Anticancer activity of triazolo-thiadiazole derivatives and inhibition of akt1 and akt2 activation

pharmaceutics

Article

Anticancer Activity of Triazolo-Thiadiazole Derivatives and

Inhibition of AKT1 and AKT2 Activation

Dimitrios T. Trafalis ^1,

*

, Soﬁa Sagredou ¹, Panayiotis Dalezis ¹, Maria Voura ², Stella Fountoulaki ²,

Nikolaos Nikoleousakos ¹, Konstantinos Almpanakis ², Maria V. Deligiorgi ¹and Vasiliki Sarli ^2,

*

1

Laboratory of Pharmacology, Faculty of Medicine, National and Kapodistrian University of Athens,

115 27 Athens, Greece; ssagredou@med.uoa.gr (S.S.); pdalezis@med.uoa.gr (P.D.);

nikosakos@hotmail.com (N.N.); mdeligiorgi@yahoo.com (M.V.D.)

Department of Chemistry, Aristotle University of Thessaloniki, University Campus,

541 24 Thessaloniki, Greece; vouram@gmail.com (M.V.); sfountou@chem.auth.gr (S.F.);

konstalmp@yahoo.gr (K.A.)

2

*

Correspondence: dtrafal@med.uoa.gr (D.T.T.); sarli@chem.auth.gr (V.S.)

Abstract: The fusion of 1,2,4-triazole and 1,3,4-thiadiazole rings results in a class of heterocycles

compounds with an extensive range of pharmacological properties. A series of 1,2,4-triazolo[3,4-

b]-1,2,4-thiadiazoles was synthesized and tested for its enzyme inhibition potential and anticancer

activity. The results show that 1,2,4-triazolo[3,4-b]-1,2,4-thiadiazoles display potent anticancer proper-

ties in vitro against a panel of cancer cells and in vivo efﬁcacy in HT-29 human colon tumor xenograft

in CB17 severe combined immunodeﬁcient (SCID) mice. Preliminary mechanistic studies revealed

that KA25 and KA39 exhibit time- and concentration-dependent inhibition of Akt Ser-473 phos-

phorylation. Molecular modeling experiments indicated that 1,2,4-triazolo[3,4-b]-1,2,4-thiadiazoles

bind well to the ATP binding site in Akt1 and Akt2. The low acute toxicity combined with in vitro

and in vivo anticancer activity render triazolo[3,4-b]thiadiazoles KA25, KA26, and KA39 promising

cancer therapeutic agents.

ꢀꢁꢂꢀꢃꢄꢅꢆꢇ

ꢀꢁꢂꢃꢄꢅꢆ

Citation: Trafalis, D.T.; Sagredou, S.;

Dalezis, P.; Voura, M.; Fountoulaki, S.;

Nikoleousakos, N.; Almpanakis, K.;

Deligiorgi, M.V.; Sarli, V. Anticancer

Activity of Triazolo-Thiadiazole

Derivatives and Inhibition of AKT1

and AKT2 Activation. Pharmaceutics

2021, 13, 493. https://doi.org/

Keywords: 1,2,4-triazolo[3,4-b]-1,2,4-thiadiazole; inhibitor of Akt phosphorylation; anticancer; MTT

assay; HT-29 human colon tumor xenograft; ATP binding site

10.3390/pharmaceutics13040493

1. Introduction

Academic Editor: Tihomir Tomašicˇ

Akt, termed protein kinase B (PKB), is a serine/threonine kinase composed of three

isoforms: Akt1, Akt2, and Akt3. All three isoforms share very similar amino acid se-

quences, with their expression level differentiated; Akt1 and Akt2 are abundantly ex-

Received: 1 March 2021

Accepted: 29 March 2021

Published: 5 April 2021

pressed, while Akt3 is particularly detected in the brain, heart, and kidneys [1,2]. All Akt

isoforms display the same basic structure comprised of three regions: (1) an amino terminal

pleckstrin homology (PH) domain that interacts with membrane phospholipids such as

phosphatidylinositol-3,4,5-triphosphate (PIP3) and phosphatidylinositol 4,5-bisphosphate

(PIP2); (2) a central kinase domain that contains the threonine regulatory residue, Thr308,

in which phosphorylation activates Akt; and (3) a carboxyl-terminal regulatory domain

that consists of a hydrophobic region of 40 amino acids including the serine regulatory

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iations.

residue (Ser473) [3]. The structural elements, PH domain, and the regulatory residues

Thr308 and Ser473 play critical roles in the activation of Akt. Two events are required

in Akt’s activation: (a) PH-domain-dependent translocation to the plasma membrane

and (b) phosphorylation at the Thr308 and Ser473 residues. The ﬁrst step includes the

interaction of the PH domain with PIP3, which is followed by the translocation of Akt to

the plasma membrane. Afterwards, Akt adopts a new conformation such that the Thr308

residue would be phosphorylated by phosphoinositide-dependent kinase-1 (PDK1). This

signaling event leads to phosphorylated Ser473 via the mechanistic targeting of rapamycin

(mTOR)C2 [4–6].

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This article is an open access article

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Attribution (CC BY) license (https://

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Pharmaceutics 2021, 13, 493. https://doi.org/10.3390/pharmaceutics13040493

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Pharmaceutics 2021, 13, 493

2 of 23

Phosphatidylinositol 3-kinase (PI3K), an upstream signaling molecule, along with Akt

constitute the PI3K/Akt signaling transduction pathway through which cellular survival

and growth are induced in response to extracellular signal [

nents of the PI3K/Akt pathway, the inhibition of Akt has been widely explored due to its as-

sociation with tumor progression and aggressiveness [ ]. Signiﬁcant alterations have been

7]. Among all protein compo-

8

demonstrated in the expression levels of Akt isoforms in certain malignancies, for instance,

Akt1 is particularly elevated in breast, prostate, and gastric tumors whilst Akt2 is overex-

pressed in prostate, ovarian, breast, pancreatic, and colorectal cancers [9–11]. Thus far, Akt

inhibitors are divided in four basic categories: (1) ATP-competitive inhibitors, (2) allosteric

inhibitors, (3) lipid-based inhibitors, and 4) PH domain inhibitors [12]. The ﬁrst category of

ATP-competitive inhibitors (CCT128930 as an Akt2 inhibitor and BAY-1125976 as an Akt1/2

inhibitor), including pan-Akt kinase inhibitors (afuresertib, GSK690693, AZD5363, GDC-

0068, and AT7867 as inhibitors of all Akt isoforms), targets the kinase domain and precisely

binds to the ATP-binding pocket [13]. The high degree of homology of the ATP-binding

site among different serine/threonine kinases as well as the extensive conservation of this

domain within the AGC kinase family (protein kinase A, G, and C families) contributes to

a low speciﬁcity of ATP-competitive inhibitors. The development of such molecules can be

further obstructed due to the lack of strong efﬁcacy against tumors in vivo, with toxicities to

normal tissues being recorded at the same time [14,15]. Regarding allosteric inhibitors, for

example, the MK-2206 compound, are associated with the Akt kinase domain. Compared

with ATP-competitive inhibitors that show efﬁcacy only against cancer cell lines with Akt

mutations, allosteric inhibitors display broader anticancer activity as compounds of this

type are potent against cancer cell lines with PI3KCA mutations or loss of phosphatase

and tensin homologue (PTEN) activity [16]. The third category of lipid-based inhibitors

(PX-866 and perifosine) blocks the interaction of Akt with PIP3 since this type of molecule

inhibits PI3K and therefore prevents the production of PIP3 from PIP2. Finally, PH domain

inhibitors (triciribine and PX-316) inactivate Akt via interactions with the PH-domain,

interrupting membrane translocation, which is required for activating Akt [17].

The fusion of 1,2,4-triazole and 1,3,4-thiadiazole rings results in a class of heterocyclic

compounds with an extensive range of pharmacological properties including antifungal, an-

tibacterial, antiviral, anti-inﬂammatory, analgesic, and anthelmintic properties [18]. Earlier

studies indicated that previous 3,6-disubstituted 1,2,4-[3,4-b]thiadiazoles induced potent

anti-inﬂammatory activity along with a minimal ulcerogenic effect and lipid peroxidation

compared to ibuprofen and ﬂurbiprofen. Some of these compounds demonstrated mod-

erate to weak antibacterial activity against Staphylococcus aureus and Escherichia coli while

their antifungal activity against Candida albicans was quite weak [19–21]. Furthermore,

1,2,4-triazolo[3,4-b][1,3,4]thiadiazole derivatives containing 3-methyl or benzyl moiety

showed moderate anti-HIV-1 activity at subcytotoxic concentrations [22]. In previous work,

we have shown that triazolo[3,4-b]thiadiazole derivatives show potent in vitro antiprolifer-

ative activities [23]. Our studies resulted in the identiﬁcation of three bioactive compounds

(KA39, KA25, and KA26) against three human colorectal cancer cell lines (Figure 1). Among

them, KA39 was the most potent anticancer agent and inhibitor of topII

at Ser-1106 as well. Additional molecular docking studies revealed that KA39 can occupy

the same binding site as etoposide and can interact with the ATPase domain of topII

but the exact mechanism of inhibition of topII phosphorylation by KA39 is still unclear.

α phosphorylation

α

,

α

As part of our ongoing work on the assessment of the biological activity of triazolo[3,4-

b][1,3,4]thiadiazoles, we report the synthesis, structural characterization, and evaluation of

inhibitory effects of ﬁfteen new derivatives. In particular, we examined the introduction of

ethyl or propyl substituents on sulfonamide combined with different substituents on C-6 of

the triazolo[3,4-b][1,3,4]thiadiazole template. In the current work, the anticancer activities

of all derivatives were primarily assessed in vitro while the most active molecules KA39,

KA25, and KA26, according to in vitro screening, were tested in vivo. Further studies

were carried out to investigate whether the most potent antitumor compound blocks the

Pharmaceutics 2021, 13, 493

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phosphorylation of Akt1 and Akt2 kinases. The molecular modeling studies indicated that

1,2,4-triazolo[3,4-b]-1,2,4-thiadiazoles bind well to the ATP binding site in Akt1 and Akt2.

Figure 1. Structures of active triazolo[3,4-b][1,3,4]thiadiazoles and overview of their activity.

2. Materials and Methods

2.1. Synthesis of Triazolo-Thiadiazole Derivatives

All reactions were carried out under an atmosphere of argon unless otherwise speciﬁed.

