Analogues of doxanthrine reveal differences between the dopamine D 1 receptor binding properties of chromanoisoquinolines and hexahydrobenzo[a]phenanthridines

Article abstract of DOI:10.1016/j.ejmech.2011.11.039

Efforts to develop selective agonists for dopamine D₁-like receptors led to the discovery of dihydrexidine and doxanthrine, two bioisosteric β-phenyldopamine-type full agonist ligands that display selectivity and potency at D₁-like receptors. We report herein an improved methodology for the synthesis of substituted chromanoisoquinolines (doxanthrine derivatives) and the evaluation of several new compounds for their ability to bind to D₁- and D₂-like receptors. Identical pendant phenyl ring substitutions on the dihydrexidine and doxanthrine templates surprisingly led to different effects on D₁-like receptor binding, suggesting important differences between the interactions of these ligands with the D₁ receptor. We propose, based on the biological results and molecular modeling studies, that slight conformational differences between the tetralin and chroman-based compounds lead to a shift in the location of the pendant ring substituents within the receptor.

Full text of DOI:10.1016/j.ejmech.2011.11.039

European Journal of Medicinal Chemistry 48 (2012) 97e107
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Analogues of doxanthrine reveal differences between the dopamine D1 receptor
binding properties of chromanoisoquinolines and hexahydrobenzo[a]
phenanthridines
Juan Pablo Cueva a, Benjamin R. Chemel b, Jose I. Juncosa Jr. c, Markus A. Lill d, Val J. Watts d,
,
David E. Nichols d
*
a Quimique Cía. Ltda., P.O. Box 17-07-9009, Quito, Ecuador
b USGS Forest and Rangeland Ecosystem Science Center, 777 NW 9th St., Suite 400, Corvallis, OR 97330, USA
c Department of Chemistry, Weinberg College of Arts and Sciences, Northwestern University, Evanston, IL 60208, USA
d Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, West Lafayette, IN 47907, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Efforts to develop selective agonists for dopamine D1-like receptors led to the discovery of dihydrexidine
and doxanthrine, two bioisosteric b-phenyldopamine-type full agonist ligands that display selectivity
Received 27 July 2011
Received in revised form
29 October 2011
Accepted 25 November 2011
Available online 3 December 2011
and potency at D1-like receptors. We report herein an improved methodology for the synthesis of
substituted chromanoisoquinolines (doxanthrine derivatives) and the evaluation of several new
compounds for their ability to bind to D1- and D2-like receptors. Identical pendant phenyl ring substi-
tutions on the dihydrexidine and doxanthrine templates surprisingly led to different effects on D1-like
receptor binding, suggesting important differences between the interactions of these ligands with the D1
receptor. We propose, based on the biological results and molecular modeling studies, that slight
conformational differences between the tetralin and chroman-based compounds lead to a shift in the
location of the pendant ring substituents within the receptor.
Keywords:
Dopamine D1 receptor
Agonist
Dihydrexidine
Doxanthrine
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
developing selective ligands for the activation of this receptor
subtype. Structureeactivity relationship studies of dopamine
Central dopamine neurotransmission has evolved to mediate
important sensory processing functions such as novelty detection,
attention, memory formation, and coding of rewarding stimuli
[1,2]. Executive and volitional functions such as reward seeking,
behavioral reinforcement, and motor control are likewise mediated
by dopaminergic pathways [3]. Furthermore, dopaminergic
systems are involved in the manifestation of CNS pathologies
including Parkinson’s disease [4], schizophrenia [5], and substance
abuse [6].
analogs have enabled the recognition of a “b-phenyldopamine
pharmacophore” [7,8] as one template for the development of
compounds displaying preference for the activation of D1-like
receptors over D2-like receptors. This predictive model has enabled
the discovery of several molecules that have shown significant
potency and selectivity for dopamine D1-like receptors [9e12]. The
prototype of this type of compound is dihydrexidine 1 (DHX, Fig. 1.)
[13], a hexahydrobenzo[a]phenanthridine developed as a con-
formationally-restricted molecule that incorporates the b-phenyl-
We have been interested for more than two decades in inves-
tigating the physiological role of the dopamine D1 receptor by
dopamine pharmacophore, and is a potent dopamine D1 full
agonist with ten-fold higher affinity for D1-like receptors over D2-
like receptors [9]. This compound demonstrated significant ther-
apeutic potential in an in vivo model of Parkinson’s disease in
African green monkeys [14] and helped to establish the importance
of D1 receptor activation in the control of motor function. Dihy-
drexidine also has been shown to increase blood flow in the
prefrontal cortex [15], and improve working memory in aged
monkeys [16].
* Corresponding author. Dept. of Medicinal Chemistry and Molecular Pharma-
cology, RHPH Pharmacy Building, Rm 506C, 575 Stadium Mall Drive, Purdue
University, West Lafayette, IN 47907-2091. Tel.: þ1 765 494 1461; fax: þ1 765 494
1414.
E-mail addresses: jpcueva@quimique.com (J.P. Cueva), dr.chemel@gmail.com (B.
R. Chemel), jjuncosa@northwestern.edu (J.I. Juncosa), mlill@purdue.edu (M.A. Lill),
wattsv@purdue.edu (V.J. Watts), drdave@pharmacy.purdue.edu, drdave@purdue.
edu (D.E. Nichols).
Given the success that has been achieved with the development
of bioisosteres of certain dopamine agonists, and in view of the
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.11.039

98
J.P. Cueva et al. / European Journal of Medicinal Chemistry 48 (2012) 97e107
2. Results and discussion
2.1. Chemistry
The synthesis of compounds 5aed (Scheme 1) was based on
a variation of the methodology employed to prepare doxanthrine, 2
[18]. The key step of this synthesis involves a Michael-type addition
of an aryl-metal reagent to nitrochromene 11 (Scheme 1).
Starting from commercially available sesamol 7, aldehyde 10
was prepared in very modest yield by Friedel-Crafts-type for-
mylation using dichloromethyl methyl ether and SnCl4 (Scheme 2).
Unfortunately, direct conversion of sesamol into the phenolic
benzaldehyde 10 was accompanied by the formation of large
amounts of insoluble, deep-blue-colored xanthylium ion 8, which
typically precipitated out of the dichloromethane reaction medium.
Xanthylium ion 8 appears to form from a chloromethoxymethyl
intermediate under the formylation conditions, which is suffi-
ciently reactive, particularly in the presence of Lewis acids, to be
attacked by the electron rich aromatic ring of another molecule of
sesamol. This heterodimerization product is then poised to undergo
intramolecular attack by the phenolic hydroxy to yield a hemi-
acetal, which can aromatize to yield a xanthydrol ether. Aided by
the electron donating oxygen atoms that flank the aromatic rings,
the xanthydrol ether species will readily eliminate methanol,
forming a resonance-stabilized xanthylium ion, 8, which displays
coloration and solubility properties consistent with similar
compounds reported previously in the literature [20,21].
To avoid formation of this xanthylium side-product, sesamol
was acetylated prior to Friedel-Crafts formylation. This simple
procedure allowed formation of aldehyde 10 in excellent yield and
essentially free from the dimerization by-product. The desired
nitrochromene 11 was then obtained in one step by treatment of 10
with nitroethylene generated in situ [22,23]. Extremely slow addi-
tion of 2-nitroethanol ensured minimization of base-catalyzed
polymerization of nitroethylene, which was nevertheless a signifi-
cant side reaction. Using this methodology, it was possible to
prepare relatively large amounts of 11.
Fig. 1. Structures of b-phenyldopamine-based dopamine D1 receptor agonists.
results of Horn et al. [17], who reported a ten-fold loss of D2 affinity
by introduction of an oxygen atom into the structure of the D2
agonist 6,7-ADTN, it was conjectured that a similar modification of
the structure of DHX might yield a ligand with enhanced D1-
selectivity. This line of reasoning led to the discovery of dox-
anthrine (þ)ꢀ2, (DOX, Fig. 1.), a chromanoisoquinoline bioisostere
of 1 that displays a greater than 300-fold selectivity for D1-like
receptors over D2-like receptors, with higher efficacy than dopa-
mine for activation of the D1 receptor [18].
To develop structure-activity relationships of compounds
derived from the new chromanoisoquinoline template, we
reviewed modifications of the hexahydrobenzo[a]phenanthridine
ring system that had resulted in compounds with improved bio-
logical properties. In the DHX series, substitution on the b-phenyl
ring at the 2-position with methyl and ethyl groups gave ligands
with increased D1-selectivity as a result of decreased D2 affinity [19]
(Fig. 2). We hypothesized that compounds with analogous substi-
tution at the 11-position of 2 would display D1-selectivity greater
than the parent compound. We also wished to investigate the
effects of electronegative substituents on the
b-phenyl ring of
doxanthrine, which had not been chemically accessible in the DHX
series. To pursue these directions, we prepared compounds bearing
methyl, ethyl, fluoro, and trifluoromethyl substituents at the 11-
position of the chromanoisoquinoline template (compounds
5aed, Fig. 2) and assessed their affinities at D1 and D2 receptors. In
addition, because the chroman oxygen of DOX appears to be
instrumental in conferring D1-selectivity to the ligand, we prepared
and tested the sulfur-containing thiochromanoisoquinoline 6.
