Synthesis and Cytoxicity of Sempervirine and Analogues

Article abstract of DOI:10.1021/acs.joc.6b00022

Sempervirine and analogues were synthesized using a route featuring Sonogashira and Larock Pd-catalyzed reactions. Structure-activity relationships were investigated using three human cancer cell lines. 10-Fluorosempervirine is the most potently cytotoxic member of the family yet described.

Full text of DOI:10.1021/acs.joc.6b00022

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Note
Synthesis and Cytoxicity of Sempervirine and Analogues
Xiaohong Pan, Chunying Yang, John L. Cleveland, and Thomas D. Bannister
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b00022 • Publication Date (Web): 01 Feb 2016
Downloaded from http://pubs.acs.org on February 2, 2016
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Synthesis and Cytoxicity of Sempervirine and Analogues
Xiaohong Pan,a Chunying Yang,b John L. Cleveland,b and Thomas D. Bannister*a
aThe Scripps Research Institute, Department of Chemistry, Jupiter, Florida, 33458, USA
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bMoffitt Cancer Center & Research Institute, Department of Tumor Biology, Tampa, Florida
33612, USA
ABSTRACT: Sempervirine and analogues were
synthesized using a route featuring Sonagashira and
Larock
Pdꢀcatalyzed
reactions.
Structureꢀactivity
relationships were investigated using three human cancer
cell lines. 10ꢀfluorosempervirine is the most potently
cytotoxic member of the family yet described.
Many natural products and their derivatives are important therapeutic agents, particularly in
the treatment of infections and cancers.1 Modern synthetic methods, especially those not relying
upon the intrinsic reactivity of groups present in the natural material, has lowered the barrier to
conducting SAR studies in natural product scaffolds to improve efficacy and therapeutic index.
We recently reported a general approach to the synthesis of tetracyclic and pentacyclic
alkaloids2 featuring highly substrateꢀtolerant Sonagashira3 and Larock4 reactions. Here we
report using the route for an SAR study in the sempervirine series.
Sempervirine has attracted significant interest since the pioneering structure elucidation5
and synthetic work6 of R. B. Woodward, who proposed its canonical forms (Figure 1). Several
elegant syntheses have been reported, notably by Swan,7 Westphal,8 Potts9 and Gribble.10 It
has been targeted due to its antiꢀtumor activity, arising from topoisomerase 1 inhibition, DNA
intercalation, and other mechanisms.11 Routes to sempervirine generally involve moderately
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long synthetic sequences, proceed with a modest overall yield, and have limitations in scope for
preparing the natural material or a small set of analogues.
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Figure 1. Sempervirine canonical forms and ring numbering; Woodward & Witkop (1949).
Previous sempervirine analogues prepared include Woodward’s indole Nꢀmethyl derivative6
and Lipińska’s three analogues wherein the E ring (Figure 1) was replaced by fused cyclopentyl,
cycloheptyl, and cyclooctyl rings, with each product obtained in six steps in about 4% overall
yield, using a route featuring an inverse electron demand Diels–Alder reaction between a 1,2,4ꢀ
triazine diene and a cyclic enamine.12 Lipińska later reported four C10 methoxy analogues
having fiveꢀ, sixꢀ, sevenꢀ, and eightꢀmembered E rings, in 2ꢀ10% yield by a route using a
Fischer indole synthesis12 and Gribble’s method10 for Cꢀring construction. Malhotra et al. used a
microwaveꢀassisted version of Westphal’s method8 to prepare the C11 methoxy analogue in
32% yield,13 a compound previously made by Huebner et al.14 Malhotra et al. also prepared a
compound wherein the E ring was replaced with two phenyl groups, in 42% yield.13 To our
knowledge the biological activity for these compounds is unreported.
Our route uses an initial regioselective semireduction of 3ꢀisoquinolone (2, Scheme 1).
Literature reduction methods required excesses of expensive and harsh reagents (SbF3 or SbF5
in CF3SO3H).15 In a solvent survey using 1 mol% PtO2, acetic acid gave unwanted pyridone ring
saturation. Adding TFA favored the desired phenyl ring reduction, with 10:1 TFAꢀtriflic acid
giving full regioselectivity. The triflate 3 was then prepared (94% yield, two steps) and used in a
Sonagashira reaction (92%) followed by a Larock indole synthesis reaction that proceeded with
high regioselectivity,16 and then a triflateꢀpromoted cyclization (91%, two steps).2 DDQ oxidation
provided sempervirine triflate 1 (96%). The route is six steps and 76% yield, with intermediate 8
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and the natural product purified by precipitation. Given the efficiency, ease of purification, and
expected substrate tolerances, we felt that an SAR study for sempervirine was feasible.
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Scheme 1. Synthesis of Sempervirine Triflate
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Br
HO
4
1) 1% PtO2, H2
50 psi, 1 h
TfOH:TFA 1:10
3% PdCl2(PPh3)2
6% CuI
6
N
N
H2N
NH
2.5% Pd(OAc)2
5% dppf, KHCO3
2) Tf2O, Et3N
CH2Cl2, 10 min
OTf
Et3N, 80 °C, 1 h
92%
O
3
2
5
DMF, 110 °C, 4 h
96%
94% (2 steps)
HO
8
OH
OTf
N
OTf
5
7
4
3
N
N
Tf2O, Et3N
DDQ
9
sempervirine triflate 1
( positionꢀnumbering
HOAc
110 °C, 4 h
96%
2
CHCl3, 0 °C
5 min
1
HN
14
HN
10
HN
13
conventions are shown)
95%
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Altering ring size within the core is a drastic change not generally possible by natural
product modification. A sevenꢀmembered ring analogue of dihydrosempervirine was prepared in
three steps and 60% yield from triflate 3 (Scheme 2). In the Larock cyclization, catalyst loading
was increased twoꢀfold relative to the Scheme 1 protocol. The lower yield seen for a longer alkyl
chain (compound 10 vs. 5) suggests hydroxyl group chelation in the alkyne palladation step.
The Larock reaction to access the 5ꢀmembered ring analogue 16 (Scheme 3), was
unsuccessful, perhaps due to poor coordination by the propargylic alcohol group and/or
gramineꢀlike instability of product 15.
