From a MMP2/CK2 multitarget approach to the identification of potent and selective MMP13 inhibitors

Article abstract of DOI:10.1039/c8ob02990c

In this article, we describe our efforts in the search of MMP2/CK2 dual targeting inhibitors. We have followed a rational drug design approach based on our experience in the selective inhibition of these two enzymes. We have successfully obtained highly a

Full text of DOI:10.1039/c8ob02990c

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From a MMP2/CK2 multitarget approach to the
identification of potent and selective MMP13
inhibitors†
Cite this: DOI: 10.1039/c8ob02990c
b
Miryam Pastor,‡a José María Zapico, ‡a Claire Coderch, a Maciej Maslyk,
a
Rostyslav Panchuk, c Beatriz de Pascual-Teresa *a and Ana Ramos
*
In this article, we describe our efforts in the search of MMP2/CK2 dual targeting inhibitors. We have fol-
lowed a rational drug design approach based on our experience in the selective inhibition of these two
enzymes. We have successfully obtained highly active MMP2 (10, IC50 = 70 nM; 11, IC50 = 100 nM) and
CK2 (16a, IC50 = 500 nM) inhibitors. However, structural fine tuning of these small molecules to simul-
taneously target both enzymes turned out to be an unattainable goal. Unexpectedly, we were lucky to
identify new and selective MMP13 inhibitors (10, IC50 = 3.7 nM and 11, IC50 = 5.6 nM) with a novel
TBB-derived scaffold. These compounds constitute an interesting starting point for further optimization.
Received 30th November 2018,
Accepted 18th December 2018
DOI: 10.1039/c8ob02990c
rsc.li/obc
We have devoted special effort in developing small mole-
cules that selectively inhibit MMP2 over MMP9, because while
Introduction
Matrix metalloproteinases (MMPs) are a family of Zn-depen- MMP2 is a validated target for cancer treatment, MMP9 is con-
dent enzymes that mediate degradation and the remodelling sidered as an anti-target in advanced stages of the disease.3
of the extracellular matrix (ECM),1 which makes them interest- Three examples of these inhibitors are shown in Fig. 1.
ing targets for therapeutic inhibitors in many inflammatory,
malignant and degenerative diseases.2
The approach we used was based on click chemistry as a
synthetic tool to connect scaffolds containing a hydroxamate
The first pre-clinical studies clearly demonstrated the invol- to act as the Zinc Binding Group (ZBG) with appropriate sub-
vement of MMPs in cancer.3 However, clinical trials were dis- units to interact with the S1′ subsite of the enzyme. We have
appointing, and MMPIs showed poor efficiency.4 The lack of performed extensive computational studies on the binding
selectivity of broad spectrum inhibitors was considered the mode of these inhibitors with both gelatinases, to rationalize
main cause of the musculoskeletal side effect (MSS) that their MMP2/MMP9 selectivity.7,8
limited their doses. For this reason, the improvement of the
We are also interested in the development of inhibitors of
MMPI selectivity is a key step toward the development of CK2,9 a serine/threonine kinase that is constitutively active
effective MMP-based drugs.5
Our research group has extensive experience in the design
and synthesis of inhibitors of MMP2 provided with high
activity and, more importantly, high selectivity over other
metalloproteinases.6
aDepartamento de Química y Bioquímica, Facultad de Farmacia, Universidad San
Pablo-CEU, CEU Universities, Urbanización Montepríncipe, 28925, Alcorcón,
Madrid, Spain. E-mail: aramgon@ceu.es, bpaster@ceu.es
bDepartment of Molecular Biology, Faculty of Biotechnology and Environment
Sciences, The John Paul II Catholic University of Lublin, 20-718 Lublin, Poland
cInstitute of Cell Biology, NAS of Ukraine, Drahomanov Str. 14/16, 79005 Lviv,
Ukraine
†Electronic supplementary information (ESI) available: Synergic effect of TBB
and compound C in Jurkat cells; 1H NMR spectra and HPLC Chromatograms of
10–15, 16a–c, 19, 22a–b and 26; MD simulations. See DOI: 10.1039/c8ob02990c
‡These authors contributed equally.
Fig. 1 Chemical structures of potent and selective MMP2 inhibitors.
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and essential for cell viability. Its proliferative and anti-apopto- MMP13).25 The two MMPs in this study belong to the medium
tic properties create a favorable cellular environment for tumor (MMP2) and large (MMP13) classification This difference in
progression and maintenance and, therefore, CK2 constitutes the loop size is the determining factor for the selective binding
an interesting target for the treatment of cancer.10 Several ATP of inhibitors of MMP13.24
competitive inhibitors of CK2 have been discovered but, only
CX-4945 has entered clinical trials for cancer therapy.11 Design
of allosteric inhibitors12 can constitute a promising strategy
to overcome the apparent lack of druggability of this consti-
tutively active kinase.13,14 Another approach is the combi-
nation of CK2 inhibitors with molecules capable of interacting
with other targets, such as multi-kinase CK2/PIM
inhibitors.15–17
Interestingly, we have demonstrated that MMP2 is a sub-
strate of CK2, and CK2-mediated phosphorylation of MMP2
Results and discussion
affects its capability of degrading gelatinase.18 Selective inhi-
Synthesis
bition of CK2 down-regulates MMP2 in vitro and the inhibition
of CK2 in human fibrosarcoma cells results in upregulation of
Alkynes 1, 2 and 3 were synthesized as previously described.19
Alkyne
4 was synthesized as depicted in Scheme 1.
MMP2. This discovery opens the possibility of new combined
therapies, or the design of molecules capable of interacting
with both enzymes. This multitarget approach is the strategy
we have explored in this work, in an effort to identify dual
MMP2/CK2 inhibitors, with potential utility in proliferative
diseases.19
We have selected two potent and selective ATP-competitive
CK2 inhibitors, TBB20,21 and DMAT,22 and we have functiona-
lized these molecules with an alkyne to connect them to other
subunits provided with a terminal azide and designed to
interact with the active site of MMP2. Previous results reported
by our research group have shown that this strategy is valid for
the design of dual HDAC/CK2 inhibitors, providing com-
pounds with IC50 values in the low micromolar range for both
enzymes and with interesting antiproliferative and antiapopto-
tic activities.19
Treatment of 1H-benzo[d]imidazole-2-thiol (5) with aqueous
HBr/Br2 in the presence of AcOH, followed by polybromination,
gave 6.
The reaction of 6 with thiourea gave 7,26 which was alkyl-
ated with 3-bromoprop-1-yne under basic conditions using
microwave irradiation to give alkyne 4.
Azides 827 and 928 were synthesized following previously
published procedures by our research group.
Copper(I) catalyzed Huisgen cycloaddition (CuAAC) between
these two azides and the four abovementioned alkynes using
tris(benzyltriazolylmethyl)amine (TBTA) as the co-catalyst, fol-
lowed by deprotection of the hydroxamate group, gave com-
pounds 10–15 with moderate to good yields and with a purity
higher than 95% (HPLC) after column chromatography
(Scheme 2).
In the course of this investigation we found a new class of
highly potent and selective MMP13 inhibitors. MMP13 is the
main protease responsible for type II collagen degradation. It
is highly over-expressed in the cartilage of osteoarthritis (OA)
patients, whereas it is not present in normal adult cartilage.23
For this reason, the development of selective MMP13 inhibi-
tors has received considerable attention in the past few
decades.24
Apart from the active site, MMPs differ specifically in the
S1′ pocket also known as the selectivity pocket. This pocket is
delimited by the Ω-loop that can vary in its length and compo-
sition. This variability allows the classification of MMPs into
three groups according to the size of the S1′ pocket: small
(MMP1, MMP7, MMP11, MMP20), medium (MMP2, MMP8,
Scheme 1 Reagents and conditions: (a) (i) HBr, AcOH, Br2, 4 h, RT, 55%;
(ii) H2O, Br2, 24 h, reflux; (b) (i) thiourea, EtOH, 48 h, reflux; 92% (ii)
NaOH, H2O (c) DMF, K2CO3, 3-bromoprop-1-yne, 20 min, 60 °C, MW,
MMP9, MMP12, MMP14, MMP16) and large (MMP3, MMP10, 18%.
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In our previous hydroxamate type MMP2 inhibitors having
a stereocenter, such as B in Fig. 1, only the R-stereoisomer was
active. To investigate whether these new hydroxamates exert
the same selectivity, 19 and 22b were synthesized as a pure R
enantiomer.
Synthesis of compound 19 is depicted in Scheme 4. Methyl
ester of D-valine was reacted with compound 6 to give 20.
Hydrolysis of 20 to the corresponding acid 21 followed by
coupling with OTHP-hydroxylamine and deprotection of the
hydroxamate gave 19.
