Probing the 'bipolar' nature of the carbonic anhydrase active site: Aromatic sulfonamides containing 1,3-oxazol-5-yl moiety as picomolar inhibitors of cytosolic CA I and CA II isoforms

Article abstract of DOI:10.1016/j.ejmech.2015.06.022

Abstract A series of potent inhibitors of human carbonic anhydrase (CA) isoforms I and II has been prepared via a direct, chemoselective sulfochlorination of a range of 1,3-oxazolyl benzenes and thiophenes, followed by primary sulfonamide synthesis. The latter functionality is a known zinc-binding group (ZBG) responsible for anchoring the inhibitors to the CA's zinc metal ion. The compound's periphery as well as the overall scaffold geometry was designed to enable optimal interactions with the two distinct sides of the enzyme's active site, one of which is lined with hydrophobic residues and while the other is predominantly hydrophilic. As a result, several compounds inhibiting the therapeutically important cytosolic CA I and CA II in picomolar range have been identified. These compounds are one of the most potent CA inhibitors identified to-date. Not only the remarkable (>10 000-fold), cytosolic CA I and CA II selectivity vs. the membrane-bound CA IX and CA XII isoforms, but also the pronounced CA II/I selectivity observed in some cases, allow considering this series as a set of isoform-selective chemical biology tools and promising starting points for drug candidate development.

Full text of DOI:10.1016/j.ejmech.2015.06.022

Accepted Manuscript
Probing the ‘bipolar’ nature of the carbonic anhydrase active site: aromatic
sulfonamides containing 1,3-oxazol-5-yl moiety as picomolar inhibitors of cytosolic CA
I and CA II isoforms
Mikhail Krasavin, Mikhail Korsakov, Mikhail Dorogov, Tiziano Tuccinardi, Nurcan
Dedeoglu, Claudiu T. Supuran
PII:
S0223-5234(15)30096-9
10.1016/j.ejmech.2015.06.022
EJMECH 7949
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 February 2015
Revised Date: 1 June 2015
Accepted Date: 8 June 2015
Please cite this article as: M. Krasavin, M. Korsakov, M. Dorogov, T. Tuccinardi, N. Dedeoglu, C.T.
Supuran, Probing the ‘bipolar’ nature of the carbonic anhydrase active site: aromatic sulfonamides
containing 1,3-oxazol-5-yl moiety as picomolar inhibitors of cytosolic CA I and CA II isoforms, European
Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.06.022.
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ACCEPTED MANUSCRIPT
Probing the ‘bipolar’ nature of the carbonic anhydrase active site: aromatic
sulfonamides containing 1,3-oxazol-5-yl moiety as picomolar inhibitors of
cytosolic CA I and CA II isoforms
Mikhail Krasavin,* Mikhail Korsakov, Mikhail Dorogov, Tiziano Tuccinardi, Nurcan Dedeoglu,
*
Claudiu T. Supuran
hydrophilic side of the active site
hCA I Ki = 41.9 nM
N
O
hCA II Ki = 0.01 nM
hCA IX Ki = 18.7 nM
hCA XII Ki = 6.7 nM
O
O
S
N
S
Zn2+
N
O
H
hydrophobic side of the active site
1

ACCEPTED MANUSCRIPT
Probing the ‘bipolar’ nature of the carbonic anhydrase active site: aromatic
sulfonamides containing 1,3-oxazol-5-yl moiety as picomolar inhibitors of
cytosolic CA I and CA II isoforms
a,*
b
b
c
Mikhail Krasavin , Mikhail Korsakov, Mikhail Dorogov, Tiziano Tuccinardi, Nurcan
d
d, *
Dedeoglu, Claudiu T. Supuran
a
Department of Chemistry, Saint Petersburg State University, 26 Universitetskii Prospect,
Peterhof, 198504 Russian Federation
b
The Ushinsky Yaroslavl State Pedagogical University, 108 Respublikanskaya St., Yaroslavl,
1
50000, Russian Federation
Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126, Pisa, Italy
c
d
Neurofarba Dept., Section of Pharmaceutical Sciences, Universita degli Studi di Firenze, Via Ugo
Schiff 6, 50019 Sesto Fiorentino, Florence, Italy
*
Corresponding authors.
E-mail: m.krasavin@spbu.ru (M. Krasavin), claudiu.supuran@unifi.it (C. T. Supuran).
ABSTRACT
A series of potent inhibitors of human carbonic anhydrase (CA) isoforms I and II has been prepared
via a direct, chemoselective sulfochlorination of a range of 1,3-oxazolyl benzenes and thiophenes,
followed by primary sulfonamide synthesis. The latter functionality is a known zinc binding group
(ZBG) responsible for anchoring the inhibitors to the CA’s zinc metal ion. The compound’s
periphery as well as the overall scaffold geometry was designed to enable optimal interactions with
the two distinct sides of the enzyme’s active site, one of which is lined with hydrophobic residues
and while the other is predominantly hydrophilic. As a result, several compounds inhibiting the
therapeutically important cytosolic CA I and CA II in picomolar range have been identified. These
compounds are one of the most potent CA inhibitors identified to-date. Not only the remarkable
(>10,000-fold), cytosolic CA I and CA II selectivity vs. the membrane-bound CA IX and CA XII
isoforms, but also the pronounced CA II/I selectivity observed in some cases, allow considering this
series as a set of isoform-selective chemical biology tools and promising starting points for drug
candidate development.
Keywords: drug discovery, carbonic anhydrase inhibitors, isoform selectivity, anti-glaucoma drugs,
1
,3-oxadiazol-5-yl aromatics, chemoselective sulfochlorination.
2

ACCEPTED MANUSCRIPT
1
. Introduction
Carbonic anhydrases (CAs) catalyse the fundamental biochemical process of carbon dioxide
hydration (a reversible reaction producing a bicarbonate anion and a proton) and are, therefore, one
1
of the principal regulators of cellular pH homeostasis. The potential of this enzyme family as an
important class of biological targets for chemotherapeutic intervention was recognized several
2
decades ago. This has led to the development of several effective drugs in areas as diverse as
ophthalmology (glaucoma), metabolic disease (diabetes) and gastroenterology (gastric and duodenal
3
ulcers).
The earlier CA inhibitors (CAIs) (examples of which include acetazolamide, methazolamide,
dorzolamide, brinzolamide – Figure 1) are almost exclusively non-selective, pan-inhibitors of all
human CAs (of which there are currently 16 isoforms known). More recent research efforts were
directed toward the discovery of isoform-selective CAIs and understanding the guiding structural
4
principles that can help achieve the desired selectivity.
5
Primary sulfonamides are the central and most prominent class of CAIs. The sulfonamide
functionality in these compounds is responsible for coordination to the enzyme’s prosthetic metal
2
+
ion (which is almost exclusively Zn across the known CAs). It is, therefore, denoted as a zinc-
binding group (ZBG). It is, however, the CAI molecule’s periphery that determines the potency and
selectivity. This is illustrated by the evolution of the weak and non-selective CA inhibitor
6
7-10
benzenesulfonamide into highly potent isoform-selective sulfonamides 1-4 (Figure 2).
In the
latter, the substituents in the benzene ring (besides the pharmacophoric sulfonamide group) are
thought to be responsible for additional contacts in the CA active site, some of which are with the
amino acid residues that are unique to specific isozymes.
The vast structural information accumulated to-date via extensive X-ray studies of numerous CA-
CAI complexes led to establishing a very notable feature that is characteristic of all the CA active
11
sites. Regardless of the species, all the known CAs possess generally rather big active sites that
are divided into two distinct sides, one of which is lined with hydrophobic residues and the other –
1
2
with polar, hydrophilic ones. While the size of the CA active site may appear puzzling in light of
the very small size of the substrates, it is the unique, ‘bipolar’ environment surrounding the catalytic
‘scene’ that allows for an efficient binding of the two closely related, yet vastly different substrate
-
13
molecules - a hydrophobic gas (CO ) and a polar, water-soluble anion (HCO ).
2
3
The information on the specific setup of the CA active site has provided substantial grounds for
designing potent and isoform-selective inhibitors. The notable examples include 4-ureidobenzene
1
4
sulfonamides recently described by McKenna and Supuran. According to the X-ray analysis, the
ureido tails in these compounds selectively occupy pockets on the hydrophobic side of the active
3

ACCEPTED MANUSCRIPT
site, leading to potent and selective inhibitors of CA II. Even more striking example of exploration
of the two halves of the CA II active site with polar as well as lipophilic moieties appended onto
1
5
acetazolamide molecule was recently disclosed by Poulsen and Supuran. In the latter work, a
thorough comparison of single- vs. dual-tail inhibitors was made, which offered a unique insight
into the importance of varying the nature of the periphery groups on the inhibitor potency toward a
particular CA isoform.
We became interested in using the synthetically accessible variants of 1,3-oxazol-5-yl aromatics as
the template for linking the pharmacophoric primary sulfonamide group and the distal periphery
motifs (polar or lipophilic). Such a template, in our view could enable several possibilities for
projecting the sulfonamide ZBG toward the ‘bottom’ of the active site while the substituents on the
1
,3-oxazole ring could not only be easily varied in nature but can also be expected to adopt a range
of conformations (due to the rotational freedom present in the diaryl linker) for optimal interactions
with both sides of the active site (Figure 3). In this paper, we present the synthesis and biological
evaluation of the new CAIs conforming to this general design idea.
2
. Results and discussion
2
.1 Chemistry
In order to confirm that the 1,3-oxadiazol-5-yl aromatic templates are suitable for the CAI design,
twenty core 5a-t were synthesized using the Robinson-Gabriel synthesis involving
cyclodehydration of α-acylaminoketones (prepared, in turn, in nearly quantitative yields from
1
6
readily available starting materials) under forcing conditions, in good yields (Table 1). The 1,3-
oxazole precursors 5a-o containing lipophilic R groups (Me, cyclopropyl, cyclobutyl) were set
aside for the subsequent introduction of the sulfonamide ZBGs (vide infra). The five ester
compounds 5p-t were promptly transformed into morpholine and pyrrolidine carboxamides 6a-j as
the ‘polar periphery’ precursors (Scheme 1). It should be noted that the immediate, direct amidation
of these 1,3-oxazole-2-carboxylic esters was mandated by the marked instability of the respective
carboxylic acids (obtained by alkaline hydrolysis) toward de-carboxylation, even at ambient
temperature.
The 1,3-oxazole substrates 5a-o and 6a-j were then subsequently subjected to direct
sulfochlorination and the respective sulfonyl chlorides 7a-o and 8a-j were produced in high yields
and as a single regioisomer, as indicated by the arrow . The 1,3-oxazole ring remained intact in the
1
7
reaction conditions, in accordance with the literature precedent. The target sulfonamides 9a-o
(lipophilic periphery) and 10a-j (polar periphery) were obtained on treatment of the respective
4

