The total synthesis of (?)-indolactam I

Article abstract of DOI:10.1016/j.tet.2019.04.069

The first total synthesis of (?)-indolactam I (1) is reported. An efficient synthetic route was established to furnish the natural product in 8 steps. This strategy employed a copper-catalyzed amino acid arylation, peptide coupling, and Lewis acid-mediate

Full text of DOI:10.1016/j.tet.2019.04.069

Accepted Manuscript
The total synthesis of (−)-indolactam I
Manuel Mendoza, Niveda Rao, UyenPhuong Tran, Catherine Castaneda, Kelvin L.
Billingsley
PII:
S0040-4020(19)30499-5
https://doi.org/10.1016/j.tet.2019.04.069
TET 30314
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 10 April 2019
Revised Date: 17 April 2019
Accepted Date: 26 April 2019
Please cite this article as: Mendoza M, Rao N, Tran U, Castaneda C, Billingsley KL, The total synthesis
of (−)-indolactam I, Tetrahedron (2019), doi: https://doi.org/10.1016/j.tet.2019.04.069.
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ACCEPTED MANUSCRIPT
The Total Synthesis of (–)-Indolactam I
Manuel Mendoza,a Niveda Rao,a UyenPhuong Tran,a Catherine Castaneda,b
and Kelvin L. Billingsleya,*
aCalifornia State University Fullerton, Department of Chemistry and Biochemistry, Fullerton, CA 92831
bDepartment of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA 94132
*Author to whom correspondence should be addressed
Correspondence to:
Prof. Kelvin Billingsley
California State University, Fullerton
Department of Chemistry and Biochemistry
McCarthy Hall 582C
Email: kbillingsley@fullerton.edu
Phone: 657-278-4252
Fax: 657-278-5316
Fullerton, CA 92831
Keywords:
natural product, total synthesis, indolactam, amino acid, protein kinase C
1

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Graphical Abstract
Abstract
The first total synthesis of (–)-indolactam I (1) is reported. An efficient synthetic route was
established to furnish the natural product in 8 steps. This strategy employed a copper-catalyzed
amino acid arylation, peptide coupling, and Lewis acid-mediated macrocyclization to yield 1
from readily available building blocks. In addition, the cell growth inhibition activity of the
natural product was examined through assays with the K562 leukemia cell line.
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1. Introduction
The teleocidins are a class of indole terpenoids that were first isolated from Streptomyces
mecliocidius over 50 years ago.1 Many of these natural products have been discovered to
effectively modulate protein kinase C (PKC) activity;2 therefore, these alkaloids have received
considerable attention in biochemical studies related to PKC-mediated cancer regulation.3
Despite the structural diversity found throughout this teleocidin family (Fig. 1), nearly all
members display the core elements represented by (–)-indolactam V (2), in which (L)-valine and
(L)-tryptophan are assembled into a 9-membered macrocycle.
Previous works4 have
demonstrated that 2 maintains the key pharmacophoric elements to promote activation of PKC
and correspondingly this natural product has been the focus of several synthetic studies.5
(–)-Indolactam I (1) was first isolated in 1990 and remains one of the only natural products in
this class that possesses a unique amino acid, (L)-isoleucine, in the indolactam macrocycle.6
Although earlier reports have suggested that increasing hydrophobicity of the teleocidins is
advantageous for PKC association,7 minimal synthetic efforts have been described to access 1.
To date, the only published strategy to prepare 1 required low yielding microbial-based
methods.8 These synthetic limitations prevent suitable biological evaluations of the natural
product and thus restrict its therapeutic potential.
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In this report, we describe the first total synthesis of (–)-indolactam I (1). The natural
product was prepared from readily available starting materials and assembled through a copper-
catalyzed arylation of (L)-isoleucine followed by subsequent zirconium-mediated cyclization.
