A one-pot synthesis and biological activity of ageladine A and analogues

Article abstract of DOI:10.1021/jm200039m

A one-pot synthesis of ageladine A and analogues is reported. The key Pictet-Spengler reaction between 2-aminohistamine and aryl aldehydes has been successfully utilized for the synthesis of the natural product and 14 analogues. These compounds were scree

Full text of DOI:10.1021/jm200039m

ARTICLE
pubs.acs.org/jmc
A One-Pot Synthesis and Biological Activity of Ageladine A and
Analogues
Sudhir R. Shengule,† Wendy L. Loa-Kum-Cheung,† Christopher R. Parish,‡
Mꢀelina Blairvacq,§ Laurent Meijer,§ Yoichi Nakao,|| and Peter Karuso*,†
†Department of Chemistry & Biomolecular Sciences, Macquarie University, Sydney NSW-2109, Australia
‡Division of Immunology and Genetics, John Curtin School of Medical Research, Australian National University,
Canberra ACT 2601, Australia
§CNRS, Protein Phosphorylation and Human Disease Group, Station Biologique, 29682 Roscoff cedex, Bretagne, France
School of Advanced Science and Engineering, Waseda University, 3-4-1, Okubo, Shinjuku-ku, Tokyo, 169-8555, Japan
S Supporting Information
b
ABSTRACT: A one-pot synthesis of ageladine A and analogues is reported. The
key PictetꢀSpengler reaction between 2-aminohistamine and aryl aldehydes has
been successfully utilized for the synthesis of the natural product and 14 analogues.
These compounds were screened for their matrix metalloprotease (MMP) and
kinase inhibition to develop the first structureꢀactivity relationship of ageladine A
analogues. One compound, which showed significant kinase activity but little MMP inhibitory activity, was found to be highly active
in an antiangiogenic screen, suggesting that the angiogenic activity of ageladine A is not associated with MMP inhibition but rather
kinase inhibitory activity. Cytotoxicity was excluded as a mode of action by the assay of ageladine A and an analogue against 60
human cell lines.
’ INTRODUCTION
formation of the imidazolopyridine moiety.4,7 Most recently,
Ando et al. reported a fourth synthesis of 1, also based on the
PictetꢀSpengler cyclization between the N-Boc-2-AH and N-
protected 2-formyl pyrroles.8 In this case, the dehydrogenation
was effected in two steps using IBX and then MnO2 instead of
choranil, which was found to improve the overall yield. Herein
we report the one-pot synthesis of 1 and a range of analogues
from 2-AH and various heterocyclic aldehydes. The analogues
synthesized were further screened for their MMP-2, MT1-
MMP, and/or kinase inhibition. A small number were further
screened for cytoxicity and their ability to inhibit angiogenesis.
Surprisingly, the 2-pyridine analogue, which displayed poor
MMP activity, was more active than the natural product in the
angiogenesis assay.
Ageladine A (1), an anticancer natural product, was recently
isolated from the marine sponge Agelas nakamurai by Fusetani
and co-workers.1 The compound is the first example of an
imidazolopyridine natural product that formally belongs to the
oroidin family of marine natural products (pyrrole-imidazole
alkaloids). 1 has shown inhibition against various matrix
metalloproteinases (MMPs) at micromolar levels and strong
antiangiogenic activity that is believed to be associated with its
MMP inhibition.2 However, 1 does not function as an MMP
inhibitor through zinc complexation and thus has a novel mode
of action.1 The important biological activity and unusual
compact structure have made 1 an important target for the
synthetic chemist. To date, four total syntheses have been
reported: Weinreb reported the first total synthesis of 1 using
a 6π-azaelectrocyclization and SuzukiꢀMiyaura coupling of N-
Boc-pyrrole-2-boronic acid and chloropyridine as a key step.3,4
Soon after, we reported the concise synthesis of 1 based on
biomimetic principles.5 The biogenesis of 1 is likely to be from
proline and histidine, which could produce 4,5-dibromo-2-
formylpyrrole and 2-aminohistamine (2-AH). Retrosynthetic
analysis suggested that an imine formed between 2-AH and
2-formylpyrrole could cyclize via a PictetꢀSpengler reaction to
yield the requisite skeleton of the 1 in one step. Utilizing this
approach, we were able to synthesize 1 in two steps from 2-AH
and N-Boc-4,5-dibromo-2-formyl pyrrole (Scheme 1).6 Wein-
reb reported the third total synthesis using a variation of his
original method to overcome the low yield of the last step but
still employing a 6π-2-azatriene electrocyclization for the
’ RESULTS
The PictetꢀSpengler reaction between the 2-AH and an
aldehyde was utilized for the synthesis of a range of tetrahy-
droageladine A analogues (Table 1). While the reaction is
accelerated by the presence of a Lewis acid such as Sc(OTf)3,6
it also proceeds under base catalysis (triethylamine, potassium
carbonate) and with no catalyst, albeit at a slower rate. Utilizing
1.5 equiv of triethylamine in ethanol was found to be the most
robust method for condensing various heterocyclic aldehydes
with 2-AH or N-boc-2-AH. Dehydrogenation was achieved in
reasonable to good yield using chloranil in chloroform for all the
Received: January 13, 2011
Published: March 17, 2011
r
2011 American Chemical Society
2492
dx.doi.org/10.1021/jm200039m J. Med. Chem. 2011, 54, 2492–2503
|

Journal of Medicinal Chemistry
ARTICLE
Scheme 1. Biomimetic Synthesis of Ageladine A
Table 1. Synthesis of Ageladine A and Analogues
a R1CHO, TEA, EtOH, rt, 3ꢀ6 h. b Chloranil, CHCl3, reflux, 12ꢀ36 h. c Pd/C, EtOH, reflux, 16ꢀ24 h. d One-pot method; R1CHO, EtOH, then Pd/C,
rt to reflux, 24 h.
analogues except those containing a pyridine (or quinoline),
where the yields were lower. The use of palladium on charcoal for
dehydrogenation gave excellent yields for the pyridine and
quinoline analogues.
2493
dx.doi.org/10.1021/jm200039m |J. Med. Chem. 2011, 54, 2492–2503

Journal of Medicinal Chemistry
ARTICLE
Table 2. MMP Inhibition (IC50) Results for Ageladine A and Analogues
a Percentage inhibition at 20 μg/mL.
A one-pot procedure, entailing stirring 2-AH and an aldehyde
in ethanol for 6 h, after which Pd/C was added and the mixture
refluxed for 24 h. This gave good yields of all the ageladine A
analogues except for pyrrole aldehydes, where the two-step
procedure gave better yields (Table 1, last column)
Biological Screening of Ageladine A Analogues. Matrix
Metalloprotease Inhibition. Compound 1 shows inhibitory activ-
ity against MMP-1, -2, -8, -9, -12, and -13 with the IC50 values of
2.0, 1.2, 0.39, 0.79, 0.33, and 0.47 μg/mL, respectively.9
Eleven analogues (2ꢀ12) were screened for their potential
inhibition of MMP-2 and MT1-MMP (MMP-14) (Table 2).
Compounds where the pyrrole ring is replaced by other five-
membered rings showed comparable or slightly reduced activity.
For example, the thiophene analogue (4) showed activity similar
to 1. When the sulfur atom is replaced by oxygen (3), however,
there is almost an order of magnitude decrease in activity. The
thiophene regioisomers (4 and 6) also showed that much better
activity is obtained if the heteroatom is in the 2-position. The
analogues where the five-membered ring is replaced with pyr-
idine (7, 8), benzothiophene (11), and quinoline (9) showed
virtually no activity, indicating that a small ring size may be
important for activity. Interestingly, the indole derivative (12)
did show selective activity against MT1-MMP, indicating that a
larger ring size might be accommodated by some MMP enzymes.
However, none of the analogues showed better activity against
MMP’s than the natural product.
Table 3 and Table S1 (Supporting Information) show the
comparative data for ageladine A and eight analogues with
positive and negative controls using the rat aorta outgrowth in
vitro assay.10
The most potent analogue in the angiogenesis assays was 7,
which is 14ꢁ more active than PI-88 (phosphomannopentaose
sulfate; 13),11 the positive control, and 3ꢁ more active than
ageladine A in the angiogenesis assay. Several related compounds
were synthesized from histidine and histamine and 2-formylpyr-
idine (14ꢀ16), which showed weaker activity but still better than
13. Adding a lipophilic bromine onto the pyridine ring (16)
reduced activity but a carboxylate on the lower pyridine (15)
seems to be tolerated.
Cytotoxicity Screening. Ageladine A and analogue 7 were
tested by the NCI against a panel of 60 human cancer cell lines
for anticancer activity (Figure S2, Supporting Information).12
Ageladine A was found to be nontoxic at 10 μM and was not
tested further. Compound 7 inhibited the growth of one cell line
(renal cancer CAKI-1) by 95% and stimulated the growth of one
cell line (Melanoma MALME-3M) by 30%. Further testing of 7
(100 μM to 10 nM) indicated that no LD50 could be found for
any cell line at concentrations used in the antiangiogenic assay
(Table S4, Supporting Information). Only five high LD50 values
(25ꢀ92 μM) could be found for NCI-H460, M14, SK-MEL-5,
IGROV1, and CAKI-1 cell lines. All cell lines showed some growth
inhibition in the low μM concentration range (1ꢀ10 μM).
Protein Kinase Inhibition. Three compounds (ageladine A, 7,
and 9) were screened against 402 protein kinases by Ambit
Biosciences (Table S2, Supporting Information), showing the
percentage of ATP binding remaining after treatment with the
Angiogenesis Inhibition. Previous studies have shown that 1
(10 μg/mL) inhibits angiogenesis from vascular progenitor cells in
3D culture using a type-I collagen gel, but its activity was not
quantified nor compared to any known antiangiogenic drug.
2494
dx.doi.org/10.1021/jm200039m |J. Med. Chem. 2011, 54, 2492–2503

