Pictet-Spengler condensations using 4-(2-aminoethyl)coumarins

Article abstract of DOI:10.1039/d0nj02664f

Androgen-deprivation therapy (ADT) is only a palliative measure, and prostate cancer invariably recurs in a lethal, castration-resistant form (CRPC). Prostate cancer resists ADT by metabolizing weak, adrenal androgens to growth-promoting 5α-dihydrotestosterone (DHT), the preferred ligand for the androgen receptor (AR). Developing small-molecule inhibitors for the final steps in androgen metabolic pathways that utilize 17-oxidoreductases required probes that possess fluorescent groups at C-3 and intact, naturally occurring functionality at C-17. Application of the Pictet-Spengler condensation to substituted 4-(2-aminoethyl)coumarins and 5α-androstane-3-ones furnished spirocyclic, fluorescent androgens at the desired C-3 position. Condensations required the presence of activating C-7 amino or N,N-dialkylamino groups in the 4-(2-aminoethyl)coumarin component of these condensation reactions. Successful Pictet-Spengler condensation, for example, of DHT with 9-(2-aminoethyl)-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinolin-11-one led to a spirocyclic androgen, (3R,5S,10S,13S,17S)-17-hydroxy-10,13-dimethyl-1,2,2′,3′,4,5,6,7,8,8′,9,9′,10,11,12,12′,13,13′,14,15,16,17-docosahydro-7′H,11′H-spiro-[cyclopenta[a]phenanthrene-3,4′-pyrido[3,2,1-ij]pyrido[4′,3′:4,5]pyrano[2,3-f]quinolin]-5′(1′H)-one. Computational modeling supported the surrogacy of the C-3 fluorescent DHT analog as a tool to study 17-oxidoreductases for intracrine, androgen metabolism. This journal is

Full text of DOI:10.1039/d0nj02664f

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Pictet-Spengler condensations using 4-(2-aminoethyl)coumarins
Vitaliy M. Sviripa, *a,b,c Michael V. Fiandalo, d Kristin L. Begley, b,e Przemyslaw Wyrebek, b,e Liliia M.
b,e
b,e
f
g
Kril, Andrii G. Balia, Sean R. Parkin, Vivekanandan Subramanian, Xi Chen, h Alexander H.
Received 00th January 20xx,
Accepted 00th January 20xx
Williams, a Chang-Guo Zhan, a,b Chunming Liu, c,e James L. Mohler, d,i and David S. Watt, b,c,e
Androgen-deprivation therapy (ADT) is only a palliative measure, and prostate cancer invariably recurs in a lethal, castration-
resistant form (CRPC). Prostate cancer resists ADT by metabolizing weak, adrenal androgens to growth-promoting 5α-
dihydrotestosterone (DHT), the preferred ligand for the androgen receptor (AR). Developing small-molecule inhibitors for
the final steps in androgen metabolic pathways that utilize 17-oxidoreductases required probes that possess fluorescent
groups at C-3 and intact, naturally occurring functionality at C-17. Application of the Pictet-Spengler condensation to
substituted 4-(2-aminoethyl)coumarins and 5-androstane-3-ones furnished spirocyclic, fluorescent androgens at the
desired C-3 position. Condensations required the presence of activating C-7 amino or N,N-dialkylamino groups in the 4-(2-
aminoethyl)coumarins component of these condensation reactions. Successful Pictet-Spengler condensation, for example,
of DHT with 9-(2-aminoethyl)-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinolin-11-one led to a spirocyclic
DOI: 10.1039/x0xx00000x
androgen,
(3R,5S,10S,13S,17S)-17-hydroxy-10,13-dimethyl-1,2,2',3',4,5,6,7,8,8',9,9',10,11,12,12',13,13',14,15,16,17-
docosahydro-7'H,11'H-spiro-[cyclopenta[a]phenanthrene-3,4'-pyrido[3,2,1-ij]pyrido[4',3':4,5]pyrano[2,3-f]quinolin]-
5'(1'H)-one. Computational modeling supported the surrogacy of the C-3 fluorescent DHT analog as a tool to study 17-
oxidoreductases for intracrine, androgen metabolism.
androgen levels during ADT that are low but sufficient to
activate the AR1,5 and promote cancer recurrence as lethal,
castration-recurrent/resistant prostate cancer (CRPC). Current
therapies for CRPC rely on inhibitors for enzymes that function
well before the final steps in the biosynthetic pathways leading
to DHT, and we sought to identify new, small-molecule
inhibitors6 for late-stage, NAD(P)H-dependent 17
hydroxysteroid dehydrogenases7.
Introduction
Prostate cancer growth and progression rely on the activation
of the androgen receptor (AR) by the circulating, testicular
androgen, testosterone (T) or its intracellular metabolite, 5α-
dihydrotestosterone (DHT), the preferred ligand for AR
transactivation1-2. Men, who present with advanced prostate
cancer or who fail potentially curative therapy, undergo
androgen-deprivation therapy (ADT) intended to lower
circulating testosterone levels, to deprive the AR of activating
ligands and to induce cancer regression.3-4 Unfortunately,
androgen-deprivation therapy is only a temporary, palliative
measure, since prostate cancer produces intratumoral
-
Prostate cancer cells utilize three, late-stage, androgen-
metabolic pathways8-12 driven by oxidoreductases to acquire
DHT (1) (Fig. 1).
a. Department of Pharmaceutical Sciences, College of Pharmacy, University of
Kentucky, Lexington, KY 40536-0596 USA.
b. Center for Pharmaceutical Research and Innovation, College of Pharmacy,
University of Kentucky, Lexington, KY 40536-0596 USA.
c. Lucille Parker Markey Cancer Center, University of Kentucky, Lexington, KY 40536-
0093 USA.
d. Department of Experimental Therapeutics, Roswell Park Comprehensive Cancer
Center, Buffalo, NY 14263 USA.
e. Department of Molecular and Cellular Biochemistry, College of Medicine,
University of Kentucky, Lexington, KY 40536-0509 USA.
f. Department of Chemistry, College of Arts and Sciences, University of Kentucky,
Lexington, KY 40506 USA
.
g. NMR Center, College of Pharmacy, University of Kentucky, Lexington, KY 40536-
0596 USA.
h. College of Chemistry and Material Science, South Central University for
Fig. 1. Late-stage metabolic pathways converging on 5α-dihydrotestosterone (DHT).
Legend: frontdoor (orange), primary backdoor (green) and secondary backdoor (purple)
pathways.
Nationalities, Wuhan 430074, People’s Republic of China
i. Department of Urology, Roswell Park Comprehensive Cancer Center, Buffalo, NY
14263 USA
.
.
† Electronic Supplementary Information (ESI) available: Copies of NMR data for
all synthesized compounds are available online. See DOI: 10.1039/x0xx00000x
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The “frontdoor” pathway converts the adrenal androgens, such 3-(trifluoroacetamido)propanoic acid with methVyilewpAortticalessOinulimne
DOI: 10.1039/D0NJ02664F
as dehydroepiandrosterone and 4-androstene-3,17-dione (
to testosterone ( ) that subsequently undergoes
4-reduction pentanoate (
to DHT ( ). The “primary backdoor” pathway converts the hydroxyphenyl)carbamate (8a), 3-(N,N-dimethylamino)phenol
penultimate 5α-androstane-3α,17β-diol ( 8b), resorcinol (8c), or 3-methoxyphenol (8d) furnished
) directly to DHT12-16
without passing through testosterone as an intermediate trifluoroacetamides; and the final, acid-catalyzed
intermediate. The “secondary backdoor” pathway converts 4- hydrolysis of these intermediates provided the 4-(2-
androstene-3,17-dione ( ) to 5α-androstane-3,17-dione ( ) that aminoethyl)coumarins 9a-9d respectively, as their
undergoes reduction of the C-17-keto group to DHT12, 14, 16-23
), hydrochloride salts. A related procedure using a benzyloxy
also without passing through testosterone as an urethane in place of the trifluoroacetamide derivative (i.e.,
2), malonate furnished methyl 3-oxo-5-(trifluoroacetamido)-
3

7); a second condensation with either methyl (3-
1
4
(
(
1
)
(3)
2
5
,
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(
1
(3)
intermediate. One commonality among these pathways
involved C-17 redox reactions mediated by the
aldo/ketoreductase24-26 (AKR1C3; HSD17B5) and 17
-
hydroxysteroid dehydrogenase8 (HSD17B3) for the conversion
of 4-androstene-3,17-dione (2) to testosterone ( ); the
conversion of 5 -androstane-3,17-dione ( ) to DHT ( ); and the
conversion of -androstan-3 -ol-17-one to
androstane-3 ,17 -diol ( ) (Fig. 1).
3

5
1
5


(
6
)
5-


4
Our focus on developing small-molecule inhibitors for various
17-oxidoreductases, alone or in combination27, that perform
these interconversions required fluorescent probes that had a
C-3 fluorophore with excitation and emission patterns in the
550-650 nm range and that retained the intact, natural
functionality at C-17. These combined challenges led us to
select coumarins as fluorophores and to explore methodology
for their attachment to the C-3 position of 5
ones28-30. We now report the scope of the Pictet-Spengler
condensation31-34
of C-7 amino-substituted 4-(2-
-androstan-3-
aminoethyl)coumarins with aldehydes and ketones, NMR and
X-ray crystallography studies that established the
diastereoselectivity in adducts derived from 5
ones, the mechanism of these reactions and computational
modeling of adducts in the active site of 17 -hydroxysteroid
dehydrogenase type 535-36 (17
-HSD5; AKR1C3). Our findings
-androstan-3-


auger well for the application of these probes in drug discovery.
Results
Variants of the Pechmann condensation37-38 provided access to
C-7 substituted 4-(2-aminoethyl)coumarins 9a-9e for this study
(
Scheme 1). Specifically, the condensation of
Scheme 2. Representative Pictet-Spengler reactions of C-7 amino-substituted 4-(2-
aminoethyl)coumarins 9. Legend: Panel A: a, 1:10 (v/v) conc. HCl acid-abs. ethanol;
Panel B: b, p-tolualdehyde; 1:10 (v/v) CH2Cl2-TFA, 25oC; c, CuBr2, O2, DBU, DMSO.
methyl-5-benzyloxycarbonylamino-3-oxopentanoate) in
condensation with 8-hydroxy-2,3,6,7-tetrahydro-1H,5H-
benzo[ij]quinolizine38
8e furnished the 4-(2-
a
Scheme 1. Pechmann synthesis of substituted 4-(2-aminoethyl)coumarins 9. Legend: a,
CH3OC(=O)CH2CO2K; b, CH3SO3H, 7; c, conc. HCl; d, TiCl(OiPr)3, 7.
(
)
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aminoethyl)coumarin 9e
ARTICLE
Scheme 1). The Pictet-Spengler Confirmation of the C-3R stereochemical assignments in the
1
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5
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8
(
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SchemeDO2AI: )10.r1e0l3ie9/dD0oNnJ02t6w64oF-
condensations of coumarins 9a, 9b or 9e that possessed C-7 spirocyclic DHT adduct 11ei
amino or C-7 N,N-dialkylamino groups with either acyclic and dimensional H-13C heteronuclear single quantum coherence
monocyclic ketones 10a-10f or 3-ketosteroids 10g-10k under (HSQC), gradient-correlation spectroscopy (gCOSY) and 2D
(
1
acidic
chromeno[3,4-c]pyridin-5-ones 11
Table 1). For example, the condensation of coumarin 9e with system appeared at
conditions
afforded
1,2,3,4-tetrahydro-5H- rotating frame NOESY (2D ROESY) experiments41-42
Scheme 2A) in good yields resonance for the protonated amine in the spirocyclic ring
9.24 ppm in the 2D ROESY spectrum and
.
The
(
(

9
5
-androstan-17

-ol-3-one (DHT) (10i) afforded a single was the starting point for this stereochemical assignment at C-
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+
diastereomer of the spirocyclic, fluorescent adduct 11ei that 3. The ammonium group (NH2 ) at C-3 was identified using a
was more compact than previously reported, bulky DHT analogs D2O exchange experiment in DMSO-d6. Correlations in the
that possessed a linker between umbelliferone and 3
-amino- ROESY spectrum between this ammonium group and
5

