Design, Synthesis, Dynamic Docking, Biochemical Characterization, and in Vivo Pharmacokinetics Studies of Novel Topoisomerase II Poisons with Promising Antiproliferative Activity

Article abstract of DOI:10.1021/acs.jmedchem.9b01760

We previously reported a first set of hybrid topoisomerase II (topoII) poisons whose chemical core merges key pharmacophoric elements of etoposide and merbarone, which are two well-known topoII blockers. Here, we report on the expansion of this hybrid mol

Full text of DOI:10.1021/acs.jmedchem.9b01760

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Article
Design, synthesis, dynamic docking, biochemical
characterization, and in vivo pharmacokinetics studies of novel
topoisomerase II poisons with promising antiproliferative activity
Jose M. Arencibia, Nicoletta Brindani, Sebastian Franco-Ulloa, Michela Nigro, JISSY
AKKARAPATTIAKAL KURIAPPAN, Giuliana Ottonello, Sine Mandrup Bertozzi, Maria
Summa, Stefania Girotto, Rosalia Bertorelli, Andrea Armirotti, and Marco De Vivo
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01760 • Publication Date (Web): 20 Mar 2020
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Design, synthesis, dynamic docking, biochemical
characterization, and in vivo pharmacokinetics studies of
novel topoisomerase II poisons with promising
antiproliferative activity
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1
Jose M. Arencibia,# Nicoletta Brindani,# Sebastian Franco-Ulloa, Michela Nigro, Jissy
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Akkarapattiakal Kuriappan, Giuliana Ottonello, Sine Mandrup Bertozzi, Maria Summa,
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Stefania Girotto, Rosalia Bertorelli, Andrea Armirotti, Marco De Vivo*
1. Molecular Modeling and Drug Discovery Lab, Istituto Italiano di Tecnologia, via Morego 30,
16163 Genova, Italy
2. Analytical Chemistry and in vivo Pharmacology, Istituto Italiano di Tecnologia, via Morego 30,
16163 Genova, Italy
Corresponding author:
Dr. Marco De Vivo – Email: marco.devivo@iit.it
#Equally contributed
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ABSTRACT
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We previously reported a first set of hybrid topoisomerase II (topoII) poisons whose chemical
core merges key pharmacophoric elements of etoposide and merbarone, which are two well-known
topoII blockers. Here, we report on the expansion of this hybrid molecular scaffold, and present
sixteen more hybrid derivatives that have been designed, synthesized, and characterized for their
ability to block topoII and for their overall drug-like profile. Some of these compounds act as
topoII poison, exhibit good solubility, metabolic (microsomal) stability, and promising
cytotoxicity in three cancer cell lines (DU145, HeLa, A549). Compound 3f (ARN24139) is the
most promising drug-like candidate, with a good pharmacokinetics profile in vivo. Our results
indicate that this hybrid new chemical class of topoII poisons deserves further exploration, and
that 3f is a favorable lead candidate as a topoII poison, meriting future studies to test its efficacy
in in vivo tumor models.
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INTRODUCTION
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Human topoisomerase II (topoII) enzymes are a validated target to treat cancer because of their
role in modifying the topology of entangled DNA strands during vital cellular processes like
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replication and transcription. Several topoII anticancer inhibitors are clinically available. One
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example is etoposide, which is used to treat a variety of cancers, including leukemia and ovarian
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cancer. However, drug resistance and the possibility of severe side effects of topoII-targeting
6, 10-17
drugs means that researchers continue to seek novel safer topoII inhibitors.
Small molecules targeting topoII are classified as either topoII poisons or topoII catalytic
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inhibitors. These two classes of topoII blockers differ in their mode of action. TopoII poisons
act by trapping the covalent topoII/DNA cleavage complex, which is formed during the catalytic
cycle required for DNA topology modification. A covalent and stable topoII/DNA cleavage
1, 12-13, 18, 21-23
complex eventually leads to the accumulation of double-strand breaks, causing cell death.
The chemotherapy drug etoposide (Figure 1) acts via this mechanism, although its
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pharmacological action can lead to severe side-effects. Additional examples of anticancer drugs
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that act as a topoII poison are doxorubicin, mitoxantrone, salvicine, and teniposide. These drugs
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are frontline therapies for a wide range of solid and hematological malignancies.
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Figure 1. Hybrid topoII poison with the scaffold A (left) was explored to discover ARN16267 as a potent
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topoII blocker. Here, the hybrid scaffold A was expanded to generate structures of type B (right above),
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with several functional groups introduced in the meta position. Structures of type C (right below) were
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generated by introducing a spacer between the thiobarbituric core and the mimic E-ring.
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TopoII catalytic inhibitors act differently than poisons and do not generate an accumulation of
topoII/DNA cleavage complex. Instead, topoII catalytic inhibitors act, for example, by inhibiting
DNA binding and/or by blocking the ATP-binding site in topoII, thus preventing ATP hydrolysis,
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which is needed for topoII function. One notable example is merbarone, which was one of the
34-37
first and most promising topoII inhibitors (Figure 1). Merbarone is a thiobarbituric derivative (6-
hydroxy-4-oxo-N- phenyl-2-thioxo-1H-pyrimidine-5-carboxamide) that blocks topoII catalysis
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and inhibits proliferation of several cancer cell lines. Notably, merbarone underwent clinical trials
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as a chemotherapy drug. These trials failed because merbarone displayed nephrotoxicity issues
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and did not generate the expected efficacy.
Recently, we used a pharmacophore hybridization strategy to realise a first set of new topoII
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poisons. They were rationally designed by combining key pharmacophoric elements of etoposide
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and merbarone to generate a new etoposide-merbarone hybrid active scaffold. In particular, we
designed, synthesized, and characterized a first set of compounds that feature the thiobarbituric
core of merbarone linked via an amide bond to the E-ring of etoposide (type A structure, Figure
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1). This design generated new N,N′-diphenyl derivatives that potently block human topoII. In
addition, our SAR studies clarified the effect of ethyl and phenyl substitutions at each nitrogen of
the thiourea moiety, as well as the influence of the number and/or position of hydroxyl and
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methoxy substituents on the mimic E–ring. Importantly, we identified compound ARN16267
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(IC50 = 30 ± 6 μM, structure in Figure 1, which was originally named compound 3 in Ref ), which
is a more potent topoII blocker than the template compounds, i.e. etoposide (IC50 = 120 ± 10 μM)
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and merbarone (IC50= 120 ±12 μM). Intriguingly, we found that ARN16267 was the most
efficient of this new chemical class in generating accumulation of topoII/DNA cleavage complex.
This suggests that ARN16267 may act as a topoII poison, although this mechanism was less
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marked than that of etoposide.
These results prompted us to investigate the SAR of these new hybrid topoII blockers. Here we
present an additional sixteen derivatives that expand the initial panel of merbarone-etoposide
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hybrid molecules. As described in Figure 1, we used ARN16267 as our best starting point for
further derivatization of its core scaffold, generating scaffolds of type B and C (Figure 1). We thus
identified a new hybrid derivative (3f, ARN24139 – see Scheme 1) with improved human topoII
inhibitory activity (7.3 ± 1.5 uM). In addition, our results confirm that this new class of hybrid
compounds acts as topoII poisons, generating accumulation of topoII/DNA cleavage complex. Our
dynamic docking simulations support binding of 3f at the cleavage complex. Additionally, 3f
showed high kinetic solubility and metabolic stability, as well as a promising antiproliferative
activity in the low μM range against DU145, HeLa, and A549 cancer cell lines. Finally, we found
3f to have a good pharmacokinetic profile in vivo. Thus, 3f can be added to the pipeline of
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compounds that are active against topoII with promising anticancer activity.
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RESULTS AND DISCUSSION
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Scheme 1. Synthesis of compounds 3a-j. Our lead compound 3f (ARN24139) is in the right column.
Exploring the structure of the new hybrid scaffold. For our new set of hybrid analogues and
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based on our previous work and results, we initially expanded our SAR studies by exploring the
effect of diverse functional groups in the meta position on the mimic E-ring (3a-j, Scheme 1).
First, we synthesized two new compounds with an ethoxy (3a) or isopropoxy (3b) group in the
meta position on the mimic E-ring. We previously found that replacing the methoxy in ARN16267
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with a hydroxyl group significantly decreased topoII inhibitory activity. We thus substituted the
original methoxy in ARN16267 with a cyano (3c), nitro (3d), or fluoro (3e) to modulate the
electron density of the mimic E-ring. Additionally, we investigated the bioisosteric replacement of
the methoxy group of ARN16267, and generated three additional new hybrid compounds, each
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bearing a trifluoromethoxy (3f), difluoromethoxy (3g), or difluoroethoxy (3h) group in the meta
position on the mimic E-ring. Similarly, we synthesized a derivative with the difluoromethyl group
in the meta position on the mimic E-aromatic ring (3i). Finally, given that a fluorine proximal to a
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methoxy can influence the overall electronic behavior of the aromatic ring, we inserted a fluoro
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in the para position of ARN16267 to obtain 3j (Scheme 1).
Scheme 2. Synthesis of compounds 6-8, 5d-f.
In the crystal structure of the ternary topoII/DNA/Etoposide complex, the drug molecule is
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stabilized by interactions with Asp463 and Arg487. To favour the formation of these interactions
for our hybrid compounds, we introduced a flexible spacer between the thiobarbituric core and the
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mimic E-ring (compounds 6-8, and 5d-f, Scheme 2).
To test this hypothesis, we generated an
additional set of six compounds with a link-mediated increased flexibility (6-8, 5d-f).