Scheme 2. Approaches to 7- and 5-membered Ring Analogues
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Structural diversity in the central ring is possible: a C7 substituent can be installed by using
hydroxyester 1717 to give tetracyclic esters 20 and 21 (Scheme 3). This core ring system and
pattern of substitution is similar to javacarboline (22).
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Scheme 3. C-ring Carboethoxy-substituted Analogues
Using electronꢀrich bromoanilines permits access to electronꢀrich sempervirine analogues
(29 and 30, Scheme 4). Here intermediates 25 and 26 were not purified and salts 27ꢀ30 were
isolated by precipitation. Compound 30 in another salt form has been described.13
Scheme 4. A-ring Methoxy-substituted Analogues
Electronꢀdeficient derivatives are accessible (37 and 38, Scheme 5). For 38 the DDQ
aromatization reaction required 7 days and excess oxidant. As another variation, isoquinolinone
2 semireduction can be omitted, providing the aromatic Eꢀring analogue 43 (Scheme 6).
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Scheme 5. Fluorinated Analogues
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Scheme 6. Aromatic Analogues
Sempervirine analogues with substituents in the A, C, and/or E ring are thus accessible.
Presumably the use of C1ꢀ or C4ꢀsubstituted analogues of isoquinoline (2) would also allow
access to D ring analogues (our previous paper described Dꢀring aza analogues).2 The B ring,
with only one site for substitution, can likely also be modified by Nꢀalkylation of an intermediate
or the final product. The ability to modify rings A-E of sempervirine by one general method
illustrates the versatility and utility of the Larock and Sonagashira reactions.
Because sempervirine has multiple mechanisms of antiꢀtumor activity,11 a single biochemical
assay is inadequate to assess potency. We conducted standard whole cell antiꢀtumor viability
MTT assays18 in three sempervirineꢀsensitive human tumor lines: Raji Burkitt lymphoma, MDAꢀ
MBꢀ231 breast cancer, and HeLa cervical cancer cells. Compounds 44ꢀ50 (Figure 2)2 were also
evaluated. Growth inhibition EC50 estimates were determined using a 6ꢀpoint dilution protocol.17
Active compounds (estimated EC50 < 3 µM) were reꢀevaluated using a 12ꢀpoint protocol.
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Figure 2. Additional analogues tested.2
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Growth inhibition curves for sempervirine and two more potent analogues are shown in
Figure 3. A 2.3ꢀfold to 3.4ꢀfold enhancement of potency for 38 relative to sempervirine (1) was
found. Compound 38 is to our knowledge the most cytotoxic sempervirine analogue known.
Enhanced potency and improved pharmacokinetic properties are likely required for animal use
of 38.19 Growth inhibition results for all other less active compounds are shown in Table 1,
based upon the 6ꢀpoint assay.17 Compounds with a nonꢀaromatic central ring were inactive at
10 µM (8, 12, 20, 27, 28, 35, 36, 42, and 44ꢀ49), thus extended planarity is essential for activity.
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Figure 3. Growth inhibition by the most potent compounds in the series.
Table 1. Estimated Growth Inhibition for all Other Compounds Tested, 6-Point MTT Assay
Compound(s)
8, 12, 20, 27, 28, 35, 36,
42, 44, 45, 46, 47, 48, 49
21, 30, 37, 43
Raji EC50
>10 µM
MDAꢀMBꢀ231 EC50
HeLa EC50
>10 µM
>10 µM
3ꢀ10 µM
3ꢀ10 µM
3ꢀ10 µM
3ꢀ10 µM
3ꢀ10 µM
>10 µM
50
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CONCLUSION
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These methods allow extensive modifications to sempervirine, with 10ꢀfluorosempervirine (38)
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being the most potently cytotoxic analogue yet described.
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EXPERIMENTAL SECTION
1
General Methods. Infrared (IR) spectra were collected on an FTꢀIR spectrometer. H NMR
spectra were recorded at 400 MHz in parts per million (ppm) downfield from an internal
standard, CHCl3 (δ 7.26) or DMSO (δ 2.54). 13C NMR spectra were recorded at 100 MHz in
parts per million (ppm) downfield from an internal standard, CHCl3 (δ 77.36) or DMSO (δ 40.45).
High resolution mass spectra (HRMS) were recorded on a TOF LC/MS spectrometer. Reagents
and anhydrous solvents used were obtained from commercial vendors. Flash column
chromatography was performed to purify compounds as indicated, using 60 Å mesh silica
columns and automated instruments.
General procedure for DDQ oxidation reactions: preparation of sempervirine triflate (1). A
solution of 8 (106 mg, 0.25 mmol) and 2,3ꢀdichloroꢀ5,6ꢀdicyanoꢀ1,4ꢀbenzoquinone (85 mg,
°
0.375 mmol) in acetic acid (1 mL) was stirred at 110 C for 4 h. After cooling to room
temperature the resulting precipitate was filtered and recrystallized with acetic acid, providing 1
(101 mg, 96%) as a yellow solid: IR (neat cmꢀ1) 3186, 1651, 1222, 1028, 745; H NMR (400
1
MHz, DMSO) δ 13.29 (s, 1H), 9.27 (s, 1H), 8.89 (d, J = 6.8 Hz, 1H), 8.72 (d, J = 6.8 Hz, 1H),
8.69 (s, 1H), 8.43 (d, J = 8.0 Hz, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.75 (t, J = 7.6 Hz, 1H), 7.51 (t, J
13
= 7.6 Hz, 1H), 3.20 (br, 2H), 3.04 (br, 2H), 1.95 (br, 4H); C NMR (100 MHz, DMSO) δ 149.9,
141.2, 135.3, 133.6, 130.8, 130.0, 129.6, 126.6, 122.4, 122.3, 121.6, 121.3, 119.9, 116.6, 113.3,
29.6, 26.6, 22.1, 22.0; HRMS (ES) m/e calc’d for C19H17N2 (MꢀOTf)+ 273.1392, found 273.1399.
