Preventing Morphine-Seeking Behavior through the Re-Engineering of Vincamine's Biological Activity

Article abstract of DOI:10.1021/acs.jmedchem.9b01924

Innovative discovery strategies are essential to address the ongoing opioid epidemic in the United States. Misuse of prescription and illegal opioids (e.g., morphine, heroin) has led to major problems with addiction and overdose. We used vincamine, an indole alkaloid, as a synthetic starting point for dramatic structural alterations of its complex, fused ring system to synthesize 80 diverse compounds with intricate molecular architectures. A select series of vincamine-derived compounds were screened for both agonistic and antagonistic activities against a panel of 168 G protein-coupled receptor (GPCR) drug targets. Although vincamine was without an effect, the novel compound 4 (V2a) demonstrated antagonistic activities against hypocretin (orexin) receptor 2. When advanced to animal studies, 4 (V2a) significantly prevented acute morphine-conditioned place preference (CPP) and stress-induced reinstatement of extinguished morphine-CPP in mouse models of opioid reward and relapse. These results demonstrate that the ring distortion of vincamine offers a promising way to explore new chemical space of relevance to opioid addiction.

Full text of DOI:10.1021/acs.jmedchem.9b01924

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Article
Preventing Morphine Seeking Behavior through the
Re-engineering of Vincamine’s Biological Activity
Verrill M. Norwood, Ariana Brice-Tutt, Shainnel O Eans, Heather Stacy, Guqin Shi, Ranjala Ratnayake, James
R. Rocca, Khalil A. Abboud, Chenglong Li, Hendrik Luesch, Jay P. McLaughlin, and Robert William Huigens III
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01924 • Publication Date (Web): 08 Jan 2020
Downloaded from pubs.acs.org on January 9, 2020
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Journal of Medicinal Chemistry
Preventing Morphine Seeking Behavior through the Re-engineering of
Vincamine’s Biological Activity
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Verrill M. Norwood IV[a,c], Ariana Brice-Tutt[b,c], Shainnel O. Eans[b,c], Heather Stacy[b,c], Guqin Shi[a,c], Ranjala
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Ratnayake[a,c], James R. Rocca[a,d], Khalil A. Abboud , Chenglong Li[a,c], Hendrik Luesch[a,c],Jay P. McLaughlin[b,c]
,
[e]
and Robert W. Huigens III*[a,c]
[a]Department of Medicinal Chemistry; College of Pharmacy, University of Florida, Gainesville, Florida 32610,
United States. [b]Department of Pharmacodynamics; College of Pharmacy, University of Florida, Gainesville,
[c]
Florida 32610, United States. Center for Natural Products, Drug Discovery & Development (CNPD3); College
of Pharmacy, University of Florida, Gainesville, Florida 32610, United States. [d]McKnight Brain Institute,
[
e]
University of Florida, Gainesville, Florida 32610, United States. Department of Chemistry, University of Florida,
Gainesville, Florida 32610, United States.
*
Corresponding author e-mail: rhuigens@cop.ufl.edu
ABSTRACT.
Innovative discovery strategies are essential to address the ongoing opioid epidemic in the United States. Misuse
of prescription and illegal opioids (e.g., morphine, heroin) has led to major problems with addiction and overdose.
We used vincamine, an indole alkaloid, as a synthetic starting point for dramatic structural alterations of its
complex, fused ring system to synthesize 80 diverse compounds with intricate molecular architectures. A select
series of vincamine-derived compounds was screened for both agonistic and antagonistic activities against a
panel of 168 GPCR drug targets. Although vincamine was without effect, the novel compound 4 (V2a)
demonstrated antagonistic activities against hypocretin (orexin) receptor 2. When advanced to animal studies, 4
(V2a) significantly prevented acute morphine-conditioned place preference (CPP) and stress-induced
reinstatement of extinguished morphine-CPP in mouse models of opioid reward and relapse. These results
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demonstrate that the ring distortion of vincamine offers a promising way to explore new chemical space of
relevance to opioid addiction.
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INTRODUCTION.
An invigorated effort to improve pain management was implemented across the United States (US) in the mid-
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990s. Hospitals and inpatient settings began to include “pain scores” as a quality measure for patients. Many
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clinicians responded to patient’s elevated pain scores by prescribing opioid medications more frequently. Initially,
opioid therapies were not believed to be highly addictive; however, we now know that there are significant
ramifications associated with opioid misuse as the US is amidst a health care crisis resulting from opioid addiction
and death related overdoses.1-4
The opioid epidemic has led to devastating public health, social and economic consequences.1-3 Several legal
and illegal opioids have played a critical role in the growing crisis and can be categorized as either prescription
pain relievers (morphine, codeine, oxycodone), synthetic opioids (fentanyl) or heroin. In 2017, more than 130
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people died every day in the US from overdosing on opioids. According to the National Survey on Drug Use
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and Health, nearly 12 million individuals in the US misused opioids in 2015. It is also known that nearly 80% of
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heroin users initially misused prescription opioids. The Center for Disease Control and Prevention estimates the
2
economic burden of prescription misuse alone to be $78.5 billion each year. In response to the unprecedented
need to treat opioid addiction, there is an increased emphasis to discover new and innovative therapeutic agents
for opioid use disorders.
Several natural products have been shown to be excellent starting points for generating diverse and complex
compound libraries, which are of importance to those involved in drug discovery.8-18 Our group is interested in
diverse indole-based compounds14,19, as many are known to bind and modulate an array of therapeutically-
relevant biological targets, from complex natural products (e.g., vincristine, binds microtubules for cancer
treatments) to structurally simple synthetic indoles (e.g., ondansetron, binds serotonin receptors for nausea
therapies).19,20 Therefore, we hypothesized that a unique library composed of architecturally diverse indole-
based compounds could lead to promising discoveries of relevance to opioid addiction.
Vincamine (1) is an indole alkaloid found in the leaves of Vinca minor and is available on an affordable deca-
gram scale (~$36/gram). This indole alkaloid is a prescription medicine (Oxybral SR) used to improve cerebral
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Journal of Medicinal Chemistry
blood flow and improve cognitive function. Despite vincamine’s interesting biological activity profile, it has no
known activities against targets relevant to opioid addiction; however, we aimed to use vincamine as a starting
point to rapidly generate complex and diverse indole-based scaffolds to explore new chemical space through
biological screens against a diverse panel of drug targets. The overarching goal of this work is to re-engineer
the biological activity of vincamine through dramatic alterations of its inherently complex molecular architecture
and identify promising new lead compounds for opioid abuse.
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A diverse library of stereochemically complex compounds, with scaffolds related to 2-9 (Fig. 1) was rapidly
generated in one to six synthetic steps from vincamine using an orchestrated series of chemoselective ring
cleavage, ring rearrangement and ring fusion reactions. Using this ring distortion approach, we synthesized an
intricate and diverse library of 80 indole-based small molecules featuring unique and complex molecular
architectures (Supp. Fig. 1 & 2). We evaluated a subset of the library against a panel of 168 GPCR drug targets,
several of which demonstrated activity profiles distinct from vincamine and other complex, indole-based small
molecules synthesized during these investigations.
CN
N
O
H
N
N
Me
N
H
2
O
O
N
N
CO2Me
I
H
9
2 Derivatives
Me
HN
3
O
Me
20 Derivatives
1
Derivative
5
Steps
CO2Me
6 Steps
4
Steps
N
H
N
H
N
N
N
N
2 Steps
N
5-6 Steps
N
MeO2C
N
MeO2C
Me
OH
O
N
H
Me
Me
8
Vincamine (1)
$36 per gram)
4 (V2a)
(
OH
1
7 Derivatives
2
Derivatives
5 Steps
1-5 Steps
5
Steps
Br
O
O
N
HN
Br
HO
CN
N
H
N
H
7
H
Me
N
N
N
5
O
OMe
6
MeO2C
Me
CO2Me
Me
25 Derivatives
6
Derivatives
2
Derivatives
Figure 1. Rapid syntheses of diverse and stereochemically complex molecules from the indole alkaloid vincamine (1).
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RESULTS AND DISCUSSION.
Ring Distortion of Vincamine to Rapidly Generate Diverse, Stereochemically Complex Indole-Based Compounds.
Following an initial query of the literature, we identified two ring cleavage transformations from vincamine that
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were useful during our ring distortion efforts.21,22 We developed a modified version of the first ring cleavage21 to
afford 12, which is a three step synthesis from vincamine using 1.) lithium aluminum hydride, 2.) sodium periodate,
then 3.) potassium hydroxide (91% yield/3 steps; ≥ 3.5 grams for each step; Scheme 1). The second ring-
cleavage reaction from vincamine utilizes methyl propiolate in methanol to afford structures similar to 17 as a
22
single diastereomer. In this reaction, the tertiary amine of vincamine undergoes a conjugate addition into methyl
propriolate, in turn, activating the C-N bond adjacent to the indole heterocycle for ring cleavage. We used various
alcohol solvents as nucleophiles to attack the carbon adjacent to the 2-position of the indole heterocycle, which
occurs with complete inversion of stereochemistry. We found sterically encumbered alcohol nucleophiles to
induce a stereoinversion of the α-hydroxyester center in vincamine during this reaction. Presumably,
stereoinversion occurs through a ring-opening and -closing pathway to furnish 18 and related analogues. An
interesting feature of 18 is the hydrogen-bonding interaction between the inverted hydroxyl and resultant
isopropyl ether oxygen atom that can be seen in the X-ray structure of 18 (Scheme 1). We believe this
stereoinversion is favored and occurs to avoid steric interactions between bulky ether groups and the methyl
ester that would be on the same face in the resulting products. In addition, this ring cleavage reaction is
applicable with several vincamine-derived substrates.
In addition, cyanogen bromide (CNBr) was utilized for regioselective C-N ring cleavage reactions, similar to our
14
previous studies with yohimbine. Compound 16 was treated with cyanogen bromide and 2-iodobenzyl alcohol
to afford indole-promoted C-N ring cleavage product 2 in 40% yield as a single diastereomer. Alternatively, a
von Braun reaction that occurs through an S 2 pathway can be employed utilizing cyanogen bromide in DMF
N
under microwave conditions to yield alternative ring-cleavage scaffold 10 (39% yield, 2 steps), which was
elaborated into a series of amide analogues 11.
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Journal of Medicinal Chemistry
During this study, we found 12 to be an excellent starting point for innovative and provocative ring distortion
reactions. One of our synthetic goals was to carry out the selective oxidation of the indole nucleus upon treatment
with meta-chloroperbenzoic acid (m-CPBA) to afford C-3 hydroxylated products (e.g., 6).19,23 The hydroxylation
of indoles at C-3 provides a synthetic opportunity to access interesting ring rearrangement scaffolds from
vincamine following a base-promoted 1,2-alkyl shift to afford spiro-3-oxindoles. During these studies, 16 was
stereoselectively hydroxylated with m-CPBA and trifluoroacetic acid (TFA; Scheme 1) in methylene chloride to
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afford 6 as a single diastereomer in 34% yield (axially protonated amine of 16 hydrogen-bond directs m-CPBA ;
β-hydroxylation affords 6, see X-ray). Subsequent exposure of 6 to sodium hydroxide induced a semi-pinacol
rearrangement to furnish spiroindoxyl 9 in 82% yield.
Vincamine Ring Distortion
H
N
N
1) p-TSA, PhMe
1) LiAlH4, THF
1) LiAlH4, THF
2) NaIO4, THF/H2O
2
) CNBr, DMF, µW
(45%, 2 steps)
2) NaIO4, THF/H2O
3) KOH (aq), EtOH
MeO2C
OH
3
) MeO2C
, MeOH
Me
(91%, 3 steps)
(76%, 3 steps)
Vincamine (1)
Br
N
Ring Cleavage
CO2Me
CN
Ring
Cleavage
N
MeI,
CN
H
N
H
N
CNBr,
H
N
K2CO3
2-I-BnOH
O
N
H
HN
O
N
H
(75%)
N
H
(40%)
O
H
CO Me
2
MeO2C
OH
OMe
Me
Me
Me
N
10
12
16
Me
Me
I
NOE
Ring Cleavage
2
17
O
OMe
Ring Cleavage
R1R2NH(a)
m-CPBA, TFA
1) NaN3, DMF, µW
CH2Cl2 (34%)
2
) PPh3, MeOH
ClCO2Et; then (a)
or EDC, DMAP
Rapid Synthesis of Diverse &
Complex Indole-Based Scaffolds
3) RCOCl, TEA, DMAP
Stereoselective
Hydroxylation
3
derivatives
18 derivatives
(21%-45%)
(Avg. Yield = 65%)
HO
O
N
R
Diversification
H
N
H
O
HN
N
O
N
H
OMe
CN
N
R1
N
Me
H
6
X-ray (6)
X-ray (17)
Me
N
13
R2
Single Diastereomer
MeO2C
NaOH, MeOH
reflux (82%)
m-CPBA,TFA
CH2Cl2
Me
11
18, 1 step
CO2Me
to synthesize
Ring Fusion
N
H
O
R
H
14
N
N
O
N
N
N
H
Me
Me
N
N
O
15
N
MeO2C
H
R
O
Me
Me
O
N
H
O
Me
13 derivatives
Me
9
R1 or R2 = H
inverted
stereochemistry
H
(Avg. Yield = 42%)
X-ray (18)
Ring Rearrangement
Scheme 1. Ring distortion of vincamine enables the rapid generation of complex compounds for biological investigations.