The differences in polarity and size of the sulfur atom in relation to
the methylene group of DHX and the oxygen atom of DOX will
allow a more complete survey of the nature of the receptor resi-
due(s) that are situated in proximity to this important position
when the ligand is bound to the D1 receptor. The results of these
studies are reported herein.
Nitrochromene 11 is known to undergo Michael-type addition
of aryl-metal reagents to provide exclusively trans adducts [18].
Thus, we anticipated that an appropriately substituted aryl-metal
reagent would allow access to 11-substituted doxanthrine analogs
5aed. In the original synthesis of DOX 2, a Grignard reagent was
employed. That approach, however, suffered severe limitations
because of the requirement for ortho/para-substituted aryl halides
that are not readily available or easily synthesized. We therefore
reasoned that para-substituted aryloxazolines 13aed (Scheme 3),
X = O, R2 = Me
X = O, R2 = Et
X = O, R2 = F
X = O, R2 = CF3
X = S, R2 = H
5a
5b
5c
5d
6
D1 Ki D2 Ki
D1/D2
R1
H (DHX)
Methyl
Ethyla
nM
6.2
8.7
29
nM Selectivitya
58
302
420
9.3
34.7
14.5
1
3
4
aTaken from Knoerzer et al [19].
Fig. 2. Structures and dopamine receptor affinities of substituted hexahydrobenzo[a]phenanthridines and proposed analogues.

J.P. Cueva et al. / European Journal of Medicinal Chemistry 48 (2012) 97e107
99
R
R
G
M
+
H
NO2
HO
HO
NH
H
O
O
O
O
OH
X
X
X = O, 5a-d
X = S,
X = O, 11
X = S, 20
7
6
M = Li or MgBr
Scheme 1. Retrosynthetic analysis for the preparation of compounds 5aed and 6.
Scheme 2. Synthesis of nitrochromene 11 and xanthylium side-product 8.
which are easily accessible from commercially available para-
substituted benzoic acids, could be ortho-lithiated, and after
treatment with nitrochromene 11, would allow exclusive produc-
tion of the appropriately substituted trans adducts 14aed. The
oxazoline moiety of these intermediates was anticipated to
undergo facile acid hydrolysis to yield the carboxylic acids, which
would allow eventual formation of the isoquinoline ring of the final
products. Thus, we reasoned that para-substituted aryloxazoline
Scheme 3. Synthesis of chromanoisoquinolines 5aed.

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J.P. Cueva et al. / European Journal of Medicinal Chemistry 48 (2012) 97e107
Scheme 4. Synthesis of thiochromanoisoquinoline 6.
reagents would function as versatile synthons in the preparation of
a variety of substituted isoquinolines 17aed.
ether at 250 ꢁC for 15 min to effect the rearrangement, obtaining
modest yields of 19. Basic hydrolysis of 19 gave a thiophenol that
proved to be relatively unstable. By immediate treatment of the
hydrolysate mixture with 2-nitroethanol, phthalic anhydride, and
dibutylamine, we were able to prepare good quantities of nitro-
thiochromene 20, isolated as bright orange crystals. The method-
ology followed in the subsequent steps was basically identical to
that employed in the synthesis of 2 [18].
The completion of the synthesis of compounds 5aed is shown in
Scheme 3. Treatment of nitrochromene 11 with organolithium
reagents prepared by ortho-lithiation of aryloxazolines 13aed,
gave, as anticipated, exclusively the trans adducts 14aed. Mild
treatment of compounds 14aed with aqueous HCl gave the amine
salts 15aed as products of partial hydrolysis of the oxazoline ring.
Reduction of the nitro groups of these compounds with powdered
zinc in acetic acid led to intermediate amine salts that upon basi-
fication and gentle heating readily cyclized to form insoluble lac-
tams 16aed, which crystallized upon formation. These lactams
were then reduced by treatment with borane in THF, and the
resulting amines 17aed were demethylated with BCl3 to yield the
catecholamine hydrochloride target compounds 5aed.
2.2. Pharmacology
The affinities of compounds 5aed and 6 for dopamine D1-like
and D2-like receptors were evaluated in a radioligand displacement
assay using porcine striatal tissue preparations. Those results are
shown in Table 1. Standard ligands for D1- and D2-like receptors,
SCH-23390 and chlorpromazine, were included in the assays for
comparison.
To prepare the thiochromanoisoquinoline analog
6 we
employed a strategy parallel to the synthesis of 2 [18], but
employing the nitrothiochromene 20 as the Michael acceptor for
the addition of aryl-Grignard reagent 21. The transformations that
led to 6 are shown in Scheme 4.
3. Conclusions
To prepare nitrothiochromene 20, we relied on the Newman-
Kwart rearrangement [24] of phenyl-O-thiocarbamate 18 into
phenyl-S-thiocarbamate 19. Thus, we prepared 18 from the previ-
ously synthesized sesamaldehyde 10, and heated it in diphenyl
Competition binding assays using porcine striatal preparations
revealed that all four of the 11-substituted analogs 5aed had
significantly lower affinity at D1-like receptors than their structural
predecessor 2, DOX. The methyl-substituted compound 5a had
a nearly six-fold decrease in D1 affinity (D1 Ki ¼ 150 nM) compared
to 2 (D1 Ki ¼ 26 nM). Given that introduction of a methyl group at
the analogous 2-position of DHX (compound 3) did not signifi-
cantly reduce the affinity of the ligand in comparison to DHX, the
loss of affinity for compound 5a was unexpected. Extension of the
hydrophobic bulk to an ethyl group (compound 5b) incurred an
even greater loss of D1 affinity (D1 Ki ¼ 185 nM) compared to 2.
Although the ethyl substituted analog of DHX (compound 4) also
displayed affinity lower than the unsubstituted compound [19], its
chroman isostere 5b clearly shows a more dramatic negative effect
of the ethyl substituent on affinity.
Table 1
Binding affinity of new compounds at D1- and D2-like receptors in porcine striatal
tissue.
Ligand
Binding in porcine striatal homogenatesa
D1-like
Ki (nM)
D2-like
Ki (nM)
Binding selectivity
(D2-like/D1-like)
DOX (2)
26 ꢂ 4.5
150 ꢂ 25
180 ꢂ 34
110 ꢂ 18
570 ꢂ 87
200 ꢂ 41
0.46 ꢂ 0.08
NA
1700 ꢂ 190
6800 ꢂ 860
6400 ꢂ 900
740 ꢂ 110
6900 ꢂ 980
2500 ꢂ 410
NA
80 ꢂ 19
55 ꢂ 13
49 ꢂ 14
8.5 ꢂ 2.1
14 ꢂ 3.1
15 ꢂ 5.6
NA
11-Me-DOX (5a)
11-Et-DOX (5b)
11-F-DOX (5c)
11-CF3-DOX (5d)
6
The disparity between the effects brought about by introduction
of identical alkyl substitutions at the 11-position of doxanthrine (2)
and the analogous 2-position of dihydrexidine, 1 must reflect crit-
ical differences between the interactions of these two isosteric
ligands within the D1 dopamine receptor. The D1 binding affinities
SCH23390
Chlorpromazine
7.6 ꢂ 2
NA
a
All results shown are the mean ꢂ SEM for 4-11 independent experiments.

J.P. Cueva et al. / European Journal of Medicinal Chemistry 48 (2012) 97e107
101
of 1 and 2 reflect very similar
D
Gꢁ values for their interaction with
differences caused by the presence of the heteroatom at the 4-
position of 2, resulting in differential interactions with residues
within the ligand binding site.
the D1 receptor, revealing that, from a thermodynamic point of
view, the net effect of replacing the 8-methylene of 1 with an
oxygen atom was insignificant. When one considers the potential
for solvation of the chroman oxygen of 2 and its analogs in an
aqueous environment, and thus, the desolvation energy that must
be overcome upon binding of 2 to the D1 receptor, the similar D1
binding affinities of 1 and 2 can most easily be explained by
invoking the formation of energetically favorable interactions
between 2 and some element(s) of the D1 binding site that can
compensate for the energetically unfavorable desolvation process.
Aside from inductive effects on the amine group, it appears likely
that the chroman oxygen of DOX and its analogs may be engaged in
a specific interaction when bound to the D1 receptor. Replacement
of the chroman oxygen of DOX with a sulfur atom resulted in
a nearly eight-fold decrease in D1 affinity.
After performing docking, molecular dynamics, and energy
minimization of both methyl-substituted derivatives, 3 and 5a, in
our previously constructed “activated” model of the D1 receptor
[25], a slight conformational difference was observed between the
DOX and DHX analogs, with the pendant phenyl ring deviating
slightly more from the catechol plane in 5a than in 3. This change
forces the DOX analog to lean more into the space occupied by TM6
(and TM7) than the DHX analog, toward residues at the extracel-
lular end of TM6.
4. Experimental
4.1. Chemistry
All reagents were commercially available (Aldrich) and were
used without further purification unless otherwise indicated. Dry
THF and diethyl ether were obtained by distillation immediately
before use from benzophenone-sodium under nitrogen. Column
chromatography was carried out using silica gel 60 (230e400
mesh). J.T. Baker flexible thin layer chromatography sheets (silica
gel IB2eF) were used to monitor reactions. Melting points were
determined using a Thomas-Hoover apparatus and are uncor-
rected. H1-NMR spectra were recorded using a 300 MHz Bruker
ARX-300 NMR spectrometer. Chemical shifts are reported in
d
values ppm relative to an internal reference (0.03%, v/v) of tet-
ramethylsilane (TMS) in CDCl3, except where noted. Chemical
ionization mass spectra (CIMS) using isobutane as a carrier gas
were obtained with a Finnigan 4000 spectrometer. Elemental
analyses were performed by the Purdue University Microanalysis
Laboratory. All the reactions were carried out under an inert
atmosphere of argon.
