Discovery of 4-Hydroxy-3-(3-(phenylureido)benzenesulfonamides as SLC-0111 Analogues for the Treatment of Hypoxic Tumors Overexpressing Carbonic Anhydrase IX

Article abstract of DOI:10.1021/acs.jmedchem.8b00770

Herein we report the 2-aminophenol-4-sulfonamide 1 and its ureido derivatives 2-23 as inhibitors of the carbonic anhydrase (CA, EC 4.2.1.1) enzymes as analogues of the hypoxic tumor phase II entering drug SLC-0111. This scaffold may determine preferential

Full text of DOI:10.1021/acs.jmedchem.8b00770

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Article
Discovery of 4-Hydroxy-3-(3-(phenylureido)benzenesulfonamides
As SLC-0111 Analogues For the Treatment of Hypoxic
Tumors Overexpressing Carbonic Anhydrase IX
Murat Bozdag, Fabrizio Carta, Mariangela Ceruso, Marta Ferraroni,
Paul C. McDonald, Shoukat Dedhar, and Claudiu T Supuran
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00770 • Publication Date (Web): 02 Jul 2018
Downloaded from http://pubs.acs.org on July 4, 2018
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Discovery of 4-Hydroxy-3-(3-(phenylureido)benzenesulfonamides as SLC-0111
Analogues for the Treatment of Hypoxic Tumors Overexpressing Carbonic
Anhydrase IX
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Murat Bozdag,1,2 Fabrizio Carta,2 Mariangela Ceruso,1 Marta Ferraroni,1 Paul C. McDonald,3
Shoukat Dedhar,3 Claudiu T. Supuran* 1
1
University of Florence, Department of Chemistry “Ugo Schiff”, Via della Lastruccia 3, 50019
Sesto Fiorentino (Florence), Italy.
2 University of Florence, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, Via Ugo Schiff
6, 50019 Sesto Fiorentino (Florence), Italy
3
Department of Integrative Oncology, BC Cancer Research Centre, BC V5Z 1L3 Vancouver,
British Columbia, Canada
Abstract. Herein we report the 2ꢀaminophenolꢀ4ꢀsulfonamide 1 and its ureido derivatives 2-23 as
inhibitors of the carbonic anhydrase (CA, EC 4.2.1.1) enzymes as analogues of the hypoxic tumor
phase II entering drug SLC-0111. This scaffold may determine preferential rotational isomers to
selectively interact within the tumorꢀassociated CAs. Most of the compounds indeed showed in
vitro selective inhibition of the tumor associated CA isoforms IX and XII. The most potent
derivative within the series was 11 (KIs of 2.59 and 7.64 nM on hCA IX and XII respectively)
which shares the 4ꢀfluorophenylureido tail with the clinical candidate. We investigated by means of
Xꢀray crystallographic studies the binding modes of 3 selected compounds of this series to CA I.
The evaluation of therapeutic efficacy of compound 11 in an orthotopic, syngeneic model of CA
IXꢀpositive breast cancer in vivo showed close matching antitumoral effects and tolerance with
SLC-0111.
Keywords: carbonic anhydrase; hypoxic tumors; ureidosulfonamides; inhibitors, SLCꢀ0111
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Introduction.
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Carbonic Anhydrases (CAs, EC 4.2.1.1) belong to the metalloenzyme superfamily and were
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1ꢀ8
identified more than seven decades ago.
To date seven genetically distinct and unrelated CA
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families have been characterized so far (i.e. αꢀ, βꢀ, γꢀ, δꢀ, ζꢀ, ηꢀ and θꢀCAs), and they all catalyze the
reversible hydration reaction of carbon dioxide to afford bicarbonate and protons.1ꢀ8 This
equilibrium is deeply involved in a plethora of processes at cellular as well as at tissue levels, and
among these, the pH homeostasis is the main one.9ꢀ12 The relevance of pH regulation in normal or in
hypoxic tumor cells is well documented. 9ꢀ13 A plethora of biological entities located on the cellular
biomembranes are directly involved in pH modulation (i.e. VꢀATPases, ion exchangers,
monocarboxylate transporters, Na+/H+ exchangers as the main ones), and among them the CA IX
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isoform recently stood up as validated drug target. CA IX is highly expressed in hypoxic tumors
(i.e. breast malignancies) and is associated with poor prognosis. 14ꢀ17 Conversely to other research
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groups which focused on the development of human (h) CA IX antibodies,
we turned our
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attention towards the identification of selective hCA IX small molecule inhibitors. Surprisingly
the insertion of the ureido moiety as a linker between the sulfonamide hCA inhibitor (CAI) warhead
and the molecular tail acted as the turning point in our extensive medicinal chemistry investigations
19, 20
within this field.
We demonstrated, by means of in vitro kinetic studies and Xꢀray crystal
adducts, that the ureido group allowed higher degrees of flexibility between the two interconnected
sections when compared to previous investigated linkers of the carboxyamido or of the sulfonamide
20
type, thus allowing the entire molecule to better allocate within the enzymatic CA cavities.
Among the large series of compounds of this type synthesized, SLC-0111 (also named U-104,
MST-104 and WBI-5111 in Figure 1) showed high affinity towards the tumor associated hCA IX
in vitro and promising antiproliferative and antimetastatic properties in in vivo breast cancer
13
models.
Such data granted SLC-0111 entering phase II clinical trials for the treatment of
advanced solid tumors.21
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Figure 1. Structure of SLC-0111.
Many contributions in the literature reported CAIs analogues of the SLC-0111 type, and among
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others it is worth mentioning the thioureido, the sulfamate and very recently the selenoureido
analogues. 24 Derivatives of SLC-0111 bearing the 19F or the 11C=O isotopes for imaging purposes
were also synthesized. 25, 26
In the search of more effective hCA IX selective inhibitors of the SLC-0111 type we report for the
first time a series of ureido containing compounds based on the 2ꢀaminophenolꢀ4ꢀsulfonamide 1. In
this study we explored their in vitro inhibition data on the physiologically relevant hCAs (i.e. I, II,
IX and XII), we determined the binding modes of selected ones by means of Xꢀray experiments on
the hCA Iꢀligand adducts, and finally we assessed their antiꢀtumor properties in vivo using an
orthotopic, syngeneic CA IXꢀpositive breast tumor model in comparison to SLC-0111.
Results and Discussion
Drug Design and Synthesis. We considered developing our ureido containing compounds
synthetic strategy on the 2ꢀaminophenolꢀ4ꢀsulfonamide 1 scaffold, whose phenolic OH was
primarily intended to form a stable intramolecular 5ꢀmembered ring with the ureido NH moiety
placed at the position 2. The introduction of such a moiety was thought to induce a rotational
restriction between C2 and N1’, which may determine preferential rotational isomers to interact
within the hCAs enzymatic sites thus resulting in enhancement of CAs enzymatic selectivity
(Figure 2).
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Figure 2. Formation of the intramolecular hydrogen bond leading to a 5ꢀmembered ring in the
target compounds may lead to C2ꢀN1’ rotational restriction.
Additionally, the formation of an intramolecular ring, as discussed above, represents a means to
selectively downtune only the C2ꢀN1’ bond frequency rotation without affecting the rotational
freedom of the remaining scaffold. Our theoretical considerations on the formation of the
intramolecular 5ꢀmembered ring were supported by a comparative 1HꢀNMR structural
characterization on compound 11 (as the structural analogue of SLC-0111) and its 4ꢀunsubstituted
analogue 11’ previously reported by our group. 27, 28 (Figure 3).
Figure 3. 1HꢀNMR 400 MHz signals at 23°C in DMSOꢀd6 of compounds 11 and 11’.
According to the 400 MHz 1HꢀNMR experiment at 23°C in DMSOꢀd6 the 1’ꢀH, 3’ꢀH and OH in 11
were assigned to the broad singlet peaks, all exchangeable with D2O, centered at δ 9.46, 8.37 and
1
10.91 respectively. The HꢀNMR experiment of 11’ in the same conditions revealed that the 1’ꢀH
and 3’ꢀH were at δ 9.04 and 8.78 respectively. The δ 0.42 downfield difference between 11 and 11’
1’ꢀH was ascribed to the formation of the intramolecular hydrogen bond interaction.
The insertion of structural modifications within small molecules greatly affect their solubility
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properties in aqueous media, which represents one of the major concerns in early stages of drug
development, as it seriously affects their bioavailability. Usually the enhancement of water
solubility properties of a drug, parallels the increase of the dissolution rate in the same medium.
This kinetic parameter has important implications mainly on firstꢀpass metabolism and
susceptibility of the drug to efflux mechanisms, which in turn affects pharmacological effectiveness
and patients’ compliance. Therefore we compared the solubility of SLC-0111 and 11 at room
temperature (r.t.) using a spectrophotometric method at the maximum absorbance of 217 nm, which
revealed that the latter was only slightly less soluble (1.66 fold) in aqueous pH 7.4 buffer solution.
