Total Synthesis and Biological Evaluation of Hybrubin A

Article abstract of DOI:10.1021/acs.joc.6b02534

Here, we report the first total synthesis of hybrubin A, a bipyrrole tetramic acid alkaloid representing a new carbon framework derived from convergent (truncated red cluster and exogenous hbn cluster) biosynthetic pathways. A highly convergent synthesis was developed, employing 4-methoxy-1,5-dihydro-2H-pyrrol-2-one (13) as a single starting material to provide hybrubin A in three steps from 13 and 20.8% overall yield. As no biological activity was prescribed to hybrubin A except for a lack of cytotoxicity, we further profiled this unique alkaloid across panels of discrete molecular targets. Interestingly, hybrubin A was found to be a ligand for a variety of GPCRs with a propensity for potent binding across therapeutically relevant adenosine receptors (A₁, A_2a, and A₃) as well as a potent activity at a kinase, FLT3. This pattern of biological activity is distinct from other related prodigiosin natural and unnatural products and is even more intriguing in the absence of cytotoxicity.

Full text of DOI:10.1021/acs.joc.6b02534

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Article
Total Synthesis and Biological Evaluation of Hybrubin A
Daniel E. Jeffries, and Craig W Lindsley
J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 05 Dec 2016
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Total Synthesis and Biological Evaluation of Hybrubin A
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ψ, †
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Daniel E. Jeffries and Craig W. Lindsley *
ψ
Department of Chemistry, Vanderbilt University, Nashville, TN 37232ꢀ6600
†
Department of Pharmacology, Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt
University, Nashville, TN 37232ꢀ6600
Corresponding Author's Eꢀmail Address ; craig.lindsley@vanderbilt.edu
Table of Contents
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Abstract
Here we report the first total synthesis of hybrubin A, a bipyrrole tetramic acid alkaloid, representing a
new carbon framework derived from convergent (truncated red cluster and exogenous hbn cluster)
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biosynthetic pathways.
A highly convergent synthesis was developed employing 4ꢀmethoxyꢀ1,5ꢀ
dihydroꢀ2Hꢀpyrrolꢀ2ꢀone (13) as a single starting material to provide hybrubin A in three steps from 13
and 20.8% overall yield. As no biological activity was prescribed to hybrubin A, except a lack of
cytotoxicity, we further profiled this unique alkaloid across panels of discrete molecular targets.
Interestingly, hybrubin A was found to be a ligand for a variety of GPCRs, with a propensity for potent
binding across therapeutically relevant adenosine receptors (A , A and A ) as well as a potent activity at
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a kinase, FLT3. This pattern of biological activity is distinct from other related prodigiosin natural and
unnatural products, and even more intriguing in the absence of cytotoxicity.
INTRODUCTION
Prodigiosin (1) and related tripyrrole natural products (Figure 1) have been the subject of renewed
biological interest by virtue of their wide breadth of activities including cytotoxicity and antibacterial
properties. In fact, the synthetic prodigiosin analogue obatoclax (2) was granted orphan drug status by the
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U.S. Food and Drug Administration for the treatment of chronic lymphocytic leukemia. Marineosin A
(3) is a novel macrocyclic spirolactam spiroiminal alkaloid structurally related to the prodigiosin family,
with potent inhibition against human colon carcinoma cell growth, with an IC of 0.5 µM in HCTꢀ116
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cells. A structurally related family, the tambjamines 4ꢀ6, are also highly cytotoxic and of interest in
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oncology. The surprising properties of these structural diverse classes of 2,2’ꢀbipyrrolic alkaloid natural
products have garnered significant attention to probe deeper into the potential applications of prodigiosinꢀ
like natural products.
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Figure 1. Structures of prodigiosin (1) and related 2,2’bipyrrole cytotoxic alkaloids obatoclax (2), marineosin A
(3), tambjamine A (4), the generic tambjamine core (5) and tambjamine K (6).
Figure 2. Structures of hybrubin A (7), hybrubin B (8) and hybrubin C (9).
To this end, structural analogs of prodigiosin, termed the hybrubins (Figure 2), and designated
hybrubin A (7), hybrubin B (8) and hybrubin C (9), were recently discovered as the result of bacterial
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artificial chromosome library screening. These “unnatural” natural products are of interest due to their
novel chimera structure which joins a 2,2’ꢀbipyrrole motif common to prodigiosin biosynthesis (truncated
red cluster) with a tetramic acid ring (exogenous hbn cluster). All three hybrubins 7ꢀ9 were isolated as
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rapidly interchanging C6’ olefin isomers, with ratios of 1:1 to 3.5:1 by H NMR, and further designated as
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hybrubin A and A , hybrubin B and B and hybrubin C and C . While both pharmacophores of these
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chimera alkaloids are known to possess biological activity, initial studies demonstrated that, surprisingly,
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the hybrubins were devoid of the cytotoxic or antibacterial properties typical of the broader class.