It is postulated that these relatively minor conformational
differences can be tolerated by the D1 receptor for the parent
ligands, DHX (2) and DOX (5), but that they result in differing
tolerance for substituents at the 2- or 11-positions of the pendant
phenyl rings, respectively. This effect is most evident in the case of
the methyl-substituted compounds, where the DHX analogue
largely retains its affinity but the DOX-derived structure does not.
Unfortunately, our molecular modeling studies have not allowed us
to identify what can only be a subtle structural basis for the
difference, but very recent mutagenesis experiments of the D1
dopamine receptor in our laboratory have shown that residues in
extracellular loop 2 (EL2), particularly L190, differentially affect the
affinity of 3 and 5a (to be published elsewhere). Specifically, mutant
L190A had significantly increased affinity for 5a, but not for 3,
suggesting a reduced steric interaction and/or increased hydro-
phobic interaction between the 11-alkyl substituent of 5a and the
smaller methyl side chain of alanine. Unfortunately, the state of the
art in modeling extracellular loops in GPCRs is not very advanced,
which may explain our inability to identify ligandereceptor inter-
actions that can account for our results, and reinforcing our
observation that the differences in the binding poses of the two
series of ligands must be subtle.
Substitution with fluoro and trifluoromethyl groups gave sharp
decreases in D1 affinity compared with the unsubstituted ligand 2.
The effect was more prominent with the bulkier trifluoromethyl
group (5d), which gave a 22-fold decrease in D1 affinity compared
to 2, and an almost four-fold decrease compared to the methyl-
substituted compound 5a. These comparisons suggest that both
steric and electronic factors may account for the decreased affinity
of compound 5d. In general, electron-withdrawing groups were
especially detrimental to affinity, suggesting that aromatic stacking
interactions may not be the driving force behind binding in this
accessory region [26].
4.2. Synthesis
4.2.1. 9H-Bis([1,3]dioxolo[4,5-b:40,50-i])xanthylium chloride (8)
In a dry two-neck flask and under an inert atmosphere, ses-
amol (50.98 g, 369 mmol) was dissolved in 600 mL of CH2Cl2. Into
this flask 52 mL (448 mmol) of SnCl4 were added and the solution
was cooled to 0 ꢁC Cl2CHOCH3 (35 mL, 387.5 mmol) was then
added dropwise and the mixture was warmed to room tempera-
ture and stirred for 3 h. The mixture was allowed to settle and the
solvent was removed by decanting, keeping a positive pressure of
nitrogen through one of the flask necks. Into the flask were then
added 200 mL of CH2Cl2, and the mixture was vigorously stirred
for 20 min. The solvent was again removed by decanting and
200 mL of CH2Cl2 were added. The mixture was filtered using
a Buchner funnel and the solids rinsed with CH2Cl2. The collected
solids were placed under vacuum to yield 51.8 g (46%) of a blue-
green solid. 1H NMR (acetone-d6):
d 5.85 (4H, OCH2O), 6.11 (1H,
CH), 6.32 (2H, ArH), 6.45 (2H, ArH); EIMS: m/z 269 (Mþ, 100);
HRMS Calculated for
287.0556, found 287.0166.
C15H11O6 (xanthylium-H2O solvate)
4.2.2. 5-Hydroxybenzo[1,3]dioxole acetate (9)
In a two-neck flask fitted with a mechanical stirrer, a suspension
of 26 g (1.086 mol) of NaH in 300 mL of dry THF was cooled and
stirred on an ice bath. A solution of 75 g (0.543 mol) of sesamol in
300 mL of dry THF was added dropwise over 1 h. After the addition,
205 mL (2.174 mol) of acetic anhydride were added, resulting in
a thick suspension. This mixture was stirred for 3 h at RT. The
reaction mixture was then cooled to 0 ꢁC and quenched by slow
careful addition of 2N HCl. Water (300 mL) was then added and the
solution was extracted with CH2Cl2 (3 ꢃ 50 mL). These extracts
were washed once with 50 mL of water, 50 mL of brine, and were
then dried over MgSO4. The mixture was filtered and the solvents
removed by rotary evaporation. Kugelrohr distillation at 80 ꢁC and
0.1 torr gave 94.89 g (97%) of the product as a clear oil. This material
had properties identical to those reported previously for this
compound [27].
In summary, development of a novel methodology for the
production of variously substituted trans-fused chromanoisoqui-
nolines has allowed comparison of the receptor binding properties
of bioisosteres and revealed important differences between the
ligand/receptor complexes of DHX 1 and DOX 2 with the D1
dopamine receptor. Explanations for the divergent SAR of the two
series appear likely to be based on subtle conformational

102
J.P. Cueva et al. / European Journal of Medicinal Chemistry 48 (2012) 97e107
4.2.3. 6-Hydroxybenzo[1,3]dioxole-5-carboxaldehyde (10)
CH3); 1.32e1.3 (2s, 6H, 2CH3). CIMS: m/z 411 (M þ Hþ, 100). Anal.
In a two-neck flask, 40 g of 9 (0.222 mol) were dissolved in
500 mL CH2Cl2 and the solution cooled to 0 ꢁC. Through a drop-
ping funnel, 52 mL (0.444 mol) of SnCl4 were added dropwise,
followed by slow addition of 22 mL (0.244 mol) of Cl2CHOCH3,
causing the formation of a precipitate. The reaction was stirred
for 2 h and was then poured over ice. The mixture was parti-
tioned and the organic layer was washed with of 2M HCl
(3 ꢃ 20 mL), and then with water (2 ꢃ 50 mL). The organic
solution was dried over Na2SO4, filtered, and the solvent removed
by rotary evaporation to afford a solid, which was triturated
under cold MeOH, filtered, and dried to yield 34.38 g (93.2%) of
10 as a tan solid: mp 120e121 ꢁC (Lit [28]. mp 125e126). 1H NMR
(C22H22N2O6) C, H, N.
4.2.6. (ꢂ)-Trans-2,3-methylenedioxy-11-methyl-6a,12b-dihydro-
6H-chromeno[3,4-c]isoquinolin-8-one (16a)
One gram of nitro-oxazoline 14a was dissolved in 60 mL THF and
20 mL of a 2N aqueous HCl solution were added. This reaction was
stirred for 48 h, and then the solution was concentrated under
reduced pressure to one-half of its initial volume. The solution was
then extracted with EtOAc (3 ꢃ 30 mL), the extracts were washed
once with water (10 mL), dried over MgSO4, filtered, and the
solvent removed under reduced pressure to yield 940 mg of the
hydrochloride salt 15a as a tan solid that was used without further
purification. Hydrochloride 15a (2.9 g, 6.237 mmol) was dissolved
in 50 mL CH3COOH, 5 g of Zn powder were added, and the mixture
was stirred under an inert atmosphere for 3 h. The reaction was
filtered through Celite, and the zinc salts on the filter were rinsed
with THF. The filtrate was concentrated under reduced pressure
and 10 mL of MeOH were added to the residue. This solution was
then basified to pH 9 by slow addition of concentrated aqueous
ammonia. This mixture was warmed to 50 ꢁC and stirred for 1 h.
After cooling, the solid was collected by filtration, washed on the
filter with cold MeOH, and dried to yield 1.45 g (67%) of fine, white,
(CDCl3):
d 9.63 (s, 1H, CHO), 6.87 (s, 1H, ArH), 6.47 (s, 1, ArH), 6.02
(s, 2H, ArOCH2O), 1.54 (s, 1H, ArOH). CIMS: m/z 167 (M þ Hþ,
100). Anal. (C8H6O4) C, H.
4.2.4. 3-Nitro-2H-6,7-methylenedioxychromene (11)
In a three-necked 1 L flask equipped with a Dean Stark trap,
condenser, and mechanical stirring, 34.38 g (0.207 mol) of ses-
amaldehyde 10 were dissolved in 500 mL of toluene containing
17.5 mL of di-n-butylamine (0.104 mmol) and 61.3 g (0.414 mol) of
phthalic anhydride. This mixture was vigorously stirred while
heating at reflux. Through a cannula, 32.65 mL (0.455 mol) of 2-
nitroethanol were added into the reaction flask over 4.5 days
using a syringe pump to maintain a rate of 0.3 mL/h. The reaction
was stirred at reflux for 12 more hours, and then cooled to RT. The
contents were filtered through Celite, and the mixture was washed
with 2 N NaOH (4 ꢃ 300 mL), brine (100 mL), and then dried over
MgSO4. After filtration, the organic solution was concentrated
under reduced pressure and the crude concentrate was passed
through a short column of silica to remove dark polar impurities.