Journal of Medicinal Chemistry
ARTICLE
Table 3. Anti-angiogenic Activity of Ageladine A and Selected Derivativesa
a Control (just DMSO) was used as a standard to calculate the percentage inhibition. 13, IC50 = 100 μg/mL was the positive control.
test compound. Values less than 50 suggest a KD in the nM range.
Ageladine A was tested at 10 μM and 1 μM and found to be very
selective, with only DYRK1A, DYRK2, TYK2 (JH2 domain), and
YSK4 showing activity of 5% or less at 10 μM (Table S2,
Supporting Information). Compound 7 inhibited the same four
kinases, but compound 9 had a different profile except that it also
inhibited DYRK1A and TYK2.
9, using 11 concentrations and a blank (in duplicate). These data
(Table 4 and Supporting Information) confirmed that a high
nanomolar dissociation constant of 7 against DYRK1A, YSK4,
and also RPS6KA1(domain 2). Analogue 9 also had a high nM
KD against DYRK1A, TYK2, PIK4CB, and PIK3C2G.
To further investigate the possible inhibitory activity of
ageladine A, and its analogues, against protein kinases, we
screened our compounds against a small panel of kinases in a
functional assay using natural or synthetic substrates and
From this primary screen, 19 kinases were selected for
quantitative dissociation constant determination against 7 and
2495
dx.doi.org/10.1021/jm200039m |J. Med. Chem. 2011, 54, 2492–2503

Journal of Medicinal Chemistry
ARTICLE
Table 4. Dissociation Constants (KD) for Compounds 7 and
9 for Selected Kinases
Palladium on carbon (Pd/C) has been used for the dehy-
drogenation of tetrahydro-β-carbolines using ethanol as a
solvent.14 This method was found to give excellent yields for
the pyridine analogues, where all other methods had failed.
Similar results were obtained for the other analogues
(Table 1). The advantage of Pd/C over chloranil for dehy-
drogenation is that the intermediates are far more soluble in
ethanol than the halogenated solvents required for chloranil
oxidation and so do not require Boc-protection and the
workup is trivial. As the PictetꢀSpengler reaction was found
not to require a catalyst, and because both steps now require
the same solvent, we investigated the possibility of a one-pot
total synthesis of ageladine A. Disappointingly, the reaction
proceeded with a very low yield for the natural product and
any analogue that contained a pyrrole. Boc protection of the
pyrrole did improve the yields. but the results were still
disappointing. Adding triethylamine or Sc(OTf)3 did not
improve the yields. However, to our delight, all other analogues
gave excellent yields of the desired product using this one-pot
procedure (Table 1). For the pyrrole containing systems, the
two-step procedure, employing Boc-protection and choranil
dehydrogenation, was found to deliver the best results, sug-
gesting that the pyrrole-NH interferes with the Pd dehydro-
genation. The success of the one-pot method allowed us to
quickly generate a range of analogues and opens the door to
industrial production of these compounds and further
analogues.
Matrix Metalloprotease Inhibition. MMPs are a small family
of zinc and calcium dependent proteases that are implicated in
many disease states. The production of excess MMPs leads to the
degradation of the extracellular matrix in connective tissue, which
leads to disease symptoms including the loss of cartilage in
osteoarthritis15 and rheumatoid arthritis,16 bone matrix degrada-
tion in osteoporosis, remodeling in Alzheimer’s disease,17 and
cancer cell invasion and metastasis.18 As the degradation of the
extracellular matrix is critical for malignant tumor growth,
invasion, metastasis, and angiogenesis,19 MMP inhibition has
become an emerging target for the treatment of cancers. To date,
there are 27 known MMPs in humans and animals. On the basis
of function and substrate specificity, they are classified as
collagenases (MMP-1, -8, and -13), gelatinases (MMP-2 and -
9), stromelysins (MMP-3, -10, and -11), membrane type (MT)
MMPs (MT1 to MT6, MMP-14 to -17, -24, and -25), matrilysin
(MMP-7), macrophage metalloelastases (MMP-12 and -19), and
enamelysin (MMP-20).20,21
compound
kinase target
7
9
(Ambit gene symbol)
KD (nM)
KD (nM)
AAK1
1500
BMPR1B
720
CAMK2G
40000a
40000
40000
7500
CAMKK1
CAMKK2
CDK7
CDKL5
40000
1200
300
CLK2
DYRK1A
290
DYRK2
970
IRAK3
8900
160
PIK3C2G
PIK4CB
350
PRKCE
40000
530
RPS6KA1(Kin.Dom.2-C-terminal)
TYK2 (JH2domain-pseudokinase)
290
ULK1
24000
220
YANK2
40000
YSK4
a 40000 indicates no binding observed.
radioactive ATP to measure any reduction in the rate of phosphor-
ylation (Table 5). 1 was found to have moderate activity against
DYRK1A and Pim 1 and weak activity against CK1 (not in the panel
of 402 kinases tested by Ambit). Interestingly, the pyridine analogue
(7) showed improved activity against CK1, which may be related to
its ability to bind metal ions (Figure 1). Furthermore, while 1 has no
activity against CDK5 and CDK1, most of the analogues did, with the
pyridine analogue (7) again showing the best activity. The quinoline
derivative (9) showed selective activity against DYRK1A.
’ DISCUSSION
Synthesis. Of the reported syntheses, the biomimetic ap-
proach has proved the most efficient.6,13 Our aim was now to
shorten the process to a one-pot reaction and expand our
methodology to the synthesis of a number of analogues, which
replace the pyrrole with other heterocycles, and thus investigate
the role of the dibromopyrrole moiety in the biological activity of
ageladine A.
The yields obtained for the tetrahydro intermediates were
excellent except for those that contained pyrrole/indole, in which
case, yellow side products were obtained, reducing the yield. Boc
protection did not help but did improve the solubility of the
product for the next step. Dehydrogenation with chloranil was
achieved in reasonable to good yield for all the analogues except
those containing a pyridine (or quinoline). Other oxidizing
agents such as S8/DMSO, permanganate, or DDQ were not
successful with any of the analogues. Similarly, solvents other
than chloroform also reduced the yield of the desired products.
We also tried the two-step (IBX, MnO2) method of Ando8 on the
2-furyl and 2-thiophenyl analogues but could only obtain low
yields of the dehydrogenated product.
Ageladine A has shown inhibitory activity against MMP-1, -2,
-8, -9, -12, and -13, suggesting they might be useful in cancer
research and therapy.9 The previous reports on the synthesis of
analogues and their biological screening against MMPs suggested
the importance of a free guanidine nucleus, pyrrole NH, and the
presence of bromine atoms on the biological function of 1.1,4,8
Collectively, these studies indicated that removal of one or both
bromines from ageladine A, or bromo-regioisomers, resulted in
loss of activity against MMPs. Similarly, methylation of one or
more nitrogens reduced or completely eliminated activity. The
authors suggested that the pyrrole NH, two bromines and
guanidine are essential for MMP inhibition. Our studies do not
support this conclusion as, for example, the thiophene analogue
(4) showed comparable activity to ageladine A. However, mov-
ing the sulfur atom (6) resulted in an order of magnitude drop in
activity, suggesting that a heteroatom in the 2-position is
important for activity.
2496
dx.doi.org/10.1021/jm200039m |J. Med. Chem. 2011, 54, 2492–2503

Journal of Medicinal Chemistry
ARTICLE
Table 5. Effect of Ageladine A and Analogues on the Activity of Five Protein Kinasesa
a Compounds were tested at various concentrations up to 100 μM and IC50 values (μM) were calculated from the doseꢀresponse curves. ꢀ, inactive
at 100 μM
Figure 1. Titration of the free base of the 2-pyridyl analogue (7) with ZnCl2 in methanol-d4.
Our results indicate that the presence of pyrrole NH and bromine
atoms is not absolutely necessary for MMP inhibitory activity and
that removal of bromines (ie 1 going to 2) results in loss of activity
that could be due to a change in lipophilicity or steric bulk.
2497
dx.doi.org/10.1021/jm200039m |J. Med. Chem. 2011, 54, 2492–2503