5,6,7,8-tetrahydroisoquinolin-3-yl trifluoromethanesulfonate (3). A solution of isoquinolinꢀ3ꢀol
(2) (2 g, 13.79 mmol) and PtO2 (30 mg, 0.132 mmol) in trifluoroacetic acid (10 mL) and triflic
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acid (1 mL) was stirred under 50 psi H2 for 1 h. The mixture was filtered and to the solution was
added ice water. The resulting mixture was made basic (pH 8) with saturated aqueous Na2CO3
and extracted with ethyl acetate. The extracts were combined and evaporated to provide
5,6,7,8ꢀtetrahydroisoquinolinꢀ3ꢀol (2 g, 98%) as a solid. To a solution of 5,6,7,8ꢀ
tetrahydroisoquinolinꢀ3ꢀol (200 mg, 1.343 mmol) and Et3N (163 mg, 1.612 mmol) in CH2Cl2 (5
mL) at 0 °C was then added trifluoromethanesulfonic anhydride（0.245 mL, 1.447
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mmol）slowly, followed by stirring for 10 min. Water was added and the CH2Cl2 layer was
separated and then filtered through a pad of silica gel. Evaporation of the CH2Cl2 provided
compound 3 (360 mg, 96%) as an oil: IR (neat cmꢀ1) 2942, 1416, 1201, 927, 834; 1H NMR (400
MHz, CDCl3) δ 8.05 (s, 1H), 6.87 (s, 1H), 2.83‒2.80 (m, 2H), 2.78‒2.75 (m, 2H), 1.87‒1.78 (m,
13
4H); C NMR (100 MHz, CDCl3) δ 154.2, 152.3, 148.9, 134.6, 120.5 (CF3), 117.4 (CF3), 115.0,
29.4, 26.1, 22.5, 22.1; HRMS (ES) m/e calc’d for C10H11F3NO3S (M+H)+ 282.0406, found
282.0408.
General procedure for Sonagashira coupling reactions: 4-(5,6,7,8-tetrahydroisoquinolin-
3-yl)but-3-yn-1-ol (5). A solution of 5,6,7,8ꢀtetrahydroisoquinolinꢀ3ꢀyl triflate (3) (1.69 g, 6.0
mmol), 3ꢀbutynꢀ1ꢀol (4) (504 mg, 7.2 mmol), PdCl2(PPh3)2 (127 mg, 0.18 mmol), CuI (69 mg,
°
0.36 mmol) in Et3N (6 mL) was stirred at 80 C for 1 h. Silica gel was added, and the resulting
mixture was evaporated and separated by flash chromatography (SiO2, EtOAc), providing
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compound 5 (1100 mg, 92%) as a solid: IR (neat cmꢀ1) 3183, 2937, 1597, 1053, 695; H NMR
(400 MHz, CDCl3) δ 8.15 (s, 1H), 7.04 (s, 1H), 3.85 (t, J = 6.4 Hz, 2H), 2.68‒2.65 (m, 6H);
1.77‒1.75 (m, 4H); 13C NMR (100 MHz, CDCl3) δ 150.3, 146.9, 140.0, 132.8, 127.1, 87.1, 81.8,
61.0, 28.7, 26.4, 24.1, 22.6, 22.4; HRMS (ES) m/e calc’d for C13H16NO (M+H)+ 202.1226, found
202.1229.
General procedure for Larock indole synthesis reactions: 2-(2-(5,6,7,8-
tetrahydroisoquinolin-3-yl)-1H-indol-3-yl)ethanol (7). 2ꢀBromoaniline (6) (52 mg, 0.3 mmol), 5
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(62 mg, 0.3 mmol), Pd(OAc)2 (1.8 mg, 0.0075 mmol), 1,1'ꢀbis(diphenylphosphino)ferrocene (8.4
mg, 0.015 mmol) and KHCO3 (90 mg, 0.9 mmol) were added to dry and degassed DMF (2 mL).
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°
The solution was heated for 4 h at 110 C, after which time the reaction was complete, as
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determined by LCMS analysis. Water was added and the mixture was extracted with ethyl
acetate. The organic extracts were combined, dried and purified by flash chromatography (SiO2,
hexanes:EtOAc=1:1), providing compound 7 (84 mg, 96%) as a solid: IR (neat cmꢀ1) 3182,
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2925, 1603, 1042, 737; H NMR (400 MHz, CDCl3) δ 9.40 (br, 1H), 8.13 (s, 1H), 7.56 (d, J = 8.0
Hz, 1H), 7.44 (s, 1H), 7.38 (d, J = 8.0 Hz, 1H), 7.21 (t, J = 7.2 Hz, 1H), 7.11 (t, J = 7.2 Hz, 1H),
13
4.05 (t, J = 6.0 Hz, 2H), 3.26 (t, J = 6.0 Hz, 2H), 2.80 (br, 2H), 2.67 (br, 2H), 1.80 (br, 4H); C
NMR (100 MHz, CDCl3) δ 148.8, 147.7, 147.4, 136.5, 134.1, 131.7, 129.3, 123.0, 121.3, 119.7,
118.9, 112.6, 111.8, 64.0, 29.2, 27.5, 26.3, 22.7, 22.5; HRMS (ES) m/e calc’d for C19H21N2O
(M+H)+ 293.1648, found 293.1656.
General procedure for triflate cyclization reactions: 7,8-dihydrosempevirine (8). To a
solution of compound 7 (44 mg, 0.15 mmol) and Et3N (24 mg, 0.225 mmol) in dry CHCl3 (1.4
°
mL) at 0 C trifluoromethanesulfonic anhydride (34 ꢁL, 0.18 mmol) was slowly added. After
stirring for an additional 5 min the resulting yellow precipitate was collected by filtration, the solid
was washed with CHCl3, and was then identified as compound 8 as a yellow solid (60.2 mg,
95%): IR (neat cmꢀ1) 3297, 1558, 1149, 1031, 748; 1H NMR (400 MHz, DMSO) δ 12.21 (s, 1H),
8.81 (s, 1H), 8.01 (s, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.40 (t, J = 7.2 Hz,
1H), 7.22 (t, J = 7.2 Hz, 1H), 4.83 (br, 2H), 3.38 (br, 2H), 3.05 (br, 2H), 2.88 (br, 2H), 1.86 (br,
4H); 13C NMR (100 MHz, DMSO) δ 158.2, 145.4, 140.2, 139.8, 134.4, 126.6, 125.9, 125.7,
121.5, 121.2, 121.0, 116.9, 113.4, 55.9, 29.8, 26.4, 21.9, 21.8, 19.8; HRMS (ES) m/e calc’d for
C19H19N2 (MꢀOTf)+ 275.1548, found 275.1553.