The solvent was removed under reduced pressure, and the
residual red powder was triturated under 2:1 hexanes/EtOAc. The
solid was collected by filtration and dried under vacuum to yield
35.56 g (77.7%) of pure nitrochromene 11 as a bright red powder:
cotton-like needles: mp >300 ꢁC (dec). 1H NMR (CDCl3):
d 7.98 (d,
1H, ArH); 7.46 (s, 1H, ArH); 6.99 (s, 1H, ArH); 6.51 (s, 1H, NH);
6.00e5.96 (2d, 2H, OCH2O); 4.30e4.26 (dd, 1H, OCH2, Jgem ¼ 9.1 Hz,
Jvic ¼ 3.6 Hz); 4.21 (d, 1H, ArCHAr, Jtrans ¼ 11.1 Hz); 3.95 (t, 1H, OCH2,
Jgem ¼ 9.1 Hz); 3.89e3.86 (dd,1H, CHN, Jtrans ¼ 11.1 Hz, Jvic ¼ 3.6 Hz);
2.41 (s, 3H, CH3). EIMS: m/z 309 (Mþ, 100). Anal. (C18H15NO4) C,
H, N.
4.2.7. (ꢂ)-Trans-2,3-methylenedioxy-11-methyl-6a,7,8,12b-
tetrahydro-6H-chromeno[3,4-c]-isoquinoline (17a)
Lactam 16a (1.3 g, 4.28 mmol) was suspended in 200 mL of dry
THF and stirred at reflux. Into this flask, 21.4 mL (21.4 mmol) of a 1M
BH3 solution in THF were added and the reaction was stirred at
reflux for 30 h. The mixture was then cooled to 0 ꢁC and water was
added carefully to quench the reaction. The mixture was concen-
trated under reduced pressure to about one-third of its original
volume and 100 mL of water were added. The mixture was then
extracted with EtOAc (2 ꢃ 15 mL) and the extracts dried over
MgSO4. The drying agent was then removed by filtration and the
filtrate concentrated under reduced pressure to afford a solid that
was dissolved in 30 mL of a 2 M solution of HCl in ethanol. This
solution was stirred at 70 ꢁC for 40 min and then cooled to 0 ꢁC
overnight to induce crystallization. The crystals were collected by
filtration to obtain 1.06 g of the HCl salt of 17a. A second batch was
obtained by crystallization from EtOH to obtain an additional 0.13 g.
The combined crops of this salt were suspended in MeOH, and
ammonia was added to pH 9. Water was added, the suspension was
extracted with CH2Cl2, the extracts dried over MgSO4, filtered, and
concentrated under reduced pressure to yield 1.05 g (85%) of the
amine. An analytical sample was obtained by recrystallization from
mp 139 ꢁC; 1H NMR (CDCl3):
d 5.20 (s, 2H, ArOCH2); 6.02 (s, 2H,
OCH2O); 6.49 (s, 1H, ArH); 6.69 (s, 1H, ArH); 7.75 (s, 1H, ArCH).
CIMS: m/z 222 (MHþ, 100). Anal. (C10H7NO5) C, H, N.
4.2.5. (ꢂ)-Trans-4-[2-(4,4-dimethyloxazolin-2-yl)-5-
methylphenyl]-6,7-methylenedioxy-3-nitrochroman (14a)
Toluyloxazoline 13a [29] (3.85 g, 20.34 mmol) was dissolved in
40 mL of dry THF and stirred at ꢀ45 ꢁC in a dry ice/chlorobenzene
bath. Using a syringe, 8.14 mL (20.34 mmol) of a 2.5 M solution of
nBuLi in hexanes was slowly added, and the solution turned bright
orange. The reaction was stirred for 1 h and was then cannulated
into a flask containing 3.0 g (13.56 mmol) of nitrochromene 11
dissolved in 200 mL of dry THF previously cooled to ꢀ78 ꢁC. This
mixture was allowed to warm to room temperature over 1 h, and
was then quenched with an aqueous solution of saturated NH4Cl.
The mixture was extracted with CH2Cl2 (4 ꢃ 30 mL), the pooled
organic extracts were rinsed with water (50 mL), and then brine
(20 mL). The extracts were dried over MgSO4, filtered, and the
solvent removed under reduced pressure to yield a dark oil. This oil
was dissolved in 40 mL of MeOH, whereupon immediate crystal-
lization occurred. The crystallizing mixture was then kept at 0 ꢁC
overnight, filtered, and the collected solids rinsed with cold
methanol to yield 3.27 g of pure product as cream-colored crystals.
A second crop was obtained from MeOH to yield a total of 3.52 g
MeOH: mp: 97e99 ꢁC. 1H NMR (MeOD):
d 7.26e7.23 (m, 2H, 2ArH);
7.16e7.13 (d,1H, ArH); 6.86 (s,1H, ArH); 6.48 (s,1H, ArH); 5.91e5.88
(2d, 2H, OCH2O); 4.46e4.39 (m, 3H, OCH2, CH2N); 4.22 (d, 1H,
ArHAr, Jtrans ¼ 11.4 Hz); 4.03 (t 1H, OCH2, Jgem ¼ 10.5 Hz); 2.30 (s, 3H,
CH3). EIMS: m/z 295 (Mþ, 100). Anal. (C18H17NO3) C, H, N.
4.2.8. (ꢂ)-Trans-11-methyl-2,3-dihydroxy-6a,7,8,12b-tetrahydro-
6H-chromeno[3,4-c]isoquinoline hydrobromide (5a)
(65.5%) of the product mp 172e175 ꢁC. 1H NMR (CDCl3):
d 7.85 (d,
1H, ArH); 7.14 (d, 1H, ArH); 6.79 (s, 1H, ArH); 6.45 (s, 1H, ArH); 6.32
(s, 1H, ArH); 5.92e5.86 (m, 2H, OCH2); 4.21 (br, 1H, ArCHAr); 4.93
(br, 1H, CHNO2); 4.65e4.59 (m, 1H, OCH2); 4.15e4.11 (dd, 1H,
Jgem ¼ 11.7 Hz, Jvic ¼ 2.4 Hz); 4.06 (s, 2H, oxazoline CH2); 2.28 (s, 3H,
Amine 17a (300 mg, 1.02 mmol) was dissolved in 15 mL of
CH2Cl2 under an inert atmosphere and the solution was cooled
to ꢀ78 ꢁC. Into this flask, 8.1 mL (8.1 mmol) of 1M BBr3 in CH2CL2
were added through a syringe. The solution was allowed to warm to

J.P. Cueva et al. / European Journal of Medicinal Chemistry 48 (2012) 97e107
103
0
ꢁC and was stirred for 4 h. To quench the reaction, 10 mL of dry
extracted with EtOAc (4 ꢃ 15 mL). The extracts were washed once
with brine, dried over MgSO4, filtered, and the solvents removed
by rotary evaporation to yield 516 mg of the crude HCl salt 15b as
a tan foam. This salt was dissolved in a mixture of 50 mL CH3COOH
and 10 mL of THF. Powdered zinc metal (2 g) was added and the
suspension was stirred overnight. The mixture was then filtered
and the zinc salts on the filter were rinsed with CH3COOH. The
filtrate was concentrated by rotary evaporation, the residue was
dissolved in 20 mL EtOH, and then basified with ammonia. This
mixture was stirred at 50 ꢁC for 4 h and was then cooled over-
night. The resulting crystals were collected by filtration, rinsed on
the filter with cold EtOH, and then dried under vacuum to yield
0.318 g (83.5%) of lactam 16b as a white fluffy product: mp
MeOH were added and the solution was stirred for another hour.
The solvents were removed under reduced pressure and 10 mL of
MeOH were again added. The solvents were then removed to yield
a yellow film, which was dissolved in 0.5 mL of isopropanol and
stored at ꢀ15 ꢁC until crystals appeared (60 days). The solvents
were then removed under reduced pressure to yield a yellow solid
that was triturated under cold EtOH and filtered to provide 221 mg
(60%) of the product, pure by NMR: mp: 195e200 ꢁC dec. 1H NMR
(D2O): d 7.30 (d,1H, ArH); 7.28 (s,1H, ArH); 7.22 (d,1H, ArH); 6.99 (s,
1H, ArH); 6.52 (s, 1H, ArH); 4.56e4.51 (dd, 1H, OCH2, Jgem ¼ 10.2 Hz,
Jvic ¼ 4.5 Hz); 4.51e4.42 (2d, 2H, CH2N); 4.22 (d, 1H, ArCHAr,
Jtrans ¼ 11.7 Hz); 4.13 (t, 1H, OCH2); 3.24 (dt, 1H, CHN, Jtrans ¼ 11.7 Hz,
Jvic ¼ 4.5 Hz). ESIMS: m/z 283 (Mþ, 100). High res. ESIMS for
>240 ꢁC 1H NMR (CDCl3):
d 8.01 (d, 1H, ArH); 7.47 (s, 1H, ArH); 7.26
C
17H19NO3 (M þ Hþ): calc. 284.1287, found 284.1289.
(m, 1H, ArH); 6.99 (s, 1H, ArH); 6.87 (s, 1H, NH); 6.51 (s, 1H, ArH);
6.00e5.96 (2d, 2H, OCH2O); 4.35e4.31 (dd, 1H, OCH2,
4.2.9. 2-(4-Ethylphenyl)-4,4-dimethyloxazoline (13b)
Jgem
¼
9.1 Hz, Jvic
¼
3.1 Hz); 4.25e4.21 (d, 1H, ArCHAr,
A
solution of 2-amino-2-methylpropanol (12.76 mL,
Jtrans ¼ 11.4 Hz); 3.99e3.93 (dd, 1H, OCH2, Jgem ¼ 9.1 Hz,
Jvic ¼ 11.1 Hz); 3.91e3.82 (dt, 1H, CHN, Jtrans ¼ 11.1 Hz, Jvic ¼ 3.1 Hz);
2.74e2.67 (q, 2H, ArCH2); 1.29e1.22 (t, 3H, CH3). EIMS: m/z 323
(Mþ, 100). Anal. (C19H17NO4) C, H, N.
