Discovery of β-Adrenergic Receptors Blocker-Carbonic Anhydrase Inhibitor Hybrids for Multitargeted Antiglaucoma Therapy

Article abstract of DOI:10.1021/acs.jmedchem.8b00625

The combination of a β-adrenergic receptors (AR) blocker and a carbonic anhydrase (CA, EC 4.2.1.1) inhibitor in eye drops formulations is one of the most clinically used treatment for glaucoma. A novel approach consisting of single-molecule, multitargeted compounds for the treatment of glaucoma is proposed here by designing compounds which concomitantly interact with the β-adrenergic and CA targets. Most derivatives of the two series of benzenesulfonamides incorporating 2-hydroxypropylamine moieties reported here exhibited striking efficacy against the target hCA II and XII, whereas a subset of compounds also showed significant modulation of β₁- and β₂-ARs. X-ray crystallography studies provided rationale for the observed hCA inhibition. The best dual-agents decreased IOP more effectively than clinically used dorzolamide, timolol, and the combination of them in an animal model of glaucoma. The reported evidence supports the proof-of-concept of β-ARs blocker-CAI hybrids for antiglaucoma therapy with an innovative mechanism of action.

Full text of DOI:10.1021/acs.jmedchem.8b00625

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Article
Discovery of #-adrenergic receptors blocker - carbonic anhydrase
inhibitor hybrids for multitargeted anti-glaucoma therapy.
Alessio Nocentini, Mariangela Ceruso, Silvia Bua, Carrie L. Lomelino, Jacob Andring, Robert
McKenna, Cecilia Lanzi, Silvia Sgambellone, Riccardo Pecori, Rosanna Matucci, Luca
Filippi, Paola Gratteri, Fabrizio Carta, Emanuela Masini, Silvia Selleri, and Claudiu T Supuran
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00625 • Publication Date (Web): 31 May 2018
Downloaded from http://pubs.acs.org on May 31, 2018
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Discovery of βꢀadrenergic receptors blocker ꢀ carbonic anhydrase inhibitor
hybrids for multitargeted antiꢀglaucoma therapy.
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Alessio Nocentini , Mariangela Ceruso , Silvia Bua , Carrie L. Lomelino , Jacob T. Andring ,
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Robert McKenna , Cecilia Lanzi , Silvia Sgambellone , Riccardo Pecori , Rosanna Matucci , Luca
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a,
a,
Filippi , Paola Gratteri , Fabrizio Carta , Emanuela Masini , Silvia Selleri *, Claudiu T. Supuran *
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Department of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, University of Florence, Polo Scientifico,
Via U. Schiff 6, 50019 Sesto Fiorentino (Firenze), Italy.
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Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Box 100245, Gainesville, FL
32610, USA
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Department of NEUROFARBAꢀPharmacology and Toxicology Section, University of Florence, Viale G. Pieraccini n.6, 50019
Florence, Italy
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Core Research Laboratory, Istituto Toscano Tumori, Florence,Italy
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Neonatal Intensive Care Unit, Medical Surgical FetoꢀNeonatal Department, “A. Meyer” University Children's Hospital, Viale G.
Pieraccini, 24,50139 Florence, Italy
Abstract
The combination of a βꢀadrenergic receptors (AR) blocker and a carbonic anhydrase (CA, EC
4.2.1.1) inhibitor in eye drops formulations is one of the most clinically used treatment for
glaucoma. A novel approach consisting of singleꢀmolecule, multiꢀtargeted compounds for the
treatment of glaucoma is proposed here by designing compounds which concomitantly interact
with the βꢀadrenergic and CA targets. Most derivatives of the two series of benzenesulfonamides
incorporating 2ꢀhydroxypropylamine moieties reported here exhibited striking efficacy against the
1
target hCA II and XII, whereas a subset of compounds also showed significant modulation of β ꢀ
and β ꢀARs. Xꢀray crystallography studies provided rationale for the observed hCA inhibition.
2
The best dualꢀagents decreased IOP more effectively than clinically used dorzolamide, timolol,
and the combination of them in an animal model of glaucoma. The reported evidence supports the
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proofꢀofꢀconcept of βꢀARs blocker ꢀ CAI hybrids for antiꢀglaucoma therapy with an innovative
mechanism of action.
Keywords: glaucoma, carbonic anhydrase; sulfonamide; βꢀadrenergic receptor; hybrids; linker.
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Introduction
Glaucoma consists of a cluster of optic neuropathies characterized by a broad spectrum of
clinical presentations and etiologies, leading to a progressive, irreversible vision loss. This causes
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blindness, affecting over 60 million people globally. An increasing number of affected people
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,4
of up to 80 million is expected by 2020 due to both population increase and aging. The diverse
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,5
types of glaucoma feature an enhanced intraꢀocular pressure (IOP). The increase in IOP results
from the malfunction of ciliary processes and the trabecular meshwork in the anterior chamber of
the eye. These tissues physiologically support an adequate pressure in the eye by regulating
aqueous humor secretion and its drainage. The main ionic constituent of the aqueous humor is
bicarbonate. This fluid is present in the region between cornea and the lens (Figure 1), and its
secretion and flow from the ciliary body to the anterior chamber leads to the homeostatic control
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of the IOP.
Glaucoma occurs when an increase in IOP occurs, which is due to either an
excessive retention of aqueous humor within the anterior chamber or to an excessive secretion of
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the fluid (Figure 1).
Openꢀangle glaucoma (OAG) and angleꢀclosure glaucoma (ACG) are the
two most common types of primary glaucoma, with OAG being the most common in the
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Americas and Europe. ACG is due to the impaired drainage of the aqueous humor from the
anterior chamber. Conversely, this angle is constitutively open in OAG, but the drainage of the
humor is diminished. The cause of the altered flow is unknown in OAG and under extensive
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investigation.
Because of the asymptomatic nature of chronic glaucoma, up to 50% of people
in the industrialized world are unaware of their diagnosis and do not receive the required
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treatment.
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Figure 1. Mechanism and available pharmacologic treatments for glaucoma.
Multiple treatments for glaucoma exist and are chiefly separated into pharmacologic, laser, and
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surgical therapies.
In most individuals, pharmacologic therapy is the first therapy of choice for
IOP reduction and generally includes the use of topically acting agents, such as eye drops, that
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reduce aqueous humor production, as well as agents that raise the outflow facility. Lowering
IOP is the cornerstone of glaucoma therapy, since each additional millimeter of mercury IOP
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increase can lead to an 11% increase in the risk of glaucoma progression.
The clinically available drugs include sympathomimetic stimulants (epinephrine),
parasympathomimetic agents (pilocarpine), βꢀblockers (timolol), carbonic anhydrase (CA)
inhibitors (CAIs, acetazolamide and dorzolamide), and prostaglandin derivatives (latanoprost and
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travoprost) (Figure 2). These classes of drugs can be used alone or in various combinations.
βꢀBlockers reduce IOP via blockade of the sympathetic nerve endings in the ciliary epithelium,
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reducing the production of aqueous humour. Among the topicallyꢀacting βꢀblockers available for
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the treatment of glaucoma, there are nonꢀselective agents, which target both β ꢀ and β ꢀ
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adrenoceptors, and cardioꢀselective drugs, which block only the β1ꢀreceptors. In the past, βꢀ
blockers were the most common first line topical glaucoma medication, however the use of more
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efficient prostaglandin analogues became the primary course of treatment during the 1990s.
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When monotherapy alone is not effective in controlling IOP, other drugs with different
mechanisms of action can replace or be added in conjunction with betaꢀblockers or prostaglandin
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analogues. Commonly used secondꢀline agents include topical CAIs. Inhibition of CAs in the
ciliary processes reduces aqueous humor secretion, probably by slowing the rate of bicarbonate
production and therefore reducing the transport of water and osmotically obligated sodium within
the fluid. As a result, the aqueous humour secretion decreases, leading to a reduction of IOP up to
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25ꢀ30%.
If necessary, CAIs such as acetazolamide, methazolamide, ethoxzolamide, and
dichlorophenamide can still be used as systemic antiglaucoma drugs, though they may show a
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wide range of undesired side effects in some patients.
Compliance is the major challenge with adding multiple drops. It has been demonstrated that
increasing the number of drop bottles to a patient’s treatment results into a negative influence on
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patient adherence. Fixed combination therapies have been developed and are currently available
in the clinic. For example, the combination of a βꢀblocker (timolol) and CAI (dorzolamide)
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represents one of the most used therapeutic options.
The current therapies are often inadequate given that topical glaucoma therapy is burdened by the
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need for multiple classes of medications to control IOP, undesired side effects, and barriers
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such as patient compliance and difficulty with proper drop instillation. New needed
pharmacotherapies for glaucoma should exhibit favorable benefitꢀrisk profiles and alternative
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mechanisms of action relative to current therapies. This can be achieved by either increasing
efficacy (ocular hypotensive efficacy), decreasing adverse events, or both.
Herein a multiꢀtargeted approach for the treatment of glaucoma is proposed by design, synthesis,
crystallography and biological in vitro / vivo evaluation of a series hybrid drugs which
concomitantly affect the βꢀadrenergic receptors and carbonic anhydrases in the eye.
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Results and Discussion
Compounds Design and Chemistry.
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Figure 2. Molecular structures of clinically used βꢀblockers and antiꢀglaucoma CAI drugs.