5-(5,6,7,8-tetrahydroisoquinolin-3-yl)pent-4-yn-1-ol (10). By the general Sonagashira coupling
procedure, 5,6,7,8ꢀtetrahydroisoquinolinꢀ3ꢀyl triflate (3) (141 mg, 0.5 mmol), 4ꢀpentynꢀ1ꢀol (9)
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(51 mg, 0.6 mmol), PdCl2(PPh3)2 (11 mg, 0.015 mmol), CuI (6 mg, 0.03 mmol) in Et3N (1 mL) at
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6
°
80 C for 1 h provided compound 10 (92 mg, 86%) as an oil after flash chromatography (SiO2,
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EtOAc): IR (neat cmꢀ1) 3280, 2931, 1595, 1473, 1060; 1H NMR (400 MHz, CDCl3) δ 8.18 (s, 1H),
7.05 (s, 1H), 3.81 (t, J = 6.0 Hz, 2H), 2.78 (br, 1H), 2.71‒2.66 (m, 4H), 2.55 (t, J = 7.2 Hz, 2H),
1.89‒1.82 (m, 2H), 1.80‒1.73 (m, 4H); 13C NMR (100 MHz, CDCl3) δ 150.6, 146.7, 140.4, 132.6,
127.2, 89.1, 81.0, 61.7, 31.5, 28.7, 26.4, 22.7, 22.5, 16.20; HRMS (ES) m/e calc’d for C14H18NO
(M+H)+ 216.1383, found 216.1385.
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3-(2-(5,6,7,8-tetrahydroisoquinolin-3-yl)-1H-indol-3-yl)propan-1-ol (11). By the general Larock
indole synthesis procedure, 2ꢀbromoaniline (6) (48 mg, 0.28 mmol), alkyne 10 (60 mg, 0.28
mmol), Pd(OAc)2 (3.2 mg, 0.014 mmol), 1,1'ꢀbis(diphenylphosphino)ferrocene (15.5 mg, 0.028
mmol), KHCO3 (84 mg, 0.84 mmol) in DMF (1.4 mL) for 2 h provided 11 (65 mg, 76%) as an oil
after flash chromatography (SiO2, hexanes:EtOAc=1:1): IR (neat cmꢀ1) 2931, 1604, 1436, 955,
734; 1H NMR (400 MHz, CDCl3) δ 9.18 (s, 1H), 8.20 (s, 1H), 7.65 (d, J = 7.6 Hz, 1H), 7.38 (d, J
= 7.6 Hz, 1H), 7.34 (s, 1H), 7.24 (td, J = 6.8 and 0.8 Hz, 1H), 7.14 (td, J = 8.0 and 0.8 Hz, 1H),
5.58 (br, 1H), 3.62 (t, J = 5.6 Hz, 2H), 3.26 (t, J = 6.4 Hz, 2H), 2.72‒2.65 (m, 4H), 2.10‒2.04 (m,
2H), 1.81‒1.73 (m, 4H); 13C NMR (100 MHz, CDCl3) δ 149.5, 148.5, 147.9, 136.6, 133.2, 131.9,
129.6, 123.2, 122.1, 119.8, 119.7, 114.4, 111.5, 60.1, 32.2, 29.2, 26.2, 22.7, 22.6, 20.6; HRMS
(ES) m/e calc’d for C20H23N2O (M+H)+ 307.1805, found 307.1804.
1,2,3,4,7,8,9,14-octahydroindolo[2',3':3,4]azepino[1,2-b]isoquinolin-6-ium triflate (12). By
the general triflate cyclization procedure, compound 11 (50 mg, 0.163 mmol), Et3N (25 mg,
0.245 mmol) and trifluoromethanesulfonic anhydride (33 ꢁL, 0.196 mmol) in dry CHCl3 (1 mL)
1
provided 12 (65 mg, 91%) as a yellow solid: IR (neat cmꢀ1) 3302, 1556, 1154, 1030, 745; H
NMR (400 MHz, DMSO) δ 11.85 (s, 1H), 8.85 (s, 1H), 8.17 (s, 1H), 7.72 (d, J = 8.0 Hz, 1H),
7.55 (d, J = 8.0 Hz, 1H), 7.42 (t, J = 7.6 Hz, 1H), 7.20 (t, J = 7.6 Hz, 1H), 4.71‒4.69 (m, 2H),
3.24 (t, J = 6.8 Hz, 2H), 3.07 (br, 2H), 2.90 (br, 2H), 2.42 (br, 2H), 1.88 (br, 4H); 13C NMR (100
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MHz, DMSO) δ 157.1, 145.6, 143.5, 138.7, 135.3, 128.8, 126.9, 126.7, 124.7, 122.1, 121.1,
121.0, 112.9, 60.3, 29.7, 26.6, 26.3, 24.9, 21.92, 21.89; HRMS (ES) m/e calc’d for C20H21N2 (Mꢀ
OTf)+ 289.1705, found 289.1709.
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3-(5,6,7,8-tetrahydroisoquinolin-3-yl)prop-2-yn-1-ol (14). By the general Sonagashira coupling
procedure, 5,6,7,8ꢀtetrahydroisoquinolinꢀ3ꢀyl triflate 3 (140 mg, 0.5 mmol), 2ꢀpropynꢀ1ꢀol 13 (34
mg, 0.6 mmol), PdCl2(PPh3)2 (11 mg, 0.015 mmol), CuI (6 mg, 0.03 mmol) in Et3N (1 mL) at 80
°C for 1 h provided 14 (81 mg, 87%) as a solid after flash chromatography (SiO2, EtOAc): IR
(neat cmꢀ1) 3143, 2934, 1041, 974, 868; 1H NMR (400 MHz, CDCl3) δ 8.21 (s, 1H), 7.13 (s, 1H),
4.51 (s, 2H), 3.84 (br, 1H), 2.73‒2.68 (m, 4H), 1.83‒1.75 (m, 4H); 13C NMR (100 MHz, CDCl3) δ
150.6, 147.1, 139.5, 133.4, 127.6, 87.6, 84.6, 51.4, 28.8, 26.5, 22.7, 22.4; HRMS (ES) m/e
calc’d for C12H14NO (M+H)+ 188.1070, found 188.1073.
