Development of certain new 2-substituted-quinazolin-4-ylaminobenzenesulfonamide as potential antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2016.01.001

Carbonic anhydrases (CA I, II, IX and XII) are known to be highly expressed in various human malignancies. CA IX is overexpressed in colorectal cancer specifically in hereditary nonpolyposis colorectal cancer. Inhibition of CA activity by small molecular CA inhibitor like sulphonamides, sulphonamide derivative (SU.D2) or HIF1a inhibitor Chetomin leads to inhibition of tumorigenesis. Eighteen new quinazolin-4-sulfonamide derivatives were prepared and characterized by means of IR, NMR and mass spectra. Certain selected derivatives were tested for their ability to inhibit four isoforms of the metalloemzyme CA, namely, CA I, CA II, CA IX and CA XII. Compound 3c was found to be highly effective in inhibiting the cancer cell proliferation. 3c decreased cell viability of human HT-29 cells in dose and time dependent manner and with IC50 of 5.45 mM. Moreover, it was tested on metastatic colon cancer cell SW-620 where it was found to be equally effective on human SW-620 cells. This novel compound inhibited the CA IX and CA XII protein expression in HT-29 cells without affecting CA I and CA II expression. These findings indicate that 3c inhibits cellular proliferation in two human colon cancer cells by specifically targeting the CA IX and CA XII expression.

Full text of DOI:10.1016/j.ejmech.2016.01.001

Accepted Manuscript
Development of certain new 2-substituted-quinazolin-4-yl-aminobenzenesulfonamide
as potential antitumor agents
Ahmed M. Alafeefy, Rehan Ahmad, Maha Abdulla, Wagdy M. Eldehna, Abdul-Malek
S. Al-Tamimi, Hatem A. Abdel-Aziz, Omar Al-Obaid, Fabrizio Carta, Abdulla A. Al-
Kahtani, Claudiu T. Supuran
PII:
S0223-5234(16)30001-0
10.1016/j.ejmech.2016.01.001
EJMECH 8291
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 November 2015
Revised Date: 18 December 2015
Accepted Date: 4 January 2016
Please cite this article as: A.M. Alafeefy, R. Ahmad, M. Abdulla, W.M. Eldehna, A.-M.S. Al-Tamimi,
H.A. Abdel-Aziz, O. Al-Obaid, F. Carta, A.A. Al-Kahtani, C.T. Supuran, Development of certain new 2-
substituted-quinazolin-4-yl-aminobenzenesulfonamide as potential antitumor agents, European Journal
of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.01.001.
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ACCEPTED MANUSCRIPT
Graphical abstract
Compound 3c noticeably decreased viability of human colorectal cancer HT-29 cells in dose and time
dependent manner with IC = 5.45 µM. It was equally effective on human colorectal cancer SW-620
5
0
cells. Moreover, it inhibited the CA IX and CA XII protein expression without affecting CA I and CA
II expression in HT-29 cells. These findings thus indicate that 3c inhibits cellular proliferation in HT-29
and SW-620 cells by selectively targeting the CA IX and CA XII expression.
O O
S
NH2
HN
N
N
3
c
Cl

ACCEPTED MANUSCRIPT
Development of certain new 2-substituted-quinazolin-4-yl-aminobenzenesulfonamide
as potential antitumor agents
a,*
b*
b
c
d
Ahmed M. Alafeefy , Rehan Ahmad , Maha Abdulla , Wagdy M. Eldehna , Abdul-Malek S. Al-Tamimi ,
e,f,*
b
g
d,h
g
Hatem A. Abdel-Aziz , Omar Al-Obaid , Fabrizio Carta , Abdulla A. Al-Kahtani , Claudiu T. Supuran
a
Department of Chemistry, Kulliyyah of Science, International Islamic University, P.O. Box 141, 25710
Kuantan, Malyasia.
b
Colorectal Research Chair, Department of Surgery, King Khalid University Hospital, King Saud University
College of Medicine, Riyadh 11451, Saudi Arabia.
c
Department of Pharmaceutical Chemistry, College of Pharmacy, Egyptian Russian University, Badr City,
Cairo, P.O. Box 11829, Egypt.
d
Department of Pharmaceutical Chemistry, College of Pharmacy, Prince Sattam Bin Abdulaziz University,
P.O. Box 173, Alkharj, 11942, Saudi Arabia.
e
Department of Applied Organic Chemistry, National Research Center, Dokki, Giza, P.O. Box 12622,
Egypt.
f
Pharmaceutical Research Department, Center of Excellence for Advanced Sciences, National Research
Centre, P.O. Box 12622, Cairo, Egypt.
g
Università degli Studi di Firenze, NEUROFARBA Department, Sezione di Scienze Farmaceutiche e
Nutraceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), Italy
h
Chemistry Department, Faculty of Science, King Saud University, P.O. Box 2455, Riyadh 11451,
Kingdom of Saudi Arabia
Keywords: Quinazoline; Sulfonamides; Sulfonylthioureas; Carbonic Anhydrase; Antitumor.
*
Corresponding authors: alafeefy@hotmail.com, Tel: + 966-50-706 9896, hatem_741@yahoo.com,
Tel: +0201009511215 and arehan@ksu.edu.sa Tel: +966540220070
1

ACCEPTED MANUSCRIPT
Abstract
Carbonic anhydrases (CA I, II, IX and XII) are known to be highly expressed in various
human malignancies. CA IX is overexpressed in colorectal cancer specifically in hereditary
nonpolyposis colorectal cancer. Inhibition of CA activity by small molecular CA inhibitor like
sulphonamides, sulphonamide derivative (SU.D2) or HIF1a inhibitor Chetomin leads to inhibition
of tumorigenesis. Eighteen new quinazolin-4-sulfonamide derivatives were prepared and
characterized by means of IR, NMR and mass spectra. Certain selected derivatives were tested for
their ability to inhibit four isoforms of the metalloemzyme CA, namely, CA I, CA II, CA IX and
CA XII. Compound 3c was found to be highly effective in inhibiting the cancer cell proliferation.
3
c decreased cell viability of human HT-29 cells in dose and time dependent manner and with IC50
of 5.45 ꢀM. Moreover, it was tested on metastatic colon cancer cell SW-620 where it was found to
be equally effective on human SW-620 cells. This novel compound inhibited the CA IX and CA
XII protein expression in HT-29 cells without affecting CA I and CA II expression. These findings
indicate that 3c inhibits cellular proliferation in two human colon cancer cells by specifically
targeting the CA IX and CA XII expression.
2

