Spirocyclic sulfonamides with carbonic anhydrase inhibitory and anti-neuropathic pain activity

Article abstract of DOI:10.1016/j.bioorg.2019.103210

A novel series of 4-oxo-spirochromane bearing primary sulfonamide group were synthetized as Carbonic Anhydrase inhibitors (CAIs) and tested for their management of neuropathic pain. Indeed, CAs have been recently validated as novel therapeutic targets in neuropathic pain. All compounds, here reported, showed strong activity against hCA II and hCA VII with K_I values in the low or sub-nanomolar range. Two compounds (6d and 6l) showed good neuropathic pain attenuating effects and longer duration than drug reference acetazolamide in an animal model of oxaliplatin induced neuropathy.

Full text of DOI:10.1016/j.bioorg.2019.103210

BioorganicChemistry92(2019)103210
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Spirocyclic sulfonamides with carbonic anhydrase inhibitory and anti-
neuropathic pain activity
⁎
Y. Kalisha Valia, Rambabu Gundlaa, Om V. Singha, Yasinalli Tambolib,
,
Lorenzo Di Cesare Manellic, Carla Ghelardinic, Abdul-Malek S. Al-Tamimid, Fabrizio Cartae,
⁎
⁎
Andrea Angelie,f, , Claudiu T. Supurane,
a Department of Chemistry, School of Technology, GITAM University, Hyderabad 502102, Telangana, India
b School of Chemical Sciences, SRTM University, Nanded 431606, Maharashtra, India
c NEUROFARBA Department, Section of Pharmacology and Toxicology, Università degli Studi di Firenze, Viale Pieraccini 6, 50139 Florence, Italy
d Department of Pharmaceutical Chemistry, College of Pharmacy, Prince Sattam Bin Abdulaziz University, P.O. Box 173, Alkharj 11942, Saudi Arabia
e University of Florence, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy
f Centre of Advanced Research in Bionanoconjugates and Biopolymers Department, “Petru Poni” Institute of Macromolecular Chemistry, Iasi, Romania
A R T I C L E I N F O
A B S T R A C T
Keywords:
A novel series of 4-oxo-spirochromane bearing primary sulfonamide group were synthetized as Carbonic
Anhydrase inhibitors (CAIs) and tested for their management of neuropathic pain. Indeed, CAs have been re-
cently validated as novel therapeutic targets in neuropathic pain. All compounds, here reported, showed strong
activity against hCA II and hCA VII with KI values in the low or sub-nanomolar range. Two compounds (6d and
6l) showed good neuropathic pain attenuating effects and longer duration than drug reference acetazolamide in
an animal model of oxaliplatin induced neuropathy.
Carbonic Anhydrases (CAs)
Carbonic Anhydrase inhibitors (CAIs)
Metalloenzymes
Neuropathic pain
1. Introduction
treatment and/or control still remains an unmet challenge [10]. Im-
portant contributions on NP were recently introduced by means of
Spirocycle containing moieties are well represented in Nature and
within the Medicinal Chemistry field [1,2,4–7] with the first report
dealing with such an interesting scaffold dated over 60 years ago [3].
The major advantages offered by spirocyclic scaffolds are mainly re-
lated to its inherent three-dimensional feature and concomitant ability
to project chemical functionalities all over the three dimensions [4].
This makes the spirocycles the ideal platform for designing ligands, able
to bind towards biological targets, far more easier and clever when
compared to planar systems [4].
biochemical evidences that accounted for the γ-aminobutyric acid
–
(GABA) receptor functionality being directly dependent from the HCO3
anion concentration [9,10]. Thus modulation of HCO3–-dependent de-
polarization of GABAA receptors, when the function of KCC2 is com-
promised after nerve injury, may be a new potential strategy to treat NP
[7,11–14]. In this context the inhibition of the Carbonic Anhydrase (CA;
EC 4.2.1.1) enzymes acts as the ideal biological targets since such en-
–
zymes are mainly involved in producing HCO3 and H+ ions as by-
products of the CO2 hydration reaction catalysis [7,11–14]. As exten-
sion of our previous contribution based on merging chemical moieties
well known for possessing anticonvulsant effects, such as the spirocycle
scaffold (i.e. gaba-pentin) with the CA inhibitor (CAI) of the benzene-
sulfonamide type [7], we report the 4-oxo-spirochromanes 6a-v and we
investigated any possible effects both in vitro and in vivo when sub-
stituents were introduced at positions 6 and 7.
In the last years spirocycle derivatives were attributed with several
biological proprieties such as antibiotics, antitumor agents, enzyme
inhibitors, receptor modulators, protein–protein interaction inhibitors
and transporters [6]. Quite recently spirocycles emerged as promising
neuropathic pain (NP) relievers [7,8], and the present study follows this
direction. NP affects approximately 6% of the adult population world-
wide [9] and despite several therapeutic options are clinically used its
⁎ Corresponding authors at: University of Florence, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence,
Italy (A. Angeli).
E-mail addresses: yasinmedchem@gmail.com (Y. Tamboli), andrea.angeli@unifi.it (A. Angeli), claudiu.supuran@unifi.it (C.T. Supuran).
https://doi.org/10.1016/j.bioorg.2019.103210
Received 31 July 2019; Received in revised form 14 August 2019; Accepted 16 August 2019
Availableonline17August2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

Y. Kalisha Vali, et al.
BioorganicChemistry92(2019)103210
primary sulfonamide group at para position (Schemes 1–4). The
planned synthesis of the compounds 6a-v involved the coupling of 2-
hydroxy acetophenone (1a-f) with N-protected-4-piperidinone and
pyrrolidine as a base in isopropanol to give the spiro intermediates 2a–f
as reported in Scheme 1 [15–19].
Treatment of free phenol 2c and 2d with commercially available
alkyl halides using K2CO3 as base in DMF afforded the spiroalkyl de-
rivatives 3a–p (Scheme 2) [15,20,21].
Scheme 1. General procedure to obtain derivatives 2a-f.
Scheme 2. General procedure to synthetized compounds 3a-p.
2. Chemistry
We designed two series of compounds with different groups at 6 and
Boc-deprotection of 2a–f and 3a–p with trifluoro acetic acid (TFA)
afforded the spiroamines 4a-f and 4g-u respectively as outlined in
Scheme 3.
7 positions of novel highly flexible 4-oxo-spirochromanes bearing the
Scheme 3. General procedure to obtain spiroamines 4a-v.
2

Y. Kalisha Vali, et al.
BioorganicChemistry92(2019)103210
Finally the desired compounds 6a-v were obtained by SNAr reaction
of amines 4a–u with 4-fluoro-3-nitrobenzenesulfonamide (5) as re-
ported in Scheme 4.
(i) The cytosolic hCA I was weakly inhibited by almost of compounds
here reported in micromolar range. On the other hand, only three
compounds showed KI values in the nanomolar range (6k, 6p, 6r
with KI of 91.1, 81.5 and 78.5, respectively). The same substituents
Scheme 4. General procedure to obtain 3-nitrobenzenesulfonamide derivatives 6a-v.
All compounds were purified by aqueous workup and trituration
using ethyl acetate to afford desired products with high purity and
yields between 50 and 90%. Degradation of compounds 6c, 6h, 6q
and 6t was observed during work-up and attempt of further pur-
ification failed. The analytical and spectroscopic data (1H NMR
and 13C NMR chemical shifts) of the purified compounds are in
agreement with the purposed structures (see experimental section
for details).
Table 1
Inhibition data of human CA isoforms I, II, VII and IX with compounds 6a, 6b,
6d-g, 6i-p, 6r, 6s, 6u, 6v and acetazolamide (AAZ) as standard drug by a
stopped-flow CO2 hydrase assay [22].
KI (nM)*
Cmp
hCA I
hCA II
hCA VII
hCA IX
6a
6b
6d
6e
6f
5500.0
790.9
7520.4
7841.0
8694.1
8940.5
8051.3
8332.4
91.1
462.7
430.0
44.6
410.1
290.5
461.3
51.8
406.8
63.7
3.8
37.0
54.5
2.8
229.3
3550.5
1557.0
2836.5
1242.0
3295.5
3745.0
4338.0
4075.0
4406.5
2750.0
2653.5
4152.5
34.6
3. Carbonic anhydrase inhibition
41.1
75.8
68.3
1.5
6g
6i
All compounds here reported were tested in vitro for their inhibitory
activity against the physiologically relevant hCA isoforms I, II, VII and
IX by means of the stopped-flow carbon dioxide hydration assay [22].
Their activities were compared to the standard CA inhibitor acet-
azolamide (AAZ).
6j
65.0
0.96
0.28
0.39
5.8
6k
6l
250.0
6392.6
8008.6
7124.1
81.5
6m
6n
6o
6p
6r
5.1
53.7
5.9
We have investigated the novel series of 4-oxo-spirochromane de-
rivatives bearing primary sulfonamide group for their interaction with
four hCAs of pharmacologic interest, using a period of 15 min of in-
cubation of the enzyme and inhibitor solutions [23–26]. The following
structure-activity-relationship (SAR) may be noted regarding the in-
hibition data of Table 1:
2.3
3.1
0.35
0.47
15.9
61.2
14.4
2.5
78.5
4.2
46.5
6s
2849.7
7240.6
4261.9
250
8.8
45.4
6u
6v
AAZ
188.1
6.1
1470.0
199.3
12.1
25.8
*Mean from 3 different assays, by a stopped flow technique (errors were in the
range of 5–10% of the reported values).
