Indolizine-phenothiazine hybrids as the first dual inhibitors of tubulin polymerization and farnesyltransferase with synergistic antitumor activity

Bioorganic Chemistry 103 (2020) 104184

Contents lists available at ScienceDirect

Bioorganic Chemistry

journal homepage: www.elsevier.com/locate/bioorg

Indolizine-phenothiazine hybrids as the ﬁrst dual inhibitors of tubulin

polymerization and farnesyltransferase with synergistic antitumor activity

a

a,⁎

b

c

a,d,e,⁎

Iuliana-Monica Moise , Elena Bîcu , Amaury Farce , Joëlle Dubois , Alina Ghinet

b

c

a

‘

Alexandru Ioan Cuza’ University of Iasi, Faculty of Chemistry, Bd. Carol I nr. 11, 700506 Iasi, Romania

Univ. Lille, Faculté des Sciences Pharmaceutiques et Biologiques, Rue du Pr Laguesse, B.P. 83, F-59006 Lille, France

Institut de Chimie des Substances Naturelles, UPR2301 CNRS, Centre de Recherche de Gif, Avenue de la Terrasse, F-91198 Gif-sur-Yvette Cedex, France

Univ. Lille, Inserm, CHU Lille, Institut Pasteur de Lille, U1167 - RID-AGE - Facteurs de risque et déterminants moléculaires des maladies liées au vieillissement, F-59000

d

Lille, France

e

Yncréa Hauts-de-France, Hautes Etudes d’Ingénieur (HEI), UCLille, Health & Environment Department, Sustainable Chemistry Team, Laboratoire de Chimie Durable et

Santé, 13 rue de Toul, F-59046 Lille, France

A R T I C L E I N F O

A B S T R A C T

Keywords:

In the incessant search for innovative cancer control strategies, this study was devoted to the design, synthesis

and pharmacological evaluation of dual inhibitors of farnesyltransferase and tubulin polymerization (FTI/MTIs).

A series of indolizine-phenothiazine hybrids 16 (amides) and 17 (ketones) has been obtained in a 4-step pro-

cedure. The combination of the two heterocycles provided potent tubulin polymerization inhibitors with similar

eﬃciency as the reference phenstatin and (-)-desoxypodophyllotoxin. Ketones 17 were also able to inhibit

human farnesyltransferase (FTase) in vitro. Interestingly, three molecules 17c, 17d and 17f were very eﬀective

against both considered biological targets. Next, nine indolizine-phenothiazine hybrids 16c, 16f, 17a-f and 22b

were evaluated for their cell growth inhibition potential on the NCI-60 cancer cell lines panel. Ketones 17a-f

were the most active and displayed promising cellular activities. Not only they arrested the cell growth of almost

all tested cancer cells, but they displayed cytotoxicity potential with GI50 values in the low nanomolar range. The

most sensitive cell lines upon treatment with indolizine-phenothiazine hybrids were NCI-H522 (lung cancer),

COLO-205 and HT29 (colon cancer), SF-539 (human glioblastoma), OVCAR-3 (ovarian cancer), A498 (renal

cancer) and especially MDA-MB-435 (melanoma). Demonstrating the preclinical eﬀectiveness of these dual in-

hibitors can be crucial. A single dual molecule could induce a synergy of antitumor activity, while increasing the

eﬀectiveness and reducing the toxicity of the classical combo treatments currently used in chemotherapy.

Indolizine

Phenothiazine

Hybrids

Dual inhibitor

Antitumor agent

Tubulin polymerization

Farnesyltransferase inhibitor

1

. Introduction

Indolizine 1 (Fig. 1), formerly named pyrroline, pyrindole, 8-pyr-

cisplatin and acts against tumors by stimulating macrophages, pro-

tecting the hematopoietic system of the toxicity of chemotherapeutic

agents [6,7]. (+)-Castanospermine (compound 3, Fig. 1) is extracted

from Castanospermum austral seeds and inhibits the multiplication of

human immunodeﬁciency virus (HIV) [8]. (-)-Ipalbidine (compound 4,

Fig. 1) is another natural compound of interest, being an analgesic

preventing addiction, as well as a leukocyte respiratory burst inhibitor

[9]. (R)-Antoﬁne 5 is also a natural indolizinic alkaloid with antitumor

potential [9–11]. (R)-Tylophorine 6 extracted from Tylophora exhibits

potential anticancer and anti-inﬂammatory activity [12]. Camptothecin

7 (Fig. 1), an important representative of alkaloids inhibiting topoi-

somerase I, contains the partially hydrogenated indolizine nucleus in

the structure [13]. (20S)-Camptothecin is extracted from the stem and

bark of the decorative tree Camptotheca acuminata, used in Chinese

traditional medicine for the treatment of cancer [14]. Moreover, many

rolopyridine and also pyrrolo[1,2-a]pyridine, is an aromatic hetero-

cycle consisting of a pyridine ring condensed with a pyrrole cycle. In-

dolizines were discovered by Angeli in 1890 and ﬁrst synthesized by

Scholtz in 1912 [1]. So far, the aromatic form has not been found in the

structure of natural products, while the hydrogenated form of in-

dolizine seems to be common, many such derivatives being isolated

from plants, microbes, marine organisms, insects or fungi [2]. For ex-

ample, (-)-swainsonine (compound 2, Fig. 1) is a natural compound

present in some plants and fungi and exploited for the antitumor po-

tential [3]. This compound has shown promising potential for the

treatment of glioma and gastric carcinoma [4,5]. (-)-Swainsonine also

enhances the in vivo eﬃcacy of chemotherapeutic agents such as

⁎

Corresponding authors at: ‘Alexandru Ioan Cuza’ University of Iasi, Faculty of Chemistry, Bd. Carol I nr. 11, 700506 Iasi, Romania (E. Bîcu and A. Ghinet).

E-mail addresses: elena@uaic.ro (E. Bîcu), alina.ghinet@yncrea.fr (A. Ghinet).

https://doi.org/10.1016/j.bioorg.2020.104184

Received 29 April 2020; Received in revised form 3 July 2020; Accepted 7 August 2020

Available online 26 August 2020

I.-M. Moise, et al.

Bioorganic Chemistry 103 (2020) 104184

Fig. 1. Structure of indolizine 1 and of natural products containing (partially)hydrogenated forms of indolizine 2–7.

analogues of this alkaloid have been synthesized in the laboratory and

are used in chemotherapy [15].

as Taxol-resistant HL60/TX1000 cell lines (Fig. 2) [23].

The use of indolizine ring in the conception and synthesis of some

analogs of phenstatin led to promising results by providing compounds

with good biological activity [21,22]. Thus, we were able to conﬁrm

that indolizine may replace successfully the cycle B of model phen-

statin. In the previously reported studies, however, the active molecules

were those presenting the classic 3,4,5-trimethoxyphenyl ring A (e.g.

compound 13, Fig. 2) [21]. In addition, our research group discovered

that other indolizines inhibited the human farnesyltransferase (FTase)

(e.g. compound 12: IC50 (FTase) = 1.3 µM [24], Fig. 2).

Due to the pronounced similarity both structurally and chemically

with the indole nucleus, the biological potential of indolizine was en-

visaged. Analogues of biologically active products in which indolizine

took the place of the indole unit were rapidly synthesized. Thus, Carbon

and Brehm [16] proposed 1-indolizinealanine as a possible anti-

metabolite of tryptophan. The literature highlights a variety of in-

dolizine derivatives with biological activity such as antimicrobial, an-

tioxidant, anti-inﬂammatory and anticancer [17]. In particular, many

indolizines have been reported for their anticancer potential. For in-

stance, such structures containing a cyclopropylketone in position 3

displayed in vitro antiproliferative activity on Hep-G2 cell lines [18],

Tubulin is a well-documented target in oncology due to its role in

chromosome segregation and cell division. This requires extremely fast

dynamics of microtubules polymerization and depolymerization, both

mediated by the tubulin. These dynamics can be blocked by diﬀerent

classes of inhibitors of tubulin that, by interfering with the dynamics of

the microtubules, stop the cancer cell in mitosis (arrest of cells in G2/M

phase of the cell cycle), eventually leading to cell death, both through

apoptosis and necrosis [25]. The FTase is another studied target in

oncology. It catalyzes the addition of a lipid group to the terminal

carboxyl of several proteins, including Ras proteins having a proto-

oncogenic role. The substrates of this enzyme undergo a process of

maturation consisting of: ﬁxation of a prenyl chain, loss of the three

terminal amino acids and methylation of the C-terminal carboxyl group.

Sometimes a palmitoylation phase ends the process. The prenylation

step is a key step, as its inhibition prevents proteins to attach to the

target membrane [26]. Inhibitors of FTase are also capable of inhibiting

bipolar spindle formation and chromosomal alignment during meta-

phase [27].

the most active in the study being compound

8 (IC50 (Hep-

G2) = 0.2 µg/mL, Fig. 2). Other products decorated with indolizine

ring are involved in the migration and proliferation of tumor cells by

preventing protein–protein interaction in which the endothelial vas-

cular growth factor (VEGF) is involved (compound 9, Fig. 2) [19].

The ﬁrst tubulin polymerization inhibitors containing an indolizine

moiety were published as phenstatin analogues, known antimitotic

agent (Fig. 2) [20]. Among these compounds, promising activity was

reported for derivatives with an indolizine ring linked to a trimethox-

yphenyl nucleus through a carbonyl group. Their cytotoxic eﬀect was

especially registered on MDA-MB-435 melanoma cells [21]. When in-

dolizine was combined with triazine and p-bromobenzoyl (compound

1

0, Fig. 2), an inhibition activity of the growth of SNB-75 SNC cancer

cells and MDA-MB-231/ATCC breast cancer cells was observed [22]. As

for indolizine-glyoxylamide 11, it displayed cytotoxic activity against

cancer cell lines which have developed resistance to chemotherapy such

In the incessant search for molecules with improved antitumor

2

I.-M. Moise, et al.

Bioorganic Chemistry 103 (2020) 104184

Fig. 2. Structure of previously described indolizine derivatives with antitumor activity 8–15 and structure of target indolizine-phenothiazine hybrids 16 and 17 and

of reference phenstatin.

activity, a new series of compounds has been conceived in this study

dual inhibitors of tubulin and FTase (MTI-FTI hybrids) can be crucial,

leading to an innovative strategy for the design of new anticancer

compounds. A single molecule may induce a synergy of antitumor ac-

tion increasing the eﬀectiveness and reducing the toxicity of the clas-

sical combo treatments.

(

target compounds, Fig. 2) where the cycle B is an indolizine unit and

the cycle A is a phenothiazine. Chemistry of the latter azaheterocycle is

part of the laboratory's expertise and proved to be a successful sub-

stitute for the classical trimethoxyphenyl group in the structure of an-

timitotic agents (e.g. phenothiazine derivative 14: IC50 (tu-

bulin) = 9.48 µM) [28]. Phenothiazine derivative 15 was not active on

tubulin but displayed activity against human FTase (IC50 = 0.6 µM

2

. Results and discussion

[

29], Fig. 2).

2.1. Chemistry

The ambition of this work was to obtain a synergy of antitumor

eﬀects using a unique compound bearing the two heterocycles that are

each present in the structure of previously identiﬁed antitumor agents

as phenstatin analogues as well as FTase inhibitors. These targeted in-

dolizine-phenothiazine hybrids may constitute dual inhibitors of tu-

bulin polymerization and farnesyltransferase. Substituents privileged

on each heterocycle are those previously identiﬁed to be beneﬁcial for

the pursued biological activity: methyl, methoxy, acetyl or bromo

In order to obtain the indolizine-phenothiazine hybrids, a four-step

synthesis described in Scheme 1 was privileged. The ﬁrst step consisted

in obtaining the pyridinium salts 19a-g in very good yields (80–93%)

by reaction of suitably substituted and commercially available pyr-

idines 18a-g with chloroacetone in THF at rt. In the second synthetic

step, salts 19a-g were subjected to a 1,3-dipolar cycloaddition in the

presence of triethylamine and ethyl propiolate as dipolarophile, in

order to create the indolizine ring. The best cycloaddition solvent in this

(

target compounds, Fig. 2).