Synthesis of 22a–b is depicted in Scheme 5. The reaction of
D-valine or glycine with 4-nitrobenzenesulfonyl chloride under
basic conditions gave the corresponding nitro compounds
23a–b.29 The nitro group was hydrogenated, and the corres-
ponding amines were transformed into their methyl esters
24a–b. N-Alkylation of the amino group with 6, followed by de-
protection, afforded carboxylic acids 25a–b. EDCI coupling of
the carboxylic acids with OTHP-hydroxylamine followed by de-
protection gave the desired hydroxamates 22a–b.
Finally, to incorporate flexibility into the linker, compound
26 was synthesized (Scheme 6). The reaction of 4,5,6,7-tetra-
bromo-1H-benzo[d]imidazole-2-thiol (7) with 2-chloroacetalde-
Scheme 2 Reagents and conditions: (a) (i) Corresponding azide, DMF,
CuSO4·5H2O, sodium ascorbate, TBTA, 24 h, RT; (ii) HCl/dioxan or acetyl
chloride/MeOH.
Sulfide derivatives 16a–c were synthesized following the
procedure described in Scheme 3. Treatment of 7 under basic
conditions with the corresponding racemic halide gave acids
17a–c, which were coupled with OTHP-hydroxylamine to give
THP-protected hydroxamates 18a–c. Final deprotection using
AcCl/MeOH gave the target hydroxamates 16a–c.
Scheme 4 Reagents and conditions: (a) D-Valine-OMe, MeOH, 20 min,
135 °C, MW; (b) THF, NaOH 1 M, overnight, RT; (c) (i) HOBT, EDCI, NMM,
OTHP-hydroxylamine, 48 h, RT; (ii) AcCl/MeOH, 0 °C.
Scheme 5 Reagents and conditions: (a); NaOH 1 M, 24 h, RT, pH > 9;
(b) (i) EtOH, Pd/C 10%, 50 p.s.i, 24 h, RT; (ii) MeOH anh, SOCl2, reflux,
overnight; (c) (i) compound 6, dry MeOH, 110 °C; (ii) THF, NaOH 1 M, RT;
(d) (i) HOBT, EDCI, NMM, OTHP-hydroxylamine, 48 h, RT; (ii) AcCl/
MeOH, 0 °C.
Scheme 3 Reagents and conditions: (a) DMF, halide derivate, K2CO3,
MW conditions; (b) DMF, HOBT, EDCI, NMM, OTHP-hydroxylamine,
48–72 h, RT; (c) AcCl/MeOH, 0 °C.
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Table 1 Inhibitory activity (IC50 values) of hydroxamates on MMP2 and
CK2
Compound
MMP2 IC50 a (μM)
CK2α IC50 a (μM)
10
11
12
13
14
15
16a
16b
16c
19
22a
22b
26
0.07
0.1
>0.3
0.3
>200
>300
>200
>300
>300
>200
0.5
2
7
10
>0.3
b
—
>100
>100
>100
>100
>100
>100
>100
90–100
90–100
90–100
a Enzymatic data are mean values from at least two independent
experiments. SD are within 10%. b IC50 for this compound is not
shown because its behavior was erratic. We observed that compound
15 slowly decomposes in DMSO solutions stored at −20 °C, and this
can be the reason for this behavior. Nevertheless, when the inhibitory
activity is measured using freshly prepared solutions, the activity is
similar to the one elicited by the most active compound 10.
Scheme 6 Reagents and conditions: (a) K2CO3, 2-chloroacetaldehyde,
MW; (b) 24a, NaBH(OAc)3, AcOH, DCM, overnight, 50 °C; (c) (i) NaOH/
THF; (ii) HOBT, EDCI, NMM, OTHP-hydroxylamine, 48 h, RT; (iii) AcCl/
MeOH, 0 °C.
hyde in the presence of potassium carbonate and under MW
conditions gave aldehyde 27. Reductive amination of 27 with
24a gave methyl ester 28. Deprotection of carboxylic acid fol-
lowed by EDC coupling and subsequent deprotection gave
hydroxamate 26.
Table 2 Inhibitory activity of compound 10 towards selected MMPs
IC50a, nM
MMP1
MMP2
MMP3
MMP7
MMP8
MMP9
MMP10
MMP12
MMP13
MMP14
>1000
70
∼500
>1000
>500
>500
434
17.8
3.7
>1000
Biological evaluation
Study of the synergic action between MMP2 and CK2
inhibition
Compound C (Fig. 1), a highly potent and selective MMP2
inhibitor, and TBB, a CK2 selective inhibitor, were combined
and their cytotoxicity in the human T-cell acute lymphoblastic
leukemia Jurkat cell line was assessed with each compound
individually or in combination. We found that dual inhibition
of both enzymes presented a synergic effect promoting cell
death. The major effect was observed when TBB and C were
a Concentration required for 50% inhibition of enzyme activity.
Standard deviations are less than 10% of mean values in three
experiments.
Disappointingly, these compounds were inactive against
added at a concentration of 20 and 25 μM, respectively (ESI CK2. Considering that the N-2 substituted hydroxamates 10
Fig. 1†). This result supports our hypothesis that dual inhi- and 11 were the most active in MMP2, the TBB fragment was
bition of MMP2 and CK2 is a promising strategy to develop substituted by a benzimidazole in the next series of com-
novel anticancer drugs.
pounds (15, 16a–c, 19, 22a–b and 26).
CK2 inhibitory activity was determined by a radiometric
On the other hand, and deduced with the help of docking
method, using a short peptide (RRRADDSDDDDD) as a sub- studies, we thought that a shorter chain could be favorable for
strate and MMP2 inhibitory activity was determined by a col- a better interaction with CK2. Therefore, we decided to elimin-
orimetric enzymatic assay. The results are collected in Table 1.
ate the triazole linkage that apparently did not bring stabiliz-
Compounds 10–14 showed good activity in MMP2, although ing interactions with the active site of CK2 (16a–c and 19).
a considerable amount of potency was lost with respect to our These compounds can be considered derivatives of TBB
previously described and structurally related MMP2 inhibi- (Ki = 0.5 μM) and DMAT (Ki = 0.04 μM).15
tors.27,28,30 However, the activity is still in the nanomolar
As expected, these compounds were active in the inhibition
range, interesting for a dual-targeting inhibitor. The selectivity of CK2, with IC50 values in the low micromolar range.
of the most active compound (10) was studied by testing its However, when the MMP2 activity was measured, we were very
inhibitory activity in a panel of ten metalloproteinases. The disappointed to see that these compounds, despite bearing an
results are collected in Table 2 and will be discussed later.
effective ZBG, were not able to inhibit this enzyme.
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As a final attempt to obtain dual activity, we enlarged the methylene linker was introduced to separate these two sub-
linker between the benzotriazole unit and the ZBG and structures. Apparently, even though the hydrophobic moieties
endowed it with a sulfonamide group to favor the hydrogen appeared to establish good interactions with the ATP binding
bond interactions with the enzyme backbone (22a–b and 26). site, the flexibility at the hydroxamic acid end of these
With these changes, not only MMP2 activity was not improved, molecules may have contributed to a destabilization of the
but also the CK2 inhibition was lost. Molecular modeling binding to CK2. Bearing all this in mind, the shorter 16a–c
methodologies were used at this point to rationalize these were designed and proposed as plausible CK2 inhibitors
unexpected results. MD simulations carried out for 26 revealed (Fig. 3). This was later confirmed by the good IC50 values
a very stable folded conformation of the free compound in a (Table 1).
water environment, which could most probably impair
binding of 26 to both enzymes (ESI Fig. 2†).
However, these changes rendered 16a–c inactive against
MMP2 as they lacked a hydrogen bond accepting moiety to
interact with the MMP2 backbone as other sulfonamide deriva-
tives previously reported presented in their structure.7,8,27,31
Compounds 22a–b and 26 were then designed with the
required sulfonamide moiety, together with the hydroxamate
group, to improve the affinity for MMP2. In addition, more
flexibility was given to the TBB end to favor the accommo-
dation within the CK2 ATP-binding site. According to our cal-
culations, 26 was the most promising compound, as it nicely
fitted the MMP2 binding site and was able to occupy the CK2
ATP-binding in the same region as in previously reported CK2
3D-structures bound to TBB and TBB-analogues (Fig. 4 and
5).9,32–34 A 100 ns MD simulation of 26 in water was carried
out to provide a rationale for the failure to inhibit MMP2 and
CK2. The addition of the flexible moiety allowed 26 to fold
itself so that the two aromatic groups establish intramolecular
π–π staking interactions. This folded conformation is very
stable, since it is maintained for 81 ns of the simulation time
and in this conformation, 26 should be unable to access the
MMP2 and CK2 binding sites (ESI Fig. 2†).