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Chemistry. The sixteen new derivatives were synthesized through regular amidation of ester 1
with amines 2a-j and 4a-f using either DMF or toluene dry as a solvent, with yields that ranged
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from 34 to 72% (Schemes 1 and 2). As previously described, in the presence of amine 2j
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substituted with a fluorine in para and a methoxy group in meta position, the formylation side
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reaction performed by DMF was preferred over the alternative and desired reaction with ester 1.
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To circumvent this problem, the reaction was conducted in toluene dry at 100 °C, obtaining 3j
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with good yield (72%). The same strategy was used to synthesize 6-8 and 5d-f (Scheme 2), where
different hydrocarbon chains were introduced between the amide and the aromatic ring. In
particular, amines 4a-c and 4f (used to prepare 6-8 and 5f) were synthesized in two steps starting
from silyl protected syringaldehyde 9 and m-anisaldehyde 12, respectively (Scheme 3). Compound
4a is a benzylamine featuring an aromatic ring with the same functionalization of the E ring of
etoposide. This was obtained through the quantitative conversion of aldehyde 9 in the related O-
methyl oxime 10, which was reduced into the desired amine 4a with NiCl2∙and NaBH4 with 48%
yield (Scheme 3, eq a).
Scheme 3. Synthesis of amines 4a-c and 4f.
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The Henry reaction between protected syringaldehyde 9 and nitromethane in the presence of
ammonium acetate gave (E)-nitrostyrene 11, with an excellent 90% yield. Compound 11 was then
completely reduced with NiCl2 and NaBH4 into phenethenamine 4b with 48% yield (Scheme 3, eq
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b). The Horner-Wadsworth-Emmons (HWE) reaction between aldehyde
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diethylcyanomethylphosphonate in the presence of potassium tert-butoxide in THF gave a 1:0.12
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mixture of (E)- and (Z)- acrylonitrile 13, in 84% yield. The chemoselective nitrile reduction with
LiAlH4 and aluminium trichloride afforded (E)-allylamine 4c after chromatography purification,
with an acceptable 35% yield (Scheme 3, eq c). The same sequential transformations of olefination
and reduction allowed the introduction of an allylic spacer for amine 4f, starting from m-
anisaldehyde 12, with a 22% overall yield (Scheme 3, eq c). In this case, too, the (E)-isomer was
obtained pure after chromatography purification. Thus, the key amidation reaction between
silylated amines 4a-c with ester 1 in toluene dry at 100 °C generated amides 5a-c, which were
deprotected using TBAF. This yielded our final targets 6-8 with a free hydroxylic group in para
position, with 22-38% yield after two steps (Scheme 2). Similarly, m-methoxy amine counterparts
5d-f were obtained with 38-62% yield using related amines 4d-f (Scheme 2).
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TopoII inhibitory activity of the novel hybrid compounds. We measured the inhibitor activity
of our new compounds against human topoIIα, using a topoII decatenation assay (Table 1).
Notably, in our assay, etoposide returned an IC50 of 47.5 ± 2.2 µM, which agrees with the activity
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reported by the manufacturer of the decatenation assay kit. Additionally, merbarone showed an
IC50 of 26.0 ± 4.7 µM, which is in line with that reported previously (IC50 = 40 μM) using a
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plasmid relaxation assay.
Interestingly, greater bulkiness of the alkyl chain on the oxygen in the meta position, as in 3a
and 3b, improved the potency of these compounds, as compared to ARN16267, which has an IC50
of 16.1 ± 2.4 µM, as measured in the decatenation assay used in this study. In fact, 3b, with the
bulkier substituent, had an IC50 of 9.7 ± 2.6 µM, while the ethoxy substitution in 3a returned an
IC50 of 12.4 ± 3.7 µM (Table 1). We then found that the electron-withdrawing nitro group, in the
meta position on the mimic E-ring in 3d, returned an IC50 of 10.2 ± 4.6 µM. Other electron-
withdrawing groups such as the cyano (3c) and the fluoro (3e) returned a comparable activity to
that of ARN16267 (Table1). This is in line with our previous demonstration of the unfavourability
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of an electron-donating group, such as a hydroxyl substitution, at this position. Similarly,
introducing a fluorine proximal to the methoxy group (3j) was detrimental for activity, with an
IC50 of 22.5 ± 5.8 µM.
We then investigated the bioisosteric replacement of the methoxy group in meta position on the
mimic E-ring. Interestingly, all the bioisosteric analogues 3f-h displayed a better activity than
ARN16267: introducing a trifluoromethoxy group in 3f returned a 2-fold increased activity with
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an IC50 of 7.3 ± 1.5 µM, while difluoroethyl in 3h returned an IC50 of 9.2 ± 0.2 µM, and the
difluoromethoxy group in 3g returned an IC50 = 11.4 ± 2.4 µM. Notably, the difluoromethyl
analogue 3i also had this improved activity, which confirms that the additional interactions
provided by fluorinated groups (also more lipophilic) can compensate the loss of the oxy-moiety
in meta position of the aromatic (mimic) E-ring.
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Table 1. Inhibitory and antiproliferative activities of sixteen new derivatives 3a-j, 6-9, 5d-f.
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Antiproliferative activity in cancer cells of etoposide and merbarone was measured in Ref .
Entry
IC50 (μM)
DU145 (μM)
HeLa (μM)
A549 (μM)
etoposide
47.5 ± 2.2
26.0 ± 4.7
16.1 ± 2.4
12.4 ± 3.7
9.7 ± 2.6
1.0 ± 0.8
18.9 ± 2.0
7.6 ± 0.8
5.7 ± 1.4
5.5 ± 0.1
14.4 ± 4.1
6.9 ± 0.3
5.6 ± 0.6
4.7 ± 0.1
6.5 ± 0.9
2.7 ± 2.5
7.8 ± 0.2
7.8 ± 0.6
7.7 ± 0.1
3.3 ± 2.5
5.0 ± 2.9
8.8 ± 0.3
13.9 ± 7.6
9.8 ± 2.1
2.4 ± 0.9
62.3 ± 6.4
5.5 ± 1.3
5.2 ± 0.9
4.2 ± 0.3
12.4 ± 1.3
11.9 ± 2.6
5.6 ± 0.5
3.8 ± 0.3
6.4 ± 0.5
2.5 ± 0.4
4.9 ± 1.4
5.3 ± 1.0
6.1 ± 1.1
3.4 ± 0.1
5.5 ± 1.1
19.1 ± 4.8
13.3 ± 1.7
9.3 ± 0.2
1.3 ± 0.1
40.0 ± 2.7
4.7 ± 0.3
3.6 ± 0.4
3.0 ± 0.3
16.9 ± 0.3
18.5 ± 3.1
4.1 ± 0.2
3.1 ± 0.1
4.8 ± 0.4
3.0 ± 1.1
4.4 ± 0.2
4.6 ± 0.1
4.8 ± 0.4
3.2 ± 1.2
4.4 ± 0.6
14.7 ± 0.3
14.8 ± 1.4
9.5 ± 1.5
merbarone
ARN16267
3a
3b
3c
3d
3e
3f
3g
3h
3i
14.4 ± 3.8
10.2 ± 4.6
15.1 ± 4.2
7.3 ± 1.5
10.2 ± 1.9
9.2 ± 0.2
11.4 ± 2.4
22.5 ± 5.8
22.5 ± 7.2
15.8 ± 3.4
8.3 ± 2.3
3j
5d
5e
5f
6
107.8 ± 10.1
74.4 ± 13.6
28.0 ± 4.4
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After evaluating the inhibitory activity of this first subset of derivatives, we assessed the activity
of the hybrid molecules with a flexible spacer connecting the thiobarbituric core and the mimic E-
ring (5d-f and 6-8, Scheme 2). We started evaluating the activity of the compound 6, where we
inserted a methylene substitution and that contains the exact E-ring of etoposide. This first
modification reduced the potency (IC50 = 107.8 ± 10.1 µM), with a 7-fold drop in activity
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compared to ARN16267 (Table 1). Conversely, inserting the same substitution in 5d only
decreased 1.4-fold the inhibitory activity (IC50 = 22.5 ± 7.2 µM) compared to the parent
compound ARN16267 (Table 1). This result confirms that a methoxy group, alone, in the mimic
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E-ring increases the potency of this hybrid scaffold, as also observed previously. Increasing the
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spacer length was also beneficial, improving the IC50 from over 100 µM for 6 to 74.4 ± 13.6 and
28.0 ± 4.4 µM for 7 and 8, respectively. This positive trend in potency could be due to a more
balanced structure where the flexibility introduced by having the hydrocarbon chain (i.e., the
spacer) is compensated by the introduced rigidity of the C=C double bond embedded in the allylic
system in 8. Within this second class of analogues, compounds are more potent when the 3-
methoxy was retained as the only substituent, with IC50 values of 22.5 ± 7.2 µM for 5d, 15.8 ±
3.4 µM for 5e, and 8.3 ± 2.3 µM for 5f. Notably, 5f showed a 2-fold increase in IC50 compared to
its template ARN16267 (which has no spacer). These data thus suggest that a flexible substituent
connecting the thiobarbituric core and the mimic E-ring in our hybrid scaffold may facilitate the
orientation of our molecules inside the active site of topoII, increasing their inhibitory potency (see
docking results, below).
Antiproliferative activity against cultured human cancer cells, metabolic stability, chemical
solubility, and topoII poisoning. The antiproliferative activity of all compounds was evaluated in
i) DU145, an androgen-independent prostate cancer cell line; ii) HeLa, a cervical cancer cell line;
and iii) A549, a lung adenocarcinoma cell line (Table 1). Notably, all new compounds showed
good antiproliferative activity with IC50 values in the low µM range. Among the most active
compounds in inhibiting topoII activity, 3f, 3h, and 5e showed cytotoxicity with IC50 values lower
than 5 µM (Table 1). Undoubtedly, this preliminary cytotoxicity data will need further
characterization, also in relation to the in vitro activity of these compounds.