The use of speciﬁc FTase inhibitors to treat diﬀerent cancers has

study was a mixture of CH

CH

3

CN and DMF (volume ratio 8/2). The use of

been disappointing in clinical trials due to the ability of the cancer cells

to circumvent the problem of the FTase inhibition and go through FTase

inhibition by using a second prenylation route mediated by structurally

related geranylgeranyltransferase. Demonstrating the eﬀectiveness of

3

CN or THF resulted in lower yields. Eight ethyl esters 20a-h were

prepared in modest to good yields (22–71%). It is to be noted that the 3-

bromopyridinium salt 19 g underwent the Huisgen cycloaddition on

two diﬀerent accessible positions on the pyridine and allowed to obtain

3

I.-M. Moise, et al.

Bioorganic Chemistry 103 (2020) 104184

i

ii

iii

R

iv

O

N

Cl

OEt

OH

18a-h

O

19a-h

20a-h

21a-h

Me

O

R

S

N

R

O

S

N

Me

R1

iv

HO

and/or

O

17a-f

16c,f,g

O

S

N

Me

N

H

and

22b

Scheme 1. Synthesis of target indolizine-phenothiazine hybrids. Reagents and conditions: (i) chloroacetone (1.2 equiv.), THF, 24–48 h, rt; (ii) ethyl propiolate (1.5

equiv.), TEA (1.4 equiv.), CH

3

CN/DMF, 24 h, 60 °C; (iii) NaOH 50% aq. solution (4 equiv.), EtOH, reﬂux, 3 h, then HCl 10% aq. solution; (iv) 10H-phenothiazine or

1

0-methyl-10H-phenothiazine (1.0 equiv.), Eaton’s reagent (4.0 equiv.) (P

2

O

5

/CH

3

SO H 1/10 w/w), 50 °C, 4–24 h.

3

two position isomers 20 g (22% yield) and 20 h (40% yield). Now, the

saponiﬁcation of the ester group furnished easily the free carboxylic

acids 21a-h in excellent yields (80–96%). The ﬁrst strategy for the ﬁnal

step was to convert carboxylic acids into corresponding acid chlorides

whose Friedel-Crafts reaction with phenothiazine or N-methylphe-

nothiazine in the presence of aluminum trichloride would generate the

indolizine-phenothiazine hybrids of general formula 16 and 17. Reac-

tion of the acid chloride of indolizine 21d was chosen as model; how-

ever, despite a good formation of the chloride, the expected acylations

failed probably due to the solubilization issues. The synthetic strategy

furnish only the bromo-indolizine 29 in 74% yield. However, while the

formation of these unexpected by-products is responsible for the modest

yields of the target compounds, very interestingly they constitute new

functionalized chemical platforms for further synthetic modulations.

2.2. Biological evaluation of the targeted products and ﬁrst SAR aspects

2

.2.1. Evaluation on tubulin polymerization and on FTase

The biological evaluation of indolizine-phenothiazine hybrids 16

and 17 started by determining their potential to inhibit the tubulin

was then modiﬁed, and the use of Eaton's reagent (10/1 MeSO

3

H/P

2

5

O )

polymerization using sheep brain tubulin. Compounds were diluted in

proved successful. This acid reagent is used without solvent and has the

advantage to be less viscous than polyphosphoric acid, enabling eﬃ-

cient magnetic stirring and easier ﬁnal workup of the crude. Eaton’s

reagent is known to promote Friedel-Crafts acylations [30–32] and

functions directly with free carboxylic acids without the need to syn-

thesize the intermediate acid chlorides. When the acylation reactions

were carried out with 10-methyl-10H-phenothiazine as substrate, only

the products acylated in the position 3 of phenothiazine were isolated

in low yields (ketones 17d’ (34%) and 17e’ (24%), Table 1). The Ea-

ton’s reagent-mediated acylation of 10H-phenothiazine provided the

acylated products in the position 3 (ketones 17a-f, Scheme 1 and

Table 1) and, in few cases, furnished also N-acylated indolizine-phe-

nothiazine hybrids (compounds 16c, 16f and 16 g, Table 1). A series of

unexpected by-products was also isolated (indolizines 23–29). The

structures of the isolated compounds are presented in Table 1. The in-

dolizine derivatives bearing a free carboxylic acid proved to have

limited stability in the presence of Eaton’s reagent and underwent

decarboxylation, acetylation of the indolizine ring upon decarboxyla-

tion and/or suppression of the acetyl unit (Table 1). The 8-bromosub-

stituted acid 21 h was the most sensitive and rapidly decarboxylated to

-

2

DMSO to obtain a 10 M concentration then diluted in the medium to

achieve the desired concentration. All compounds were ﬁrst tested at a

concentration of 100 µM to calculate their inhibition ratio. DMSO was

used as a negative control. Phenstatin and (-)-desoxypodophyllotoxin,

known strong tubulin polymerization inhibitors, were used as positive

references. Only compounds that induced more than 60% inhibition of

tubulin polymerization at 100 µM underwent the IC50 evaluation and

were further evaluated on tubulin at eight diﬀerent concentrations. The

results are described in Table 2.

The N-acylation of the phenothiazine ring which provided com-

pounds 16c, 16f and 16 g was not an advantageous pharmacomodu-

lation, none of these molecules showing notable antimitotic activity. On

the contrary, the acylation of phenothiazines in position 3 was toler-

ated. Ketones 17c, 17d and 17f displayed IC50 values on tubulin

polymerization in the micromolar range. The best tubulin inhibitors in

the current study were compounds 17d and 17f with similar eﬃciency

as the reference phenstatin and (-)-desoxypodophyllotoxin. Ketones

1

7d and 17f have very similar chemical structure, the only diﬀerence

being the nature of their substituent in the position 7 of the indolizine

ring. Compound 17d is decorated with a methyl substituent while the

4

I.-M. Moise, et al.

Bioorganic Chemistry 103 (2020) 104184

Table 1

Isolated products and secondary products obtained by Eaton’s reagent-mediated acylation of phenothiazine substrates.

Phenothiazine derivative

Carboxylic Acid

Product (isolated yield)

Secondary product (isolated yield)

–

compound 17f is substituted by a methoxy group. The slightly increased

antitubulin activity of compound 17f (IC50 (tubulin) = 1.11 µM,

Table 2) compared to 17d (IC50 (tubulin) = 3.32 µM, Table 2) is

probably due to the methoxy substituent, the classic group found in the

para position on the B cycle of reference molecules such as phenstatin

or combretastatin A-4. A diminished antimitotic activity was noted for

other ketones (17a, 17b and 17e) while molecules 17d’ and 17e’

bearing

a N-methylphenothiazine unit were inactive (Table 2).

5

I.-M. Moise, et al.

Bioorganic Chemistry 103 (2020) 104184

Table 2

Evaluation of synthesized molecules on tubulin polymerization and on human FTase in vitro.

a,b

b

2c

% FTI^b^,^e

IC50 (FTase) (µM)^b

R²

Molecule

% TPI

IC50 (tubulin) (µM)

R

d

1

2

6c

46

32

19

43

41

84

77

0

-

–

49

37

65

95

73

89

96

82

95

81

86

79

–

6f

–

6 g

7a

7b

7c

7d

7d’

7e

7e’

7f

–

26.48

0.35

4.80

0.81

0.25

14.56

0.68

6.46

0.39

2.90

0.9902

0.9179

0.8332

0.9034

0.8982

0.9662

0.8829

0.8632

0.8938

0.9970

–

27.7

3.32

–

0.9046

0.8844

–

56

9

–

100

44

99

100

1.11

–

0.8929

–

2b

Phenstatin

3.43

1.76

0.9378

0.9740

(

-)-Desoxypodophyllotoxin

Chaetomellic acid A

100

0.183

0.9898

a

b

c

d

e

Inhibition of tubulin polymerization at a 100 µM concentration

Values represent mean of two experiments

Coeﬃcient of determination.

Not determined

Inhibition of human FTase at a 100 µM concentration

Consequently, the nitrogen atom of phenothiazine must imperatively

remain unsubstituted in the series of ketones in order to ensure good

inhibition of tubulin.

in Table 3). The cell lines on which tested compounds were the most

active were NCI-H522 (lung cancer), COLO-205 and HT29 (colon

cancer), SF-539 (human glioblastoma), MDA-MB-435 (melanoma),

OVCAR-3 (ovarian cancer) and A498 (renal cancer) (Table 3). Com-

pound 17b proved non-eﬀective in cell growth inhibition. This result

corroborated well with its activity registered on the biological targets

(Table 2). Only a moderate eﬃciency was registered on HCT-116 colon

cancer cells (Table 3). The bulky derivative 22b displayed limited ef-

ﬁciency probably due to solubility issues and diﬃculty to cross cell

membranes. The log P value of compounds was next estimated with

ACD software. The predicted values were: 16c (Log P = 5.06 ± 1.34),

16f (Log P = 4.52 ± 1.52), 17a (Log P = 5.11 ± 1.32), 17b (Log

Indolizine-phenothiazine hybrids were next subjected to a biolo-

gical evaluation against human FTase. DMSO and chaetomellic acid A

were used as negative and positive controls, respectively. The same

threshold inhibition criteria as for the tubulin polymerization assay

were applied to select active molecules for IC50 calculation. Ten of the

twelve considered compounds were able to inhibit the protein. In the

series of N-acylated phenothiazines, only derivative 16 g showed a

weak inhibitory activity against human FTase (IC50 = 26.48 µM,

Table 2). All tested ketones 17a-f and 22b displayed activity against the

protein. Again, N-methylated phenothiazines were less active compared

to nitrogen-free congeners (compare IC50 values on FTase of com-

pounds 17d vs 17d’ and 17e vs 17e’, Table 2). In terms of substitution

of the indolizine ring, small groups are tolerated, especially in the po-

sition 7 of indolizine. Five derivatives displayed submicromolar IC50

values and the best FTase inhibitors in this study were 17a, 17d and 17f

P = 5.57

±

1.32), 17c (Log P = 5.57

1.32), 17e (Log P = 6.03

±

1.32), 17d (Log

1.32), 17f (Log

±

P = 5.02 ± 1.52), 22b (Log P = 7.54 ± 1.45). As expected, com-

pound 22b had the biggest log P value in the series. This compound has

low aqueous solubility, compromising bioavailability.

Only compounds which satisﬁed pre-determined threshold inhibi-

tion criteria in a minimum number of cell lines progressed to the full 5-

dose assay (Table 4). Five phenothiazines acylated in position 3 (17a,c-

f) were thus submitted to GI50 calculation. These experimental drugs

exhibited signiﬁcant growth inhibition in the nanomolar range

(Table 4). All these molecules also displayed great eﬃciencies on both

studied targets. Besides, compounds 17c, 17d and 17f are the dual

inhibitors identiﬁed in this report (Table 2). It is to be noted that the

same ranking in terms of biological activity is observed for these dual

inhibitors as previously seen in the protein assays. Compound 17f was

the most active against tubulin and had the same inhibitory eﬃciency

on FTase as the structurally close compound 17d, and induced the best

antitumor activity against the 60-cell panel. Compound 17d was the

second most active in the dual inhibitors’ series followed by compound

(

Table 2). The bisacylated derivative 22b displayed unexpected mi-

cromolar activity against FTase, weaker however compared to the other

3

-acylated derivatives (Table 2).