ACCEPTED MANUSCRIPT
1
. Introduction
Cancer as a dreadful disease was responsible for about 13% of the total worldwide mortality. It
is the second major cause of death, and is projected to become the world’s single leading cause of
morbidity by 2030. Colorectal cancer is the second leading cause of cancer related deaths
rd
worldwide and the 3 most common cancer in USA among men and women. According to
American Cancer Society data, 93,090 colon cancer and 39610 rectal cancers are diagnosed in 2015
[
1]. In spite of the substantial progress and efforts in many aspects of cancer research, there is very
little progress as indicated by the scarcity of candidates reaching the clinical trials phase [2].
Several problems delay the reach to an ideal drug that could effectively combat cancer. The
complexity of tumors, the lack of discrimination between healthy and tumor cell in addition to the
development of drug resistance are some of such obstacles [3].
Tumor growth depends on multiple factors, like activation of various signaling pathway
including the physiological process of angiogenesis. There are two major research directions in this
respect, the first one rely on the opportunities for tumor inhibition with mono-targeted therapies
and are currently being evaluated for clinical efficacy. In this approach, a single pathway is blocked
with a specific agent in combination regimens with cytotoxins [4].
However, the other approach suggested that optimal therapy may require inhibition of several
signaling pathways including angiogenesis. This understanding directed researchers to speculate
that compounds which simultaneously inhibit multiple pathways are preferred to achieve maximal
clinical efficacy. The feasibility of identifying these multiplex inhibitors was demonstrated with the
entry of several such inhibitors into clinical trials [5].
In many types of hypoxic tumors, two isoforms of the metalloenzyme carbonic anhydrase (CA)
are highly overexpressed, CA IX and XII, but they lack from normal tissues [6]. They are involved
in tumor acidification, metastasis and invasion and their inhibition leads to a profound antitumor
effect and are commonly referred to as the tumor associated isoforms [7]. Promising model
antitumor agents, based on our search for mono-targeted agents, that hopefully are anticipated to
inhibit CA IX and/or CA XII, with limited or no noticeable inhibitory effect on CA I and/or CA II,
so as to minimize the untoward effect are receiving a considerable interest.
Based on the fact that various sulfonamide derivatives are potent CA inhibitors, this became
clear from the numerous research publications, patents and candidates in clinical trials reporting
their ability to concentrate in hypoxic tumors and effectiveness to combat cancer [8-11]. Herein, we
propose to synthesize novel sulfonamides linked to the quinazoline scaffold for testing their ability
to inhibit CA I, II, IX and XII expression.
3

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2
. Results and discussion
2
.1. Chemistry
The target quinazoline derivatives 3a-p were prepared via a generalized route are depicted in
Scheme 1. The key intermediates 4-chloroquinazolines 2a-h, were prepared via heating the
quinazoline derivatives 1a-h under reflux temperature with phosphorus oxychloride in presence of
catalytic amount of N,N-dimethylaniline which act as acid binder [11-15].
The target compounds 3a-p was synthesized by condensing sulfanilamide or sulfathiazole
with corresponding 4-chloroquinazolines 2a-h in refluxing 2-propanol. The I.R. spectra of
-1
compounds 3a-p showed two absorption bands at 1370 and 1190 cm for SO in addition to the
2
-1
absorption bands of NH and NH groups in the region 3400-2330 cm for compounds 3a-h and
2
-1
absorption bands of 2 NH groups in the region δ 3300-3200 cm for compounds 3i-p whereas,
1
their H NMR showed D O exchangeable signals of NH and NH groups in the expected regions δ
2
2
1
1.0-12.0 and δ 7.30-7.50, respectively. Their mass spectra revealed, in each case, a peak
corresponding to the molecular ion.
“
Please Insert Scheme 1 Here”
Furthermore, the reaction of compounds 3c, e with phenyl isothiocyanate in the presence of
dry potassium carbonate in refluxing dry acetone afforded sulfonylthioureas derivatives 4a, b.
-1
-1
The IR spectra of these compounds exhibited two bands at 1380 cm and 1190 cm due to SO
2
-1
1
group as well as thiourea carbonyl absorption at 1715 cm . The H NMR of these compounds
revealed 3 D O exchangeable signals of 3 NH protons around 8.80, 9.70 and 11.80.
2
2
.2 Effect of compounds on cell viability
All of the eighteen compounds were tested for cell viability on human colon cancer cell line HT-29.
Compound 3c was found to inhibit cell viability significantly to 12% (Fig 1A). Effect of various
concentration of 3c was tested on the cell viability of HT-29. Compound 3c decreased cell viability
in a dose dependent (Fig 1B) and time dependent (Fig 1C) manner. The IC50 for compound 3c was
found to be 5.45µM. The effect of 3c was also assessed on another colon cancer cell line SW-620.
Similar to earlier finding with HT-29, 3c also inhibited cell viability significantly in SW-620
metastatic colon cancer cells (Fig 1D). Taken together these findings thus indicate that 3c inhibits
cell proliferation in human colon cancer cell lines.
4