This data suggested that the structural modification introduced in our series (i.e. 2ꢀaminophenolꢀ4ꢀ
sulfonamide 1 scaffold) did not significantly affect one of the main parameters of druggability, at
least for these closely related SLC-0111 derivative.
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The synthetic plan adopted for the synthesis of compounds 2-23 consisted of coupling of 1 with
commercially available isocyanates using acetonitrile as solvent and according to our previously
reported procedures (Scheme 1). 19, 20, 27, 28
Scheme 1. General synthesis of 2ꢀureidosubstituted benzenesulfonamides 2-23. 19, 20, 27, 28
1
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All compounds obtained were properly characterized by means of H, C, 19FꢀNMR spectroscopy,
HRMS and were ≥ 95% HPLC pure (see Experimental Section for details).
CA Inhibition. We investigated here the CA inhibitory properties of our reported compounds 1–23
and the obtained results were compared to the clinically used drug acetazolamide (AAZ), used as
standard CAI, against four physiological relevant isoforms, (i.e. hCAs I, II, IX and XII) by means of
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the stopped flow CO2 hydrase assay. 29
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Table 1: Inhibition data of human CA isoforms hCA I, II, IX and XII with sulfonamides 1-23 and
the acetazolamide (AAZ) by a stopped flow CO2 hydrase assay. 29
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_______________________________________________________________________________
KI (nM)*
Compound
R
hCA I
hCA II
hCA IX
hCA XII
________________________________________________________________________________
1
5354.1
354.0
409.3
378.6
273.3
825.3
309.3
1434.0
377.1
323.1
441.0
2493.0
2079.8
2476.5
3333.8
>10000
1647.7
2343.9
667.8
26.1
254.1
231.4
216.1
262.5
169.0
19.9
205.6
2.9
6.0
5.5
6.9
5.5
5.7
44.1
43.5
6.6
7.1
3.0
7.6
8.0
7.8
5.8
2
cyclopentyl
phenethyl
C6H5CH2
3
4
5
4ꢀMeC6H4CH2
C6H5
6
1394.7
136.9
7
2ꢀMeC6H4
4ꢀClC6H4
3ꢀClC6H4
2ꢀClC6H4
4ꢀFC6H4
8
997.6
9
1253.5
368.7
10
11
12
13
14
14.2
2.6
1107.5
2103.1
>10000
7290.5
4ꢀNO2C6H4
2ꢀNO2ꢀC6H4
3,5ꢀMe2C6H3
13.9
2.6
25.8
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2,5ꢀMe2C6H3
2ꢀiꢀPrC6H4
3ꢀMeSC6H4
4ꢀEtC6H4
306.1
345.8
739.0
665.6
551.8
255.1
654.3
2262.9
344.5
5080
94.3
329.5
179.7
23.6
48.0
44.7
35.9
57.8
5.2
16
762.3
728.5
771.7
3406.2
951.3
373.2
4416.5
842.3
960
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26.3
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19
4ꢀnꢀBuC6H4
2ꢀMeOC6H4
2ꢀEtOC6H4
4ꢀPhOC6H4
1ꢀnaphthyl
157.0
23.2
20
7.3
21
134.1
31.0
9.6
22
69.0
8.7
23
12.3
SLC-0111**
AAZ
45.1
4.5
250.0
12.0
25.0
5.7
*
Mean from 3 different assays, by a stopped flow technique (errors were in the range of ±
5ꢀ10 % of the reported values). ** From ref. 19
The following structureꢀactivityꢀrelationship (SAR) can be drawn from the data reported in Table 1:
(i) The cytosolic and kinetically slowest hCA I isoform was poorly inhibited by all compounds in
the series (KIs 255.1ꢀ5354.1 nM). The most effective inhibitor among all was the 2ꢀ
methoxyphenylureido derivative 20 which showed comparable potency with the reference CAI
AAZ (KIs of 255.1 and 250.0 nM respectively). Weaker inhibition potencies were observed for
derivatives 2ꢀ5, 7, 9ꢀ11, 15, 16 and 23 whose KIs were comprised between 273.3 to 441.0 nM,
whereas the remaining compounds resulted ineffective against the hCA I (KIs between 551.8 to
5354.1 nM). It should be noted that the weakest inhibitor among the series was the amino
unsubstituted compound 1 (KI of 5354.1 nM).
(ii) In analogy to the previous one, the second cytosolic isoform (i.e. hCA II) was weakly inhibited
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by most of the compounds reported. Among the series the 2,5ꢀdimethylphenylureido derivative 15
was the most potent inhibitor (KI of 94.3 nM). A modest decrease of potency (1.5 fold) was
obtained with the 2ꢀmethylphenylureido derivative 7 (KI of 137.0 nM). All remaining compounds
were ineffective inhibitors against the hCA II (see Table 1).
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(iii) Better inhibition results were obtained for the transmembrane and tumor associated isoform
hCA IX (Table 1). As reported above, derivatives 2ꢀ6, 8, 15, 16, 19 and 21 showed the lowest
inhibitory potencies among the compounds tested with KI values in the range of 134.1ꢀ329.5 nM,
thus far less potent when compared to the reference CAI AAZ (KI 25.0 nM). Interestingly the
unsubstituted 1 as well as its derivatives 7, 14, 17, 18, 20 and 22 showed remarkable potencies and
comparable to the standard AAZ (KI values comprised between 19.9 and 26.1 nM). The
introduction of the 2ꢀchloro, 4ꢀnitro and 1ꢀnaphthylureido substituents into the 3ꢀaminoꢀ4ꢀ
hydroxybenzenesulfonamide 1 scaffold (compounds 10, 12 and 23) determined a clear enhancement
of the inhibitory potencies against the hCA IX (KIs of 14.2, 13.9 and 12.3 nM respectively). Finally
compounds 9, 11 and 13 were the most potent inhibitors within the series tested against the IX
isoform (KIs 2.9, 2.6 and 2.6 nM respectively) and they deserve deeper SAR considerations. Among
the chlorophenylureido substituted derivatives 8-10, the compound 9 (which is the most active)
possess the halogen in meta position, whereas the introduction of the chloro in ortho or in para
clearly reduced the inhibition potencies against the hCA IX of 4.9 and 70.9 fold respectively.
Fluctuations of the kinetic properties in a regioisomer dependent manned were also observed for
compounds 12 and 13. Shift of the nitro moiety from para (compound 12) to ortho to afford the
derivative 13 resulted in a 5.3 fold enhancement of the inhibitory potencies, and thus obtaining the
second most potent inhibitor against the hCA IX within the entire series (see Table 1).
As reported above in Table 1 compounds 11 and 13 match each other in term of inhibition potency
against the hCA IX (KIs 2.6 nM) but presented quite different substitution patterns. Specifically
compound 11 contains the 4ꢀfluorophenylureido moiety in analogy to SLC-0111. Interestingly the
latter was 17.3 fold less potent against the hCA IX when compared to 11.
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(iv) The second tumor associated isoform (i.e. hCA XII) was weakly inhibited by derivatives 6, 7,
15ꢀ18 and 22 which showed KIs spanning between 35.9 and 69.0 nM. Conversely the remaining
derivatives in the series were far more potent, with KIs in the low nanomolar range comparable to
the standard AAZ. As reported in Table 1 the inhibition data of these compounds were similar to
each other, thus not allowing to draw detailed SAR data.
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In summary we reported first time the in vitro inhibitory activity of 2ꢀaminophenolꢀ4ꢀsulfonamide 1
and its phenylureido derivatives 2-23. Overall the entire series showed preferential inhibition of the
tumor associated isoforms hCA IX and XII over the cytosolic, and herein considered, offꢀtarget
hCA isoforms I and II. Only compound 15 resulted to be an inhibitor of hCA II even if the KI value
was rather high (94.3 nM). Of particular interest in this study is a comparative analysis of the
inhibition values relative to compound 11 over SLC-0111 which share the same 4ꢀ
fluorophenylureido tail. As reported in Table 1, 11 was 11.5 and 17.4 fold more effective when
compared to SLC-0111 in inhibiting hCA I and IX respectively, whereas it showed KI values 11.5
and 1.7 fold lower for the cytosolic hCA II and for the second tumor associated XII. The calculated
selectivity indexes (KIhCAII/KIhCAIX and KIhCAII/KIhCAXII) for SLC-0111 were 21.3 and 213.3
respectively whereas for its structural analogue 11 were of 426 and 145.7. Thus, our new compound
was more effective in inhibiting the tumor associated isoform IX when compared to SLC-0111.
Therefore in consideration of the inhibition data reported above we consider compound 11 worth to
be further investigated, as a SLC-0111 analogue.