After this initial evaluation of the new set of hybrid compounds for their inhibitory activity
against topoII in vitro and for their biological cytotoxicity, we selected 3b, 3f-i, and 5e for further
evaluation. We assessed their metabolic stability using mouse serum and mouse liver microsomes,
and we assessed their kinetic solubility in neutral buffer. These compounds had excellent plasma
and microsomal stability with half-time values greater than 120 and 60 min, respectively.
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Additionally, 3f, 3g, and 3i displayed excellent solubility in aqueous buffer (pH 7.4), with values
greater than 200 µM (Table 2).
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Table 2. Kinetic solubility of 3b, 3f-h, 5e.
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Entry
Kinetic solubility (μM)
ARN16267
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3b
3f
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3g
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5e
In view of these results, 3f, 3g, and 3i were tested in a cleavage complex formation assay to
further ascertain their mode of action as topoII poisons. As shown in Figure 2, all these hybrid
molecules were confirmed to be poisons and thus able to generate an accumulation of topoII/DNA
cleavage complex. In particular, 3f had the greatest poison efficacy, being about 1.5-fold better
than the template ARN16267, at 200 µM concentration. Given 3f’s promising in vitro activity as
a topoII poison and its overall drug-like profile, we examined its binding mode to topoIIα and its
in vivo pharmacokinetic profile in mice.
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Figure 2. Poison activity of the hybrid compounds. Agarose gel electrophoresis of plasmid DNA incubated
in the absence (no enzyme) or presence of 1 U topoII containing either 1% DMSO as control vehicle (no
enzyme and topoII lanes) or 200 μM of the compound. Labels are shown above each lane. Numbers in the
bottom correspond to the normalized intensity of the linear form (FIII). Plasmid forms are indicated by the
arrow-points on the right: supercoiled (FI), nicked (FII), and linear (FIII).
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Docking and molecular simulations of 3f bound to the target We used docking and atomistic
force-field-based molecular dynamics (MD) simulations to model 3f bound to the topoII/DNA
cleavage site.52-55 First, the crystal structure of the topoIIα isoform (PDBID: 5GWK) was used for
the docking studies.25, 56 As seen in Figure S2, when the compound was first docked into the
cleavage site, the mimic E-ring slightly shifted relative to the position of the E-ring of etoposide
in the crystal.25 Our calculations revealed several key contacts between the ligand and vicinal
residues that confer the system a stable, inhibited conformation, thus endorsing the compound’s
action as a topoII poison. The protein aids the ligand’s anchoring within the pocket by a cation-π
link formed between Arg487 and the E-ring. The neighbouring DNA bases also contribute to the
stabilization of the complex. Specifically, the G+5 and C-1 bases display π-stacking interactions with
the heterocycle inherited from merbarone (Figure 3). Similarly, the T+1 and C-1 bases align to the
phenyl substituents at the thiobarbituric core in a perpendicular fashion resembling a T-shaped π-
interaction. Finally, an H-bond between T+1 and the ligand’s N-H moiety was also identified.
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Figure 3. Interaction diagram of the lead compound 3f bound to the DNA/topoII complex as derived
from docking calculations. The computational study flags a cation-π interaction with Arg487, π-stacking
with various DNA bases, and an H-bond with T+1, as the main drivers for ligand binding.
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Once the general interaction pattern was established, we proceeded to perform equilibrium
molecular dynamics (MD) simulations (~200 ns) to analyze the evolution and stability of the
ternary topoII/DNA/3f model system.14, 57-58 For this, distances representative of the interactions
described above were tracked (Figure 4A). The simulations corroborate the role of Arg487 in
stabilizing the drug at the cleaved site. Indeed, we monitored the distance between the carbon atom
of the guanidinium group and the centroid of the E-ring and found that it remains under 6 Å for
99% of the simulation (Figure 4B).
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The H-bonds between the ligand and the cleaved complex were also examined. It is worth to
note that the –NH linker present in compound 3f adds to the rigidity of the molecule. In fact, it
enables the formation of a six-membered intramolecular pseudo-cycle. The cycled configuration
was present for ~79% of the overall simulation time. Nonetheless, the –NH group was found to
intermittently invert in order to form an H-bond with the oxygen from the deoxyribose ring of G+5.
The latter bond persisted for a total of ~17% of the production run, thus making a complementary
interaction for the stabilization of the cleaved complex holo-form.
Similarly, we examined the staggered π-stacking formed between the thiobarbituric cycle and
the G+5 and C-1 bases (Figure 4B). Here, the distance to the G+5 base remains consistently smaller at
a stable value of 3.6 ± 0.3 Å, whereas the distance to C-1 extends to 4.4 ± 0.4 Å. A similar pattern
is observed between the phenyl groups and the vicinal base pairs. In summary, the binding mode
most frequently visited is stable in our MD simulations too, with key cation-p, H-bonds, and
stacking interactions formed with the surrounding DNA/protein complex.
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Figure 4. Dynamic description for the binding of 3f to the in the DNA cleavage/religation active site of
topoIIα (PDBID: 5GWK). A) The lead compound in its binding mode. The residues directly interacting
with the drug are shown in orange carbons, the rest of the DNA in pink, and the protein in white. The insets
zoom into the distances of interest, particularly, those of Arg487 with the E-ring, and the merbarone core
with the G+5 and C-1 bases. B) Evolution of the distances of interest over time.
In vivo pharmacokinetics. Finally, based on the overall results and drug-like profile, 3f was
selected as our lead for in vivo pharmacokinetics studies, as a preparatory characterization for
future analyses of in vivo drug efficacy in animal models of cancer. We tested two different routes
of administration: i) intravenous (I.V.) injection at a concentration of 3 mg/Kg (n=3 for each time
point) and oral (P.O.) treatment at a dose of 10 mg/Kg (n=3 for each time point). Despite the
relatively low thermodynamic solubility of 3f (30µM in PBS), the compound reached the target
concentration in the formulation used for the in vivo experiments. The mean plasma concentration
versus time profile of 3f is shown in Figure 5, and the corresponding pharmacokinetic parameters
are summarized in the inserted table. During the PK studies, via either I.V. or P.O. administration,
3f was well tolerated by all the animals, and no treatment-related clinical signs were observed.
The peak plasma 3f concentration for I.V. was observed at the earliest time point (5min after
administration), and the concentration of 3f in plasma was above the lower limit of quantification
throughout the sampling period. The I.V. profile of 3f presents a very fast distribution phase with
a Cmax of 7366 ng/mL, followed by a slower exposure phase. The compound was still detectable
after 2 h at a concentration of 551 ng/mL, with Clp value of 0.004 L/min/Kg. After oral
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administration (10 mg/Kg), plasma concentration of about 400 ng/mL was reached relatively fast
(1 h) and it was stably maintained for at least 6 h. The maximum concentration was achieved at
approximately 2 h post oral administration, 428 ng/mL. These data indicate that the compound 3f
is well tolerated. Indeed, the animal behavior and the obtained PK profiles indicate that the dose
of 3f could be increased. This may be beneficial, given the observed very high protein binding of
this compound (>99% in both mouse and human plasma) that may limit target engagement.
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Figure 5. Mouse PK profiles of 3f following intravenous (I.V.) and oral administration (P.O.) at 3 and
10mg/Kg, respectively. The observed and calculated PK parameters following intravenous (I.V.) and oral
administration (P.O.). The bioavailability F was calculated to 8% based on the AUC (area under curve)
from t=0 to 240 min.
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CONCLUSION
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Based on our previous results on a novel hybrid scaffold with structural elements of merbarone
39
and etoposide, we have here reported the design, synthesis, and extensive experimental-
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computational characterization of new hybrid molecules that act as topoII poisons. The resulting
SAR elucidated the key structural features that enhanced the potency and antiproliferative activity
of our new etoposide-merbarone hybrid compounds. These new compounds were often equipotent
and sometimes more potent relative to the reference compounds merbarone and etoposide.
Inhibitory activity was improved by introducing a bulkier group in meta position of the mimic E-
ring (3a and 3b, Scheme 1). Incorporating electron-withdrawing groups preserved or slightly
improved the inhibitory activity (3c-e, Scheme 1), while the bioisosteric substitution with fluorine-
embedding groups (3f-h, Scheme 1) was highly favorable. Furthermore, in the structural design of
these new bioactive hybrid molecules, the combined functionalization of both the aromatic E-ring
and the hydrocarbon spacer was essential to fine-tuning the drug-target interactions, as proven by
the activity of more flexible hybrid topoII poisons (6-8, and 5d-f, Scheme 2). Taken together, the
inhibitory activity and extensive analyses of the drug-likeness profile indicate the novel derivative
3f (ARN24139) as the most drug-like topoII poison of this novel chemical series. This lead
compound has also shown promising antiproliferative activity against cancer cell lines and a
favorable pharmacokinetic profile, which are promising features for future in vivo efficacy studies.