ACCEPTED MANUSCRIPT
sulfonyl chlorides with aqueous ammonia (Scheme 2; yields are provided in Section 4 of this
1
article). The regiochemistry of the sulfochlorination step (initially determined based on H NMR
spectra of the sulfochlorides) was further confirmed by the through-space interactions observed in
the NOESY spectra of representative sufonamides 9i, 9l and 9o (see Supplementary Data). Notably,
the choice of the aromatic groups for direct sulfochlorination determined the wide range of
possibilities for the ZBG sulfonamide to be orientated relative to the linear diaryl linker, in
accordance with the general CAI design idea articulated depicted in Figure 3.
2
.2 Biological activity
The biological activity of the twenty-five compounds 9a-o and 10a-j as CA inhibitors was
evaluated against the human CA I, II, IX and XII. The cytosolic isoforms I and II are the usual off-
targets for the membrane-bound isoforms IX and XII. While CA I and II are both involved in
1
8-19
20
edema
(and CA II is a classical target for treating glaucoma ), CA IX and XII are the highly
2
1
pursued targets for the development of new cancer treatment, with one such compound (SLC-
2
2
0
111) recently in Phase I clinical trials for the treatment of advanced, metastatic solid tumors. The
inhibition data are presented in Table 2 and are grouped according to the geometry of the ZBG
sulfonamide relative to the diaryl core, for easier analysis of the structure-activity relationships
(SAR).
The p-phenylidene-linked subseries is an apparent source of some really potent and selective CA II
inhibitors: the picomolar inhibition of the enzyme by 9a-c appeared very promising, particularly
when considering the 100-1000-fold selectivity vs. other three enzymes (including CA I) were
consistently observed. Interestingly, the CA II/I selectivity was completely lost in the ‘polar tail’,
carboxamide compounds 10a-b (the trend is rationalized by in silico docking – see Section 2.3).
In the m-phenylidene-linked subseries, the inhinitory potency across all four isoforms appeared to
be depleted for the ‘lipophilic tail’ compounds (9d-i). However, in the ‘polar tail’ counterparts (10c-
f), the inhibitory effect toward CA II (and, to some extent, toward CA IX) is abruptly restored,
leading to the best CA II/I and CA II/XII selectivities (> 100,000-fold) in the entire series
investigated here.
The 2,5-thiophene and 2,4-thiophene-linked subseries displayed a very interesting SAR pattern,
illustrating, in our view, the intricacy of the hydrophobic/hydrophilic ‘tail’ contributions to the
observed compounds’ potency and selectivity. The compounds in question (9j-o and 10g-j)
generally appear biased toward CA II. It also appears, in case of the 2,5-thiophene-linked
compounds, that small-alkyl substitutions (except cyclobutyl) in the 1,3-oxazole ring produce
5

ACCEPTED MANUSCRIPT
weaker CA II inhibitors compared to the carboxamide substitutions in the same position. These
compounds also display a 1000-fold CA II/I selectivity. This pattern becomes completely scrambled
when it comes to the 2,4-thiophene-linked counterparts: the selectivity seems to be lost and the 2-
methyl-1,3-oxazole compound (9m) is nearly equipotent toward CA I and CA II. Additionally, the
CA I / CA II selectivity of latter subseries appears to be particularly sensitive to the variations in the
carboxamide portion. Indeed, 10i is >880-fold less potent than 10 j toward CA I.
Overall, the analysis of the biological activity data (with respect to potency and selectivity) allowed
us to nominate several front-runners compounds for further investigation as CA II inhibitor drug
leads (9a, 10a, 10e, 10h, 10i). The analysis of the SAR also resulted in several important
observations that we further attempted to rationalize via in silico docking experiments using
currently available crystal structures of several CA isoforms.
2
.3 Docking studies
When the inhibitory activity of compound 9a toward the panel of four CAs was compared with that
of compound 10a, a striking, nearly 10,000-fold difference in the inhibition of CA I was noted. The
two compounds differ only with regard to the substitution in the 1,3-oxazole ring and display
similar activity patterns on the other three enzymes. This observation led us to perform in silico
docking experiments in order to determine what a likely binding pose and the crucial interactions of
compound 9a in the CA II active site could be (vs. the other three isoforms) and what causes the
drastic change in the CA I potency when the methyl group is replaced with pyrrolidinocrbonyl
substituent (to give 10a).
Figure 4 shows the docking of compound 9a into the active sites of CA I, CA II, CA IX and CA
XII. In all four cases, the sulfonamide group acts as a ZBG and forms hydrogen bonds to the protein
backbone and the hydroxy group of T196 (CA II numbering), whereas the phenyl ring shows
lipophilic interactions with the conserved A/V119, L138, V140, L195. In the CA I, IX and XII
isoforms, which are weakly inhibited by 9a, the 1,3-oxazole ring itself is not involved in any
important interactions and the methyl group is directed toward the solvent-exposed region of the
binding site. Contrary to this picture, in the CA II, the methyl group is disposed into a lipophilic
cleft delimited by the non-conserved I89 and T128. It is this interaction that could be responsible
for the acitivity bias of 9a toward CA II.
When docked into CA II, compound 10a shows a very similar pose to that of compound 9a, with
the pyrrolidine moiety forming strong lipophilic interactions with I89 and F128. Contrary to this
situation, due to the mutation of I89 and F128 with F89 and L129, respectively, the pyrrolidine
6

ACCEPTED MANUSCRIPT
group is rotated about 90 ° and forms lipophilic interactions with L129 and A130 in CA I. This is
the likely cause for the observed 10 pM inhibition of the latter isoform. In CA IX, the pyrrolidine
moiety is directed toward the solvent-exposed area, while in CA XII it is in proximity to the
hydroxy group of S128, displaying no important interaction (Figure 5).
The next question we sought to address using in silico docking, was the drastic loss of potency
(specifically, vs. CA II) generally observed in the 1,3-phenylidene-linked compounds compared to
their 1,4-phenylidene-linked counterparts (cf. 9a and 9g). As shown in Figure 6A, 9g displays an
unusual coordination to the zinc ion with a loss of the hydrogen bond to the nitrogen backbone of
T196, due to the ‘bent’ shape of the 1,3-phenylidene linker and the presence of the methoxy group.
The difference in the crucial anchoring to the zinc ion between 9g and 9a (Figure 6B) is the likely
cause of the ~100,000-fold difference in the activity of these compounds toward CA II.
Further, we were interested in comparing the p-phenylidene linked compounds to their 2,5-
1
8
thiophene counterparts (the two aromatic groups are considered isosteres of each other). The
pronounced difference in CA inhibition profile becomes apparent if compounds 10a and 10g are
compared. 10a (a p-phenylidene-linked compound) is a potent inhibitor of both CA I and CA II.
However, 10g (the 2,5-thiophene-linked analog) is >4000 times less potent toward CA I while
continuing to provide picomolar inhibition of CA II. The docking of 10g to the active site of the two
enzymes (Figure 7) shows that the pyrrolidine portion in the compound still maintains the lipophilic
interactions with I89 and F128 (the same interactions were observed for 10a – see Figure 5).
Differently, in the CA I active site, the analogous lipophilic interactions with L129 and A130 are
lost, which is the likely cause for the observed drop in potency.
Finally, we sought to understand the reason for the utter sensitivity of the CA I inhibitory potency
observed in the 2,4-thiophene-linked subseries. Indeed, compounds 10i and 10j provide nearly
identical inhibition of CA II while their CA I potency differs >800-fold. The CA I docking pose of
compound 10i (Figure 8A) turned out to be very similar to that of compound 10g (Figure 7A), with
the pyrrolidine ring pointing toward the solvent-exposed region of the binding site, which correlates
with similar CA I potencies of the two compounds. When the pyrrolidine motif in 10i is replaced
with morpholine (to give 10j), it doesn’t alter the docking pose of the compound (10j); however, a
weak hydrogen bond between the morpholine oxygen and Y202 (Figure 8B), which is the likely
reason for the observed restoration of CA I potency.
3
. Conclusions
7

ACCEPTED MANUSCRIPT
In this article, we presented the design, synthesis and biological evaluation of novel diaryl
sulfonamides as inhibitors of human carbonic anhydrases. The twenty five compounds employed in
this study were designed to enable a variation in the topology of the sulfonamide zinc-binding
group (relative to the diaryl moiety). More importantly, the compounds incorporated a set of
lipophilic as well as polar side chains in order to act as probes for the ‘bipolar’ architecture of the
CA active site. Some of the 1,3-oxazole containing compounds described herein, displayed a
remarkable, picomolar inhibitory potency toward CA II and CA I isoforms and an informative SAR
pattern, which will be very useful in designing the next generation, optimized CA inhibitors around
this chemotype (and, potentially, other diaryl scaffolds). Several front-runner compounds (9a, 10a,
1
0e, 10h, 10i) were selected for further studies as drug leads, based on their potency (mostly,
against CA II, which is a clinically validated target) as well as selectivity. The results of these
studies as well as further SAR-guided optimization of this series will be reported in due course.
4
. Experimental protocols
All reactions were carried out in oven-dried glassware in atmosphere of nitrogen. Melting points
were measured with a BuchiВ-520 melting point apparatus and are uncorrected. Thin-layer
chromatography was carried out on Silufol UV-254 silica gel plates using an appropriate mixture of
1
ethyl acetate and hexane. Compounds were visualized with short-wavelength UV light. H NMR
1
3
and C NMR spectra were recorded on Bruker MSL-300 spectrometers in DMSO-d using TMS as
6
an internal standard. Elemental analyses were obtained at Research Institute for Chemical Crop
Protection (Moscow, Russia) using Carlo ErbaStrumentazione 1106 analyzer. Mass spectra were
recorded using Shimadzu LCMS-2020 system with electron impact (EI) ionization. All and reagents
and solvents were obtained from commercial sources and used without purification.
4
4
.1 Synthesis
.1.1. General Procedure 1 (GP1): preparation of 1,3-oxazoles 5a-f and 5j-o
An appropriate α-acylamino precursor (13.0 mmol) was added to 94% sulphuric acid. The reaction
mixture was heated at 80 °C under stirring for 1.5 h, cooled to room temperature and poured into
ice water (100 mL). The solution was neutralized with 28% aqueous ammonia and extracted with
ethyl acetate (150 mL). The organic extract was dried over anhydrous Na SO , filtered, and
2
4
concentrated under reduced pressure to afford desired analytically pure 1,3-oxazoles 5.
.1.2. General Procedure 2 (GP2): preparation of 1,3-oxazoles 5g-i
4
8

ACCEPTED MANUSCRIPT
An appropriate α-acylamino precursor (10.0 mmol) was added to phosphorus (V) oxychloride (130
mol). The reaction mixture was heated at 80 °C under stirring for 1.5 h, cooled to room temperature
and poured over crushed ice. The precipitate formed was filtered off, washed with water, air-dried
and further crystallized from acetonitrile to give desired analytically pure 1,3-oxazoles 5.
4
.1.3. 2-Methyl-5-phenyl-1,3-oxazole (5a)
1
Yellow solid, m.p. 126-128 °С, yield 82 %; H NMR (400 MHz, DMSO-d ) δ ppm 8.21 (s, 1H,
6
Hoxazole), 7.80 (d, J = 7.2 Hz, 2H, 2,6-H ), 7.43 (t, J = 7.2 Hz, 2H, 3,5-H ), 7.40 (t, J = 7.2 Hz, 1H,
Ar
Ar
+
4
1
-H ), 2.47 (s, 3H, CH ); MS m/z (relative intensity) 159 (M , 100), 118 (15), 117 (68), 116 (38),
Ar 3
05 (47), 89 (46), 90 (50), 76 (36), 77(42), 78(22), 63 (34), 43 (29), 39 (42), 32 (12); anal. calcd for
C H NO (159.19): C, 75.45; H, 5.70; N, 8.80; found: C, 75.31; H, 5.60; N, 8.83.
10
9
4
.1.4. 2-Cyclopropyl-5-phenyl-1,3-oxazole (5b)
1
Yellow solid, m.p. 123-125 °С, yield 79 %; H NMR (400 MHz, DMSO-d ) δ ppm 7.64 (d, J = 7.2
6
Hz, 2H, 2,6-H ), 7.49 (s, 1H, H
), 7.43 (t, J = 7.2 Hz, 2H, 3,5-H ), 7.32 (t, J = 7.2 Hz, 1H, 4-
oxazole Ar
Ar
H ), 2.16 (m, 1H, CHcyclopropyl), 1.05 (m, 2H, Hcyclopropyl), 1.00 (m, 2H, Hcyclopropyl); MS m/z (relative
Ar
+
intensity) 185 (M , 100), 118 (15), 117 (42), 116 (23), 105 (62), 89 (60), 90 (73), 76 (12), 77(62),
7
8(23), 63 (14), 62 (23), 51 (45), 50 (42), 43 (26), 39 (42), 32 (32); anal. calcd for C H NO
12 11
(185.23): C, 77.81; H, 5.99; N, 7.56; found: C, 77.73; H, 6.08; N, 7.53.
4
.1.5. 2-Cyclobutyl-5-phenyl-1,3-oxazole (5c)
1
Yellow solid, m.p. 105-107 °С, yield 73 %; H NMR (400 MHz, DMSO-d ) δ ppm 7.66 (d, J = 7.2
6
Hz, 2H, 2,6-H ), 7.56 (s, 1H, H
), 7.44 (t, J = 7.2 Hz, 2H, 3,5-H ), 7.34 (t, J = 7.2 Hz, 1H, 4-
oxazole Ar
Ar
H ), 3.69 (m, 1H, CHcyclobutyl), 2.36 (m, 2H, Hcyclobutyl), 2.33 (m, 2H, Hcyclobutyl), 2.07 (m, 1H,
Ar
+
Hcyclobutyl), 1.92 (m, 1H, Hcyclobutyl); MS m/z (relative intensity) 199 (M , 100), 145 (12), 118 (75), 89
(35), 90 (75), 76 (47), 77(86), 78(24), 63 (35), 62 (43), 51 (47), 50 (62), 43 (33), 39 (39), 32 (21);
anal. calcd for C H NO (199.25): C, 78.36; H, 6.58; N, 7.03; found: C, 78.30; H, 6.53; N, 7.05.
1
3
13
4
.1.6. 2-Methyl-5-(4-methylphenyl)-1,3-oxazole (5d)
1
Yellow solid, m.p. 110-112 °С, yield 65 %; H NMR (400 MHz, DMSO-d ) δ ppm 7.90 (s, 1H,
6
Hoxazole), 7.69 (d, J = 8.2 Hz, 2H, H ), 7.29 (d, J = 8.2 Hz, 2H, H ), 2.34 (s, 3H, CH ), 1.33 (s, 3H,
Ar
Ar
3
+
CH ); MS m/z (relative intensity) 173 (M , 100), 131 (30), 130 (54), 119 (42), 132 (62), 116 (42),
3
1
15 (35), 104 (42), 103 (48), 77 (58), 77 (41), 67 (32), 65 (32), 43 (42), 39 (34), 32 (42); anal. calcd
for C H NO (173.22): C, 76.27; H, 6.40; N, 8.09; found: C, 76.19; H, 6.41; N, 8.11.
1
1
11
9