X-Ray crystallography. We assessed the binding modes of compounds 5, 15 and 20 within the
hCA I active site by means of Xꢀray crystallographic experiments. Among the series reported, these
compounds were the most active in vitro in inhibiting the hCA I isoform. In this study we
particularly focused on the hCA isoform I since the largest majority of Xꢀray crystal adducts
reported in this field is related to the ubiquitous hCA II or to the tumor associated IX mimic, 30 but
hCA I is very abundant in red blood cells (at 1 µM concentrations level), and may influence the
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pharmacokinetics of drugs interacting with it or other CA isoforms of pharmacological interest.
As reported below in Figure 4 all compounds were coordinated to the Zn (II) ion by means of the
deprotonated sulfonamide moiety, which in turn is also engaged in a strong Hꢀbond with the OH of
Thr199 residue. One of the sulfonamide oxygen is also involved in a hydrogen interaction with the
amide nitrogen of Thr199. This coordination pattern, which is typical for all primary sulfonamides,
is respected also in these cases. 30
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Figure 4. Active site region in the hCA I−5 (A), 15 (B) and 20 (C) complexes. The omit Fo − Fc
electron density maps (contoured at 2.0 σ) for the three inhibitors are also shown. PDB accession
codes 6F3B, 6FAF and 6FAG respectively.
Slight differences of the allocated inhibitors within the enzymatic cavity were observed when a
superimposition of the three structures was performed (Figure 5).
Figure 5. Superimposition of hCA I−5 (light blue ), 15 (green) and 20 (pink) adducts.
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As reported above 15 and 20 superimpose almost perfectly, whereas 5 (the only one with an
additional CH2 moiety in the tail) showed a different orientation. Surprisingly the insertion of the
small CH2 spacer forces the inhibitor 5 to interact mostly with the hydrophobic part of the CA I
active site cavity.
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Deeper structural considerations of the hCA Iꢀ15 and 20 adducts revealed the aromatic rings of the
sulfamoylphenyl heads making hydrophobic contacts with residues Phe91, Ala121, Leu 198 and a
Tꢀshaped stacking with His94. Additional hydrogen bonding interactions occurred between the
phenoxy and the N1’ꢀureido moieties with Gln92 and His67 respectively. As for the inhibitor 20,
(the most active in inhibiting the hCA I) the 2’ꢀmethoxy substituent resulted engaged in additional
hydrophobic interaction with Leu198.
The main interactions for the sulfamoylphenyl head of compound 5 were the hydrophobic contacts
with Leu198 and a parallel πꢀstacking with His200. The OH on the aromatic ring formed a waterꢀ
bridged linkage with Pro201 and N1’ of the ureido group with Gln92. The 4’ꢀmethylphenyl moiety
of the same inhibitor was also engaged by means of hydrophobic contacts with Ala135 and Pro202.
Although the tumor associated hCA IX active site differs from the offꢀtarget hCAs I and II isoforms
for the higher hydrophobic aminoacidic content, it is interesting to note that the KI values of 5
relative to these isoforms are all of the same order of magnitude, whereas compound 15 and 20
showed high affinity for the hCA II and IX isoform respectively. This is somehow unexpected since
the crystallography revealed that the extra methylene group in 5 pushed the molecule towards the
hydrophobic section of the CAs cleft. Superpositions of the active sites of the complex hCA Iꢀ5
with those of hCA II and IX also revealed that important mutations such as Val131/Phe131 (hCA
IX/II), Leu91/Phe91 and Ala204/Tyr204 (hCA IX/I) are present (see Figure 6).
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Figure 6. Superimposition of the active site of the complex hCAIꢀ5 (pale brown) with those of
hCAII (green) and hCAIX (salmon).
Effect of Compound 11 on Primary Breast Tumor Growth and Comparison to SLC-0111. The
effect of sulfonamide compound 11 on tumor growth in vivo was investigated using the orthotopic,
syngeneic 4T1 model of breast cancer and obtained results were compared to treatment of similar
tumors with SLC-0111, which shares the same 4ꢀfluorophenyl)ureido tail. Importantly, 4T1 mouse
tumors demonstrate robust hypoxiaꢀinduced expression of CA IX and this model has been used
previously to evaluate CA IX inhibitors, including sulfonamides 13a and glycosyl coumarins.13b 4T1
murine breast tumor cells were inoculated into the mammary fat pad of BALB/c mice and
treatments were initiated after tumors were established (arrows in Figure 7). Both the observed
tumor volumes and the volume range at which treatments were initiated are similar to those
13
reported in published studies evaluating CA IX inhibitors in this tumor model. Compounds were
administered daily by intraperitoneal injection and caliper measurements were used to monitor
tumor growth. As reported below in Figure 7A treatment of animals with increasing concentrations
of 11 evoked dose dependent inhibition of tumor growth, with a maximum effect at 100 mg/kg. A
comparison of the reduction in tumor growth in animals treated with equivalent doses of 11 or
SLC-0111 showed nearly superimposable responses (Figure 7B), demonstrating that both
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compounds were similarly efficacious in vivo.
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Figure 7. Inhibition of tumor growth after treatment of mice harboring 4T1 tumors with compound
11 and SLC-0111 at diverse dosages. Data show the mean ± SEM. N = 6 to 8 animals/group.
**P<0.01. (B). Data show the mean ± SEM. N = 6 to 7 animals/group. **P<0.01, ***P<0.001.
Additionally, no significant weight reduction was observed for any of the treated animals at the
concentrations and the dosing schedules of compound 11 in our experiments, thus demonstrating
that, in analogy to SLC-0111, compound 11 is wellꢀtolerated in vivo at doses that are
therapeutically active (Figure 8).
Figure 8. Spider plots showing body weights for individual animals treated with vehicle, 11 and
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SLC-0111 as indicated. Both compounds were wellꢀtolerated by the animals at the doses tested.
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Conclusions.
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We report here a series of sulfonamides, compound 1 and its derivatives 2-23 possessing aromatic/
aliphatic ureido tails, and they were assayed in vitro on hCA I, II, IX and XII isoforms. Almost all
compounds showed selective inhibitory potencies against the tumor associated isoforms hCA IX
and XII except for the compound 15 which showed a 3.49 fold selectivity on hCA II (KI of 94.29
nM) over the IX. The most effective inhibitors (i.e. with KIs in the low nanomolar range) of hCA IX
were the 4ꢀfluorophenyl 11 (KI of 2.59 nM), 2ꢀnitrophenyl 13 (KI of 2.61 nM) and the 3ꢀ
chlorophenyl 9 (KI of 2.85 nM). In particular the SLC-0111 structural analogue (i.e. compound 11)
showed a better selectivity profile against the hCA IX thus giving support to our design strategy for
the obtainment of compounds with controlled degrees of tail flexibility. We reported the binding
modes of selected compounds 5, 15 and 20 in adduct with hCA I, which revealed the classical
coordination pattern within the hCA cavity. Superposition of the three structures showed that the 5
inhibitor tail, contrarily to compounds 15 and 20, was oriented towards the hydrophobic part of the
cavity. Finally, we investigated the antiꢀtumor activity of compound 11 in vivo using an orthotopic
syngeneic breast tumor model that robustly expresses hypoxiaꢀinducible CA IX. We found that
compound 11 doseꢀdependently inhibited tumor growth to levels that matched those observed with
SLC-0111 treatment, and was also associated with an absence of adverse effects similar to SLC-
0111.
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Experimental Section.
Materials and Reagents. All anhydrous solvents and reagents used in this study were purchased
from Alfa Aesar, TCI and SigmaꢀAldrich. The synthetic reactions involving airꢀ or moistureꢀ
sensitive chemicals were carried out under a nitrogen atmosphere using dried glassware and
syringes techniques in order to transfer the solutions. Nuclear magnetic resonance (1HꢀNMR, 13Cꢀ
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NMR, 19FꢀNMR) spectra were recorded using a Bruker Advance III 400 MHz spectrometer using
DMSOꢀd6 as solvent. The chemical shifts are reported in parts per million (ppm) and the coupling
constants (J) are expressed in Hertz (Hz). The splitting patterns are designated as: s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet; brs, broad singlet; dd, doublet of doublets. The correct
assignment of exchangeable protons (i.e. OH and NH) was carried out by means of the addition of
D2O. Analytical thinꢀlayer chromatography (TLC) was done on Merck silica gel Fꢀ254 plates. Flash
chromatography was performed on Merck Silica gel 60 (230ꢀ400 mesh ASTM) as the stationary
phase and appropriate mixtures of ethyl acetate/nꢀhexane were the eluents. Melting points (m.p.)
