Design, synthesis, and biological evaluation of stable colchicine binding site tubulin inhibitors as potential anticancer agents

Article abstract of DOI:10.1021/jm500764v

To block the metabolically labile sites of novel tubulin inhibitors targeting the colchicine binding site based on SMART, ABI, and PAT templates, we have designed, synthesized, and biologically tested three focused sets of new derivatives with modifications at the carbonyl linker, the para-position in the C ring of SMART template, and modification of A ring of the PAT template. Structure-activity relationships of these compounds led to the identification of new benzimidazole and imidazo[4,5-c]pyridine-fused ring templates, represented by compounds 4 and 7, respectively, which showed enhanced antitumor activity and substantially improved the metabolic stability in liver microsomes compared to SMART. MOM group replaced TMP C ring and generated a potent analogue 15, which showed comparable potency to the parent SMART compound. Further modification of PAT template yielded another potent analogue 33 with 5-indolyl substituent at A ring.

Full text of DOI:10.1021/jm500764v

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Article
Design, Synthesis, and Biological Evaluation of Stable Colchicine
Binding Site Tubulin Inhibitors as Potential Anticancer Agents
Yan Lu, Jianjun Chen, Jin Wang, Chien-Ming Li, Sunjoo Ahn,
Christina M Barrett, James T Dalton, Wei Li, and Duane D Miller
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500764v • Publication Date (Web): 14 Aug 2014
Downloaded from http://pubs.acs.org on August 17, 2014
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Design, Synthesis, and Biological Evaluation of
Stable Colchicine Binding Site Tubulin Inhibitors as
Potential Anticancer Agents
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Yan Lu,† Jianjun Chen,† Jin Wang,† Chien-Ming Li,‡ Sunjoo Ahn,‡ Christina M. Barrett,‡
James T. Dalton,‡ Wei Li*,†, Duane D. Miller*,†
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Department of Pharmaceutical Sciences, University of Tennessee, Health Science Center,
Memphis, TN 38163
‡ GTx Inc, Preclinical R&D, Memphis, TN, 38163
ABSTRACT: To block the metabolically labile sites of novel tubulin inhibitors targeting the
colchicine binding site based on SMART, ABI, and PAT templates, we have designed,
synthesized, and biologically tested three focused sets of new derivatives with modifications at
the carbonyl linker, the paraꢀposition in the C ring of SMART template, and modification of A
ring of the PAT template. Structure–activity relationships of these compounds led to the
identification of new benzimidazole and imidazo[4,5ꢀc]pyridine ꢀfused ring templates,
represented by compounds 4 and 7, respectively, which showed enhanced antitumor activity and
substantially improved the metabolic stability in liver microsomes compared to SMART. MOM
group replaced TMP C ring generated a potent analogue 15, which showed comparable potency
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to the parent SMART compound. Further modification of PAT template yielded another potent
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analogue 33 with 5ꢀindolyl substituent at A ring.
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KEYWORDS: Tubulin polymerization inhibitor; Melanoma; Prostate cancer; Antiproliferative
activity; Structureꢀactivity relationships; Multidrug resistance; colchicine binding site, liver
microsome stability.
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Introduction
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Tubulin/microtubuleꢀinteracting drugs are used successfully for treatment of a wide variety of
human cancers. They are commonly classified into two major categories: microtubuleꢀstabilizing
(e.g., taxanes, epothilones) and microtubuleꢀdestabilizing drugs (e.g., vinca alkaloids,
colchicine). Three major binding sites on α,βꢀtubulin subunits have been identified as taxanesꢀ,
vinca alkaloidꢀ and colchicineꢀbinding sites.1 While antimitotic agents interacting with the
taxanesꢀ or vinca alkaloidꢀbinding sites in tubulin are tremendously successful in clinical
oncology, there are no Food and Drug Administration (FDA)–approved colchicineꢀbinding site
drugs currently available for cancer treatment. Most of the colchicineꢀbinding agents have high
potency, relatively simple chemical structures for optimization, selective toxicity towards tumor
vasculature, and show promising ability to overcome Pꢀglycoprotein (Pꢀgp) efflux pump
mediated multidrug resistance.2 Therefore, the colchicineꢀbinding site compounds have attracted
great interest from medicinal chemists in recent years.
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Several outstanding agents for such an approach are listed in Figure 1. Combretastatin Aꢀ4
(CAꢀ4) is the most active member of the combretastatin family, isolated from the African tree
Combretum caffrum. CAꢀ4 exhibits strong antitubulin activity by binding to the colchicineꢀsite
and underwent Phase II and Phase III studies in clinical.3 The replacement of the olefinic bridge
of CAꢀ4 with a carbonyl group yields phenstatin,4 which has similar potency and mechanism of
actions with CAꢀ4. BPR0L0755 and Oxiꢀ61966 are 2ꢀaroylindole and dihydronaphthalene
analogues of CAꢀ4, which show strong inhibition on tubulin polymerization. Methylated
chalcone SD400, which has an IC50 value of 0.21 nM against K562 human leukemia cells, is a
potent tubulin inhibitor.7 Podophyllotoxin is a nonꢀalkaloid toxin lignin, and it also possesses an
anticancer property that can be attributed to the inhibition of tubulin polymerization through
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binding to the colchicine binding site.8 All these agents shared a common 3,4,5ꢀ
trimethoxyphenyl (TMP) moiety in their chemical structures. This TMP moiety, common to the
above mentioned antiꢀtubulin agents, has been shown to be crucial for inhibiting the growth of
tumor cells.9
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Our research group discovered 4ꢀsubstituted methoxybenzoylꢀarylꢀthiazole10 (SMART, Figure
2), 2ꢀarylꢀ4ꢀbenzoylꢀimidazole (ABI)11, and phenyl amino thiazole (PAT)12 templates as
potential antiꢀcancer agents targeting tubulin by binding to the colchicine binding site. These
agents show low nanomolar inhibition on various cancer cell lines and can effectively overcome
a number of clinically relevant multidrug resistant mechanisms that are often associated with the
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use of existing tubulin inhibitors. Considering the structure similarity, the TMP ring plays an
important role in these templates to enhance cytotoxic activity, whereas pharmacokinetic (PK)
studies showed that these compounds have low bioavailability mainly due to two major
metabolic processes in human liver microsomes: the carbonyl reduction and demethylation in the
TMP ring.14 As a result, the halfꢀlife of the SMART compound 10 is only 17 min.15 These data
highlight the need for modifications of carbonyl linker and TMP ring that could reduce metabolic
liability at these sites and potentially increase the bioavailability of these agents. Previously we
have replaced the carbonyl with a variety of linkers (sulfonyl, sulfinyl, hydrazide, etc), but those
modifications had limited success in overcoming the metabolic stability problem while
maintaining the high potency.12 In this article, we presented our latest approach of cyclizing the
carbonyl with the B ring (Figure 2), which yielded a new “D” ring on the top of “B” ring and
thus blocked the metabolic reduction of the ketone linker to a secondary alcohol. In a separate
approach, we focused on the modification of the TMP “C” ring of SMART to specifically block
the known sites of demethylation metabolism while maintaining or improving the in vitro
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antiproliferative activity profile. Based on these initial studies, we made a series of modifications
focused in TMP C ring at the paraꢀposition and produced derivatives with comparable or
increased activity. Several strategies including the incorporation of an alkylating group, a
hydrogen bond donor, or a hydrophobic group were examined. Finally, we further modified the
PAT template and obtained another highly active analogue bearing a 5ꢀindolyl moiety at “A”
ring position.
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Chemistry Modification
We focused our efforts in preparing three series of modifications. The first two series of
compounds were designed based on overcoming two major metabolismꢀrelated liabilities: ketone
reduction and demethylation in the C ring. As an alternative approach to replacing the carbonyl
with other function groups, 12 we designed new ringꢀbioisosteres of the ketone carbonyl (Figure
2). Five analogues in this new series were synthesized as shown in Scheme 1 and their activities
were evaluated against both prostate cancer and melanoma cell lines. The synthesis approach
included the aldehydeꢀamine condensation, in which the intermediate imidazolidine was
oxidized to the imidazoline (1), and this was followed by the Suzuki coupling of (3,4,5ꢀ
trimethoxyphenyl)boronic acid using Pd(PPh3)4 as a catalyst to give 2.
