Sea Urchin Embryo Model As a Reliable in Vivo ... - ACS Publications

Subscriber access provided by Kaohsiung Medical University

Article

The sea urchin embryo model as a reliable in vivo phenotypic screen to characterize selective antimitotic molecules. Comparative evaluation of combretapyrazoles, -isoxazoles, -1,2,3-triazoles, and -pyrroles as tubulin binding agents. Marina N. Semenova, Dmitry V. Demchuk, Dmitry V. Tsyganov, Natalia B. Chernysheva, Alexander V. Samet, Eugenia A. Silyanova, Victor P. Kislyi, Anna S. Maksimenko, Alexander E. Varakutin, Leonid D. Konyushkin, Mikhail M. Raihstat, Alex S. Kiselyov, and Victor Vladimirovich Semenov ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.8b00113 • Publication Date (Web): 19 Nov 2018 Downloaded from http://pubs.acs.org on November 19, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Combinatorial Science

The sea urchin embryo model as a reliable in vivo phenotypic screen to characterize selective antimitotic molecules. Comparative evaluation of combretapyrazoles, -isoxazoles, -1,2,3-triazoles, and -pyrroles as tubulin binding agents Marina N. Semenova,† Dmitry V. Demchuk,‡ Dmitry V. Tsyganov,‡ Natalia B. Chernysheva,‡ Alexander V. Samet,‡ Eugenia A. Silyanova,‡ Victor P. Kislyi,‡ Anna S. Maksimenko,‡ Alexander E. Varakutin,‡ Leonid D. Konyushkin,‡ Mikhail M. Raihstat,‡ Alex S. Kiselyov,§ Victor V. Semenov,*,‡ †

N. K. Koltzov Institute of Developmental Biology RAS, 26 Vavilov Street, 119334 Moscow,

Russian Federation ‡ N.

D. Zelinsky Institute of Organic Chemistry RAS, 47 Leninsky Prospect, 119991 Moscow,

Russian Federation § Genea

Biocells US Inc., Suite 210, 11099 North Torrey Pines Rd., La Jolla, CA 92037, USA

Corresponding author: Victor V. Semenov Address: N. D. Zelinsky Institute of Organic Chemistry, RAS, Leninsky Prospect, 47, 119991, Moscow, Russian Federation. Tel.: +7 916 620 9584; fax: +7 499 137 2966. E-mail: [email protected] E-mail addresses: Marina N. Semenova

[email protected]

Dmitry V. Demchuk

[email protected]

Dmitry V. Tsyganov

[email protected]

Natalia B. Chernysheva

[email protected]

Alexander V. Samet

[email protected]

Eugenia A. Silyanova

[email protected]

Victor P. Kislyi

[email protected]

Anna S. Maksimenko

[email protected]

Alexander E. Varakutin

[email protected]

Leonid D. Konyushkin

[email protected]

Mikhail M. Raihstat

[email protected]

Alex S. Kiselyov

[email protected]

Victor V. Semenov

[email protected]

1 ACS Paragon Plus Environment

ACS Combinatorial Science 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 57

ABSTRACT

A series of both novel and reported combretastatin analogues including diarylpyrazoles, isoxazoles, -1,2,3-triazoles and -pyrroles were synthesized via improved protocols in order to evaluate their antimitotic antitubulin activity using in vivo sea urchin embryo assay and a panel of human cancer cells. A systematic comparative structure-activity relationship studies of these compounds were conducted. Pyrazoles 1i and 1p, isoxazole 3a, and triazole 7b were found to be the most potent antimitotics across all tested compounds causing cleavage alteration of the sea urchin embryo at 1 nM, 0.25 nM, 1 nM, and 0.5 nM, respectively. These agents exhibited comparable cytotoxicity against human cancer cells. Structure-activity relationship studies revealed that compounds substituted with 3,4,5-trimethoxyphenyl ring A and 4-methoxyphenyl ring B displayed the highest activity. 3-Hydroxy group in the ring B was essential for the antiproliferative activity in the diarylisoxazole series, whereas it was not required for potency of diarylpyrazoles. Isoxazoles 3 with 3,4,5-trimethoxy substituted ring A and 3-hydroxy-4-methoxy substituted ring B were more active than the respective pyrazoles 1. Of the azoles substituted with the same set of other aryl pharmacophores, diarylpyrazoles 1, 4,5-diarylisoxazoles 3, and 4,5-diaryl-1,2,3-triazoles 7 displayed similar strongest antimitotic antitubulin effect followed by 3,4-diarylisoxazoles 5, 1,5-diaryl-1,2,3-triazoles 8, and pyrroles 10 that showed the lowest activity. Introduction of the amino group into the heterocyclic core decreased the antimitotic antitubulin effect of pyrazoles, triazoles, and to a lesser degree of 4,5-diarylisoxazoles, whereas potency of the respective 3,4-diarylisoxazoles was increased. Keywords: Combretastatins Diarylazoles Sea urchin embryo Antimitotic Microtubule destabilization Abbreviations: CA2, combretastatin A-2 CA4, combretastatin A-4 SAR, structure-activity relationship


Page 3 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Cytostatic agents that affect structure and/or dynamics of microtubules attract considerable attention in the development of novel antitumor agents that are potent, selective, address cancer resistance, and display more favorable therapeutic window. An appropriate integration of target-based and phenotypic screening is a very promising approach in drug discovery.1 In this regard a choice of proper model is of a paramount importance in the phenotypic screening. Ideally, a model organism should be readily available, accessible, and feasible. It should also furnish data relevant to human physiology. While searching for novel microtubule dynamics modulating compounds, we selected a sea urchin embryo model for the phenotypic assay. This organism is well characterized and widely used in experimental biology to study various aspects of embryology, developmental and molecular biology, genetics and ecology.2–8 There are multiple advantages to a sea urchin embryo over alternative biological models. These include a) simple access to a large number of gametes, b) facile artificial fertilization and rearing, c) quick and synchronous cell division during early embryogenesis, d) well studied differentiation and morphogenesis, e) direct relevance to mammalian and human signaling networks. Relatively large size and optical transparency of sea urchin eggs, embryos and larvae allows for convenient monitoring of the assay with conventional microscope. Sea urchin embryos can be reliably reared in filtered seawater without need for specialized equipment, culture media, or sterile conditions. Sea urchin and human genomes share more than 7,000 genes including orthologs associated with a number of human disorders.9, 10 From the evolutionary point of view, sea urchins are more related to humans than other model organisms including fruit fly Drosophila melanogaster and nematode Caenorhabditis elegans.9 A number of fundamental aspects of embryogenesis, regulatory pathways and signalling macromolecules, which play key roles in all animal and human cells, were first discovered in the sea urchin embryos.2–4 As a result, these represent a robust and versatile model organism for developmental biology and biomedical research.10–13 Furthermore, experimental settings using the sea urchin embryos are ethical since the artificial spawning does not result in animal death, embryos develop outside the female organism, and both post spawned adult sea urchins and the excess of intact embryos can be released back to the sea, their natural habitat. Several research teams have been using sea urchin embryos to identify small molecules that modulate cell division.14–20 Following both literature evidence as well as our internal experience with the organism, we applied the sea urchin embryos to identification of antimitotics with microtubule destabilizing mode of action. This decision was based on the unique developmental features of sea urchin embryos that rely on microtubule dynamics, namely, two 3 ACS Paragon Plus Environment


Page 4 of 57

distinct phases of embryogenesis that can be easily observed in situ. These phases include a) microtubule-dependent blastomere divisions during cleavage stage that occur every 35–40 min (Figure 1, A) and b) active swimming of hatched embryos that is mediated by the coordinated beating of numerous cilia comprised of microtubules. We reasoned that an antitubulin agent would affect both cell division and embryo motility thus making the sea urchin embryo to be a convenient phenotypic model. Notably, in our hands treatment of the embryos with diverse microtubule destabilizing agents including colchicine, nocodazole, podophyllotoxin, combretastatins, vinblastin, and dolastatin 15 selectively affect cleavage and alter swimming behavior. While intact embryos move rapidly at the surface of the seawater with animal pole forward, slowly rotating around animal-vegetal axis, microtubule destabilizers cause the organism to stop swimming forward, settle on the bottom of the vessel, and spin.21 Intriguingly, while a microtubule stabilizer paclitaxel (Figure 1, G) along with several non-tubulin acting antimitotics do alter cellular division, these agents fail to induce spinning of the hatched embryos.21 In addition, eggs arrested by microtubule destabilizers acquire specific tuberculate shape (Figure 1, E), which could also be considered as an indirect indication of their antitubulin effect. Combination of these phenotypic features resulted in development of simple, reliable, and efficient in vivo phenotypic assay that identifies microtubule-specific antimitotics.21 Importantly, the sea urchin embryo assay combines both phenotypic and target-based screening as it shows test molecule effect on the dividing cells of the developing organism (Figure 1) and provides evidence for the microtubule destabilizing mode of action. The protocol was further validated using conventional purified tubulin polymerization and cell-based assays22–24 to show good correlation of data for the reported actives. It should be noted that the sea urchin embryo system also allows for the identification of compounds that display non-tubulin antiproliferative activity, selective effects on distinct morphogenetic events, and systemic toxicity. Next, we established the optimized rearing conditions for the assay. Specifically, the best concentration of the sea urchin early embryos was found to be in the 400–2500 eggs (embryos)/mL interval to provide an even monolayer suitable for reproducible and tractable screening. Increasing embryo count could result in nonselective absorbtion of the tested molecule by the embryo. Moreover, 6-well plates offered favorable topology/volume (5 mL). Large diameter and small well depth were of particular importance to secure normal development of the sea urchin embryos, sufficient aeration and monolayer in particular. In contrast, test runs using 24-well plates and 1 mL well volume were unsuccessful primarily due to the observed embryo malformations. It was a combination of both specific culture conditions and lack of an automation to track specific changes in the embryo’s motility (spinning instead of forward swimming) that limited our ability to improve the assay throughput. Despite of these 4 ACS Paragon Plus Environment

Page 5 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


challenges, the sea urchin embryo model offers considerable advantages in the phenotypic screening. These include: i) facile and rapid access to a large number of gametes and eggs immediately suitable for screening; ii) efficient incubation with no need for chemicals and/or specific media, sterility or specialized equipment; iii) feasible monitoring of the phenotypic effects that could be accomplished via a conventional low magnification light microscope; iv) sound reproducibility.

Figure 1. Typical effects of tubulin/microtubule targeting compounds on the sea urchin Paracentrotus lividus eggs and embryos. (A–C) Control. (A) Intact egg with normal bipolar mitotic spindle (light spots), the first cleavage anaphase. (B) Eight-cell embryo. (C) Early blastula. (D–F) Effects of the microtubule destabilizer combretastatin A-4 at 20 nM (D and E) and 5 nM (F). (D) Arrested egg without mitotic apparatus. (E) Tuberculate arrested eggs. (F) Cleavage alteration. (G) Aberrant multipolar mitotic apparatus in arrested egg in the presence of the microtubule stabilizer paclitaxel (5 M). Compounds were added to zygotes at 8–15 min postfertilization. Samples were incubated at 21 C. Eggs/embryos were observed at 1 h (A), 2.5 h (B, D, E, G), and 6 h (C, F) postfertilization. The average egg/embryo diameter is 115 m.



Page 6 of 57

Considering potential for inadequate compound solubility that often hampers screening procedures and could resulted in inconsistent data, we streamlined sample preparation by diluting the initial 10 mM DMSO compound solutions by 10 fold with 96% EtOH. In our hands, this step dramatically enhanced solubility of the test molecules in seawater, as evidenced by the microscopy examination of samples. For the sea urchin embryos, maximal tolerated concentrations of solvents and surfactants commonly used in medicinal chemistry were determined as well (Table 1) (Semenova M.N., unpublished). Table 1. Toxicity of Selected Solvents and Surfactants on Sea Urchin Paracentrotus lividus Embryos (% Concentration) effects developmental

systemic toxicity/

no effect

delay/alteration

mortality

Ethanol

0.5

1

1.5

Dimethylsulfoxide

0.2

0.5

>1

Dimethylformamide

0.2

0.5

1

Dimethylacetamide

0.02

0.04–0.5

1

2-Pyrrolidone

0.1

0.2–0.5

1

N-Methyl-pyrrolidone

0.02

0.05–0.2

0.5-1

Cremophor EL

0.002

0.005–0.02

>0.02

Sorbitan monolaurate

0.002

0.005-0.01

0.013

PEG 300

0.2

NAa

0.5-1

PEG 6000

0.12

0.3

0.6

solvent/surfactant

a NA:

not available. Isosteric replacement of the labile cis-double bond in a highly potent natural antimitotic

combretastatin A-4 (CA4, Figure 2) is a feasible strategy to access novel tubulin modulators with enhanced physiochemical properties.25–29 The ethene bridge furnishes critical cis-configuration of the biaryl template necessary for the optimized interaction of a molecule with the colchicine binding site of tubulin.30, 31 Five-membered N-containing heterocycles were reported to provide a non-isomerizable and metabolically stable isosteric replacement for the cis-styrene.25–29 Several research groups including our team designed expeditious synthetic routes to respective fivemembered N-heterocycles termed combretazoles that recapitulate cis-biaryl topology for rings A and B of the parent CA4 and display activity both in vitro and in vivo.32–55 However, the direct comparison of compounds activities and/or general structure-activity relationship (SAR) across 6 ACS Paragon Plus Environment

Page 7 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


multiple classes of comretazoles are challenging primarily due to the formidable controversies associated with their cytotoxicity and in vitro inhibition of purified tubulin polymerization, as reported by multiple authors. For example, published ITP50 (concentration causing 50% inhibition of purified tubulin polymerization) values for the parent CA4 range between 0.175,56 6.0,57 and 12.7 M.58 There are also considerable discrepancies in the reported GI50 values for human cancer cell lines (Table 2), which are likely the result of different experimental protocols, improper identification and/or contamination of cancer cell lines.59

Table 2. Human Cancer Cell Growth Inhibition by CA4 GI50, nM NCI cancer cell linea

ref. 60

ref. 42

ref. 39

10

46

ref. 61

screen data

K-562

1–2.5

HCT-116

1–3.2

HT29

100–1600

PC-3

1

MCF7

5

a K562:

ref. 35

350 270

4200 5400

220

1000

myelogenous leukemia; HCT-116: colon carcinoma; HT29: colon adenocarcinoma; PC-

3: prostate adenocarcinoma; MCF7: breast adenocarcinoma. Moreover, inconsistent data persistent in the literature on the biological activity of orthobiaryl substituted pyrazoles, isoxazoles and triazoles also may have resulted from the inaccurate determination of chemical structure, insufficient purity55, 62 or solubility of the tested molecules. In our hands some azole derivatives of CA4 precipitated in the assay medium (seawater) upon dilution of respective DMSO stocks to afford fine crystals visible under a microscope. Considering these discrepancies, we decided to conduct a systematic head-to-head structureactivity relationship (SAR) studies of the reported and novel active pyrazole, isoxazole, 1,2,3triazole-, and pyrrole-based analogues of CA4 and combretastatin A-2 (CA2) (Figure 2) using in vivo phenotypic sea urchin embryo assay and in vitro cancer cell line cytotoxicity data. As a part of the project, we synthesized CA4 and CA2 analogues that exhibit relevant substitution pattern in the rings A and B as well as their derivatives with amino group in the N-heterocycle.



Bridge

Page 8 of 57

O N

HN N

R3

R3

RO

R1

A RO OCH3

R1

CA2: R = -CH2CA4: R = CH3

R3 R1

R2

B OH OCH3

N O

R2

R2

Pyrazoles 1: R3 = H

4,5-Diaryl-isoxazoles 3: R3 = H

3,4-Diaryl-isoxazoles 5: R3 = H

2: R3 = NH2

4: R3 = NH2

6: R3 = NH2 NH

N N

N N NH

N

R3

R1

R1

R2

R2

R2 4,5-Diaryl-1,2,3-triazoles 7

R1

1,5-Diaryl-1,2,3-triazoles 8: R3 = H 9: R3 = NH2

Pyrroles 10

Figure 2. Structures of combretastatins A-4 and A-2 and their cis-restricted azole analogues. RESULTS AND DISCUSSION Chemistry. While the respective direct analogues of CA4 including combretapyrazoles 1a, 1c53 and combretaisoxazoles 3a, 3b,33, 34 (Schemes 1 and 2) have been described in the literature, their synthesis was rather convoluted. In order to streamline the approach to relevant combretapyrazoles 1a and 1b we introduced a robust synthetic protocol that included iodosodiacetate-mediated rearrangement of easily accessible chalcones 13a,b into ketals 14a,b followed by their in situ cyclization to furnish the targeted pyrazoles (Scheme 1). Scheme 1. Synthesis of 4,5-Diarylpyrazoles 1 and 4,5-Diarylisoxazoles 3a


Page 9 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


O OH O

O

O R

R

a

OCH3

R

OH

Isovanillin H3CO

H3CO

R

R

H3CO

b

OCH3 R

12a,b

11a,b

13a,b 56-61% c OCH3

O R

R H3CO

R

d

OH

H3CO

O

H3CO

OCH3

OCH3 R

14a,b in situ 70-80% e

O

O

13-Ac a,b 72-81%

f O N

HN N H3CO

R H3CO R

1a: 72% 1b: 63% aReagents

OH OCH3

H3CO H3CO

3a 55%

OH

a: R = OCH3 b: R = H

OCH3

and conditions: (a) ref. 63; (b) NaOH–EtOH, rt, 24 h; (c) AcCl–Pyridine, CH2Cl2, rt, 3

h; (d) PhI(OAc)2, MeOH–H+, rt, 8 h; (e) EtOH–H+, N2H42HCl, reflux, 1 h; (f) EtOH–H+, NH2OHHCl, reflux, 2 h. The corresponding combretapyrazole isomer 1c and respective analogues were obtained via similar approach from guaiacol and respective benzaldehydes. Conveniently, the protective acetate functionalities were successfully removed during the isomerization of 13 (Scheme 2). Scheme 2. Synthesis of 4,5-Diarylpyrazoles 1 and 4,5-Diarylisoxazoles 3a



Page 10 of 57

R1

O

R2 O

O OH

R3

O

a

R4

OCH3

R3 R4

12

Guaiacol

OH

R2

b

OCH3

OCH3

O

R1

11

13 c

R2 R3

R1

O

R1 OH

R4 H3CO

d

OCH3

OCH3

R1 R2

O

R2

O

R3

OCH3 R4

14 in situ 84% e

O

13-Ac 88% f

N NH

R1 R2

N O

R3

R3 R4

R4

OH

OH OCH3

OCH3

1c: R1 = H, R2 = R3 = R4 = OCH3 (12%)

3b: R1 = H, R2 = R3 = R4 = OCH3 (26%)

1d: R1 = R2 = R4 = H, R3 = OCH3 (48%)

3c: R1 = H, R4 = OCH3, R2, R3 = -OCH2O- (18%) 3d: R1 = R4 = OCH3, R2, R3= -OCH2O- (23%) 3e: R1 = R2 = R4 = H, R3 = OCH3 (52%)

1e: R1 = R4= OCH3, R2, R3 = -OCH2O- (18%)

aReagents

and conditions: (a) ref. 64; (b) NaOH–EtOH, rt, 24 h; (c) AcCl–Pyridine, CH2Cl2, rt, 3

h; (d) PhI(OAc)2, MeOH–H+, rt, 8 h; (e) EtOH–H+, N2H42HCl, reflux, 1 h; (f) EtOH–H+, NH2OHHCl, reflux, 2 h. Combretaisoxazole-based isomers 3a and 3b were synthesized following the same synthetic route (Schemes 1 and 2). In general, the absence of –OH functionalities in the Ph-ring facilitated access to the intermediate chalcones 13 (Scheme 3) that rearranged in situ to afford ketoacetals 14 (path A) or an epoxy-intermediate 15 to yield ketoaldehydes 16 (path B). Importantly, in many instances neither 14 nor 16 were isolated. Instead, the reaction mixture was treated with NH2NH2 or NH2OH to result in pyrazoles 1 or isoxazoles 3. Scheme 3. Synthesis of 4,5-Diarylpyrazoles 1a 10 ACS Paragon Plus Environment

Page 11 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


R1

O

O

R1

R2

R5

+

R3

a

R6

R4

R7

11

12

Path B

O

R2

R5

R3

R6 R4

e

O

R1

R5

R2

R6

R3

R7

R7

R4

15 65-89%

13 62-89%

Path A

O

f

b R2 R1

R3

R1

O R5

R4 H3CO

OCH3

c

N O

R2

R1

g

R3

R6

14 67% d

R1

N NH

R2

h R3 R4 R 7

NH2

1f,g,j-n

R5

R2

R3

R4

R5

R6

1f:

H

NO2

OCH3

H

OCH3

OCH3

1g:

H

OCH3

OCH3

NO2

OCH3

1j:

H

OCH2C6H5 OCH3

H

1k:

H

OCH2C6H5 OCH3

H

1l:

H

OCH3

OCH3

OCH3

OCH3 OCH3 H

N NH

H3CO H3CO

1i Path A, B

R6

R1

OCH3

OCH3

-OCH2CH2O-

R7

Yield

OCH3 62% H

23%

AB

OCH3

52%

A

H

62%

A

H

17%

B

H

OCH3

H

46%

A

OCH3

H

OCH3

H

61%

B

OCH3 OCH3

H

56%

A

3g: OCH3 3h: OCH3

-OCH2O-

OCH3

H

H

54%

A

-OCH2O-

OCH3

H

H

75%

A

OCH3 H

3i:

H

-NHCOCH2O-

OCH3

H

OCH3

H

70%

A

3j:

H

OCH3

OCH3

-OCH2CH2O-

H

18%

B

aReagents

OCH3

A

OCH3 OCH3

NH2

Path

1m: OCH3 -OCH2O1n: H -NHCOCH2O3f: OCH3 -OCH2O-

OCH3

R7

H3CO

h

OCH3 1h Path A

16

R6

d

HN N

H3CO H3CO

O

R4

R5 R6

3h-j

H3CO

R5

R3

R4 R 7

R7

O

R2

and conditions: (a) NaOH–EtOH, rt, 24 h; (b) PhI(OAc)2, MeOH–H+, rt, 8 h; (c)

EtOH–H+, NH2OHHCl, reflux, 2 h; (d) EtOH–H+, N2H42HCl, reflux, 1 h; (e) 30% H2O2,



Page 12 of 57

NaOH–EtOH, rt, 48 h; (f) Et2OBF3, CH2Cl2, 0–20 C, 3h; (g) EtOH–H+, reflux, 2 h; (h) NiCl26H2O–NaBH4, MeOH–THF, rt, 12 h. Additional analogues of 1 and 3 that did not feature unprotected hydroxy moiety in the benzene ring were prepared via a high-yield formylation of diarylethanones 22 followed by condensation of the resulting intermediate 23 with NH2NH2 or NH2OH (Scheme 4). Of note, starting compounds 18 and 20 were efficiently prepared from the same benzaldehydes 11 (60– 90% yields). Although the described procedure included more stages, it allowed for both scale up and improved total yields of the targeted compounds due to the isolation and purification of intermediates 22 and 23. Scheme 4. Synthesis of 4,5-Diarylpyrazoles 1 and 4,5-Diarylisoxazoles 3a R1

O

O

R2(R5)

R2

a

R3(R6)

O R2

b

OH

R3

R3

R4(R7)

R4

R4

11 R1 = H

17

18

R5

HO

Cl

R5

Cl

d

R6

e

R6 R7

R7

20

21

R4

R5

R6

R7

Yield

H

OCH3

H

75%

1o: OCH3

OCH3

1p:

OCH3

H

OCH3

Br

H

OCH3

H

79%

-OCH2O-

H

OCH3

H

81%

H

OCH3

H

46%

H

1q: OCH3 OCH3

1s: OCH3

OCH3

-OCH2CH2O-

OCH3

OCH3

H

OCH3

H

OCH3

-OCH2O-

1u:

H

OCH3

H

OCH3

H

3k: OCH3

OCH3

3l:

OCH3

H

OCH3

OCH3

3m: OCH3

OCH3

H

OCH3 OCH3

R7

R4

22 60-90% g O

R5

R2 R3

R6

N

R7

R4

23 100% h

76%

i

OCH3 66% H

87%

OCH3 69%

Br

H

OCH3

H

64%

3n: OCH3

-OCH2O-

H

OCH3

H

74%

3o: OCH3

-OCH2CH2O-

H

OCH3

H

3p:

H

OCH3

H

OCH3

-OCH2O-

3q:

H

OCH3

H

OCH3

H

aReagents

R6

R3

OCH3 70%

1t:

H

f

R6

R7

R3

R5

R2

R5

Zn

19 R2

O

+

c

1r:

Cl

72% 81%

O N

HN N R2

R2

R3

R3 R4 R 7

1o-u

R5 R6

R4 R 7

3k-q

R5 R6

OCH3 78%

and conditions: (a) CO(NH2)2H2O2, CH3OH, reflux, 1.5 h;65 (b) SOCl2, CH2Cl2, drops

DMF, rt, overnight; (c) NaBH4, MeOH, rt, 6–7 h;48 (d) SOCl2, C6H6, 45–50 C, 40 min;48 (e) Mg, ZnCl2, LiCl, THF, rt, 2h;66 (f) CuCN2LiCl, THF, -20 C, 1 h, then rt overnight;66 (g) 12 ACS Paragon Plus Environment

Page 13 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


DMFDMA, without solvent, 60 C, 3h; (h) MeOH–H+, N2H42HCl, reflux, 1 h; (i) MeOH–H+, NH2OHHCl, reflux, 2 h. The same chemical route (Scheme 4) was reported in the literature to afford both polymethoxy-4,5-diarylisoxazoles 3 and their 3,4-diarylsubstituted analogues 5 (Figure 2),67 however in our assessment the respective structures were not assigned properly.68 Considerably less accessible 3,4-diaryl-5-unsubstituted isoxazoles 5a–c were selectively synthesized from nitrostilbenes 25 and carbethoxypyridinium bromide.68 3-Amino-4,5-diaryl- and isomeric 5amino-3,4-diarylisoxazoles 4 and 6 were accessed via the synthetic procedure published by our team earlier.48, 51 Isoxazoles modified with the ortho-OH group (3r and 3s) were commercially available from Chemical Block Ltd. Their structural integrity was confirmed prior to biological testing. To expand on a series of combretazoles, we prepared several reported combreta-1,2,3triazoles 7a–c.50 A more expeditious route to these molecules involving treatment of cyanocombretastatins with NaN3 was published recently.52 The corresponding N-substituted isomers 8a–c and 9a,b were synthesized as described earler.50 Compound structures are presented in Table 5. Finally, several combreta-3,4-diarylpyrroles 10a–e (Table 6) featuring substituents found in the respective active analogues of pyrazole-, isoxazole- and 1,2,3-triazoles were synthesized under conditions of the Barton-Zard reaction. Hydrolysis of the pyrrole-2carboxylates 25 followed by decarboxylation of the resulting pyrrole-2-carboxylic acids 26 afforded target 3,4-diarylpyrroles 10a–e (Scheme 5). Scheme 5. Synthesis of 3,4-Diarylpyrroles 10a–ea



EtO 2C

-

O

R1

R1

+

R2

N

O

+

EtO 2CCH2 N

R3

C

HOOC

-

R1

NH

R2

b

R3

a

R4

24

Page 14 of 57

R3 R4

R5

NH

R2

25

R4

26

R5

R5

c R1

R2

R3

R4

10a:

H

OCH3

OCH3

OCH3

OCH3

73%

10b:

H

OCH3

H

OCH3

OCH3

46%

-OCH2O-

OCH3

OCH3

60%

10c: OCH3 10d:

H

10e: OCH3 aReagents

OCH3

OCH3

-OCH2O-

R5

Yield

OCH3

H

69%

OCH3

H

60%

H N

R1 R2 R3 R4

10a-e

R5

and conditions: (a) K2CO3, EtOH, rt, 12–36 h; (b) (i) NaOH, EtOH–H2O, reflux; 3–5

h; (ii) 18% HCl; (c) heating at mp, 1–2 min. Biological Evaluation: SAR Studies The phenotypic assessment of pyrazole, isoxazole, 1,2,3-triazole, and pyrrole derivatives is summarized in Tables 3–6. Compounds CA4 and CA2 served as positive controls. In the sea urchin embryo assay, diarylpyrazoles 1a, 1c, 1e, 1g–j, 1l, 1o–u, and 2a, diarylisoxazoles 3a–e, 3j–r, 4a–c, 5a, and 6, 4,5-diaryl-1,2,3-triazoles 7a and 7b, 1,5-diaryl-1,2,3-triazoles 8a and 8b, and 3,4-diarylpyrroles 10a and 10b were determined to be potent antimitotic microtubule destabilizing agents exhibiting profound cleavage alteration/arrest and embryo spinning. Multiple derivatives including 1g–i, 1l, 1o, 1q–s, 1u, 3a–c, 3e, 3k, 3l, 3n, 3o, 6, and 7a showed activity comparable to the parent CA4 and CA2. The most potent pyrazole 1p and triazole 7b displayed higher antimitotic activity than that of combretastatins. Although compounds 1d, 1f, 1m, 1n, 2b, 3i and 5b failed to induce embryo spinning, they were still classified as weak microtubule destabilizing agents since the arrested post-treatment eggs aquired tuberculate shape. Molecules 1b, 2c–e, 3g, 3h, 5c, 7c, 8c, and 9a showed antimitotic effects likely mediated by non-tubulin mechanisms, whereas 1k, 9b, and 10c exhibited marginal antiproliferative activity. Compounds 2f, 3f, 3s, 4d, 10d, and 10e failed to produce any activity up to 4 M concentration.


Page 15 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 3. Effect of Diarylpyrazoles 1 and 2 on Sea Urchin Embryos and Human Cancer Cells sea urchin embryo effects, EC (M)b

NCI60 screen

cleavage

cleavage

embryo

mean GI50,

alteration

arrest

spinning

Mc

CA4

0.002

0.01

0.5

0.0032e

CA2

0.002

0.01

0.5

2.66f

0.01

0.02

0.5

>5g

0.1

2

>10

0.05

0.2

2

0.1

2 TEi

>10

NDh

0.05

0.2

4

NDh

0.05

0.5 TEi

>10

NDh

0.005

0.05

2

NDh

compda

structure

mean GI, %d

HN N H3CO

1aa

H3CO H3CO

OH OCH3 HN N

1b

H3CO

NDh

OH OCH3

H3CO

1ca

H3CO H3CO

N NH

0.007g

OH OCH3

N NH

1d

H3CO OH OCH3

H3CO O

1e

N NH

O H3CO

OH OCH3 HN N

H3CO

1fa

H3CO H3CO

H3CO

1g

H3CO H3CO

NO2 OCH3 N NH

NO2 OCH3



Page 16 of 57

HN N H3CO

1ha

H3CO H3CO

0.005

0.025

0.5

0.020

77.1

0.001

0.005

0.1

0.5

2

1

4

>4

>4

NDh

0.005

0.02

0.5

NDh

0.1

1 TEi

>5

NDh

0.2

1 TEi

>5

NDh

0.002

0.05

0.5

0.033

85.0

0.00025

0.005

0.05

0.012

92.2

0.005

0.02

0.1

NH2 OCH3 N NH

H3CO

1i

H3CO H3CO

NDh

NH2 OCH3

HN N H3CO

1j

H3CO H3CO

NDh

73.0

O OCH3 HN N

1k

H3CO O OCH3

N NH

H3CO

1l

H3CO H3CO

O O

H3CO O

1m

N NH

O H3CO OCH3 O

1n

N NH

H N O H3CO

OCH3

HN N H3CO

1o

H3CO H3CO OCH3 N NH

H3CO

1p

H3CO H3CO OCH3

HN N H3CO

1q

H3CO

NDh

Br OCH3


Page 17 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


HN N H3CO

1r

O

0.005

0.05

0.5

0.309

87.2

0.005

0.05

0.5

0.182

77.0

0.01

0.2

2

NDh

0.002

0.1

0.2

NDh

0.01

0.05

1

0.046

89.2

0.1

1 TEi

>10

0.302

75.8

0.2

>4

>10

4.365

54.0

0.5

>4

>5

NDh

18.1

1

>4

>5

NDh

32.0

O OCH3 HN N H3CO

1s

O O OCH3 N NH

H3CO

1t

O O OCH3

N NH

H3CO

1u H3CO OCH3 H2N

N NH

H3CO

2aj

H3CO H3CO OCH3

HN N H3CO

2bj

NH2

H3CO H3CO OCH3

H2N

N NH

H3CO

2cj

O O OCH3 HN N H3CO

2dj

NH2

O O OCH3

HN N H3CO

2ej

NH2

O O OCH3



H2N H3CO O

2fj

Page 18 of 57

N NH

>4

O

>4

NDh

>4

H3CO OCH3

a Previously

published compounds: 1a and 1c,53 1f and 1h,69 2a–f.48

b Hereinafter,

the sea urchin embryo assay was conducted as described previously.21 Fertilized

eggs and hatched blastulae were exposed to 2-fold decreasing concentrations of compounds. Duplicate measurements showed no differences in effective threshold concentration (EC) values. c Hereinafter,

GI50: concentration required for 50% cell growth inhibition.

d Hereinafter,

GI %: single dose inhibition of cell growth at 10 M concentration.

e Data

from ref. 70.

f Mean g

value for 7 human cancer cell lines.71

GI50 for HUVEC.53

h Hereinafter,

ND: not determined.

i

Hereinafter, TE: tuberculate eggs typical of microtubule destabilizing agents.

j

Synthesis, EC values and NCI60 screen data from ref. 48.

Table 4. Effect of Diarylisoxazoles 3–6 on Sea Urchin Embryos and Human Cancer Cells sea urchin embryo effects, EC (M) compda

structure

cleavage

cleavage

embryo

alteration

arrest

spinning

NCI60 screen mean GI50, M

mean GI, %

0.0009 HeLab O N

0.0014 HepG2b

H3CO

3aa

H3CO H3CO

0.001

0.005

0.02

OH OCH3

0.0005 OVCAR-3b 3.0 HT29c 8.5 SVECc

N O

H3CO

3ba

H3CO H3CO

0.002

0.005

0.05

OH OCH3

0.0339 HepG2b 0.0148 OVCAR-3b

N O

H3CO

3c

0.0294 HeLab

0.002

O O

0.02

0.05

ND

OH OCH3


Page 19 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


H3CO O

3d

N O

O H3CO

0.05

0.2

4

ND

0.002

0.01

0.5

ND

>4

>4

>4

ND

1

>4

>5

ND

4

>4

>4

ND

0.2

4 TE

>5

ND

0.02

0.1

2

ND

OH OCH3 N O

3e

H3CO OH OCH3

H3CO O

3f

N O

O H3CO

OCH3 OCH3

H3CO O

3g

N O

O H3CO OCH3 N O

H3CO O

3h

O H3CO

O

3i

N O

H N O H3CO

OCH3

H3CO

3j

N O

H3CO H3CO

O O

4

>4

>4

ND

4.3

0.004

0.02

0.2

0.5

4

5

Br OCH3

O N H3CO

3n

O O OCH3 O N H3CO

3o

O O OCH3 N O

H3CO

3p

O O OCH3

N O

H3CO

3q

ND

H3CO OCH3 N O

3rd

OH

H3CO

OCH3

N O

H3CO

3sd

OH

H3CO OH H2N H3CO

4ae

N O

H3CO H3CO

ND

OCH3

H2N H3CO

4be

N O

O

2.951

72.7

O OCH3


Page 21 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


O N H3CO

4ce

NH2

O O

0.005

0.02

0.5

ND

>4

>4

>4

ND

2.7

0.01

0.1

1

0.166

75.9

0.2

2 TE

>10

ND

2

>4

>4

ND

0.002

0.01

0.1

ND

OCH3

H3CO O

4de

H2N

N O

O H3CO OCH3 O N

H3CO

5af

H3CO H3CO OCH3 O N

H3CO

5bf H3CO OCH3

O N

H3CO O

5cf

O H3CO H2N H3CO

6e

O N

H3CO H3CO OCH3

a Previously b Data

published compounds: 3a,33, 34 3b, 3k and 3l,34 4a–d and 6,51 5a–c.55

from ref. 34; HeLa: human cervical carcinoma, HepG2: human liver carcinoma, OVCAR-

3: human ovarian carcinoma. c Data

from ref. 33; HT29: human colon adenocarcinoma, SVEC: primary mouse seminal vesicle

epithelial cells. d Purchased

from Chemical Block Ltd.

e Synthesis,

EC values and NCI60 screen data from ref. 51.

f

Synthesis, EC values and NCI60 screen data from ref. 55.



Page 22 of 57

Table 5. Effect of 1,2,3-Triazoles 7–9 on Sea Urchin Embryos and Human Cancer Cells50 sea urchin embryo effects, EC (M)

compd

structure

cleavage cleavage

embryo

mean GI50,

alteration

arrest

spinning

M

mean GI, %

0.005

0.02

0.1

0.013

83.5

0.0005

0.002

0.05

0.034

92.7

0.5

>4

>5

0.813

75.1

0.02

0.2

1

0.01

0.1

0.5

1

>4

>4

ND

0.1

>4

>5

ND

4

>4

>4

HN N N

H3CO

7a

NCI60 screen

H3CO H3CO OCH3 HN N N

H3CO

7b

O O OCH3

H3CO O

7c

HN N N

O H3CO OCH3 N N N

H3CO

8a

H3CO H3CO

0.027 K562a

OCH3 N N N

H3CO

8b

O

0.178

83.8

O OCH3 H3CO O

8c

N N N

O H3CO OCH3

N N N

H3CO

9a

NH2

H3CO H3CO OCH3 H2N H3CO

9b

N N N

H3CO H3CO

ND

9.9

OCH3

a Data

from ref. 37; K562: human myelogenous leukemia. 22 ACS Paragon Plus Environment

Page 23 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 6. Effect of Diarylpyrroles 10 on Sea Urchin Embryos sea urchin embryo effects, EC (M) compd

structure

cleavage

cleavage

embryo

alteration

arrest

spinning

0.05

0.2

4

0.5

1

5

4

>4

>4

>4

>4

>4

>4

>4

>4

NH H3CO

10a

H3CO H3CO OCH3

NH H3CO

10b H3CO OCH3 H3CO O

10c

NH

O H3CO OCH3

NH H3CO

10d

H3CO H3CO

H3CO O

10e

NH

O H3CO

In the series of pyrazoles featuring the 2,3,4-trimethoxyphenyl ring A next to the N atom of pyrazole linker antitubulin activity decreased in the following order: 1o (4-methoxyphenyl ring B) > 1h (3-amino-4-methoxyphenyl ring B) > 1a (3-hydroxy-4-methoxyphenyl ring B) > 1f (3-nitro-4-methoxyphenyl ring B) > 1j (3-O-benzyl-4-methoxyphenyl ring B). Compounds with trisubstituted rings A (1o, 1q, 1r, and 1s) displayed similar or equal effect, suggesting that the presence of 3,4,5-trimethoxyphenyl ring A was not critical to the overall antimitotic effect. In contrast, pyrazoles exhibiting 3-hydroxy-4-methoxyphenyl ring B next to the N atom (ex., 1c and 1e) and tri- or tetrasubstituted ring A, exhibited more potent antitubulin effect as compared to monosubstituted derivative 1d. According to the literature, the replacement of 3-hydroxy group in the ring B of CA4 and CA2 with 3-amino substituent afforded molecules with antiproliferative activity higher than the parent CA2.71-73 In the sea urchin embryo assay, pyrazole analogues substituted with 3-amino group in the ring B (1h, EC = 5 nM and 1i, EC=1 nM) exhibited activity similar to that of the 23 ACS Paragon Plus Environment