An approach consisting of singleꢀmolecule, multiꢀtargeted compounds was chosen over the coꢀ
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administration of single drugs due to potential therapeutic benefits.
Indeed, it is likely that
single target agents cannot sufficiently affect the intricate biochemical processes involved in disease
pathology, such as the above depicted for glaucoma. Most biological targets interact in a complex
network of enzymes/receptors whose equilibrium could be more likely influenced by drugs that
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affect multiple targets.
Moreover, combinatorial drug therapies could be replaced by multiꢀ
functional molecules, minimizing the pharmacokinetic and metabolic issues that arise from multiple
drug intake. Patients are more likely to remember to take a single drug; thus, compliance is
enhanced. Finally, chemical or metabolic drugꢀdrug interactions are avoided by means of multiꢀ
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targeted molecules.
The hybrid drug strategy has been previously applied to target glaucoma by carbonic anhydrase
inhibition. CAI – nitric oxide (NO) donor hybrids stood out amongst the most effective topically
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active agents.
Since hypertensive glaucoma patients show a decreased content of NO/cGMP in
the aqueous humor, it has been shown that NOꢀdonors can decrease IOP in normal and pathological
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conditions. One such compounds was twofold more efficient than dorzolamide to reduce high IOP
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characteristic of this disease in an animal model.
The herein reported derivatives feature a benzene sulfonamide moiety, representing the CA
inhibitory fragment, and the aryloxyꢀ2ꢀhydroxypropylamine portion of βꢀblockers such as
propranolol and timolol (Figure 2, 3).
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Sulfonamides are the most effective zinc binding group (ZBG) commonly used to design CAIs.
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Primary sulfonamides (Figure 2), such as acetazolamide (AAZ) or brinzolamide (BRZ),
represent the firstꢀgeneration CAIs and have been clinically used for almost 70 years as antiꢀ
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glaucoma agents, diuretics, antiꢀepileptics, or antiꢀobesity drugs.
Dorzolamide (DZA), a
secondꢀgeneration CAI, was the first topically acting sulfonamide used clinically as an antiꢀ
glaucoma medication. It is indicated for the reduction of elevated IOP in patients with openꢀangle
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glaucoma or ocular hypertension that do not sufficiently respond to βꢀblockers.
Figure 3. Design of aryloxyꢀ2ꢀhydroxypropylamine sulfonamides dualꢀtargeted agents.
Modulation of the βꢀARs was investigated by swapping the substituents appended at the aryloxyꢀ2ꢀ
hydroxypropylamine moiety, which is a rather common hallmark of βꢀblockers and thus maintained
in the hybrids structure (Figure 2, 3). The aryloxyꢀ2ꢀhydroxypropylamine portion was directly
appended at the benzenesulfonamide scaffold (derivatives 22ꢀ29) or alternatively detached by
means of an ethylbenzamide spacer (derivatives 9ꢀ18). In this second case, uniquely small aliphatic
amines were considered in the βꢀblocker portion, owing to the hydrophobic nature of the spacer.
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The incorporation of such 2ꢀhydroxypropilamine moieties at the benzenesulfonamide scaffold (both
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direct and spaced) represents an application of the CAI “tail approach”,
the most common
method to develop isoform selective inhibitors within the zincꢀbinders class. This approach explores
the modulation of moieties appended at the aromatic/heterocyclic ring present in the scaffold of the
CAIs in order to selectively promote interactions with isoform unique residues at the entrance of the
active site cavity.
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Scheme 1. General synthetic procedure for compounds 9ꢀ18.
Two synthetic strategies were planned to obtain the hybrid compounds. Preparation of the first
series molecules 9ꢀ18 shared a common key epoxy intermediate 8, for which the synthetic route is
illustrated in Scheme 1. Coupling of ethylamminobenzenesulfonamide 2 with freshly prepared 4ꢀ
acetoxybenzoyl chloride 2 generated amide 4, which was therefore deacetylated with sodium
methoxide and protected on the sulfonamide group with N,Nꢀdimethylformamide dimethyl acetal.
Reaction of the resulting phenol 6 with allyl bromide followed by epoxidation of the olefinic
function in presence of Oxone and acetone provided for intermediate 8. Epoxide ring opening (8) to
afford 2ꢀhydroxypropylamine derivatives 9ꢀ18 was achieved by treatment with an excess of varied
primary and secondary amines. Reaction with primary amines occurred with an additional and
unexpected cleavage of the sulfonamide protecting group, resulting directly in the 2ꢀ
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hydroxypropylamine derivatives 9ꢀ16. Because secondary amines were not able to carry out the
deprotection process, treatment in acidic media was necessary to free the sulfonamide group and
generate compounds 17ꢀ18.
In a similar manner, preparation of the second series of molecules 22ꢀ29 (Scheme 2) shared the
common epoxy intermediate 21, which was obtained by reacting the Nꢀprotected 4ꢀ
hydroxybenzenesulfonamide 20 with epibromohydrin in presence of Cs CO . Epoxide 21 was
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reacted with different amines and subsequently treated in acidic media in the case of secondary
amines to give the 2ꢀhydroxypropylamine derivatives 22ꢀ29. These synthetic strategies were
adopted to provide 9ꢀ18 and 22ꢀ29 as racemates, since the mixture of both enantiomers was deemed
more suitable to initially seek for biological activity.
Scheme 2. General synthetic procedure for compounds 22ꢀ29.
Carbonic Anhydrase Inhibition.
The CA inhibition profiles of compounds 9ꢀ18 and 22ꢀ29 were evaluated, in addition to
acetazolamide (AAZ) as standard inhibitor, against four physiologically relevant isoforms, hCA I
II, IX, and XII. The choice of these isoforms was based upon: hCA II and XII are upregulated in the
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eyes of glaucoma patients
and might be responsible for increased blood flow and thus the
oxygen supply in hypoxic neovascular retinic tissues; hCA IX has been found to be upregulated in
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the hypoxiaꢀsuffering cells of the retinal pigment epithelium;
and hCA I is the main offꢀtarget
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isoform for the therapeutic application of CAIs in the reported ocular diseases. The following
structure–activity relationship (SAR) can be gathered from the inhibition data reported in Table 1:
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Table 1. Inhibition data of human CA isoforms hCA I, II, IX and XII with sulfonamides 9ꢀ18, 22ꢀ
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CO2 hydrase assay.
_
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hCAIX
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23.8
2.4
CH
CH
3
2
3
11
12
13
CH
3
ꢀCH(CH
ꢀ CH(CH) CH CH
ꢀC(CH3)3
ꢀCH Ph
ꢀCH
ꢀ(CH
)
2
3
2
3
14
15
16
17
2
2
CH
2
OH
16.6
1.5
27.7
4.1
2
CH
2
)O(CH
2
CH
2
)ꢀ
7.0
18
ꢀ CH(CH
ꢀCH
ꢀCH(CH
ꢀ CH(CH)
ꢀC(CH
3
)
2
ꢀ CH(CH
3
)
2
6.6
1.2
6.0
1.6
4.9
22
23
24
3
H
H
750.8
350.4
74.5
234.3
85.4
145.6
183.1
230.6
250
1174.3
155.5
240.6
174.1
44.1
75.0
15.2
48.9
12
23.4
47.6
107.7
95.4
93.1
59.2
84.6
126.5
25
3
)
2
44.4
58.6
41.9
72.4
39.8
82.2
164.1
5.7
3
CH
2
CH
3
H
H
H
H
H
25
3 3
)
26
27
28
ꢀCH
ꢀCH
ꢀCH
2
2
2
Ph
CH
CH
2
2
Ph
OPh
29
AAZ
DZA
ꢀ(CH
2
CH
2
)N(CH
3
2 2
)(CH CH )ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
b
50000
9
52
3.5
______________________________________________________________________________
a. Mean from 3 different assays, by a stopped flow technique (errors were in the range of ± 5ꢀ10 % of the reported values).
b. From ref. 27.
(i) The cytosolic isoform hCA I was effectively inhibited by most of the sulfonamides of the first
series with inhibition constants (K s) ranging in the low nanomolar range, between 6.6 and 59.1
I
nM. Conversely, the benzenesulfonamides directly bearing the 2ꢀhydroxypropylamine (22ꢀ29)
moieties were found to act as weaker inhibitors with K
I
s spanning between 74.5 and 750.8 nM.
The secꢀbutyl and benzyl bearing derivatives 24 and 26 stood out from the others with K
I
s less
than 100 nM. All the first series compounds and 24, 26ꢀ28 from the second series were stronger
inhibitors compared to the clinically used AAZ (K value of 250 nM).
I
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
4
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4
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5
5
5
5
5
5
5
5
5
5
6
(
ii) The physiologically dominant isoform hCA II was strongly inhibited by all reported
sulfonamides (K values ranging between 1.5 and 75.0 nM, Table 1), apart from the second series
I
compounds incorporating small substituents (methyl, isopropyl, secꢀbutyl and tertꢀbutyl) on the
I
amine moiety (22ꢀ25), whose K s spanned from 155.5 to 1174.3 nM. The hCA II inhibition
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
profiles showed the efficacy of the spacerꢀcontaining compounds. It is worth mentioning that the
bulkiest derivatives of the first series 15 (benzylamine, K 1.9 nM), 17 (morpholine, K 1.5 nM)
I
I
and 18 (diisopropylamine, K 1.2 nM) as well as those of the second series 26ꢀ29 (K s ranging
I
I
between 15.2 and 75.0 nM) exhibited the strongest hCA II inhibition profiles within each subset.