Journal of Medicinal Chemistry
ARTICLE
Most of the MMP inhibitors reported so far inhibit this family
of enzyme by chelation of the zinc(II) ion at the proteases active
site and are thus of restricted medicinal utility. Ageladine A is of
interest because it has been shown to inhibit MMPs without
binding zinc.1 In contrast, 7 strongly complexes zinc, as deter-
mined by NMR spectroscopy (Figure 1). Adding 0.05 equiv of
zinc chloride to a solution of 7 in CD3OD resulted in splitting of
the aromatic signals into two groups, consistent with the forma-
tion of a organometallic complex. Further addition of zinc led to
considerable broadening of all signals. As 7 contains a 2,20-
dipyridyl moiety, this is not a totally unexpected result but
interesting because this compound showed little inhibition of
either MMP tested (Table 2), confirming that ageladine A and its
analogues do not inhibit MMPs through zinc complexation.
Angiogenesis Inhibition. Angiogenesis plays a critical role in
cancer growth and metastasis by providing tumors with an
adequate blood supply.22,23 An attractive recent strategy for
new anticancer drugs is to target angiogenesis rather than directly
eliminate tumor cells with highly cytotoxic agents.22 The anti-
angiogenic activity of ageladine A was originally demonstrated by
experiments carried out in vitro in a vascular organization model
using mouse ES cells.1
At 10 μg/mL, ageladine A showed inhibition of vascular
formation from aggregates of vascular progenitor cells in 3D
culture using a type-I collagen gel, but its activity was not
quantified nor compared to any known antiangiogenic drug. In
this study, advantage was taken of a novel in vitro angiogenesis
assay10,24 to quantify the antiangiogenic activity of ageladine A
and eight related compounds (Table 3). The assay involves
culturing rat aorta vessels in a fibrin gel and measuring the effect
of different substances on the growth of microvessels from the
severed ends of the vessel fragment into the surrounding fibrin
gel. This assay has been successfully used to screen for anti-
angiogenic compound in the past and was used to discover the
antiangiogenic sulfated oligosaccharide, 13,11 which has under-
gone extensive clinical trials in cancer patients,25 and was used as
a positive control. Using this assay, we have quantified the
antiangiogenic activity of ageladine A (IC50) and 11 analogues
and compared these to the known drug 13 (Table 3, Table S1,
Supporting Information). The most striking feature is that there
seems to be no correlation between MMP inhibition and
inhibition of angiogenesis. For example, 11 showed virtually no
MMP inhibition but antiangiogenic activity better than the
natural product. Similarly, 4 showed the best MMP inhibition
of the analogues yet showed only weak inhibition of angiogenesis.
This complete lack of correlation suggests that the antiangiogenic
activity of ageladine A is not closely related to its MMP inhibition
or, at least, that MMP inhibition is not the natural product’s
primary mode of activity. Compounds 14ꢀ16 also showed that
the guanidine, previously reported to be required for MMP
inhibition, is not essential for antiangiogenic activity.
against all cell lines. These data indicated that while these
compounds might inhibit growth of cancer cell lines they are
not cytotoxic.
Kinase Screening. Protein kinases play an important role in
the regulation of numerous cell functions including cell division
cycle, gene expression, exocytosis, neuronal functions, mem-
brane transport, and cellular differentiation. The human genome
encodes 518 protein kinases comprising 90 tyrosine kinases, 338
serine/threonine kinases, and 50 sequences lacking a functional
catalytic site.26ꢀ29 Deregulation of kinase activities is frequently
observed during the development of human diseases such as
cancer, diabetes, Alzheimer’s disease, inflammation, etc. This has
stimulated an intensive search for low molecular weight pharma-
cological inhibitors (drugs) for protein kinases.30ꢀ33 As the
strong antiangiogenic activity of ageladine A cannot be fully
explained by moderate MMP inhibition, we postulated that, as
yet unidentified, protein kinases might constitute additional
targets. In addition, the structure of ageladine A is reminiscent
of hymenialdisine, a marine natural product that is a potent
inhibitor of CDKs, Mek1, GSK3β, CK1, and Chk1 kinases.34,35
An initial screen of 402 kinases showed moderate but highly
selective activity. The assay, developed by Ambit Biosciences
(San Diego, CA), uses phage-displayed natural and mutant
kinases to assess KD values against immobilized ATP.36 The
assay has been extensively validated and relies on the ability for a
small molecule to bind to the ATP binding site of the tested
kinases.37 The selectivity scores (Table S3, Supporting In-
formation) of greater than 35% inhibition at 1 μM for ageladine
A (0.008), 7 (0.022), and 9 (0.008) are better than the most
selective kinase drug (lapatinib S = 0.010).37 Interestingly, the
natural product inhibited mainly Ser/Thr kinases (70% of the top
10) and 7 inhibited almost exclusively these kinases (90% of top
10), while 9 inhibited mostly Tyr or inositol kinases (60% of top
10). Functional screening against a small panel of disease related
kinases (Table 5) showed moderate activity against a small
number of kinases, particularly DYRK1A.
These data, taken together, show moderate inhibition of
selective kinases with DYRK1A being a common thread. Agela-
dine A and its antiangiogenic analogues may be inhibitors of
DYRK1A or a related kinase. DYRK1A is a member of the dual-
specificity tyrosine phosphorylation-regulated kinase family that
contains a nuclear targeting signal sequence, a protein kinase
domain, a leucine zipper motif, and a highly conservative 13-
consecutive-histidine repeat. It catalyzes its own autophosphor-
ylation on serine/threonine and tyrosine residues. It may play a
significant role in a signaling pathway regulating cell proliferation
and has recently been found to be involved in reduced tumor
angiogenesis in Down’s Syndrome.38
’ CONCLUSION
Cytotoxicity Screening. The observed antiangiogenic activity
could be related to general cytotoxicity resulting in reduced
vascular outgrowths in the angiogenesis assay due to cell death.
To test this, ageladine A and 7 were tested against a panel of 60
human cancer cell lines. Little if any cytotoxicity was observed,
but some growth inhibition was apparent for most cell lines at
low μM concentration (Figure S2, Supporting Information). The
five-dose assay over 48 h (Table S4, Supporting Information) for
7 differentiated growth inhibition (retardation of growth but no
cell death) from cytotoxicity (retardation of growth due to cell
death) and confirmed that 7 was noncytotoxic at <100 μM
We have successfully developed an industrially useful, one-pot
method for the synthesis of ageladine A and analogues using a
biomimetic PictetꢀSpengler cyclization between 2-aminohista-
mine/histamine/histadine and various heterocyclic aldehydes.
The method is concise, robust, relatively cheap, and fast com-
pared to previously published methods and can be used to make a
diverse range of analogues by simply changing the starting
materials. The robustness of the methodology has been demon-
strated by the successful synthesis of 14 ageladine A analogues
that differ only in the pendant heterocycle and the substitution
pattern on the imidazole and pyridine rings. These analogues
2498
dx.doi.org/10.1021/jm200039m |J. Med. Chem. 2011, 54, 2492–2503