133.1 mmol) in 100 mL CH2Cl2 was cooled to 0 ꢁC. Into this flask, 4-
ethylbenzoyl chloride (11.22 g, 66.59 mmol) was introduced
dropwise. This mixture was stirred for
4 h, then 10.2 mL
(139.8 mmol) of SOCl2 were introduced dropwise. The mixture was
allowed to warm to room temperature overnight. The solvents
were removed by rotary evaporation, and water (50 mL) was added.
The aqueous mixture was washed twice with 15 mL CH2Cl2, and
then basified with aqueous ammonia. The cloudy mixture was
extracted with CH2Cl2 (3 ꢃ 30 mL), and the pooled organic extracts
were washed once with 20 mL of water and dried over MgSO4. The
mixture was then filtered and the solvents removed under reduced
pressure to yield 7.5 g (55%) of oxazoline 13b as a colorless oil. 1H
4.2.12. (ꢂ)-Trans-2,3-methylenedioxy-11-ethyl-6a,12b-tetrahydro-
6H-chromeno[3,4-c]isoquinoline hydrochloride (17b)
In a 500 mL flask, 1.97 g (6.09 mmol) of lactam 16b was sus-
pended in 200 mL of dry THF. Into this suspension, 61 mL
(60.87 mmol) of a 1 M solution of BH3 in THF were introduced,
and the reaction was stirred and heated at reflux for 48 h. The
clear solution was then reduced to about one-third volume by
rotary evaporation, cooled to 0 ꢁC, and quenched with water. This
mixture was extracted with 3 ꢃ 30 mL CH2Cl2, and the organic
extracts were washed once with 10 mL water and once with
brine. The pooled extracts were then dried over MgSO4, filtered,
and the solvents removed by rotary evaporation to yield a viscous
oil. This oil was stirred for 4 h at 40ꢁ C with 25 mL of 2 M HCl in
EtOH, whereupon the product crystallized. The crystals were
collected by filtration and dried to yield 1.97 g (94%) of HCl salt
NMR (CDCl3):
d 7.93 (d, 2H, ArH); 7.25 (d, 2H, ArH); 4.18 (s, 2H,
OCH2); 2.68 (q, 2H, CH2); 1.43 (s, 6H, 2CH3); 1.22 (t, 3H, CH3).
4.2.10. (ꢂ)-Trans-4-[2-(4,4-dimethyl-oxazolin-2-yl)-5-
ethylphenyl]-6,7-methylenedioxy-3-nitrochroman (14b)
Under dry nitrogen, 7.50 g (36.89 mmol) of oxazoline 13b were
dissolved in 15 mL of dry THF, and cooled to ꢀ78 ꢁC in a dry ice/
acetone bath. Into this flask,15 mL (37.5 mmol) of a 2.5M solution of
nBuLi in hexanes were introduced through a syringe. The mixture
was stirred for 4 h, then a solution of 5.44 g (34.59 mmol) of
nitrochromene 11 in 40 mL of dry THF, previously to cooled
to ꢀ78 ꢁC, was introduced through a cannula. The reaction was
removed from the dry ice bath and stirred for 30 min, and then was
quenched with a saturated solution of NH4Cl. The mixture was then
extracted with 3 ꢃ 30 mL CH2Cl2, washed once with water, once
with brine, and dried over MgSO4. The mixture was then filtered
and the filtrate was concentrated by rotary evaporation to yield
a dark oil. This oil was then dissolved in hot EtOH and left to cool
overnight in a freezer. The resulting crystals were collected by
filtration and air-dried to yield 5.29 g of the product as yellowish
crystals. A second crop provided an additional 0.35 g (50.6% total
17b as a white powder: mp >240 ꢁC 1H NMR (DMSO-d6):
d 10.07
(br, 2H, þNH2); 7.36 (d, 1H, ArH); 7.22 (br, 2H, 2ArH); 6.98 (s, 1H,
ArH); 6.67 (s, 1H, ArH); 6.05 (2d, 2H, OCH2O); 4.49e4.31 (m, 4H,
OCH2, CH2N, ArCHAr); 4.16 (dd, 1H, OCH2, Jgem ¼ Jvic ¼ 10.5 Hz);
3.16 (dt, 1H, CHN, Jtrans ¼ Jvic ¼ 10.8 Hz, Jvic2 ¼ 4.5 Hz); 2.61 (q, 2H,
ArCH2); 1.16 (t, 3H, CH3). EIMS: m/z 309 (Mþ, 100). Anal.
(C19H19NO3) C, H, N.
4.2.13. (ꢂ)-Trans-2,3-dihydroxy-11-ethyl-6a,7,8,12b-tetrahydro-
6H-chromeno[3,4-c]isoquinoline hydrobromide (5b)
In a 50 mL flask, 150 mg (0.485 mmol) of amine 17b (free base)
was dissolved in 10 mL CH2Cl2. Under an inert, dry atmosphere, this
solution was cooled to ꢀ78 ꢁC and 2 mL (2 mmol) of a 1M solution
of BBr3 in CH2Cl2 were added. The flask was then placed in an ice-
water bath, stirred overnight at 0 ꢁC, and then 5 mL of dry MeOH
were added. The reaction flask was allowed to warm to RT and the
solvents were removed by rotary evaporation. Dry MeOH (5 mL)
was added to the residue and the solvents were removed under
reduced pressure. Again, 5 mL of MeOH were added and the
solvents were removed to yield a yellow foam that was pure by
NMR, and was crystallized from EtOAc/EtOH to yield 19 mg (12%) of
5b as light yellow powder: mp 188e203 ꢁC dec. 1H NMR (D2O):
yield): mp 138 ꢁC. 1H NMR (CDCl3):
d 7.85 (dd, 1H, ArH); 7.17 (d, 1H,
ArH); 6.80 (s, 1H, ArH); 6.45 (s, 1H, ArH); 6.33 (s, 1H, ArH);
5.92e5.86 (2d, 2H, OCH2O); 5.66 (br, 1H, ArCHAr); 4.95 (br, 1H,
CHNO2); 4.66e4.60 (m, 1H, OCH2); 4.14e4.05 (m, 3H, OCH2, oxa-
zoline CH2); 2.60 (q, 2H, ArCH2); 1.29 (2s, 6H, 2CH3); 1.12 (t, 3H,
CH3). ESIMS: m/z (rel. intensity) 424 (Mþ, 100). Anal. (C23H24NO2) C,
H, N.
4.2.11. (ꢂ)-Trans-2,3-methylenedioxy-11-ethyl-6a,12b-dihydro-6H-
chromeno[3,4-c]isoquinolin-8-one (16b)
In a 100 mL flask, 500 mg (1.18 mmol) of nitro-oxazoline 14b
were dissolved in 20 mL of THF and 20 mL 2 M HCl and stirred
overnight at room temperature. The solution was concentrated
under reduced pressure to one-half of its original volume and
d 7.37 (d, 1H, ArH); 7.36 (s, 1H, ArH); 7.31 (d, 1H, ArH); 7.05 (s, 1H,
ArH); 6.59 (s, 1H, ArH); 4.61e4.56 (dd, 1H, OCH2, Jvic ¼ 4.2 Hz);
4.55e4.43 (2d, 2H, CH2N); 4.32e4.28 (d, 1H, ArCHAr,
Jtrans ¼ 11.4 Hz); 4.22e4.15 (dd, 1H, OCH2, Jgem ¼ Jvic ¼ 10.8 Hz);
3.36e3.26 (dt, 1H, CHN, Jtrans ¼ 11.4 Hz, 4.72e4.52, Jvic ¼ 4.2 Hz);
2.66e2.63 (q, 2H, ArCH2); 1.18 (t, 3H, CH3) ESIMS: m/z 297 (Mþ,

104
J.P. Cueva et al. / European Journal of Medicinal Chemistry 48 (2012) 97e107
100). High res. ESIMS for C18H21NO3 (M þ Hþ): calc. 297.1365, found
45 min with 10 mL of a 2 M solution of HCl in EtOH, to produce
a white precipitate. The mixture was filtered and the collected
solids dried to yield 1.05 g of the HCl salt as a white powder. Water
(30 mL) was added to the filtrates and the mixture was washed
with 20 mL of ether, basified with NH4OH and extracted with
CH2Cl2 (10 mL x 3). The extracts were dried over MgSO4, filtered
and the solvent removed under reduced pressure. The residue was
then crystallized from cold EtOH to yield an additional 170 mg of
the free base as white crystals (88.7% total yield): mp 132e135 ꢁC.
297.1369.