The second aim was focused on modification of the para-position at the C ring. One purpose
of this approach is to bypass the potential metabolic problems caused by demethylation.
Preliminary modifications of the TMP ring were not successful in our initial studies since the
potency was totally lost when single substituted methoxyphenyl (at o-, m-, p-positions,
respectively) or 3,4ꢀdimethoxy substituted phenyl replaced the TMP moiety in the SMART
template. Interestingly, 3,5ꢀdimethoxy substituted phenyl maintained a certain level of activity in
the 200ꢀ400 nM range.10 It indicated that the paraꢀOMe of TMP might be a potential location for
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further chemical modification. Another important reason for modification at C ring is based on
the hypothesis that introducing different functional groups at the para-position of the TMP ring
will likely form stronger interactions with Cysꢀ241 in the βꢀtubulin subunit (Figure 3), and thus
increasing the potency of inhibition of tubulin polymerization. Furthermore, the modification of
the C ring can help us better understand the potential metabolic demethylation mechanism. We
introduced both hydrophobic (OBn 14, OMOM 15) and hydrophilic (OCH2CH2NH2, 18) groups
as shown in Scheme 2. Another strategy is coming from the hypothesis that if an alkylating
group was introduced at the paraꢀposition of the TMP ring, it may form an irreversible covalent
bond with the mercapto group of Cysꢀ241 in the colchicine binding domain and induce
irreversible mitotic blocks. A wellꢀdescribed mechanism for inhibiting microtubule assembly is
small molecule binding to tubulin via a covalent interaction with a tubulin amino residue. Bai et
al.16 reported that 2ꢀ and 3ꢀchloroacetyl analogues of dimethylthiocolchicine bound irreversibly
to the colchicine binding site primarily with Cysꢀ241 and prevented colchicine binding agents
from binding to the same site. The covalent interaction of 2,4ꢀdichlorobenzyl thiocyanate
(Figure 3) with tubulin occurs at multiple cysteine residues, especially Cysꢀ241of βꢀtubulin.17
Formation of the covalent bond between tubulin and the 2, 4ꢀdichlorobenzyl mercaptan moiety
appeared to be reversible. 2ꢀFluoroꢀ1ꢀmethoxyꢀ4ꢀ(pentafluorophenylꢀsulfonamido)benzene
(T138067, Figure 3) irreversibly bound βꢀtubulin by the thiol group of Cysꢀ241 displacing the
para-F atom. It recruits unmodified tubulin dimers into large, amorphous aggregates, and thus
quickly depletes the pool of tubulin available for microtubule formation.18 Based on the above
reports, we proposed to modify the template of our tubulin inhibitors by introducing an
alkylating function group to form a covalent bond or enhance the interaction between Cysꢀ241
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and TMP ring. Thus, chloroacetic analogue (12) and trifluoroacetate (13) in Scheme 2 were also
synthesized and tested.
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The last aim of modification is based on the “A” ring of PAT template, which we discovered
from SMART agents by inserting an amino linker between the “B” and “C” rings. This PAT
template increased the oral bioavailability from 3% (SMART series) to 21%.12 Based on our
extensive studies on the SMART template, we selected 5ꢀindolyl to be introduced into the
bottom A ring and synthesized 33 (Scheme 3). Meanwhile, we replaced the thiazole B ring with
imidazole (27) to compare with the parental ABI compound as illustrated in Scheme 3. Nꢀ
Phenylꢀ1Hꢀimidazolꢀ2ꢀamine (21) was prepared from aminoꢀacetaldehyde diethyl acetal after
three steps. The protections of the imidazole ring with PhSO2 or Boc groups followed by 3,4,5ꢀ
trimethoxybenzoyl lithium attacking cannot afford the desired target compound while two byꢀ
products 22 and 23 were obtained. Then we chose triphenylmethyl (i.e., trityl or Tri) as a
protecting group for the imidazole and prepared two products protected at 4ꢀ and Nꢀposition (24
and 25) of imidazole. Then the reaction of 24 with 3,4,5ꢀtrimethoxybenzoyl lithium followed by
deprotection of the trityl generated 27, the imidazole analog of the PAT template.
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Results and Discussion
Blocking Ketone Reduction by Introducing a new “D” Ring. In our previous studies,12, 15
we made attempts to introduce alternatives to the carbonyl linker in order to avoid potential
metabolic problems but those approaches were unsuccessful. The replacements of the carbonyl
linker in the SMART template ncluded double bonds, amides, oximes, hydrazide, acrylonitriles,
cyanoimine, sulfonyl amide, sulfur ether, and sulfonyl/sulfinyl compounds but we obtained only
limited success. The oxime and hydrazide derivatives demonstrated a 2ꢀ to 3ꢀfold improved halfꢀ
life in human liver microsomes, indicating that metabolic stability of SMART can be extended
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by blocking ketone reduction. However, these derivatives had less potent antiproliferative
activities (micromolar range of IC50). In the current approaches, we designed a new template
with the fused D ring (Table 1) on top of the B ring, which maintains the conjugated structure,
mimics the carbonyl group, but could potentially bypass the ketone reduction. From the
proliferative activity data as compared to SMART compound 10, most of the benzoꢀimidazole 3-
6 showed only moderate activity, except 4, which has a 5ꢀindolyl at the A ring position, showed
comparable potency against tested melanoma and prostate cancer cell lines. For further
modification, we retained this 5ꢀindolyl at the A ring, utilized pyridineꢀfused to the imidazole to
replace the benzoꢀimidazole and yielded 7. This compound showed increased potency compared
to both parent SMART compound 10 and 4. The IC50 values improved by at least 5ꢀfold against
melanoma A375 cells and androgen sensitive prostate cancer LNCaP cells. These novel fused
ring templates represented new chemotypes for further optimizing our colchicine binding site
inhibitors, which is also expected to remove the potential phase I metabolic reactions caused by
ketone reduction.
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When 4 and 7 were docked into the colchicine binding site in tubulin (Figure 4, PDB code:
1SA0), they showed very similar binding poses and overlapped with the native ligand reasonably
well. As anticipated, the TMP moiety in 4 or 7 occupied the pocket of the trimethoxy moiety in
the native ligand (DAMAꢀcolchicine), but showed some shifting in its position. This slight shift
positioned the oxygen atoms in two methoxy groups of the TMP close to Cysꢀ241 of the βꢀ
subunit and allowed the formation of two hydrogen bonds (yellow dotted lines). The imidazole
NH moiety in 4 or 7 formed another hydrogen bond to Thrꢀ179 in the αꢀsubunit as shown in
Figure 4. Interestingly, due to the formation of the new Dꢀring which forced a planar structure in
the middle portion of 4 or 7, the 5ꢀindolyl moiety changed orientation to reach toward the GTP in
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the αꢀsubunit. The glide docking scores for compounds 4 (ꢀ8.58) and 7 (ꢀ8.10) were comparable
with that of the native ligand, DAMAꢀcolchicine (ꢀ9.26) based on this modeling calculation,
suggesting they may have comparable effects in tubulin binding.
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C Ring Modification: H-Bonding and Alkylation of the Colchicine Binding Site. The
methoxy groups in the TMP ring were known to interact with Cysꢀ241 residue of DAMAꢀ
colchicine in the coꢀcrystallized tubulin structure (PDB code 1SA0). We hypothesized that a
series of functional groups (R1, R2) attached to the 4ꢀoxygen atom of the “C” ring will bind to
Cysꢀ241 in βꢀtubulin. Thus, we designed and synthesized a template which may interact with
Cysꢀ241 through either hydrogen bonding or by alkylating at Cꢀring paraꢀposition as shown in
Scheme 2.
The synthesized new analogues were tested against both melanoma and prostate cancer cells
for their antiproliferative activity. Compound 15 showed improved activity compared to the
parent SMART compound (i.e., 55 nM (15) vs. 19 nM (SMART 10) against B16ꢀF1 cells;
Table 2). This discovery encouraged the hypothesis that the para position of C ring is a tolerant
location for further modification. The two atoms extension (i.e., ꢀOCH2ꢀ) of 15 was potent.