Journal of Medicinal Chemistry
ARTICLE
have been tested for their MMP inhibitory effect. While none are
as active as the natural product, several have comparable activity
that has allowed a preliminary structureꢀactivity relationship to
be developed that can be used to synthesize second generation
analogues. We have also shown, for the first time, that ageladine A
also has kinase inhibitory activity and that some of the analogues
are more active than the parent natural product in angiogenesis
and kinase assays. These results suggest that ageladine A may
target a kinase rather than an MMP to achieve its reported
antiangiogenic activity. Testing of the most active kinase-inhibi-
tory analogues showed that the 2-pyridine analogues (7, 14, 15)
were highly active as antiangiogenic compounds. Surprisingly,
the regioisomer 3-pyridine analogue (8) showed no kinase nor
angiogenic activity. These data suggest that ageladine A may
constitute a new kinase-inhibitory scaffold from which more
potent and selective inhibitors might be designed.
20 mL), as many of these compounds were found to have a high affinity
for charcoal. The combined filtrates were concentrated under reduced
pressure and subject to flash chromatography over silica gel using a
gradient of 5:95ꢀ20:80 (MeOH:DCM saturated with ammonia),
yielding the ageladine A analogues (Table 1), which were further
purified by HPLC as their TFA salts.
General Procedures for the One-Pot Synthesis of Agela-
dine A and Analogues. d. A mixture of 2-aminohistamine (0.11
mmol) and aldehyde (Table 1; 0.13 mmol) was stirred in ethanol at
room temperature for 6 h, after which 10% Pd/C (24 mg) was added and
the reaction mixture refluxed for a further 24 h. The mixture was then
filtered through a Celite pad and washed with methanol (3 ꢁ 25 mL)
and toluene:methanol (50:50; 2 ꢁ 20 mL). The combined filtrates were
concentrated under reduced pressure and chromatographed over silica
gel using a gradient of 5:95ꢀ20:80 (MeOH:DCM saturated with
ammonia), giving 1ꢀ12 (Table 1) respectively.
e. To a stirred solution of histamine or histidine (0.180 mmol) in
ethanol (2 mL) and aldehyde (0.180 mmol) was added potassium
carbonate (2.16 mmol) and molecular sieves (3 Å; 200 mg). The
reaction mixture was refluxed for 24 h, after which 10% Pd/C (60
mg) and cyclopentene (120 μL) were added. The reaction mixture was
allowed to reflux for 2 days. The mixture was then filtered through a pad
of Celite and washed with methanol (3 ꢁ 25 mL) and toluene:methanol
(50:50; 2 ꢁ 20 mL). The combined filtrates were concentrated under
reduced pressure and chromatographed over silica using a gradient of
2:98ꢀ15:85 (methanol:chloroform saturated with ammonia) to yield
14ꢀ16.
’ GENERAL METHODS
Chloroform, dichloromethane, ethanol, and methanol were obtained
from Froline Australia and were distilled before use. Merck Silica Gel 60
(230ꢀ400 mesh) was used for flash chromatography. All the reagents
were obtained from commercial sources and used without further
purification. NMR spectra were recorded in 5 mm Pyrex tubes
(Wilmad, USA) on either a DPX-400 400 MHz or DPX-600 600
MHz spectrometer (Bruker, Germany). IR spectra were recorded on a
Paragon PE1000 FTIR spectrophotometer (Perkin-Elmer, USA). Che-
mical shifts of 1H NMR and 13C NMR were referenced to the solvents
peaks: δH 3.30 and δC 49.0 for CD3OD and δH 2.49 and δC 39.2 for
DMSO-d6. A Phenomenex reverse phase HPLC column (Gemini, 5 μm,
150 mm ꢁ 2 mm) was used for LC-MS (LCMS-2010 EV equipped with
a photodiode array detector of electrospray source) using water/
acetonitrile/0.1% formic acid as mobile phase. All test compounds were
purified to >95% purity by reverse phase HPLC using a 600E multi-
solvent delivery system and a 490 programmable multiwavelength
detector (Waters, USA), using a Phenomenex Gemini column (10 μ,
250 mm ꢁ 10 mm) using a gradient from 5% acetonitrile/0.05% TFA to
80% acetonitrile/0.05% TFA. Purity was confirmed by 1H and 13C NMR
spectroscopy (pp S20ꢀS44, Supporting Information). All structures of
all final compounds and intermediates were confirmed by 13C and/or 1H
NMR spectroscopy and high-resolution mass spectrometry, measured
on Bruker Apex 4.7T FTICR-MS. Ageladine A and all the analogues
were characterized and stored as their TFA salts after HPLC.
Zinc Titration. The free base of analogue 7 was generated from the
HPLC purified TFA salt by filter chromatography on silica gel
(methanol/dichloromethane/ammonia). The free base (4.9 mg, 22
μmol) was then dissolved in CD3OD (300 μL), followed by the addition
of 0, 10, and 30 μL of ZnCl2 (0.11 M in CD3OD). 1H NMR spectra were
recorded on a Bruker DPX400 400 MHz NMR spectrometer.
MMP Inhibition Assays. Recombinant human MMP-239 and
MT1ꢀMMP40 were prepared following the literature procedures. The
fluorescent substrate; MOCAc-Pro-Leu-Gly-Leu-A2pr(Dnp)-Ala-Arg-
NH2 was purchased from Peptide Institute Inc., Osaka. Inhibition assays
were carried out by a modified procedure of Knight et al.41 Test samples
(2 μL), including positive (Batimostat, BB-94; IC50 0.29, 1.45 μg/mL
for MMP-2 and MT1-MMP respectively)42 and negative (1% DMSO)
controls, were added to wells of 96-well microtiter plates, each of which
contained 100 μL of TNC buffer (50 mM Tris-HCl pH 7.5, 150 mM
NaCl, 10 mM CaCl2, 0.02% NaN3 as a preservative and 0.05% Brij-35
detergent). Aliquots (50 μL) of enzyme solution (5 ng/mL for MMP-2
and 40 ng/mL for MT1-MMP) were added to this solution and
preincubated at 37 °C for 10 min. After preincubation, 50 μL of
substrate solution (10 μM) was added to the mixture to begin the
reaction. Fluorescence values were measured at an excitation of 328 nm
and an emission of 393 nm after incubation at 37 °C for 3 h. As all the
ageladine A analogues are also fluorescent at 390 nm, a reference sample
with all components except the enzyme was used as a blank and
subtracted from all assay readings.
Angiogenesis Assay. An in vitro angiogenesis assay was used in
this study to assess the antiangiogenic activity of the different com-
pounds. The procedure, which has been described in detail previously,24
used rat aorta fragments and is based on a procedure previously
described by Brown et al.10 using human placenta vessel fragments.
Briefly, thoracic aortas were excised from 3ꢀ9 month-old female Fischer
rats, cross-sectioned at 1 mm intervals and embedded individually in
0.5 mL of a fibrin gel containing 5 μg/mL of aprotinin (to prevent
fibrinolysis) in each well of a 48-well culture plate. Immediately after
vessel embedding, 0.5 mL/well of medium M199, supplemented with
20% fetal calf serum, 0.1% ε-aminocaproic acid, 1% L-glutamine,, and
antibiotics, was added. Each test compound (10 mg/mL, dissolved in
General Procedure for PictetꢀSpengler Cyclization of
2-Aminohistamine. a. To a stirred solution of 2-AH (0.11 mmol)
or Boc-2AH (0.11 mmol) in ethanol (5 mL) was added triethylamine
(0.16 mmol) and aldehyde (0.13 mmol). The reaction mixture was
stirred at rt for 3ꢀ6 h, after which the solvent was removed under
reduced pressure and the reaction mixture was further subjected to flash
chromatography over silica gel using a gradient of 5:95ꢀ30:70 (MeOH:
DCM saturated with ammonia) yielding the tetrahydro-intermediates
(56ꢀ92%).
General Procedures for the Dehydrogenation of Tetrahy-
dropyridine Intermediate. b. To a stirred solution of the tetrahy-
dro-intermediate (0.15 mmol) in chloroform (10 mL) was added
chloranil (0.30 mmol) and the reaction mixture refluxed for 15ꢀ24 h.
Solvent was removed on a rotary evaporator and the residue obtained
further purified by flash chromatography over silica gel with a gradient of
5:95ꢀ15:85 (MeOH:DCM saturated with ammonia), giving ageladine
A analogues (Table 1).
c. To a stirred solution of tetrahydro-intermediate (0.15 mmol) in
ethanol was added 10% Pd/C (30 mg) and the reaction mixture refluxed
for 18ꢀ24 h. The mixture was then filtered through a Celite pad and
washed with methanol (3 ꢁ 25 mL) and toluene:methanol (50:50; 2 ꢁ
2499
dx.doi.org/10.1021/jm200039m |J. Med. Chem. 2011, 54, 2492–2503