Three natural products in the teleocidin family including 1 were then evaluated for their cell
growth inhibitory properties in a human leukemia cell line in order to ascertain the functional
effects of increasing hydrophobicity at key positions of the scaffold.
2. Results and Discussion
2.1 Synthesis of 1
Several factors establish 1 as an attractive synthetic target for therapeutic applications. The
natural product (1) maintains the core structural elements to promote efficient activation of PKC,
(2) possesses an increased hydrophobicity relative to 2, and (3) lacks the C6 and C7 alkyl
substitution found in many teleocidins, which have been prepared traditionally by lengthy
synthetic routes.9 In an effort to further elucidate the biological profile of the teleocidin-based
natural products, we sought to implement a synthetic route to access 1 that relied upon a modular
approach to prepare indolactam alkaloids.5m This strategy required efficient C4-nitrogen bond
formation with the appropriate amino acid and successful application of methods to establish the
(L)-tryptophan subunit of the indolactam macrocycle.
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The synthesis of 1 was initiated from commercially available 4-bromoindole (Scheme 1).
Indole protection was performed with tosyl chloride on a multi-gram scale. Recrystallization of
the crude product cleanly afforded 3 in an 88% yield. Heteroaryl bromide 3 was then subjected
to a copper-catalyzed Ullmann coupling with amino acid (L)-isoleucine to install the C4-nitrogen
bond.10 Although minimal product was observed in the absence of an indole-protecting group,
N-tosyl protected 3 smoothly afforded N-aryl amino acid 4 under the standard reaction
conditions. However, rapid degradation of 4 was observed upon isolation of the intermediate.
To obviate these issues, the crude product from the metal-catalyzed coupling was simply filtered
through a pad of silica, concentrated, and immediately transitioned to the next transformation.
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Therefore, 4 was directly subjected to reductive amination conditions using formaldehyde and
NaBH3CN to afford 5. Because air sensitivity was also found for 5, the material was again
advanced to the subsequent step without further isolation. Intermediate 5 underwent peptide
coupling with (L)-serine methyl ester to afford 6 as a stable and isolable solid. This protocol for
the conversion of 3 to dipeptide 6 resulted in a 25% yield over the 3-step sequence. Although
slower reaction rates were observed and decarboxylation11 of 5 was detected as a major
byproduct, this procedure successfully addressed the challenges associated with the instability of
intermediates 4 and 5. In addition, this 3-step protocol has been conducted on a >600 mg scale
to yield 6 in short order.
Macrocycle formation requires activation of the C8-alcohol to promote nucleophilic attack
by the indole moiety. With dipeptide 6 in hand, we attempted a magnesium-promoted
deprotection–dehydration reaction, which would remove the N-tosyl group and simultaneously
eliminate the C8-alcohol, to form enoate 7 in one pot. The standard conditions for this
transformation were to allow 20 equivalents of magnesium turnings to react with dipeptide 6 for
7 h at room temperature. This procedure, although successful for the production of 7, afforded
the desired product in a modest 21% yield. Through optimization of this process, it was
discovered that deprotection occurred efficiently at 0 °C with 10 equivalents of magnesium
turnings in 4 h, and no concurrent dehydration was observed. Once complete, the reaction was
warmed to 40 °C and elimination of the C8-alcohol was detected over 3 h. This new protocol
furnished the desired product 7 in an improved 65% yield.
Enoate 7 was employed in the Lewis acid-mediated closure of the 9-membered indolactam
ring.12 An excess of ZrCl4 and rigorously dried 7 were necessary to promote the transformation.