Docking
Given the flexibility of the Ω-loop that delimits the S1′ pocket
of MMPs and the limitations of docking calculations in which
full backbone flexibility of the protein is not considered, the
preliminary docking studies were carried out using an MMP2
structure that was issued from a conformational analysis8 to
ensure a more opened conformation of the S1′ pocket. Ligands
10–15 were all promising candidates as they all fitted into the
MMP2 structure in a manner similar to that previously
described for other structurally related reported compounds.
That is, while the deprotonated hydroxamic acid chelated the
Zn2+ ion, the rest of the molecules entered the S1′ pocket
(Fig. 2).7,8,27,31
Additionally, these ligands fitted within the ATP-binding
site of CK2. Although the binding mode did not look as prom-
ising as for MMP2, we hoped that the interactions of the TBB
moiety combined with the hydrogen bonds of the hydroxamic
acid could stabilize enough the binding mode to achieve inhi-
bition at least in the micromolar range.
MMP13 inhibitors
However, with IC50 values in our hands, we concluded that,
despite being predicted to be good MMP2 binders, all ligands
seemed to be too long and rigid due to the simultaneous pres-
ence of the TBB and the triazole-phenyl moieties, even if a
The study of the selectivity of compound 10 in a panel of ten
metalloproteinases (Table 2) showed that this compound exhi-
bits an extraordinary inhibitory activity in MMP13 (IC50 = 3.7
Fig. 2 PyMOL stick and cartoon representation of the docking pose of
15 in MMP2. Hydrogen bonds and chelating interactions are shown in
dashed lines, the zinc ion is shown as a grey sphere and only polar
hydrogens are shown for clarity.
Fig. 3 PyMOL stick and cartoon representation of the docking pose of
16a in CK2. Hydrogen bonds are shown in dashed lines and only polar
hydrogens are shown for clarity.
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Fig. 6 Left: Inhibitory activity of the synthesized compounds. Right:
Inhibitory activity of related compounds.27,28
of the TBB moiety and the larger size of the Ω-loop, this com-
pound may be better stabilized by MMP13 (Fig. 7).
Fig. 4 PyMOL stick and cartoon representation of the docking pose of
26 in MMP2. Hydrogen bonds and chelating interactions are shown in
dashed lines, the zinc ion is shown as a grey sphere and only polar
hydrogens are shown for clarity.
This was further assessed by means of 20 ns MD simu-
lations of apo MMP2 and MMP13 after which the most popu-
lated conformers of the Ω-loops were extracted. The shorter
loop of MMP2 confers more rigidity and therefore less varia-
bility to the Ω-loop conformation, whereas the longer loop of
MMP13 gives rise to much more opened and different possible
conformations (ESI Fig. 3†).
MMP13 (also known as collagenase 3) is a member of the
matrix metalloproteinase family, considered as an important
target for the treatment of osteoarthritis (OA) because of the
key role it plays in the degradation of type II collagen in carti-
lage. OA, also known as a degenerative joint disease, is charac-
terized by cartilage degeneration and osseous overgrowth and,
to date, there is no cure for it. Treatment consists basically of
reducing pain and improving function of the affected joints.35
Therefore, there is high interest in developing potent and
selective inhibitors of MMP13, which may avoid undesirable
Fig. 5 PyMOL stick and cartoon representation of the docking pose of
26 in CK2. Hydrogen bonds are shown in dashed lines and only polar
hydrogens are shown for clarity.
nM). Moreover, it was about 20-fold more active in MMP13
than in MMP2, a highly homologous enzyme, and 5-fold more
active than in MMP12. For the rest of enzymes tested, the com-
pound was inactive. Compound 11 presented a similar activity
in MMP13, and selectivity towards MMP2 (Fig. 6).
This result was surprising, because in our previous studies
we had never observed such a degree of discrimination
between MMP2 and MMP13 in compounds with related struc-
tures (some examples in Fig. 6).27,28
This different selectivity may arise from the different size of
the Ω-loop amongst the different MMPs.8 MMP-13 has a
bigger and therefore slightly more flexible Ω-loop compared to
Fig. 7 PyMOL stick and cartoon representation of the docking pose of
10 in MMP13. Hydrogen bonds and chelating interactions are shown in
dashed lines, the zinc ion is shown as a grey sphere and only polar
that of MMP2. Given the larger size of 10 due to the presence hydrogens are shown for clarity.
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side effects presented by the inhibition of other 10.6 Hz, 1H), 3.04–2.95 (m, 1H), 2.87–2.80 (m, 1H), 1.72–1.41
metalloproteinases.36
(m, 6H).
Deprotection was performed following method A: starting
Further studies will be performed to study the potential of
these types of compounds and other analogs for the treatment from OTHP-10 (40 mg, 0.043 mmol) afforded 10 (36 mg, 100%)
1
of OA and other inflammatory diseases.
as a white solid (100%). Mp 185 °C (dec.). H NMR (400 MHz,
DMSO) δ 10.67 (s, 1H), 8.87 (brs, 1H), 8.68 (s, 1H), 8.43 (d, J =
8.9 Hz, 1H), 7.83 (d, J = 8.8 Hz, 2H), 7.67 (d, J = 8.7 Hz, 2H),
7.14–6.92 (m, 5H), 5.19 (t, J = 7.0 Hz, 2H), 3.82 (td, J = 9.1,
5.6 Hz, 1H), 3.59 (t, J = 7.0 Hz, 2H), 2.78 (dd, J = 13.6, 5.5 Hz,
1H), 2.63 (dd, J = 13.6, 9.3 Hz, 1H). HPLC purity 98%.
Experimental
4,5,6,7-Tetrabromo-2-(prop-2-yn-1-ylthio)-1H-benzo[d]
imidazole (4)
N-Hydroxy-4-((4-(4-(2-(perbromo-2H-benzo[d][1,2,3]triazol-2-yl)
ethyl)-1H-1,2,3-triazol-1-yl)phenyl)sulfonyl)tetrahydro-2H-
pyran-4-carboxamide (11)
A solution of 726 (230 mg, 0.494 mmol), K2CO3 (204 mg,
1.48 mmol) and 3-bromoprop-1-yne (96 μL, 0.59 mmol) in
DMF (14 mL) was heated under MW conditions for 20 min at
60 °C. The product was precipitated with a mixture of ice/H2O. Following the general procedure for CuAAC, starting from
The compound was used in the next reaction without further azide
9 (100 mg, 0.24 mmol) and alkyne 1 (154 mg,
purification. Mp 209 °C (dec.). 1H NMR (400 MHz, DMSO) 0.317 mmol), and after purifications by column chromato-
δ 13.53 (s, 1H), 4.21 (d, J = 2.6 Hz, 2H), 3.26 (t, J = 2.6 Hz, 1H). graphy on silica gel (DCM/MeOH 0.3–0.6%), OTHP-11 (188,
1
MS (ES+): [M]+ = 504.
86%) was obtained as a white solid. Mp 138–141 °C. H NMR
(400 MHz, CDCl3) δ 9.37 (s, 1H), 8.03 (d, J = 8.8 Hz, 2H), 7.92
(d, J = 8.8 Hz, 2H), 7.89 (s, 1H), 5.18 (t, J = 7.0 Hz, 2H), 5.02
(brs, 1H), 4.04–3.99 (m, 3H), 3.74–3.71 (m, 3H), 3.49 (td, J =
12.1, 5.4 Hz, 2H), 2.33 (td, J = 12.7, 4.8 Hz, 2H), 2.08 (d, J =
13.3 Hz, 2H), 1.91–1.60 (m, 6H). MS (ES+): [M + H]+ = 898.
Deprotection was performed following method A: starting
from OTHP-11 (170 mg, 0.193 mmol) afforded 11 (80 mg, 50%)
General procedure for copper-catalyzed azide–alkyne
cycloaddition (CuAAC) reactions
A mixture of 1.1–1.3 eq. of the corresponding alkyne, 1 eq. of
the corresponding azide, 0.3 eq. of freshly made aqueous
0.1 M CuSO4·5H2O solution, 0.6 eq. of 0.5 M sodium ascorbate
solution and 0.01 eq. of TBTA in DMF was stirred at RT until
completion of the reaction (TLC). The reaction mixture was
then concentrated under vacuum, H2O was added to the
mixture and the formed precipitate was filtered and purified.