However, the idea of alkylation at p-position did not work as expected on the inhibition of cancer
cell growth. From the result shown in Table 2, the potency of alkylating agent 12 dropped
significantly against both melanoma and prostate cancer cells. 13 and 14 showed similar trends
on activity as 12. 17, with a phthalimide protection group, showed micromolar range potency.
Introducing an ethyl amine (18) at the pꢀposition remained moderate activity with hundreds of
nanomolar IC50s, but it still was less potent than the parent compound SMART.
Modifications of the PAT Template: The PAT template was obtained by inserting an amino
linker between the “A” ring and the “B” ring of the SMART template. This template maintained
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the potency and improved the oral bioavailability (>30%) compared to SMART (F=3.3%). The
ABI template also showed high potency and improved bioavailability. Thus we designed to
integrate the ABI imidazole ring into the PAT template and obtained 27. However, this new
imidazole “B” ring variant of the PAT compound did not demonstrate activity against any of the
tested cell lines. In contrast, the 5ꢀindolyl 4 and 7, showed excellent potency in the first “D” ring
fused analogues, was also introduced into the PAT template to generate 33 (Figure 5). This
analogue showed excellent growth inhibition for both prostate cancer and melanoma cells in
vitro. The IC50s were increased 2ꢀ3 fold on prostate cancer cells compared to the parent PAT
compound.
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Molecular modeling studies with 33 (Figure 5) showed three hydrogen bonding interactions
between this ligand and the tubulin α,βꢀdimer, similar to those observed between 4 or 7 and
tubulin. However, the 5ꢀindolyl moiety in 33 did not seem to reach the GTP moiety as in 4 or 7,
possibly due to the fact that the ketone moiety was not forced into a ring system as seen in 4 or 7.
Thus, 33 mainly stays within the βꢀsubunit of tubulin dimer, and shows a slightly better glide
docking score (ꢀ8.70).
In vitro Metabolic Stability Studies. To determine whether the metabolism of the labile
carbonyl linker may be reduced by incorporation into a cyclic structure, we measured the
metabolic stability in liver microsomes for two potent compounds (4 and 7). The carbonyl linker
in the SMART compound was susceptible to ketone reduction and was replaced by a new D
ring in these two newly designed compounds. This modification preserved the potency while
improving metabolic stability about 2ꢀ3 fold (17 min vs. 45 and 51 min in human microsomes,
Table 4) compared to the parent SMART compound. Furthermore, the potency of the cyclic D
ring compounds 4 and 7 increased. Another active analogue 15 with an extended methoxymethyl
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(MOM) tail at the paraꢀC ring did not improve its metabolic stability in any of the tested liver
microsomes. Another substituent, the aminoethyl of 18, at the same paraꢀO position blocked the
metabolic liability of the TMP ring (T1/2 ranged from 110 ~ 225 min in the tested liver
microsomes species). This result confirmed our hypothesis that the paraꢀposition of the C ring
could be a modifiable place for improvement of compound stability. However, the selection of
functional groups is very important and it is worth further investigating in future studies.
In vitro Metabolic Pathways of compounds 4, 7, 15, and 18. In order to understand why
these new analogues demonstrated different metabolic patterns in the liver microsomes, we
performed additional experiments using a higher concentration (50 µM) of the tested
compounds. We utilized a high resolution mass spectrometer for the identification of the
metabolites with a mass error of less than 2 ppm generally. The detailed information regarding
the mass spectrum and the chromatogram of each metabolites are presented in the supplementary
materials. For compound 15 (Figure 6), the removal of the MOM group to form M1 is the major
metabolic pathway (Figure 10A), followed by oꢀdemethylation of the 3’ꢀ or 5’ꢀmethly group to
generate M2. This result is consistent with the short halfꢀlife (<10 min) of this compound, as the
MOM group seems to be unstable after exposure to liver microsomes. M3 is also the oꢀ
demethylation product, however, we were unable to pinpoint the exact site for this demethylation
due to limited information available at this stage. M4 is the product resulted from ketone
reduction and it was further hydroxylated to M5 at a position that is currently unidentifiable due
to limited information. For compound 18 (Figure 7), M6 (deꢀalkylation) and M8 (ketone
reduction) are the major metabolites (Figure 10B), M7 (deamination) is a minor product. For
compound 4 (Figure 8), oꢀdemethylation (M9) and monoꢀhydroxylation (M10) are the major
products (Figure 10C). M9 and M10 have more than one possible structures as indicated in the
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chromatograms (supplementary materials). For compound 7 (Figure 9), various metabolites
including oꢀdemethylation (M12), monoꢀhydroxylation (M14), oꢀdemethylation followed by
monoꢀhydroxylation (M11), and dihydroxylation (M13) were detected. M14 is the major
metabolite (Figure 10D). All of these metabolites have multiple isomeric forms as indicated in
the chromatograms (supplementary materials).
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Compounds Inhibit in vitro Tubulin Polymerization. We investigated the inhibition of
tubulin polymerization of selected potent compounds 4 and 7 with improved metabolic
properties and compared them with positive control colchicine and negative control taxol.
DMSO was used as a blank control. Bovine brain tubulin (> 97% pure) was incubated with the
individual compounds (5 or 10 ꢀM) to test their effect on tubulin polymerization (Figure 11).
After a 20 min incubation, tubulin polymerization was inhibited to the extent of 30% and 60% by
4 at 5 and 10 ꢀM, respectively (Figure 11A), as compared to vehicle. While about 33% and 81%
inhibition was observed for 7 at 5 and 10 ꢀM, respectively (Figure 11B). Both 4 and 7 showed
stronger inhibition than colchicine at the two tested concentrations. These data suggest that these
compounds exhibit strong antiꢀtubulin polymerization activity that corresponds well with their
cytotoxicity.
Conclusion
In this report, three series of new derivatives targeting modifications of the carbonyl linker, the
C ring para-position of the SMART template, and the PAT template were synthesized and
screened for their antiproliferative activities. Structure–activity relationships (SAR) of these
compounds led to the identification of lead analogues 4 and 7, which showed enhanced
anticancer activity in vitro compared to SMART 10 while increasing the metabolic stability on
human liver microsomes. Utilizing the MOM group to replace the paraꢀposition methoxy on the
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C ring, which is considered nonꢀreplaceable in many reports, also generated a potent analogue
15, which showed comparable potency to the parent compound 10. Further modification of the
PAT template yielded a potent analogue 33 with a 5ꢀindolyl substituent at the “A” ring.
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Experimental Section.
General. All reagents were purchased from SigmaꢀAldrich Chemical Co., Fisher Scientific
(Pittsburgh, PA), AK Scientific (Mountain View, CA), Oakwood Products (West Columbia, SC),
etc. and were used without further purification. Moistureꢀsensitive reactions were carried under
an argon atmosphere. Routine thin layer chromatography (TLC) was performed on aluminum
backed Uniplates. (Analtech, Newark, DE). Melting points were measured with FisherꢀJohns
melting point apparatus (uncorrected). NMR spectra were obtained on a Bruker AX 300
(Billerica, MA) spectrometer or Varian Inovaꢀ500 spectrometer. Chemical shifts are reported as
parts per million (ppm) relative to TMS in CDCl3. Mass spectral data was collected on a Bruker
ESQUIRE electrospray/ion trap instrument in positive and negative ion modes. Elemental
analyses were performed by Atlantic Microlab Inc., (Norcross, GA). Unless specified, all the
tested compounds described in the article present > 95% purity established through combustion
analysis.
General Procedure for the preparation of 3-7. Different aldehydes, 3ꢀbromobenzeneꢀ1,2ꢀ
diamine (3 mmol), pꢀtoluenesulfonic acid (0.3 mmol), and 15 mL of EtOH were refluxed for 24
h under argon atmosphere. The solvent was removed, 25 mL of water was added, and the
mixture was extracted with EtOAc (3× 50 mL). The combined organic layers were dried on
MgSO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography to
give the desired 4ꢀbromoꢀ2ꢀsubstitutedꢀ1Hꢀbenzo[d]imidazole.