4.2.14. (ꢂ)-Trans-4-[2-(4,4-dimethyloxazolin-2-yl)-5-
fluorophenyl]-6,7-methylenedioxy-3-nitrochroman (14c)
Under a dry N2 atmosphere,1.747 g (9.04 mmol) of oxazoline 13c
[30] were dissolved in 15 mL of dry THF, and cooled to ꢀ45 ꢁC in
a dry ice/chlorobenzene bath. Into this flask, 3.6 mL (9.04 mmol) of
a 2M solution of nBuLi in hexanes were introduced through
a syringe. The mixture was stirred for 1.25 h and then a solution of
1.0 g (4.52 mmol) of nitrochromene 11 in 40 mL of dry THF,
previously cooled to ꢀ78 ꢁC, was introduced through a cannula. The
reaction was removed from the dry ice bath and stirred for 30 min,
and then 20 mL of a saturated solution of NH4Cl were added. The
mixture was extracted with CH2Cl2 (3 ꢃ 30 mL), washed once with
water, and once with brine. The organic extracts were dried over
MgSO4, filtered, and the solvents removed by rotary evaporation to
yield a crude oil. This oil was then dissolved in hot MeOH, where-
upon immediate crystallization occurred. The mixture was cooled,
and the crystals were collected and dried to afford 1.22 g of the
product as yellowish crystals. A second crop yielded 0.06 g of
1H NMR (CDCl3):
d 7.21 (dd, 1H, ArH); 7.14 (dd, 1H, ArH); 6.95 (dt,
1H, ArH); 6.89 (s, 1H, ArH); 6.52 (s, 1H, ArH); 5.95 (s, 2H, OCH2O);
4.31 (dd, 1H, OCH2, Jgem ¼ 10.2 Hz, Jvic ¼ 5.1 Hz); 4.05 (s, 2H,
ArCH2N); 3.87 (d, 1H, ArCHAr, Jtrans ¼ 10.8 Hz); 3.88 (t, 1H, OCH2,
Jvic ¼ 10.2 Hz); 3.03e2.94 (dt, 1H, CHN, Jtrans ¼ 10.8 Hz). EIMS: m/z
299 (Mþ, 100). High res. EIMS for: C17H15FNO3 calc. 299.2964,
found 299.2966.
4.2.17. (ꢂ)-Trans-2,3-dihydroxy-11-fluoro-6a,7,8,12b-tetrahydro-
6H-chromeno[3,4-c]isoquinoline hydrochloride (5c)
In a 50 mL flask, 100 mg (0.334 mmol) of amine 17c was dis-
solved in 10 mL CH2Cl2. The solution was cooled to ꢀ78 ꢁC and
1.33 mL (1.33 mmol) of a 1 M solution of BCl3 in CH2Cl2 were added.
The reaction flask was then placed into an ice-water bath, stirred
for 6 h, and then 5 mL of dry MeOH were added. The reaction flask
was allowed to warm to RT and the solvents were removed by
rotary evaporation. Another 5 mL of MeOH were added and the
solution was concentrated again by rotary evaporation, leaving
a yellow foam that was crystallized from EtOAc/EtOH to yield 31 mg
(28%) of 5c as a light yellow powder: mp 181e200 ꢁC dec. 1H NMR
crystals (68% total yield): mp 183 ꢁC 1H NMR (CDCl3):
d 7.85 (dd, 1H,
ArH); 7.08e7.01 m, 1H, ArH); 6.75 (dd, 1H, ArH); 6.48 (s, 1H, ArH);
6.35 (s, 1H, ArH); 5.95e5.91 (2d, 2H, OCH2O); 5.75 (s, 1H, ArCHAr);
4.98 (br, 1H, CHNO2); 4.72e4.66 (m, 1H, OCH2); 4.10 (dd, 1H, OCH2,
Jgem ¼ 12.6 Hz); 4.06 (s, 2H, oxazoline CH2); 1.31 (d, 6H, 2CH3).
ESIMS: m/z 415 (Mþ, 100). Anal. (C21H19FN2O6) C, H, N.
4.2.15. (ꢂ)-Trans-2,3-methylenedioxy-11-fluoro-6a,12b-dihydro-
6H-chromeno[3,4-c]isoquinolin-8-one (16c)
(DMSO-d6):
d
10.18 (br, 2, þNH2); 9.43 (s, 1H, OH); 9.10 (s, 1H, OH);
A solution of 500 mg (1.21 mmol) of oxazoline 14c were dis-
solved in 10 mL of THF and 10 mL 2M HCl. This solution was stirred
for 4 h at 60 ꢁC, allowed to cool, and extracted four times with
15 mL EtOAc. The extracts were washed once with brine. Upon
standing, the organic layer produced crystals, which were filtered
and rinsed with EtOAc to yield 549 mg (97%) of HCl salt 15c. These
crystals were then dissolved in 15 mL CH3COOH, and 0.5 g of
powdered zinc metal were added. This suspension was stirred
overnight and then filtered, rinsing the zinc salts on the filter with
CH3COOH. The filtrate was then concentrated by rotary evapora-
tion. The residue was dissolved in 5 mL EtOH and then basified
with ammonia to produce crystallization. This suspension was
heated at 50 ꢁC and stirred for 4 h, then cooled and stirred over-
night. The crystals were collected by filtration, and rinsed on the
filter with EtOAc/EtOH. Drying under vacuum afforded 265 mg
(72.3%) of lactam 16c as a white fluffy product: mp >240 ꢁC 1H
7.77 (t, 1H, ArH); 7.46 (m, 2H, 2ArH); 7.07 (s, 1H, ArH); 6.63 (s, 1H,
ArH); 4.72e4.52 (m, 4H, ArCHAr, ArCH2N, OCH2,); 4.32 (t, 1H,
OCH2); 3.36 (dt, 1H, CHN, Jtrans ¼ 11.1 Hz). ESIMS: m/z 288 (Mþ, 100).
High res. ESIMS for (M þ): C16H16FNO3 calc. 288.1036, found
288.1036.
4.2.18. 2-(4-trifluoromethylphenyl)-4,4-dimethyloxazoline (13d)
This compound was prepared using the methodology of Meyers
et al. [31] and had properties identical to those reported previously
[32]. Its NMR spectrum has not been previously reported. 1H NMR
(CDCl3):
d 8.03 (d, 2H, ArH); 7.63 (d, 2H, ArH); 4.12 (s, 2H, OCH2);
1.47 (s, 6H, 2CH3); 1.22 (t, 3H, CH3).
4.2.19. (ꢂ)-Trans-4-[2-(4,4-dimethyl-oxazolin-2-yl)-5-
trifluoromethylphenyl]-6,7-methylenedioxy-3-nitrochroman (14d)
Under a dry atmosphere, 2.20 g (9.04 mmol) of oxazoline 13d
were dissolved in 35 mL of dry THF and cooled to ꢀ78 ꢁC. Into this
flask, 3.8 mL (9.5 mmol) of a 2M solution of nBuLi in hexanes were
introduced through a syringe. The mixture was stirred for 1 h, at
which point a solution of 1 g (4.52 mmol) of nitrochromene 11 in
150 mL of dry THF, previously cooled to ꢀ78 ꢁC, was introduced
through a cannula. The reaction was removed from the dry ice bath
and stirred for 30 min, and was then quenched by adding 10 mL of
a saturated solution of NH4Cl. The solution was then extracted with
3 ꢃ 50 mL CH2Cl2, the extracts washed once with water, once with
brine, dried over MgSO4, filtered, and the filtrate was then
concentrated by rotary evaporation to yield a crude oil. This oil was
dissolved in MeOH and cooled to RT overnight. The resulting
crystals were collected by filtration and dried to yield 1.45 g (69%)
NMR (DMSO-d6):
d 7.95 (dd, 1H, ArH); 7.29 (m, 2H, 2ArH); 7.17 (s,
1H, ArH); 6.60 (s, 1H, ArH); 6.02 (d, 2H, OCH2O); 4.31 (dd, 1H, OCH2,
Jgem ¼ 9.9 Hz, Jvic ¼ 3.6 Hz); 4.25 (d, 1H, ArCHAr, Jtrans ¼ 11.4 Hz);
3.87 (dd, 1H, Jgem ¼ Jtrans ¼ 10.5 Hz); 3.67 (dt, 1H, CHN,
Jtrans ¼ 11.4 Hz, Jvic ¼ 3.9 Hz). EIMS: m/z 313 (Mþ, 100). Anal.
(C17H12FNO4) C, H, N.
4.2.16. (ꢂ)-Trans-2,3-methylenedioxy-11-fluoro-6a,7,8,12b-
tetrahydro-6H-chromeno[3,4-c]-isoquinoline (17c)
In a 250 mL flask, 1.3 g (4.86 mmol) of lactam 16c were sus-
pended in 40 mL of dry THF. Into this suspension, 20 mL
(20 mmol) of a 1M solution of BH3 in THF were introduced, and
the solution was stirred at reflux overnight. The clear solution was
then cooled to 0 ꢁC, quenched with water, and reduced to one-
third of its volume by rotary evaporation. This mixture was
extracted with CH2Cl2 (3 ꢃ 15 mL), and the extracts were washed
once with 10 mL water and once with brine. This solution was
then dried over MgSO4, filtered, and the filtrate was concentrated
by rotary evaporation to yield a viscous oil. This oil was stirred for
of the product as yellow crystals: mp 176 ꢁC. 1H NMR (CDCl3):
d 8.07
(d, 1H, ArH); 7.63 (d, 1H, ArH); 7.30 (s, 1H, ArH); 6.50 (s, 1H, ArH);
6.30 (s, 1H, ArH); 5.96e5.91 (2d, 2H, OCH2O); 5.76 (s, 1H, ArCHAr);
4.96 (br, 1H, CHNO2); 4.72e4.62 (m, 1H, OCH2); 4.12e4.08 (s, m, 3H,
oxazoline CH2, OCH2); 1.39e1.33 (m, 6H, 2CH3). ESIMS: m/z 464
(Mþ, 100). Anal. (C22H19F3N2O6) C, H, N.