Similar to previous studies5j,5m with related indolactam precursors, only one stereoisomer (8) was
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isolated from the cyclization reaction. Under these conditions, tricycle 8 was produced in a 53%
yield with 28% of starting material 7 recovered (74% yield based upon recovered starting
material). From 8, the synthesis of 1 was completed in two final transformations. C9-ester
undergoes a relatively slow epimerization when treated with weak base (NaHCO3) at 40 °C for
72 h to produce diastereomer 9 in a 32% isolated yield. Because of a challenging separation of
stereoisomers, the mass balance from the purification was a mixture of diastereomers 8 and 9
(33% isolated yield) as well as unreacted starting material 8 (28% yield), which could be isolated
and re-subjected to the epimerization conditions. Attempts to further optimize this reaction were
unsuccessful, as higher temperatures resulted in decomposition. Fortunately, when ester 9 was
treated with LiBH4, natural product 1 was cleanly isolated in a 91% yield. The spectral
information for 1 was consistent with the report6a of the natural product isolation.13 This 8-step
sequence represents the first total synthesis of alkaloid 1.
2.2 Biological evaluation of PKC activators
Teleocidin-based natural products have been shown to associate with the C1 regulatory
domain of conventional and novel PKCs and thereby induce intracellular PKC signal
transduction pathways.2 Because PKC activation also requires a membrane component, it is
believed that hydrophobic groups on the small molecule ligand (e.g., teleocidin) help promote
PKC activation by facilitating the interaction of the membrane with the enzyme.14 Binding
affinities of various teleocidins with PKC isozymes provide support for this hypothesis.7 In this
work, we sought to employ cell-based analyses to determine the functional effects of increasing
hydrophobicity for the indolactam scaffold.
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Cell growth inhibition assays were conducted using the K562 human leukemia cell line.
Table 1 provides a summary of the effective concentrations for three natural products: 1, 2 and
teleocidin A-1. Nearly all members of the teleocidin family possess the core elements
represented by (–)-indolactam V (2), and this compound proved to be highly potent in vitro
(EC50 = 143 ± 75 nM). (–)-Indolactam I (1) is more hydrophobic than 2 with Log P values15 of
1.7 and 1.3, respectively. Despite a difference in hydrophobicity, 1 displayed a similar activity
as 2, and an EC50 value of 157 ± 56 nM for 1 was found. Concurrent to these studies with 1 and
2, (–)-teleocidin A-1 was also subjected to the cancer cell-based assay. This natural product
possesses a branched geranyl chain at C7, which increases the overall hydrophobicity of the
structure (Log P = 4.7). However, a significant decrease in in vitro activity was observed for (–)-
teleocidin A-1 (EC50 = 423 ± 49 nM) versus 2 (ρ = 0.047, t-test) or 1 (ρ = 0.021, t-test). These
preliminary results suggest that increasing hydrophobicity for teleocidin analogues may not
directly translate to improved in vitro activity in specific cancer cell lines.
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3. Conclusions
In summary, we have described a concise synthetic route to access (–)-indolactam I (1). This
work represents the first total synthesis of the natural product, and we have further demonstrated
that 1 exhibits comparable in vitro potency to (–)-indolactam V (2) in a human leukemia cell
line. Both of these indolactam alkaloids displayed lower effective concentrations than the more
hydrophobic (–)-teleocidin A-1. These studies will guide our future efforts towards the
development of novel therapeutics that have applications in PKC-dependent diseases.
4. Experimental Section
4.1 General
Unless otherwise noted, all reactions were performed under an air atmosphere in oven-dried
glassware. (–)-indolactam V (2) and (–)-teleocidin A-1 were purchased from Adooq Bioscience
and Cayman Chemical, respectively. Commercially available reagents were purchased from
Sigma-Aldrich and used without further purification. Reactions were stirred using Teflon-coated
magnetic stirrer bars. Reactions were monitored via gas chromatography and/or thin layer silica
gel chromatography (TLC) using 0.25 mm silica gel 60F plates with fluorescent indicator. Plates
were visualized under UV light without further staining. Products were purified via column
chromatography using Silica gel 60, 230-400 mesh and the solvent system(s) indicated.