Journal of Medicinal Chemistry
ARTICLE
DMSO) was added to the medium (maximum final concentration of test
compound 100 μg/mL), and each treatment was performed in six
replicates. Control cultures received medium containing 0.1% DMSO
but without any test compound. The antiangiogenic compound, 13 (100
μg/mL), was included as a positive control. Vessels were cultured at
37 °C in 5% CO2 in air for 5 days and the medium, with or without test
compound, was changed on day 4. Vessel growth was quantified
manually under 40ꢁ magnification on day 5, with growth being
estimated as the percentage of the field (ꢁ40) around the vessel
fragment that was occupied by vessel outgrowths. Inhibition of 100%
indicates that no vessel outgrowth was observed, and 0 indicates the
same amount as control, allowing the assay to be used for angiogenic as
well as antiangiogenic compounds. The IC50 values were calculated by
fitting the data to a standard Hill plot, using the blank as the zero value
(nominally 0.01 μg/mL) and constraining the top value to be 100
(GraphPad Prism version 4.0).
phosphocellulose paper and, 20 s later, the filters were washed five times
(for at least 5 min each time) in a solution of 10 mL of phosphoric acid/L
of water. The wet filters were counted in the presence of 1 mL ACS
(Amersham) scintillation fluid.
CK1 (porcine brain, native) was assayed as described for CDK1 but
using the CK1-specific peptide substrate RRKHAAIGpSAYSITA,44
obtained from Millegen (Labege, France).
DYRK1A (rat, recombinant, expressed in E. coli as a GST fusion
protein) was purified by affinity chromatography on glutathione-agarose
and assayed as described for CDK1/cyclin B using myelin basic protein
(1 mg/mL) as a substrate.
Pim1 (human recombinant) was assayed with histone H1 as de-
scribed for CDK1 and CDK5.
N0-tert-Butoxycarbonyl-5-(2-amino-4,5,6,7-tetrahydroimi-
dazo[4,5-c]pyridine-4-yl)-2,3-dibromopyrrole. To a stirred so-
lution of 2-aminohistamine (10 mg, 79 μmol) in ethanol (5 mL) was
added N-Boc-4,5-dibromo-2-formylpyrrole (27.9 mg, 79 μmol) and
scandium triflate (3.8 mg, 7.9 μmol), and the reaction mixture stirred at
room temperature for 5 h. The solvent was then removed in vacuo
leaving a brown residue, which was purified by column chromatography
on silica gel using a gradient of 5:95ꢀ15:85 (MeOH:DCM saturated
with ammonia) to give tetrahydroageladine A as a yellow solid (16 mg,
Cytoxicity Screening. Was carried out by the NCI (Cancer Drug
Discovery and Development Program). The screening is a two-stage
process, beginning with the evaluation of all compounds against the 60
cell lines at a single dose of 10 μM. Compounds which exhibit significant
growth inhibition are evaluated against the 60 cell panel at five
concentration levels where LD50 and GI50 are quantified.43
1
44%). UV (acetonitrile/water) λmax 220 nm, H NMR (400 MHz,
Kinase Screening. Was conducted at Ambit Biosciences, San
Diego, CA.36 For most assays, kinase-tagged T7 phage strains were
grown in parallel in 24-well blocks in an Escherichia coli host derived from
the BL21 strain. E. coli were grown to log-phase and infected with T7
phage from a frozen stock (multiplicity of infection = 0.4) and incubated
with shaking at 32 °C until lysis (90ꢀ150 min). The lysates were
centrifuged (6000g) and filtered (0.2 μm) to remove cell debris. The
remaining kinases were produced in HEK-293 cells and subsequently
tagged with DNA for qPCR detection. Streptavidin-coated magnetic
beads were treated with biotinylated small molecule ligands for 30 min at
room temperature to generate affinity resins for kinase assays. The
liganded beads were blocked with excess biotin and washed with
blocking buffer (SeaBlock (Pierce), 1% BSA 0.05% Tween 20, 1 mM
DTT), to remove unbound ligand and to reduce nonspecific phage
binding. Binding reactions were assembled by combining kinases,
liganded affinity beads, and test compounds in 1ꢁ binding buffer
(20% SeaBlock, 0.17ꢁ PBS, 0.05% Tween 20, 6 mM DTT). Test
compounds were prepared as 40ꢁ stocks in 100% DMSO and directly
diluted into the assay. All reactions were performed in polypropylene
384-well plates in a final volume of 0.04 mL. The assay plates were
incubated at room temperature with shaking for 1 h, and the affinity
beads were washed with wash buffer (1ꢁ PBS, 0.05% Tween 20). The
beads were then resuspended in elution buffer (1ꢁ PBS, 0.05% Tween
20, 0.5 mM nonbiotinylated affinity ligand) and incubated at room
temperature with shaking for 30 min. The kinase concentration in the
eluates was measured by qPCR.
DMSO-d6) δ 11.61 (bs, 1H), 10.18 (bs, 1H), 5.97 (bs, 0.5H), 5.78 (bs,
0.5H), 5.52 (bs, 0.5H), 5.06 (bs, 2H), 4.11 (bs, 0.5H), 3.97 (bs, 1H),
3.06 (m, 1H), 2.5 (m, 1H), 2.33 (bm, 1H), 1.42 (s, 9H). 13C NMR (100
MHz, DMSO-d6) δ 153.8, 149.2, 134.1, 125.0, 120.8, 109.6, 98.6, 96.5,
79.2, 49.1, 48.3, 39.1, 28.1 (3C), 22.0; m/z 360 (48%), 362 (100) 364
(52). HRMS found [M þ Naþ] 483.9777, C15H19N5O279Br81BrNa
requires 483.9777.
Ageladine A (1). To a stirred solution of tetrahydroageladine A (10
mg, 21.6 μmol) in chloroform (5 mL) was added chloranil (15.9 mg, 65
μmol), and the reaction mixture was heated at 80 °C (reflux) for 8 h. The
solvent was removed in vacuo and the yellow residue subjected to
column chromatography over silica using a gradient of 5:95ꢀ15:85
(MeOH:DCM saturated with ammonia) to yield pure ageladine A (1) as
a yellow solid (5 mg, 65%). Ageladine A was characterized as its TFA salt
(HPLC gradient 1:9ꢀ9:1 accetonitrile/water/0.1% TFA) for compar-
ison to the original natural product. UV (acetonitrile/water) λmax 210,
220 (sh), 248, 275 (sh), 290, 363, 380 (sh) nm. 1H NMR (400 MHz,
MeOD) δ 8.06 (d, J = 6.4 Hz, 1H), 7.42 (d, J = 6.4 Hz, 1H), 7.17 (s, 1H).
13C NMR (100 MHz, MeOD) δ 160.8, 147.2, 136.7, 133.0, 128.5, 125.6,
115.1, 107.8, 105.4, 102.3; m/z 356 (53%), 358 (100) 360 (48).
N0-tert-Butyl-2-(2-amino-4,5,6,7-tetrahydro-1H-imidazo-
1
[4,5-c]pyridine-4-yl)1H-pyrrole-1-carboxylate. H NMR (400
MHz, CDCl3) δ 7.15 (dd, J = 3.3, 1.8 Hz, 1H), 5.99 (t, J = 3.3 Hz, 1H),
5.82 (dd, J = 2.6, 1.8 Hz, 1H), 5.3 (s, 1H), 4.76 (bs, 2H), 3.01ꢀ2.80 (m,
2H), 2.54ꢀ2.28 (m, 2H), 1.58 (s, 9H). 13C NMR (100 MHz, CDCl3) δ
149.6, 148.1, 135.3, 125.8, 124.2, 121.9, 115.2, 109.9, 84.1, 49.0, 38.8,
28.0 (3C), 23.2. IR (KBr) 3345, 2978, 1736, 1622, 1478, 1331, 1158,
1119, 1061, 845, 729 cmꢀ1. HRMS found [M þ H]þ 304.1781,
C15H21N5O2 requires 304.1774.
Kinase Assay. Buffer A: 10 mM MgCl2, 1 mM EGTA, 1 mM DTT,
25 mM Tris-HCl pH 7.5, 50 μg heparin/mL. Buffer C: 60 mM β-
glycerophosphate, 15 mM p-nitrophenylphosphate, 25 mM Mops (pH
7.2), 5 mM EGTA, 15 mM MgCl2, 1 mM DTT, 1 mM sodium vanadate,
1 mM phenyl phosphate.
1
4-(Pyrrole-2-yl)-1H-imidazo[4,5-c]pyridin-2-amine (2). H
Kinase activities were assayed in buffer A or C, at 30 °C, at a final ATP
concentration of 15 μM. Blank values were subtracted and activities
expressed as percent of the maximal activity, i.e. in the absence of
inhibitors. Controls were performed with appropriate dilutions of
DMSO, which was used to dissolve all test compounds.
CDK1/cyclin B (native M phase starfish oocytes) and CDK5/p25
(recombinant human) were prepared as previously described.35 Kinase
activity was assayed in buffer C, with 1 mg histone H1/mL, in the
presence of 15 μM [γ-32P] ATP (3000 Ci/mmol, 10 mCi/mL) in a final
volume of 30 μL. After 30 min incubation at 30 °C, 25 μL aliquots of
supernatant were spotted onto 2.5 cm ꢁ 3 cm pieces of Whatman P81
NMR (400 MHz, CD3OD) δ 7.93 (d, J = 6.5 Hz, 1H), 7.34 (d, J = 6.5
Hz, 1H), 7.23 (dd, J = 2.6, 1.4 Hz, 1H), 7.18 (dd, J = 4.0, 1.4 Hz, 1H),
6.42 (dd, J = 3.9, 2.6 Hz, 1H). 13C NMR (100 MHz, CD3OD) δ 160.2,
146.1, 136.4, 131.6, 131.5, 125.5, 123.4, 112.5, 111.8, 104.2. HRMS
found [M þ H]þ 200.0932, C10H9N5 requires 200.0936.
N00-tert-Butyl-4-(furan-2-yl)-4,5,6,7-tetrahydro-1H-imidazo-
1
[4,5-c]pyridine-2-ylcarbamate. H NMR (400 MHz, CDCl3) δ
7.36 (dd, J = 1.7, 0.8 Hz, 1H), 6.26 (dd, J = 3.1, 1.8 Hz, 1H), 5.89ꢀ5.87
(m, 1H), 5.22 (s, 1H), 2.97 (ddd, J = 13.0, 6.0, 1.7 Hz, 1H), 2.84 (ddd, J =
13.0, 10.7, 4.6 Hz, 1H), 2.60ꢀ2.49 (m, 1H), 2.34 (ddd, J = 16.2, 4.5, 1.2
Hz, 1H), 2.24 (bs, 1H), 2.23 (bs, 1H), 1.29 (s, 9H). 13C NMR (100
2500
dx.doi.org/10.1021/jm200039m |J. Med. Chem. 2011, 54, 2492–2503