The carboxylic acid of 12 readily undergoes amidation reactions using ethyl chloroformate, or 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC) with catalytic 4-dimethylaminopyridine (DMAP), followed by the
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addition of diverse amines to afford 13 (18 amide derivatives, 65% average yield; Scheme 1). In contrast to the
hydroxylation of the indole nucleus (e.g., 16 to 6), we discovered that amide analogues (13) undergo an intriguing
oxidative ring fusion reaction upon treatment with m-CPBA and TFA (Scheme 1 & 2). Surprisingly, no C-3
hydroxylated products were observed upon treatment of amides 13 with m-CPBA and TFA; however, this
transformation was robust and afforded 13 unique ring-fused analogues 15 in 31-56% yield (42% average yield).
Rationalizing the reaction mechanism for the oxidative ring fusion was an initial challenge. However, we
hypothesized that m-CPBA initially reacts with the indole of 13 to give the expected 3-hydroxyindolenine
intermediate. Then, we proposed that TFA (17 equivalents) under refluxing conditions leads to a subsequent
elimination (dehydration), resulting in intermediate 14, which undergoes a final 5-exo-trig cyclization between
the amide nitrogen (nucleophile) and the resonance-stabilized carbocation adjacent to the C-2 position of the
indole heterocycle would rationally lead to the observed ring-fused scaffolds (15).
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Indole Oxidation
H
in Acid
N
Oxidative Ring-
Fusion Method
R
m-CPBA
O
N
N
N
O
1
7 eq. TFA, CH Cl
2
2
N
H
Me
H
R
o
N
(Avg. Yield = 42%)
0
C
reflux
Me
5-exo-trig
24 hr
14
H
15
1
3 derivatives
3
X-ray of 25
H
N
O
m-CPBA, TFA method results
2
N
1
H
yield (%)
entry
R
HN
R
Me
TFA, CH Cl
2 2
Validation of Reaction Pathway
Conversion to 24 (74% yield)
0 o
C
reflux
.5 hr
1
3
20
41
56
2
(R = alkyl, benzyl)
21
positions 1-3 of the
indole indicated
NOE
correlations
2
2
3
Me 43
37
H
HO
H HN
H
O
H
H
N
2
PIFA, MeCN, H O
2
N
O
O
N
N
H
24
44
Indole Oxidation under
Mild Conditions
N
Me
H
Me
25
OMe 41
19 (26% isolated yield, direct hydroxylation at C-3)
Scheme 2. Novel oxidative cyclization method from amides (13) to ring fusion analogues of 15 with mechanistic validation
involving critical compound 19.
To test our hypothesis regarding the mechanism of the oxidative ring fusion reaction (13 to 15), we utilized the
hypervalent iodine reagent [bis(trifluoroacetoxy)iodo]benzene (PIFA) and water as a milder oxidizing system24
to isolate desired C-3 hydroxylated 19 (Scheme 2) in 26% yield. 19 is an important intermediate regarding our
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proposed reaction mechanism that leads to the formation of ring fusion analogues (15). Upon exposure of 19 to
TFA (without m-CPBA) in refluxing methylene chloride, we were delighted to see the rapid formation of ring-
fusion compound 24 in 74% yield in 2.5 hours (Scheme 2; the X-ray of analogue 25 confirms C-N bond formation
upon ring-closure). These findings support our proposed mechanism regarding the conversion of amides (13) to
novel ring fusion analogues (15).
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Biological Evaluation against a Diverse Panel of GPCR Drug Targets.
Following synthesis, seven unique vincamine-derived compounds were selected to represent a maximal
chemical diversity from the synthesized library (analogues 3-9; one from each of the distinct scaffold-types from
vincamine; chemoinformatic analyses of vincamine-derived analogues can be seen in Supp. Fig. 6) and
screened against a panel of 168 G protein-coupled receptor (GPCR) drug targets in cell-based assays (Fig. 2;
Supp. Fig. 3 & 4).25 Each compound was screened at 20 µM for agonist and antagonist activities against the
26
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GPCR panel, which contained targets relevant to opioid addiction (e.g., CNR1 , HCRTR2 ). In addition, parent
vincamine and comparator indole alkaloid yohimbine (26) were screened alongside the new synthetic analogues.
We hypothesized that indole-based compounds with diverse molecular architectures would lead to new and
exciting hit compounds that demonstrate GPCR activity profiles distinct from each other and the parent
vincamine.
Although minimal agonistic activity was found in this screen, differential profiles were obtained in the antagonism
mode. The GPCR antagonists were indeed distinct from vincamine’s activity profile (≥80% inhibition was
considered to be a “hit” in this screen; Fig. 2). Vincamine demonstrated no antagonistic activities against this
panel of GPCRs; however, five of the seven diverse, ring-distorted compounds reported at least one GPCR “hit”,
including: 3 (6 hits, 3.6% hit rate), 4 (1 hit, 0.6% hit rate), 5 (8 hits, 4.8% hit rate), 7 (4 hits, 2.4% hit rate), 8 (1
hit, 0.6% hit rate). As a comparator in this screen, yohimbine (26; indole alkaloid) was shown to antagonize 7
GPCRs (4.2% hit rate) that include adrenergic, dopamine and serotonin receptors, which has previously been
reported in the literature.28
The “gain-of-function” GPCR antagonistic activities regarding 3, 4, 5, 7 and 8 compared to vincamine was the
initial proof-of-concept result that our ring distortion approach could lead to molecules with re-engineered
biological activities (based on >70% differential antagonistic activity in screen comparing vincamine to ring
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Page 8 of 52
distortion analogues). Concentration-response experiments were subsequently performed with select
compounds (4, 5, 7) for antagonistic activities against specific GPCRs. These experiments allowed for the
determination of potency for analogues 4, 5 and 7 against relevant GPCRs, resulting in several IC50 values in
the low micromolar range.
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Following initial screens, we turned our attention to analogue 4 (V2a), which demonstrates antagonistic activity
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against hypocretin (orexin) receptor 2 (HCRTR2; IC = 2.26 µM; Fig. 2). We were encouraged by initial results,
50
as hypocretin receptor 2 antagonism recently demonstrated dose-dependent reduction of heroin self-
27
administration in rats. In addition, analogue 4 demonstrated a good selectivity profile for targeting HCRTR2 in
the GPCR panel.
GPCR
1
3
4
5
6
7
8
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26
ADCYAP1R1
ADORA3
ADRA1B
ADRA2A
ADRA2B
ADRA2C
ADRB1
ADRB2
AGTR1
AGTRL1
AVPR1A
AVPR1B
AVPR2
BDKRB1
BDKRB2
BRS3
A
B
C3AR1
C5AR1
C5L2
CALCR
CALCRL-RAMP1
CALCRL-RAMP2
CALCRL-RAMP3
CALCR-RAMP2
CALCR-RAMP3
CCKAR
CCKBR
CCR10
CCR1
CCR2
CCR3
CCR4
CCR5
CCR6
CCR7
CCR8
CCR9
CHRM1
CHRM2
CHRM3
CHRM4
CHRM5
CMKLR1
CNR1
CNR2
CRHR1
CRHR2
CRTH2
CX3CR1
CXCR1
CXCR2
CXCR3
CXCR4
CXCR5
CXCR6
CXCR7
DRD1
DRD2L
DRD2S
DRD3
DRD4
DRD5
EBI2
EDG1
EDG3
EDG4
EDG5
EDG6
EDG7
EDNRA
EDNRB
F2R
C
F2RL1
F2RL3
FFAR1
FPR1
FPRL1
FSHR
GALR1
GALR2
GCGR
Hit Scaffolds with Differential
Antagonistic Activity Against
GPCR Drug Targets
H
N
N
GHSR
GIPR
GLP1R
GLP2R
GPR1
GPR103
GPR109A
GPR109B
GPR119
GPR120
GPR35
GPR92
GRPR
HCRTR1
HCRTR2
HRH1
MeO C
2
OH
Me
Vincamine (1)
Br
Br
HRH2
HRH3
HRH4
HTR1A
HTR1B
HTR1E
HTR1F
HTR2A
HTR2C
HTR5A
KISS1R
LHCGR
LTB4R
MC1R
H
N
O
N
MC3R
MC4R
MC5R
N
O
MCHR1
MCHR2
MLNR
MRGPRX1
MRGPRX2
MTNR1A
NMBR
NMU1R
NPBWR1
NPBWR2
N
N
N
H
N
CN
N
H
H
O
NPFFR1
Me
N
N
N
7
NPSR1B
NPY1R
NPY2R
NTSR1
OPRD1
OPRK1
OPRL1
OPRM1
OXER1
OXTR
P2RY1
P2RY11
P2RY12
P2RY2
P2RY4
P2RY6
PPYR1
PRLHR
PROKR1
PROKR2
PTAFR
PTGER2
PTGER3
PTGER4
PTGFR
PTGIR
PTHR1
PTHR2
RXFP3
SCTR
SSTR1
SSTR2
SSTR3
SSTR5
TACR1
TACR2
TACR3
TBXA2R
TRHR
TSHR(L)
UTR2
VIPR1
N
H
O
N
3
Me
NH
MeO C
2
5
H
O
OMe
Me
Me
4
Type-2 Diabetes (CNR1)
Gliomas (FPR1)
Heroin Addiction
(HCRTR2)
CNR1: IC50 = 4.95 µM
CNR2: IC50 = 25.7 µM
GPR119: IC50 = 4.62 µM
HRH1: IC50 = 4.16 µM
NPSR1B: IC50 = 3.90 µM
PRLHR: IC50 = 13.8 µM
PTGIR: IC50 = 12.5 µM
Non-small Cell Lung
Carcinoma
CCR8: IC50 = 5.20 µM
FPR1: IC50 = 1.08 µM
NPSR1B: IC50 = 5.77 µM
PRLHR: IC50 = 2.43 µM
HCRTR2: IC50 = 2.26 µM
CHRM4: IC50 = 12.5 µM
DRD2L: 98% inhibition
(20 µM)
Gain-of-Function Biological Activities
Relevant to Human Health and Disease
VIPR2
0%
50%
100%
Figure 2. GPCR screening results (heatmaps) with therapeutic and probe applications for vincamine ring distortion
compounds 3, 4, 5, 7 and 8. (A) Antagonistic activity heatmap of 168 GPCR panel against vincamine, yohimbine and tested
vincamine ring distortion compounds. (B) Focused heatmap of hit compounds and antagonistic activities against GPCRs of
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interest in medicine. (C) Active compounds 3, 4, 5 and 7 display differential GPCR activity profiles compared to vincamine
1) and other ring distortion compounds.
Journal of Medicinal Chemistry
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(
3
Stability concerns regarding the potential acid labile N,N’-carbon (sp ) center adjacent to the 2-position of the
indole nucleus of 4 were probed experimentally. Encouragingly, we found that 4 could be readily interconverted
to the corresponding HCl salt (of the basic amine) and free base forms under mild experimental conditions (see
Supporting Information for HCl salt and free base of 4, including NMR spectra). In addition, analogue 4 proved
to have robust acid stability following 24 hour exposure to acidic media at 37 °C (92.1% recovery in pH 2.0;
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9.2% recovery in pH 4.88; Supp. Info.). With the selective HCRTR2 antagonistic activity and chemical stability
results in hand, we were curious to know if 4 could attenuate opioid-seeking in mouse models using behavioral
pharmacological approaches.
Animal Studies to Determine the Efficacy of 4 (V2a) in Morphine-Seeking Mouse Models.
We evaluated 4 (V2a) to determine the potential for this novel compound to prevent acute morphine conditioned
place preference (CPP) and stress-induced reinstatement of extinguished morphine CPP in mice. The major
problem in treating individuals with opioid dependence is the occurrence of relapse, which is often caused by a
stressful event.29-31 For humans, examples of stressful life events that could lead to relapse (reinstatement)
include the loss of employment, or a relationship; however, in reinstatement experiments in the lab, mice are
subjected to a forced swim test that triggers a stress response that leads to subsequent morphine-seeking
behaviors.