Despite a generally comparable efficacy with the standard AAZ (K
I
value of 12 nM), only
compounds 12, 15, 17 and 18 exhibited a stronger effectiveness.
(iii) The data in Table 1 depicted similar inhibitory trends against the membraneꢀassociated
isoforms hCA IX and XII, which were strongly inhibited by most derivatives. In detail, the K s for
I
the first series of derivatives against hCA IX spanned from 1.7 (9, methylamine) to 83.1 nM (14,
tertꢀbutylamine), whereas the efficacy of compounds 22ꢀ29 was weaker with inhibition constants
ranging between 23.4 (22, methylamine) and 126.5 nM (29, Nꢀmethylpiperazine), not permitting
a rationale for a SAR. Again, only compounds 9ꢀ11, 16ꢀ18 among the benzamideꢀbearing
derivatives and compound 22 in series two were found to possess at least comparable efficacy to
the AAZ (K
iv) Most of the spacerꢀsupplied compounds demonstrated strong hCA XII inhibitory
effectiveness (K s ranging between 1.5 and 4.1 nM), except for 12 (isopropylamine), 14 (tertꢀ
I
of 25 nM).
(
I
butylamine) and 16 (ethanolamine), which showed a 10ꢀfold diminished activity (K s of 18.1ꢀ27ꢀ8
I
nM). Among the spacerꢀdevoid derivatives, compound 22, that incorporated a methyl group on
I
the amine moiety, exhibited low nanomolar efficacy (K of 4.9 nM), whereas the remaining
compounds were significantly weaker with inhibition constants ranging between 39.8 and 164.1
nM.
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(
v) Noteworthy, the first series of derivatives were shown to be generally more efficacious against
the hCAs in comparison to the directly linked dualꢀderivatives, to a greater extent against the
cytosolic isozymes hCA I and II. The second series compounds 22 and 23, which bear a methyl
and isopropyl group appended at the amine moiety, were able to target hCA IX and XII with
selectivity over the cytosolic isozymes (selectivity ratio hCA II / IX of 50.2 and 3.2 and hCA II /
XII of 240.5 and 3.5, respectively). The inhibition profiles of compounds 22 and 23 indicate a
potential to target several CAsꢀassociated diseases without causing the typical sideꢀeffects of nonꢀ
selective inhibitors in systematically administered treatments.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
βꢀADR Binding assay
The βꢀADRs binding properties of compounds 9ꢀ18 and 22ꢀ29 were evaluated, in addition to
3
4
propranolol and atenolol
as standard βꢀblockers, against the human cloned β ꢀ and β ꢀADRs
1 2
receptors expressed in HEK293T cell membranes. The following remarks can be drawn from the
data reported in Table 2:
Table 2. Inhibition binding constants (pK
ADRs expressed in HEK293T cell membranes.
_______________________________________________________________________________
i 1 2
) of the tested compounds for human cloned β ꢀ and β ꢀ
_
a
Compound
pK
i
R
R
1
β
1
ꢀADR
2
β ꢀADR
________________________________________________________________________________
9
1
1
ꢀCH
ꢀCH
ꢀCH
3
2
2
H
H
H
H
H
H
H
H
<4
<4
<4
<4
<4
<4
<4
<4
<4
<4
<4
4.98±0.06
<4
<4
<4
<4
<4
<4
<4
<4
<4
<4
<4
4.50±0.05
0
1
CH
CH
3
2
3
CH
3
12
ꢀCH(CH
)
2
13
14
15
ꢀ CH(CH)
ꢀC(CH
ꢀCH Ph
ꢀCH CH
ꢀ(CH CH
3
CH
2
CH
3
3
)
3
2
16
2
2
OH
)O(CH
17
18
22
2
2
2
CH
2
)ꢀ
ꢀ CH(CH3)2 ꢀ CH(CH3)2
ꢀCH3
ꢀCH(CH3)2
H
H
23
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
25
4
ꢀ CH(CH)
ꢀC(CH
ꢀCH Ph
3
CH
2
CH
3
H
H
H
H
H
4.81±0.03
4.87±0.06
4.10±0.03
4.57±0.02
5.18±0.05
<4
7.93±0.03
5.82±0.14
8.27±0.08
4.52±0.13
5.30±0.08
<4
4.40±0.02
4.55±0.03
<4
8.76±0.02
5.02±0.20
9.68±0.02
3 3
)
26
27
28
2
ꢀCH CH Ph
2
2
ꢀCH CH OPh
2
2
29
ꢀ(CH
2
CH
2
3 2 2
)N(CH )(CH CH )ꢀ
b
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Propranolol
Atenolol
Timolol
ꢀ
ꢀ
b
c
______________________________________________________________________________________________
a. Values are reported as mean ± SEM of 3ꢀ5 experiments, each one performed in duplicate.
b. Racemate.
c. From ref. 34
The competition binding experiments did not show striking affinities of the multiꢀtargeted
derivatives. The data in Table 2 highlight the absence of binding affinity reported towards both the
β and β ꢀADRs for the spacerꢀendowed derivatives 9ꢀ18, which were not able to compete with
1
2
3
[
i
H]ꢀCGP12177 at the receptor subtypes below 100 µM. Conversely, interesting pK values can be
gathered from the binding curves measured for the second series compounds (22ꢀ29). Most
derivatives were found to exhibit low micromolar affinities for both βꢀADRs subtypes (except 22
and 29).
(a)
(
b)
1
00
β
1
ꢀADR
100
50
0
β
1
ꢀADR
β2ꢀADR
β ꢀADR
2
5
0
0
ꢀ8
ꢀ7
ꢀ6
ꢀ5
ꢀ4
ꢀ3
ꢀ2
ꢀ8
ꢀ7
ꢀ6
ꢀ5
ꢀ4
ꢀ3
ꢀ2
log [25]
log [28]
3
Figure 4. Inhibition of [ H]ꢀCGP12177 specific binding to membrane homogenates of HEK293T cells
which stably express the β
1 2
ꢀ and β ꢀadrenergic receptors by increasing concentration of compound (a)
2
5 and (b) 28. The curves were fitted using the standard four parameter logistic equation and are the mean ±
S.E.M. from 3ꢀ4 independent experiments. Nonꢀ specific binding was determined in the presence of 10 µM
propranolol. Yꢀaxis: normalized Bound/Total bound.
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When the tertꢀbutylamine bearing 25 showed approximately a threeꢀfold β /β selectivity (below 10
2
1
µM for the β ꢀAR subtype), the remaining derivatives showed preferential affinity for the β ꢀAR,
2
1
with the highest selectivity (over fourꢀfold) reported for 28. The incorporation of a simple methyl
group or ꢀmethylpiperazine moiety in the 2ꢀhydroxylpropyl portion deprives respectively 22 and
9 of any affinity for both receptor subtypes up to 100 µM.
The K values measured for most compounds 23ꢀ28 were comparable to the racemic standard βꢀ
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
i
3
4
blocker atenolol (low micromolar range). Moreover, the K of 25 against the β ꢀsubtype exceed
i
2
that reported for the clinically used drug. Such results are of interest in terms of single enantiomer
binding constants evaluation for future developed derivatives.
Although the similarity between derivatives 22ꢀ29 (sulfonamideꢀbearing) and clinically used βꢀ
blockers, such as atenolol, practolol and celiprolol (which possess an amide or urea group appended
at the same position of the aromatic scaffold) indicates analogue functional tendency, further
investigations with this respect are currently ongoing.
Xꢀray Crystallography
The Xꢀray crystal structure of hCA II was determined in complex with compounds 12 and 23 (Table
3). Compounds 12 (R enantiomer) and 23 (S enantiomer) were observed to bind directly to the
active site zinc and displace the catalytic water, as is expected for sulfonamideꢀbased compounds
(Figure 5A, 5B). Hydrogen bonds were observed between the nitrogen and oxygen atoms of the
sulfonamide and Thr199 (2.7ꢀ2.8 and 3.0Å , respectively) (Figure 6A, 6B).
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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5
5
5
5
5
6
A.
B.
L204
V135
L204
V135
P202
P202
V143
L141
V143
L141
L198
L198
T199
T200
V207
T199
T200
V207
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
F131
F131
H64
N62
H64
N62
V121
I91
N67
V121
I91
N67
Q92
Q92
Figure 5. Surface representation of hCA II in complex with inhibitors. A. hCA II in complex
with compound 12 (represented as green sticks). B. hCA II in complex with compound 23
(
represented as cyan sticks). Hydrophobic and hydrophilic residues are labeled and coloured
orange and purple, respectively).
(
The presence of the spacer in compound 12 resulted in the formation of an additional hydrogen
bond between the carbonyl and Q92 side chain (3.0Å ). This observation rationalizes the stronger
binding affinity of compound 12 for CA II in relation to the direct compound 23 (Figure 6A). Both
inhibitors were further stabilized by van der Waals interactions with active site residues I91, V121,
F131, L141, and L198 in the case of compound 12 and residues Q92, V121, F131, V135, L198, and
P202 for compound 23 (Figure 6A, 6B).