ACCEPTED MANUSCRIPT
4
.1.7. 2-Cyclopropyl-5-(4-methylphenyl)-1,3-oxazole (5e)
1
Yellow solid, m.p. 122-124 °С, yield 48 %; H NMR (400 MHz, DMSO-d ) δ ppm 7.52 (d, J = 8.2
6
Hz, 2H, H ), 7.41 (s, 1H, H
), 7.24 (d, J = 8.2 Hz, 2H, H ), 2.32 (s, 3H, CH ), 2.14 (m, 1H,
oxazole Ar 3
Ar
+
CHcyclopropyl), 1.05 (m, 2H, Hcyclopropyl), 1.00 (m, 1H, Hcyclopropyl); MS m/z (relative intensity) 199 (M ,
00), 131 (21), 130 (42), 119 (45), 117 (23), 116 (45), 115 (62), 104 (41), 103 (42), 77 (41), 77
32), 67 (12), 65 (42), 43 (45), 39 (62), 32 (62); anal. calcd for C H NO (199.25): C, 78.37; H,
1
(
1
3
13
6
.58; N, 7.03; found: C, 78.28; H, 6.58; N, 7.00.
4
.1.8. 2-Cyclobutyl-5-(4-methylphenyl)-1,3-oxazole (5f)
1
Yellow solid, m.p. 82-84 °С, yield 56 %; H NMR (400 MHz, DMSO-d ) δ ppm 7.56 (d, J = 8.2
6
Hz, 2H, H ), 7.48 (s, 1H, H
), 7.26 (d, J = 8.2 Hz, 2H, H ), 3.68 (m, 1H, CHcyclobutyl), 3.32 (s,
oxazole Ar
Ar
3
H, CH ), 2.35 (m, 2H, Hcyclobutyl), 2.33 (m, 2H, Hcyclobutyl), 2.05 (m, 1H, Hcyclobutyl), 1.93 (m, 1H,
3
+
Hcyclobutyl); MS m/z (relative intensity) 213 (M , 100), 159 (57), 131 (35), 130 (46), 119 (47), 117
86), 116 (36), 115 (75), 104 (37), 77 (24), 67 (13), 65 (53), 43 (59), 39 (64), 32 (50); anal. calcd for
C H NO (213.28): C, 78.84; H, 7.09; N, 6.57; found: C, 78.80; H, 7.20; N, 6.56.
(
14
15
4
.1.9. 5-(4-Methoxyphenyl)-2-methyl-1,3-oxazole (5g)
1
Yellow solid, m.p. 115-117 °С, yield 83 %; H NMR (400 MHz, DMSO-d ) δ ppm 7.97 (s, 1H,
6
Hoxazole), 7.57 (d, J = 8.9 Hz, 2H, H ), 7.03 (d, J = 8.9 Hz, 2H, H ), 3.79 (s, 3H, OCH ), 2.45 (s,
Ar
Ar
3
+
3
H, CH ); MS m/z (relative intensity) 189 (M , 100), 160 (53), 146 (24), 135 (86), 133 (24), 132
3
(
67), 77 (24), 76 (54), 39 (54), 32 (24); anal. calcd for C H NO (189.22): C, 69.82; H, 5.86; N,
11 11 2
7
.40; found: C, 69.88; H, 5.91; N, 7.41.
4
.1.10. 2-Cyclopropyl-5-(4-methoxyphenyl)-1,3-oxazole (5h)
1
Yellow solid, m.p. 102-104 °С, yield 75 %; H NMR (400 MHz, DMSO-d ) δ ppm 7.55 (d, J = 8.9
6
Hz, 2H, H ), 7.41 (s, 1H, H
), 7.01 (d, J = 8.9 Hz, 2H, H ), 3.70 (s, 3H, OCH ), 2.13 (m, 1H,
oxazole Ar 3
Ar
+
CHcyclopropyl), 1.05 (m, 2H, Hcyclopropyl), 1.00 (m, 2H, Hcyclopropyl); MS m/z (relative intensity) 215 (M ,
1
00), 160 (35), 146 (42), 135 (16), 133 (76), 132 (83), 104 (28), 103 (29), 91 (31), 77 (21), 76 (23),
9 (27), 32 (41); anal. calcd for C H NO (215.25): C, 72.54; H, 6.09; N, 6.51; found: C, 72.53;
3
1
3
13
2
H, 6.02; N, 6.47.
4
.1.11. 2-Cyclobutyl-5-(4-methoxyphenyl)-1,3-oxazole (5i)
1
Yellow solid, m.p. 113-115 °С, yield 72 %; H NMR (400 MHz, DMSO-d ) δ ppm 7.58 (d, J = 8.9
6
Hz, 2H, H ), 7.41 (s, 1H, H
), 7.11 (d, J = 8.9 Hz, 2H, H ), 3.60 (s, 3H, OCH ), 3.65 (m, 1H,
oxazole Ar 3
Ar
1
0

ACCEPTED MANUSCRIPT
CHcyclobutyl), 2.34 (m, 2H, Hcyclobutyl), 2.32 (m, 2H, Hcyclobutyl), 2.07 (m, 1H, Hcyclobutyl), 1.92 (m, 1H,
+
Hcyclobutyl); MS m/z (relative intensity) 229 (M , 100), 175 (41), 160 (24), 146 (41), 135 (15), 133
(52), 132 (25), 104 (31), 103 (25), 91 (42), 77 (24), 76 (63), 65 (57), 43 (12), 39 (54), 32 (12); anal.
calcd for C H NO (229.28): C, 73.34; H, 6.59; N, 6.11; found: C, 73.30; H, 6.55; N, 6.01.
1
4
15
2
4
.1.12. 2-Methyl-5-(thiophen-2-yl)-1,3-oxazole (5j)
1
Brown solid, m.p. 98-100 °С, yield 64 %; H NMR (400 MHz, DMSO-d ) δ ppm 7.61 (dd, J =4.9
6
3-4
Hz, J =1.3 Hz, 1H, 3-H
), 7.39 (dd, J =3.6 Hz, J =1.3 Hz, 1H, 5-Hthiophene), 7.33 (s, 1H,
thiophene 4-5 3-5
3
-5
Hoxazole), 7.14 (dd, J =4.9 Hz, J =3.6 Hz, 1H, 4-H
), 2.45 (s, 3H, CH ); MS m/z (relative
thiophene 3
3-4
4-5
+
intensity) 165 (M , 100), 123 (32), 122 (42), 111 (56), 109 (21), 108 (6), 97 (15), 70 (16), 69 (42),
5
8 (17), 57 (18), 45 (42), 39 (62), 38 (32), 32 (42), 30 (32); anal. calcd for C H NOS (165.22): C,
8 7
5
8.16; H, 4.27; N, 8.48; found: C, 58.02; H, 4.23; N, 8.45.
4
.1.13. 2-Cyclopropyl-5-(thiophen-2-yl)-1,3-oxazole (5k)
1
Brown solid, m.p. 99-102 °С, yield 73 %; H NMR (400 MHz, DMSO-d ) δ ppm 7.60 (dd, J =4.9
6
3-4
Hz, J =1.3 Hz, 1H, 3-H
), 7.36 (dd, J =3.6 Hz, J =1.3 Hz, 1H, 5-Hthiophene), 7.30 (s, 1H,
thiophene 4-5 3-5
3
-5
Hoxazole), 7.13 (dd, J =4.9 Hz, J =3.6 Hz, 1H, 4-Hthiophene), 2.14 (m, 1H, CHcyclopropyl), 1.05 (m, 2H,
3-4
4-5
+
Hcyclopropyl), 0.97 (m, 2H, Hcyclopropyl); MS m/z (relative intensity) 191 (M , 100), 151 (52), 123 (12),
09 (39), 108 (46), 97 (35), 96 (53), 70 (12), 69 (35), 45 (76), 39 (34), 38 (97), 32 (65), 30 (45);
anal. calcd for C H NOS (191.25): C, 62.80; H, 4.74; N, 7.32; found: C, 62.71; H, 4.80; N, 7.35.
1
1
0
9
4
.1.14. 2-Cyclobutyl-5-(thiophen-2-yl)-1,3-oxazole (5l)
1
Brown solid, m.p. 92-105 °С, yield 74 %; H NMR (400 MHz, DMSO-d ) δ ppm 7.61 (dd, J =4.9
6
3-4
Hz, J =1.3 Hz, 1H, 3-H
), 7.40 (dd, J =3.6 Hz, J =1.3 Hz, 1H, 5-Hthiophene), 7.37 (s, 1H,
thiophene 4-5 3-5
3
-5
Hoxazole), 7.14 (dd, J =4.9 Hz, J =3.6 Hz, 1H, 4-Hthiophene), 3.66 (m, 1H, CHcyclobutyl), 2.34 (m, 2H,
3-4
4-5
Hcyclobutyl), 2.32 (m, 2H, Hcyclobutyl), 2.05 (m, 1H, Hcyclobutyl), 1.90 (m, 1H, Hcyclobutyl); MS m/z
+
(
relative intensity) 205 (M , 100), 151 (36), 123 (56), 122 (76), 111 (69), 109 (40), 69 (35), 58 (97),
5
7 (59), 45 (36), 39 (28), 38 (42), 32 (47), 30 (53); anal. calcd for C H NOS (205.28): C, 64.37;
11 11
H, 5.40; N, 6.82; found: C, 64.32; H, 5.33; N, 6.85.
4
.1.15. 2-Methyl-5-(thiophen-3-yl)-1,3-oxazole (5m)
1
Brown solid, m.p. 100-102 °С, yield 68 %; H NMR (400 MHz, DMSO-d ) δ ppm 7.74 (d, J =2.4
6
2-4
Hz, 1H, 2-Hthiophene), 7.67 (dd, J =5.1 Hz, J =2.4 Hz, 1H, 4-H
), 7.41 (d, J =2.4 Hz, 1H, 5-
2-4
4-5
thiophene
4-5
+
Hthiophene), 7.32 (s, 1H, Hoxazole), 2.45 (s, 3H, CH ); MS m/z (relative intensity) 165 (M , 100), 123
3
(42), 122 (41), 111 (23), 109 (21), 108 (42), 97 (16), 83 (22), 84 (23), 70(41), 69 (23), 45 (41), 39
1
1