3

Y. Kalisha Vali, et al.
BioorganicChemistry92(2019)103210
in position 6 or 7 of 4-oxo-spirochromane scaffold did not effec-
6. Experimental part
tively influence the inhibition profile against this isoform.
(ii) The second dominant cytosolic human isoform, hCA II, on the
other hand, was effectively inhibited by these compounds. In
particular, this time, substituents in 7 position of 4-oxo-spir-
ochromane scaffold showed a better constant inhibition values
than derivatives with substituents in 6 position leading to low
nanomolar range inhibition such as 6l, 6m, 6o, 6p, 6r and 6s with
KI that spanning among 3.1–8.8 nM. One exception was showed by
compounds 6u and 6v where bromine atom increased the potency
when located in 6 position of 4-oxo-spirochromane scaffold (KI
6.1 nM than 188.1 nM of 6v).
6.1. General
All reactions were carried out in an oven-dried glassware under
inert atmosphere (N2). Solvents and all the reagents were purchased
from Sigma- Aldrich (India), Alfa Aesar (India) and TCI (India).
Analytical thin-layer chromatography (TLC) was carried out on silica
gel F-254 plates (Merck). Nuclear magnetic resonance spectra (1H NMR:
400 MHz and 13C NMR: 100 MHz) were recorded in CDCl3 or DMSO‑d6
using an Avance III 400 MHz spectrometer (Bruker). 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; t, triplet; q, quadruplet; m, multiplet; br s, broad
singlet; dd, double of doublets. The LC mass spectra were obtained
using a 1200 l triple quadrupole system (Varian, Palo Alto, CA)
equipped by electron spray source (ESI) operating in both positive and
negative ions and the compounds resulted > 95% pure. Melting points
(m.p.) were carried out in open capillary tubes on a Büchi Melting Point
B-540 apparatus and were uncorrected. Elemental composition of
compounds was calculated on the basis of their measured accurate
masses, accepting only results with an attribution error less than 5 ppm
and not integer RDB (double bond/ring equivalents). Stock solutions of
analytes were prepared using acetone (1.0 mg mL−1) and stored at 4 °C.
Then working solutions of each analyte were prepared by dilution of
the stock solutions using mQ H2O/CAN 1/1(v/v) up to a concentration
(iii) The last cytosolic isoform here investigated, hCA VII, was strongly
inhibited by all compounds here reported showing inhibition
constants in low nanomolar range and reaching for several of them
such as 6k-m subnamolar range (KI 0.28–0.96 nM). These com-
pounds even if more active against this isoform showed the same
features of hCA II where substituents in 7 position of 4-oxo-spir-
ochromane scaffold prove to be more efficaciously than 6 position
except for bromine atom of derivatives 6u and 6v.
(iv) The transmembrane tumor-associate isoform hCA IX, contrarily to
the other cytosolic human isoforms, was weakly inhibited by these
derivatives with constant of inhibition values spanning in the mi-
cromolar range except for compounds 6p, 6r and 6s where the
potency increase drastically reaching to medium nanomolar range
of inhibition (KI 34.6–46.5 nM). For this isoform, anyway, the
position of substituents did not play an important role for the in-
hibition activity.
of 1.0 μg mL−1
.
6.2. General procedure for the synthesis of compounds 2a to 2f
4. Biological assays
To a stirred solution of 2-hydroxyacetophenone derivative (1 eq)
and Boc-4-piperidone (1.5 eq) in Isopropanol (10 mL for 1 mmol of
substrate), pyrrolidine (2 eq) was added drop wise at RT and stirred for
15 min. Reaction mass heated to reflux for 24 h. TLC showed comple-
tion of starting material. The reaction mixture was cooled to RT and
concentrated under reduced pressure. The crude compound was diluted
with water, pH adjusted to ~6 using 1 N HCl and extracted with ethyl
acetate. Combined organic layer washed with brine, dried over Na2SO4,
filtered and concentrated to get crude compound. The crude compound
was purified by flash column chromatography using 10–30% (v/v)
ethyl acetate in pet ether as eluent to get desired compound 2.
Tert-butyl 4-oxospiro[chroman-2,4′-piperidine]-1′-carboxylate (2a)
[15,16]:
Kipping in mind the inhibition pattern of the compounds here re-
ported and the not a precise knowledge which CA isoforms are involved
in the neuropathic pain response. We focused the attention on inhibi-
tion of physiologically cytosolic relevant CA isoforms hCA II where it is
expressed in the central/peripheral nervous system and hCA VII pre-
dominantly expressed in the brain and being absent in most other tis-
sues. In this context, we investigated 4 different 4-oxo-spirochromane
derivatives (6d, 6i, 6l and 6m) and compared with acetazolamide
(AAZ) as possible pain relievers in a mice model of neuropathic pain
induced by oxaliplatin (Fig. 1).
Compounds 6d and 6l showed a dose dependent pain-relieving ef-
fect (30 and 100 mg kg−1). Moreover, all derivatives, here tested, lead
the maximum of the pain threshold at 30 min of administration and
their effects finished at 60 min. In particular, 6d and 6l at 100 mg kg−1
reaching the same maximum pain-relieving effect when the same model
was treated with acetazolamide (AAZ). The position of OH group in 4-
oxo-spirochromane scaffold did not play a crucial role for the maximum
of the efficacy, indeed, derivatives 6d and 6l showed the same peak at
30 min. On the other hand, hydroxyl group in 7 position (6l) increased
the potency at 30 mg kg−1. Different substituents from OH group such
as for compounds 6i or 6m lead to decrease on the potency of pain-
relieving effects.
Following the above general procedure using compound 1a and N-
Boc-4-piperidone yielded compound 2a in 85% yield as a white solid.
M.p: 217–219 °C; TLC: Rf: 0.4 (30% EtOAc in pet ether). 1H NMR in
CDCl3: δ 7.85 (dd, J = 2 Hz, 8.4 Hz, 1H), 7.51–7.46 (m, 1H), 7.02–6.96
(m, 2H), 3.86 (m, 2H), 3.21 (br t, 2H), 2.71 (s, 2H), 2.02 (d, J = 8 Hz,
2H), 1.64–1.55 (m, 2H) and 1.46 (s, 9H). LCMS (ESI positive) m/
z = 218.2 [M+H-100]+ and m/z = 262.1 [M+H-56 (tert-butyl)]+
.
Tert-butyl
6-methyl-4-oxospiro[chroman-2,4′-piperidine]-1′-carbox-
ylate (2b) [17]:
Following the above general procedure using compound 1b and N-
Boc-4-piperidone yielded compound 2b in 70% yield as a white solid.
M.p: 218–220 °C; TLC: Rf: 0.3 (30% EtOAc in pet ether). 1H NMR in
CDCl3: δ 7.65 (d, J = 2 Hz, 1H), 7.29 (dd, J = 2 Hz, 8 Hz, 1H), 6.68 (d,
J = 8 Hz, 1H), 3.85 (m, 2H), 3.23–3.20 (m, 2H), 2.68 (s, 2H), 2.29 (s,
3H), 2.01 (d, J = 8 Hz, 2H), 1.58–1.55 (m, 2H) and 1.46 (s, 9H). LCMS
(ESI positive) m/z = 232.2 [M+H-100]+ and m/z = 276.1 [M+H-56
5. Conclusions
In conclusion, we have synthetized a novel series of 4-oxo-spir-
ochromanes bearing the primary sulfonamide group as CAIs and po-
tentially useful for the management of neuropathic pain. All com-
pounds here reported, showed strong activity against the hCA II and
hCA VII isoforms with KI values in the low or sub-nanomolar range.
Compounds 6d and 6l showed comparable pain threshold level to AAZ
in an in vivo model of the disease. Quite interestingly 6d and 6l showed
the maximum effects at 30 min after administration and lasted up to
60 min. Taken together, these data strongly suggest the translational
utility of these inhibitors as novel pain relievers.
(tert-butyl)]+
.
Tert-butyl 6-hydroxy-4-oxospiro[chroman-2,4′-piperidine]-1′-car-
boxylate (2c) [15]:
Following the above general procedure using compound 1c and N-
Boc-4-piperidone yielded compound 2c in 72% yield as an off white
solid. M.p: 222–224 °C; TLC: Rf: 0.2 (40% EtOAc in pet ether). 1H NMR
in CDCl3: δ 7.29 (d, J = 3.2 Hz, 1H), 7.07 (dd, J = 3.2 Hz, 8.8 Hz, 1H),
4

Y. Kalisha Vali, et al.
BioorganicChemistry92(2019)103210
Fig. 1. Effect of acute administration of carbonic anhydrase inhibitors 6d, 6i, 6l and 6m and AAZ on oxaliplatin induced neuropathic pain in the mouse Cold plate
test **P < 0.01 vs vehicle + vehicle treated animals; ^P < 0.05 and ^^P < 0.01 vs oxaliplatin + vehicle treated animals. Each value represents the mean
of 10 mice performed in 2 different experimental sets.