Commercial reagents of high purity were purchased and used without further puriﬁcation,

unless otherwise noted. Reactions were monitored by thin-layer chromatography (TLC)

and by using UV light as a visualizing agent and ethanolic p-anisaldehyde solution or using

aqueous ceric sulfate/phosphomolybdic acid and heat as developing agents. The ¹H and

¹³C NMR spectra were recorded at 500 and 126 MHz, and tetramethylsilane was used as an

internal standard. Chemical shifts are indicated in δ values (ppm) from internal reference

peaks (Tetramethylsilane TMS ¹H 0.00; CDCl₃¹H 7.26, ¹³C 77.00; DMSO-d₆¹H 2.50, and

¹³C 39.51). Melting points (m.p.) are uncorrected. The LC-MS spectra were recorded on a

LC20AD Shimadzu connected to Shimadzu LCMS-2010EV equipped with C18 analytical

column (SUPELCO Discovery (C18, 25 cm

and KA39 were synthesized according to a previously reported procedure [23].

Methyl 2-(3,4-dimethoxyphenyl)acetate, S1: 2-(3,4-dimethoxyphenyl)acetic acid (1 g

×

4.6 mm, 5 µm)). Compound 2, KA25, KA26,

,

5.1 mmol) was dissolved in 24 mL dry dichloromethane (DCM), a catalytic amount of

◦

dry dimethylformamide (DMF) was added, and the mixture was cooled at 0 C. Then,

1.32 mL (15.31 mmol) of oxalyl chloride was added dropwise and the reaction was left

stirring at room temperature for 0.5 h. The addition of MeOH (20 mL) in portions at 0 ^◦C

was followed, and the reaction was stirred at room temperature for 10 min. The reaction

was quenched with 20 mL of H₂O, and the mixture was extracted with dichloromethane

(3 × 20 mL). The organic extracts were dried over Na₂SO₄, ﬁltered, and concentrated

under reduced pressure. The residue was puriﬁed by silica gel column chromatography

(eluent; hexane/ethyl acetate, 4/1) to afford S1 as a yellow oil in 99% yield. The spectral

1

data were in accordance with those reported in the literature [24]. S1: H NMR (500 MHz,

CDCl₃) δ 6.81 (s, 3H), 3.88 (s, 3H), 3.86 (s, 3H), 3.69 (s, 3H), 3.57 (s, 2H).

Methyl 2-(2-(chlorosulfonyl)-4,5-dimethoxyphenyl)acetate, 2: to a solution of S1 (3.7 g

,

17.64 mmol) in 35 mL CHCl₃, 5.28 mL (79.4 mmol) of chlorosulfonic acid was added

dropwise at 0 ^◦C, and the reaction was stirred at room temperature for 1 h. The reaction

was quenched with H₂O (35 mL), and the mixture was extracted with dichloromethane

( 3). The organic layers were combined, dried over Na₂SO₄, ﬁltered, and concentrated

×

under reduced pressure. The residue was puriﬁed by silica gel column chromatography

(eluent; hexane/ethyl acetate = 2/1) to yield compound 2 as a white solid (88%). The

1

spectral data were in accordance with those reported in the literature [23]. 2: H NMR

(

500 MHz, CDCl₃)

(s, 3H)

¹³C NMR (126 MHz, CDCl₃)

56.4, 52.4, 38.0.

δ

7.52 (s, 1H), 6.88 (s, 1H), 4.11 (s, 2H), 3.97 (s, 3H), 3.95 (s, 3H), 3.73

;

δ

170.5, 154.1, 148.0, 134.6, 127.8, 115.3, 111.3, 56.4,

Methyl 2-(2-(N,N-diethylsulfamoyl)-4,5-dimethoxyphenyl)acetate, 3a: to the solution

of

2

(450 mg, 1.46 mmol) in 1.8 mL tetrahydrofuran (THF), 0.3 mL diethylamine (2.92 mmol)

◦

was added at 0 C. The reaction mixture was stirred for 1 h at room temperature. The

mixture was concentrated under reduced pressure to obtain 3a as a yellow solid (99% yield).

Pharmaceutics 2021, 13, 493

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3a: m.p. = 108–110 ^◦C; ¹H NMR (500 MHz, CDCl₃) δ 7.45 (s, 1H), 6.82 (s, 1H), 3.99 (s, 2H),

3.93 (s, 3H), 3.92 (s, 3H), 3.71 (s, 3H), 3.25 (q, J = 7.1 Hz, 4H), 1.12 (t, J = 7.1 Hz, 6H); ¹³

C

NMR (126 MHz, CDCl₃)

δ 171.6, 151.9, 147.5, 130.0, 127.0, 115.2, 112.9, 56.3, 56.1, 52.0, 40.5,

37.6, 13.4; ESI–MS m/z for C₁₅H₂₃NNaO₆S [M + Na]⁺calcd 368.11, found 367.95.

Methyl 2-(2-(N,N-diisopropylsulfamoyl)-4,5-dimethoxyphenyl)acetate, 3b: 450 mg

(1.46 mmol) of 2 was dissolved in 1.8 mL of THF, and 0.41 mL of diisopropylamine

◦

(2.92 mmol) was added at 0 C. The reaction was stirred for 1 h at room temperature. THF

and amine excess were concentrated under reduced pressure to afford 3b as a yellow solid

◦

in 99% yield. 3b: m.p. = 105–107 C; ¹H NMR (500 MHz, CDCl₃)

δ

7.44 (s, 1H), 6.76 (s, 1H),

4.08 (s, 2H), 3.92 (s, 3H), 3.90 (s, 3H), 3.70 (s, 3H), 3.58 (m, 2H), 1.31 (d, J = 6.8 Hz, 12H); ¹³

NMR (126 MHz, CDCl₃) 171.7, 151.7, 147.5, 131.1, 127.3, 114.8, 112.7, 56.1, 56.1, 52.0, 48.6,

37.8, 21.8; ESI–MS m/z for C₁₇H₂₇NNaO₆S [M + Na]⁺calcd 396.15, found 395.95.

2-(2-hydrazinyl-2-oxoethyl)-N,N-diethyl-4,5-dimethoxybenzenesulfonamide, 4a: a mix-

ture of compound 3a (185 mg, 0.513 mmol) in hydrazine hydrate 80% (0.22mL, 4.56 mmol

was reﬂuxed for 0.5 h. The mixture was extracted with dichloromethane (3 8 mL),

C

δ

)

×

and the combined organic extracts were dried over Na₂SO₄, ﬁltered, and concentrated

under reduced pressure to afford 161 mg of compound 4a (87% yield, white solid). 4a:

1

m.p. = 121–123 ^◦C; H NMR (500 MHz, CDCl₃)

δ 7.82 (s, 1H), 7.23 (s, 1H), 6.89 (s, 1H),

3.79 (s, 3H), 3.76 (s, 3H), 3.66 (s, 2H), 3.14 (q, J = 7.1 Hz, 4H), 0.98 (t, J =7.1 Hz, 6H); ¹³

NMR (126 MHz, CDCl₃) 170.3, 151.8, 147.1, 129.1, 127.4, 114.2, 112.0, 55.9, 40.4, 37.7, 13.2

1 carbon missing due to overlapping); ESI–MS m/z for C₁₄H₂₃N₃NaO₅S [M + Na]⁺calcd

C

δ

(

368.13, found 367.95.

2-(2-hydrazinyl-2-oxoethyl)-N,N-diisopropyl-4,5-dimethoxybenzenesulfonamide, 4b:

a mixture of compound 3b (250 mg, 0.67 mmol) in hydrazine hydrate 80% (0.28 mL,

5.66 mmol) was reﬂuxed for 0.5 h. The mixture was extracted with dichloromethane

(3

×

10 mL), and the combined organic extracts were dried over Na₂SO₄, ﬁltered, and

concentrated under reduced pressure to afford 230 mg of compound 4b in 92% yield as

a yellow solid. 4b: m.p. = 121–123 C; ¹H NMR (500 MHz, CDCl₃) 7.90 (s,1H), 7.43 (s,

◦

1H), 7.01 (s, 1H), 3.93 (s, 3H), 3.90 (s,3H), 3.89 (s, 2H), 1.32 (d, J = 6.8 Hz, 12H); ¹³C NMR

(126 MHz, CDCl₃) δ 171.0, 152.2, 147.5, 130.1, 128.0, 114.1, 112.7, 56.3, 56.2, 49.0, 37.8, 21.9;

ESI–MS m/z for C₁₆H₂₇N₃NaO₅S [M + Na]⁺calcd 396.16, found 396.00.

2-((4-amino-5-mercapto-4H-1,2,4-triazol-3-yl)methyl)-N,N-diethyl-4,5-dimethoxybenzene

sulfonamide, 5a: to a ﬂask containing 400 mg (1.16 mmol) of 4a, abs EtOH (16 mL) and

KOH (97.6 mg, 1.74 mmol) were successively added. Then, 105 µL (1.74 mmol) of CS₂was

added dropwise at 0 ^◦C and the reaction mixture was allowed to warm to room temper-

ature and was stirred for 24 h. Subsequently, 16 mL of diethyl ether was added, and the

solid product was ﬁltered and washed with diethyl ether. The product was dried and used

in the next reaction without further puriﬁcation. To a sealed vial, the previous salt and

hydrazine hydrate 80% (4.49 mL, 92.41 mmol) was added and the mixture was reﬂuxed

for 2 h. Acidiﬁcation with 10% aq. HCl was followed, and the pH was adjusted to 7. The

product was formed, ﬁltered through a Gooch funnel, washed with cold water, and dried

to afford 326 mg of compound 5a in 70% yield as a white solid. 5a: m.p. = 187–189 ^◦C

¹H NMR (500 MHz, DMSO-d₆)

13.42 (s, 1H), 7.30 (s, 1H), 6.97 (s, 1H), 5.58 (s, 2H), 4.28

(s, 2H), 3.82 (s, 3H), 3.78 (s, 3H), 3.16 (q, J = 7.0, 4H), 0.99 (t, J =7.0 Hz, 6H); ¹³C NMR

;

δ

(

126 MHz, DMSO-d₆) δ 165.9, 151.5, 151.3, 147.0, 129.6, 127.1, 115.4, 112.3, 55.8, 40.5, 27.7,

13.5 (1 carbon missing due to overlapping); ESI–MS m/z for C₁₅H₂₃N₅NaO₄S₂[M + Na]⁺

calcd 424.11, found 423.90.