were measured in open capillary tubes with a Gallenkamp MPD350.BM3.5 apparatus and are
uncorrected. The HPLC was performed by using a Waters 2690 separation module coupled with a
photodiode array detector (PDA Waters 996) using a NovaꢀPak C18 4 ꢁm 3.9 mm × 150 mm
(Waters) silicaꢀbased reverse phase column. The sample was dissolved in 10% acetonitrile/H2O and
an injection volume of 45 ꢁL. The mobile phase (flow rate 1.0 mL/min) was a gradient of H2O +
trifluoroacetic acid (TFA) 0.1% (A) and acetonitrile + TFA 0.1% (B), with steps as follows:
(A%:B%), 0−10 min 90:10, 10−25 min gradient to 60:40, 26:28 min isocratic 20:80, 29−35 min
isocratic 90:10. TFA 0.1% in water as well in acetonitrile was used as counterion. All compounds
reported here were ≥ 95% HPLC pure. The solvents used in MS measures were acetone, acetonitrile
(Chromasolv grade) and mQ water 18 MU. The high resolution mass spectrometry (HRMS)
analysis were performed with a Thermo Finnigan LTQ Orbitrap mass spectrometer coupled with an
electrospray ionization source (ESI). Analysis were carried out in positive ion mode [M+H]+, and it
was used a proper dwell time acquisition to achieve 60,000 units of resolution at Full Width at Half
Maximum (FWHM). Elemental composition of compounds were calculated on the basis of their
measured accurate masses, accepting only results with an attribution error less than 5 ppm and a not
integer RDB (double bond/ring equivalents) value. 31 Stock solutions of analytes were prepared
using acetone (1.0 mg mLꢀ1) and stored at 4 °C. Then working solutions of each analyte were
prepared by dilution of the stock solutions using mQ H2O/acetonitrile 1/1 (v/v) up to a concentration
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of 1.0 ꢁg mLꢀ1 The HRMS analysis was performed by introducing the analyte working solution via
syringe pump at 10 ꢁL minꢀ1.
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Synthesis of Compounds.
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General procedure for the synthesis of compounds 2-23. A solution of 2ꢀaminophenolꢀ4ꢀ
sulfonamide 1 (0.2 g, 1.0 equiv) in dry acetonitrile (3ꢀ4 mL) was treated with a proper aromatic/
aliphatic isocyanate (1.0 equiv). The reaction mixture was stirred at rt until the consumption of
starting materials (TLC monitoring). Reaction was quenched with H2O and treated accordingly to
afford the compounds 2-23.
Synthesis of 3-(3-cyclopentylureido)-4-hydroxybenzenesulfonamide (2). A solution of 2ꢀ
aminophenolꢀ4ꢀsulfonamide 1 (0.2 g, 1.0 equiv) in dry acetonitrile (4 mL) was treated with
cyclopentyl isocyanate (1.0 equiv) according to the reported general procedure. The reaction was
quenched with H2O (1.0 mL) to give precipitates which were washed with diethyl ether (3 x 5 mL),
filtered and dried under vacuum to afford the titled product 2 as a pale brown solid. Yield 44%;
silica gel TLC Rf 0.28 (MeOH/DCM 10% v/v); mp 195ꢀ196 °C; δH (400 MHz, DMSOꢀd6) 1.40 (2H,
m, Cp), 1.59 (2H, m, Cp), 1.67 (2H, m, Cp), 1.87 (2H, m, Cp), 3.97 (1H, m, Cp), 6.90 (1H, d, J 8.4,
ArꢀH), 6.99 (1H, d, J 6.8, exchangeable with D2O, NH), 7.07 (2H, s, exchangeable with D2O,
SO2NH2), 7.25 (1H, dd, J 2.4, 8.4, ArꢀH), 7.98 (1H, s, exchangeable with D2O, NH), 8.59 (1H, d, J
2.4, ArꢀH), 10.70 (1H, s, exchangeable with D2O, OH); δC (100 MHz, DMSOꢀd6) 24.1, 33.7, 51.8,
114.4, 116.5, 119.9, 129.6, 135.7, 148.8, 155.6; m/z (ESI positive) 300.0 [M+H]+.
Synthesis of 4-hydroxy-3-(3-phenethylureido)benzenesulfonamide (3). A solution of 2ꢀ
aminophenolꢀ4ꢀsulfonamide 1 (0.2 g, 1.0 equiv) in dry acetonitrile (3 mL) was treated with
phenethyl isocyanate (1.0 equiv) according to the reported general procedure. The reaction was
quenched with H2O (1.0 mL) to give precipitates which were washed with diethyl ether (3 x 5 mL),
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filtered and dried under vacuum to afford the titled product 3 as a light brown solid. Yield 42%;
silica gel TLC Rf 0.37 (MeOH/DCM 10% v/v); mp 199ꢀ200 °C; δH (400 MHz, DMSOꢀd6) 2.79 (2H,
t, J 6.4, CH2), 3.38 (2H, m, CH2), 6.91 (1H, d, J 8.4, ArꢀH), 6.99 (1H, t, J 6.4, exchangeable with
D2O, NH), 7.07 (2H, s, exchangeable with D2O, SO2NH2), 7.33 (6H, m, ArꢀH), 8.13 (1H, s,
exchangeable with D2O, NH), 8.60 (1H, d, J 2.4, ArꢀH), 10.68 (1H, s, exchangeable with D2O,
OH); δC (100 MHz, DMSOꢀd6) 36.7, 41.5, 114.6, 116.8, 120.1, 127.0, 129.3, 129.5, 129.6, 135.7,
140.5, 148.9, 156.0; m/z (ESI negative) 334.0 [MꢀH]ꢀ.
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Synthesis of 3-(3-benzylureido)-4-hydroxybenzenesulfonamide (4).
A
solution of 2ꢀ
aminophenolꢀ4ꢀsulfonamide 1 (0.2 g, 1.0 equiv) in dry acetonitrile (3 mL) was treated with benzyl
isocyanate (1.0 equiv) according to the reported general procedure. The reaction was quenched with
H2O (1.0 mL) to give precipitates which were washed with diethyl ether (3 x 5 mL), filtered and
dried under vacuum to afford the titled product 4 as a pale brown solid. Yield 76%; silica gel TLC
Rf 0.26 (MeOH/DCM 10% v/v); mp 196ꢀ197 °C; δH (400 MHz, DMSOꢀd6) 4.34 (2H, d, J 6.0, CH2),
6.91 (1H, d, J 8.4. ArꢀH), 7.09 (2H, s, exchangeable with D2O, SO2NH2), 7.26ꢀ7.40 (6H, m, ArꢀH),
7.44 (1H, t, J 6.0, exchangeable with D2O, NH), 8.21 (1H, s, exchangeable with D2O, NH), 8.62
(1H, d, J 2.4, ArꢀH), 10.74 (1H, s, exchangeable with D2O, OH); δC (100 MHz, DMSOꢀd6) 43.6,
114.5, 116.7, 120.2, 127.7, 128.1, 129.3, 129.4, 135.7, 141.1, 149.0, 156.1; m/z (ESI positive) 322.0
[M+H]+.
Synthesis of 4-hydroxy-3-(3-(4-methylbenzyl)ureido)benzenesulfonamide (5). A solution of 2ꢀ
aminophenolꢀ4ꢀsulfonamide 1 (0.2 g, 1.0 equiv) in dry acetonitrile (3 mL) was treated with 4ꢀ
methylbenzyl isocyanate (1.0 equiv) according to the reported general procedure. The reaction was
quenched with H2O (1.0 mL) to give precipitates which were washed with diethyl ether (3 x 5 mL),
filtered and dried under vacuum to afford the titled product 5 as a pale brown solid. Yield 75%;
silica gel TLC Rf 0.28 (MeOH/DCM 10% v/v); mp 190ꢀ191 °C; δH (400 MHz, DMSOꢀd6) 2.32 (3H,
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s, CH3), 4.29 (2H, d, J 5.6, CH2), 6.91 (1H, d, J 8.4, ArꢀH), 7.09 (2H, s, exchangeable with D2O,
SO2NH2), 7.18 (2H, d, J 8.0, ArꢀH), 7.22 (2H, d, J 8.0, ArꢀH), 7.26 (1H, dd, J 2.4, 8.4, ArꢀH), 7.38
(1H, t, J 5.6, exchangeable with D2O, NH), 8.19 (1H, s, exchangeable with D2O, NH), 8.61 (1H, d,
J 2.4, ArꢀH), 10.74 (1H, s, exchangeable with D2O, OH); δC (100 MHz, DMSOꢀd6) 21.6, 43.4,
114.5, 116.8, 120.2, 128.1, 129.4, 129.8, 135.7, 136.7, 138.0, 149.0, 156.0; m/z (ESI negative)
334.0 [MꢀH]ꢀ.
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Synthesis of 4-hydroxy-3-(3-phenylureido)benzenesulfonamide (6).