-androstan-17 linker between individually the C-1α C-2α, C-4α and C-5α protons (Fig. 2)

-ol28 or possessed
a
fluorescein isothiocyanate and either a C-3 hydrazone or O- confirmed the 3α-orientation of the ammonium group in 11ei
.
carboxymethyloxime derivative of DHT29-30. The hygroscopic
nature of some of the hydrochloride and trifluoroacetate salts
of the adducts 11 necessitated their isolation as N-acetyl
derivatives, as noted in Table 1
.
Table 1. Synthesis conditions and yields of Pictet-Spengler adducts 11 formed from C-7
amino-substituted 4-(2-aminoethyl)coumarins 9 and ketones 10.
Fig. 2. 2D ROESY spectrum of DHT analog 11ei. Spectrum recorded using Agilent 400
MHz at 25°C. Dotted lines show region expanded that displays the NOE connectivity
+
between NH2 and ring A protons (i.e., H-5 (and H-1 overlapping), H-4, H-2, and CH2
adjoining NH2+).
The Pictet-Spengler reactions of coumarins
9 and various
Legend: 1Condition A: 1:10 (v/v) acetone-TFA, reflux; condition B: 1:10 (v/v) TFA-CH2Cl2,
25oC; condition C: 1:10 (v/v) conc. HCl acid-abs. ethanol, reflux; 2isolated as N-acetyl
derivative; 3isolated as hydrochloride salt; 4isolated as a trifluoroacetate salt.
ketones employed three different conditions that depended on
the solubility of the ketone component and the desire, in the
case of steroid condensations, to precipitate the products from
acidic, ethanol solutions using water. A mixture of acetone-
trifluoroacetic acid at reflux sufficed for reactions with acetone
(condition A); trifluoracetic acid in dichloromethane at 25oC;
A range of steroidal and non-steroidal carbonyl compounds
served to define the scope of these Pictet-Spengler reactions
with coumarins
methylpiperidin-4-one (10f) with 9b and the condensation of
-androstan-17 -ol-3-one (10i) with coumarins 9a and 9e
Scheme 2A) In general, condensations with unhindered
9 as illustrated by the condensation of 1-
(condition B) effected the condensations of
9 with monocyclic
ketones 10b-10f, and 1:10 concentrated hydrochloric acid in
ethanol at reflux (condition C) promoted successful reactions
with steroidal ketones 10g-10k to give the desired adducts 11
5


(
.
ketones varying from acyclic to monocyclic ketones were
successful in yields of 60-90% (Table 1). In contrast, the
condensations of the coumarin 9e either with hindered ketones
(
Table 1). Depending on conditions, the acid-catalyzed
condensation of 17α-methyl-5α-androstan-17 -ol-3-one (10j

)
such as 5
such as testosterone (
reactivity proved advantageous in the regiospecific
modification of 5 -androstane-3,17-dione (10h) with coumarin
-androstan-17-one or with
,-unsaturated ketones
3
) were unsuccessful. This differential

9e that led exclusively to the C-3 adduct 11eh in 57% yield.
Contrary to a prior report of a successful Pictet-Spengler
reaction of an amino-substituted coumarin with
formaldehyde39, condensations of aldehydes with coumarins
9
led to poor yields of isolated products, presumably because of
adventitious air-oxidation. Deliberate efforts to oxidize the
intermediate 1,2,3,6-tetrahydropyridines from condensations
with aldehydes led to poor yields of pyridines. For example, a
Pictet-Spengler reaction of coumarin 9e with p-tolualdehyde
followed by cupric bromide-catalyzed air oxidation40 of the
intermediate tetrahydropyridine 12 led to only a 20% yield of
Fig. 3. Wagner-Meerwein Rearrangment. Panel A: Products of Pictet-Spengler
condensation of coumarin 9e and 17-methyl-5-androstan-17-ol-3-one (10j). Panel
B: An ellipsoid plot (50% probability) for the single-crystal structure of rearrangement
product (14). Hydrogen atoms were omitted to enhance clarity.
the 5H-chromeno[3,4-c]pyridin-5-one 13 (Scheme 2B).
This journal is © The Royal Society of Chemistry 20xx
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with coumarin 9e led not only to the expected product 11ej but either an aldehyde or a ketone to give an intermediate
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also to a Wagner-Meerwein rearrangement43 product 14
(Fig. imminium salt and a subsequent cyclization via a spirocyclic
DOI: 10.1039/D0NJ02664F
3A). The rearrangement product 14 provided suitable crystals intermediate to provide a substituted 2,3,4,9-tetrahydro-1H-
for an X-ray crystallographic structure determination (CSD pyrido[3,4-b]indole. Superficially, the Pictet-Spengler reaction
deposition number CCDC 1888376) that again confirmed the C- of coumarins
9 was a vinylogous extension of the classic
3α orientation of the ammonium group in accord with the reaction involving 3-(2-aminoethyl)-1H-indoles, as displayed in
aforementioned NMR-based stereochemical assignments (Fig. a skeletal format (Fig. 4), in which the iminium carbon linked to
3B).
the  carbon in the indole case and to the −carbon in the
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coumarin case.
A mechaism for these acid-catalyzed Pictet-Spengler reactions
of coumarins 9a, 9b and 9e with ketones 10 must take into
account the following observations: [1] the requirement for
highly acidic, aqueous conditions; [2] the rapid formation of the
products from the intermediate, iminium salts derived from the
condensation of the C-7 amino- or N,N-dialkylamino-
Discussion
In the course developing small-molecule therapies for CRPC, we
required fluorescent analogs for the androgens that appear in
the penultimate, intratumoral pathways converging on 5-
dihydrotestosterone (DHT). Specifically, we focused our
interest on inhibitors for the 17-oxidoreductases that appear in
three places in the late-stage metabolism of androgens to DHT,
and we required fluorescent androgen probes with fluorescent
groups at C-3 and natural functionality at C-17. The acid-
substituted coumarins 9a, 9b and 9e with ketones 10; and [3]
the failure of the 7-hydroxy- and 7-methoxy substituted 4-(2-
aminoethyl)coumarins 9c and 9d, respectively, to participate in
these reactions.
We propose a mechanism for the successful Pictet-Spengler
condensations
aminoethyl)coumarins 9a, 9b and 9e with ketones 10 that
involves an initial, rapid condensation leading to an
intermediate imine and a rate-determining cyclization to a
tetrahydropyridine product 11. For example, the acid-catalyzed
catalyzed Pictet-Spengler reaction of coumarins 9a
bearing C-7 amino or C-7 N,N-dimethylamino substituents with
3-ketosteroids, such as 5 -androstane-3,17-dione (10h) and
-androstan-17 -ol-3-one (10i), met these objectives and
, 9b and 9e
of
C-7
amino-substituted
4-(2-

5


provided the adducts 11 with an intensely fluorescent,
spirocyclic group attached at C-3 in good yield (Table 1). These
reactions required either a 1:10 (v/v) mixture of trifluoroacetic
acid and dichloromethane for ketones 10a-10f or a 1:10 (v/v)
mixture of concentrated hydrochloric acid and absolute ethanol
for steroidal ketones 10g-10k. A combination of sophisticated
NMR experiments and an X-ray structure of a Wagner-
condensation of coumarin 9e with 5

-androstan-17
10i) furnishes an intermediate, biscationic iminium ion 14a
Scheme 3). The acidic conditions (est. pH 1) used for these
-ol-3-one
(
(
Meerwein rearrangement43 product 14
(Fig. 3) established the
C-3R-diastereoselectivity of these reactions with steroid
substrates. Similar Pictet-Spengler reactions, however, of
coumarins 9c and 9d bearing C-7 hydroxyl or methoxy
substituents, respectively, in place of C-7 amino substituents
were unsuccessful. Likewise, extension of this Pictet-Spengler
reaction to the condensations of coumarins
was largely unsuccessful, even after attempts to effect the
deliberate oxidation of the intermediate adduct to a pyridine
9 with aldehydes
(
Scheme 2B).
The classic Pictet-Spengler reaction31-32 involved an acid-catalyzed
condensation of activated 2-(1H-indol-3-yl)ethan-1-amine with
Scheme 3. Mechanism of Pictet-Spengler reactions of 4-(2-aminoethyl)coumarins 9 with
ketones (10).
condensations and the calculated pKa values for 7-(N,N-
dimethylamino)coumarin
(pKa
3.48)
and
N-
methylcyclohexanimine (pKa 10.14), using the ChemAxon
software (version 19.18; ChemAxon, Inc., Cambridge, MA),
support a biscationic structure involving protonation of both
the imine and aniline moieties in 14a. The agreement between
calculated pKa values for N,N-dimethylaniline (pKa 5.02) and
experimental values (pKa 5.0644-5.0745) supported the veracity
of these calculated pKa values. This suggestion for a biscationic
Fig. 4. Contrasting skeletons of key participants in Pictet-Spengler reactions.
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species finds precedent a similar species generated from p-
aminostyrene with strong acids having Hammett acidities H0
greater than one46. In our case, protonation followed by
tautomerization leads to the biscationic, enolic form of the
coumarin 14b that undergoes rate-limiting condensation to
furnish the iminoquinone methide 14c. Addition of the si-face
ARTICLE
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DOI: 10.1039/D0NJ02664F
The BCD rings of 11eh were inserted into the SP1 binding pocket
in an identical fashion as seen for
angular methyl groups of and 11ei projected into the oxyanion
hole of 17
-HSD5 bounded by Y55, H117 and NADP+. Hydrogen
bonding with S129 further stabilizes the observed binding mode
with 11eh These binding features indicated that the
5, and the C-18 and C-19
5