Ethyl 2-hydroxy-5-(5,6,7,8-tetrahydroisoquinolin-3-yl)pent-4-ynoate (18). By the general
Sonagashira coupling procedure, 5,6,7,8ꢀtetrahydroisoquinolinꢀ3ꢀyl triflate (3) (160 mg, 0.569
mmol), ethyl 2ꢀhydroxypentꢀ4ꢀynoate (17) (97 mg, 0.683 mmol), PdCl2(PPh3)2 (12 mg, 0.0171
mmol), CuI (6.5 mg, 0.0341 mmol) in Et3N (1 mL) at 80 °C for 2 h provided 18 (145 mg, 94%) as
an oil after flash chromatography (SiO2, hexanes:EtOAc=1:1): IR (neat cmꢀ1) 2934, 1734, 1195,
1
1098, 1033; H NMR (400 MHz, CDCl3) δ 8.13 (s, 1H), 6.98 (s, 1H), 4.64 (br, 1H), 4.41 (t, J =
5.6 Hz, 1H), 4.23‒4.15(m, 2H), 2.92(qd, J = 16.8 and 4.8 Hz, 2H), 2.63‒2.58 (m, 4H),
13
1.74‒1.68 (m, 4H), 1.24 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, CDCl3) δ 173.1, 150.3, 146.7,
139.5, 132.8, 127.3, 84.5, 82.7, 69.3, 61.8, 28.5, 26.3, 26.1, 22.5, 22.3, 14.3; HRMS (ES) m/e
calc’d for C16H20NO3 (M+H)+ 274.1438, found 274.1439.
Ethyl 2-hydroxy-3-(2-(5,6,7,8-tetrahydroisoquinolin-3-yl)-1H-indol-3-yl)propanoate (19). By the
general Larock indole synthesis procedure, 2ꢀbromoaniline (6) (102 mg, 0.593 mmol), alkyne 18
(135 mg, 0.494 mmol), Pd(OAc)2 (5.6 mg, 0.0247 mmol), 1,1'ꢀbis(diphenylphosphino)ferrocene
(27.4 mg, 0.0494 mmol), KHCO3 (148 mg, 1.482 mmol) in DMF (2.5 mL) for 2 h provided 19
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(120 mg, 67%) as an oil after flash chromatography (SiO2, hexanes:EtOAc=1:1): IR (neat cmꢀ1)
2929, 1729, 1603, 1205, 739; 1H NMR (400 MHz, CDCl3) δ 9.28 (s, 1H), 7.96 (s, 1H), 7.43‒7.40
(m, 1H), 7.16 (s, 1H), 7.06‒7.03 (m, 1H), 6.99‒6.94 (m, 2H), 4.66 (t, J = 5.6 Hz, 1H), 4.36‒4.28
(m, 1H), 4.21‒4.13 (m, 1H), 3.45 (d, J = 5.6 Hz, 2H), 2.74‒2.59 (m, 4H), 1.82‒1.71 (m, 4H),
1.39 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 175.8, 148.2, 147.1, 146.6, 136.6, 134.6,
131.8, 128.9, 122.9, 121.7, 119.4, 118.5, 112.1, 110.1, 73.0, 61.3, 29.3, 29.1, 26.4, 22.8, 22.6,
14.6; HRMS (ES) m/e calc’d for C22H25N2O3 (M+H)+ 365.1860, found 365.1861.
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7-(ethoxycarbonyl)-2,3,4,7,8,13-hexahydro-1H-indolo[2',3':3,4]pyrido[1,2-b]isoquinolin-6-ium
triflate (20). By the general triflate cyclization procedure, compound 19 (110 mg, 0.302 mmol),
Et3N (46 mg, 0.453 mmol) and trifluoromethanesulfonic anhydride (62 ꢁL, 0.362 mmol) in dry
CHCl3 (2.0 mL) at room temperature for 1 h provided 20 (119 mg, 80%) as a yellow solid: IR
1
(neat cmꢀ1) 3228, 1744, 1156, 1029, 768; H NMR (400 MHz, DMSO) δ 12.32 (s, 1H), 8.82 (s,
1H), 8.13 (s, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.41 (t, J = 7.2 Hz, 1H), 7.22
(t, J = 7.6 Hz, 1H), 6.14 (d, J = 6.0 Hz, 1H), 4.17‒4.06 (m, 2H), 3.94 (d, J = 17.2 Hz, 1H), 3.74
(dd, J = 17.2 and 6.8 Hz, 1H), 3.19‒3.06 (m, 2H), 2.98‒2.83 (m, 2H), 1.97‒1.83 (m, 4H), 1.10 (t,
J = 6.8 Hz, 3H); 13C NMR (100 MHz, DMSO) δ 169.0, 160.3, 146.1, 140.1, 140.0, 134.7, 127.0,
125.7, 125.5, 121.8, 121.4, 121.3, 114.3, 113.6, 66.9, 63.7, 30.1, 26.5, 23.3, 21.8, 21.7, 14.7;
HRMS (ES) m/e calc’d for C22H23N2O2 (MꢀOTf)+ 347.1760, found 347.1763.