2-((4-amino-5-mercapto-4H-1,2,4-triazol-3-yl)methyl)-N,N-diisopropyl-4,5-dimethoxy

benzene sulfonamide, 5b: to a ﬂask containing 300 mg (0.803 mmol) of 4b, abs EtOH (12 mL)

and KOH (67.3 mg, 1.2 mmol) were successively added. Then, 73

µ

L (1.2 mmol) of CS₂

◦

was added dropwise at 0 C and the reaction mixture was allowed to warm to room

temperature and was stirred for 24 h. Subsequently, 12 mL of diethyl ether was added,

and the solid product was ﬁltered and washed with diethyl ether. The product was dried

and used in the next reaction without further puriﬁcation. To a sealed vial, the previous

Pharmaceutics 2021, 13, 493

5 of 23

salt and hydrazine hydrate 80% (3.11 mL, 63.97 mmol) was added and the mixture was

reﬂuxed for 2 h. Acidiﬁcation with 10% aq. HCl was followed, and the pH was adjusted to

7. The product was formed, ﬁltered through a Gooch funnel, washed with cold water, and

dried to afford 258 mg of 5b as a white solid (75% yield). 5b: m.p. = 162–164 ^◦C; ¹H NMR

(500 MHz, DMSO-d₆)

δ 13.42 (s, 1H), 7.31 (s, 1H), 6.88 (s, 1H), 5.55 (s, 2H), 4.38 (s, 2H),

3.80 (s, 3H), 3.77 (s, 3H), 3.65–3.53 (m, 2H), 1.21 (d, J = 6.7 Hz, 12H); ¹³C NMR (126 MHz,

DMSO-d₆) 165.9, 151.4, 151.4, 147.1, 130.6, 127.6, 114.8, 112.2, 55.8, 55.7, 48.0, 27.7, 21.6;

ESI–MS m/z for C₁₇H₂₇N₅NaO₄S₂[M + Na]⁺calcd 452.14, found 452.00.

General procedure for the preparation of 1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazoles: the

mixture of 0.268 mmol of 5a,b and 0.268 mmol of carboxylic acids in phosphorous oxy-

chloride (0.4 mL) was stirred while heated under reﬂux for 2 h. The reaction mixture was

cooled to room temperature and then poured into ice. Then, the pH was adjusted to 8

with aqueous K₂CO₃. The mixture was extracted with dichloromethane, and the organic

extracts were dried over Na₂SO₄, ﬁltered, and concentrated under reduced pressure. The

residue was puriﬁed by silica gel column chromatography.

2-((6-(3-chlorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)methyl)-_◦N,N-diethyl-

4,5-dimethoxybenzenesulfonamide, 6a: beige solid, 55% yield, m.p. = 203–205 C; ¹H NMR

(500 MHz, CDCl₃)

(d, J = 8.0 Hz, 1H), 7.32 (s, 1H), 7.14 (s, 1H), 4.73 (s, 2H), 3.81 (s, 3H), 3.77 (s, 3H), 3.17

(d, J = 7.1 Hz, 4H), 0.99 (t, J = 7.1 Hz, 6H); ¹³C NMR (126 MHz, CDCl₃)

164.6, 152.6,

δ 7.95 (s, 1H), 7.91 (d, J = 7.7 Hz, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.66

δ

151.6, 147.2, 146.2, 134.3, 132.6, 131.7, 131.0, 129.5, 127.3, 126.3, 125.9, 115.5, 112.3, 55.9, 40.5,

27.4, 13.4 (1 carbon is missing due to overlapping); ESI–MS m/z for C₂₂H₂₄ClN₅NaO₄S₂

[M + Na]⁺calcd 544.09, found 543.95.

2-((6-(4-chlorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)methyl)-_◦N,N-diethyl-

4,5-dimethoxybenzenesulfonamide, 6b: beige solid, 63% yield, m.p. = 128–130 C; ¹H NMR

(500 MHz, CDCl₃)

(s, 3H), 3.84 (s, 3H), 3.27 (d, J = 7.1 Hz, 4H), 1.10 (t, J = 7.1 Hz, 6H); ¹³C NMR (126 MHz,

CDCl₃) 165.4, 152.2, 147.6, 139.1, 129.8, 128.3, 127.8, 127.4, 114.1, 113.0, 56.3, 56.2, 40.7,

δ 7.80 (d, J = 8.3 Hz, 2H), 7.49–7.51 (m, 3H), 6.93 (s, 1H), 4.87 (s, 2H), 3.92

δ

27.7, 13.6, 13.4; (3 carbons missing due to overlapping) ESI–MS m/z for C₂₂H₂₄ClN₅NaO₄S₂

[M + Na]⁺calcd 544.09, found 543.95.

N,N-diethyl-2-((6-(4-ﬂuorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)methyl)-

4,5-dimethoxybenzenesulfonamide, 6c: yellow solid, 58% yield, m.p. = 169–171 ^◦C; ¹H

NMR (500 MHz, DMSO-d₆)

δ 8.01 (m, 2H), 7.48 (t, J = 8.7 Hz, 2H), 7.33 (s, 1H), 7.15 (s, 1H),

4.74 (s, 2H), 3.83 (s, 3H), 3.78 (s, 3H), 3.18 (d, J = 7.1 Hz, 4H), 0.98 (t, J = 7.1 Hz, 6H); ¹³

C

NMR (126 MHz, DMSO-d₆) δ 165.0, 164.6, 152.6, 151.6, 147.2, 146.2, 129.7, 129.4, 127.4, 125.7,

117.0, 115.5, 112.3, 55.9, 55.9, 40.5, 27.4, 13.4; ESI–MS m/z C₂₂H₂₄FN₅NaO₄S₂[M + Na]⁺

calcd 528.12, found 527.85.

N,N-diethyl-4,5-dimethoxy-2-((6-(4-nitrophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-

3-yl) methyl)benzenesulfonamide, 6d: red solid, 58% yield, m.p. = 192–194 ^◦C; ¹H NMR

(500 MHz, CDCl₃)

4.77 (s, 2H), 3.84 (s, 3H), 3.79 (s, 3H), 3.19 (q, J = 7.1 Hz, 4H), 0.99 (t, J = 7.1 Hz, 6H); ¹³

NMR (126 MHz, CDCl₃) 164.2, 151.6, 149.6, 147.2, 134.6, 130.7, 129.5, 128.5, 127.3, 124.8,

δ 8.44 (d, J = 8.7 Hz, 2H), 8.22 (d, J = 8.7 Hz, 2H), 7.34 (s, 1H), 7.16 (s, 1H),

C

δ

123.7, 115.5, 112.3, 55.9, 40.5, 27.4, 13.5 (1 carbon is missing due to overlapping); ESI–MS

m/z for C₂₂H₂₄N₆NaO₆S₂[M + Na]⁺calcd 555.11, found 554.90.

2-((6-(2,5-dinitrophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)methyl)-N,N-diethyl-

4,5-dimethoxybenzenesulfonamide, 6e: orange solid, 32% yield, m.p. = 201–203 ^◦C; ¹H

NMR (500 MHz, DMSO-d₆)

7.33 (s, 1H), 7.08 (s, 1H), 4.70 (s, 2H), 3.83 (s, 3H), 3.78 (s, 3H), 3.12 (q, J = 7.0, 4H), 0.94 (t,

J = 7.0 Hz, 6H); ¹³C NMR (126 MHz, DMSO-d₆)

161.2, 157.6, 151.7, 150.6, 149.4, 147.2,

δ 8.73 (s, 1H), 8.70 (d, J = 8.8 Hz, 1H), 8.50 (d, J = 8.8 Hz, 1H),

δ

129.4, 128.2, 127.7, 127.1, 127.0, 124.4, 115.3, 112.5, 55.9, 55.8, 40.3, 27.3, 13.3 (1 carbon is

missing due to overlapping); ESI–MS m/z for C₂₂H₂₃N₇NaO₈S₂[M + Na]⁺calcd 600.09,

found 599.90.

2-((6-(3-chlorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)methyl)-N,N-diisopropyl-

4,5-dimethoxybenzenesulfonamide, 7a: yellow solid, 53% yield, m.p. = 160–162 ^◦C; ¹H

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NMR (500 MHz, DMSO-d₆)

δ

7.96 (s, 1H), 7.90 (d, J = 7.6 Hz, 1H), 7.76 (d, J = 7.9 Hz, 1H),

7.65 (t, J = 7.9 Hz, 1H), 7.37 (s, 1H), 7.09 (s, 1H), 4.85 (s, 2H), 3.82 (s, 3H), 3.77 (s, 3H),

3.59–3.65 (m, 2H), 1.23 (d, J = 6.7 Hz, 12H); ¹³C NMR (126 MHz, DMSO-d₆)

δ

164.6, 151.9,

151.6, 147.2, 146.3, 134.3, 132.5, 131.6, 130.9, 130.4, 127.6, 126.3, 125.9, 115.0, 112.4, 55.8, 55.8,

48.1. 27.3, 21.5; ESI–MS m/z for C₂₄H₂₉ClN₅O₄S₂[M + H]⁺calcd 550.13, found 549.85.

2-((6-(4-fluorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)methyl)-N,N-diisopropyl-

◦

4,5-dimethoxybenzenesulfonamide, 7b: beige solid, 65% yield, m.p. = 177–179 C; ¹H NMR

(500 MHz, DMSO-d₆)

δ 8.00 (dd, J = 8.8, 5.2 Hz, 2H), 7.47 (t, J = 8.8 Hz, 2H), 7.37 (s, 1H), 7.09

(s, 1H), 4.84 (s, 2H), 3.82 (s, 3H), 3.77 (s, 3H), 3.36–3.64 (m, 2H), 1.22 (d, J = 6.8, 12H); ¹³

C

NMR (126 MHz, DMSO-d₆) δ 165.0, 164.5, 152.6, 151.6, 147.2, 146.3, 130.4, 129.7, 127.6, 125.7,

117.0, 115.0, 112.5, 55.8, 55.8, 48.1, 27.3, 21.5; ESI–MS m/z for C₂₄H₂₉FN₅NaO₄S₂[M + Na]⁺

calcd 556.15, found 555.95.

N,N-diisopropyl-4,5-dimethoxy-2-((6-(4-nitrophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-

3-yl)methyl)benzenesulfonamide, 7c: beige solid, 54% yield, m.p. = 212–214 ^◦C; ¹H NMR

(500 MHz, CDCl₃)

δ 8.36 (d, J = 8.8, 5.2 Hz, 2H), 8.07 (d, J = 8.8 Hz, 2H), 7.49 (s, 1H), 6.89 (s,

1H), 5.00 (s, 2H), 3.90 (s, 3H), 3.84 (s, 3H), 3.59–3.66 (m, 2H), 1.33 (d, J = 6.8Hz, 12H); ¹³

C

NMR (126 MHz, CDCl₃) δ 163.8, 153.3, 152.2, 150.1, 147.9, 147.4, 135.1, 131.3, 128.3, 127.5,

124.7, 113.9, 113.0, 56.3, 56.3, 48.9, 27.9, 22.1; ESI–MS m/z C₂₄H₂₈N₆NaO₆S₂[M + Na]⁺calcd

583.14, found 582.95.

2-((6-(2,5-dinitrophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)methyl)-N,N-diisopropyl-

4,5-dimethoxybenzenesulfonamide, 7d: yellow solid, 35% yield, m.p. = 180–182 ^◦C; ¹H

NMR (500 MHz, DMSO-d₆)

1H), 6.98 (s, 1H), 4.81 (s, 2H), 3.81 (s, 3H), 3.76 (s, 3H), 3.55 (2H, overlapping with H₂O),

1.19 (d, J = 6.7 Hz, 12H); ¹³C NMR (126 MHz, DMSO-d₆)

161.2, 151.6, 150.5, 149.3, 147.2,

δ 8.71 (dd, J = 15.3, 5.5 Hz, 2H), 8.50 (d, J = 8.8 Hz, 1H), 7.35 (s,

δ

146.1, 130.3, 128.2, 127.7, 127.4, 127.0, 124.5, 114.6, 112.5, 55.8, 55.7, 48.1, 27.2, 21.5; (1 carbon

is missing due to overlapping) ESI–MS m/z for C₂₄H₂₇N₇KO₈S₂[M + H]⁺calcd 606.14,

found 605.90.