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Therefore the need for independent synthesis to further probe for biological activity was apparent. Here
we report the first total synthesis of hybrubin A via a highly convergent synthetic approach that enabled
detailed biological evaluation and the discovery of a unique and unexpected pharmacological profile.
RESULTS AND DISCUSSION
Our first generation retrosynthetic analysis of 7 (Figure 3) begins with an elimination step to provide tertꢀ
butyl ether 10. A strategic disconnection across the C3’ꢀC6’ olefin then affords known bipyrrole 11 and
the corresponding tetramic acid 12, which can be joined through aldol chemistry. The synthesis began
with efforts to generate bipyrrole 11, which conveniently had been synthesized en route to marineosins A
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and B previously reported by our laboratory (Scheme 1). The core 2,2’ꢀbipyrrole of 11 has been the
subject of numerous other syntheses and methodologies spanning over five decades both in the context
6ꢀ11
presented here as well as in the context of porphyrinoids, among others.
Conversion of the
commercially available 13
Figure 3. First generation retrosynthetic analysis of hybrubin A (1).
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began with modified Vilsmeier conditions to formylate the pyrrolidine with concurrent installation of a
bromine atom to afford 14. Treatment of 14 with standard Suzuki coupling conditions with the
commercially available NꢀBocꢀ2ꢀpyrrole boronic acid yielded desired bipyrrole 11 in 44% yield, after the
desired coupling and hydrolysis of enamine were achieved with complete conversion of 14. Upon
purification, a small amount of NꢀBoc deprotected 11 could be collected; further explanation of the
modest yield is possible homocoupling or protodeboronation occasionally seen in crossꢀcouplings of
heteroaryl halides. To address the latter, facile transformations utilizing potassium
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heteroaryltrifluoroborates or MIDA boronates as opposed to standard boronic acids have been reported
which help avoid these side reactions. As seen with our previously reported synthesis using this route (see
ref. 2) yields were generally low; therefore this sequence was performed on gram scale to supply material
for the completion of the total synthesis. Of note is the brevity of the sequence in which four distinct
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chemical transformations are made in only two sets of reaction conditions.
Scheme 1. Synthesis of the key bipyrrole 11.
With bipyrrole 11 in hand, our attention now turned to syntheses of the tetramic acid motif and
ultimately hybrubin A (Scheme 2). Initially it was envisioned that the tetramic acid moiety 12 could be
prepared in a single step from the amino acid
cyclization reaction which has found use in several related syntheses.
condensation and removal of protecting groups a mixture of hybrubins A (7) and B (8) would be obtained.
Lꢀthreonine and Meldrum’s acid using a previously reported
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After a subsequent aldol
15 was smoothly transformed to 12, however it was quickly realized 12 was prone to decomposition from
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silica gel chromatography, and as such, was taken on crude into the next step. Since 15 was used directly
from a commercial source as the depicted diastereomer and the desired final product is devoid of the two
stereogenic centers d.r. ratios of proceeding products were not obtained. The following Aldol
condensation with 11 was the first reaction in the sequence which required significant optimization
(Table 1). The use of stoichiometric amounts of heterogeneous and homogenous bases in various solvents
and temperatures was unable to afford the desired condensation to deliver 10 in acceptable yields. Here it
is worthwhile to mention that sodium hydride, when employed in two one equivalent portions, resulted in
chiefly recovered starting materials presumably on basis of the reversibility of the aldol reaction. When
employing catalytic reagents, yields increased noticeably; however, when exposed to reaction
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temperatures above 80 C, the labile Boc protecting group was also removed. This observation was later
used to our advantage when the transformation was carried out in refluxing ethanol with piperidine to
elicit both the desired aldol condensation and removal of the labile pyrrole Boc group in good yields
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68% for two steps). With the carbon skeleton of the hybrubins in place, completion of the synthesis
required only deprotection of the tertꢀbutyl ether moiety and the Boc group on the tetramic acid, followed
by dehydration to give 7 and 8.
Table 1. Reaction optimization for Aldol condensation to produce 10.
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Entry
Base
Temp ( C)
Solvent
THF
1,4 dioxane
1,4 dioxane
THF
Yield
3.2%
10%
3.1%
3%
7.8%
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23.4%
32%
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NEt (3 eq)
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NEt (3 eq)
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N,N-Diisopropylethylamine (3 eq)
Cs CO (2 eq)
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NaH (2 eq)
NaOtBu (2.5 eq)
L-proline (0.05 eq)
THF
MeOH
DMSO
DMSO
DMSO
L-proline (0.20 eq)
(S)-5-(pyrrolidine-2-yl)-1H-tetrazole)
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Scheme 2. First generation approach to the tetramic acid moiety and the synthesis of hybrubins A (7) and B (8).
