Sydnone Cycloaddition Route to Pyrazole-Based Analogs of Combretastatin A4

Article abstract of DOI:10.1021/acs.jmedchem.6b01128

The combretastatins are an important class of tubulin-binding agents. Of this family, a number of compounds are potent tumor vascular disrupting agents (VDAs) and have shown promise in the clinic for cancer therapy. We have developed a modular synthetic r

Full text of DOI:10.1021/acs.jmedchem.6b01128

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Article
A Sydnone Cycloaddition Route to Pyrazole-
Based Analogs of Combretastatin A4
Andrew W. Brown, Matthew Fisher, Gillian M. Tozer, Chryso Kanthou, and Joseph P.A. Harrity
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01128 • Publication Date (Web): 02 Oct 2016
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A Sydnone Cycloaddition Route to Pyrazole-Based Analogs of Combretastatin A4
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Andrew W. Brown,†,‡ Matthew Fisher,‡ Gillian M. Tozer,*,‡ Chryso Kanthou,*,‡ Joseph P.
A. Harrity*,†
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† Department of Chemistry, Dainton Building, Brook Hill, University of Sheffield, S3 7HF
‡
Department of Oncology & Metabolism, The Medical School, University of Sheffield,
Beech Hill Road, S10 2RX
Abstract
The combretastatins are an important class of tubulinꢀbinding agents. Of this family, a
number of compounds are potent tumor Vascular Disrupting Agents (VDAs) and have shown
promise in the clinic for cancer therapy. We have developed a modular synthetic route to
combretastatin analogs based on a pyrazole core through highlyꢀregioselective alkyne
cycloaddition reactions of sydnones. These compounds show modest to high potency against
human umbilical vein endothelial cell proliferation. Moreover, evidence is presented that
these novel VDAs have the same mode of action as CA4P and bind reversibly to βꢀtubulin –
believed to be a key feature in avoiding toxicity. The most active compound from in vitro
studies was taken forward to an in vivo model and instigated an increase in tumor cell
necrosis.
Introduction
Tumor growth and metastasis are dependent upon a functional blood vessel network1 and,
since tumor blood vessels are abnormal, they represent an attractive target for cancer
therapy.2,3 Tumor Vascular Disrupting Agents (VDAs) cause a rapid and selective collapse of
tumor blood vessels leading to widespread tumor cell death via necrosis.4 The most wellꢀ
studied class of VDAs are the tubulin binding microtubule depolymerizing agents. The tumor
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vascular disrupting properties of colchicine, the classic tubulin binding agent of this type,
have been known since the 1930s, but these effects are only seen at near to the maximum
tolerated dose (MTD) in mice.5,6 The structurally related stilbene compounds, the
combretastatins, were isolated from the Cape Bush Willow tree, Combretum caffrum in the
late 1980s.7 These compounds were initially studied for their potential as antimitotic agents.8
However, it later became apparent that the combretastatins had great potential as VDAs.9 The
lead compound, combretastatin Aꢀ4 (CA4), was shown to be effective at causing selective
tumor vascular collapse, within minutes, at nonꢀtoxic doses.10 It was swiftly developed into
the waterꢀsoluble, sodium phosphate salt prodrug, disodium combretastatin Aꢀ4 3ꢀOꢀ
phosphate (CA4P).11 CA4P is not active as a tubulin binding agent, but is rapidly cleaved in
plasma to CA4.12 The lower toxicity (and thus higher MTD) of CA4 compared to colchicine
is attributed to CA4 binding reversibly to the βꢀtubulin subunit, as opposed to colchicine that
binds pseudoꢀirreversibly.13 CA4P is currently undergoing Phase II/III clinical trials in
combination with bevacizumab.14
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Although CA4P is a promising clinical candidate, it does have a number of issues associated
with it. Firstly, the cis orientation of the olefinic linker is key to activity, with the trans
isomer being essentially inactive15 – an issue further emphasized by the tendency of cis
stilbenes to undergo cisꢀtrans isomerization in heat, light and protic media.16 A promising
strategy to overcome this issue has been the incorporation of heterocyclic motifs to replace
the olefinic linker.17,18,19,20,21,22,23 This strategy allows the cis configuration of the
combretastatins to be “fixed”, inhibiting metabolic decomposition (Figure 1). However, any
compound design would have to take into account the result of molecular docking studies that
clearly indicate the need for appropriately disposed electron rich aryl rings, such as those in
colchicine and CA4, which are key pharmacophores for tubulin polymerization
inhibition.24,25,26
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Figure 1. Tubulinꢀbinding VDAs
Although a number of significant contributions have been made in the field of rigid
combretastatin analogs, only limited examples of this approach using a pyrazole core have
been documented.16,20,27,28 To the best of our knowledge, there are no examples of simple
(without extra substitution of the pyrazole core), pyrazoleꢀbridged CA4 analogs reported and
there has not been a systematic evaluation into the effect of substitution pattern. The pyrazole
motif is an important fragment in the pharmaceutical industry and is present in a number of
commercial medicines and agrichemicals.29,30 However, synthetic routes to these structurally
important diazoles are often linear or suffer from regioselectivity issues during the
heterocycle forming step. In this regard, we felt that sydnones could represent convenient and
potentially versatile starting materials to access these desirable therapeutic analogs. Sydnones
belong to the mesoionic family of compounds and they undergo [3+2] cycloadditionꢀ
retrocycloaddition reactions with alkynes to form pyrazoles.31 These cycloadditions can be
highly regioselective and offer access to a wide range of pyrazoles.32,33,34,35,36,37 In the context
of developing novel VDAs, we envisaged that the sydnone cycloaddition strategy could be
exploited to access a broad range of pyrazole templated combretastatin analogs from similar
starting materials (Scheme 1). However, we recognized that a number of synthetic challenges
needed to be met in order to achieve this goal: (1) access to 1,5ꢀsubstituted pyrazoles
theoretically required high temperature cycloaddition with acetylene gas; (2) 4,5ꢀdisubstituted
pyrazoles required the innate regioselectivity of the cycloaddition with terminal alkynes to be
reversed (typically, the largest substituent is incorporated at the C3); (3) the cycloaddition of
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biaryl alkynes for the preparation of 3,4ꢀsubstituted pyrazoles was unlikely to proceed with
regiocontrol, thereby delivering isomeric mixtures.
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Scheme 1. Sydnone cycloaddition strategy to pyrazoleꢀbased analogs
Results and Discussion
Preparation of 1,5-Disubstituted Analogs
Our investigations began with the preparation of 1,5ꢀdisubstituted pyrazoles. We proposed a
strategy that exploited our previously developed direct arylation of sydnones to furnish the
key diaryl intermediates (Scheme 2).38 We then envisaged that trimethylsilylacetylene could
act as a highꢀboiling point acetylene equivalent in the cycloaddition to provide the target
pyrazoles after protodesilylation.