Journal of Medicinal Chemistry
ARTICLE
MHz, MeOD) δ 155.3, 150.8, 149.9, 141.4, 134.8, 116.2, 110.2, 108.0,
85.2, 50.3, 38.3, 27.6 (3C), 25.5. IR (KBr) 3443, 1724, 1647, 1389, 1350,
1145, 1110, 837, 729 cmꢀ1. HRMS found [M þ H]þ 305.1612,
C15H20N4O3 requires 305.1614.
8.43 (dd, J = 4.9, 1.6 Hz, 1H), 7.75ꢀ7.71 (m, 1H), 7.38 (ddd, J = 7.8, 4.9,
0.8 Hz, 1H), 4.89 (t, J = 1.7 Hz, 1H), 3.12ꢀ3.04 (m, 1H), 3.01ꢀ2.93 (m,
1H), 2.68ꢀ2.59 (m, 1H), 2.57ꢀ2.48 (m, 1H). 13C NMR (100 MHz,
CD3OD) δ 150.8, 150.5, 149.1, 139.9, 138.4, 127.1, 125, 124.7, 55.9,
42.2, 23.8. IR (KBr) 3317, 1620, 1574, 1475, 1425, 1094, 1028, 801,
713 cmꢀ1. HRMS found [M þ H]þ 216.1241, C11H13N5 requires
216.1249.
4-(Pyridine-3-yl)-1H-imidazo[4,5-c]pyridin-2-amine (8).
1H NMR (400 MHz, CD3OD) δ 9.28 (d, J = 1.9 Hz, 1H), 8.79 (dd,
J = 5.0, 1.5 Hz, 1H), 8.59ꢀ8.55 (m, 1H), 8.30 (d, J = 6.6 Hz, 1H), 7.76
(ddd, J = 8.0, 5.0, 0.8 Hz, 1H), 7.60 (d, J = 6.6 Hz, 1H). 13C NMR (100
MHz, CD3OD) δ 162.0, 151.5, 150.0, 149.7, 138.8, 137.6, 135.4, 133.8,
129.1, 125.7, 107.9. HRMS found [M þ H]þ 212.0928, C11H9N5
requires 212.0936.
4-(Quinoline-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]-
pyridine-2-amine. 1H NMR 400 MHz, CD3OD) δ 8.82 (d, J = 2.1
Hz, 1H), 8.17 (d, J = 2.1 Hz, 1H), 8.01 (dd, J = 8.5, 0.9 Hz, 1H), 7.89 (dd,
J = 8.1, 1.4 Hz, 1H), 7.75ꢀ7.70 (m, 1H), 7.60ꢀ7.55 (m, 1H), 5.00 (t, J =
1.8 Hz, 1H), 3.16ꢀ3.08 (m, 1H), 3.05ꢀ2.98 (m, 1H), 2.73ꢀ2.64 (m,
1H), 2.61ꢀ2.52 (m, 1H). 13C NMR (100 MHz, CD3OD) δ 152.4,
150.7, 148.2, 137.5, 136.5, 130.9, 129.4, 129.3, 128.9, 128.2, 126.8, 124.6,
55.9, 42.2, 23.8. IR (KBr) 3422, 2925, 1620, 1573, 1473, 1124, 861,
752 cmꢀ1. HRMS found [M þ H]þ 266.1406, C15H15N5 requires
266.1406.
4-(Quinoline-3-yl)-1H-imidazo[4,5-c]pyridin-2-amine (9).
1H NMR (400 MHz, CD3OD) δ 9.53 (d, J = 2.2 Hz, 1H), 9.11 (d,
J = 2.2 Hz, 1H), 8.33 (d, J = 6.4 Hz, 1H), 8.18ꢀ8.12 (m, 2H), 7.98ꢀ7.92
(m, 1H), 7.80ꢀ7.75 (m, 1H), 7.62 (d, J = 6.3 Hz, 1H). 13C NMR (100
MHz, CD3OD) δ 161.9, 149.7, 148.3, 139.7, 137.7, 135.8 (br), 135.5,
133.7 (br), 133.5, 130.2, 129.5, 128.8, 128.7, 125.5, 107.9. HRMS found
[M þ H]þ 262.1091, C15H11N5 requires 262.1093.
4-(Furan-2-yl)-1H-imidazo[4,5-c]pyridin-2-amine (3). 1H
NMR (400 MHz, CD3OD) δ 8.10 (d, J = 6.4 Hz, 1H), 7.92 (dd, J =
1.8, 0.7 Hz, 1H), 7.67 (dd, J = 3.6, 0.6 Hz, 1H), 7.44 (d, J = 6.4 Hz, 1H),
6.80 (dd, J = 3.5, 1.8 Hz, 1H). 13C NMR (100 MHz, CD3OD) δ 161.8,
150.3, 147.5, 145.4, 133.9, 133.4, 127.1, 116.9, 114.3, 107.1. HRMS
found [M þ H]þ 201.0777, C10H8N4O requires 201.0776.
N00-tert-Butyl-4-(thiophen-2-yl)-4,5,6,7-tetrahydro-1H-imi-
dazo[4,5-c]pyridine-2-ylcarbamate. 1H NMR (400 MHz,
CDCl3) δ 7.20 (dd, J = 5.0, 1.2 Hz, 1H), 6.89 (dd, J = 5.0, 3.5 Hz,
1H), 6.70ꢀ6.68(m, 1H), 5.80 (bs, 1H), 5.40 (s, 1H), 3.00 (ddd, J = 13.3,
6.2, 2.1 Hz, 1H), 2.91 (ddd, J = 13.3, 10.2, 4.8 Hz, 1H), 2.56 (dddd, J =
16.4, 10.1, 6.3, 1.9 Hz, 1H), 2.30 (dddd, J = 16.4, 4.8, 2.1, 0.6 Hz, 1H),
2.23 (bs, 1H), 1.29 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 150.6,
149.9, 147.6, 134.5, 126.2, 125.7, 124.5, 118.5, 85.4, 51.8, 38.4, 27.5
(3C), 25.6. IR (KBr) 3470, 2929, 1714, 1647, 1388, 1346, 1146, 1102,
838, 809, 729 cmꢀ1. HRMS found [M þ H]þ 321.1393, C15H20N4O2S
requires 321.1385.
4-(Thiophen-2-yl)-1H-imidazo[4,5-c]pyridin-2-amine (4).
1H NMR (400 MHz, CD3OD) δ 8.10 (d, J = 6.4 Hz, 1H), 8.09 (dd,
J = 3.8, 1.1 Hz, 1H), 7.88 (dd, J = 5.0, 1.1 Hz, 1H), 7.48 (d, J = 6.4 Hz,
1H), 7.32 (dd, J = 5.0, 3.8 Hz, 1H). 13C NMR (100 MHz, CD3OD) δ
161.3, 148.7, 136.9, 133.7, 133.0, 132.5, 132.2, 131.1, 129.3, 106.4.
HRMS found [M þ H]þ 217.0539, C10H8N4S requires 217.0548.
4-(Furan-3-yl)-1H-imidazo[4,5-c]pyridin-2-amine (5). 1H
NMR (400 MHz, CD3OD) δ 8.74 (m, 1H), 8.10 (d, J = 6.4 Hz, 1H),
7.81 (m, 1H), 7.49 (d, J = 6.4 Hz, 1H), 7.23 (m, 1H). 13C NMR (100
MHz, CD3OD) δ 161.4, 148.5, 147.5, 146.2, 137.8, 132.8, 131.2, 118.0,
108.7, 106.4; m/z 201 (100; M þ Hþ). HRMS found [M þ H]þ
201.0771, C10H8N4O requires 201.0776.
4-Phenyl-1H-imidazo[4,5-c]pyridin-2-amine (10). 1H NMR
(400 MHz, CD3OD) δ 8.13 (d, J = 5.4 Hz, 1H), 7.90 (d, J = 7.4 Hz, 2H),
7.54ꢀ7.48 (m, 2H), 7.46ꢀ7.41 (m, 1H), 7.19 (d, J = 5.5 Hz, 1H). 13C
NMR (100 MHz, CD3OD) δ 148.8, 136.3, 134.9, 131.9, 129.7, 129.4,
128.9, 120.0, 117.1, 114.2, 111.4, 107.5; m/z 211 (100; M þ Hþ).
HRMS found (M þ H)þ 211.0987, C12H10N4 requires 211.0984.
N00-tert-Butyl-4-(benzo[b]thiophen-3-yl)-4,5,6,7-tetrahy-
dro-1H-imidazo[4,5-c]pyridine-2-ylcarbamate. 1H NMR (400
MHz, CDCl3) δ 7.94 (d, J = 7.7 Hz, 1H), 7.85ꢀ7.81 (m, 1H),
7.42ꢀ7.29 (m, 2H), 6.83 (s, 1H), 5.86 (bs, 1H), 5.57 (s, 1H), 2.96
(ddd, J = 13.1, 5.9, 2.1 Hz, 1H), 2.82 (ddd, J = 13.1, 10.3, 4.7 Hz, 1H),
2.63ꢀ2.53 (m, 1H), 2.43ꢀ2.35 (m, 1H), 2.17 (bs, 1H), 1.02 (s, 9H).
13C NMR (100 MHz, CDCl3) δ 150.8, 150.0, 140.9, 137.9, 134.9, 124.4,
124.2, 124.1, 123.0, 122.2, 117.8, 85.1, 50.8, 38.6, 27.4 (3C), 25.8. IR
(KBr) 3453, 2928, 1722, 1627, 1369, 1326, 1263, 1138, 835, 765,
734 cmꢀ1. HRMS found [M þ H]þ 371.1547, C19H22N4O2S requires
371.1542.
4-(Benzo[b]thiophen-3-yl)-1H-imidazo[4,5-c]pyridin-2-amine
(11). 1H NMR (400 MHz, CD3OD) δ 8.29 (s, 1H), 8.27 (d, J = 6.5 Hz,
1H), 8.10ꢀ8.07 (m, 1H), 7.79ꢀ7.75 (m, 1H), 7.60 (d, J = 6.5 Hz, 1H),
7.55ꢀ7.48 (m, 2H). 13C NMR (100 MHz, CD3OD) δ 159.2, 158.8,
151.7, 141.7, 137.8, 135.8, 133.3, 126.9, 126.6, 124.2, 123.5, 117.4, 114.6,
109.0. HRMS found [M þ H]þ 267.0706, C14H10N4S requires
267.0704.
N00-tert-Butyl-3-(2-amino-4,5,6,7-tetrahydro-1H-imidazo-
[4,5-c]pyridine-4-yl)1H-indole-1-carboxylate. 1H NMR (400
MHz, CDCl3) δ 8.03 (d, J = 8.3 Hz, 1H), 7.42 (d, J = 7.7 Hz, 1H),
7.36 (s, 1H), 7.24ꢀ7.19 (m, 1H), 7.13ꢀ7.08 (m, 1H), 4.99 (s, 1H),
3.11ꢀ3.00 (m, 1H), 2.93ꢀ2.82 (m, 1H), 2.50ꢀ2.27 (m, 2H), 1.59 (s,
9H). 13C NMR (100 MHz, CDCl3) δ 149.7, 148.1, 135.8, 129.4, 124.5,
124.4, 122.6, 121.9, 119.7, 115.2, 83.7, 49.9, 49.0, 41.6, 28.2 (3C), 23.7.
IR (KBr) 3334, 2977, 1735, 1635, 1562, 1453, 1371, 1308, 1255, 1154,
N00-tert-Butyl-4-(thiophen-3-yl)-4,5,6,7-tetrahydro-1H-imi-
dazo[4,5-c]pyridine-2-ylcarbamate. 1H NMR (400 MHz, CDCl3)
δ 7.27ꢀ7.24 (m, 1H), 7.02 (dd, J = 4.9, 1.3 Hz, 1H), 6.82ꢀ6.80 (m, 1H),
5.80 (bs, 1H), 5.23 (s, 1H), 2.99ꢀ2.92 (m, 1H), 2.86ꢀ2.78 (m, 1H),
2.60ꢀ2.50 (m, 1H), 2.41ꢀ2.33 (m, 1H), 2.20 (bs, 1H), 1.24 (s, 9H). 13C
NMR (100 MHz, CDCl3) δ 150.5, 150, 144.6, 134.2, 127.7, 125.4, 122.4,
118.8, 85.1, 52.0, 38.5, 27.4, (3C) 25.8. IR (KBr) 3447, 3106, 1724, 1647,
1388, 1346, 1259, 1142, 1107, 839, 748 cmꢀ1. HRMS found [M þ H]þ
321.1397, C15H20N4O2S requires 321.1385.
4-(Thiophen-3-yl)-1H-imidazo[4,5-c]pyridine-2-amine (6).
1H NMR (400 MHz, CD3OD) δ 8.60 (dd, J = 2.9, 1.3 Hz, 1H), 8.11 (d,
J = 6.4 Hz, 1H), 7.90 (dd, J = 5.2, 1.3 Hz, 1H), 7.73 (dd, J = 5.1, 2.9 Hz,
1H), 7.51 (d, J = 6.4 Hz, 1H). 13C NMR (100 MHz, CD3OD) δ 161.9,
150.3, 137.0, 133.2, 132.7, 131.9, 130.8, 128.9, 127.2, 107.0. HRMS
found [M þ H]þ 217.0545, C10H8N4S requires 217.0548.
4-(Pyridine-2-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyri-
dine-2-amine. 1H NMR 400 MHz, CD3OD) δ 8.51 (ddd, J = 4.9, 1.7,
0.9 Hz, 1H), 7.79ꢀ7.73 (m, 1H), 7.36ꢀ7.26 (m, 2H), 4.92 (t, J = 1.8 Hz,
1H), 3.14ꢀ3.07 (m, 1H), 3.01ꢀ2.93 (m, 1H), 2.65ꢀ2.50 (m, 2H). 13C
NMR (100 MHz, CD3OD) δ 162.1, 150.7, 150.1, 138.4, 126.5, 124.9,
124.3, 124.0, 59.0, 42.0, 24.0. IR (KBr) 3404, 1620, 1573, 1473, 1435,
1092, 752 cmꢀ1. HRMS found [M þ H]þ 216.1246, C11H13N5 requires
216.1249.
1
4-(Pyridine-2-yl)-1H-imidazo[4,5-c]pyridin-2-amine (7). H
NMR (400 MHz, CD3OD) δ 8.89ꢀ8.80 (m, 2H), 8.28 (d, J = 6.2 Hz,
1H), 8.07ꢀ8.02 (m, 1H), 7.57ꢀ7.52 (m, 2H). 13C NMR (100 MHz,
CD3OD) δ 161.6, 160.5, 150.8, 150.0, 148.9, 139.0, 135.9, 133.8, 126.5,
125.0, 107.7. HRMS found [M þ H]þ 212.0932, C11H9N5 requires
212.0936.
4-(Pyridine-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyr-
idine-2-amine. 1H NMR 400 MHz, CD3OD) δ 8.49ꢀ8.46 (m, 1H),
2501
dx.doi.org/10.1021/jm200039m |J. Med. Chem. 2011, 54, 2492–2503

