 2.52 ("t", J = 7.3 Hz, 2H), 2.21
1
3
(bs, 1H), 1.94 (bs, 1H), 1.18+1.26+1.41 (m, 3H); CꢀNMR (75 MHz, CD OD) ppm δ
3
1
74.74, 155.43, 152.85, 136.84, 127.09, 125.83, 124.97, 114.95, 52.56, 52.33, 45.21,
+
+
+
41.75, 34.72, 34.46, 32.15, 29.99, 12.62, 13.47; MS (CI ) m/z 219.100 ([M ꢀC H O ] ,
4
7
2
+
+
+
6
2.63), 306.157 (M ,100); HRMS calcd. for C H N O (M , DCI /CH ) 306.1580
16 22 2 4 4
Found 306.1567.
3ꢀ(6ꢀ(Ethyl(methyl)carbamoyloxy)indolinꢀ3ꢀyl)propanoic acid hydrochloride, (99).
Synthesized from 66 by Procedure D, was stirred at room temperature for 2 h, was
o
1
isolated as a white crystals in 23% yield, mp 117ꢀ119 C. HꢀNMR (300 MHz, D O) ppm
2
δ 7.51 (bd, J = 8.1 Hz, 1H), 7.31 (bd, 1H), 7.24 (dd, J = 8.1, 1.8 Hz, 1H), 4.05ꢀ4.15 (m,
1
H), 3.59+3.68 (m, 2H), 3.30ꢀ3.48 (m, 1H), 3.06+2.94 (s, 3H), 2.50 ("t", J = 7.7 Hz, 2H),
1
3
2.11ꢀ2.16 (m, 1H), 1.87ꢀ1.94 (m, 1H), 1.11ꢀ1.36 (m, 3H); CꢀNMR (75 MHz, D O) ppm
2
δ 177.44, 155.79, 151.27, 150.97, 135.95, 126.34, 124.04, 113.73, 51.35, 44.23; MS
+
+
+
+
(CI ) m/z 219.096 ([M ꢀC H O ] , 17.24), 292.142 (M , 18.23); HRMS calcd. for
3 5 2
+
+
C H N O (M , DCI /CH ) 292.1423 found 292.1424.
15
20
2
4
4
3
ꢀ(2ꢀ(Methoxy
carbonyl)ethyl)ꢀ1ꢀindolineꢀ6ꢀyl
butyl(methyl)carbamate
hydrochloride, (100). Synthesized from 67 by Procedure D, was stirred at room
temperature for 1 h, was isolated as a white hygroscopic crystals in 60% yield, mp
1
hygroscopic salt. HꢀNMR (300 MHz, D O) ppm δ 7.53 (d, J = 8.0 Hz, 1H), 7.28 (d, J =
2
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