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Corresponding bromides obtained from last step (1 eq), 3,4,5ꢀtrimethoxyphenylboronic acid (1
eq), THF (3 ml) / water (0.3 ml) solution of sodium carbonate (2 eq), and tetrakistriphenyl
phosphinepalladium (0.1 eq) was refluxed overnight. After adding water to a reaction mixture,
extracted with ethyl acetate. The organic layer was dried on MgSO4, filtered and concentrated in
vacuo. The residue was purified by flash chromatography to give desired fused “D” ring
benzoimidazole compounds 3-6, or imidazo[4,5ꢀc]pyridine (7).
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2-Phenyl-4-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazole (3). H NMR (CDCl3): 3.78,
3.93 (s, s, 6H), 3.91, 3.98 (s, s, 3H), 6.10, 6.82 (s, s, 2H), 7.29ꢀ8.08 (m, 8H), 9.70, 9.84 (s, br,
1H). MS (ESI) m/z 359.1 [MꢀH]ꢀ, 361.4 [M+H]+. Anal. (C22H20N2O3) C, H, N.
2-(1H-Indol-3-yl)-4-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazole (4). 1H NMR (DMSOꢀ
d6): 3.76, 3.97 (s, s, 3H), 3.90, 3.97 (s, s, 6H), 6.96ꢀ7.62 (m, H), 7.62, 7.72 (s, s, 2H), 8.16, 8.42
(s, br, 1H), 8.58, 8.66 (d, d, 1H), 11.57, 11.64 (s, s, 1H), 12.16, 12.60 (s, s, 1H). MS (ESI) m/z
398.1 [MꢀH]ꢀ, 400.3 [M+H]+. Anal. (C24H21N3O3) C, H, N.
2-(1H-Indol-5-yl)-4-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazole (5). 1H NMR (DMSOꢀ
d6): 3.73, 3.87 (s, s, 6H), 3.75, 3.92 (s, s, 3H), 5.70, 5.75 (s,s, 2H), 6.32, 6.49 (s, s, 1H), 6.54,
6.85 (d, d, 1H), 7.22ꢀ7.65 (m, 4H), 8.01, 8.42 (br, s, 2H), 11.09, 11.37(s, s, 1H), 12.36, 12.84 (s,
s, 1H). MS (ESI) m/z 398.1 [MꢀH]ꢀ, 400.1 [M+H]+. Anal. (C24H21N3O3) C, H, N.
3-(4-(3,4,5-Trimethoxyphenyl)-1H-benzo[d]imidazol-2-yl)-1H-indazole (6). 1H NMR
(DMSOꢀd6): 3.76 (s, 3H), 3.95 (s, 6H), 6.65 (s, 1H), 6.93 (t, 1H), 7.34ꢀ7.34 (m, 2H), 7.45ꢀ7.57
(m, 4H), 7.63 (s, 2H), 7.70 (d, 1H), 8.75 (d, 1H), 12.96 (s, 1H), 13.77 (s, 1H). MS (ESI) m/z
399.1 [MꢀH]ꢀ, 401.3 [M+H]+. Anal. (C23H20N4O3) C, H, N.
2-(1H-Indol-5-yl)-4-(3,4,5-trimethoxyphenyl)-1H-imidazo[4,5-c]pyridine (7). 1H NMR
(DMSOꢀd6): 3.77 (s, 3H), 3.96 (s, 6H), 6.61 (s, 1H), 7.46ꢀ7.48 (m, 2H), 7.59 (d, 1H), 8.08 (dd,
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1H), 8.36 (d, 1H), 8.41 (s, 2H), 8.49 (s, 1H), 11.44(s, 1H), 13.26 (s, 1H). MS (ESI) m/z 399.0
[MꢀH]ꢀ, 401.3 [M+H]+. Anal. (C23H20N4O3) C, H, N.
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N-(2,2-Diethoxyethyl)carbodiimide (19). At 0oC, to a solution of the aminoacetaldehyde
diethyl acetal (5.32 g, 40 mmol) in a mixture of diethyl ether (20 mL) and hexane (20 mL) was
added BrCN (4.22 g, 40 mmol). A solid precipitated from solution. The reaction mixture was
magnetically mixed overnight at room temperature. The solid was removed by filtration, and the
reaction mixture was concentrated. Flash chromatography of the concentrated residue afforded
2.82 g of the reagent (44.6 %). MS (ESI) m/z 156.8 [MꢀH]ꢀ, 180.9 [M+Na]+.
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(4-Hydroxy-3,5-dimethoxyphenyl)(2-phenylthiazol-4-yl)methanone
(11).
SMART
intermediates 8-10 were prepared from benzonitrile and cysteine following the same procedure
as described in our previous publication.10 10 (500 mg, 1.4 mmol) was dissolved in CH2Cl2 (50
mL) at RT under argon protection. Anhydrous AlCl3 (374 mg, 2.8 mmol) was added, and the
reaction mixture stirred for 12 h. The reaction was quenched with H2O (30 mL), the organic
phase separated, and the aqueous phase extracted with CH2Cl2 (2 × 20 mL). The combined
organic phases were washed with brine, dried over Mg2SO4, filtered, and concentrated to dryness
under reduced pressure. Compound 11 (410 mg, 85.9 % yield) was obtained after flash column
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purification using hexanesꢀEtOAc system. H NMR (CDCl3): 4.00 (s, 6H), 6.02 (s, 1H), 7.47ꢀ
7.48 (m, 3H), 7.91 (s, 2H), 8.01ꢀ8.03 (m, 2H), 8.27 (s, 1H). MS (ESI) m/z 339.9 [MꢀH]ꢀ, 364.1
[M+Na]+. Anal. (C18H15NO4S) C, H, N.
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2,6-Dimethoxy-4-(2-phenylthiazole-4-carbonyl)phenyl 2-chloroacetate (12). At 0 C, 2ꢀ
chloroacetyl chloride (100 mg, 0.9 mmol) was added to a solution of 11 (100 mg, 0.29 mmol) in
CH2Cl2 (30 mL). Then triethylamine (44 mg, 0.44 mmol) was charged in the mixture and stirred
until starting material disappeared on TLC. The reaction mixture was quenched with H2O (10
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mL), the organic phase separated, and the aqueous phase extracted with CH2Cl2 (2 × 10 mL).
The combined organic phases were washed with brine, dried over Mg2SO4, filtered, and
concentrated to dryness under reduced pressure. Compound 12 (99 mg, 81.7 % yield) was
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obtained after flash column purification using hexanesꢀEtOAc system. M. p. 147ꢀ148 C. H
NMR (CDCl3): 3.92 (s, 6H), 4.42 (s, 2H), 7.47ꢀ7.49 (m, 3H), 7.82 (s, 2H), 8.00ꢀ8.02 (m, 2H),
8.32 (s, 1H). MS (ESI) m/z 418.1 [MꢀH]ꢀ. Anal. (C20H16ClNO5S) C, H, N.
2,6-Dimethoxy-4-(2-phenylthiazole-4-carbonyl)phenyl 2,2,2-trifluoroacetate (13). At 0 oC,
trifluoroacetyl anhydride (189 mg, 0.9 mmol) was added to a solution of 11 (100 mg, 0.29 mmol)
in CH2Cl2 (10 mL). Then DMAP (54 mg, 0.44 mmol) was charged in the mixture and stirred at
RT until starting material disappeared on TLC. The reaction mixture was quenched with H2O (10
mL), the organic phase separated, and the aqueous phase extracted with CH2Cl2 (2 × 10 mL).
The combined organic phases were washed with brine, dried over Mg2SO4, filtered, and
concentrated to dryness under reduced pressure. Compound 13 (89 mg, 70.2 % yield) was
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obtained after flash column purification using hexanesꢀEtOAc system. M. p. 151ꢀ153 C. H
NMR (CDCl3): 3.94 (s, 6H), 7.48ꢀ7.49 (m, 3H), 7.84 (s, 2H), 8.00ꢀ8.02 (m, 2H), 8.34 (s, 1H).
MS (ESI) m/z 438.1 [M+H]+. Anal. (C20H164F3NO5S) C, H, N.