ACCEPTED MANUSCRIPT
2
.3. Inhibition of Carbonic anhydrase IX and XII expression
Human colon cancer cell line HT-28 was known to express high level of CA IX [16]. To assess the
effect of compound 3c on the CA IX expression, we treated human HT-29 cells with increasing
amount of compound 3c. Treatment with compound 3c was associated with a concentration
dependent inhibition in CA IX expression (Fig 2). Similar inhibition was also found for CA XII.
However this compound has no effect on CA I and CA II expression. Equal loading was confirmed
by immunoblotting with antibody against β-actin (Fig 2). These findings indicate that compound 3c
inhibits CA IX and CA XII expression. Compound 3c was found to be very effective in reducing
CA IX and CA XII expression in human colon cancer cells. CA IX plays important role in
producing and maintaining an intracellular pH favorable for tumor cell growth and survival along
with increasing the acidic extracellular space thereby enhancing cancer cell invasion [17]. Blocking
CA IX expression would disrupt pH regulation and impair tumor growth and metastasis. Depletion
of CA IX in breast cancer xenografts resulted in primary tumor growth attenuation. Colorectal
cancer cell xenografts also regressed with CA IX and CA XII depletion [18]. CA IX and CA XII
expression in cancer cells is regulated by the activation of PI3K and component of MAPK pathway
during hypoxia [19, 20]. The possible mechanism for downregulation of CA expression might
result from inhibition of PI3K and MAPK pathway. Another principal regulator of CA transcription
is HIF1α [21]. The inhibition of CA IX and CA XII protein expression might result from decrease
in HIF1α binding to HRE (hypoxia responsive element) present in CA promoter and thereby
inhibiting CA transcription.
3
. Conclusion
The impaired removal of CO during increased metabolic production and accumulation of lactic
2
acid can result in a highly acidic microenvironment. Adaptation of tumor cells to this acidic
environment is important for cancer cell survival. This adoption to acidic environment is being
coordinated by increased expression of CA specifically CA IX and CA XII. Both of them are
known to express highly in numerous cancer types. CA IX is a biomarker for poor clinical
outcome. CA is an important target for anticancer drug discovery. Most of biochemical data point
to targeting CA IX by developing small molecule inhibitors. These inhibitors inhibit tumor-
associated CA IX for hypoxic tumors. Targeting CA would provide therapeutic agents that might
alter the extracellular acidification induced by CA IX and CA XII. Many small molecule inhibitors
5

ACCEPTED MANUSCRIPT
of CA IX have been developed and pharmalogical evaluation is still going on. This study
demonstrates that this new derivative of sulphonamide inhibits cellular proliferation of colon cancer
cells. This inhibitor also demonstrates its specificity by targeting CA IX and CA XII protein
expression not all the CAs like CAI and CA II. In this regard CA IX and CA XII specific inhibitors,
such as 3c would provide greater selectivity. This inhibition in CA expression might be at the
transcription level by involving HIF1α as well as by blocking PI3K and MAPK pathway. This
compound may be developed for treating colon tumor or other solid tumor which are
overexpressing CA IX and CA XII. This new derivative of sulphonamide needs to be tested for its
efficacy in combination studies along with doxorubicin and other known anticancer agents.
4
. Experimental
4
4
.1. Chemistry
.1.1. General
Melting points (ºC, uncorrected) were determined in open capillaries on a Gallenkamp melting
point apparatus (Sanyo Gallenkamp, Southborough, UK) and were uncorrected. Precoated silica gel
plates (silica gel 0.25 mm, 60G F254; Merck, Germany) were used for thin layer chromatography,
dichloromethane/methanol (9.5:0.5) mixture was used as a developing solvent system and the spots
were visualized by ultraviolet light and/or iodine. Infra-red spectra were recorded in KBr discs
1
using IR-470 Shimadzu spectrometer (Shimadzu, Tokyo, Japan). H NMR spectra were recorded
on Bruker AC-300 Ultra Shield NMR spectrometer (δ ppm; Bruker, Flawil, Switzerland) at 300
1
13
MHz for H and 75 MHz for C, using TMS as internal standard and peak multiplicities were
designed as follows: s, singlet; d, doublet; t, triplet; m, multiplet. Electron Impact Mass Spectra
were recorded on a Shimadzu GC-MS-QP 5000 instrument (Shimadzu, Tokyo, Japan). Elemental
analyses were performed, on Carlo Erba 1108 Elemental Analyzer (Heraeus, Hanau, Germany), at
the Micro-analytical Unit, Faculty of Science, Cairo University, Cairo, Egypt, and the found results
were within ±0.4% of the theoretical values. During the synthesis, reaction progress was monitored
using analytical thin layer chromatography (TLC) on silica gel plates. All solvents and reagents
were purified and dried by standard techniques.
4
.1.2. General procedure for preparation of 4-chloroquinazolines 2a-h.
6

ACCEPTED MANUSCRIPT
To a magnetically stirred solution of phosphorus oxychloride (20 mL) and N,N-
o
dimethylaniline (1 mL) at 0 C was added portion wise 2-(un-substituted/substituted) quinazolin-
4
(3H)-one 1a-h (3.0 g). The reaction mixture was refluxed for 8 h. The reaction mixture was then
poured onto ice water and the pH adjusted to alkaline with 2 N NaOH. The aqueous solution was
extracted three times with dichloromethane. The combined organic layers were dried over Na SO4
2
and filtered, and the solvent was removed by distillation. The obtained solid was recrystallized
from ethanol to give 2a-h [11-15].
4
.1.3. General procedure for preparation of compounds 3a-p.
To a stirred solution of 4-chloroquinazolines 2a-h (1 mmol) in refluxing 2-propanol (15 mL),
a solution of sulfanilamide or sulfathiazole (1 mmol) in 2-propanol was added. The reaction
mixture was refluxed for 3 h. The precipitate formed was collected by filtration while hot and
washed with hot 2-propanol and then with water and crystallized from ethanol to give compounds
3
a-p, respectively.
4
.1.3.1. 4-(Quinazolin-4-ylamino)benzenesulfonamide (3a). Yield (66%); m.p. >300 °C; IR ν 3409,
−
1 1
3
377, 3231 (NH, NH ), 3114 (CH arom.), 1366, 1207 (SO ), cm ; H NMR (DSMO-d ) δ 7.37
2 2 6
(
brs, 2H, NH , D O exchange.), 7.62-7.65 (m, 2H, Ar-H), 7.95-7.98 (m, 2H, Ar-H), 8.05-8.15 (m,
2 2
1
H, Ar-H), 8.39-8.42 (m, 2H, Ar-H), 8.80-8.82 (m, 2H, Ar-H), 11.10 (s, 1H, NH, D O, exchange.);
2
1
3
C NMR (DSMO-d ) δ 113.6, 119.6, 124.9, 125.1, 126.3, 128.8, 136.5, 138.6, 139.6, 141.6, 150.9,
6
+
1
60.1; MS m/z (Rel. Int.) 300 (M , 34). Anal. (C H N O S 300.07). C, 55.99 (56.26); H, 4.03
14
12
4
2 ,
(4.28); N, 18.66 (18.43); S, 10.67 (10.83).
4
.1.3.2. 4-((2-Phenylquinazolin-4-yl)amino)benzenesulfonamide (3b). Yield (66%); m.p. 292-294
−1 1
°
C; IR ν 3416, 3375, 3226 (NH, NH ), 3108 (CH arom.), 1371, 1197 (SO ), cm ; H NMR
2 2
(
DSMO-d ) δ 7.45 (brs, 2H, NH , D O, exchange.), 7.67-7.64 (m, 3H, Ar-H), 7.72-7.69 (t, J=7.2
6 2 2
Hz 2H, Ar-H), 7.98-7.97 (d, J=8.6 Hz, 2H, Ar-H), 8.26-8.24 (d, J=8.3 Hz, 2H, Ar-H), 8.39-8.37 (d,
1
3
J=7.7 Hz, 2H, Ar-H), 8.91-8.90 (d, J=8.2 Hz, 2H, Ar-H), 11.54 (s, 1H, NH, D O, exchange.); C
2
NMR (DSMO-d ) δ 113.1, 116.5, 120.3, 123.8, 126.4, 126.5, 127.5, 128.7, 129.0, 131.5, 133.7,
6
+
1
35.4, 139.1, 155.3, 163.6, 171.1; MS m/z (Rel. Int.) 376 (M , 22). Anal. (C H N O S 376.10).
20
16
4
2 ,
C, 63.81 (63.65); H, 4.28 (4.02); N, 14.88 (14.69); S, 8.52 (8.38).
7