(E)-2-((6-(4-chlorostyryl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)methyl)-N,N-diethyl-

4,5-dimethoxybenzenesulfonamide, 9a: orange solid, 55% yield, m.p. = 196–198 ^◦C; ¹H

NMR (500 MHz, DMSO-d₆)

7.33 (s, 1H) 7.10 (s, 1H), 4.69 (s, 2H), 3.83 (s, 3H), 3.78 (s, 3H), 3.16 (q, J = 7.1 Hz, 4H), 0.98

(t, J = 7.1 Hz, 6H); ¹³C NMR (126 MHz, DMSO-d₆)

165.7, 151.6, 147.1, 146.1, 139.4, 134.8,

δ 7.84 (d, J = 8.6 Hz, 2H), 7.64 (s, 2H), 7.53 (d, J = 8.6 Hz, 2H),

δ

133.4, 129.8, 129.4, 129.1, 127.3, 119.0, 115.4, 112.3, 55.9, 55.9, 40.5, 27.3, 13.4, 13.4 (1 carbon is

missing due to overlapping); ESI–MS m/z for C₂₄H₂₆ClN₅NaO₄S₂[M + Na]⁺calcd 570.10,

found 569.95.

(E)-N,N-diethyl-2-((6-(4-fluorostyryl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)methyl)-

4,5-dimethoxybenzenesulfonamide, 9b: yellow solid, 67% yield, m.p. = 118–120 ^◦C, ¹H

NMR (500 MHz, DMSO-d₆)

J = 16.4 Hz, 1H), 7.31 (s, 1H) 7.30 (d, J = 8.6 Hz, 2H), 7.10 (s, 1H), 4.69 (s, 2H), 3.83 (s, 3H),

3.78 (s, 3H), 3.17 (q, J = 7.1 Hz, 4H), 0.98 (t, J =7.1 Hz, 6H); ¹³C NMR (126 MHz, DMSO-d₆)

δ 7.87 (dd, J = 8.6, 5.6 Hz, 2H), 7.65 (d, J = 16.4 Hz, 1H), 7.57 (d,

δ

165.9, 163.2, 151.6, 147.2, 146.1, 139.6, 131.6, 130.5, 129.4, 127.4, 118.1, 116.1, 115.4, 112.3, 55.8,

40.5, 27.3, 13.5; (2 carbons are missing due to overlapping) ESI–MS m/z for C₂₄H₂₇FN₅O₄S₂

[M + H]⁺calcd 531.15, found 532.00.

(E)-N,N-diethyl-4,5-dimethoxy-2-((6-(3-nitrostyryl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-

◦

3-yl)methyl)benzenesulfonamide, 9c: brown solid, 37% yield, m.p. = 110–112 C; ¹H NMR

(500 MHz, CDCl₃)

7.58 (dd, J = 8.4, 4.2 Hz, 1H), 7.47 (s, 1H), 7.11 (d, J = 16.2 Hz, 1H), 6.88 (s, 1H), 4.81 (s, 2H),

3.90 (s, 3H), 3.82 (s, 3H), 3.25 (q, J = 7.1 Hz, 4H), 1.08 (t, J = 7.1 Hz, 6H); ¹³C NMR (126 MHz

CDCl₃) 164.6, 152.6, 152.1, 147.7, 147.5, 146.5, 136.0, 133.9, 130.6, 130.0, 129.6, 128.8, 127.4,

δ 8.09 (d, J = 8.2 Hz, 1H), 7.85 (d, J = 16.2 Hz, 1H), 7.70 (d, J = 4.1 Hz, 2H),

,

δ

125.3, 122.6, 113.9, 113.0, 56.2, 56.1, 40.6, 27.5, 13.4; ESI–MS m/z for C₂₄H₂₆N₆NaO₆S₂

[M + Na]⁺calcd 581.13, found 581.10.

(E)-N,N-diethyl-4,5-dimethoxy-2-((6-(4-nitrostyryl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-

◦

3-yl)methyl)benzenesulfonamide, 9d: brown solid, 26% yield, m.p. = 126–128 C; ¹H NMR

(500 MHz, CDCl₃)

δ 8.28 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 8.4 Hz, 2H), 7.48 (s, 1H), 7.33 (d,

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J = 12.8 Hz, 2H), 6.89 (s, 1H), 4.83 (s, 2H), 3.91 (s, 3H), 3.83 (s, 3H), 3.26 (q, J = 7.1 Hz, 4H),

1.11 (t, J = 7.1 Hz, 6H); ¹³C NMR (126 MHz, CDCl₃)

δ 164.4, 152.5, 152.1, 148.5, 147.6, 146.7,

139.9, 137.6, 129.7, 128.3, 127.3, 124.4, 122.1, 114.0, 113.0, 56.3, 56.2, 40.7, 27.6, 13.5. ESI–MS

m/z for C₂₄H₂₆N₆NaO₆S₂[M + Na]⁺calcd 581.13, found 580.90.

(E)-2-((6-(4-chlorostyryl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)methyl)-N,N-diisopro-

pyl-4,5-dimethoxybenzenesulfonamide, 10a: white solid, 54% yield, m.p. = 135–137 ^◦C;

¹H NMR (500 MHz, DMSO-d₆)

δ

7.83 (d, J = 8.6 Hz, 2H), 7.63 (d, J = 2.5 Hz, 2H), 7.52 (d,

J = 8.5 Hz, 2H), 7.35 (s, 1H), 7.03 (s, 1H), 4.78 (s, 2H), 3.82 (s, 3H), 3.77 (s, 3H), 3.63–3.56

(m, 2H), 1.21 (d, J = 6.8 Hz, 12H); ¹³C NMR (126 MHz, DMSO-d₆)

165.7, 151.5, 147.2,

139.3, 134.8, 133.4, 130.4, 129.8, 129.0, 127.7, 119.0, 114.8, 112.4, 55.8, 55.8, 48.1, 27.3, 21.5;

2 carbons are missing due to overlapping) ESI–MS m/z for C₂₆H₃₀ClN₅O₄S₂[M + H]⁺

δ

(

calcd 576.15, found 575.95.

(E)-2-((6-(4-fluorostyryl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)methyl)-N,N-diisopro-

pyl-4,5-dimethoxybenzenesulfonamide, 10b: yellow solid, 66% yield, m.p. = 127–129 ^◦C;

¹H NMR (500 MHz, DMSO-d₆)

δ 7.88 (dd, J = 8.6, 5.6 Hz, 2H), 7.65(d, J = 16.4 Hz, 1H),

7.56 (d, J = 16.4 Hz, 1H), 7.36 (s, 1H), 7.31 (d, J = 8.6, 2H), 7.08 (s, 1H), 4.78 (s, 2H), 3.82

(s, 3H), 3.77 (s, 3H), 3.57–3.62 (m, 2H), 1.22 (d, J = 6.7 Hz, 12H); ¹³C NMR (126 MHz,

DMSO-d₆)

δ 165.8, 163.2, 151.5, 147.2, 146.2, 139.5, 131.1, 130.5, 130.4, 127.7, 118.1, 116.1,

114.8, 112.4, 55.8, 55.8, 48.1, 27.3, 21.5; (1 carbon is missing due to overlapping) ESI–MS m/z

for C₂₆H₃₁FN₅O₄S₂[M + H]⁺calcd 560.18, found 560.05.

The NMR spectra of triazolo[3,4-b]thiadiazoles derivatives are presented in the

Supplementary Materials.

2.2. Cell Lines and Culture Conditions

The in vitro antiproliferative activities of the novel triazolo[3,4-b]thiadiazole deriva-

tives were estimated in ten well-established human cancer cell lines including three ovarian

(SKOV-3, UWB 1.289, and UWB 1.289+BRCA1), ﬁve colorectal adenocarcinoma (DLD-1,

HT-29, LS174T, LoVo, and SW403), and two prostate cancer cell lines (PC-3 and DU-145).

All cancer cell lines were obtained from the American Type Culture Collection (ATCC) and

cultivated in different culture media according to the instructions. The required growth

mediums were supplemented with 10% fetal bovine serum and 1% streptomycin/penicillin.

◦

All cancer cell lines were cultured as monolayers and maintained at 37 C, 5% CO₂in

humidiﬁed air.

2.3. Drug Preparation for In Vitro Studies

Stock solutions of all tested compounds were prepared by dissolving the required

amount of substances in the DMSO solvent to reach the desired concentration of 15 mM.

The ﬁnal volume of DMSO did not exceed 1% in the culture medium so that no cytotoxic

effects could be revealed.

2.4. In Vitro Antiproliferative Assay

To evaluate the in vitro antiproliferative activity of the triazolo[3,4-b]thiadiazole

derivatives, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

was assessed. According to the protocol, cells were ﬁrstly seeded into a 96-well plate

◦

at a density of 8

×

10³cells and grown for 24 h at 37 C, 5% CO₂in humidiﬁed air.

Subsequently, cells were treated with the tested compounds in a speciﬁc range of drug

concentrations (1–100 µM). Following 48 h of drug exposure, the culture media were

discarded and replaced with 100 µL of fresh medium. Afterwards, 50 µL of dissolved MTT

in phosphate-buffered saline (PBS) solution (5 mg/mL) were added in each well and cells

were incubated for 3 h at 37 ^◦C, 5% CO₂in humidiﬁed air. Incubation time was followed

by MTT discard and the addition of 100 µL of DMSO solvent so that the formazan crystals

could be dissolved. The absorbance of the converted dye was immediately measured at

a wavelength of 540 nm on an ELISA reader (VersaMax Microplate Reader, Orleans, CA,

USA) [25].

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The in vitro anticancer effect of a tested compound on cell viability was determined

by three dose–response parameters termed GI₅₀, TGI, and IC₅₀. More speciﬁcally, the GI₅₀

value indicates the required concentration of the compound that impels 50% cell growth

inhibition, the TGI value corresponds to the concentration that induces 100% cell growth

inhibition, and the IC₅₀value is the concentration that causes 50% cell death. The two

parameters GI₅₀and TGI represent the cytostatic activity, whereas IC₅₀reﬂects the cytotoxic

effect of a drug [26,27]. To estimate the three parameters, nine absorbance measurements

were required including control 24 h (Ct24), control 72 h (Ct72), and the mean of cell

survival in all seven drug concentrations (Tt72). The percentage growth inhibition was

calculated according to National Cancer Institute’s (NCI) protocol: [(Tt72x)

−

(Ct24)/(Ct72)

(Ct24)/Ct24]

−

(Ct24)]

×

100 for concentrations for which Tt72x > Ct24 and [(Tt72x)

−

×

100 for concentrations for which Tt72x < Ct24. GI₅₀was calculated from [(Tt72x)

−

(Ct24)/(Ct72)

(Ct24)]

−

(Ct24)]

×

100 = 50, TGI was calculated from [(Tt72x)

(Ct24)/Ct24]

−

(Ct24)/(Ct72)

−

×

100 = 0, and IC₅₀was calculated from [(Tt72x)

−

×

100 = 50. All the

experiments were carried out in triplicate.