Scheme 2. Strategy for the synthesis of 1,5ꢀdisubstituted pyrazoles
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Sydnone starting materials were readily prepared by established routes of Nꢀnitrosation of
amino acids, followed by cyclodehydration with trifluoroacetic anhydride (TFAA).31,38 The
synthesis began with the coupling of sydnone 1 affording diaryl sydnone 2 in moderate yield
9
(Scheme 3). Pleasingly, sydnone
2
underwent successful cycloaddition with
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trimethylsilylacetylene to afford TMSꢀsubstituted pyrazoles 3a and 3b in good yield and
excellent (but inconsequential) regioselectivity. Previous experience had suggested that 3ꢀ
silyl pyrazoles could be particularly resistant to hydrolysis. Fortunately however, a refluxing
solution of TBAF in THF afforded the desilated product 4 in useful yield. Finally, simple
hydrogenation of the benzyl ether afforded CA4 analog 5.
Scheme 3. Synthesis of pyrazole 5
Preparation of the isomeric 1,5ꢀpyrazole 10 required a slight modification to the route.
Sydnone 6 was subjected to direct arylation conditions, followed by benzyl protection of the
phenol (Scheme 4). Surprisingly, benzyl protection of the phenol prior to direct arylation
resulted in a sydnone that decomposed when subjected to various direct arylation conditions.
Sydnone 7 underwent cycloaddition in very good yield and regioselectivity. TBAF
deprotection of the trimethylsilyl group proceeded in moderate yield. The resulting pyrazole
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9 was contaminated with a small amount of an inseparable, unknown byꢀproduct.
Fortunately, analog 10 was isolated cleanly after benzyl hydrogenation.
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Scheme 4. Synthesis of pyrazole 10
Preparation of 4,5-Disubstituted Analogs
We next turned our attention to the preparation of 4,5ꢀdisubstituted pyrazoles. Elegant work
by Taran et al. demonstrated that copper catalysis can reverse the natural regioselectivity of
sydnone cycloadditions.33 Recently, the chemistry has been improved to tolerate bromide
substituents at the sydnone C4 position, which could then be further elaborated by crossꢀ
coupling.32 However, this chemistry is currently limited to Nꢀaryl sydnones. As our interest
lay with Nꢀalkyl sydnones, a new methodology was needed. We proposed to extend the
strategy used for the synthesis of 1,5ꢀsubstituted pyrazoles to borylated
trimethylsilylacetylene (Scheme 5). We have previously reported the cycloaddition of
alkynylboranes with sydnones to be highly regioselective and importantly, tolerant of Nꢀ
alkylsydnones.35 In the case of disubstituted alkynes, the boron substituent is generally
incorporated at the pyrazole C4 position.
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Scheme 5. Strategy for the synthesis of 4,5ꢀdisubstituted pyrazoles
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The synthesis began with the direct arylation of Nꢀmethylꢀ and Nꢀbenzylsydnone, which
proceeded smoothly on gram scale affording C4ꢀarylated sydnones 11-14 (Scheme 6). We
were delighted to find all substrates successfully underwent cycloaddition and the
corresponding pyrazoles were isolated as single regioisomers, 15-18, albeit contaminated
with varying amounts of protodeborylated byꢀproduct. The SuzukiꢀMiyaura reaction required
some optimization, but was successfully achieved using Buchwald’s XPhosPdG2 precatalyst
to afford 19-22. The products were again contaminated with varying amounts of
protodeborylated byꢀproduct. Pleasingly, subjection of 23-26 to refluxing TBAF successfully
resulted in silyl deprotection, and 23-26 were readily separable from their corresponding byꢀ
products at this stage. Benzyl hydrogenation afforded the 4,5ꢀdisubstituted analogs of CA4,
27-32. In the case of Nꢀbenzylpyrazoles 24 and 26, incomplete hydrogenation afforded a
separable mixtures of N-H pyrazoles 28 and 31 and Nꢀbenzylpyrazoles 29 and 32
respectively.
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R2-Br
R3-Br
XPhosPdG2 (10%)
Na2CO3,
TMS
N
N
R1
O
O
PinB
R2
O
N
O
N
TMS
BPin
Pd(OAc)2 (5%)
XPhos (10%)
K2CO3, DMF
80 C, 16 h
N
N
R2
Xylenes
R1
R1
DME:H2O (1:1)
80 C, 16 h
180 C, 48 h
11: R1=Me; R2=Ar1; 86%
12: R1=Bn; R2=Ar1; 59%
13: R1=Me; R2=Ar2; 71%
14: R1=Bn; R2=Ar2; 55%
15: R1=Me; R2=Ar1; 92%
16: R1=Bn; R2=Ar1; 66%
17: R1=Me; R2=Ar2; 73%
18: R1=Bn; R2=Ar2; 64%
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Ar1
=
TMS
R3
MeO
Ar2
OMe
OMe
R3
H2, Pd/C, MeOH
R.T. 16-72 h
TBAF
N
N
N
R1
=
N
R2
THF
R2
R1
Reflux, 24 h
23: R1=Me; R2=Ar1; R3=Ar2; 55%
24: R1=Bn; R2=Ar1; R3=Ar2; 64%
25: R1=Me; R2=Ar2; R3=Ar1; 56%
26: R1=Bn; R2=Ar2; R3=Ar1; 74%
19: R1=Me; R2=Ar1; R3=Ar2; 69%
20: R1=Bn; R2=Ar1; R3=Ar2; 72%
21: R1=Me; R2=Ar2; R3=Ar1; 60%
22: R1=Bn; R2=Ar2; R3=Ar1; 49%
OBn
OMe
OH
OH
OMe
OMe
MeO
MeO
MeO
MeO
MeO
HO
MeO
N
N
N
N
N
N
MeO
MeO
MeO
MeO
N
N
HO
R
Me
R
Me
MeO
MeO
OMe
OMe
27: 58%
R=H, 28: 30%
(R=Bn, 29 22%)
30: 67%
R=H, 31: 27%
(R=Bn, 32: 27%)
Scheme 6. Synthesis of 4,5ꢀdisubstituted analogs
Preparation of 3,4-Disubstituted Analogs
The final analogs to prepare were the 3,4ꢀdisubstituted pyrazoles. It was originally anticipated
that we could utilize alkynylboronate cycloaddition chemistry to access these compounds
(Scheme 7, A).36 However, we found that alkynes bearing these particularly electron rich
aromatic rings were prone to protodeborylation, and pyrazole boronic esters could not be
obtained under a variety of conditions. We proposed an alternative route whereby the innate
regioselectivity of the sydnone cycloaddition was exploited, followed by bromination and
subsequent crossꢀcoupling (Scheme 7, B).