of the enol to the
-face of the iminium ion in 14b (i.e.,
9
.
equatorial addition) produces an iminoquinone methide 14c
with C-3R and C-3’R stereochemistry. The alternative, re-face
addition provides the C-3’S epimer but proceeds through a
transition state that is more sterically hindered than that from
the re-face based on MM2 calculations for the relative stability
of the 3R,3’R and the 3R,3’S products. Final, irreversible
deprotonation of 14c at C-3’ affords the observed product 11ei
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
fluorescent ASD-based adduct 11eh possessed a binding mode
that matched that of ASD itself. In a similar fashion, the ASD
adduct 11eh occupied the same binding pocket seen for a
previously described inhibitor, 3-carboxamido-1,3,5-(10)-
estratrien-17R-spiro-2-(5,5-dimethyl-6-oxo)tetrahydropyran
(EM1404) that bound to17
-HSD5 (PDB:1ZQ5)36.
(
Scheme 3).
Heating the adduct 11ef with tetrahydro-4H-thiopyran-4-one
10e) in dichloromethane-trifluoroacetic acid (condition B) for 3
Conclusions
(
days failed to provide any of the spirocyclic ketone-exchange
product 11ee. In summary, an electron-donating partner in the
coumarin-based Pictet-Spengler reaction is a vinylogous analog
of the indole partner in the classic Pictet-Spengler reaction. The
activating C-7 amino substituent in the coumarin moiety 9e
counterbalances the deactivating carbonyl group and leads to a
In summary, the Pictet-Spengler condensation of substituted 4-
(2-aminoethyl)coumarins and ketones furnished fluorescent
(4aS,14bS)-1,2,3,4,4a,8,9,12,13,14b-decahydro-5H,7H,11H-
pyrido[3,2,1-ij]pyrido[4',3':4,5]pyrano[2,3-f]quinolin-5-ones.
This work describes the scope of this variant of the Pictet-
Spengler reaction with various coumarins, proposes a
mechanism consistent with the substituents in the coumarin
moiety, and defines the stereochemistry at C-3 in spirocyclic
products derived from 3-ketosteroids by a combination of
detailed NMR studies and an X-ray structure. Computational
modeling supported the surrogacy of a C-3 fluorescent
biscationic enol intermediate, such as 14b
leads to successful condensations. Analogous reactions of 4-(2-
aminoethyl)coumarins 9c and 9d Scheme 1) that possess either
(Scheme 3) that then
(
a C-7 hydroxy or C-7 methoxy group fail to generate a bicationic
enol intermediate and hence, fail to produce adducts 11 just as
benzofuran cases fail in the classic Pictet-Spengler reactions.
Computational modeling of the binding of the fluorescent,
spirocyclic adduct 11eh in the ligand-binding domain of human
17-hydroxysteroid dehydrogenase-5 (17-HSD5) indicated
that the compact nature of this fluorescent androgen 11eh did
not interfere with binding to the active site. The adduct 11eh
derivative of 5-androstan-17-ol-3-one as a tool to study 17-
oxidoreductases for intracrine, androgen metabolism in
prostate cancer. Future studies will describe applications of
these fluorescent androgens for image flow cytometry and will
elucidate the effects of these fluorescent androgens on the
prevention of prostate cancer growth promotion during ADT
and on the stimulation of androgen receptor-regulated gene
expression.
adopted the same pose as the naturally occurring ligand, 5
androstane-3,17-dione (
ASD)35 (Fig. 5).
-
5
;
Experimental
Chemicals were purchased from Millipore Sigma (St. Louis, MO,
USA) or Fisher Scientific (Hampton, NH, USA) or were
synthesized according to literature procedures. Solvents were
used from commercial vendors without further purification
unless otherwise noted. Nuclear magnetic resonance spectra
were acquired on a Varian (1H at 400MHz and 13C at 100MHz or
1H at 500MHz and 13C at 125MHz) instruments. High resolution
electrospray ionization (ESI) mass spectra were recorded on an
LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher
Scientific, Waltham, MA, USA). The FT resolution was set at
100,000 (at 400 m/z). Samples were introduced through direct
infusion using a syringe pump with a flow rate of 5μL/min.
Purity of compounds was established using combustion
analyses (Atlantic Microlabs, Inc. Norcross, GA, USA).
Compounds were chromatographed on preparative layer Merck
silica gel F254 (Fisher Scientific) plates unless otherwise
indicated.
Fig. 5. Computational modeling. Panel A: 5-Androstane-3,17-dione (5) in the SP1
binding site of AKR1C3 (PDB: 1XF0) containing proximal NADP+35
Panel B: Pictet-
Spengler adduct (11eh) derived from 5-androstane-3,17-dione (5) in the SP1 binding
site of AKR1C3 (PDB: 1XF0) containing proximal NADP+35
.
.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
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New Journal of Chemistry
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Page 6 of 15
ARTICLE
Journal Name
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2
3
4
5
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8
9
Methyl 3-Oxo-5-((2,2,2-trifluoroacetyl)amino)pentanoate (7). oxo-4-(2-(2,2,2-trifluoroacetamido)ethyl)-2H-chromen-7-
View Article Online
1
To a solution of 22.3 g (250 mmol) of  
-alanine in 125 mL of yl)carbamate: mp 215-216°C. H NMR (D4O00I: 1M0.H10z3,9D/DM0NSJO0-2d666)4F
methanol at 25oC was added 35 mL (250 mmol) of 10.19 (s, 1H), 9.57 (t, J = 5.2 Hz, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.57
triethylamine. After 5 min, 37 mL (312 mmol, 1.25 eq) of ethyl (d, J = 1.6 Hz, 1H), 7.4 (dd, J = 8.4 Hz, 1.6 Hz, 1H), 6.19 (s, 1H),
trifluoroacetate was added, and the mixture was allowed to stir 3.71 (s, 3H), 3.51 (q, J = 6.8 Hz, 2H), 2.99 (t, J = 6.8 Hz, 2H). 13C
2
for 24 h at 25oC. The solvent was evaporated under reduced NMR (100 MHz, DMSO-d6)

159.9, 156.4 (q, JCF = 35.7 Hz),
pressure, and the residue was diluted with 50 mL of H2O and 154.2, 153.8, 153.1, 142.8, 125.6, 115.9 (q, 1JCF = 287 Hz), 114.3,
acidified with concentrated hydrochloric acid. The mixture was 113.4, 112.2, 104.6, 52.1 (CH3), 38 (CH2), 30.1 (CH2). HRMS (ESI)
extracted with ethyl acetate, and the combined organic layers Calcd for C15H14F3N2O5 [MH+]: 359.085. Found: 359.085. Anal.
were washed with brine and dried over anhydrous MgSO4. Calcd. for C15H13F3N2O5: C, 50.29; H, 3.66; N, 7.82. Found: C,
After filtration, the solvent was evaporated at reduced pressure 50.53; H, 3.75; N, 7.80. A solution of 5 g (14 mmol) of methyl
to give 42.1 g (91 %) of 3-(2,2,2-trifluoroacetamido)propanoic (2-oxo-4-(2-(2,2,2-trifluoroacetamido)ethyl)-2H-chromen-7-
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
acid47 as a white solid: mp 114-116oC (lit47 mp 114-116oC). H yl)carbamate was refluxed in 60 mL of concentrated
NMR (400 MHz, DMSO-d6) δ 12.3 (s, 1H), 9.47 (br s, 1H), 3.42- hydrochloric acid for 42 h. The solvent was removed in vacuo.
3.3 (m, 2H), and 2.54-2.46 (m, 2H). 13C NMR (100 MHz, DMSO- The residue was recrystallized from methanol to give 3.63 g
1
d6) δ 172.5, 156.4 (q, 2JCF = 35.7 Hz), 116 (q, 1JCF = 286.9 Hz), 35.6, (94%) of 9a as a bishydrochloride salt: H NMR (400 MHz, D2O)
32.8. A suspension of 3.08 g (32.3 mmol) of magnesium chloride
and 7.2 g (46.2 mmol, 1.4 eq) of monomethyl monopotassium = 7.2 Hz, 2H), 3.19 (t, J = 7.2 Hz, 2H). H NMR (400 MHz, DMSO-

7.61 (d, J = 9.2 Hz, 1H), 6.83-6.91 (m, 2H), 6.2 (s, 1H), 3.37 (t, J
1
malonate in 45 mL of anhydrous THF was stirred for 4 h at 50°C d6)
under a nitrogen atmosphere. In a second flask, 6 g (37 mmol, 6.46 (s, 1H), 5.98 (s,1H), 3.2-3 (m, 4H). 13C NMR (100 MHz, D2O)
1.2 eq) of 1.1‘-carbonyldiimidazole was added portionwise to a 162.9, 153.6, 152.5, 138.5, 126.2, 117.9, 116.1, 113.2, 109,
solution of 5.7 (30.8 mmol) of 3-[(2,2,2- 37.4, 28.6. HRMS (ESI) Calcd for C11H13N2O2 [MH+]: 205.0972.
 8.12 (s, 1H), 7.52 (d, J = 8.4 Hz, 1H), 6.6 (d, J = 8.4 Hz, 1H),