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105
4.2.20. (ꢂ)-Trans-2,3-methylenedioxy-11-trifluoromethyl-6a,12b-
dihydro-6H-chromeno[3,4-c]-isoquinolin-8-one (16d)
ArCH2N, Jgem ¼ 15.1 Hz); 4.18 (t, 1H, OCH2); 3.26 (dt, 1, CHN,
Jtrans ¼ 10.8 Hz, Jvic ¼ 3.6 Hz). ESIMS: m/z (rel. intensity) 337 (Mþ,
100). High res. ESIMS for C17H16F3NO3 (M þ Hþ): calc. 338.1004,
found 338.1000.
In a 250 mL flask, 2.57 g (5.56 mmol) of oxazoline 14d were
dissolved in a mixture of 40 mL of THF and 40 mL 2M HCl. This
solution was stirred overnight, 100 mL of water were added, and
the mixture was then extracted with 4 ꢃ 30 mL CH2Cl2. The pooled
organic extracts were washed twice with 10 mL of water and dried
over MgSO4. The mixture was filtered, and the filtrate was
concentrated by rotary evaporation to afford 2.57 g (89.5%) of
a yellow foam that could be crystallized from iPrOH, but was used
without further purification. In a 250 mL flask, 2.79 g of HCl salt 15d
were dissolved in 150 mL of CH3COOH. Powdered Zn (1 g) was
added and the suspension was stirred overnight. The reaction was
filtered, the solids on the filter were rinsed with CH3COOH, and the
filtrate was reduced to dryness under vacuum. The residue was
dissolved in 15 mL EtOH, and ammonia was added to basify (pH 10)
the mixture. This solution was cooled to 0 ꢁC overnight. The
resulting solid was collected by filtration, washed on the filter with
cold EtOH, and then dried under vacuum to yield 1.77 g (90.6%) of
lactam 16d as a white, fluffy product: mp >240 ꢁC. 1H NMR (DMSO-
4.2.23. 6-(O-Dimethylthiocarbamoyl)-1,3-benzodioxole (18)
Under N2, NaH (0.32 g, 13.25 mmol) was slurried with stirring
in 20 mL of dry THF at ꢀ78 ꢁC. A solution of sesamaldehyde 10
(2 g, 12.05 mmol) dissolved in 50 mL of dry THF was then added
dropwise to the suspension, followed by a solution of 1.638 g
(13.25 mmol) of dimethylthiocarbamoyl chloride dissolved in
15 mL of dry THF. The reaction was allowed to warm to RT, was
stirred for 12 h, and was then carefully quenched with water and
extracted with CH2Cl2 (3 ꢃ 15 mL). The pooled extracts were
washed with brine, dried over MgSO4, filtered, and the solvent
removed under reduced pressure to afford an orange solid,
which was recrystallized from EtOH to yield 2.30 g (75%) of pure
thiocarbamate 18 as lightbrown flakes: mp 137e140 ꢁC. 1H NMR
(CDCl3):
d 9.89 (s, 1H, CHO); 7.31 (s, 1H, ArH); 6.60 (s, 1H, ArH);
6.10 (s, 2H, OCH2O); 3.48 (s, 3H, CH3); 3.41 (s, 3H, CH3). CIMS:
d6):
d
8.85 (s, 1, NH); 8.10 (d, 1H, ArH); 7.82 (d, 1H, ArH); 7.79 (s, 1H,
254 (M þ Hþ, 100). Anal. (C11H11NO4S) C, H, N.
ArH); 7.19 (s, 1H, ArH); 6.63 (s, 1H, ArH); 6.04 (2d, 2H, OCH2O,
Jgem ¼ 14.1 Hz); 4.33 (d, 1H, ArCHAr, Jtrans ¼ 11.7 Hz); 4.32 (dd, 1H,
4.2.24. 6-(S-Dimethylthiocarbamoyl)-1,3-benzodioxole (19)
In a single-necked flask attached to a condenser, and under an
inert atmosphere, 2.27 g (8.96 mmol) of thiocarbamate 18 were
dissolved in 35 mL of diphenyl ether. This solution was heated at
250 ꢁC for 15 min and then immediately cooled to RT with an ice
bath. Hexanes (100 mL) was added to the solution, which was
stored overnight at 0 ꢁC to cause precipitation. The solid was
collected by filtration, washed repeatedly on the filter with
hexanes, and purified by column chromatography to yield 1.27 g
(56%) of the product as a brown solid. This compound was used
without further characterization.
OCH2, Jgem
¼
10.2 Hz, Jvic
¼
3.6 Hz); 3.89 (t, 1H, OCH2,
Jgem ¼ 10.2 Hz), 3.73 (dt, 1H, NCH, Jtrans ¼ 11.7 Hz, Jvic ¼ 3.6 Hz).
ESIMS: m/z 363 (Mþ, 100). Anal. (C18H12F3NO4) C, H, N.
4.2.21. (ꢂ)-Trans-2,3-methylenedioxy-11-trifluoromethyl-
6a,7,8,12b-tetrahydro-6H-chromeno[3,4-c]isoquinoline
hydrochloride (17d)
In a 250 mL flask, 1.765 g (4.86 mmol) of lactam 16d were sus-
pended in 50 mL of dry THF. Into this suspension, 24.3 mL
(24.3 mmol) of a 1 M solution of BH3 in THF were introduced, and
the reaction was heated at reflux and stirred overnight. The clear
solution was then reduced to about one-third of its volume by
rotary evaporation, cooled to 0 ꢁC, and diluted with H2O. This
mixture was extracted with 3 ꢃ 25 mL CH2Cl2, and the extracts
were washed once with 10 mL water and once with brine. This
solution was then dried over MgSO4, filtered, and the solvents
removed by rotary evaporation to yield a viscous oil. A 2M solution
of HCl in EtOH (10 mL) was added to this oil, and the mixture was
stirred for 45 min to produce, after 10 min, a white precipitate. The
suspension was cooled to 0 ꢁC for 2 h and filtered. The solid on the
filter was rinsed with cold EtOH and dried to afford 1.57 g (84%) of
4.2.25. 3-Nitro-2H-6,7-methylenedioxythiochromene (20)
In a single-neck flask, 2.96 g of thiocarbamate 19 (11.70 mmol)
were dissolved in 400 of mL of MeOH and heated to 60 ꢁC. Next,
35 mL of a 2M solution of NaOH in MeOH were added, and the
solution was heated at reflux for 2.5 h until TLC indicated
complete hydrolysis. The mixture was acidified to pH 7 with 2 M
HCl in EtOH. The solvents were then removed under reduced
pressure, avoiding exposure to air. Toluene (400 mL) was added to
the mixture, followed by 1 mL (5.85 mmol) of dibutylamine, 3.46 g
(23.39 mmol) of phthalic anhydride, and 0.3 mL (4.19 mmol) of 2-
nitroethanol. This mixture was heated at reflux for 3 h, at which
time addition of 2-nitroethanol was begun at a rate of about 1
drop every 15 min, for a total of 1.6 mL (22.33 mmol) of the
reagent added over 8 h. The reaction was stirred at reflux over-
night and was then cooled and filtered through Celite. The filtrate
was extracted several times with 2 M NaOH, washed once with
water, dried over Na2SO4, filtered, and reduced to dryness by
rotary evaporation. The crude nitrochromene was purified by
column chromatography to yield 1.78 g (64%) of a bright red
the HCl salt as a white powder: mp >240 ꢁC. 1H NMR (CDCl3):
d 7.78
(d, 1, ArH); 7.71 (d, 2H, ArH); 7.63 (s, 1H, ArH); 7.02 (s, 1H, ArH); 6.69
(s, 1H, ArH); 6.05 (2d, 2H, OCH2O); 4.61e4.44 (m, 4H, OCH2, NCH2,
ArCHAr); 4.18 (t, 1H, OCH2); 3.26 (dt, 1H, J1 ¼ 10.8 Hz, J2 ¼ 3.9 Hz).
ESIMS: m/z 349 (Mþ, 100). Anal. (C18H14F3NO3) C, H, N.
4.2.22. (ꢂ)-Trans-2,3-dihydroxy-11-trifluoromethyl-6a,7,8,12b-
tetrahydro-6H-chromeno[3,4-c]-isoquinoline hydrochloride (5d)
In a 50 mL flask, 702 mg (2.01 mmol) of amine 17d was dis-
solved in 20 mL CH2Cl2 under a dry N2 atmosphere. The flask was
cooled to ꢀ78 ꢁC and 8 mL (8 mmol) of a 1 M solution of BCl3 in
CH2Cl2 were added. The reaction flask was then placed in an ice-
water bath, and stirred for 4.5 h, at which time 4 mL of dry
MeOH were added. The reaction was allowed to warm to RT and
the solvents were removed by rotary evaporation. Addition of 5 mL
of dry MeOH and concentration by rotary evaporation was
repeated twice more, to leave a tan-orange foam, which was
crystallized from CH3CN/MeOH to afford a light orange powder:
powder: mp: 128e131 ꢁC. 1H NMR (CDCl3):
d 7.82 (s, 1H, ArCH);
6.82 (s, 1H, ArH); 6.81 (s, 1H, ArH); 6.03 (s, 2H, OCH2O); 4.07 (s, 2H,
OCH2). EIMS: 237 (Mþ, 100). High res. EIMS for C10H8NO4S (M þ):
calc. 237.0096, found 237.0099.