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Scheme 7. Strategy for the synthesis of 3,4ꢀdisubstituted pyrazoles
The route began with the cycloaddition of Nꢀmethyl and Nꢀbenzylsydnone with
arylacetylenes. The reactions proceeded in good yield and high regioselectivity to afford
pyrazoles 33-36 (Scheme 8). We were pleased to find that Nꢀbromosuccinamide (NBS) in
acetonitrile rapidly and selectively brominated the pyrazoles. After basic workꢀup, the
brominated products could be subjected without further purification to SuzukiꢀMiyaura
coupling, affording products 37 and 39 in good yield over 2 steps. Products 38 and 40 were
isolated after subjection of the crude SuzukiꢀMiyaura products to silyl deprotection
conditions of potassium carbonate in refluxing methanol in useful yield over 3 steps. In the
case of Nꢀmethyl analogs 37 and 39, this route represents rapid access to the compounds in
effectively 2 pots. Compounds 38 and 40 were subjected to hydrogenation to afford the free
NHꢀpyrazole analogs 28 and 31, thus providing an alternative, shorter route to these analogs
from the alkynylboronate strategy above.
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Scheme 8. Synthesis of 3,4ꢀdisubstituted analogs
Biological Evaluation
With a library of twelve pyrazoleꢀbased analogs of CA4 in hand, attention turned to
evaluating their potential as anticancer agents. Studies within our laboratories have
previously shown that for CA4P, vascular disruptive effects dominate rather than direct
targeting of cancerous cells.39 Therefore, we proposed that the primary target of VDAs,
endothelial cells, would provide an accurate insight into the activity of the pyrazoleꢀbased
analogs.
Initial Screens
CA4 is a tubulin binding agent, therefore we proposed that a convenient method for the rapid
assessment of activity was to study the effects of compounds on endothelial cell proliferation,
which is inhibited by disruption of the microtubuleꢀrich mitotic spindle. It is important to
note that the action of CA4 as a VDA is not due to effects on HUVEC proliferation or
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prevention of mitotic spindle formation. However, the analysis of drugꢀinduced effects cell
proliferation provide a useful insight into microtubule targeting potential. Screening results
are depicted in Table 1.
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Entry
1
Compound R1
R2
Ar2
Ar1
Ar1
Ar1
Ar1
Ar2
Ar2
Ar2
H
R3
H
R4
H
GI50
Ar1
28 nM
5
2
Ar2
H
H
>750 nM
68 nM
10
3
Me
Ar2
Ar2
Ar2
Ar1
Ar1
Ar1
Ar2
Ar2
Ar1
Ar1
ꢀ
H
27
4
H
H
>5000 nM
>2500 nM
118 nM
7 nM
28
5
Bn
H
29
6
Me
H
30
7
H
H
31
8
Bn
H
>750 nM
>5000 nM
>5000 nM
256 nM
>2000 nM
8 nM
32
9
Me
Ar1
Ar1
Ar2
Ar2
ꢀ
37
10
11
12
13
14
Bn
H
38
Me
H
39
Bn
H
40
CA4P
ꢀ
ꢀ
ꢀ
Colchicine
ꢀ
ꢀ
ꢀ
25 nM
Table 1. Values for growth inhibition of HUVECs by pyrazole anologs vs. CA4P and
colchicine
The variation observed in the activities of the compounds was quite remarkable given that all
of the compounds screened were direct analogs of CA4, differing only in their location on the
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pyrazole core. The most active compounds 5, 27, 30 and 31 (entries 1, 3, 6 and 7) exhibited
low nanomolar activity. The most active compound 31, showed similar activity to CA4P
(entry 7 vs 13). However, a number of compounds only showed low micromolar activities
(entries 4, 9 and 12). Further studies were undertaken on the two most active compounds 5
and 31 (Figure 2).
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Immunofluorescence Studies
To confirm that the drug effects were due to microtubule disruption, drugꢀinduced changes in
the endothelial cell cytoskeleton and morphology were studied via immunofluorescence for
compounds 5 and 31. The microtubule disruption associated with CA4P treatment is also
accompanied by changes in cell shape and morphology brought about by the rapid
remodelling of the actin cytoskeleton.40 Therefore, endothelial cells in culture were stained
for actin filaments using phalloidin, microtubules using an antibody to β−tubulin and nuclei
stained with 4',6ꢀdiamidinoꢀ2ꢀphenylindole (DAPI).
In cells that are in interphase, polymers of α−tubulin and β−tubulin originate from the
centrosome and radiate to the edge of the cell as tubular filaments referred to as microtubules
(Figure 3, A), whereas actin is mostly pericellular (Figure 3, B). Upon treatment with 250 nM
CA4P for 30 minutes, interphase microtubules became disrupted (Figure 3, C), leading to
changes in cell morphology. Disruption of microtubules led to the formation of actin stress
fibres across the cells (Figure 3, D). Interestingly, a longer (90 minutes) treatment at a higher
dose of 1 ꢀM was required for similar effects to be observed with colchicine (Figure 3, E and
F). A 250 nM treatment with 5 resulted in the disruption of interphase microtubules and
significant changes in endothelial cell shape and morphology (Figure 3, G and H). Similarly,
a 250 nM treatment with 31 also caused pronounced effects on endothelial cell shape and
morphology (Figure 3, I and J).
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Figure 3. Drug effects on HUVEC cytoskeletal structures. Cells were treated with a single
dose of either vehicle (A, B), 250 nM CA4P (C, D), 1 ꢀM colchicine (E, F), 250 nM drug 5
(G, H) or 250 nM drug 31 (I, J). Drug treatments were for 30 min with CA4P, drug 5 and
drug 31 and 90 minutes with colchicine. Cells were fixed and stained with an antibody to βꢀ
tubulin (A, C, E, G, I) and Fꢀactin (B, D, F, H, J) and nuclei stained with DAPI.
Recovery Experiments
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CA4P is thought to be a potent VDA at well below its maximum tolerated dose due to the
reversibility of its binding to tubulin.12 Therefore, we tested the reversibility of the tubulin
binding of compounds 5 and 31 using immunofluorescence in parallel with CA4P and
colchicine.
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As shown in Figure 3, a 30 minute treatment with 250 nM of either CA4P, 5 or 31 was
sufficient to produce a profound effect on cellular morphology. A 30 minute treatment with
CA4P, followed by removal of the drug and 60 minutes incubation led to significant recovery
of the cells (Figure 4, A and B). There was a clear recovery of microtubules and a reduction
in actin stress fibres. Importantly, cells treated with colchicine did not show signs of recovery
within the same time frame (Figure 4, C and D). Indeed, recovery was not observed, even
after a 120 minute incubation upon removal of the drug (Figure 4, E and F). Our
interpretation of these data is as follows. Firstly, that the binding of colchicine was indeed
pseudoꢀirreversible whereas CA4 bound reversibly, and secondly that its action was slower
than that of CA4. It is also interesting to note that this slower action was not due to a
decreased rate of diffusion through the cell membrane. If it were the case, removal of the
drug after thirty minutes would prevent microtubule disruption. Furthermore, 5 appeared to
have a similar profile to CA4P with the cells also showing full recovery after 60 minutes
(Figure 4, G and H). Intriguingly, cells treated with 31 did not recover significantly 60
minutes after drug removal (Figure 4, I and J). However, early signs of recovery were
apparent at this time point, because microtubules were beginning to radiate from the
centrosome (I). Full recovery of the cytoskeleton was nevertheless seen 120 minutes after
drug removal (Figure 4, K and L). This slower recovery than CA4P was particularly
interesting because we felt it could be an indicator to either a wider therapeutic window or
toxicity. Either result would add value to this assay in assessing the viability of compounds as
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VDAs in vitro. Such a model has been quite elusive thus far. Therefore, we sought to further
probe the potential of 31 as a VDA.