Interestingly, as imagined in the design of these indolizine-phe-

nothiazine hybrids, dual inhibitors of tubulin and FTase were identi-

ﬁed. Three molecules 17c, 17d and 17f were active against both con-

sidered biological targets in vitro with IC50 values in the same range as

for reference compounds.

2

.2.2. Evaluation on cancer cell lines

Nine indolizine-phenothiazine hybrids 16c, 16f, 17a-f and 22b

were selected by the National Cancer Institute (NCI) for evaluation of

their cell growth inhibition potential on the NCI-60 cancer cell lines

panel. All compounds submitted to the NCI were tested initially at a

single high dose (10 µM) in the full NCI panel (Table 3).

1

7c. As expected, compound 17e, more speciﬁc as FTase inhibitor,

conserved cell growth inhibition potential, but diminished compared to

dual inhibitors.

The Cell One-Dose Screen conﬁrmed that N-acylated phenothiazines

The most unexpected excellent antitumor activity was that induced

by phenothiazine-ketone 17a, the best compound of the series in the

NCI evaluation. Compound 17a displayed cell proliferation inhibition

at low nanomolar concentrations (GI50 (NCI-H522) = 1.8 nM and (GI50

1

6c and 16f had very limited eﬃciency in inhibiting the growth of

cancer cell lines. Compound 16c, inactive in the tubulin polymerization

and FTase assays, was also devoid of activity on cancer cells while

compound 16f showed modest cell growth inhibition on SR (50%), NCI-

H522 (53%) and PC-3 (48%) cells. These results are in line with the

results of the tests carried out on the two targets of interest.

(

MDA-MB-435) = 1.8 nM, Table 4). The fact that the most active

product was not the best inhibitor of tubulin polymerization and/or

FTase in the family shows that it can penetrate the cell membrane

better or reach other biological target(s), one of which is FTase

Ketones 17a-f were more active and displayed promising cellular

activities. Not only they arrested the cell growth of almost all tested

cancer cells, but they displayed cytotoxicity potential (negative values

(

IC50 = 0.35 µM, Table 2). The NCI Compare algorithm, a comparison

6

I.-M. Moise, et al.

Bioorganic Chemistry 103 (2020) 104184

Table 3

Results of the in vitro human cancer cell growth inhibition for selected indolizine-phenothiazine hybrids 16c, 16f, 17a-f and 22b ^a^,^b^.

Compound

16c

16f

17a

17b

17c

17d

17e

17f

22b

a,b

Cell type

Cell line

SR

GI%

11

12

11

7

Leukemia

50

32

12

25

53

4

91

86

95

24

23

17

27

29

14

29

0

87

n.t.

97

95

11

17

0

HL-60(TB)

CCRF-CEM

K-562

−5

84

−13

n.t.

96

−10

92

89

c

n.t.

−29

97

92

95

93

3

d

Non-Small Cell Lung Cancer

Colon Cancer

NCI-H522

NCI-H460

A549/ATCC

COLO 205

HT29

32

0

87

−48

99

−31

99

−21

96

13

0

94

9

23

29

35

39

13

18

26

n.t.

5

89

80

86

96

89

2

0

−55

−13

99

−44

88

−62

−57

−24

−20

83

−37

−15

96

−41

−1

99

0

47

43

4

11

42

2

17

15

0

HCT-116

MDA-MB-435

M14

94

Melanoma

−10

80

−27

83

–22

90

–33

90

11

0

16

10

0

1

SK-MEL-5

A498

n.t.

−12

93

92

−27

−16

51

−37

−27

66

−40

−18

48

3

Renal cancer

0

−5

−8

43

0

RXF 393

TK-10

0

41

0

5

0

41

0

−26

–32

−2

91

65

70

SNC cancer

SF-539

0

10

7

−38

91

0

−28

96

−3

−1

−2

−26

n.t.

79

−12

86

11

26

22

0

SF-295

12

0

2

SNB-75

16

0

87

0

76

79

Ovarian Cancer

Breast cancer

OVCAR-3

OVCAR-8

SK-OV-3

MDA-MB-231/ATCC

MCF-7

−17

93

0

−27

81

−14

96

−48

n.t.

81

0

n.t.

3

12

0

2

90

75

83

0

7

34

18

37

48

0

82

21

19

11

14

80

97

−14

85

−17

79

1

11

1

75

89

6

MDA-MB-468

PC-3

96

86

−3

82

86

−4

76

0

Prostate cancer

a

19

0

78

73

n.t.

98

12

0

DU-145

98

86

99

−18

Data obtained from NCI’s in vitro 60-cell one dose screen (experiments conducted with 10 µM concentration of tested molecule).

GI% is the percentage of growth inhibition of tumor cells.

b

c

d

Not tested.

A value of -x means x% cancer cells lethality of preexisting cells (cytotoxic eﬀect).

tool that analyzes the proﬁle of cellular growth inhibition from the NCI-

These interactions may explain the antitubulin potential of this mole-

cule.

6

0 cell line panel of tested compounds to assist in the identiﬁcation of

molecules with similar activity proﬁles, was used to try to obtain other

clues about the potential targets achieved. The results indicated that

molecule 17a has a very similar proﬁle as known cytotoxic agents used

in clinics: doxorubicin (DNA intercalant), vinblastine and taxol (tubulin

inhibitors).

Only one conformation well overlapped with the previous molecule

was found for indolizine-phenothiazine 17d’, N-methylated analogue of

17d (Fig. 3(d)). The additional methylation of phenothiazine could

provide better hydrophobic contact with the binding site. However, the

bulkier structure of 17d’ compared to 17d seems to prevent the correct

positioning in the active site.

The most sensitive cancer cell line to all the tested experimental

drugs was MDA-MB-435 melanoma cells but tested compounds have

also shown great potential on other cancer cell lines such as HL-60 (TB)

and K-562 (leukemia), NCI-H522 (lung cancer), and OVCAR-3 (ovarian

cancer), respectively (Table 4).

The behavior of the same molecules 17a, 17d and 17d’ was next

studied in the active site of FTase (Fig. 3(e)-(h)). Derivative 17a dis-

played 25 conformations that conveniently overlapped, with the phe-

nothiazine tricycle slightly oﬀset in front of the zinc cation of FTase

(

Fig. 3(e)). Its analog 17d with an additional methyl grafted on the

indolizine heterocycle was positioned in the same way, having 27

overlapping conformations out of 30 and better site compatibility

2.3. Docking

(

Fig. 3(f)). As a reminder, molecule 17d was identiﬁed as a dual in-

To investigate how the indolizine-phenothiazine hybrids interact

hibitor of both tubulin and FTase. The results of molecular modeling

agree with the experimental results. The addition of one methyl to the

molecule on the phenothiazine nitrogen atom in compound 17d’

with the biological targets, molecular modeling studies were performed

on selected compounds: a speciﬁc strong inhibitor of FTase (17a), a

dual tubulin/FTase inhibitor (17d) and a modest FTase inhibitor

(

Fig. 3(g) and (h)), aﬀected the positioning of the compound in the

(

17d’). First, docking studies were conducted in the colchicine binding

FTase site. For this molecule, there were two possibilities of binding:

with the phenothiazine close to the zinc cation such as the preceding

molecules (Fig. 3(g)) or vice versa, with a weak or even absent inter-

action with the metal cation (Fig. 3(h)).

site of tubulin, compared to Combretastatin A-4 (CA-4) as reference

compound (Fig. 3).

Compound 17a displayed a single conformation with the tricycle on

the left side of the binding site while the indolizine ring occupied the

entry into the smaller side pocket which is generally occupied by the

cycle B (3′-hydroxy-4′-methoxyphenyl) of CA-4 (Fig. 3(a)). Since com-

pound 17a has rather rigid structure, this was the only possibility to ﬁt

in the active site, although the contact between the carbonyl group and

the protein surface is not optimal (Fig. 3(b)). This is in accordance with

the experimental results obtained in the tubulin polymerization assay.

Compound 17d had a behavior like 17a. The additional methyl on the

indolizine ﬁtted into the side pocket, thus adding a positive hydro-

phobic contact and partially mimicking the B-ring of CA-4 (Fig. 3(c)).

3. Conclusions

A new series of indolizine-phenothiazine hybrids has been designed,

synthesized and evaluated on tubulin polymerization and human FTase.

An NCI screen was performed on nine synthesized derivatives. This

screen utilized 60 diﬀerent human tumor cell lines, representing leu-

kemia, melanoma and cancers of the lung, colon, brain, ovary, breast,

prostate, and kidney cancers.

7

I.-M. Moise, et al.

Bioorganic Chemistry 103 (2020) 104184

Table 4

of the phenothiazine through a carbonyl bridge; ii) maintaining the

phenothiazine nitrogen atom unsubstituted and iii) the substitution on

the indolizine ring by a methyl or a methoxy group on positions 6, 7

and 8.

Results of the NCI 60 Cell Five-Dose Screen for selected compounds 17a and

a,b.

1

7c-f

Compound

17a

17c 17d 17e 17f

Thus, small molecular weight tubulin-FTase inhibitors were ob-

tained (molecules 17c, 17d and 17f). To the best of our knowledge this

is the ﬁrst time when dual inhibitors (MTI-FTI hybrids) acting on tu-

bulin polymerization and on FTase is realized. This duality of actions

oﬀers new perspectives and highlights the importance of such inhibitors

in the design of new anticancer drugs. As expected, they were active in

the screening on the 60 tumor cell lines performed at NCI especially on

MDA-MB-435 melanoma cell lines.

a,b

Cell type

Cell line

SR

GI50 (nM)

c

Leukemia

< 3.3 118 38.6 302 56.7

HL-60(TB)

CCRF-CEM

K-562

< 2.4 296 47.1 263 26

< 3.3 314 257 394 39

< 3.3 53.7 41

206 46.2

Non-Small Cell Lung

Cancer

NCI-H522

1.8

34.1 41.2 197 48.7

NCI-H460

A549/ATCC

COLO 205

HT29

< 3.3 133 46.5 301 35.4

< 3.3 206 64.8 310 42.4

Colon Cancer

Melanoma

3.3

147 59.8 202 24.5

The most promising antitumor activity in the NCI evaluation was

that induced by phenothiazine-ketone 17a, displaying cell proliferation

inhibition at low nanomolar concentrations on multiple cancer cell

lines (e.g. GI50 (NCI-H522) = 1.8 nM; GI50 (MDA-MB-435) = 1.8 nM).

This compound was less eﬀective against tubulin compared to dual

inhibitors. The excellent cellular activity may be due to binding to

another biological target(s) and/or better cell penetration. All these

aspects require further investigation.

< 3.3 252 62.7 325 33

< 3.3 183 41.4 332 28.5

HCT-116

MDA-MB-435

M14

1.8

31.9 29.7 46.8 19

< 3.3 146 90.6 267 30.9

< 3.3 77.7 122 406 46.2

SK-MEL-5

OVCAR-3

OVCAR-8

SK-OV-3

A498

Ovarian Cancer

Renal Cancer

3.7

62.5 32.3 222 26.2

< 3.3 361 451 379 50.3

4.4

2.3

3.3

3.7

194 189 279 37.2

99.8 43.1 66.4 30.2

RXF 393

CAKI-1

227 41.1 162 28.2

d

4. Experimental section

n.t.