1
All new compounds were characterized by H NMR, 13C NMR and high-resolution mass
spectrometry. Known compounds were characterized by 1H NMR and melting points (for solids)
and compared to their literature values. NMR spectra were measured on a Bruker 400 (1H at 400
MHz, 13C at 100 MHz) magnetic resonance spectrometer. 1H chemical shifts are reported relative
to the residual solvent peak (chloroform = 7.26 ppm) as follows: chemical shift (δ), (proton ID,
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multiplicity (s = singlet, bs = broad singlet, d = doublet, bd = broad doublet, ddd = doublet of
doublet of doublets, dddd = doublet of doublet of… etc., ddddd = doublet of doublet of… etc., t
13
= triplet, q = quartet), integration, coupling constant(s) in Hz.). C chemical shifts are reported
13
relative to the residual deuterated solvent C signals (chloroform = 77 ppm). High-resolution
mass spectra were obtained using a high-resolution liquid chromatography mass spectrometer.
Melting points were obtained on a Mel-Temp capillary melting point apparatus.
4.2 4-bromo-1-tosyl-1H-indole (3)
An oven-dried round bottom flask equipped with a magnetic stir bar was charged with 4-
bromoindole (2.50 g, 12.8 mmol). The flask was sealed with a septum, and a balloon with needle
was inserted through the septum. DMF (25 mL) was added via syringe through a septum and the
reaction was cooled to 0 °C with an ice bath. NaH (60%) (245 mg, 15.3 mmol) was added
directly to the flask in one portion. The reaction was allowed to warm to room temperature over
45 min followed by re-cooling to 0 °C. Tosyl chloride (3.60 g, 19.1 mmol) in DMF (15 mL) was
added via syringe over 2 min. The reaction was allowed to warm to room temperature overnight
(15 h). The crude reaction mixture was diluted with H2O (25 mL). The resulting solution was
extracted with EtOAc (100 mL). The organic layer was separated. The aqueous layer was
extracted with EtOAc (100 mL, ×2). The combined organic layers were dried with MgSO4,
filtered and concentrated to yield a brown oil. Recrystallization (EtOAc/hexanes) provided the
desired product 3 as a light brown solid (3.94 g) in 88% yield. Characterization data matched
previously reported information.5m 1H-NMR (500 MHz, CDCl3): δ 7.96 (d, J = 8.27 Hz, 1H),
7.77 (d, J = 8.35 Hz, 2H), 7.64 (d, J = 2.14 Hz, 1H), 7.41 (d, J = 7.80 Hz, 1H), 7.26 (m, 2H), 7.19
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(d, J = 8.15 Hz, 1H), 6.75 (d, J = 3.40 Hz, 1H), 2.37 (s, 3H) ppm. 13C-NMR (125 MHz, CDCl3):
δ 145.3, 135.1, 131.4, 130.0, 126.9, 126.8, 126.2, 125.9, 125.5, 115.0, 112.6, 108.8, 21.6 ppm.
4.3 Methyl N-methyl-N-(1-tosyl-1H-indol-4-yl)-L-isoleucyl-L-serinate (6)
An oven-dried schlenk flask equipped with magnetic stir bar was charged with CuI (34.7 mg,
0.18 mmol), (L)-isoleucine (286 mg, 2.18 mmol), 3 (640 mg, 1.82 mmol) and Cs2CO3 (889 mg,
2.73 mmol). The flask was sealed with a septum and then evacuated and backfilled with argon
(this sequence was carried out three times). DMSO (3.64 mL) was added via syringe through the
septum. Under an argon atmosphere, the septum was replaced and sealed with a Teflon
screwcap. The reaction was heated to 90 °C for 20 h and then cooled to room temperature. The
solution was filtered through a pad of silica eluting with methanol and then concentrated to yield
brown oil 4, which was used directly in the subsequent transformation.