In counterbalanced conditioned place preference experiments in mice32-36, intracerebroventricular (i.c.v.)
treatment of 4 was without significant effect alone, but a 20 minute pretreatment prior to conditioning prevented
acute morphine-CPP (two-way ANOVA, F(3,170)=4.65, p=0.004; Fig. 3A). A similar pretreatment with the parent
vincamine was without significant effect on morphine-CPP (final preference, 219±74s, p=0.998 versus morphine-
CPP, Tukey’s post hoc test), further suggesting the effects of 4’s in vivo efficacy is due to HCRTR2 antagonism.
Pretreatment with 4 also significantly prevented stress-induced reinstatement of extinguished morphine-seeking
behavior (one-way ANOVA, F(6,281)=9.76, p<0.0001; Fig. 3B) compared to vehicle control in mice subjected to a
forced swim test, whereas vincamine did not. Combined, this data clearly indicates that ring distortion can be
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Page 10 of 52
used to re-engineer vincamine’s biological activity and 4 holds promise for further exploration as a potential
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treatment for opioid abuse.
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Figure 3. Results of 4 (V2a) and vincamine in morphine-CPP studies in mouse models of opioid reward and relapse. (A) 4
pretreatment prevents morphine conditioned place preference (CPP) in mice. In place conditioning, mice exhibited
significant morphine-CPP (left bars), but 4 (60 nmol, i.c.v.) did not significantly change initial preferences (center bars). 4,
but not vincamine, pretreatment 20 minutes prior to morphine significantly prevented morphine-CPP. n=16-30 mice/group.
†
*
, p<0.05 vs preconditioning response; , p<0.05 vs morphine-CPP, Tukey post-hoc test. (B) Stress-induced reinstatement
of morphine CPP in mice prevented by 4 pretreatment. Mice exhibited significant morphine CPP, with extinction occurring
by 7 weeks (left bars). Forced swim stress (right bars) reinstated place preference in vehicle or vincamine-treated mice. 4
pretreatment prevented stress-induced reinstatement. n=16-21 mice/group; 58 mice total. *p<0.05 vs preconditioning
response (leftmost bar); †, p<0.05 vs. post CPP (second bar on left); ‡, p<0.05 vs. stress-induced reinstatement (striped
red bar, center), Tukey post-hoc test.
Molecular Modelling of 4 (V2a) in HCRTR2.
We used molecular modelling to determine a binding mode of 4 (V2a) in the antagonistic binding site of HCRTR2.
Our goal was to establish a rational design pathway for future development efforts. Initially, we docked HCRTR2
37
antagonists, suvorexant and EMPA, to their respective binding conformations (suvorexant to PDB ID 4s0v ;
38
EMPA to PDB ID 5wqc ). The re-docking of these antagonists using Glide XP and AutoDock4 generated binding
poses close to crystallographic binding modes both with root-mean-square deviation (rmsd) less than 1.0Å,
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Journal of Medicinal Chemistry
whereas re-docking using Glide SP could not find the crystallographic binding modes both with rmsd larger than
.0Å. Therefore, we found our customized docking protocols with Glide XP and AutoDock4 to be better than
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Glide SP scoring, which was reasonable as the binding site of HCRTR2 is relatively closed with a deep binding
cleft. When compound 4 was docked to both modelled conformations, we found the 4s0v conformation to be
significantly better than 5wqc, since using 1.5Å rmsd threshold, 4s0v yields 11% conformational clustering with
0
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-10.2 kcal/mol calculated binding energy and 5wqc yields 4% conformational clustering with -9.1 kcal/mol. This
result made sense, as suvorexant has structural similarities to 4.
The binding mode of 4 (V2a) to HCRTR2 matches that of suvorexant at several corresponding functional
groups/fragments, which include (1) the indole group of 4 to the benzoxazole group of suvorexant, (2)
azacyclohexyl to diazepam, and (3) triazole to triazole (see Supp. Fig. 5). A major difference is the terminal
benzyl group of 4 binds deeply into an extra subpocket of HCRTR2 (binding model seen in Fig. 4). The indole of
4 binds at a major hydrophobic region (major residues of Pro131, Cys107 and Val138, not shown for clarity).
The triazole of 4 has an aromatic interaction with Tyr317, the same for terminal benzene with Phe227.
Collectively, our data suggests that 4 is an ideal template to develop improved HCRTR2 antagonists using
molecular modelling to rationally design new agents to treat opioid abuse.
Figure 4. Molecular modelling of 4 (V2a) bound to HCRTR2. (Note. PDB ID 4s0v)
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CONCLUSIONS.
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4
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4
4
4
4
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5
5
5
5
5
5
5
6
In conclusion, we have synthesized a diverse collection of complex compounds from vincamine bearing distinct
molecular architectures. During these investigations, we discovered a novel oxidative ring fusion reaction that
led to the discovery of 4 (V2a), which demonstrates HCRTR2 antagonistic activities. As HCRTR2 was recently
27
reported to be relevant in heroin addiction , 4 was advanced to animal studies and demonstrated in vivo efficacy
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in acute and reinstatement mouse models of morphine reward and abuse. In addition, we established a model
for 4 binding to HCRTR2 using molecular docking, which will be utilized to direct future analogue design and
synthesis. These findings further support the therapeutic potential of HCRTR2 antagonists to treat opioid abuse.
Finally, this work demonstrates the significant potential ring distortion has in re-engineering the biological activity
of natural products, such as vincamine, for new applications of critical importance to human health.
EXPERIMENTAL SECTION.
General Information. The purities of all compounds evaluated in biological assays were confirmed to be ≥95%
by LC-MS using a Shimadzu Prominence HPLC system, AB Sciex 3200 QTRAP spectrometer, and a Kinetex
EVO C18 column (50 mm × 2.1 mm × 2.6 µm) with a 30-minute linear gradient from 30-95% acetonitrile in 5 mM
ammonium bicarbonate at a flow rate of 0.25 mL/min. All GPCR screens and concentration-dependent
experiments were conducted at DiscoverX (https://www.discoverx.com/arrestin). All animal experiments were
carried out at the University of Florida and authorized by the Institutional Animal Care and Use Committee
(IACUC) under protocol 201910772. The University of Florida’s accreditation number for animal welfare work is
A3377-01. University of Florida also holds continuous accreditation from AAALAC since 1960.
Chemistry. All reactions were carried out under an atmosphere of argon unless otherwise specified. Chemical
reagents were purchased from commercial sources and used without further purification. Vincamine was
purchased from AK Scientific at >98% purity (~$36 per gram during these studies). Anhydrous solvents were
transferred via syringe to flame-dried glassware, which was cooled under a stream of dry argon. All microwave
reactions were carried out in sealed tubes in an Anton Paar Monowave 300 Microwave Synthesis Reactor. A
constant power was applied to ensure reproducibility. Temperature control was automated via IR sensor and all
indicated temperatures correspond to the maximal temperature reached during each experiment. Analytical thin
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Journal of Medicinal Chemistry
layer chromatography (TLC) was performed using 250 μm Silica Gel 60 F254 pre-coated plates (EMD Chemicals
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5
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6
Inc.). Flash column chromatography was performed using 230-400 Mesh 60Å Silica Gel (Sorbent Technologies).
1
NMR experiments were recorded on the following instruments: Varian Unity spectrometer (400 MHz for H NMR;
13
1
13
1
00 MHz for C NMR), Bruker Avance III spectrometer (500 MHz for H NMR; 125 MHz for C NMR), Bruker
Avance II spectrometer (600 MHz for H NMR; 150 MHz for 13C NMR), and Agilent Systems VNMRS
1
0
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13
spectrometer (600 MHz for H NMR; 150 MHz for C NMR). All spectra are presented using MestReNova 11.0
(Mnova) software and are displayed without the use of the signal suppression function. Spectra were obtained
1
13
1
13
in the following solvents (reference peaks included for H and C NMRs): CDCl ( H NMR: 7.26 ppm; C NMR:
3
1
13
1
13
7
7.23 ppm), d -DMSO ( H NMR: 2.50 ppm; C NMR: 39.52 ppm), CD CN ( H NMR: 1.94 ppm; C NMR: 1.32
6
3
ppm) and d -CD OD ( H NMR: 3.31 ppm; 13C NMR: 49.00 ppm). NMR samples where the respective solvent
1
4
3
1
peaks were buried in the sample signals referenced TMS at 0.00 ppm for H NMR experiments. NMR
experiments were performed at room temperature unless otherwise indicated. Chemical shift values (δ) are
1
13
1
reported in parts per million (ppm) for all H NMR and C NMR spectra. H NMR multiplicities are reported as:
s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, m = multiplet, br = broad.
Select compounds are presented in this section with key signals used for characterization. Compound structures
were converted into IUPAC names using MarvinSketch (ChemAxon; Cambridge, MA). Melting points were
obtained on a Mel-Temp II capillary melting point apparatus and were uncorrected. High-resolution mass spectra
were obtained from the Mass Spectrometry Facility in the Chemistry Department at the University of Florida (UF).
Synthesis procedure for the generation of 2, Methyl 2-[(7S,8R)-3-cyano-7-ethyl-8-[(2-iodophenyl)methoxy]-
1H,2H,3H,4H,5H,6H,7H,8H,9H-azecino[5,4-b]indol-7-yl]acetate. Compound 16 (179 mg, 0.549 mmol) was
added to a round-bottom flask and dissolved in chloroform (11 mL). To the resulting solution was added 2-
iodobenzyl alcohol (374 mg, 1.60 mmol) and then a 3.0 M solution of cyanogen bromide (0.137 mL, 0.412 mmol
in dichloromethane) was added dropwise at room temperature. The reaction was allowed to stir for 25 hours
and, upon completion (by TLC), was quenched with brine, extracted with dichloromethane, and the organic layer
was dried with sodium sulfate. The organic layer was then filtered, concentrated in vacuo, and crude material
purified via column chromatography 100% hexanes to 3:2 hexanes:ethyl acetate to afford 2 (130 mg, 40%) as a
1
o
yellow-brown residue. H NMR: (500 MHz, CDCl at 50 C) δ 10.07 (s, 1H), 7.80 (d, J = 7.9 Hz, 1H), 7.51 (d, J =
3
7
.9 Hz, 1H), 7.48 (d, J = 8.2 Hz, 1H), 7.30 (dd, J = 8.2, 7.5 Hz, 1H), 7.26 – 7.18 (m, 2H), 7.13 (dd, J = 8.3, 7.5
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Hz, 1H), 6.96 (td, J = 7.6, 1.8 Hz, 1H), 4.54 (s, 1H), 4.24 (d, J = 11.6 Hz, 1H), 4.13 (d, J = 11.6 Hz, 1H), 3.70 (m,
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H), 3.58 (m, 1H), 3.54 (s, 3H), 3.21 (dt, J = 15.4, 3.3 Hz, 1H), 3.14 (dt, J = 14.2, 4.9 Hz, 1H), 3.01 – 2.92 (m,
H), 2.45 (d, J = 13.3 Hz, 1H), 2.37 (m, 1H), 2.31 (d, J = 13.4 Hz, 1H), 1.65 – 1.40 (m, 4H), 1.31 (m, 1H), 0.99
2
13
o
(
t, J = 7.5 Hz, 3H). C NMR: (100 MHz, CDCl at 50 C) δ 174.7, 140.3, 139.6, 136.4, 132.9, 130.4, 129.7, 128.2,
3
127.4, 122.8, 119.7, 119.6, 118.4, 112.2, 111.9, 99.3, 78.8, 75.2, 53.6, 53.0, 51.8, 46.0, 38.3, 30.7, 26.9, 26.2,
0
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+
20
o
23.7, 8.2. HRMS (ESI): calc. for C H IN O Na [M + Na] : 432.2258, found: 432.2261. [] : -23 (c 0.10 g/100
28
32
3
3
D
mL, MeOH). Note: The stereochemistry of 2 was determined using 1-D NOESY (see NMR spectrum; Supporting
Information).