g
trifluoroacetyl)amino]propanoic acid in 30 mL of anhydrous THF Found: 205.0977. Anal. Calcd. for C11H14Cl2N2O2: C, 47.67; H,
at 0-5°C. The mixture in the second flask was stirred for 1 h at 5.09; N, 10.11. Found: C, 47.41; H, 4.94; N, 9.95.
25oC and was added dropwise to the methylmagnesium 4-(2-Aminoethyl)-7-N,N-dimethylamino-2H-chromen-2-one
malonate suspension at 25oC. The mixture was stirred for 16 h, Hydrochloride (9b). To a stirred suspension of 1.37 g (10 mmol)
concentrated, and diluted with ethyl acetate. The ethyl acetate of 3-(N,N-dimethyamino)phenol and 2.41 g (10 mmol) of methyl
solution was washed with saturated, aqueous NaHCO3 solution 3-oxo-5-[(2,2,2-trifluoroacetyl)amino]-pentanoate (
7) in 30 mL
and brine. After drying over anhydrous MgSO4, the mixture was of toluene was added 20 mL of (20 mmol)
1
M
filtered and concentrated. The product was chromatographed chlorotriisopropyloxytitanium(IV) in hexanes. The mixture was
on silica gel using 2% methanol-dichloromethane to give 6.1 g refluxed for 10 h under a nitrogen atmosphere, cooled and
of
7
as a colorless oil (82%). 1H NMR (400 MHz, CDCl3) δ 7.13 diluted with 40 mL of hexane. The precipitate was collected by
(br s, 1H), 3.27 (s, 3H), 3.64-3.56 (m, 2H), 3.48 (s, 2H), 2.87 (t, J filtration and washed with 40 mL of hexane. The product was
= 5.7 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 202.1, 167.4, purified by recrystallization from methanol to give 2.2 g (67%)
1
157.4 (q, 2JCF = 37 Hz), 115.8 (q, 1JCF = 287.6 Hz), 52.7, 48.8, 41.5, of a yellow solid: mp 195-196°C. H NMR (400 MHz, DMSO-d6)
34.5. HRMS (ESI) Calcd for C8H11O4NF3 [MH+]: 242.0635. δ 9.57 (t, J = 5.6 Hz, 1H, NH), 7.58 (d, J = 9.2 Hz, 1H), 6.71 (dd, J
Found: 242.0636.
= 9.2, 2.4 Hz, 1H), 6.52 (d, J = 2.4 Hz, 1H), 5.91 (s, 1H), 3.49 (dt,
Methyl N-(3-Hydroxyphenyl)carbamate (8a). A solution of 10 J = 6.8, 5.6 Hz, 2H), 3.01 (s, 6H), 2.93 (t, J = 6.8 Hz, 2H). 13C NMR
2
g (91.6 mmol) of 3-aminophenol in 35 mL of ethyl acetate was (100 MHz, DMSO-d6) δ 160.6, 156.2 (q, JCF = 35.7 Hz), 155.5,
refluxed for 30 min. To the clear solution was added 4 mL (45.8 153.5, 152.8, 125.4, 115.9 (q, JCF = 286.5 Hz), 109.1, 108.1,
1
mmol, 0.5 eq) of methyl chloroformate dropwise over a period 107.8, 97.6, 39.7, 38.2, 30.1. HRMS (ESI) Calcd for C15H16F3N2O3
of 30 min. The mixture was cooled to 25oC. The white solid was [MH+]: 329.1108. Found: 329.1108. Anal. Calcd. for
collected by vacuum filtration and washed with 1:1 ethyl C15H15F3N2O3: C, 54.88; H, 4.61; N, 8.53. Found: C, 55.01; H,
1
acetate-hexanes to give 7.67 g (50%) of 8a: H NMR (400 MHz, 4.43; N, 8.58. A mixture of 2.2 g (6.7 mmol) of N-(2-(7-(N,N-
DMSO-d6) δ 9.5 (s, 1H, NH), 9.34 (s, 1H, OH), 6.98-7.05 (m, 2H), dimethylamino)-2-oxo-chromen-4-yl)ethyl)-2,2,2-trifluoroacet-
6.82-6.84 (m, 1H), 6.36-6.39 (m, 1H), 3.63 (s, 3H, CH3). 13C NMR amide in 4.5 mL of concentrated hydrochloric acid was refluxed
(100 MHz, DMSO-d6) δ 158.1, 154.3, 140.6, 129.8, 109.9, 109.4, for 4 h. After cooling, the product was concentrated in vacuo.
105.7, 51.9.
7-Amino-4-(2-aminoethyl)-2H-chromen-2-one
The residue was recrystallized from methanol to give 1.14 g
(63%) of 9b: 1H NMR (400 MHz, D2O) δ 7.54 (d, J = 8.8 Hz, 1H),
Dihydrochloride (9a). To a stirred solution of 4.94 g (20.5 6.83 (dd, J = 8.8, 2.8 Hz, 1H), 6.53 (d, J = 2.8 Hz, 1H), 6.03 (s, 1H),
mmol) of methyl 3-oxo-5-[(2,2,2-trifluoroacetyl)amino]- 3.37 (t, J = 7.2 Hz, 2H), 3.14 (t, J = 7.2 Hz, 2H), 3.04 (s, 6H). H
1
pentanoate (
7
) in 60 mL of methanesulfonic acid was added NMR (400 MHz, DMSO-d6) δ 8.18 (s, 3H), 7.64 (d, J = 9 Hz, 1H),
eq) of methyl N-(3- 6.75 (dd, J = 8.8, 2.4 Hz, 1H), 6.58 (d, J = 2.4 Hz, 1H), 6.03 (s, 1H),
3.42 (20.5 mmol,
g
1
hydroxyphenyl)carbamate (8a) portion-wise at 0°C. The 3.2-2.98 (m, 10H). 13C NMR (100 MHz, D2O) δ 163.8, 154.4,
mixture was stirred at 25oC for 16 h and quenched by pouring 153.1, 150.1, 125.7, 112.6, 112, 110.1, 102.1, 41.8, 37.7, 28.7.
into cold water. The precipitate was collected by filtration and 13C NMR (100 MHz, DMSO-d6) δ 160.6, 155.5, 152.4, 152.3,
recrystallized from methanol to give 5.59 g (76%) of methyl (2- 125.7, 109.8, 109.4, 108.4, 98.6, 40.2, 37.8, 28.8. HRMS (ESI)
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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Page 7 of 15
New Journal of Chemistry
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Journal Name
ARTICLE
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8
9
Calcd for C13H17 N2O2 [MH+]: 233.1285. Found: 233.1283. Anal. N-(2-(7-methoxy-2-oxo-chromen-4-yl)ethyl)acetamide
Calcd. for C13H17ClN2O2: C, 58.10; H, 6.38; N, 10.42; Cl, 13.19. refluxed in 25 mL of concentrated hydrochloric acid for 3 h.
was
View Article Online
DOI: 10.1039/D0NJ02664F
Found: C, 58.08; H, 6.45; N, 10.45; Cl, 13.26.
After cooling, the product was concentrated in vacuo. The
4-(2-Aminoethyl)-7-hydroxy-2H-chromen-2-one
Hydrochloride (9c). To a stirred solution of 3.3 g, (13.7 mmol) of 9d. H NMR (400 MHz, DMSO-d6) δ 8.02 (s, 3H), 7.79 (d, J =
residue was recrystallized from methanol to give 1.67 g (76%)
1
of methyl 3-oxo-5-((2,2,2-trifluoroacetyl)amino)pentanoate (
7
)
8.4 Hz, 1H), 7.08-6.96 (m, 2H), 6.28 (s, 1H), 3.87 (s, 3H), 3.2-3
1
in 25 mL of methanesulfonic acid was added 1.37 g (12.4 mmol) (m, 4H). H NMR (400 MHz, D2O) δ 7.72 (d, J = 8.8 Hz, 1H), 7.05
of resorcinol portionwise at 0-5°C. The mixture was stirred for (dd, J = 8.8, 2.4 Hz, 1H), 7.02 (d, J = 2.4 Hz, 1H), 6.29 (s, 1H), 3.91
3 h at this temperature and was quenched by pouring into 100 (s, 3H), 3.39 (t, J = 7.4 Hz, 2H), 3.22 (t, J = 7.4 Hz, 2H). 13C NMR
mL of ice water. The precipitate was collected by filtration and (100 MHz, DMSO-d6) δ 162.4, 160, 155.2, 151.9, 126.1, 112.2
recrystallized from methanol to give 2.32 g (62%) of the 2,2,2- (two C), 112, 101.1, 56, 37.5, 28.8. HRMS (ESI) Calcd for
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
trifluoro-N-(2-(7-hydroxy-2-oxo-2H-chromen-4-yl)ethyl)acet-
amide: mp 219-220°C. H NMR (400 MHz, DMSO-d6) δ 10.56 (s, C12H14ClNO3: C, 56.37; H, 5.52; N, 5.48. Found: C, 56.32; H,
1H, OH), 9.57 (t, J = 5.4 Hz, 1H, NH), 7.68 (d, J = 8.8 Hz, 1H), 6.81 5.59; N, 5.54.
C12H14NO3 [MH+]: 220.0968. Found 220.0976. Anal. Calcd. for
1
(dd, J = 8.8, 2.4 Hz, 1H), 6.72 (d, J = 2.4 Hz, 1H), 6.1 (s, 1H), 3.5 8-(2-Aminoethyl)-2,3,4,5-tetrahydro-1H,4H-11-oxa-3a-aza-
(dt, J = 7, 5.4 Hz, 2H), 2.97 (t, J = 7 Hz, 2H). 13C NMR (100 MHz, benzo[de]anthracen-10-one Hydrochloride (9e).
The
2
CDCl3) δ 161.2, 160.2, 156.4 (q, JCF = 36.1 Hz), 155.2, 153.5, procedure of Wirtz and Kazmaier38 was repeated using 8.33 g
126.2, 115.9 (q, 1JCF = 287.8 Hz), 113, 111.2, 110.5, 102.5, 38.1, (44
30.2. HRMS (ESI) Calcd for C13H11 F3NO4 [MH+]: 302.0635. benzo[ij]quinolizine, 10.8
mmol)
of
8-hydroxy-2,3,6,7-tetrahydro-1H,5H-
(44 mmol) of methyl-5-
g
Found: 302.0635. Anal. Calcd. for C13H10F3NO4: C, 51.84; H, (benzyloxycarbonylamino)-3-oxopentanoate, and 88 mL (88
3.35; N, 4.65. Found: C, 51.98; H, 3.50; N, 4.62. A mixture of mmol, 2 eq) of a 1M solution of chlorotriisopropyloxy-
1.51 g (5 mmol) of 2,2,2-trifluoro-N-(2-(7-hydroxy-2-oxo- titanium(IV) in hexanes to afford 13 g (77%) of a 1:9 mixture of
chromen-4-yl)ethyl)acetamide in 20 mL of concentrated the benzyl and isopropyl [2-(10-oxo-2,3,5,6-tetrahydro-
hydrochloric acid was refluxed for 2 h. After cooling, the 1H,4H,10H-11-oxa-3a-aza-benzo[de]anthracen-8-yl)ethyl]-
product was concentrated in vacuo. The residue was carbamates as a yellow solid. To 5.05 g (13.6 mmol) of this
recrystallized from methanol to give 945 mg (78%) of 9c: 1H mixture of esters was added 12 mL of concentrated HCl. The
NMR (400 MHz, D2O) δ 7.67 (d, J = 8.8 Hz, 1H), 6.95 (dd, J = 8.8, solution was heated at 95oC for 9 h, cooled, and concentrated
2 Hz, 1H), 6.81 (d, J = 2 Hz, 1H), 6.26 (s, 1H), 3.43 (t, J = 7.6 Hz, in vacuo. The residue was suspended in a mixture of methanol-
1
2H), 3.23 (t, J = 7.6 Hz, 2H). H NMR (400 MHz, DMSO-d6) δ 10.7 acetone and was filtered to afford 4.3 g (98%) of 9e as a yellow
(s, 1H), 8.11 (s, 3H), 7.7 (d, J = 8.8 Hz, 1H), 6.84 (dd, J = 8.8, 1.6 hydrochloride salt: mp 238-239oC (lit.38 mp 114-118oC for free
Hz, 1H), 6.76 (d, J = 1.6 Hz, 1H), 6.2 (s, 1H), 3.2-3 (m, 4H). 13C base). 1H NMR (400 MHz, DMSO-d6) δ 8 (s, 3H), 7.22 (s, 1H),
NMR (100 MHz, D2O) δ 163.4, 159.9, 153.9, 153.2, 125.6 (CH), 5.93 (s, 1H), 3.3-3.16 (m, 4H), 3.12-2.92 (m, 4H), 2.8-2.7 (m, 4H),
113.4 (CH), 111, 109.7 (CH), 102.5 (CH), 37.4 (CH2), 28.5 (CH2). 1.94-1.84 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 160.7, 152.2,
13C NMR (100 MHz, DMSO-d6) δ 161.4, 160.2, 155.2. 152, 126.2 150.9, 145.6, 121.7, 118, 107.5, 106.8, 105.7, 49.2, 48.7, 37.9,
(CH), 113.1 (CH), 111.2 (CH), 110.8, 102.6 (CH), 37.5 (CH2), 28.8 28.9, 27, 21, 20.1, 20. HRMS (ESI) Calcd for C17H21N2O2 [MH+]:
(CH2). HRMS (ESI) Calcd for C11H12O3N [MH+]: 206.0812. 285.1598. Found 285.1597. Anal. Calcd. for C17H21ClN2O2∙H2O:
Found: 206.0813. Anal. Calcd. for C11H12ClNO3: C, 54.67; H, C, 60.26; H, 6.84; N, 8.27. Found: C, 60.46; H, 6.57; N, 8.19.
5.01; N, 5.80. Found: C, 54.81; H, 4.97; N, 5.80.
4-(2-Aminoethyl)-7-methoxy-chromen-2-one Hydrochloride Aminoethyl)coumarins
(9d). To a stirred solution of 2.65 g (11 mmol) of methyl 3-oxo- Dimethylamino)-4,4-dimethyl-1,2,3,4-tetrahydro-5H-
5-[(2,2,2-trifluoroacetyl)amino]pentanoate ( ) in 20 mL of chromeno[3,4-c]pyridin-5-one (11ba). To a stirred solution of
Condition
A
for the Pictet-Spengler Reaction of 4-(2-
9
with Acetone. 8-(N,N-
7
methanesulfonic acid was added 1.24 g (10 mmol) of 3- 50 mg (0.19 mmol) of 9b in 0.5 mL of trifluoroacetic acid were
methoxyphenol portionwise at 0-5°C. The mixture was stirred added 0.5 mL of acetone. The mixture was stirred under reflux
for 30 min at this temperature and diluted with ice water. The for 30 min. After cooling, the mixture was neutralized with a
precipitate was collected by filtration and recrystallized from saturated, aqueous solution of NaHCO3 and stirred for 2 h at
methanol to give 2.28 g (72%) of 2,2,2-trifluoro-N-(2-(7- 25oC. A precipitate was collected by filtration and purified by
methoxy-2-oxo-2H-chromen-4-yl)ethyl)acetamide: mp 156- chromatography using 1:25 methanol-dichloromethane (Rf
158°C. 1H NMR (400 MHz, DMSO-d6) δ 9.58 (t, J = 4.4 Hz, 1H, 0.29) to provide 45 mg (88%) of 11ba. mp 179-181°C. H NMR
1
NH), 7.78 (d, J = 8.8 Hz, 1H), 7.02 (d, J = 2.4 Hz, 1H), 6.98 (dd, J = (400 MHz, DMSO-d6) δ 7.47 (d, J = 9 Hz, 1H), 6.71 (dd, J = 9, 2.6
8.8, 2.4 Hz, 1H), 6.17 (s, 1H), 3.86 (s, 3H), 3.51 (dt, J = 7, 4.4 Hz, Hz, 1H), 6.5 (d, J = 2.6 Hz, 1H), 2.99 (s, 6H), 2.96 (t, J = 5.8 Hz,
2H), 3 (t, J = 7 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 162.4, 2H), 2.67 (t, J = 5.7 Hz, 2H), 1.38 (s, 6H). 13C NMR (100 MHz,
2
160, 156.4 (q, JCF = 35.7 Hz), 155.1, 153.3, 126 (CH), 115.9 (q, DMSO-d6) δ 159.27, 153.73, 152.04, 148.14, 124.98, 123.54,
1JCF = 286.9 Hz), 112.2 (quaternary C and CH), 111.4 (CH), 101 109.02, 108.6, 96.88, 51.42, 36.69, 26.68, 26.03. HRMS (ESI)
(CH), 55.9 (CH3), 38 (CH2), 30.2 (CH2). HRMS (ESI) Calcd for Calcd for C16H21N2O2 [MH+]: 273.1598. Found: 273.1600.
C14H13F3NO4 [MH+]: 316.0791. Found: 316.0793. Anal. Calcd. Condition
B
for the Pictet-Spengler Reaction of 4-(2-
for C14H12F3NO4: C, 53.34; H, 3.84; N, 4.44. Found: C, 53.44; H, Aminoethyl)coumarins with Monocyclic Ketones.
3.97; N, 4.29. A mixture of 2.69 g (8.54 mmol) of 2,2,2-trifluoro- 2,2',3,3',5,6,8',9',12',13'-Decahydro-7'H,11'H-spiro[pyran-4,4'-
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ARTICLE
Journal Name
7.03 (s, 1H),
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pyrido[3,2,1-ij]pyrido[4',3':4,5]pyrano[2,3-f]quinolin]-5'(1'H)- 11ei as a free base: H NMR (400 MHz, DMSO-d6)