2.5. Animals

CB17 severe combined immunodeﬁcient (SCID) male and female mice were used

for toxicity and antitumor activity studies. Mice were obtained from National Center for

Scientiﬁc Research (NCSR) Demokritos, Institute of Biology, Athens, Greece. CB17 SCID

mice were fed water and an irradiated standard rodent diet ad libitum and housed under

speciﬁc pathogen-free conditions. For ethical reasons, the weight and tumor volumes of the

animals were measured twice a week during the experiment. In case of cachexia, suffering,

or increase in the tumor volume up to 2000 mm³, the animals were sacriﬁced by cervical

disruption. The protocol was approved by the ethical committee of the National and

Kapodistrian University of Athens and was conducted according to the European Directive

86/609/EEC guidelines for the care and use of laboratory animals. The study received a

permit from the Veterinary Directorate of the Prefecture of Athens (approval #: 2023/2017)

according to the Greek legislation conforming to the 2010/53/ EU Council Directive.

2.6. In Vivo Acute Toxicity

For intraperitoneal (i.p.) treatment, stock solutions of the 3 tested compounds were

prepared immediately before use. They were suspended in corn oil in the desired concen-

trations following initial dissolution in 10% dimethyl sulfoxide (DMSO). This concentration

by itself produced no observable toxic effect. Brieﬂy, the acute toxicity of the tested

compounds was assessed for lethality in CB17 SCID mice and determined as previously

described [28,29]. The three derivatives (KA25, KA26, and KA39) were tested at four

dosages: 50, 100, 150, and 250 mg/kg. Each dosage comprised a group of ten mice, in-

cluding males and females, that received single i.p. injections. Overall, forty mice were

required to estimate the acute toxicity for each compound (10 mice/dosage). All mice were

observed for 30 days. The therapeutic dose of the compounds is usually deﬁned as LD10

(lethal dose for 10% of animals), while the short-term poisoning potential of a substance

is termed LD50 (lethal dose for 50% of animals). Both doses were estimated graphically

(30-day curves).

2.7. HT-29 Colorectal Adenocarcinoma

HT-29 (human colorectal adenocarcinoma) tumor volume was developed by subcuta-

neously implanting 2

×

10⁶HT-29 cells/mouse in the right hind ﬂank. In vivo antitumor

activity was investigated in 22 mice, of which 7 mice served as the common control group

and the 15 remaining mice were divided into three groups of 5 mice. Therefore, each deriva-

tive was administered in a group of ﬁve mice. The animals were randomized into test

groups on day 28 when their tumor volumes were in the 150–200 mm³range. After random-

ization, the mice were treated intraperitoneally (day 1) with the tested compounds (KA25,

KA26, and KA39) as a single dose LD10. The control group received saline buffer (0.2 mL)

.

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2.8. Estimation of Tumor Growth

The tumor volumes were measured using an analogue caliper twice a week starting

on the ﬁrst day of treatment. To determine the volume (mm³) of each tumor in an animal,

the following formula was used: (D

×

d²/2), where D and d refer to the larger and smaller

perpendicular dimensions collected at each measurement, respectively. The diameters were

determined from two orthogonal measurements on each tumor. The mean tumor volume

was determined by average all tumor volumes in each group [30]. The weekly mean tumor

area change was determined and tumor inhibition (TI) was calculated by the formula:

TI (%) = [1

−

(TWT

−

TWZ)/[(TWC

−

TWZ)]

×

100, where TWT is determined as the

tumor area (mm³) in treated animals at the time of evaluation, TWZ is determined as the

tumor area (mm³) at the time of initiation of treatment (zero time), and TWC is determined

as the tumor volume (mm³) in untreated animals (controls) at the time of evaluation. The

animals were sacriﬁced when the tumor volume reached 2000 mm³(endpoint).

2.9. Detection of Akt1 and Akt2 Phosphorylation by Intracellular Flow Cytometry

To investigate whether KA25 and KA39 triazolo[3,4-b]thiadiazole derivatives inhibit

the phosphorylation of Akt1 and Akt2 isoforms, three colorectal cancer cell lines including

HT-29, LoVo, and SW403 were intracellularly stained upon treatment with the two referred

compounds. Firstly, cells were seeded into a 6-well plate at a density of 7

and maintained at 37 C in a 5% humidiﬁed air. After 24 h of cell growth, the culture

medium was discarded and replaced with 2 mL of fresh medium. Then, the cells were

×

10⁵cells/well

◦

treated with KA25 and KA39 derivatives at GI50 and TGI concentrations (

24 h. The required GI50 and TGI concentrations ( M) of the KA25 and KA39 derivatives

in HT-29 and LoVo cells have been determined in previous studies [23]. To estimate the

TGI concentration ( M) of the KA25 compound in SW403 cells, further MTT assays were

µM) for 3 and

µ

carried out as previously described. Following drug exposure at the mentioned treatment

conditions, the cells were collected after being washed with ice-cold PBS (pH = 7.4) and

detached enzymatically with standard trypsinization. Two centrifugations were followed

at 1500 rpm for 5 min, including two washing steps with 2 mL of cell staining buffer (Bi-

oLegend, San Diego, CA, USA). Cell pellets were ﬁxed with 500 µL of pre-warmed ﬁxation

buffer (BioLegend, San Diego, CA, USA) and incubated at 37 ^◦C for 15 min. Afterwards,

one centrifugation was carried out at 1000 rpm for 5 min, followed by supernatant discard

and resuspension of cell pellet through vortex. Then, the cells were washed with 2 mL

of cell staining buffer twice, including two centrifugations at 1000 rpm for 5 min at room

temperature. After the cell pellet was resuspended with gentle pipetting, cells were perme-

abilized by adding 1000 µL of pre-chilled True-Phos Permeabilization buffer (BioLegend,

San Diego, CA, USA) while vortexing. Following overnight incubation at −20 ^◦C, the cells

were centrifuged at 2000 rpm for 5 min and the supernatant was discarded. After the cell

pellet was resuspended by vortexing, two centrifugations were performed at 2000 rpm for

5 min including two washes with 2 mL of cell staining buffer. The cell pellet was resus-

pended with 200

appropriate tubes. To detect total Akt1 and Akt2, 2

(BioLegend, San Diego, CA, USA) was added, respectively. Additionally, 2

anti-Akt phospho(Ser473) (BioLegend, San Diego, CA, USA) was added for the detection of

either phosphorylated AKT1 or AKT2. Furthermore, a cocktail of isotype controls (5 L of

puriﬁed mouse IgG1, 5 L of puriﬁed mouse IgG2b, and 1 L of puriﬁed rabbit polyclonal

µ

L of cell staining buffer, and equal volumes were transferred into the

L of puriﬁed anti-Akt1 and anti-Akt2

L of puriﬁed

µ

isotype control antibody) was used to differentiate nonspeciﬁc background signals from

speciﬁc antibody signals. The cells were incubated for 30 min at room temperature in the

dark and then stained with 5

µL of phycoerythrin (PE) donkey anti-rabbit IgG and 2 µL of

Alexa Fluor 488 Goat anti mouse IgG (BioLegend, San Diego, CA, USA). After 15 min of

incubation in the dark, the cells were resuspended with 1000 µL of cell staining buffer and

analyzed in the ﬂow cytometer (CyFlow^®, SL, Partec, GmbH, Muenster, Germany). Flow

cytometric analysis was carried out in triplicate and performed by Partec Flomax software.

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The inhibitory impact of the two triazolo[3,4-b]thiadiazole derivatives KA25 and KA39

on the phosphorylation of either Akt1 (p-Akt1) or Akt2 (p-Akt2) was determined by the

following formulas: p-Akt1(%) or p-Akt2 (%) = [(mean-xtreated

−

isotypetreated)]/[(mean-

xuntreated isotypeuntreated)] 100, where the isotype value is subtracted from the

−

×

mean-x value of the corresponding condition, including treated and untreated cells. As

the formulas indicate, the difference in value of treated cells is divided by the difference in

value of untreated cells (control group) to estimate the extent of inhibition. In addition to

the above formulas, the ratios phosphoAkt1/total Akt1 as well as phphoAkt2/total Akt2

were calculated and correlated to the corresponding ratios of untreated cells (control).

2.10. In Silico Studies

Molecular docking studies were carried out using Autodock Vina package [31], and

crystal structures of Akt1 (Protein Data Bank (PDB) ID: 4GV1) and Akt2 (PDB ID: 2JDR)

were taken from Protein Data Bank. For all PDB structures, during docking, co-crystallized

inhibitors and water molecules were removed while polar hydrogens and Gasteiger charges

were added using Auto Dock Tools 1.5.6. The triazolo[3,4-b]thiadiazole derivatives were

built and optimized through energy minimization in ChemOfﬁce 15.0. Prior to docking

simulation, a grid box with spacing 1 Å and size of 20

×

20

×

20 (Å) around the ATP

binding sites of all kinases was deﬁned, and the “exhaustiveness” was set to 100. The

docking poses with the lowest binding afﬁnity were selected for presentation. PyMOL 2.1.1

viewer (Schrödinger, LLC) and BIOVIA/Discovery Studio were used for visualization of

the docked complexes.

2.11. Statistical Analysis

Student’s t-test was used to compare the level of signiﬁcance between the experimental

groups. Differences with p-valued less than 0.05 were considered signiﬁcant (p < 0.05, two

tailed paired t-test). Microsoft Excel (Microsoft Hellas, Athens, Greece) was used.

3. Results

3.1. Synthesis of triazolo[3,4-b]thiadiazole Derivatives

3.1.1. Synthesis of 3,6-disubstituted 1,2,4-triazolo-[3,4-b]-[1,3,4]-thiadiazoles

Synthesis of the 1,2,4-triazolo-[3,4-b]-[1,3,4]-thiadiazoles was accomplished according

to the synthetic methodology developed by our group (Schemes 1 and 2) [23]. After a

reaction with oxalyl chloride, 2-(3,4-dimethoxyphenyl)acetic acid

the corresponding methyl ester reacted with chlorosulfonic acid in CHCl₃to give methyl

2-(2-(chlorosulfonyl)-4,5-dimethoxyphenyl)acetate in 88% yield. Subsequently, sulfonyl

chloride furnished the corresponding sulfonamides 3a,b after reaction with Et₂NH or

1 was esteriﬁed and

2

iPr₂NH. Furthermore, these compounds reacted with hydrazine hydrate to give hydrazides

4a,b that reacted with KOH and CS₂in EtOH to give the potassium thiocarbamates. The

latter products were cyclized in the presence of hydrazine hydrate to triazoles 5a,b [32].