A solution of 2ꢀ
aminophenolꢀ4ꢀsulfonamide 1 (0.2 g, 1.0 equiv) in dry acetonitrile (4 mL) was treated with phenyl
isocyanate (1.0 equiv) according to the reported general procedure. The reaction was quenched with
H2O (1.0 mL) to give precipitates which were washed with diethyl ether (3 x 5 mL), filtered and
dried under vacuum to afford the titled product 6 as a pale brown solid. Yield 67%; silica gel TLC
Rf 0.25 (MeOH/DCM 10% v/v); mp 223ꢀ224 °C (dec); δH (400 MHz, DMSOꢀd6) 6.99 (2H, m, Arꢀ
H), 7.13 (2H, s, exchangeable with D2O, SO2NH2), 7.32 (3H, m, ArꢀH), 7.50 (2H, d, J 8.0, ArꢀH),
8.39 (1H, s, exchangeable with D2O, NH), 8.68 (1H, d, J 2.4, ArꢀH), 9.41 (1H, s, exchangeable with
D2O, NH), 10.87 (1H, s, exchangeable with D2O, OH); δC (100 MHz, DMSOꢀd6) 114.5, 116.9,
118.9, 120.8, 122.8, 128.8, 129.8, 135.8, 140.6, 149.2, 153.2; m/z (ESI negative) 306.0 [MꢀH]ꢀ.
Synthesis of 4-hydroxy-3-(3-(o-tolyl)ureido)benzenesulfonamide (7). A solution of 2ꢀ
aminophenolꢀ4ꢀsulfonamide 1 (0.2 g, 1.0 equiv) in dry acetonitrile (3 mL) was treated with 2ꢀ
methylphenyl isocyanate (1.0 equiv) according to the reported general procedure. The reaction was
quenched with H2O (1.0 mL) to give precipitates which were washed with diethyl ether (3 x 5 mL),
filtered and dried under vacuum to afford the titled product 7 as a beige solid. Yield 85%; silica gel
TLC Rf 0.33 (MeOH/DCM 10% v/v); mp 201ꢀ202 °C; δH (400 MHz, DMSOꢀd6) 2.29 (3H, s, CH3),
6.99 (2H, m, ArꢀH), 7.18 (4H, m, 2H exchangeable with D2O, SO2NH2), 7.32 (1H, dd, J 2.4, 8.4,
ArꢀH), 7.87 (1H, d, J 8.0, ArꢀH), 8.64 (1H, s, exchangeable with D2O, NH), 8.69 (1H, d, J 2.4, Arꢀ
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H), 8.84 (1H, s, exchangeable with D2O, NH), 10.85 (1H, s, exchangeable with D2O, OH); δC (100
MHz, DMSOꢀd6) 19.0, 114.6, 117.2, 120.7, 122.4, 123.7, 127.0, 128.8, 129.0, 131.1, 135.7, 138.2,
149.3, 153.6; m/z (ESI positive) 321.9 [M+H]+.
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Synthesis of 3-(3-(4-chlorophenyl)ureido)-4-hydroxybenzenesulfonamide (8). A solution of 2ꢀ
aminophenolꢀ4ꢀsulfonamide 1 (0.2 g, 1.0 equiv) in dry acetonitrile (3 mL) was treated with 4ꢀ
chlorophenyl isocyanate (1.0 equiv) according to the reported general procedure. The reaction was
quenched with H2O (1.0 mL) to give precipitates which were washed with diethyl ether (3 x 5 mL),
filtered and dried under vacuum to afford the titled product 8 as a beige solid. Yield 82%; silica gel
TLC Rf 0.29 (MeOH/DCM 10% v/v); mp 223ꢀ224 °C (dec); δH (400 MHz, DMSOꢀd6) 6.98 (1H, d,
J 8.4, ArꢀH), 7.15 (2H, s, exchangeable with D2O, SO2NH2), 7.33 (1H, dd, J 2.4, 8.4, ArꢀH), 7.37
(2H, d, J 8.8, ArꢀH), 7.52 (2H, d, J 8.8, ArꢀH), 8.42 (1H, s, exchangeable with D2O, NH), 8.67 (1H,
d, J 2.4, ArꢀH), 9.55 (1H, s, exchangeable with D2O, NH), 10.93 (1H, s, exchangeable with D2O,
OH); δC (100 MHz, DMSOꢀd6) 114.6, 116.9, 120.4, 121.0, 126.3, 128.6, 129.7, 135.8, 139.6, 149.3,
153.1; m/z (ESI negative) 339.9 [MꢀH]ꢀ.
Synthesis of 3-(3-(3-chlorophenyl)ureido)-4-hydroxybenzenesulfonamide (9). A solution of 2ꢀ
aminophenolꢀ4ꢀsulfonamide 1 (0.2 g, 1.0 equiv) in dry acetonitrile (3 mL) was treated with 3ꢀ
chlorophenyl isocyanate (1.0 equiv) according to the reported general procedure. The reaction was
quenched with H2O (1.0 mL) to give precipitates which were washed with diethyl ether (3 x 10
mL), filtered and dried under vacuum to afford the titled product 9 as a pale brown solid. Yield
58%; silica gel TLC Rf 0.28 (MeOH/DCM 10% v/v); mp 235ꢀ236 °C (dec); δH (400 MHz, DMSOꢀ
d6) 6.99 (1H, d, J 8.4, ArꢀH), 7.06 (1H, m, ArꢀH), 7.16 (2H, s, exchangeable with D2O, SO2NH2),
7.25 (1H, m, ArꢀH), 7.35 (2H, m, ArꢀH), 7.80 (1H, t, J 2.0, ArꢀH), 8.45 (1H, s, exchangeable with
D2O, NH), 8.67 (1H, d, J 2.4, ArꢀH), 9.62 (1H, s, exchangeable with D2O, NH), 10.96 (1H, s,
exchangeable with D2O, OH); δC (100 MHz, DMSOꢀd6) 114.6, 117.0, 117.3, 118.2, 121.1, 122.4,
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128.5, 131.4, 134.2, 135.8, 142.1, 149.3, 153.1; m/z (ESI positive) 341.9 [M+H]+.
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Synthesis of 3-(3-(2-chlorophenyl)ureido)-4-hydroxybenzenesulfonamide (10). A solution of 2ꢀ
aminophenolꢀ4ꢀsulfonamide 1 (0.2 g, 1.0 equiv) in dry acetonitrile (4 mL) was treated with 2ꢀ
chlorophenyl isocyanate (1.0 equiv) according to the reported general procedure. The reaction was
quenched with H2O (1.0 mL) to give precipitates which were washed with diethyl ether (3x 10 mL),
filtered and dried under vacuum to afford the titled product 10 as a beige solid. Yield 80%; silica
gel TLC Rf 0.33 (MeOH/DCM 10% v/v); δH (400 MHz, DMSOꢀd6) 6.99 (1H, d, J 8.4; ArꢀH), 7.08
(1H, dt, J 1.2, 8.0, ArꢀH), 7.15 (2H, s, exchangeable with D2O, SO2NH2), 7.34 (2H, m, ArꢀH), 7.49
(1H, dd, J 1.2, 8.0, ArꢀH), 8.15 (1H, dd, J 1.2, 8.0, ArꢀH), 8.66 (1H, d, J 2.4, ArꢀH), 9.10 (1H, s,
exchangeable with D2O, NH), 9.22 (1H, exchangeable with D2O, NH), 10.88 (1H, exchangeable
with D2O, OH); δC (100 MHz, DMSOꢀd6) 114.8, 117.7, 121.3, 123.2, 123.6, 124.6, 128.4, 128.6,
130.3, 135.7, 137.0, 149.8, 153.4; m/z (ESI negative) 339.9 [MꢀH]ꢀ.