ACCEPTED MANUSCRIPT
4
.1.3.3. 4-((2-(4-Chlorophenyl)quinazolin-4-yl)amino)benzenesulfonamide (3c). Yield (61%); m.p.
−
1 1
2
81-285 °C; IR ν 3415, 3372, 3229 (NH, NH ), 3114 (CH arom.), 1375, 1196 (SO ) cm ; H NMR
2 2
(DSMO-d ) δ 7.49 (brs, 2H, NH , D O, exchange.), 7.66-7.68 (d, J= 8.5 Hz, 2H, Ar-H), 7.77-7.80
6 2 2
(t, J=7.6 Hz, 2H, Ar-H), 7.96-7.98 (d, J=8.6 Hz, 2H, Ar-H), 8.04-8.09 (m, 2H, Ar-H), 8.31-8.33 (d,
J=7.7 Hz 2H, Ar-H), 8.36-8.38 (d, J=7.9 Hz, 1H, Ar-H), 8.98-9.0 (d, J=7.9 Hz, 1H, Ar-H), 11.77
1
3
(
s, 1H, NH, D O, exchange.); C NMR (DSMO-d ) δ 112.8, 116.5, 121.3, 123.9, 124.6, 126.3,
2
6
+
1
2
28.2, 129.0, 130.8, 135.9, 138.1, 140.1, 140.9, 156.4, 158.8, 165.3; MS m/z (Rel. Int.) 412 (M +
+
, 11), 410 (M , 34). Anal. (C H ClN O S 410.06). C, 58.46 (58.31); H, 3.68 (3.49); N, 13.64
2
0
15
4
2 ,
(13.85); S, 7.80 (8.04).
4
.1.3.4. 4-((2-(4-(Dimethylamino)phenyl)quinazolin-4-yl)amino)benzenesulfonamide (3d). Yield
(
59%); m.p. >300°C; IR ν 3412, 3379, 3224 (NH, NH ), 3113 (CH arom.), 2986, 2869 (CH aliph.),
2
−
1 1
1
371, 1192 (SO ) cm ; H NMR (DSMO-d ) δ 2.50 (s, 6H, 2CH ), 6.86-6.88 (d, J= 8.9 Hz, 2H,
2 6 3
Ar-H), 7.48 (brs, 2H, NH , D O, exchange.), 7.73-7.76 (t, J=7.6 Hz, 2H, Ar-H), 7.97-8.10 (m, 2H,
2
2
Ar-H), 8.21-8.28 (m, 4H, Ar-H), 8.81-8.82 (d, J=8.3 Hz 2H, Ar-H), 11.50 (s, 1H, NH, D O,
2
1
3
exchange.); C NMR (DSMO-d ) δ 40.8 (2CH ), 111.5, 115.9, 121.2, 124.5, 126.3, 128.1, 129.4,
6
3
+
1
30.8, 131.0, 135.6, 138.2, 140.6, 141.7, 155.7, 158.3, 173.5; MS m/z (Rel. Int.) 419 (M , 22).
Anal. (C H N O S 419.14). C, 62.99 (63.27); H, 5.05 (4.83); N, 16.69 (16.83); S, 7.64 (7.40).
2
2
21
5
2 ,
4
.1.3.5. 4-((2-(4-Methoxyphenyl)quinazolin-4-yl)amino)benzenesulfonamide (3e). Yield (71%);
m.p. 295-297 °C; IR ν 3418, 3379, 3221 (NH, NH ), 3116 (CH arom.), 2985, 2874 (CH aliph.),
2
−1 1
1
366, 1189 (SO ) cm ; H NMR (DSMO-d ) δ 3.75 (s, 3H, OCH ), 7.49 (brs, 2H, NH , D O,
2 6 3 2 2
exchange.), 7.78-7.81 (t, J=7.7 Hz, 2H, Ar-H), 7.97-7.99 (d, J=7.1 Hz, 2H, Ar-H), 8.06-8.14 (m,
H, Ar-H), 8.32-8.40 (m, 2H, Ar-H), 8.93-8.95 (d, J=7.0 Hz, 2H, Ar-H), 11.73 (s, 1H, NH, D O,
4
2
1
3
exchange.); C NMR (DSMO-d ) δ 55.7 (OCH ), 112.5, 114.6, 120.4, 121.3, 124.2, 124.6, 126.3,
6
3
+
1
27.8, 131.4, 136.0, 139.9, 141.0, 156.7, 158.8, 163.6, 170.1; MS m/z (Rel. Int.) 406 (M , 15).
Anal. (C H N O S 406.11). C, 62.06 (61.85); H, 4.46 (4.65); N, 13.78 (13.95); S, 7.89 (7.62).
2
1
18
4
3 ,
4
.1.3.6. 4-((2-(3,4-Dimethoxyphenyl)quinazolin-4-yl)amino)benzenesulfonamide (3f). Yield (63%);
m.p. 294-296 °C; IR ν 3421, 3378, 3216 (NH, NH ), 3114 (CH arom.), 2981, 2875 (CH aliph.),
2
−
1 1
1
362, 1188 (SO ) cm ; H NMR (DSMO-d ) δ 3.77 (s, 6H, 2OCH ), 7.19-7.21 (d, J=8.7 Hz, 2H,
2 6 3
Ar-H), 7.49 (brs, 2H, NH , D O, exchange.), 7.79-7.82 (t, J=7.6 Hz, 2H, Ar-H), 7.96-7.98 (d,
2
2
J=8.6 Hz, 2H, Ar-H), 8.02-8.13 (m, 1H, Ar-H), 8.39-8.40 (d, J=8.4 Hz, 2H, Ar-H), 8.86-8.88 (d,
8