View Article Online
one (11ed). To a stirred suspension of 100 mg (0.31 mmol, 1 4.4 (d, J = 4.8 Hz, 1H), 3.48-3.37 (m, 1H),D3O.2I: (1q0,.10J3=9/5D.60NHJz0,2646H4)F,
eq) of 9e in 2 mL dichloromethane was added 0.2 mL of 2.84 (t, J = 5.7 Hz, 2H), 2.7 (q, J = 6 Hz, 4H), 2.58 (t, J = 5.6 Hz,
trifluoroacetic acid followed by 125 mg (1.25 mmol, 4 eq) of 2H), 2.52-2.43 (m, 1H), 2.32 (t, J = 13 Hz, 1H), 1.95-1.76 (m, 6H),
tetrahydro-4H-pyran-4-one. The mixture was stirred at 25oC for 1.75-1.67 (m, 1H), 1.66-1.54 (m, 2H), 1.54-1.43 (m, 2H), 1.39-
2-5 days with monitoring by TLC for the disappearance of 1.26 (m, 4H), 1.25-1.03 (m, 5H), 1-0.75 (m, 7H), 0.74-0.64 (m,
starting material. Diethyl ether (ca. 5 mL) was added to the 1H), 0.62 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
mixture. A precipitate was collected by filtration to provide 149.08, 144.58, 122.52, 121.11, 117.68, 108.18, 104.87, 80.11,
 159.5, 149.38,
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2,2',3,3',5,6,8',9',12',13'-decahydro-7'H,11'H-spiro[pyran-4,4'-
pyrido[3,2,1-ij]pyrido[4',3':4,5]pyrano[2,3-f]quinolin]-5'(1'H)-
one 2,2,2-trifluoroacetate that was treated with saturated, 26.3, 23.11, 21.16, 20.28, 20.22, 19.88, 11.61, 11.39. HRMS
aqueous solution of NaHCO3 to give a free base that was (ESI) Calcd for C36H49N2O3 [MH+]: 557.3738. Found: 557.3738.
purified by chromatography using 1:10 methanol- Anal. Calcd for C36H48N2O3: C, 77.66; H, 8.69; N, 5.03. Found:
53.89, 53.82, 50.79, 49.16, 48.67, 42.59, 39.94, 36.76, 36.13,
35.69, 35.28, 35.25, 33.32, 31.48, 29.88, 28.18, 27.84, 27.13,
dichloromethane (Rf 0.51) to furnish 90 mg (79%) of 11ed. mp C, 77.41; H, 8.95, N, 4.99.
1
204-206

C (decomp). H NMR (400 MHz, DMSO-d6) δ 7.06 (s, 4,4-Dimethyl-1,2,3,4,8,9,12,13-octahydro-5H,7H,11H-
1H), 3.87-3.71 (m, 2H), 3.57 (dd, J = 10.8, 4.9 Hz, 2H), 3.21 (q, J pyrido[3,2,1-ij]pyrido[4',3':4,5]pyrano[2,3-f]quinolin-5-one
= 5.6 Hz, 4H), 2.89 (t, J = 5.6 Hz, 2H), 2.76-2.68 (m, 4H), 2.68- 2,2,2-trifluoroacetate (11ea). The procedure described under
2.59 (m, 4H), 1.95-1.8 (m, 4H), 1.2 (d, J = 13.1 Hz, 2H). 13C NMR Condition A was repeated using 60 mg (0.19 mmol) of 9-(2-
(100 MHz, DMSO-d6) δ 159.36, 149.97, 149.19, 144.75, 121.2, aminoethyl)-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-
117.78, 107.99, 104.85, 62.46, 51.34, 49.15, 48.66, 36.09, 31.91, f]pyrido[3,2,1-ij]quinolin-11-one hydrochloride (9e) in 0.5 mL of
27.12, 26.06, 21.11, 20.24, 19.85. HRMS (ESI) Calcd for trifluoroacetic acid and 0.5 mL of acetone. The mixture was
C22H27N2O3 [MH+]: 367.2016. Found: 367.2020.
cooled and diluted with 3 mL of diethyl ether. A precipitate was
Condition
C
for the Pictet-Spengler Reaction of 4-(2- collected by filtration to provide 77 mg (94%) of analytically
1
Aminoethyl)coumarins
9
with
Steroidal
Ketones. pure 11ea as trifluoroacetate salt: mp 230-231°C (decomp). H
(3R,5S,10S,13S,17S)-17-Hydroxy-10,13-dimethyl-
NMR (400 MHz, DMSO-d6)  9.35 (s, 2H), 7.15 (s, 1H), 3.43 (t, J
= 6.4 Hz, 2H), 3.25 (q, J = 6.4 Hz, 4H), 3.02 (t, J = 6.1 Hz, 2H), 2.79-
2.64 (m, 4H), 1.95-1.8 (m, 4H), 1.68 (s, 6H). 13C NMR (100 MHz,
1,2,2',3',4,5,6,7,8,8',9,9',10,11,12,12',13,13',14,15,16,17-
docosahydro-7'H,11'H-spiro[cyclopenta[a]phenanthrene-
3,4'-pyrido[3,2,1-ij]pyrido[4',3':4,5]pyrano[2,3-f]quinolin]-
5'(1'H)-one Hydrochloride (11ei). To a suspension of 66 mg 115.21, 106.08, 104.88, 55.14, 49.17, 48.65, 34.93, 27.07, 23.8,
(0.21 mmol, 1.2 eq) of 9e hydrochloride in 2 mL of absolute 22.28, 20.9, 20, 19.74. HRMS (ESI) Calcd for C20H25N2O2 [MH+]:
ethanol was added 50 mg (0.17 mmol, 1 eq) of 5α-androstan- 325.1911. Found: 325.1911. Anal. Calcd for C22H25F3N2O4: C,
DMSO-d6)
 158.26, 149.27, 146.13, 145.65, 121.61, 118.42,
17-ol-3-one (10i). To this suspension in a sealed tube was 60.27; H, 5.75; N, 6.39. Found: C, 59.99; H, 5.63, N, 6.29.
added 0.2 mL of concentrated HCl, and the mixture was stirred 3'-Acetyl-2',3',8',9',12',13'-hexahydro-7'H,11'H-spiro[cyclo-
under reflux for 24 h. The suspension became a clear solution pentane-1,4'-pyrido[3,2,1-ij]pyrido[4',3':4,5]pyrano[2,3-
within the first hour of heating, and a precipitate of the desired f]quinolin]-5'(1'H)-one (11eb). To 123 mg (0.26 mmol) of
product then appeared. The reaction was quenched by the 2',3',8',9',12',13'-hexahydro-7'H,11'H-spiro[cyclopentane-1,4'-
addition of ca. 3 mL of water, and the precipitate was collected pyrido[3,2,1-ij]pyrido[4',3':4,5]pyrano[2,3-f]quinolin]-5'(1'H)-
by filtration to provide 73 mg (71%) of 11ei
purification was achieved by recrystallization from methanol: of dichloromethane at 0°C was added 59 mg (0.58 mmol, 2.2 eq)
1H NMR (400 MHz, DMSO-d6)
9.41-9.13 (m, 2H), 7.14 (s, 1H), of triethylamine followed by 25 mg (0.32 mmol, 1.2 eq) of acetyl
.
Additional one 2,2,2-trifluoroacetate obtained under Condition B in 2 mL

4.43 (br s, 1H), 3.45 (t, 2H), 3.29-3.22 (m, 4H), 3.12-3 (m, 2H), chloride. The mixture was stirred at 25oC for 2 h, poured into
2.82-2.64 (m, 4H), 2.57 (t, J = 14.2 Hz, 1H), 1.96-1.8 (m, 4H), 1.8- water, and extracted with dichloromethane. The organic layers
1.7 (m, 2H), 1.7-1.55 (m, 4H), 1.56-1.41 (m, 4H), 1.42-1.3 (m, were dried over anhydrous MgSO4 filtered, and concentrated.
3H), 1.28-1.06 (m, 5H), 1.05-0.77 (m, 7H), 0.65 (s, 3H). 13C NMR The product was purified by chromatography using 1:50
(100 MHz, DMSO-d6)

158.61, 149.23, 147.42, 145.69, 121.59, methanol-dichloromethane (Rf = 0.37 after two developments)
1
118.47, 114.52, 106.22, 104.7, 80.04, 58.69, 52.91, 50.82, 49.16, to provide 71 mg of 11eb (68%). mp 234-236°C (decomp). H
48.65, 42.59 (two C), 36.7, 35.26, 35.05, 34.83, 32.41, 31.64, NMR (400 MHz, DMSO-d6) δ 7.06 (s, 1H), 3.52 (t, J = 5.5 Hz, 2H),
31.17, 29.84, 27.47, 27.09, 25.4, 23.05, 22.54, 20.88, 20.12, 3.23 (q, J = 5.9 Hz, 4H), 2.83 (t, J = 5.5 Hz, 2H), 2.72 (q, J = 6.2 Hz,
19.96, 19.69, 11.42, 11.37. HRMS (ESI) Calcd for C36H49N2O3 4H), 2.28-2.12 (m, 4H), 2.1 (s, 3H), 2.08-1.98 (m, 2H), 1.89 (m,
[MH+]: 557.3738. Found: 557.3744. Anal. Calcd for 6H). 13C NMR (100 MHz, DMSO-d6) δ 169.51, 159.14, 148.84,
C36H49ClN2O3 • H2O: C, 70.74; H, 8.41; N, 4.58; Cl, 5.80. Found: 147.32, 144.91, 121.89, 121.26, 117.92, 107.16, 104.81, 70,
C, 70.51; H, 8.43, N, 4.66; Cl, 5.72. The hydrochloride salt of the 49.15, 48.65, 42.21, 38.77, 27.63, 27.13, 25.78, 25.01, 21.05,
desired product was suspended in dichloromethane and 20.16, 19.81. HRMS (ESI) calcd for C24H29N2O3 [MH+]: 393.2173.
washed with an aqueous, saturated solution of NaHCO3. The Found: 393.2158.
dichloromethane layer was dried over anhydrous Na2SO4, 3'-Acetyl-2',3',8',9',12',13'-hexahydro-7'H,11'H-spiro[cyclo-
filtered, concentrated and purified by chromatography on silica hexane-1,4'-pyrido[3,2,1-ij]pyrido[4',3':4,5]pyrano[2,3-
gel using 1:10 methanol-dichloromethane (Rf 0.55) to afford f]quinolin]-5'(1'H)-one (11ec). To 110 mg (0.23 mmol) of
8 | J. Name., 2012, 00, 1-3
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Journal Name
ARTICLE
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2',3',8',9',12',13'-hexahydro-7'H,11'H-spiro[cyclohexane-1,4'-
pyrido[3,2,1-ij]pyrido[4',3':4,5]pyrano[2,3-f]quinolin]-5'(1'H)-
yield 21%. H NMR (500 MHz, DMSO-d6) δ 9.22 (Vbierw.dA,rtJicl=e O3n8lin.9e
DOI: 10.1039/D0NJ02664F
Hz, 3H), 7.45 (d, J = 8.8 Hz, 1H), 6.61 (dd, J = 8.8, 2.2 Hz, 1H),
one 2,2,2-trifluoroacetate in 2 mL of dichloromethane at 0°C 6.40 (d, J = 2.2 Hz, 1H), 6.26 (s, 2H), 3.13-2.93 (m, 2H), 2.76-2.57
was added 51 mg (0.51 mmol, 2.2 eq) of triethylamine followed (m, 2H), 1.97-1.78 (m, 2H), 1.78-1.72 (m, 1H), 1.71-1.56 (m, 3H),
by 22 mg (0.28 mmol, 1.2 eq) of acetyl chloride. The mixture 1.57-1.43 (m, 6H), 1.44-1.03 (m, 8H), 0.97 (s, 3H), 0.95-0.74 (m,
was stirred at 25oC for 2 h, poured into water, and extracted 4H), 0.70 (s, 3H). Calcd for C30H41N2O2 [MH+]: 461.3163. Found:
with dichloromethane. The organic layers were dried over 461.3167.
5
6
7
8
9
anhydrous MgSO4 and concentrated. The product was purified (4R,5'S,8'S,9'S,10'S,13'S,14'S)-8-(Dimethylamino)-10',13'-
by chromatography using 1:20 methanol-dichloromethane (Rf = dimethyl-1',2,2',3,4',5',6',7',8',9',10',11',12',13',14',15',16',17'-
0.37 after two developments) to provide 59 mg of 11ec (63%). octadecahydrospiro[chromeno[3,4-c]pyridine-4,3'-
1H NMR (400 MHz, CDCl3) δ 6.83 (s, 1H), 3.81 (t, J = 6.2 Hz, 2H), cyclopenta[a]phenanthren]-5(1H)-one hydrochloride (11bg).
3.29-3.17 (m, 4H), 2.84 (q, J = 6.7 Hz, 4H), 2.74 (t, J = 6.4 Hz, 2H), Condition C: yield 70%; purified by trituration with hot
2.67-2.53 (m, 4H), 2.24 (s, 3H), 2.06-1.9 (m, 4H), 1.77-1.62 (m, methanol. 1H NMR (400 MHz, CF3COOD) δ 7.96 (d, J = 8.8 Hz,
2H), 1.51-1.38 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 173.15, 1H), 7.87 (br. s, 1H), 7.77 (d, J = 2.3 Hz, 1H), 7.73 (dd, J = 8.9, 2.1
160.01, 149.92, 146.37, 145.21, 123.36, 120.58, 118.01, 108.62, Hz, 1H), 3.86-3.69 (m, 2H), 3.45 (s, 6H), 3.11 (t, J = 13.8 Hz, 1H),
106.3, 62.36, 50.01, 49.66, 39.76, 33.83, 27.97, 26.71, 25.42, 2.94 (t, J = 13.9 Hz, 1H), 1.98-1.9 (m, 1H), 1.9-1.77 (m, 2H), 1.72
25.41, 22.56, 21.86, 20.9, 20.46. HRMS (ESI) calcd for (d, J = 12.6 Hz, 1H), 1.67-1.48 (m, 5H), 1.47-1.32 (m, 5H), 1.32-
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C25H31N2O3 [MH+]: 407.2329. Found: 407.2314.
2,2',3,3',5',6',8,9,12,13-Decahydro-7H,11H-spiro[pyrido[3,2,1- 0.78 (dt, J = 11.7, 11, 3.8 Hz, 1H), 0.69 (s, 3H). 13C NMR (100
ij]pyrido[4',3':4,5]pyrano[2,3-f]quinoline-4,4'-thiopyran]- MHz, CF3COOD) δ 162.54, 154.54, 149.99, 146.98, 129.77,
5(1H)-one 2,2,2-Trifluoroacetate (11ee). Condition B: mp > 126.7, 122.58, 120.00, 111.85, 64.46, 56.5, 49.28, 43.36, 42.64,
220
C; yield 81%. 1H NMR (400 MHz, DMSO-d6) δ 9.7 (br. s, 41.94, 40.43, 38.2, 37.98, 37.9, 35.14, 34.58, 33.80, 29.71,
1.23 (m, 3H), 1.22-1.1 (m, 3H), 1.09 (s, 3H), 1.04-0.87 (m, 2H),