Page 24 of 57

parent CA4 (EC = 2 nM). Furthermore, these compounds were more potent than 3hydroxyphenyl derivatives 1a (EC = 10 nM) and 1c (EC = 50 nM), but less active than the respective mono-methoxysubstituted pyrazoles 1o (EC = 2 nM) and 1p (EC = 0.25 nM). 3Nitrophenyl derivatives 1f (EC = 50 nM) and 1g (EC = 5 nM) were less potent.than their 3aminophenyl analogues 1h and 1i. Moreover, the removal of 3-hydroxy group from the ring B of 1a and 1c furnished more potent compounds 1o, 1p. Notably, pyrazole 1l with disubstituted ring B was more active than 1c. The data further that the antimitotic activity of pyrazoles was not influenced by the 3-OH group in the ring B similar to the data reported in the literature.26, 27, 30 However, for less active compounds containing tetrasubstituted ring A, namely 1e and 1m, lack of 3-hydroxy group afforded less active molecule, 1e > 1m. Following these findings, pyrazole derivatives substituted with 4-methoxyphenyl group (the B ring) were studied in more detail. Compound 1p containing 3,4,5-trimethoxyphenyl ring A showed the best potency with the EC value of 0.25 nM. The antimitotic antitubulin effect of compounds with different substitution pattern in the A ring decreased in the following order: 1p > 1u (3,5dimethoxyphenyl ring A > 1t (3-methoxy-4,5-methylenedioxyphenyl ring A) > 1m (2,5dimethoxy-3,4-methylenedioxyphenyl ring A)  1b (3-hydroxy-4-methoxyphenyl ring A) > 1n (1,4-benzoxazinone ring A) > 1k (3-O-benzyl- 4-methoxyphenyl ring A). Comparison of activity for diarylpyrazole pairs featuring interchanged rings A and B (1a vs 1c, 1b vs 1d, 1f vs 1g, 1h vs 1i, 1o vs 1p, and 1r vs 1t, respectively) did not reveal any obvious trends: 1a > 1c (3,4,5trimethoxyphenyl and 3-hydroxy-4-methoxyphenyl rings); 1b = 1d (4-methoxyphenyl and 3hydroxy-4-methoxyphenyl rings); 1f 3n (3-methoxy-4,5-methylenedioxyphenyl ring A) > 3m (3,4-dimethoxy-5-Br ring A). Notably, compounds containing the 3,4,5trimethoxysubstituted ring A (3k, 3n, and 3o) showed consistently high activity with the EC values of 2–5 nM for cleavage alteration. A replacement of 5-methoxy group of the ring A with Br (3m) yielded a 10-fold reduction in potency. On the contrary, for isoxazoles featuring 3hydroxy-4-methoxyphenyl ring B next to the O atom as exemplified by 3b (3,4,5trimethoxyphenyl ring A), 3c (3-methoxy-4,5-methylenedioxyphenyl ring A) and 3e (4methoxyphenyl ring A) the EC values were similar (ca. 2–5 nM), however 3d (2,5-dimethoxy3,4-methylenedioxyphenyl ring A) was less active. We concluded that 2,3,4-tri- and 4monosubstituted ring A pharmacophores were well tolerated in these series, whereas tetrasubstituted aromatic ring yielded reduced antimitotic activity. For 4,5-diarylisoxazoles with 4-methoxyphenyl pharmacophore for the ring B the antimitotic activity decreased in the following order: 3l (3,4,5-trimethoxyphenyl ring A) > 3p (3methoxy-4,5-methylenedioxyphenyl ring A) > 3q (3,5-dimethoxyphenyl ring A) > 3i (1,4benzoxazinone ring A) > 3g (2,5-dimethoxy-3,4-methylenedioxyphenyl ring A). In our hands, the derivative 3l with 3,4,5-trisubstituted ring A displayed the highest effect. Changing positions of rings A and B in 4,5-diarylisoxazoles suggested that for the best antimitotic activity, 3,4,5-trimethoxyphenyl or relevant group should be connected to position 5 of the isoxazole ring. Several representative examples included 3a > 3b (3,4,5-trimethoxyphenyl and 3-hydroxy-4-methoxyphenyl rings), 3k > 3l (3,4,5-trimethoxyphenyl and 4-methoxyphenyl rings), 3n > 3p (3-methoxy-4,5-methylenedioxyhenyl and 4-methoxyphenyl rings). Shuffling N and O atoms in the isoxazole ring consistently led to a less active compound (3l vs 5a; 3q vs 5b) indicating that 4,5-diarylisoxazoles 3 were more potent than their 3,4analogues 5. In both diarylisoxazole series 3 and 5, 3,4,5-trimethoxyphenyl substituent for the ring A furnished the best activity. Molecules with 3,5-dimethoxyphenyl ring A were markedly less potent than their respective 3,4,5-trimethoxyphenyl analogues, as shown by 3l vs 3q and 5a vs 5b. Molecules 3h and 5c with unsubstituted ring B exhibited modest antiproliferative effect in vivo. Introducing unsubstituted NH2 group into the isoxazole ring (Figure 3) resulted in a mixed outcome. For example, whereas amino-compounds of 4,5-diarylisoxazole series 4b (EC = 500 nM), 4c (EC = 5 nM), and 4d (EC > 4 M) were less potent than the respective analogues 25 ACS Paragon Plus Environment


Page 26 of 57

3p (EC = 20 nM), 3o (EC = 2 nM), and 3g (EC = 1 M), compound 4a showed higher activity than that of 3l. In contrast, 3,4-diarylisoxazole 6 (EC = 2 nM) exhibited enhanced activity compared to 5a (EC = 10 nM). It is worth noting that for diarylisoxazoles introduction of the amino group gave diverse results. In summary, in the diarylisoxazole series the most active antimitotic compounds featured 3,4,5-trisubstituted phenyl pharmacophore for the ring A connected to position 5 of the isoxazole ring. 3-Hydroxy-4-methoxyphenyl group for the ring B was essential. 4,5-Diarylisoxazoles were more potent than their 3,4-analogues. Introduction of 3-amino group into 4,5-diarylisoxazoles tended to reduce the activity whereas similar modification of the respective 3,4-diarylisoxazoles improved antimitotic effect (6 vs 5a). R H3CO

N O

H3CO H3CO

R H3CO

R H3CO O

N O

O

O OCH3 3p: R = H 4b: R = NH2

R

O N H3CO

R

O

OCH3 3g: R = H 4d: R = NH2

O N

H3CO H3CO H3CO

O

H3CO

O OCH3

3l: R = H 4a: R = NH2

N O

OCH3 3o: R = H 4i: R = NH2

OCH3 5a: R = H 6: R = NH2

Figure 3. Structures of aminoisoxazoles 4a-i, 6 and their unsubstituted analogues 3l, 3p, 3g, 3o, and 5a. In the diaryl-1,2,3-triazole series, the most potent molecules contained 3,4,5trimethoxyphenyl (7a and 8a) or 3-methoxy-4,5-methylenedioxyphenyl (7b and 8b ) pharmacophore as the ring A and 4-methoxyphenyl group as the ring B. Compounds with tetramethoxy substituted ring A (7c and 8c) exhibited weak antiproliferative effect. The introduction of 4-NH2 group into triazole linker of 1,5-diaryl-1,2,3-triazoles (8a vs 9a and 8b vs 9b) markedly decreased antitubulin activity. 4,5-Diaryl-1,2,3-triazoles 7a and 7b (C–C geometry) were found to be considerably more active than the respective 1,5-diaryl-1,2,3triazoles 8a and 8b (N–C geometry). We concluded that for the 1,2,3-triazole series, 3,4,5trimethoxyphenyl substituted the ring A enhanced the antitubulin effect, whereas 4-amino group in the 1,2,3-triazole ring led to decreased activity. 4,5-Diaryl (C–C geometry) substitution yielded more potent compounds. 3,4-Diarylpyrroles were less potent than the parent CA2 and CA4. Compound 10a with 3,4,5-trimethoxyphenyl ring A and 4-methoxyphenyl ring B was the most active derivative displaying strong antimitotic microtubule destabilizing effect. Similar to isoxazoles, removal of 4-methoxy substituent from the ring A resulted in less active molecule 10b. Tetrasubstituted ring A was not tolerated as compound 10c showed only marginal antiproliferative activity. 26 ACS Paragon Plus Environment

Page 27 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Diarylpyrroles with unsubstituted ring B (10d and 10e) were inactive. In summary, for diarylpyrrole series 3,4,5-trimethoxy substituted ring A and 4-methoxy group in the B ring were essential for antitubulin activity. Of combretazoles containing 3,4,5-trimethoxy substituted ring A and 3-hydroxy-4methoxy substituted ring B, isoxazoles 3 were more active than the respective pyrazoles 1 following the sequence 3a >> 1a; 3b > 1c; 3e >> 1d. Molecules exhibiting tetrasubstituted ring A (ex., isoxazole 3d and pyrazole 1e) were equally potent. In a series of CA4 analogues substituted with 3,4,5-trimethoxyphenyl pharmacophore as the ring A and 4-methoxyphenyl group as the ring B the observed anti-mitotic activity varied as follows: 1p > 3k  1o > 7a  3l > 5a > 8a > 10a (Figure 4). Het H3CO H3CO N

NH

1p

>

O

N



3k

HN

N

B

A

>

OCH3

OCH3 N

1o

N

7a

NH



N

O

3l

>

O

5a

N

>

N N N

NH

>

8a

10a

Figure 4. Comparative activity of combretazoles with 3,4,5-trimethoxyphenyl ring A and 4methoxyphenyl ring B. In general, 3,4,5-trimethoxyphenyl ring A was the optimal substituent, whereas introducing tetra-substituted functionalities (2,5-dimethoxy-3,4-methylenedioxyphenyl) significantly decreased the activity. In the respective series of combretazoles, 3,4,5trimethoxyphenyl-substituted molecules consistently displayed better antimitotic activity as summarized below: pyrazoles: 1p >> 1m; 2a >> 2f; isoxazoles: 3l >> 3q; 4a >> 4d; triazoles: 7a >> 7c; 8a >> 8c; pyrroles: 10a >> 10c. A summary of antimitotic activity for the combretazole derivatives substituted with alternative pharmacophores as the ring A and 4-methoxyphenyl group as the ring B is shown in Figure 5. As expected, both structure of the actual azole as well as nature of the ring A showed a major impact on the relative activity of respective derivatives. In most cases, pyrazoles 1 were more active than the corresponding isoxazoles 3. The same was observed for derivatives with 3,4,5-trimethoxyphenyl pharmacophore as the ring A and ethylenedioxyphenyl group as the ring B: pyrazole 1l (EC = 5 nM) was more potent than the corresponding isoxazole 3j (EC = 20 nM). 4,5(C–C)-Diaryltriazoles 7 displayed significant activity, whereas 1,5(N–C)-diaryltriazoles 8 27 ACS Paragon Plus Environment


Page 28 of 57

were less active, and pyrroles 10 showed minimal effect. Of particular note was triazole 7b endowed with 3-methoxy-4,5-methylenedioxy substituted ring A (EC = 0.5 nM). 4-Methoxy group in the ring A of pyrazoles 1, isoxazoles 3 and 5, and pyrroles 10 was important to the activity as follows: 1p > 1u; 3l > 3q; 5a > 5b; 10a > 10b. Het

R

B

A

OCH3 Het

Ring A: H3CO

N

NH

1u

H3CO H3CO

N

O

N

NH

H3CO O

N

N

>

HN

H3CO Br H N

NH

O H3CO

N

>

N

N

1n

NH

NH

7c

>

1q N

N

>

5b



O

NH

10b

N

>

N N N

3n



8b

N

NH

>

1t

N

O

3p

N

HN

1m

H3CO

N

HN

O

1s

OCH3 O

O

>

3o

H3CO

>

1r

O O

O

3q

7b

O

O

N

>

O

>

N

O



N N N

3g

8c

>

NH

10c

N

3m



N

O

3i

Figure 5. Comparative activity of combretazoles with 4-methoxyphenyl group as the ring B. In general, introduction of amino substituent into pyrazole and triazole fragment of the combretazoles and to a lesser extent into isoxazole ring of 4,5-diarylisoxazoles decreased the antimitotic antitubulin effect. However, 3,4-aminoisoxazole 6 with 3,4,5-trimethoxyphenyl ring A and 4-methoxyphenyl ring B was as active as CA4 (EC = 2 nM), and 4,5diarylaminoisoxazole 4a showed comparable effect (EC = 4 nM). Similarly, introduction of the amino group into the isoxazole ring of 4,5-diarylisoxazole 3o (EC = 2 nM) resulted in a modest 28 ACS Paragon Plus Environment

Page 29 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


change in activity (4c, EC = 5 nM). For aminoazole derivatives, isoxazoles 4 and 6 were more active than respective pyrazoles 2 and 1,5-diaryltriazoles 9 (Figure 6). NH2 Het A

B

R

NH2

OCH3

Het

Ring A: H3CO

O

H2N

H3CO H3CO H3CO

N

>

6 H2N

O

N

O

H3CO O

N

O

4c

H2N

>

4a N

NH

>

NH2

N

NH

2a N

H2N

2c

O

O

H2N

>

HN

N

NH2



2b

N N N

N

NH2 H2N

N N

>

9a

9b

O

4b

>>

HN

N

NH2

2e

Figure 6. Comparative activity of combretazoles with amino group in N-heterocycle. The sea urchin embryo data for the antitubulin compounds correlated well with the respective assays using human cancer cell lines (Tables 3–5). For example, the most potent pyrazole derivative identified in vivo (1p) showed the highest cytotoxicity in NCI60 screening. Similarly, compounds 1h, 1o, 1r, and 1s displaying good activity in the sea urchin embryos also exhibited sub-M GI50 values in the cell-based screening. Pyrazole 1h with 3-amino group in the ring B altered cleavage at 5 nM concentration in the sea urchin assay while exhibiting the IC50 value of 8.4 nM in murine adenocarcinoma colon 26 cells.69 In a similar fashion, compounds 2c, 2d, and 2e identified as weaker hits in vivo were also less cytotoxic against human cancer cells. Aminopyrazoles were generally less cytotoxic than their unsubstituted analogues (2a < 1p; 2b < 1o; 2d < 1r; 2e < 1s). In the sea urchin embryo model, pyrazole 1a with trimethoxyphenyl ring A next to the N1 atom showed enhanced activity compared to its analogue 1c (EC values of 10 and 50 nM, respectively) (Table 3). In contrast, 1a failed to inhibit HUVEC (human umbilical vein endothelial cells) growth up to 5 M concentration, although the reported HUVEC cytotoxicity 29 ACS Paragon Plus Environment


Page 30 of 57

for 1c was significant with GI50 value of 7 nM.53 It should be noted that our melting points for 1a (171–173 C) and 1c (232–234 C) did not match the reported literature data (198–200 C and 90–93 C, respectively).53 Possible reasons of the discrepancy will be discussed in details elsewhere.74 Diarylisoxazoles with 3,4,5-trimethoxyphenyl substituent for the ring A (3a, 3b, 3k, and 3l) were highly potent showing EC values of 1–5 nM in the sea urchin embryo assay. These molecules exhibited strong cytotoxicity against human cancer cell lines including HeLa (cervical carcinoma), HepG2 (liver carcinoma), and OVCAR-3 (ovarian carcinoma) in the nanomolar IC50 range (Table 4).34 These cytotoxicity data correlated well with our observations on the importance of 3-hydroxy group in the ring B of diarylisoxazoles (3a > 3k, 3b > 3l) and preferable position of the ring A close to O atom of the isoxazole ring (3a > 3b; 3k > 3l). Both sea urchin embryo tests and NCI60 cytotoxicity screen identified 3o as the highly potent antimitotic compound followed by less active 5a, 3p, 3r, and 4b. Diarylisoxazoles 3s and 4d were inactive in either assay. In 1,2,3-triazole series, compounds 7a and 7b showed the highest effects both in the sea urchin embryo tests and NCI60 screen. Compounds 8b and 7c were less potent, whereas 9b displayed only marginal activity. 1,5-Diaryl-1,2,3-triazole 8a also exhibited strong cytotoxicity against K562 human myelogenous leukemia cells.35 Notably, several treatises mentioned methylenedioxyphenyl functionality to be a metabolic and safety liability in vivo.75 However, considering our sea urchin embryo data we think that antimitotics featuring polyalkoxyphenyl moieties deserve a more detailed pharmacokinetics and toxicology insight in a relevant animal models. For example, compounds 2f, 3f, 3s, 4d, 10d, and 10e were inactive in the sea urchin embryo test, and 3s and 4d failed to affect growth of human cancer cells (see Tables 3, 4, and 6). CONCLUSIONS The effect of diverse reported and novel pyrazole, isoxazole, 1,2,3-triazole, and pyrrole derivatives of combretastatins as microtubule destabilizing agents was studied using the sea urchin embryo model. The model provided both reliable and reproducible results on antimitotic and microtubule destabilizing activity of the tested molecules. Notably, data obtained from the in vivo phenotypic experiments showed excellent correlation with the respective cell-based cytotoxicity values. We further introduced reliable and reproducible dilution procedure for molecules to address the salt-containing medium (seawater) solubility issues. Several synthetic protocols were streamlined and validated to afford shorter and more efficient access to the key combretastatin analogues. 30 ACS Paragon Plus Environment

Page 31 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Multiple heterocyclic derivatives of combretastatins (combretazoles) exhibited antimitotic effects comparable to the parent molecules. Of note, 4,5-pyrazoles 1i (EC = 1 nM) and 1p (EC = 0.25 nM), 4,5-isoxazole 3a (EC = 1 nM), and 1,2,3-triazole 7b (EC = 0.5 nM) were more active than the parent CA4 (EC = 2 nM). A comparative analysis of combretazoles revealed that the overall antimitotic activity was defined by the substitution pattern of phenyl pharmacophores and N-containing linker. Of particular significance were 3,4,5-trimethoxyphenyl moiety for the ring A and 4-methoxyphenyl group for the ring B. 3-Hydroxy group in the ring B was not essential for antimitotic activity in diarylpyrazoles 1, whereas it was critical to the antitubulin effect in the diarylisoxazole series 3. Of the similarly substituted active azoles, diarylpyrazoles 1, 4,5-diarylisoxazoles 3, and 4,5diaryl-1,2,3-triazoles 7 displayed high antitubulin effect. 3,4-Diarylisoxazoles 5 showed lesser activity followed by 1,5-diaryl-1,2,3-triazoles 8, whereas pyrroles 10 were identified to be the weakest antimitotic agents. Introduction of the amino group into pyrazole and triazole moieties, and to a lesser extent into isoxazole ring of 4,5-diarylisoxazoles inevitably decreased the overall antimitotic effect of the resultant molecules. EXPERIMENTAL PROCEDURES Chemistry. Materials and Methods. Melting points were measured using a Boetius melting point apparatus and were uncorrected. 1H NMR and 13C NMR spectra were recorded on a Bruker DRX-500 instrument [working frequencies of 500.13 MHz (1H) and 125.76 MHz (13C), respectively]. Chemical shifts were stated in parts per million (ppm) and referenced to the appropriate NMR solvent peaks. Spin-spin coupling constants (J) were reported in Hertz (Hz). NMR spectra were prepared using the custom software designed at N. D. Zelinsky Institute of Organic Chemistry RAS (Moscow, Russian Federation) (http://nmr.ioc.ac.ru:8080/SDF2PDF.kl1). Low resolution mass spectra (m/z) were recorded on a Finnigan MAT/INCOS 50 mass spectrometer at 70 eV using direct probe injection. High resolution mass spectra (HRMS) were measured on a Bruker maXis and micrOTOF II instruments using electrospray ionization (ESI). Elemental analysis was performed on the automated Perkin-Elmer 2400 CHN micro-analyzer. Flash chromatography was accomplished using Silica (Acros, 0.035–0.070 mm, 60 Å). Thin layer chromatography (TLC) analysis was performed using Merck 60 F254 plates. Solvents, benzaldehydes, and ethyl bromoacetate were purchased from Acros Organics (Belgium) at the highest commercial quality and used as received. Synthesis of aldehydes, acids, benzylic alcohols, benzylchlorides, benzoylchlorides, and acetophenones was described earlier.48, 64, 65 3-Amino-4,5-diarylpyrazoles 2,48 3-amino-4,531 ACS Paragon Plus Environment


Page 32 of 57

diarylisoxazoles 4, 5-amino-3,4-diarylisoxazoles 6,51 3,4-diarylisoxazoles 5,55 and diaryl-1,2,3triazoles 7–950 were synthesized according to published procedures. For safe handling of compounds, the recommendations described in CA4 MSDS were used, including description of first aid measures, extinguishing media, accidental release measures, handling, storage, exposure controls, and personal protection.76 General Procedure for the Preparation of Chalcones 13.64, 77 NaOH (60 mmol) was added to a vigorously stirred solution of benzaldehyde 11 (20 mmol) and arylacetophenone 12 (20 mmol) in EtOH (150 mL) at +5 C (ice bath). The reaction mixture was stirred at room temperature for 24 h, concentrated in vacuo, the residue was treated with distilled water (130 mL), neutralized with 15% HCl and extracted with CH2Cl2 (3  80 mL). Organic extracts were washed with brine (2  80 mL), dried over anhydrous Na2SO4 and concentrated in vacuo. The resulting residue was crystallized from EtOAc-petroleum (1:1) and dried to afford chalcones 13. General Procedure for the Preparation of Chalcone Acetates (13-Ac). A mixture of AcCl (1.18 g, 15 mmol) and pyridine (1.19 g, 15 mmol) in abs. CH2Cl2 (15 mL) was added by small portions to a stirred solution of chalcone 13 (10 mmol) and pyridine (0.79 g, 10 mmol) in abs. CH2Cl2 (50 mL) at 20 C. The reaction mixture was stirred at room temperature for 3 h, diluted with water (50 mL), and the organic layer was separated. Water layer was extracted by CH2Cl2 (30 mL). Combined organic extracts were washed with water (3  50 mL), dried with Na2SO4 and evaporated. The residue was crystallized from EtOAc/petroleum ether, 1:1, and dried to afford chalcone acetates 13a-Ac. General Procedure for the Preparation of Dimethylketoacetals 14 with Simultaneous Removal of Acetyl Group (Schemes 1 and 2). A solution of H2SO4 (50%, 5 mL) in MeOH was added dropwise to a vigorously stirred suspension of chalcone 13-Ac (20 mmol) and PhI(OAc)2 (30 mmol) in MeOH (100 mL). The mixture was stirred for 8 h at room temperature and kept overnight at 2–6 C. The reaction mixture was diluted with water (300 mL) and extracted with CH2Cl2 (3  100 mL). The organic layer was washed with brine (2  80 mL), water (50 mL) and dried over anhydrous Na2SO4. Solvent was removed in vacuo and the resulting oil was purified using flash-chromatography (EtOAc/hexane, 1:4) to afford 14 used for subsequent synthetic steps without further purification. General Procedure for the Preparation of Dimethylketoacetals 14 (Scheme 3). A solution of H2SO4 (50%, 5.5 mL) in MeOH was added dropwise to the vigorously stirred suspension of chalcone 13 (6.57g, 20 mmol), PhI(OAc)2 (6.86 g, 21.3 mmol) in MeOH (93 mL) and stirred for 4 h at room temperature. The reaction solution was diluted with water (150 mL) and extracted with CH2Cl2 (2  100 mL). The organic layer was washed with brine, water (2  50 32 ACS Paragon Plus Environment