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EXPERIMENTAL SECTION
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CHEMISTRY
General considerations. All the commercially available reagents and solvents were used as
purchased from vendors without further purification. Dry solvents were purchased from Sigma-
Aldrich. Automated column chromatography purifications were done using a Teledyne ISCO
apparatus (CombiFlash Rf) with pre-packed silica gel columns of different sizes (from 4 g up to
24 g) and mixtures of increasing polarity of cyclohexane and ethyl acetate (EtOAc) or
dichloromethane (DCM) and methanol (MeOH). NMR data were collected on 400 MHz or 600
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MHz (1H) and 100 MHz or 150 MHz ( C). Spectra were acquired at 300 K, using deuterated
1
dimethylsulfoxide (DMSO–d6) or deuterated chloroform (CDCl3) as solvents. For H-NMR, data
are reported as follows: chemical shift, multiplicity (s= singlet, d= doublet, dd= double of doublets,
t= triplet, q= quartet, m= multiplet), coupling constants (Hz), and integration. UPLC/MS analyses
were run on a Waters ACQUITY UPLC/MS instrument consisting of an SQD (Single Quadrupole
Detector). The analyses were performed on an ACQUITY UPLC BEH C18 column (50x2.1mmID,
particle size 1.7µm) with a VanGuard BEH C18 pre-column (5x2.1mmID, particle size 1.7µm)
(LogD>1). The mobile phase was 10mM NH4OAc in H2O at pH 5 adjusted with AcOH (A) and
10mM NH4OAc in MeCN-H2O (95:5) at pH 5 (B). Electrospray ionization in positive and negative
mode was applied in the mass scan range 100-500Da. Depending on the analysis method used, a
different gradient increasing the proportion of mobile phase B was applied. For analysis method
A, the mobile-phase B proportion increased from 5% to 95% in 3 min. For analysis method B, the
mobile-phase B proportion increased from 50% to 100% in 3 min. High-resolution mass
spectrometry (HRMS) was carried out on a Waters Synapt G2 Quadrupole-Tof Instrument
equipped with an ESI ion source. The analyses were run on an ACQUITY UPLC BEH C18 column
(50x2.1mmID, particle size 1.7µm), using H2O + 0.1% formic acid (A) and MeCN + 0.1% formic
acid as mobile phase. All final compounds displayed ≥ 95% purity as determined by NMR and
UPLC/MS analysis.
3-(1,1-Difluoroethyl)aniline (2h). A solution of 3-nitroacetophenone (165 mg, 1 mmol) in CH2Cl2
dry (4 ml) was treated with [Bis(2-methoxyethyl)amino]sulphur trifluoride 50wt% solution in
toluene (2.5 mmol) at room temperature under argon. EtOH (24μL, 0.4 mmol) was added and the
reaction mixture was stirred at room temperature for 48h. After which time, the solution was
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poured into NaHCO3 saturated solution, extracted with CH2Cl2 (2x5mL). Combined organic layers
were dried with Na2SO4, filtered and concentrated under vacuum. Purification by silica gel flash
chromatography (elution by gradient from 100 to 95/5 Cyclohexane/EtOAc) afforded pure 1-(1,1-
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Difluoroethyl)-3-nitrobenzene (73 mg, 39% yield). H NMR (400MHz, CDCl3): δ 8.38 (bs, 1H),
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8.31 (d, J = 7.7 Hz, 1H), 7.85 (d, J = 7.7 Hz, 1H), 7.64 (dd, J = 7.9, 7.9 Hz, 1H), 1.98 (t, JH-F = 18.2
Hz, 3H). Then tin chloride dihydrate (440 mg, 1.95 mmol) was added to a solution of compound
1-(1,1-Difluoroethyl)-3-nitrobenzene (73 mg, 0.39 mmol) in 3 mL of ethanol. The reaction mixture
was refluxed for one hour. The mixture was slowly poured on cooled water. The pH was adjusted
to 7 by addition of an aqueous 5N solution of sodium hydroxide, then adjusted to 9 by addition of
an aqueous NaHCO3 satured solution. The product was extracted with EtOAc (3x5mL). The
organic phases were combined, dried over Na2SO4, filtered and concentrated under vacuum.
Purification by silica gel flash chromatography (elution by gradient from 100 to 80/20
Cyclohexane/EtOAc) afforded pure compound 3h (58 mg, 95% yield). UPLC/MS: Rt = 1.75 min
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+
1
(method A), MS (ESI) m/z: 158.0 [M+H] , C8H10F2N [M+H] calculated: 158.1. H NMR (400MHz,
CDCl3): δ 7.19 (dd, J = 7.9, 7.9 Hz, 1H), 6.89 (d, J = 7.8 Hz, 1H), 6.82 (bs, 1H), 6.73 (d, J = 7.5
3
Hz, 1H), bs (2H), 1.89 (t, JH-F = 18.2 Hz, 3H).
General procedure 1: method A for amide formation
A solution 0.5M of ethyl 6-hydroxy-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate 1 (1 equiv) and an appropriate aniline (1 equiv) in DMF dry was stirred at 100 °C for
30 min, then cooled to room temperature, and the product was precipitated with water, filtered,
and rinsed with MeOH, yielding pure compound.
General procedure 2: method B for amide formation
A solution 0.5M of ethyl 6-hydroxy-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate 1 (1 equiv) and an appropriate amine (1.2 equiv) in toluene dry was stirred at 100 °C
for 2 h. After completion of reaction, the solvent was removed under vacuum. The product was
purified by flash chromatography and/or by trituration with MeOH, yielding pure compound.
N-(3-ethoxyphenyl)-6-hydroxy-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-
5-carboxamide (3a). Title compound was prepared according to general procedure 1 using: 3-
ethoxyaniline 2a (31 mg, 0.23 mmol), ester 1 (86 mg, 0.23 mmol) in anhydrous DMF (0.46 mL).
Then, water (3 mL) was added, the resulting precipitate was filtered and rinsed with water (2 mL)
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and MeOH (2 mL), yielding 3a (70 mg, 66% yield). UPLC/MS: Rt = 1.99 min (method A), MS
+
(ESI) m/z: .460.4 [M+H] , C25H22N3O4S [M+H] calculated: 460.5. HRMS (AP-ESI) m/z calc for
+
+
1
C25H22N3O4S [M+H] 460.1331, found 460.1331. H NMR (400MHz, DMSO-d6): δ 11.63 (s, 1H,
NH), 7.51-7.33 (m, 10H, Ph), 7.29 (dd, J = 8.1, 8.1 Hz, 1H, Ar), 7.14 (dd, J = 2.2, 2.2 Hz, 1H, Ar),
7.10 (dd, J = 8.0, 1.9 Hz, 1H, Ar), 6.80 (dd, J = 8.2, 2.3 Hz, 1H, Ar), 4.02 (q, J = 7.0 Hz, 2H), 1.30
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(t, J = 6.9 Hz, 3H). C (100MHz, DMSO-d6): δ 178.3 (CS), 168.7 (Cq), 159.0 (Cq), 139.0 (Cq),
136.4 (Cq), 130.1 (CH), 129.1 (CH), 129.0 (CH), 128.4 (CH), 114.0 (CH), 112.1 (CH), 108.1
(CH), 84.0 (Cq), 63.3 (CH2), 14.6 (CH3).
6-hydroxy-N-(3-isopropoxyphenyl)-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxamide (3b). Title compound was prepared according to general
procedure 1 using: 3-isopropiloxyaniline 2b (45 mg, 0.30 mmol), ester 1 (110 mg, 0.30 mmol) in
anhydrous DMF (0.6 mL). Then, water (3 mL) was added, the resulting precipitate was filtered
and rinsed with water (2 mL) and MeOH (2 mL), yielding 3b (50 mg, 35% yield). UPLC/MS: Rt
−
−
= 2.10 min (method A), MS (ESI) m/z: 472.4 [M−H] , C26H22N3O4S [M−H] calculated: 472.5.
+
1
HRMS (AP-ESI) m/z calc for C26H24N3O4S [M+H] 474.1488, found 474.1494. H NMR (400MHz,
CDCl3): δ 11.85 (s, 1H, NH), 7.50-7.60 (m, 6H, Ph), 7.36-7.33 (m, 4H, Ph), 7.27 (dd, J = 8.0, 5.0
Hz, 1H, Ar), 7.08-7.04 (m, 2H, Ar), 6.79 (dd, J = 8.3, 2.4 Hz, 1H, Ar), 4.54 (quint, J = 6.0 Hz,
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1H), 1.36 (d, J = 6.0 Hz, 6H). C (100MHz, CDCl3): δ 178.6 (CS), 169.3 (Cq), 167.8 (Cq), 162.3
(Cq), 158.7 (Cq), 139.2 (Cq), 138.1 (Cq), 136.2 (Cq), 130.2 (CH), 129.8 (CH), 129.7 (CH), 129.3
(CH), 129.1 (CH), 128.8 (CH), 128.6 (CH), 114.2 (CH), 114.0 (CH), 109.5 (CH), 83.6 (Cq), 70.3
(CH), 22.1 (CH3).
N-(3-cyanophenyl)-6-hydroxy-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carboxamide (3c). Title compound was prepared according to general procedure 1 using: 3-
cianoaniline 2c (48 mg, 0.41 mmol), ester 1 (150 mg, 0.41 mmol) in anhydrous DMF (0.82 mL).
Then, water (4 mL) was added, the resulting precipitate was filtered and rinsed with water (4 mL)
and MeOH (4 mL), yielding 3c (122 mg, 68% yield) as light pink amorphous solid. UPLC/MS: Rt
+
= 1.75 min (method A), MS (ESI) m/z: 441.4 [M+H] , C24H17N4O3S [M+H] calculated: 441.4.