1
as a white solid. Mp 217–221 °C. H NMR (400 MHz, DMSO)
δ 11.04 (brs, 1H), 9.24 (brs, 1H), 8.82 (s, 1H), 8.10 (t, J = 8.6 Hz,
2H), 7.92 (d, J = 8.6 Hz, 2H), 5.17 (t, J = 6.9 Hz, 2H), 3.89 (dd,
J = 11.0, 2.9 Hz, 2H), 3.58 (t, J = 6.9 Hz, 2H), 3.16 (t, J = 11.6 Hz,
2H), 2.24 (d, J = 13.2 Hz, 2H), 1.96 (td, J = 13.6, 4.8 Hz, 2H). MS
(ES+): [M + H]+ = 813.7. HPLC purity 95%. Elemental analysis
C22H19Br4N7O5S requires; C, 32.50; H, 2.36; N, 12.06; S, 3.94
found; C: 32,37; H: 2,64; N: 11,76; S: 3,86.
General procedure for deprotection of hydroxamates
Method A. A suspension of the corresponding protected hydro-
xamate (1.00 eq.) in 4 M HCl dioxane solution (2 mL) was
stirred at RT under argon. After completion, the reaction
mixture was quenched by adding saturated NaHCO3, and the (R)-N-Hydroxy-2-(4-(4-(2-(perbromo-1H-benzo[d][1,2,3]triazol-1-
precipitate was filtered, and washed with water. The product yl)ethyl)-1H-1,2,3-triazol-1-yl)phenylsulfonamido)-3-
was purified in case it was needed.
phenylpropanamide (12)
Method B. HCl was prepared by adding acetyl chloride
(10 eq.) to dry MeOH (1 mL) at 0 °C. The solution was added
to a suspension of the corresponding protected hydroxamate
in MeOH at 0 °C. After the same work-up described in method
A, the desired compound was obtained.
Following the general procedure for CuAAC, starting from
azide 8 (71.30 mg, 0.160 mmol) and alkyne 2 (100 mg,
0.2083 mmol), and after purification by column chromato-
graphy on silica gel (DCM/MeOH 0.5%), gave OTHP-12 (80 mg,
54%) as a white solid. Mp 215 °C (dec.). This compound is
1
present as a diastereoisomeric A : B mixture (50 : 50), H NMR
(R)-N-Hydroxy-2-(4-(4-(2-(perbromo-2H-benzo[d][1,2,3]triazol-2-
yl)ethyl)-1H-1,2,3-triazol-1-yl)phenylsulfonamido)-3-
phenylpropanamide (10)
(400 MHz, CDCl3) δ 7.85 (s, 1H), 7.79–7.71 (m, 4H), 7.20–7.18
(m, 3H), 7.06–7.04 (m, 2H), 5.38 (t, J = 7.1 Hz, 2H), 4.72 (s,
isomer A, 0.5H), 4.68 (s, isomer B, 0.5H), 3.94–3.82 (m, 2H),
Following the general procedure for CuAAC, starting from 3.67–3.49 (m, 3H), 3.05 (dd, J = 14.1, 6.5 Hz, 1H), 2.93 (dd, J =
azide (95 mg, 0.21 mmol) and alkyne (114 mg, 13.8, 8.1 Hz, 1H), 1.80–1.54 (m, 6H).
0.234 mmol), and after purification by column chromato- Deprotection was performed following method A: starting
8
1
graphy on silica gel (DCM/MeOH 0.5–0.7%) followed by recrys- from OTHP-12 (70 mg, 0.075 mmol) afforded 12 (50 mg, 79%)
1
tallization (EtOAc) OTHP-10 (70 mg, 36%) was obtained as a as a white solid. Mp 219–222 °C. H NMR (400 MHz, DMSO)
1
white solid. Mp 210–213 °C. H NMR (400 MHz, CDCl3) δ 7.90 δ 10.66 (brs, 1H), 8.70 (s, 1H), 8.41 (d, J = 8.9 Hz, 1H), 7.84 (d, J
(s, 1H), 7.75–7.73 (m, 2H), 7.69–7.66 (m, 2H), 7.16–6.99 (m, = 8.8 Hz, 2H), 7.68 (d, J = 8.8 Hz, 2H), 7.17–6.94 (m, 5H), 5.28
5H), 5.13 (t, J = 7.0 Hz, 2H), 4.57 (brs, 1H), 3.90 (t, J = 7.1 Hz, (t, J = 7.1 Hz, 2H), 3.82 (td, J = 9.1, 5.8 Hz, 1H), 3.47 (t, J =
1H), 3.85–3.74 (m, 1H), 3.67 (t, J = 7.0 Hz, 2H), 3.48 (t, J = 7.1 Hz, 2H, under H2O peak), 2.79 (dd, J = 13.6, 5.5 Hz, 1H),
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2.65 (dd, J = 13.6, 9.3 Hz, 1H). HPLC purity 94%. Elemental (147 mg, 46%) as a white solid. This compound is present as a
analysis C25H20Br4N8O4S requires; C, 35.40; H, 2.38; N, 13.21; diastereoisomeric A : B mixture (45 : 55), mp 167–170 °C. 1H
S, 3.78 found C: 35.20; H: 2.53; N: 13.01; S: 3.71.
NMR (400 MHz, DMSO-d6) δ 13.53 (s, 1H), 11.26 (s, isomer A,
0.45H), 11.18 (s, isomer B, 0.55H), 8.86 (s, 1H), 8.51 (d, J =
9.3 Hz isomer A, 0.45H), 8.47 (d, J = 8.9 Hz, isomer B,
0.55H),7.94–7.90 (m, 2H), 7.74 (d, J = 7.6 Hz, 2H), 7.17–6.99
(m, 5H), 4.76 (s, 2H), 4.53 (s, isomer B, 0.55H), 4.24 (s, isomer
N-Hydroxy-4-(4-(4-(2-(perbromo-1H-benzo[d][1,2,3]triazol-1-yl)
ethyl)-1H-1,2,3-triazol-1-yl)phenylsulfonamido)tetrahydro-2H-
pyran-4-carboxamide (13)
Following the general procedure for CuAAC, starting from A, 0.45H), 3.94–3.82 (m, 1H), 3.80–3.64 (m, 1H), 2.78 (dd, J =
azide (100 mg, 0.24 mmol) and alkyne (154 mg, 13.8, 5.4 Hz, 2H), 2.70–2.63 (m, 1H), 1.50–1.32 (m, 6H). MS
0.310 mmol), and after purification by column chromato- (ES+): [M]+ = 949.6.
9
2
graphy on silica gel (DCM/MeOH 0.3–0.4%) gave OTHP-13
Deprotection was performed following method B: starting
(108 mg, 50%) as a white solid. Mp 150–153 °C. 1H NMR from OTHP-15 (100 mg, 0.105 mmol) afforded 15 (40 mg, 44%)
1
(400 MHz, CDCl3) δ 9.38 (s, 1H), 8.03 (d, J = 7.1 Hz, 2H), 7.93 as a white solid. Mp 205–206 °C. H NMR (400 MHz, DMSO)
(d, J = 8.8 Hz, 2H), 7.88 (s, 1H), 5.38 (t, J = 8.8 Hz, 2H), 5.03 δ 10.68 (s, 1H), 8.86 (s, 1H), 8.45 (d, J = 9.0 Hz, 1H), 7.88 (d, J =
(brs, 1H), 4.06–3.94 (m, 3H), 3.74 (d, J = 11.3 Hz, 1H), 3.61 (t, 8.8 Hz, 2H), 7.68 (d, J = 8.8 Hz, 2H), 7.18–6.87 (m, 5H), 4.77 (s,
J = 7.1 Hz, 2H), 3.51 (td, J = 11.9, 4.4 Hz, 2H), 2.33 (td, J = 12.8, 2H), 3.89–3.72 (m, 1H), 2.78 (dd, J = 13.6, 5.6 Hz, 1H),
4.8 Hz, 2H), 2.09 (d, J = 13.2 Hz, 2H), 1.92–1.58 (m, 6H).
2.70–2.56 (m, 1H). MS (ES+): [M + H]+ = 865.4. HPLC purity
Deprotection was performed following method A: starting 97%.
from OTHP-13 (140 mg, 0.153 mmol) afforded 13 (120 mg,
General procedure for the synthesis of 17a–c
96%) as a white solid. Mp 214–217 °C. 1H NMR (400 MHz,
DMSO) δ 11.05 (s, 1H), 9.25 (s, 1H), 8.83 (s, 1H), 8.12 (d, J = To a solution of 7 (1 eq.) and K2CO3 (3–5 eq.) in DMF (5 mL) at
8.8 Hz, 2H), 7.93 (d, J = 8.8 Hz, 2H), 5.27 (t, J = 7.2 Hz, 2H), 0 °C, the corresponding halide (1.2–1.4 eq.) was added and the
3.89 (dd, J = 11.5, 3.4 Hz, 2H), 3.47 (t, J = 7.1 Hz, 2H), 3.23–3.03 mixture was heated at 60 °C under MW conditions until reac-
(m, 2H), 2.24 (d, J = 13.0 Hz, 2H), 1.97 (td, J = 12.9, 4.4 Hz, 2H). tion completion (TLC). HCl 1 M was added until a white pre-
HPLC purity >98%. MS (ES+): [M + H]+ = 813.5.
cipitate was formed, which was filtered and used in the next
step without further purification.