The desired 1,2,4-triazolo[3,4-b]-[1,2,4]-thiadiazoles (6a–e, 7a–d, 9a–d, and 10a,b) were

ﬁnally prepared via the reaction of triazoles 5a,b with POCl₃and various benzoic or

cinnamic acids.

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Scheme 1. Synthesis of benzoic acid derivatives.

Scheme 2. Synthesis of cinnamic acid derivatives.

3.1.2. In Vitro Anticancer Activity

The ﬁfteen novel triazolo-thiadiazole derivatives and the previously described KA25,

KA26, and KA39 were tested in vitro in two well-established human cancer cell lines:

PC-3 and SKOV-3 cells. According to the ﬁrst drug screening results, the 13 derivatives

were inactive at the tested concentrations while the most active compounds (KA25, KA26,

KA39, 6e, and 7d) were further tested in vitro in colorectal, ovarian, and prostate cancer

cell lines. As Tables 1 –3 show, the most potent anticancer activities in all human can-

cer cell lines were induced by the KA39, 6e, and 7d compounds (p < 0.001, two tailed

paired t-test). All these compounds bear the 2,5-dinitrophenyl substituent on C-6 of the

triazolo[3,4-b][1,3,4]thiadiazole core. Nevertheless, the triazolo[3,4-b]thiadiazole KA39

was signiﬁcantly more active than 6e and 7d, which displayed less anticancer potency

(Tables 1–3). Concerning KA25 and KA26 derivatives, both exhibited cytostatic activity in

all human cancer cell lines, whereas KA25 induced cytotoxic effects only in ovarian and

prostate cancer cells.

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Table 1. Growth inhibition/cytostatic (GI50 and TGI) and cytocidal/cytotoxic (IC50) effects induced by triazolo[3,4-

b]thiadiazole derivatives KA25, KA26, and KA39 on nine human cancer cell lines. GI₅₀: 50% growth inhibition; TGI: Total

growth inhibition; IC₅₀: the concentration that causes 50% cell death.

KA25

KA26

KA39

Cell Line

GI₅₀

TGI

IC₅₀

GI₅₀

TGI

IC₅₀

GI₅₀

TGI

IC₅₀

(µM)

SKOV-3

15 ± 0.5

2 ± 0.5

50 ± 1.5

5 ± 0.7

85 ± 2.1

62 ± 1.2

20 ± 0.5

29 ± 0.7

>100

7 ± 0.2

6 ± 0.2

12 ± 0.8

10 ± 0.8

25 ± 1.4

22 ± 1.2

UWB 1.289

53 ± 1

70 ± 1.4

UWB1.289+

BRCA1

4.0 ± 0.5

13 ± 0.8

38 ± 1.1

42 ± 0.5

65 ± 0.7

>100

6 ± 0.5

11 ± 0.7

94 ± 2.3

HT-29

LS174T

SW403

LoVo

1 ± 0.3

9.3 ± 2.0

4.8 ± 0.8

8 ± 0.76

14 ± 1.0

12.8 ± 1.3

>100

100 ± 0.1

>100

2 ± 0.76

8 ± 1.5

11 ± 0.8

15 ± 0.8

42.5 ± 1.5

7 ± 1.0

>100

11.5 ± 0.8

12 ± 1.5

5.2 ± 0.2

2.2 ± 0.2

5 ± 0.15

5.8 ± 0.2

15.9 ± 0.55

17 ± 1.3

7.9 ± 0.7

5.5 ± 0.1

8 ± 0.1

19.5 ± 0.9

21.5 ± 1.5

10 ± 0.76

10.5 ± 0.15

12 ± 0.1

>100

13.8 ± 0.52

22 ± 0.8

13.2 ± 0.5

>100

PC-3

>100

DU-145

27 ± 0.3

70 ± 1.3

8 ± 0.4

10.3 ± 1.8

Table 2. Growth inhibition/cytostatic (GI50 and TGI) and cytocidal/cytotoxic (IC50) induced by the

thirteen newly synthesized triazolo[3,4-b]thiadiazoles on two human cancer cell lines.

PC-3

SKOV-3

Compound

GI₅₀

TGI

IC₅₀

GI₅₀

TGI

IC₅₀

(µM)

9b

10b

9a

42 ± 1.0

31 ± 1.1

11 ± 1.0

2 ± 0.5

32 ± 0.4

25 ± 1.4

20 ± 0.8

90 ± 2.0

25 ± 0.5

50 ± 0.5

23 ± 0.8

38 ± 2.0

30 ± 0.5

>100

66 ± 2.5

>100

50 ± 2.0

84 ± 2.0

76 ± 6.0

36 ± 0.1

6 ± 0.3

16 ± 0.8

>100

10a

6b

6a

84 ± 2.0

56 ± 0.6

112 ± 2.5

>100

104 ± 0.8

>100

60 ± 0.8

14 ± 2.5

46 ± 1.5

>100

7a

>100

6c

>100

7b

6d

7c

80 ± 1

>100

80 ± 0.8

80 ± 0.5

15 ± 1.4

>100

102 ± 1.2

>100

28 ± 2.2

>100

9c

>100

9d

>100

Table 3. Growth inhibition/cytostatic (GI50 and TGI) and cytocidal/cytotoxic (IC50) induced by

tri-azolo[3,4-b]thiadiazole derivatives 6e and 7d on ﬁve human cancer cell lines.

6e

7d

Cell Lines

GI₅₀

TGI

IC₅₀

GI₅₀

TGI

IC₅₀

(µM)

SKOV-3

PC-3

18 ± 0.7

11 ± 1.0

13 ± 0.5

25 ± 2.0

18 ± 1.0

24 ± 1.2

17 ± 1.0

18.3 ± 0.2

37 ± 2.0

29 ± 0.5

38 ± 1.8

26.5 ± 0.9

28 ± 0.2

53.6 ± 1.05

44 ± 1.0

13 ± 0.5

8 ± 1.0

6.8 ± 0.8

7.3 ± 0.75

17.9 ± 2.51

17 ± 1.0

11 ± 1.04

14 ± 0.5

12 ± 0.5

26 ± 1.52

23 ± 1.4

22 ± 0.3

21.3 ± 0.9

22 ± 1.0

40 ± 1.0

DU-145

DLD-1

HT-29

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3.1.3. In Vivo Acute Toxicity

The triazolo[3,4-b]thiadiazole derivatives (KA25, KA26, and KA39) were also eval-

uated for their in vivo acute toxicity, and such values are illustrated in Table 4 . More

speciﬁcally, the data showed that the LD10 values for KA25, KA26, and KA39 were 200,

100, and 70 mg/kg, respectively. The low acute toxicity in correlation with the in vitro

anticancer activity provided evidence that the triazolo[3,4-b]thiadiazoles KA25, KA26, and

KA39 are of high interest for cancer therapeutics since they promise high therapeutic ratios.

Table 4. Acute toxicity of compounds in CB17 severe Combined Immunodeﬁciency (SCID) mice.

Compound

KA39

LD₁₀(mg/kg)

LD₅₀(mg/kg)

70

100

360

160

KA25

200

100

KA26

3.1.4. In Vivo Anticancer Activity

We also examined the antitumor activity of compounds KA25, KA39, and KA26 using

the LD10 dose in an HT-29 human tumor (colorectal adenocarcinoma) xenograft in CB17

SCID mice. The results are presented in Figure 2A,B. As it is shown, the treatments with

KA25, KA39, and KA26 caused statistically signiﬁcant (p < 0.001) reductions in the size of

HT-29 tumors in mice (Figure 2A). Eighteen days after treatment, the volume size of the

tumors was 1003

group that received a single dose of KA39, to 528.5

a single dose of KA26, and to 521.6

101.6 mm³for the group that received a single dose

±

287.8 mm³for the control group compared to 489.17

±

81.5 mm³for the

±

167.7 mm³for the group that received

±

of KA25 at the respective LD10 concentration levels (Table 4 and Figure 2B). With respect

to tumor inhibition (%), the KA39, KA26, and KA25 derivatives displayed 67.6%, 62.4%,

and 62.1%, respectively. As it is indicated by the TI% values, all compounds were equally

effective in the actual reduction in HT-29 tumor volume and rate of tumor growth.

Figure 2. The in vivo antitumor activity of triazolo[3,4-b]thiadiazoles derivatives, KA39, KA26, and KA25 in mice bearing

that HT-29 tumor. ( In vivo growth inhibition of HT-29 human colorectal adenocarcinoma induced by KA39, KA26, and

A

)

KA25 intraperitoneal administration. (B) The best response of tumor was induced by KA39 on day 18. Two-tailed paired

t-test; * p < 0.05.

3.1.5. Phosphorylation of Akt1 and Akt2 Isoforms

Today, it is well-established that the PI3K/Akt pathway regulates both repair and

response to genotoxic damage. Therefore, we aimed to investigate the effect of KA39 and

KA25 on Akt1 and Akt2 phosphorylation. Three colorectal cancer cell lines (HT-29, LoVo,

and SW403) were treated with KA39 and KA25 at GI50 and TGI concentrations (µM) for 3

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and 24 h. With respect to HT-29 cancer cells, treatment with KA39 at a TGI concentration of

15.9

µM for 3 and 24 h induced signiﬁcant inhibition of Akt1 phosphorylation, decreasing

the Akt1 phospho-form by 72.9% and 88.5%, respectively (p < 0.01) (Figure 3A).