53

529 52.8

Prostate Cancer

CNS Cancer

PC-3

< 3.3 259 50.6 321 47.4

< 3.3 386 188 407 42.6

DU-145

4.1. Materials and methods

SF-539

2.8

2.0

6.5

3.4

2.6

203 58.1 185 21.2

SF-295

93.7 36

187 25.5

Starting materials are commercially available and were used

SNB-75

158 41.6 134 25.9

351 348 472 57.8

49.7 34.5 209 36.7

102 66.3 487 33.1

Breast Cancer

MDA-MB-231/ATCC

MCF7

without further puriﬁcation (suppliers: Carlo Erba Reagents S.A.S.,

Tokyo Chemical Industry Co. Ltd. and Acros Organics). Melting points

were measured on a MPA 100 OptiMelt® apparatus and are un-

corrected. Nuclear Resonance Magnetic (NMR) were acquired at

MDA-MB-468

a

b

Data obtained from NCI’s in vitro 60 cell 5-dose screen.

1

13

400 MHz for H NMR and at 100 MHz for C NMR, on a Varian 400-

GI50 is the molar concentration of synthetic compound causing 50% growth

1

13

MR spectrometer or at 500 MHz for H NMR and at 125 MHz for

C

inhibition of tumor cells.

c

-5

-6

-

NMR, on a Bruker Avance III 500 MHz spectrometer with tetra-

methylsilane (TMS) as internal standard, at 25 °C. Chemical shifts (δ)

are expressed in ppm relative to TMS. Splitting patterns are designed: s,

singlet; d, doublet; dd, doublet of doublet; t, triplet; m, multiplet; quint,

quintuplet; br s, broaden singlet; br t, broaden triplet. Coupling con-

stants (J) are reported in Hertz (Hz). Thin layer chromatography (TLC)

was realized on Macherey Nagel silica gel plates with ﬂuorescent in-

dicator and were visualized under a UV-lamp at 254 nm and 365 nm.

Compound 17a has been tested at concentrations 5.10 M, 5.10 M, 5.10

7

-8

-9

M, 5.10 M and 5.10 M. On some cell lines, the GI50 could not be calculated

since the compound exhibited an inhibitory activity still greater than 50% in-

hibition (see SI for full dose–response curves).

d

Not tested.

Compounds were obtained in a 4-step procedure, and Eaton's re-

agent was essential to obtain target hybrids in the ﬁnal step. It gener-

ated reaction products that were in some cases diﬀerent depending on

the nature of indolizine substituents (phenothiazine-ketones 17a-f and

Flash chromatography was performed with

a CombiFlash Rf

Companion (Teledyne-Isco System) using RediSep packed columns. IR

spectra were recorded on a Varian 640-IR FT-IR or on a FTIR Bruker

Tensor 27 spectrometer. Elemental analyses (C, H, N) of new com-

pounds were determined on a Thermo Electron apparatus by “Pole

Chimie Moleculaire-Welience”, Faculte des Sciences Mirande, Dijon,

France.

2

2b or phenothiazine-amides 16c, 16f-g). It also led to the formation of

numerous by-products 23–29 that explained the low yields of the

compounds of interest. The structures of by-products obtained were

elucidated. These indolizine derivatives constitute new functionalized

chemical platforms that may interest the medicinal chemistry com-

munity for further modulations.

4.2. General procedure for the synthesis of pyridinium salts 19a-g

The ﬁnal compounds are characterized by a new cycle A (phe-

nothiazine substituted in position 3) and a new cycle B (acet-

ylindolizine) linked by the characteristic carbonyl connector as the

reference phenstatin. The combination of the two heterocycles provided

phenothiazine-ketones as inhibitors of tubulin polymerization with the

same activity as the parent drug. The condition to maintain the anti-

tubulin potential was to keep the phenothiazine unsubstituted on the

nitrogen atom and the indolizine substituted with a methyl or methoxy

group in position 7. The grafting of a methyl group on the nitrogen

atom of phenothiazine suppressed the activity. The obtained phe-

nothiazine-amides 16c, 16f and 16 g did not exhibit any antitubulin

activity.

Chloroacetone (1.2 equiv.) was added to a solution of pyridine 18a-

g (1 equiv.) in THF. The resulting mixture was stirred at rt for 24–48 h.

The precipitate formed was ﬁltered, washed with cold acetone and

dried to provide pure pyridinium salt 19a-g in very good yields.

The physico-chemical characterization of salts 19a-h corresponded

to that described in the literature [33].

4

.3. General 1,3-dipolar cycloaddition procedure for the synthesis of

indolizines 20a-h

The suitably substituted pyridinium salt 19a-g (1 equiv.) was added

The hybrids of this series have also inhibited the FTase activity and

emphasized similar structure–activity relationships. However, this en-

zyme was less sensitive to the substituents present on the indolizine. It

can be concluded that the optimal structural changes for the activity on

FTase were: i) the placing of the indolizine heterocycle in the position 3

to a mixture of CH CN/DMF (volume ratio 8/2). Triethylamine (1.4

3

equiv.) was then added to the resulting suspension. After few minutes of

magnetic stirring, ethyl propiolate (1.5 equiv.) has been added and the

mixture was stirred under heating at 60 °C for 24 h. The volatiles were

then evaporated. The crude was then dissolved in a mixture of ethyl

8

I.-M. Moise, et al.

Bioorganic Chemistry 103 (2020) 104184

Fig. 3. Structure and docking of indolizine-phenothiazine hybrids in the active site of: tubulin (colchicine binding site) (a) combretastatin A-4, (b) compound 17a, (c)

compound 17d, (d) compound 17d’ and FTase (e) compound 17a, (f) compound 17d, (g) compound 17d’ and (h) compound 17d’.

9

I.-M. Moise, et al.

Bioorganic Chemistry 103 (2020) 104184

Fig. 3. (continued)

acetate and distilled water. The organic layer was collected, and the

aqueous layer was extracted twice with ethyl acetate. The combined

precipitate formed was ﬁltered, washed with cold absolute ethanol and

dried to provide pure carboxylic acid 21a-h.

organic layers were dried over anhydrous Na

2

SO . After the ﬁltration of

4

hydrated Na

2

SO

4

, the ﬁltrate was concentrated and then puriﬁed by

4.4.1. 3-Acetylindolizin-1-yl-carboxylic acid (21a)

ﬂash chromatography on silica prepacked column (chromatographic

eluent: gradient n-heptane/EtOAc) to obtain pure indolizines 20a-h.

Indolizines 20a-f presented the same physico-chemical properties as

previously reported [33].

The general procedure was used with 3.0 g (12.97 mmol) of in-

dolizine ethyl ester 20a in 50 mL of absolute EtOH. White solid; 80%

1

yield (2.1 g); mp (EtOH) 252–253 °C; H NMR (DMSO‑d , 500 MHz) δ

6

(ppm): 2.55 (s, 3H, CH ), 7.21 (t, J = 7.0 Hz, 1H, ArH), 7.53 (t,

3

J = 8.0 Hz, 1H, ArH), 8.11 (s, 1H, ArH), 8.28 (d, J = 7.5 Hz, 1H, ArH),

1

3

4

.3.1. 3-Acetyl-1-carboxyethyl-6-bromoindolizine (20 g)

9.78 (d, J = 7.0 Hz, 1H, ArH), 12.49 (br s, 1H, COOH); C NMR

The general procedure was used with 4.9 g (19.56 mmol) of N-

(DMSO‑d

6

, 125 MHz) δ (ppm): 27.3 (CH ), 105.6 (C), 115.6 (CH), 118.9

3

(

methylcarbonylmethyl)-3-bromopyridinium chloride 19 g in a mixture

(CH), 122.2 (C), 126.4 (CH), 127.6 (CH), 128.3 (CH), 138.5 (C), 164.8

−1

of 24 mL CH

3

CN and 6 mL DMF. Yellow solid; 22% yield (1.27 g); mp

(C), 187.7 (C); IR ν (cm ): 3120, 3110, 1674, 1637, 1519, 1489, 1466,

1442, 1370, 1336, 1284, 1245, 1202, 1161, 1046, 1001, 932, 911, 784,

748, 693, 636, 562, 457, 422.

1

(

EtOAc:n-heptane) 159–161 °C; H NMR (CDCl

3

, 500 MHz) δ (ppm):

1

.43 (t, J = 7.0 Hz, 3H, CH

3

), 2.59 (s, 3H, CH

3

), 4.40 (q, J = 7.0 Hz,

2

H, CH ), 7.45 (d, J = 9.0, 2.0 Hz, 1H, ArH), 7.95 (s, 1H, ArH), 8.41 (d,

2

1

3

J = 9.5 Hz, 1H, ArH), 10.08 (s, 1H, ArH); C NMR (CDCl

3

, 125 MHz) δ

4.4.2. 3-Acetyl-5-methylindolizin-1-yl-carboxylic acid (21b)

(

ppm): 14.7 (CH

3

), 24.5 (CH

3

2

), 60.5 (CH ), 106.7 (C), 110.4 (C), 120.0

The general procedure was used with 2.3 g (9.38 mmol) of in-

dolizine ethyl ester 20b in 30 mL of absolute EtOH. White solid; 91%

CH), 122.9 (C), 126.0 (CH), 129.2 (CH), 130.4 (CH), 137.4 (C), 163.8

−

1

C), 188.1 (C); IR ν (cm ): 3115, 2985, 1708, 1687, 1643, 1514, 1476,

yield (1.85 g); mp (EtOH) 219–224 °C; H NMR (DMSO‑d , 500 MHz) δ

6

1

7

426, 1362, 1255, 1213, 1185, 1047, 1018, 989, 978, 932, 869, 821,

73, 728, 642, 629, 576, 443, 424.

(ppm): 2.51 (s, 3H, CH ), 2.62 (s, 3H, CH ), 7.06 (t, J = 7.0 Hz, 1H,

3

ArH), 7.50 (dd, J = 7.0, 2.0 Hz, 1H, ArH), 8.14 (s, 1H, ArH), 8.24 (d,

1

3

J = 7.0 Hz, 1H, ArH), 12.41 (br s, 1H, COOH); C NMR (DMSO‑d ,

6

4

.3.2. 3-Acetyl-1-carboxyethyl-8-bromoindolizine (20 h)

125 MHz) δ (ppm): 27.3 (CH ), 105.6 (C), 115.6 (CH), 118.9 (CH),

3

Product obtained in the same reaction with 20 g; yellow solid; 40%

122.2 (C), 126.4 (CH), 127.6 (CH), 128.3 (CH), 138.5 (C), 164.8 (C),

1

−1

yield (2.31 g); mp (EtOAc:n-heptane) 105–107 °C; H NMR (CDCl

3

,

187.7 (C); IR ν (cm ): 2742, 2607, 1672, 1651, 1629, 1519, 1485,

5

00 MHz) δ (ppm): 1.43 (t, J = 7.0 Hz, 3H, CH

3

), 2.60 (s, 3H, CH

3

),

1450, 1432, 1388, 1346, 1318, 1261, 1188, 1093, 1077, 1020, 945,

883, 867, 790, 774, 733, 703, 635, 588, 546, 521, 446.