(4-(Benzyloxy)-3,5-dimethoxyphenyl)(2-phenylthiazol-4-yl)methanone (14). Under an
argon atmosphere, potassium carbonate (49 mg, 0.352 mmol) and benzyl bromide (33 mg, 0.194
mmol) were added to a solution of 11 (60 mg, 0.176 mmol) in 10 mL of dry DMF. The mixture
was stirred for 1 h at 100oC and then transferred into water (10 mL). The compound 14 was
extracted with EtOAc, washed with distilled water, dried on magnesium sulfate, and
concentrated under vacuum using a rotary evaporator. The crude oily product was purified by
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flash column and white solid 14 (51 mg) was obtained. Yield = 67.2%. M. p. 119ꢀ120 C. H
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NMR (CDCl3): 3.92 (s, 6H), 5.15 (s, 2H), 7.29ꢀ7.37 (m, 3H), 7.48ꢀ7.51 (m, 5H), 7.79 (s, 2H),
8.01ꢀ8.02 (m, 2H), 8.28 (s, 1H). MS (ESI) m/z 432.1 [M+H]+. Anal. (C25H21NO4S) C, H, N.
(3,5-Dimethoxy-4-(methoxymethoxy)phenyl)(2-phenylthiazol-4-yl)methanone (15). At 0
oC, MOMCl (27 mg, 0.33 mmol) was added to a solution of 11 (75 mg, 0.22 mmol) in CH2Cl2
(10 mL). Then Hunig’s base (57 mg, 0.44 mmol) was charged in the mixture and stirred at RT
until starting material disappeared on TLC. The reaction mixture was quenched with H2O (10
mL), the organic phase separated, and the aqueous phase extracted with CH2Cl2 (2 × 10 mL).
The combined organic phases were washed with brine, dried over Mg2SO4, filtered, and
concentrated to dryness under reduced pressure. Compound 15 (83 mg, 98.0 % yield) was
obtained as yellow crystals after flash column purification using hexanesꢀEtOAc system. M. p.
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103ꢀ104 C. H NMR (CDCl3): 3.62 (s, 3H), 3.95 (s, 6H), 5.26 (s, 2H), 7.47ꢀ7.49 (m, 3H), 7.80
(s, 2H), 8.01ꢀ8.03 (m, 2H), 8.28 (s, 1H). MS (ESI) m/z 408.1 [M+Na]+. Anal. (C20H19NO5S) C,
H, N.
2-(2-(2,6-Dimethoxy-4-(2-phenylthiazole-4-carbonyl)phenoxy)ethyl)isoindoline-1,3-dione
(17). To a solution of 11 (200 mg, 0.59 mmole) and 2ꢀ(2ꢀbromoethyl)isoindolineꢀ1,3ꢀdione (223
mg, 0.88 mmol) in DMF (2.5 ml) was added K2CO3 (97 mg, 0.7 mmol) and stirred the reaction
mixture at 120oC for overnight. Then the reaction mixture was quenched in water and extracted
with ethyl acetate. The organic layer was concentrated and further purified by column
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chromatography to get 132 mg of pure desired product 17. Yield = 43.5%. M. p. 148ꢀ150 oC. H
NMR (CDCl3) δ 3.71 (s, 6H), 4.14 (t, 2H, J=5.5 Hz), 4.41 (t, 2H, J=5.5 Hz), 7.49ꢀ7.51 (m, 3H),
7.70 (s, 2H), 7.75 (q, 2H, J=3.0 Hz), 7.91 (q, 2H, J=3.0 Hz), 8.01ꢀ8.03 (m, 2H), 8.27(s, 1H). MS
(ESI) m/z 537.1 [M+Na]+. Anal. (C28H22N2O6S) C, H, N.
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(4-(2-Aminoethyl)-3,5-dimethoxyphenyl)(2-phenylthiazol-4-yl)methanone (18). To
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solution of 11 (23 mg, 0.07 mmole) and tertꢀbutyl (2ꢀbromoethyl)carbamate (23 mg, 0.1 mmol)
in DMF (2.5 ml) was added Cs2CO3 (46 mg, 0.2 mmol) and the reaction mixture was stirred for
3 days at RT until TLC showing the reaction had finished. Then the reaction mixture was
quenched in ice cold water and extracted with ethyl acetate. The organic layer was concentrated
and further purified by column chromatography to get 22 mg of pure desired product tert-butyl
(2ꢀ(2,6ꢀdimethoxyꢀ4ꢀ(2ꢀphenylthiazoleꢀ4ꢀcarbonyl)phenoxy)ethyl)carbamate 16. Yield = 65.1%.
MS (ESI) m/z 483.9 [MꢀH]ꢀ, 485.1 [M+H]+. Boc protected compound 16 was added to a solution
of HCl in dioxane (4M) and stirred for overnight. The precipitate was collected and washed with
diethyl ether to afford HCl salts of compound 18. 1H NMR (Acetoneꢀd6): 3.09ꢀ3.13 (q, 2H,
J=5.5 Hz), 3.79 (br, 2H), 3.90 (s, 6H), 4.17 (t, 2H, J=5.5 Hz), 7.55ꢀ7.58 (m, 3H), 7.66 (s, 2H),
8.02ꢀ8.04 (m, 2H), 8.68 (s, 1H). MS (ESI) m/z 385.1 [M+H]+. Anal. (C20H20N2O3S) C, H, N.
N-Phenyl-1H-imidazol-2-amine (21). At 0oC, to a solution of the aminoꢀacetaldehyde diethyl
acetal (2.66 g, 20 mmol) in diethyl ether/hexane mixture (20 mL, 1:1) was added BrCN (2.11 g,
20 mmol) in small portions. The reaction mixture was stirred at RT overnight. The solid is
removed by filtration and washed with ether. The combined filtrate is concentrated. Purification
by flash column chromatography (silica gel, eluting with dichloromethane to 5% methanol in
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dichloromethane, gradient) affords Nꢀ(2,2ꢀdiethoxyethyl)carbodiimide 19. H NMR 500 MHz
(CDCl3): 1.23 (t, 6H, J=7.0 Hz), 3.16 (t, 2H, J=6.0 Hz), 3.56 (dt, 2H), 3.64 (br, s, 1H), 3.73(dt,
2H), 4.58 (t, J=5.0 Hz, 1H). MS (ESI) m/z 156.8 [MꢀH]ꢀ, 180.9 [M+Na]+. Aniline (1.66 g, 17.8
mmol) was dissolved in ethanol (50 mL), and a solution of 19 (2.82 g, 17.8 mmol) in 5 mL
diethyl ether was added dropwise. Methanesulfonic acid (1.71 g, 17.8 mmol) was then added,
and the mixture was refluxed for 24 h. The reaction mixture was poured into NaOH (0.5 M) and
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extracted with CH2Cl2. Drying with MgSO4 and concentrated in vacuo afforded a product that
was subjected to flash chromatography to give the intermediate guanidine 20 (3.3 g, 73.8%). The
guanidine (3g, 12 mmoL) was dissolved in HCl (5 mL, 6 M) at 0 °C and then stirred for 2 h.
After the starting material was consumed, NaOH (25%) was added until a precipitate formed (pH
14). This mixture was stirred for 30 min. The reaction was then poured into NaOH (0.5 M),
extracted with CH2Cl2, dried, and concentrated. Flash chromatography afforded 21 (1.16 g,
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61%). H NMR (DMSOꢀd6): 6.68 (s, 2H), 6.75 (m, 1H), 7.17 (m, 2H), 7.34 (m, 2H), 8.58 (s,
1H). MS (ESI) m/z 157.6 [MꢀH]ꢀ, 160.0 [M + H]+.
3,4,5-Trimethoxy-N-phenyl-N-(1-(phenylsulfonyl)-1H-imidazol-2-yl)benzamide (22). To a
solution of Nꢀphenylꢀ1Hꢀimidazolꢀ2ꢀamine 21 (40 mg, 0.25 mmol) in CH2Cl2 (10 mL) was
added benzenesulfonyl chloride (441 mg, 2.5 mmoL) and triethylamine (252 mg, 2.5 mmol).