Steric hindrance between the benzene ring in spacerꢀcontaining compound 12 and F131 shifted the
tail of this compound toward the interface between the hydrophobic and hydrophilic regions of the
active site, allowing a second molecule to bind between monomers within the crystal lattice
(
Supplemental Figure 1A). This secondary inhibitor molecule interacts directly with hCA II through
hydrogen bonding with D72 (2.6 and 2.7Å ) and is further stabilized through an interaction with the
NH group of the active siteꢀbound 12 (2.9Å ) (Supplemental Figure 2). Similarly, two additional
molecules of compound 23 were observed to bind outside the hCA II active site, thought to bind
due to crystal packing (Supplemental Figure 1B).
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9
A.
B.
P202
V135
L198
L141
F131
F131
L198
3
.0
3
.0
T199
T199
3
.0
D72
2.8
2
.7
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
I91
V121
V121
Q92
Q92
H119
H119
H94
H94
H96
H96
Figure 6. Active site binding interactions. hCA II in complex with A. compound 12 (green) and
B. compound 23 (cyan). Hydrogen bonds are represented as black dashes with distances labelled in
angstroms.
Table 3. Xꢀray crystallography statistics.
Sample
PDB Accession Code
CA II_12
5WLV
CA II_23
5WLT
P2
1
P2
a = 42.6, b = 41.6, c = 72.5; a = 42.7, b = 41.6,c = 72.7;
β= 104.3
30.8 – 1.4 (1.42 – 1.40)
69,038
1
Space Group
Cell Dimensions (Å;°)
β= 104.5
40.3 – 1.6 (1.60 – 1.57)
53,038
Resolution (Å)
Total Reflections
I/Iσ
Redundancy
Completeness (%)
Rcryst (%)
21.3 (2.8)
3.2 (3.1)
17.1 (2.2)
4.0 (4.0)
99.4 (99.5)
14.5 (22.3)
16.7 (25.5)
5.2 (40.4)
3.4 (27.6)
2099
90.2 (92.7)
14.5 (18.9)
17.6 (25.0)
6.8 (54.8)
3.5 (28.7)
2094
Rfree (%)
Rsym (%)
Rpim
#
of Protein Atoms
of Water Molecules
of Ligand Atoms
Ramachandran stats
(%): Favored, allowed,
generouslyallowed
Avg. B factors (Å2):
Main-chain, Side-
chain, Ligand
#
234
60
213
#
76
97.3, 2.7, 0
96.9,3.1, 0
15.2, 19.5, 40.0
14.4, 18.7, 41.3
0.006,0.918
rmsd for bond lengths,
angles (Å,°)
0
.006, 0.939
Antiglaucoma Activity (IOP Lowering Activity)
In spite of not striking βꢀADRꢀbinding affinities, several molecules within the second series
demonstrated a multiꢀtargeted action and excellent and suitable water solubility to be formulated as
1
% eye drops at the neutral pH value. We investigated the intraocular pressure (IOP) lowering
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1
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
4
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5
5
5
5
5
5
6
properties of some of these compounds, more precisely, 23 (isoꢀpropylamine, for which the Xꢀray
crystal structure in complex with hCA II was reported), 25 (tertꢀbutylamine) and 27
(phenethylamine) in a rabbit model of glaucoma. The morpholineꢀbearing compound 17 of the first
subset was included in the study to evaluate the IOP lowering efficacy of a uniquely CA inhibitory
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
35,36
derivative.
DZA hydrochloride and timolol, as well as their combination (1% + 0.25%, in the clinically used
ratio) were used for comparison as a standard drugs, with a control using the vehicle
(hydroxypropylcellulose at 0.05%). The 1% compounds eye drops were administered to rabbits
with high IOP, induced by the injection of 0.1 mL of hypertonic saline solution (5% in distilled
water) into the vitreous of both eyes. The benzamide derivative 17 was a strong, low nanomolar
inhibitor for hCA II, IX and XII (K
the contrary, compounds 23, 25 and 27 showed a significant, low micromolar action against the β
and β ꢀADR, at the expense of a weaker efficacy against target hCAs (Figure 7).
I
s of 1.5, 7.0 and 4.1 nM), but lacked activity on the βꢀADR. On
1
ꢀ
2
Figure 7. Drop of intraocular pressure (ꢁIOP, mmHg) versus time (min) in hypertonic salineꢀinduced ocular
hypertension in rabbits, treated with 50 ꢂL of 1 % solution of compounds 17, 23, 25, 27. Timolol 0.25%,
DRZ 1% and their association (DRZ+timolol) were used as reference drugs. Data are analyzed with 2way
Anova followed by Dunnett’s multiple comparison test. * p<0.05 17 and DRZ vs vehicle at 60'; ** p<0.005
timolol and DRZ+timolol vs vehicle at 60'; *** p<0.001 27 vs vehicle at 60'; ****p< 0.0001 23 and 25 vs
vehicle at 60'.
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The dual agents 23, 25, 27 were more effective than standard DRZ and timolol with IOP decreases
of 8.25, 10.75 and 6.75 mmHg at 60 min postꢀadministration, respectively (Figure 7). Compound
25 stood out as the most efficient one, with a twofold enhanced efficacy in comparison to DRZ and
timolol, which caused an IOP decrease of 4.75 mmHg and 5.73 at 60 min postꢀadministration,
respectively. Noteworthy, 1% eye drops of the multiꢀtargeted derivatives were more effective than
the combination of the CAI and βꢀblocker leads in the ratio 1% + 0.25%. CAI 17 showed to lower
IOP at 60 min postꢀadministration in a comparable manner to the standards. Compounds 17, 23 and
25 showed similar IOP lowering effectiveness after 2 hr postꢀadministration in the range 6.33ꢀ6.75
mmHg, with 27 excelling among the others producing a IOP drop of 8.0 mmHg. The IOP drops
produced by all assayed derivatives were greater that those occurring using the standard drugs and
their combination (3.0, 3.54, 5.0 mmHg, respectively). When compounds 23 and 25 were more
active after 60 min postꢀadministration, compounds 17 and 27 reached the greater efficacy after 120
min. Derivatives 17, 23 and 25 maintained IOP lowering action at 4 hr postꢀadministration (3.25ꢀ
3.67 mmHg), when timolol and the drugs combination were barely active and DRZ lost its efficacy.
A significant enhancement in the IOP lowering efficacy of dualꢀagents 23, 25, 27 with respect to 17
1
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9
0
(
that uniquely inhibit the CAs) was observed at 60 min postꢀadministration, whereas the range
thinned after 2ꢀhours, with the multiꢀtarget 27 standing out anyhow as the most efficient compound.
It is worth stressing that 17 inhibits hCA II and XII much more potently (K s of 1.5 and 4.1 nM)
than the 23, 25, 27 (K s in the range 75.0ꢀ174.1 and 39.8ꢀ44.4 nM, respectively). As a result, it is
I
I
reasonable to ascribe the powerful IOP lowering efficacy of 17, that did not possess a dual action, to
the orders of magnitude greater hCA II and XII inhibition than 23, 25, 27. Hence, the more effectual
IOP lowering action of the hybrids compounds, in spite of a remarkably weaker CA inhibition than
derivative 17, witnesses the concomitant, although unbalanced (nanomolar vs micromolar)
modulation of two physiological systems, namely CAs and βꢀADRs.
Conclusions
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With the incidence of glaucoma steadily growing because of both demographic expansion and
population aging, new pharmacologic therapies that possess more favourable benefitꢀrisk profiles
are needed. These can be achieved by either increasing efficacy (ocular hypotensive efficacy),
decreasing adverse events, or both. The combination of a βꢀblocker and a CAI included in eye drops
is one of the clinicallyꢀavailable options to treat glaucoma and is extensively used. In the present
work, a novel single molecule, multiꢀtargeted approach was chosen over the coꢀadministration of
single drugs due to varied potential therapeutic benefits, such as an improved effect onto the
intricate biochemical processes involved in disease pathology. Concomitant modulation of βꢀ
adrenergic and CA systems present in the eye was achieved by combining benzenesulfonamide
fragments of classical CAIs with 2ꢀhydroxypropylamine fragments of known, clinically used βꢀ
blockers, such as propranolol or timolol. The resulting two series of molecules, which differ by the
spacer incorporated between the pharmacophores, were investigated for their inhibitory activity
against target (II, IX and XII) and offꢀtarget (I) hCAs as well as for their effectiveness to modulate
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0
1 2
the β ꢀ and β ꢀARs. A first subset of derivatives reported no multiꢀtargeted modulatory efficacy,
with a remarkable CA inhibitory potency at the expense of a void affinity to βꢀARs. The second
subset of hybrid molecules exhibited a slightly worsening of CA inhibition profiles, with the
affinity to βꢀARs raising to micromolar range, which is comparable to racemic βꢀblocker atenolol.
The Xꢀray crystal structure of hCA II was determined in complex with compounds 12 and 23,
namely the isopropylꢀsubstituted derivatives of both series. Multiꢀtarget derivatives 23, 25, 27
were shown to possess more effective IOP lowering properties than the lead, clinically used
dorzolamide and timolol, and their combination based onto the balanced multiꢀtargeted
modulation. The reported evidence supports the proofꢀofꢀconcept of adrenergic receptors blocker
ꢀ
carbonic anhydrase inhibitor hybrids for antiꢀglaucoma therapy with an innovative mechanism
of action. These spread the way of the research of more effective antiꢀglaucoma agents acting on
the two systems in a more powerful and balanced manner. Identification of eutomers within the
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racemic mixtures will be of help in enhancing efficacy onto βꢀARs without significantly affecting
the compounds CA inhibitory activity.