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Synthesis of 3-(3-(4-fluorophenyl)ureido)-4-hydroxybenzenesulfonamide (11). A solution of 2ꢀ
aminophenolꢀ4ꢀsulfonamide 1 (0.2 g, 1.0 equiv) in dry acetonitrile (3 mL) was treated with 4ꢀ
fluorophenyl isocyanate (1.0 equiv) according to the reported general procedure. The reaction was
quenched with H2O (1.0 mL) and extracted with ethyl acetate (3 x 15 mL). The combined organic
layers were washed with H2O (3 x 20 mL), dried over Na2SO4, filtered and concentrated under
vacuum to give the titled product 11 as a beige solid. Yield 86%; silica gel TLC Rf 0.28
(MeOH/DCM 10% v/v); mp 230ꢀ231 °C (dec); δH (400 MHz, DMSOꢀd6) 6.97 (1H, d, J 8.4, ArꢀH),
7.16 (4H, m, 2H exchangeable with D2O, SO2NH2), 7.32 (1H, dd, J 2.4, 8.4, ArꢀH), 7.50 (2H, m,
ArꢀH), 8.37 (1H, s, exchangeable with D2O, NH), 8.66 (1H, d, J 2.4, ArꢀH), 9.46 (1H, s,
exchangeable with D2O, NH), 10.91 (1H, s, exchangeable with D2O, OH); δC (100 MHz, DMSOꢀd6)
2
3
4
114.6, 116.3 (d, JCꢀF 22), 116.9, 120.5 (d, JCꢀF 8), 120.9, 128.8, 135.8, 137 (d, JCꢀF 2), 149.2,
1
153.3, 158.3 (d, JCꢀF 237); δF (376 MHz, DMSOꢀd6) ꢀ121.4 (1F, s); m/z (ESI negative) 324.0 [Mꢀ
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Synthesis of 4-hydroxy-3-(3-(4-nitrophenyl)ureido)benzenesulfonamide (12). A solution of 2ꢀ
aminophenolꢀ4ꢀsulfonamide 1 (0.2 g, 1.0 equiv) in dry acetonitrile (4 mL) was treated with 4ꢀ
nitrophenyl isocyanate (1.0 equiv) according to the reported general procedure. The reaction was
quenched with H2O (1.0 mL) to give precipitates which were washed with diethyl ether (3 x 10
mL), filtered and dried under vacuum to afford the titled product 12 as a pale brown solid. Yield
75%; silica gel TLC Rf 0.33 (MeOH/DCM 10% v/v); mp 258ꢀ259 °C (dec); δH (400 MHz, DMSOꢀ
d6) 6.99 (1H, d, J 8.4, ArꢀH), 7.17 (2H, s, exchangeable with D2O, SO2NH2), 7.37 (1H, dd, J 2.4,
8.4, ArꢀH), 7.74 (2H, d, J 9.2, ArꢀH), 8.24 (2H, d, J 9.2, ArꢀH), 8.62 (1H, s, exchangeable with
D2O, NH), 8.68 (1H, d, J 2.4, ArꢀH), 10.13 (1H, s, exchangeable with D2O, NH), 11.04 (1H, s,
exchangeable with D2O, OH); δC (100 MHz, DMSOꢀd6) 114.8, 117.2, 118.2, 121.6, 126.2, 128.1,
135.8, 142.0, 147.2, 149.5, 152.7; m/z (ESI negative) 350.9 [MꢀH]ꢀ.
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Synthesis of 4-hydroxy-3-(3-(2-nitrophenyl)ureido)benzenesulfonamide (13). A solution of 2ꢀ
aminophenolꢀ4ꢀsulfonamide 1 (0.2 g, 1.0 equiv) in dry acetonitrile (3 mL) was treated with 2ꢀ
nitrophenyl isocyanate (1.0 equiv) according to the reported general procedure previously reported.
The reaction was quenched with H2O (1.0 mL) to give precipitates which were washed with diethyl
ether (3 x 10 mL), filtered and dried under vacuum to afford the titled product 13 as a yellow solid.
Yield 77%; silica gel TLC Rf 0.31 (MeOH/DCM 10% v/v); δH (400 MHz, DMSOꢀd6) 7.00 (1H, d, J
8.4, ArꢀH), 7.16 (2H, s, exchangeable with D2O, SO2NH2), 7.29 (1H, t, J 7.2, ArꢀH), 7.37 (1H, dd, J
2.4, 8.4, ArꢀH), 7.72 (1H, t, J 7.2, ArꢀH), 8.06 (2H, m, ArꢀH), 8.56 (1H, d, J 2.4, ArꢀH), 9.27 (1H, s,
exchangeable with D2O, NH), 9.87 (1H, s, exchangeable with D2O, NH), 10.90 (1H, s,
exchangeable with D2O, OH); δC (100 MHz, DMSOꢀd6) 115.0, 118.2, 121.8, 124.0, 124.9, 126.1,
128.1, 134.4, 135.3, 135.7, 140.6, 150.1, 153.0; m/z (ESI negative) 350.9 [MꢀH]ꢀ.
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Synthesis of 3-(3-(3,5-dimethylphenyl)ureido)-4-hydroxybenzenesulfonamide (14). A solution
of 2ꢀaminophenolꢀ4ꢀsulfonamide 1 (0.2 g, 1.0 equiv) in dry acetonitrile (3 mL) was treated with
3,5ꢀdimethlyphenyl isocyanate (1.0 equiv) according to the reported general procedure. The
reaction was quenched with H2O (1.0 mL) to give precipitates which were washed with diethyl
ether (3 x 10 mL), filtered and dried under vacuum to afford the titled product 14 as a pale brown
solid. Yield 81%; silica gel TLC Rf 0.39 (MeOH/DCM 10% v/v); mp 228ꢀ229 °C (dec); δH (400
MHz, DMSOꢀd6) 2.27 (6H, s, 2 x CH3), 6.66 (1H, s, ArꢀH), 6.97 (1H, d, J 8.4, ArꢀH), 7.13 (4H, m,
2H exchangeable with D2O, SO2NH2), 7.32 (1H, dd, J 2.4, 8.4, ArꢀH), 8.38 (1H, s, exchangeable
with D2O, NH), 8.69 (1H, d, J 2.4, ArꢀH), 9.27 (1H, s, exchangeable with D2O, NH), 10.88 (1H,
brs; exchangeable with D2O, OH); δC (100 MHz, DMSOꢀd6) 22.1, 114.5, 116.6, 116.8, 120.7,
124.4, 128.9, 135.8, 138.7, 140.5, 149.2, 153.2; m/z (ESI positive) 336.0 [M+H]+.
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Synthesis of 3-(3-(2,5-dimethylphenyl)ureido)-4-hydroxybenzenesulfonamide (15). A solution
of 2ꢀaminophenolꢀ4ꢀsulfonamide 1 (0.2 g, 1.0 equiv) in dry acetonitrile (3 mL) was treated with
2,5ꢀdimethylphenyl isocyanate (1.0 equiv) according to the reported general procedure. The
reaction was quenched with H2O (1.0 mL) to give precipitate which were washed with diethyl ether
(3 x 10 mL), filtered and dried under vacuum to afford the titled product 15 as a beige solid. Yield
95%; silica gel TLC Rf 0.22 (MeOH/DCM 10% v/v); mp 245ꢀ246 °C (dec); δH (400 MHz, DMSOꢀ
d6) 2.24 (3H, s, CH3), 2.29 (3H, s, CH3), 6.80 (1H, d, J 8.0, ArꢀH), 6.98 (1H, d, J 8.4, ArꢀH), 7.08
(1H, d, J 8.0, ArꢀH), 7.13 (2H, s, exchangeable with D2O, SO2NH2), 7.31 (1H, dd, J 2.4, 8.4, ArꢀH),
7.73 (1H, s, ArꢀH), 8.58 (1H, s, exchangeable with D2O, NH), 8.70 (1H, d, J 2.4, ArꢀH), 8.83 (1H,
s, exchangeable with D2O, NH), 10.83 (1H, s, exchangeable with D2O, OH); δC (100 MHz, DMSOꢀ
d6) 18.6, 21.9, 114.6, 117.1, 120.7, 122.9, 124.3, 125.6, 129.0, 131.0, 135.8, 135.9, 138.1, 149.3,
153.6; m/z (ESI negative) 334.0 [MꢀH]ꢀ.
Synthesis of 4-hydroxy-3-(3-(2-isopropylphenyl)ureido)benzenesulfonamide (16). A solution of
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2ꢀaminophenolꢀ4ꢀsulfonamide 1 (0.2 g, 1.0 equiv) in dry acetonitrile (3 mL) was treated with 2ꢀ
isopropylphenyl isocyanate (1.0 equiv) according to the general procedure previously reported. The
reaction was quenched with H2O (1.0 mL) to give precipitates which were washed with diethyl
ether (3 x 10 mL), filtered and dried under vacuum to afford the titled product 16 as a beige solid.
Yield 11%; silica gel TLC Rf 0.42 (MeOH/DCM 10% v/v); mp 218ꢀ219 °C; δH (400 MHz, DMSOꢀ
d6) 1.23 (6H, d, J 6.8, 2 x CH3), 3.25 (1H, m, CH), 6.98 (1H, d, J 8.4, ArꢀH), 7.14 (4H, m, 2H
exchangeable with D2O, SO2NH2), 7.32 (2H, m, ArꢀH), 7.67 (1H, dd, J 1.6, 8.0, ArꢀH), 8.66 (1H, s,
exchangeable with D2O, NH), 8.68 (1H, d, J 2.4, ArꢀH), 8.77 (1H, s, exchangeable with D2O, NH),
10.86 (1H, s, exchangeable with D2O, OH); δC (100 MHz, DMSOꢀd6) 24.2, 27.6, 114.6, 117.1,
120.7, 125.0, 125.0, 126.2, 126.6, 129.1, 135.7, 136.3, 140.8, 149.3, 154.1; m/z (ESI negative)
348.0 [MꢀH]ꢀ.