Synthesis procedure for the generation of 3, 2-[(1S,12bS)-1-Ethyl-1H,2H,3H,4H,6H,7H,12H,12bH-indolo[2,3-
a]quinolizin-1-yl]-N-cyclopentylacetamide: Compound 12 (138 mg, 0.441 mmol) was added to a flame-dried
o
round-bottom flask and dissolved in anhydrous dichloromethane (4.5 mL). The reaction was cooled to 0 C and
triethylamine (89 µL, 0.882 mmol) was added dropwise. After stirring for five minutes, ethyl chloroformate (47
µL, 0.485 mmol) was added and the reaction continued to stir for an additional 30 minutes. Cyclopentylamine
(0.435 mL, 4.41 mmol; 0.3 M in dichloromethane) and 4-dimethylaminopyridine (DMAP, one crystal) in
anhydrous dichloromethane were then added to the reaction mixture, which was allowed to slowly warm to room
temperature. The reaction continued to stir for three hours and, upon completion (monitored by thin layer
chromatography, TLC), was quenched with brine. The crude reaction mixture was extracted with
dichloromethane, dried with sodium sulfate, filtered, and the organic layer was concentrated in vacuo. The crude
product was then purified via column chromatography using a gradient of 99:1 hexanes:triethylamine to
1
74.5:24.5:1 hexanes:ethyl acetate:triethylamine to afford 3 (152 mg, 91%) as a yellow-white solid. H NMR: (600
MHz, CDCl ) δ 7.93 (s, 1H), 7.46 (d, J = 7.9 Hz, 1H), 7.33 (d, J = 7.7 Hz, 1H), 7.15 (dd, J = 8.3, 7.5 Hz, 1H), 7.09
3
(
dd, J = 8.2, 7.2 Hz, 1H), 5.41 (s, 1H), 3.84 (m, 1H), 3.35 (s, 1H), 3.05 – 2.90 (m, 2H), 2.89 (m, 1H), 2.72 – 2.53
m, 2H), 2.40 (dd, J = 14.4, 11.7 Hz, 1H), 2.12 – 2.01 (m, 2H), 2.00 – 1.87 (m, 3H), 1.81 (m, 1H), 1.68 – 1.36 (m,
(
8
1
2
H), 1.18 (t, J = 7.6 Hz, 3H), 1.15 (m, 1H), 0.97 (m, 1H). 13C NMR: (150 MHz, CDCl ) δ 172.0, 136.2, 133.3,
3
26.8, 121.9, 119.7, 118.1, 111.9, 111.1, 66.8, 57.1, 54.3, 51.2, 41.0, 40.6, 33.5, 33.1, 32.8, 31.7, 23.8, 23.8,
2.4, 22.1, 8.3. HRMS (ESI): calc. for C H N O [M + H] : 380.2696, found: 380.2710. MP: 64-66 C. []D20: -
+
o
24
34
3
o
45 (c 0.11 g/100 mL, MeOH). Note: N-cyclopentylbenzamide was synthesized to assist with the characterization
of the aliphatic region of 3 (Supporting Information).
ACS Paragon Plus Environment

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Journal of Medicinal Chemistry
N3
1
2
3
4
5
6
7
8
9
N
N
N
N
,
CuSO4
N
O
N
N
H
Me
Sodium Ascorbate
N
O
N
(
49%)
H
Me
21
4
Synthesis procedure for the generation of 4, (1S,17S)-20-[(1-Benzyl-1H-1,2,3-triazol-4-yl)methyl]-17-ethyl-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
,
,
,
3
,13,20-triazapentacyclo[11.7.0.0¹ ¹⁷.0² ¹⁰.0⁴ ⁹]icosa-2(10),4,6,8-tetraen-19-one: Anhydrous copper sulfate (66.4
mg, 0.42 mmol) and sodium ascorbate (258 mg, 1.3 mmol) were added to a round-bottom flask and dissolved
in a solution of tert-butanol:water (1:2, 30 mL). Alkyne-containing compound 21 (307 mg, 0.884 mmol) was
added, then benzyl azide (332 µL, 2.7 mmol) as a solution in dichloromethane (2 mL) was added and the reaction
was stirred for 1 hour at room temperature, before heating to 30 °C and allowed to stir for an additional 14 hours.
Upon completion (by TLC), the reaction was quenched with brine, extracted with dichloromethane, and the
organic layer was dried with sodium sulfate. The organic layer was filtered, concentrated in vacuo, and crude
material purified via column chromatography using a gradient of 99:1 hexanes:triethylamine to 24.5:74.5:1
hexanes:dichloromethane:triethylamine. Following the column, the compound was dissolved in chloroform and
1
washed with deionized water to afford 4 (210 mg, 49%) as a yellow solid. H NMR: (500 MHz, CDCl ) δ 9.13 (s,
3
1
H), 7.40 (d, J = 8.9 Hz, 2H), 7.21 – 7.14 (m, 2H), 7.10 – 7.04 (m, 3H), 6.78 (s, 1H), 6.73 (d, J = 6.7 Hz, 2H),
4.68 (d, J = 14.7 Hz, 1H), 4.55 (d, J = 15.1 Hz, 1H), 4.09 (d, J = 15.1 Hz, 1H), 4.05 (d, J = 14.9 Hz, 1H), 3.36 (dt,
J = 12.0, 6.3 Hz, 1H), 3.27 (dt, J = 11.8, 4.8 Hz, 1H), 3.02 – 2.88 (m, 3H), 2.70 – 2.58 (m, 2H), 2.24 (d, J = 16.9
Hz, 1H), 2.03 (dt, J = 13.8, 4.0 Hz, 1H), 1.77 (m, 1H), 1.57 (sextet, J = 7.4 Hz, 1H), 1.44 (td, J = 13.1, 3.7 Hz,
1
1
3
8
H), 1.37 – 1.20 (m, 2H), 0.80 (t, J = 7.4 Hz, 3H). 13C NMR: (100 MHz, CDCl ) δ 173.7, 143.1, 137.2, 134.7,
3
33.0, 128.9, 128.6, 127.9, 126.2, 122.4, 121.6, 119.4, 118.7, 114.0, 111.9, 82.7, 52.9, 50.2, 49.8, 44.7, 43.6,
+
5.3, 32.4, 25.9, 21.8, 17.8, 9.6. HRMS (ESI): calc. for C H N O [M + H] : 481.2710, found: 481.2731. MP: 83-
29
33
6
o
20
o
5 C. [] : +102 (c 0.10 g/100 mL, MeOH).
D
O
Br
H
N
O
Br
N
Cl
TEA, DMAP
59%)
H
Me
H
N
Me
N
(
OMe
O
OMe
O
7
4
5
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Journal of Medicinal Chemistry
Page 16 of 52
Synthesis procedure for the generation of 5, (11S,19R)-15-(3-Bromobenzoyl)-11-ethyl-19-methoxy-8,15-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
,
,
diazatetracyclo[9.6.2.0² ⁷.0⁸ ¹⁸]nonadeca-1(18),2,4,6-tetraen-9-one: Compound 74 (18.2 mg, 0.056 mmol) was
dissolved in dichloromethane (0.5 mL) before triethylamine (16.0 µL, 0.112 mmol) was added. The mixture was
o
cooled to 0 C before 3-bromobenzoyl chloride (8.0 µL, 0.061 mmol, 0.3 M in dichloromethane) and 4-
dimethylaminopyridine (DMAP, one crystal) were added as a solution in dichloromethane. The reaction was
allowed to stir for three hours as it slowly warmed to room temperature. Upon completion (by TLC), the reaction
was quenched with brine, extracted with dichloromethane, and dried with sodium sulfate. The resulting organic
layer was then filtered, concentrated in vacuo, and crude material was purified via column chromatography using
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
a gradient of 100% hexanes to 3:1 hexanes:ethyl acetate to afford 5 (17 mg, 59%) as a white-brown solid. H
o
NMR: (400 MHz, CD CN at 65 C) δ 8.38 (dt, J = 8.2, 0.8 Hz, 1H), 7.52 – 7.44 (m, 3H), 7.39 (td, J = 7.7, 1.1 Hz,
3
1
1
4
1
1
H), 7.27 (td, J = 7.5, 1.0 Hz, 1H), 7.13 (m, 1H), 7.10 (dd, J = 8.0, 7.8 Hz, 1H), 6.74 (d, J = 8.0 Hz, 1H), 4.54 (s,
H), 3.64 (m, 1H), 3.52 – 3.39 (m, 2H), 3.29 (s, 3H), 3.16 (m, 1H), 2.94 (d, J = 17.6 Hz, 1H), 2.71 (dt, J = 14.0,
.9 Hz, 1H), 2.36 (dd, J = 17.6, 1.3 Hz, 1H), 1.90 (m, 1H), 1.68 – 1.57 (m, 4H), 0.89 (t, J = 7.6 Hz, 3H), 0.78 (m,
H). 13C NMR: (100 MHz, CD CN at 65 C) δ 172.7, 170.4, 141.1, 136.4, 134.9, 133.0, 131.4, 130.6, 130.4,
o
3
26.6, 126.1, 125.0, 123.0, 120.6, 119.5, 117.2, 75.8, 56.8, 51.6, 50.2, 44.4, 41.8, 31.6, 27.3, 23.5, 22.5, 8.2.
+
o
20
o
HRMS (ESI): calc. for C H BrN O [M + H] : 509.1434, found: 509.1447. MP: 68-70 C. [] : +52 (c 0.12
27
30
2
3
D
g/100 mL, MeOH).
Synthesis procedure for the generation of 6, Methyl 2-[(1S,7aR,12bS)-1-ethyl-7a-hydroxy-
1H,2H,3H,4H,6H,7H,7aH,12bH-indolo[2,3-a]quinolizin-1-yl]acetate: Compound 16 (1.18 g, 3.61 mmol) was
added to a round-bottom flask and dissolved in anhydrous dichloromethane (132 mL). The resulting solution was
o
cooled to -41 C (acetonitrile/dry ice), and trifluoroacetic acid (5.0 mL, 61.5 mmol) was added. The reaction stirred
for 5 minutes, then m-chloroperbenzoic acid (623 mg, 3.61 mmol) was added as a 0.3 M solution in anhydrous
dichloromethane (12.0 mL) and the reaction stirred for 12 hours. Then, the reaction was quenched with 3 M
aqueous ammonium hydroxide. The neutralized solution was partitioned between dichloromethane and brine,
then the organic layer was dried with sodium sulfate. The organic layer was filtered, concentrated in vacuo, and
crude material purified via column chromatography using a gradient of 99:1 hexanes:triethylamine to 82.5:16.5:1
1
hexanes:ethyl acetate:triethylamine to afford 6 (294 mg, 34%) as a white crystalline solid. H NMR: (400 MHz,
CDCl ) δ 7.49 (d, J = 7.6 Hz, 1H), 7.41 (d, J = 7.0 Hz, 1H), 7.31 (dd, J = 8.2, 7.7 Hz, 1H), 7.18 (dd, J = 8.2, 7.3
3
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Page 17 of 52
Journal of Medicinal Chemistry
Hz, 1H), 4.48 (m, 1H), 3.55 (s, 3H), 3.12 (s, 1H), 3.06 – 2.97 (m, 2H), 2.88 (d, J = 13.5 Hz, 1H), 2.62 (ddd, J =
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
14.4, 9.8, 6.4 Hz, 1H), 2.39 (d, J = 13.5 Hz, 1H), 2.27 (ddd, J = 12.2, 7.8, 6.4 Hz, 1H), 2.10 – 1.95 (m, 3H), 1.92
1
(
m, 1H), 1.74 (sextet, J = 7.5 Hz, 1H), 1.63 (m, 1H), 1.60 – 1.42 (m, 2H), 1.05 (t, J = 7.5 Hz, 3H). Note: H
13
spectrum referenced TMS at 0.00 ppm. C NMR: (100 MHz, CDCl ) δ 183.8, 173.6, 154.1, 139.9, 129.3, 126.2,
3
122.3, 121.0, 82.5, 71.5, 55.6, 51.5, 50.8, 40.7, 38.9, 32.2, 31.3, 29.7, 21.4, 8.1. HRMS (ESI): calc. for
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
o
20
o
C H N O [M + H] : 343.2046, found: 343.2036. MP: 122-124 C. [] : -51 (c 0.12 g/100 mL, MeOH).