S.E.M.
5

Y. Kalisha Vali, et al.
BioorganicChemistry92(2019)103210
6.87 (d, J = 8.8 Hz, 1H), 6.03 (s, 1H), 3.85 (s, 2H), 3.21–3.19 (m, 2H),
2.67 (s, 2H), 2.00 (d, J = 8 Hz, 2H), 1.61–1.54 (m, 2H) and 1.46 (s,
9H). LCMS (ESI positive) m/z = 234 [M+H-100]+ and m/z = 278 [M
J = 3 Hz, 3H). 13C NMR in DMSO‑d6: δ 190.8, 159.7, 156.0, 151.7,
119.8, 119.4, 114.9, 113.6, 79.7, 68.8, 64.6, 44.4, 34.2, 28.4, 14.8.
LCMS (ESI positive) m/z = 262.2 [M+H-100]+ and m/z = 306.1 [M
+H-56 (tert-butyl)]+
.
+H-56 (tert. butyl)]+
.
Tert-butyl 7-hydroxy-4-oxospiro[chroman-2,4′-piperidine]-1′-car-
boxylate (2d) [15]
Tert-butyl 4-oxo-6-propoxyspiro[chroman-2,4′-piperidine]-1′-car-
boxylate (3c):
Following the above general procedure using compound 1d and N-
Boc-4-piperidone yielded compound 2d in 60% yield as an off white
solid. M.p: 221–223 °C; TLC: Rf: 0.22 (40% EtOAc in pet ether). 1H NMR
in CDCl3: δ 8.16 (brs, 1H, –OH), 7.77 (d, J = 8.8 Hz, 1H), 6.52 (d d,
J = 2.4 Hz, 8.8 Hz, 1H), 6.36 (d, J = 2.4 Hz, 1H), 3.90–3.87 (m, 2H),
3.20 (t, 2H), 2.65 (s, 2H), 2.03 (d, J = 8 Hz, 2H), 1.63–1.55 (m, 2H) and
1.47 (s, 9H). LCMS (ESI positive) m/z = 234.1 [M+H-100]+ and m/
Following the above general procedure using compound 2c and n-
propyl iodide yielded compound 3c in 80% yield as an off white solid.
M.p: 222–224 °C; TLC: Rf: 0.3 (15% EtOAc in pet ether). 1H NMR in
CDCl3: δ 7.28 (d, J = 3.2 Hz, 1H), 7.10 (d d, J = 3.2 Hz, 8.8 Hz, 1H),
6.90 (d, J = 8.8 Hz, 1H), 3.89 (t, 2H), 3.85 (s, 2H), 3.19 (t, 2H), 2.68 (s,
2H), 2.01 (d, 2H), 1.81–1.74 (m, 2H), 1.61–1.53 (m, 2H), 1.45 (s, 9H)
and 1.01 (t, J = 3 Hz 3H). 13C NMR in DMSO‑d6: δ 190.9, 159.6, 156.0,
151.7, 119.8, 119.4, 114.8, 113.5, 79.8, 69.3, 68.7, 44.5, 34.2, 28.4,
22.6, 10.4. LCMS (ESI positive) m/z = 276.1 [M+H-100]+ and m/
z = 278 [M+H-56 (tert-butyl)]+
.
Tert-butyl 6-bromo-4-oxospiro[chroman-2,4′-piperidine]-1′-carboxylate
(2e) [18]:
z = 320.1 [M+H-56 (tert. butyl)]+
.
Following the above general procedure using compound 1e and N-
Boc-4-piperidone yielded compound 2e in 83% yield as a white solid.
M.p: 220–222 °C; TLC: Rf: 0.4 (30% EtOAc in pet ether). 1H NMR in
CDCl3: δ 7.97 (s, 1H), 7.55 (d, J = 8.8 Hz, 1H), 6.82 (d, J = 8 Hz, 1H),
3.85 (m, 2H), 3.2 (t, 2H), 2.7 (s, 2H), 2.00 (d, J = 8 Hz, 2H), 1.62–1.58
(m, 2H) and 1.44 (s, 9H). LCMS (ESI positive) m/z = 296, 298 [M+H-
100, Bromo pattern]+ and m/z = 339.8, 341.9 [M+H-56 (tert-butyl,
Tert-butyl
6-isopropoxy-4-oxospiro[chroman-2,4′-piperidine]-1′-car-
boxylate (3d) [15]:
Following the above general procedure using compound 2c and
isopropyl iodide yielded compound 3d in 82% yield as an off white
solid. M.p: 220–222 °C; TLC: Rf: 0.3 (15% EtOAc in pet ether). 1H NMR
in CDCl3: δ 7.29 (d, J = 3.2 Hz, 1H), 7.08 (dd, J = 3.2 Hz, 9.2 Hz, 1H),
6.89 (d, J = 8.8 Hz, 1H), 4.52–4.46 (m, 1H), 3.86 (s, 2H), 3.20 (t, 2H),
2.68 (s, 2H), 2.01 (d, J = 8 Hz, 2H), 1.57–1.54 (m, 2H), 1.45 (s, 9H)
and 1.31 (d, 6H). 13C NMR in DMSO‑d6: δ 190.9, 159.6, 151.7, 151.0,
119.8, 119.4, 114.9, 113.6, 79.8, 75.8, 68.7, 44.5, 34.2, 28.4, 22.0.
LCMS (ESI positive) m/z = 276.1 [M+H-100]+ and m/z = 320.1 [M
bromo pattern)]+
.
Tert-butyl 7-bromo-4-oxospiro[chroman-2,4′-piperidine]-1′-carboxylate
(2f) [17,19]:
Following the above general procedure using compound 1f and N-
Boc-4-piperidone yielded compound 2f in 80% yield as a white solid.
M.p: 220–222 °C; TLC: Rf: 0.4 (30% EtOAc in pet ether). 1H NMR in
CDCl3: δ 7.71 (d, J = 8.8 Hz, 1H), 7.20 (d, J = 2 Hz, 1H), 7.15 (d d,
J = 2 Hz, 8.4 Hz, 1H), 3.96–3.86 (m, 2H), 3.23–3.19 (m, 2H), 2.70 (s,
2H), 2.04–1.95 (m, 4H) and 1.46 (s, 9H). LCMS (ESI positive) m/
z = 296.1, 298.1 [M+H-100, Bromo pattern]+ and m/z = 339.8,
341.9 [M+H-56 (tert-butyl, bromo pattern)]+
+H-56 (tert. butyl)]+
.
Tert-butyl 6-(cyanomethoxy)-4-oxospiro[chroman-2,4′-piperidine]-
1′-carboxylate (3e):
Following the above general procedure using compound 2c and
bromo acetonitrile yielded compound 3e in 86% yield as an off white
solid. M.p: 220–222 °C; TLC: Rf: 0.22 (30% EtOAc in pet ether). 1H NMR
in CDCl3: δ 7.40 (d, J = 3.2 Hz, 1H), 7.20 (dd, J = 3.2 Hz, 8.8 Hz, 1H),
6.99 (d, J = 9.2 Hz, 1H), 4.75 (s, 2H), 3.87 (s, 2H), 3.19 (t, 2H), 2.71 (s,
2H), 2.01 (d, J = 8 Hz, 2H), 1.64–1.56 (m, 2H) and 1.45 (s, 9H). 13C
NMR in DMSO‑d6: δ 190.9, 159.6, 152.4, 149.2, 120.2, 119.3, 115.7,
115.3, 113.5, 79.8, 68.7, 60.3, 43.0, 34.2, 28.4. LCMS (ESI positive) m/
6.3. General procedure for the synthesis of compounds 3a to 3p
To a stirred solution of compound 2 (c and d) (1 eq) in anhydrous
DMF (5 mL for 1 mmol of substrate), K2CO3 (3 eq) was added and
stirred for 15 min. Appropriate alkyl halide (1.5 eq) was added drop-
wise at RT and stirred for 20 h. TLC showed completion of reaction. The
reaction mixture was quenched in ice cold water and extracted with
diethyl ether. The combined organic layer was washed with brine, dried
over Na2SO4, filtered and concentrated to get crude compound. The
crude compound was purified by flash column chromatography using
10–30% EtOAc in pet ether as eluent to get desired compound 3.
Tert-butyl 6-methoxy-4-oxospiro[chroman-2,4′-piperidine]-1′-carbox-
ylate (3a) [15]:
z = 272.9 [M+H-100]+
.
Tert-butyl
6-(2-hydroxyethoxy)-4-oxospiro[chroman-2,4′-piper-
idine]-1′-carboxylate (3f):
Following the above general using procedure compound 2c and
bromo ethanol yielded compound 3f in 64% yield as an off white solid.