Experimental protocols
Chemistry
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Anhydrous solvents and all reagents were purchased from SigmaꢀAldrich, Alfa Aesar and TCI. All r
eactions involving airꢀ or moistureꢀsensitive compounds were performed under a nitrogen atmosphe
re using dried glassware and syringes techniques to transfer solutions. Nuclear magnetic resonance (
1
13
HꢀNMR, CꢀNMR) spectra were recorded using a Bruker Advance III 400 MHz spectrometer in D
MSOꢀd6. Chemical shifts are reported in parts per million (ppm) and the coupling constants (J) are
expressed in Hertz (Hz). Splitting patterns are designated as follows: s, singlet; d, doublet; sept, sept
et; t, triplet; q, quadruplet; m, multiplet; brs, broad singlet; dd, double of doubles. The assignment o
f exchangeable protons (OH and NH) was confirmed by the addition of D O. Analytical thinꢀlayer c
2
hromatography (TLC) was carried out on Sigma Aldrich silica gel Fꢀ254 plates. Flash chromatograp
hy purifications were performed on Sigma Aldrich Silica gel 60 (230ꢀ400 mesh ASTM) as the statio
nary phase and ethyl acetate/nꢀhexane or MeOH/DCM were used as eluents. Melting points (mp) w
ere measured in open capillary tubes with a Gallenkamp MPD350.BM3.5 apparatus and are uncorre
cted. HPLC was performed by using a Waters 2690 separation module coupled with a photodiode ar
ray detector (PDA Waters 996) and as column a NovaꢀPak C18 4 ꢂm 3.9 mm × 150 mm (Waters), s
ilicaꢀbased reverse phase column. Sample was dissolved in acetonitrile 10%, and an injection volum
e of 45 ꢂL was used. The mobile phase, at a flow rate of 1 mL/min, was a gradient of water + trifluo
roacetic 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 wer
e >96% HPLC pure. Derivatives 9ꢀ18 and 22ꢀ29 were obtained and evaluated as racemates. None of
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the screened derivatives reported PAINS alerts determined by SwissADME server (www.swissadm
e.ch).
37
4ꢀAcetoxybenzoic acid (2).
A suspension of 4ꢀhydroxy benzoic acid 1 (1.0 g, 1.0 eq) in Ac
four drops of H SO 95% and the obtained solution was stirred 3h at 60°C under a nitrogen
atmosphere. The reaction mixture was cooled to r.t., quenched with slush (50g) and then stirred for
.5 h to obtain a white precipitate, that was filtered and washed with water to afford the titled
compound 2. 82% yield; silica gel TLC 0.23 (EtOAc/ ꢀHex 50 % v/v); δ (400 MHz, DMSOꢀ ):
2.34 (s, 3H, C ), 7.30 (d, J = 8.8, 2H, Ar), 8.02 (d, J = 8.8, 2H, Ar), 13.05 (bs, 1H, exchange with
O, COO ); δ (100 MHz, DMSOꢀ ): 21.8, 123.0, 129.3, 131.8, 154.9, 167.6, 169.8.
Experimental in agreement with reported data.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
O (5 ml, 7.3 eq) was treated with
2
4
1
R
f
n
H
d
6
H
3
D
2
H
C
d
6
37
38
4
ꢀ(2ꢀ(4ꢀAcetoxybenzamido)ethyl)benzenesulfonamide (4).
Oxalyl chloride (4.15 eq) and DMF (0.02 eq) were added to a solution 4ꢀacetoxy benzoic acid 2 (0.9
g, 1.0 eq) in CH Cl (18 ml), under a nitrogen atmosphere. The reaction mixture was stirred 0.5h at
2
2
r.t. and at reflux temperature for 2.5h. The volatiles were removed under vacuum and the obtained
viscous oil is dissolved in DMA (4 ml). Pyridine (10.0 eq) and 4ꢀ(2ꢀaminoethyl)ꢀ
benzensulfonamide 3 (1.05 eq) were added to the obtained solution under a nitrogen atmosphere,
the reaction mixture was stirred for 1h at r.t. and then was quenched with slush (30g). The pH was
taken to 2 with HCl 6M, at ice bath temperature to obtain a precipitate that was filtered and washed
with water and Et O to afford the titled compound 7. 83% yield; silica gel TLC Rf 0.30
2
(MeO
H
/DCM 10 % v/v); δ
.55 (q, J = 7.2 2H, C ), 7.25 (d, J = 8.4, 2H, Ar), 7.32 (s, 2H, exchange with D
.47 (d, J = 8.0, 2H, Ar), 7.78 (d, J = 8.0, 2H, Ar), 7.89 (d, J = 8.4, 2H, Ar), 8.64 (t,
CO); δ (100 MHz, DMSOꢀ ): 21.8, 35.7, 41.4, 122.6, 126.6, 129.5, 130.1, 133.0, 143.0, 144.7,
H
(400 MHz, DMSOꢀ
d
6
): 2.32 (s, 3H, C
H
3
), 2.97 (t, J = 7.2, 2H, C
H
2
),
3
7
,
H
2
2
O, SO
2
N
H
2
),
J
= 5.2, 1H,
NH
C
d
6
38
153.5, 166.4, 169.9. Experimental in agreement with reported data.
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4
ꢀHydroxyꢀNꢀ(4ꢀsulfamoylphenethyl)benzamide (5).
4ꢀ(2ꢀ(4ꢀAcetoxybenzamido)ethyl) benzenesulfonamide 4 (1.35 g, 1.0 eq) was added to a freshly
prepared solution of sodium (1.0 eq) in dry methanol ( 40 ml) under a nitrogen atmosphere and the
resulting solution was stirred for 2h at r.t. The reaction mixture was quenched with slush (40 g) and
acidified to pH 1ꢀ2 with HCl 6M, to obtain a precipitate that was filtered and washed with water
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
4
4
5
5
5
5
5
5
5
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5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
and Et O to afford the titled compound 4. 82% yield; silica gel TLC Rf 0.22 (MeOH/DCM 10 %
2
v/v); δ (400 MHz, DMSOꢀd6): 2.94 (t, J = 7.2, 2H, CH2), 3.51 (q, J = 7.2
,
2H, CH2), 6.81 (d, J =
), 7.45 (d, J = 8.8, 2H, Ar), 7.71 (d, J = 8.8,
= 5.2, 1H, N CO), 9.97 (s, 1H, exchange with D O,
6
d ): 35.9, 41.3, 115.7, 126.2, 126.7, 130.0, 130.1, 143.0, 144.9, 161.0,
H
8.8, 2H, Ar), 7.31 (s, 2H, exchange with D
2H, Ar), 7.77 (d, J = 8.8, 2H, Ar), 8.35 (t,
); δ (100 MHz, DMSOꢀ
66.9.
Experimental in agreement with reported data.
2 2 2
O, SO NH
J
H
2
O
H
C
1
38
Nꢀ{2ꢀ[4ꢀ(1ꢀDimethylaminoꢀethylidenesulfamoyl)ꢀphenyl]ꢀethyl}ꢀ4ꢀhydroxyꢀbenzamide (6) .
N,NꢀDimethylformamide dimethyl acetal (2.0 eq) was added to a solution of 4ꢀ(2ꢀ(4ꢀ
hydroxybemzamido)ethyl)benzenesulfonamide 5 (0.94 g, 1.0 eq) in DMF (5 ml) cooled to 0°C. The
reaction mixture was stirred 20’ at r.t. and then quenched with H O (40 ml) to obtain a precipitate,
2
that was filtered and washed with water and Et
gel TLC 0.30 (MeOH/DCM 10 % v/v); δ
3.17 (s, 3H, C ), 3.50 (q, = 6,8 , 2H, C ), 6.81 (d, J = 8.8, 2H, Ar), 7.41 (d, J = 8.8, 2H, Ar),
7.71 (m, 4H, Ar), 8.23 (s, 1H), 8.35 (t, = 5.2, 1H, N CO), 9.97 (s, 1H, exchange with D O, O );
δC (100 MHz, DMSOꢀd6): 35.8, 35.9, 41.3, 41.8, 115.7, 126.2, 126.9, 129.9, 130.1, 141.8, 144.8,
2
O to afford the titled compound 6. 84% yield; silica
R
f
H
(400 MHz, DMSOꢀ ): 2.92 (m, 5H, C + C ),
d
6
H
2
H
3
H
3
J
H
2
J
H
H
2
160.6, 161.0, 166.9.
4
ꢀAllyloxyꢀNꢀ{2ꢀ[4ꢀ(1ꢀdimethylaminoꢀethylidenesulfamoyl)ꢀphenyl]ꢀethyl}ꢀbenzamide (7).
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2
2
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5
6
K CO (2.0 eq) and allyl bromide (1.2 eq) were added to a solution of Nꢀ{2ꢀ[4ꢀ(1ꢀdimethylaminoꢀ
2
3
ethylidenesulfamoyl)ꢀphenyl]ꢀethyl}ꢀ4ꢀhydroxyꢀbenzamide 6 (1.0 g, 1.0 eq) in dry DMA (4 ml)
under a nitrogen atmosphere. The suspension was stirred for 1.5h at 50°C and then quenched with
water (40 ml). The obtained solid was filtered and purified by silica gel chromatography eluting
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
with 10% MeOH/DCM to afford the titled compound 7. 74% yield; silica gel TLC
R
f
0.50
(
MeOH/DCM 10 % v/v); δ (400 MHz, DMSOꢀd6): 2.93 (m, 5H, CH2 + CH3), 3.17 (s, 3H, CH3),
H
3
.51 (q,
J
= 6,8 , 2H, CH2), 4.65 (d,
J
= 5.2, 2H, CH2), 5.31 (dd,
), 7.02 (d, J = 8.4, 2H, Ar), 7.42 (d, J = 8.8, 2H, Ar), 7.72 (d,
8.4, 2H, Ar), 7.82 (d, J = 8.8, 2H, Ar), 8.24 (s, 1H), 8.46 (t, = 5.2, 1H, N CO); δ
DMSOꢀ ): 35.8, 35.9, 41.3, 41.8, 69.2, 115.1, 118.6, 126.9, 127.8, 129.8, 130.1, 134.3, 141.8,
44.7, 160.6, 161.3, 166.6.