The oil 4 was transferred to a round bottom flask equipped with magnetic stir bar. The flask
was sealed with a septum, and a balloon with needle was inserted through the septum. CH3CN
(17.5 mL) followed by formaldehyde (2.2 mL; 38% solution) were added consecutively to the
stirring solution via syringe. After 5 min, NaBH3CN (525 mg, 8.35 mmol) was added directly to
the flask followed by the dropwise addition of acetic acid (0.56 mL) over one minute. The
reaction was allowed to stir for 1 h at room temperature. The crude reaction mixture was diluted
with H2O (10 mL). The resulting solution was extracted with EtOAc (15 mL). The organic
layer was separated. The aqueous layer was extracted with EtOAc (15 mL, ×2). The combined
organic layers were dried with MgSO4, filtered and concentrated to yield a light brown oil 5 that
was used directly in the subsequent transformation.
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L-serine methyl ester hydrochloride (425 mg, 2.73 mmol) was charged into an oven-dried
round bottom flask equipped with magnetic stir bar. The flask was sealed with a septum, and a
balloon with needle was inserted through the septum. CH2Cl2 (14 mL) followed by
triethylamine (1.00 mL, 726 mg, 4.21 mmol) were added consecutively to the stirring solution
via syringe. The reaction was allowed to stir at room temperature for 30 min and then cooled to 0
°C with an ice bath. Hydroxybenzotriazole hydrate (332 mg, 2.17 mmol) was added directly to
flask followed by the addition of 5 dissolved in 4.00 mL of CH2Cl2 (an additional 0.50 mL used
to
ensure
all
material
transferred
to
reaction
flask).
1-Ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (418 mg, 2.18 mmol) was then added directly
to the flask. The reaction was allowed to stir at room temperature overnight (15 h). The crude
reaction mixture was diluted with H2O (10 mL). The resulting solution was extracted with
CH2Cl2 (10 mL). The organic layer was separated. The aqueous layer was extracted with
CH2Cl2 (10 mL, ×2). The combined organic layers were dried with MgSO4, filtered and
concentrated. Purification by flash column chromatography (55% EtOAc/hexanes) on silica gel
1
afforded the desired product 6 (236 mg) in a 25% yield as a colorless solid. MP: 44-46 °C. H-
NMR (400 MHz, CDCl3): δ 7.74 (d, J = 8.44 Hz, 2H), 7.65 (d, J = 8.30 Hz, 1H), 7.55 (d, J = 3.76
Hz, 1H), 7.19-7.23 (m, 3H), 6.80 (d, J = 7.87 Hz, 1H), 6.75 (d, J = 3.77 Hz, 1H), 6.03 (bs, 1H),
4.53 (dt, J = 7.39, 3.55 Hz, 1H), 3.70-3.79 (m, 2H), 3.66 (s, 3H), 3.57 (m, 1H), 2.82 (s, 3H), 2.35
(s, 3H), 2.10 (bs, 1H), 1.94 (bs, 1H), 1.25 (m, 1H), 0.96 (d, J = 6.45 Hz, 3H), 0.93 (t, J = 7.34 Hz,
3H) ppm. 13C-NMR (100 MHz, CDCl3): δ 170.4, 169.6, 145.0, 136.2, 135.2, 129.9, 126.9, 126.8,
125.4, 125.1, 124.0, 113.0, 108.1, 107.0, 71.7, 63.3, 54.0, 52.5, 35.1, 33.6, 25.8, 21.5, 16.1, 11.8
ppm. HRMS-ESI+: calculated for C26H33N3O6S+H: 516.2168; Found: 516.2159.