N-Hydroxy-4-((4-(4-((perbromo-1H-benzo[d][1,2,3]triazol-1-yl)
methyl)-1H-1,2,3-triazol-1-yl)phenyl)sulfonyl)tetrahydro-2H- 2-((4,5,6,7-Tetrabromo-1H-benzo[d]imidazol-2-yl)thio)acetic acid
pyran-4-carboxamide (14) (17a)
Following the general procedure for CuAAC, starting from Following the general procedure and starting from 7 (50 mg,
azide 9 (70 mg, 0.17 mmol) and alkyne 3 (88.70 mg, 0.107 mmol), K2CO3 (74 mg, 0.535 mmol) and 2-chloroacetic
0.19 mmol), and after purification by column chromatography acid (12.19 mg, 0.129 mmol) gave 17a (47 mg, 84%) as a white
on silica gel (DCM/MeOH 0.75–1%) gave OTHP-14 (23 mg, solid. Mp 257–260 °C (in accordance with lit data).37 MS (ES+):
1
16%) as a white solid. Mp 218.6–220.5 °C. H NMR (40 MHz, [M + H]+ = 524.7.
CDCl3) δ 9.35 (s, 1H), 8.01 (d, J = 8.8 Hz, 2H), 7.91 (d, J =
2-((4,5,6,7-Tetrabromo-1H-benzo[d]imidazol-2-yl)thio)propanoic
8.7 Hz, 2H), 7.88 (s, 1H), 6.44 (s, 2H), 5.00 (brs, 1H), 4.02–3.94
acid (17b)
(m, 3H), 3.76–3.69 (m, 1H), 3.47 (tdd, J = 11.5, 5.0, 1.3 Hz, 2H),
2.36–2.25 (m, 2H), 2.06 (d, J = 13.2 Hz, 2H), 1.88–1.60 (m, 6H).
Following the general procedure and starting from 7 (50 mg,
Deprotection was performed following method A: starting 0.107 mmol), K2CO3 (44 mg, 0.321 mmol) and 2-bromopropa-
from OTHP-14 (67 mg, 0.078 mmol) afforded 14 (40 mg, 67%) noic acid (19 mg, 0.129 mmol) gave 17b (40 mg, 69%) as a
1
as a white solid. Mp 222–223 °C. H NMR (400 MHz, DMSO) white solid. Mp 125–128 °C. MS (ES−): [M − H]− = 536.7.
δ 10.98 (brs, 1H), 9.21 (brs, 1H), 8.92 (s, 1H), 8.13 (d, J = 8.8
Hz, 2H), 7.90 (d, J = 8.8 Hz, 2H), 6.39 (s, 2H), 3.88 (dd, J = 11.0,
3-Methyl-2-((4,5,6,7-tetrabromo-1H-benzo[d]imidazol-2-yl)thio)
butanoic acid (17c)
3.5 Hz, 2H), 3.15 (t, J = 11.6 Hz, 2H), 2.22 (d, J = 13.1 Hz, 2H),
2.03–1.87 (m, 2H). HPLC purity 97%. MS (ES+): [M + H]+
=
Following the general procedure and starting from 7 (50 mg,
799.6. Elemental analysis C21H17Br4N7O5S required; C, 31.56; 0.107 mmol), K2CO3 (44.3 mg, 0.321 mmol) and 2-bromo-3-
H, 2.14; N, 12.27; S, 4.01 Found C: 31.61; H: 2.20; N: 12.00; methylbutanoic acid (27.1 mg, 0.14 mmol) gave 17c (40 mg,
S: 3.88.
66%) as a white solid. Mp 162.3–165.2 °C. MS (ES−): [M − H]− =
564.7.
(R)-N-Hydroxy-3-phenyl-2-(4-(4-(((4,5,6,7-tetrabromo-1H-benzo
[d]imidazol-2-yl)thio)methyl)-1H-1,2,3-triazol-1-yl)
phenylsulfonamido)propanamide (15)
General procedure for amide coupling
To a solution of the corresponding carboxylic acid (1 eq.) in
Following the general procedure for CuAAC, starting from DMF HOBT hydrate (1.2–5 eq.), EDCI (1.44–6.78 eq.) and
azide 8 (116.4 mg, 0.261 mmol), and alkyne 4 (98.86 mg, O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (4.0–9.4 eq.) were
0.443 mmol), and after purification by column chromato- added. In some cases, Et3N was also added. The reaction
graphy on silica gel (7 : 3 hexane/EtOAc) gave OTHP-15 mixture was stirred from 24–72 h at RT. Water was added to
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1
the reaction mixture and the formed precipitate was filtered solid. Mp 189 °C (dec.). H NMR (400 MHz, DMSO) δ 10.89 (s,
and purified by column chromatography.
1H), 4.02 (s, 2H). MS (ES+): [M + H]+ = 539.7. HPLC purity 97%.
2-((4,5,6,7-Tetrabromo-1H-benzo[d]imidazol-2-yl)thio)-N-
((tetrahydro-2H-pyran-2-yl)oxy)acetamide (18a)
N-Hydroxy-2-((4,5,6,7-tetrabromo-1H-benzo[d]imidazol-2-yl)
thio)propanamide (16b)
Following the general procedure for amide coupling, starting Deprotection was performed following method B (General pro-
from 17a (140 mg, 0.267 mmol), HOBT (43 mg, 0.321 mmol), cedure for deprotection to hydroxamates): starting from 18b
EDCI (59.72 mg, 0.385 mmol), H2N-OTHP (62.61 mg, (50 mg, 0.076 mmol), 16a (27 mg, 65%) was obtained as a
0.5352 mmol) and Et3N (112 μL, 0.80 mmol) after purification white solid. Mp 207–208 °C. 1H NMR (400 MHz, DMSO) δ
by column chromatography on silica gel (8 : 2 hexane/EtOAc) 13.55 (brs, 1H), 11.01 (s, 1H), 9.18 (brs, 1H), 4.53 (q, J = 6.7 Hz,
followed by recrystallization (hexane/EtOAC) gave 18a (113 mg, 1H), 1.60 (d, J = 6.8 Hz, 3H). MS (ES+): [M + H]+ = 553.6. HPLC
68%) as a white solid. Mp 199–202 °C. MS (ES+): [M + Na]+ = purity 95%.
645.7. Elemental analysis C14H13Br4N3O3S requires: C, 26.99;
H, 2.10; N, 6.75; S, 5.15; found: C, 26.97; H, 2.11; N, 6.76; S,
5.12.
N-Hydroxy-3-methyl-2-((4,5,6,7-tetrabromo-1H-benzo[d]
imidazol-2-yl)thio)butanamide (16c)
Deprotection was performed following method B (General pro-
cedure for deprotection to hydroxamates): starting from 18c
(57 mg, 0.085 mmol), 16c (20 mg, 40%) was obtained as a
2-((4,5,6,7-Tetrabromo-1H-benzo[d]imidazol-2-yl)thio)-N-
((tetrahydro-2H-pyran-2-yl)oxy)propanamide (18b)
Following the general procedure for amide coupling, starting white solid. Mp 182–185 °C. 1H NMR (400 MHz, DMSO)
from 17b (248 mg, 0.46 mmol), HOBT (351 mg, 2.60 mmol) δ 13.59 (brs, 1H), 11.00 (s, 1H), 9.16 (brs, 1H), 4.28 (d, J =
EDCI (483 mg, 6.78 mmol), and H2N-OTHP (507 mg, 7.5 Hz, 1H), 2.25–2.16 (m, 1H), 1.02 (d, J = 6.8 Hz, 3H), 1.00 (d,
4.34 mmol) after column chromatography on silica gel (7 : 3 to J = 6.7 Hz, 3H). MS (ES−): [M − H]− = 579.4. HPLC purity
1 : 1 hexane/EtOAc) followed by a second chromatography >98%.