ACCEPTED MANUSCRIPT
1
3
J=8.4 Hz, 2H, Ar-H), 11.69 (s, 1H, NH, D O, exchange.); C NMR (DSMO-d ) δ 55.7, 55.8
2
6
(
2OCH ), 112.4, 114.7, 120.1, 121.4, 124.1, 124.4, 126.2, 127.7, 132.0, 135.9, 139.4, 148.6, 155.2,
3
+
1
58.5, 163.8, 165.3; MS m/z (Rel. Int.) 436 (M , 25). Anal. (C H N O S 436.12). C, 60.54
2
2
20
4
4 ,
(60.37); H, 4.62 (4.81); N, 12.84 (13.05); S, 7.35 (7.67).
4
.1.3.7. 4-((2-(Benzo[d][1,3]dioxol-5-yl)quinazolin-4-yl)amino)benzenesulfonamide (3g). Yield
(
65%); m.p. 278-281 °C; IR ν 3427, 3373, 3227 (NH, NH ), 3118 (CH arom.), 2985, 2878 (CH
2
−
1 1
aliph.), 1365, 1183 (SO ) cm ; H NMR (DSMO-d ) δ 6.19 (s, 2H, OCH O), 7.14-7.15 (d, J=8.3
2
6
2
Hz, 2H, Ar-H), 7.48 (brs, 2H, NH , D O, exchange.), 7.61-7.63 (m, 2H, Ar-H), 7.75-7.77 (t, J=7.7
2
2
Hz, 2H, Ar-H), 7.87-8.0 (m, 3H, Ar-H), 8.24-8.26 (d, J=8.3 Hz, 2H, Ar-H), 11.58 (s, 1H, NH, D O,
2
1
3
exchange.); C NMR (DSMO-d ) δ 102.3, 108.3, 108.7, 112.7, 114.6, 120.3, 121.6, 124.0, 124.4,
6
+
1
25.2, 126.3, 127.7, 132.0, 135.7, 140.1, 147.9, 158.6, 163.7, 165.1; MS m/z (Rel. Int.) 420 (M ,
1). Anal. (C H N O S 420.09). C, 59.99 (60.18); H, 3.84 (4.05); N, 13.33 (13.52); S, 7.63
2
2
1
16
4
4 ,
(7.79).
4
.1.3.8. 4-((2-(3,4,5-Trimethoxyphenyl)quinazolin-4-yl)amino)benzenesulfonamide (3h). Yield
(
60%); m.p. 275-277 °C; IR ν 3423, 3375, 3232 (NH, NH ), 3112 (CH arom.), 2983, 2876 (CH
2
−
1 1
aliph.), 1365, 1189 (SO ) cm ; H NMR (DSMO-d ) δ 3.78 (s, 9H, 3OCH ), 6.81 (s, 2H, Ar-H),
2
6
3
7
.48 (brs, 2H, NH , D O, exchange.), 7.51-7.82 (d, J=7.6 Hz, 2H, Ar-H), 7.62-7.64 (d, J=7.8 Hz,
2 2
2
H, Ar-H), 8.44-8.46 (d, J=8.4 Hz, 2H, Ar-H), 8.82-8.84 (d, J=8.3 Hz, 2H, Ar-H), 12.06 (s, 1H,
1
3
NH, D O, exchange.);; C NMR (DSMO-d ) δ 56.2, 60.3 (2OCH ), 106.7, 112.5, 114.2, 120.2,
2
6
3
1
21.5, 124.4, 124.6, 126.1, 127.6, 132.3, 135.8, 141.2, 152.9, 159.1, 163.6, 165.5; MS m/z (Rel.
+
Int.) 466 (M , 27). Anal. (C H N O S 466.13). C, 59.22 (58.96); H, 4.75 (4.92); N, 12.01
2
3
22
4
5 ,
(12.25); S, 6.87 (7.07).
4
2
6
.1.3.9. 4-(Quinazolin-4-ylamino)-N-(thiazol-2-yl)benzenesulfonamide (3i). Yield (69%); m.p. 275-
−
1 1
77 °C; IR ν 3354, 3321 (2NH), 3117 (CH arom.), 1372, 1186 (SO ) cm ; H NMR (DSMO-d ) δ
2
6
.67-6.68 (d, J=8.4 Hz, 2H, Ar-H), 6.85-6.86 (d, J=4.6 Hz, 1H, Ar-H), 7.26-7.27 (d, J=4.5 Hz, 1H,
Ar-H), 7.85-7.89 (d, J=7.6 Hz, 1H, Ar-H), 7.94-7.96 (d, J=8.6 Hz, 1H, Ar-H), 8.01-8.02 (d, J=8.3
Hz, 2H, Ar-H), 8.01-8.02 (d, J=8.3 Hz, 2H, Ar-H), 8.09-8.13 (t, J=7.60 Hz, 1H, Ar-H), 9.04 (brs,
1
3
1
1
H, NH, D O, exchange.), 12.0 (s, 1H, NH, D O, exchange.); C NMR (DSMO-d ) δ 108.4, 113.6,
2 2 6
19.6, 124.5, 124.8, 125.1, 126.4, 128.7, 136.4, 138.6, 139.7, 139.8, 150.8, 160.0, 168.8; MS m/z
9