+
+
HRMS (AP-ESI) m/z calc for C24H17N4O3S [M+H] 441.1021, found 441.1025. H NMR (400MHz,
1
DMSO-d6): δ 11.72 (s, 1H, NH), 8.10 (bs, 1H, Ar), 7.86 (d, J = 8.2 Hz, 1H, Ar), 7.66 (d, J = 7.7
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Hz, Ar), 7.58 (dd, J = 8.0, 8.0 Hz, 1H, Ar), 7.51-7.33 (m, 10H, Ph). C (100MHz, DMSO-d6): δ
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178.2 (CS), 168.7 (Cq), 164.1 (Cq), 139.1 (Cq), 138.8 (Cq), 130.5 (CH), 129.1 (CH), 129.0 (CH),
128.9 (CH), 126.8 (CH), 125.1 (CH), 118.3 (Cq), 111.9 (Cq), 84.7 (Cq).
6-hydroxy-N-(3-nitrophenyl)-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carboxamide (3d). Title compound was prepared according to general procedure 1 using: 3-
nitroaniline 2d (38 mg, 0.27 mmol), ester 1 (100 mg, 0.27 mmol) in anhydrous DMF (0.54 mL).
Then, water (4 mL) was added, the resulting precipitate was filtered and rinsed with water (4 mL)
and MeOH (4 mL), yielding 3d (70 mg, 57% yield) as pale yellow amorphous solid. UPLC/MS:
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Rt = 1.86 min (method A), MS (ESI) m/z: 461.5 [M+H] , C23H17N4O5S [M+H] calculated: 461.5.
+
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HRMS (AP-ESI) m/z calc for C23H17N4O5S [M+H] 461.0920, found 461.0924. H NMR (400MHz,
DMSO-d6): δ 11.84 (s, 1H, NH), 8.59 (dd, J = 2.2, 2.2 Hz, 1H, Ar), 8.01 (ddd, J = 8.2, 2.3, 0.8 Hz,
1H, Ar), 7.89 (ddd, J = 8.2, 2.1, 0.8 Hz, 1H, Ar), 7.64 (dd, J = 8.2, 8.2 Hz, 1H, Ar), 7.50-7.31 (m,
13
10H, Ph). C NMR (100MHz, CDCl3): δ 178.2 (CS), 169.9 (Cq), 168.1 (Cq), 162.2 (Cq), 148.9
(Cq), 138.9 (Cq), 137.6 (Cq), 136.8 (Cq), 130.3 (CH), 129.9(CH), 129.8 (CH), 129.6 (CH), 129.3
(CH), 128.6 (CH), 128.5 (CH), 127.0 (CH), 120.6 (CH), 116.6 (CH), 84.1 (Cq).
N-(3-fluorophenyl)-6-hydroxy-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carboxamide (3e). Title compound was prepared according to general procedure 1 using: 3-
fluoroaniline 2e (48 μL, 0.50 mmol), ester 1 (184 mg, 0.50 mmol) in anhydrous DMF (1 mL).
Then, water (5 mL) was added, the resulting precipitate was filtered and rinsed with water (5 mL)
and MeOH (5 mL), yielding 3e (82mg, 38% yield) as white amorphous solid. UPLC/MS: Rt =
+
+
1.86 min (method A), MS (ESI) m/z: 434.3 [M+H] , C23H17FN3O3S [M+H] calculated: 434.5. HRMS
+
1
(AP-ESI) m/z calc for C23H17FN3O3S [M+H] 434.0975, found 434.0974. H NMR (600MHz,
CDCl3): δ 11.91 (s, 1H, NH), 7.59-7.56 (m, 4H, Ar), 7.53-7.49 (m, 2H, Ar), 7.41 (ddd, J = 10.4,
2.3, 2.3 Hz, 1H, Ar), 7.35 (m, 5H), 7.18 (dd, J = 8.8, 2.0 Hz, 1H, Ar), 6.94 (ddd, J = 8.2, 8.2, 2.5
13
Hz, 1H, Ar). C NMR (150 MHz, CDCl3): δ 178.4 (CS), 169.5 (Cq), 167.9 (Cq), 163.8 (Cq), 163.0
1
3
(d, JCF = 246.3 Hz, Cq), 139.1 (Cq), 137.9 (Cq), 136.8 (d, JCF = 10.8 Hz, Cq), 130.6 (d, JCF = 9.1
3
4
Hz, CH), 129.9 (CH), 129.8 (CH), 129.4 (CH), 129.2 (CH), 128.7 (CH), 128.5 (CH), 117.2 (d, JCF
2
= 3.0 Hz, CH), 113.2 ( JCF = 21.5 Hz, CH), 109.4 (CH, JCF = 21.4 Hz, CH), 83.8 (Cq). F NMR (565
2
19
MHz): δ −110.3 (s).
6-hydroxy-4-oxo-1,3-diphenyl-2-thioxo-N-(3-(trifluoromethoxy)phenyl)-1,2,3,4-
tetrahydropyrimidine-5-carboxamide (3f). Title compound was prepared according to general
procedure 1 using: 3-trifluoromethoxyaniline 2f (54 μL, 0.41 mmol), ester 1 (150 mg, 0.41 mmol)
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in anhydrous DMF (0.82 mL). Then, water (4 mL) was added, the resulting precipitate was filtered
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6
7
and rinsed with water (4 mL) and MeOH (4 mL), yielding 3f (79 mg, 39% yield) as white
+
amorphous solid. UPLC/MS: Rt = 2.05 min (method A), MS (ESI) m/z: 500.3 [M+H] ,
8
9
+
C24H17F3N3O4S [M+H] calculated: 500.5. HRMS (AP-ESI) m/z calc for C24H17F3N3O4S [M+H]
+
10
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500.0892, found 500.0883. H NMR (400MHz, DMSO-d6): δ 11.70 (s, 1H, NH), 7.71 (s, 1H, Ar),
13
7.54-7.31 (m, 12H, Ar), 7.19 (d, J = 7.7 Hz, 1H, Ar). C (150MHz, CDCl3): δ 178.4 (CS), 169.6
(Cq), 168.0 (Cq), 162.2 (Cq), 149.7 (Cq), 139.0 (Cq), 137.8 (Cq), 136.8 (Cq), 130.6 (CH), 129.9
1
(CH), 129.8 (CH), 129.5 (CH), 129.2 (CH), 128.7 (CH), 128.5 (CH), 120.5 (q, JCF = 258 Hz, Cq),
19
119.9 (CH), 118.4 (CH), 114.6 (CH), 83.8 (Cq). F NMR (565 MHz): δ −57.8 (s).
N-(3-(difluoromethoxy)phenyl)-6-hydroxy-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxamide (3g). Title compound was prepared according to general
procedure 1 using: 3-(difluoromethoxy)aniline 2g (51 μL, 0.41 mmol), ester 1 (150 mg, 0.41
mmol) in anhydrous DMF (0.82 mL). Then, water (4 mL) was added, the resulting precipitate was
filtered and rinsed with water (4 mL) and MeOH (4 mL), yielding 3g (67 mg, 34% yield) as white
+
amorphous solid. UPLC/MS: Rt = 1.95 min (method A), MS (ESI) m/z: 482.4 [M+H] ,
+
C24H18F2N3O3S [M+H] calculated: 482.5. HRMS (AP-ESI) m/z calc for C24H18F2N3O3S [M+H]
+
1
482.0986, found 482.0984. H NMR (600MHz, CDCl3): δ 11.9 (s, 1H, NH), 7.58-7.50 (m, 6H, Ar),
13
7.38-7.31 (m, 7H), 7.00 (d, J = 8.1 Hz, 1H, Ar), 6.52 (t, J = 73.4 Hz, 1H, CHF2). C (150MHz,
CDCl3): δ 178.4 (CS), 169.6 (Cq), 167.9 (Cq), 162.2 (Cq), 151.6 (Cq), 139.1 (Cq), 137.9 (Cq),
136.7 (Cq), 130.6 (CH), 129.9 (CH), 129.8 (CH), 129.5 (CH), 129.2 (CH), 128.7 (CH), 128.5
1
19
(CH), 118.6 (CH), 117.1 (CH), 115.7 (t, JCF = 260.5 Hz, CH), 113.3 (CH), 83.8 (Cq). F NMR (565
MHz): δ −81.2 (s).
N-(3-(1,1-difluoroethyl)phenyl)-6-hydroxy-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxamide (3h). Title compound was prepared according to general
procedure 1 using: 3-(1,1-difluoroethyl)aniline 2h (50 mg, 0.32 mmol), ester 1 (117 mg, 0.32
mmol) in anhydrous DMF (0.64 mL). Then, water (3 mL) was added, the resulting precipitate was
filtered and rinsed with water (3 mL) and MeOH (3 mL), yielding 3h (104 mg, 68% yield) as white
+
amorphous solid. UPLC/MS: Rt = 2.08 min (method B), MS (ESI) m/z: 480.1 [M+H] ,
+
C25H20F2N3O3S [M+H] calculated: 480.5. HRMS (AP-ESI) m/z calc for C25H20F2N3O3S [M+H]
+
1
480.1193, found 480.1194. H NMR (400MHz, CDCl3): δ 11.95 (s, 1H, NH), 7.63-7.47 (m, 8H,
Ar), 7.44 (dd, J = 7.9, 7.9 Hz, 1H, Ar), 7.37 (d, J = 7.9 Hz, 1H, Ar), 7.33-7.29 (m, 4H), 1.90 (t, J
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= 18.2 Hz, 3H). C (100MHz, CDCl3): δ 178.5 (CS), 169.5 (Cq), 167.9 (Cq), 162.3 (Cq), 139.7 (t,
2
JCF = 27.3 Hz, Cq), 139.1 (Cq), 137.9 (Cq), 135.6 (Cq), 129.9 (CH), 129.8 (CH), 129.5 (CH), 129.2
3
(CH), 128.7 (CH), 122.9 (CH), 122.5 (t, JCF = 5.9 Hz, CH), 121.3 (t, JCF = 239.5 Hz, CF2), 118.2 (t,
1
7
8
9
3
2 19
JCF = 6.4 Hz, CH), 83.7 (Cq), 26.0 (t, JCF = 29.5 Hz, CH3). F NMR (565 MHz): δ −87.1 (s).