J
= 1.6, 10.4, 1H, C
H
), 5.44 (dd,
J
= 2, 17.4, 1H, CH), 6.08 (m, 1H, CH
J
=
J
H
C
(100 MHz,
d
6
1
Nꢀ{2ꢀ[4ꢀ(1ꢀDimethylaminoꢀethylidenesulfamoyl)ꢀphenyl]ꢀethyl}ꢀ4ꢀoxiranylmethoxyꢀ
39
benzamide (8).
NaHCO (3.5 eq) and oxone (1.0 eq) were added to a solution of 4ꢀallyloxyꢀNꢀ{2ꢀ[4ꢀ(1ꢀ
3
dimethylaminoꢀethylidenesulfamoyl)ꢀphenyl]ꢀethyl}ꢀbenzamide 7 (1.60 g, 1.0 eq) in a mixture
aceton/water 3:2 (40 ml) at 0°C and the resulting suspension was stirred 15’ at the same
temperature. Additional NaHCO
raised to r.t. The reaction mixture was stirred at r.t. for 4h, then the temperature was reduced again
to 0°C and other two portions of NaHCO (3.5 eq x 2) and oxone (1.0 eq x 2) were added, the
3
(3.5 eq) and oxone (1.0 eq) were added and the temperature was
3
second 15’ after the first. The reaction mixture stirred at r.t. for 18h, then filtered to remove the
undissolved salts and treated with an aqueous solution of Na S O (5.0 eq). The obtained
2
2
4
suspension was concentrated in vacuum and then extracted with ethyl acetate (4 x 25 ml). The
combined organic layers were dried over anhydrous Na SO and concentrated under reduced
pressure to give a residue purified by silica gel chromatography eluting with 10% MeOH/DCM to
2
4
afford the titled compound 8. 76% yield; silica gel TLC Rf 0.50 (MeOH/DCM 10 % v/v); δ (400
H
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MHz, DMSOꢀd6): 2.76 (dd, J = 2.6, 5.2, 1H, C
H
), 2.89 (t,
), 3.52 (q, J = 6,8 , 2H, CH2), 3.93 (dd,
), 7.05 (d, J = 8.8, 2H, Ar), 7.42 (d, J = 8.4, 2H, Ar), 7.72 (d,
8.4, 2H, Ar), 7.82 (d, J = 8.8, 2H, Ar), 8.24 (s, 1H), 8.46 (t, = 5.2, 1H, N CO); δ (100 MHz,
): 35.8, 35.9, 41.3, 41.8, 44.7, 50.5, 70.0, 114.9, 126.9, 128.0, 129.8, 130.1, 141.8, 144.7,
60.6, 161.4, 166.6.
J
= 4.8, 1H, C
H
), 2.92 (m, 5H, CH3
+
C
C
=
H2), 3.17 (s, 3H, CH3), 3.38 (m, 1H, C
H
J
= 6.6, 11.4, 1H,
H
), 4.43 (dd, = 2.7, 11.4, 1H, C
J
H
J
J
H
C
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
DMSOꢀd
6
1
General synthetic procedure of compounds 4ꢀ(3ꢀalkylaminoꢀ2ꢀhydroxyꢀpropoxy)ꢀNꢀ[2ꢀ(4ꢀ
4
0,41
sulfamoylꢀphenyl)ꢀethyl]ꢀbenzamide 9ꢀ16, 22ꢀ29.
Procedure 1
The proper epoxide 8 or 21 (0.25 g, 1.0 eq) was suspended in variable volume of primary amine or,
alternatively in EtOH and treated with the appropriate primary amine (10 eq), and the reaction
mixture was stirred at r.t. or under heating until starting materials were consumed (TLC
monitoring). EtOH and excess of amine were removed under vacuum and the residue was triturated
with water (9ꢀ16) or with Et
2
O (22ꢀ26). A solution of the obtained deprotected free base in HCl
1.25M in MeOH was stirred at r.t. for 20’ and then concentrated under vacuum to give a residue
that was triturated with aceton to give the titled compounds 9ꢀ16, 22ꢀ26.
Procedure 2
The proper secondary amine (3 eq) was added to a suspension of the proper epoxide 8 or 21 (0.25 g,
1.0 eq) in EtOH (8 ml) and reaction mixture stirred at 70°C until starting materials were consumed
(TLC monitoring). EtOH and excess of amine were removed under vacuum and the obtained
residue was triturated with water (17ꢀ18) or with Et O (27ꢀ29). A solution of the resulting protected
2
free base in HCl 1.25 in MeOH was stirred at 80 °C in a sealed tube until starting materials were
consumed (TLC monitoring). The solvent was removed under vacuum and the obtained residue was
triturated with aceton to give the titled compounds 17ꢀ18, 27ꢀ29.
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4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4ꢀ(2ꢀHydroxyꢀ3ꢀmethylaminoꢀpropoxy)ꢀNꢀ[2ꢀ(4ꢀsulfamoylꢀphenyl)ꢀethyl]ꢀbenzamide
hydrochloride (9).
Compound 9 was obtained according the general procedure 1 earlier reported. Nꢀ{2ꢀ[4ꢀ(1ꢀ
Dimethylaminoꢀethylidenesulfamoyl)ꢀphenyl]ꢀethyl}ꢀ4ꢀoxiranylmethoxyꢀbenzamide 8 (0.25 g, 1.0
eq) was suspended in EtOH and treated with a 35% aqueous solution of methylamine overnight at
r.t. and then the obtained free base was treated with HCl 1.25M in MeOH (10 ml) to afford the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
corresponding hydrochloride salt 9. 55% yield; silica gel TLC
v/v); δ (400 MHz, DMSOꢀ ): 2.64 (m, 3H, C ), 2.95 (t, J = 6.8, 2H, C
.15 (m, 1H, C ), 3.53 (q, J = 7.2, 2H, C ), 4.05 (d, J = 5.2, 2H, C ), 4.21 (m, 1H, C
bs, 1H, exchange with D O, O ), 7.04 (d, J = 8.8, 2H, Ar), 7.33 (s, 2H, exchange with D O,
R
f
0.10 (TFA/MeO
), 3.03 (m, 1H, C
), 5.95
H
/DCM 3/15/82%
H
d
6
H
3
H
2
H
),
3
H
H
2
H
2
H
(
H
2
2
SO NH2), 7.46 (d, J = 8.4, 2H, Ar), 7.78 (d, J = 8.4, 2H, Ar), 7.85 (d, J = 8.8, 2H, Ar), 8.53 (t, J =
2
+
2
5.6, 1H, CON
H
), 8.73 (bd, exchange with D
2
O, 2H, N
H
); δ
C
(100 MHz, DMSOꢀ
d
6
): 33.93, 35.78,
41.31, 51.79, 65.62, 70.78, 114.98, 126.61, 127.98, 129.89, 130.06, 142.95, 144.79, 161.42, 166.56;
ꢀ
+
m/z (ESI positive) 408.2 [MꢀCl ]
4ꢀ(2ꢀHydroxyꢀ3ꢀ(methylamino)propoxy)ꢀNꢀ(4ꢀsulfamoylphenethyl)benzamide hydrochloride
(10).
Compound 10 was obtained according the general procedure 1 earlier reported. Nꢀ{2ꢀ[4ꢀ(1ꢀ
Dimethylaminoꢀethylidenesulfamoyl)ꢀphenyl]ꢀethyl}ꢀ4ꢀoxiranylmethoxyꢀbenzamide 8 (0.25 g, 1.0
eq) was treated with ethylamine in EtOH overnight at r.t. and then the obtained free base was
treated with HCl 1.25M in MeOH (10 ml) to afford the corresponding hydrochloride salt 10. 54%
yield; silica gel TLC
R
f
0.15 (TFA/MeO
), 2.95 (t, J = 6.8, 2H, C
), 4.06 (d, J = 5.2, 2H, C
H
/DCM 3/15/82% v/v);δ
), 3.03 (m, 3H, C + C
), 4.22 (m, 1H, C
H
(400 MHz, DMSOꢀ ): 1.27 (t,
d
6
J
J
=
=
O
7.2, 3H, C
H
3
H
2
H
H
2
), 3.17 (m, 1H, CH), 3.52 (q,
7.2, 2H, C
H
2
H
2
H
), 5.95 (bs, 1H, exchange with D
2
O,
H
), 7.04 (d, J = 8.8, 2H, Ar), 7.33 (s, 2H, exchange with D O, SO NH2), 7.46 (d, J = 8.4, 2H, Ar),
2
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.78 (d, J = 8.4 Hz, 2H, Ar), 7.85 (d, J = 8.8, 2H, Ar), 8.51 (t, J = 5.6, 1H, exchange with D O,
2
+
CON
H
), 8.71 (bd, 2H, exchange with D O, NH2 ); δ (100 MHz, DMSOꢀd6): 11.70, 35.75, 41.28,
2
C
43.30, 49.80, 65.77, 70.78, 114.95, 126.59, 127.95, 129.87, 130.03, 142.93, 144.76, 161.40, 166.52;
ꢀ +
m/z (ESI positive) 422.2[MꢀCl ]
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4ꢀ(2ꢀHydroxyꢀ3ꢀpropylaminoꢀpropoxy)ꢀNꢀ[2ꢀ(4ꢀsulfamoylꢀphenyl)ꢀethyl]ꢀbenzamide
hydrochloride (11).