ACCEPTED MANUSCRIPT
(
69), 38 (15), 32 (41), 30 (15); anal. calcd for C H NOS (165.22): C, 58.16; H, 4.27; N, 8.48;
8 7
found: C, 58.09; H, 4.22; N, 8.47.
4
.1.16. 2-Cyclopropyl-5-(thiophen-3-yl)-1,3-oxazole (5n)
1
Brown solid, m.p. 78-80 °С, yield 67 %; H NMR (400 MHz, DMSO-d ) δ ppm 7.72 (dd, J =3.0
6
2-4
Hz, J =1.2 Hz, 1H, 2-H
), 7.62 (dd, J =5.0 Hz, J =3.0 Hz, 1H, 4-Hthiophene), 7.40 (dd, J4-
thiophene 2-4 4-5
2
-5
5=
5.0 Hz, J =1.2 Hz, 1H, 5-Hthiophene), 7.29 (s, 1H, Hoxazole), 2.13 (m, 1H, CHcyclopropyl), 1.04 (m,
2-5
+
2
H, Hcyclopropyl), 0.98 (m, 2H, Hcyclopropyl); MS m/z (relative intensity) 191 (M , 100), 151 (35), 123
(35), 108 (83), 97 (13), 83 (45), 84 (64), 70 (34), 69 (75), 45 (35), 39 (65), 32 (44), 30 (35); anal.
calcd for C H NOS (191.25): C, 62.80; H, 4.74; N, 7.32; found: C, 62.65; H, 4.79; N, 7.26.
1
0
9
4
.1.17. 2-Cyclobutyl-5-(thiophen-3-yl)-1,3-oxazole (5o)
1
Brown solid, m.p. 92-105 °С, yield 81 %; H NMR (400 MHz, DMSO-d ) δ ppm 7.76 (dd, J =3.0
6
2-4
Hz, J =1.2 Hz, 1H, 2-H
), 7.67 (dd, J =5.0 Hz, J =3.0 Hz, 1H, 4-Hthiophene), 7.43 (dd, J4-
thiophene 2-4 4-5
2
-5
5=
5.0 Hz, J =1.2 Hz, 1H, 5-Hthiophene), 7.36 (s, 1H, Hoxazole), 4.61 (m, 1H, CHcyclobutyl), 2.34 (m, 4H,
2-5
+
Hcyclobutyl), 2.03 (m, 1H, Hcyclobutyl), 1.92 (m, 1H, Hcyclobutyl); MS m/z (relative intensity) 205 (M ,
1
00), 151 (53), 123 (35), 122 (53), 111 (24), 84 (35), 70(36), 69 (46), 45 (64), 39 (83), 38 (14), 32
(
46), 30 (68); anal. calcd for C H NOS (205.28): C, 64.36; H, 5.40; N, 6.82; found: C, 64.20; H,
11
11
5
.32; N, 6.79.
.1.18. General Procedure 3 (GP3): preparation of 1,3-oxazoles 6a-j
4
The cyclodehydration step was carried out as described in GP3. The 1,3-oxazoles 5p-t were
promptly added to pyrrolidine or morpholine (100 mmol). The reaction mixture was heated at 50 °C
under stirring for 2.5 h and cooled to room temperature. Water (150 mL) was added. The precipitate
formed was filtered off, washed with water, air-dried and further crystallized from isopropyl alcohol
to give desired analytically pure 1,3-oxazoles 6.
4
.1.19 (5-Phenyl-1,3-oxazol-2-yl)(pyrrolidin-1-yl)methanone (6a)
1
Brown solid, m.p. 127-130 °С, yield 85 % (amidation step); H NMR (400 MHz, DMSO-d ) δ ppm
6
7
.80 (s, 1H, Hoxazole), 7.79 (d, J = 7.2 Hz, 2H, 2,6-H ), 7.49 (t, J = 7.2 Hz, 2H, 3,5-H ), 7.41 (t, J
Ar Ar
=
7.2 Hz, 1H, 4-H ), 3.96 (t, J = 6.7 Hz, 2H, Hpyrrolidine), 3.55 (t, J = 6.7 Hz, 2H, Hpyrrolidine), 1.94(t,
Ar
+
J = 6.7 Hz, 4H, Hpyrrolidine); MS m/z (relative intensity) 242 (M , 28), 116 (31), 105 (14), 102 (30),
9
8 (17), 89 (19), 77 (28), 70 (100), 56 (33), 55 (40), 42 (33), 41 (38), 39 (25), 29 (11); anal. calcd
for C H N O (242.28): C, 69.40; H, 5.82; N, 11.56; found: C, 69.36; H, 5.76; N, 11.49.
1
4
14
2
2
1
2

ACCEPTED MANUSCRIPT
4
.1.20 Morpholin-4-yl(5-phenyl-1,3-oxazol-2-yl)methanone (6b)
1
Brown solid, m.p. 93-96 °С, yield 79 % (amidation step); H NMR (400 MHz, DMSO-d ) δ ppm
6
7
7
2
7
.81 (s, 1H, Hoxazole), 7.79 (d, J = 7.2 Hz, 2H, 2,6-H ), 7.50 (t, J = 7.2 Hz, 2H, 3,5-H ), 7.42 (t, J =
Ar A
.2 Hz, 1H, 4-H ), 4.12 (m, 2H, Hmorpholine), 3.69 (m, 6H, Hmorpholine); MS m/z (relative intensity)
Ar
+
58 ( M , 18), 172 (31), 171(25), 116 (57), 114 (12), 105 (19), 102 (30), 89 (23), 86 (100), 77 (35),
0 (30), 63 (12), 56 (57), 42 (42), 41 (12), 39 (13), 30 (16), 29 (38); anal. calcd for C H N O
3
1
4
14
2
(258.28): C, 65.11; H, 5.46; N, 10.85; found: C, 65.01; H, 5.46; N, 10.87.
4
.1.21. [5-(4-Methylphenyl)-1,3-oxazol-2-yl](pyrrolidin-1-yl)methanone (6c)
1
Yellow solid, m.p. 168-170 °С, yield 82 % (amidation step); H NMR (400 MHz, DMSO-d ) δ ppm
6
7
.83 (s, 1H, Hoxazole), 7.68 (d, J = 8.2 Hz, 2H, H ), 7.32 (d, J = 8.2 Hz, 2H, H ), 3.91 (m, 2H,
Ar Ar
Hpyrrolidine), 3.51 (m, 2H, Hpyrrolidine), 2.35 (s, 3H, CH ), 1.89 (m, 4H, Hpyrrolidine); MS m/z (relative
3
+
intensity) 256 (M , 10), 130 (20), 119 (8), 116 (15), 115 (12), 98 (11), 91 (15), 77 (10), 70 (100), 56
(
24), 55 (27), 42 (40), 41 (42), 39 (22), 29 (7); anal. calcd for C H N O (256.31): C, 70.29; H,
15 16 2 2
6
.29; N, 10.93; found: C, 70.20; H, 6.35; N, 10.94.
4
.1.22. [5-(4-Methylphenyl)-1,3-oxazol-2-yl](morpholin-4-yl)methanone (6d)
1
Yellow solid, m.p. 157-159 °С, yield 83 % (amidation step); H NMR (400 MHz, DMSO-d ) δ ppm
6
7
.83 (s, 1H, Hoxazole), 7.68 (d, J = 7.9 Hz, 2H, H ), 7.33 (d, J = 8.2 Hz, 2H, H ), 4.07 (m, 2H,
Ar Ar
+
Hmorpholine), 3.67 (m, 6H, Hmorpholine), 2.35 (s, 3H, CH ); MS m/z (relative intensity) 272 ( M , 10),
3
1
86 (34), 185(34), 130 (60), 119 (17), 116 (33), 103 (19), 89 (9), 86 (100), 77 (17), 70(32), 63 (9),
6 (49), 42 (40), 41 (12), 39 (13), 30 (13), 29 (28); anal. calcd for C H N O (272.31): C, 66.16;
5
1
5
16
2
2
H, 5.92; N, 10.29; found: C, 66.08; H, 5.97; N, 10.28.
4
.1.23. [5-(4-Methoxyphenyl)-1,3-oxazol-2-yl](pyrrolidin-1-yl)methanone (6e)
1
Yellow solid, m.p. 110-112 °С, yield 79 % (amidation step); H NMR (400 MHz, DMSO-d ) δ ppm
6
7
.72 (d, J = 8.5 Hz, 2H, H ), 7.64 (s, 1H, H
), 7.03 (d, J = 8.5 Hz, 2H, H ), 3.95 (m, 2H,
oxazole Ar
Ar
Hpyrrolidine), 3.83 (s, 3H, OCH ), 3.54 (m, 2H, Hpyrrolidine), 1.94 (m, 4H, Hpyrrolidine); MS m/z (relative
3
+
intensity) 272 (M , 21), 146 (15), 132 (15), 98 (13), 77 (6), 70 (100), 56 (19), 55 (25), 42 (23), 41
(
22), 39 (10), 29 (7); anal. calcd for C H N O (272.31): C, 66.16; H, 5.92; N, 10.29; found: C,
15 16 2 3
6
5.99; H, 5.92; N, 10.32.
.1.24. [5-(4-Methoxyphenyl)-1,3-oxazol-2-yl](morpholin-4-yl)methanone (6f)
4
1
3