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Figure 4. Recovery of cytoskeletal structures after drug removal. HUVECs were treated
with a single dose of either 250 nM CA4P (A, B), 1 ꢀM colchicine (C, D, E, F), 250 nM drug
5 (G, H) or 250 nM drug 31 (I, J, K, L). Drug treatments were for 30 min. Cells were then
washed with serum containing medium and allowed to recover for 60 min (A, B, C, D, G, H,
I, J) or 120 min (E, F, K, L). Cells were fixed and stained with an antibody to βꢀtubulin (A,
C, E, G, I, K) and Fꢀactin (B, D, F, H, J, L) and nuclei stained with DAPI.
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Analysis of Rho-GTPase/Rho Kinase Signalling Pathway Activation.
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We focused on the most active compound from the initial screens, 31 and compared its
activity to CA4P in assays linked to mode of action and vascular disruption. Previous studies
had revealed that activation of the RhoAꢀGTPase signal pathway was involved in actin
remodelling and monolayer disruption caused by CA4P.40,41 Activated RhoꢀGTPase causes
contractility and stress fibre formation through polymerization of actin and the
phosphorylation of myosin light chain (MLC). The latter activity is dictated through the
activation of serine/threonine Rho kinases (ROCKs). Thus, we investigated both 31 and
CA4P induced effects on the phosphorylation of MLC (pMLC) in HUVECs. As shown in
Figure 5, both compounds instigated significant increases in pMLC over a range of
concentrations, indicating that both compounds were acting via a similar pathway.
Compound 31 induced significant phosphorylation of MLC at the lower tested concentration
of 50 nM while CA4P was less active at this dose. To further confirm the involvement of
ROCK in the induction of MLC phosphorylation by compound 31, cells were treated with the
specific ROCK inhibitor Yꢀ27632. Similar to what was previously published for CA4P,40,41
the induction of MLC phosphorylation by compound 31 was abrogated by Yꢀ27632,
confirming the involvement of ROCK in this process (Figure 5C).
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Figure 5. Drugꢀinduced induction of phosphorylation of Rho kinase target MLC. HUVECs
were treated with increasing concentrations of CA4P or drug 31 for 15 minutes after which
proteins were extracted and analyzed for phosphorylation of ROCK target MLC (pMLC) by
western blotting using an antibody specific to the phosphorylated form of the protein (A).
Immunoblots were reprobed with an antibody to actin to confirm equal loading. In B, pMLC
band intensities were analyzed by ImageJ and results expressed as foldꢀchange over control
cells treated with vehicle alone. Each column represents the mean of 3ꢀ4 independent cell
culture experiments±SEM. * represents P < 0.05 for the significance of differences between
drug treatment groups and controls (oneꢀway ANOVA followed by a Tukey postꢀtest). In C,
HUVECs were incubated with Rho kinase inhibitor Yꢀ27632 (5 ꢀM) for 5 minutes and then
treated with 500 nM drug 31 for 15 minutes. Proteins were extracted and analyzed for pMLC
as in A.
Endothelial Monolayer Permeability Experiments
Rapid disruption of the endothelial cell monolayer is key to the activity of VDAs.40
Therefore, studies on the effects of compound 31 and CA4P on cell monolayer permeability
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to fluorescent dextran were undertaken. Confluent HUVEC monolayers were treated for 30
minutes before removal of the drug and addition of fluorescent dextran for a further 30
minutes. Since 31 was shown above to be effective at triggering induction of Rho signalling
at a concentration of 50 nM (see Figure 5), both CA4P and compound 31 were tested at this
dose. Both drugs instigated a significant increase in monolayer permeability at this low dose
(Figure 6). Importantly however, 31 led to a significantly larger increase in monolayer
permeability than CA4P at this dose. This correlated with the increased activity of compound
31 over CA4P in stimulating pMLC phosphorylation.
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Figure 6. Drugꢀinduced changes in endothelial monolayer permeability comparing CA4P and
31. Confluent monolayers of cells grown on microporous filter inserts were treated with
vehicle control, CA4P (50 nM, 30 min) or compound 31 (50 nM, 30 min). Drugs were
removed and replaced with FITC–dextran for a further 30 min. The passage of FITCꢀdextran
through the monolayer was quantified and expressed as a percentage of FITC that passed
through a filter without cells. Results are a mean of 3 independent experiments ± SEM. **
represents P < 0.01 and *** represents P < 0.001 for the significance of differences between
groups (oneꢀway ANOVA followed by a Tukey postꢀtest).
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In Vivo Studies
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The maximum tolerated dose (MTD) of 31 in SCID (severe combined immunodeficiency)
mice was found to be greater than 162 mg/kg (0.454 mmol/kg; data not shown). We next
investigated whether 31 showed efficacy in a tumor model. SCID mice were implanted with
5 x 106 SW1222 (human colorectal adenocarcinoma) cells via subcutaneous injection. Tumors
were allowed to reach 8 mm in diameter and then treated with either vehicle (50%
Na2CO3/NaCl), CA4P (100 mg/kg, 0.227 mmol/kg) or a solution of 31 (81 mg/kg, 0.227
mmol/kg). Tumors were excised 24 hours later and necrosis levels calculated in H&E stained
5 µm thick sections by a random point scoring microscopy method using a ‘Chalkley’
eyepiece graticule.42,43 As shown in Figure 7, CA4P treated tumors were significantly more
necrotic than the untreated tumors, with % necrosis very similar for CA4P and 31 treated
tumors. However, the tendency for necrosis to increase with 31 treatment just failed to reach
significance with the animal numbers used (Figure 7). CA4P appeared to exhibit greater
variability in necrosis levels with some tumors showing no response upon treatment.
Maximum response was higher for CA4P, but over one third of the treatment group failed to
respond. In contrast, treatment with 31 resulted in much less variation, with 11% (a single
tumor) failing to respond. It is of particular note that 31 showed signs of efficacy in vivo
without further derivatisation for water solubility and appeared to remain active in the
circulation.