4

.41 (q, J = 7.0 Hz, 2H, CH ), 6.85(t, J = 7.0 Hz, 1H, ArH), 7.60 (d,

2

J = 7.5 Hz, 1H, ArH), 7.94 (s, 1H, ArH), 9.98 (d, J = 7.0 Hz, 1H, ArH);

1

3

C NMR (CDCl

3

, 125 MHz) δ (ppm): 14.5 (CH

3

), 27.7 (CH

3

), 61.1

4.4.3. 3-Acetyl-8-methylindolizin-1-yl-carboxylic acid (21c)

(

CH ), 109.6 (C), 112.2 (C), 114.8 (CH), 122.4 (C), 127.3 (CH), 128.0

2

The general procedure was used with 2.7 g (11.01 mmol) of in-

−1

CH), 131.5 (CH), 135.0 (C), 163.9 (C), 187.8 (C); IR ν (cm ): 3120,

dolizine ethyl ester 20c in 50 mL of absolute EtOH. White solid; 96%

1

2

1

981, 1708, 1643, 1520, 1471, 1422, 1382, 1367, 1344, 1221, 1204,

166, 1120, 1048, 987, 938, 865, 780, 764, 651, 620, 576, 560.

yield (2.3 g); mp (EtOH) 182–185 °C; H NMR (DMSO‑d

6

, 500 MHz) δ

(ppm): 2.55 (s, 3H, CH

3

), 2.71 (s, 3H, CH

3

), 7.11 (t, J = 7.1 Hz, 1H,

ArH), 7.29 (d, J = 7.1 Hz, 1H, ArH), 8.11 (s, 1H, ArH), 9.79 (d,

1

3

4

.4. General procedure for the saponiﬁcation of ethyl esters and generation

J = 7.2 Hz, 1H, ArH), 12.20 (br s, 1H, COOH); C NMR (DMSO‑d ,

6

of carboxylic acids 21a-h

125 MHz) δ (ppm): 21.7 (CH ), 27.8 (CH ), 108.4 (C), 115.7 (CH),

3

1

21.7 (C), 126.4 (CH), 128.6 (2CH), 129.6 (C), 137.6 (C), 165.2 (C),

−1

Indolizine ethyl ester 20a-h (1 equiv.) was suspended in absolute

ethanol. NaOH 50% aqueous solution (4 equiv.) was added and the

mixture was reﬂuxed for 3 h. The medium became homogenous (this

corresponded to the complete formation of the sodium carboxylate;

187.7 (C); IR ν (cm ): 2936, 2716, 1662, 1632, 1512, 1480, 1438,

1350, 1267, 1234, 1223, 1148, 1027, 995, 946, 867, 767, 738, 706,

681, 646, 568, 487, 434.

1

reaction monitored by H NMR). After cooling to rt, HCl 10% aqueous

4.4.4. 3-Acetyl-7-methylindolizin-1-yl-carboxylic acid (21d)

solution was added to the crude to obtain a pH slightly acid. The

The general procedure was used with 1.5 g (6.11 mmol) of

10

I.-M. Moise, et al.

Bioorganic Chemistry 103 (2020) 104184

indolizine ethyl ester 20d in 20 mL of absolute EtOH. White solid; 91%

carboxylic acid 21a-h (1 equiv.) and 10H-phenothiazine or 10-methyl-

10H-phenothiazine (1 equiv.) were then added to the Eaton’s reagent.

The medium was stirred at 50 °C for 4–24 h. After cooling to rt, the

Eaton’s reagent from the crude has been carefully neutralized by adding

1

yield (1.2 g); mp (EtOH) 248–250 °C; H NMR (DMSO‑d

6

, 500 MHz) δ

(

ppm): 2.51 (s, 3H, CH

3

), 2.58 (s, 3H, CH

3

), 7.14 (dd, J = 7.2, 1.6 Hz,

1

H, ArH), 8.12 (s, 1H, ArH), 8.14 (s, 1H, ArH), 9.73 (d, J = 7.2 Hz, 1H,

1

3

ArH), 12.47 (br s, 1H, COOH); C NMR (DMSO‑d

6

, 125 MHz) δ (ppm):

slowly an aqueous solution of NaHCO

3

(exothermic reaction). Ethyl

SO

, the ﬁltrate was concentrated

2

1.0 (CH

3

), 27.0 (CH

3

), 104.4 (C), 117.5 (CH), 117.8 (CH), 121.8 (C),

acetate was then added. The organic layer was over anhydrous Na

2

4

.

1

26.7 (CH), 127.7 (CH), 138.6 (C), 139.0 (C), 164.8 (C), 187.3 (C); IR ν

After the ﬁltration of hydrated Na

2

SO

4

−

1

(

cm ): 1659, 1627, 1514, 1485, 1464, 1364, 1338, 1294, 1277, 1236,

and then puriﬁed by ﬂash chromatography on silica prepacked column

(chromatographic eluent: gradient n-heptane/EtOAc 100/0 to 0/100)

to obtain pure indolizine-phenothiazine hybrids 16c,f,g and 17a-f.

1

6

195, 1182, 1148, 1117, 1044, 1013, 939, 875, 816, 778, 749, 669,

22, 552, 507, 419.

4

.4.5. 3-Acetyl-6,8-dimethylindolizin-1-yl-carboxylic acid (21e)

4.5.1. 1-[(10H-Phenothiazin-3-yl)carbonyl]-3-acetylindolizine (17a)

The general procedure was used with 0.91 g (4.47 mmol) of car-

boxylic acid 21a and 0.89 g (4.47 mmol) of 10H-phenothiazine. Orange

The general procedure was used with 1.5 g (5.78 mmol) of in-

dolizine ethyl ester 20e in 20 mL of absolute EtOH. White solid; 83%

1

yield (1.1 g); mp (EtOH) 209–211 °C; H NMR (DMSO‑d

6

, 500 MHz) δ

solid; 46% yield (0.92 g); mp (EtOAc/n-heptane) 228–229 °C; H NMR

(

ppm): 2.32 (s, 3H, CH

3

), 2.54 (s, 3H, CH

3

), 2.71 (s, 3H, CH

3

), 7.19 (s,

), 27.4

(CDCl

3

, 400 MHz) δ (ppm): 2.59 (s, 3H, CH ), 6.04 (s, 1H, NH), 6.57 (d,

3

1

H, ArH), 8.07 (s, 1H, ArH), 9.63 (s, 1H, ArH), 12.30 (br s, 1H, COOH);

J = 8.0 Hz, 1H, ArH), 6.62 (d, J = 8.0 Hz, 1H, ArH), 6.87 (t,

J = 7.7 Hz, 1H, ArH), 6.99 (t, J = 8.8 Hz, 2H, ArH), 7.11 (t,

J = 7.7 Hz, 1H, ArH), 7.48–7.54 (m, 3H, ArH), 7.77 (s, 1H, ArH), 8.53

1

3

C NMR (DMSO‑d

6

, 125 MHz) δ (ppm): 17.3 (CH ), 21.2 (CH

3

(

CH

3

), 107.7 (C), 121.0 (C), 123.8 (CH), 124.7 (C), 128.0 (CH), 128.5

−

1

13

C), 131.1 (CH), 136.1 (C), 164.8 (C), 187.1 (C); IR ν (cm ): 1667,

(d, J = 8.8 Hz, 1H, ArH), 9.96 (d, J = 6.8 Hz, 1H, ArH); C NMR

1

632, 1514, 1482, 1442, 1418, 1367, 1344, 1236, 1209, 1177, 1086,

024, 982, 949, 926, 851, 781, 760, 688, 661, 584, 556, 505, 423.

(CDCl

3

, 100 MHz) δ (ppm): 27.37 (CH ), 112.9 (CH), 116.4 (CH), 116.7

3

(CH), 117.1 (CH), 119.6 (C), 120.2 (CH + C), 120.5 (C), 122.4 (CH),

1

22.8 (C), 127.0 (C), 128.6 (CH), 129.2 (CH), 131.3 (CH), 132.8 (CH),

4.4.6. 3-Acetyl-7-methoxyindolizin-1-yl-carboxylic acid (21f)

133.0 (CH), 133.5 (CH), 135.8 (C), 138.8 (C), 140.4 (C), 187.4 (C),

−1

The general procedure was used with 1.93 g (7.39 mmol) of in-

187.9 (C); IR ν (cm ): 3302, 2360, 1560, 1497, 1463, 1357, 1309,

dolizine ethyl ester 20f in 30 mL of absolute EtOH. White solid; 91%

1267, 1226, 1194, 1156, 1106, 1035, 860, 744, 637, 535, 424. Elem.

1

yield (1.56 g); mp (EtOH) 245–246 °C; H NMR (DMSO‑d

6

, 500 MHz) δ

Analysis calcd. for C23

H

16

N

2

O S: C, 71.86; H, 4.20; N, 7.29. Found: C,

2

(

ppm): 2.52 (s, 3H, CH

3

), 3.92 (s, 3H, CH

3

), 6.93 (dd, J = 7.6, 2.8 Hz,

72.18; H, 4.13; N, 7.15%.

1

H, ArH), 7.62 (d, J = 2.8 Hz, 1H, ArH), 8.03 (s, 1H, ArH), 9.65 (d,

1

3

J = 7.6 Hz, 1H, ArH), 12.36 (br s, 1H, COOH); C NMR (DMSO‑d

6

,

4.5.2. 1,3-Bisacetylindolizine (23)

1

3

25 MHz) δ (ppm): 26.3 (CH ), 55.3 (CH

3

), 96.7 (CH), 103.4 (C), 108.5

By-product from the synthesis of ketone 17a. Yellow solid; 26%

1

(

CH), 121.0 (C), 126.7 (CH), 129.4 (CH), 140.8 (C), 158.3 (C), 164.5

yield (0.17 g); mp (EtOAc/n-heptane) 110–112 °C; H NMR (CDCl

3

,

−

1

C), 186.4 (C); IR ν (cm ): 1669, 1635, 1524, 1497, 1472, 1434, 1352,

400 MHz) δ (ppm): 2.49 (s, 3H, CH

3

), 2.61 (s, 3H, CH ), 7.05 (td,

3

1

7

309, 1282, 1232, 1190, 1105, 1045, 1019, 942, 889, 839, 814, 775,

59, 669, 644, 628, 532, 484, 435.

J = 6.8, 1.2 Hz, 1H, ArH), 7.44 (td, J = 6.8, 1.2 Hz, 1H, ArH), 7.99 (s,

1H, ArH), 8.45 (d, J = 8.8 Hz, 1H, ArH), 9.88 (d, J = 8.8 Hz, 1H, ArH);

1

3

C NMR (CDCl

3

, 100 MHz) δ (ppm): 8.5 (CH

3

), 24.8 (CH ), 110.6 (C),

3

4.4.7. 3-Acetyl-6-bromoindolizin-1-yl-carboxylic acid (21 g)

113.3 (CH), 117.1 (CH), 120.3 (C), 121.9 (CH), 125.8 (CH), 126.5 (CH),

−1

The general procedure was used with 0.63 g (2.03 mmol) of in-

135.0 (C), 182.6 (C), 185.3 (C); IR ν (cm ): 1622, 1509, 1481, 1440,

dolizine ethyl ester 20 g in 15 mL of absolute EtOH. White solid; 80%

1411, 1359, 1333, 1306, 1260, 1205, 1167, 1106, 1017, 935, 876, 843,

1

yield (0.58 g); mp (EtOH) 204–205 °C; H NMR (DMSO‑d

6

, 500 MHz) δ

759, 672, 634, 561, 421. Elem. Analysis calcd. for C12

H

11NO : C, 71.63;

2

(

ppm): 2.57 (s, 3H, CH

3

), 7.67 (dd, J = 9.6, 1.6 Hz, 1H, ArH), 8.13 (s,

H, 5.51; N, 6.96. Found: C, 71.91; H, 5.42; N, 7.08%.

1

H, ArH), 8.24 (d, J = 9.2 Hz, 1H, ArH), 9.94 (s, 1H, ArH), 12.66 (br s,

1

3

H, COOH); C NMR (DMSO‑d

6

, 125 MHz) δ (ppm): 27.7 (CH

3

), 107.2

4.5.3. 1-[(10H-Phenothiazin-3-yl)carbonyl]-3-acetyl-5-methylindolizine

(17b)

(

C), 110.0 (C), 120.4 (CH), 122.8 (C), 126.7 (CH), 128.2 (CH), 130.3

−

1

CH), 137.0 (C), 164.9 (C), 188.7 (C); IR ν (cm ): 2937, 2755, 2600,

The general procedure was used with 2.31 g (10.64 mmol) of car-

boxylic acid 21b and 2.12 g (10.64 mmol) of 10H-phenothiazine.