7-(ethoxycarbonyl)-2,3,4,13-tetrahydro-1H-indolo[2',3':3,4]pyrido[1,2-b]isoquinolin-6-ium
triflate (21). By the general DDQ oxidation procedure, 20 (50 mg, 0.1 mmol) and 2,3ꢀdichloroꢀ
°
5,6ꢀdicyanoꢀ1,4ꢀbenzoquinone (34 mg, 0.15 mmol) in acetic acid (1.0 mL) at 100 C for 8 h
1
provided 21 (40.2 mg, 82%) as a yellow solid: IR (neat cmꢀ1) 2938, 1722, 1220, 1158, 756; H
NMR (400 MHz, DMSO) δ 13.42 (s, 1H), 9.88 (s, 1H), 9.28 (s, 1H), 8.59 (s, 1H), 8.46 (d, J = 8.0
Hz, 1H), 7.76‒7.69 (m, 2H), 7.51 (td, J = 8.0 and 1.2 Hz, 1H), 4.65 (q, J = 7.2 Hz, 2H), 3.16 (br,
13
2H), 3.05 (br, 2H), 2.00‒1.93 (m, 4H), 1.59 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, DMSO) δ
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162.6, 151.4, 141.6, 134.8, 134.6, 132.6, 131.9, 130.1, 124.2, 123.4, 123.3, 122.9, 122.1, 121.2,
119.6, 113.8, 63.9, 29.6, 27.3, 22.1, 22.0, 14.9; HRMS (ES) m/e calc’d for C22H21N2O2 (MꢀOTf)+
345.1603, found 345.1601.
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9-methoxy-2,3,4,7,8,13-hexahydro-1H-indolo[2',3':3,4]pyrido[1,2-b]isoquinolin-6-ium
triflate
(27). A solution of 2ꢀbromoꢀ3ꢀmethoxyaniline (23) (53 mg, 0.26 mmol), alkyne 5 (41 mg, 0.2
mmol), Pd(OAc)2 (4.5 mg, 0.02 mmol), 1,1'ꢀbis(diphenylphosphino)ferrocene (22 mg, 0.04
°
mmol) and KHCO3 (60 mg, 0.6 mmol) in DMF (1.0 mL) was degassed and stirred at 110 C for
10 h. After cooling to room temperature water was added and the mixture was extracted with
ethyl acetate. The organic extracts were combined, washed with water, filtered through a pad of
silica gel and dried to provide the crude intermediate 25. Et3N (31 mg, 0.3 mmol) and CHCl3 (1.5
mL) were then added to this crude intermediate, and then trifluoromethanesulfonic anhydride
°
(34 ꢁL, 0.2 mmol) was slowly added to the mixture at 0 C. After stirring for 5 min the resulting
precipitate was filtered and washed with CHCl3 to provide 27 (52 mg, 57%) as a yellow solid: IR
1
(neat cmꢀ1) 3246, 1513, 1247, 1032, 745; H NMR (400 MHz, DMSO) δ 12.15 (s, 1H), 8.76 (s,
1H), 7.92 (s, 1H), 7.29 (t, J = 8.0 Hz, 1H), 7.11 (d, J = 8.4 Hz, 1H), 6.64 (d, J = 8.0 Hz, 1H), 4.79
(t, J = 6.8 Hz, 2H), 3.93 (s, 3H), 3.49 (t, J = 6.8 Hz, 2H), 3.01 (br, 2H), 2.84 (br, 2H), 1.85 (br,
4H); 13C NMR (100 MHz, DMSO) δ 158.1, 155.8, 145.1, 141.2, 140.1, 134.1, 128.0, 124.6,
120.7, 116.8, 116.6, 106.3, 101.3, 56.3, 55.8, 29.8, 26.4, 22.0, 21.9, 21.5; HRMS (ES) m/e
calc’d for C20H21N2O (MꢀOTf)+ 305.1654, found 305.1652.
11-methoxy-2,3,4,7,8,13-hexahydro-1H-indolo[2',3':3,4]pyrido[1,2-b]isoquinolin-6-ium triflate
(28). A solution of 2ꢀbromoꢀ5ꢀmethoxyaniline (24) (51 mg, 0.25 mmol), 5 (51 mg, 0.25 mmol),
Pd(OAc)2 (5.6 mg, 0.025 mmol), 1,1'ꢀbis(diphenylphosphino)ferrocene (28 mg, 0.05 mmol) and
K2CO3 (87 mg, 0.625 mmol) in DMF (1.5 mL) was degassed and stirred at 110 °C for 4 h. After
cooling to room temperature water was added and the mixture was extracted with ethyl acetate.
The organic extracts were combined, washed with water, filtered through a pad of silica gel and
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dried to provide the crude intermediate 26. Et3N (38 mg, 0.375 mmol) and CHCl3 (2 mL) were
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then added to this crude intermediate, and then trifluoromethanesulfonic anhydride (42 ꢁL, 0.25
7
8
°
mmol) was slowly added to the mixture at 0 C. After stirring for 5 min the resulting precipitate
9
was filtered and washed with CHCl3 to provide 28 (79 mg, 70%) as a yellow solid: IR (neat cmꢀ1)
3293, 1554, 1249, 1031, 810; 1H NMR (400 MHz, DMSO) δ 11.98 (s, 1H), 8.71 (s, 1H), 7.87 (s,
1H), 7.62 (d, J = 8.8 Hz, 1H), 6.95 (d, J = 2.0 Hz, 1H), 6.86 (dd, J = 8.8 and 2.0 Hz, 1H), 4.79 (t,
J = 7.2 Hz, 2H), 3.88 (s, 3H), 3.34 (t, J = 7.2 Hz, 2H), 3.01 (br, 2H), 2.84 (br, 2H), 1.85 (br, 4H);
13C NMR (100 MHz, DMSO) δ 159.8, 157.9, 144.9, 141.2, 140.3, 133.5, 124.8, 122.2, 120.3,
120.2, 117.8, 112.9, 95.1, 56.3, 55.7, 29.8, 26.3, 22.0, 21.9, 19.9; HRMS (ES) m/e calc’d for
C20H21N2O (MꢀOTf)+ 305.1654, found 305.1653.