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After screening numerous conditions for the removal of both protecting groups in 10
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simultaneously it became evident a twoꢀstep procedure would be more advantageous. Owing to the high
aqueous solubility of 16 it was further desired to perform these transformations in absence of aqueous
workups. With these goals in mind, a deprotection sequence which entailed thermal removal of the Boc
group followed directly by treatment of crude material with TMSI was developed which furnished 16 in
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5% yield across both steps.
Subsequent attempts at dehydration of the free alcohol were once again
subject to optimization. Traditional acidic or basic conditions (i.e. HCl in dioxanes, TFA in CHCl , and
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NaOMe in MeOH) failed to affect the desired dehydration. Dehydration reagents such as SOCl , POCl ,
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and Martin Sulfurane were also unable to efficiently dehydrate 16, and provided only unreacted starting
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material. It was not until 16 was treated with acetic anhydride and heated to 110 C that the desired
conversion of 16 to a mixture of 7 and 8 was achieved, albeit in low yields (19%). The route presented in
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Scheme 3 is capable of successful total synthesis of 7 and 8, but is dominated by protecting group
manipulations and culminates in harsh, low yielding dehydration conditions. Additionally, compounds 7
and 8 were unable to be separated via HPLC despite following analogous conditions to those reported by
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Tao and coworkers.
Thus, a second generation approach would be required to access 7 in high yield and purity for
biological study. While developing the first approach, a secondary strategy towards hybrubin A (7) was
simultaneously explored (Figure 4). This approach utilized the same starting material 13 as the
complementary arm of the convergent synthesis for key fragments 11 and 17, and prioritized the
Figure 4. Second generation retrosynthetic analysis of hybrubin A (7).
installation of the exocyclic olefin. Therefore, treatment of 13 (Scheme 3) with acetaldehyde and sodium
tertꢀbutoxide to furnish 18 provided an appealing alternative to the multiꢀstep deprotection and
dehydration sequence previously developed. The anticipated transformation proceeded smoothly and
purification of the crude reaction mixture on silica gel yielded fractions of one pure olefin isomer and
fractions of complex mixtures. NOESY experiments showed the pure isomer thus obtained was the
desired Zꢀolefin; proton NMR experiments were never able to confirm noticeable presence of the E
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olefin. This observation is supported by A1,3 strain considerations which would favor the Zꢀolefin.
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While the collection of only a single olefin geometry at this stage denies access to hybrubin B, it also
provides a complementary approach to the isolation of hybrubin A from B via HPLC performed by Tao
and coworkers. After facile transformation of 18 to 17 by brief treatment with concentrated HCl in THF
and subsequent aldol condensation with the previously optimized conditions, 7 was obtained in 70%
yield. In summary, hybrubin A (7) was selectively obtained in a convergent fashion from a shortest linear
three step reaction sequence from 13. The second generation sequence provides 7 in 20.8% overall yield
in only 20.5 total hours of reaction time, without need for chromatographic separation of the exocylic
olefins or discrete protecting group manipulations from a single starting material 13. The synthetic 7 was
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identical to the natural product in all aspects.
Scheme 3. Second generation approach to the tetramic acid moiety and the synthesis of hybrubin A (7).
With sufficient quantities of 7 in hand, we initiated biological studies. First, we evaluated
cytotoxicity, as related prodigiosin alkaloids 1ꢀ6 displayed robust cytotoxicity against HCTꢀ116 colon
cancer cell lines. To our surprise, 7 (at concentrations up to 10 µM) was devoid of cytotoxicity after
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incubation in HCTꢀ116 cells for 24 and 48 hours as measured by cell viability (WSTꢀ1 metabolism).
Thus not only does 7 differentiate structurally from 1ꢀ6, but also differs in terms of general bioactivity.
Based on this outcome, we then evaluated 7 in radioligand binding assays against 68 discrete targets
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(GPCRs, ion channels and transporters) at an initial concentration of 10 µM.
We were excited to see
that 7 afforded significant activity (Table 2) at only 4 of the 68 targets (>70% inhibition@10 µM), with
the greatest activity against the adenosine receptors (A , A and A ) as well as the serotonin 5ꢀHT
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receptor. Followꢀup K determinations showed that 7 was a potent ligand to these GPCRs, affording
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Table 2. Summary of significant results of 7 against a panel of GPCRs, ion channels and transporters.
a
Target
Species
human
human
human
human
% inhibiton@10 µM
K (nM)
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550
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Adenosine A3
Serotonin 5ꢀHT2B
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potency at A (2.6 µM) and 5ꢀHT (1.05 µM) and nanomolar binding to A2A (550 nM) and A (54 nM).