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Figure 7. In vivo study of drugꢀinduced effects on tumor cell necrosis comparing CA4P and
31. Each point represents the combined data from a single tumor. 5 sections (cut at different
tumor depths) were analyzed per tumor using a x20 objective on a Nikon Eclipse TS100
microscope microscope. The total section was analyzed and % necrosis calculated in each
field from the relative number of points in a Chalkley eyepiece graticule coꢀincident with
necrotic versus viable tumor tissue. Bars represent the mean±SEM of the combined data for
each tumor. * represents P < 0.05 for the significance of differences between groups (oneꢀ
way ANOVA followed by a Tukey postꢀtest).
Conclusion
In conclusion, we have successfully utilized highlyꢀregioselective cycloadditions of sydnones
to access twelve direct pyrazoleꢀbased analogs of combretastatin A4. Two of the compounds
display similar efficacy to CA4P in vitro. We have established that both compounds exhibit
reversible binding and that the most active compound activates the RhoAꢀROCK pathway, a
characteristic of a CA4Pꢀlike mode of action. The most active compound also significantly
increases cell monolayer permeability relative to CA4P at doses of 50 nM. Finally, evaluation
of compound efficacy in vivo showed a tendency toward increased necrosis in a similar
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manner to CA4P at an equivalent dose, without the need for the incorporation of water
solubilizing groups.
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Experimental
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1H NMR spectra were recorded on a Bruker AVIII HD 400 (400 MHz), Bruker AVI 400 (400
MHz), or DPXꢀ400 (400 MHz) supported by an Aspect 3000 data system. Chemical shifts are
reported in parts per million (ppm) from tetramethylsilane with the residual protic solvent
resonance as the internal standard (CHCl3: δ 7.26, DMSO: δ 2.50). 13C NMR spectra were
recorded on a Bruker AVIII HD 400 (100.6 MHz), Bruker AVI 400 (100.6 MHz), or DPXꢀ
400 (100.6 MHz) with complete proton decoupling. Chemical shifts are reported in ppm from
tetramethylsilane with the solvent as the internal reference (CDCl3: δ 77.16, DMSO:
δ 39.52). Highꢀresolution mass spectra (HRMS), recorded for accurate mass analysis, were
performed on electrospray mode (TOF ES+). Infrared (IR) spectra were recorded on a Perkin
Elmer Paragon 100 FTIR spectrophotometer, νmax in cmꢀ1. Samples were recorded as solids
using a solid probe. Bands were characterised as broad (br), strong (s), medium (m), and
weak (w). All solvents and reagents obtained from Acros, Fisher, Sigma, Alfa or Fluorochem
and used without purification unless specified. Flash chromatography was performed on
silica gel (BDH Silica Gel 60 43ꢀ60). Thin layer chromatography (TLC) was performed on
aluminium backed plates preꢀcoated with silica (0.2 mm, Merck DCꢀalufolien Kieselgel 60
F254) which were developed using ultraviolet (UV). HPLC chromatograms were obtained on
a Perkin Elmer Series 200 HPLC system using a Waters XBridge C18 250 x 4.6 mm column
and a gradient of 5ꢀ95% MeCN/H2O over 20 min. All compounds submitted for biological
testing were recrystallized to consistent melting point and judged as >95% pure by HPLC and
1H and 13C NMR spectroscopy.
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NꢀMethylꢀ, Nꢀbenzylꢀ and Nꢀ(3,4,5ꢀtrimethoxyphenyl)sydnone (1)38 were prepared by
standard methods (cyclodehydration of Nꢀnitroso amino acid with trifluoroacetic anhydride)
similarly to Nꢀ(3ꢀhydroxyꢀ4ꢀmethoxyphenyl)sydnone (see below).
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General Procedure A: Direct Arylation of Sydnones. A mixture of sydnone (1 eq.), aryl
halide (1.5 eq.), palladium acetate (5 mol %), XPhos (10 mol %) and potassium carbonate (2ꢀ
3 eq.) in DMF (0.1 – 0.5 M) under an atmosphere of nitrogen was heated at 80 ꢀ 120 °C for
14 hours before the reaction was allowed to cool to ambient temperature and water was
added. The resulting mixture was extracted with ethyl acetate:40ꢀ60 petroleum ether (9:1)
and the combined organic layers dried over MgSO4 and concentrated in vacuo. Flash silica
chromatography (eluting solvent 20%ꢀ100% ethyl acetate in 40ꢀ60 petroleum ether) afforded
the target 3,4ꢀdisubstituted sydnones. The compounds could be further purified by
recrystallization from ethanol or dichloromethane/petrol.
General Procedure B: Cycloaddition of Sydnones with Terminal Alkynes.
A Schlenk tube was charged with sydnone (1 eq.), alkyne (2ꢀ4 eq.) and xylenes (1 M). The
ο
tube was then sealed and heated at 160 C for 24 hours. The mixture was allowed to cool to
ambient temperature and loaded onto a short plug of silica and washed with 40ꢀ60 petroleum
ether before elution with ethyl acetate. Volatiles were removed in vacuo and the crude
residue purified by flash silica chromatography (gradient starting with 100% 40ꢀ60 petroleum
ether and ending with 40% ethyl acetate in 40ꢀ60 petroleum ether) affording the target
trisubstituted pyrazoles as mixtures of regioisomers. Only characterization for the major
regioisomer is reported.
General Procedure C: Preparation of Pyrazole Boronic Esters. A Schlenk tube was
charged with sydnone (1 eq.), alkyne (2 eq.) and xylenes (1 M). The tube was then sealed and
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heated at 180 C for 48 hours. The mixture was allowed to cool to ambient temperature and
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loaded onto a short plug of silica and washed with 40ꢀ60 petroleum ether before elution with
ethyl acetate. Volatiles were removed in vacuo and the crude residue purified by flash silica
chromatography (gradient starting with 100% 40ꢀ60 petroleum ether and ending with 40%
ethyl acetate in 40ꢀ60 petroleum ether) affording the target pyrazole boronic esters as an
inseparable mixture of the target material and the product arising from protodeboronation.
Only characterization for the target material is reported. 13C NMR signals for CꢀB were not
observed due to T2 broadening.
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General Procedure D: Suzuki-Miyaura Coupling of Pyrazole Boronic Esters. A flask
equipped with a reflux condenser was charged with pyrazole boronic ester (1 eq.),
XPhosPdG2 (0.1 eq.), sodium carbonate (2 eq.) and degassed 1,2ꢀdimethoxyethane:water
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(1:1, 0.1 M) and heated at 80 C under an inert atmosphere of nitrogen for 14 hours. The
mixture was allowed to cool to ambient temperature, poured into water and extracted with
ethyl acetate. The combined organic layers were dried over MgSO4 and concentrated in
vacuo. Flash silica chromatography (gradient starting with 100% 40ꢀ60 petroleum ether and
ending with 40% ethyl acetate in 40ꢀ60 petroleum ether) afforded the target crossꢀcoupled
pyrazoles as an inseparable mixture of the target material and the product arising from
protodeboronation. Only characterization for the target material is reported.