4.2.26. (ꢂ)-Trans-2,3-methylenedioxy-6a,12b-dihydro-6H-
thiochromeno[3,4-c]isoquinoline (22)
In a dry flask, 2.99 g (12.30 mmol) of 2-(2-bromophenyl)-1,3-
dioxane were dissolved in 25 mL of dry THF and 597 mg
(26.61 mmol) of magnesium powder were added, along with 1
drop of 1,2-dibromoethane. This solution was heated at reflux and
stirred for 1 h until formation of the Grignard reagent was
mp >187 ꢁC dec. 1H NMR (DMSO-d6): 10.03 (br, 2, þNH2); 9.25 (s,
d
1H, OH); 8.92 (s, 1H, OH); 7.77 (d, 1H, ArH); 7.71 (s, 1H, ArH); 7.69
(d, 1H, ArH); 6.81 (s, 1H, ArH); 6.41 (s, 1H, ArH); 4.59e4.45 (2d, 2H,

106
J.P. Cueva et al. / European Journal of Medicinal Chemistry 48 (2012) 97e107
complete. The mixture was then cooled to 0 ꢁC and, through
a dropping funnel, a solution of 972 mg (4.10 mmol) of nitro-
thiochromene 20 in 35 mL of dry THF were added dropwise over
0.5 h. The reaction was stirred for 10 min and was then quenched
with a satd solution of NH4Cl. The mixture was then filtered and
extracted several times with EtOAc. The combined organic extracts
were washed once with water, once with brine, dried over MgSO4,
and filtered. The filtrate was reduced to dryness to yield an oil that
was purified using silica gel flash column chromatography, eluting
with a 4:1 mixture of hexanes:EtOAc to provide 867 mg (52.7%) of
1H, ArH); 4.43 (t, 2H, ArCH2N); 4.26e4.22 (d, 1H, ArCHAr,
Jtrans ¼ 10.5 Hz); 3.45e3.35 (m, 1H, NCH2); 3.28e3.19 (m, 1H, SCH2);
3.10e3.03 (t, 1H, SCH2, J ¼ 10.5 Hz). ESIMS: 286 (M þ Hþ, 100). Anal.
(C16H16ClNO2S) C, H, N.
4.3. Pharmacology methods
4.3.1. Materials
[3H]spiperone (95 Ci/mmol) and [3H]SCH-23390 (81 Ci/mmol)
were purchased from Amersham Biosciences (Piscataway, NJ, USA).
[3H]Cyclic AMP (30 Ci/mmol) was purchased from PerkinElmer
(Boston, MA, USA). Chlorpromazine, SCH-23390, butaclamol, and
most other reagents were purchased from SigmaeAldrich Chemical
Company (St. Louis, MO, USA).
pure 22 as a white powder: mp 154 ꢁC 1H NMR (CDCl3):
d 7.22 (m,
1H, ArH); 7.27e7.24 (m, 2H, ArH); 6.60 (s, 1H, ArH); 6.48 (s, 1H,
ArH); 5.85 (s, 2H, OCH2O); 5.80 (d, 1H, ArCHAr, J ¼ 3.3 Hz); 5.62 (s,
1H, OCHO); 5.28e5.25 (m, 1H, CHNO2); 4.27e4.18 (m, 2H, OCH2C);
3.98e3.90 (m, 2H, OCH2C); 3.43 (m, 2H, SCH2); 2.26e2.20 (m, 1H,
CCH2C); 1.40 (bd, 1H, CCH2C). EIMS: 401 (Mþ, 100). Anal.
(C20H19NO6S) C, H, N.
4.3.2. Competition binding experiments
Porcine striatal tissue was obtained fresh from the Purdue
Butcher Block and prepared as previously described [18]. Briefly,
striatal tissue was homogenized using a potter-type homogenizer,
suspended in homogenization buffer (20 mM Hepes, 0.32 M
sucrose, pH 7.4), and centrifuged at 1000 ꢃ g for 10 min at 4 ꢁC. The
pellet (P1) was discarded and the supernatant was centrifuged at
30,000 ꢃ g for 10 min at 4 ꢁC. The resulting pellet (P2) was resus-
pended in 50 mM Tris buffer (pH 7.4) by briefly using a Kinematica
homogenizer, and then centrifuged at 30,000 ꢃ g for 30 min at 4 ꢁC.
This pellet was resuspended again in 50 mM Tris buffer, dispensed
into 1 mL aliquots, and centrifuged for 10 min at 13,000 ꢃ g and
4.2.27. (ꢂ)-Trans-2,3-methylenedioxy-6a,7,8,12b-tetrahydro-6H-
thiochromeno[3,4-c]isoquinoline (24)
Into a solution of 550 mg (1.37 mmol) of compound 22 in 10 mL
of THF and 30 mL CH3COOH were added 2 g of powdered zinc. This
suspension was vigorously stirred for 6 h at 70 ꢁC and was then
cooled to RT, filtered, and the filtrate was concentrated under
reduced pressure. To the residue was added 30 mL of 2 M ethanolic
HCl and this solution was stirred for 30 min. The solvents were
removed once again and 30 mL of a 2 M solution of NaOH and
30 mL of CH2Cl2 were added. This biphasic mixture was stirred
overnight. The organic layer was then separated, washed with
water, dried over Na2SO4, filtered, and the solvent removed to leave
418 mg of a light brown solid (23), which was used without further
purification.
To a solution of 400 mg of the crude imine 23 (1.356 mmol) in
10 mL of dry THF and 30 mL of EtOH was added 85 mg of NaCNBH3
(1.36 mmol) followed by dropwise addition of 4 mL of a 2M solu-
tion of HCl in EtOH over 10 min. The suspension was stirred for 4 h
and was then reduced under vacuum to about one-third of its
original volume. Into this suspension, 10 mL of a 2M solution of
NaOH were added and the mixture was extracted with CH2Cl2
(3 ꢃ 10 mL). The pooled organic extracts were washed once with
water, dried over Na2SO4, filtered, and the solvent was removed.
The resulting residue was purified by column chromatography,
eluting with EtOAc to yield 316 mg (78%) of amine 24 as a tan solid.
An analytical sample was prepared by crystallizing the HCl salt
4
ꢁC. A BCA protein assay was used to determine the final protein
concentration of the pellets. Supernatant was aspirated and pellets
were frozen at ꢀ80ꢁ C until use.
Radioligand binding assays were performed as previously
described [18], with only minor modifications. Pellets were resus-
pended (1 mL/mg) in receptor binding buffer (50 mM Hepes, 4 mM
MgCl2, pH 7.4) and 75 mg protein was used per assay tube. Receptor
isotherms were performed with [3H]SCH-23390 and [3H]spiperone
to determine Bmax and Kd for D1-like and D2-like receptor sites,
respectively (760 fmol/mg and 0.44 nM for [3H]SCH-23390, and
250 fmol/mg and 0.075 nM for [3H]spiperone). All D2-like receptor
binding assays were performed in the presence of 50 nM ketanserin
to mask 5-HT2A sites. Nonspecific binding was defined with 5 mM
butaclamol. Drug dilutions for competitive binding experiments
were made in receptor binding buffer and added to assay tubes
containing 75
m
g protein and either 1 nM [3H]SCH-23390 or
0.15 nM [3H]spiperone. All binding experiments were incubated for
30 min at 37 ꢁC, and were terminated by filtration with ice cold
wash buffer using a 96-well Packard Filtermate cell harvester. After
from EtOH/Et2O: mp >260 ꢁC dec. 1H NMR (DMSO-d6):
d 10.11 (bs,
1H, þNH); 9.69 (bs, 1H, þNH); 7.49e7.40 (m, 4H, ArH); 7.07 (s, 1H,
ArH); 6.48 (s, 1H, ArH); 6.00 (d, 2H, OCH2O); 4.41 (bs, 2H, ArCH2N);
4.27 (d, 1H, ArCHAr); 3.45e3.42 (m, 2H, SCH2, CHN); 3.11 (t, 1H,
SCH2). High res. ESIMS for C17H16NO2S (M þ H þ): calc. 298.0902,
found 298.0904.
the samples were dried, 30 mL of Packard Microscint O was added to
each well. Radioactivity was counted using a Packard Topcount
scintillation counter.
Acknowledgments
4.2.28. (ꢂ)-2,3-Dihydroxy-6a,7,8,12b-tetrahydro-6H-thiochromeno
[3,4-c]isoquinoline hydro-chloride (6)
This work was supported by NIH grants MH MH42705 (DEN),
MH60973 (VJW), and GM085604 (MAL)
A solution of 300 mg (1.01 mmol) of amine 24 in 8 mL of CH2Cl2
was placed into a flask, cooled to ꢀ78 ꢁC, and 4 mL (4.0 mmol) of
a 1M solution of BBr3 in CH2Cl2 were added through a syringe. This
solution was allowed to warm to 0 ꢁC in an ice bath and was stirred
for 3 h. Dry EtOH (5 mL) was then added to quench the reaction,
and the solution was stirred for 30 min at RT. The solvents were
removed by rotary evaporation, and 5 mL of dry EtOH were again
added. Removal of the solvent yielded a white powder that was
collected by filtration to afford a total of 104 mg (34%) of pure
catecholamine 6 as a tan powder, in two crops: mp 288e294 ꢁC dec.
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