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Thus, 7 represents a new chemotype for a selective A ligand, with 10ꢀfold selectivity over A2A and 48ꢀ
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fold selectivity over A1. We also screened 7 against a panel of 369 wildtype and mutant kinases at a
concentration of 10 µM, and noted very limited kinase activity (significant reduction of enzyme activity
at only 7 of the 369 kinases) and upon IC50 determinations, 7 proved to only inhibit FLT3 with
submicromolar potency (IC = 505 nM), as the other six kinase hits had IC values in the 1.6 to 7.9 µM
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range (and the remaining 362 kinases had IC s > 10 µM).
Coupled with the lack of cytotoxicity of 7
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attractive lead compound derived from nature.
These data also suggest that additional FLT3 ligands
might be mined from screening known A ligands, and vice versa. Overall, the biological profile of 7 is
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unique and very exciting, and argues for the synthesis of unnatural analogs to develop SAR and new in
vivo tool compounds.
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In conclusion, we performed the first total synthesis of hybrubin A (7) via a rapid and highly
convergent approach (3 steps, 20.8% overall yield from 13) employing a single starting material to access
two chemically distinct, advanced intermediates. Extensive biological studies showed that 7 lacked
general cytotoxicity, and was a potent ligand at a number of therapeutically relevant GPCRs, with
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selective binding to the A receptor. In addition, a broad kinase panel found 7 to be a selective inhibitor of
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FLT3, a therapeutic target for AML.
Based on these data, we are currently evaluating the in vivo
efficacy of 7 in animal models as well as developing routes to access hybrubins B and C selectively, as
well as unnatural analogs. Studies are underway and additional results and refinements will be reported
in due course.
EXPERIMENTAL SECTION
General. All reagents and solvents were commercial grade and purified prior to use when
necessary. Thin layer chromatography (TLC) was performed on glassbacked silica gel.
Visualization was accomplished with UV light, and/or the use ninhydrin or potassium
permanganate solution followed by brief heating with a heat gun. Flash chromatography on silica
gel was performed using Silica Gel 60 (230ꢀ400 mesh). Preparative reversedꢀphase HPLC was
run with a system containing a C18 column (30 x 100mm). IR spectra were acquired from
samples which were applied to NaCl plates as DCM solutions followed by generous air drying.
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13
Melting points were obtained with an automated melting point system. H and C NMR spectra
were recorded on either 400 MHz or 600MHz instruments. Chemical shifts are reported in ppm
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relative to residual solvent peaks as an internal standard at the following chemical shifts ( H and
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C respectively): 7.26 and 77.1 ppm for CDCl3
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2.50 and 39.5 ppm for DMSOꢀd , 3.31 and 49.2
;
ppm for CD OD. Data are reported as follows: chemical shift, multiplicity (s = singlet, d =
3
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doublet, t = triplet, q = quartet, p = pentet, qd = quartet of doublets, br = broad, m = multiplet),
coupling constant (Hz), integration. Low resolution mass spectra (LCMS) were obtained using a
+
+
quadrupole with electrospray ionization generating [M+H] or [M+Na] ions. High resolution
mass spectra (HRMS) were obtained using a QꢀTOF with electrospray ionization generating
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[M+H] or [M+Na] ions.
(5Z)-5-ethylidene-3-((4-methoxy-1H,1'H-[2,2'-bipyrrol]-5-yl)methylene)pyrrolidine-2,4-
dione (7). To a stirred solution of (5Z)ꢀ5ꢀethylidenepyrrolidineꢀ2,4ꢀdione 17 (47.1 mg, 0.380
mmol) in ethanol (1.07 mL) and piperidine (86.8 µL, 0.540 mmol) was added 11 (78.0 mg, 0.270
o
mmol) and the resulting mixture warmed to 82 C and allowed to stir for 8 hours. Upon reaction
completion as determined by LC/MS the mixture was cooled to ambient temperature and all
volatiles were removed in vacuo. The resulting deep red gel was dissolved in a 1 mL of
methanol and purified over a strong cation exchange column. Desired material eluted after
addition of 2M ammonia solution in MeOH; the organics collected from this method were
brought to dryness under a steady stream of air before being resuspended in a 15% MeOH in
DMSO solution for purification using Gilson reverseꢀphase chromatography system (30 x 100
mm column, 32ꢀ51% MeCN / 0.1% trifluoroacetic acid (aq), 9 min gradient, 300 nm
wavelength) to afford hybrubin A, (5Z)ꢀ5ꢀethylideneꢀ3ꢀ((4ꢀmethoxyꢀ1H,1'Hꢀ[2,2'ꢀbipyrrol]ꢀ5ꢀ
yl)methylene)pyrrolidineꢀ2,4ꢀdione, as a red solid (56.2 mg, 0.189 mmol, 70% yield). m.p. 246ꢀ
o
1
248 C; H NMR (400.1 MHz, DMSOꢀd major regioisomer) δ (ppm): 13.58 (br.s, 1H), 12.01
6
,
(br.s, 1H), 9.89 (s, 1H), 7.1924 (s, 1H), 7.1919 (s, 1H), 6.79 (br.s, 1H), 6.58 (d, J= 2.4 Hz, 1H),
1
3
1
6.35 (m, 1H), 5.55 (q, J= 7.4 Hz, 1H), 3.98 (s, 3H), 1.78 (d, J= 7.4 Hz, 3H).