20
27
2
3
D
Br
O
O
H N
2
Cl
HN
CN
N
H
Br
TEA, cat. DMAP
75%)
CN
N
N
H
N
(
MeO C
2
Me
MeO C
2
84
Me
7
General
amidation
procedure
for
the
synthesis
of
7,
Methyl
(4aS,12bS)-12-{2-[(4-
bromophenyl)formamido]ethyl}-1-cyano-4a-ethyl-1H,2H,3H,4H,4aH,12bH-indolo[1,2-h]1,7-naphthyridine-6-
carboxylate. Compound 84 (33.5 mg, 0.089 mmol) was added to a flame-dried round-bottom flask and dissolved
o
in dichloromethane (2.0 mL). The solution was then cooled to 0 C, before triethylamine (25.0 µL, 0.177 mmol)
was added dropwise. 4-Bromobenzoyl chloride (21.4 mg, 0.097 mmol) and 4-dimethylaminopyridine (DMAP,
one crystal) in dichloromethane were added dropwise to the reaction. The reaction was allowed to warm to room
temperature and proceeded for 5 hours. Upon completion (by TLC), the reaction was quenched with saturated
aqueous sodium bicarbonate and extracted with dichloromethane. The resulting organic layer was washed with
brine, dried with sodium sulfate, filtered, concentrated in vacuo, and finally purified via column chromatography
1
using a gradient of 100% hexanes to 1:1 hexanes:ethyl acetate to yield 7 (33.8 mg, 75%) as a white solid. H
NMR: (400 MHz, CDCl ) δ 7.63 – 7.57 (m, 3H), 7.51 – 7.46 (m, 2H), 7.24 – 7.17 (m, 2H), 7.13 (td, J = 7.3, 1.4
3
Hz, 1H), 6.84 (t, J = 6.2 Hz, 1H), 6.13 (d, J = 1.7 Hz, 1H), 4.04 (d, J = 1.7 Hz, 1H), 3.97 (s, 3H), 3.90 – 3.75 (m,
2H), 3.35 (ddd, J = 14.6, 7.5, 5.7 Hz, 1H), 3.18 (dt, J = 12.7, 2.1 Hz, 1H), 3.03 – 2.88 (m, 2H), 2.09 (d, J = 14.1
Hz, 1H), 1.87 – 1.62 (m, 2H), 1.40 (td, J = 14.1, 4.0 Hz, 1H), 1.11 – 0.93 (m, 2H), 0.71 (t, J = 7.5 Hz, 3H). 13
C
NMR: (100 MHz, CDCl ) δ 167.0, 163.4, 134.8, 133.2, 131.7, 130.3, 129.1, 128.9, 127.6, 127.2, 126.2, 124.1,
3
121.3, 119.6, 118.0, 117.0, 112.7, 56.5, 52.9, 49.4, 41.1, 39.6, 32.3, 31.4, 24.2, 21.9, 8.0. HRMS (ESI): calc. for
+
o
20
o
C H BrN O Na [M + Na] : 583.1315, found: 583.1299. MP: 83-85 C. [] : -73 (c 0.10 g/100 mL, MeOH).
29
29
4
3
D
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Journal of Medicinal Chemistry
Page 18 of 52
CO2Me
CO Me
2
CO Me
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
N
H
N
H
N
H
H
OMe
N
N
Me
+
Me
+
Me
N
N
N
MeO
OH
MeO2C
MeO C
OH
2
MeO C
O
Me
2
O
8
1
8
OH
54
55
General procedure for the synthesis of 8, 54 and 55: Compound 81 (462 mg, 1.37 mmol) was added to a
flame-dried round-bottom flask and dissolved in chloroform (137 mL). Then, phenol (5.10 g, 55.0 mmol) was
added to the reaction mixture before methyl propiolate (204 µL, 2.06 mmol), which was added dropwise. The
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
o
reaction was heated to 62 C for 7 hours. Upon completion (as monitored by TLC), the reaction was cooled to
room temperature, quenched with saturated aqueous brine, and extracted with dichloromethane. The organic
layer was collected and dried with sodium sulfate, filtered and concentrated in vacuo. The resulting crude material
was then purified via column chromatography using a gradient of 100% hexanes to 3:2 hexanes:ethyl acetate to
afford 8 (247 mg, 35%) as a white solid, 54 (96.2 mg, 14%) as a white solid and 55 (191 mg, 27%) as a white
solid. Note: This reaction gave various product distributions. For example, this reaction was scaled to 3.4 grams
of 81 (10.3 mmol) and, following the same procedure (methyl propriolate: 1.4 mL, 14.4 mmol; phenol: 19.4 g,
2
06 mmol; chloroform, 206 mL), we isolated 2.5 g of 8 (47% yield), 267 mg of 54 (5%) and 637 mg of 55 (12%).
Analogue 8, Methyl (11S,19S)-11-ethyl-19-(4-hydroxyphenyl)-15-[(1E)-3-methoxy-3-oxoprop-1-en-1-yl]-8,15-
,
,
1
diazatetracyclo[9.6.2.0² ⁷.0⁸ ¹⁸]nonadeca-1(18),2,4,6,9-pentaene-9-carboxylate: H NMR: (400 MHz, CDCl at 50
3
oC) δ 7.67 (d, J = 13.3 Hz, 1H), 7.38 (d, J = 7.6 Hz, 1H), 7.24 – 7.09 (m, 3H), 6.93 (d, J = 8.3 Hz, 2H), 6.67 (d, J
=
8.3 Hz, 2H), 6.16 (s, 1H), 5.93 (s, 1H), 4.83 (d, J = 13.3 Hz, 1H), 4.11 (s, 1H), 4.00 (s, 3H), 3.77 (s, 3H), 3.74
m, 1H), 3.52 (dd, J = 14.2, 7.1 Hz, 1H), 3.16 – 2.93 (m, 2H), 2.66 (d, J = 13.6 Hz, 1H), 2.53 (dd, J = 14.8, 8.3
Hz, 1H), 1.78 (dd, J = 14.8, 10.5 Hz, 1H), 1.61 (m, 1H), 1.44 – 1.17 (m, 2H), 1.08 – 0.85 (m, 2H), 0.79 (t, J = 7.3
(
13
o
Hz, 3H). C NMR: (100 MHz, CDCl at 50 C) δ 170.6, 164.6, 155.5, 151.8, 138.0, 135.1, 130.5, 130.3, 130.2,
3
1
29.6, 128.7, 122.6, 121.1, 118.0, 115.5, 112.8, 112.2, 86.8, 59.1, 56.4, 52.8, 51.1, 45.6, 43.3, 34.4, 27.2, 21.8
(
2), 8.2. Note: Characterization of overlapping carbon signals at 21.8 ppm is based on HSQC studies of 58
+
(related analogue; Supporting Information). HRMS (ESI): calc. for C H N O [M + H] : 515.2540, found:
31
35
2
5
o
20
o
5
15.2547. MP: 157-159 C. [] : -134 (c 0.15 g/100 mL, MeOH).
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Page 19 of 52
Journal of Medicinal Chemistry
Analogue 54, Methyl (11S,19S)-11-ethyl-19-(2-hydroxyphenyl)-15-[(1E)-3-methoxy-3-oxoprop-1-en-1-yl]-8,15-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
,
,
1
diazatetracyclo[9.6.2.0² ⁷.0⁸ ¹⁸]nonadeca-1(18),2,4,6,9-pentaene-9-carboxylate: H NMR: (400 MHz, CDCl at 50
3
oC) δ 7.67 (d, J = 13.4 Hz, 1H), 7.38 (d, J = 7.3 Hz, 1H), 7.25 – 7.16 (m, 2H), 7.13 (dd, J = 7.2, 1.3 Hz, 1H), 7.02
–
6.96 (m, 2H), 6.75 (dd, J = 7.9, 0.7 Hz, 1H), 6.72 (dd, J = 8.2, 7.7 Hz, 1H), 6.29 (m, 1H), 6.25 (s, 1H), 5.11 (s,
H), 4.76 (d, J = 13.4 Hz, 1H), 4.02 (s, 3H), 3.74 (s, 3H), 3.71 (m, 1H), 3.61 (dd, J = 14.0, 7.5 Hz, 1H), 3.19 (dd,
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
J = 15.2, 13.8 Hz, 1H), 2.98 (dd, J = 14.1, 13.0 Hz, 1H), 2.67 (d, J = 14.7 Hz, 1H), 2.58 (dd, J = 14.3, 8.4 Hz,
13
1H), 1.93 – 1.79 (m, 3H), 1.40 (m, 1H), 1.10 (m, 1H), 0.94 (m, 1H), 0.82 (t, J = 7.7 Hz, 3H). C NMR: (125 MHz,
o
CDCl at 50 C) δ 170.7, 164.7, 154.0, 152.3, 138.2, 135.3, 130.6, 130.0, 129.9 (2), 128.9, 127.6, 126.3, 122.6,
3
121.0, 118.1, 115.6, 113.4, 112.2, 87.2, 59.5, 57.0, 52.7, 51.0, 44.2, 36.7, 34.7, 24.6, 22.6, 21.7, 8.2. Note:
Carbon signals at 59.5, 57.0, 22.6 and 21.7 ppm are broad and challenging to observe; however, these can be
seen in the HSQC and HSQC-TOCSY spectra (these signals are labeled in each of these corresponding
+
o
spectra). HRMS (ESI): calc. for C H N O [M + H] : 515.2540, found: 515.2549. MP: 240-242 C, decomposed.
31
35
2
5
o
[
]D20: -74 (c 0.10 g/100 mL, CH Cl ).
2
2
Analogue
55,
Methyl
(11S,19R)-11-ethyl-15-[(1E)-3-methoxy-3-oxoprop-1-en-1-yl]-19-phenoxy-8,15-
,
,
1
diazatetracyclo[9.6.2.0² ⁷.0⁸ ¹⁸]nonadeca-1(18),2,4,6,9-pentaene-9-carboxylate: H NMR: (400 MHz, CDCl at 50
3
oC) δ 7.59 (d, J = 13.4 Hz, 1H), 7.41 (dd, J = 7.8, 1.3 Hz, 1H), 7.29 (m, 1H), 7.23 (td, J = 6.9, 1.2 Hz, 1H), 7.20
–
7.12 (m, 3H), 6.96 (m, 1H), 6.88 – 6.83 (m, 2H), 6.35 (d, J = 1.5 Hz, 1H), 5.04 (d, J = 1.5 Hz, 1H), 4.78 (d, J =
3.4 Hz, 1H), 4.00 (s, 3H), 3.77 (s, 3H), 3.67 (dt, J = 13.6, 3.0 Hz, 1H), 3.39 (dd, J = 14.4, 8.3 Hz, 1H), 2.87 (dd,
1
J = 14.1, 12.6 Hz, 1H), 2.77 (dd, J = 15.2, 13.1 Hz, 1H), 2.65 (d, J = 14.4 Hz, 1H), 2.48 (ddd, J = 14.1, 7.8, 2.4
Hz, 1H), 2.00 (sextet, J = 7.5 Hz, 1H), 1.88 (sextet, J = 7.5 Hz, 1H), 1.78 (ddd, J = 14.1, 7.8, 2.4 Hz, 1H), 1.56
13
o
(
m, 1H), 1.06 (t, J = 7.5 Hz, 3H), 0.99 – 0.89 (m, 2H). C NMR: (100 MHz, CDCl at 50 C) δ 169.6, 164.2, 158.1,
3
1
51.3, 135.8, 133.3, 129.8, 129.5, 129.3, 128.0, 123.8, 123.0, 121.0, 119.9, 118.7, 116.0, 112.9, 87.5, 76.0,
9.2, 56.1, 52.6, 50.8, 43.9, 31.8, 26.7, 21.8 (2), 8.2. Note: Characterization of overlapping carbon signals at
5
21.8 ppm is based on HSQC studies of 58 (related analogue; Supporting Information). HRMS (ESI): calc. for
+
o
20
o
C H N O Na [M + Na] : 537.2360, found: 537.2365. MP: 108-110 C. [] : +46 (c 0.21 g/100 mL, CH Cl ).
31
34
2
5
D
2
2
Synthesis
procedure
for
the
generation
of
9,
(1R,12S,19S)-12-Ethyl-9,16-
,
,
,
diazapentacyclo[10.6.1.0¹ ⁹.0³ ⁸.0¹⁶ ¹⁹]nonadeca-3,5,7-triene-2,10-dione. Compound 6 (12.9 mg, 0.038 mmol)
was added to a round-bottom flask and dissolved in methanol (1.5 mL). Sodium hydroxide (8.0 mg, 0.19 mmol)
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 52
o
was added and the reaction was heated to 64 C for 3 hours after being equipped with a reflux condenser. Upon
completion, the reaction was quenched with brine, extracted with chloroform, and the organic layer was dried
with sodium sulfate. The organic layer was filtered, concentrated in vacuo, and crude material purified via column
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
chromatography using
a
gradient of 99:1 hexanes:triethylamine to 74.5:24.5:1 hexanes:ethyl
1
acetate:triethylamine to afford 9 (9.7 mg, 82%) as a white-brown solid. H NMR: (400 MHz, CDCl ) δ 8.29 (d, J
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
=
8.2 Hz, 1H), 7.75 (d, J = 7.7 Hz, 1H), 7.66 (td, J = 7.8, 1.4 Hz, 1H), 7.24 (m, 1H), 3.44 (d, J = 15.6 Hz, 1H),
3.11 (m, 1H), 3.03 (dd, J = 8.8, 7.4 Hz, 1H), 2.53 (s, 1H), 2.49 (m, 1H), 2.30 (td, J = 11.4, 3.1 Hz, 1H), 2.17 (dd,
J = 12.4, 5.5 Hz, 1H), 2.11 (dd, J = 15.6, 2.1 Hz, 1H), 1.95 (td, J = 11.3, 7.8 Hz, 1H), 1.79 – 1.67 (m, 2H), 1.61
(
m, 1H), 1.21 (m, 1H), 0.91 (m, 1H), 0.78 – 0.69 (m, 4H). 13C NMR: (100 MHz, CDCl ) δ 200.9, 170.9, 152.3,
3
136.9, 125.0, 124.5, 122.7, 119.9, 75.5, 68.8, 51.6, 51.0, 40.9, 38.4, 38.3, 32.3, 30.1, 22.3, 7.1. HRMS (ESI):
+
o
20
o
calc. for C H N O [M + H] : 311.1754, found: 311.1756. MP: 115-117 C. [] : +363 (c 0.27 g/100 mL,
19
23
2
2
D
MeOH).