Page 33 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


mL) and dried over anhydrous Na2SO4. The solvent was removed in vacuo and the resulting oil was treated with MeOH (20 mL) at reflux to yield a clear solution and kept overnight at 2–6 C. The resulting crystals were filtered, washed with cold EtOH (2  3 mL), petroleum ether (3 mL) and dried in vacuo to afford dimethylacetal 14. General Procedure for the Preparation of Epoxychalcones 15 (Scheme 3).63 Hydrogen peroxide (30% aq, 0.6 mL) was added to a vigorously stirred suspension of chalcone 13 (4 mmol) in EtOH (15 mL) and NaOH (1N, 1.9 mL) at room temperature. The reaction mixture was stirred at 30 C for 3 h and left for 24 h at room temperature followed by addition of the second portion of NaOH (1N, 1.9 mL) and H2O2 (30%, 0.6 mL). The resulting mixture was stirred for 6 h at room temperature. Subsequently, the third portion of NaOH (1N, 1.9 mL) and H2O2 (30%, 0.6 mL) was added and stirred for 24 h at room temperature. The residue was filtered, washed with EtOH and H2O and dried in vacuo to afford epoxychalcones 15. General Procedures for the Rearrangement of Epoxychalcones 15 to Ketoaldehydes 16. Most of the intermediates 16 were used for the next stage without further purification. 3-(4-Methoxy-3-nitrophenyl)-3-oxo-2-(3,4,5-trimethoxyphenyl)propanal (16g). BF3Et2O (1.3 mL, 10.1 mmol) in CH2Cl2 (3 mL) was added dropwise for 5 min to a stirred solution of (4methoxy-3-nitrophenyl)[3-(3,4,5-trimethoxyphenyl)oxiran-2-yl]methanone (3.89 g, 10 mmol) in dry CH2Cl2 (30 ml) at -8 C (ice-salt bath) under Ar to result in temperature raising to -3 C. The solution was kept at -8 C for 10 min followed by addition of 5% aq. NaHCO3 to pH 8 (~30 mL), the mixture was extracted with CH2Cl2 (2  15 mL), combined organic layers were washed with water (3  10 mL), dried over anhydrous Na2SO4 and concentrated in vacuo to result in 3.5 g of brownish-yellow solid which was used without further purification. Analytical sample (TLC, EtOAc/hexane, 1:1, Rf = 0.41) was prepared by preparative LC (200 mg of the crude product; 95% MeCN, 4.9% water, 0.1% HCOOH) to furnish bright-yellow crystals of the targeted product (1.47 g, 38% yield); mp 135–136 C; 1H NMR (CDCl3):  15.77 (1H, d, J = 5.7 Hz, OH), 8.59 (1H, d, J = 5.7 Hz, H-1), 8.04 (1H, d, J = 2.3 Hz, H-2''), 7.56 (1H, dd, J = 8.9 Hz, J = 2.3 Hz, H-6''), 6.92 (1H, d, J = 8.9 Hz, H-5''), 6.36 (2H, s, H-2',6'), 3.96 (3H, s, OCH3), 3.88 (3H, s, OCH3), 3.76 (6H, s, 2OCH3); 13C NMR (CDCl3):  183.7, 181.6, 154.9, 153.8, 139.0, 138.0, 135.1, 130.5, 127.5, 127.2, 115.9, 112.6, 107.6, 61.1, 56.8, 56.3 (pure enol form). HRMS (ESI/QTOF) m/z: 412.3407 [M + Na]+ (100%), 428.4493 [M + K]+ (60%). Anal. Calcd. for C19H19NO8: C, 58.61; H, 4.92; N 3.60. Found: C, 58.79; H, 4.80; N 3.76. 3-(2,3-Dihydro-1,4-benzodioxin-6-yl)-3-oxo-2-(3,4,5-trimethoxyphenyl)propanal (16l). BF3Et2O (0.38 mL, 2.96 mmol) was added dropwise via syringe to a suspension of epoxide 15 (2.69 mmol) in CH2Cl2 (15 mL) at 0 C under Ar. The mixture was stirred at 20 C for 3 h, 33 ACS Paragon Plus Environment


Page 34 of 57

treated with NaHCO3 (5% solution) and extracted with CHCl3 (2  15 mL). The combined extracts were washed with water (2  15 mL) and concentrated in vacuo. The product was isolated by column chromatography (EtOAc/petroleum ether, 1:4, Rf = 0.6) to yield the respective ketoaldehyde 16 as a mixture of the enol- and aldehyde tautomers. Intermediates 16 were used for the next stage without further purification. General Procedure for the Preparation of 1,2-Diarylethanones 22. Compounds were synthesized according to the published procedure66 by acylation of respective Zn derivatives prepared in situ from benzoyl chlorides 18 with benzyl chlorides 20. General Procedure for the Preparation of 3-(Dimethylamino)-1,2-diarylprop-2-en-1ones 23. The targeted 3-(dimethylamino)-1,2-diarylprop-2-en-1-ones 23 were prepared according to a modified published procedure78 by reaction of 1,2-diarylethanones 22 with N,Ndimethylformamide dimethyl acetal (DMFDMA). A mixture of 22 (3.6 mmol) and DMFDMA (1 g, 8.4 mmol) was stirred under argon at 60 C for 3 h. The mixture was concentrated in vacuo to yield yellow crystalline enaminone 23 (3.6 mmol, quantitative yield) that was used without further purification for the synthesis of pyrazoles 1 and isoxazoles 3. General Procedure for the Preparation of 4,5-Diarylpyrazoles 1 (Schemes 1–4). NH2NH22HCl (0.410 g, 3.9 mmol) was added to a solution of respective ketoaldehyde (dimethylketoacetal 14, ketoaldehyde 16 or enaminone 23, 3.6 mmol) in methanol (20 mL), the mixture was refluxed for 1 h, concentrated in vacuo and the resulting residue was dissolved in ethylacetate (50 mL), washed with 5% aq. NaHCO3 (50 mL), distilled water (50 mL), dried over MgSO4 and concentrated in vacuo to furnish crude pyrazoles 1. 4-(3-Hydroxy-4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrazole (1a) (Scheme 1). Light brown solid; 140 mg (72%); mp 171–173 C (lit.53 198–200 C); 1H NMR (DMSO-d6)

 13.1 (0.5H, s, NH), 12.9 (0.5H, s, NH), 8.95 (1H, s, OH), 7.80 (0.5H, s, H-3), 7.56 (0.5H, s, H3), 6.90 (1H, d, J = 8.3 Hz, H-5'), 6.76 (2H, s, H-2'',6''), 6.75-6.66 (2H, m, H-2',6'), 3.75 (3H, s, OCH3-4'), 3.67 (6H, s, OCH3-3'',5''), 3.63 (3H, s, OCH3-4''); 13C NMR (DMSO-d6)  152.6, 146.4, 146.3, 139.4, 137.1, 128.5, 126.2, 119.6, 119.3, 118.9, 115.8, 112.3, 104.9, 60.0, 56.0, 55.6; EIMS m/z 357 [M+1]+ (21), 356 [M]+ (100), 341 (28), 162 (13), 155 (12), 140 (10), 133 (10), 127 (10), 119 (49), 105 (14), 91 (21), 77 (15). Anal. Calcd for C19H20N2O5: C 64.04; H 5.66; N 7.86. Found: C 64.12; H 5.70; N 7.78. 4-(3-Hydroxy-4-methoxyphenyl)-5-(4-methoxyphenyl)-1H-pyrazole (1b) (Scheme 1). White solid; 38 mg (32%); mp 151–153 C; 1H NMR (DMSO-d6)  12.97 (0.5H, s, NH), 12.83 (0.5H, s, NH), 8.88 (1H, s, OH), 7.77 (0.5H, s, H-3), 7.54 (0.5H, s, H-3), 7.34 (2H, d, J = 7.6 Hz, H-2'',6''), 6.97 (1H, d, J = 7.9 Hz, H-3'',5''), 6.88 (1H, d, J = 7.9 Hz, H-3'',5''), 6.85 (1H, d, J = 8.3 34 ACS Paragon Plus Environment

Page 35 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Hz, H-5'), 6.70–6.64 (2H, m, H-2',6'), 3.77 (3H, s, OCH3), 3.75 (3H, s, OCH3); 13C NMR (DMSO-d6)  158.4, 146.2, 139.2, 128.6, 126.4, 19.4, 118.6, 117.9, 115.7, 115.2, 114.4, 113.5, 112.3, 55.5, 55.0; EIMS m/z 297 [M+1]+ (21), 296 [M]+ (100), 281 (22), 263 (12), 253 (12), 235 (16), 118 (10). Anal. Calcd for C17H16N2O3: C 68.91; H 5.44; N 9.45. Found: C 69.02; H 5.47; N 9.42. 5-(3-Hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrazole (1c) (Scheme 2). Gray solid; 32 mg (12%); mp 232–234 C (lit.53 90–93 C); 1H NMR (DMSO-d6)  13.08 (0.5H, s, NH), 13.00 (0.5H, s, NH), 9.04 (0.5H, br, OH), 8.99 (0.5H, br, OH), 7.30 (1H, m, H6''), 7.21 (0.5H, s, H-3), 7.13 (2H, s, H-2',6'), 7.04 (0.5H, s, H-3), 7.02 (1H, s, H-2''), 6.98 (1H, m, H-5''), 3.86 (6H, s, OCH3-3',5'), 3.81 (3H, s, OCH3), 3.69 (3H, s, OCH3); EIMS m/z 357 [M+1]+ (20), 356 [M]+ (100), 342 (6), 341 (40), 327 (1), 313 (7), 118 (11), 91 (16). Anal. Calcd for C19H20N2O5: C 64.04; H 5.66; N 7.86. Found: C 64.15; H 5.70; N 7.78. 5-(3-Hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl)-1H-pyrazole (1d) (Scheme 2). White solid; 65 mg (48%); mp 166–168 C; 1H NMR (DMSO-d6, 500 MHz)  12.93 and 12.85 (1H, br, NH), 9.08 and 8.92 (1H, br, OH), 7.76 and 7.60 (1H, br, H-3), 7.19 (2H, d, J = 8.4 Hz, H-2',6'), 6.88 (2H, m, H-2'',6''), 6.88 (2H, d, J = 8.4 Hz, H-3',5'), 6.79 (1H, d, J = 8.0 Hz, H-5''), 3.77 (3H, s, OCH3), 3.74 (3H, s, OCH3); 13C NMR (DMSO-d6)  157.6, 147.3, 146.2, 139.1, 128.9, 128.4, 125.8, 122.5, 118.8, 117.7, 115.1, 113.8, 112.0, 55.5, 55.0; EIMS m/z 297 [M+1]+ (18), 296 [M]+ (100), 281 (7), 263 (8), 253 (6), 235 (12), 221 (11), 127 (12), 118 (12), 91 (12), 77 (30). Anal. Calcd for C17H16N2O3: C 68.91; H 5.44; N 9.45. Found: C 68.98; H 5.46; N 9.39. 4-(4,7-Dimethoxy-1,3-benzodioxol-5-yl)-5-(3-hydroxy-4-methoxyphenyl)-1H-pyrazole (1e) (Scheme 2). Dark gray solid; 25 mg (18%); mp 128–132 C; 1H NMR (DMSO-d6)  12.95 (0.5H, br, NH), 12.83 (0.5H, br, NH), 8.98 (1H, br, OH), 7.70 (0.5H, br, H-3), 7.48 (0.5H, br, H3), 6.88 (2H, m, H-2'',6''), 6.77 (1H, d, J = 7.6 Hz, H-5''), 6.02 (2H, s, OCH2O), 3.74 (3H, s, OCH3), 3.66 (3H, s, OCH3), 3.48 (3H, s, OCH3); 13C NMR (DMSO-d6)  146.1, 138.8, 138.5, 135.6, 135.2, 119.9, 117.9, 114.4, 113.6, 111.9, 109.6, 101.4, 59.2, 56.3, 55.4; EIMS m/z 371 [M+1]+ (20), 370 [M]+ (100), 325 (5), 171 (13), 170 (11), 169 (33), 140 (12), 127 (11). Calcd for C19H18N2O6: C 61.62; H 4.90; N 7.56. Found: C 61.74; H 4.93; N 7.47. 4-(4-Methoxy-3-nitrophenyl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrazole (1f) (Scheme 3, path B). Yellow solid; 180 mg (62%); mp 189–191 C (MeOH); 1H NMR (CDCl3)  12.17 (1H, br, NH), 7.88 (1H, s, H-3), 7.93 (0.5H, s, H-2'), 7.79 (0.5H, s, H-2'), 7.49 (1H, d, J = 8.6 Hz, H6'), 7.10 (1H, d, J = 8.4 Hz, H-5'), 6.80 and 6.78 (2H, s, H-2'',6''), 3.99 (3H, s, OCH3-4'), 3.90 (3H, s, OCH3-4''), 3.77 and 3.75 (6H, s, OCH3-3'',5''); 13C NMR (DMSO-d6)  153.0, 152.7, 150.3, 147.1, 139.4, 138.4, 137.6, 137.0, 134.0, 133.5, 129.2, 126.0, 124.5, 124.0, 116.4, 115.9, 35 ACS Paragon Plus Environment


Page 36 of 57

114.3, 105.2, 60.0, 56.6, 55.7, 55.6; EIMS m/z 386 [M+1]+ (19), 385 [M]+ (100), 371 (7), 370 (36), 140 (10), 139 (12), 77 (6). Anal. Calcd for C19H19N3O6: C 59.22; H 4.97; N 10.90. Found: C 59.29; H 5.01; N 10.85. 5-(4-Methoxy-3-nitrophenyl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrazole (1g) (Scheme 3, path A, B). Path B: NH2NH2H2O (0.55 mL, 17.2 mmol) was added to a solution of crude ketoaldehyde 12 (17 mM) in CH3CN (25 mL), the resulting mixture was stirred for 24 h at room temperature, concentrated in vacuo, treated with CH2Cl2 (10 mL) followed by distilled water (2 15 mL). The organic phase was dried over MgSO4, concentrated in vacuo to furnish crude 1f that was recrystallized from MeOH followed by CH3CN to afford pure pyrazole product (benzene/EtOAc, 1:1, Rf = 0.5). Respective yields were 0.64g (58%) (path B), 0.24 g (23%) (path A); mp 202–203 C (CH3CN); 1H NMR (DMSO-d6)  13.30 (0.5H, s, NH), 13.14 (0.5H, s, NH), 8.02 (1H, s, H-2''), 7.97 (1H, s, H-3), 7.74 (1H, d, J = 8.7 Hz, H-6''), 7.38 (1H, d, J = 8.7 Hz, H-5''), 6.58 (2H, s, H-2',6'), 3.93 (3H, s, OCH3-4''), 3.67 (9H, s, OCH3-3',4',5'); 13C NMR (DMSO-d6)  152.8, 151.2, 144.7, 138.8, 136.2, 133.5, 129.3, 128.5, 126.4, 123.7, 119.1, 114.2, 105.7, 105.2, 60.0, 56.7, 55.7; EIMS m/z 386 [M+1]+ (20), 385 [M]+ (100), 371 (16), 370 (91), 251 (15), 235 (19), 221 (20), 220 (23), 209 (24), 208 (30), 195 (29), 188 (62), 181 (27), 180 (36), 179 (35), 155 (28), 154 (62), 153 (36), 152 (29), 140 (49), 139 (51), 111 (52), 98 (85), 77 (50). Anal. Calcd for C19H19N3O6: C 59.22; H 4.97; N 10.90. Found: C 59.32; H 5.00; N 10.83%. 4-(3-Amino-4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrazole hydrochloride (1h) (Scheme 3, path A).69 Hydrogenation procedure described for the synthesis of 1i was used (see below) to obtain 1h as a light-brown powder; (110 mg, 65% yield); mp 145–147 C (benzene/EtOAc, 1:1, Rf = 0.30); 1H NMR (DMSO-d6)  9.95 (2H, br.s., NH), 7.77 (1H, s, H-3), 7.37 (1H, d, J = 1.6 Hz, H-2'), 7.32 (1H, dd, J = 8.5 Hz, J = 1.6 Hz, H-6'), 7.21 (1H, d, J =8.5 Hz, H-5'), 6.72 (2H, s, H-2'',6''), 4.10 (2H, br, NH2), 3.90 (3H, s, OCH3), 3.68 (3H, s, OCH3), 3.65 (6H, s, OCH3-3'',5''); 13C NMR (DMSO-d6)  152.7, 151.0, 142.3, 137.0, 134.1, 129.1, 126.7, 126.1, 123.8, 120.5, 117.3, 112.6, 104.8, 60.0, 56.2, 55.6; EIMS m/z 356 [M+1]+ (21), 355 (100), 341 (11), 340 (56), 162 (15), 155 (12), 140 (10), 127 (10), 119 (14), 105 (12), 91 (17). Anal. Calcd for C19H22ClN3O4: C 58.24; H 5.66; Cl 9.05, N 10.72. Found: C 58.32; H 5.69; Cl 9.14, N 10.79. 5-(3-Amino-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrazole hydrochloride (1i) (Scheme 3). Synthesized according to the general hydrogenation procedure described in ref. 79. NaBH4 powder (0.04 g, 1.04 mmol) was added portion-wise to a mixture of pyrazole 1e (0.1 g, 0.2 mmol) and NiCl2H2O (0.12 g, 0.52 mM) in MeOH/THF (6 mL, 1:2) at 0 C. The reaction mixture was stirred at room temperature for 1 hr, concentrated, the residue was treated with 5% 36 ACS Paragon Plus Environment

Page 37 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


aq. NH3 (20 mL) followed by CH2Cl2 (3  15 mL). Combined organic extracts were washed with distilled water (2  10 mL), concentrated and purified via column chromatography (EtOAc/petroleum ether, 2:1, Rf = 0.3) to yield the respective amine that was dissolved in 3 mL of MeOH, treated with 1 mL of concentrated aq. HCl and triturated with acetone (5 mL) to yield the desired HCl salt. White solid; 37 mg (36%); mp 158–162 C; 1H NMR (DMSO-d6)  10.08 (2H, br, NH), 7.95 (1H, s, H-3), 7.60 (1H, d, J = 2.1 Hz, H-2''), 7.45 (1H, dd, J = 8.6 Hz, J = 2.1 Hz, H-6''), 7.34 (1H, d, J = 8.6 Hz, H-5''), 6.54 (2H, s, H-2',6'), 5.9 (2H, br, NH2), 3.91 (3H, s, OCH3-4''), 3.65 (6H, s, OCH3-3',5'), 3.66 (3H, s, OCH3-4'); 13C NMR (DMSO-d6)  152.8, 151.8, 136.0, 132.2, 128.9, 128.4, 125.3, 123.6, 120.4, 118.7, 112.5, 112.3, 105.2, 60.0, 56.2, 55.6; EIMS m/z 356 [M+1]+ (21), 355 [M]+ (100), 341 (15), 340 (62), 265 (14), 250 (10), 239 (10), 211 (10), 162 (19), 155 (15), 154 (11), 140 (12), 127 (16), 120 (13), 106 (11), 105 (10). Anal. Calcd for C19H22ClN3O4: C 58.24; H 5.66; Cl 9.05, N 10.72. Found: C 58.36; H 5.71; Cl 8.98, N 10.66. 4-[3-(Benzyloxy)-4-methoxyphenyl]-5-(3,4,5-trimethoxyphenyl)-1H-pyrazole (1j) (Scheme 3, path A). White solid; 86 mg (52%); mp 132-134 C; 1H NMR (DMSO-d6)  13.11 (0.5H, s, NH), 12.95 (0.5H, s, NH), 7.88 (0.5H, s, H-3), 7.63 (0.5H, s, H-3), 6.99 (1H, s, H-2'), 7.38-7.29 (5H, m, Ph), 6.96 (1H, d, J = 8.2 Hz, H-5'), 6.89 (0.5H, d, J = 8.5 Hz, H-6'), 6.86 (0.5H, d, J = 8.5 Hz, H-6'), 6.73 (2H, s, H-2'',6''), 4.95 (2H, s, OCH2), 3.76 (3H, s, OCH3-4'), 3.66 (6H, s, OCH3-3'',5''), 3.62 (3H, s, OCH3-4''); 13C NMR (DMSO-d6)  152.8, 152.5, 147.8, 147.5, 147.0, 139.3, 137.0, 129.6, 128.4, 128.3, 128.1, 127.7, 127.5, 126.0, 125.7, 121.1, 120.6, 118.7, 114.3, 113.8, 112.2, 105.1, 69.9, 56.0, 55.6; EIMS m/z 447 [M+1]+ (30), 446 [M]+ (100), 445 (5), 387 (6), 386 (19), 356 (10), 355 (15), 340 (14), 295 (9), 292 (13), 263 (9), 91 (90). Anal. Calcd for C26H26N2O5: C 69.94; H 5.87; N 6.27. Found: C 70.07; H 5.90; N 6.18. 4-[3-(Benzyloxy)-4-methoxyphenyl]-5-(4-methoxyphenyl)-1H-pyrazole (1k) (Scheme 3, path A). Dark yellow oil; 123 mg (62%); 1H NMR (DMSO-d6)  12.92 (1H, br.s, NH), 7.75 (0.5H, s, H-3), 7.73 (0.5H, s, H-3), 7.38-7.29 (5H, m, Ph), 7.36 (2H, d, J = 8.4 Hz, H-2'',6''), 6.95 (1H, d, J = 7.7 Hz, H-5'), 6.93 (1H, d, J = 1.8 Hz, H-2'), 6.92 (2H, d, J = 8.4 Hz, H-3'',5''), 6.81 (1H, dd, J = 8.2 Hz, J = 1.8 Hz, H-6'), 4.93 (2H, s, OCH2), 3.77 (3H, s, OCH3), 3.76 (3H, s, OCH3); 13C NMR (DMSO-d6)  158.8, 147.6, 147.5, 137.0, 130.6, 129.1, 128.3, 128.1, 127.7, 127.6, 127.5, 126.0, 120.4, 118.0, 113.8, 112.2, 69.9, 55.5, 55.0; EIMS m/z 387 [M+1]+ (27), 386 [M]+ (100), 357 (3), 296 (16), 295 (29), 267 (15), 263 (20), 236 (6), 235 (10), 91 (67). Anal. Calcd for C24H22N2O3: C 74.59; H 5.74; N 7.25. Found: C 74.65; H 5.78; N 7.18. 5-(2,3-Dihydro-1,4-benzodioxin-6-yl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrazole hydrochloride (1l) (Scheme 3, path B). White solid; 23 mg (16%); mp 108–110 C; 1H NMR 37 ACS Paragon Plus Environment