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Synthesis of 4-hydroxy-3-(3-(3-(methylthio)phenyl)ureido)benzenesulfonamide (17). A solution
of 2ꢀaminophenolꢀ4ꢀsulfonamide 1 (0.2 g, 1.0 equiv) in dry acetonitrile (3 mL) was treated with 3ꢀ
(methylthio)phenyl isocyanate (1.0 equiv) according to the reported general procedure. The reaction
was quenched with H2O (1.0 mL) to give precipitates which were washed with diethyl ether (3 x 10
mL), filtered and dried under vacuum to afford the titled product 17 as a pale brown solid. Yield
56%; silica gel TLC Rf 0.20 (MeOH/DCM 10% v/v); mp 220ꢀ221 °C (dec); δH (400 MHz, DMSOꢀ
d6) 2.50 (3H, s, CH3), 6.90 (1H, d, J 8.0, ArꢀH), 6.98 (1H, d, J 8.4, ArꢀH), 7.15 (3H, m, 2H
exchangeable with D2O, SO2NH2), 7.26 (1H, t, J 8.0, ArꢀH), 7.33 (1H, dd, J 2.4, 8.4, ArꢀH), 7.55
(1H, m, ArꢀH), 8.40 (1H, s, exchangeable with D2O, NH), 8.67 (1H, d, J 2.4, ArꢀH), 9.47 (1H, s,
exchangeable with D2O, NH), 10.90 (1H, s, exchangeable with D2O, OH); δC (100 MHz, DMSOꢀd6)
15.5, 114.6, 115.4, 115.8, 116.9, 120.2, 120.9, 128.7, 130.3, 135.8, 139.7, 141.2, 149.2, 153.1; m/z
(ESI negative) 352.0 [MꢀH]ꢀ.
Synthesis of 3-[3-(4-ethyl-phenyl)-ureido]-4-hydroxy-benzenesulfonamide (18). A solution of 2ꢀ
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aminophenolꢀ4ꢀsulfonamide 1 (0.2 g, 1.0 equiv) in dry acetonitrile (3 mL) was treated with 4ꢀ
ethylphenyl isocyanate (1.0 equiv) according to the reported general procedure. The reaction was
quenched with H2O (1.0 mL) to give precipitates which were washed with diethyl ether (3 x 10
mL), filtered and dried under vacuum to afford the titled product 18 as a beige solid. Yield 78%;
silica gel TLC Rf 0.29 (MeOH/DCM 10% v/v); mp 210ꢀ211 °C; δH (400 MHz, DMSOꢀd6) 1.20 (3H,
t, J 7.6, CH3), 2.59 (2H, m, CH2), 6.96 (1H, d, J 8.4, ArꢀH), 7.14 (2H, s, exchangeable with D2O,
SO2NH2), 7.16 (2H, d, J 8.4, ArꢀH), 7.32 (1H, dd, J 2.4, 8.4, ArꢀH), 7.40 (2H, d, J 8.4, ArꢀH), 8.36
(1H, s, exchangeable with D2O, NH), 8.68 (1H, d, J 2.4, ArꢀH), 9.33 (1H, s, exchangeable with
D2O, NH), 10.87 (1H, s, exchangeable with D2O, OH); δC (100 MHz, DMSOꢀd6) 16.7, 28.4, 114.5,
116.8, 119.0, 120.7, 128.9, 129.0, 135.8, 138.2, 138.3, 149.2, 153.3; m/z (ESI negative) 334.0 [Mꢀ
H]ꢀ.
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Synthesis of 3-(3-(4-butylphenyl)ureido)-4-hydroxybenzenesulfonamide (19). A solution of 2ꢀ
aminophenolꢀ4ꢀsulfonamide 1 (0.2 g, 1.0 equiv) in dry acetonitrile (4 mL) was treated with 4ꢀ
butylphenyl isocyanate (1.0 equiv) according to the reported general procedure. The reaction was
quenched with H2O (1.0 ml) to give precipitates which were washed with diethyl ether (3 x 10 mL),
filtered and dried under vacuum to afford the titled product 19 as a beige solid. Yield 65%; silica
gel TLC Rf 0.74 (ethyl acetate 100% v/v); mp 204ꢀ205 °C (dec); δH (400 MHz, DMSOꢀd6) 0.93
(3H, t, J 7.3, CH3), 1.33 (2H, m, CH2), 1.56 (2H, quint, J 7.3, CH2), 2.56 (2H, m, CH2), 6.96 (1H, d,
J 8.4, ArꢀH), 7.13 (4H, m, 2H exchangeable with D2O, SO2NH2), 7.31 (1H, dd, J 2.4, 8.4, ArꢀH),
7.39 (2H, d, J 8.4, ArꢀH), 8.36 (1H, s, exchangeable with D2O, NH), 8.68 (1H, d, J 2.4, ArꢀH), 9.33
(1H, s, exchangeable with D2O, NH), 10.89 (1H, brs, exchangeable with D2O, OH); δC (100 MHz,
DMSOꢀd6) 14.7, 22.6, 34.2, 35.1, 114.5, 116.8, 119.0, 120.7, 128.9, 129.5, 135.8, 136.7, 138.3,
149.2, 153.3; m/z (ESI negative) 362.0 [MꢀH]ꢀ.
Synthesis of 4-hydroxy-3-(3-(2-methoxyphenyl)ureido)benzenesulfonamide (20). A solution of
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2ꢀaminophenolꢀ4ꢀsulfonamide 1 (0.2 g, 1.0 equiv) in dry acetonitrile (3 mL) was treated with 2ꢀ
methoxyphenyl isocyanate (1.0 equiv) according to the reported general procedure. The reaction
was quenched with H2O (1.0 mL) to give precipitates which were washed with diethyl ether (3 x 10
mL), filtered and dried under vacuum to afford the titled product 20 as a beige solid. Yield 44%;
silica gel TLC Rf 0.28 (MeOH/DCM 10% v/v); mp 209ꢀ210 °C (dec); δH (400 MHz, DMSOꢀd6)
3.90 (3H, s, OCH3), 6.90ꢀ7.05 (4H, m, ArꢀH), 7.13 (2H, s, exchangeable with D2O, SO2NH2), 7.32
(1H, dd, J 2.4, 8.4, ArꢀH), 8.15 (1H, dd, J 1.2, 8.0, ArꢀH), 8.66 (1H, d, J 2.4, ArꢀH), 9.03 (1H, s,
exchangeable with D2O, NH), 9.10 (1H, s, exchangeable with D2O, NH), 10.75 (1H, s,
exchangeable with D2O, OH); δC (100 MHz, DMSOꢀd6) 56.6, 111.8, 114.7, 117.7, 120.0, 120.9,
121.4, 123.0, 128.9, 129.6, 135.7, 149.2, 149.8, 153.6; m/z (ESI negative) 336.0 [MꢀH]ꢀ.
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Synthesis of 3-(3-(2-ethoxyphenyl)ureido)-4-hydroxybenzenesulfonamide (21). A solution of 2ꢀ
aminophenolꢀ4ꢀsulfonamide 1 (0.2 g, 1.0 equiv) in dry acetonitrile (4 mL) was treated with 2ꢀ
ethoxyphenyl isocyanate (1.0 equiv) according to the reported general procedure. The reaction was
quenched with H2O (1.0 mL) to give precipitates which were washed with diethyl ether (3 x 10
mL), filtered and dried under vacuum to afford the titled product 21 as a beige solid. Yield 80%;
silica gel TLC Rf 0.72 (ethyl acetate 100% v/v); mp 208ꢀ209 °C (dec); δH (400 MHz, DMSOꢀd6)
1.45 (3H, t, J 7.0, CH3), 4.16 (2H, q, J 7.0, OCH2), 6.91 (1H, dt, J 1.6, 8.0, ArꢀH), 6.97 (2H, m, Arꢀ
H), 7.03 (1H, dd, J 1.6, 8.0, ArꢀH), 7.13 (2H, s, exchangeable with D2O, SO2NH2), 7.33 (1H, dd, J
2.4, 8.4, ArꢀH), 8.13 (1H, dd, J 1.6, 8.0, ArꢀH), 8.64 (1H, d, J 2.4, ArꢀH), 8.83 (1H, s, exchangeable
with D2O, NH), 9.11 (1H, s, exchangeable with D2O, NH), 10.77 (1H, s, exchangeable with D2O,
OH); δC (100 MHz, DMSOꢀd6) 15.7, 64.8, 112.9, 114.8, 118.0, 120.4, 121.0, 121.3, 123.1, 128.8,
129.8, 135.7, 148.3, 149.9, 153.6; m/z (ESI negative) 350.0 [MꢀH]ꢀ.