General Procedure E: Trimethylsilyl Group Cleavage. A flask equipped with a reflux
condenser was charged with pyrazole (1 eq.), and TBAF (10 eq. 1 M solution in THF) and
heated at reflux under an inert atmosphere of nitrogen for 24 hours. The mixture was allowed
to cool to ambient temperature, poured into water, neutralized with NaHCO3 and extracted
with ethyl acetate. The combined organic layers were dried over MgSO4 and concentrated in
vacuo. Flash silica chromatography (gradient starting with 100% 40ꢀ60 petroleum ether and
ending with 60% ethyl acetate in 40ꢀ60 petroleum ether) afforded the target desilated
pyrazoles.
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General Procedure F: Bromination and Subsequent Suzuki-Miyaura Coupling of
Pyrazoles. i) To a flask containing pyrazole was added a solution of Nꢀbromosuccinamide (1
eq.) in acetonitrile (0.2 M). The resulting mixture was stirred for one hour at ambient
temperature, before being poured into water. The mixture was extracted with ethyl acetate
and the combined organic layers washed with aqueous sodium hydroxide (1 M). The organic
layer was dried over MgSO4, filtered and volatiles removed in vacuo. The crude material was
concentrated into a flask. The flask was then charged with aryl boronate (1.5 eq.),
XPhosPdG2 (10 mol %), sodium carbonate (2ꢀ3.5 eq.) and thoroughly degassed 1,2ꢀ
dimethoxyethane:water (1:1, 0.1 M). The mixture was heated at 80 οC for 14 hours, cooled to
ambient temperature, poured into water and extracted with ethyl acetate. The combined
organic layers were dried over MgSO4, filtered and volatiles removed in vacuo. Flash silica
chromatography (gradient starting with 100% 40ꢀ60 petroleum ether and ending with 100%
ethyl acetate in 40ꢀ60 petroleum ether) afforded the target pyrazoles. ii) In cases where the
products obtained were the TBSꢀprotected phenols, the crude Suzuki product was subjected
to potassium carbonate (3 eq.) in refluxing methanol for 3 hours, before the mixture was
poured into water and extracted with ethyl acetate. The organic layers were dried over
MgSO4, filtered and volatiles removed in vacuo, followed by purification.
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General Procedure G: Benzyl Deprotection. A flask was charged with pyrazole and Pd/C
(10% wt./wt., 100 mg/mmol) and methanol (0.1 M). The flask was flushed with H2 (balloon)
and stirred for 24ꢀ72 hours. The mixture was filtered through celiteTM and concentrated in
vacuo. Flash silica chromatography (gradient starting with 100% 40ꢀ60 petroleum ether and
ending with 100% ethyl acetate in 40ꢀ60 petroleum ether) afforded the target debenzylated
pyrazoles.
4-(3-Phenylmethoxy-4-methoxyphenyl)-N-(3,4,5-trimethoxyphenyl)sydnone (2). Nꢀ3,4,5ꢀ
Trimethoxyphenylsydnone (500 mg, 1.98 mmol) and 1ꢀbromoꢀ3ꢀphenylmethoxyꢀ4ꢀ
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methoxybenzene (872 mg, 2.97 mmol) were subjected general procedure A, affording 2 as a
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colourless solid (542 mg, 59%). M.p.: 144ꢀ146 °C; H NMR (400 MHz, CDCl3) δ 3.78 (6H,
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s), 3.87 (3H, s), 3.92 (3H, s), 4.97 (2H, s), 6.64 (2H, s), 6.81 (1H, d, J = 8.5 Hz), 6.92 (1H,
dd, J = 8.5, 2.0 Hz), 6.98 (1H, d, J = 2.0 Hz), 7.27ꢀ7.35 (5H, m); 13C NMR (101 MHz,
CDCl3) δ 56.1, 56.7, 61.3, 71.0, 102.6, 108.0, 111.8, 112.6, 117.0, 120.9, 127.8, 128.1, 128.7,
130.0, 136.6, 140.7, 148.2, 150.2, 154.2, 167.1; FTIR: νmax 2939 (w), 1738 (m), 1602 (m),
1260 (s), 1234 (s), 1132 (s), 1016 (m), 993 (m); HRMS (ESIꢀTOF) m/z [M+H]+ calculated for
C25H25N2O7: 465.1656. Found: 465.1654.
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1-(3,4,5-Trimethoxyphenyl)-3-(trimethylsilyl)-5-(3-phenylmethoxy-4-methoxyphenyl)-
pyrazole
(3a,
major)
and
1-(3,4,5-trimethoxyphenyl)-4-(trimethylsilyl)-5-(3-
phenylmethoxy-4-methoxyphenyl)-pyrazole (3b, minor). Sydnone 2 (136 mg, 0.293
mmol) and trimethylsilylacetylene (115 mg, 1.17 mmol) were subjected to general procedure
B, affording an inseparable mixture of pyrazoles 3a and 3b as an orange oil (113 mg, 75%,
95:5). 1H NMR (400 MHz, CDCl3) δ 0.36 (9H, s), 3.69 (6H, s), 3.82 (3H, s), 3.87 (3H, s),
4.95 (2H, s), 6.51 (1H, s), 6.52 (2H, s), 6.76 (1H, d, J = 1.5 Hz), 6.81ꢀ6.88 (2H, m), 7.24ꢀ
7.35 (5H, m); 13C NMR (101 MHz, CDCl3) δ ꢀ0.9, 56.0, 56.1, 61.0, 71.1, 103.3, 111.5, 113.4,
114.6, 122.0, 123.3, 127.0, 127.9, 128.6, 136.1, 136.8, 137.3, 142.9, 147.8, 149.5, 153.2,
153.7; FTIR: νmax 2954 (w), 2836 (w), 1597 (m), 1505 (s), 1229 (s), 1125 (s), 1023 (m), 1005
(m), 979 (m); HRMS (ESIꢀTOF) m/z [M+H]+ calculated for C29H35N2O5Si: 519.2310. Found:
519.2291.
1-(3,4,5-Trimethoxyphenyl)-5-(3-phenylmethoxy-4-methoxyphenyl)-pyrazole
(4).
Pyrazoles 3a and 3b (86 mg, 0.166 mmol, 95:5) and TBAF in THF (1.7 mL, 1.7 mmol) were
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subjected to general procedure E, affording pyrazole 4 as a yellow oil (50 mg, 67%). H
NMR (400 MHz, CDCl3) δ 3.67 (6H, s), 3.84 (3H, s), 3.88 (3H, s), 4.97 (2H, s), 6.39 (1H, d,
J = 2.0 Hz), 6.49 (2H, s), 6.77 (1H, s), 6.83ꢀ6.85 (2H, m), 7.26ꢀ7.34 (5H, m), 7.66 (1H, d, J =
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2.0 Hz); 13C NMR (101 MHz, CDCl3) δ 56.1, 56.2, 61.1, 71.3, 102.9, 107.3, 111.6, 114.8,
122.2, 123.1, 127.1, 128.1, 128.7, 135.9, 136.8, 137.3, 140.1, 142.9, 148.0, 149.9, 153.2;
FTIR: νmax 2936 (w), 2836 (w), 1598 (m), 1508 (s), 1454 (m), 1415 (m), 1230 (s), 1118 (s),
1019 (m), 1002 (m); HRMS (ESIꢀTOF) m/z [M+H]+ calculated for C26H27BN2O5: 447.1914.