Br
N
CN
N
H
H
N
CNBr, µW
47%)
N
(
MeO C
MeO C
2
2
Me
Me
81
10
Synthesis procedure for the generation of 10, Methyl (4aS,12bS)-12-(2-bromoethyl)-1-cyano-4a-ethyl-
H,2H,3H,4H,4aH,12bH-indolo[1,2-h]1,7-naphthyridine-6-carboxylate. Compound 81 (503 mg, 1.50 mmol) was
1
added to a flame-dried microwave vial and dissolved in N,N-dimethylformamide (15 mL). Then, a 3.0 M solution
of cyanogen bromide in dichloromethane (1.5 mL, 4.50 mmol) was added dropwise to the reaction, which was
o
subsequently subjected to microwave irradiation for six minutes at 100 C. Upon completion of the reaction
(monitored by TLC), the reaction was cooled to room temperature, diluted with ethyl acetate, and quenched with
brine (3 x 100 mL). The organic layer was collected and dried with sodium sulfate, then filtered, and finally
concentrated in vacuo. The crude material was purified via column chromatography using a gradient of 100%
1
chloroform to 99:1 chloroform:acetone to yield 10 (310 mg, 47%) as an orange-white solid. H NMR: (400 MHz,
CDCl ) δ 7.59 (d, J = 7.6, 1H), 7.28 – 7.14 (m, 3H), 6.15 (d, J = 1.5 Hz, 1H), 4.11 (d, J = 1.5 Hz, 1H), 3.93 (s,
3
3H), 3.76 (td, J = 9.5, 7.1 Hz, 1H), 3.63 (td, J = 9.5, 7.1 Hz, 1H), 3.48 – 3.29 (m, 3H), 3.07 (td, J = 12.1, 3.0 Hz,
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Journal of Medicinal Chemistry
1
H), 2.01 (d, J = 13.4 Hz, 1H), 1.79 – 1.56 (m, 2H), 1.30 (td, J = 13.1, 3.9 Hz, 1H), 1.11 – 0.96 (m, 2H), 0.69 (t,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
13
J = 7.5 Hz, 3H). Note: H spectrum referenced TMS at 0.00 ppm. C NMR: (100 MHz, CDCl ) δ 163.3, 134.6,
3
130.0, 128.0, 127.9, 127.6, 123.9, 121.2, 119.2, 117.5, 116.6, 112.7, 56.6, 52.8, 49.9, 39.4, 32.2, 31.5, 31.4,
+
o
28.6, 21.7, 8.1. HRMS (ESI): calc. for C H BrN O Na [M + Na] : 464.0944, found: 464.0959. MP: 115-117 C.
22
24
3
2
]D20: -76 (c 0.24 g/100 mL, CH Cl ).
o
[
2 2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Key Signals and Correlations for 10
3
3
.76 (td, 1H)
.63 (td, 1H)
COSY Correlations
HSQC-TOCSY Correlations
Br
3.35 (m, 2H)
Br
2
Br
Key
3.41 (m, 1H)
3.07 (td, 1H)
CN
1
H
N
CN
CN
N
H
H
N
N
1.70 (m, 2H)
N
N
3
MeO C
2
4
5
Me
MeO2C
2
1
.01 (d, 1H)
.30 (td, 1H)
MeO2C
Me
4.11 (d, 1H)
6.6
Me
5
Key 1H Signals (Black) / Key 13C Signals (Blue)
HMBC Correlations
Br
Br
117.5 ppm
N
C
N
C
N
116.6 ppm
N
Key
N
N
H
H
MeO C
2
MeO C
2
Me
Me
H
H
N
N
KOH, µW
N
O
N
H
(99%)
CO H
2
Me
Me
80
12
Synthesis procedure for the generation of 12, 2-[(1S,12bS)-1-Ethyl-1H,2H,3H,4H,6H,7H,12H,12bH-
indolo[2,3-a]quinolizin-1-yl]acetic acid. Lactam 80 (3.53 g, 12.0 mmol) was reacted with potassium hydroxide in
7
equal portion in a flame-dried microwave vial. Each portion of 80 was dissolved in ethanol (8.5 mL) and a 5.0
M aqueous potassium hydroxide solution (5 mL) was added. The resulting reaction mixture was then subjected
o
to microwave irradiation for 20 minutes at 180 C. The 7 individually reacted portions were then combined in an
Erlenmeyer flask and acidified to pH 5-6. The resulting solution was then extracted with dichloromethane. The
organic layer was collected and then dried with sodium sulfate, filtered, and concentrated in vacuo. The crude
material was purified via column chromatography using a 99:1 dichloromethane:triethylamine to 94:5:1
1
dichloromethane:methanol:triethylamine solvent system to elute, affording 12 (3.80 g, 99%) as a white solid. H
NMR: (400 MHz, d -DMSO) δ 12.59 (s, 1H), 9.86 (s, 1H), 7.46 (d, J = 8.1 Hz, 1H), 7.36 (d, J = 7.5 Hz, 1H), 7.03
6
(dd, J = 8.7, 7.5 Hz, 1H), 6.95 (dd, J = 8.4, 7.4 Hz, 1H), 3.40 (s, 1H), 3.02 – 2.97 (m, 2H), 2.81 – 2.72 (m, 2H),
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Journal of Medicinal Chemistry
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2
.60 – 2.48 (m, 2H), 2.40 (td, J = 11.7, 2.7 Hz, 1H), 2.04 (sextet, J = 7.5 Hz, 1H), 1.95 – 1.82 (m, 2H), 1.77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(sextet, J = 7.5 Hz, 1H), 1.66 (d, J = 14.2 Hz, 1H), 1.59 – 1.48 (m, 2H), 1.06 (t, J = 7.5 Hz, 3H). Note: TMS was
13
referenced at 0.00 ppm for this spectrum. C NMR: (100 MHz, d -DMSO) δ 174.1, 136.6, 132.1, 126.1, 120.8,
6
118.5, 117.2, 111.8, 110.6, 66.1, 56.0, 53.2, 39.2, 38.9, 31.4, 31.1, 21.7, 21.5, 8.4. HRMS (ESI): calc. for
+
o
o
21
20
o
C H N O [M + H] : 313.1911, found: 313.1918. MP: 204-206 C, lit. 203-206 C. [] : -207 (c 0.13 g/100
19 25
2
2
D
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
mL, CH Cl ).
2
2
Synthesis procedure for the generation of 16, Methyl 2-[(1S,12bS)-1-ethyl-1H,2H,3H,4H,6H,7H,12H,12bH-
indolo[2,3-a]quinolizin-1-yl]acetate. Compound 12 (1.51 g, 4.84 mmol) was added to a round-bottom flask and
o
dissolved in anhydrous N,N-dimethylformamide (48 mL). The solution was cooled to 0 C, then anhydrous
potassium carbonate (1.34 g, 9.69 mmol) and iodomethane (0.362 mL, 5.81 mmol) were added sequentially.
The resulting reaction mixture warmed slowly to room temperature and reacted for 3 hours. Upon completion by
TLC, the reaction was quenched with brine, extracted with ethyl acetate, and the organic layer was washed with
deionized water three times (~ 1 L). The organic layer was dried with sodium sulfate, filtered, concentrated in
vacuo, and crude material purified via column chromatography using a gradient of 99:1 hexanes:triethylamine to
1
82.5:16.5:1 hexanes:ethyl acetate:triethylamine to afford 16 (1.18 g, 75%) as a brown solid. H NMR: (400 MHz,
CDCl ) δ 7.83 (s, 1H), 7.47 (d, J = 7.8 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.15 (td, J = 8.0, 1.3 Hz, 1H), 7.09 (td,
3
J = 8.0, 1.1 Hz, 1H), 3.49 (s, 3H), 3.38 (m, 1H), 3.09 – 2.97 (m, 3H), 2.89 (m, 1H), 2.68 – 2.53 (m, 2H), 2.40 (td,
13
J = 12.5, 2.8 Hz, 1H), 2.06 – 1.86 (m, 3H), 1.82 (m, 1H), 1.67 – 1.55 (m, 3H), 1.18 (t, J = 7.7 Hz, 3H). C NMR:
(100 MHz, CDCl ) δ 173.7, 136.1, 132.6, 126.8, 121.7, 119.4, 117.9, 112.4, 110.9, 66.3, 56.9, 54.0, 51.2, 40.4,
3
+
38.1, 32.3, 31.4, 22.2, 22.1, 8.2. HRMS (ESI): calc. for C H N O [M + H] : 327.2067, found: 327.2074. MP:
20 27 2 2
o
20
o
127-129 C. []D : -52 (c 0.74 g/100 mL, CH Cl ).
2
2
CO Me
2
O
N
H
H
OMe
MeOH
N
N
Me
N
O
Me
OMe
O
80
17
Synthesis procedure for the generation of 17, Methyl (2E)-3-[(11S,19R)-11-ethyl-19-methoxy-9-oxo-8,15-
,
,
diazatetracyclo[9.6.2.0² ⁷.0⁸ ¹⁸]nonadeca-1(18),2,4,6-tetraen-15-yl]prop-2-enoate. Lactam 80 (38.9 mg, 0.132
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Page 23 of 52
Journal of Medicinal Chemistry
mmol) was added to a round-bottom flask and dissolved in methanol (13 mL). Methyl propiolate (18 µL, 0.198
mmol) was then added dropwise and the reaction stirred at room temperature for 5 minutes before heating to
reflux for 22 hours. Upon completion (by TLC), the reaction was quenched with saturated aqueous brine and
extracted with ethyl acetate. The organic layer was collected, dried with sodium sulfate, filtered and concentrated
in vacuo. The crude material was purified via column chromatography using a gradient of 100% hexanes to 2:1
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
o
hexanes:ethyl acetate to afford 17 (44.9 mg, 83%) as a white solid. H NMR: (400 MHz, CDCl at 50 C) δ 8.46
3
(d, J = 7.4 Hz, 1H), 7.55 (d, J = 13.3 Hz, 1H), 7.48 (dd, J = 7.4, 0.7 Hz, 1H), 7.39 (td, J = 7.4, 0.7 Hz, 1H), 7.32
(td, J = 7.4, 0.7 Hz, 1H), 4.80 (d, J = 13.4 Hz, 1H), 4.14 (s, 1H), 3.72 (m, 1H), 3.71 (s, 3H), 3.55 (dd, J = 14.2,
6
1
7
1
.8 Hz, 1H), 3.21 (s, 3H), 3.14 (td, J = 14.1, 2.6 Hz, 1H), 3.01 (d, J = 17.3 Hz, 1H), 2.93 (m, 1H), 2.38 (d, J =
7.4 Hz, 1 H), 2.33 (m, 1H), 1.94 (sextet, J = 7.6 Hz, 1H), 1.88 – 1.69 (m, 3H), 1.62 – 1.52 (m, 2H), 0.87 (t, J =
.7 Hz, 3H), 0.84 (m, 1H). 13C NMR: (100 MHz, CDCl at 50 C) δ 169.6, 169.4, 150.4, 135.5, 133.7, 128.9,
o
3
26.0, 124.1, 118.9, 118.2, 117.2, 87.9, 75.5, 58.9, 56.5, 55.8, 50.9, 43.6, 40.9, 34.1, 26.6, 22.4, 22.0, 7.9. HRMS
+
o
20
o
(
ESI): calc. for C H N O [M + H] : 411.2278, found: 411.2269. MP: 116-118 C. [] : +37 (c 0.15 g/100 mL,
24
31
2
4
D
CH Cl ). Note: X-ray data of 17 can be found in the Supporting Information.