ACCEPTED MANUSCRIPT
1
Yellow solid, m.p. 88-90 °С, yield 79 % (amidation step); H NMR (400 MHz, DMSO-d ) δ ppm
6
7
.72 (d, J = 8.5 Hz, 2H, H ), 7.67 (s, 1H, H
), 7.04 (d, J = 8.5 Hz, 2H, H ), 4.13 (m, 2H,
Ar
oxazole
Ar
+
Hmorpholine), 3.89 (s, 3H, OCH ), 3.68 (m, 6H, H
); MS m/z (relative intensity) 288 ( M , 28),
3
morpholine
2
5
5
02 (14), 201 (18), 145 (39), 135 (11), 132 (29), 114 (11), 89 (7), 86 (100), 77 (11), 70 (31), 63 (6),
6 (47), 42 (28), 41 (7), 32 (16), 30 (9), 29 (26); anal. calcd for C H N O (288.31): C, 62.49; H,
1
5
16
2
4
.59; N, 9.72; found: C, 62.43; H, 5.63; N, 9.75.
4
.1.25. Pyrrolidin-1-yl[5-(thiophen-2-yl)-1,3-oxazol-2-yl]methanone (6g)
1
Brown solid, m.p. 121-123 °С, yield 80 % (amidation step); H NMR (400 MHz, DMSO-d ) δ ppm
6
7
.74 (dd, J =4.9 Hz, J =1.0 Hz, 1H, 3-Hthiophene), 7.72 (s, 1H, Hoxazole), 7.58 (dd, J =3.6 Hz, J
3-4 3-5 4-5 3-
5=
1.0 Hz, 1H, 5-Hthiophene), 7.21 (dd, J =4.9 Hz, J =3.6 Hz, 1H, 4-Hthiophene), 3.90 (m, 2H,
3-4 4-5
+
Hpyrrolidine), 3.50 (m, 2H, Hpyrrolidine), 1.88 (m, 4H, Hpyrrolidine); MS m/z (relative intensity) 248 (M ,
1), 122 (30), 111 (15), 108 (15), 98 (19), 70 (100), 56 (38), 55 (39), 45 (18), 42 (52), 41 (58), 39
42), 29 (12); anal. calcd for C H N O S (248.31): C, 58.05; H, 4.87; N, 11.28; found: C, 57.97;
2
(
1
2
12
2
2
H, 4.92; N, 11.30.
4
.1.26. Morpholin-4-yl[5-(thiophen-2-yl)-1,3-oxazol-2-yl]methanone (6h)
1
Brown solid, m.p. 60-70 °С, yield 73 % (amidation step); H NMR (400 MHz, DMSO-d ) δ ppm
6
7
.74 (dd, J =4.9 Hz, J =1.0 Hz, 1H, 3-Hthiophene), 7.72 (s, 1H, Hoxazole), 7.59 (dd, J =3.6 Hz, J
3-4 3-5 4-5 3-
5=
1.0 Hz, 1H, 5-Hthiophene), 7.21 (dd, J =4.9 Hz, J =3.6 Hz, 1H, 4-Hthiophene), 4.06 (m, 2H,
3-4 4-5
+
Hmorpholine), 3.66 (m, 6H, Hmorpholine); MS m/z (relative intensity) 264 (M , 19), 178 (21), 177 (21),
1
22 (69), 114 (18), 111 (22), 108 (26), 95 (13), 86 (100), 70 (51), 69 (23), 58(15), 57(12), 56 (82),
5 (34), 42 (62), 41 (20), 39 (25), 30 (23), 29 (55); anal. calcd for C H N O S (264.31): C, 54.53;
4
1
2
12
2
3
H, 4.58; N, 10.60; found: C, 54.41; H, 4.65; N, 10.53.
4
.1.27. Pyrrolidin-1-yl[5-(thiophen-3-yl)-1,3-oxazol-2-yl]methanone (6i)
1
Brown solid, m.p. 134-136 °С, yield 78 % (amidation step); H NMR (400 MHz, DMSO-d ) δ ppm
6
7
.97 (s, 1H, Hthiophene), 7.73 (s, 1H, Hthiophene), 7.71 (s, 1H, Hoxazole), 7.52 (s, 1H, Hthiophene), 3.90 (m,
2
H, Hpyrrolidine), 3.51 (m, 2H, Hpyrrolidine), 1.88 (m, 4H, Hpyrrolidine); MS m/z (relative intensity) 248 (
+
M , 19), 122 (25), 111 (16), 108 (17), 98 (16), 70 (100), 56 (29), 55 (31), 45 (20), 42 (36), 41 (40),
3
9 (30), 29 (9); anal. calcd for C H N O S (248.31): C, 58.05; H, 4.87; N, 11.28; found: C, 58.00;
12 12 2 2
H, 4.92; N, 11.19.
4
.1.28. Morpholin-4-yl[5-(thiophen-3-yl)-1,3-oxazol-2-yl]methanone (6j)
1
4

ACCEPTED MANUSCRIPT
1
Brown solid, m.p. 103-105 °С, yield 80 % (amidation step); H NMR (400 MHz, DMSO-d ) δ ppm
6
7
.98 (s, 1H, Hthiophene), 7.74 (s, 1H, Hthiophene), 7.71 (s, 1H, Hoxazole), 7.52 (s, 1H, Hthiophene), 4.05 (m,
+
2
H, Hmorpholine), 3.66 (m, 6H, Hmorpholine); MS m/z (relative intensity) 264 (M , 26), 178 (32), 177
(29), 122 (57), 114 (15), 111 (23), 108 (33), 95 (8), 86 (100), 70 (53), 69 (9), 58 (11), 57(7), 56
(
44), 45 (37), 42 (56), 41 (11), 39 (24), 30 (13), 29 (27); anal. calcd for C H N O S (264.31): C,
12
12
2
3
5
4.53; H, 4.58; N, 10.60; found: C, 54.50; H, 4.53; N, 10.66.
.1.29 General Procedure 4 (GP4): preparation of sulfonamides 9a-o and 10a-j
4
The starting 1,3-oxazole 5 or 6 (6.0 mmol) was added portion-wise to a cooled mixture of
chlorosulfonic acid (60 mmol) and thionyl chloride (6.0 mmol). The resulting mixture was heated at
6
0 °С for 2 hours, cooled to room temperature and poured over crushed ice. The precipitate formed
was filtered off, washed with water and dissolved in chloroform (100 mL). The solution was
washed with 5% aqueous potassium carbonate, dried over anhydrous calcium chloride, filtered and
concentrated. The residue was flash-chromatographed on silica gel using an appropriate gradient of
ethyl acetate in hexanes as eluent. The intermediate sulfonyl chloride (7 or 8) was obtained as a
viscous, slowly crystallizing oil. A portion of it (1.25 mmol) was dissolved in 1,4-dioxane (2.5 mL)
and treated with 25% aqueous ammonia (0.56 mL, 7.5 mmol). The resulting mixture was heated at
7
5 °C for 2 hours. The volatiles were remove in vacuo and the residue was treated with water (10
mL). The resulting precipitate was filtered off and crystallized from isopropyl alcohol to give
sulfonamides 9 or 10. The yield of sulfonamides is reported for two consecutive steps
(sulfochlorination and sulfonamide synthesis).
4
.1.30. 4-(2-Methyl-1,3-oxazol-5-yl)benzenesulfonamide (9a)
1
White solid, m.p. 221-224 °С (i-PrOH), yield 72 %; H NMR (400 MHz, DMSO-d ) δ ppm 7.88
6
(
d, J=8.8 Hz, 2H, H ), 7.84 (d, J=8.8 Hz, 2H, H ), 7.70 (s, 1H, H
), 7.40 (s, 2H, NH ), 2.49 (s,
oxazole 2
Ar
Ar
1
3
3
1
H, CH ); C NMR (126 MHz, DMSO-d ) δ ppm 162.64, 149.96, 144.04, 131.42, 127.39, 125.54,
3 6
+
24.71, 14.60; MS m/z (relative intensity) 238 (M , 100), 174 (21), 158 (19), 130 (10), 120 (5), 103
(
7), 77 (7), 76 (7), 63 (7), 54 (9), 50 (5), 43 (5), 27 (5); anal. calcd for C H N O S (238.27): C,
10 10 2 3
5
0.41; H, 4.23; N, 11.76; found: C, 50.36; H, 4.23; N, 11.80.
4
.1.31. 4-(2-Cyclopropyl-1,3-oxazol-5-yl)benzenesulfonamide (9b)
1
White solid, m.p. 181-184 °С (i-PrOH), yield 83 %; H NMR (400 MHz, DMSO-d ) δ ppm 7.87 (d,
6
J=8.8 Hz, 2H, H ), 7.84 (d, J=8.8 Hz, 2H, H ), 7.66 (s, 1H, H
), 7.39 (s, 2H, NH ), 2.18 (m,
oxazole 2
Ar
Ar
1
3
1
H, CHcyclopropyl), 1.06 (m, 4H, Hcyclopropyl); C NMR (126 MHz, DMSO-d ) δ ppm 162.46, 150.04,
6
+
1
44.16, 131.48, 127.39, 125.50, 124.66, 21.10, 9.90, 9.84; MS m/z (relative intensity) 264 (M ,
1
5

ACCEPTED MANUSCRIPT
1
00), 263 (18), 238 (12), 200 (6), 184 (11), 129 (13), 128 (9), 89 (18), 80 (14), 77 (5), 76 (8), 75
(
(
5), 63 (6), 53 (11), 52 (5), 51 (5), 50 (5), 41 (7), 39 (8), 27 (7); anal. calcd for C H N O S
12
12
2
3
264.31): C, 54.53; H, 4.58; N, 10.60; found: C, 54.48; H, 4.58; N, 10.62.
4
.1.32. 4-(2-Cyclobutyl-1,3-oxazol-5-yl)benzenesulfonamide (9c)
1
White solid, m.p. 192-195 °С (i-PrOH), yield 64 %; H NMR (400 MHz, DMSO-d ) δ ppm 7.89 (d,
6
J=8.8 Hz, 2H, H ), 7.86 (d, J=8.8 Hz, 2H, H ), 7.73 (s, 1H, H
), 7.40 (s, 2H, NH ), 3.72 (quin,
2
Ar
Ar
oxazole
J=8.3 Hz, 1H, CHcyclobutyl), 2.18 (m, 4H, Hcyclobutyl), 2.06 (m, 1H, Hcyclobutyl), 1.94 (m, 1H, Hcyclobutyl);
1
3
C NMR (126 MHz, DMSO-d ) δ ppm 167.98, 149.76, 144.09, 131.48, 127.38, 125.48, 124.81,
6
+
3
8.30, 33.20, 27.69, 18.99; MS m/z (relative intensity) 278 (M , 30), 252 (6), 251 (16), 250 (100),
1
86 (9), 170 (13), 115 (8), 89 (15), 39 (8), 27 (5); anal. calcd for C H N O S (278.33): C, 56.10;
1
3
14
2
3
H, 5.07; N, 10.06; found: C, 56.02; H, 5.08; N, 10.09.
4
.1.33. 2-Methyl-5-(2-methyl-1,3-oxazol-5-yl)benzenesulfonamide (9d)
1
White solid, m.p. 195-198 °С (i-PrOH), yield 71 %; H NMR (400 MHz, DMSO-d ) δ ppm 8.10 (s,
6
1
H, Х-H ), 7.77 (d, J=7.7 Hz, 1H, B-H ), 7.56 (s, 1H, H
), 7.47 (s, 2H, NH ), 7.46 (d, J=7.7
oxazole 2
Ar
Ar
1
3
Hz, 1H, A-H ), 2.60 (s, 3H, CH ); C NMR (126 MHz, DMSO-d ) δ ppm 161.96, 150.07, 143.74,
Ar
3
6
+
1
36.56, 133.95, 127.56, 126.52, 123.91, 122.86, 20.54, 14.54; MS m/z (relative intensity) 252 (M ,
6), 172 (27), 171 (100), 143 (10), 116 (23), 115 (21), 103 (17), 102 (25), 89 (16), 77 (21), 63 (11),
4 (14), 51 (10), 43 (11), 27 (9); anal. calcd for C H N O S (252.29): C, 52.37; H, 4.79; N, 11.10;
7
5
1
1
12
2
3
found: C, 52.31; H, 4.79; N, 11.12.
4
.1.34. 5-(2-Cyclopropyl-1,3-oxazol-5-yl)-2-methylbenzenesulfonamide (9e)
1
White solid, m.p. 175-177 °С (i-PrOH), yield 80 %; H NMR (400 MHz, DMSO-d ) δ ppm 8.07 (d,
6
J =1.1 Hz, 1H, X-H ), 7.75 (dd, J =8.1 Hz, J =1.1 Hz, 1H, B-H ), 7.53 (s, 1H, Hoxazole), 7.47
BX
Ar
AB
BX
Ar
(s, 2H, NH ), 7.45 (d, J =8.1 Hz, 1H, A-H ), 2.60 (s, 3 H, CH ), 2.18 (m, 1H, CHcyclopropyl), 1.08
2 AB Ar 3
1
3
(
m, 2H, Hcyclopropyl), 1.01 (m, 2H, Hcyclopropyl); C NMR (DMSO-d ) δ ppm 166.32, 149.40, 143.74,
6
1
36.39, 133.92, 127.45, 126.52, 123.93, 122.74, 67.25, 20.55, 9.40, 9.04; MS m/z (relative
+
intensity) 278 (M , 100), 277 (10), 198 (21), 197 (91), 168 (13), 142 (13), 141 (24), 128 (15), 115
(11), 103 (16), 102 (19), 90 (12), 89 (26), 80 (24), 77 (26), 63 (15), 53 (26), 52 (13), 51 (14), 41
(
20), 39 (21), 27 (19); anal. calcd for C H N O S (278.33): C, 56.10; H, 5.07; N, 10.06; found: C,
1
3
14
2
3
5
6.00; H, 5.07; N, 10.10.
.1.35. 5-(2-Cyclobutyl-1,3-oxazol-5-yl)-2-methylbenzenesulfonamide (9f)
4
1
6