Page 38 of 57

(DMSO-d6)  7.87 (1H, s, H-3), 6.97 (1H, d, J = 2.0 Hz, H-8''), 6.94 (0.5H, d, J = 2.0 Hz, H-7''), 6.92 (0.5H, d, J = 2.0 Hz, H-7''), 6.89 (0.5H, s, H-5''), 6.87 (0.5H, s, H-5''), 6.57 (2H, s, H-2',6'), 4.24 (4H, s, H-2'',3''), 3.66 (3H, s, OCH3-4'), 3.56 (6H, s, OCH3-3',5'); EIMS m/z 369 [M+1]+ (10), 368 (65), 353 (52), 239 (20), 211 (17), 195 (23), 183 (23), 168 (24), 163 (31), 155 (41), 140 (31), 139 (33), 128 (21), 127 (27), 126 (23), 107 (20), 89 (21), 79 (34), 77 (48), 43 (100). Anal. Calcd for C20H21ClN2O5: C 59.34; H 5.23; Cl 8.76, N 6.92. Found: C 59.26; H 5.18; Cl 8.84; N 7.01. 4-(4,7-Dimethoxy-1,3-benzodioxol-5-yl)-5-(4-methoxyphenyl)-1H-pyrazole (1m) (Scheme 3, path A). White solid; 120 mg (46%); mp 79–81 C; 1H NMR (DMSO-d6)  13.05 (0.5H, s, NH), 12.86 (0.5H, s, NH), 7.73 (0.5H, s, H-3), 7.48 (0.5H, s, H-3), 7.32 (2H, s, H-2'',6''), 6.94 (2H, s, H-3'',5''), 6.36 (1H, c, H-6'), 6.02 (2H, s, OCH2O), 3.74 (3H, s, OCH3), 3.65 (3H, s, OCH3), 3.47 (3H, s, OCH3); 13C NMR (DMSO-d6)  158.6, 140.4, 138.9, 138.6, 135.7, 135.4, 129.4, 128.3, 128.1, 127.0, 119.9, 113.7, 109.6, 101.5, 59.2, 56.3, 55.0; EIMS m/z 355 [M+1]+ (24), 354 [M]+ (100), 339 (8), 309 (6), 281 (11), 161 (15), 155 (12), 140 (7), 77 (6). Anal. Calcd for C19H18N2O5: C 64.40; H 5.12; N 7.91. Found: C 64.52; H 5.16; N 7.81. 8-Methoxy-6-[5-(4-methoxyphenyl)-1H-pyrazol-4-yl]-2H-1,4-benzoxazin-3(4H)-one (1n) (Scheme 3, path B). White solid; 170 mg (61%); mp 192–195 C; 1H NMR (DMSO-d6)  10.62 (1H, s, NH), 7.79 (1H, s, H-3), 7.38 (2H, d, J = 8.8 Hz, H-2'',6''), 6.97 (2H, d, J =8.8 Hz, H3'',5''), 6.56 (1H, d, J = 1.8 Hz, H-5'), 6.45 (1H, d, J = 1.8 Hz, H-7'), 4.76 (1H, br.s, NH), 4.51 (2H, s, H-2'), 3.78 (3H, s, OCH3-4''), 3.65 (3H, s, OCH3-8'); 13C NMR (DMSO-d6)  165.0, 159.4, 148.1, 142.2, 134.1, 131.3, 129.4, 128.2, 126.8, 122.6, 118.3, 114.1, 108.1, 107.4, 66.7, 55.7, 55.2; EIMS m/z 352 [M+1]+ (28), 351 (100), 322 (6), 308 (3), 280 (1), 266 (3), 250 (3), 239 (2), 211 (1). Anal. Calcd for C19H17N3O4: C 64.95; H 4.88; N 11.96. Found C 64.87; H 4.82; N 12.08. 4-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrazole (1o) (Scheme 4). White solid; 182 mg (75%); mp 142–143 C; 1H NMR (CDCl3)  10.5 (1H, br, NH), 7.65 (1H, s, H-3), 7.27 (2H, d, J = 8.2 Hz, H-2',6'), 6.88 (2H, d, J = 8.2 Hz, H-3',5'), 6.70 (2H, s, H-2'',6''), 3.87 (3H, s, OCH3), 3.81 (3H, s, OCH3), 3.70 (6H, s, OCH3-3'',5''); 13C NMR (DMSO-d6)  157.9, 152.6, 146.8, 139.5, 137.0, 129.7, 129.4, 128.6, 125.6, 118.5, 113.8, 104.8, 60.0, 55.5, 55.0; EIMS m/z 341 [M+1]+ (23), 340 [M]+ (100), 326 (10), 325 (46), 265 (11), 250 (16), 211 (11), 168 (11), 155 (15), 154 (21), 140 (11), 139 (13), 119 (10), 77 (10). Anal. Calcd for C19H20N2O4: C 67.05; H 5.92; N 8.23. Found: C 67.14; H 5.97; N 8.15. 5-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrazole (1p) (Scheme 4). White solid; 160 mg (70%); mp 141–142 C; 1H NMR (CDCl3)  11.0 (1H, br, NH), 7.68 (1H, s, H-3), 38 ACS Paragon Plus Environment

Page 39 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


7.41 (2H, d, J = 8.2 Hz, H-2'',6''), 6.89 (2H, d, J = 8.2 Hz, H-3'',5''), 6.52 (2H, s, H-2',6'), 3.86 (3H, s, OCH3), 3.82 (3H, s, OCH3), 3.72 (6H, s, OCH3-3',5'); 13C NMR (DMSO-d6)  159.2, 158.6, 152.6, 147.1, 139.3, 135.9, 131.7, 130.9, 129.3, 126.5, 113.8, 113.5, 105.4, 104.9, 103.8, 59.9, 55.5, 55.3; EIMS m/z 341 [M+1]+ (22), 340 [M]+ (100), 326 (17), 325 (85), 265 (11), 250 (16), 211 (15), 168 (12), 154 (20), 140 (11), 139 (12), 119 (12), 77 (12). Anal. Calcd for C19H20N2O4: C 67.05; H 5.92; N 8.23. Found: C 67.12; H 5.96; N 8.17. 5-(3-Bromo-4,5-dimethoxyphenyl)-4-(4-methoxyphenyl)-1H-pyrazole (1q) (Scheme 4). Yellowish solid; 230 mg (79%); mp 130–132 C; 1H NMR (CDCl3)  10.46 (1H, br, NH), 7.64 (1H, s, H-3), 7.26 (1H, s, H-2''), 7.24 (2H, d, J = 8.6 Hz, H-2',6'), 6.92 (1H, s, H-6''), 6.89 (2H, d, J = 8.6 Hz, H-3',5'), 3.87 (3H, s, OCH3), 3.82 (3H, s, OCH3), 3.66 (3H, s, OCH3); 13C NMR (DMSO-d6)  158.0, 152.9, 145.2, 144.7, 139.6, 131.4, 129.7, 129.3, 128.9, 125.4, 122.8, 118.8, 116.3, 113.9, 111.6, 60.0, 55.6, 55.0; EIMS m/z 391 [M+2]+ (20), 390 [M+1]+ (100), 389 [M]+ (20), 388 (100), 375 (22), 373 (22), 279 (15), 266 (12), 251 (17), 208 (15), 192 (11), 180 (12), 179 (15), 154 (30), 152 (15), 126 (16), 77 (15). Anal. Calcd for C18H17BrN2O3: C 55.54; H 4.40; Br 20.53; N 7.20. Found: C 55.46; H 4.36; Br 20.65; N 7.15. 5-(7-Methoxy-1,3-benzodioxol-5-yl)-4-(4-methoxyphenyl)-1H-pyrazole (1r) (Scheme 4). Yellowish solid; 140 mg (81%); mp 151–152 C; 1H NMR (DMSO-d6)  13.05 (0.5H, s, NH), 12.91 (0.5H, s, NH), 7.83 (0.5H, s, H-3), 7.59 (0.5H, s, H-3), 7.23 and 7.19 (2H, d, J = 8.4 Hz, H-2',6'), 6.92 (2H, d, J = 8.7 Hz, H-3',5'), 6.71 (0.5H, s, H-4''), 6.69 (0.5H, s, H-4''), 6.57 (1H, d, J = 1.4 Hz, H-6''), 6.02 (1H, s, OCH2O), 5.98 (1H, s, OCH2O), 3.75 (3H, s, OCH3-4'), 3.73 and 3.68 (3H, s, OCH3-7''); 13C NMR (DMSO-d6)  157.9, 148.3, 146.8, 143.1, 139.4, 129.6, 129.2, 128.5, 125.7, 118.6, 113.9, 107.5, 101.5, 101.1, 56.0, 55.0; EIMS m/z 325 [M+1]+ (25), 324 [M]+ (100), 323 (7), 309 (14), 251 (5), 208 (6). Anal. Calcd for C18H16N2O4: C 66.66; H 4.97; N 8.64. Found: C 6.57; H 4.93; N 8.70. 5-(8-Methoxy-2,3-dihydro-1,4-benzodioxin-6-yl)-4-(4-methoxyphenyl)-1H-pyrazole (1s) (Scheme 4). White solid; 180 mg (46%); mp 100–102 C; 1H NMR (CDCl3)  10.7 (1H, br, NH), 7.64 (1H, s, H-3), 7.26 (2H, d, J = 8.1 Hz, H-2',6'), 6.87 (2H, d, J = 8.1 Hz, H-3',5'), 6.67 (1H, s, H-5''), 6.56 (1H, s, H-7''), 4.33 (2H, s, OCH2-3''), 4.26 (2H, s, OCH2-2''), 3.81 (3H, s, OCH3-4'), 3.56 (3H, s, OCH3-8''); 13C NMR (DMSO-d6)  157.8, 148.4, 143.5, 139.6, 139.3, 129.4, 128.5, 125.7, 118.0, 113.8, 108.6, 104.1, 63.9, 63.7, 55.4, 55.0; EIMS m/z 339 [M+1]+ (21), 338 [M]+ (100), 337 (2), 323 (5), 309 (2), 250 (5). Anal. Calcd for C19H18N2O4: C 67.45; H 5.36; N 8.28. Found: C 67.53; H 5.39; N 8.19. 4-(7-Methoxy-1,3-benzodioxol-5-yl)-5-(4-methoxyphenyl)-1H-pyrazole (1t) (Scheme 4). White solid; 173 mg (76%); mp 130–132 C; 1H NMR (CDCl3)  10.5 (1H, br, NH), 7.64 (1H, s, 39 ACS Paragon Plus Environment


Page 40 of 57

H-3), 7.38 (2H, d, J = 8.8 Hz, H-2'',6''), 6.89 (2H, d, J = 8.8 Hz, H-3'',5''), 6.48 (1H, d, J = 1.5 Hz, H-4''), 6.47 (1H, d, J = 1.5 Hz, H-6''), 5.96 (2H, s, OCH2O), 3.83 (3H, s, OCH3), 3.77 (3H, s, OCH3); 13C NMR (DMSO-d6)  159.2, 158.5, 148.3, 143.1, 139.3, 133.2, 129.2, 128.6, 127.9, 122.0, 118.4, 114.1, 113.6, 107.8, 107.3, 102.1, 101.6, 101.0, 56.0, 55.0; EIMS m/z 325 [M+1]+ (24), 324 [M]+ (100), 323 (5), 295 (2), 251 (6). Anal. Calcd for C18H16N2O4: C 66.66; H 4.97; N 8.64. Found: C 66.73; H 5.01; N 8.58. 4-(3,5-Dimethoxyphenyl)-5-(4-methoxyphenyl)-1H-pyrazole (method C) (1u) (Scheme 4). White solid; 680 mg (66%); mp 124–126 C; 1H NMR (CDCl3)  10.92 (1H, br, NH), 7.68 (1H, s, H-3), 7.41 (2H, d, J = 8.8 Hz, H-2'',6''), 6.88 (2H, d, J = 8.8 Hz, H-3'',5''), 6.46 (2H, d, J = 2.3 Hz, H-2',6'), 6.37 (1H, t, J = 2.3 Hz, H-4'), 3.82 (3H, s, OCH3-4''), 3.70 (6H, s, OCH3-3',5'); 13C NMR (CDCl3)  161.2, 160.1, 143.5, 136.0, 135.6, 130.1, 123.8, 119.7, 114.4, 106.8, 99.3, 55.7; EIMS m/z 311 [M+1]+ (20), 310 [M]+ (100), 309 (42), 281 (9), 279 (6), 237 (10), 235 (11), 223 (13), 221 (11), 209 (16), 193 (17), 192 (16), 181 (17), 165 (16), 155 (31), 152 (27), 139 (20), 126 (21), 91 (28), 77 (23). Anal. Calcd for C18H18N2O3: C 69.66; H 5.85; N 9.03. Found: C 69.72; H 5.89; N 8.96. General Procedure for the Preparation of 4,5-Diarylisoxazoles 3 (Schemes 1–4). 4,5Diarylisoxazoles 3 were prepared according to a modified procedure published in ref. 80. NH2OHHCl (3.9 mmol) was added to a solution of ketoaldehyde derivative (dimethylketoacetal 14, ketoaldehyde 16 or enaminone 22, 3.6 mmol) in methanol (20 mL). The resulting mixture was refluxed for 2 h, concentrated in vacuo, the residue was treated with ethylacetate (50 mL), and the organic phase was washed with distilled water (2  25 mL), dried over MgSO4 and concentrated to afford crude isoxazoles 3. 4-(3-Hydroxy-4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)isoxazole (3a) (Scheme 1). White crystals; 710 mg (55%); mp 158–160 C (lit.33 mp 167–168 C ); 1H NMR (DMSO-d6)  9.17 (1H, s, OH), 8.78 (1H, s, H-3), 7.0 (1H, d, J = 8.0 Hz, H-5'), 6.92 (2H, s, H-2'',6''), 6.90 (1H, d, J = 8.0 Hz, H-6'), 6.88 (1H, s, H-2'), 3.79 (3H, s, OCH3-4'), 3.72 (3H, s, OCH3-4''), 3.69 (6H, s, 2OCH3-3',5'); 13C NMR (DMSO-d6)  162.2 (C-5), 153.1 (2C, C-3'',5''), 152.3 (C-3), 147.7 (C4'), 146.8 (C-3'), 139.0 (C-4''), 122.5 (C-1'), 121.9 (C-1''), 119.7 (C-6'), 115.7 (C-4), 115.6 (C-2'), 112.6 (C-5'), 104.5 (2C, C-2'',6''), 60.2 (OCH3), 55.9 (2OCH3), 55.7 (OCH3); EIMS m/z 357 [M]+ (100), 358 (21), 342 (11), 314 (10), 195 (56). Anal. Calcd for C19H19NO6: C 63.86; H 5.36; N 3.92. Found: C 63.74; H 5.29; N 3.97. 5-(3-Hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)isoxazole (3b) (Scheme 2). White crystals; 335 mg (26%); mp 171–173 C (lit.34 no mp value and NMR spectra); 1H NMR (DMSO-d6)  9.40 (1H, s, OH), 8.83 (1H, s, H-3), 7.11 (1H, d, J = 2.1 Hz, H-2''), 7.10 40 ACS Paragon Plus Environment

Page 41 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(1H, dd, J = 6.6 Hz, J = 2.1 Hz, H-6''), 7.03 (1H, d, J = 6.6 Hz, H-5''), 6.74 (2H, s, H-2',6'), 3.81 (3H, s, OCH3-4''), 3.71 (6H, s, 2OCH3-3',5'), 3.70 (3H, s, OCH3-4'); EIMS m/z 357 [M]+ (100), 358 (20), 342 (14), 329 (12), 283 (17), 151 (44). Anal. Calcd for C19H19NO6: C 63.86; H 5.36; N 3.92. Found: C 63.74; H 5.28; N 4.12. 4-(7-methoxy-1,3-benzodioxol-5-yl)-5-(3-hydroxy-4-methoxyphenyl)-isoxazole (3c) (Scheme 2). Light yellow crystals; 220 mg (18%); mp 166 C; 1H NMR (DMSO-d6)  9.4 (1H, s, OH), 8.77 (1H, s, H-3), 7.04 (3H, m, H-2'',5'',6''), 6.72 (1H, d, J = 1.5 Hz, H-4'), 6.64 (1H, d, J = 1.5 Hz, H-6'), 6.03 (2H, s, OCH2O), 3.81 (3H, s, OCH3), 3.78 (3H, s, OCH3); 13C NMR (DMSOd6)  163.1 (C-5), 152.3 (C-3), 149.5 (C-4''), 148.8 (C-3''), 146.7 (C-3a'), 143.5 (C-7'), 134.6 (C7a'), 123.8 ( C-5'), 119.6 (C-1''), 118.8 (C-6''), 114.6 (C-4), 113.9 (C-2''), 112.4 (C-5''), 108.4 (C6'), 102.4 (C-4'), 101.5 (C-2'), 56.3 (OCH3), 55.6 (OCH3); EIMS m/z 341 [M]+ (24), 342 (5), 152 (11), 151 (100), 123 (12). Anal. Calcd for C18H15NO6: C 63.34; H 4.43; N 4.10. Found: C 63.42; H 4.47; N 4.03. 4-(4,7-Dimethoxy-1,3-benzodioxol-5-yl)-5-(3-hydroxy-4-methoxyphenyl)-isoxazole (3d) (Scheme 2). White crystals; 307 mg (23%); mp 138–140 C; 1H NMR (DMSO-d6)  9.43 (1H, s, OH), 8.56 (1H, s, H-3), 7.01-6.99 (3H, m, H-2',5',6'), 6.53 (1H, s, H-6'), 6.08 (2H, s, OCH2O), 3.79 (3H, s, OCH3), 3.72 (3H, s, OCH3), 3.62 (3H, s, OCH3); 13C NMR (DMSO-d6)  163.6 (C5), 153.1 (C-3), 149.3 (C-4''), 146.6 (C-3''), 139.1 (C-3a'), 138.8 (C-7'), 136.8 (C-7a'), 135.9 (C4'), 119.9 (C-1''), 118.1 (C-5'), 115.7 (C-5'), 113.3 (C-2''), 112.2 (C-5''), 110.3 (C-4), 109.6 (C6'), 102.0 (C-2'), 59.7 (OCH3), 56.6 (OCH3), 55.6 (OCH3). EIMS m/z 371 [M]+ (18), 232 (10), 152 (11), 151 (100), 123 (13), 108 (10). Anal. Calcd for C19H17NO7: C 61.45; H 4.61; N 3.77. Found: C 61.54; H 4.64; N 3.64. 5-(3-Hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl)isoxazole (3e) (Scheme 2). White crystals; 560 mg (52%); mp 147–149 C (lit.81 no mp value); 1H NMR (DMSO-d6)  9.37 (1H, s, OH), 8.73 (1H, s, H-3), 7.35 (2H, d, J = 8.7 Hz, H-2',6'), 7.01 (3H, br.s, H-2'',5'',6''), 6.99 (2H, d, J = 8.7 Hz, H-3'',5''), 3.80 (3H, s, OCH3), 3.79 (3H, s, OCH3); 13C NMR (DMSO-d6)  162.8 (C5), 158.9 (C-4'), 152.3 (C-3), 149.4 (C-4''), 146.7 (C-3''), 129.7 (2C, C-2',6'), 121.9 (C-1'), 119.8 (C-1'), 118.6 (C-6''), 114.4 (2C, C-3',5'), 113.8 (C-4, C-2''), 112.3 (C-5''), 55.6 (OCH3), 55.2 (OCH3); EIMS m/z 297 [M]+ (100), 298 (19), 254 (39), 227 (13), 151 (65). Anal. Calcd for C17H15NO4: C 68.68; H 5.09; N 4.71. Found: C 68.83; H 5.19; N 4.47. 4-(4,7-Dimethoxy-1,3-benzodioxol-5-yl)-5-(3,4-dimethoxyphenyl)isoxazole (3f) (Scheme 3, path A). White crystals; 780 mg (56%); mp 122–124 C; 1H NMR (DMSO-d6)  8.60 (1H, d, J = 1.1 Hz, H-3), 7.11 (2H, m, H-2'',6''), 7.04 (1H, d, J = 8.3 Hz, H-5'), 6.58 (1H, s, H-6'), 6.08 (2H, s, OCH2O), 3.78 (3H, s, OCH3), 3.73 (3H, s OCH3), 3.68 (3H, s, OCH3), 3.63 (3H, s, 41 ACS Paragon Plus Environment