2
2
CO Me
CO Me
2
2
O
N
H
N
H
H
OMe
OH
Me
N
N
Me
Me
+
Me
Me
MeO
N
N
MeO C
HO
OH
2
O
Me
Me
O
OH
O
MeO C
2
Me
Me
1
18
63
Synthesis procedure for the generation of 18 and 63. Vincamine 1 (100 mg, 0.282 mmol) was added to a
flame-dried round-bottom flask and dissolved in isopropanol (28 mL). Methyl propiolate (41 µL, 0.462 mmol) was
then added dropwise to the mixture and the reaction was heated to reflux for 2 hours. Upon completion, the
reaction was cooled, then concentrated in vacuo. Finally, the crude material was purified via column
chromatography using a gradient of 100% hexanes to 3:1 hexanes:ethyl acetate to afford 18 (84.4 mg, 60%) as
a colorless solid and V3m (35.7 mg, 25%) as a clear-brown residue.
Analogue 18, Methyl (9R,11S,19R)-11-ethyl-9-hydroxy-15-[(1E)-3-methoxy-3-oxoprop-1-en-1-yl]-19-(propan-2-
,
,
1
yloxy)-8,15-diazatetracyclo[9.6.2.0² ⁷.0⁸ ¹⁸]nonadeca-1(18),2,4,6-tetraene-9-carboxylate: H NMR: (400 MHz,
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o
CDCl at 50 C) δ 7.56 – 7.50 (m, 2H), 7.20 – 7.11 (m, 2H), 7.08 (m, 1H), 4.95 (s, 1H), 4.75 (d, J = 13.3 Hz, 1H),
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
.41 (s, 1H), 3.83 (s, 3H), 3.73 (m, 1H), 3.67 (s, 3H), 3.51 (septet, J = 5.9 Hz, 1H), 3.41 (dd, J = 14.0, 8.1 Hz,
H), 3.11 (m, 1H), 3.04 – 2.94 (m, 2H), 2.50 (ddd, J = 14.1, 8.1, 2.0 Hz, 1H), 2.44 (d, J = 14.7 Hz, 1H), 2.27 (d,
1
J = 14.6 Hz, 1H), 1.87 (sextet, J = 7.5 Hz, 1H), 1.70 – 1.53 (m, 3H), 1.39 (m, 1H), 1.27 (m, 1H), 1.15 (d, J = 5.9
13
o
Hz, 3H), 0.92 (d, J = 5.9 Hz, 3H), 0.86 (t, J = 7.5 Hz, 3H). C NMR: (100 MHz, CDCl at 50 C) δ 172.0, 169.6,
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
151.3, 135.4, 134.6, 127.6, 123.0, 120.3, 118.5, 111.9, 111.0, 86.7, 82.3, 71.8, 68.9, 58.0, 56.1, 53.4, 50.6, 46.5,
41.8, 34.1, 29.4, 23.4, 22.3 (2), 21.2, 8.1. Note: The presence of two carbon signals at 22.3 ppm was elucidated
+
by a HSQC experiment. HRMS (ESI): calc. for C H N O Na [M + Na] : 521.2622, found: 521.2628. MP: 158-
28
38
2
6
o
20
o
160 C. [] : +62 (c 0.27 g/100 mL, CH Cl ).
D
2
2
Key Signals and Correlations for 18
3
3
.73 (m, 1H)
.00 (m, 1H)
CO Me 3.41 (dd, 1H)
2
2.50 (ddd, 1H)
3
3
.11 (m, 1H)
.00 (m, 1H)
N
H
1.61 (m, 1H)
1.39 (m, 1H)
1.59 (m, 1H)
1.27 (m, 1H)
Me
N
MeO C
2
4
71.8
.41 (s, 1H)
i
O Pr
OH
Key 1H Signals (Black or Red), Key 13C Signals (Blue)
COSY Correlations
NOE Correlation
HSQC Correlations
CO Me
2
CO Me
2
N
N
H
H
Me
N
Me
MeO C
N
2
Me
Me
MeO C
2
O
HO
NOE
i
O Pr
HO
Analogue 63, Methyl (9S,11S,19R)-11-ethyl-9-hydroxy-15-[(1E)-3-methoxy-3-oxoprop-1-en-1-yl]-19-(propan-2-
,
,
1
yloxy)-8,15-diazatetracyclo[9.6.2.0² ⁷.0⁸ ¹⁸]nonadeca-1(18),2,4,6-tetraene-9-carboxylate: H NMR: (400 MHz,
o
CDCl at 50 C) δ 7.61 (d, J = 13.3 Hz, 1H), 7.51 (m, 1H), 7.22 – 7.12 (m, 3H), 4.82 (d, J = 13.3 Hz, 1H), 4.55 (s,
3
1
H), 4.40 (s, 1H), 3.80 (s, 3H), 3.72 (m, 1H), 3.71 (s, 3H), 3.59 – 3.47 (m, 2H), 3.16 (dd, J = 15.7, 13.8 Hz, 1H),
.00 – 2.81 (m, 2H), 2.85 (d, J = 13.7 Hz, 1H), 2.30 (dd, J = 13.8, 9.2 Hz, 1H), 1.93 – 1.70 (m, 4H), 1.60 – 1.49
3
(m, 2H), 1.22 (dd, J = 15.9, 7.9 Hz, 1H), 1.14 (d, J = 6.0 Hz, 3H), 0.90 (d, J = 6.1 Hz, 3H), 0.86 (t, J = 7.6 Hz,
13
o
3H). C NMR: (100 MHz, CDCl at 50 C) δ 175.0, 169.7, 150.6, 135.6, 135.2, 128.3, 122.9, 120.5, 118.5, 112.6,
3
112.3, 87.3, 83.2, 70.3, 67.5, 59.4, 56.7, 54.0, 50.7, 41.0, 40.9, 33.2, 27.3, 23.7, 22.0 (2), 21.3, 8.1. Note:
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Journal of Medicinal Chemistry
Justification for the overlapping carbon signals at 22.0 ppm is based on HSQC studies of 18. HRMS (ESI): calc.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
20
o
for C H N O Na [M + Na] : 521.2622, found: 521.2630. [] : +58 (c 0.10 g/100 mL, CH Cl ).
28
38
2
6
D
2
2
Key Signals and Correlations for 63
CO Me 3.55 (m, 1H)
3.72 (m, 1H)
2
2.30 (dd, 1H)
2.89 (m, 1H)
1.74 (m, 1H)
.52 (m, 1H)
3
.16 (dd, 1H)
N
H
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2.91 (m, 1H)
1
.85 (m, 1H)
1.22 (dd, 1H)
Me
N
HO
i
O Pr
4.40 (s, 1H)
67.5
MeO C
2
Key 1H Signals (Black)
Key 13C Signals (Blue)
CO Me
2
CO Me
2
N
H
N
H
Me
N
Me
HO
N
Me
Me
HO
O
i
O
O Pr
MeO C
O
2
Me
COSY Correlations
2-D NOE Correlations (red)
HSQC Correlations
HO
H N
H N
PIFA
O
MeCN:H O (2:1)
O
N
H
2
N
Ph
Ph
(26%)
Me
HN
19 Me
HN
28
Synthesis
procedure
for
the
generation
of
19,
2-[(1S,7aR,12bS)-1-Ethyl-7a-hydroxy-
1H,2H,3H,4H,6H,7H,7aH,12bH-indolo[2,3-a]quinolizin-1-yl]-N-benzylacetamide: Compound 28 (403 mg, 1.00
mmol) was added to a round-bottom flask and dissolved in acetonitrile:water (2:1, 30 mL). The resulting solution
o
was cooled to 0 C, and a 0.3 M solution of [bis(trifluoroacetoxy)iodo]benzene (PIFA; 860 mg, 2.00 mmol) in
o
acetonitrile was added slowly via syringe over 5 minutes. The reaction stirred for 3 hours at 0 C, then the reaction
was quenched with cold saturated aqueous sodium bicarbonate. The mixture was then extracted with
dichloromethane, washed with brine, and the organic layer was dried with sodium sulfate. The organic layer was
filtered, concentrated in vacuo, and crude material purified via column chromatography using a gradient of 99:1
hexanes:triethylamine to 49.5:49.5:1 hexanes:ethyl acetate:triethylamine to afford 19 (109 mg, 26%) as a brown
1
residue. H NMR: (500 MHz, CDCl ) δ 7.49 (d, J = 7.5 Hz, 1H), 7.41 (d, J = 7.2 Hz, 1H), 7.32 (dd, J = 8.2, 7.7
3
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Journal of Medicinal Chemistry
Page 26 of 52
Hz, 1H), 7.29 – 7.16 (m, 4H), 7.12 (d, J = 7.4 Hz, 2H), 6.09 (t, J = 5.3 Hz, 1H), 4.36 (dd, J = 14.6, 5.9 Hz, 1H),
.87 (dd, J = 14.4, 5.0 Hz, 1H), 3.12 (s, 1H), 3.08 – 2.99 (m, 2H), 2.65 (m, 1H), 2.60 (d, J = 14.5 Hz, 1H), 2.40
d, J = 13.6 Hz, 1H), 2.30 (ddd, J = 12.2, 7.1, 1.8 Hz, 1H), 2.13 – 1.82 (m, 5H), 1.65 (m, 1H), 1.54 (ddd, J = 13.9,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
(
13
9
.9, 3.3 Hz, 1H), 1.47 (td, J = 13.1, 3.9 Hz, 1H), 1.09 (t, J = 7.4 Hz, 3H). C NMR: (100 MHz, CDCl ) δ 184.1,
3
171.9, 154.1, 139.9, 138.3, 129.3, 128.7, 128.0, 127.4, 126.2, 122.3, 121.0, 82.6, 72.0, 55.7, 50.9, 43.6, 41.0,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
40.8, 32.9, 32.2, 29.9, 21.4, 8.3. HRMS (ESI): calc. for C H N O [M + H] : 418.2489, found: 418.2484. []D20:
+
26
32
3
2
o
+16 (c 0.37 g/100 mL, CH Cl ). NOTE: COSY, 1-D ROESY were used to determine the relative stereochemistry
2
2
of 19 (see Supporting Information).
Key Correlations for 19
COSY Correlations
1-D ROESY Correlations
D
HO
H
E
HO
N
H
H
N
D
H
H
C
N
MeB
HN
H
O
N
A
H E
H
N
Me
O
A
O
O
H
NH
N
HC
NH
MeB
Side-View (19)
D
A
B
Chem3D Minimized Model of 19
E
C
H
N
N
N
m-CPBA
N
H
O
N
H
Me
TFA
O
Me
HN
30
20
General procedure for the oxidative ring fusion synthesis of 20-25 and 46-52 from corresponding amide
precursors (e.g., 30): Amide 30 (44.6 mg, 0.127 mmol) was added to a flame-dried round-bottom flask and
o
dissolved in anhydrous dichloromethane (5.1 mL). The reaction was cooled to 0 C, and trifluoroacetic acid
(0.175 mL, 2.16 mmol) was added. The resultant solution stirred for five minutes, then m-chloroperoxybenzoic
acid (21.9 mg, 0.127 mmol) was added as a 0.3 M solution in anhydrous dichloromethane. The reaction was
then refluxed for 24 hours. Upon completion, the reaction was quenched with 3 M aqueous ammonium hydroxide.