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N-(3-(difluoromethyl)phenyl)-6-hydroxy-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxamide (3i). Title compound was prepared according to general
procedure 1 using: 3-(difluoromethyl)aniline 2i (43 mg, 0.30 mmol), ester 1 (110 mg, 0.30 mmol)
in anhydrous DMF (0.60 mL). Then, water (3 mL) was added, the resulting precipitate was filtered
and rinsed with water (3 mL) and MeOH (3 mL), yielding 3i (45 mg, 32% yield) as white
+
amorphous solid. UPLC/MS: Rt = 1.94 min (method A), MS (ESI) m/z: 466.4 [M+H] ,
+
C24H18F2N3O3S [M+H] calculated: 466.5. HRMS (AP-ESI) m/z calc for C24H18F2N3O3S [M+H]
+
1
466.1037, found 466.1041. H NMR (600MHz, CDCl3): δ 11.97 (s, 1H, NH), 7.68 (s, 1H), 7.61-
13
7.47 (m, 8H, Ar), 7.38-7.31 (m, 5H, Ar), 6.63 (t, J = 56.5 Hz, 1H, CHF2). C (150MHz, CDCl3): δ
2
178.5 (CS), 169.6 (Cq), 167.9 (Cq), 162.2 (Cq), 139.1 (Cq), 137.9 (Cq), 135.9 (Cq), 135.8 (t, JCF
= 22.6 Hz, Cq), 130.0 (CH), 129.9 (CH), 129.8 (CH), 129.5 (CH), 129.2 (CH), 128.7 (CH), 128.5
3
(CH), 123.8 (CH), 123.3 (t, JCF = 6.1 Hz, CH), 119.0 (t, JCF = 6.1 Hz, CH), 119.0 (t, JCF = 6.2 Hz,
3
3
1
19
CH), 114.0 (t, JCF = 240.8 Hz, CH), 83.8 (Cq). F NMR (565 MHz): δ −111.5 (s).
N-(4-fluoro-3-methylphenyl)-6-hydroxy-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxamide (3j). Title compound was prepared according to general
procedure 2 using: 4-fluoro-3-methoxy-aniline 2j (61 mg, 0.43 mmol), ester 1 (132 mg, 0.36
mmol) in anhydrous toluene (0.72 mL). Then, the solvent was removed under vacuum, the residue
was treated with water (3 mL), the resulting precipitate was filtered and rinsed with MeOH (3 mL),
yielding 3j (126 mg, 72% yield) as white amorphous solid. UPLC/MS: Rt = 1.94 min (method A),
+
+
MS (ESI) m/z: 464.4 [M+H] , C24H19FN3O4S [M+H] calculated: 464.5. HRMS (AP-ESI) m/z calc
+
1
for C24H19FN3O4S [M+H] 464.1080, found 464.1084. H NMR (600MHz, CDCl3): δ 11.84 (s, 1H,
13
NH), 7.59-7.49 (m, 6H, Ar), 7.34-7.31 (m, 4H), 7.09-7.06 (m, 3H). C (150MHz, CDCl3): δ 178.5
1
(CS), 169.1 (Cq), 167.8 (Cq), 162.3 (Cq), 150.4 ( JCF = 245.4 Hz, Cq), 148.1 ( JCF = 11.3 Hz, Cq),
2
4
139.1 (Cq), 138.0 (Cq), 131.5 (d, JCF = 3.3, Cq), 129.9 (CH), 129.8 (CH), 129.4 (CH), 129.1 (CH),
2
128.7 (CH), 128.5 (CH), 116.5 (d, JCF = 19.6 Hz, CH), 114.1 (d, JCF = 6.8 Hz, CH), 107.6 (CH),
3
19
83.6 (Cq), 56.4 (OCH3). F NMR (565 MHz): δ −136.7.
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[4-[tert-butyl(dimethyl)silyl]oxy-3,5-dimethoxy-phenyl]methanamine (4a). A suspension of
compound 10 (250 mg, 0.77 mmol), in 7 mL of MeOH dry, was cooled to 0 °C and treated with
NiCl2·6H2O (734.0 mg, 3.09 mmol). The resulting mixture was stirred at the same temperature for
5 min before the addition of NaBH4 (290 mg, 7.66 mmol). After 30 min, the reaction mixture was
quenched with saturated aqueous NH4Cl (10 mL) solution and extracted with EtOAc (3×15 mL).
The combined extracts were dried over Na2SO4 and concentrated under vacuum. Flash
chromatographic purification (elution by gradient from 100 to 80/20 DCM/MeOH∙NH3 1N)
afforded compound 4a (110 mg, 48% yield) as a viscous oil. UPLC/MS: Rt = 0.94 min (Method
10
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B), MS (ESI) m/z: 281 of main fragment. H NMR (400MHz, CDCl3): δ 6.51 (s, 2H), 3.80 (bs,
2H), 3.79 (s, 6H), 1.00 (s, 9H, tBu TBS), 0.12 (s, 6H, CH3 TBS).
2-[4-[tert-butyl(dimethyl)silyl]oxy-3,5-dimethoxy-phenyl]ethanamine (4b). A suspension of
compound 11 (250 mg, 0.74 mmol), in 7 mL of MeOH dry, was cooled to 0 °C and treated with
NiCl2·6H2O (703.6 mg, 2.96 mmol). The resulting mixture was stirred at the same temperature for
5 min before the addition of NaBH4 (279.9 mg, 7.40 mmol). After 30 min, the reaction mixture
was quenched with saturated aqueous NH4Cl (10 mL) solution and extracted with EtOAc (3×15
mL). The combined extracts were dried over Na2SO4 and concentrated under vacuum. Flash
chromatographic purification (elution by gradient from 100 to 80/20 DCM/MeOH∙NH3 1N)
afforded compound 4b (110 mg, 48% yield) as a viscous oil. UPLC/MS: Rt = 1.16 min (method
+
+
1
B), MS (ESI) m/z: 312.2 [M+H] , C16H30NO3Si [M+H] calculated: 312.2. H NMR (400MHz,
DMSO-d6): δ 6.45 (s, 2H), 3.75 (s, 6H), 2.75 (m, 2H), 2.55 (m, 2H), 1.00 (s, 9H, tBu TBS), 0.12
(s, 6H, CH3 TBS).
(E)-3-[4-[tert-butyl(dimethyl)silyl]oxy-3,5-dimethoxy-phenyl]prop-2-en-1-amine (4c).
A 2M solution of LiALH4 in THF (1.37 mL, 2.73 mmol) was added to a suspension of AlCl3 (363
mg, 2.73 mmol) in THF dry (6 mL) at 0°C under argon. After 10 min, a solution of intermediate
13 (774 mg, 0.78 mmol) in 5 mL of THF dry was added dropwise. The mixture was stirred for 30
min at 50°C, and then cooled at 0°C, quenched with ice water (5 mL). The pH was adjusted to 9-
10 with NaOH 2M solution. The mixture was extracted with EtOAc (3x20 mL). The combined
1
organic extracts were dried over Na2SO4 and concentrated under vacuum. The H-NMR of the crude
of the reaction showed the presence of E/Z isomers in ratio 1:0.12. Flash chromatographic
purification (elution by gradient from 100 to 80/20 DCM/MeOH-NH3 1N) afforded compound (E)-
4c (90 mg, 35% yield) as a viscous oil. UPLC/MS: Rt = 1.26 min (method B), MS (ESI) m/z:
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307.2 main fragment. H NMR (400MHz, CDCl3): δ 6.58 (s, 2H), 6.41 (dd, J = 16.0, 1.5 Hz, 1H),
6.20 (ddd, J = 15.8, 6.0, 6.0 Hz, 1H), 3.80 (s, 6H), 3.47 (dd, J = 6.0, 1.4 Hz, 2H), 1.00 (s, 9H, tBu
TBS), 0.12 (s, 6H, CH3 TBS).
7
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(E)-2-(3-methoxyphenyl)ethenamine (4f). A 2M solution of LiAlH4 in THF (1.75 mL, 3.5 mmol)
was added to a suspension of AlCl3 (467 mg, 3.5 mmol) in anhydrous THF (8 mL) at 0°C under
argon. After 10 min, a solution of intermediate 14 (159 mg, 1.0 mmol) in 6 mL of anhydrous THF
was added dropwise. The mixture was stirred for 30 min at 50°C, and then cooled at 0°C, quenched
with ice water (7 mL). The pH was adjusted to 9-10 with NaOH 2M solution. The mixture was
extracted with EtOAc (3x20 mL). The combined organic extracts were dried over Na2SO4 and
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concentrated under vacuum. The H-NMR of the crude of the reaction showed the presence of E/Z
isomers in ratio 1:0.10. Flash chromatographic purification (elution by gradient from 100 to 80/20
DCM/MeOH-NH3 1N) yielded title compound (E)-4f (45 mg, 27% yield) as yellow viscous oil.
1
UPLC/MS: Rt = 1.15 min (method A), MS (ESI) m/z: 147.0 main fragment. H NMR (400MHz,
CDCl3): δ 7.22 (dd, J = 7.9, 7.9 Hz, 1H), 6.97 (d, J = 7.8 Hz, 1H), 6.91 (dd, J = 2.0, 2.0 Hz, 1H),
6.78 (dd, J = 8.1, 2.6 Hz, 1H), 6.48 (ddd, J = 15.9, 1.7, 1.7 Hz, 1H), 6.32 (ddd, J = 15.8, 5.8, 5.8
Hz, 1H), 3.81 (s, 3H), 3.49 (dd, J = 5.8, 1.3 Hz, 2H).