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4.4
Methyl 2-((2S,3S)-2-((1H-indol-4-yl)(methyl)amino)-3-methylpentanamido)acrylate (7)
An oven-dried reaction tube equipped with magnetic stir bar was charged with 6 (108 mg,
0.21 mmol). Methanol (5.3 mL) was added directly to tube followed by the addition of
magnesium turning (51 mg, 2.10 mmol). The tube was sealed and cooled to 0 °C with an ice
bath and allowed to stir for 4 h. After this time, the reaction mixture was warmed to 40 °C and
stirred for an additional 3 h. The crude reaction mixture was cooled to room temperature and
diluted with aq. NH4Cl (6 mL). The resulting solution was extracted with EtOAc (10 mL). The
organic layer was separated. The aqueous layer was extracted with EtOAc (10 mL, ×2). The
combined organic layers were dried with MgSO4, filtered and concentrated. The crude product
was further purified by flash column chromatography (25% EtOAc/hexanes) on silica gel to
1
afford the product 7 (46.5 mg) in a 65% yield as a colorless oil. H-NMR (400 MHz, CDCl3):
8.21-8.34 (m, 2H), 7.17 (dd, J = 3.26, 2.48 Hz, 1H), 7.07-7.11 (m, 2H), 6.69 (s, 1H), 6.61-6.65
(m, 1H), 6.58 (s, 1H), 5.83 (d, J = 1.48 Hz, 1H), 4.18 (d, J = 7.60 Hz, 1H), 3.71 (s, 3H), 2.89 (s,
3H), 2.07-2.19 (m, 1H), 1.86 (dd, J = 9.47, 7.52 Hz, 1H), 1.22-1.33 (m, 1H), 0.98 (d, J = 6.63
13
Hz, 3H), 0.94 (t, J = 7.42 Hz, 3H). C-NMR (100 MHz, CDCl3): 170.3, 164.1, 144.5, 137.4,
130.8, 123.0, 122.7, 121.2, 110.0, 108.6, 106.1, 101.1, 71.5, 52.7, 37.0, 34.4, 26.4, 16.3, 12.1
ppm. HRMS-ESI+: calculated for C19H25N3O3+H: 344.1974; Found: 344.1964.
4.5
Methyl
(2S,5R)-2-((S)-sec-butyl)-1-methyl-3-oxo-2,3,4,5,6,8-hexahydro-1H-
[1,4]diazonino[7,6,5-cd]indole-5-carboxylate (8)
An oven-dried reaction tube equipped with magnetic stir bar was charged with 7 (45.6 mg,
0.13 mmol). The reaction tube was transferred into a glove box. ZrCl4 (465 mg, 2.0 mmol) and
then CH2Cl2 (1.25 mL) were added. The reaction tube was sealed, transferred out of the glove
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box and then heated to 34 °C. After 24 h, the reaction mixture was cooled to room temperature
and then added dropwise to saturated aqueous NaHCO3 (10 mL) at 0 °C. The resulting solids
were removed by filtration over celite (EtOAc eluent). The layers were separated and the
aqueous layer was extracted with EtOAc (10 mL, ×3). The combined organic layers were dried
over MgSO4. Evaporation under reduced pressure afforded the crude product, which was further
purified by flash chromatography (40% EtOAc/hexanes) to afford the desired product 8 (24.2
mg, 53% yield) as a colorless solid and recovered starting material 7 (12.8 mg, 28% recovered).
MP: 122-124 °C. 1H-NMR (400 MHz, CDCl3): δ 8.08 (s, 1H), 7.06 (t, J = 7.84 Hz, 1H), 7.01 (d,
J = 2.45 Hz, 1H), 6.97 (d, J = 8.06 Hz, 1H), 6.77 (d, J = 7.57 Hz, 1H), 6.68 (d, J = 7.51 Hz, 1H),
4.50 (dt, J = 7.15, 3.62 Hz, 1H), 3.94 (d, J = 8.45 Hz, 1H), 3.93 (s, 3H), 3.46 (dd, J = 15.36, 3.25
Hz, 1H), 3.31 (dd, J = 15.48, 3.90 Hz, 1H), 3.13 (s, 3H), 2.52 (m, 1H), 1.51 (m, 1H), 1.26 (m,
13
1H), 0.71 (d, J = 6.58 Hz, 3H), 0.48 (t, J = 7.39 Hz, 3H) ppm. C-NMR (100 MHz, CDCl3): δ
172.5, 172.4, 147.9, 138.4, 122.8, 122.6, 120.3, 113.2, 109.3, 105.2, 67.0, 57.3, 53.1, 35.2, 34.0,
32.5, 24.8, 16.0, 9.5 ppm. HRMS-ESI+: calculated for C19H25N3O3+H: 344.1974; Found:
344.1967.