1

6

640, 1511, 1481, 1361, 1280, 1237, 1185, 1036, 993, 933, 810, 657,

30, 479, 422.

1

Orange solid; 25% yield (1.0 g); mp (EtOAc/n-heptane) 212–214 °C; H

NMR (DMSO‑d

6

, 400 MHz) δ (ppm): 2.50 (s, 3H, CH ), 2.63 (s, 3H,

3

4

.4.8. 3-Acetyl-8-bromoindolizin-1-yl-carboxylic acid (21 h)

CH ), 6.72 (d, J = 8.2 Hz, 1H, ArH), 6.77 (d, J = 8.2 Hz, 1H, ArH),

3

The general procedure was used with 2.13 g (6.87 mmol) of in-

6.88 (d, J = 7.2 Hz, 1H, ArH), 7.16 (d, J = 3.2 Hz, 1H, ArH), 7.25 (dd,

J = 9.1, 7.1 Hz, 1H, ArH), 7.28 (d, J = 1.5 Hz, 1H, ArH), 7.44 (d,

J = 2.0 Hz, 1H, ArH), 7.47 (d, J = 1.6 Hz, 1H, ArH), 7.50 (d,

J = 3.1 Hz, 1H, ArH), 7.60 (dd, J = 7.8, 2.0 Hz, 1H, ArH), 8.21 (d,

dolizine ethyl ester 20 h in 25 mL of absolute EtOH. White solid; 93%

1

yield (1.8 g); mp (EtOH) 196–197 °C; H NMR (DMSO‑d

6

, 500 MHz) δ

(

ppm): 2.58 (s, 3H, CH

3

), 7.07 (t, J = 8.7 Hz, 1H, ArH), 7.77 (d,

1

3

J = 8.4 Hz, 1H, ArH), 8.13 (s, 1H, ArH), 9.86 (d, J = 6.8 Hz, 1H, ArH),

J = 8.9 Hz, 1H, ArH), 9.45 (s, 1H, NH); C NMR (DMSO‑d , 100 MHz)

6

1

3

1

2.66 (br s, 1H, COOH); C NMR (DMSO‑d

6

, 125 MHz) δ (ppm): 28.1

δ (ppm): 18.8 (CH

3

), 26.6 (CH ), 111.9 (CH), 112.4 (CH), 113.5 (C),

3

(

CH ), 110.5 (C), 111.7 (C), 115.7 (CH), 122.3 (C), 127.6 (CH), 127.7

3

114.5 (CH), 114.6 (CH), 116.3 (C), 116.5 (C), 117.6 (CH), 117.9 (CH),

125.0 (CH), 126.9 (CH), 127.2 (CH), 129.4 (CH), 129.7 (CH), 131.7 (C),

135.5 (C), 135.6 (C), 136.9 (C), 143.0 (C), 145.3 (C), 186.9 (C), 195.6

−

1

CH), 131.7 (CH), 134.0 (C), 165.0 (C), 188.2 (C); IR ν (cm ): 2924,

2

9

569, 1672, 1641, 1515, 1477, 1429, 1345, 1246, 1209, 1183, 1044,

96, 938, 867, 766, 727, 691, 653, 635, 477.

−

1

(C); IR ν (cm ): 3272, 1665, 1589, 1557, 1482, 1434, 1347, 1322,

1261, 1226, 1205, 1148, 958, 917, 807, 781, 742, 716, 580, 442. Elem.

Analysis calcd. for C24 S: C, 72.34; H, 4.55; N, 7.03. Found: C,

72.70; H, 4.84; N, 6.98%.

4

.5. General procedure for the synthesis of acylphenothiazines 16c,f,g and

H

18

N O

2 2

1

7a-f

Eaton’s reagent was freshly prepared (4 equiv.) by mixing phos-

4.5.4. Bis-(5-methylindolizin-1-yl)carbonyl(10H-phenothiazin-3,7-diyl)

(22b)

phorus pentoxide with methanesulfonic acid (1:10, w:w) under ni-

trogen atmosphere and magnetic stirring for 10 min at 60 °C. The

By-product from the synthesis of ketone 17b. Orange solid; 14%

11

I.-M. Moise, et al.

Bioorganic Chemistry 103 (2020) 104184

1

yield (0.7 g); mp (EtOAc/n-heptane) 215–218 °C; H NMR (DMSO‑d

6

,

6.92 (d, J = 6.8 Hz, 1H, ArH), 7.49 (d, J = 4.4 Hz, 1H, ArH), 9.73 (d,

1

3

4

00 MHz) δ (ppm): 2.55 (s, 6H, 2CH

3

), 6.80 (d, J = 8.4 Hz, 2H, ArH),

J = 7.2 Hz, 1H, ArH); C NMR (CDCl

3

, 100 MHz) δ (ppm): 18.1 (CH ),

3

6

.89 (dt, J = 6.9, 1.1 Hz, 2H, ArH), 7.18 (d, J = 3.3 Hz, 2H, ArH), 7.25

27.1 (CH

3

), 100.3 (CH), 113.6 (CH), 122.9 (CH), 123.2 (C), 123.3 (CH),

(

dd, J = 9.1, 6.7 Hz, 2H, ArH), 7.31 (d, J = 2.4 Hz, 2H, ArH), 7.48 (dd,

126.4 (CH), 127.6 (C), 139.4 (C), 186.5 (C). Elem. Analysis calcd. for

J = 8.2, 1.8 Hz, 2H, ArH), 7.51 (d, J = 3.3 Hz, 2H, ArH), 8.22 (d,

C H11NO: C, 76.28; H, 6.40; N, 8.09. Found: C, 76.54; H, 6.64; N,

11

8.18%.

1

3

J = 9.1 Hz, 2H, ArH), 9.38 (s, 1H, NH); C NMR (DMSO‑d

6

, 100 MHz)

δ (ppm): 18.8 (2CH

3

), 112.0 (2CH), 112.4 (2CH), 113.5 (2C), 114.4

(

2CH), 116.3 (2CH), 117.6 (2CH), 117.9 (2CH), 124.9 (2CH), 127.3

4.5.9. 1-[(10H-Phenothiazin-3-yl)carbonyl]-3-acetyl-7-methylindolizine

(17d)

(

2C), 129.7 (2C), 135.1 (2CH), 135.6 (2C), 136.9 (2C), 143.7 (2C),

−

1

4

86.9 (2C); IR ν (cm ): 3301, 1633, 1585, 1555, 1479, 1427, 1352,

The general procedure was used with 0.63 g (2.91 mmol) of car-

boxylic acid 21d and 0.58 g (2.91 mmol) of 10H-phenothiazine. Green

287, 1193, 1151, 1124, 1069, 916, 829, 781, 734, 704, 680, 630, 575,

1

37. Elem. Analysis calcd. for C32

H

23

N

3

O

2

S: C, 74.83; H, 4.51; N, 8.18.

solid; 44% yield (0.44 g); mp (EtOAc/n-heptane) 122–125 °C; H NMR

Found: C, 75.20; H, 4.75; N, 8.36%.

(DMSO‑d

6

, 400 MHz) δ (ppm): 2.52 (s, 3H, CH

3

), 2.54 (s, 3H, CH ),

3

6

.70–6.82 (m, 3H, ArH), 6.93 (d, J = 8.3 Hz, 1H, ArH), 7.01 (t,

4

.5.5. 5-Methylindolizin-1-carboxylic acid (24)

J = 8.9 Hz, 1H, ArH), 7.17 (d, J = 8.9 Hz, 1H, ArH), 7.34 (s, 1H, ArH),

By-product from the synthesis of ketone 17b. Orange solid; 6% yield

7.52 (d, J = 8.9 Hz, 1H, ArH), 7.98 (s, 1H, ArH), 8.26 (s, 1H, ArH), 9.06

1

13

(

0.11 g); mp (EtOAc/n-heptane) 181–184 °C;

H

NMR (CDCl

3

,

(s, 1H, NH), 9.74 (d, J = 7.7 Hz, 1H, ArH); C NMR (DMSO‑d

6

,

4

00 MHz) δ (ppm): 2.59 (s, 3H, CH

3

), 6.63 (d, J = 6.4 Hz, 1H, ArH),

100 MHz) δ (ppm): 21.0 (CH

3

), 27.1 (CH ), 111.3 (C), 113.5 (CH),

3

7

.10 (t, J = 6.8 Hz, 1H, ArH), 7.19 (d, J = 2.8 Hz, 1H, ArH), 7.40 (d,

114.7 (CH), 115.9 (CH), 116.3 (CH), 118.1 (CH), 118.6 (C), 121.7 (C),

122.5 (CH), 126.2 (C), 126.8 (C), 127.4 (C), 127.7 (CH), 129.5 (C),

132.8 (CH), 139.6 (C), 139.8 (CH), 140.4 (2CH), 145.0 (C), 186.5 (C),

1

3

J = 2.8 Hz, 1H, ArH), 8.21 (d, J = 8.8 Hz, 1H, ArH); C NMR (CDCl

3

,

1

00 MHz) δ (ppm): 17.7 (CH

3

), 103.2 (C), 109.5 (CH), 110.8 (CH),

−

1

15.5 (CH), 116.5 (CH), 121.4 (CH), 133.0 (C), 135.4 (C), 166.4 (C); IR

187.5 (C); IR ν (cm ): 3275, 2919, 1634, 1591, 1563, 1503, 1462,

1420, 1362, 1336, 1289, 1228, 1197, 1146, 1083, 1013, 932, 883, 827,

799, 774, 740, 701, 620, 550, 422. Elem. Analysis calcd. for

−1

ν (cm ): 1645, 1523, 1475, 1452, 1365, 1290, 1259, 1192, 1151,

1

110, 1048, 989, 910, 790, 763, 737, 706, 570, 513, 439. Elem.

NO : C, 68.56; H, 5.18; N, 8.00. Found: C,

8.72; H, 5.34; N, 8.19%.

Analysis calcd. for C10

H

9

2

C

24

7.36%.