Reaction mixture was stirred overnight at room temperature. The reaction mixture was quenched
by sat. NH4Cl and extracted with CH2Cl2. Drying with MgSO4 and concentrated in vacuo
afforded a product that was subjected to flash chromatography to give a benzenesulfonyl
protected intermediate (79 mg, 72%). This intermediate was dissolved in THF and cooled down
to ꢀ78oC, and then tꢀBuLi (1.7M) was charged under Ar2. After stirred for an hour, 3,4,5ꢀ
trimethoxybenzoyl chloride (47 mg, 0.26 mmoL) was added and stirred overnight. The reaction
mixture was poured into NH4Cl (Sat.) and extracted with ethyl acetate. Drying with MgSO4 and
concentrated in vacuo afforded a crude product that was purified by flash chromatography to
give 22 (35%). 1H NMR (CDCl3): 3.78 (s, 6H), 3.87 (s, 3H), 6.91 (s, 2H), 6.97 (s, 1H), 7.18 (m,
2H), 7.20 (d, 1H), 7.25 (m, 2H), 7.38 (m, 2H), 7.40 (d, 1H), 7.54 (br, 1H), 7.59 (t, 2H). MS (ESI)
m/z 491.9 [MꢀH]ꢀ, 516.1 [M + Na]+.
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N-(1H-imidazol-2-yl)-3,4,5-trimethoxy-N-phenylbenzamide (23). To a solution of Nꢀ
phenylꢀ1Hꢀimidazolꢀ2ꢀamine 21 (900 mg, 5.66 mmoL) in dioxane and water (30 mL, 3:1) was
added Boc2O (2.68 g, 12.3 mmol) and NaOH (0.6 g, 15 mmol) and stirred for 4 h. The mixture
was concentrated in vacuo and the residue was purified by flash chromatography to obtain the
Boc protected intermediate. This intermediate (130 mg, 0.502 mmol) was dissolved in THF and
cooled down to ꢀ78oC, and then tꢀBuLi (0.65 mL, 1.7M, 1.1 mmol) was charged under Ar2. After
stirred for an hour, 3,4,5ꢀtrimethoxybenzoyl chloride (116 mg, 0.502 mmoL) was added and
stirred overnight. The reaction mixture was poured into NH4Cl (Sat.) and extracted with ethyl
acetate. Drying with MgSO4 and concentrated in vacuo afforded a crude product that was
purified by flash chromatography to give 23 (35%). 1H NMR (CDCl3): 3.65 (s, 6H), 3.79 (s, 3H),
6.56 (s, 2H), 6.90 (m, 2H), 7.27ꢀ7.39 (m, 5H), 11.17 (br, 1H). MS (ESI) m/z 351.8 [MꢀH]ꢀ, 376.3
[M + Na]+.
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N-Phenyl-4-trityl-1H-imidazol-2-amine (24) and N-phenyl-1-trityl-1H-imidazol-2-amine
(25). To a solution of Nꢀphenylꢀ1Hꢀimidazolꢀ2ꢀamine (159 mg, 10 mmoL) in triethylamine and
CH2Cl2 stirring under an inert atmosphere at 0°C, was added (chloromethanetriyl)tribenzene (5
eq). The solution was allowed to warm to RT and stir until complete by TLC. The reaction
mixture was then concentrated in vacuo, quenched with saturated aqueous sodium bicarbonate
and extracted with ethyl acetate. Then dried with magnesium sulfate, and concentrated in vacuo.
The resulting residue is purified by flash chromatography to give two different protected
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products. 24: H NMR (DMSOꢀd6): 6.0 (s, 1H), 6.75 (m, 1H), 7.29ꢀ7.62 (m, 19H), 8.65 (s, 1H),
1
10.62 (s, 1H). MS (ESI) m/z 399.9 [MꢀH]ꢀ, 403.1 [M+H]+. 25: H NMR (DMSOꢀd6): 6.08 (s,
1H), 6.41 (s, 1H), 6.85 (s, 1H), 7.13ꢀ7.52 (m, 20H), 8.65 (s, 1H), 10.62 (s, 1H). MS (ESI) m/z
399.8 [MꢀH]ꢀ, 402.8 [M+H]+.
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(2-(Phenylamino)-1-trityl-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (26). To
a solution of Nꢀphenylꢀ1ꢀtritylꢀ1Hꢀimidazolꢀ2ꢀamine 25 (116 mg, 0.289 mmol) in THF (10 mL)
stirring under an inert atmosphere at ꢀ78°C, was added tꢀBuLi (0.34 mL, 1.7M, 0.58 mmol) and
trimethoxybenzoyl chloride (66.5 mg, 0.289 mmoL). The reaction mixture was reacted for
overnight, then quenched by NH4Cl (Sat.) and extracted with ethyl acetate. Drying with MgSO4
and concentrated in vacuo afforded a crude product that was purified by flash chromatography to
give 26 (75 mg, 43.7 %). 1H NMR (DMSOꢀd6): 3.71, (s, 3H), 3.78 (s, 6H), 5.87 (s, 1H), 6.94 (s,
2H), 7.18ꢀ7.58 (m, 21H). MS (ESI) m/z 594.2 [MꢀH]ꢀ, 596.3 [M+H]+.
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(2-(Phenylamino)-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl)methanone
(27).
(2ꢀ
(Phenylamino)ꢀ1ꢀtritylꢀ1Hꢀimidazolꢀ4ꢀyl)(3,4,5ꢀtrimethoxyphenyl)methanone was dissolved in
a solution of HCl in diethyl ether (2M) and stirred overnight. Saturated NaHCO3 solution is then
added and the reaction mixture is extracted three times with ether. The combined organic layers
are dried (sodium sulfate), filtered and concentrated in vacuo. The residue is purified by flash
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chromatography to give pure 27. H NMR (DMSOꢀd6): 3.73 (s, 3H), 3.82 (s, 6H), 6.62 (s, 2H),
7.02 (s, 2H), 7.33 (d, 2H), 7.43ꢀ7.51 (m, 3H), 7.54 (br, 1H). MS (ESI) m/z 352.1 [MꢀH]ꢀ, 354.3
[M+H]+. Anal. (C19H19N3O4) C, H, N.
N-((1H-indol-5-yl)carbamothioyl)benzamide (28). A mixture of 5ꢀnitroꢀ1Hꢀindole (11 g,
67.9 mmol) and Pd/C (5%; 1 g), dissolved in ethanol (50 mL), was hydrogenated for 3 h at 40
psi. The reaction mixture was filtered and the excess of ethanol was evaporated under reduced
pressure. Solid product was recrystallized from hexane to obtain the pure compound 5ꢀ
aminoindole. Yield: 92.5%. 1H NMR (500 MHz, CDCl3): δ 3.50 (s, 2 H), 6.37 (s, 1 H), 6.67 (dd,
1 H), 6.95 (s, 1 H), 7.13 (s, 1 H), 7.20 (d, 1 H), 7.96 (br, 1 H). MS (ESI) m/z 133.0 (M+H)+. A
solution of 5ꢀaminoindole (8 g, 60.6 mmol) in acetone (150 mL) was reacted with
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benzoylisothiocyanate (9.88 g, 60. mmol) at RT for about 4 h until TLC showed reaction
finished to yield compound 28. 1H NMR (300 MHz, CDCl3): δ 6.61 (br, 1 H),7.26ꢀ7.28 (d, 1H),
7.38ꢀ7.45 (m, 2H), 7.54ꢀ7.59 (m, 2H), 7.65ꢀ7.70 (m, 1 H), 7.91ꢀ7.94 (m, 2 H), 7.98 (s, 1 H), 8.27
(s, br, 1 H), 9.12 (s, 1 H), 12.51 (s, 1 H). MS (ESI) m/z 318.1 [M + Na]+.
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2-(1H-Indol-5-ylamino)-N-methoxy-N-methylthiazole-4-carboxamide (32). The resulting
solid 28 was filtered and treated with 2 N NaOH in THF (120 mL). The mixture was refluxed for
about 6 h and allowed to warm to RT. The solvent was evaporated off under vacuum. The
residue was diluted with water (20 mL) and neutralized to pH 7 with 1N HCl. The resulting solid
was filtered and dried under vacuum to afford 5ꢀindolylthiourea (29). Compound 29 (0.01 mol)
and ethyl bromopyruvate (0.011 mol) were dissolved in 3 mL ethanol and held at reflux for 2 h.