M.p: 218–220 °C; TLC: Rf: 0.3 (40% EtOAc in pet ether). 1H NMR in
CDCl3: δ 7.30 (d, J = 2.7 Hz, 1H), 7.14 (dd, J = 2.8 Hz, 8.8 Hz, 1H),
6.93 (d, J = 8.8 Hz, 1H), 4.07 (t, 2H), 3.96 (s, 2H), 3.86 (s, 2H), 3.18 (t,
2H), 2.69 (s, 2H), 2.01 (d, J = 8 Hz, 2H), 1.62–1.56 (m, 2H) and 1.46
(s, 9H). 13C NMR in DMSO‑d6: δ 190.9, 159.6, 156.0, 151.7, 119.8,
119.4, 114.9, 113.6, 79.8, 69.5, 68.7, 60.9, 44.5, 43.0, 34.2, 28.4.LCMS
(ESI positive) m/z = 278.2 [M+H-100]+ and m/z = 322.1 [M+H-56
Following the above general procedure using compound 2c and
methyl iodide yielded compound 3a in 83% yield as an off white solid.
M.p: 218–220 °C; TLC: Rf: 0.3 (15% EtOAc in pet ether). 1H NMR in
CDCl3: δ 7.29 (d, J = 2.4 Hz, 1H), 7.09 (d d, J = 2 Hz, 8.4 Hz, 1H), 6.91
(d, J = 8 Hz, 1H), 3.85–3.79 (m, 2H), 3.78 (s, 3H), 3.22–3.16 (m, 2H),
2.68 (s, 2H), 2.02–1.99 (m, 2H), 1.63–1.54 (m, 2H) and 1.45 (s, 9H).
13C NMR in DMSO‑d6: δ 190.9, 159.6, 152.4, 152.1, 120.2, 119.3,
115.3, 113.5, 79.8, 68.7, 55.8, 44.5, 43.0, 34.2, 28.4. LCMS (ESI po-
sitive) m/z = 248.1 [M+H-100]+ and m/z = 292.1 [M+H-56 (tert.
(tert. butyl)]+
.
Tert-butyl 6-(2-methoxyethoxy)-4-oxospiro[chroman-2,4′-piperidine]-
1′-carboxylate (3 g): Following the above general using procedure
compound 2c and methoxy ethyl bromide yielded compound 3 g in
80.5% yield as an off white solid. M.p: 220–222 °C; TLC: Rf: 0.4 (30%
EtOAc in pet ether). 1H NMR in CDCl3: δ 7.30 (d, J = 2.7 Hz, 1H), 7.17
(dd, J = 2.9 Hz, 8.8 Hz, 1H), 6.91 (d, J = 8.8 Hz, 1H), 4.10 (t, 2H), 3.86
(s, 2H),3.74 (t, 2H), 3.44 (s, 3H), 3.20 (t, 2H), 2.69 (s, 2H), 2.01(d,
J = 8 Hz, 2H), 1.60–1.57 (m, 2H) and 1.46 (s, 9H). 13C NMR in
DMSO‑d6: δ 190.9, 159.6, 156.0, 151.7, 119.8, 119.4, 114.9, 113.6,
79.8, 72.2, 69.0, 68.7, 59.3, 44.5, 43.0, 34.2, 28.4. LCMS (ESI positive)
butyl)]+
.
Tert-butyl
6-ethoxy-4-oxospiro[chroman-2,4′-piperidine]-1′-carbox-
ylate (3b) [15]:
Following the above general procedure using compound 2c and
ethyl bromide yielded compound 3b in 80% yield as an off white solid.
M.p: 219–221 °C; TLC: Rf: 0.3 (15% EtOAc in pet ether). 1H NMR in
CDCl3: δ 7.28 (d, J = 3.2 Hz, 1H), 7.10 (dd, J = 3.2 Hz, 8.8 Hz, 1H),
6.90 (d, J = 8.8 Hz, 1H), 4.00 (q, 2H), 3.85 (s, 2H), 3.19 (t, 2H), 2.68 (s,
2H), 2.01 (d, J = 8 Hz, 2H), 1.6–1.57 (m, 2H), 1.45 (s, 9H) and 1.39 (t,
m/z = 292.1 [M+H-100]+ and m/z = 336.1 [M+H-56 (tert. butyl)]+
.
Tert-butyl
6-acetoxy-4-oxospiro[chroman-2,4′-piperidine]-1′-car-
boxylate (3 h):
Following the above general procedure using compound 2c and
acetic anhydride yielded compound 3 h in 70% yield as a white solid.
6

Y. Kalisha Vali, et al.
BioorganicChemistry92(2019)103210
220–222 °C; TLC: Rf: 0.40 (30% EtOAc in pet ether). 1H NMR in CDCl3:
δ 7.55 (d, J = 2.4 Hz, 1H), 7.22 (dd, J = 2 Hz, 8.8 Hz, 1H), 6.99 (d,
J = 8.8 Hz, 1H), 3.88 (s, 2H), 3.18 (t, 2H), 2.7 (s, 2H), 2.29 (s, 3H), 2.02
(d, J = 8 Hz, 2H), 1.7–1.6 (m, 2H) and 1.46 (s, 9H). 13C NMR in
DMSO‑d6: δ 190.9, 169.0, 159.6, 156.9, 142.8, 126.6, 122.5, 119.6,
114.7, 79.8, 68.7, 44.5, 43.0, 34.2, 28.4, 20.3. LCMS (ESI positive) m/
piperidine]-1′-carboxylate (3n):
Following the above general procedure using compound 2d and
bromo ethanol yielded compound 3n in 80% yield as a white solid. M.p:
220–222 °C; TLC: Rf: 0.3 (30% EtOAc in pet ether). 1H NMR in CDCl3: δ
7.81 (d, J = 8.8 Hz, 1H), 6.59 (d, J = 7.2 Hz, 1H), 6.45 (s, 1H),
4.12–4.11 (m, 2H), 3.99–3.95 (m, 2H), 3.87 (s, 2H), 3.19 (t, 2H), 2.66
z = 276.1 [M+H-100]+ and m/z = 320.1 [M+H-56 (tert. butyl)]+
.
(s, 2H), 2.01 (d, J = 8 Hz, 2H), 1.60–1.57 (m, 2H) and 1.46 (s, 9H). 13
C
Tert-butyl 7-methoxy-4-oxospiro[chroman-2,4′-piperidine]-1′-carbox-
ylate (3i) [20,21]:
NMR in DMSO‑d6: δ 190.9, 164.4, 162.2, 159.6, 130.0, 113.3, 105.9,
101.5, 79.8, 69.5, 68.7, 60.9, 44.5, 43.0, 34.2, 28.4. LCMS (ESI posi-
tive) m/z = 278.2 [M+H-100]+ and m/z = 322.1 [M+H-56 (tert.
Following the above general procedure using compound 2d and
methyl iodide yielded compound 3i in 90% yield as a white solid. M.p:
221–223 °C; TLC: Rf: 0.3 (15% EtOAc in pet ether). 1H NMR in CDCl3: δ
7.80 (d, J = 8.8 Hz, 1H), 6.57 (d, J = 8.8 Hz, 1H), 6.43 (s, 1H), 3.84 (s,
5H), 3.20 (t, 2H), 2.66 (s, 2H), 2.02 (d, J = 8 Hz, 2H), 1.59–1.57 (m,
2H) and 1.46 (s, 9H). 13C NMR in DMSO‑d6: δ 190.9, 165.6, 162.6,
159.6, 130.4, 114.0, 105.8, 101.4, 79.8, 68.7, 55.8, 44.5, 43.0, 34.2,
28.4. LCMS (ESI positive) m/z = 248.1 [M+H-100]+ and m/z = 292.1
butyl)]+
.
Tert-butyl
7-(2-methoxyethoxy)-4-oxospiro[chroman-2,4′-piper-
idine]-1′-carboxylate (3o):
Following the above general procedure using compound 2d and
methoxy ethyl bromide yielded compound 3o in 83% yield as a white
solid. M.p: 221–223 °C; TLC: Rf: 0.4 (30% EtOAc in pet ether). 1H NMR
in CDCl3: δ 7.80 (d, J = 8.8 Hz, 1H), 6.60 (d, J = 8 Hz, 1H), 6.45 (s,
1H), 4.16–4.15 (m, 2H), 3.86 (s, 2H), 3.77 (s, 2H), 3.45 (s, 3H), 3.19 (t,
2H), 2.66 (s, 2H), 2.01 (d, J = 8 Hz, 2H), 1.64–1.57 (m, 2H) and 1.46
(s, 9H). 13C NMR in DMSO‑d6: δ 190.9, 164.4, 162.2, 159.6, 130.0,
113.3, 105.9, 101.5, 79.8, 72.2, 69.0, 68.7, 59.3, 44.5, 43.0, 34.2, 28.4.
LCMS (ESI positive) m/z = 292.2 [M+H-100]+ and m/z = 337.1 [M
[M+H-56 (tert. butyl)]+
.
Tert-butyl
7-ethoxy-4-oxospiro[chroman-2,4′-piperidine]-1′-carbox-
ylate (3j) [20]:
Following the above general procedure using compound 2d and
ethyl bromide yielded compound 3j in 94% yield as a white solid. M.p:
220–222 °C; TLC: Rf: 0.3 (15% EtOAc in pet ether). 1H NMR in CDCl3: δ
7.79 (d, J = 8.8 Hz, 1H), 6.55 (d, J = 8.8 Hz, 1H), 6.40 (s, 1H), 4.06 (q,
2H), 3.86 (s, 2H), 3.19 (t, 2H), 2.65 (s, 2H), 2.02 (d, J = 8 Hz, 2H),
1.62–1.57 (m, 2H), 1.46 (s, 9H) and 1.42 (t, 3H). 13C NMR in DMSO‑d6:
δ 190.9, 164.4, 162.2, 159.6, 130.0, 113.3, 105.9, 101.5, 79.8, 68.7,
64.6, 44.5, 43.0, 34.2, 28.4, 14.8. LCMS (ESI positive) m/z = 262.1 [M
+H-56 (tert. butyl)]+
.