2H), 7.15 (s, 1H), 3.44-3.33 (m, 2H), 3.26 (q, J = 6.1 Hz, 4H), 3.19 28.46, 26.96, 25.12, 22.68, 21.76, 18.15, 12.68. HRMS (ESI)
(t, J = 13.2 Hz, 2H), 3.09 (t, J = 6.2 Hz, 2H), 2.93 (td, J = 14.3, 4 Calcd for C32H45N2O2 [MH+]: 489.3476. Found: 489.3478.
Hz, 2H), 2.73 (q, J = 6.9 Hz, 4H), 2.57-2.48 (m, 2H), 2.15 (br. d, J (3R,5S,8S,9S,10S,13S,14S)-10,13-Dimethyl-1,2,2',3',4,5,6,7,
= 14.6 Hz, 2H), 1.87 (q, J = 5.4, 5 Hz, 4H). 13C NMR (100 MHz, 8,8',9,9',10,11,12,12',13,13',14,15,16,17-docosahydro-
DMSO-d6) δ 158.49, 149.32, 147.75, 145.72, 121.7, 118.47, 7'H,11'H-spiro[cyclopenta[a]phenanthrene-3,4'-pyrido[3,2,1-
106.25, 104.71, 64.91, 49.17, 48.66, 34.51, 30.23, 27.09, 21.32, ij]pyrido[4',3':4,5]pyrano[2,3-f]quinolin]-5'(1'H)-one
20.9, 19.99, 19.72, 15.17. HRMS (ESI) Calcd for C22H27N2O2S hydrochloride (11eg). Condition C: yield 86%; purified by
1
[MH+]: 383.1788. Found: 383.1791.
8-(Dimethylamino)-1'-methyl-2,3-dihydrospiro[chromeno-
trituration with hot methanol. H NMR (400 MHz, CF3COOD) δ
7.98-7.43 (m, 1H), 3.94-3.69 (m, 6H), 3.5 (t, J = 6.2 Hz, 2H), 3.25
[3,4-c]pyridine-4,4'-piperidin]-5(1H)-one (11bf). Condition B: (t, J = 6.9 Hz, 4H), 3.05 (t, J = 13.7 Hz, 1H), 2.61-2.41 (m, 4H),
1
mp > 220

C; yield 73%. H NMR (400 MHz, DMSO-d6) δ 7.48 (d, 2.03 (d, J = 14 Hz, 1H), 1.98-1.86 (m, 2H), 1.81 (d, J = 12.6 Hz,
J = 9.2 Hz, 1H), 6.72 (dd, J = 9, 2.4 Hz, 1H), 6.49 (d, J = 2.4 Hz, 1H), 1.77-1.57 (m, 5H), 1.57-1.43 (m, 5H), 1.43-1.31 (m, 3H), 1.3-
1H), 3 (s, 6H), 2.9 (t, J = 6.8 Hz, 2H), 2.73-2.66 (m, 4H), 2.62-1.58 1.2 (m, 3H), 1.19 (s, 3H), 1.12-0.96 (m, 2H), 0.87 (dt, J = 12.3,
(m, 4H), 2.3 (s, 3H), 1.36 (d, J = 12.4 Hz, 2H). 13C NMR (100 MHz, 11.8, 4.5 Hz, 1H), 0.79 (s, 3H). 13C NMR (100 MHz, CF3COOD) δ
DMSO-d6) δ 159.22, 153.86, 152.16, 150.31, 125.05, 122.14, 162.99, 150.42, 150.15, 137.1, 131.52, 126.21, 123.77, 121.25,
109.1, 108.67, 96.71, 50.97, 49.93, 44.9, 35.8, 30.32, 26. HRMS 64.47, 56.55, 56.27, 43.42, 42.67, 41.98, 40.47, 38.29, 38, 37.94,
(ESI) Calcd for C19H26N3O2 [MH+]: 328.2020. Found: 328.2023. 35.25, 34.63, 33.84, 29.74, 28.5, 27, 26.84, 25.12, 22.71, 21.8,
1-Methyl-2',3',8',9',12',13'-hexahydro-7'H,11'H-
spiro[piperidine-4,4'-pyrido[3,2,1-
21.55, 21.36, 20.57, 18.19, 12.7. HRMS (ESI) Calcd for
C36H49N2O2 [MH+]: 541.3789. Found: 541.3790.
ij]pyrido[4',3':4,5]pyrano[2,3-f]quinolin]-5'(1'H)-one (11ef). (3R,5S,8S,9S,10S,13S,14S)-10,13-Dimethyl-
Condition B: mp = 144-146 C (decomp); yield 84%; 1,2',3',4,5,6,7,8,8',9,9',10,11,12,12',13,13',14,15,16-

chromatographic solvent: 1:10 methanol-dichloromethane (Rf icosahydro-7'H,11'H-spiro[cyclopenta[a]phenanthrene-3,4'-
1
0.13). H NMR (400 MHz, DMSO-d6) δ 9.41 (s, 1H), 7.1 (s, 1H), pyrido[3,2,1-ij]pyrido[4',3':4,5]pyrano[2,3-f]quinoline]-
3.31-3.11 (m, 8H), 2.9 (t, J = 6.6 Hz, 2H), 2.84 (dt, J = 14.8, 13.8, 5',17(1'H,2H)-dione (11eh). Condition C: yield 57%. 1H NMR
4.5 Hz, 2H), 2.76 (s, 3H), 2.75-2.69 (m, 4H), 2.67 (t, J = 5.6 Hz, (400 MHz, DMSO-d6) δ 7.04 (s, 1H), 3.21 (q, J = 5.5 Hz, 4H), 2.85
2H), 2.02-1.8 (m, 4H), 1.6 (d, J = 14.2 Hz, 2H). 13C NMR (100 (t, J = 5.8 Hz, 2H), 2.71 (q, J = 6.1 Hz, 4H), 2.58 (t, J = 5.7 Hz, 2H),
MHz, DMSO-d6) δ 159.5, 157.92, 157.62, 151.09, 149.26, 2.46-2.3 (m, 3H), 2.08-1.95 (m, 1H), 1.93-1.78 (m, 5H), 1.74 (dd,
145.05, 121.32, 119.13, 118.87, 118.03, 115.87, 107.72, 104.82, J = 12.7, 3.3 Hz, 1H), 1.7-1.59 (m, 2H), 1.59-1.42 (m, 3H), 1.4-
49.96, 49.16, 48.66, 35.97, 27.12, 25.74, 21.05, 20.17, 19.83. 1.32 (m, 2H), 1.31-1.18 (m, 4H), 1.18-1.08 (m, 3H), 1.02-0.96 (m,
HRMS (ESI) Calcd for C23H30N3O2 [MH+]: 380.2333. Found: 1H), 0.95 (s, 3H), 0.79 (s, 3H), 0.79-0.7 (m, 1H). 13C NMR (100
380.2337.
MHz, DMSO-d6) δ 219.89, 159.52, 149.46, 149.08, 144.61,
122.46, 121.13, 117.71, 108.15, 104.87, 53.87, 53.64, 50.83,
49.16, 48.66, 47.14, 36.12, 35.77, 35.32, 34.63, 33.23, 31.45,
30.65, 28.01, 27.8, 27.12, 26.28, 21.38, 21.15, 20.27, 19.86,
(4R,5'S,8'S,9'S,10'S,13'S,14'S)-8-Amino-10',13'-dimethyl-
1',2,2',3,4',5',6',7',8',9',10',11',12',13',14',15',16',17'-
octadecahydrospiro[chromeno[3,4-c]pyridine-4,3'-
cyclopenta[a]phenanthren]-5(1H)-one (11ag). Condition C:
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13.49, 11.57. HRMS (ESI) Calcd for C36H47N2O3 [MH+]: [a]phenanthrene-3,4'-pyrido[3,2,1-ij]pyrido[4',3':4,5]-
View Article Online
555.3581. Found: 555.3584.
4R,5'S,8'R,9'S,10'S,13'S,14'S,17'S)-8-Amino-17'-hydroxy-
10',13'-dimethyl-1',2,2',3,4',5',6',7',8',9',10',11',12',13',14',
15',16',17'-octadecahydro-spiro[chromeno[3,4-c]pyridine-
4,3'-cyclopenta[a]phenanthrene]-5(1H)-one (11ai).
pyrano[2,3-f]quinolin]-5'(1'H)-one (11ej)DOI: 10.1039/D0NJ02664F
and (3R,5S,8R,
(
9S,10S)-10,17,17-trimethyl-1,2,2',3',4,5,6,7,8,8',9,9',10,
11,12,12',13',15,16,17-icosahydro-7'H,11'H-spiro[cyclopenta-
[a]phenanthrene-3,4'-pyrido-[3,2,1-ij]pyrido[4',3':4,5]pyrano-
The [2,3-f]quinolin]-5'(1'H)-one (14). A suspension of 200 mg (0.62
procedure described under Condition C was repeated using 87 mmol, 1.2 eq) of 9e and 160 mg (0.52 mmol, 1 eq) of 10j in 2 mL
mg (0.3 mmol, 1 eq) of 5 -androstan-17 -ol-3-one and 100 mg of absolute ethanol was stirred under reflux for 48 h to afford


10
11
12
13
14
15
16
17
18
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20
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26
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47
48
49
50
51
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53
54
55
56
57
58
59
60
(0.36 mmol, 1.2 eq) of the dihydrochloride salt of 9a in 3 mL of 240 mg of a crude as a mixture of 11ej and 14 that was
absolute ethanol and 0.3 mL of concentrated HCl to afford 94 suspended in water and dichloromethane and washed with a
1
mg (57%) of 11ai as an dihydrochloride salt: H NMR (400 MHz, saturated NaHCO3 solution. The organic layers were dried over
DMSO-d6)