The reaction was cooled, the crystalline ethyl 2ꢀ(1Hꢀindolꢀ5ꢀylamino)thiazoleꢀ4ꢀcarboxylate (30)
was collected by filtration and washed with ethanol. Refluxing the mixture of ethyl esters with
the NaOHꢀethanol solution gave 2ꢀ(1Hꢀindolꢀ5ꢀylamino)thiazoleꢀ4ꢀcarboxylic acid (31) which
was used directly in the next steps. To a mixture of the crude acid (2.5 mmol), HBTU (2.6 mmol)
and NMM (5.3 mmol) in CH2Cl2 (30 mL) was added HCl salt of HNCH3OCH3 (2.6 mmol) and
stirring continued at RT for overnight. The reaction mixture was diluted with CH2Cl2 (20 mL)
and sequentially washed with water, satd. NaHCO3, brine and dried over MgSO4. The solvent
was removed under reduced pressure to yield a crude product, which was purified by column
chromatography to obtain pure Weinreb amide 2ꢀ(1Hꢀindolꢀ5ꢀylamino)ꢀNꢀmethoxyꢀNꢀ
1
methylthiazoleꢀ4ꢀcarboxamide (32) (45.6% yield for overall 5 steps). H NMR (CDCl3): 3.42 (s,
3H), 3.77 (s, 3H), 6.54 (m, 1H), 7.26 (m, 1H), 7.29 (m, 2H), 7.40 (d, 2H), 7.61 (m, 1H), 8.30 (br,
1H). MS (ESI) m/z 303.0 [M+H]+.
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(2-((1H-Indol-5-yl)amino)thiazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (33). At ꢀ78°C, to
a solution of 5ꢀbromoꢀ1,2,3ꢀtrimethoxybenzene (1.235 g, 5.0 mmol) in 30 mL THF was charged
n-BuLi in hexane (2.5N, 2.4 mL, 6 mmol) under Ar2 protection and stirred for 10 min. Weinreb
amide 32 (1 mmol) in 10 mL THF was added to lithium reagent and allowed to stir at RT for 2 h.
The reaction mixture was quenched with satd. NH4Cl, extracted with ethyl ether, dried with
MgSO4 The solvent was removed under reduced pressure to yield a crude product, which was
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purified by column chromatography to obtain pure compound 33 (51.7% yield). H NMR (300
MHz, CDCl3) δ 3.89 (s, 6 H), 3.93 (s, 3 H), 6.55 (m, 1 H), 7.15ꢀ7.12 (m, 1 H), 7.28ꢀ7.26 (m, 1
H), 7.36 (s, 1 H), 7.39 (s, 1 H), 7.46 (s, 2 H), 7.68 (d, 1 H), 8.29 (br, 1 H). MS (ESI) m/z 432.1
(M + Na)+, 408.0 (M ꢀ H)ꢀ. Anal. (C23H19N3O4S) C, H, N.
Molecular Modeling. The molecular modeling studies were performed with the published
crystal structures of the α,βꢀtubulin dimer in complex with DAMAꢀcolchicine (Protein Data
Bank code 1SA0). Schrodinger Molecular Modeling Suite 2013 (Schrodinger Inc., Portland, OR)
11, 19
was used for the modeling studies with procedures similar to those described before
.
Briefly, the structures of the proteinꢀligand complexes were prepared using the Protein
Preparation module, and the active ligand binding sites were defined based on the native ligand.
Both native ligand DAMAꢀcolchicine and the designed tubulin inhibitors described in this study
were built and prepared for docking using the Ligprep module before they were docked into
1SA0. The Glide docking score obtained from this modeling approach is an estimation of the
binding energy (kcal/mol) when a ligand binds to the tubulin dimer. A lower (more negative)
number suggests more favorable binding interaction between a ligand and the receptor. Data
analyses were performed using the Maestro interface of the software.
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Cell Culture and Cytotoxicity Assay of Prostate Cancer and Melanoma. All cell lines were
obtained from ATCC (American Type Culture Collection, Manassas, VA, USA), while cell
culture supplies were purchased from Cellgro Mediatech (Herndon, VA, USA). We examined
the antiproliferative activity of our antiꢀtubulin compounds in four human prostate cancer cell
lines (LNCaP, DU 145, PCꢀ3, and PPCꢀ1) and three melanoma cell lines (A375, B16ꢀF1 and
WMꢀ164). All prostate cancer cell lines were cultured in RPMI 1640, supplemented with 10%
fetal bovine serum (FBS). Melanoma cells were cultured in DMEM, supplemented with 5%
FBS, 1% antibiotic/antimycotic mixture (SigmaꢀAldrich, Inc., St. Louis, MO, USA) and bovine
insulin (5 ꢁg/ml; SigmaꢀAldrich). The cytotoxic potential of the antiꢀtubulin compounds was
evaluated using the sulforhodamine B (SRB) assay after 96 h of treatment.
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In Vitro Tubulin Polymerization Assay. Bovine brain tubulin (0.4 mg, >97% pure)
(Cytoskeleton, Denver, CO) was mixed with 10 ꢀM of the test compounds and incubated in 100
ꢀl of general tubulin buffer (80 mM PIPES, 2.0 mM MgCl2, 0.5 mM EGTA, and 1 mM GTP) at
pH 6.9. The absorbance of wavelength at 340 nm was monitored every 1 min for 20 min by the
SYNERGY 4 Microplate Reader (BioꢀTek Instruments, Winooski, VT). The spectrophotometer
was set at 37 °C for tubulin polymerization.
Microsomal stability assay. Metabolic stability studies were performed by incubating the test
compounds (0.5 ꢁM) in a total reaction volume of 1.2 mL containing 1 mg/mL microsomal
protein in reaction buffer [0.2 M of phosphate buffer solution (pH 7.4), 1.3 mM NADP+, 3.3 mM
glucoseꢀ6ꢀphosphate, and 0.4 U/mL glucoseꢀ6ꢀphosphate dehydrogenase] at 37 °C in a shaking
incubator 12. Pooled human liver microsomes were utilized to examine metabolic stability. The
NADPH regenerating system (solution A and B) was obtained from Xenotech, LLC (Lenexa,
KS). Aliquots (100 ꢀL) from the reaction mixtures to determine metabolic stability were
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sampled at 5, 10, 20, 30, 60, and 90 min. Acetonitrile (200 ꢀL) containing 200 nM of the internal
standard was added to quench the reaction and to precipitate the proteins. Samples were then
centrifuged at 10,000 rpm for 15 min at RT, and the supernatant was analyzed directly by LCꢀ
MS/MS (AB Sciex API4500). For metabolite identification, the reaction mixture was incubated
for 2 h with 50 ꢁM test compound concentration under the previously described conditions.20
The supernatants were analyzed using a Water Xevo G2ꢀS high resolution mass spectrometer.
Acknowledgment. This research was supported by the Van Vleet Endowed Professorship
(D.D.M.), and NIH grant R01CA148706, 1S10RR026377ꢀ01, 1S10OD010678ꢀ01 (W.L.). The
content is solely the responsibility of the authors and does not necessarily represent the official
views of the National Institutes of Health. We thank Dr. Christopher Coss for his help with the
data collection at GTx, Inc. We thank Dr. Michael Mohler at GTx, Inc. for his proofreading and
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editorial assistance.
Supporting information: Supporting Information is available for high resolution mass spectra of the
metabolites. This material is available free of charge via the Internet at http://pubs.acs.org.
*
Corresponding authors: Duane D. Miller, Eꢀmail: dmiller@uthsc.edu. Phone: (901)448ꢀ6026.