Tert-butyl
7-acetoxy-4-oxospiro[chroman-2,4′-piperidine]-1′-car-
boxylate (3p):
Following the above general procedure using compound 2d and
acetic anhydride yielded compound 3p in 93% yield as a white solid.
M.p: 220–222 °C; TLC: Rf: 0.41 (30% EtOAc in pet ether). 1H NMR in
CDCl3: δ 7.88 (d, J = 8.8 Hz, 1H), 6.78 (s, 1H), 6.76 (d, J = 8.0 Hz, 1H),
3.87 (s, 2H), 3.19 (t, 2H), 2.70 (s, 2H), 2.31 (s,3H), 2.02 (d, J = 8 Hz,
2H), 1.61–1.57 (m, 2H) and 1.46 (s, 9H). 13C NMR in DMSO‑d6: δ
190.9, 170.5, 169.0, 162.0, 159.6, 129.8, 118.5, 113.1, 110.1, 79.8,
68.7, 44.5, 43.0, 34.2, 28.4, 20.3. LCMS (ESI positive) m/z = 276.2 [M
+H-100]+ and m/z = 306.1 [M+H-56 (tert. butyl)]+
.
Tert-butyl 4-oxo-7-propoxyspiro[chroman-2,4′-piperidine]-1′-car-
boxylate (3k)
Following the above general procedure using compound 2d and n-
propyl iodide yielded compound 3k in 92% yield as a white solid. M.p:
223–226 °C; TLC: Rf: 0.3 (15% EtOAc in pet ether). 1H NMR in CDCl3: δ
7.79 (d, J = 8.8 Hz, 1H), 6.56 (d, J = 8.8 Hz, 1H), 6.41 (s, 1H), 3.95 (t,
2H), 3.86 (s, 2H), 3.18 (t, 2H), 2.65 (s, 2H), 2.02 (d, J = 8 Hz, 2H),
1.84–1.79 (m, 2H), 1.59–1.57 (m, 2H), 1.46 (s, 9H) and 1.03 (t, 3H).
13C NMR in DMSO‑d6: δ 190.9, 164.4, 162.2, 159.6, 130.0, 113.3,
105.9, 101.5, 79.8, 69.3, 68.7, 44.5, 43.0, 34.2, 28.4, 22.6, 10.4. LCMS
(ESI positive) m/z = 276.1 [M+H-100]+ and m/z = 320.1 [M+H-56
+H-100]+ and m/z = 320.1 [M+H-56 (tert. butyl)]+
.
6.4. General procedure for the synthesis of compounds 4a to 4b
To an ice cold stirred solution of compound 2a-2f or 3a-3p (1 eq) in
anhydrous DCM (5 mL for 1 mmol of substrate), Trifluoroacetic acid
(0.5 mL for 1 mmol of substrate) was added drop wise under N2 at-
mosphere and stirred for 30 min. The reaction mixture slowly warmed
to RT and stirred for 16 h. TLC showed completion of reaction. The
reaction mixture was concentrated under reduced pressure, co-distilled
with DCM (2 × 5 mL) and dried under high vacuum to get desired
compound 4a to 4u as thick brown liquid. This was used in next step
without any purification.
(tert. butyl)]+
Tert-butyl
.
7-isopropoxy-4-oxospiro[chroman-2,4′-piperidine]-1′-
carboxylate (3l):
Following the above general procedure using compound 2d and
isopropyl iodide yielded compound 3l in 90% yield as a white solid.
M.p: 221–223 °C; TLC: Rf: 0.3 (15% EtOAc in pet ether). 1H NMR in
CDCl3: δ 7.79 (d, J = 8.8 Hz, 1H), 6.52 (d, J = 8.8 Hz, 1H), 6.39 (s, 1H),
4.61–4.58 (m, 1H), 3.86 (s, 2H), 3.19 (t, 2H), 2.65 (s, 2H), 2.02 (d,
J = 8 Hz, 2H), 1.58–1.56 (m, 2H), 1.46 (s, 9H) and 1.36 (d, 6H). 13C
NMR in DMSO‑d6: δ 190.9, 164.3, 162.2, 159.6, 130.0, 113.3, 105.9,
101.5, 79.8, 75.8, 68.7, 44.5, 43.0, 34.2, 28.4, 22.0. LCMS (ESI posi-
tive) m/z = 276.1 [M+H-100]+ and m/z = 320.1 [M+H-56 (tert.
6.5. General procedure for the synthesis of compounds 6a-v
To an ice cold stirred solution of compound 4a-4u (1 eq) in anhy-
drous DCM (10 mL for 1 mmol of substrate), TEA (3 eq) was added drop
wise and stirred for 10 min. Compound 5 (1 eq) was added slowly and
stirred at RT for 16 h (precipitation was observed during the reaction
progress). TLC showed completion of reaction. The reaction mixture
was concentrated under reduced pressure. Water was added to the
crude material and stirred for 30 min. Obtained solid was filtered,
washed with water and dried under vacuum. The solid was triturated
with ethyl acetate, filtered and dried under high vacuum to afford
compound 6a-v.
butyl)]+
.
Tert-butyl 7-(cyanomethoxy)-4-oxospiro[chroman-2,4′-piperidine]-
1′-carboxylate (3 m):
Following the above general procedure using compound 2d and
bromo acetonitrile yielded compound 3 m in 92% yield as a white solid.
M.p: 222–224 °C; TLC: Rf: 0.2 (30% EtOAc in pet ether). 1H NMR in
CDCl3: δ 7.87 (d, J = 8.8 Hz, 1H), 6.63 (dd, J = 2.2 Hz, 8.8 Hz, 1H),
6.53 (d, J = 2.0 Hz, 1H), 4.81 (s, 2H), 3.88 (s, 2H), 3.19 (t, 2H), 2.69 (s,
2H), 2.02 (d, J = 8 Hz, 2H), 1.64–1.59 (m, 2H) and 1.46 (s, 9H). 13C
NMR in DMSO‑d6: δ 190.9, 163.6, 162.6, 159.6, 130.4, 115.7, 114.0,
105.8, 101.4, 79.8, 75.8, 68.7, 60.3, 44.5, 43.0, 34.2, 28.4. LCMS (ESI
positive) m/z = 273.1 [M+H-100]+ and m/z = 317.1 [M+H-56 (tert.
3-nitro-4-(4-oxospiro[chroman-2,4′-piperidin]-1′-yl)benzenesulfo-
namide (6a):
48% yield as a yellow solid. TLC: Rf: 0.25 (30% Acetone in pet
ether). m.p. 125 °C; 1H NMR in DMSO‑d6: δ 8.22 (d, J = 2.4 Hz, 1H, H-
1), 7.88 (dd, J = 2 Hz, 8.8 Hz, 1H, H-3), 7.74 (dd, J = 1.6 Hz, 8.8 Hz,
1H, H-7), 7.61–7.57 (m, 1H-H-8),7.48 (d, J = 8.8 Hz, 1H, H-10), 7.41
(s, 2H, SO2NH2), 7.11–7.04 (m, 2H, H-2,8), 3.30–3.27 (m, 2H, H-4),
butyl)]+
.
Tert-butyl
7-(2-hydroxyethoxy)-4-oxospiro[chroman-2,4′-
7

Y. Kalisha Vali, et al.
BioorganicChemistry92(2019)103210
3.22–3.18 (m, 2H, H-4′), 2.9 (s, 2H, H-6), 2.02 (m, 2H, H-5) and
1.90–1.83 (m, 2H, H-5′). 13C NMR in DMSO‑d6: δ 191.8, 159.0, 147.8,
139.0, 136.9, 135.0, 131.2, 126.3, 124.9, 121.7, 121.6, 120.9, 118.8,
147.2, 138.4, 134.4, 130.7, 124.9, 124.3, 121.2, 120.3, 119.6, 107.9,
77.1, 69.4, 46.7, 46.0, 33.1 and 21.9. LCMS (ESI positive) m/z = 476.3
[M+H]+: Elemental Analysis Calculated, 55.57; H, 5.30; N, 8.84; S,
6.74. Found, 55.58; H, 5.32; N, 8.82; S, 6.77.
77.9, 47.2, 46.6 and 33.7. LCMS (ESI positive) m/z = 418.1 [M+H]+
;
Elemental Analysis Calculated C, 54.67; H, 4.59; N, 10.07; S, 7.68.
Found C, 54.69; H, 4.58; N, 10.06; S, 7.70.