H

18

N

2

O S: C, 72.34; H, 4.55; N, 7.03. Found: C, 72.62; H, 4.55; N,

2

6

4

.5.6. 1-[(10H-Phenothiazin-3-yl)carbonyl]-3-acetyl-5-methylindolizine

4.5.10. 1-[(10-Methyl-10H-phenothiazin-3-yl)carbonyl]-3-acetyl-7-

methylindolizine (17d’)

(

17c)

The general procedure was used with 1.99 g (9.18 mmol) of car-

The general procedure was used with 1.8 g (8.30 mmol) of car-

boxylic acid 21d and 1.77 g (8.30 mmol) of 10-methyl-10H-phe-

boxylic acid 21c and 1.83 g (9.18 mmol) of 10H-phenothiazine. Yellow

1

solid; 36% yield (1.2 g); mp (EtOAc/n-heptane) 200–202 °C; H NMR

nothiazine. Orange solid; 34% yield (1.13 g); mp (EtOAc/n-heptane)

1

(

DMSO‑d

6

, 400 MHz) δ (ppm): 2.27 (s, 3H, CH

3

), 2.53 (s, 3H, CH

3

), 6.72

144–145 °C; H NMR (CDCl

3

, 500 MHz) δ (ppm): 2.50 (s, 3H, CH ),

3

m, J = 8.4, 8.0, 1.6 Hz, 2H, ArH), 6.80 (t, J = 8.0 Hz, 1H, ArH), 6.92

d, J = 7.6 Hz, 1H, ArH), 7.01 (t, J = 8.0 Hz, 1H, ArH), 7.18 (t,

2.54 (s, 3H, CH

3

), 3.46 (s, 3H, CH ), 6.87 (d, J = 7.5 Hz, 1H, ArH), 6.90

3

(d, J = 8.5 Hz, 1H, ArH), 6.95 (dd, J = 7.0, 2.0 Hz, 1H, ArH), 6.99 (td,

J = 7.5, 1.0 Hz, 1H, ArH), 7.17 (dd, J = 7.5, 1.5 Hz, 1H, ArH), 7.21 (td,

J = 8.0, 1.5 Hz, 1H, ArH), 7.50 (d, J = 1.5 Hz, 1H, ArH), 7.69 (dd,

J = 8.0, 2.0 Hz, 1H, ArH), 7.71 (s, 1H, ArH), 8.39 (t, J = 1.5 Hz, 1H,

J = 6.8 Hz, 1H, ArH), 7.31 (d, J = 6.8 Hz, 1H, ArH), 7.40 (br s, 1H,

ArH), 7.52 (dd, J = 8.0, 2.0 Hz, 1H, ArH), 7.89 (s, 1H, ArH), 9.16 (br s,

1

3

1

H, NH), 9.80 (d, J = 7.6 Hz, 1H, ArH); C NMR (DMSO‑d , 100 MHz)

6

1

3

δ (ppm): 20.7 (CH

3

), 27.8 (CH

3

), 113.9 (CH), 114.0 (C), 115.4 (CH),

ArH), 9.83 (d, J = 7.0 Hz, 1H, ArH); C NMR (CDCl

3

, 125 MHz) δ

1

15.7 (CH), 116.0 (C), 116.4 (CH), 116.7 (CH), 121.6 (C), 123.2 (CH),

(ppm): 21.7 (CH

3

), 27.3 (CH

3

), 35.8 (CH

3

), 112.5 (C), 113.5 (CH),

26.3 (C), 126.7 (CH), 127.5 (CH), 128.2 (CH), 128.2 (C), 129.4 (CH),

31.5 (CH), 132.7 (C), 137.5 (C), 140.5 (C), 146.3 (C), 187.8 (C), 188.0

114.6 (CH), 118.7 (CH), 119.3 (CH), 122.5 (C), 123.1 (C), 123.3 (CH),

123.6 (C), 127.5 (CH), 127.8 (CH), 127.9 (CH), 128.1 (CH), 128.7(CH),

129.3 (CH), 134.5 (C), 140.4 (C), 140.9 (C), 144.9 (C), 148.9 (C), 187.6

−

1

(

C); IR ν (cm ): 3315, 1610, 1591, 1561, 1504, 1470, 1425, 1397,

−1

1

304, 1214, 1145, 1072, 949, 927, 836, 753, 703, 635, 582. Elem.

S: C, 72.34; H, 4.55; N, 7.03. Found: C,

(C), 188.7 (C); IR ν (cm ): 1704, 1614, 1573, 1505, 1462, 1334, 1286,

Analysis calcd. for C24

H

18

N

2

O

2

1251, 1231, 1197, 1141, 1105, 1013, 931, 885, 832, 805, 763, 745,

7

2.11; H, 4.25; N, 6.77%.

619, 55, 425. Elem. Analysis calcd. for C25

H

20

N

2

O S: C, 72.79; H, 4.89;

2

N, 6.79. Found: C, 72.93; H, 5.10; N, 6.98%.

4

.5.7. 1-[(10H-Phenothiazin-10-yl)carbonyl]-3-acetyl-8-methylindolizine

(

16c)

4.5.11. 1-[(10H-Phenothiazin-3-yl)carbonyl]-3-acetyl-6,8-

dimethylindolizine (17e)

By-product from the synthesis of ketone 17c. Yellow solid; 14%

1

yield (0.49 g); mp (EtOAc/n-heptane) 203–206 °C; H NMR (DMSO‑d

00 MHz) δ (ppm): 2.48 (s, 3H, CH ), 2.59 (s, 3H, CH ), 6.26 (d,

J = 8.1 Hz, 2H, ArH), 6.78–6.92 (m, 6H, ArH), 7.00 (dd, J = 7.3,

6

,

The general procedure was used with 1.8 g (7.78 mmol) of car-

boxylic acid 21e and 1.55 g (7.78 mmol) of 10H-phenothiazine. Orange

4

3

1

solid; 27% yield (0.91 g); mp (EtOAc/n-heptane) 199–202 °C; H NMR

1

3

1

.9 Hz, 2H, ArH), 7.59 (s, 1H, ArH), 9.89 (d, J = 6.5 Hz, 1H, ArH);

C

(DMSO‑d

6

, 500 MHz) δ (ppm): 2.35 (s, 6H, 2CH

3

), 2.50 (s, 3H, CH ),

3

NMR (DMSO‑d

6

, 100 MHz) δ (ppm): 18.0 (CH ), 27.4 (CH ), 114.4

3

6.71 (d, J = 8.0 Hz, 1H, ArH), 6.72 (d, J = 8.5 Hz, 1H, ArH), 6.80 (t,

J = 7.5 Hz, 1H, ArH), 6.91 (d, J = 7.5 Hz, 1H, ArH), 7.00 (t,

J = 7.5 Hz, 1H, ArH), 7.20 (s, 1H, ArH), 7.38 (d, J = 2.0 Hz, 1H, ArH),

7.50 (dd, J = 8.0, 2.0 Hz, 1H, ArH), 7.82 (s, 1H, ArH), 9.15 (s, 1H, NH),

(

CH + C), 115.3 (C), 115.9 (2CH), 119.8 (2C), 121.3 (C), 122.5 (2CH),

1

24.8 (CH), 125.2 (CH), 126.5 (CH), 126.6 (2CH), 127.0 (2CH), 128.2

−1

(

2C), 135.6 (C), 144.5 (C), 186.6 (C); IR ν (cm ): 1629, 1587, 1460,

1

3

1

4

433, 1337, 1306, 1278, 1238, 1066, 1035, 951, 773, 745, 651, 562,

50. Elem. Analysis calcd. for C24 S: C, 72.34; H, 4.55; N, 7.03.

9.65 (s, 1H, ArH); C NMR (DMSO‑d

20.2 (CH ), 27.4 (CH

116.0 (C), 116.2 (C), 120.9 (C), 122.8 (CH), 123.7 (CH), 125.0 (C),

26.3 (CH), 127.0 (CH), 127.7 (CH), 128.4 (C), 130.6 (CH), 131.0 (CH),

6

, 125 MHz) δ (ppm): 17.9 (CH

3

),

H

18

N

2

O

2

3

), 113.5 (CH), 114.9 (CH), 115.1 (C), 115.9 (CH),

Found: C, 72.49; H, 4.58; N, 6.93%.

1

4.5.8. 3-Acetyl-8-methylindolizine (25)

132.3 (C), 135.9 (C), 140.1 (C), 145.8 (C), 187.2 (C), 187.4 (C); IR ν

−1

By-product from the synthesis of ketone 17c. Orange oil; 32% yield

(cm ): 3275, 2919, 1634, 1591, 1563, 1503, 1462, 1420, 1362, 1336,

1289, 1228, 1197, 1146, 1083, 1013, 932, 883, 827, 799, 774, 740,

1

(

0.5 g); H NMR (CDCl

3

, 400 MHz) δ (ppm): 2.48 (s, 3H, CH ), 2.55 (s,

3

H, CH

3

), 6.48 (d, J = 4.4 Hz, 1H, ArH), 6.79 (t, J = 7.2 Hz, 1H, ArH),

701, 620, 550, 422. Elem. Analysis calcd. for C25

H

20

N

2

O S: C, 72.79; H,

2

12

I.-M. Moise, et al.

Bioorganic Chemistry 103 (2020) 104184

4

.89; N, 6.79. Found: C, 73.08; H, 5.01; N, 6.99%.

624, 535, 437. Elem. Analysis calcd. for C24

H

18

N

2

O S: C, 69.55; H,

3

4

.38; N, 6.76. Found: C, 69.94; H, 4.60; N, 7.13%.

4.5.12. 1,3-Bisacetyl-6,8-dimethylindolizine (26)

By-product from the synthesis of ketone 17e. Orange solid; 6% yield

4.5.16. 1-[(10H-Phenothiazin-10-yl)carbonyl]-3-acetyl-7-

1

(

0.1 g); mp (EtOAc/n-heptane) 141–144 °C; H NMR (CDCl

3

, 500 MHz)

methoxyindolizine (16f)

δ (ppm): 2.35 (s, 3H, CH

3

), 2.46 (s, 3H, CH

3

), 2.59 (s, 3H, CH

3

), 2.63 (s,

By-product from the synthesis of ketone 17f. Pink solid; 32% yield

1

3

1

3

H, CH

3

), 7.04 (s, 1H, ArH), 7.98 (s, 1H, ArH), 9.69 (s, 1H, ArH);

C

),

(0.5 g); mp (EtOAc/n-heptane) 222–225 °C; H NMR (DMSO‑d

6

,

NMR (CDCl

3

, 100 MHz) δ (ppm): 12.4 (CH

3

), 18.4 (CH

3

), 21.4 (CH

3

400 MHz) δ (ppm): 2.14 (s, 3H, CH

3

), 3.91 (s, 3H, CH ), 6.65 (s, 1H,

3

2

1

2

1

7.6 (CH

3

), 115.3 (C), 121.9 (C), 125.0 (CH), 125.6 (C), 126.8 (CH),

ArH), 6.87 (dd, J = 7.9, 2.8 Hz, 1H, ArH), 7.30–7.33 (m, 4H, ArH), 7.53

(d, J = 2.8 Hz, 1H, ArH), 7.61–7.64 (m, 4H, ArH), 9.52 (d, J = 7.9 Hz,

−1

29.3 (C), 132.0 (CH), 135.3 (C), 186.5 (C), 187.4 (C). IR ν (cm ):

918, 1667, 1639, 1513, 1478, 1432, 1404, 1357, 1277, 1225, 1176,

142, 1101, 1040, 982, 950, 886, 855, 798, 754, 661, 575, 524, 481.

1

3

1H, ArH); C NMR (DMSO‑d

6

, 100 MHz) δ (ppm): 26.7 (CH ), 56.2

3

(CH

3

), 97.6 (CH), 105.8 (CH), 109.5 (C), 120.9 (CH), 126.6 (CH), 127.2

Elem. Analysis calcd. for C14

H

15NO : C, 73.34; H, 6.59; N, 6.11. Found:

2

(2CH), 127.6 (2CH), 127.7 (2CH), 128.0 (2CH), 129.9 (C), 132.3 (2C),

−

1

C, 73.50; H, 6.88; N, 6.39%.

139.7 (2C), 141.9 (C), 158.8 (C), 162.9(C), 186.5 (C); IR ν (cm ):

622, 1519, 1455, 1425, 1362, 1303, 1238, 1190, 1128, 1082, 1023,

931, 837, 808, 757, 725, 690, 661, 629, 526, 477. Elem. Analysis calcd.

for C24 S: C, 69.55; H, 4.38; N, 6.76. Found: C, 69.90; H, 4.53;

N, 7.01%.