(DCM/MeOH 0.5 to 1%) and recrystallization (hexane/EtOAc)
gave 18b (79 mg, 27%) as a white solid. This compound is
(R)-Methyl 3-methyl-2-((4,5,6,7-tetrabromo-1H-benzo[d]
imidazol-2-yl)amino)butanoate (20)
present as a diastereoisomeric A : B mixture (60 : 40), mp
1
208–209 °C. H NMR (400 MHz, DMSO) δ 13.54 (s, 1H), 11.60 To a solution of 6 (300 mg, 0.58 mmol) in dry MeOH, D-valine-
(s, Isomer A, 0.6H), 11.54 (s, Isomer B, 0.4H), 4.87 (s, Isomer B, OMe (767 mg, 5.85 mmol) was added and the reaction mixture
0.4H), 4.85 (s, Isomer A, 0.6H), 4.60–4.54 (m, 1H), 3.91–3.83 was heated at 110 °C for 24–48 h in a sealed tube until com-
(m, 1H), 3.54–3.43 (m, 1H), 1.65–1.51 (m, 9H). Elemental ana- pletion of the reaction was seen by TLC. The formed precipi-
lysis C15H15Br4N3O3S requires: C, 28.28; H, 2.37; N, 6.60; S, tate was collected by filtration to give 20 (178 mg, 55%) as a
5.03; found: C, 28.32; H, 2.37; N, 6.61; S, 4.98.
light-yellow solid which was used in the next reaction without
further purification. Mp 298 °C (dec.). 1H NMR (400 MHz,
CDCl3) δ 8.67 (s, 1H), 6.05 (d, J = 9.3 Hz, 1H), 4.68 (dd, J = 9.1,
5.5 Hz, 1H), 3.91 (s, 3H), 2.36–2.25 (m, 1H), 1.07 (d, J = 6.8 Hz,
3-Methyl-2-((4,5,6,7-tetrabromo-1H-benzo[d]imidazol-2-yl)
thio)-N-((tetrahydro-2H-pyran-2-yl)oxy)butanamide (18c)
Following the general procedure for amide coupling, starting 6H). 1.00 (d, J = 6.9 Hz, 6H). MS (ES+): [M + H]+ = 563.8.
from 17c (180 mg, 0.318 mmol), HOBT (154.54 mg,
1.145 mmol), EDCI (212.9 mg, 1.37 mmol) and H2N-OTHP
(223 mg, 1.90 mmol) after column chromatography on silica
(R)-3-Methyl-2-((4,5,6,7-tetrabromo-1H-benzo[d]imidazol-2-yl)
amino)butanoic acid (21)
gel (DCM/MeOH 1%) and recrystallization (hexane/EtOAc) gave Ester 20 (258 mg, 0.46 mmol) was dissolved in 1 : 1 THF NaOH
18c (120 mg, 57%) as a white solid. This compound is present 1 M (20 eq.). The reaction mixture was stirred at RT and con-
as a diastereoisomeric A : B mixture (45 : 55), mp 176–178 °C. trolled by TLC until no starting material was seen. The reac-
1H NMR (400 MHz, CDCl3) δ 11.21 (s, Isomer A, 0.45H), 11.14 tion crude product was then washed with EtOAc and the
(s, Isomer B, 0.55H), 10.43 (s, Isomer A, 0.45H), 10.30 (s, aqueous phase was acidified. The formed precipitate was fil-
Isomer B, 0.55H), 5.06 (s, Isomer B, 0.55H), 4.97 (s, Isomer A, tered to give 21 (230 mg, 92%) as a white solid which was used
0.45H), 3.96–3.82 (m, 2H), 3.65 (d, J = 11.7 Hz, Isomer B, in the next reaction without further purification. Mp
1
0.55H), 3.31 (d, J = 11.8 Hz, Isomer A, 0.45H), 2.38–2.29 (m, 253–255 °C. H NMR (400 MHz, DMSO) δ 7.30 (d, J = 6.6 Hz,
1H), 1.86–1.53 (m, 6H), 1.20–1.08 (m, 6H). Elemental analysis 1H), 4.49–4.45 (m, 1H), 2.30–2.22 (m, 1H), 0.98 (d, J = 6.9 Hz,
C17H19Br4N3O3S requires: C, 30.70; H, 2.88; N, 6.32; S, 4.82; 6H), 0.96 (d, J = 6.9 Hz, 6H).
found: C, 30.50; H, 2.83; N, 6.31; S, 4.79.
(R)-N-Hydroxy-3-methyl-2-((4,5,6,7-tetrabromo-1H-benzo[d]
N-Hydroxy-2-((4,5,6,7-tetrabromo-1H-benzo[d]imidazol-2-yl)
imidazol-2-yl)amino)butanamide (19)
thio)acetamide (16a)
Following the general procedure for amide coupling, starting
Deprotection was performed following method B (General pro- from 21 (230 mg, 0.419 mmol) gave, after purification by
cedure for deprotection of hydroxamates): starting from 18a column chromatography on silica gel (8 : 2 hexane/EtOAc), fol-
(50 mg, 0.08 mmol), 16a (34 mg, 79%) was obtained as a white lowed by recrystallization from hexane/EtOAC, OTHP-19
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(60 mg, 22%) as a white solid. This compound is present as a 8.9 Hz, 2H), 7.74 (d, J = 8.9 Hz, 2H), 3.68 (d, J = 6.2 Hz, 2H),
1
diastereoisomeric A : B mixture (35 : 65), mp 200 °C (dec.). H 3.54 (s, 3H). MS (ES+): [M + H]+ = 676.7. Following the same
NMR (400 MHz, CDCl3) δ 10.07 (brs, 1H), 9.15 (brs, 1H), 6.85 procedure described before for the hydrolysis of 20, this ester
(brs, isomer A, 0.35H), 6.69 (brs, isomer B, 0.65H), 5.26 (s, (150 mg, 0.221 mmol, 1 eq.) gave 25a (120 mg, 75%) as a white
isomer A, 0.35H), 5.19 (s, isomer B, 0.65H), 4.35–3.90 (m, 2H), solid. Mp 251 °C (dec.). 1H NMR (400 MHz, DMSO) δ 12.61
3.67 (d, J = 11.3 Hz, 1H), 2.29–2.25 (m, 1H), 1.96–1.58 (m, 6H), (brs, 1H), 12.22 (brs, 1H), 9.92 (brs, 1H), 7.91 (d, J = 8.8 Hz,
1.12–1.07 (m, 6H).
2H), 7.82 (brs, 1H), 7.76 (d, J = 8.8 Hz, 2H), 3.56 (d, J = 6.0 Hz,
Deprotection was performed following method B: starting 2H). MS (ES+): [M + H]+ = 662.7.
from OTH-19 (60 mg, 0.09 mmol) afforded 19 (50 mg, 80%).
(R)-3-Methyl-2-(4-((4,5,6,7-tetrabromo-1H-benzo[d]imidazol-2-yl)
amino)phenylsulfonamido)butanoic acid (25b)
Mp 182–183 °C. 1H NMR (400 MHz, DMSO) δ 10.85 (s, 1H),
6.65 (d, J = 9.3 Hz, 1H), 4.19 (dd, J = 9.0, 6.4 Hz, 1H), 2.08–1.92
(m, 1H), 0.93–0.91 (m, 6H). MS (ES+): [M + H]+ = 564.7. HPLC Following the same procedure described for the synthesis of
purity 98%.
20, starting from 6 (273 mg, 0.523 mmol) and 24b (300 mg,
1.047 mmol), the corresponding ester (273 mg, 72%) was
obtained as a yellowish solid. Mp 271 °C (dec.). 1H NMR
Methyl 2-(4-aminophenylsulfonamido)acetate (24a)
A mixture of 23a29 (4.48 g, 17.23 mmol) in absolute EtOH (400 MHz, DMSO) δ 12.05 (s, 1H), 9.69 (s, 1H), 8.12 (d, J =
(3 mL per mmol) and Pd/C 10% (35 mg per mmol) was intro- 9.3 Hz, 1H), 7.90 (d, J = 8.8 Hz, 2H), 7.71 (d, J = 8.8 Hz, 2H),
duced into a Parr shaker apparatus at 50 p.s.i. of hydrogen for 3.52 (dd, J = 9.2, 7.1 Hz, 1H), 3.39 (s, 3H), 1.92–1.84 (m, 1H),
24–72 h, until completion of the reaction was seen by TLC. 0.82 (d, J = 6.7 Hz, 3H). 0.78 (d, J = 6.8 Hz, 3H). MS (ES+):
The catalyst was filtered and the solvent was removed under [M + H]+ = 718.6. Following the same procedure described
vacuum to give the corresponding amino compound (3.4 g, before for the hydrolysis of 20, this ester was hydrolysed
86%) as a light-yellow solid. Mp 169–171 °C. 1H NMR (447 mg, 0.62 mmol, 1 eq.) to give 25b (335 mg, 77%) as a
1
(400 MHz, DMSO) δ 12.50 (s, 1H), 7.40 (d, J = 8.7 Hz, 2H), 6.58 white solid. Mp 195–198 °C. H NMR (400 MHz, DMSO) δ 9.88
(d, J = 8.7 Hz, 2H), 5.92 (brs, 2H), 3.44 (d, J = 5.7 Hz, 2H). This (s, 1H), 7.99 (d, J = 8.7 Hz, 2H), 7.84 (d, J = 8.5 Hz, 1H), 7.53 (d,
solid (1.22 g, 5.3 mmol) was added to a solution of SOCl2 at J = 8.8 Hz, 2H), 3.50–3.43 (m, 1H), 1.90–1.80 (m, 1H), 0.80 (d,
0 °C. The mixture was heated at reflux for 24 h, the solvent was J = 6.7 Hz, 6H), 0.77 (d, J = 6.8 Hz, 6H). MS (ES+): [M + H]+ =
removed under vacuum and 24a (1.09 g, 84%) was obtained as 704.6.