N-(4-((tert-butyldimethylsilyl)oxy)-3,5-dimethoxybenzyl)-6-hydroxy-4-oxo-1,3-diphenyl-2-
thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide (5a). Title compound was prepared
according to general procedure 2 using: amine 4a (80 mg, 0.27 mmol), ester 1 (83 mg, 0.22 mmol)
in anhydrous toluene (0.44 mL). Then, the solvent was removed under vacuum. Flash
chromatographic purification (elution by gradient from 100 to 85/15 Cyclohexane/EtOAc)
afforded 5a (95 mg, 70% yield) as a viscous oil. UPLC/MS: Rt = 2.08 min (method B), MS (ESI)
+
+
1
m/z: 620.3 [M+H] , C32H38N3O6SSi [M+H] calculated: 620.8. H NMR (400MHz, CDCl3): δ 10.13
(dd, J = 5.8, 5.8 Hz, 1H, NH), 7.55-7.42 (m, 6H, Ph), 7.30-7.23 (m, 4H, Ph), 6.44 (s, 2H, Ar), 4.48
13
(d, J = 5.9 Hz, 2H), 3.76 (s, 6H), 1.00 (s, 9H), 0.12 (s, 6H). C (100MHz, CDCl3): δ 179.0 (CS),
170.4 (CONH), 167.5 (Cq), 162.2 (Cq), 152.0 (Cq), 128.7 (CH), 127.8 (CH), 105.8 (CH), 83.1
(Cq), 56.0 (OCH3), 45.0 (CH2), 25.9 (CH3, TBS), 18.8 (Cq, TBS), −4.5 (CH3, TBS).
N-(4-((tert-butyldimethylsilyl)oxy)-3,5-dimethoxyphenethyl)-6-hydroxy-4-oxo-1,3-diphenyl-
2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide (5b). Title compound was prepared
according to general procedure 2 using: amine 4b (40 mg, 0.12 mmol), ester 1 (37 mg, 0.10 mmol)
in anhydrous toluene (0.50 mL). Then, the solvent was removed under vacuum. Flash
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chromatographic purification (elution by gradient from 100 to 75/25 Cyclohexane/EtOAc)
5
6
afforded 5b (27 mg, 42% yield) as a viscous oil. UPLC/MS: Rt = 2.38 min (method B), MS (ESI)
+
+
1
m/z: 634.2 [M+H] , C33H40N3O6SSi [M+H] calculated: 634.8. H NMR (400MHz, CDCl3): δ 10.04
(dd, J = 5.7, 5.7 Hz, 1H, NH), 7.54-7.43 (m, 6H, Ph), 7.29-7.22 (m, 4H, Ph), 6.35 (s, 2H, Ar), 3.71
(s, 6H), 3.65 (ddd, J = 6.8, 6.8, 6.8 Hz, 2H), 2.80 (dd, J = 6.8, 6.8 Hz, 2H), 1.00 (s, 9H), 0.10 (s,
6H).
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(E)-N-(3-(4-((tert-butyldimethylsilyl)oxy)-3,5-dimethoxyphenyl)allyl)-6-hydroxy-4-oxo-1,3-
diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide (5c). Title compound was
prepared according to general procedure 2 using: amine 4c (65 mg, 0.20 mmol), ester 1 (63 mg,
0.17 mmol) in anhydrous toluene (0.34 mL). Then, the solvent was removed under vacuum. Flash
chromatographic purification (elution by gradient from 100 to 75/25 Cyclohexane/EtOAc)
afforded 5c (43 mg, 39% yield) as a viscous oil. UPLC/MS: Rt = 2.30 min (method B), MS (ESI)
+
+
1
m/z: 646.3 [M+H] , C34H39N3O6SSi [M+H] calculated: 646.8. H NMR (400MHz, CDCl3): δ 10.06
(dd, J = 5.3, 5.3 Hz, 1H, NH), 7.55-7.42 (m, 6H, Ph), 7.30-7.26 (m, 4H, Ph), 6.53 (s, 2H, Ar), 6.47
(ddd, J = 15.7, 1.4, 1.4 Hz, 1H), 6.02 (ddd, J = 15.7, 6.5, 6.5 Hz, 1H), 4.17 (ddd, J = 6.5, 6.5, 1.4
13
Hz, 2H), 3.79 (s, 6H), 1.00 (s, 9H), 0.12 (s, 6H). C (100MHz, CDCl3): δ 179.0 (CS), 170.6
(CONH), 167.4 (Cq), 162.3 (Cq), 151.8 (Cq), 139.4 (Cq), 138.4 (Cq), 134.7 (CH), 129.7 (CH),
129.2 (CH), 128.9 (CH), 128.6 (CH), 103.9 (CH, 2C), 83.1 (Cq), 55.9 (OCH3), 42.6 (CH2), 25.9
(CH3, TBS), 18.9 (Cq, TBS), −4.5 (CH3, TBS)
6-hydroxy-N-(3-methoxybenzyl)-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-
5-carboxamide (5d). Title compound was prepared according to general procedure 2 using: 3-
methoxybenzylamine 4d (21 μL, 0.16 mmol), ester 1 (50 mg, 0.14 mmol) in anhydrous toluene
(0.28 mL). Then, the solvent was removed under vacuum. Flash chromatographic purification
(elution by gradient from 100 to 75/25 Cyclohexane/EtOAc) afforded 5d (39 mg, 62% yield) as
+
an amorphous white solid. UPLC/MS: Rt = 2.26 min (method A), MS (ESI) m/z: 460.2 [M+H] ,
+
+
C25H22N3O4S [M+H] calculated: 460.5. HRMS (AP-ESI) m/z calc for C25H22N3O4S [M+H] 460.1331,
1
found 460.1325. H NMR (400MHz, DMSO-d6): δ 10.26 (dd, J = 6.0, 6.0 Hz, 1H, NH), 7.47-7.35
(m, 6H, Ar), 7.30-7.25 (m, 5H, Ar), 6.91-6.85 (m, 3H, Ar), 4.55 (d, J = 6.2 Hz, 2H), 3.73 (s, 3H).
13
C (100MHz, DMSO-d6): δ 178.6 (CS), 169.7 (Cq), 159.4 (Cq), 139.3 (Cq), 138.8 (Cq), 129.8
(CH), 129.0 (CH), 128.2 (CH), 119.7 (CH), 113.6 (CH), 112.7 (CH), 82.8 (Cq), 55.1 (OCH3), 43.7
(CH2).
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6-hydroxy-N-(3-methoxyphenethyl)-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-
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tetrahydropyrimidine-5-carboxamide (5e). Title compound was prepared according to general
procedure 2 using: 3-methoxyphenethylamine 4e (38 μL, 0.26 mmol), ester 1 (80 mg, 0.22 mmol)
in anhydrous toluene (0.44 mL). Then, the solvent was removed under vacuum. Flash
chromatographic purification (elution by gradient from 100 to 75/25 Cyclohexane/EtOAc)
afforded 5e (43 mg, 41% yield) as an amorphous white solid. UPLC/MS: Rt = 2.37 min (method
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+
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A), MS (ESI) m/z: 474.1 [M+H] , C26H24N3O4S [M+H] calculated: 474.1. HRMS (AP-ESI) m/z calc
+
1
for C26H24N3O4S [M+H] 474.1488, found 474.1489. H NMR (400MHz, CDCl3): δ 10.03 (dd, J =
6.0, 6.0 Hz, 1H, NH), 7.54-7.43 (m, 6H, Ar), 7.29-7.20 (m, 5H, Ar), 6.79-6.72 (m, 3H, Ar), 3.76
13
(s, 3H), 3.67 (ddd, J = 6.9, 6.9, 6.9 Hz, 2H), 2.87 (dd, J = 7.2 Hz, 2H). C (150MHz, CDCl3): δ
179.0 (CS), 170.8 (Cq), 167.3 (Cq), 162.2 (Cq), 159.9 (Cq), 139.4 (Cq), 139.1 (Cq), 138.4 (Cq),
129.9 (CH), 129.6 (CH), 129.1 (CH), 128.9 (CH), 128.8 (CH), 128.7 (CH), 121.0 (CH), 114.4
(CH), 112.5 (CH), 83.0 (Cq), 55.3 (OCH3), 41.9 (CH2), 35.5 (CH2).