ACCEPTED MANUSCRIPT
+
(
Rel. Int.) 383 (M , 32). Anal. (C H N O S 383.05). C, 53.25 (53.41); H, 3.42 (3.69); N, 18.26
17 13 5 2 2,
(18.47); S, 16.72 (16.89).
4
.1.3.10. 4-((2-Phenylquinazolin-4-yl)amino)-N-(thiazol-2-yl)benzenesulfonamide (3j). Yield
−1 1
(
67%); m.p. 295-297 °C; IR ν 3326, 3259 (2NH), 3114 (CH arom.), 1373, 1180 (SO ) cm ; H
2
NMR (DSMO-d ) δ 6.87-6.88 (d, J=4.6 Hz, 2H, Ar-H), 7.27-7.28 (m, 3H, Ar-H), 7.57-7.58 (m,
6
4
H, Ar-H), 7.85-7.89 (d, J=7.6 Hz, 1H, Ar-H), 7.94-7.96 (d, J=8.6 Hz, 1H, Ar-H), 7.99-8.13 (d,
J=8.4 Hz, 2H, Ar-H), 8.33-8.37 (d, J=8.3 Hz, 2H, Ar-H), 8.96 (brs, 1H, NH, D O, exchange.),
2
1
3
1
1.78 (s, 1H, NH, D O, exchange.); C NMR (DSMO-d ) δ 108.3, 112.8, 119.9, 124.2, 124.5,
2 6
1
24.6, 126.5, 128.2, 129.0, 129.2, 133.2, 136.0, 139.2, 140.3, 155.3, 159.1, 159.4, 168.8; MS m/z
+
(
Rel. Int.) 459 (M , 00). Anal. (C H N O S 459.08). C, 60.11 (60.38); H, 3.73 (3.95); N, 15.24
23
17
5
2 2,
(12.41); S, 13.95 (14.17).
4
.1.3.11. 4-((2-(4-Chlorophenyl)quinazolin-4-yl)amino)-N-(thiazol-2-yl)benzenesulfonamide (3k).
Yield (69%); m.p. 276-277 °C; IR ν 3341, 3255 (2NH), 3121 (CH arom.), 2981, 2885 (CH aliph.),
−
1 1
1
2
374, 1187 (SO ) cm ; H NMR (DSMO-d ) δ 6.86-6.88 (m, 2H, Ar-H), 7.26-7.27 (d, J=7.7 Hz,
2 6
H, Ar-H), 7.69-7.71 (d, J=8.5 Hz, 2H, Ar-H), 7.78-7.80 (d, J=7.6 Hz, 2H, Ar-H), 7.93-7.95 (d,
J=8.6 Hz, 2H, Ar-H), 8.15-8.17 (d, J=8.4 Hz, 2H, Ar-H), 8.37-8.38 (d, J=8.4 Hz, 2H, Ar-H), 8.83
1
3
(
brs, 1H, NH, D O, exchange.), 11.25 (s, 1H, NH, D O, exchange.); C NMR (DSMO-d ) δ 108.2,
2 2 6
1
1
12.7, 119.8, 123.2, 124.5, 125.6, 126.6, 128.2, 129.0, 129.2, 130.6, 130.6, 134.2, 139.1, 140.5,
+
+
54.8, 157.3, 159.2, 166.5; MS m/z (Rel. Int.) 495 (M + 2, 4), 493 (M , 13). Anal.
(
C H ClN O S 493.04). C, 55.92 (56.17); H, 3.26 (2.98); N, 14.18 (14.35); S, 12.98 (12.75).
23 16 5 2 2,
4
.1.3.12.
4-((2-(4-(Dimethylamino)phenyl)quinazolin-4-yl)amino)-N-(thiazol-2-yl)benzene
sulfonamide (3l). Yield (61%); m.p. 253-255 °C; IR ν 3341, 3256 (2NH), 3120 (CH arom.), 2983,
−
1 1
2
887 (CH aliph.), 1375, 1182 (SO ) cm ; H NMR (DSMO-d6) δ 3.08 (s, 6H, 2CH ), 6.86-6.88
2 3
(
m, 2H, Ar-H), 7.26-7.27 (d, J=7.7 Hz, 2H, Ar-H), 7.69-7.71 (d, J=8.5 Hz, 2H, Ar-H), 7.78-7.80
d, J=7.6 Hz, 2H, Ar-H), 7.93-7.95 (d, J=8.6 Hz, 2H, Ar-H), 8.15-8.17 (d, J=8.4 Hz, 2H, Ar-H),
(
8
.37-8.38 (d, J=8.4 Hz, 2H, Ar-H), 8.83 (brs, 1H, NH, D O, exchange.), 11.25 (s, 1H, NH, D O,
2 2
1
3
exchange.); C NMR (DSMO-d ) δ 108.2, 112.7, 119.8, 123.2, 124.5, 125.6, 126.6, 128.2, 129.0,
6
+
1
29.2, 130.6, 130.6, 134.2, 139.1, 140.5, 154.8, 157.3, 159.2, 166.5; MS m/z (Rel. Int.) 502 (M ,
7). Anal. (C H N O S 502.12). C, 59.74 (60.01); H, 4.41 (4.69); N, 16.72 (16.95); S, 12.76
3
2
5
22
6
2 2,
(12.55).
1
0