ACCEPTED MANUSCRIPT
1
White solid, m.p. 170-173 °С (i-PrOH), yield 75 %; H NMR (500 MHz, DMSO-d ) δ ppm 8.12 (d,
6
JBX =1.2 Hz, 1H, X-H ), 7.82 (dd, J =7.9, J =1.2 Hz, 1H, B-H ), 7.63 (s, 1H, Hoxazole), 7.51 (m,
Ar
AB
BX
Ar
2
4
H, NH ), 7.48 (d, J =7.9 Hz, 1H, A-H ), 3.73 (m, 1H, CH
), 2.62 (s, 3H, CH ), 2.38 (m,
2
AB
Ar
cyclobutyl 3
1
3
H, Hcyclobutyl), 2.07 (m, 1H, Hcyclobutyl), 1.95 (m, 1H, Hcyclobutyl); C NMR (126 MHz, DMSO-d ) δ
6
ppm 165.91, 149.75, 143.55, 136.59, 133.48, 127.62, 126.31, 123.94, 122.52, 33.00, 26.98, 20.55,
+
1
1
9.01; MS m/z (relative intensity) 292 (M , 43), 291 (9), 266 (6), 265 (15), 264 (100), 184 (16),
83 (79), 154 (6), 128 (13), 103 (5), 102 (7), 89 (6), 77 (8), 39 (7); anal. calcd for C H N O S
1
4
16
2
3
(292.36): C, 57.52; H, 5.52; N, 9.58; found: C, 57.48; H, 5.52; N, 9.60.
4
.1.36. 2-Methoxy-5-(2-methyl-1,3-oxazol-5-yl)benzenesulfonamide (9g)
1
White solid, m.p. 215-217 °С (i-PrOH), yield 75 %; H NMR (400 MHz, DMSO-d ) δ ppm 7.90 (d,
6
J =1.9 Hz, 1H, X-H ), 7.83 (dd, J =8.6, Hz, J =1.9 Hz, 1H, B-H ), 7.41 (s, 1H, Hoxazole), 7.31
BX
Ar
AB
BX
Ar
1
3
(
d, J =8.6 Hz, 1H, A-H ), 7.17 (s, 2H, NH ), 3.94 (s, 3H, OCH ), 2.50 (c, 3H, CH ); C NMR
AB Ar 2 3 3
(
126 MHz, DMSO-d ) δ ppm 165.74, 156.54, 149.38, 132.76, 129.79, 124.14, 122.00, 120.77,
6
+
1
14.45, 57.38, 12.46; MS m/z (relative intensity) 268 (M , 100), 190 (5), 189 (12), 162 (6), 161 (6),
1
31 (7), 119 (5), 118 (6), 104 (7), 103 (5), 91 (5), 89 (17), 77 (6), 76 (11), 75 (6), 63 (8), 54 (8), 43
(
10), 36 (7), 27 (5), 15 (7); anal. calcd for C H N O S (268.29): C, 49.25; H, 4.51; N, 10.44;
11 12 2 4
found: C, 49.15; H, 4.51; N, 10.46.
4
.1.37. 5-(2-Cyclopropyl-1,3-oxazol-5-yl)-2-methoxybenzenesulfonamide (9h)
1
White solid, m.p. 211-215 °С (i-PrOH), yield 68 %; H NMR (400 MHz, DMSO-d ) δ ppm 7.94 (d,
6
J =1.8 Hz, 1H, X-H ), 7.84 (dd, J =8.6, Hz, J =1.8 Hz, 1H, B-H ), 7.45 (s, 1H, Hoxazole), 7.29
BX
Ar
AB
BX
Ar
(d, J =8.6 Hz, 1H, A-H ), 7.18 (s, 2H, NH ), 3.94 (s, 3H, OCH ), 2.17 (m, 1H, CHcyclopropyl), 1.06
AB Ar 2 3
1
3
(
m, 2H, Hcyclopropyl), 0.99 (m, 2H, Hcyclopropyl); C NMR (126 MHz, DMSO-d ) δ ppm 165.82,
6
1
56.51, 149.37, 132.72, 129.77, 123.65, 122.74, 120.71, 114.40, 67.23, 57.24, 9.37, 8.95; MS m/z
+
(
relative intensity) 294 (M , 100), 293 (7), 279 (5), 215 (6), 129 (5), 128 (8), 115 (7), 89 (7), 80 (7),
7
9
6 (7), 53 (7), 41 (6), 39 (6), 27 (6); anal. calcd for C H N O S (294.33): C, 53.05; H, 4.79; N,
13 14 2 4
.52; found: C, 52.96; H, 4.80; N, 9.54.
4
.1.38. 5-(2-Cyclobutyl-1,3-oxazol-5-yl)-2-methoxybenzenesulfonamide (9i)
1
White solid, m.p. 175-177 °С (i-PrOH), yield 69 %; H NMR (400 MHz, DMSO-d ) δ ppm 7.97 (d,
6
J =2.0 Hz, 1H, X-H ), 7.88 (dd, J =8.7, J =2.0 Hz, 1H, B-H ), 7.52 (s, 1H, Hoxazole), 7.31 (d,
BX
Ar
AB
BX
Ar
J =8.7 Hz, 1H, A-H ), 7.18 (s, 2H, NH ), 3.94 (s, 3H, OCH ), 3.70 (m,1H, CHcyclobutyl), 2.35 (m,
AB
Ar
2
3
1
3
4
H, Hcyclobutyl), 2.04 (m, 1H, Hcyclobutyl), 1.93 (m, 1H, Hcyclobutyl); C NMR (126 MHz, DMSO-d ) δ
6
ppm 166.84, 156.63, 149.84, 132.70, 129.97, 123.80, 122.68, 120.72, 114.42, 57.24, 33.14, 27.69,
1
7

ACCEPTED MANUSCRIPT
+
1
8.99; MS m/z (relative intensity) 308 (M , 25), 282 (5), 281 (14), 280 (100), 225 (6), 201 (7), 115
(
5), 89 (11), 76 (7), 55 (6), 39 (9), 27 (7); anal. calcd for C H N O S (308.36): C, 54.53; H, 5.23;
1
4
16
2
4
N, 9.08; found: C, 54.49; H, 5.23; N, 9.08.
4
.1.39. 5-(2-Methyl-1,3-oxazol-5-yl)thiophene-2-sulfonamide (9j)
1
White solid, m.p. 154-157 °С (i-PrOH), yield 76 %; H NMR (400 MHz, DMSO-d ) δ ppm 7.78 (s,
6
2
2
1
H, NH ), 7.55 (s, 1H, Hoxazole), 7.54 (d, J=4.0 Hz, 1H, Hthiophene), 7.38 (d, J=4.0 Hz, 1H, Hthiophene),
2
1
3
.48 (s, 3H, CH ); C NMR (126 MHz, DMSO-d ) δ ppm 162.26, 145.31, 134.09, 131.76, 125.03,
3
6
+
24.57, 14.48; MS m/z (relative intensity) 244 (M , 100), 189 (25), 180 (12), 164 (7), 152 (16), 136
(
12), 111 (6), 110 (8), 109 (59), 95 (37), 93 (7), 83 (6), 82 (15), 69 (12), 64 (7), 54 (20), 53 (7), 52
5), 45 (16), 43 (25), 39 (6), 38 (5), 27 (11), 26 (6), 15 (5); anal. calcd for C H N O S (244.29): C,
(
8
8
2
3 2
3
9.33; H, 3.30; N, 11.47; found: C, 39.28; H, 3.30; N, 11.45.
4
.1.40. 5-(2-Cyclopropyl-1,3-oxazol-5-yl)thiophene-2-sulfonamide (9k)
1
White solid, m.p. 140-142 °С (i-PrOH), yield 69 %; H NMR (400 MHz, DMSO-d ) δ ppm 7.79 (s,
6
2
H, NH ), 7.55 (s, 1H, Hoxazole), 7.53 (s, 1H, Hthiophene), 7.45 (s, 1H, Hthiophene), 2.16 (m, 1H,
2
1
3
CHcyclopropyl), 1.08 (m, 2H, Hcyclopropyl), 1.02 (m, 2H, Hcyclopropyl); C NMR (126 MHz, DMSO-d ) δ
6
ppm 162.19, 145.34, 133.97, 131.24, 125.24, 124.20, 65.83, 10.49, 10.05; MS m/z (relative
+
intensity) 270 (M , 100), 269 (16), 268 (16), 244 (10), 190 (19), 162 (7), 135 (55), 134 (19), 123
(5), 98 (7), 95 (14), 91 (10), 82 (16), 80 (21), 69 (7), 54 (5), 53 (23), 52 (9), 51 (10), 45 (11), 41
(
16), 39 (17), 38 (10), 29 (9), 27 (23), 26 (7), 15 (7); anal. calcd for C H N O S (270.33): C,
1
0
10
2
3 2
4
4.43; H, 3.73; N, 10.36; found: C, 44.35; H, 3.73; N, 10.32.
4
.1.41. 5-(2-Cyclobutyl-1,3-oxazol-5-yl)thiophene-2-sulfonamide (9l)
1
White solid, m.p. 149-152 °С (i-PrOH), yield 78 %; H NMR (400 MHz, DMSO-d ) δ ppm 7.78 (s,
6
2
3
1
1
2
H, NH ), 7.59 (s, 1H, Hoxazole), 7.54 (d, J=4.0 Hz, 1H, Hthiophene), 7.40 (d, J=4.0 Hz, 1H, Hthiophene),
2
.69 (quin, J=8.33 Hz, 1H, CHcyclobutyl), 2.35 (m, 4H, Hcyclobutyl), 2.04 (m, 1H, Hcyclobutyl), 1.93 (m,
1
3
H, Hcyclobutyl); C NMR (126 MHz, DMSO-d ) δ ppm 167.59, 145.37, 145.11, 134.12, 131.72,
6
+
24.96, 124.69, 33.02, 27.66, 18.98; MS m/z (relative intensity) 284 (M , 33), 283 (5), 258 (12),
57 (14), 256 (100), 201 (12), 192 (5), 164 (7), 148 (7), 121 (18), 95 (15), 94 (5), 66 (6), 64 (5), 55
(
11), 45 (7), 39 (11), 29 (6), 27 (10); anal. calcd for C H N O S (284.36): C, 46.46; H, 4.25; N,
11 12 2 3 2
9
.85; found: C, 46.43; H, 4.25; N, 9.87.
.1.42. 4-(2-Methyl-1,3-oxazol-5-yl)thiophene-2-sulfonamide (9m)
4
1
8