a light-yellow solid which was used in the next step without
N-Hydroxy-2-(4-((4,5,6,7-tetrabromo-1H-benzo[d]imidazol-2-yl)
further purification. Mp 187–193 °C. 1H NMR (400 MHz,
amino)phenylsulfonamido)acetamide (22a)
DMSO) δ 7.79 (s, 1H), 7.47 (d, J = 8.7 Hz, 2H), 6.72 (d, J =
8.6 Hz, 2H), 5.72 (brs, 2H), 3.59 (s, 2H), 3.55 (s, 3H).
Following the general procedure for amide coupling, starting
from 25a (100 mg, 0.151 mmol) gave after purification by
column chromatography on silica gel (hexane/EtOAc from 1/1
(R)-Methyl-2-(4-aminophenylsulfonamido)-3-methylbutanoate
(24b)
to 0/1) OTHP-protected 22a (80 mg, 70%) as a white solid. Mp
1
Following the same procedure for hydrogenation as that for 311–313 °C. H NMR (400 MHz, DMSO) δ 12.10 (s, 1H), 11.12
24a, starting from 23b29 (6.1 gr, 0.019 mol) gave after recrystal- (s, 1H), 9.73 (s, 1H), 7.93 (d, J = 8.6 Hz, 2H), 7.87 (t, J = 6.2 Hz,
lization (EtOH/H2O) a yellow solid (5.20 gr, 100%). Mp 1H), 7.75 (d, J = 8.6 Hz, 2H), 4.71 (bs, 1H), 3.92–3.83 (m, 1H),
172–175 °C. 1H NMR (400 MHz, DMSO) δ 12.50 (s, 1H), 7.37 3.50–3.47 (m, 1H), 3.38 (d, J = 6.2 Hz, 2H), 1.68–1.39 (m, 6H).
(d, J = 8.7 Hz, 2H), 6.55 (d, J = 8.7 Hz, 2H), 5.87 (s, 2H), 3.35 MS (ES+): [M + H]+ = 677.6.
(dd, J = 8.3, 6.0 Hz, 1H), 1.96–1.76 (m, 1H), 0.78 (dd, J = 13.5,
Deprotection was performed following method B: starting
6.8 Hz, 6H).
from OTHP-22a (108 mg, 0.142 mmol) afforded 22a as a white
This solid (400 mg, 1.47 mmol) was esterified as in the pre- solid (80 mg, 84%). Mp 225–228 °C. 1H NMR (400 MHz,
vious example, giving 24b (420 mg, quantitative conversion) as DMSO) δ 12.08 (s, 1H), 10.53 (s, 1H), 9.70 (s, 1H), 8.86 (s, 1H),
a light yellow solid. Mp 168–169 °C. 1H NMR (400 MHz, 7.92 (d, J = 8.8 Hz, 2H), 7.81–7.76 (m, 3H), 3.32 (under the
DMSO) δ 7.69 (d, J = 9.3 Hz, 1H), 7.35 (d, J = 8.7 Hz, 2H), 6.56 water peak, 2H). MS (ES+): [M + H]+ = 677.6. HPLC purity 98%.
(d, J = 8.7 Hz, 2H), 5.90 (s, 2H), 3.39 (s, 3H), 3.39–3.36 (m, 1H),
(R)-N-Hydroxy-3-methyl-2-(4-((4,5,6,7-tetrabromo-1H-benzo[d]
1.87–1.79 (m, 1H), 0.80 (d, J = 6.7 Hz, 3H). 0.76 (d, J = 6.8 Hz,
imidazol-2-yl)amino)phenylsulfonamido)butanamide (22b)
3H). MS (ES+): [M + Na]+ = 309.0.
Following the general procedure for amide coupling, starting
2-(4-((4,5,6,7-Tetrabromo-1H-benzo[d]imidazol-2-yl)amino)
from 25b (338 mg, 0.48 mmol) gave after purification by
phenylsulfonamido)acetic acid (25a)
column chromatography on silica gel (hexane/EtOAc 1/1 to 0/1)
Following the same procedure described before for the syn- OTHP-22b (256 mg, 66%) as a white solid. This compound is
thesis of 20, starting from 6 (300 mg, 0.58 mmol) and 24a present as a diastereoisomeric A : B mixture (55 : 45), mp
(285 mg, 1.17 mmol), a light-yellow solid ester (200 mg, 51%) 234 °C (dec.). 1H NMR (400 MHz, DMSO) δ 12.02 (s, 1H), 11.14
was obtained. Mp 266 (dec.). 1H NMR (400 MHz, DMSO) (s, isomer A, 0.55H), 11.05 (s, isomer B, 0.45H), 9.64 (s, 1H),
δ 12.10 (s, 1H), 9.73 (s, 1H), 8.05 (t, J = 6.2 Hz, 1H), 7.91 (d, J = 7.92–7.89 (m, 2H), 7.81–7.70 (m, 3H), 4.72 (bs, isomer B,
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0.45H), 4.32 (m, isomer A, 0.55H), 3.87–3.80 (m, 1H), 3.44–3.38 Biological activity
(m, 2H), 1.80–1.72 (m, 1H), 1.60–1.30 (m, 6H), 0.83–0.72 (m,
MMP inhibition assays. MMP activity measurements were
performed using a MMP Inhibitor Profiling Kit purchased
from Enzo Life Science International, Inc., and following the
manufacturer’s protocol with slight modifications. Proteolytic
activity was measured using a thiopeptide substrate (Ac-PLG-
[2-mercapto-4-methylpentanoyl]-LG—OC2H5)38,39 where the
MMP cleavage site peptide bond has been replaced by a thio-
ester bond. Hydrolysis of this bond by MMPs produces a sulf-
hydryl group that reacts with DTNB to form 2-nitro-5-thioben-
zoic acid, which was detected by its absorbance at 414 nm
(microplate photometer 90 Thermo Scientific Multiscan FC).
Enzyme reactions were carried out at 37 °C in a 100 μL final
volume of solutions, where the catalytic domains of the corres-
ponding MMP were incubated in triplicate with at least seven
concentrations of inhibitors. The assay buffer contained the
following components: 50 mM HEPES, 10 mM CaCl2 95,
0.05% Brij-35 and 1 mM DTNB at pH 7.5. After addition of the
substrate, the increase of absorbance was recorded in 1 min
time intervals for 30 min. Data were plotted as OD versus time
for each sample, to obtain the reaction velocity (V) in OD per
min. The percentage of residual activity for each compound
was calculated using the following formula: % of remaining
activity = (V in the presence of inhibitor/Vcontrol) × 100. An
inhibitor, NNGH, was included as a prototypic control inhibi-
tor. The concentration of the compound that provided 50%
inhibition of enzymatic activity (IC50) was determined by using
semi-logarithmic dose–response plots (Graph Pad Prism 5.0
for Windows, Graph Pad Software Inc., San Diego, California,
USA, 2007).
CK2 inhibition assays. Recombinant human protein kinase
GST-CK2 a was produced by bacteria and purified as described
elsewhere.40 The phosphorylation reactions were conducted at
37 °C for 5 min in 50 μl samples each containing 1 pmol of
human recombinant CK2α, 40 mM of peptide substrate
(RRRADDSDDDDD), and an appropriate concentration of the
tested compounds (0.1–200 µM). The reaction buffer contained
20 µM [γ-32P]ATP (specific radioactivity 300–1000 cpm
pmol−1), 15 mM Mg2+, 20 mM Tris–HCl pH 7.5, and 6 mM
2-mercaptoethanol. 10 µl of the assay mixture was spotted
onto a square (1 cm/1 cm) of Whatman P81 paper and allowed
to dry. Each square was immersed in cold 0.5% phosphoric
acid, and washed 3 times for 10 min, allowed to dry and trans-
ferred into a vessel containing Opti-Phase liquid (scintillation
solution) and radioactivity was quantified using a scintillation
counter (MicroBeta, PerkinElmer) by Cerenkov counting. All
experiments were performed in triplicate. IC50 values were cal-
culated using GraphPad Prism (version 4.0) software.
6H).