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9-methoxy sempervirine (29). By the general DDQ oxidation procedure, 27 (46 mg, 0.1 mmol)
and 2,3ꢀdichloroꢀ5,6ꢀdicyanoꢀ1,4ꢀbenzoquinone (30 mg, 0.13 mmol) in acetic acid (1.0 mL) at
100 °C for 30 min provided 29 (42 mg, 93%) as a yellow solid: IR (neat cmꢀ1) 3171, 1352, 1240,
1
1026, 733; H NMR (400 MHz, DMSO) δ 13.17 (s, 1H), 9.21 (s, 1H), 8.78 (d, J = 6.8 Hz, 1H),
8.52 (s, 1H), 8.41 (d, J = 6.8 Hz, 1H), 7.66 (t, J = 8.0 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 6.96 (d, J
= 8.0 Hz, 1H), 4.12 (s, 3H), 3.17‒3.12 (m, 2H), 3.02‒2.98 (m, 2H), 1.97‒1.89 (m, 4H); 13C NMR
(100 MHz, DMSO) δ 156.8, 149.7, 143.0, 135.5, 133.5, 131.4, 131.0, 129.8, 127.2, 121.3,
120.0, 117.9, 111.7, 106.1, 102.8, 56.8, 29.8, 26.8, 22.2, 22.2; HRMS (ES) m/e calc’d for
C20H19N2O (MꢀOTf)+ 303.1497, found 303.1497.
11-methoxy sempervirine (30). By the general DDQ oxidation procedure, 28 (68 mg, 0.15
mmol) and 2,3ꢀdichloroꢀ5,6ꢀdicyanoꢀ1,4ꢀbenzoquinone (52 mg, 0.225 mmol) in acetic acid (1.0
°
mL) at 100 C for 2 h provided 30 (64 mg, 94%) as a yellow solid: IR (neat cmꢀ1) 1626, 1251,
1158, 1033, 799; 1H NMR (400 MHz, DMSO) δ 13.06 (s, 1H), 9.15 (s, 1H), 8.81 (d, J = 5.2 Hz,
1H), 8.57 (d, J = 6.0 Hz, 1H), 8.52 (s, 1H), 8.24 (d, J = 8.8 Hz, 1H), 7.16 (s, 1H), 7.08 (d, J = 8.4
Hz, 1H), 3.98 (s, 3H), 3.15 (br, 2H), 3.00 (br, 2H), 1.93 (br, 4H); 13C NMR (100 MHz, DMSO) δ
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161.8, 149.4, 143.4, 135.2, 133.0, 130.9, 130.1, 127.1, 123.7, 122.7, 119.8, 116.2, 115.5, 113.2,
95.4, 56.6, 29.7, 26.7, 22.2, 22.2; HRMS (ES) m/e calc’d for C20H19N2O (MꢀOTf)+ 303.1497,
found 303.1500.
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7,8-dihydro-12-fluorosempervirine (35). A solution of 2ꢀbromoꢀ6ꢀfluoroaniline (31) (46 mg,
0.24 mmol), alkyne 5 (41 mg, 0.2 mmol), Pd(OAc)2 (2.3 mg, 0.01 mmol), 1,1'ꢀ
bis(diphenylphosphino)ferrocene (11 mg, 0.02 mmol) and KHCO3 (60 mg, 0.6 mmol) in DMF
°
(1.0 mL) was degassed and stirred at 110 C for 2 h. After cooling to room temperature water
was added and the mixture was extracted with ethyl acetate. The organic extracts were
combined, washed with water, filtered through a pad of silica gel and dried to provide the crude
intermediate 33. Et3N (31 mg, 0.3 mmol) and CHCl3 (1.5 mL) were then added to this crude
intermediate, and then trifluoromethanesulfonic anhydride (34 ꢁL, 0.2 mmol) was slowly added
°
to the mixture at 0 C. After stirring for 5 min the resulting precipitate was filtered and washed
with CHCl3 to provide 35 (65 mg, 74%) as a yellow solid: IR (neat cmꢀ1) 3247, 1557, 1239, 1029,
787; 1H NMR (400 MHz, DMSO) δ 12.58 (s, 1H), 8.84 (s, 1H), 8.15 (s, 1H), 7.58 (d, J = 8.0 Hz,
1H), 7.25‒7.14 (m, 2H), 4.86 (t, J = 7.2 Hz, 2H), 3.40 (t, J = 7.2 Hz, 2H), 3.03 (br, 2H), 2.88 (br,
2H), 1.86 (br, 4H); 13C NMR (100 MHz, DMSO) δ 158.4, 151.4 (d, 1JCꢀF = 243.5 Hz), 145.6,
139.8, 135.1, 129.4, 129.3, 127.9, 127.7, 127.1, 121.9, 121.9, 121.5, 117.5, 117.5, 117.5, 111.0,
110.9, 56.0, 29.9, 26.5, 21.9, 21.8, 19.8; HRMS (ES) m/e calc’d for C19H18FN2 (MꢀOTf)+
293.1454, found 293.1450.
7,8-dihydro-10-fluorosempervirine (36). A solution of alkyne 5 (70 mg, 0.348 mmol), Pd(OAc)2
(2 mg, 0.0087 mmol), 1,1'ꢀbis(diphenylphosphino)ferrocene (10 mg, 0.0174 mmol) and KHCO3
°
(104 mg, 1.04 mmol) in DMF (1.6 mL) was degassed and stirred at 110 C. A solution of 2ꢀ
bromoꢀ4ꢀfluoroaniline (32) (199 mg, 1.04 mmol) in DMF (0.4 mL) was then added slowly to this
solution, and the mixture was maintained at 110 °C for 4 h. After cooling to room temperature the
resulting mixture was quenched with water and extracted with ethyl acetate. The organic
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extracts were combined, washed with water, filtered through a pad of silica gel, and dried to
provide the crude intermediate. Et3N (53 mg, 0.522 mmol) and CHCl3 (1 mL) were then added
to this crude intermediate , and trifluoromethanesulfonic anhydride (60 ꢁL, 0.348 mmol) was
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°
then added slowly to this mixture at 0 C. After stirring for 10 min the resulting precipitate was
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filtered and washed with CHCl3 to provide 36 (115 mg, 75%) as a yellow solid: IR (neat cmꢀ1)
3285, 1561, 1282, 1034, 813; 1H NMR (400 MHz, DMSO) δ 12.28 (s, 1H), 8.80 (s, 1H), 7.96 (s,
1H), 7.57‒7.53 (m, 2H), 7.24 (td, J =9.2 and 2.0 Hz, 1H), 4.84 (t, J = 7.2 Hz, 2H), 3.36 (t, J = 7.2
Hz, 2H), 3.03 (br, 2H), 2.87 (br, 2H), 1.86 (br, 4H); 13C NMR (100 MHz, DMSO) δ 159.5 (d,
1JCꢀF = 233.4 Hz), 158.3, 145.5, 139.9, 136.4, 134.9, 127.6, 125.9, 125.8, 121.2, 116.7, 116.7,
115.4, 115.1, 114.8, 114.7, 105.8, 105.6, 56.0, 29.9, 26.5, 21.9, 21.8, 19.8; HRMS (ES) m/e
calc’d for C19H18FN2 (MꢀOTf)+ 293.1454, found 293.1450.