Journal of Medicinal Chemistry
ARTICLE
1091, 855, 747 cmꢀ1. HRMS found [M þ H]þ 354.1940, C19H23N5O2
requires 354.1930.
buffer; MOCAcꢀ, 7-methoxycoumarin-4-acetylꢀ;NCI, National
Cancer Institute; ES cells, embryonic stem cells
4-(1H-Indole-3-yl)-1H-imidazo[4,5-c]pyridine-2-amine (12).
1H NMR (400 MHz, CD3OD) δ 8.15 (s, 1H), 8.12 (d, J = 6.59 Hz, 1H),
7.87ꢀ7.83 (m, 1H), 7.79ꢀ7.55 (m, 1H), 7.46 (d, J = 6.6 Hz, 1H), 7.46
(d, J = 6.6 Hz, 1H), 7.34ꢀ7.23 (m, 2H). 13C NMR (100 MHz, CD3OD)
δ 162.0, 138.4, 134.3, 133.5, 130.2, 125.7, 124.4, 122.5, 120.1, 113.4,
107.3, 106.1. HRMS found [M þ H]þ 250.1087, C14H11N5 requires
250.1093.
’ REFERENCES
(1) Fujita, M.; Nakao, Y.; Matsunaga, S.; Seiki, M.; Itoh, Y.;
Yamashita, J.; van Soest, R. W. M.; Fusetani, N. Ageladine A: an
antiangiogenic matrixmetalloproteinase inhibitor from the marine
sponge Agelas nakamurai. J. Am. Chem. Soc. 2003, 125, 15700–15701.
(2) Nakao, Y.; Fusetani, N. Enzyme inhibitors from marine inverte-
brates. J. Nat. Prod. 2007, 70, 689–710.
(3) Meketa, M. L.; Weinreb, S. M. Total synthesis of ageladine A, an
angiogenesis inhibitor from the marine sponge Agelas nakamurai. Org.
Lett. 2006, 8, 1443–1446.
(4) Meketa, M. L.; Weinreb, S. M.; Nakao, Y.; Fusetani, N. Applica-
tion of a 6π-1-azatriene electrocyclization strategy to the total synthesis
of the marine sponge metabolite ageladine A and biological evaluation of
synthetic analogues. J. Org. Chem. 2007, 72, 4892–4899.
(5) Andrade, P.; Willoughby, R.; Pomponi, S. A.; Kerr, R. G. Biosynthetic
studies of the alkaloid, stevensine, in a cell culture of the marine sponge
Teichaxinella morchella. Tetrahedron Lett. 1999, 40, 4775–4778.
(6) Shengule, S. R.; Karuso, P. Concise total synthesis of the marine
natural product ageladine A. Org. Lett. 2006, 8, 4083–4084.
(7) Meketa, M. L.; Weinreb, S. M. A new total synthesis of the zinc
matrixmetalloproteinase inhibitor ageladine a featuring a biogenetically
patterned 6 π-2-azatriene electrocyclization. Org. Lett. 2007, 9, 853–855.
(8) Ando, N.; Terashima, S. Synthesis and matrix metalloproteinase
(MMP)-12 inhibitory activity of ageladine A and its analogs. Bioorg. Med.
Chem. Lett. 2007, 17, 4495–4499.
4-(Pyridin-2-yl)-3H-imidazo[4,5-c]pyridine (14). 1H NMR
(400 MHz, CD3OD) δ 9.52 (d, J = 7.2 Hz, 1H), 8.98 (bs, 1H), 8.91
(s, 1H), 8.61 (d, J = 5.9 Hz, 1H), 8.23 (t, J = 7.7 Hz, 1 H), 8.19 (d, J = 5.9
Hz, 1H), 7.75 (bs, 1H). 13C NMR (100 MHz, CDCl3) δ 150.1, 148.9,
147.4, 146.5, 140.0, 138.6, 135.9, 134.7, 127.0, 126.5, 111.99. HRMS
found [M þ H]þ 197.0824, C11H9N4 requires 197.0822.
4-(Pyridin-2-yl)-3H-imidazo[4,5-c]pyridine-6-carboxylic Acid
(15). Obtained as the free base after column chromatography as colorless
crystals (48%). 1H NMR (400 MHz, DMSO-d6) δ 8.91ꢀ8.89 (m, 2H),
8.76 (s, 1H), 8.48 (s, 1H), 8.20 (dt, J= 7.8, 1.6 Hz, 1H), 7.67 (dd, J= 7.0, 5.3
Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 167.1, 154.8, 149.4, 149.3,
148.7, 140.8, 140.7, 139.5, 125.7, 123.1, 116.7. HRMS found [M þ H]þ
241.0714, C12H9N4O2 requires 241.0720.
4-(6-Bromopyridin-2-yl)-3H-imidazo[4,5-c]pyridine-6-car-
boxylic Acid (16). Obtained as a pale-yellow solid after HPLC (71%).
1H NMR (400 MHz, DMSO-d6) δ 12.68 (bs, 1H), 8.76 (d, J = 7.72 Hz,
1H), 8.68 (s, 1H), 8.48 (s, 1H), 8.05 (t, J = 7.8 Hz, 1H), 7.83 (dd, J = 7.8,
0.78 Hz, 1H). 13C NMR (150 MHz, DMSO-d6 at ꢀ10 °C) δ 168.7,
158.3, 152.3, 149.6, 142.7, 141.8, 140.6, 132.4, 130.5, 122.9, 119.7.
LRMS (ESI) m/z 319 (100%), 321 (98%). HRMS found [M þ H]þ
318.9828, C12H8N4O279Br requires 318.9825
(9) Mannello, F.; Tonti, G.; Papa, S. Matrix metalloproteinase
inhibitors as anticancer therapeutics. Curr. Cancer Drug Targets 2005,
5, 285–298.
(10) Brown, K. J.; Maynes, S. F.; Bezos, A.; Maguire, D. J.; Ford,
M. D.; Parish, C. R. A novel in vitro assay for human angiogenesis. Lab.
Invest. 1996, 75, 539–555.
(11) Parish, C. R.; Freeman, C.; Brown, K. J.; Francis, D. J.; Cowden,
W. B. Identification of sulfated oligosaccharide-based inhibitors of tumor
growth and metastasis using novel in vitro assays for angiogenesis and
heparanase activity. Cancer Res. 1999, 59, 3433–3441.
(12) Shoemaker, R. H. The NCI60 human tumour cell line anti-
cancer drug screen. Nature Rev. Cancer 2006, 6, 813–823.
(13) Ando, N.; Terashima, S. Synthesis and matrix metalloprotei-
nase (MMP)-12 inhibitory activity of ageladine A and its analogs. Bioorg.
Med. Chem. Lett. 2007, 17, 4495–4499.
’ ASSOCIATED CONTENT
S
Supporting Information. Raw data for the screening of
b
a subset of compounds against 402 human kinases and calculation
of IC50 values, selectivity values, and treespot diagrams. Calculations
for antiangiogenic activity and cytoxicity data against 60 human cell
lines. All 1H and 13C spectra for synthetic products. This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
(14) Mahboobi, S.; Wiegrebe, W.; Popp, A. Synthesis of enantio-
merically pure (ꢀ)-(S)-brevicolline. J. Nat. Prod. 1999, 62, 577–579.
(15) Lohmander, L. S.; Hoerrner, L. A.; Lark, M. W. Metalloprotei-
nases, tissue inhibitor, and proteoglycan fragments in knee synovial fluid
in human osteoarthritis. Arthritis Rheum. 1993, 36, 181–189.
(16) Murphy, G.; Hembry, R. M. Proteinases in rheumatoid arthritis.
J. Rheumatol. 1992, 19, 61–64.
Corresponding Author
*Phone: þ612 9850 8290. Fax: þ612 9850 8313. E-mail: peter.
karuso@mq.edu.au.
’ ACKNOWLEDGMENT
(17) Peress, N.; Perillo, E.; Zucker, S. Localization of tissue inhibitor
of matrix metalloproteinases in Alzheimer's disease and normal brain.
J. Neuropathol. Exp. Neurol. 1995, 54, 16–22.
(18) Chang, C.; Werb, Z. The many faces of metalloproteases: cell
growth, invasion, angiogenesis and metastasis. Trends Cell Biol. 2001,
11, S37–S43.
(19) Tosetti, F.; Ferrari, N.; De Flora, S.; Albini, A. “Angiopreven-
tion”: angiogenesis is a common and key target for cancer chemopre-
ventive agents. FASEB J. 2002, 16, 2–14.
(20) Pendꢀas, A. M.;Kn€auper, V.; Puente, X. S.; Llano, E.; Mattei, M.-G.;
Apte, S.; Murphy, G.; Lꢀopez-Otín, C. Identification and characterization
of a novel human matrix metalloproteinase with unique structural charac-
teristics, chromosomal location, and tissue distribution. J. Biol. Chem. 1997,
272, 4281–4286.
We thank Macquarie University for an iMURS scholarship (SRS).
The authors are indebted to Professor Motoharu Seiki (University of
Tokyo) for providing the MMP enzymes used in this study and Anna
Bezos for performing the angiogenesis assays. This research was
supported in part, by grants from the EEC (FP6-2002-Life Sciences
and Health, PRO-KINASE Research Project) (L.M., J.E.), the
Canceropole Grand-Ouest grant (L.M.), the Association France-
Alzheimer Finistere (L.M.), and the Ligue Nationale contre le
Cancer (L.M.). CRP is supported by a NHMRC Program grant.
’ ABBREVIATIONS USED
2-AH, 2-aminohistamine; MMP, matrix metalloprotease; MT1-
MMP, membrane type 1 metalloprotease; IBX, 2-iodoxy-
benzoic acid; DCM, dichloromethane; DDQ, 2,3-dichloro-5,6-dicya-
nobenzoquinone; TNC, tris/sodium chloride/calcium chloride
(21) Matter, H.; Schudok, M. Recent advances in the design of
matrix metalloprotease inhibitors. Curr. Opin. Drug Discovery Dev. 2004,
7, 513–535.
2502
dx.doi.org/10.1021/jm200039m |J. Med. Chem. 2011, 54, 2492–2503