Fax: (901)448ꢀ3446. Wei Li, Eꢀmail: wli@uthsc.edu. Phone: 901ꢀ448ꢀ7532. Fax: 901ꢀ448ꢀ6828.
a
Abbreviations: ABI, 2ꢀarylꢀ4ꢀbenzoylꢀimidazole; Boc2O, tertꢀbutyl dicarbonate; CAꢀ4,
combretastatin Aꢀ4; DMF, dimethylformamide ; DMSO, dimethyl sulfoxide; EDCI, 1ꢀethylꢀ3ꢀ
(3ꢀdimethylaminopropyl)carbodiimide; HOBt, hydroxybenzotriazole; MOM, methoxymethyl;
NMM, Nꢀmethylmorpholine; NMR, nuclear magnetic resonance; PAT, phenyl amino thiazole;
Pꢀgp, Pꢀglycoprotein; PK, pharmacokinetic; RT, room temperature; Phth, phthalimide; SMART,
4ꢀsubstituted
methoxybenzoylꢀarylꢀthiazole;
THF,
tetrahydrofuran;
TMP,
3,4,5ꢀ
trimethoxyphenyl; TsOH, pꢀtoluenesulfonic acid.
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References:
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9
1. Jordan, M. A.; Wilson, L., Microtubules as a target for anticancer drugs. Nature reviews.
Cancer 2004, 4 (4), 253ꢀ265.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
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39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2. Lu, Y.; Chen, J.; Xiao, M.; Li, W.; Miller, D. D., An overview of tubulin inhibitors that
interact with the colchicine binding site. Pharmaceutical research 2012, 29 (11), 2943ꢀ2971.
3. Nam, N. H., Combretastatin Aꢀ4 analogues as antimitotic antitumor agents. Curr Med
Chem 2003, 10 (17), 1697ꢀ1722.
4. Pettit, G. R.; Toki, B.; Herald, D. L.; VerdierꢀPinard, P.; Boyd, M. R.; Hamel, E.; Pettit,
R. K., Antineoplastic agents. 379. Synthesis of phenstatin phosphate. J Med Chem 1998, 41 (10),
1688ꢀ1695.
5. Kuo, C. C.; Hsieh, H. P.; Pan, W. Y.; Chen, C. P.; Liou, J. P.; Lee, S. J.; Chang, Y. L.;
Chen, L. T.; Chen, C. T.; Chang, J. Y., BPR0L075, a novel synthetic indole compound with
antimitotic activity in human cancer cells, exerts effective antitumoral activity in vivo. Cancer
Res 2004, 64 (13), 4621ꢀ4628.
6. Sriram, M.; Hall, J. J.; Grohmann, N. C.; Strecker, T. E.; Wootton, T.; Franken, A.;
Trawick, M. L.; Pinney, K. G., Design, synthesis and biological evaluation of
dihydronaphthalene and benzosuberene analogs of the combretastatins as inhibitors of tubulin
polymerization in cancer chemotherapy. Bioorg Med Chem 2008, 16 (17), 8161ꢀ8171.
7. Liu, X.; Go, M. L., Antiproliferative properties of piperidinylchalcones. Bioorg Med
Chem 2006, 14 (1), 153ꢀ163.
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Page 28 of 42
1
2
3
4
5
6
8. Burns, R. G., Analysis of the colchicineꢀbinding site of betaꢀtubulin. FEBS letters 1992,
297 (3), 205ꢀ208.
7
8
9
9. (a) Alvarez, C.; Alvarez, R.; Corchete, P.; PerezꢀMelero, C.; Pelaez, R.; Medarde, M.,
Exploring the effect of 2,3,4ꢀtrimethoxyꢀphenyl moiety as a component of indolephenstatins. Eur
J Med Chem 2010, 45 (2), 588ꢀ97; (b) Brancale, A.; Silvestri, R., Indole, a core nucleus for
potent inhibitors of tubulin polymerization. Med Res Rev 2007, 27 (2), 209ꢀ38; (c) Ray, K.;
Bhattacharyya, B.; Biswas, B. B., Role of Bꢀring of colchicine in its binding to tubulin. J Biol
Chem 1981, 256 (12), 6241ꢀ6244.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
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36
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
10. Lu, Y.; Li, C. M.; Wang, Z.; Ross, C. R., 2nd; Chen, J.; Dalton, J. T.; Li, W.; Miller, D.
D., Discovery of 4ꢀsubstituted methoxybenzoylꢀarylꢀthiazole as novel anticancer agents:
synthesis, biological evaluation, and structureꢀactivity relationships. J Med Chem 2009, 52 (6),
1701ꢀ1711.
11. Chen, J.; Li, C. M.; Wang, J.; Ahn, S.; Wang, Z.; Lu, Y.; Dalton, J. T.; Miller, D. D.; Li,
W., Synthesis and antiproliferative activity of novel 2ꢀarylꢀ4ꢀbenzoylꢀimidazole derivatives
targeting tubulin polymerization. Bioorg Med Chem 2011, 19 (16), 4782ꢀ4795.
12. Li, C. M.; Chen, J.; Lu, Y.; Narayanan, R.; Parke, D. N.; Li, W.; Ahn, S.; Miller, D. D.;
Dalton, J. T., Pharmacokinetic optimization of 4ꢀsubstituted methoxybenzoylꢀarylꢀthiazole and
2ꢀarylꢀ4ꢀbenzoylꢀimidazole for improving oral bioavailability. Drug metabolism and disposition:
the biological fate of chemicals 2011, 39 (10), 1833ꢀ1839.
13. Wang, Z.; Chen, J.; Wang, J.; Ahn, S.; Li, C. M.; Lu, Y.; Loveless, V. S.; Dalton, J. T.;
Miller, D. D.; Li, W., Novel tubulin polymerization inhibitors overcome multidrug resistance and
reduce melanoma lung metastasis. Pharmaceutical research 2012, 29 (11), 3040ꢀ3052.
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Page 29 of 42
Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
14. Li, C. M.; Wang, Z.; Lu, Y.; Ahn, S.; Narayanan, R.; Kearbey, J. D.; Parke, D. N.; Li,
W.; Miller, D. D.; Dalton, J. T., Biological activity of 4ꢀsubstituted methoxybenzoylꢀarylꢀ
thiazole: an active microtubule inhibitor. Cancer Res 2011, 71 (1), 216ꢀ224.
10
11
12
13
14
15
16
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18
19
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21
22
23
24
25
26
27
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43
44
45
46
47
48
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50
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52
53
54
55
56
57
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60
15. Li, C. M.; Lu, Y.; Narayanan, R.; Miller, D. D.; Dalton, J. T., Drug metabolism and
pharmacokinetics of 4ꢀsubstituted methoxybenzoylꢀarylꢀthiazoles. Drug metabolism and
disposition: the biological fate of chemicals 2010, 38 (11), 2032ꢀ2039.
16. Bai, R.; Covell, D. G.; Pei, X. F.; Ewell, J. B.; Nguyen, N. Y.; Brossi, A.; Hamel, E.,
Mapping the binding site of colchicinoids on beta ꢀtubulin. 2ꢀChloroacetylꢀ2ꢀ
demethylthiocolchicine covalently reacts predominantly with cysteine 239 and secondarily with
cysteine 354. J Biol Chem 2000, 275 (51), 40443ꢀ40452.
17. Bai, R. L.; Lin, C. M.; Nguyen, N. Y.; Liu, T. Y.; Hamel, E., Identification of the
cysteine residue of betaꢀtubulin alkylated by the antimitotic agent 2,4ꢀdichlorobenzyl
thiocyanate, facilitated by separation of the protein subunits of tubulin by hydrophobic column
chromatography. Biochemistry 1989, 28 (13), 5606ꢀ12.
18. Shan, B.; Medina, J. C.; Santha, E.; Frankmoelle, W. P.; Chou, T. C.; Learned, R. M.;
Narbut, M. R.; Stott, D.; Wu, P.; Jaen, J. C.; Rosen, T.; Timmermans, P. B.; Beckmann, H.,
Selective, covalent modification of betaꢀtubulin residue Cysꢀ239 by T138067, an antitumor agent
with in vivo efficacy against multidrugꢀresistant tumors. Proceedings of the National Academy of
Sciences of the United States of America 1999, 96 (10), 5686ꢀ5691.
19. (a) Slominski, A. T.; Kim, T. K.; Li, W.; Yi, A. K.; Postlethwaite, A.; Tuckey, R. C., The
role of CYP11A1 in the production of vitamin D metabolites and their role in the regulation of
epidermal functions. The Journal of steroid biochemistry and molecular biology 2013, in press,
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