C { H} NMR
.
(150 MHz, DMSOꢀd , major regioisomer) δ (ppm): 182.0, 169.0, 161.6, 137.9, 136.5, 124.1,
6
123.5, 122.5, 121.1, 111.0, 110.4, 105.5, 103.1, 93.3, 58.8, 12.1. FTIR: (neat) υ 2916, 2848,
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ꢀ1
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356, 2335, 1670, 1613, 1549, 1504, 1454, 1365, 1212, 1133, 952, 808 cm . LCMS: R : 0.883
T
+
min., m/z = 298.1 [M + H] , >99% abs @ 215, 254, and 300n, HRMS-ESI: calcd for
+
C H N O Na [M + Na] m/z 320.1013; found m/z 320.1003.
16
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3
3
10
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tert-butyl (S)-2-((R)-1-(tert-butoxy)ethyl)-4-((4-methoxy-1H,1'H-[2,2'-bipyrrol]-5-yl)methylene)-
,5-dioxopyrrolidine-1-carboxylate (10). Piperidine (389.2 µL, 2.41 mmol) was added to a
3
stirring solution of 12 (613.55 mg, 2.05 mmol) in ethanol (4.82 mL). The resulting solution was
allowed to briefly stir before 11 (350 mg, 1.21 mmol) was introduced and the reaction warmed
o
8
2 C. After 6 hours of stirring LC/MS confirmed reaction completion and all volatiles were
removed in vacuo to produce crude product as a pitch colored oil. Upon purification using
Teledyne ISCO CombiꢀFlash system (silica gel loading, 12G column, 15ꢀ40% EtOAc, 30 min
run) tertꢀbutyl (S)ꢀ2ꢀ((R)ꢀ1ꢀ(tertꢀbutoxy)ethyl)ꢀ4ꢀ((4ꢀmethoxyꢀ1H,1'Hꢀ[2,2'ꢀbipyrrol]ꢀ5ꢀ
yl)methylene)ꢀ3,5ꢀdioxopyrrolidineꢀ1ꢀcarboxylate was obtained as a red solid and mixture of
o
1
interchanging C6’ olefin isomers (388 mg, 0.820 mmol, 68% yield). m.p. 166ꢀ168 C H NMR
(400.1 MHz, CDCl , integrations based off more abundant C6’ isomer) δ (ppm): 13.36 (s, 1H),
3
9
2
1
1
.64 (s, 1H), 7.39 (s, 1H), 7.09ꢀ7.07 (m, 1H), 6.75ꢀ6.73 (m, 1H), 6.38ꢀ6.36 (m, 1H), 6.07 (d, J=
.4Hz, 1H), 4.43 (q.d, 1H, J = 6.6, 1.5 Hz), 4.10 (d, 1H, J= 1.5Hz), 3.92 (s, 3H), 1.60 (s, 9H),
1
3
1
.16 (s, 9H), 1.10 (s, 3H). C { H} NMR .(100.6 MHz, CDCl ) δ (ppm): 192.4, 170.0, 163.2,
3
50.3, 140.4, 124.4, 124.1, 123.8, 122.7, 112.8, 111.2, 106.1, 92.9, 82.4, 74.6, 68.2, 66.7, 58.5,
+
28.5, 28.3, 20.1. LCMS: R : 1.098 min., m/z = 472.3 [M + H] , >99% abs @ 215 and 254nm.
T
+
HRMS-ESI: calcd for C H N O [M + H] m/z 472.2449; found m/z 472.2443.
2
5
34
3
6
tert-butyl 5'-formyl-4'-methoxy-1H,1'H-[2,2'-bipyrrole]-1-carboxylate (11).