ACCEPTED MANUSCRIPT
4
.1.3.13.
4-((2-(4-Methoxyphenyl)quinazolin-4-yl)amino)-N-(thiazol-2-yl)benzenesulfonamide
(3m). Yield (73%); m.p. 283-285 °C; IR ν 3342, 3257 (2NH), 3123 (CH arom.), 2985, 2891 (CH
−
1 1
aliph.), 1379, 1187 (SO ) cm ; H NMR (DSMO-d ) δ 3.79 (s, 3H, OCH ), 6.87-6.88 (d, J=6.5 Hz,
2
6
3
2
H, Ar-H), 7.16-7.18 (d, J=8.7 Hz, 2H, Ar-H), 7.28-7.29 (d, J=4.5 Hz, 2H, Ar-H), 7.77-7.80 (t,
J=7.6 Hz, 2H, Ar-H), 8.05-8.11 (d, J=8.3 Hz, 2H, Ar-H), 8.31-8.33 (d, J=8.3 Hz, 2H, Ar-H), 8.37-
.39 (d, J=8.7 Hz, 2H, Ar-H), 8.92 (brs, 1H, NH, D O, exchange.), 11.69 (brs, 1H, NH, D O,
8
2
2
1
3
exchange.); C NMR (DSMO-d ) δ 55.7 (OCH ), 108.4, 112.5, 114.5, 124.2, 124.5, 126.5, 127.1,
6
3
1
28.0. 128.5, 129.8, 133.6, 139.3, 145.6, 150.0, 151.7, 156.7, 158.8, 163.6 168.8; MS m/z (Rel.
+
Int.) 489 (M , 36). Anal. (C H N O S 489.09). C, 58.88 (58.73); H, 3.91 (4.18); N, 14.31
24
19
5
3 2,
(14.55); S, 13.10 (13.28).
4
.1.3.14. 4-((2-(3,4-Dimethoxyphenyl)quinazolin-4-yl)amino)-N-(thiazol-2-yl)benzenesulfonamide
(3n). Yield (64%); m.p. 270-271 °C; IR ν 3347, 3251 (2NH), 3129 (CH arom.), 2986, 2892 (CH
−
1 1
aliph.), 1385, 1190 (SO ) cm ; H NMR (DSMO-d ) δ 3.78 (s, 6H, 2OCH ), 6.88-6.89 (d, J=8.4
2
6
3
Hz, 2H, Ar-H), 7.14-7.16 (d, J=8.2 Hz, 2H, Ar-H), 7.29-7.30 (d, J=4.3 Hz, 2H, Ar-H), 7.75-7.95
m, 4H, Ar-H), 8.02-8.06 (m, 3H, Ar-H), 8.85 (brs, 1H, NH, D O, exchange.), 11.71 (brs, 1H, NH,
(
2
1
3
D O, exchange.); C NMR (DSMO-d ) δ 55.7 (OCH ), 108.3, 111.7, 112.4, 120.1, 120.8, 124.5,
2
6
3
1
26.3, 127.8, 128.8, 129.5, 135.9, 139.3, 146.3, 148.5, 150.3, 156.2, 158.6, 168.8, 171.3; MS m/z
+
(
Rel. Int.) 519 (M , 16). Anal. (C H N O S 519.10). C, 57.79 (57.53); H, 4.07 (4.38); N, 13.48
2
5
21
5
4 2,
(13.64); S, 12.34 (12.51).
4
.1.3.15.
4-((2-(Benzo[d][1,3]dioxol-5-yl)quinazolin-4-yl)amino)-N-(thiazol-2-
yl)benzenesulfonamide (3o). Yield (69%); m.p. 279-281 °C; IR ν 3326, 3281 (2NH), 3121 (CH
−
1 1
arom.), 2988, 2875 (CH aliph.), 1369, 1182 (SO ) cm ; H NMR (DSMO-d ) δ 6.19 (s, 2H,
2
6
OCH O), 6.77-6.79 (m, 3H, Ar-H), 7.12-7.14 (d, J=8.3 Hz, 2H, Ar-H), 7.53-7.58 (d, J=7.6 Hz, 2H,
2
Ar-H), 7.67-7.69 (d, J=7.6 Hz, 2H, Ar-H), 7.83-7.84 (d, J=7.4 Hz, 2H, Ar-H), 8.01-8.02 (d, J=7.1
1
3
Hz, 2H, Ar-H), 8.88 (brs, 1H, NH, D O, exchange.), 11.58 (s, 1H, NH, D O, exchange.); C NMR
2
2
(
DSMO-d ) δ 101.3, 108.1, 112.5, 115.6, 116.1, 116.7, 120.2, 120.6, 124.0, 126.3, 125.1, 128.6,
6
+
1
3
29.3, 133.8, 138.3, 146.0, 148.1, 149.4, 150.1, 160.2, 164.5, 168.3; MS m/z (Rel. Int.) 503 (M ,
2). Anal. (C H N O S 503.07). C, 57.25 (56.92); H, 3.40 (3.65); N, 13.91 (14.08); S, 12.73
2
4
17
5
4 2,
(12.93).
1
1

ACCEPTED MANUSCRIPT
4
.1.3.16.
N-(Thiazol-2-yl)-4-((2-(3,4,5-trimethoxyphenyl)quinazolin-4-
yl)amino)benzenesulfonamide (3p). Yield (72%); m.p. 287-290 °C; IR ν 3330, 3208 (2NH), 3122
−
1 1
(
CH arom.), 2978, 2876 (CH aliph.), 1380, 1192 (SO ) cm ; H NMR (DSMO-d ) δ 3.78 (s, 9H,
2 6
3
2
2
OCH ), 6.81-6.83 (m, 3H, Ar-H), 7.47 (brs, 2H, NH , D O, exchange.), 7.51-7.52 (d, J=7.2 Hz,
3
2
2
H, Ar-H), 7.63-7.65 (m, 3H, Ar-H), 8.28-8.30 (d, J=7.3 Hz, 2H, Ar-H), 8.65-8.67 (d, J=7.6 Hz,
1
3
H, Ar-H), 9.25 (brs, 1H, NH, D O, exchange.), 12.06 (s, 1H, NH, D O, exchange.); C NMR
2
2
(
DSMO-d ) δ 56.2, 56.7 (3OCH ), 107.2, 111.3, 115.9, 116.5, 120.1, 125.3, 126.1, 128.0, 128.6,
6 3
+
1
29.4, 133.5, 138.5, 140.1, 146.2, 150.7, 151.6, 158.0, 160.1, 162.9; MS m/z (Rel. Int.) 549 (M ,
4). Anal. (C H N O S 549.11). C, 56.82 (57.03); H, 4.22 (4.47); N, 12.74 (13.02); S, 11.67
2
2
6
23
5
5 2,
(11.82).
4
.1.4. General procedure for preparation of compounds 4a, b.
A solution of phenylisothiocyanate (0.14 g, 1 mmol) in dry acetone was added to a stirred
mixture of 3c, e (1.7 g, 5 mmol) and anhydrous potassium carbonate (1.38 g, 10 mmol) in dry
acetone (25 mL). The reaction mixture was heated under reflux for 12 h. The solvent was removed
under reduced pressure and the remaining residue was dissolved in water. The obtained mixture
was filtered and the filtrate acidified with glacial acetic acid. The solid product was collected by
filtration, washed with water, dried and crystallized from ethanol.
4
.1.4.1
4-((2-(4-Chlorophenyl)quinazolin-4-yl)amino)-N-(phenylcarbamothioyl)
benzenesulfonamide (4a). Yield (69%); m.p. 175-177 °C; IR ν 3329, 3305, 3294 (3NH), 3117 (CH
−1 1
arom.), 1715 (C=O), 1386, 1192 (SO ) cm ; H NMR (DSMO-d ) δ 6.59-6.61 (m, 3H, Ar-H),
2
6
6
.97-7.01 (d, J=8.40 Hz, 2H, Ar-H), 7.22-7.23 (d, J=7.9 Hz, 2H, Ar-H), 7.36-7.38 (m, 3H, Ar-H),
.48-7.51 (m, 2H, Ar-H), 7.56-7.58 (m, 3H, Ar-H), 7.88-7.89 (d, J=7.9 Hz, 2H, Ar-H), 8.88-9.01
7
(
brs, 1H, NH, D O, exchange.), 10.21-10.30 (brs, 1H, NH, D O, exchange.), 11.84-11.89 (brs, 1H,
2
2
1
3
NH, D O, exchange.); C NMR (DSMO-d ) δ 114.0, 118.5, 120.6, 123.1, 126.3, 128.1, 128.6,
2
6
1
29.6, 133.5, 135.2, 136.8, 137.3, 146.3, 150.3, 157.7, 157.9, 165.8, 171.3; MS m/z (Rel. Int.) 548
+
+
(
M + 2, 11), 546 (M , 32). Anal. (C H ClN O S 545.07) C, H, N. C, 59.39 (59.56); H, 3.69
27 20 5 2 2,
(3.85); N, 12.83 (12.66); S, 11.74 (11.91).
4
.1.4.2
4-((2-(4-Methoxyphenyl)quinazolin-4-yl)amino)-N-(phenylcarbamothioyl)
benzenesulfonamide (4b). Yield (75%); m.p. 186-188 °C; IR ν 3331, 3286 (3NH), 3121 (CH
−
1 1
arom.), 2981, 2893 (CH aliph.), 1715 (C=O), 1375, 1188 (SO ) cm ; H NMR (DSMO-d ) δ 3.75
2
6
1
2