4-(6-isopropoxy-4-oxospiro[chroman-2,4′-piperidin]-1′-yl)-3-ni-
trobenzenesulfonamide (6 g):
4-(6-methyl-4-oxospiro[chroman-2,4′-piperidin]-1′-yl)-3-ni-
trobenzenesulfonamide (6b):
76% yield as a yellow solid. TLC: Rf: 0.18 (30% Acetone in pet
ether). m.p. 98 °C 1H NMR in DMSO‑d6: δ 8.20 (s, 1H, H-1), 7.87 (d,
J = 8.8 Hz, 1H, H-3), 7.46 (d, J = 8.8 Hz , 1H, H-9), 7.41 (br s, 2H,
SO2NH2), 7.19–7.15 (m, 2H, H-7, H-10), 7.02 (d, J = 8.4 Hz, 1H, H-2),
4.52 (m, 1H), 3.31–3.28 (m, 2H, H-4), 3.19–3.16 (m, 2H, H-4′), 2.85 (s,
2H, H-6), 2.01–1.98 (m, 2H, H-5), 1.84–1.83 (m, 2H, H-5′) and 1.23 (d,
J = 8 Hz, 6H). 13C NMR in DMSO‑d6: δ 191.3, 152.7, 151.4, 147.3,
138.4, 134.4, 130.7, 126.1, 124.4, 121.2, 120.4, 119.7, 109.7, 77.1,
70.0, 46.7, 46.1, 33.1 and 21.70. LCMS (ESI positive) m/z = 476.2 [M
+H]+; Elemental Analysis Calculated, C, 55.57; H, 5.30; N, 8.84; S,
6.74. Found, C, 55.59; H, 5.31; N, 8.83; S, 6.75.
65% yield as a yellow solid. TLC: Rf: 0.25 (30% Acetone in pet
ether). m.p. 112 °C. 1H NMR in DMSO‑d6: δ 8.20 (s, 1H, H-1), 7.87 (d,
J = 8.8 Hz, 1H, H-3), 7.52 (s, 1H, H-7), 7.46 (d, J = 8.8 Hz , 1H, H-9),
7.39 (m, 1H, H-2), 6.99 (d, J = 8.4 Hz, 1H, H-10), 3.30–3.25 (m, 2H, H-
4), 3.19–3.13 (m, 2H, H-4′), 2.85 (s, 2H, H-6), 2.26 (s, 3H), 1.99 (m, 2H,
H-5) and 1.84–1.81 (m, 2H, H-5′). 13C NMR in DMSO‑d6: δ 192.1,
156.5, 147.3, 138.4, 137.3, 134.4, 130.7, 130.1, 125.4, 124.3, 121.2,
120.0, 118.2, 77.1, 46.8, 46.0, 33.1 and 19.8. LCMS (ESI positive) m/
z = 432.2 [M+H]+; Elemental Analysis Calculated C, 55.68; H, 4.91;
N, 9.74; S, 7.43. Found C, 55.69; H, 4.89; N, 9.73; S, 7.41.
4-(6-hydroxy-4-oxospiro[chroman-2,4′-piperidin]-1′-yl)-3-ni-
trobenzenesulfonamide (6c):
4-(6-(2-hydroxyethoxy)-4-oxospiro[chroman-2,4′-piperidin]-1′-yl)-
3-nitrobenzenesulfonamide (6i):
57% yield as a yellow solid. TLC: Rf: 0.15 (40% Acetone in pet
ether). m.p. 137 °C 1H NMR in DMSO‑d6: δ 8.20 (s, 1H, H-1), 7.87 (d,
J = 8 Hz, 1H, H-3), 7.46 (d, J = 8.8 Hz , 1H, H-9), 7.40 (br s, 2H,
SO2NH2), 7.24–7.18 (m, 2H, H-7, H-10), 7.04 (d, J = 8 Hz, 1H, H-2),
3.96 (brs, 2H), 3.68 (brs, 2H), 3.30–3.27 (m, 2H, H-4), 3.19–3.13 (m,
2H, H-4′), 2.86 (s, 2H, H-6), 2.00 (m, 2H, H-5) and 1.84–1.82 (m, 2H,
H-5′). 13C NMR in DMSO‑d6: δ 191.2, 152.8, 147.3, 138.4, 134.4, 130.7,
125.0, 124.3, 121.2, 120.3, 119.6, 108.1, 77.1, 70.1, 59.4, 46.7, 46.1
and 33.1. LCMS (ESI positive) m/z = 478.2 [M+H]+ ; Elemental
Analysis Calculated, C, 52.82; H, 4.86; N, 8.80; S, 6.71. Found, C, 52.81;
H, 4.85; N, 8.82; S, 6.69.
80% yield as an orange solid. TLC: Rf: 0.1 (30% Acetone in pet
ether). m.p. 130 °C. 1H NMR in DMSO‑d6: δ 9.36 (s, 1H, –OH), 8.20 (s,
1H, H-1), 7.86 (d, J = 8 Hz, 1H, H-3), 7.46 (d, J = 8 Hz, 1H, H-9), 7.40
(brs, 2H, SO2NH2), 7.06–7.00 (m, 2H. H-2, H-7), 6.94 (m, 1H, H-10),
3.31–3.27 (m, 2H, H-4), 3.18–3.16 (m, 2H, H-4′), 2.81 (s, 2H-H-6), 2.01
(m, 2H, H-5) and 1.84–1.81 (m, 2H, H-5′). 13C NMR in DMSO‑d6: δ
191.45, 151.57, 151.34, 147.3, 138.39, 134.38, 130.74, 124.6, 124.37,
121.19, 120.58, 119.3, 109.65, 76.82, 46.86, 46.10 and 33.17. LCMS
(ESI positive) m/z = 434.3 [M+H]+; Elemental Analysis Calculated C,
52.65; H, 4.42; N, 9.69; S, 7.40. Found C, 52.67; H, 4.40; N, 9.70; S,
7.38.
4-(6-(2-methoxyethoxy)-4-oxospiro[chroman-2,4′-piperidin]-1′-yl)-
3-nitrobenzenesulfonamide (6j):
4-(6-methoxy-4-oxospiro[chroman-2,4′-piperidin]-1′-yl)-3-ni-
trobenzenesulfonamide (6d):
76% yield as a yellow solid. TLC: Rf: 0.28 (40% Acetone in pet
ether). m.p. 109 °C. 1H NMR in DMSO‑d6: δ 8.20 (s, 1H, H-1), 7.87 (d,
J = 8.4 Hz, 1H, H-3), 7.45 (d, J = 8.8 Hz , 1H, H-9), 7.40 (br s, 2H,
SO2NH2), 7.25–7.18 (m, 2H, H-7, H-10), 7.04 (d, J = 8 Hz, 1H, H-2),
4.06 (brs, 2H), 3.63 (brs, 2H), 3.33–3.29 (m, 5H), 3.18–3.16 (m, 2H, H-
4′), 2.86 (s, 2H, H-6), 2.01 (m, 2H, H-5) and 1.83–1.81 (m, 2H, H-5′).
13C NMR in DMSO‑d6: δ 191.2, 152.9, 152.6, 147.2, 138.4, 134.4,
130.7, 125.0, 124.3, 121.2, 120.3, 119.7, 108.0, 77.1, 70.2, 67.4, 58.0,
67% yield as a yellow solid. TLC: Rf: 0.15 (30% Acetone in pet
ether). m.p. 142 °C. 1H NMR in DMSO‑d6: δ 8.21 (s, 1H, H-1), 7.87 (d,
J = 8 Hz, 1H, H-3), 7.46 (d, J = 8 Hz , 1H, H-9), 7.41 (brs, 2H,
SO2NH2),7.21–7.17 (m, 2H, H-7, H-10), 7.03 (d, J = 8 Hz, 1H, H-2),
3.74 (s, 3H), 3.28–3.25 (m, 2H, H-4), 3.19–3.16 (m, 2H,H-4′), 2.86 (s,
2H, H-6), 1.99 (m, 2H, H-5) and 1.86–1.83 (m, 2H, H-5′). 13C NMR in
DMSO‑d6: δ 191.3, 153.4, 152.8, 147.3, 138.4, 134.4, 130.7, 124.6,
124.4, 121.2, 120.3, 119.7, 107.1, 77.1, 55.5, 46.7, 46.1 and 33.1.
46.6, 46.0 and 33.1. LCMS (ESI positive) m/z = 492.3 [M+H]+
;
LCMS (ESI positive) m/z = 448.2 [M+H]+
;
Elemental Analysis
Elemental Analysis Calculated, C, 53.76; H, 5.13; N, 8.55; S, 6.52.
Found, C, 53.74; H, 5.11; N, 8.56; S, 6.51.
Calculated C, 53.69; H, 4.73; N, 9.39; S, 7.16. Found C, 53.72; H, 4.72;
N, 9.38; S, 7.14.