4.6
Methyl
(2S,5S)-2-((S)-sec-butyl)-1-methyl-3-oxo-2,3,4,5,6,8-hexahydro-1H-
[1,4]diazonino[7,6,5-cd]indole-5-carboxylate (9).
An oven-dried reaction tube equipped with magnetic stir bar was charged with 8 (10.6 mg,
0.031 mmol). Methanol (2.07 mL) was added to the reaction followed by NaHCO3 (71.4 mg,
0.85 mmol). The reaction tube was sealed and heated to 40 °C for 72 h. After this time, the
reaction mixture was cooled to room temperature. The solution was concentrated and then
partitioned between EtOAc (5.3 mL) and water (5.3 mL). The organic layer was removed, and
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the aqueous layer was extracted with EtOAc (6 mL, ×3). The combined organic layers were
dried with MgSO4, filtered and concentrated. The crude product was purified by flash column
chromatography (10% CH3CN/benzene) on silica gel to afford the epimer 9 (3.4 mg, 32% yield)
as a colorless solid. A mixture of 8 and 9 (3.6 mg, 33% yield) and recovered starting material 8
1
(3.0 mg, 28% recovered) was also obtained. 9 exists as two rotamers at room temperature. H-
NMR (400 MHz, CDCl3), signals corresponding to major rotamer shown: δ 8.20 (bs, 1H), 7.15
(t, J = 7.75 Hz, 1H), 7.07 (t, J = 7.91 Hz, 1H), 7.02 (d, J = 7.40 Hz, 1H), 6.91 (d, J = 2.47 Hz,
1H), 5.24 (d, J = 10.54 Hz, 1H), 5.13 (dd, J = 10.57, 4.74 Hz, 1H), 3.69 (s, 3H), 3.62 (dd, J =
17.80, 5.24 Hz, 1H), 3.13-3.35 (m, 2H), 2.72 (s, 3H), 2.43 (m, 1H), 2.19 (m, 1H), 2.04 (m, 1H),
13
1.07 (t, J = 7.45 Hz, 3H), 0.90 (d, J = 6.46 Hz, 3H) ppm. C-NMR (100 MHz, CDCl3), signals
corresponding to both major and minor rotamers shown: δ 172.7, 171.5, 171.4, 170.2,147.6,
145.1, 139.4, 139.1, 128.3, 127.9, 124.2, 123.1, 123.0, 122.9, 121.4, 114.5, 110.2, 108.8, 107.0,
104.4, 75.0, 69.8, 58.1, 55.6, 52.9, 52.0, 36.3, 35.8, 34.3, 30.6, 30.3, 29.7, 25.2, 24.6, 17.4, 15.7,
11.1, 10.3 ppm. HRMS-ESI+: calculated for C19H25N3O3+H: 344.1974; Found: 344.1963.
4.7 (–)-Indolactam I (1).
9 (3.4 mg, 0.010 mmol) was charged into an oven-dried reaction tube equipped with
magnetic stir bar. The reaction tube was transferred into a glove box. LiBH4 (2.2 mg, 0.10
mmol) and THF (100 µL) were added. The reaction tube was sealed, transferred out of the glove
box and then allow to stir at room temperature for 5 h. After this time, the reaction was added
dropwise to water (0.20 mL) at 0 °C. The resulting solution was extracted with CH2Cl2 (0.50
mL). The organic layer was separated. The aqueous layer was extracted with CH2Cl2 (0.50 mL,
×2). The combined organic layers were dried with MgSO4, filtered and concentrated.