(E)-6-hydroxy-N-(3-(3-methoxyphenyl)allyl)-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxamide (5f). Title compound was prepared according to general
procedure 2 using: amine 4f (45 mg, 0.28 mmol), ester 1 (85 mg, 0.23 mmol) in anhydrous toluene
(0.46 mL). Then, the solvent was removed under vacuum, flash chromatographic purification
(elution by gradient from 100 to 70/30 Cyclohexane/EtOAc) afforded 5f (42 mg, 38% yield) as an
+
amorphous white solid. UPLC/MS: Rt = 2.33 min (method A), MS (ESI) m/z: .486.1 [M+H] ,
+
1
C27H24N3O4S [M+H] calculated: 486.5. H NMR (600MHz, CDCl3): δ 10.11 (dd, J = 5.5, 5.5 Hz,
1H, NH), 7.56-7.48 (m, 7H, Ar), 7.33-7.30 (m, 3H, Ar), 7.25 (dd, J = 8.0, 8.0 Hz, 1H, Ar), 6.95
(d, J = 7.7 Hz, 1H, Ar), 6.89 (dd, J = 2.0, 2.0 Hz, 1H, Ar), 6.84 (dd, J = 8.0, 2.2 Hz, 1H), 6.56 (d,
J = 15.8 Hz, 1H), 6.18 (ddd, J = 15.8, 6.4, 6.4 Hz, 1H), 4.22 (ddd, J = 6.1, 6.1, 1.4 Hz, 2H), 3.83
13
(s, 3H). C NMR (150MHz, CDCl3): δ 179.0 (CS), 170.7 (CONH), 167.4 (Cq), 162.2 (Cq), 159.9
(Cq), 139.3 (Cq), 138.4 (Cq), 137.4 (Cq), 134.1 (CH), 129.8 (CH), 129.7 (CH), 129.2 (CH), 128.9
(CH), 128.8 (CH), 128.6 (CH, 2C), 122.8 (CH), 119.3 (CH), 114.0 (CH), 111.9 (CH), 83.1 (Cq),
55.4 (OCH3), 42.5 (CH2).
General procedure 3: TBS deprotection. A solution 0.5M of silylated precursor (1 equiv) was
treated with TBAF 1M solution in THF (1.5 equiv). The reaction mixture stirred for 3 h. Then, the
mixture was diluted with EtOAc, washed with water, and concentrated under vacuum. The crude
material was purified by flash chromatography.
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Journal of Medicinal Chemistry
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6-hydroxy-N-(4-hydroxy-3,5-dimethoxybenzyl)-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxamide (6). Title compound was prepared according to general
procedure 3 using: intermediate 5a (90 mg, 0.14 mmol), TBAF 1M in THF (220 μL, 0.27 mmol)
in anhydrous THF (0.28 mL). The crude was purified by silica gel flash chromatography (elution
by gradient from 100 to 60/40 Cyclohexane/EtOAc) to yield 6 (42 mg, 54%) as pale yellow
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−
amorphous solid. UPLC/MS: Rt = 2.08 min (method A), MS (ESI) m/z: 504.2 [M−H] , C26H22N3O6S
−
[M−H] calculated: 504.5. HRMS (AP-ESI) m/z calc for C26H24N3O6S [M+H] 506.1386, found
+
1
506.1373. H NMR (400MHz, DMSO-d6): δ 10.18 (dd, J = 6.2, 6.2 Hz, 1H, NH), 8.37 (s, 1H, OH),
7.48-7.35 (m, 6H, Ph), 7.30-7.23 (m, 4H, Ph), 6.65 (s, 2H, Ar), 4.46 (d, J = 6.1 Hz, 2H), 3.73 (s,
13
6H). C (100MHz, DMSO-d6): δ 178.6 (CS), 169.4 (Cq), 150.0 (Cq), 139.2 (Cq), 135.3 (Cq), 128.9
(CH), 128.1 (CH), 126.7 (CH), 106.1 (CH), 82.8 (Cq), 56.1 (OCH3), 43.7 (CH2).
6-hydroxy-N-(4-hydroxy-3,5-dimethoxyphenethyl)-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxamide (7). Title compound was prepared according to general
procedure 3 using: intermediate 5b (26 mg, 0.04 mmol), TBAF 1M in THF (60 μL, 0.06 mmol) in
anhydrous THF (0.1 mL). The crude was purified by silica gel flash chromatography (elution by
gradient from 100 to 60/40 Cyclohexane/EtOAc) to yield 7 (11 mg, 53%) as pale yellow
−
amorphous solid. UPLC/MS: Rt = 2.11 min (method A), MS (ESI) m/z: 518.1 [M−H] , C27H24N3O6S
−
+
1
[M−H] calculated: 518.6. HRMS (AP-ESI) m/z calc for C27H26N3O6S [M+H] 520.1542. H NMR
(400MHz, CDCl3): δ 10.07 (dd, J = 5.8, 5.8 Hz, 1H, NH), 7.55-7.42 (m, 6H, Ph), 7.29-7.22 (m,
4H, Ph), 6.40 (s, 2H, Ar), 5.41 (s, 1H, OH), 3.8 (s, 6H), 3.65 (ddd, J = 6.1, 6.1, 6.1 Hz, 2H), 2.81
13
(dd, J = 6.8 Hz, 6.8 Hz, 2H). C (150MHz, CDCl3): δ (CS), 179.0 (CS), 170.6 (CONH), 167.3
(Cq), 162.2 (Cq), 147.3 (Cq), 139.4 (Cq), 138.4 (Cq), 133.8 (Cq), 129.7 (CH), 129.6 (CH), 129.2
(CH), 128.9 (CH), 128.8 (CH), 128.6 (CH), 105.5 (CH), 83.0 (Cq), 56.4 (OCH3), 42.2 (CH2), 35.5
(CH2).
(E)-6-hydroxy-N-(3-(4-hydroxy-3,5-dimethoxyphenyl)allyl)-4-oxo-1,3-diphenyl-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxamide (8). Title compound was prepared according to
general procedure 3 using: intermediate 5c (40 mg, 0.06 mmol), TBAF 1M in THF (90 μL, 0.09
mmol) in anhydrous THF (0.12 mL). The crude was purified by silica gel flash chromatography
(elution by gradient from 100 to 60/40 Cyclohexane/EtOAc) to yield 8 (29 mg, 88%) as pale
−
yellow amorphous solid. UPLC/MS: Rt = 2.11 min (method A), MS (ESI) m/z: 530.3 [M−H] ,
−
C28H24N3O6S [M−H] calculated: 530.6. HRMS (AP-ESI) m/z calc for C28H26N3O6S [M+H] 532.1542,
+
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found 532.1524. H NMR (400MHz, DMSO-d6): δ 10.01 (dd, J = 6.2, 6.2 Hz, 1H, NH), 8.44 (s,
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1H, OH), 7.47-7.28 (m, 11H, Ph, OH), 6.68 (m, 2H, Ar), 6.46 (d, J = 15.8 Hz, 1H), 6.17 (ddd, J =
13
15.8, 6.2, 6.2 Hz, 1H), 4.13 (dd, J = 6.0, 6.0, 1.4 Hz, 2H), 3.75 (s, 6H). C (150 MHz, DMSO-d6):
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δ 178.7 (CS), 169.6 (CONH), 148.1 (Cq), 139.4 (Cq), 135.7 (Cq), 132.7 (Cq), 129.1 (CH), 128.3
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(CH), 126.7 (Cq), 121.7 (CH), 104.0 (CH), 83.2 (Cq), 58.0 (OCH3), 41.9 (CH2).
1-[4-[tert-butyl(dimethyl)silyl]oxy-3,5-dimethoxy-phenyl]-N-methoxy-methanimine
(10).
Sodium acetate (138 mg, 1.69 mmol) and N-Methylhydroxylamine hydrochloride (141 mg, 1.69
50
mmol) were added to a solution of compound 9 (250 mg, 0.844 mmol) in MeOH dry (5 ml) under
argon. The reaction mixture was stirred at 50°C for 5h until completion of reaction. The solvent
was removed under vacuum, and the residue was suspended in water (5 mL) and the product was
extracted with EtOAc (5x3 mL). Collected organic layers were died with Na2SO4, filtered and,
concentrated under vacuum affording desired product 10 as a mixture of E/Z isomers (260 mg,
95% yield). The product was used as such without further purification. UPLC/MS: Rt = 2.42 min
+
+
1
(method B), MS (ESI) m/z: 326.2 [M+H] , C16H27NO3Si [M+H] calculated: 326.2. H NMR
(400MHz, CDCl3) of major isomer: δ 7.96 (s, 1H), 6.78 (s, 2H), 3.95 (s, 3H), 3.82 (s, 6H), 1.00 (s,
9H, tBu TBS), 0.13 (s, 6H, CH3 TBS).
1-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]-2,6-dimethoxy-4-[(1E)-2-nitroethenyl]benzene
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(11). Nitromethane (2.7 ml, 50.5 mmol) was carefully added to a mixture of aldehyde 9 (300 mg,
1.01 mmol) and ammonium acetate (77.1 mg, 1.01 mmol) in toluene dry (10 mL) under argon.
The reaction mixture was stirred for 20 hours at reflux under argon. Then the reaction mixture was
cooled at room temperature, quenched with water (10 mL), and extracted with EtOAc (2x10 mL).
Collected organic layers were dried over Na2SO4, filtered and concentrated under vacuum. Flash
chromatographic purification (elution by gradient from 100 to 95/5 Cyclohexane/EtOAc) afforded
compound (E)-11 (308 mg, 90% yield) as amorphous yellow solid. UPLC/MS: Rt = 2.41 min
+
+
1
(method B), MS (ESI) m/z: 340.2 [M+H] , C16H26NO5Si [M+H] calculated: 340.1. H NMR
(400MHz, CDCl3): δ 7.93 (d, J = 13.5 Hz, 1H), 7.52 (d. J = 13.5 Hz, 1H), 6.73 (s, 2H), 3.84 (s,
6H), 1.01 (s, 9H, tBu TBS), 0.15 (s, 6H, CH3 TBS).
3-[4-[tert-butyl(dimethyl)silyl]oxy-3,5-dimethoxy-phenyl]prop-2-enenitrile 13. To a solution
of diethylcyanomethyl phosphonate (180 μL, 1.1 mmol) in THF (8 mL) was added t-BuOK (125
50
mg, 1.1 mmol) at ice-water bath temperature with stirring for 30 min. After that, aldehyde 9 (300
mg, 1.0 mmol) in THF (3 mL) was added dropwise into the above mixture at room temperature
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