1

4.5.13. 3-Acetyl-6,8-dimethylindolizine (27) [34]

By-product from the synthesis of ketone 17e. Yellow solid; 35%

H

18

N O

2 3

1

yield (0.51 g); mp (EtOAc/n-heptane) 57–59 °C; H NMR (CDCl

3

,

4

00 MHz) δ (ppm): 2.31 (s, 3H, CH

3

), 2.44 (s, 3H, CH

3

), 2.53 (s, 3H,

CH

3

), 6.42 (d, J = 4.4 Hz, 1H, ArH), 6.79 (s, 1H, ArH), 7.41 (d,

4.5.17. 1,3-Bisacetyl-7-methoxyindolizine (28)

1

3

J = 4.8 Hz, 1H, ArH), 9.56 (s, 1H, ArH); C NMR (CDCl

3

, 100 MHz) δ

By-product from the synthesis of ketone 17f. White-oﬀ solid; 22%

1

(

ppm): 17.7 (CH

3

), 18.3 (CH

3

), 26.9 (CH

3

), 99.8 (CH), 122.0 (C), 122.7

yield (0.2 g); mp (EtOAc/n-heptane) 117–118 °C; H NMR (CDCl

3

,

CH), 123.1 (C), 124.1 (CH), 126.0 (CH), 126.5 (C), 138.0 (C), 186.1

400 MHz) δ (ppm): 2.48 (s, 3H, CH

CH ), 6.71 (d, J = 8.0 Hz, 1H, ArH), 7.79 (s, 1H, ArH), 7.90 (s, 1H,

ArH), 9.71 (d, J = 8.0 Hz, 1H, ArH); C NMR (CDCl

(ppm): 11.0 (CH ), 26.9 (CH ), 55.8 (CH ), 97.6 (CH), 109.6 (CH),

111.6 (C), 122.3 (C), 125.1 (CH), 130.4 (CH), 140.3 (C), 160.4 (C),

3

), 2.56 (s, 3H, CH

3

), 3.93 (s, 3H,

−

1

C). IR ν (cm ): 2920, 1613, 1511, 1464, 1434, 1381, 1343, 1286,

3

1

3

1

6

7

224, 1197, 1169, 1135, 1105, 1035, 970, 948, 912, 841, 780, 744,

72, 582, 479, 443. Elem. Analysis calcd. for C12 13NO: C, 76.98; H,

.00; N, 7.48. Found: C, 77.21; H, 7.13; N, 7.26%.

3

, 100 MHz) δ

H

3

−

1

85.2 (C), 187.2 (C). IR ν (cm ): 1648, 1615, 1511, 1459, 1427, 1361,

4

.5.14. 1-[(10H-Phenothiazin-3-yl)carbonyl]-3-acetyl-6,8-

1303, 1266, 1239, 1195, 1154, 1122, 1087, 1023, 944, 908, 840, 815,

dimethylindolizine (17e’)

741, 683, 632, 599, 510, 428. Elem. Analysis calcd. for C13

H

13NO : C,

3

The general procedure was used with 0.58 g (2.51 mmol) of car-

boxylic acid 21e and 0.54 g (2.53 mmol) of 10-methyl-10H-phe-

67.52; H, 5.67; N, 6.06. Found: C, 67.67; H, 6.03; N, 6.41%.

nothiazine. Green solid; 24% yield (0.24 g); mp (EtOAc/n-heptane)

4.5.18. 1-[(10H-Phenothiazin-10-yl)carbonyl]-3-acetyl-6-bromoindolizine

(16 g)

1

2

50–151 °C; H NMR (CDCl

3

, 400 MHz) δ (ppm): 2.37 (s, 3H, CH

3

),

.47 (s, 3H, CH

3

), 2.50 (s, 3H, CH

3

), 3.44 (s, 3H, CH

3

), 6.85 (d,

The general procedure was used with 0.37 g (1.31 mmol) of car-

J = 8.5 Hz, 2H, ArH), 6.97 (td, J = 7.8, 1.3 Hz, 1H, ArH), 7.04 (s, 1H,

ArH), 7.13 (dd, J = 7.8, 1.6 Hz, 1H, ArH), 7.18 (td, J = 7.8, 1.6 Hz, 1H,

ArH), 7.54 (s, 1H, ArH), 7.71 (d, J = 2.0 Hz, 1H, ArH), 7.75 (dd,

boxylic acid 21 g and 0.26 g (1.31 mmol) of 10H-phenothiazine. White-

1

pink solid; 47% yield (0.27 g); mp (EtOAc/n-heptane) 107–108 °C;

H

NMR (CDCl

3

, 400 MHz) δ (ppm): 2.31 (s, 3H, CH ), 6.81 (t, J = 7.6 Hz,

3

1

3

J = 8.3, 2.0 Hz, 1H, ArH), 9.73 (s, 1H, ArH); C NMR (CDCl

3

,

1H, ArH), 7.02 (s, 1H, ArH), 7.13–7.15 (m, 4H, ArH), 7.42–7.44 (m, 2H,

1

9

00 MHz) δ (ppm): 18.4 (CH

3

), 20.9 (CH

3

), 27.4 (CH

3

), 35.7 (CH ),

3

ArH), 7.46–7.51 (m, 2H, ArH), 7.55 (d, J = 8.6 Hz, 1H, ArH), 9.81 (d,

1

3

13.2 (CH), 114.5 (CH), 115.5 (C), 121.4 (C), 122.8 (C), 123.2 (C),

23.3 (CH), 124.9 (CH), 125.4 (C), 127.3 (CH), 127.3 (CH), 127.6 (CH),

27.7 (CH), 129.0 (CH), 129.2 (C), 131.3 (CH), 133.7 (C), 137.2 (C),

J = 7.8 Hz, 1H, ArH); C NMR (CDCl

3

, 100 MHz) δ (ppm): 27.3 (CH ),

3

111.8 (C), 112.8 (C), 114.3 (CH), 122.1 (C), 123.5 (CH), 126.6 (2CH),

127.0 (2CH), 127.2 (2CH), 127.5 (2CH), 127.6 (CH), 129.3 (CH), 132.3

−

1

−1

44.6 (C), 149.5 (C), 187.2 (C), 188.8 (C); IR ν (cm ): 2920, 1620,

572, 1505, 1462, 1414, 1335, 1253, 1234, 1207, 1174, 1139, 1039,

80, 948, 925, 849 822, 770, 740, 664, 648, 584, 546, 470, 436. Elem.

(2C), 134.4 (C), 139.1 (2C), 164.1 (C), 187.4 (C); IR ν (cm ): 1668,

1650, 1631, 1530, 1460, 1428, 1395, 1363, 1339, 1311, 1282, 1253,

1198, 1180, 1126, 1084, 1005, 943, 825, 754, 729, 688, 657, 630, 453.

Analysis calcd. for C26

H

22

N

2

O

2

S: C, 73.21; H, 5.20; N, 6.57. Found: C,

Elem. Analysis calcd. for C23

H

15BrN

2

O S: C, 59.62; H, 3.26; N, 6.05.

2

7

3.44; H, 5.50; N, 6.75%.

Found: C, 59.78; H, 3.43; N, 6.09%.

4

.5.15. 1-[(10H-Phenothiazin-3-yl)carbonyl]-3-acetyl-7-

4.5.19. 3-Acetyl-8-bromoindolizine (29) [34]

methoxyindolizine (17f)

The general procedure was used with 0.91 g (3.22 mmol) of car-

boxylic acid 21 h and 0.64 g (3.22 mmol) of 10H-phenothiazine. White

The general procedure was used with 0.9 g (3.86 mmol) of car-

boxylic acid 21f and 0.77 g (3.86 mmol) of 10H-phenothiazine. Green

1

solid; 74% yield (0.57 g); mp (EtOAc/n-heptane) 74–75 °C; H NMR

1

solid; 31% yield (0.48 g); mp (EtOAc/n-heptane) 202–205 °C; H NMR

(CDCl

3

, 400 MHz) δ (ppm): 2.55 (s, 3H, CH ), 6.64 (d, J = 4.6 Hz, 1H,

3

(

CDCl

3

, 400 MHz) δ (ppm): 2.51 (s, 3H, CH

3

), 3.94 (s, 3H, CH

3

), 6.02

ArH), 6.68 (t, J = 7.4 Hz, 1H, ArH), 7.29 (d, J = 7.4 Hz, 1H, ArH), 7.49

13

br s, 1H, NH), 6.55 (dd, J = 7.9, 1.0 Hz, 1H, ArH), 6.60 (d, J = 8.3 Hz,

(d, J = 4.6 Hz, 1H, ArH), 9.77 (d, J = 7.0 Hz, 1H, ArH); C NMR

(CDCl , 100 MHz) δ (ppm): 27.3 (CH ), 103.3 (CH), 112.6 (C), 113.3

1

H, ArH), 6.75 (dd, J = 7.5, 2.8 Hz, 1H, ArH), 6.86 (td, J = 7.4, 1.2 Hz,

H, ArH), 6.95–7.00 (m, 2H, ArH), 7.46 (d, J = 1.7 Hz, 1H, ArH), 7.48

3

(CH), 123.6 (CH), 124.1 (C), 125.8 (CH), 127.3 (CH), 137.4 (C), 187.2

−1

(

dd, J = 7.9, 1.7 Hz, 1H, ArH), 7.67 (s, 1H, ArH), 7.96 (d, J = 2.7 Hz,

(C). IR ν (cm ): 1633, 1506, 1463, 1423, 1378, 1358, 1327, 1281,

1

3

1

H, ArH), 9.76 (d, J = 7.9 Hz, 1H, ArH); C NMR (CDCl

3

, 100 MHz) δ

1234, 1187, 1166, 1048, 1014, 932, 885, 780, 756, 708, 663, 568.

(

ppm): 27.2 (CH

3

), 56.0 (CH

3

), 98.5 (CH), 109.7 (CH), 111.4 (CH),

Elem. Analysis calcd. for C10

8

H BrNO: C, 50.45; H, 3.39; N, 5.88. Found:

1

13.8 (CH), 115.1 (CH), 116.6 (C), 117.0 (C), 121.8 (CH), 122.6 (CH),

26.4 (C), 127.1 (C), 127.8 (CH), 127.9 (CH), 129.4 (CH), 130.2 (CH),

33.5 (C), 141.0 (C), 142.6 (C), 145.4 (C), 160.1 (C), 187.0 (C), 187.1

C, 50.89; H, 3.72; N, 6.13%.

4.6. Tubulin polymerization assay

−

1

(

C); IR ν (cm ): 3264, 1643, 1616, 1569, 1493, 1470, 1427, 1362,

1

334, 1301, 1274, 1217, 1189, 1113, 1073, 1028, 944, 822, 741, 688,

Sheep brain tubulin was puriﬁed according to the method of

13

I.-M. Moise, et al.

Bioorganic Chemistry 103 (2020) 104184

Shelanski [35] by two cycles assembly–disassembly and then dissolved

Author contributions

in the assembly buﬀer containing 0.1 M MES, 0.5 mM MgCl , 1 mM

2

EGTA, and 1 mM of GTP (pH 6.6) to give a tubulin concentration of

about 2–3 mg/mL. Tubulin assembly was monitored by ﬂuorescence

according to reported procedure [36] using DAPI as ﬂuorescent mole-

cule. Assays were realized on 96-well plates prepared with Biomek

NKMC and Biomek 3000 from Beckman coulter and read at 37 °C on

Wallac Victor ﬂuorimeter from Perkin–Elmer. Each measurement was

reproduced twice (two independent experiments on diﬀerent 96-well

plates) in duplicate. The IC50 value of each compound was determined

as tubulin polymerization inhibition by 50% compared to the rate in the

absence of compound. The IC50 values for all compounds were com-

pared to the IC50 of phenstatin and (-)-desoxypodophyllotoxin and

measured the same day under the same conditions.

A.G. wrote the manuscript, performed experiments on tubulin

polymerization and on FTase in vitro and analyzed the data; I.-M. M.

performed the organic synthesis and characterization of molecules; J.D.

performed experiments on tubulin polymerization; A.F. performed

molecular modeling on tubulin and FTase; E.B. supervised the organic

synthesis and contributed to the writing of the manuscript.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://

doi.org/10.1016/j.bioorg.2020.104184 .

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15

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Indolizine-phenothiazine hybrids as the first dual inhibitors of tubulin polymerization and farnesyltransferase with synergistic antitumor activity

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