Page 42 of 57

OCH3); 13C NMR (DMSO-d6)  163.6 (C-5), 153.1 (C-3), 150.3 (C-4''), 148.7 (C-3''), 139.1 (C3a'), 138.8 (C-7'), 136.8 (C-7a'), 135.9 (C-4'), 119.8 (C-1'), 119.6 (C-6''), 115.6 (C-5'), 111.9 (C5''), 110.60 (C-4), 109.7 (C-2''), 109.6 (C-6'), 102.0 (C-2'), 59.8 (OCH3), 56.7 (OCH3), 55.6 (OCH3), 55.3 (OCH3); EIMS m/z 386 [M+1]+ (8),385 [M]+ (38), 232 (12), 166 (10), 165 (100). Anal. Calcd for C20H19NO7: C 62.33; H 4.97; N 3.63. Found: C 62.50; H 5.10; N 3.53. 4-(4,7-Dimethoxy-1,3-benzodioxol-5-yl)-5-(4-methoxyphenyl)isoxazole (3g) (Scheme 3, path A). White crystals; 690 mg (54%); mp 116–117 C; 1H NMR (DMSO-d6)  8.58 (1H, s, H3), 7.50 (2H, d, J = 8.9 Hz, H-2'',6''), 7.02 (2H, d, J = 8.9 Hz, H-3'',5''), 6.55 (1H, s, H-6'), 6.08 (2H, s, OCH2O), 3.79 (3H, s, OCH3), 3.72 (3H, s, OCH3), 3.62 (3H, s, OCH3); 13C NMR (DMSO-d6)  163.6 (C-5), 160.6 (C-4''), 153.1 (C-3), 139.1 (C-3a'), 138.8 (C-7'), 136.8 (C-7a'), 135.9 (C-4'), 128.0 (2C, C-2'',6''), 119.9 (C-1''), 115.5 (C-5'), 114.5 (2C, C-3'',5''), 110.4 (C-4), 109.6 (C-6'), 102.0 (C-2'), 59.7 (OCH3), 56.6 (OCH3), 55.3 OCH3); EIMS m/z 355 [M]+ (16), 136 (9), 135 (100), 107 (9). Anal. Calcd for C19H17NO6: C 64.22; H 4.82; N 3.94. Found: C 64.34; H 4.91; N, 3.74. 4-(4,7-Dimethoxy-1,3-benzodioxol-5-yl)-5-phenylisoxazole (3h) (Scheme 3, path A). White crystals; 880 mg (75%); mp 144–146 C; 1H NMR (DMSO-d6)  8.65 (1H, d, J = 2.4 Hz, H-3), 7.57 (2H, m, H-2'',6''), 7.47 (3H, m, H-3'',4'',5''), 6.56 (1H, s, H-6'), 6.08 (2H, s, OCH2O), 3.71 (3H, s, OCH3), 3.60 (3H, s, OCH3); 13C NMR (DMSO-d6)  163.6 (C-5), 153.2 (C-3), 139.1 (C-3a'), 138.8 (C-7'), 136.9 (C-7a'), 135.8 (C-4'), 130.2 (C-4''), 129.0 (2C, C-2'',6''), 127.4 (C-1''), 126.4 (2C, C-3'',5''), 115.2 (C-5'), 111.8 (C-4), 109.5 (C-6'), 102.0 (C-2'), 59.6 (OCH3), 56.6 (OCH3); EIMS m/z 326 [M+1]+ (12), 325 [M]+ (66), 324 (10), 310 (11), 220 (10), 163 (10), 106 (11), 105 (100). Anal. Calcd for C18H15NO5: C, 66.46; H, 4.65; N, 4.31. Found: C, 66.32; H, 4.55; N, 4.45. 8-Methoxy-6-[5-(4-methoxyphenyl)-1,2-oxazol-4-yl]-2H-1,4-benzoxazin-3(4H)-one (3i) (Scheme 3, path B). White solid; 890 mg (70%); mp 238–242 C; 1H NMR (DMSO-d6)  10.69 (1H, s, NH), 8.79 (1H, s, H-3), 7.58 (2H, d, J = 8.9 Hz, H-2'',6''), 7.06 (2H, d, J = 8.9 Hz, H3'',5''), 6.77 (1H, d, J = 1.9 Hz, H-5'), 6.57 (1H, d, J = 1.9 Hz, H-7'), 4.58 (2H, s, OCH2), 3.81 (3H, s, OCH3), 3.75 (3H, s, OCH3); 13C NMR (DMSO-d6)  164.9 (C-3'), 163.1 (C-5), 160.7 (C4'), 152.3 (CH-3), 148.6 (C-8'), 132.1 (C-8a'), 128.7 (2C, C-2'', 6''), 128.5 (NH-C-4a'), 123.6 (C1''), 119.4 (C-6'), 114.6 (2C, C-3'', 5''), 114.5 (C-4), 108.2 (C-5'), 107.6 (C-7'), 66.7 (C-2'), 55.9 (OCH3), 55.4 (OCH3); EIMS m/z 353 [M+1]+ (24), 352 [M]+ (100), 136 (6), 135 (66). Anal. Calcd for C19H16N2O5: C 64.77; H 4.58; N 7.95. Found: C 64.97; H 4.67; N 7.75. 5-(2,3-Dihydro-1,4-benzodioxin-6-yl)-4-(3,4,5-trimethoxyphenyl)-1,2-oxazole (3j) (Scheme 3, path B). Light-yellow crystals; 213 mg (16%); mp 118–120 C; 1H NMR (DMSO-d6) 42 ACS Paragon Plus Environment

Page 43 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


 8.86 (1H, s, H-3), 7.13 (2H, m, H-5'',7''), 6.94 (2H, d, J = 8.9 Hz, H-8''), 6.75 (2H, s, H-2',6'), 4.29 (2H, m, OCH2), 4.27 (2H, m, OCH2), 3.72 (6H, s, OCH3), 3.70 (3H, s, OCH3); 13C NMR (DMSO-d6)  163.7 (C-5), 154.3 (2C, C-3',5'), 153.5 (C-3), 146.4 (C-4a''), 144.7 (C-8a''), 138.5 (C-4'), 126.1 (C-6''), 121.7 (C-7''), 121.3 (C-1'), 118.9 (C-5''), 116.9 (C-8''), 116.4 (C-4), 107.1 (2C, C-2',6'), 65.5 (C-2''), 65.3 (C-''), 61.3 (OCH3), 57.1 (2C, 2OCH3); EIMS m/z 370 [M+1]+ (9), 369 [M]+ (390), 354 (8), 218 (7), 213 (6), 164 (10), 163 (100), 135 (12), 107 (21), 79 (17), 77 (13). Anal. Calcd for C20H19NO6: C 65.03; H 5.18; N 3.79. Found: C 65.15; H 5.27; N 3.57. 4-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)isoxazole (3k) (Scheme 4). White solid; 1.07 g (87%); mp 117–118 °C (lit.34 no NMR spectra and mp value; lit.67 1H NMR spectrum; no mp value); 1H NMR (CDCl3)  8.30 (1H, s, H-3), 7.35 (2H, d, J = 8.9 Hz, H-2',6'), 6.95 (2H, d, J = 8.9 Hz, H-3',5'), 6.89 (2H, s, H-2'',6''), 3.88 (3H, s, OCH3), 3.74 (3H, s, OCH3), 3.73 (6H, s, OCH3); 13C NMR (CDCl3)  163.2 (C-5), 159.5 (C-4'), 153.3 (C-3), 152.0 (2C, C-3'',5''), 139.3 (C-4''), 130.1 (2C, C-2', 6'), 122.9 (C-1'), 122.3 (C-1''), 115.4 (C-4), 114.3 (2C, C-3', 5'), 104.3 (2C, C-2'',6''), 60.9 (OCH3), 56.0 (2C, 2OCH3), 55.3 (OCH3); EIMS m/z 342 [M+1]+ (21), 341 [M]+ (100), 326 (27), 298 (20), 195 (56), 168 (32), 153 (24). Anal. Calcd for C19H19NO5: C 66.85; H 5.61; N 4.10. Found: C 66.95; H 5.67; N, 3.95. 5-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)isoxazole (3l) (Scheme 4). White solid; 850 mg (69%); mp 143–144 °C (MeOH) (lit.34 no mp value and NMR spectra); 1H NMR (CDCl3)  8.30 (1H, s, H-3), 7.62 (2H, d, J = 8.8 Hz, H-''2,6''), 6.91 (2H, d, J = 8.8 Hz, H-3'',5''), 6.58 (2H, s, H-3',5'), 3.90 (3H, s, OCH3), 3.84 (3H, s, OCH3), 3.79 (6H, s, OCH3); 13C NMR (CDCl3)  163.9 (C-5), 160.9 (C-4''), 153.6 (2C, C-3',5''), 151.7 (C-3), 137.8 (C-4'), 128.8 (2C, C-2'', 6''), 125.8 (C-1'), 120.1 (C-1''), 115.0 (C-4), 114.1 (2C, C-3'', 5''), 105.9 (2C, C-2',6'), 60.9 (OCH3), 56.2 (2C, 2OCH3), 55.3 (OCH3); EIMS m/z 341 [M]+ (60), 136 (21), 135 (100), 77 (26). Anal. Calcd for C19H19NO5: C 66.85; H 5.61; N 4.10. Found: C 66.94; H 5.71; N 3.90. 5-(3-Bromo-4,5-dimethoxyphenyl)-4-(4-methoxyphenyl)isoxazole (3m) (Scheme 4). White solid; 900 mg (64%); mp 72–73 °C; 1H NMR (CDCl3)  8.31 (1H, s, H-3), 7.45 (1H, d, J = 2.0 Hz, H-2''), 7.32 (2H, d, J = 8.8 Hz, H-2',6'), 7.12 (1H, d, J = 2.0 Hz, H-6''), 6.96 (2H, d, J = 8.8 Hz, H-3',5'), 3.89 (3H, s, OCH3), 3.85 (3H, s, OCH3), 3.72 (3H, s, OCH3); 13C NMR (DMSO-d6)

 160.8 (C-5), 159.3 (C-4'), 153.6 (C-5''), 152.4 (C-3), 147.1 (C-4''), 129.8 (2C, C-2',6'’), 124.4 (C-3''), 122.7 (C-2''), 121.1 (C-1'), 117.1 (C-1''), 116.2 (C-4), 114.5 (2C, C-3',5'), 111.1 (C-6''), 60.3 (OCH3), 56.1 (OCH3), 55.3 (OCH3); EIMS m/z 391 [M]+ (100), 389 [M]+ (100), 310 (50), 255 (43), 245 (88), 243 (87), 169 (44). Anal. Calcd for C18H16BrNO4: C 55.40; H 4.13; N 3.59. Found: C 55.29; H 4.18; N 3.47.



Page 44 of 57

5-(7-Methoxy-1,3-benzodioxol-5-yl)-4-(4-methoxyphenyl)isoxazole (3n) (Scheme 4). White crystals; 870 mg (74%); mp 91–92 °C; 1H NMR (DMSO-d6)  8.79 (1H, s, H-3), 7.37 (2H, d, J = 8.7 Hz, H-2',6'), 7.01 (2H, d, J = 8.7 Hz,, H-3',5'), 6.88 (1H, d, J = 1.5 Hz, H-4''), 6.73 (1H, d, J = 1.5 Hz, H-6''), 6.07 (2H, s, OCH2O), 3.79 (3H, s, OCH3), 3.75 (3H, s, OCH3); 13C NMR (DMSO-d6)  162.3 (C-5), 159.1 (C-4''), 152.4 (C-3), 148.8 (C-3a''), 143.5 (C-7''), 136.5 (C-7a''), 129.8 (2C, C-2', 6'), 121.6 (C-5''), 121.2 (C-1'), 115.2 (C-4), 114.5 (2C, C-3', 5'), 107.4 (C-4''), 102.1 (C-6''), 100.9 (C-2''), 56.3 (OCH3), 55.2 (OCH3); EIMS m/z 325 [M]+ (80), 179 (100), 152 (56), 151 (38). Anal. Calcd for C18H15NO5: C 66.46; H 4.65; N 4.31. Found: C, 66.53; H, 4.69; N, 4.23. 5-(8-Methoxy-2,3-dihydro-1,4-benzodioxin-6-yl)-4-(4-methoxyphenyl)isoxazole (3o) (Scheme 4). White solid; 880 mg (72%); mp 99–100 °C; 1H NMR (CDCl3)  8.27 (1H, s, H-3), 7.32 (2H, d, J = 8.8 Hz, H-2',6'), 6.94 (2H, d, J = 8.8 Hz, H-3',5'), 6.86 (1H, s, H-5''), 6.80 (1H, s, H-7''), 4.34 (2H, m, OCH2), 4.26 (2H, m, OCH2), 3.84 (3H, s, OCH3), 3.75 (3H, s, OCH3); 13C NMR (DMSO-d6)  162.4 (C-5), 159.1 (C-4'), 152.4 (C-3), 149.0 (C-4a''), 143.9 (C-8''), 134.8 (C-8a''), 129.9 (2C, C-2',6'), 121.7 (C-6''), 119.0 (C-1'), 115.0 (C-4), 114.4 (2C, C-3',5'), 108.4 (C-5''), 103.3 (C-7''), 64.1 (C-3''), 64.0 (C-2''), 55.7 (OCH3), 55.2 (OCH3); EIMS m/z 340 [M+1]+ (23), 339 [M]+ (100), 240 (27), 193 (58), 166 (44). Anal. Calcd for C19H17NO5: C 67.25; H 5.05; N 4.13. Found: C 67.34; H 5.12; N 3.98. 4-(7-Methoxy-1,3-benzodioxol-5-yl)-5-(4-methoxyphenyl)isoxazole (3p) (Scheme 4). White crystals; 950 mg (81%); mp 87–88 °C (MeOH); 1H NMR (CDCl3)  8.27 (1H, s, H-3), 7.60 (2H, d, J = 8.9 Hz, H-2'',6''), 6.91 (2H, d, J = 8.9 Hz, H-3'',5''), 6.54 (2H, s, H-4',6'), 6.01 (2H, s, OCH2O), 3.84 (6H, s, OCH3); 13C NMR (DMSO-d6)  163.0 (C-5), 160.7 (C-4''), 152.3 (C-3), 148.8 (C-3a'), 143.5 (C-7'), 134.7 (C-7a'), 128.8 (2C, C-2'', 6''), 123.7 (C-5'), 119.5 (C1''), 114.7 (C-4), 114.6 (2C, C-3'', 5''), 108.3 (C-6'), 102.3 (C-4'), 101.6 (C-2'), 56.4 (OCH3), 55.4 (OCH3); EIMS m/z 325 [M]+ (60), 135 (100), 92 (12), 77 (21). Anal. Calcd for C18H15NO5: C 66.46; H 4.65; N 4.31. Found: C 66.37; H 4.60; N 4.42. 4-(3,5-Dimethoxyphenyl)-5-(4-methoxyphenyl)isoxazole (3q) (Scheme 4). Light-yellow crystals; 875 mg (78%); mp 92–94 C; 1H NMR (CDCl3)  8.31 (1H, s, H-3), 7.62 (2H, d, J = 8.7 Hz, H-2'',6''), 6.90 (2H, d, J = 8.7 Hz, H-3'',5''), 6.52 (2H, d, J = 2.0 Hz, H-2',6'), 6.46 (1H, d, J = 2.0 Hz, H-4'), 3.83 (3H, s, OCH3), 3.76 (6H, s, OCH3); 13C NMR (DMSO-d6)  164.6 (C-5), 161.6 (2C, C-3',5'), 161.4 (C-4''), 152.2 (C-3), 132.7 (C-1'), 129.4 (2C, C-2'',6''), 120.6 (C-1''), 115.4 (C-4), 114.6 (2C, C-3'',5''), 107.1 (2C, C-2',6'), 100.4 (C-4'), 55.9 (2C, OCH3), 55.8 (OCH3); EIMS m/z 311 [M]+ (91), 312 (19), 204 (14), 139 (16), 135 (100). Anal. Calcd for C18H17NO4: C 69.44; H 5.50; N 4.50. Found: C 69.49; H 5.53; N 4.45. 44 ACS Paragon Plus Environment

Page 45 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


General Procedure for the Preparation of Ethyl 3,4-Diarylpyrrole-2-carboxylates 25. Dry K2CO3 (138 mg, 1 mmol) was added at once to the solution of 1,2-diarylnitroethylene (0.5 mmol) and ethyl isocyanoacetate (57 mg, 0.5 mmol) in ethanol (2 mL) at room temperature. The mixture was stirred for additional 12–36 h (TLC, toluene/petroleum ether, 2:1), diluted with distilled water (13 mL) and extracted with ethylacetate (3  3 mL). The combined organic layers were dried by filtration through a cotton plug, concentrated in vacuo and concentrated to afford 25. General Procedure for the Preparation of 3,4-Diarylpyrrole-2-carboxylic Acids 26. A solution of ethyl ester 25 (0.15 mmol) and NaOH (12 mg, 0.3 mmol) in EtOH/water (5 mL, 10:1) was refluxed until the starting 25 was consumed as evidenced by TLC (ethyl acetate/heptane, 1:2) and treated with 1M aq. NaOH (9 mL) followed by reflux for 30 min. The resulting hot suspension was filtered, the pH of the filtrate was adjusted to 1 with 20% aq HCl to afford the precipitate that was collected, washed with water (2  5 mL) and dried in vacuo. General procedure for the preparation of 3,4-diarylpyrroles 10. Pyrrole-2-carboxylic acid (50 mg) was heated in a glass flask to melting point and the heating was continued until evolution of gas stopped (1 min). The resulting residue was cooled to room temperature and purified by column chromatography (Silica, EtOAc/hexanes, 1:3) to afford the respective 3,4diarylpyrrole 10. 3-(3,4,5-Trimethoxyphenyl)-4-(4-methoxyphenyl)-1H-pyrrole (10a). Yellow crystals; 32 mg (73%); mp 142–143 C; 1H NMR (DMSO-d6)  11.02 (1H, s, NH), 7.15 (2H, d, J = 8.4 Hz, H-2'',6''), 6.98 (1H, s, H-5), 6.86 (2H, d, J = 8.4 Hz, H-3'',5''), 6.84 (1H, s, H-2), 6.46 (2H, s, H2',6'), 3.73 (3H, s, OCH3), 3.63 (3H, s, OCH3), 3.58 (6H, s, OCH3); 13C NMR (DMSO-d6)  158.5, 153.6, 136.5, 133.1, 130.6, 129.9, 122.9, 122.7, 118.6, 118.4, 114.7, 106.3, 61.2, 56.7, 56.2; EIMS m/z 340 [M+1]+ (22), 339 [M]+ (100), 325 (16), 324 (72), 249 (6). Anal. Calcd for C20H21NO4: C 70.78; H 6.24; N 4.13. Found: C 70.87; H 6.31; N 3.89. 3-(3,5-Dimethoxyphenyl)-4-(4-methoxyphenyl)-1H-pyrrole (10b). Yellow crystals; 23 mg (46%); mp 127–128 °C; 1H NMR (DMSO-d6)  11.09 (1H, s, NH), 7.12 (2H, d, J = 8.6 Hz, H2'',6''), 6.98 (1H, s, H-5), 6.84 (2H, d, J = 8.6 Hz, H-3'',5''), 6.83 (1H, s, H-2), 6.33 (2H, d, J = 2.2 Hz, H-2',6'), 6.26 (1H, t, J = 2.2 Hz, H-4'), 3.73 (3H, s, OCH3), 3.61 (6H, s, OCH3); 13C NMR (DMSO-d6)  160.1, 157.3, 138.3, 131.5, 129.4, 128.7, 121.7, 121.6, 117.8, 117.4, 107.3, 105.8, 97.2, 55.2, 55.0, 54.8; HRMS (ESI/QTOF) m/z: 332.3491 [M + Na]+ (100%), 348.4576 [M + K]+ (53%). Anal. Calcd for C19H19NO3, C 73.77; H 6.19; N 4.53%. Found: C 73.92; H 6.25; N 4.36%.