Journal of Medicinal Chemistry
ARTICLE
(22) Folkman, J. Angiogenesis: an organizing principle for drug
discovery? Nature Rev. Drug Discovery 2007, 6, 273–286.
(41) Knight, C. G.; Willenbrock, F.; Murphy, G. A novel coumarin-
labeled peptide for sensitive continuous assays of the matrix metallo-
proteinases. FEBS Lett. 1992, 296, 263–266.
(42) Davies, B.; Brown, P. D.; East, N.; Crimmin, M. J.; Balkwill,
F. R. A synthetic matrix metalloproteinase inhibitor decreases tumor
burden and prolongs survival of mice bearing human ovarian carcinoma
xenografts. Cancer Res. 1993, 53, 2087–2091.
(43) Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.;
Czerwinski, M. J.; Fine, D. L.; Abbott, B. J.; Mayo, J. G.; Shoemaker,
R. H.; Boyd, M. R. Feasibility of drug screening with panels of human
tumor cell lines using a microculture tetrazolium assay. Cancer Res. 1988,
48, 589–601.
(44) Reinhardt, J.; Ferandin, Y.; Meijer, L. Purification of CK1 by
affinity chromatography on immobilised axin. Protein Expression Purif.
2007, 54, 101–109.
(23) Folkman, J. Angiogenesis. Annu. Rev. Med. 2006, 57, 1–18.
(24) Sydnes, M. O.; Bezos, A.; Burns, C.; Kruszelnicki, I.; Parish,
C. R.; Su, S.; Rae, A. D.; Willis, A. C.; Banwell, M. G. Synthesis and
biological evaluation of some enantiomerically pure C8c-C15 monoseco
analogues of the phenanthroquinolizidine-type alkaloids cryptopleurine
and julandine. Aust. J. Chem. 2008, 61, 506–520.
(25) Khachigian, L. M.; Parish, C. R. Phosphomannopentaose
sulfate (PI-88): heparan sulfate mimetic with clinical potential in
multiple vascular pathologies. Cardiovasc. Drug Rev. 2004, 22, 1–6.
(26) Ohya, T. Identification of 4-methylspinaceamine, a Pictetꢀ
Spengler condensation reaction product of histamine with acetaldehyde,
in fermentated foods and its metabolite in human urine. J. Agric. Food.
Chem. 2006, 54, 6909–6915.
(27) Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam,
S. The protein kinase complement of the human genome. Science 2002,
298, 1912–1934.
(28) Kostich, M.; English, J.; Madison, V.; Gheyas, F.; Wang, L.; Qiu,
P.; Greene, J.; Thomas, M. Human members of the eukaryotic protein
kinase family. Genome Biol. 2002, 3, research0043.1–research0043.12.
(29) Neidner, R. H.; Buzko, O. V.; Hatse, N. M.; Taylor, A.;
Gribskov, M.; Taylors, S. S. Protein kinase resource: an integrated
environment for phosphorylation research. Proteins 2006, 63, 78–86.
(30) Fischer, P. M. The design of drug candidate molecules as
selective inhibitors of therapeutically relevant protein kinases. Curr.
Med. Chem. 2004, 11, 1563–1583.
(31) Weinmann, H.; Metternich, R. Drug discovery process for
kinase inhibitors. ChemBioChem 2005, 6, 455–459.
(32) Smith, P. J.; Yue, E. CDK Inhibitors of Cyclin-Dependent Kinases
As Anti-tumor Agents; Monographs on Enzyme Inhibitors; CRC Press,
Taylor & Francis: Boca Raton, FL, 2006; Vol. 2, p 448.
(33) Meijer, L.; Flajolet, M.; Greengard, P. Pharmacological inhibitors of
glycogen synthase kinase 3. Trends Pharmacol. Sci. 2004, 25, 471–480.
(34) Wan, Y. Q.;Hur, W. Y.;Cho, C. Y.;Liu, Y.;Adrian, F. J.;Lozach, O.;
Bach, S.; Mayer, T.; Fabbro, D.; Meijer, L.; Gray, N. S. Synthesis and target
identification of hymenialdisine analogs. Chem. Biol. 2004, 11, 247–259.
(35) Meijer, L.; Thunnissen, A. M. W. H.; White, A. W.; Garnier, M.;
Nikolic, M.; Tsai, L. H.; Walter, J.; Cleverley, K. E.; Salinas, P. C.; Wu,
Y. Z.; Biernat, J.; Mandelkow, E. M.; Kim, S. H.; Pettit, G. R. Inhibition of
cyclin-dependent kinases, GSK-3β and CK1 by hymenialdisine, a marine
sponge constituent. Chem. Biol. 2000, 7, 51–63.
(36) Fabian, M. A.; Biggs, W. H., III; Treiber, D. K.; Atteridge, C. E.;
Azimioara, M. D.; Benedetti, M. G.; Carter, T. A.; Ciceri, P.; Edeen,
P. T.; Floyd, M.; Ford, J. M.; Galvin, M.; Gerlach, J. L.; Grotzfeld, R. M.;
Herrgard, S.; Insko, D. E.; Insko, M. A.; Lai, A. G.; Lelias, J. M.; Mehta,
S. A.; Milanov, Z. V.; Velasco, A. M.; Wodicka, L. M.; Patel, H. K.;
Zarrinkar, P. P.; Lockhart, D. J. A small molecule-kinase interaction map
for clinical kinase inhibitors. Nature Biotechnol. 2005, 23, 329–336.
(37) Karaman, M. W.; Herrgard, S.; Treiber, D. K.; Gallant, P.;
Atteridge, C. E.; Campbell, B. T.; Chan, K. W.; Ciceri, P.; Davis, M. I.;
Edeen, P. T.; Faraoni, R.; Floyd, M.; Hunt, J. P.; Lockhart, D. J.; Milanov,
Z. V.; Morrison, M. J.; Pallares, G.; Patel, H. K.; Pritchard, S.; Wodicka,
L. M.; Zarrinkar, P. P. A quantitative analysis of kinase inhibitor
selectivity. Nature Biotechnol. 2008, 26, 127–132.
(38) Baek, K.-H.; Zaslavsky, A.; Lynch, R. C.; Britt, C.; Okada, Y.;
Siarey, R. J.; Lensch, M. W.; Park, I.-H.; Yoon, S. S.; Minami, T.;
Korenberg, J. R.; Folkman, J.; Daley, G. Q.; Aird, W. C.; Galdzicki, Z.;
Ryeom, S. Down’s syndrome suppression of tumour growth and the role
of the calcineurin inhibitor DSCR1. Nature 2009, 459, 1126–1130.
(39) Itoh, Y.; Takamura, A.; Ito, N.; Maru, Y.; Sato, H.; Suenaga, N.;
Aoki, T.; Seiki, M. Homophilic complex formation of MT1-MMP
facilitates proMMP-2 activation on the cell surface and promotes tumor
cell invasion. EMBO J. 2001, 20, 4782–4793.
(40) Kinoshita, T.; Sato, H.; Takino, T.; Itoh, M.; Akizawa, T.; Seiki, M.
Processing of a precursor of 72-kilodalton type IV collagenase/gelatinase A
by a recombinant membrane-type 1 matrix metalloproteinase. Cancer Res.
1996, 56, 2535–2538.
2503
dx.doi.org/10.1021/jm200039m |J. Med. Chem. 2011, 54, 2492–2503