(A)

(B)

(C)

(D)

Figure 3. Inhibition of Akt1 and Akt2 phosphorylation in HT-29 cancer cells treated with the triazolo[3,4-b]thiadiazole

derivatives KA25 and KA39 at GI50 and TGI concentrations for 3 and 24 h. ( ) The diagram illustrates the KA25- and

KA39-induced inhibition of Akt1 phosphorylation expressed as absolute values (%). ( ) The diagram shows the induced

inhibition of Akt1 phosphorylation by KA25 and KA39 according to the ratio phosphoAkt1/total Akt1 correlated with

control (untreated cells). ( ) The diagram displays the KA25- and KA39-induced inhibition of Akt2 phosphorylation

A

B

C

expressed as absolute values (%). (D) The diagram presents the induced inhibition of Akt2 phosphorylation by KA25 and

KA39 according to the ratio phosphoAkt2/total Akt2 correlated with the control (untreated cells). * p < 0.05, ** p < 0.01, and

*** p < 0.001.

Moreover, the phosphorylated form of Akt1 was reduced by 73.66% and 75.71% upon

cell treatment with the referred derivative at a GI50 concentration of 11.5 µM for 3 and 24 h,

respectively (p < 0.01) (Figure 3A). As Figure 3B presents, the ratio phosphoAkt1/total Akt1,

correlated with control, is in line with the above-referenced absolute values (%), indicating

that the Akt1 phospho-form was declined to a greater extent when cells were treated with

KA39 at TGI concentration (

signiﬁcantly diminished after HT-29 cells were treated with KA25 at a TGI concentration of

120 M for 3 and 24 h, as the Akt1 phospho-form was decreased by 86.45% and 87.73%,

respectively (p < 0.01) (Figure 3A). It is noteworthy that cell exposure to the same derivative

at a GI50 concentration of 1 M for 3 and 24 h impeded Akt1 phosphorylation (83.38% and

79.8%, respectively) to the same extent as TGI concentration ( M) at both treatment times

µM) for 24 h (p < 0.01). The phosphorylated form of Akt1 was

µ

(p < 0.01) (Figure 3A). The KA25-induced inhibition of Akt1 phosphorylation, estimated by

the ratio phosphoAkt1/total Akt1, supports that the Akt1 phospho-form was signiﬁcantly

reduced under all treatment conditions whereas the greatest decrease was impelled after

treatment at TGI concentration (µM) for 3 h (p < 0.01) (Figure 3B).

Phosphorylation of the Akt2 isoform was also inhibited by the two derivatives KA39

and KA25. More speciﬁcally, following treatment with KA39, at TGI (TGI = 15.9

µM) and

GI50 concentrations (GI50 =11.5 M) for 24 h, the Akt2 phospho-form was reduced by 86%

µ

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and 83.55%, respectively (p < 0.01) (Figure 3C). Contrary to 24 h, Akt2 phosphorylation was

less decreased when HT-29 cells were treated at TGI and GI50 concentrations (µM) for 3 h,

diminishing the phosphorylated form of Akt2 by 58.87% and 60.76%, respectively (p < 0.01)

(Figure 3C). The KA39-induced inhibition of Akt2 phosphorylation was also evaluated

by the ratio phosphoAkt2/total Akt2, according to which the Akt2 phospho-form was

signiﬁcantly decreased under all treatment conditions, with no remarkable ﬂuctuations

arising (p < 0.01) (Figure 3D). However, according to the ratio phosphoAkt2/total Akt2,

KA39 impelled a greater inhibition of Akt2 phosphorylation after cell exposure a TGI con-

centration (

µM) for 24 h (Figure 3D). Furthermore, KA25 resulted in signiﬁcant inhibition

of Akt2 phosphorylation after cells were treated at TGI concentration (TGI = 120

µM) for

3 and 24 h, reducing the Akt2 phospho-form by 82.6% and 86.4%, respectively (p < 0.01)

(Figure 3C). The inhibition of Akt2 phosphorylation, induced by KA25, was also recorded

when cells were treated at GI50 concentration (GI50 =1 µM) for 3 and 24 h, diminishing the

Akt2 phospho-form by 73.74% and 76.9%, respectively (p < 0.01) (Figure 3C). According to

the ratio phosphoAkt2/total Akt2, KA25 declined the phosphorylated form of Akt2 to a

similar extent, though cell exposure at GI50 concentration (µM) for 3 h exhibited a rather

low inhibitory effect on Akt2 phosphorylation (p < 0.01) (Figure 3D).

Further investigation showed that phosphorylation of the two Akt isoforms (Akt1

and Akt2) was signiﬁcantly inhibited by KA39 and KA25 in SW403 and LoVo cancer cells

cancer cells (See the Supplementary Materials, Figures S47A–D and S48A–D). Even though

KA39 inhibited Akt1 phosphorylation to a similar extent, the greatest reduction in Akt1

phospho-form (%) was observed upon treatment at TGI concentration (

three cancer cell lines. On the other hand, KA39 particularly decreased the phosphorylated

form of Akt2 (%) when the cells were treated at both concentrations ( M) for 24 h. The

µM) for 24 h in all

µ

KA39-induced inhibition of Akt1 and Akt2 phosphorylation, expressed as absolute values

(%), also corresponds to the ratios phopshoAkt1/total Akt1 and phosphoAkt2/total Akt2,

respectively. In relation to KA25, the Akt1 and Akt2 phospho-forms were remarkably

reduced (%) under all treatment conditions, though cell exposure at TGI concentration

(

µ

M) for 3 and 24 h impeded phosphorylation of the two Akt isoforms to a greater extent.

Nevertheless, according to the ratio phosphoAkt1/total Akt1, Akt1 phosphorylation was

signiﬁcantly inhibited upon cell treatment at TGI concentration ( M) for 3 h in all three

µ

cancer cell lines. On the other hand, as the ratio phosphoAkt2/total Akt2 suggests, the

KA25-induced inhibition of Akt2 phosphorylation was similar in HT-29 and SW403 cells

whereas the greatest decrease in the Akt2 phospho-form was found in LoVo cells after

treatment at TGI concentrations (µM) for 3 and 24 h.

3.1.6. In Silico Studies

In a following step, in order to understand the interactions of KA25 and KA39 with

Akt1 and Akt2 kinases, molecular docking studies were performed using the Autodock

Vina software 1.1.2 [31]. Prior to docking, the docking protocol was validated. The co-

crystallized inhibitors were removed and re-docked into the catalytic sites of Akt1 (PDB ID:

4GV1) and Akt2 (PDB ID: 2JDR) to determine the reliability of the applied docking protocol.

As illustrated in Figure S1, the docking predictions of ligands in both crystal structures

using Autodock Vina had acceptable root-mean-square deviation (RMSD) values. The data

from the docking experiments of KA25 and KA39 are demonstrated below. The selected

derivatives (KA25 and KA39) have high binding energies (< 8.5 kcal/mol) on the two Akt

−

isoforms (Table 5, Table S1 and Table S2). Both compounds were found to occupy the ATP

binding site of the tested kinases, as presented in Figure 4, and the most stable binding

modes among the top docking poses are illustrated in Figures 5 and 6. The docking analysis

revealed that the Akt1–KA25 complex had the most promising results. Compound KA25

was stabilized by various van der Waals interactions and one hydrogen bond with a Lys158

(3.64 Å) residue of Akt1. It also displayed electrostatic interactions with Asp292 and Lys179

residues as well as hydrophobic interactions with Phe161, Leu156, Val164, and Ala177. The

2,5-dinitrophenyl group of KA39 is oriented towards the hinge region of Akt1, but does

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not interact with it, while the 4-ﬂuorophenyl group of KA25 interacts with the hinge region

of Akt1 at Ala230 and Tyr229. The interactions between KA39 and the binding site of

Akt1 contain electrostatic interactions with Lys179, Glu191, and Asp292 and hydrophobic

interactions with Val164. The opposite is observed for Akt2; KA39 displays hydrophobic

interactions with Phe443, Ala179, Val166, Met282, Phe163, and Thr292; and KA25 stands

outside the hinge and interacts with Phe443, Phe163, Val166, and Lys181. According to

the selected poses, it is clear that the synthesized derivatives are oriented in the catalytic

pocket of Akt kinases and may act as ATP pocket binders.

(a)

(b)

Figure 4. Structures of KA25 (magenta) and KA39 (green) occupying the ATP binding site of (a) 4GV1

(Akt1, pink) and (b) 2JDR (Akt2, blue).

(A)

(B)

left) in the active site of (A) Akt1 (4GV1) and (B) Akt2 (2JDR).

Figure 5. Binding mode of KA25

(

Dotted lines in different colors reﬂect various types of interaction such as hydrogen bonding, charge

or polar interactions, and van der Waals and π-sigma interactions.

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(A)

(B)

Figure 6. Binding mode of KA39 (right) in the active site of (A) Akt1 (4GV1) and (B) Akt2 (2JDR). Dotted lines in different

colors reﬂect various types of interaction such as hydrogen bonding, charge or polar interactions, and van der Waals and

π-sigma interactions.

Table 5. Molecular docking results showing the binding energy between Akt1 and Akt2 with the

synthesized derivatives KA25 and KA39.

Akt1

Akt2

Compounds

Docking Score (Kcal/mol)

KA25

KA39

−9.5

−8.7

−8.9

−8.6

4. Discussion

Aberrations in the PI3K/Akt signal transduction pathway are a very common phe-

nomenon in human carcinogenesis, with over half of tumors showing irregular Akt ac-

tivation [33]. More precisely, the overexpression of certain oncogenes or a lack of tumor

suppressor genes inﬂuences the biological activity of the PI3K/Akt network, stimulat-

ing cancer cell growth. For instance, mutations of Epidermal Growth Factor Receptor,

EGFR/PI3K, loss of the tumor suppressor protein phosphatase and tensin homologue

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(PTEN), and mutations or ampliﬁcations of Akt itself enhance Akt’s signaling in cancer

cells [34,35]. Ampliﬁed and mutated Akt isoforms play crucial roles in tumorigenesis, while

Akt2 is particularly involved in EMT transition and metastasis. Regarding mutations, Akt1

is the most frequently mutated isoform compared with Akt2, in which mutations do not

occur at high frequency and cannot be considered as activating [36,37]. The most prevalent

mutation in Akt1, E17K, is located within the PH domain and is found in several solid

tumors including breast (5.9%), colorectal (1.6%), lung (0.6%), and ovarian cancers (0.8%)

as well as in melanoma (0.5%) [38,39]. Akt1E17K mutation, induced by the substitution of

lysine to glutamic acid at amino acid 17, leads to a pathological translocation of Akt1 to

the plasma membrane and to alterations in PIP speciﬁcity as binding to PIP2 seems to be

highly increased. Given that Akt1 normally binds to PIP3, further utilization of PIP2 in the

case of Akt1E17K enhances Akt1⁰s signaling [39–41].

Tumor aggressiveness as well as poor survival rates are particularly linked to Akt2⁰s

ampliﬁcation and overexpression [9]. According to earlier studies, mutations in the Akt2

isoform are quite rare whilst ampliﬁcations in the isoform have been detected in 16% of

pancreatic cancers; 16% of uterine cancers; 13% of breast cancers; and 5–10% of ovarian,

lung, and bladder cancers. The role of Akt2 in oncogenesis is reﬂected by a reduction in

cell growth and invasiveness induced by siRNA depletion of Akt2 in cells that overexpress

Akt. On the other hand, an ampliﬁed Akt1 is less frequent since it has been found only in

20% of neuroendocrine prostate cancers, 10% of pancreatic cancers, and 3–5% of breast and

serous ovarian cancers [42]. Altogether, Akt is strongly implicated in many types of cancer

and therefore has been validated as a therapeutic target nearly two decades ago [43].