Found: 447.1925.
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1-(3,4,5-Trimethoxyphenyl)-5-(3-hydroxy-4-methoxyphenyl)-pyrazole (5). Pyrazole 4
(139 mg, 0.311 mmol) and Pd/C (31 mg) were subjected to general procedure G, affording
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pyrazole 5 as a colourless solid (75 mg, 68%). M.p.: 102ꢀ103 °C; H NMR (400 MHz,
CDCl3) δ 3.68 (6H, s), 3.83 (3H, s), 3.86 (3H, s), 5.99 (1H, br), 6.42 (1H, d, J = 2.0 Hz), 6.53
(2H, s), 6.69 (1H, dd, J = 8.5, 2.0 Hz), 6.76 (1H, d, J = 8.5, Hz), 6.87 (1H, d, J = 2.0, Hz),
7.65 (1H, d, J = 2.0 Hz); 13C NMR (101 MHz, CDCl3) δ 56.0, 56.2, 61.1, 102.9, 107.9, 110.6,
115.2, 121.0, 123.8, 135.9, 137.2, 140.1, 142.9, 145.6, 146.8, 153.1; FTIR: νmax 3133 (br),
2933 (w), 2840 (w), 1601 (m), 1499 (s), 1458 (m), 1420 (m), 1272 (m), 1232 (s), 1118 (s),
1010 (m), 1002 (m), 933 (m); HRMS (ESIꢀTOF) m/z [M+H]+ calculated for C19H21N2O5:
357.1445. Found: 357.1446; HPLC: Ret. Time: 13.65 min, Purity 96.3%.
N
-(3-Hydroxy-4-methoxyphenyl)sydnone (6). 2ꢀ((3ꢀHydroxyꢀ4ꢀmethoxyphenyl)amino)
acetic acid (0.97 g, 4.9 mmol) was dissolved in 1,2ꢀdimethoxethane (20 mL) and
isoamylnitrite (0.58 g, 4.9 mmol) was added and the reaction stirred for 3 hours at room
ο
temperature. Volatiles were removed in vacuo at <30 C and the crude material triturated
with petroleum ether:diethyl ether (20:1). Caution nitrosamines are highly toxic and
carcinogenic. The crude material was suspended in dichloromethane (20 mL) and cooled at 0
οC. Trifluoracetic anhydride (1.56 g, 7.41 mmol) was added and the reaction stirred for 1.5
hours at r.t. A small amount of water was added and the mixture basified with sodium
bicarbonate and extracted with dichloromethane. The combined organic layers were dried
over MgSO4 and volatiles removed in vacuo. 6 was isolated as a brown solid (0.29 g, 28%).
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The product could be further purified by recrystallization from ethanol. M.p.: 205ꢀ209 °C
(dec.); 1H NMR (400 MHz, DMSOꢀd6): δ 3.87 (3H, s), 7.18 (1H, d, J = 9.0 Hz), 7.30 (1H, d,
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J = 2.5 Hz), 7.36 (1H, dd, J = 2.5, 9.0 Hz), 7.66 (1H, s), 9.99 (1H, s). C NMR (101 MHz,
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DMSOꢀd6): δ 56.0, 94.3, 108.1, 112.2, 112.5, 127.4, 147.4, 150.8, 168.5; FTIR: νmax 3248
(br), 2924 (w), 1755 (s), 1720 (s), 1517 (s), 1285 (s), 1031 (m); HRMS calculated for
C9H9N2O4 (ES+)(+H+): 209.0562. Found: 209.0553.
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4-(3,4,5-Trimethoxyphenyl)-
methoxyphenyl)sydnone (473 mg, 2.27 mmol) and 5ꢀbromoꢀ1,2,3ꢀtrimethoxybenzene (842
mg, 3.41 mmol) were subjected to general procedure affording, 4ꢀ(3,4,5ꢀ
N-(3-hydroxy-4-methoxyphenyl)sydnone. Nꢀ(3ꢀHydroxyꢀ4ꢀ
A
trimethoxyphenyl)ꢀNꢀ(3ꢀhydroxyꢀ4ꢀmethoxyphenyl)sydnone as a colourless solid (402 mg,
47%). M.p.: 196ꢀ197 °C; 1H NMR (400 MHz, CDCl3) δ 3.67 (6H, s), 3.83 (3H, s), 4.00 (3H,
s), 5.94 (1H, s), 6.59 (2H, s), 6.92ꢀ7.05 (2H, m), 7.09 (1H, s); 13C NMR (101 MHz, CDCl3) δ
56.1, 56.5, 61.0, 104.7, 108.0, 111.0, 111.7, 117.2, 119.9, 127.8, 138.5, 147.0, 149.5, 153.3,
167.2; FTIR: νmax 3295 (br), 3082 (w), 2940 (w), 2836 (w), 1710 (s), 1600 (w), 1581 (m),
1509 (s), 1229 (s), 1125 (s) 1014 (m), 998 (m); HRMS calculated for C18H19N2O7
(ES+)(+H+): 375.1192. Found: 375.1181.
4-(3,4,5-Trimethoxyphenyl)-
N
-(3-phenylmethoxy-4-methoxyphenyl)sydnone
(7).
A
mixture of 4ꢀ(3,4,5ꢀtrimethoxyphenyl)ꢀNꢀ(3ꢀhydroxyꢀ4ꢀmethoxyphenyl)sydnone (402 mg,
1.07 mmol),benzyl bromide (368 mg, 2.15 mmol) and Hünigs base (278 mg, 2.15 mmol)
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were heated at 130 C for 2 hours. The reaction was allowed to cool to ambient temperature
and water was added. The mixture was extracted with dichloromethane and the combined
organic layers washed with saturated ammonium chloride and brine, before drying over
MgSO4 and concentrating in vacuo. Flash silica chromatography (gradient starting with 100%
40ꢀ60 petroleum ether and ending with 40% ethyl acetate in 40ꢀ60 petroleum ether) afforded
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sydnone 7 as a yellow solid (296 mg, 60%). M.p.: 58ꢀ60 °C; H NMR (400 MHz, CDCl3) δ
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3.62 (6H, s), 3.83 (3H, s), 3.96 (3H, s), 5.09 (2H, s), 6.51 (2H, s), 6.98ꢀ7.03 (2H, m), 7.09
(1H, dd, J = 8.5, 2.5 Hz), 7.29ꢀ7.36 (5H, m); 13C NMR (101 MHz, CDCl3) δ 56.1, 56.6, 61.1,
71.6, 104.5, 107.8, 110.6, 111.7, 118.3, 119.9, 127.3 (x2 C), 128.6, 128.9, 135.5, 138.5,
149.1, 152.5, 153.4, 167.1; FTIR: νmax 2934 (w), 2836 (w), 1728 (s), 1581 (m), 1512 (s),
1226 (s), 1123 (s), 999 (s); HRMS (ESIꢀTOF) m/z [M+H]+ calculated for C25H25N2O7:
465.1656. Found: 465.1668.
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1-(3-Phenylmethoxy-4-methoxyphenyl)-3-(trimethylsilyl)-5-(3,4,5-trimethoxyphenyl)-
pyrazole (8a, major) and 1-(3-phenylmethoxy-4-methoxyphenyl)-4-(trimethylsilyl)-5-
(3,4,5-trimethoxyphenyl)-pyrazole (8b, minor). Sydnone 7 (290 mg, 0.624 mmol) and
trimethylsilylacetylene (245 mg, 2.50 mmol) were subjected to general procedure B,
affording an inseparable mixture of pyrazoles 8a and 8b as an orange oil (256 mg, 79%,
95:5). 1H NMR (400 MHz, CDCl3) δ 0.36 (9H, s), 3.65 (6H, s), 3.84 (3H, s), 3.87 (3H, s),
5.02 (2H, s), 6.38 (2H, s), 6.56 (1H, s), 6.83 (1H, d, J = 8.5 Hz), 6.88 (1H, dd, J = 8.5, 2.5
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Hz), 6.95 (1H, d, J = 2.5 Hz), 7.27ꢀ7.34 (5H, m); C NMR (101 MHz, CDCl3) δ ꢀ0.9, 56.1,
56.4, 61.0, 71.3, 106.0, 111.5, 112.0, 113.3, 118.6, 126.3, 127.3, 128.1, 128.6, 133.6, 136.6,
137.8, 143.0, 148.3, 149.2, 153.1, 153.6; FTIR: νmax 2951 (w), 2834 (w), 1584 (m), 1514 (s),
1236 (s), 1126 (s), 1005 (m), 978 (m); HRMS (ESIꢀTOF) m/z [M+H]+ calculated for
C29H35N2O5Si: 519.2310. Found: 519.2314.
1-(3-Phenylmethoxy-4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-pyrazole
(9).
Pyrazoles 8a and 8b (256 mg, 0.494 mmol, 95:5) and TBAF in THF (5 mL, 5 mmol) were
subjected to general procedure E, affording pyrazole 9 as a yellow oil (137 mg, 62%).
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Product was contaminated with ~10% unknown byꢀproduct. H NMR (400 MHz, CDCl3) δ
3.64 (6H, s), 3.83 (3H, s), 3.86 (3H, s), 5.01 (2H, s), 6.38 (2H, s), 6.45 (1H, d, J = 2.0 Hz),
6.81ꢀ6.84 (2H, m), 6.93ꢀ6.97 (1H, m), 7.25ꢀ7.36 (5H, m), 7.65 (1H, d, J = 2.0 Hz); 13C NMR
(101 MHz, CDCl3) δ 56.0, 56.3, 61.0, 71.2, 106.0, 107.0, 111.3, 111.6, 118.3, 125.9, 127.2,
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127.3, 128.0, 128.6, 133.3, 136.5, 139.9, 142.9, 148.2, 149.1, 153.0; FTIR: νmax 2934 (w),
2836 (w), 1584 (m), 1512 (s), 1414 (m), 1236 (s), 1121 (s), 1002 (m), 910 (m); HRMS (ESIꢀ
TOF) m/z [M+H]+ calculated for C26H27BN2O5: 447.1914. Found: 447.1914.
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1-(3-Hydroxy-4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-pyrazole (10). Pyrazole 9
(137 mg, 0.307 mmol) and Pd/C (31 mg) for 24 hours were subjected to general procedure G,
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affording pyrazole 10 as a colourless solid (76 mg, 69%). M.p.: 55ꢀ58 °C; H NMR (400
MHz, CDCl3) δ 3.68 (6H, s), 3.85 (3H, s), 3.88 (3H, s), 5.98 (1H, br), 6.43 (2H, s), 6.46 (1H,
d, J = 2.0 Hz), 6.75 (1H, dd, J = 8.5, 2.5 Hz), 6.78 (1H, d, J = 8.5, Hz), 6.97 (1H, d, J = 2.5,
Hz), 7.66 (1H, d, J = 2.0 Hz); 13C NMR (101 MHz, CDCl3) δ 56.1, 56.3, 61.1, 106.1, 107.0,
110.4, 112.7, 117.5, 126.0, 133.9, 138.0, 139.9, 143.1, 145.9, 146.3, 153.1; FTIR: νmax 3146
(br), 2941 (w), 2836 (w), 1585 (m), 1511 (s), 1498 (s), 1416 (m), 1239 (s), 1120 (s), 1000
(m); HRMS (ESIꢀTOF) m/z [M+H]+ calculated for C19H21N2O5: 357.1445. Found: 357.1444;
HPLC: Ret. Time: 13.54 min, Purity 95.2%.
4-(3,4,5-Trimethoxyphenyl)-N-methylsydnone (11). NꢀMethylsydnone (250 mg, 2.50
mmol) and 5ꢀbromoꢀ1,2,3ꢀtrimethoxybenzene (926 mg, 3.75 mmol) were subjected to general
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procedure A, affording 11 as a colourless solid (571 mg, 86%). M.p.: 123ꢀ124 °C; H NMR
(400 MHz, CDCl3) δ 3.86 (3H, s), 3.87 (6H, s), 4.12 (3H, s), 6.74 (2H, s); 13C NMR (101
MHz, CDCl3) δ 39.0, 56.5, 61.1, 105.7, 108.2, 119.6, 139.2, 153.9, 167.3; FTIR: νmax 2932
(w), 2858 (w), 1726 (s), 1585 (m), 1241 (m), 1225 (m), 1124 (s), 998 (m); HRMS (ESIꢀTOF)
m/z [M+H]+ calculated for C12H15N2O5: 267.0981. Found: 267.0970.
4-(3,4,5-Trimethoxyphenyl)-N-benzylsydnone (12). NꢀBenzylsydnone (500 mg, 2.84
mmol) and 5ꢀbromoꢀ1,2,3ꢀtrimethoxybenzene (1.05 g, 4.26 mmol) were subjected to general
procedure A, affording 12 as a colourless solid (570 mg, 59%). M.p.: 101 °C; 1H NMR (400
MHz, CDCl3) δ 3.69 (6H, s), 3.84 (3H, s), 5.52 (2H, s), 6.52 (2H, s), 7.18ꢀ7.24 (2H, m), 7.37ꢀ
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