Deprotection was performed following method B: starting
from OTHP-22b (150 mg, 0.24 mmol) gave 22b (87 mg, 50%)
1
as a white solid. Mp 197–200 °C. H NMR (400 MHz, DMSO)
δ 10.50 (brs, 1H), 9.83 (s, 1H), 7.89 (d, J = 8.9 Hz, 2H),
7.74–7.72 (m, 3H), 3.28 (t, J = 8.3 Hz, 1H), 1.82–1.69 (m, 1H),
0.77–0.73 (m, 6H). MS (ES+): [M + H]+ = 719.7. HPLC purity
98%.
2-((4,5,6,7-Tetrabromo-1H-benzo[d]imidazol-2-yl)thio)acetaldehyde
(27)
Following the same procedure described before for 17a–c,
starting from 7 (100 mg, 0.215 mmol), K2CO3 (89 mg,
0.65 mmol) and 2-chloroacetaldehyde (27.29 μL, 0.22 mmol)
gave 27 (103 mg, 93%) as a white solid. Mp 201.7 °C (dec.). MS
(ES−): [M − H]− = 506.4.
Methyl 2-(4-((2-((4,5,6,7-tetrabromo-1H-benzo[d]imidazol-2-yl)
thio)ethyl)amino)phenylsulfonamido)acetate (28)
27 (586 mg, 1.15 mmol), NaBH(OAc)3 (485 mg, 2.29 mmol),
AcOH (1.1 mL) and 24a (286 mg, 1.045 mmol) were added to
6 mL of anh. DCM and stirred at 50 °C overnight. The reaction
mixture was diluted with 20 mL of DCM and washed with satu-
rated NaCl, and the organic phase was dried with MgSO4, fil-
tered and dried under vacuum. The crude product was purified
by column chromatography (hexane/EtOAc from 7 : 3 to 1 : 1)
to afford 28 (165 mg, 21%) as a light yellow solid. Mp
225–228 °C. 1H NMR (400 MHz, DMSO) δ 13.52 (s, 1H), 7.76 (t,
J = 6.2 Hz, 1H), 7.50 (d, J = 8.7 Hz, 2H), 6.96 (t, J = 5.4 Hz, 1H),
6.90 (d, J = 8.6 Hz, 2H), 3.60–3.50 (m, 7H), 3.46–3.39 (m, 2H).
MS (ES+): [M + Na]+ = 758.6.
N-Hydroxy-2-(4-((2-((4,5,6,7-tetrabromo-1H-benzo[d]imidazol-
2-yl)thio)ethyl)amino)phenylsulfonamido)acetamide (26)
Following the same procedure described above for the
synthesis of 21, starting from 28 (339 mg, 0.46 mmol) the
corresponding carboxylic acid (164 mg, 49%) was obtained as
an oil and was used in the next step without further
purification.
Following the general procedure for amide coupling, start-
ing from this carboxylic acid (164 mg, 0.227 mmol), gave after
purification by column chromatography on silica gel (hexane/
EtOAc 1 : 9 to 0 : 10) OTHP-26 (70 mg, 37%) as a light yellow
solid. 1H NMR (400 MHz, DMSO) δ 13.50 (s, 1H), 11.05 (s, 1H),
7.52 (d, J = 8.5 Hz, 2H), 6.90 (m, 3H), 4.71 (brs, 1H), 3.92–3.85
(m, 1H), 3.59–3.40 (m, 3H), 3.35–3.25 (m, 4H), 1.65–1.45 (m,
6H).
Deprotection was performed following method B: starting
from OTHP-26 (50 mg, 0.061 mmol) gave 26 as a white solid
In vitro studies on mammalian cell lines
(25 mg, 45%). Mp 189–192 °C. 1H NMR (400 MHz, DMSO) Human Jurkat T-leukemia cells were kindly provided by Prof.
δ 10.49 (brs, 1H), 7.52 (d, J = 8.3 Hz, 2H), 7.46 (brs, 1H), 6.90 Walter Berger (cell culture collection at Vienna Medical
(d, J = 8.6 Hz, 2H), 3.65–3.51 (m, 2H), 3.50–3.37 (m, 2H), 3.19 University, Institute of Cancer Research). Cells were cultured
(brs, 2H).
MS (ES−): [M − H]− = 735.4 HPLC purity 95%.
in the RPMI medium supplemented with 10% fetal calf serum
(Sigma-Aldrich, St Louis, USA), 50 µg mL−1 streptomycin
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(Sigma-Aldrich, St Louis, USA), and 50 units per mL penicillin were applied, and electrostatic interactions were treated using
(Sigma-Aldrich, St Louis, USA) under a 5% CO2-containing the smooth particle mesh Ewald method52 with a grid spacing
humidified atmosphere at 37 °C. For experiments, cells (5 × of 1 Å. The cutoff distance for the non-bonded interactions
105 per well) were seeded into 24-well tissue culture plates was 9 Å, the SHAKE53 algorithm was applied to all bonds, and
(Greiner Bio-one, Germany). The short-term (24 h) cytotoxic an integration step of 2.0 fs was used throughout. After an
effect of antitumor drugs was studied under an Evolution 300 initial energy minimization of the water molecules and
Trino microscope (Delta Optical, Poland) after cell staining counter-ions, the system was heated to 300 K in 25 ps after
with Trypan blue (0.1%). Statistical analysis of data was con- which the solvent was allowed to redistribute around the posi-
ducted in GraphPad Prisms Software (GraphPad Software, Inc.) tionally restrained solute for 220 ps. After this time, the solute
using Student’s t test. Statistical significance was set at was allowed to move freely so as to explore all the plausible
P ≤ 0.05.
conformations in water. Snapshots of the 100 ns MD trajectory
were collected every 20 ps for further analysis carried out with
the cpptraj module of AMBER to extract the conformers.
Molecular modelling
To build the models we selected a structure of MMP2 issued
from a previous conformational analysis of the flexibility of the
Ω-loop,8 the X-Ray structure of MMP13 bound to an inhibitor
(PDB code 3KEK),41 crystal structure of CK2 (PDB code 4KWP),
bound to a benzimidazole inhibitor.16 The two proteins were
prepared with the Protein Preparation Wizard module of the
Schrödinger software after elimination of the bound inhibitors
and water molecules.42,43 In this protein preparation the side
chain of the catalytic Glu121 of MMP2 and Glu223 of MMP13
was considered to be protonated as this residue is known to
deprotonate different inhibitors.44 The Glide software was
used for the 20 Å size grid preparation that englobed the
whole binding site with the grid centre located on the catalytic
Zinc ion for MMP2 and MMP13 and the center of the ATP-
binding site in CK2.45–48 Ligands were prepared using the
LigPrep module of the Schrödinger suite.49 The hydroxamate
form of the hydroxamic acid moiety was used for the docking
calculations in MMP2 and MMP13 as it is supposed to be
deprotonated by Glu121 and Glu223 respectively, whereas the
non-protonated form of the hydroxamic acid was used for the
dockings in CK2. These calculations were performed using the
extra precision mode (XP) of the glide docking protocol to
ensure the best possible binding pose in MMP2 and CK2.45–48
The induced-fit docking protocol was applied for MMP13 as
performed in the literature50 to ensure ligand accommodation
in the binding site. For the conformational analysis of 26 and
the Ω-loop of apo MMP2 (model 1 extracted from the NMR
obtained structure under PDB code 1HOV) and MMP13 (PDB
code 3KEK) the charge distribution of the compound was
obtained by fitting the quantum mechanically calculated
(RHF/6-31G**//RHF/3-21G*) molecular electrostatic potential
(MEP) of the geometry-optimized molecules to a point charge
model, as implemented in Gaussian 03 (Gaussian, Inc.,
Wallingford, CT). The general AMBER (http://ambermd.org/)
force field (GAFF) was used to assign bonded and nonbonded
parameters (parm03) to the ligand atoms. The compound and
the two proteins were then immersed in truncated octahedra
containing ∼10 000 TIP3P and ∼13 000 TIP3P water molecules,
respectively.51 To achieve system electroneutrality 4Na+ and
3Cl− were added to the MMP2 and MMP13, respectively. The
sander and pmemd modules of the AMBER14 suite (http://
ambermd.org/) were used for the restrained and unrestrained
MD simulations, respectively. Periodic boundary conditions
Conclusions
From the study carried out, we can conclude that the design of
compounds capable of interacting simultaneously with MMP2
and CK2 has proven to be an unattainable goal in our hands.
Nevertheless, it has allowed us to describe a series of com-
pounds with a remarkable activity against MMP2 and another
series of CK2 inhibitors, both of interest in tumor processes.
Moreover, we have found an interesting hit for the future
development of potent and selective MMP13 inhibitors with
potential application in osteoarthritis.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was supported by project CTQ2014-52604-R
(MINECO/FEDER, UE).
Notes and references
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