Page 46 of 57

3-(4,7-Dimethoxy-2H-1,3-benzodioxol-5-yl)-4-(4-methoxyphenyl)-1H-pyrrole (10c). Yellow crystals; 27 mg (60%); mp 150–151 °C; 1H NMR (DMSO-d6)  10.89 (1H, s, NH), 7.07 (2H, d, J = 8.2 Hz, H-2'',6''), 6.88 (1H, s, H-5), 6.80 (2H, d, J = 8.2 Hz, H-3',5'), 6.77 (1H, s, H2), 6.28 (1H, s, H-6'), 5.98 (2H, s, OCH2O), 3.69 (3H, s, OCH3), 3.61 (3H, s, OCH3), 3.39 (3H, s, OCH3); 13C NMR (DMSO-d6)  158.2, 140.1, 139.5, 136.9, 135.7, 130.5, 129.2, 124.1, 123.7, 119.5, 118.4, 116.9, 114.7, 111.1, 102.5, 60.2, 57.4, 56.1; EIMS m/z 354 [M+1]+ (23), 353 [M]+ (100), 338 (10), 308 (3), 280 (3), 250 (3). Anal. Calcd for C20H19NO5: C 67.98; H 5.42; N 3.96. Found: C 68.15; H 5.54; N 3.69. 3-Phenyl-4-(3,4,5-trimethoxyphenyl)-1H-pyrrole (10d). Yellow crystals; 30 mg (69%); mp 128–130 °C; 1H NMR (CDCl3)  8.30 (1H, s, NH), 7.32–7.27 (4H, m, H-2',3',5',6'), 7.19 (1H, t, J = 7.1 Hz, H-4'), 6.94 (1H, t, J = 2.4 Hz, H-2), 6.92 (1H, t, J = 2.4 Hz, H-5), 6.48 (2H, s, H-2''6''), 3.84 (3H, s, OCH3), 3.67 (6H, s, OCH3); 13C NMR (DMSO-d6)  153.6, 137.6, 136.5, 132.9, 129.5, 129.2, 126.5, 123.1, 123.0, 118.9, 118.8, 106.4, 61.2, 56.7; EIMS m/z 310 [M+1]+ (20), 309 [M]+ (100), 295 (20), 294 (94), 279 (7), 251 (7), 234 (24), 219 (23), 204 (12), 191 (17), 180 (31), 152 (26), 139 (22). Anal. Calcd for C19H19NO3: C 73.77; H 6.19; N 4.53. Found: C 73.89; H 6.25; N 4.32. 3-(4,7-Dimethoxy-2H-1,3-benzodioxol-5-yl)-4-phenyl-1H-pyrrole (10e). Yellow crystals; 26 mg (60%); mp 136–137 °C; 1H NMR (DMSO-d6)  10.95 (1H, s, NH), 7.21 (2H, t, J = 7.5 Hz, H-3'',5''), 7.16 (2H, d, J = 7.5 Hz, H-2'',6''), 7.08 (1H, t, J = 7.1 Hz, H-4'), 6.97 (1H, s, H-5), 6.79 (1H, s, H-2), 6.29 (1H, s, H-6'), 5.98 (2H, s, OCH2O), 3.61 (3H, s, OCH3), 3.36 (3H, s, OCH3); 13C NMR (DMSO-d6)  140.1, 139.5, 138.1, 136.8, 135.8, 129.2, 128.2, 126.0, 124.0, 123.9, 119.8, 118.6, 117.5, 111.1, 102.5, 60.1, 57.4; EIMS m/z 324 [M+1]+ (22), 323 [M]+ (100), 308 (16), 250 (4), 178 (4), 139 (5). Anal. Calcd for C19H17NO4: C 70.58; H 5.30; N 4.33. Found: C 70.73; H 5.39; N 4.15. Biology. Phenotypic Sea Urchin Embryo Assay.21 Adult sea urchins, Paracentrotus lividus L. (Echinidae), were collected from the Mediterranean Sea on the Cyprus coast and kept in an aerated seawater tank. Gametes were obtained by intracoelomic injection of 0.5 M KCl. Eggs were washed with filtered seawater and fertilized by adding drops of diluted sperm. Embryos were cultured at room temperature under gentle agitation with a motor-driven plastic paddle (60 rpm) in filtered seawater. The embryos were observed with a Biolam light microscope (LOMO, St. Petersburg, Russia). For treatment with the test compounds, 5 mL aliquots of embryo suspension were transferred to six-well plates and incubated as a monolayer at a concentration up to 2000 embryos/mL. Stock solutions of tested molecules were prepared in DMSO at 10 mM concentration followed by a 10-fold dilution with 96% EtOH. This procedure 46 ACS Paragon Plus Environment

Page 47 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


enhanced the solubility of the test compounds in the salt-containing medium (seawater), as evidenced by microscopic examination of the samples. The maximal tolerated concentrations of DMSO and EtOH in the in vivo assay were determined to be 0.2% and 0.5%, respectively. Higher concentrations of either DMSO (0.5%) or EtOH (1%) caused nonspecific alteration and retardation of the sea urchin embryo development independent of the treatment stage. Combretastatins A-4 and A-2 (synthesized as reported previously65, 70) served as reference compounds. The antiproliferative activity was assessed by exposing fertilized eggs (8–15 min after fertilization, 45–55 min before the first mitotic cycle completion) to 2-fold decreasing concentrations of the compound. Cleavage alteration and arrest were clearly detected at 2.5 h and 5.5 h after fertilization, when control embryos reached 8-cell and early blastula stages, respectively. The effects were estimated quantitatively as an effective threshold concentration, resulting in cleavage alteration and embryo death before hatching or full mitotic arrest. At these concentrations all tested microtubule destabilizers caused 100% cleavage alteration and embryo death before hatching, whereas at 2-fold lower concentrations the compounds failed to produce any effect. For microtubule-destabilizing activity, the compounds were tested on free-swimming blastulae just after hatching (8–10 h after fertilization), which originated from the same embryo culture. Embryo spinning was observed after 15 min to 20 h of treatment, depending on the structure and concentration of the compound. Both spinning and lack of forward movement were interpreted to be the result of the microtubule-destabilizing activity of a molecule. Video illustrations are available at http://www.chemblock.com. Sea urchin embryo assay data are available at http://www.zelinsky.ru. Experiments with the sea urchin embryos fulfill the requirements of biological ethics. The artificial spawning does not cause animal death, embryos develop outside the female organism, and both post spawned adult sea urchins and the excess of intact embryos are returned to the sea, their natural habitat. SUPPORTING INFORMATION Analytical data for compounds 3r, 3s, 11n, 13, 13-Ac, 14–16, 22 and 1H and 13C NMR spectra of compounds 1a–u, 3a–q, and 10a–e. This material is available free of charge via the Internet at ....... AUTHOR INFORMATION Corresponding Author. Professor Victor V. Semenov, N. D. Zelinsky Institute of Organic Chemistry RAS, 47 Leninsky Prospect, 119991 Moscow, Russian Federation. Email: [email protected] 47 ACS Paragon Plus Environment


Page 48 of 57

Author Contributions. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources. The Russian Scientific Foundation, Grant no. 18-13-00044. AKNOWLEDGMENTS We thank the National Cancer Institute (NCI) (Bethesda, MD, USA) for screening compounds by the Developmental Therapeutics Program at NCI (Anti-cancer Screening Program; http://dtp.cancer.gov). We also wish to thank Dr. Ju. A. Strelenko from N. D. Zelinsky Institute of Organic Chemistry RAS for the development of NMR spectra presentation software. REFERENCES 1.

Moffat, J. G.; Rudolph, J.; Bailey, D. Phenotypic Screening in Cancer Drug Discovery —

Past, Present and Future. Nat. Rev. Drug Discov. 2014, 13, 588–602. 2.

Pederson, T. The Sea Urchin's Siren. Dev. Biol. 2006, 300, 9–14.

3.

McClay, D. R. Evolutionary Crossroads in Developmental Biology: Sea Urchins.

Development (Cambridge, England) 2011, 138, 2639–2648. 4.

Ernst, S. G. Offerings from an Urchin. Dev. Biol. 2011, 358, 285–294.

5.

Matranga, V.; Corsi, I. Toxic Effects of Engineered Nanoparticles in the Marine

Environment: Model Organisms and Molecular Approaches. Mar. Environ. Res. 2012, 76, 32– 40. 6.

Gambardella, C.; Ferrando, S.; Gatti, A. M.; Cataldi, E.; Ramoino, P.; Aluigi, M. G.;

Faimali, M.; Diaspro, A.; Falugi, C. Review: Morphofunctional and Biochemical Markers of Stress in Sea Urchin Life Stages Exposed to Engineered Nanoparticles. Environ. Toxicol. 2016, 31, 1552–1562. 7.

Agnello, M. Introduction Chapter: Sea Urchin-Knowledge and Perspectives. In Sea

Urchin - from Environment to Aquaculture and Biomedicine, Agnello, M., Ed. InTech: Rijeka, Croatia, 2017; pp 3–16. 8.

Guida, M.; Guida, M.; De Felice, B.; Santafede, D.; Alessandro, R.; Di Spiezio Sardo, A.;

Scognamiglio, M.; Ferrara, C.; Bifulco, G.; Nappi, C. Assessment of DNA Damage by Rapd in Paracentrotus lividus Embryos Exposed to Amniotic Fluid from Residents Living Close to Waste Landfill Sites. J. Biomed. Biotechnol. 2010, 2010, 1–7. 48 ACS Paragon Plus Environment

Page 49 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


9.

Sodergren, E.; Weinstock, G. M.; Davidson, E. H.; Cameron, R. A.; Gibbs, R. A.;

Angerer, R. C.; Angerer, L. M.; Arnone, M. I.; Burgess, D. R.; Burke, R. D.; Coffman, J. A.; Dean, M.; Elphick, M. R.; Ettensohn, C. A.; Foltz, K. R.; Hamdoun, A.; Hynes, R. O.; Klein, W. H.; Marzluff, W.; McClay, D. R.; Morris, R. L.; Mushegian, A.; Rast, J. P.; Smith, L. C.; Thorndyke, M. C.; Vacquier, V. D.; Wessel, G. M.; Wray, G.; Zhang, L.; Elsik, C. G.; Ermolaeva, O.; Hlavina, W.; Hofmann, G.; Kitts, P.; Landrum, M. J.; Mackey, A. J.; Maglott, D.; Panopoulou, G.; Poustka, A. J.; Pruitt, K.; Sapojnikov, V.; Song, X.; Souvorov, A.; Solovyev, V.; Wei, Z.; Whittaker, C. A.; Worley, K.; Durbin, K. J.; Shen, Y.; Fedrigo, O.; Garfield, D.; Haygood, R.; Primus, A.; Satija, R.; Severson, T.; Gonzalez-Garay, M. L.; Jackson, A. R.; Milosavljevic, A.; Tong, M.; Killian, C. E.; Livingston, B. T.; Wilt, F. H.; Adams, N.; Bellé, R.; Carbonneau, S.; Cheung, R.; Cormier, P.; Cosson, B.; Croce, J.; Fernandez-Guerra, A.; Genevière, A.-M.; Goel, M.; Kelkar, H.; Morales, J.; Mulner-Lorillon, O.; Robertson, A. J.; Goldstone, J. V.; Cole, B.; Epel, D.; Gold, B.; Hahn, M. E.; Howard-Ashby, M.; Scally, M.; Stegeman, J. J.; Allgood, E. L.; Cool, J.; Judkins, K. M.; McCafferty, S. S.; Musante, A. M.; Obar, R. A.; Rawson, A. P.; Rossetti, B. J.; Gibbons, I. R.; Hoffman, M. P.; Leone, A.; Istrail, S.; Materna, S. C.; Samanta, M. P.; Stolc, V.; Tongprasit, W.; Tu, Q.; Bergeron, K.-F.; Brandhorst, B. P.; Whittle, J.; Berney, K.; Bottjer, D. J.; Calestani, C.; Peterson, K.; Chow, E.; Yuan, Q. A.; Elhaik, E.; Graur, D.; Reese, J. T.; Bosdet, I.; Heesun, S.; Marra, M. A.; Schein, J.; Anderson, M. K.; Brockton, V.; Buckley, K. M.; Cohen, A. H.; Fugmann, S. D.; Hibino, T.; Loza-Coll, M.; Majeske, A. J.; Messier, C.; Nair, S. V.; Pancer, Z.; Terwilliger, D. P.; Agca, C.; Arboleda, E.; Chen, N.; Churcher, A. M.; Hallböök, F.; Humphrey, G. W.; Idris, M. M.; Kiyama, T.; Liang, S.; Mellott, D.; Mu, X.; Murray, G.; Olinski, R. P.; Raible, F.; Rowe, M.; Taylor, J. S.; TessmarRaible, K.; Wang, D.; Wilson, K. H.; Yaguchi, S.; Gaasterland, T.; Galindo, B. E.; Gunaratne, H. J.; Juliano, C.; Kinukawa, M.; Moy, G. W.; Neill, A. T.; Nomura, M.; Raisch, M.; Reade, A.; Roux, M. M.; Song, J. L.; Su, Y.-H.; Townley, I. K.; Voronina, E.; Wong, J. L.; Amore, G.; Branno, M.; Brown, E. R.; Cavalieri, V.; Duboc, V.; Duloquin, L.; Flytzanis, C.; Gache, C.; Lapraz, F.; Lepage, T.; Locascio, A.; Martinez, P.; Matassi, G.; Matranga, V.; Range, R.; Rizzo, F.; Röttinger, E.; Beane, W.; Bradham, C.; Byrum, C.; Glenn, T.; Hussain, S.; Manning, G.; Miranda, E.; Thomason, R.; Walton, K.; Wikramanayke, A.; Wu, S.-Y.; Xu, R.; Brown, C. T.; Chen, L.; Gray, R. F.; Lee, P. Y.; Nam, J.; Oliveri, P.; Smith, J.; Muzny, D.; Bell, S.; Chacko, J.; Cree, A.; Curry, S.; Davis, C.; Dinh, H.; Dugan-Rocha, S.; Fowler, J.; Gill, R.; Hamilton, C.; Hernandez, J.; Hines, S.; Hume, J.; Jackson, L.; Jolivet, A.; Kovar, C.; Lee, S.; Lewis, L.; Miner, G.; Morgan, M.; Nazareth, L. V.; Okwuonu, G.; Parker, D.; Pu, L.-L.; Thorn, R.; Wright, R. The Genome of the Sea Urchin Strongylocentrotus purpuratus. Science (New York, N.Y.) 2006, 314, 941–952. 49 ACS Paragon Plus Environment


10.

Page 50 of 57

Di Bernardo, M.; Di Carlo, M. The Sea Urchin Embryo: A Model for Studying Molecular

Mechanisms Involved in Human Diseases and for Testing Bioactive Compounds. In Sea Urchin - from Environment to Aquaculture and Biomedicine, Agnello, M., Ed. InTech: Rijeka, Croatia, 2017; pp 119–144. 11.

Falugi, C.; Lammerding-Koppel, M.; Aluigi, M. G. Sea Urchin Development: An

Alternative Model for Mechanistic Understanding of Neurodevelopment and Neurotoxicity. Birth Defects Res. C 2008, 84, 188–203. 12.

Kumburegama, S.; Wikramanayake, A. H. Wnt Signaling in the Early Sea Urchin

Embryo. Methods Mol. Biol. 2008, 469, 187–199. 13.

Range, R. C.; Martinez-Bartolome, M.; Burr, S. D. The Power of Simplicity: Sea Urchin

Embryos as in Vivo Developmental Models for Studying Complex Cell-to-Cell Signaling Network Interactions. J. Vis. Exp. 2017, (120), e55113. 14.

de Andrade Cunha, G. M.; Fontenele, J. B.; Nobre Júnior, H. V.; de Sousa, F. C. M.;

Silveira, E. R.; Nogueira, N. A. P.; de Moraes, M. O.; Viana, G. S. B.; Costa‐Lotufo, L. V. Cytotoxic Activity of Chalcones Isolated from Lonchocarpus sericeus (Pocr.) Kunth. Phytother. Res. 2003, 17, 155–159. 15.

Madari, H.; Panda, D.; Wilson, L.; Jacobs, R. S. Dicoumarol: A Unique Microtubule

Stabilizing Natural Product That Is Synergistic with Taxol. Cancer Res. 2003, 63, 1214–1220. 16.

Nishioka, D.; Marcell, V.; Cunningham, M.; Khan, M.; Von Hoff, D. D.; Izbicka, E. The

Use of Early Sea Urchin Embryos in Anticancer Drug Testing. In Novel Anticancer Drug Protocols, Buolamwini, J. K.; Adjei, A. A., Eds. Humana Press: Totowa, NJ, 2003; pp 265–276. 17.

Militão, G. C. G.; Jimenez, P. C.; Wilke, D. V.; Pessoa, C.; Falcão, M. J.; Lima, M. A. S.;

Silveira, E. R.; de Moraes, M. O.; Costa-Lotufo, L. V. Antimitotic Properties of Pterocarpans Isolated from Platymiscium floribundum on Sea Urchin Eggs. Planta Med. 2005, 71, 683–685. 18.

de Lima, D. P.; Rotta, R.; Beatriz, A.; Marques, M. R.; Montenegro, R. C.; Vasconcellos,

M. C.; Pessoa, C.; de Moraes, M. O.; Costa-Lotufo, L. V.; Frankland Sawaya, A. C. H.; Eberlin, M. N. Synthesis and Biological Evaluation of Cytotoxic Properties of Stilbene-Based Resveratrol Analogs. Eur. J. Med. Chem. 2009, 44, 701–707. 19.

Magalhães, H. I. F.; Bezerra, D. P.; Cavalcanti, B. C.; Wilke, D. V.; Rotta, R.; de Lima,

D. P.; Beatriz, A.; Alves, A. P. N. N.; Bitencourt, F. d. S.; de Figueiredo, I. S. T.; Alencar, N. M. N.; Costa-Lotufo, L. V.; Moraes, M. O.; Pessoa, C. In Vitro and in Vivo Antitumor Effects of (4Methoxyphenyl)(3,4,5-Trimethoxyphenyl)Methanone. Cancer Chemoth. Pharm. 2011, 68, 45– 52.


Page 51 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


20.

Macedo, D.; Mendonca Junior, F. J.; de Moura, R. O.; Marques-Santos, L. F. Antimitotic

Activity of the Pyrimidinone Derivative Py-09 on Sea Urchin Embryonic Development. Toxicol. In Vitro 2016, 31, 72–85. 21.

Semenova, M. N.; Kiselyov, A. S.; Semenov, V. V. Sea Urchin Embryo as a Model

Organism for the Rapid Functional Screening of Tubulin Modulators. BioTechniques 2006, 40, 765–774. 22.

Semenova, M. N.; Kiselyov, A. S.; Titov, I. Y.; Raihstat, M. M.; Molodtsov, M.;

Grishchuk, E.; Spiridonov, I.; Semenov, V. V. In Vivo Evaluation of Indolyl Glyoxamides in the Phenotypic Sea Urchin Embryo Assay. Chem. Biol. Drug Des. 2007, 70, 485–490. 23.

Kiselyov, A. S.; Semenova, M. N.; Chernyshova, N. B.; Leitao, A.; Samet, A. V.; Kislyi,

K. A.; Raihstat, M. M.; Oprea, T.; Lemcke, H.; Lantow, M.; Weiss, D. G.; Ikizalp, N. N.; Kuznetsov, S. A.; Semenov, V. V. Novel Derivatives of 1,3,4-Oxadiazoles Are Potent Mitostatic Agents Featuring Strong Microtubule Depolymerizing Activity in the Sea Urchin Embryo and Cell Culture Assays. Eur. J. Med. Chem. 2010, 45, 1683–1697. 24.

Semenova, M. N.; Kiselyov, A. S.; Tsyganov, D. V.; Konyushkin, L. D.; Firgang, S. I.;

Semenov, R. V.; Malyshev, O. R.; Raihstat, M. M.; Fuchs, F.; Stielow, A.; Lantow, M.; Philchenkov, A. A.; Zavelevich, M. P.; Zefirov, N. S.; Kuznetsov, S. A.; Semenov, V. V. Polyalkoxybenzenes from Plants. 5. Parsley Seed Extract in Synthesis of Azapodophyllotoxins Featuring Strong Tubulin Destabilizing Activity in the Sea Urchin Embryo and Cell Culture Assays. J. Med. Chem. 2011, 54, 7138–7149. 25.

Nam, N. H. Combretastatin A-4 Analogues as Antimitotic Antitumor Agents. Curr. Med.

Chem. 2003, 10, 1697–722. 26.