Compound 11 was obtained according the general procedure 1 earlier reported. Nꢀ{2ꢀ[4ꢀ(1ꢀ
Dimethylaminoꢀethylidenesulfamoyl)ꢀphenyl]ꢀethyl}ꢀ4ꢀoxiranylmethoxyꢀbenzamide 8 (0.25 g, 1.0
eq) was treated with propylamine (8 ml) for 8h at 50°C and then the obtained free base was treated
with HCl 1.25M in MeOH (10 ml) to afford the corresponding hydrochloride salt 11. 64% yield;
silica gel TLC Rf 0.24 (TFA/MeO
.0, 3H, C ), 1.68 (m, 2H, C ), 2.94 (m, 4H, 2 x C
3.53 (m, 2H, C ), 4.06 (d, J = 5.2, 2H, C ), 4.21 (m, 1H, C
2 2
), 7.04 (d, J = 8.8, 2H, Ar), 7.33 (s, 2H, exchange with D O, SO NH
H/DCM 3/15/82% v/v); δ (400 MHz, DMSOꢀd6): 0.95 (t, J =
H
6
H
3
H
2
H
2
), 3.03 (m, 1H, C
), 5.94 (bs, 1H, exchange with D
), 7.46 (d, J = 8.4, 2H, Ar),
H
), 3.17 (m, 1H, C
H
),
H
2
H
2
H
2
O,
O
H
2
7.77 (d, J = 8.4, 2H, Ar), 7.84 (d, J = 8.8, 2H, Ar), 8.50 (t, J = 5.6, 1H, CONH), 8.61 (bs, 2H,
+
exchange with D O, NH2 ); δ (100 MHz, DMSOꢀd6): 11.87, 19.68, 35.76, 41.30, 49.71, 50.28,
2
C
65.74, 70.80, 114.98, 126.61, 127.95, 129.89, 130.05, 142.94, 144.78, 161.44, 166.58; m/z (ESI
ꢀ
+
positive) 436.2 [MꢀCl ]
4ꢀ(2ꢀHydroxyꢀ3ꢀisopropylaminoꢀpropoxy)ꢀNꢀ[2ꢀ(4ꢀsulfamoylꢀphenyl)ꢀethyl]ꢀbenzamide
hydrochloride (12).
Compound 12 was obtained according the general procedure 1 earlier reported. Nꢀ{2ꢀ[4ꢀ(1ꢀ
dimethylaminoꢀethylidenesulfamoyl)ꢀphenyl]ꢀethyl}ꢀ4ꢀoxiranylmethoxyꢀbenzamide 8 (0.25 g, 1.0
eq) was treated with isopropylamine (5 ml) for 6h at 50°C and then the obtained free base was
treated with HCl 1.25M in MeOH (10 ml) to afford the corresponding hydrochloride salt 12. 54%
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
yield; silica gel TLC Rf 0.40 (TFA/MeO
6.0, 6H, 2 x CH3,), 2.96 (t, J = 7.2, 2H, CH2,), 3.01 (m, 1H, C
.4, 2H, C ,), 4.08 (d, J = 5.2, 2H, C ,), 4.25 (m, 1H, C ), 5.95 (d, J = 5.2, 1H, exchange with
), 7.05 (d, J = 8.8, 2H, Ar), 7.33 (s, 2H, exchange with D O, SO ), 7.46 (d, J = 8.4,
H/DCM 3/15/82% v/v); δ (400 MHz, DMSOꢀd6): 1.29 (t, J
H
=
H), 3.16 (m, 1H, CH), 3.53 (q, J =
6
H
2
H
2
H
D
2
O, OH
2
2 2
NH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2H, Ar), 7.78 (d, J = 8.4, 2H, Ar), 7.85 (d, J = 8.8, 2H, Ar), 8.52 (t, J = 5.6, 1H, exchange with
+
D O, CON
2
H
), 8.70 (bd, 2H, exchange with D O, NH2 ); δ (100 MHz, DMSOꢀd6): 24.01, 35.94,
2
C
49.27, 50.99, 69.42, 72.04, 115.05, 126.75, 126.97, 127.69, 129.96, 130.19, 143.09, 144.93, 162.07,
ꢀ
+
166.78; m/z (ESI positive) 436.2 [MꢀCl ] .
4ꢀ(2ꢀHydroxyꢀ3ꢀsecꢀbutylaminoꢀpropoxy)ꢀNꢀ[2ꢀ(4ꢀsulfamoylꢀphenyl)ꢀethyl]ꢀbenzamide
hydroclhoride (13).
Compound 13 was obtained according the general procedure 1 earlier reported. Nꢀ{2ꢀ[4ꢀ(1ꢀ
Dimethylaminoꢀethylidenesulfamoyl)ꢀphenyl]ꢀethyl}ꢀ4ꢀoxiranylmethoxyꢀbenzamide 8 (0.25 g, 1.0
eq) was treated with secꢀbutylamine (8 ml) overnight at r.t. and then the obtained free base was
treated with HCl 1.25M in MeOH (10 ml) to afford the corresponding hydrochloride salt 13. 49%
yield; silica gel TLC Rf 0.31 (TFA/MeO
H/DCM 3/15/82% v/v); δ (400 MHz, DMSOꢀd6): 0.94 (td,
H
J = 2.0, 7.6, 3H, CH3), 1.27 (t, J = 6.4, 3H, CH3), 1.51 (m, H, C
H
), 1.82 (m, 1H, C
), 3.53 (q, J = 6.4, 2H, C ), 4.08 (d, J = 5.2,
), 5.94 (d, J = 2.4, 1H, exchange with D O, O ), 7.05 (d, J = 8.8, 2H,
H), 2.96 (t, J =
7.2, 2H, C
H
2
), 3.03 (m, 1H, C
H
), 3.17 (m, 2H, C
H
2
H
2
2
H, C ), 4.26 (m, 1H, C
H
2
H
2
H
Ar), 7.33 (s, 2H, exchange with D
2
O, SO
2
N
H
2
), 7.46 (d, J = 8.4, 2H, Ar), 7.77 (d, J = 8.4, 2H, Ar),
+
7.84 (d, J = 8.8, 2H, Ar), 8.52 (t, J = 5.6, 1H, CON
H
), 8.77 (bt, exchange with D O, 2H, NH2 ); δC
2
(100 MHz, DMSOꢀd6): 10.66, 15.89, 25.90, 35.77, 41.31, 47.51, 55.88, 65.97, 70.80, 114.98,
ꢀ +
1
26.61, 127.94, 129.90, 130.05, 142.95, 144.79, 161.45, 166.55; m/z (ESI positive) 450.2 [MꢀCl ]
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4ꢀ(2ꢀHydroxyꢀ3ꢀtꢀbutylaminoꢀpropoxy)ꢀNꢀ[2ꢀ(4ꢀsulfamoylꢀphenyl)ꢀethyl]ꢀbenzamide
hydrochloride (14).
Compound 14 was obtained according the general procedure 1 earlier reported. Nꢀ{2ꢀ[4ꢀ(1ꢀ
Dimethylaminoꢀethylidenesulfamoyl)ꢀphenyl]ꢀethyl}ꢀ4ꢀoxiranylmethoxyꢀbenzamide 8 (0.25 g, 1.0
eq) was treated with tꢀbutylamine (8 ml) overnight at r.t. and then the obtained free base was treated
with HCl 1.25M in MeOH (10 ml) to afford the corresponding hydrochloride salt 14. 47% yield;
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
silica gel TLC
R
f
0.25 (TFA/MeO
), 1.35 (s, 9H, 3 x C
), 4.10 (d, J = 5.2 , 2H, C
H), 7.06 (d, J = 8.8, 2H, Ar), 7.33 (s, 2H, exchange with D O, SO NH2), 7.46 (d, J = 8.4,
H
/DCM 3/15/82% v/v); δ
), 2.96 (m, 3H, C + C
), 4.26 (m, 1H, C ), 5.95 (d, J = 4.8, 1H, exchange with
H
(400 MHz, DMSOꢀ
d
6
): 0.94 (td, J =
2.0, 7.6, 3H, C
H
3
H
3
H
H
2
), 3.17 (m, 1H, C
H), 3.52 (q, J =
6.4, 2H, C
H
2
H
2
H
D O, O
2
2
2
2H, Ar), 7.78 (d, J = 8.4, 2H, Ar), 7.85 (d, J = 8.8, 2H, Ar), 8.53 (t, J = 5.6, 1H, CON
H
), 8.74 (bt,
+
2
H, exchange with D O, NH ); δ (100 MHz, DMSOꢀd ): 25.90, 35.76, 41.29, 44.91, 57.31, 66.35,
2 2 C 6
70.76, 114.99, 126.60, 127.94, 129.88, 130.04, 142.94, 144.77, 161.44, 166.54; m/z (ESI positive)
ꢀ
+
450.2 [MꢀCl ]
4ꢀ(2ꢀHydroxyꢀ3ꢀbenzylaminoꢀpropoxy)ꢀNꢀ[2ꢀ(4ꢀsulfamoylꢀphenyl)ꢀethyl]ꢀbenzamide (15).