The resulting mixture was then extracted with dichloromethane, washed with brine, and the organic layer was
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Journal of Medicinal Chemistry
dried with sodium sulfate. The organic layer was filtered, concentrated in vacuo, and crude material purified via
column chromatography using a gradient of 99:1 hexanes:triethylamine to 79.5:19.5:1 hexanes:ethyl
acetate:triethylamine to afford ring-fusion product 20 (18.5 mg, 42%) as a green-yellow residue.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
,
,
,
Analogue
20,
(1S,17S)-20-Cyclopropyl-17-ethyl-3,13,20-triazapentacyclo[11.7.0.0¹ ¹⁷.0² ¹⁰.0⁴ ⁹]icosa-
1
2
(10),4,6,8-tetraen-19-one. Yield: 42%; 18.5 mg 20; green-yellow residue. H NMR: (400 MHz, CDCl ) δ 9.47
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(s, 1H), 7.55 (d, J = 8.2 Hz, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.17 (td, J = 7.4, 1.0 Hz, 1H), 7.09 (dd, J = 8.2, 7.5 Hz,
1
H), 3.53 (ddd, J = 11.6, 9.1, 4.5 Hz, 1H), 3.21 – 3.10 (m, 2H), 3.04 (m, 1H), 3.03 (d, J = 16.7 Hz, 1H), 2.89 –
.72 (m, 2H), 2.17 (d, J = 17.1 Hz, 1H), 2.14 (m, 1H), 1.94 (m, 1H), 1.81 (m, 1H), 1.59 – 1.35 (m, 3H), 1.13
2
(sextet, J = 7.5 Hz, 1H), 0.80 (m, 1H; buried under triplet), 0.78 (t, J = 7.5 Hz, 3H), 0.55 (m, 1H), 0.39 – 0.22 (m,
2H). 13C NMR: (100 MHz, CDCl ) δ 175.5, 136.9, 133.3, 126.2, 122.1, 119.2, 118.3, 113.5, 112.2, 84.6, 50.4,
3
+
49.9, 44.5, 43.7, 33.1, 26.7, 24.7, 22.6, 20.0, 9.4, 6.8, 5.4. HRMS (ESI): calc. for C H N O [M + H] : 350.2227,
22
28
3
found: 350.2213. []D20: +92 (c 0.22 g/100 mL, CH Cl ).
o
2
2
,
,
,
Analogue
21,
(1S,17S)-17-Ethyl-20-(prop-2-yn-1-yl)-3,13,20-triazapentacyclo[11.7.0.0¹ ¹⁷.0² ¹⁰.0⁴ ⁹]icosa-
1
2
1
8
(10),4,6,8-tetraen-19-one. Yield: 56%; 27.5 mg 21; off-white residue. H NMR: (400 MHz, CDCl ) δ 9.63 (s,
3
H), 7.66 (dt, J = 8.1, 0.8 Hz, 1H), 7.53 (d, J = 7.9 Hz, 1H), 7.20 (ddd, J = 7.9, 6.9, 1.2 Hz, 1H), 7.11 (ddd, J =
.1, 6.9, 0.8 Hz, 1H), 3.94 (dd, J = 17.3, 2.6 Hz, 1H), 3.61 (dd, J = 17.3, 2.6 Hz, 1H), 3.47 – 3.34 (m, 2H), 3.25
(m, 1H), 3.16 (d, J = 17.0 Hz, 1H), 3.01 (dt, J = 12.8, 4.2 Hz, 1H), 2.94 – 2.79 (m, 2H), 2.31 (d, J = 17.0 Hz, 1H),
2.05 (m, 1H), 1.98 (t, J = 2.6 Hz, 1H), 1.88 (m, 1H), 1.68 – 1.52 (m, 2H), 1.47 (m, 1H), 1.33 (sextet, J = 7.6 Hz,
1H), 0.86 (t, J = 7.6 Hz, 3H). 13C NMR: (100 MHz, CDCl ) δ 174.4, 137.5, 131.6, 126.0, 122.4, 119.2, 118.2,
3
114.4, 112.4, 83.6, 78.9, 70.8, 50.1, 49.6, 43.9, 43.7, 32.8, 29.8, 27.1, 22.2, 18.0, 9.5. HRMS (ESI): calc. for
+
20
o
C H N O [M + H] : 348.2070, found: 348.2075. [] : +22 (c 0.23 g/100 mL, CH Cl ).
22
26
3
D
2
2
,
,
,
Analogue
22,
(1S,17S)-20-Butyl-17-ethyl-3,13,20-triazapentacyclo[11.7.0.0¹ ¹⁷.0² ¹⁰.0⁴ ⁹]icosa-2(10),4,6,8-
1
tetraen-19-one. Yield: 43%; 27.0 mg 22; light green solid. H NMR: (400 MHz, CDCl ) δ 9.41 (s, 1H), 7.54 –
3
7
.47 (m, 2H), 7.16 (td, J = 8.2, 1.1 Hz, 1H), 7.08 (td, J = 7.9, 0.9 Hz, 1H), 3.39 (m, 1H), 3.25 – 3.10 (m, 2H), 3.10
2.91 (m, 4H), 2.88 – 2.74 (m, 2H), 2.19 (d, J = 16.8 Hz, 1H), 2.02 (m, 1H), 1.82 (m, 1H), 1.62 – 1.45 (m, 2H),
–
13
1.45 – 1.11 (m, 4H), 1.06 – 0.92 (m, 2H), 0.80 (t, J = 7.5 Hz, 3H), 0.59 (t, J = 7.3 Hz, 3H). C NMR: (100 MHz,
CDCl ) δ 174.6, 137.3, 133.1, 126.2, 122.2, 119.2, 118.2, 113.5, 112.1, 83.5, 50.3, 50.0, 44.4, 43.7, 41.3, 32.8,
3
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Journal of Medicinal Chemistry
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+
3
0.0, 26.6, 22.4, 20.7, 18.3, 13.7, 9.6. HRMS (ESI): calc. for C H N O [M + H] : 366.2540, found: 366.2547.
23 32 3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
o
20
o
MP: 170-172 C. [] : +48 (c 0.17 g/100 mL, CH Cl ).
D
2
2
,
,
,
Analogue
23,
(1S,17S)-20-Cyclopentyl-17-ethyl-3,13,20-triazapentacyclo[11.7.0.0¹ ¹⁷.0² ¹⁰.0⁴ ⁹]icosa-
1
2
(10),4,6,8-tetraen-19-one. Yield: 37%; 24.0 mg 23; off-white solid. H NMR: (400 MHz, CDCl ) δ 9.18 (s, 1H),
3
7
.50 (d, J = 7.8 Hz, 1H), 7.45 (d, J = 8.1 Hz, 1H), 7.17 (dd, J = 8.3, 7.2 Hz, 1H), 7.08 (dd, J = 8.1, 7.3 Hz, 1H),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3.66 (p, J = 8.9, 1H), 3.58 (ddd, J = 11.7, 8.1, 2.2 Hz, 1H), 3.25 – 3.12 (m, 2H), 3.00 – 2.86 (m, 2H), 2.94 (d, J =
16.7 Hz, 1H), 2.78 (dt, J = 15.2, 4.2 Hz, 1H), 2.46 (m, 1H), 2.09 (d, J = 16.5 Hz, 1H), 2.01 (d, J = 13.8, 1H), 1.90
–
3
5
1.71 (m, 4H), 1.66 – 1.44 (m, 3H), 1.43 – 1.29 (m, 2H), 1.23 – 1.04 (m, 2H), 0.82 (m, 1H), 0.79 (t, J = 7.4 Hz,
H). 13C NMR: (125 MHz, CDCl ) δ 173.4, 137.2, 133.3, 126.3, 122.3, 119.3, 118.3, 113.5, 111.9, 83.6, 52.9,
3
0.0, 49.8, 45.3, 43.3, 32.4, 28.7, 28.0, 26.8, 25.3, 24.8, 22.2, 17.1, 9.7. HRMS (ESI): calc. for C H N O [M +
2
4
32
3
+
o
20
o
H] : 378.2540, found: 378.2546. MP: 210-212 C. [] : +22 (c 0.20 g/100 mL, CH Cl ).
D
2
2
Key Signals and
Correlations for 24
3.32 (ddd, 1H)
3.13 (m, 1H)
2
.63 (m, 1H)
2.53 (td, 1H)
2
2
.86 (ddd, 1H)
.78 (dt, 1H)
N
N
O
N
H
1
.74 (m, 1H)
1.26 (m, 1H)
84.5
Key 1H Signals (Black)
Key 13C Signals (Blue)
Me
1
1
.99 (d, 1H)
.49 (td, 1H)
N
N
N
N
N
N
O
O
O
N
N
N
H
Me
H
H
Me
Me
COSY Correlations
HMBC Correlations
HSQC Correlations
,
,
,
Analogue 24, (1S,17S)-20-Benzyl-17-ethyl-3,13,20-triazapentacyclo[11.7.0.0¹ ¹⁷.0² ¹⁰.0⁴ ⁹]icosa-2(10),4,6,8-
1
tetraen-19-one. Yield: 44%; 20.0 mg 24; light green solid. H NMR: (400 MHz, CDCl ) δ 8.52 (s, 1H), 7.46 (m,
3
1H), 7.21 – 7.17 (m, 3H), 7.15 – 6.98 (m, 5H), 4.43 (d, J = 14.6 Hz, 1H), 3.87 (d, J = 14.7 Hz, 1H), 3.32 (ddd, J
= 11.6, 6.5, 5.1 Hz, 1H), 3.13 (m, 1H), 2.92 (d, J = 17.0 Hz, 1H), 2.86 (ddd, J = 14.8, 7.9, 5.5 Hz, 1H), 2.78 (dt,
J = 15.2, 4.6 Hz, 1H), 2.63 (m, 1H), 2.53 (ddd, J = 13.3, 10.2, 3.2 Hz, 1H), 2.31 (d, J = 16.9 Hz, 1H), 1.99 (m,
13
1
H), 1.72 (m, 1H), 1.57 (sextet, J = 7.2 Hz, 1H), 1.49 (m, 1H), 1.33 – 1.15 (m, 2H), 0.81 (t, J = 7.2 Hz, 3H). C
NMR: (100 MHz, CDCl ) δ 175.2, 137.8, 137.4, 132.5, 128.7, 128.1, 126.8, 126.0, 122.3, 119.1, 118.2, 114.3,
3
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Journal of Medicinal Chemistry
+
1
12.1, 84.5, 49.9, 49.6, 44.8, 44.3, 43.6, 32.8, 27.2, 22.3, 17.7, 9.6. HRMS (ESI): calc. for C H N O [M + H] :
26 30 3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
o
20
o
4
00.2383, found: 400.2393. MP: 90-92 C. [] : +24 (c 0.20 g/100 mL, CH Cl ).
D
2
2
,
Analogue
25,
(1S,17S)-17-Ethyl-20-(2-methoxyethyl)-3,13,20-triazapentacyclo[11.7.0.0¹ ¹⁷.0² ¹⁰.0⁴ ⁹]icosa-
,
,
1
2
(10),4,6,8-tetraen-19-one. Yield: 41%; 18.5 mg 25; white solid. H NMR: (500 MHz, CDCl ) δ 8.60 (s, 1H), 7.49
3
(
d, J = 7.8 Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.18 (ddd, J = 8.2, 7.0, 1.2 Hz, 1H), 7.09 (dd, J = 8.1, 7.5 Hz, 1H),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3.54 (dt, J = 13.9, 7.1 Hz, 1H), 3.48 (ddd, J = 11.8, 7.2, 4.9 Hz, 1H), 3.21 – 3.03 (m, 5H), 2.97 (s, 3H), 2.95 (m,
1H), 2.89 – 2.75 (m, 2H), 2.87 (d, J = 16.8 Hz, 1H), 2.19 (d, J = 16.7 Hz, 1H), 2.00 (dt, J = 14.0, 3.7 Hz, 1H),
1
.83 (m, 1H), 1.57 (sextet, J = 7.4 Hz, 1H), 1.48 (td, J = 13.4, 3.7 Hz, 1H), 1.35 (dp, J = 13.3, 4.2 Hz, 1H), 1.17
13
(
sextet, J = 7.4 Hz, 1H), 0.78 (t, J = 7.4 Hz, 3H). C NMR: (150 MHz, CDCl )δ 174.6, 137.5, 133.6, 126.6, 122.7,
3
1
19.6, 118.5, 113.9, 112.0, 82.9, 69.4, 58.6, 49.9, 49.9, 44.4, 43.8, 39.9, 32.4, 26.7, 22.2, 17.6, 9.6. HRMS (ESI):
+
o
20
o
calc. for C H N O [M + H] : 368.2333, found: 368.2343. MP: 194-196 C, decomposed. [] : +61 (c 0.20
22
30
3
2
D
g/100 mL, CH Cl ). X-ray: Structure and supporting data can be viewed in the Supporting Information.
2
2
Key Signals and
Correlations for 25
3
3
.48 (ddd, 1H)
.18 (m, 1H)
OMe
3
2
.09 (m, 1H)
.95 (m, 1H)
2.84 (m, 1H)
.78 (m, 1H)
2
N
N
O
N
H
1
1
.83 (m, 1H)
.35 (dp, 1H)
8
2.9
Key 1H Signals (Black)
Key 13C Signals (Blue)
Me
2.00 (dt, 1H)
1.48 (td, 1H)
COSY/TOCSY
Correlations
HMBC Correlations
OMe
HSQC Correlations
OMe
OMe
N
N
N
N
N
N
O
O
O
N
H
Me
N
H
N
H
Me
Me
Synthesis procedure for the generation of 24 from 19: Compound 19 (26.0 mg, 0.062 mmol) was added to
a flame-dried round-bottom flask and dissolved in anhydrous dichloromethane (2.5 mL). The reaction was cooled
o
to 0 C, and trifluoroacetic acid (0.085 mL, 1.05 mmol) was added. The reaction was slowly warmed to reflux
and allowed to react for 2 hours. Upon completion, the reaction was quenched with 3 M aqueous ammonium
hydroxide. The resulting mixture was then extracted with dichloromethane, washed with brine, and the organic
layer was dried with sodium sulfate, filtered and concentrated in vacuo. The crude product was then purified via
column chromatography using a gradient of 99:1 hexanes:triethylamine to 79.5:19.5:1 hexanes:ethyl
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