“A Novel Conjugated Oxa-spiro Derivative Compound”.

Abstract

ABSTRACT: The present invention relates to a novel conjugated Oxa-spiro Derivative Compound of formula I and salts thereof. More particularly, the present invention relates to a process for preparation of a novel conjugated Oxa-spiro Derivative Compound of formula I. Further, the present invention relates to novel conjugated Oxa-spiro Derivative with potent anti-cancer activity as pharmaceutical agents and their pharmaceutically acceptable salts and their pharmaceutically acceptable solvents or mixture thereof.

Specification

Description:FIELD OF INVENTION:

The present invention relates to a novel conjugated Oxa-spiro Derivative Compound of formula I and salts thereof. More particularly, the present invention relates to a process for preparation of a novel conjugated Oxa-spiro Derivative Compound of formula I. Further, the synthesis of the novel conjugated oxa-spiro derivative compound of formula I, R1 and R3 underwent a strategic cyclization reaction to form a six-membered ring structure. The present invention relates to novel conjugated Oxa-spiro Derivative with potent anti-cancer activity as pharmaceutical agents and their pharmaceutically acceptable salts and their pharmaceutically acceptable solvents or mixture thereof.

Formula I
wherein, R1 and R3 combined with each other to form a six-membered ring
R1 = -H, -Br, -Ar, -COOH, -NO2
R2 = -H or -OCH3
R3 = -H and
x = -CH2-, -CH2-CH2-

BACKGROUND OF INVENTION:

Saturated monocyclic units cyclohexane, cyclopentane, piperidine, etc. dominated in chemistry and in drug discovery for a long time. The situation started changing at the beginning of this century. In 2009, Lovering introduced the concept of "escape from flatland", which already changed the way medicinal chemists think. Today, scientists tend to use small F(sp3)-rich molecules in their research. In 2010, saturated spirocycles were shown to possess improved physico-chemical characteristics over their common monocyclic counterparts. Since that time, spirocyclic molecules have been playing an important role in chemistry.

Spirobibenzopyran is a spiroketal molecule first reported by Borsche and Geyer in Justus Liebigs Annalen der Chemie (1913), 393, 29-60. They have reported the synthesis of the molecules of the type (II):


Fatih M. Uckun et al have disclosed novel tubulin binding compounds (SPIKETS, III) with potent tubulin depolymerization activity and inhibitory activity against tubulin polymerization. in US 2002/0151580 A1. The compounds were stated to be effective agents for inhibiting cellular proliferation, for example, in cancer cells. The compounds are adapted to interact favourably with a novel binding pocket on tubulin. The general structure of the compounds were given as under.

Below are some of the examples and the structure:

Most recently, M. Livendahl et al have explored the structures of the type X and XI for their interaction with G-quadruplex (G4) structures which are involved in many important biological processes and can be linked to several human diseases. Some of the synthesized spirocyclic compounds were found to efficiently stabilize G4 structures without inducing structural changes by binding the first G-tetrad in the G4 structure ( Organic & Biomolecular Chemistry (2017), 15(15), 3265-3275)


Since then the spiroketals have been explored majorly as synthetic challenge. Spiropyrans have also been explored because of their photochromic and thermochromic properties. They have found good applications in display and storage of information. Many of the spiropyrans were explored for printing and photocopying purposes where upon acidic treatment coloured material was obtained. However, biological and pharmacological properties of this class of compounds have not been reported extensively.

Oxa-spiro compounds are characterized by their unique structural framework, which typically exhibits interesting chemical and physical properties. However, the synthesis of novel derivatives that can enhance their functionality remains a challenge in the field. There is a need for new compounds that can exhibit improved biological activity and utility in various applications.

Oxa-spiro derivatives constitute a distinctive class of organic compounds defined by their intricate spirocyclic architectures, which integrate oxygen atoms into the molecular framework. These derivatives demonstrate significant chemical versatility, positioning them as promising candidates for diverse applications, particularly within the realms of pharmaceuticals and materials science. The spiro center introduces enhanced molecular rigidity, facilitating targeted interactions with biomolecular entities, which may lead to improved therapeutic efficacy. Furthermore, the presence of conjugated π-systems in oxa-spiro derivatives can endow them with advantageous optical and electronic characteristics, thereby enabling advancements in organic electronics and optoelectronic applications. Ongoing research into their multifaceted functionalities underscores the potential of oxa-spiro derivatives to contribute substantially to the development of innovative materials and novel therapeutic agents.