1′-(2-nitro-4-sulfamoylphenyl)-4-oxospiro[chroman-2,4′-piperidin]-
6-yl acetate (6k):
4-(6-ethoxy-4-oxospiro[chroman-2,4′-piperidin]-1′-yl)-3-ni-
trobenzenesulfonamide (6e):
60% yield as a yellow solid. TLC: Rf: 0.2 (40% Acetone in pet ether).
m.p. 133 °C 1H NMR in DMSO‑d6: δ 8.21 (s, 1H, H-1), 7.87 (d,
J = 8.8 Hz, 1H, H-3), 7.50–7.35 (m, 5H), 7.14 (d, J = 8 Hz, 1H, H-2),
3.32–3.30 (m, 2H, H-4), 3.21–3.19 (m, 2H, H-4′), 2.92 (s, 2H, H-6),
2.24 (s, 3H), 2.05 (m, 2H, H-5) and 1.87–1.85 (m, 2H, H-5′). 13C NMR
in DMSO‑d6: δ 190.9, 169.3, 156.0, 147.2, 144.1, 138.4, 134.5, 130.7,
130.2, 124.3, 121.2, 120.4, 119.5, 118.1, 77.7, 46.4, 46.0, 33.1 and
20.7. LCMS (ESI positive) m/z = 476.2 [M+H]+; Elemental Analysis
Calculated, C, 53.05; H, 4.45; N, 8.84; S, 6.74. Found, C, 53.03; H, 4.46;
N, 8.85; S, 6.76.
75% yield as a yellow solid. TLC: Rf: 0.15 (30% Acetone in pet
ether). m.p. 138 °C. 1H NMR in DMSO‑d6: δ 8.21 (s, 1H, H-1), 7.85 (d,
J = 8 Hz, 1H, H-3), 7.46 (d, J = 8 Hz , 1H, H-9), 7.41 (br s, 2H,
SO2NH2), 7.15 (m, 2H, H-7, H-10), 7.04 (d, J = 8.4 Hz, 1H, H-2), 3.98
(brs, 2H), 3.32–3.28 (m, 2H, H-4), 3.19 (m, 2H, H-4′), 2.86 (s, 2H, H-6),
2.00 (m, 2H, H-5), 1.83 (m, 2H, H-5′) and 1.29 (brs, 3H). 13C NMR in
DMSO‑d6: δ 191.3, 152.7, 152.6, 147.3, 138.4, 134.4, 130.7, 125.0,
124.4, 121.2, 120.3, 119.7, 107.8, 77.1, 63.5, 46.7, 46.1, 33.1 and 14.5.
LCMS (ESI positive) m/z = 462.2 [M+H]+
; Elemental Analysis
Calculated C, 54.66; H, 5.02; N, 9.11; S, 6.95. Found C, 54.64; H, 5.00;
N, 9.13; S, 6.97.
4-(7-hydroxy-4-oxospiro[chroman-2,4′-piperidin]-1′-yl)-3-ni-
trobenzenesulfonamide (6 l):
3-nitro-4-(4-oxo-6-propoxyspiro[chroman-2,4′-piperidin]-1′-yl)ben-
zenesulfonamide (6f):
61% yield as a yellow solid. TLC: Rf: 0.15 (40% Acetone in pet
ether). m.p. 144 °C. 1H NMR in DMSO‑d6: δ 10.59 (br s, 1H), 8.21 (s,
1H, H-1), 7.87 (d, J = 8.4 Hz, 1H, H-3), 7.59–7.43 (m, 4H), 6.47–6.36
(m, 2H), 3.27–3.14 (m, 4H, H-4-4′), 2.75 (s, 2H, H-6), and 1.99–1.83
(m, 4H, H5-5′). 13C NMR in DMSO‑d6: δ 189.3, 164.8, 160.5, 147.3,
138.4, 134.4, 130.7, 128.0, 124.4, 121.2, 113.2, 110.2, 103.1, 77.4,
72% yield as a yellow solid. TLC: Rf: 0.16 (30% Acetone in pet
ether). m.p. 145 °C. 1H NMR in DMSO‑d6: δ 8.20 (s, 1H, H-1), 7.87 (d,
J = 8.4 Hz, 1H, H-3), 7.46 (d, J = 8.8 Hz , 1H, H-9), 7.40 (br s, 2H,
SO2NH2), 7.19–7.16 (m, 2H, H-7, H-10), 7.03 (d, J = 8.4 Hz, 1H, H-2),
3.89 (brs, 2H), 3.32–3.28 (m, 2H, H-4), 3.19 (m, 2H, H-4′), 2.86 (s, 2H,
H-6), 2.00 (d, J = 8 Hz, 2H), 1.84–1.83 (m, 2H, H-5), 1.70–1.69 (m, 2H,
H-5′) and 0.96 (t, J = 6 Hz, 3H). 13C NMR in DMSO‑d6: δ 191.2, 152.7,
46.5, 46.1 and 33.4. LCMS (ESI positive) m/z = 434.2 [M+H]+
;
Elemental Analysis Calculated, C, 52.65; H, 4.42; N, 9.69; S, 7.40.
Found, C, 52.64; H, 4.40; N, 9.71; S, 7.38.
8

Y. Kalisha Vali, et al.
BioorganicChemistry92(2019)103210
4-(7-methoxy-4-oxospiro[chroman-2,4′-piperidin]-1′-yl)-3-ni-
trobenzenesulfonamide (6m):
3-nitrobenzenesulfonamide (6 s):
73% yield as a yellow solid. TLC: Rf: 0.28 (40% Acetone in pet
ether). m.p. 105 °C. 1H NMR in DMSO‑d6: δ 8.21 (s, 1H, H-1), 7.87 (d,
J = 8 Hz, 1H, H-3), 7.65 (d, J = 8 Hz, 1H, H-7), 7.47 (d, J = 8 Hz , 1H,
H-2), 7.41 (br s, 2H, SO2NH2), 6.63 (m, 2H, H-8,H-10), 4.16 (t,
J = 6 Hz, 2H), 3.64 (t, J = 6 Hz, 2H), 3.31 (m, 2H, H-4), 3.29 (s, 3H),
3.20 (m, 2H, H-4′), 2.8 (s, 2H), 2.00 (m, 2H, H-5) and 1.84 (m, 2H, H-
5′). 13C NMR in DMSO‑d6: δ 189.6, 165.0, 160.5, 147.3, 138.3, 134.4,
130.7, 127.6, 124.4, 121.1, 114.2, 109.8, 102.0, 77.7, 70.0, 67.5, 58.1,
74% yield as a yellow solid. TLC: Rf: 0.15 (30% Acetone in pet
ether). m.p. 110 °C. 1H NMR in DMSO‑d6: δ 8.20 (s, 1H, H-1), 7.87 (d,
J = 8.4 Hz, 1H, H-3), 7.66 (d, J = 8 Hz , 1H, H-7), 7.46 (d, J = 8 Hz,
1H, H-2), 7.40 (br s, 2H, SO2NH2), 6.61 (m, 2H, H-8, H-10), 3.81 (s,
3H), 3.30–3.28 (m, 2H, H-4), 3.20–3.18 (m, 2H, H-4′), 2.80 (s, 2H, H-
6), 2.02 (m, 2H, H-5) and 1.87–1.84 (m, 2H, H-5′). 13C NMR in
DMSO‑d6: δ 189.6, 165.8, 160.5, 147.2, 138.3, 134.4, 130.7, 127.5,
124.3, 121.1, 114.1, 109.5, 101.5, 77.7, 55.7, 46.4, 46.1 and 33.3.
46.5, 46.1 and 33.3. LCMS (ESI positive) m/z = 492.2 [M+H]+
;
LCMS (ESI positive) m/z = 448.2 [M+H]+
;
Elemental Analysis
Elemental Analysis Calculated C, 53.76; H, 5.13; N, 8.55; S, 6.52.
Found, C, 53.75; H, 5.12; N, 8.56; S, 6.51.
Calculated, C, 53.69; H, 4.73; N, 9.39; S, 7.16. Found, C, 53.67; H, 4.72;
N, 9.40; S, 7.18.
4-(7-bromo-4-oxospiro[chroman-2,4′-piperidin]-1′-yl)-3-ni-
trobenzenesulfonamide (6u):
4-(7-ethoxy-4-oxospiro[chroman-2,4′-piperidin]-1′-yl)-3-ni-
trobenzenesulfonamide (6n):
73% yield as a yellow solid. TLC: Rf: 0.3 (30% Acetone in pet ether).
m.p. 99 °C. 1H NMR in DMSO‑d6: δ 8.20 (s, 1H, H-1), 7.88 (d,
J = 7.2 Hz, 1H, H-3), 7.65 (d, J = 8.4 Hz, 1H, H-7), 7.47 (d, J = 8.8 Hz
, 1H, H-2), 7.41 (m, 3H), 7.25 (d, J = 8.4 Hz, 1H, H-8), 3.19–3.16 (m,
2H, H-4), 3.09–3.07 (m, 2H, H-4′), 2.91 (s, 2H, H-6), 2.00 (m, 2H, H-5)
and 1.88–1.85 (m, 2H, H-5′). 13C NMR in DMSO‑d6: δ 190.6, 159.0,
147.2, 138.5, 134.5, 130.7, 129.5, 127.6, 124.4, 124.3, 121.2, 119.5,
78.4, 46.0, 45.7 and 33.1. LCMS (ESI positive) m/z = 495.9 & 497.9 [Br
pattern, M+H]+; Elemental Analysis Calculated C, 45.98; H, 3.66; N,
8.47; S, 6.46. Found, C, 45.97; H, 3.65; N, 8.45; S, 6.48.