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Evaporation under reduced pressure afforded the crude product, which was further purified by
flash chromatography (70% EtOAc/hexanes) to afford the 1 (2.8 mg, 91% yield) as a colorless
solid. As previously reported,6a 1 exists as two rotamers at room temperature, and the
characterization data matched the previous report. 1H NMR (400 MHz, CDCl3), signals
corresponding to major rotamer shown: δ 7.99 (bs, 1H), 7.08 (t, J = 7.94 Hz, 1H), 6.84-6.98 (m,
3H), 6.51 (d, J = 7.68 Hz, 1H), 4.52 (d, J = 10.30 Hz, 1H), 4.30 (m, 1H), 3.76 (d, J = 11.16 Hz,
1H), 3.56 (m, 1H), 3.21 (d, J = 17.57 Hz, 1H), 3.03 (dd, J = 17.37, 3.74 Hz, 1H), 2.93 (s, 3H),
2.42 (m, 1H), 2.36 (m, 1H), 1.34 (m, 1H), 0.91 (d, J = 6.41 Hz, 3H), 0.58-0.61 (m, 4H) ppm.
13C-NMR (100 MHz, CDCl3), signals corresponding to major rotamer shown: δ 173.6, 147.8,
139.3, 123.0, 121.3, 117.8, 114.4, 106.1, 103.8, 69.4, 65.2, 55.5, 34.2, 33.1, 29.7, 24.6, 17.2, 10.3
ppm. HRMS-ESI+: calculated for C18H25N3O2+H: 316.2025; Found: 316.2015.
4.6 Cell growth inhibition assay: MTT viability readout
K562 cells (ATCC) were cultured in RPMI medium 1640 (Gibco) supplemented with FBS
(10% v/v), penicillin (100 units/ml) and streptomycin (50 units/mL) at 37 °C, in 5% CO2 in air in
a humidified incubator. Cells were subcultured every 72-96 h (once cellular concentration
reaches >1,000,000 cells/mL) to a starting concentration of 200,000 cells/mL. Cell concentration
was determined with a cell counter.
To assay compounds, cells were plated in plasma-treated polystyrene 96-well plates at a
density of 10,000-15,000 cells/well in 50 ꢀL of culture media, and allowed to incubate for 30
min while compound dilutions were prepared. Compounds were diluted from a 10 mM DMSO
stock solution with culture media to a concentration of 20 ꢀM. Colchicine was used as a positive
control in all experiments. Triplicate serial dilutions were then prepared in culture media from
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40 ꢀM to 160 pM across 10 wells of a 96 well plate (diluted by factors of 4). Plates containing
cells were dosed with compound (50 uL/well) to afford final assay concentrations of 20 ꢀM to 80
pM. The maximum DMSO concentration was 0.25%. Cells were then incubated at 37 °C in 5%
CO2 in a humidified incubator for 48 h, at which time 10 ꢀL of a 5 mg/mL solution of thiazolyl
blue tetrazolium bromide (Aldrich) in cell culture media was added to each well. The cells were
allowed to incubate an additional 2.5 h, at which time they were lysed with 100 ꢀL of a detergent
solvent (20 mL Triton-X 100 (Aldrich) in 180 mL of 0.1 N HCl in isopropanol solution).
Plates were analyzed with a VERSAmax tunable microplate reader (Molecular Devices)
using SOFTmax Pro® version 3.1.1, reading at 570 nm and subtracting at 690 nm. The
sigmoidal dose response curves generated by plotting corrected signal vs. log (drug
concentration) were analyzed (least squares regression) using Prism® by GraphPad to generate
EC50 values. An average of several experiments is reported as indicated; experimental error
indicates standard error of the mean.
Acknowledgements
This research was financially supported by startup funds granted by the College of Natural
Sciences and Mathematics at California State University, Fullerton. Instrumentation support was
provided by the National Science Foundation MRI (CHE1726903) for acquisition of an UPLC-
MS.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.org/10.1016/.
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