12-fluorosempervirine (37). By the general DDQ oxidation procedure, 35 (74 mg, 0.167
mmol) and 2,3ꢀdichloroꢀ5,6ꢀdicyanoꢀ1,4ꢀbenzoquinone (76 mg, 0.335 mmol) in acetic acid (1.8
°
mL) at 100 C for 4 h provided 37 (66 mg, 90%) as a yellow solid: IR (neat cmꢀ1) 2948, 1406,
1222, 1026, 776; 1H NMR (400 MHz, DMSO) δ 13.40 (s, 1H), 9.17 (s, 1H), 8.79 (d, J = 7.2 Hz,
1H), 8.65 (s, 1H), 8.61 (d, J = 7.2 Hz, 1H), 8.15 (d, J = 7.6 Hz, 1H), 7.58 (dd, J = 11.2 and 8.0
Hz, 1H), 7.44‒7.39 (m, 1H), 3.13 (br, 2H), 3.00 (br, 2H), 1.94 (br, 4H); 13C NMR (100 MHz,
DMSO) δ 151.0 (d, 1JCꢀF = 244.5 Hz), 150.7, 136.0, 134.4, 131.7, 131.0, 129.6, 129.5, 127.5,
125.1, 125.1, 123.0, 122.9, 121.9, 121.8, 120.5, 118.8, 116.9, 114.3, 114.2, 29.8, 26.8, 22.13,
22.09; HRMS (ES) m/e calc’d for C19H16FN2 (MꢀOTf)+ 291.1298, found 291.1299.
10-fluorosempervirine (38). By the general DDQ oxidation procedure, 36 (66 mg, 0.15 mmol)
and 2,3ꢀdichloroꢀ5,6ꢀdicyanoꢀ1,4ꢀbenzoquinone (340 mg, 1.5 mmol) in acetic acid (1.0 mL) at
°
100 C for 7 d provided 38 (34 mg, 52%) as a yellow solid: IR (neat cmꢀ1) 2943, 1651, 1248,
1
1153, 820; H NMR (400 MHz, DMSO) δ 13.66 (br, 1H), 9.25 (s, 1H), 8.82 (d, J = 6.8 Hz, 1H),
8.76 (s, 1H), 8.62 (d, J = 6.8 Hz, 1H), 8.24 (dd, J = 8.8 and 2.0 Hz, 1H), 7.85 (dd, J = 8.8 and
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4.0 Hz, 1H), 7.60 (td, J = 9.2 and 2.0 Hz, 1H), 3.17 (br, 2H), 3.03 (br, 2H), 1.94 (br, 4H); 13C
NMR (100 MHz, DMSO) δ 159.6 (d, 1JCꢀF = 234.9 Hz), 150.5, 138.4, 136.0, 134.2, 131.9,
131.8, 126.7, 122.1, 122.0, 121.6, 121.5, 120.8, 118.3, 118.1, 117.0, 115.3, 115.2, 107.8, 107.6,
29.8, 26.8, 22.2, 22.1; HRMS (ES) m/e calc’d for C19H16FN2 (MꢀOTf)+ 291.1298, found
291.1295.
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13H-indolo[2',3':3,4]pyrido[1,2-b]isoquinolin-6-ium triflate (43). By the general DDQ oxidation
procedure, 421 (42 mg, 0.1 mmol) and 2,3ꢀdichloroꢀ5,6ꢀdicyanoꢀ1,4ꢀbenzoquinone (34 mg, 0.15
mmol) in acetic acid (1.0 mL) at 100 °C for 30 min provided 43 (33 mg, 79%) as a yellow solid:
1
IR (neat cmꢀ1) 3215, 1221, 1027, 885, 733; H NMR (400 MHz, DMSO) δ 13.40 (s, 1H), 10.48
(s, 1H), 9.44 (s, 1H), 9.04 (d, J = 6.0 Hz, 1H), 8.70 (d, J = 6.4 Hz, 1H), 8.44 (d, J = 8.0 Hz, 1H),
8.34 (d, J = 10.0 Hz, 1H), 8.32 (d, J = 8.4 Hz, 1H), 8.15 (t, J = 7.2 Hz, 1H), 8.00 (t, J = 7.2 Hz,
13
1H), 7.78 (d, J = 8.4 Hz, 1H), 7.62 (t, J = 7.2 Hz, 1H), 7.42 (t, J = 7.6 Hz, 1H); C NMR (100
MHz, DMSO) δ 141.3, 140.7, 135.5, 135.4, 131.2, 130.9, 130.0, 129.3, 128.8, 127.9, 126.8,
125.8, 122.4, 122.2, 122.1, 119.4, 118.2, 117.7, 113.5; HRMS (ES) m/e calc’d for C19H13N2 (Mꢀ
OTf)+ 269.1079, found 269.1077.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI:
xx.xxxx/acs.joc.xxxxxxx. 1H and 13C NMR spectra for new compounds (pdf)
AUTHOR INFORMATION
Corresponding Author
* phone 561ꢀ228ꢀ2206, email tbannist@scripps.edu
Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGEMENTS
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Partial salary support was provided by grants from the U.S. National Institutes of Health: U54
MH084512ꢀ05ꢀ21755, R01 DA035056 and R01 CA154739.
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DEDICATION
This paper is dedicated to the memory of John Michael “Mike” Kane, an inspirational organic
and medicinal chemist, mentor, and friend to T.D.B.
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