ꢀBocꢀpyrroleꢀ2ꢀboronic acid (3.13 g, 14.8 mmol) and 14 (2.56 g, 9.88 mmol) were dissolved in
1
degassed 1,4ꢀdioxane (74.1 mL) and water (8.2 mL). Sodium carbonate (3.14 g, 29.6 mmol) was
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then added and the mixture purged with argon for one minute before
Tetrakis(triphenylphosphine)palladium(0) (1.14 g, 0.988 mmol) was added and the reaction
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vessel sealed and heated to 85 C for 3.5 hours. The mixture was then cooled to ambient
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temperature and poured into water (130 mL) followed by the slow addition of 1 N aq. HCl to
achieve pH = 7. The solution was next transferred to a separatory funnel and extracted with
DCM (3 x 70 mL). The resulting organic layers were dried over magnesium sulfate, filtered, and
condensed directly onto silica gel for purification using Teledyne ISCO Combi Flash system
(solid loading, 40G column, 0ꢀ30% EtOAc, 35 min run) to afford tertꢀbutyl 5'ꢀformylꢀ4'ꢀ
methoxyꢀ1H,1'Hꢀ[2,2'ꢀbipyrrole]ꢀ1ꢀcarboxylate as a light orange solid (1.27 g, 4.37 mmol, 44%
2
,6 1
yield).
H NMR (400.1 MHz, CDCl ) δ (ppm): 10.74 (br.s, 1H), 9.51 (s, 1H), 7.32 (m, 1H),
3
1
3
1
6.65 (m, 1H), 6.23 (t, J= 3.6Hz, 1H). 6.06 (d, J= 2.6 Hz, 1H), 3.87 (s, 3H), 1.59 (s, 9H). C { H}
NMR (100.6 MHz, CDCl ) δ (ppm): 174.3, 157.7, 149.7, 130.3, 126.0, 124.5, 118.3, 116.9,
3
+
1
11.5, 94.8, 85.7, 57.9, 27.9 LCMS: R : 0.988 min., m/z = 291.1 [M + H] . HRMS-ESI: calcd
T
+
for C H N O [M + H] m/z 291.1347; found m/z 291.1340.
1
5
19
2
4
tert-butyl (S)-2-((R)-1-(tert-butoxy)ethyl)-3,5-dioxopyrrolidine-1-carboxylate (12). To a solution
of 2,2ꢀdimethylꢀ1,3ꢀdioxaneꢀ4,6ꢀdione (Meldrum’s acid, 1.73 g, 12.0 mmol) and 4ꢀ
o
dimethylaminopyridine (1.86 g, 15.3 mmol) in DCM (72.6 mL) at 0 C was added 15 (3.00 g,
10.9 mmol) and then Nꢀ(3ꢀdimethylaminopropylꢀN`ꢀethylcarbodiimide hydrochloride (EDC,
2.53 g, 13.1 mmol). The resulting mixture was warmed to ambient temperature and allowed to
stir for 21 hours. At this time all solvent was removed in vacuo and the resulting yellow oil
resuspended in EtOAc (175 mL) and washed with brine (2 x 80 mL), 1M citric acid (3 x 100
mL), and again brine (100 mL). The resulting organic layer was passed through a phase
o
separator and heated to 77 C for 90 minutes. Upon cooling all volatiles were removed in vacuo
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to afford crude tertꢀbutyl (S)ꢀ2ꢀ((R)ꢀ1ꢀ(tertꢀbutoxy)ethyl)ꢀ3,5ꢀdioxopyrrolidineꢀ1ꢀcarboxylate as
1
a yellow foamy oil (2.93 g, 9.78 mmol, 89% yield). H NMR (400.1 MHz, CDCl ) δ (ppm):
3
4.59 (p, J = 6.2Hz, 1H), 4.34 (d, J= 5.2 Hz, 1H), 3.60 (s, 2H), 1.50 (s, 9H), 1.27 (s, 9H). 0.99 (d,
10
11
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59
60
+
J= 6.4 Hz, 3H) LCMS: R : 0.919 min., m/z = 621.2 [2M + Na] , >99% abs @ 215 and 254nm.
T
+
HRMS-ESI: calcd for C H NO Na [M + Na] m/z 322.1633; found m/z 322.1627.