Synthesis of 4-hydroxy-3-(3-(4-phenoxyphenyl)ureido)benzenesulfonamide (22). A solution of
2ꢀaminophenolꢀ4ꢀsulfonamide 1 (0.2 g, 1.0 equiv) in dry acetonitrile (3 mL) was treated with 4ꢀ
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phenoxyphenyl isocyanate (1.0 equiv) according to the reported general procedure. The reaction
was quenched with H2O (1.0 mL) to give precipitates which were washed with diethyl ether (3 x 10
mL), filtered and dried under vacuum to afford the titled product 22 as a beige solid. Yield 70%;
silica gel TLC Rf 0.20 (MeOH/DCM 10% v/v); mp 225ꢀ226 °C; δH (400 MHz, DMSOꢀd6) 7.00 (5H,
m, ArꢀH), 7.14 (3H, m, 2H exchangeable with D2O, SO2NH2), 7.33 (1H, dd, J 2.4, 8.4, ArꢀH), 7.40
(2H, m, ArꢀH), 7.52 (2H, d, J 8.8, ArꢀH), 8.37 (1H, s, exchangeable with D2O, NH), 8.68 (1H, d, J
2.4, ArꢀH), 9.44 (1H, s, exchangeable with D2O, NH), 10.89 (1H, exchangeable with D2O, OH); δC
(100 MHz, DMSOꢀd6) 114.6, 116.9, 118.7, 120.6, 120.8, 120.9, 123.8, 128.9, 130.9, 135.8, 136.7,
149.2, 151.7, 153.3, 158.6; m/z (ESI negative) 398.0 [MꢀH]ꢀ.
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Synthesis of 4-hydroxy-3-(3-(naphthalen-1-yl)ureido)benzenesulfonamide (23). A solution of 2ꢀ
aminophenolꢀ4ꢀsulfonamide 1 (0.2 g, 1.0 equiv) in dry acetonitrile (3 mL) was treated with 1ꢀ
naphthyl isocyanate (1.0 equiv) according to the reported general procedure. The reaction was
quenched with H2O (1.0 mL) to give precipitates which were washed with diethyl ether (3 x 10
mL), filtered and dried under vacuum to afford the titled product 23 as a beige solid. Yield 84%;
silica gel TLC Rf 0.35 (MeOH/DCM 10% v/v); mp 235ꢀ236 °C (dec); δH (400 MHz, DMSOꢀd6)
7.01 (1H, d, J 8.4, ArꢀH), 7.16 (2H, s, exchangeable with D2O, SO2NH2), 7.35 (1H, dd, J 2.4, 8.4,
ArꢀH), 7.51 (1H, t, J 8.0, ArꢀH), 7.60 (2H, m, ArꢀH), 7.67 (1H, d, J 8.0, ArꢀH), 7.96 (1H, d, J 8.0,
ArꢀH), 8.11 (1H, d, J 8.0, ArꢀH), 8.26 (1H, d, J 8.0, ArꢀH), 8.75 (1H, d, J 2.4, ArꢀH), 9.00 (1H, s,
exchangeable with D2O, NH), 9.44 (1H, s, exchangeable with D2O, NH), 10.93 (1H, s,
exchangeable with D2O, OH); δC (100 MHz, DMSOꢀd6) 114.9, 117.3, 118.5, 121.1, 122.6, 124.1,
126.7, 126.9, 127.0, 129.1, 129.5, 134.8, 135.4, 135.9, 149.6, 154.0; m/z (ESI negative) 356.0 [Mꢀ
H]ꢀ.
In vitro enzyme assays.
Carbonic Anhydrase Inhibition. The CAꢀcatalyzed CO2 hydration activity was performed on an
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Applied Photophysics stoppedꢀflow instrument using phenol red (at a concentration of 0.2 mM) as a
pH indicator with 20 mM Hepes (pH 7.5) as the buffer, 20 mM Na2SO4, and following the initial
rates of the CAꢀcatalyzed CO2 hydration reaction for a period of 10−100 s and working at the
maximum absorbance of 557 nm.29 The CO2 concentrations ranged from 1.7 to 17 mM. For each
inhibitor six traces of the initial 5−10 % of the reaction have been used in order to determine the
initial velocity. The uncatalyzed reaction rates were determined in the same manner and subtracted
from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled water,
and dilutions up to 0.01 nM were prepared. Solutions containing inhibitor and enzyme were
preincubated for 15 min at room temperature prior to assay in order to allow the formation of the
E−I complex. The inhibition constants were obtained as nonlinear least squares protocols using
PRISM 3, 31ꢀ33 and are the mean from at three different measurements. All hCAs were recombinant
ones and were obtained in house. 31ꢀ33
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X-Ray crystallography.
Crystallization and data collection. Crystals of hCA I complexed with compounds 5, 15 and 20
were obtained using the sitting drop vapor diffusion method. 2 µl of 10 mg/ml solution of hCA I in
TrisꢀHCl 20 mM pH 9.0 were mixed with 2 µl of a solution of 28ꢀ31% PEG4000, 0.2 M Sodium
acetate, 0.1 M Tris pH 8.5ꢀ9.0 and were equilibrated against the same solution at 296 K. Crystals of
the protein grew in fifteen days. Afterwards hCA I crystals were soaked in 5 mM inhibitor solutions
for 1 day. The crystals were flashꢀfrozen at 100K using a solution obtained by adding 15% (v/v)
glycerol to the mother liquor solution as cryoprotectant. Data on crystals of the complexes were
collected using synchrotron radiation at the ID30B beamline at ESRF (Grenoble, France) with a
wavelength of 0.973 Å and a PILATUS3 6M Dectris CCD detector. Data were integrated and
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scaled using the program XDS. Data processing statistics are showed in Supporting Information
Table 1.
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Structure determination. The crystal structure of hCA I (PDB accession code: 1JV0) without
solvent molecules and other heteroatoms was used to obtain initial phases for the structures using
Refmac5. 35 5% of the unique reflections were selected randomly and excluded from the refinement
data set for the purpose of Rfree calculations. The initial |Fo ꢀ Fc| difference electron density maps
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unambiguously shown the inhibitor molecules. Atomic models for inhibitors were calculated and
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energy minimized using the program JLigand 1.0.40.
Refinements proceeded using normal
protocols of positional, isotropic atomic displacement parameters alternating with manual building
of the models using COOT. 37 Solvent molecules were introduced automatically using the program
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ARP.
The quality of the final models were assessed with COOT and RAMPAGE.39 Crystal
parameters and refinement data are summarized in Table 1 in Supporting Informations. Atomic
coordinates were deposited in the Protein Data Bank (PDB accession code: 6F3B, 6FAF and
6FAG). Graphical representations were generated with Chimera. 40
Biological assays.
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Cell culture. The 4T1 murine breast cancer cell line was cultured as previously described.
Briefly, cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) medium
containing 25 mM glucose (Gibco cat # 11995ꢀ065, Burlington, Ontario) and supplemented with
10% fetal bovine serum (FBS; Gibco, Burlington, Ontario) and nonꢀessential amino acids (1x
NEAA). Cells were routinely tested for mycoplasma contamination using the LookOut Mycoplasma
PCR detection kit (Sigma; Cat. No. MP0035) The 4T1 cells have been authenticated using short
tandem repeat DNA profiling (DNA fingerprinting) by a commercial testing facility (Genetica,
Burlington, NC, USA). In addition, the cell lines were routinely tested for viability, morphology,
hypoxiaꢀinduced endogenous CA IX expression and in vivo tumor growth.
Orthotopic syngeneic breast tumor model. All animal studies and procedures were performed in
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accordance with protocols approved by the Institutional Animal Care Committee at the BC Cancer
Research Centre and University of British Columbia (Vancouver, BC, Canada). 4T1 murine
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mammary tumor cells (1.0 x 106 cells/animal) were implanted orthotopically into the mammary fat
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pad of BALB/c mice (Simonsen Laboratories, Gilroy, CA) as described previously.
Tumors
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were measured 3x/week using digital calipers and volumes were calculated using the modified
ellipsoid formula ((length x width x width)π/6). Immediately prior to initiation of treatment, mice
were randomized and sorted into groups of similar average tumor volume. Treatment was initiated
when mean tumor volumes reached approximately 60 mm3.
Drug Treatment. Compound 11 and SLC-0111 were solubilized at the indicated concentrations in
the same vehicle (37.5% PEGꢀ400/12.5% ethanol/50%PBS), divided into daily aliquots and frozen
at ꢀ80°C until use. Compounds were administered by intraperitoneal injection daily until the
experimental endpoint. Control animals were administered the vehicle alone in a similar fashion.
The investigators were not blinded to the treatment groups.
Statistical Analysis. For tumor growth data, statistical analyses were performed using GraphPad
Prism 7. Data are reported as the mean ± SEM. Outliers were identified using Grubb’s test (alpha =
0.05) and excluded from further analysis. For comparison of three or more data sets, a oneꢀway
ANOVA was performed and a HolmsꢀSidak’s test was used to correct of multiple comparisons.
Statistical significance was set at P< 0.05.
Ancillary Informations.
Supporting Information is available free of charge on the ACS Publications website: SMILES
representation for compounds (CSV), summary of data collection and atomic model refinement
statistics in Supporting Information Table 1.
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