9.33 (br s, 3H), 7.43 (d, J = 8.6 Hz, 1H), 6.6 (dd, J = anhydrous
Na2SO4,
filtered,
concentrated,
and
8.8, 1.9 Hz, 1H), 6.4 (d, J = 2.1 Hz, 1H), 6.22 (br. s, 2H), 4.43 (d, J chromatographed using 1:10 methanol-dichloromethane (Rf
1
= 4.8 Hz, 1H), 3.57-3.4 (m, 1H), 3.13-2.93 (m, 2H), 2.76-2.57 (m, 0.25) to provide 57 mg (19%) of 11ej: H NMR (400 MHz, DMSO-
2H), 1.9-1.78 (m, 2H), 1.78-1.72 (m, 1H), 1.71-1.56 (m, 3H), 1.57- d6)
 7.1-6.98 (m, 1H), 4.03 (s, 1H), 3.2 (q, J = 5.6 Hz, 4H), 2.85
1.43 (m, 4H), 1.44-1.03 (m, 8H), 0.96 (s, 3H), 0.95-0.74 (m, 4H), (t, J = 5 Hz, 2H), 2.7 (q, J = 6 Hz, 4H), 2.58 (t, J = 5.5 Hz, 2H), 2.48-
0.64 (s, 3H). HRMS (ESI) Calcd for C30H41N2O3 [MH+]: 477.3112. 2.43 (m, 1H), 2.33 (t, J = 13 Hz, 1H), 1.93-1.78 (m, 5H), 1.72 (t, J
Found: 477.3093. The procedure described previously for the = 10.7 Hz, 1H), 1.66-1.56 (m, 1H), 1.56-1.28 (m, 8H), 1.29-1.09
conversion of hydrochloride salts to free bases was repeated to (m, 7H), 1.07 (s, 3H), 1.02-0.94 (m, 1H), 0.93 (s, 3H), 0.9-0.8 (m,
afford, after chromatography on silica gel using 1:10 methanol- 1H), 0.74 (s, 3H), 0.71-0.62 (m, 1H). 13C NMR (100 MHz, DMSO-
dichloromethane (Rf 0.38), 11ai as a free base: 1H NMR (400 d6)
MHz, CDCl3) 7.31 (d, J = 8.5 Hz, 1H), 6.55 (dd, J = 8.6, 2.3 Hz, 104.87, 79.72, 53.94, 53.71, 50.45, 49.16, 48.67, 45.21, 38.38,
 159.49, 149.36, 149.08, 144.6, 121.12, 117.7, 108.15,

1H), 6.52 (d, J = 2.2 Hz, 1H), 4.05 (br s, 2H), 3.63 (t, J = 8.5 Hz, 36.13, 36.07, 35.68, 35.22, 33.32, 31.65, 31.55, 28.22, 27.8,
1H), 3.08 (t, J = 5.9 Hz, 2H), 2.74-2.65 (m, 2H), 2.58 (t, J = 13.3 27.13, 26.27, 26.19, 23.1, 21.15, 20.27, 19.87, 14.25, 11.61.
Hz, 1H), 2.13-1.97 (m, 2H), 1.84-1.76 (m, 1H), 1.69-1.61 (m, 1H), HRMS (ESI) Calcd for C37H51N2O3 [MH+]: 571.3894. Found:
1.57-1.39 (m, 4H), 1.38-1.34 (m, 1H), 1.34-1.3 (m, 1H), 1.3-1.26 571.3902. In addition to 11ej, chromatography on silica gel
(m, 2H), 1.26-1.22 (m, 4H), 1.19-1.12 (m, 2H), 1.11-1.06 (m, 1H), using 1:10 methanol-dichloromethane (Rf 0.48) afforded 58 mg
1
1.04 (s, 3H), 1.01-0.86 (m, 3H), 0.82-0.73 (m, 1H), 0.74 (s, 3H). (20%) of 14: H NMR (400 MHz, CDCl3)

6.91 (s, 1H), 3.27-3.14
13C NMR (100 MHz, CDCl3)
 160.1, 154.37, 149.31, 148.26, (m, 4H), 3.09-3 (m, 2H), 2.84 (t, J = 6.5 Hz, 2H), 2.75 (t, J = 12.5
125.34, 125, 111.77, 100.78, 82.22, 55.1, 54.48, 51.37, 43.18, Hz, 2H), 2.69-2.65 (m, 2H), 2.6 (t, J = 13.3 Hz, 1H), 2.28-2.15 (m,
41.3, 37.02, 36.62, 36.31, 35.88, 35.76, 34.07, 31.83, 30.72, 1H), 2.12-1.98 (m, 2H), 1.99-1.88 (m, 5H), 1.88-1.72 (m, 2H),
29.86, 28.63, 28.34, 27.1, 23.54, 20.71, 11.98, 11.33. HRMS 1.67-1.62 (m, 1H), 1.61-1.57 (m, 3H), 1.56-1.48 (m, 2H), 1.4-1.27
(ESI) Calcd for C30H41N2O3 [MH+]: 477.3112. Found: 477.3112. (m, 3H), 1.27-1.16 (m, 2H), 1.16-1.08 (m, 1H), 1.08-1 (m, 2H), 1
(4R,5'S,8'R,9'S,10'S,13'S,14'S,17'S)-8-(Dimethylamino)-17'-
hydroxy-10',13'-dimethyl-1',2,2',3,4',5',6',7',8',9',10',11',12',
13',14',15',16',17'-octadeca-hydrospiro[chromeno[3,4-
c]pyridine-4,3'-cyclopenta[a]phenanthren]-5(1H)-one
(s, 3H), 0.98-0.96 (m, 1H), 0.93 (s, 3H), 0.92 (s, 3H). 13C NMR
(100 MHz, CDCl3) 160.71, 149.87, 148.5, 144.99, 141.18,
136.52, 123.18, 120.93, 118, 109.11, 106.38, 54.99, 51.95,
50.05, 49.68, 45.49, 41.33, 39.73, 36.86, 36.79, 36.25, 36.07,

hydrochloride (11bi).
Condition C was repeated using 59 mg (0.2 mmol, 1 eq) of 5
androstan-17 -ol-3-one and 65 mg (0.24 mmol, 1.2 eq) of the C37H49N2O2 [MH+]: 553.3789. Found: 553.3789. Anal. Calcd
The procedure described under 33.96, 31.6, 29.89, 29.2, 28.26, 27.96, 27.19, 26.94, 26.64,
-
22.63, 22.55, 21.93, 21.01, 20.43, 11.4. HRMS (ESI) Calcd for

hydrochloride salt of 9b in 2 mL of absolute ethanol and 0.2 mL for C37H50N2O3: C, 80.39; H, 8.75; N, 5.07. Found: C, 80.17; H,
of concentrated HCl to provide 94 mg (85%) of 11bi. This 8.94, N, 5.13.
product was triturated with hot methanol to achieve additional (3R,5S,8R,9S,10S,13R,14S,17R)-10,13-Dimethyl-17-((R)-6-
1
purification: H NMR (400 MHz, DMSO-d6)

9.64-9.27 (m, 2H), methylheptan-2-yl)-1,2,2',3',4,5,6,7,8,8',9,9',10,11,12,12',
7.55 (d, J = 9 Hz, 1H), 6.77 (dd, J = 9.1, 2.5 Hz, 1H), 6.56 (d, J = 13,13',14,15,16,17-docosahydro-7'H,11'H-spiro[cyclopenta-
2.5 Hz, 1H), 4.44 (d, J = 4.8 Hz, 1H), 3.49-3.4 (m, 1H), 3.12-3.04 [a]phenanthrene-3,4'-pyrido[3,2,1-ij]pyrido[4',3':4,5]pyrano-
(m, 2H), 3.02 (s, 6H), 2.71-2.58 (m, 2H), 1.92-1.78 (m, 1H), 1.78- [2,3-f]quinolin]-5'(1'H)-one hydrochloride (11ek). Condition
1.71 (m, 1H), 1.7-1.58 (m, 3H), 1.56-1.4 (m, 5H), 1.4-1.26 (m, C: mp > 230
3H), 1.26-1.09 (m, 5H), 0.96 (s, 3H), 0.93-0.76 (m, 4H), 0.64 (s, methanol. 1H NMR (500 MHz, CF3COOD) δ 7.57 (s, 1H), 3.84-
3H). 13C NMR (100 MHz, DMSO-d6)
159.09, 154.34, 153.2, 3.76 (m, 1H), 3.76-3.61 (m, 4H), 3.42 (t, J = 6.9 Hz, 2H), 3.15 (t, J
C; yield 78%. Purified by trituration with hot

125.94, 109.94, 97.07, 80.48, 53.32, 51.2, 43.03, 37.13, 35.75, = 6.6 Hz, 4H), 3.13-3.07 (m, 1H), 3-2.9 (m, 1H), 2.52-2.34 (m, 4H),
35.53, 31.62, 30.28, 28.01, 23.49, 20.58, 11.95, 11.82. HRMS 3.48-3.38 (m, 1H), 2.11-2.01 (m, 3H), 1.98-1.73 (m, 2H), 1.64-
(ESI) Calcd for C32H45N2O3 [MH+]: 505.3425. Found: 505.3405. 1.54 (m, 4H), 1.53-1.43 (m, 7H), 1.42-1.3 (m, 3H), 1.19-1.11 (m,
(3R,5S,8R,9S,10S,13S,14S,17S)-17-Hydroxy-10,13,17-
trimethyl-1,2,2',3',4,5,6,7,8,8',9,9',10,11,12,12',13,13',14,
15,16,17-docosahydro-7'H,11'H-spiro[cyclopenta-
4H), 1.1 (s, 3H), 1.07-0.91 (m, 4H), 0.89 (d, J = 6.4 Hz, 3H), 0.8
(dd, J = 6.7, 1.5 Hz, 6H), 0.78-0.72 (m, 2H), 0.68 (s, 3H). 13C NMR
(126 MHz, CF3COOD) δ 163.02, 150.43, 150.18, 137.13, 131.54,
126.26, 126.23, 123.8, 121.28, 64.51, 58.58, 58.53, 56.61, 56.29,
10 | J. Name., 2012, 00, 1-3
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44.6, 43.44, 41.89, 41.46, 38.32, 38.09, 37.99, 37.85, 37.66, NMR Spectroscopy
View Article Online
DOI: 10.1039/D0NJ02664F
35.27, 34.61, 33.52, 29.9, 29.76, 28.52, 26.87, 25.78, 25.66,
NMR experiments were carried out at 25oC using DMSO-d6 on
an Agilent 400 MHz spectrometer using a 5 mm probe equipped
with a z-gradient optimized for 1H detection. Chemical shifts
were referenced relative to the residual protonated solvent
signal set at 2.54 ppm51. A normal one-dimensional 1H and 13C
(proton decoupled) and DEPT-135 (Distorsionless Enhancement
of Polarization Transfer) spectra52 were acquired and two-
dimensional homonuclear (1H-1H) correlation spectroscopy
(COSY and ROESY)41-42 2D-heteronuclear correlations (1H-13C)
spectra (HSQC and HMBC) were acquired using standard pulse
sequences from the Agilent library. Spectral widths of 4340 Hz
for both dimensions with 1024 complex data points in t2 and
256 t1 increments were used for ROESY and COSY experiments.
The relaxation delay between successive pulse cycles was set to
1s and ROESY mixing time of 400 millisec was used. Forty
transients for each increment were collected for ROESY, and 16
transients for each increment were collected for COSY
experiments. Phase sensitive two-dimensional spectra were
obtained using the time-proportional phase incrementation
method (TPPI). Spectra were zero-filled to 2048x1024 real data
25.15, 23.51, 23.27, 22.72, 21.59, 21.39, 20.61, 19.6, 12.9,
12.71. HRMS (ESI) Calcd for C44H65N2O2 [MH+]: 653.5041.
Found: 653.5043.
4-(p-Tolyl)-8,9,12,13-tetrahydro-5H,7H,11H-pyrido[3,2,1-
ij]pyrido[4',3':4,5]pyrano[2,3-f]quinolin-5-one (13).
The
procedure described under Condition B was repeated using 200
mg (0.62 mmol) of 9e and 0.29 mL (2.49 mmol) of 4-
toluylaldehyde to afford crude 4-(p-tolyl)-1,2,3,4,8,9,12,13-
octahydro-5H,7H,11H-pyrido[3,2,1-
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60
ij]pyrido[4',3':4,5]pyrano[2,3-f]quinolin-5-one
2,2,2-
trifluoroacetate in 58% yield. To 100 mg (0.2 mmmol) of this
trifluoracetate salt in 6 mL of dimethyl sulfoxide was added 100
L (0.7 mmol, 3.5 eq) of 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) and 94 mg (0.42 mmol, 2.1 eq) of cupric bromide. The
solution was stirred at 25°C for 16 h. The mixture was poured
into 20 mL of aqueous ammonia solution (5% w/w) and
extracted with ethyl acetate. The organic layers were washed
with brine dried over anhydrous MgSO4, filtered and
concentrated. The product was purified by chromatography
using 1:50 methanol-dichloromethane (Rf 0.26) to provide 15
points along f2 and f1, respectively. Sine-bell
/3 window
mg (20%) of 13 as a yellow powder: mp 216-218
C. 1H NMR
functions were used in both dimensions. HSQC adiabatic NMR
spectrum was acquired with spectral widths of 4595 Hz (1H) and
20100 Hz (13C) with 1s of recycled delay to show direct H-13C
(400 MHz, DMSO-d6) δ 8.67 (d, J = 5.6 Hz, 1H), 7.96 (d, J = 5.7
Hz, 1H), 7.73 (s, 1H), 7.38 (d, J = 8.1 Hz, 2H), 7.19 (d, J = 7.9 Hz,
2H), 3.31-3.22 (m, 4H), 2.79 (t, J = 6.3 Hz, 2H), 2.74 (t, J = 6.4 Hz,
2H), 2.37 (s, 3H), 1.9 (d, J = 6.8 Hz, 4H). 13C NMR (101 MHz,
DMSO-d6) δ 162.9, 158.91, 151.24, 149.32, 146.12, 144.32,
138.54, 137.33, 128.87, 127.89, 121.57, 118.18, 113.2, 110.85,
105.61, 102.94, 49.22, 48.67, 27.03, 20.96, 20.93, 20.07, 19.99.
HRMS (ESI) Calcd for C26H23N2O2 [MH+]: 383.1754. Found:
383.1756.
1
connectivity and C-H correlation.
Computational Modeling
The X-ray structure35 of the androgen-ligand binding domain in
human 17
-hydroxysteroid dehydrogenase type 5 (PDB: 1XF0)
with 5 -androstane-3,17-dione was selected as the template to