1
5
25
5
N-((5-bromo-3-methoxy-2H-pyrrol-2-ylidene)methyl)-N-ethylethanamine (14). A 250 mL round
bottom flask containing a stir bar was charged with N,Nꢀdiethylformamide (4.72 mL, 42.4 mmol)
and DCM (35 mL). The mixture was then cooled in an ice bath and a solution of phosphorus (V)
oxybromide (15.2 g, 53.1 mmol) in DCM (10.6 mL) was added dropwise over the course of
several minutes. After addition was complete the reaction was allowed to stir for twenty
additional minutes before a solution of 4ꢀmethoxyꢀ3ꢀpyrrolinꢀ2ꢀone (3.00 g, 26.5 mmol) in DCM
(
26 mL) was introduced dropwise over the course of several minutes. Once the addition was
complete the reaction mixture was transferred directly from the ice bath to metal round bottom
o
warmer preheated to 42 C and allowed to stir for 3.5 hours. Upon cooling of the reaction
mixture to ambient temperature the material was transferred to a 1 L round bottom flask, cooled
in an ice bath, and quenched with the dropwise addition of water (30 mL). Then, a solution of
aqueous sodium hydroxide (15% w/w, 350 mL) was carefully added and the resulting mixture
allowed to stir at ambient temperature for 20 minutes. The material was then transferred to a
separatory funnel and extracted with DCM (3 x 120 mL). The combined organic layers were
dried over magnesium sulfate, filtered, and condensed directly onto silica gel for purification
using Teledyne ISCO Combi Flash system (solid loading, 40G column, 0ꢀ20% EtOAc, 25 min
run) to afford Nꢀ((5ꢀbromoꢀ3ꢀmethoxyꢀ2Hꢀpyrrolꢀ2ꢀylidene)methyl)ꢀNꢀethylethanamine as pale
2,6 1
yellow crystals (3.33 g, 12.8 mmol, 49% yield).
H NMR (400.1 MHz, CDCl ) δ (ppm): 6.99
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(
s, 1H), 5.59 (s, 1H), 4.11 (q, J= 7.1Hz, 2H), 3.75 (s, 3H), 3.39 (q, J= 7.2Hz, 2H), 1.28 (m, 6H)
1
3
1
C { H} NMR (100.6 MHz, CDCl ) δ (ppm): 165.2, 138.6, 133.4, 120.7, 96.3, 57.9, 51.1, 44.5,
3
+
1
4.5, 12.4. LCMS: R : 0.552 min., m/z = 259.2 [M + H] >99% abs @ 215 and 254nm. HRMS-
T
0
1
2
3
4
5
6
7
8
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0
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+
ESI: calcd for C H BrN O [M + H] m/z 259.0448; found m/z 259.0439.
1
0
16
2
(S)-5-((R)-1-hydroxyethyl)-3-((4-methoxy-1H,1'H-[2,2'-bipyrrol]-5-yl)methylene)pyrrolidine-
2
,4-dione (16). Solid 10 (59.5 mg, 0.160 mmol) was heated neat in a two dram vial fully
o
immersed in a pieꢀblock at 180 C for 90 minutes. The resulting crude solid was cooled and
immediately dissolved chloroform (1.62 mL) and treated with trimethylsilyl iodide (47.6 µL,
o
0
.320 mmol) and allowed to stir at 45 C for one hour. The reaction mixture was then cooled to
ambient temperature and diluted with MeOH (1 mL) before all volatiles were removed in vacuo.
The resulting deep red solid was dissolved in MeOH (1 mL) and purified over a strong cation
exchange cartridge with product eluting following addition of 2M ammonia in MeOH solution
after several rinses of pure MeOH. In this manner, (S)ꢀ5ꢀ((R)ꢀ1ꢀhydroxyethyl)ꢀ3ꢀ((4ꢀmethoxyꢀ
1H,1'Hꢀ[2,2'ꢀbipyrrol]ꢀ5ꢀyl)methylene)pyrrolidineꢀ2,4ꢀdione was obtained as a fine red solid
o
1
(43.3 mg, 0.137 mmol, 85.7%) after complete removal of all volatiles. m.p 242ꢀ244 C H NMR
(400.1 MHz, CD OD, inseparable mixture of olefins) δ (ppm): 7.30 (s, 2H), 7.10 (s, 2H), 6.88 (s,
3
2
H), 6.35ꢀ6.33 (m, 3H), 6.33 (s, 1H), 4.16 (br.s, 2H), 4.01 (s, 3H), 3.99 (s, 3H), 3.77 (d, J=3.3
13
1
Hz, 1H), 1.34 (d, J=6.5 Hz, 3H), 1.12 (d, J=6.4 Hz, 3H). C { H} NMR (150 MHz, DMSOꢀd ,
6
inseparable mixture of olefins) δ (ppm):195.8, 195.3, 173.3, 171.3, 161.5, 161.0, 137.4, 137.1,
1
23.9, 123.3, 123.1, 122.1, 120.8, 120.4, 117.7, 117.4, 110.8, 110.5, 109.8, 108.8, 107.7, 107.2,
9
3.2, 92.4, 67.2, 66.9, 66.7, 65.0, 58.6, 58.5, 20.6, 17.1. LCMS: R : 0.833 min., m/z = 316.1 [M
T
+
+
+ H] , >99% abs @ 215, 254, and 300nm. HRMS-ESI: calcd for C H N O [M + H] m/z
1
6
18
3
4
3
16.1299; found m/z 316.1292.