56% yield as a yellow solid. TLC: Rf: 0.15 (30% Acetone in pet
ether). m.p. 131 °C. 1H NMR in DMSO‑d6: δ 8.21 (s, 1H, H-1), 7.87 (d,
J = 8 Hz, 1H, H-3), 7.65 (d, J = 8 Hz, 1H, H-7), 7.46 (d, J = 8.8 Hz ,
1H, H-2), 7.41 (br s, 2H, SO2NH2), 6.59 (m, 2H, H-8,H-10), 4.09 (q,
J = 1.5 Hz, 2H), 3.29 (m, 2H, H-4), 3.20 (m, 2H, H-4′), 2.79 (s, 2H, H-
6), 2.00 (m, 2H, H-5), 1.84 (m, 2H, H-5′) and 1.31 (t, J = 6 Hz, 3H). 13
C
NMR in DMSO‑d6: δ 189.6, 165.1, 160.5, 147.3, 138.3, 134.3, 130.7,
127.6, 124.4, 121.1, 114.0, 109.8, 101.9, 77.7, 63.8, 46.5, 46.1, 33.3
and 14.3. LCMS (ESI positive) m/z = 462.2 [M+H]+; Elemental
Analysis Calculated, C, 54.66; H, 5.02; N, 9.11; S, 6.95. Found, C, 54.68;
H, 5.01; N, 9.12; S, 6.97.
4-(6-bromo-4-oxospiro[chroman-2,4′-piperidin]-1′-yl)-3-ni-
trobenzenesulfonamide (6v):
3-nitro-4-(4-oxo-7-propoxyspiro[chroman-2,4′-piperidin]-1′-yl)ben-
zenesulfonamide (6o):
85% yield as an orange solid. TLC: Rf: 0.3 (30% Acetone in pet
ether). m.p. 103 °C. 1H NMR in DMSO‑d6: δ 8.21 (s, 1H, H-1), 7.93–7.77
(m, 3H), 7.47–7.41 (m, 3H), 7.11 (s, 1H, H-10), 3.39–3.31 (m, 2H, H-4),
3.13–3.08 (m, 2H, H-4′), 2.93 (s, 2H, H-6), 2.00 (m, 2H, H-5) and
1.88–1.85 (m, 2H, H5′). 13C NMR in DMSO‑d6: δ 190.3, 157.6, 147.2,
138.7, 138.4, 134.5, 130.7, 127.8, 124.3, 121.8, 121.2, 121.1, 112.8,
78.0, 47.2, 47.0 and 36.0. LCMS (ESI positive) m/z = 496.2 & 498.2 [Br
pattern, M+H]+; Elemental Analysis Calculated C, 45.98; H, 3.66; N,
8.47; S, 6.46. Found, C, 45.97; H, 3.64; N, 8.45; S, 6.47.
70% yield as a yellow solid. TLC: Rf: 0.15 (30% Acetone in pet
ether). m.p. 145 °C. 1H NMR in DMSO‑d6: δ 8.21 (s, 1H, H-1), 7.87 (d,
J = 8.4 Hz, 1H, H-3), 7.65 (d, J = 8 Hz, 1H, H-7), 7.46 (d, J = 8.8 Hz ,
1H, H-2), 7.41 (br s, 2H, SO2NH2), 6.60 (m, 2H, H-8,H-10), 3.99 (q,
J = 2.6 Hz, 2H), 3.29 (m, 2H, H-4), 3.32 (m,2H, H-4′), 3.19 (m, 2H),
2.79 (s, 2H, H-6), 2.00 (m, 2H, H-5), 1.84 (m, 2H, H-5′), 1.71 (m, 2H)
and 0.95 (t, J = 6 Hz, 3H). 13C NMR in DMSO‑d6: δ 189.6, 165.3, 160.5,
147.3, 138.3, 134.4, 130.7, 127.5, 124.4, 121.1, 114.0, 109.8, 101.9,
77.7, 69.5, 46.5, 46.1, 33.3, 21.7 and 10.2. LCMS (ESI positive) m/
z = 476.3 [M+H]+; Elemental Analysis Calculated, C, 55.57; H, 5.30;
N, 8.84; S, 6.74. Found, C, 55.56; H, 5.28; N, 8.83; S, 6.76.
4-(7-isopropoxy-4-oxospiro[chroman-2,4′-piperidin]-1′-yl)-3-ni-
trobenzenesulfonamide (6p):
7. Carbonic anhydrase inhibition
An Applied Photophysics stopped-flow instrument has been used for
assaying the CA catalyzed CO2 hydration activity [20]. Phenol red (at a
concentration of 0.2 mM) has been used as indicator, working at the
absorbance maximum of 557 nm, with 20 mMHepes (pH 7.5) as buffer,
and 20 mM Na2SO4 (for maintaining constant the ionic strength), fol-
lowing the initial rates of the CA-catalyzed CO2 hydration reaction for a
period of 10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM
for the determination of the kinetic parameters and inhibition con-
stants. For each inhibitor at least six traces of the initial 5–10% of the
reaction have been used for determining the initial velocity. The un-
catalyzed 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 0.01 nM
were done thereafter with the assay buffer. Inhibitor and enzyme so-
lutions 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 [23–26], and represent the mean from at least three different
determinations. All CA isofoms were recombinant ones obtained in-
house as reported earlier [23–26].
87% yield as a yellow solid. TLC: Rf: 0.18 (30% Acetone in pet
ether). m.p. 88 °C. 1H NMR in DMSO‑d6: δ 8.21 (s, 1H, H-1), 7.87 (d,
J = 8 Hz, 1H, H-3), 7.64 (d, J = 8 Hz , 1H, H-7), 7.47 (d, J = 8.8 Hz,
1H, H-2), 7.41 (br s, 2H, SO2NH2), 6.58–6.56 (m, 2H, H-8,H-10),
4.73–4.70 (m, 1H), 3.32–3.28 (m, 2H, H-4), 3.20–3.17 (m, 2H, H-4′),
2.78 (s, 2H, H-6), 2.01–1.98 (m, 2H, H-5), 1.86–1.83 (m, 2H, H-5′) and
1.27 (d, J = 4 Hz, 6H). 13C NMR in DMSO‑d6: δ 189.5, 164.2, 160.5,
147.3, 138.3, 134.3, 130.7, 127.6, 124.4, 121.1, 113.8, 110.3, 102.5,
77.6, 70.0, 46.5, 46.1, 33.3 and 21.6. LCMS (ESI positive) m/z = 476.2
[M+H]+; Elemental Analysis Calculated, C, 55.57; H, 5.30; N, 8.84; S,
6.74. Found, C, 55.55; H, 5.31; N, 8.83; S, 6.75.
4-(7-(2-hydroxyethoxy)-4-oxospiro[chroman-2,4′-piperidin]-1′-yl)-
3-nitrobenzenesulfonamide (6r):
52% yield as a yellow solid. TLC: Rf: 0.15 (40% Acetone in pet
ether). m.p. 74 °C. 1H NMR in DMSO‑d6: δ 8.21 (s, 1H, H-1), 7.87 (d,
J = 8 Hz, 1H, H-3), 7.65 (d, J = 8.4 Hz, 1H, H-7), 7.47 (d, J = 8.4 Hz ,
1H, H-2), 7.41 (br s, 2H, SO2NH2), 6.61 (m, 2H, H-8,H-10), 4.9 (br s,
1H, –OH), 4.05 (t, J = 6 Hz, 2H), 3.70 (m, 2H, H-4), 3.32 (m, 2H, H-4′),
3.20 (m, 2H), 2.8 (s, 2H, H-6), 2.00 (m, 2H, H-5) and 1.84 (m, 2H, H-
5′). 13C NMR in DMSO‑d6: δ 189.6, 165.3, 160.5, 147.2, 138.3, 134.3,
130.7, 127.5, 124.4, 121.1, 114.1, 109.8, 102.0, 77.7, 70.1, 59.2, 46.5,
46.1 and 33.3. LCMS (ESI positive) m/z = 478.2 [M+H]+; Elemental
Analysis Calculated, C, 52.82; H, 4.86; N, 8.80; S, 6.71. Found, C, 52.80;
H, 4.85; N, 8.81; S, 6.73.
8. Biological assays
Animals. Male CD-1 albino mice (Envigo, Varese, Italy) weighing
approximately 22–25 g at the beginning of the experimental procedure,
were used. Animals were housed in Ce.S.A.l (Centro Stabulazione
Animali da Laboratorio, University of Florence) and used at least
4-(7-(2-methoxyethoxy)-4-oxospiro[chroman-2,4′-piperidin]-1′-yl)-
9

Y. Kalisha Vali, et al.
BioorganicChemistry92(2019)103210
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26 × 41 cm). Animals were fed a standard laboratory diet and tap
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the Directive 2010/63/EU of the European parliament and of the
European Union council (22 September 2010) on the protection of
animals used for scientific purposes. The ethical policy of the University
of Florence complies with the Guide for the Care and Use of Laboratory
Animals of the US National Institutes of Health (NIH Publication No.
85-23, revised 1996; University of Florence assurance number: A5278-
01). Formal approval to conduct the experiments described was ob-
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Florence. Experiments involving animals have been reported according
to ARRIVE guideline. All efforts were made to minimize animal suf-
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Declaration of Competing Interest
The authors state no conflict of interests.
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