Tron, G. C.; Pirali, T.; Sorba, G.; Pagliai, F.; Busacca, S.; Genazzani, A. A. Medicinal

Chemistry of Combretastatin A4: Present and Future Directions. J. Med. Chem. 2006, 49, 3033– 3044. 27.

Mikstacka, R.; Stefanski, T.; Rozanski, J. Tubulin-Interactive Stilbene Derivatives as

Anticancer Agents. Cell. Mol. Biol. Lett. 2013, 18, 368–397. 28.

Jaroch, K.; Karolak, M.; Gorski, P.; Jaroch, A.; Krajewski, A.; Ilnicka, A.; Sloderbach,

A.; Stefanski, T.; Sobiak, S. Combretastatins: In Vitro Structure-Activity Relationship, Mode of Action and Current Clinical Status. Pharmacol. Rep. 2016, 68, 1266–1275. 29.

Rajak, H.; Dewangan, P. K.; Patel, V.; Jain, D. K.; Singh, A.; Veerasamy, R.; Sharma, P.

C.; Dixit, A. Design of Combretastatin a-4 Analogs as Tubulin Targeted Vascular Disrupting Agent with Special Emphasis on Their Cis-Restricted Isomers. Curr. Pharm. Des. 2013, 19, 1923–1955. 51 ACS Paragon Plus Environment


30.

Page 52 of 57

Cushman, M.; Nagarathnam, D.; Gopal, D.; Chakraborti, A. K.; Lin, C. M.; Hamel, E.

Synthesis and Evaluation of Stilbene and Dihydrostilbene Derivatives as Potential Anticancer Agents That Inhibit Tubulin Polymerization. J. Med. Chem. 1991, 34, 2579–2588. 31.

Pettit, G. R.; Rhodes, M. R.; Herald, D. L.; Hamel, E.; Schmidt, J. M.; Pettit, R. K.

Antineoplastic Agents. 445. Synthesis and Evaluation of Structural Modifications of (Z)- and (E)-Combretastatin A-4. J. Med. Chem. 2005, 48, 4087–4099. 32.

Pati, H., N.; Wicks, M.; Holt Jr. Herman, L.; LeBlanc, R.; Weisbruch, P.; Forrest, L.;

Lee, M. Synthesis and Biological Evaluation of Cis-Combretastatin Analogs and Their Novel 1,2,3-Triazole Derivatives. Heterocyclic Commun. 2005, 11, 117–120. 33.

Kaffy, J.; Pontikis, R.; Carrez, D.; Croisy, A.; Monneret, C.; Florent, J.-C. Isoxazole-

Type Derivatives Related to Combretastatin a-4, Synthesis and Biological Evaluation. Bioorg. Med. Chem. 2006, 14, 4067–4077. 34.

Sun, C.-M.; Lin, L.-G.; Yu, H.-J.; Cheng, C.-Y.; Tsai, Y.-C.; Chu, C.-W.; Din, Y.-H.;

Chau, Y.-P.; Don, M.-J. Synthesis and Cytotoxic Activities of 4,5-Diarylisoxazoles. Bioorg. Med. Chem. Lett. 2007, 17, 1078–1081. 35.

Zhang, Q.; Peng, Y.; Wang, X. I.; Keenan, S. M.; Arora, S.; Welsh, W. J. Highly Potent

Triazole-Based Tubulin Polymerization Inhibitors. J. Med. Chem. 2007, 50, 749–754. 36.

Cafici, L.; Pirali, T.; Condorelli, F.; Del Grosso, E.; Massarotti, A.; Sorba, G.; Canonico,

P. L.; Tron, G. C.; Genazzani, A. A. Solution-Phase Parallel Synthesis and Biological Evaluation of Combretatriazoles. J. Comb. Chem. 2008, 10, 732–740. 37.

Odlo, K.; Hentzen, J.; dit Chabert, J. F.; Ducki, S.; Gani, O. A. B. S. M.; Sylte, I.; Skrede,

M.; Flørenes, V. A.; Hansen, T. V. 1,5-Disubstituted 1,2,3-Triazoles as Cis-Restricted Analogues of Combretastatin A-4: Synthesis, Molecular Modeling and Evaluation as Cytotoxic Agents and Inhibitors of Tubulin. Bioorg. Med. Chem. 2008, 16, 4829–4838. 38.

Xue, N.; Yang, X.; Wu, R.; Chen, J.; He, Q.; Yang, B.; Lu, X.; Hu, Y. Synthesis and

Biological Evaluation of Imidazol-2-One Derivatives as Potential Antitumor Agents. Bioorg. Med. Chem. 2008, 16, 2550–2557. 39.

Liu, T.; Dong, X.; Xue, N.; Wu, R.; He, Q.; Yang, B.; Hu, Y. Synthesis and Biological

Evaluation of 3,4-Diaryl-5-Aminoisoxazole Derivatives. Bioorg. Med. Chem. 2009, 17, 6279– 6285. 40.

Biersack, B.; Effenberger, K.; Knauer, S.; Ocker, M.; Schobert, R. Ru(Η6-Arene)

Complexes of Combretastatin-Analogous Oxazoles with Enhanced Anti-Tumoral Impact. Eur. J. Med. Chem. 2010, 45, 4890–4896.


Page 53 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


41.

Burja, B.; Čimbora-Zovko, T.; Tomić, S.; Jelušić, T.; Kočevar, M.; Polanc, S.; Osmak,

M. Pyrazolone-Fused Combretastatins and Their Precursors: Synthesis, Cytotoxicity, Antitubulin Activity and Molecular Modeling Studies. Bioorg. Med. Chem. 2010, 18, 2375–2387. 42.

Odlo, K.; Fournier-Dit-Chabert, J.; Ducki, S.; Gani, O. A.; Sylte, I.; Hansen, T. V. 1,2,3-

Triazole Analogs of Combretastatin A-4 as Potential Microtubule-Binding Agents. Bioorg. Med. Chem. 2010, 18, 6874–6885. 43.

Romagnoli, R.; Baraldi, P. G.; Cruz-Lopez, O.; Lopez Cara, C.; Carrion, M. D.; Brancale,

A.; Hamel, E.; Chen, L.; Bortolozzi, R.; Basso, G.; Viola, G. Synthesis and Antitumor Activity of 1,5-Disubstituted 1,2,4-Triazoles as Cis-Restricted Combretastatin Analogues. J. Med. Chem. 2010, 53, 4248–4258. 44.

Liu, T.; Cui, R.; Chen, J.; Zhang, J.; He, Q.; Yang, B.; Hu, Y. 4,5-Diaryl-3-

Aminopyrazole Derivatives as Analogs of Combretastatin A-4: Synthesis and Biological Evaluation. Arch. Pharm. (Weinheim) 2011, 344, 279–286. 45.

Akselsen, Ø. W.; Odlo, K.; Cheng, J.-J.; Maccari, G.; Botta, M.; Hansen, T. V. Synthesis,

Biological Evaluation and Molecular Modeling of 1,2,3-Triazole Analogs of Combretastatin A1. Bioorg. Med. Chem. 2012, 20, 234–242. 46.

Beale, T. M.; Bond, P. J.; Brenton, J. D.; Charnock-Jones, D. S.; Ley, S. V.; Myers, R. M.

Increased Endothelial Cell Selectivity of Triazole-Bridged Dihalogenated A-Ring Analogues of Combretastatin A–1. Bioorg. Med. Chem. 2012, 20, 1749–1759. 47.

Romagnoli, R.; Baraldi, P. G.; Salvador, M. K.; Preti, D.; Aghazadeh Tabrizi, M.;

Brancale, A.; Fu, X. H.; Li, J.; Zhang, S. Z.; Hamel, E.; Bortolozzi, R.; Basso, G.; Viola, G. Synthesis and Evaluation of 1,5-Disubstituted Tetrazoles as Rigid Analogues of Combretastatin A-4 with Potent Antiproliferative and Antitumor Activity. J. Med. Chem. 2012, 55, 475–488. 48.

Tsyganov, D. V.; Konyushkin, L. D.; Karmanova, I. B.; Firgang, S. I.; Strelenko, Y. A.;

Semenova, M. N.; Kiselyov, A. S.; Semenov, V. V. Cis-Restricted 3-Aminopyrazole Analogues of Combretastatins: Synthesis from Plant Polyalkoxybenzenes and Biological Evaluation in the Cytotoxicity and Phenotypic Sea Urchin Embryo Assays. J. Nat. Prod. 2013, 76, 1485–1491. 49.

Zaninetti, R.; Cortese, S. V.; Aprile, S.; Massarotti, A.; Canonico, P. L.; Sorba, G.; Grosa,

G.; Genazzani, A. A.; Pirali, T. A Concise Synthesis of Pyrazole Analogues of Combretastatin A1 as Potent Anti-Tubulin Agents. ChemMedChem. 2013, 8, 633–643. 50.

Demchuk, D. V.; Samet, A. V.; Chernysheva, N. B.; Ushkarov, V. I.; Stashina, G. A.;

Konyushkin, L. D.; Raihstat, M. M.; Firgang, S. I.; Philchenkov, A. A.; Zavelevich, M. P.; Kuiava, L. M.; Chekhun, V. F.; Blokhin, D. Y.; Kiselyov, A. S.; Semenova, M. N.; Semenov, V. V. Synthesis and Antiproliferative Activity of Conformationally Restricted 1,2,3-Triazole 53 ACS Paragon Plus Environment


Page 54 of 57

Analogues of Combretastatins in the Sea Urchin Embryo Model and against Human Cancer Cell Lines. Bioorg. Med. Chem. 2014, 22, 738–755. 51.

Tsyganov, D. V.; Khrustalev, V. N.; Konyushkin, L. D.; Raihstat, M. M.; Firgang, S. I.;

Semenov, R. V.; Kiselyov, A. S.; Semenova, M. N.; Semenov, V. V. 3-(5-)-Amino-ODiarylisoxazoles: Regioselective Synthesis and Antitubulin Activity. Eur. J. Med. Chem. 2014, 73, 112–125. 52.

Madadi, N. R.; Penthala, N. R.; Howk, K.; Ketkar, A.; Eoff, R. L.; Borrelli, M. J.;

Crooks, P. A. Synthesis and Biological Evaluation of Novel 4,5-Disubstituted 2h-1,2,3-Triazoles as Cis-Constrained Analogues of Combretastatin A-4. Eur. J. Med. Chem. 2015, 103, 123–132. 53.

Brown, A. W.; Fisher, M.; Tozer, G. M.; Kanthou, C.; Harrity, J. P. A. Sydnone

Cycloaddition Route to Pyrazole-Based Analogs of Combretastatin A4. J. Med. Chem. 2016, 59, 9473–9488. 54.

Romagnoli, R.; Baraldi, P. G.; Prencipe, F.; Oliva, P.; Baraldi, S.; Tabrizi, M. A.; Lopez-

Cara, L. C.; Ferla, S.; Brancale, A.; Hamel, E.; Ronca, R.; Bortolozzi, R.; Mariotto, E.; Basso, G.; Viola, G. Design and Synthesis of Potent in Vitro and in Vivo Anticancer Agents Based on 1-(3',4',5'-Trimethoxyphenyl)-2-Aryl-1H-Imidazole. Sci. Rep. 2016, 6, 26602. 55.

Chernysheva, N. B.; Maksimenko, A. S.; Andreyanov, F. A.; Kislyi, V. P.; Strelenko, Y.

A.; Khrustalev, V. N.; Semenova, M. N.; Semenov, V. V. Regioselective Synthesis of 3,4Diaryl-5-Unsubstituted Isoxazoles, Analogues of Natural Cytostatic Combretastatin A4. Eur. J. Med. Chem. 2018, 146, 511–518. 56.

Gaukroger, K.; Hadfield, J. A.; Lawrence, N. J.; Nolan, S.; McGown, A. T. Structural

Requirements for the Interaction of Combretastatins with Tubulin: How Important Is the Trimethoxy Unit? Org. Biomol. Chem. 2003, 1, 3033–3037. 57.

Duan, J.-X.; Cai, X.; Meng, F.; Lan, L.; Hart, C.; Matteucci, M. Potent Antitubulin

Tumor Cell Cytotoxins Based on 3-Aroyl Indazoles. J. Med. Chem. 2007, 50, 1001–1006. 58.

Ghinet, A.; Rigo, B.; Hénichart, J.-P.; Le Broc-Ryckewaert, D.; Pommery, J.; Pommery,

N.; Thuru, X.; Quesnel, B.; Gautret, P. Synthesis and Biological Evaluation of Phenstatin Metabolites. Bioorg. Med. Chem. 2011, 19, 6042–6054. 59.

Horbach, S. P. J. M.; Halffman, W. The Ghosts of Hela: How Cell Line Misidentification

Contaminates the Scientific Literature. PLOS ONE 2017, 12, e0186281. 60.

Zhou, J.; Jin, J.; Zhang, Y.; Yin, Y.; Chen, X.; Xu, B. Synthesis and Antiproliferative

Evaluation of Novel Benzoimidazole-Contained Oxazole-Bridged Analogs of Combretastatin A4. Eur. J. Med. Chem. 2013, 68, 222–232. 61.

Vilanova, C.; Díaz-Oltra, S.; Murga, J.; Falomir, E.; Carda, M.; Redondo-Horcajo, M.;

Díaz, J. F.; Barasoain, I.; Marco, J. A. Design and Synthesis of Pironetin Analogue/Colchicine 54 ACS Paragon Plus Environment

Page 55 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Hybrids and Study of Their Cytotoxic Activity and Mechanisms of Interaction with Tubulin. J. Med. Chem. 2014, 57, 10391–10403. 62.

Rajitha, C.; Dubey, P. K.; Sunku, V.; Piedrafita, F. J.; Veeramaneni, V. R.; Pal, M.

Synthesis and Pharmacological Evaluations of Novel 2H-Benzo[B][1,4]Oxazin-3(4h)-One Derivatives as a New Class of Anti-Cancer Agents. Eur. J. Med. Chem. 2011, 46, 4887–4896. 63.

Semenov, V. V.; Tsyganov, D. V.; Semenova, M. N.; Chuprov-Netochin, R. N.; Raihstat,

M. M.; Konyushkin, L. D.; Volynchuk, P. B.; Marusich, E. I.; Nazarenko, V. V.; Leonov, S. V.; Kiselyov, A. S. Efficient Synthesis of Glaziovianin A Isoflavone Series from Dill and Parsley Extracts and Their in Vitro/in Vivo Antimitotic Activity. J. Nat. Prod. 2016, 79, 1429–1438. 64.

Chernyshova, N. B.; Tsyganov, D. V.; Khrustalev, V. N.; Raihstat, M. M.; Konyushkin,

L. D.; Semenov, R. V.; Semenova, M. N.; Semenov, V. V. Synthesis and Antimitotic Properties of Ortho-Substituted Polymethoxydiarylazolopyrimidines. ARKIVOC 2017, 2017 Part (iii), 151–165. 65.

Semenov, V. V.; Kiselyov, A. S.; Titov, I. Y.; Sagamanova, I. K.; Ikizalp, N. N.;

Chernysheva, N. B.; Tsyganov, D. V.; Konyushkin, L. D.; Firgang, S. I.; Semenov, R. V.; Karmanova, I. B.; Raihstat, M. M.; Semenova, M. N. Synthesis of Antimitotic Polyalkoxyphenyl Derivatives of Combretastatin Using Plant Allylpolyalkoxybenzenes. J. Nat. Prod. 2010, 73, 1796–1802. 66.

Metzger, A.; Piller, F. M.; Knochel, P. Polyfunctional Benzylic Zinc Chlorides by the

Direct Insertion of Magnesium into Benzylic Chlorides in the Presence of LiCl and ZnCl2. Chem. Commun. 2008, 5824–5826. 67.

Sun, L.; Borella, C.; Li, H.; Jiang, J.; Chen, S.; Koya, K.; Inoue, T.; Du, Z.; Foley, K.;

Wu, Y.; Zhang, M.; Ying, W. Isoxazole Combretastatin Derivatives for the Treatment of Disorders. WO2006/089177, 2006. 68.

Chernysheva, N. B.; Maksimenko, A. S.; Andreyanov, F. A.; Kislyi, V. P.; Strelenko, Y.

A.; Khrustalev, V. N.; Semenova, M. N.; Semenov, V. V. Synthesis of 3,4-Diaryl-5-Carboxy4,5-Dihydroisoxazole 2-Oxides as Valuable Synthons for Anticancer Molecules. Tetrahedron 2017, 73, 6728–6735. 69.

Ohsumi, K.; Hatanaka, T.; Fujita, K.; Nakagawa, R.; Fukuda, Y.; Nihei, Y.; Suga, Y.;

Morinaga, Y.; Akiyama, Y.; Tsuji, T. Syntheses and Antitumor Activity of Cis-Restricted Combretastatins: 5-Membered Heterocyclic Analogues. Bioorg. Med. Chem. Lett. 1998, 8, 3153–3158. 70.

Pettit, G. R.; Singh, S. B.; Boyd, M. R.; Hamel, E.; Pettit, R. K.; Schmidt, J. M.; Hogan,

F. Antineoplastic Agents. 291. Isolation and Synthesis of Combretastatins A-4, A-5, and A-6. J. Med. Chem. 1995, 38, 1666–1672. 55 ACS Paragon Plus Environment


71.

Page 56 of 57

Pettit, G. R.; Anderson, C. R.; Herald, D. L.; Jung, M. K.; Lee, D. J.; Hamel, E.; Pettit, R.

K. Antineoplastic Agents. 487. Synthesis and Biological Evaluation of the Antineoplastic Agent 3,4-Methylenedioxy-5,4‘-Dimethoxy-3‘-Amino-Z-Stilbene and Derived Amino Acid Amides. J. Med. Chem. 2003, 46, 525–531. 72.

Ohsumi, K.; Nakagawa, R.; Fukuda, Y.; Hatanaka, T.; Morinaga, Y.; Nihei, Y.; Ohishi,

K.; Suga, Y.; Akiyama, Y.; Tsuji, T. Novel Combretastatin Analogues Effective against Murine Solid Tumors: Design and Structure−Activity Relationships. J. Med. Chem. 1998, 41, 3022– 3032. 73.

Monk, K. A.; Siles, R.; Hadimani, M. B.; Mugabe, B. E.; Ackley, J. F.; Studerus, S. W.;

Edvardsen, K.; Trawick, M. L.; Garner, C. M.; Rhodes, M. R.; Pettit, G. R.; Pinney, K. G. Design, Synthesis, and Biological Evaluation of Combretastatin Nitrogen-Containing Derivatives as Inhibitors of Tubulin Assembly and Vascular Disrupting Agents. Bioorg. Med. Chem. 2006, 14, 3231–3244. 74.

Tsyganov, D. V.; Semenova, M. N.; Konyushkin, L. D.; Ushkarov, V. I.; Raihstat, M. M.;

Semenov, V. V. Convenient Synthesis of Cis-Restricted Pyrazole and Isoxazole Analogues of Combretastatins. Mendeleev Commun. 2019, 29, N2, DOI: 10.1016/j.mencom.2019.03.015. 75.

Blagg, J. Structure–Activity Relationships for in Vitro and in Vivo Toxicity. In Annual

Reports in Medicinal Chemistry, Wood, A., Ed. Academic Press: 2006; Vol. 41, pp 353–368. 76.

Combretastatin A4 MSDS. http://www.molbase.com/en/msds_117048-59-6-moldata-

89575.html#tabs. 77.

Saxena, H. O.; Faridi, U.; Srivastava, S.; Kumar, J. K.; Darokar, M. P.; Luqman, S.;

Chanotiya, C. S.; Krishna, V.; Negi, A. S.; Khanuja, S. P. S. Gallic Acid-Based Indanone Derivatives as Anticancer Agents. Bioorg. Med. Chem. Lett. 2008, 18, 3914–3918. 78.

SanMartín, R.; de Marigorta, E. M.; Domínguez, E. A Convenient Alternative Route to

Β-Aminoketones. Tetrahedron 1994, 50, 2255–2264. 79.

Nose, A.; Kudo, T. Reduction with Sodium Borohydride-Transition Metal Salt Systems.

I. Reduction of Aromatic Nitro Compounds with the Sodium Borohydride-Nickelous Chloride System. Chem. Pharm. Bull. 1981, 29, 1159–1161. 80.

Domínguez, E.; Ibeas, E.; Martínez de Marigorta, E.; Palacios, J. K.; SanMartín, R. A

Convenient One-Pot Preparative Method for 4,5-Diarylisoxazoles Involving Amine Exchange Reactions. J. Org. Chem. 1996, 61, 5435–5439. 81.

Lee, S.; Kim, J. N.; Lee, H. K.; Yoon, K. S.; Shin, K. D.; Kwon, B.-M.; Han, D. C.

Biological Evaluation of KRIBB3 Analogs as a Microtubule Polymerization Inhibitor. Bioorg. Med. Chem. Lett. 2011, 21, 977–979. 56 ACS Paragon Plus Environment

Page 57 of 57 1 2 3 4 5 6 7


ACS Paragon Plus Environment

Sea Urchin Embryo Model As a Reliable in Vivo ... - ACS Publications

Recommend Documents