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(
Z)-5-ethylidenepyrrolidine-2,4-dione (17). To a stirred solution of (Z)ꢀ5ꢀethylideneꢀ4ꢀmethoxyꢀ
1
,5ꢀdihydroꢀ2Hꢀpyrrolꢀ2ꢀone 18 (451 mg, 3.24 mmol) in THF (64.5 mL) at ambient temperature
was carefully added concentrated HCl (38 mL). The resulting faint yellow solution was stirred at
ambient temperature for 90 minutes at which time TLC confirmed reaction completion.
Saturated aqueous sodium bicarbonate (40 mL) was then slowly added to quench the reaction.
The reaction mixture was transferred to a separatory funnel and extracted with EtOAc (3 x 50
mL) and the resulting pooled organics were dried over magnesium sulfate. Crude product was
purified using Teledyne ISCO CombiꢀFlash system (solid loading on silica gel, 4G column, 20ꢀ
10
11
12
13
14
15
16
17
18
19
20
21
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23
24
25
26
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40
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44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7
0% EtOAc, 25 min run) to afford (Z)ꢀ5ꢀethylidenepyrrolidineꢀ2,4ꢀdione as a light yellow solid
(269 mg, 2.15 mmol, 66% yield). Spectroscopic data of this compound reported in compliance
2
7
o
1
with previously reported data. m.p 184ꢀ186 C H NMR (400.1 MHz, CDCl ) δ (ppm): 9.32
3
13
1
(s, 1H), 5.76 (q, J=7.5 Hz, 1H), 3.10 (s, 2H x 1/2), 1.82 (d, J=7.5Hz, 3H). C { H} NMR
.(100.6 MHz, CDCl ) δ (ppm): 192.4, 170.7, 136.5, 106.7, 40.6, 12.0. LCMS: R : 0.116 min.,
3
T
+
+
m/z = 126.2 [M + H] , >99% abs @ 215 and 254 nm. HRMS-ESI: calcd for C H NO [M + H]
6
8
2
m/z 126.0557; found m/z 126.0548.
(
Z)-5-ethylidene-4-methoxy-1,5-dihydro-2H-pyrrol-2-one (18). To a 100 mL round bottom flask
equipped with a magnetic stir bar was added ethanol (44 mL), 4ꢀmethoxyꢀ3ꢀpyrrolinꢀ2ꢀone (1.00
g, 8.84 mmol), sodium tertꢀbutoxide (1.70 g, 17.7 mmol), and lastly acetaldehyde (2.00 mL, 35.4
o
mmol). The resulting mixture was warmed to 40 C and allowed to stir for four hours.
Following this, all volatiles were removed via rotovaporation. The resulting orange oil was
condensed directly onto silica gel for purification using Teledyne ISCO CombiꢀFlash system
(12G column, 30ꢀ90% EtOAc, 45 min run). Careful TLC analysis of the resulting column
fractions was used to selectively collect (Z)ꢀ5ꢀethylideneꢀ4ꢀmethoxyꢀ1,5ꢀdihydroꢀ2Hꢀpyrrolꢀ2ꢀ
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one which upon concentration was afforded as a white solid (551 mg, 3.96 mmol, 45%). m.p
o
1
1
37ꢀ139 C; H NMR (400.1 MHz, CDCl ) δ (ppm): 9.01 (s, 1H), 5.46 (q, J= 7.5 Hz, 1H), 5.09
3
1
3
1
(
d, 1H, J= 1.5 Hz), 3.81 (s, 3H), 1.86 (d, J= 7.5Hz, 3H). C { H} NMR .(100.6 MHz, CDCl ) δ
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
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4
5
6
7
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9
0
(
ppm): 172.8, 166.0, 134.1, 105.9, 92.4, 57.8, 12.4. LCMS: R : 0.112 min., m/z = 140.1 [M +
T
+
+
H] , >99% abs @ 215 nm. HRMS-ESI: calcd for C H NO [M + H] m/z 140.0713; found m/z
7
10
2
140.0703.
ACKNOWLEDGEMENT
The authors warmly thank the Department of Pharmacology and the Warren Family and Foundation,
through the William K. Warren, Jr. Chair in Medicine, for support of our programs and training in basic
organic chemistry, and to Dr. Lawrence Marnett and Mrs. Brenda Crews for the HCTꢀ116 viability assay
data.
1
13
Supporting Information. Supplemental Tables, data and copies of all H and C NMR spectra. This
material is available free of charge via the Internet at http://pubs.acs.org.
References
1
.
Kim, P.S.; Jochems C.; Grenga, I.; Donahue, R.N.; Tsang, K.W.; Gulley, J.L.; Schlom, J.; Farsaci, B.
J.Immunol. 2014, 192, 2622ꢀ2633.
2
3
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