ACCEPTED MANUSCRIPT
1
White solid, m.p. 182-185 °С (i-PrOH), yield 81 %; H NMR (300 MHz, DMSO-d ) δ ppm 7.98 (d,
6
J=1.3 Hz, 1H, Hthiophene), 7.84 (d, J=1.3 Hz, 1H, Hthiophene), 7.76 (s, 2H, NH ), 7.47 (s, 1H, Hoxazole),
2
1
3
2
1
1
.46 (s, 3H, CH ); C NMR (126 MHz, DMSO-d ) δ ppm 165.80, 148.18, 146.39, 129.21, 127.57,
3
6
+
25.27, 123.84, 9.01; MS m/z (relative intensity) 244 (M , 100), 216 (8), 190 (7), 189 (28), 175 (9),
73 (16), 164 (6), 136 (14), 125 (11), 123 (7), 110 (7), 109 (22), 108 (7), 97 (7), 95 (32), 93 (7), 83
(5), 82 (19), 81 (5), 69 (12), 64 (9), 54 (26), 53 (8), 52 (7), 51 (7), 50 (5), 45 (26), 43 (18), 39 (8),
3
8 (8), 27 (16), 26 (7), 15 (5); anal. calcd for C H N O S (244.29): C, 39.33; H, 3.30; N, 11.47;
8 8 2 3 2
found: C, 39.32; H, 3.30; N, 11.50.
4
.1.43. 4-(2-Cyclopropyl-1,3-oxazol-5-yl)thiophene-2-sulfonamide (9n)
1
White solid, m.p. 150-153 °С (i-PrOH), yield 75 %; H NMR (500 MHz, DMSO-d ) δ ppm 7.98 (d,
6
J=1.2 Hz, 1H, Hthiophene), 7.85 (d, J=1.2 Hz, 1H, Hthiophene), 7.79 (s, 2H, NH ), 7.46 (s, 1H, Hoxazole),
2
1
3
2
.17 (m, 1H, CHcyclopropyl), 1.08 (m, 2H, Hcyclopropyl), 1.01 (m, 2H, Hcyclopropyl); C NMR (126 MHz,
DMSO-d ) δ ppm 165.80, 148.18, 146.39, 129.21, 127.57, 125.27, 123.84, 67.23, 9.37, 9.01; MS
6
+
m/z (relative intensity) 270 (M , 100), 269 (16), 268 (13), 244 (11), 241 (5), 190 (14), 189 (5), 162
(
6), 136 (5), 135 (41), 134 (14), 133 (6), 123 (6), 110 (5), 98 (5), 95 (18), 91 (11), 83 (5), 82 (15),
1 (5), 80 (19), 69 (12), 64 (8), 54 (11), 53 (26), 52 (14), 51 (11), 50 (7), 45 (19), 43 (6), 41 (19), 39
25), 38 (8), 29 (11), 27 (26), 26 (6), 15 (6); anal. calcd for C H N O S (270.33): C, 44.43; H,
8
(
1
0
10
2
3 2
3
.73; N, 10.36; found: C, 44.40; H, 3.73; N, 10.35.
4
.1.44. 4-(2-Cyclobutyl-1,3-oxazol-5-yl)thiophene-2-sulfonamide (9o)
1
White solid, m.p. 169-173 °С (i-PrOH), yield 70 %; H NMR (400 MHz, DMSO-d ) δ ppm 8.00 (d,
6
J=1.5 Hz, 1H, Hthiophene), 7.85 (d, J=1.5 Hz, 1H, Hthiophene), 7.76 (s, 2H, NH ), 7.51 (s, 1H, Hoxazole),
2
3
.68 (m, 1H, CHcyclobutyl), 2.34 (m, 4H, Hcyclobutyl), 2.04 (m, 1H, Hcyclobutyl), 1.92 (m, 1H, Hcyclobutyl);
1
3
C NMR (126 MHz, DMSO-d ) δ ppm 166.85, 148.22, 146.85, 129.25, 127.67, 125.61, 123.77,
6
+
3
3.10, 27.66, 18.98; MS m/z (relative intensity) 284 (M , 31), 283 (7), 258 (12), 257 (14), 256
(100), 228 (6), 201 (18), 148 (7), 121 (21), 95 (13), 82 (8), 66 (8), 64 (6), 55 (9), 53 (5), 45 (11), 41
(
5), 39 (17), 29 (8), 27 (11); anal. calcd for C H N O S (284.36): C, 46.46; H, 4.25; N, 9.85;
1
1
12
2
3 2
found: C, 46.45; H, 4.26; N, 9.84.
4
.1.45. 4-[2-(Pyrrolidin-1-ylcarbonyl)-1,3-oxazol-5-yl]benzenesulfonamide (10a)
1
White solid, m.p. 265-267 °С (i-PrOH), yield 65 %; H NMR (400 MHz, DMSO-d ) δ ppm 8.06 (s,
6
1
H, Hoxazole), 7.99 (d, J=8.6 Hz, 2H, H ), 7.84 (d, J=8.6 Hz, 2H, H ), 7.46 (s, 2H, NH ), 3.93 (t,
Ar Ar 2
J=6.7 Hz, 2H, Hpyrrolidine), 3.53 (t, J=6.7 Hz, 2H, Hpyrrolidine), 1.93 (m, 2H, Hpyrrolidine), 1.87 (m, 2H,
1
3
Hpyrrolidine); C NMR (75 MHz, DMSO-d ) δ ppm 155.50, 154.24, 150.77, 144.79, 130.06, 127.10,
6
1
9

ACCEPTED MANUSCRIPT
+
1
25.77, 125.43, 49.03, 47.38, 26.39, 23.83; MS m/z (relative intensity) 321 (M , 22), 98 (11), 70
(
100), 56 (14), 55 (20), 42 (5), 41 (5); anal. calcd for C H N O S (321.36): C, 52.33; H, 4.70; N,
14
15
3
4
1
3.08; found: C, 52.32; H, 4.71; N, 13.10.
4
.1.46. 4-[2-(Morpholin-4-ylcarbonyl)-1,3-oxazol-5-yl]benzenesulfonamide (10b)
1
White solid, m.p. 190-193 °С (i-PrOH), yield 70 %; H NMR (400 MHz, DMSO-d ) δ ppm 8.05 (s,
6
1
H, Hoxazole), 7.98 (d, J=8.8 Hz, 2H, H ), 7.94 (d, J=8.8 Hz, 2H, H ),7.45 (br. s., 2H, NH ), 4.05
Ar Ar 2
1
3
(
m, 2H, Hmorpholin), 3.67 (m, 6H, Hmorpholine); C NMR (126 MHz, DMSO-d ) δ ppm 155.58, 154.83,
6
1
51.26, 145.23, 130.35, 127.51, 125.86, 125.76, 67.19, 66.84, 47.93, 43.53; MS m/z (relative
+
intensity) 337 (M , 5), 251 (16), 250 (21), 181 (8), 131 (6), 115 (6), 114 (12), 87 (6), 86 (100), 72
(
5), 70 (24), 56 (18), 42 (16), 29 (5); anal. calcd for C H N O S (337.36): C, 49.85; H, 4.48; N,
14 15 3 5
1
2.46; found: C, 49.80; H, 4.49; N, 12.40.
4
.1.47. 2-Methyl-5-[2-(pyrrolidin-1-ylcarbonyl)-1,3-oxazol-5-yl]benzenesulfonamide (10c)
1
White solid, m.p. 247-250 °С (i-PrOH), yield 63 %; H NMR (500 MHz, DMSO-d ) δ ppm 8.24 (d,
6
J =1.2 Hz, 1H, X-H ), 7.98 (s, 1H, H
), 7.94 (dd, J =7.9, Hz, J =1.2 Hz, 1H, B-H ), 7.56
oxazole AB BX Ar
BX
Ar
(
s, 2H, NH ), 7.55 (d, J =8.6 Hz, 1H, A-H ), 3.94 (t, J=6.7 Hz, 2H, Hpyrrolidin), 3.55 (t, J=6.7 Hz,
2 AB Ar
13
2
H, Hpyrrolidin), 2.66 (s, 3H, OCH ), 1.95 (m, 2H, Hpyrrolidine), 1.89 (m, 2H, Hpyrrolidine); C NMR (126
3
MHz, DMSO-d ) δ ppm 155.73, 153.99, 151.37, 143.69, 137.81, 134.38, 128.58, 125.47, 124.16,
6
+
1
23.79, 49.12, 47.07, 26.56, 24.23, 20.69; MS m/z (relative intensity) 335 (M , 23), 98 (13), 70
(
100), 56 (12), 55 (20), 44 (5); anal. calcd for C H N O S (335.38): C, 53.72; H, 5.11; N, 12.53;
15 17 3 4
found: C, 53.58; H, 5.11; N, 12.55.
4
.1.48. 2-Methyl-5-[2-(morpholin-4-ylcarbonyl)-1,3-oxazol-5-yl]benzenesulfonamide (10d)
1
White solid, m.p. 239-242 °С (i-PrOH), yield 61 %; H NMR (400 MHz, DMSO-d ) δ ppm 8.21 (d,
6
J =1.7 Hz, 1H, X-H ), 7.92 (s, 1H, H
), 7.91 (dd, J =7.9, Hz, J =1.2 Hz, 1H, B-H ), 7.54
oxazole AB BX Ar
BX
Ar
(
d, J =8.6 Hz, 1H, A-H ), 7.53 (s, 2H, NH ), 4.07 (m, 2H, Hmorpholine), 3.67 (m, 6H, Hmorpholine),
AB Ar 2
1
3
2
1
.63 (s, 3H, CH ); C NMR (126 MHz, DMSO-d ) δ ppm 155.58, 154.36, 151.44, 143.93, 138.07,
3 6
34.17, 128.58, 125.47, 124.38, 123.79, 67.19, 66.85, 47.93, 43.53, 20.66; MS m/z (relative
+
intensity) 351 (M , 19), 266 (6), 265 (24), 264 (35), 252 (6), 130 (8), 128 (6), 114 (20), 87 (5), 86
(
100), 70 (23), 56 (13), 42 (12); anal. calcd for C H N O S (351.38): C, 51.27; H, 4.88; N, 11.96;
15 17 3 5
found: C, 51.25; H, 4.88; N, 11.97.
4
.1.49. 2-Methoxy-5-[2-(pyrrolidin-1-ylcarbonyl)-1,3-oxazol-5-yl]benzenesulfonamide (10e)
2
0