Compound 15 was obtained according the general procedure 1 earlier reported. Nꢀ{2ꢀ[4ꢀ(1ꢀ
Dimethylaminoꢀethylidenesulfamoyl)ꢀphenyl]ꢀethyl}ꢀ4ꢀoxiranylmethoxyꢀbenzamide 8 (0.25 g, 1.0
eq) was treated with benzylamine in EtOH for 7h at 50°C to afford the titled compound 15 as a free
base. 68% yield; silica gel TLC Rf 0.35 (TFA/MeO
d6): 2.19 (bs, 1H, exchange with D O, N ), 2.62 (m, 2H, CH2), 2.95 (m, 2H, CH2), 3.53 (q, J = 6.4,
), 3.97 (m, 2H, C ), 4.07 (m, 1H, C ), 5.04 (d, J = 4.4, 1H, exchange
), 7.01 (d, J = 8.8, 2H, Ar), 7.32 (m, 7H, partial exchange with D O, Ar + SO ),
.46 (d, J = 8.4, 2H, Ar), 7.78 (d, J = 8.4, 2H, Ar), 7.81 (d, J = 8.8, 2H, Ar), 8.46 (t, J = 5.6, 1H,
H/DCM 3/15/82% v/v);δ (400 MHz, DMSOꢀ
H
H
2
2H, C
H
2
), 3.76 (s, 2H, C
H
2
H
2
H
with D
2
O , O
H
2
2 2
NH
7
CON
H
); δ (100 MHz, DMSOꢀd6): 35.79, 41.31, 52.62, 54.01, 69.07, 71.82, 114.90, 126.61,
C
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
27.43, 127.55, 128.80, 129.02, 129.82, 130.04, 141.82, 142.94, 144.79, 161.92, 166.64; m/z (ESI
+
positive) 484.2 [M+H] .
4ꢀ(2ꢀHydroxyꢀ3ꢀ((2ꢀhydroxyethyl)amino)propoxy)ꢀNꢀ(4ꢀsulfamoylphenethyl)benzamide
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
hydrochloride (16).
Compound 16 was obtained according the general procedure 1 earlier reported. Nꢀ{2ꢀ[4ꢀ(1ꢀ
dimethylaminoꢀethylidenesulfamoyl)ꢀphenyl]ꢀethyl}ꢀ4ꢀoxiranylmethoxyꢀbenzamide 8 (0.25 g, 1.0
eq) was treated with ethanolamine in EtOH overnight at r.t. and then the obtained free base was
treated with HCl 1.25M in MeOH (10 ml) to afford the corresponding hydrochloride salt 16. 46%
yield; silica gel TLC
MHz, DMSOꢀd6): 2.96 (t, J = 6.8, 2H, CH2), 3.03 (m, 3H, C
7.2, 2H, CH2), 3.74 (m, 2H, CH2), 4.06 (d, J = 4.8, 2H, CH2 ), 4.27 (m, 1H, C
exchange with D O, O ), 5.93 (bs, 1H, exchange with D O, OH,), 7.04 (d, J = 8.8, 2H, Ar), 7.33
(s, 2H, exchange with D O, SO ), 7.46 (d, J = 8.4, 2H, Ar), 7.78 (d, J = 8.4, 2H, Ar), 7.85 (d,
8.8, 2H, Ar), 8.53 (t, J = 5.6, 1H, exchange with D O, CON ), 8.80 (bd, 2H, exchange with D O,
R
f
0.09 (TFA/MeO
H
/DCM 3/15/82% v/v) (MeOH/DCM 10 % v/v); δ
+ CH2 ), 3.17 (m, 1H, C ), 3.52 (q,
), 5.30 (bs, 1H,
H
(400
H
H
J
=
H
2
H
2
2
2
N
H
2
J
=
2
H
2
+
N
H2 ); δ (100 MHz, DMSOꢀd6): 35.75, 41.28, 50.27, 50.42, 65.65, 70.84, 114.96, 126.59, 127.93,
C
ꢀ +
129.87, 130.03, 142.93, 114.76, 161.41, 166.53; m/z (ESI positive) 438.2 [MꢀCl ] .
4ꢀ(2ꢀHydroxyꢀ3ꢀmorpholinopropoxy)ꢀNꢀ(4ꢀsulfamoylphenethyl)benzamide
hydrochloride
(17).
Compound 17 was obtained according the general procedure 2 earlier reported. Nꢀ{2ꢀ[4ꢀ(1ꢀ
Dimethylaminoꢀethylidenesulfamoyl)ꢀphenyl]ꢀethyl}ꢀ4ꢀoxiranylmethoxyꢀbenzamide 8 (0.25 g, 1.0
eq) was treated with morpholine in EtOH for 5h at 70°C and then the obtained protected free base
was treated with HCl 1.25M in MeOH in sealed tube to afford the titled compound 17 as
hydrochloride salt. 43% yield; silica gel TLC
R
f
0.20 (TFA/MeO
H
/DCM 3/15/82% v/v); δ
H
(400
MHz, DMSOꢀd6): 2.96 (t, J = 6.8, 2H, CH2), 2.28 (m, 4H, 2 x CH2), 3.17 (m, 1H, C
H), 3.53 (m,
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H, 2 x CH2), 3.85 (m, 2H, CH2), 4.03 (m, 2H, CH2), 4.06 (d, J = 4.8, 2H, CH2), 4.43 (m, 1H, CH),
6.04 (bs, 1H, exchange with D O, OH), 7.04 (d, J = 8.8, 2H, Ar), 7.33 (s, 2H, exchange with D O,
2
2
SO
2
N
H
2
), 7.46 (d, J = 8.4, 2H, Ar), 7.78 (d, J = 8.4, 2H, Ar), 7.85 (d, J = 8.8, 2H, Ar), 8.51 (t, J =
+
5
.6, 1H, exchange with D
DMSOꢀ
30.05, 142.95, 144.78, 161.41, 166.54; m/z (ESI positive) 464.2 [MꢀCl ] .
2
O, CON
H
), 10.37 (bs, 1H, exchange with D
2
O, N
H
); δ
C
(100 MHz,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
d ): 35.76, 51.93, 53.56, 59.69, 63.98, 64.32, 71.10, 115.01, 126.61, 128.02, 129.91,
ꢀ
+
1
4ꢀ(2ꢀHydroxyꢀ3ꢀdiisopropylaminoꢀpropoxy)ꢀNꢀ[2ꢀ(4ꢀsulfamoylꢀphenyl)ꢀethyl]ꢀbenzamide
hydrochloride (18).
Compound 18 was obtained according the general procedure 2 earlier reported. Nꢀ{2ꢀ[4ꢀ(1ꢀ
Dimethylaminoꢀethylidenesulfamoyl)ꢀphenyl]ꢀethyl}ꢀ4ꢀoxiranylmethoxyꢀbenzamide 8 (0.25 g, 1.0
eq) was treated with diisopropylamine in EtOH for 24h at 70°C and then the obtained protected free
base was treated with HCl 1.25M in MeOH in sealed tube to afford the titled compound 18 as
hydrochloride salt. 41% yield; silica gel TLC
MHz, DMSOꢀ ): 1.30 (m, 12H, 4 x C ), 2.96 (t, J = 6.8, 2H, C
), 3.53 (m, 2H, CH2), 3.74 (m, 2H, 2 x C ), 4.13 (m, 2H, CH2), 4.27 (m, H, C
), 7.06 (d, J = 8.8, 2H, Ar), 7.33 (s, 2H, exchange with D O,
R
f
0.36 (TFA/MeO
H
/DCM 3/15/82% v/v); δ
), 3.19 (m, 1H, C ), 3.33 (m,
), 5.93 (d, J
H
(400
d
6
H
3
H
2
H
1H, C
H
H
H
=
4.8, 1H, exchange with D O, O
H
2
2
SO
.6, 1H, exchange with D
6
DMSOꢀd ): 17.07, 17.94, 35.76, 51.08, 55.70, 56.55, 66.53, 70.49, 114.95, 126.60, 128.01, 129.90,
2 2
NH ), 7.46 (d, J = 8.4, 2H, Ar), 7.78 (d, J = 8.4, 2H, Ar), 7.85 (d, J = 8.8, 2H, Ar), 8.51 (t, J =
+
5
2
2 C
O, CONH), 9.01 (bs, 1H, exchange with D O, NH ); δ (100 MHz,
ꢀ
+
130.03, 142.95, 144.76, 161.32, 166.52; m/z (ESI positive) 478.2 [MꢀCl ] .
42
N,NꢀDimethylaminomethyleneꢀ4ꢀhydroxyꢀbenzenesulfonamide (20).
N,NꢀDimethylformamide diethyl acetal (1.2eq) was added to
a
solution of 4ꢀ
hydroxybenzenesulfonamide 19 (1.5 g, 1.0 eq) in DMF (1.5 ml) at 0°C and that was stirred for 0.5h
at r.t. The reaction mixture was treated with EtOAc (40ml) and the obtained solid was filtered and
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