The present study attempts to explore the inhibitory impact of KA25 and KA39

derivatives on Akt1 and Akt2 phosphorylation in three colorectal cancer cell lines (HT-29,

LoVo, and SW403) with speciﬁc PIK3CA and KRAS statuses (Table 6). More speciﬁcally,

two of the referred cell lines, HT-29 and SW403, harbor mutations in PIK3CA, p.P449T and

p.Q546K, respectively, while LoVo cells retain a wild-type PIK3CA (https://cancer.sanger.

ac.uk/cosmic, accessed on 4 September 2020). With regard to PIK3CA mutations, most of

them are concentrated in hotspots within two domains: the helical (exon 9) and kinase (exon

20) [44]. Nonetheless, four additional missense mutations have been detected including

p.P449T, which belongs to gain-of-function mutations and contributes to enhanced PIK3CA

kinase activity (>2-fold) compared to the wild-type PIK3CA. In general terms, gain-of-

function mutations in PIK3CA are known for triggering the PI3K kinase activity and

consequently activating its downstream molecules such as Akt [45]. Further studies have

indicated that rare PIK3CA mutants, including p.Q546K, possess a high oncogenic potency

as well as hyperactivate PI3K/Akt downstream signaling cascade [46]. Due to the fact

that HT-29 and SW403 cell lines contain gain-of-function mutations in PIK3CA, the levels

of phosphorylated Akt may be elevated in both cell lines in comparison to the wild-type

PIK3CA expressed by the LoVo cell line.

Table 6. Description of histotypes and special characteristics of the three human colorectal cancer

cell lines in which the inhibition of Akt (Akt1/2) phosphorylation, induced by KA25 and KA39,

was investigated.

Human Cell Line

Designation

Cancer Type

KRAS Status

wild-type

p.G13D

PIK3CA Status

p.P449T

References

[47,48]

Colorectal adenocarcinoma

HT-29

Colorectal adenocarcinoma,

Duke’s type C, grade IV

LoVo

wild-type

[47,48]

Colorectal adenocarcinoma

Dukes’ type C, grade III

SW403

p.G12V

p.Q546K

[49–51]

With respect to KRAS, two of the three cell lines, LoVo and SW403, are KRAS-mutant-

bearing p.G13D and p.G12V mutations, respectively (Table 6). Experimental studies using

these cell lines showed that neither p.G12V nor p.G13D KRAS mutations stimulate the

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phosphorylation of Akt to a greater extent than wild-type KRAS cells. Moreover, it has

been also demonstrated that Akt phosphorylation was decreased in KRAS-G12V cells

compared to KRAS wild-type cell lines [52–54]. Consequently, the LoVo and SW403 cell

lines (KRAS-mutant) probably express similar or lower levels of phosphorylated Akt than

HT-29 cells (KRAS wild-type). As far as cell sensitivity to Akt inhibitors is concerned,

recent studies support that cell lines with PI3K and/or PTEN mutations display a higher

susceptibility to this type of inhibitor than cells with KRAS and/or v-raf murine sarcoma

viral oncogene homolog B (BRAF) mutations [55]. More speciﬁcally, Akt inhibitors show

greater selectivity and potency in cells with increased Akt kinase activity resulting from

mutations in PI3K or PTEN. Later studies implied that cell sensitivity to allosteric Akt1/2

inhibitors is signiﬁcantly associated with Akt phosphorylation in human cancer cell lines,

leading to the conclusion that cell lines with elevated levels of phosphorylated Akt are

more susceptible to the pharmacologic inhibition of Akt1/2. However, the cell lineage may

affect the pharmacologic sensitivity to Akt1/2 inhibition, for instance, PIK3CA-mutant

breast cancer cell lines are more sensitive than PIK3CA-mutant colon cancer cell lines.

Altogether, high levels of phosphorylated Akt signify AKT dependency in PIK3CA-mutant

cancer cells [56,57].

On the other hand, Akt inhibitors, utilized in the inhibition of downstream effector

pathways, have not shown an impressive antitumor efﬁcacy on KRAS-mutant tumors [58].

As observed, cells with mutated KRAS and/or BRAF are less sensitive to Akt inhibitors

since no alterations in the activation of MAPK pathway have arisen [55]. A relevant

example that correlates PIK3CA and RAS mutations with sensitivity to Akt inhibitors

concerns the AZD5363 compound; PIK3CA and PTEN mutations are strongly associated

with sensitivity to this Akt inhibitor, regardless of RAS status [59]. Additionally, tumors

bearing an overactivated PI3K/Akt pathway without any EGFR, RAS, or BRAF mutations

depend more on this pathway and become more sensitive to selective Akt inhibitors [60].

It is noteworthy that both of the referred to KRAS mutations (p.G12V and p.G13D) are

associated with resistance to anti-EGFR therapy via intracellular pathways, restricting

the administration of anti-EGFR monoclonal antibodies (mAbs) to patients bearing KRAS

wild-type tumors [61,62]. Based on PIK3CA and KRAS statuses, we could put forward the

hypothesis that KA25- and KA39-induced inhibition of Akt1 and Akt2 phosphorylation

might be more effective in HT-29 (PIK3CA-mutant; KRAS wild-type) than those of LoVo

cells (PIK3CA wild-type; KRAS-mutant). Regarding SW403 cells, in which PIK3CA and

KRAS mutations concur, Akt1 and Akt2 phosphorylation could be impeded similarly

to either HT-29 or LoVo cells. Nevertheless, both methods of estimation (% inhibition

and ratios correlated with control) indicate that both triazolo[3,4-b]thiadiazole derivatives

exhibited signiﬁcant inhibitory activity against Akt1and Akt2 phosphorylation in all three

cancer cell lines. It is notable that the inhibition of Akt1 and Akt2 phosphorylation, induced

by KA25 and KA39, was fairly similar in all three cell lines carrying PIK3CA and/or KRAS

mutations. Nonetheless, it could be suggested that the reduction in phosphorylated Akt

(Akt1/2) was slightly greater in LoVo (mutated KRAS) and SW403 (mutated KRAS and

PIK3CA) cells compared to the HT-29 cell line (mutated PIK3CA).

Furthermore, pharmacokinetic studies on the tested compounds are currently under

investigation. Premature experimental data showed that these compounds have poor water

solubility (log mol/L =

−

4.4 to

−

3.5), high CaCO₂permeability (log Papp in 10⁻⁶cm/s

=

−

0.135 to 0.965), and high intestinal absorption (94–100%). These compounds present

low distribution volumes with high binding to serum proteins (72–92%), are unable to

penetrate the CNS, and are poorly distributed to the brain. They are not substrates of

P-glycoprotein but they inhibit both glycoproteins I and II. They metabolize in liver, and

they are substrates of CYP3A4 but inhibit CYP2C19 and CYP2C19. Their total clearance in

rats was 2.2–5.7 mL/min/kg.

It is interesting to note that a large number of small molecules are proven to inhibit

Akt in vitro and in vivo, though only a limited number of these have entered clinical

trials [48]. Ongoing clinical studies indicate that the tested Akt inhibitors are not effective

Pharmaceutics 2021, 13, 493

20 of 23

as single agents, displaying rather low therapeutic responses. Furthermore, even if there is

increasing knowledge concerning Akt functions and activation, there are no Akt inhibitors

approved for oncologic use. For example, miltefosine, the only Akt inhibitor approved, is

intended for treating visceral and cutaneous leishmaniasis and not cancer disease [38,48].

Therefore, the discovery of drugs such as KA39 constitutes a valuable tool in the research

area of Akt inhibitors due to its high anticancer potency and multitarget behavior.

5. Conclusions

In this work, preliminary mechanistic studies showed that KA25 and KA39 exhibit

time- and concentration-dependent inhibition of Akt Ser-473 phosphorylation while in

silico studies pointed out the potentials of KA25 and KA39 to act as ATP binders in the

catalytic sites of Akt1 and Akt2. Among all the tested triazolo[3,4-b]thiadiazole derivatives,

KA39 is the most potent antitumor agent since it induced the most signiﬁcant cytostatic

and cytotoxic activities against all cancer cell lines. According to the current ﬁndings and

prior studies that displayed triazolo[3,4-b]thiadiazole derivatives as topoisomerase II

α

inhibitors, it can be assumed that this particular class of chemical compounds behave

as multitarget anticancer agents. More precisely, KA39 exhibited a signiﬁcant inhibitory

impact on the activities of other biological systems such as those of topoisomerase II

(topII ). Phosphorylation at the Ser1106 site, closely associated with the catalytic activity

of topII , triggers the decatenation function of the enzyme. However, treatment with the

KA39 compound induced a signiﬁcant inhibition of the phosphorylation at Ser1106 and,

as a consequence, attenuated topII ’s enzymatic activity [23]. Furthermore, it has been

shown that KA39 interferes with the catalytic cycle of topII , leading to the formation

α

of supercoiled DNA, either by blocking the interaction of the enzyme with its plasmid

substrate or by strangling its ATPase action. Further investigations are currently being

carried out in order to explore new targets of triazolo[3,4-b]thiadiazole compounds.

Supplementary Materials: The following are available online at https://ww w .mdpi.com/article/10

.3390/pharmaceutics13040493/s1, Figure S1, Tables S1 and S2 Docking Studies; ¹H-NMR, ¹³C-NMR

and ESI-MS spectra for the synthesized compounds Figure S2–S46, Tables S3–S24 analytical methods

for LC-MS; Figure S47, Figure S48 Inhibition of Akt1 and Akt2; Figure S49–S75 ﬂow cytometric

analysis data.

Author Contributions: Conceptualization, D.T.T., P.D. and V.S.; methodology, V.S. and M.V.; software;

formal analysis, P.D., V.S. and D.T.T.; investigation, D.T.T., P.D., S.S., M.V., S.F., N.N., M.V.D., K.A.

and V.S.; resources, D.T.T. and V.S.; data curation, P.D., S.S., D.T.T., V.S. and S.F.; writing—original

draft preparation, D.T.T., S.S. and V.S.; writing—review and editing, S.S., D.T.T. and V.S.; supervision,

D.T.T. and V.S.; project administration, D.T.T.; funding acquisition, D.T.T. All authors have read and

agreed to the published version of the manuscript.

Funding: This work was ﬁnancially supported by the GALENICA S.A. and ENERGON BIO TECH-

NOLOGIES S.A. and granted by the Hellenic Society of Medical Oncology (HESMO) and Athena

Institute of Biomedical Sciences, Greece.

Institutional Review Board Statement: The study was approved by the ethical committee of the

National & Kapodistrian University of Athens and was conducted according to the European

Directive 86/609/EEC guidelines for the care and use of laboratory animals. The study received a

permit from the Veterinary Directorate of the Prefecture of Athens (Approval #: 2023/2017) according

to the Greek legislation conforming to the 2010/53/EU Council Directive.

Informed Consent Statement: Not applicable.

Data Availability Statement: The data presented in this study are available in supplementary material

.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

Pharmaceutics 2021, 13, 493

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Article Doi

Anticancer activity of triazolo-thiadiazole derivatives and inhibition of akt1 and akt2 activation

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