model the binding of the fluorescent adduct 11eh. The initial
enzyme structure was downloaded from the RCSB Protein Data
Bank and was subsequently prepared for docking via Autodock
X-Ray Crystallography
X-ray diffraction data were collected at 90.0(2) K on a Bruker D8 Tools.53 The adduct 11eh was docked using Autodock Vina54
Venture -axis diffractometer using MoK(α) X-rays and using into the position occupied by 5 -androstane-3,17-dione in 17
well-established, low-temperature crystal-handling HSD5. The binding poses of 11eh obtained from Vina that


-
techniques48. Raw data were integrated, scaled, merged and contained high overlap with that of 11eh. The binding pose was
corrected for Lorentz-polarization effects using the APEX3 further refined by performing a series of energy minimization
package (Bruker-AXS Inc., Madison, WI, US. Corrections for processes. Briefly, the AMBER14SB force field55 and the second
absorption were applied using SADABS49. The structure was generation of the general AMBER force field (gaff2) were used
solved by direct methods (SHELXT50) and refinement was for the proteins and ligands, respectively. Partial charges for
carried out against F2 by weighted full-matrix least-squares 11eh were generated via the Antechamber56 program in AMBER
(SHELXL50). Hydrogen atoms were found in difference maps 18 using the AM1-BCC model.57 Two courses of minimization
placed at calculated positions and refined using riding models. were conducted using a hybrid protocol of twenty five-hundred
Non-hydrogen atoms were refined with anisotropic steps of steepest descent minimization followed by a conjugate
displacement parameters. Absolute configuration was known gradient minimization until a maximum twenty five-hundred
due to the unchanging stereochemistry of the steroid ring iteration steps was reached or the convergence criterion (the
system. Atomic scattering factors were taken from the root-mean-square of the energy gradient is less than 1 × 10–4
International Tables for Crystallography.
Experimental kcal/mol·Å) was satisfied. During the first step of minimization,
conditions, atomic coordinates, bond lengths and angles, as a force constant of 100 kcal/mol·Å2 was applied on the protein
well as diffraction data and the crystallographic model were atoms. The second minimization step consisted of one
archived in the CIF, available free of charge from the Cambridge thousand steps of steepest descent minimization, followed by
Crystallographic Data Centre (CCDC), structure code CCDC fifteen hundred steps of conjugate gradient minimization, and
1888376.
this course of minimization had no restraints for either the
ligand or the protein atoms.
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Clinical Implications of Elevated Blood LevelsV.ieEwnAdrtoicclerOnRleinve
Author contributions
2017, 38 (3), 220-254.
DOI: 10.1039/D0NJ02664F
3
Huggins, C.; Scott, W. W., Bilateral Adrenalectomy in Prostatic
Cancer: Clinical Features and Urinary Excretion of 17-
Ketosteroids and Estrogen. Annals of surgery 1945, 122 (6),
1031-41.
Isaacs, J. T., Antagonistic effect of androgen on prostatic cell
death. The Prostate 1984, 5 (5), 545-57.
Mohler, J. L.; Gregory, C. W.; Ford, O. H., 3rd; Kim, D.; Weaver,
C. M.; Petrusz, P.; Wilson, E. M.; French, F. S., The androgen
axis in recurrent prostate cancer. Clinical cancer research : an
official journal of the American Association for Cancer
Research 2004, 10 (2), 440-8.
Frasinyuk, M. S.; Zhang, W.; Wyrebek, P.; Yu, T.; Xu, X.; Sviripa,
V. M.; Bondarenko, S. P.; Xie, Y.; Ngo, H. X.; Morris, A. J.;
Mohler, J. L.; Fiandalo, M. V.; Watt, D. S.; Liu, C., Developing
antineoplastic agents that target peroxisomal enzymes:
cytisine-linked isoflavonoids as inhibitors of hydroxysteroid
17-beta-dehydrogenase-4 (HSD17B4). Org Biomol Chem
2017, 15 (36), 7623-7629.
Selles Vidal, L.; Kelly, C. L.; Mordaka, P. M.; Heap, J. T., Review
of NAD(P)H-dependent oxidoreductases: Properties,
engineering and application. Biochim Biophys Acta Proteins
Proteom 2018, 1866 (2), 327-347.
Stuchbery, R.; McCoy, P. J.; Hovens, C. M.; Corcoran, N. M.,
Androgen synthesis in prostate cancer: do all roads lead to
Rome? Nat Rev Urol 2017, 14 (1), 49-58.
Pippione, A. C.; Boschi, D.; Pors, K.; Oliaro-Bosso, S.; Lolli, M.
L., Androgen-AR axis in primary and metastatic prostate
cancer: chasing steroidogenic enzymes for therapeutic
intervention. J. Cancer Metastasis Treat. 2017, 3, 328-361.
James L. Mohler, Michael V. Fiandalo, Chunming Liu, Vitaliy M.
Sviripa and David S. Watt conceived the overall experimental
question, assisted with and contributed to the experimental
design, and supervised data acquisition and analysis and
manuscript preparation. Vitaliy M. Sviripa, Kristin L. Begley,
Przemyslaw Wyrebek, Liliia Kril and David S. Watt planned and
executed the synthetic chemistry. Vivekanandan Subramanian
performed the 2D NMR studies. Sean R. Parkin performed the
X-ray crystallography study. Xi Chen, Alexander H. Williams and
Chang-Guo Zhan performed the pKa calculations and the
computational modeling studies. All authors reviewed the
manuscript.
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Conflicts of interest
7
CL and DSW have partial ownership in a for-profit venture,
Epionc, Inc., that seeks to develop small-molecule inhibitors for
cancer treatment. In accord with University of Kentucky
policies, CL and DSW have disclosed this work to the University
of Kentucky’s Intellectual Property Committee and to a Conflict
of Interest Oversight Committee.
8
9
Acknowledgements
10 Fiandalo, M. V.; Stocking, J. J.; Pop, E. A.; Wilton, J. H.;
Mantione, K. M.; Li, Y.; Attwood, K. M.; Azabdaftari, G.; Wu,
Y.; Watt, D. S.; Wilson, E. M.; Mohler, J. L., Inhibition of
dihydrotestosterone synthesis in prostate cancer by
combined frontdoor and backdoor pathway blockade.
Oncotarget 2018, 9 (13), 11227-11242.
11 Mohler, J. L.; Titus, M. A.; Wilson, E. M., Potential prostate
cancer drug target: bioactivation of androstanediol by
conversion to dihydrotestosterone. Clin Cancer Res 2011, 17
(18), 5844-9.
12 Mohler, J. L.; Titus, M. A.; Bai, S.; Kennerley, B. J.; Lih, F. B.;
Tomer, K. B.; Wilson, E. M., Activation of the androgen
receptor by intratumoral bioconversion of androstanediol to
dihydrotestosterone in prostate cancer. Cancer Res 2011, 71
(4), 1486-96.
13 Mohler, J.; Titus, M., Tissue levels of androgens in castration-
recurrent prostate cancer. In Androgen Action in Prostate
Cancer, Mohler, J. L.; Tindall, D., Eds. Springer Science: New
York, NY, 2009; pp 175-185.
14 Chang, K. H.; Li, R.; Papari-Zareei, M.; Watumull, L.; Zhao, Y.
D.; Auchus, R. J.; Sharifi, N., Dihydrotestosterone synthesis
bypasses testosterone to drive castration-resistant prostate
cancer. Proceedings of the National Academy of Sciences of
the United States of America 2011, 108 (33), 13728-33.
15 Auchus, R. J., The backdoor pathway to dihydrotestosterone.
Trends in endocrinology and metabolism: TEM 2004, 15 (9),
432-8.
VMS was aided in part by an institutional grant from the
American Cancer Society (IRG-16-182-28). DSW was supported
by NIH R01 CA172379, the Office of the Dean of the College of
Medicine, the Markey Cancer Center, the Center for
Pharmaceutical Research and Innovation (CPRI) in the College
of Pharmacy, Department of Defense Idea Development Award
PC150326P2, and NIH P20 RR020171 from the National Institute
of General Medical Sciences (to L. Hersh). CL was supported by
NCI (R01 CA172379). JLM was supported by NCI (P01 CA77739,
P20 RR020171, R21 CA2051, and P30 GM110787); the
Department of Defense (DoD Prostate Cancer Research
Program Award W81XWH-16-1-0635 and Post-doctoral
Training Award W81XWH-15-1-0409); the National Science
Foundation for the D8 Venture diffractometer (MRI award
CHE1625732); the Shared Instrument Award (NIH
1S10D018048); NCI research Specialist Award (R50CA211108)
and the NCI Cancer Center Support Grant to Roswell Park
Comprehensive Cancer Center (P30 CA016056) for the Flow and
Image, Pathology Network, Biostatistics and Bioinformatics
Shared Resources. This manuscript’s contents are solely the
responsibility of the authors and do not necessarily represent
the official views of the NIH, NSF, NIGMS or the DoD.
16 Bauman, D. R.; Steckelbroeck, S.; Williams, M. V.; Peehl, D. M.;
Penning, T. M., Identification of the major oxidative 3alpha-
hydroxysteroid dehydrogenase in human prostate that
converts 5alpha-androstane-3alpha,17beta-diol to 5alpha-
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Amino-substituted coumarins undergo Pictet-Spengler condensations with 3-ketosteroids to
afford spirocyclic androgens for the study 17-oxidoreductases for intracrine, androgen
metabolism.
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C-7 amino-substituted
4-(2-aminoethyl)coumarin
5-dihydrotestosterone
OH
N
O
H
O
H
NH
H