ACCEPTED MANUSCRIPT
1
White solid, m.p. 275-278 °С (i-PrOH), yield 69 %; H NMR (400 MHz, DMSO-d ) δ ppm 8.09 (s,
6
1
H, X-H ), 8.00 (d, J=8.4 Hz, 1H, B-H ), 7.89 (s, 1H, Hoxazole), 7.38 (d, J=8.4 Hz, 1H, A-HAr),
Ar Ar
7
.24 (br. s., 2H, NH ), 3.97 (s, 3H, OCH ), 3.92 (t, J=6.4 Hz, 2H, Hpyrrolidine), 3.52 (t, J=6.4 Hz, 2H,
2
3
1
3
Hpyrrolidine), 1.89 (m, 4H, Hpyrrolidine); C NMR (75 MHz, DMSO-d ) δ ppm 157.10, 154.68, 154.29,
6
1
51.04, 132.55, 130.52, 124.34, 123.32, 119.19, 114.22, 56.95, 49.03, 47.34, 26.42, 23.82; MS m/z
+
(
relative intensity) 351 (M , 10), 211 (6), 98 (15), 70 (100), 56 (13), 55 (25), 29 (5); anal. calcd for
C H N O S (351.38): C, 51.27; H, 4.88; N, 11.96; found: C, 51.21; H, 4.88; N, 11.95.
15
17
3
5
4
.1.50. 2-Methoxy-5-[2-(morpholin-4-ylcarbonyl)-1,3-oxazol-5-yl]benzenesulfonamide (10f)
1
White solid, m.p. 198-201 °С (i-PrOH), yield 72 %; H NMR (400 MHz, DMSO-d ) δ ppm 8.09 (d,
6
J =2.2 Hz, 1H, X-H ), 8.00 (dd, J =8.7, J =2.2 Hz, 1H, B-H ), 7.89 (s, 1H, Hoxazole), 7.38 (d,
BX
Ar
AB
BX
Ar
J =8.77 Hz, 1H, A-H ), 7.23 (br. s., 2H, NH ), 4.08 (m, 2H, H
), 3.97 (s, 3H, OCH ), 3.67
morpholine 3
AB
Ar
2
1
3
(
m, 6H, Hmorpholine); C NMR (126 MHz, DMSO-d ) δ ppm 157.53, 155.56, 153.96, 151.52, 132.90,
6
1
30.95, 124.75, 123.30, 119.46, 114.62, 67.19, 66.87, 57.36, 47.91, 43.52; MS m/z (relative
+
intensity) 367 (M , 31), 282 (7), 281 (26), 280 (49), 268 (8), 254 (7), 225 (14), 211 (16), 114 (16),
8
7 (5), 86 (100), 70 (28), 56 (11), 44 (5), 42 (11); anal. calcd for C H N O S (367.38): C, 49.04;
15 17 3 6
H, 4.66; N, 11.44; found: C, 49.00; H, 4.67; N, 11.45.
4
.1.51. 5-[2-(Pyrrolidin-1-ylcarbonyl)-1,3-oxazol-5-yl]thiophene-2-sulfonamide (10g)
1
White solid, m.p. 226-229 °С (i-PrOH), yield 70 %; H NMR (400 MHz, DMSO-d ) δ ppm 7.92 (s,
6
1
3
H, Hoxazole), 7.86 (s, 2 H, NH ), 7.60 (d, J=3.8 Hz, 1H, Hthiophene), 7.58 (d, J=3.8 Hz, 1H, Hthiophene),
2
13
.90 (t, J=6.7 Hz, 2H, Hpyrrolidine), 3.51 (t, J=6.7 Hz, 2H, Hpyrrolidine), 1.90 (m, 4H, Hpyrrolidine); C
NMR (126 MHz, DMSO-d ) δ ppm 155.27, 154.34, 147.03, 146.63, 132.82, 131.83, 126.72,
6
+
1
25.62, 49.45, 47.82, 26.81, 24.23; MS m/z (relative intensity) 327 (M , 12), 98 (24), 71 (7), 70
(
100), 56 (24), 55 (39), 42 (7), 41 (6); anal. calcd for C H N O S (327.38): C, 44.03; H, 4.00; N,
12 13 3 4 2
1
2.84; found: C, 43.98; H, 4.01; N, 12.85.
4
.1.52. 5-[2-(Morpholin-4-ylcarbonyl)-1,3-oxazol-5-yl]thiophene-2-sulfonamide (10h)
1
White solid, m.p. 188-191 °С (i-PrOH), yield 73 %; H NMR (400 MHz, DMSO-d ) δ ppm 7.91 (s,
6
1
4
1
H, Hoxazole), 7.86 (s, 2 H, NH ), 7.60 (d, J=3.8 Hz, 1H, Hthiophene), 7.58 (d, J=3.8 Hz, 1H, Hthiophene),
2
1
3
.03 (m, 2H, Hmorpholine), 3.66 (m, 6H, Hmorpholine); C NMR (126 MHz, DMSO-d ) δ ppm 155.27,
6
54.22, 147.09, 146.70, 132.67, 131.82, 126.81, 125.19, 67.14, 66.82, 47.93, 43.53; MS m/z
+
(
relative intensity) 343 (M , 12), 258 (6), 257 (17), 256 (30), 201 (7), 187 (11), 137 (6), 114 (22),
8
6 (100), 72 (6), 70 (40), 64 (8), 56 (21), 45 (6), 42 (25), 29 (6); anal. calcd for C H N O S
12 13 3 5
2
(343.38): C, 41.97; H, 3.82; N, 12.24; found: C, 41.95; H, 3.82; N, 12.26.
2
1

ACCEPTED MANUSCRIPT
4
.1.53. 4-[2-(Pyrrolidin-1-ylcarbonyl)-1,3-oxazol-5-yl]thiophene-2-sulfonamide (10i)
1
White solid, m.p. 268-271 °С (i-PrOH), yield 64 %; H NMR (400 MHz, DMSO-d ) δ ppm 8.20 (d,
6
J=1.2 Hz, 1H, Hthiophene), 7.96 (d, J=1.2 Hz, 1H, Hthiophene), 7.81 (s, 1H, Hoxazole), 7.86 (s, 2H, NH2),
13
3
.91 (t, J=6.7 Hz, 2H, Hpyrrolidine), 3.51 (t, J=6.7 Hz, 2H, Hpyrrolidine), 1.89 (m, 4H, Hpyrrolidine); C
NMR (126 MHz, DMSO-d ) δ ppm 154.94, 154.59, 148.63, 148.21, 127.94, 124.75, 49.47, 47.79),
6
+
2
6.82, 24.24; MS m/z (relative intensity) 327 (M , 1), 187 (9), 98 (14), 70 (100), 56 (17), 55 (25),
4
2 (6), 41 (5); anal. calcd for C H N O S (327.38): C, 44.03; H, 4.00; N, 12.84; found: C, 44.01;
12
13
3
4 2
H, 4.00; N, 12.82.
4
.1.54. 4-[2-(Morpholin-4-ylcarbonyl)-1,3-oxazol-5-yl]thiophene-2-sulfonamide (10j)
1
White solid, m.p. 226-229 °С (i-PrOH), yield 68 %; H NMR (400 MHz, DMSO-d ) δ ppm 8.20 (d,
6
J=1.2 Hz, 1H, Hthiophene), 7.96 (d, J=1.2 Hz, 1H, Hthiophene), 7.81 (s, 1H, Hoxazole), 7.86 (s, 2H, NH2),
1
3
4
1
.05 (m, 2H, Hmorpholine), 3.67 (m, 6H, Hmorpholine); C NMR (126 MHz, DMSO-d ) δ ppm 155.50,
6
53.85, 148.66, 148.27, 128.11, 127.94, 124.31, 67.19, 66.85, 47.91, 43.52; MS m/z (relative
+
intensity) 343 (M , 8), 258 (6), 257 (21), 256 (28), 201 (16), 187 (13), 114 (14), 86 (100), 70 (33),
5
6 (18), 45 (7), 42 (21), 29 (5); anal. calcd for C H N O S (343.38): C, 41.97; H, 3.82; N, 12.24;
12 13 3 5 2
found: C, 41.92; H, 3.82; N, 12.28.
4
.2 Carbonic anhydrase inhibition assay
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed
2
3
CO hydration activity. Phenol red (at a concentration of 0.2 mM) has been used as indicator,
2
working at the absorbance maximum of 557 nm, with 20 mM Tris (pH 8.3) as buffer, and 20 mM
Na SO (for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed
2
4
CO hydration reaction for a period of 10-100 s. The CO concentrations ranged from 1.7 to 17 mM
2
2
for the determination of the kinetic parameters and inhibition constants. For each inhibitor at least
six traces of the initial 5-10% of the reaction have been used for determining the initial velocity.
The uncatalyzed 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-deionized water and dilutions
up to 0.005 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were
preincubated together for 15 min at room temperature prior to assay, in order to allow for the
formation of the E-I complex. The inhibition constants were obtained by non-linear least-squares
methods using PRISM 3 and the Cheng-Prusoff equation, as reported earlier, and represent the
mean from at least three different determinations. All CA isoforms were recombinant ones obtained
2
4
in-house.
2
2

ACCEPTED MANUSCRIPT
4
.3 Docking studies
2
5
26
The crystal structures of hCA I (pdb code 1AZM), hCA II (pdb code 2AW1), hCA IX (pdb code
2
7
28
29
3
IAI), and hCA XII (pdb code 1JD0) were taken from the Protein Data Bank. After adding
hydrogen atoms and removing complexed ligands, the four proteins were minimized using Amber
3
0
1
1 software and parm03 force field at 300 K. The four proteins were placed in a rectangular
parallelepiped water box, an explicit solvent model for water, TIP3P, was used and the complexes
were solvated with a 10 Å water cap. Sodium ions were added as counter ions to neutralize the
system. Two steps of minimization were then carried out; in the first stage, we kept the protein
2
fixed with a position restraint of 500 kcal/mol•Å and we solely minimized the positions of the
water molecules. In the second stage, we minimized the entire system through 5000 steps of
steepest descent followed by conjugate gradient until a convergence of 0.05 kcal/Å•mol. The region
of interest used by the docking program GOLD version 5.1 was defined in order to contain the
residues within 15 Å from the original position of the ligand in the X-ray structure. Metal
coordination in GOLD is modeled as ‘pseudohydrogen bonding’ in which metals can be considered
to bind to H-bond acceptors and the metal will compete with H-bond donors for interaction. The
catalytic zinc ion was set to have a tetrahedral coordination geometry. The ‘allow early termination’
option was deactivated, while the possibility for the ligand to flip ring corners was activated. The
remaining GOLD default parameters were used, and the ligands were submitted to 30 genetic
algorithm runs. The docking analysis was carried out using the ChemScore fitness function
imposing the formation of an H bond between the ligands and the hydroxy group of the highly
3
1
conserved residue T192 (CA II sequence numbering). Cluster analysis was performed on the
results using an rmsd tolerance of 2.0 Å and the best docked conformation was taken into account.
Acknowledgements
This research was supported by the Russian Scientific Fund (project grant 14-50-00069).
Appendix A. Supplementary data.
Supplementary data related to this article can be found at http://dx.doi.org/....
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2
5

ACCEPTED MANUSCRIPT
Figure 1. Examples of the early pan-CA inhibitors – clinically used drugs.
HN
HN
O
O
O
N
N
N
N
O
O
O
S NH2
O
S NH2
O
S NH
O
2
S NH2
N
N
S
S
S
N
S
S
O O
S
O
H
O O
O
acetazolamide
methazolamide
dorzolamide
brinzolamide
Figure 2. Examples 1-4 of isoform-selective benzenesulfonamides.
NH2
O
S
O
NH2
O
NH2
O
S
O
NH2
O
S
O
S
O
N
Cl
N
H N
2
HN
MeO
MeO
N
O
N
O
N
NH
S
EtO
N
F
OEt
3, hCAIX K = 0.34 nM9
2, hCAIX K = 2.2 nM8
10
7
1
, hCAXII K = 1.9 nM
i
4, hCAVII K = 4.6 nM
i
i
i
Figure 3. Scaffold geometry and periphery projection for optimal inhibitor anchoring and probing
of the two distinct sides of the CA active site.
hydrophilic side of the active site
benzene or
thiophene linker
hydrophilic or
lipophilic group
O
several possibilities
for anchoring to Zn2
+
N
rotational
movement
hydrophobic side of the active site
2
6

ACCEPTED MANUSCRIPT
Scheme 1. Synthesis of 1,3-oxazol-5-yl aromatic substrates 5a-t and 6a-j for subsequent
sulfochlorination.
R''
O
R'
O
N
a
O
b
O
d
c
Br
H
R
O
NH2
N
R
O
R = COOEt
N
N
(5p-t)
O
6a-j
5a-t
S
S
=
*
*
MeO
*
*
*
Reagents and conditions: (a) i. urotropine, EtOH, 2h, ii. HCl, EtOH, 24 h; (b) RCOCl, py, 80 °C,
1
h; (c) H SO , 80 °C, 1.5 h (2- and 3-thienyl, 4-tolyl) or POCl , 80 °C, 2h (4-MeOC H ); (d)
2
4
3
6
4
R′NHR′′ (neat), 50 °C, 2h.
Scheme 2. Preparation of sulfonamides 9a-o and 10a-j studied in this work.
R
O
R
a
ClO S
O
b
R
2
H NO S
O
2
2
N
N
N
5
a-o
7
a-o
9a-o
R''
O
R'
O
R''
O
R'
R''
O
N
R'
N
N
a
ClO S
O
2
b
H NO S
O
2
2
N
N
N
6
a-j
8
a-j
1
0a-j
S
S
=
*
*
MeO
*
*
*
Reagents and conditions: (a) HSO Cl, SOCl , 60 °C, 2 h; (b) NH , 1,4-dioxane, 75 °C, 2 h.
3
2
3
2
7

ACCEPTED MANUSCRIPT
Figure 4. Docking of compound 9a into CA I (A), CA II (B), CA IX (C), and CA XII (D).
Figure 5. Docking of compound 10a into CA I (A), CA II (B), CA IX (C), and CA XII (D).
2
8

ACCEPTED MANUSCRIPT
Figure 6. Docking of compound 9g into CA II (A) and the overlay of 9g (sky blue) and 9a (green)
in the active site of CA II showing the difference in Zn ion coordination.
Figure 7. Docking of compound 10g into CA I (A) and CA II (B).
Figure 8. Docking of compounds 10i (A) and 10j (B) into CA I.
2
9