ACCEPTED MANUSCRIPT
(
s, 3H, OCH ), 6.79-6.80 (m, 3H, Ar-H), 7.07-7.08 (d, J=8.7 Hz, 2H, Ar-H), 7.10-7.11 (d, J=7.8
3
Hz, 2H, Ar-H), 7.13-7.14 (m, 3H, Ar-H), 7.31-7.33 (m, 2H, Ar-H), 7.48-7.50 (m, 3H, Ar-H), 7.53-
.55 (d, J=7.7 Hz, 2H, Ar-H), 8.85-8.87 (s, 1H, NH, D O, exchange.), 9.80-9.83 (s, 1H, NH, D O,
7
2
2
1
3
exchange.), 11.63-11.69 (brs, 1H, NH, D O, exchange.); C NMR (DSMO-d ) δ 55.3 (OCH ),
2
6
3
1
1
6
13.5, 113.9, 121.2, 121.5, 123.2, 123.6, 124.4, 126.1, 126.3, 128.4, 128.5, 129.1, 129.8, 133.9,
+
39.4, 157.7, 158.3, 161.6; MS m/z (Rel. Int.) 541 (M , 18). Anal. (C H N O S 541.12). C,
2
8
23
5
3 2,
2.09 (61.85); H, 4.28 (4.53); N, 12.93 (12.77); S, 11.84 (11.67).
4
.2. Biology
4
.2.1. Cell Culture
Human colon cancer cell line HT-29 and SW620 were grown in DMEM (Invitrogen) containing
0% heat-inactivated fetal bovine serum, 100 µg/ml streptomycin, 100 units/ ml penicillin and 2
1
mmol/l L-glutamine. For cell viability assay cells treated with the compounds for 24 hour and for
protein expression the cells were treated for 18 hour.
4
.2.2. Cell Viability assay
Cell viability was determined using Trypan blue on Vi-Cell XR instrument (Beckman Coulter).
Briefly, after culturing cells for 24 h, they were treated with 10 µM of the compound tested for 24
h. All the cells were collected and live cell number was determined using cell viability analyzer
(Vi-Cell XR, Beckman Coulter). In certain experiment, cells were treated with various
concentration of compound 3c along with different time points.
4
.2.3. Western Blotting
Whole cell lysates were prepared as described [22]. Soluble proteins were analyzed by
immunoblotting with anti-CAIX, CA XII, CAI, CAII (Dilution 1:1000; Santa Cruz Biotechnology)
and anti-β-actin (Sigma). Reactivity was detected with horseradish peroxidase-conjugated
secondary antibodies and chemiluminescence (GE healthcare). Membranes were developed using
C-Digit Blot Scanner (LI-COR, Hamburg, Germany).
1
3

ACCEPTED MANUSCRIPT
Acknowledgement
This project was supported by the Deanship of Scientific Research at Prince Sattam bin
Abdulaziz University under the research project number "2014/03/2048".
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Figure Legends
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Figure 1. A) HT-29 cells were treated with 10 µM of various compound for 24 h. Cell viability was
determined using Trypan Blue on Vi-Cell XR instrument.
Figure 1. B) HT-29 cells were treated with different concentration of 3c for 24 h, total cells were
collected and live cell count was determined using cell viability analyzer.
Figure 1. C) HT-29 cells were treated with compound 3c for different time point and viable cell
number was determined using cell viability analyzer.
Figure 1. D) SW620 cells were treated with 5 and 10 µM of 3c compound, live cell number was
determined using Vi-Cell XR cell viability analyzer.
Figure 2. HT-29 cells were treated with various concentration of 3c compound, cells were
harvested and incubated with cell lysis buffer. Total cell lysates were run on precast TGX gels and
immunoblotted with the CA IX, CA XII, CA I, CA II and β-actin antibodies.
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Scheme 1. Synthesis of compounds 3a-p and 4a, b

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Fig 1A
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8
6
4
2
0
0
0
0
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C 3a 3b 3c 3d 3e 3f 3g 3h 3i 3j 3k 3l 3m 3n 3o 3p 4a 4b

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Fig 1B
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1
20
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80
60
40
20
0
1
C
0.5
1
1.5
2.5
5
7.5
10
Concentration (µM)

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Fig 1C
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C
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Time (h)

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Fig 1D
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Control
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Concentration (µM)

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Fig 2
C
2.5
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10 (µM)
CA IX
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0
3
IB: Anti-CA IX
CA XII
4
IB: Anti-CA XII
IB: Anti-CA I
CA I
2
2
9
9
CA II
IB: Anti-CA II
IB: Anti-β-actin
β-actin
4
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Research Highlights
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•
•
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•
Eighteen new compounds were developed and tested against colorectal cancer.
Compound 3C was noticeably effective as potential antitumor agent.
It selectively inhibited protein expression of CA IX and CA XII.
Compound 3c neither affected the expression of CA I nor that of CA II.
This candidate deserves further studies to develop potential antitumor agent.