In the above prior art no reports of a conjugated oxa-spiro derivative compound of formula I has not been disclosed.

SUMMARY OF INVENTION:

The principal objective of the present invention is to synthesize a novel conjugated oxa-spiro derivative compound of formula I.

wherein, R1 and R3 combined with each other to form a six-membered ring
R1 = -H, -Br, -Ar, -COOH, -NO2
R2 = -H or -OCH3
R3 = -H and
x = -CH2-, -CH2-CH2-

Another objective of the present invention is to provide a process for preparation of a novel conjugated oxa-spiro derivative compound of formula I comprises: reaction of Hydroxybenzaldehydes and cycloalkanones in presence of solvent with secondary amines and acid.

Another objective of the present invention is to provide preparation of conjugated oxa-spiro derivative compound of formula I, wherein a Hydroxybenzaldehydes are selected from 2-hydroxybenzaldehyde, 5-bromo-2-hydroxybenzaldehyde, 2-hydroxy-3-methoxybenzaldehyde, 2-hydroxy-1-naphthaldehyde, 5-nitro-2-hydroxybenzaldehyde, 3-formyl-4-hydroxybenzoic acid.

Another objective of the present invention is to provide preparation of conjugated oxa-spiro derivative compound of formula I, wherein solvent are selected from Methanol, Absolute alcohol, DCM, acetonitrile, petroleum ether, water, Ethyl acetate, isopropyl alcohol, acetone, hexane, chloroform, diethyl ether, Dimethylformamide, n-propanol.

Another objective of the present invention is to provide preparation of conjugated oxa-spiro derivative compound of formula I, wherein acids are selected from glacial Acetic acid, Con. Hydrochloric acid, acetic acid, sulphuric acid, citric acid, formic acid.

Another objective of the present invention is to provide preparation of conjugated oxa-spiro derivative compound of formula I, wherein secondary amines are selected from pyrrolidine, piperidine, morpholine, diethyl amine.

DETAILED DESCRIPTION OF INVENTION:
In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented here. The present technology is also illustrated by the examples herein, which should not be construed as limiting in any way.

As used herein, the terms below have the meanings indicated.

The singular forms "a," "an," and "the" may refer to plural articles unless specifically stated otherwise.

The term "about," as used herein, is intended to qualify the numerical values which it modifies, denoting such a value as variable within a margin of error. When no particular margin of error, such as a standard deviation to a mean value given in a chart or table of data, is recited, the term "about" should be understood to mean that range which would encompass the recited value and the range which would be included by rounding up or down to that figure as well, taking into account significant figures.

The embodiments, illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed technology. Additionally, the phrase "consisting essentially of" will be understood to include those elements specifically recited and those additional elements that do not materially affect the basic and novel characteristics of the claimed technology. The phrase "consisting of" excludes any element not specified.

The compounds, compositions, articles, devices, and methods described herein can be understood more readily by reference to the following detailed description of specific aspects of the disclosed subject matter and the Examples and Figures.

Likewise, many modifications and other embodiments of the compositions and methods described herein will come to mind to one of skill in the art to which the invention pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which the invention pertains. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.

The term "pharmaceutically acceptable" as used herein includes reference to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. This term includes acceptability for both human and veterinary purposes.

The present invention is to the strategic fusion of substituents R1 and R3 facilitates the cyclization process to yield a six-membered ring structure within the conjugated oxa-spiro derivative compound denoted as formula I. This transformation is critical as it enhances the compound's electronic delocalization and steric configuration, thereby optimizing its structural integrity and reactivity profiles. The formation of this six-membered ring introduces a unique interplay of electronic interactions and steric effects, which are pivotal in modulating the compound's properties. Subsequent characterization via NMR and mass spectrometry confirmed the formation of the six-membered ring, validating the structural integrity and the successful integration of R1 and R3 into the compound of interest.

The present invention relates to synthesize a novel conjugated oxa-spiro derivative compound of formula I.

wherein, R1 and R3 combined with each other to form a six-membered ring
R1 = -H, -Br, -Ar, -COOH, -NO2
R2 = -H or -OCH3
R3 = -H and
x = -CH2-, -CH2-CH2-

Another embodiment of the present invention is to provide a process for preparation of a novel conjugated oxa-spiro derivative compound of formula I comprises: reaction of Hydroxybenzaldehydes and cycloalkanone in presence of solvent with secondary amine and acid.

According to one embodiment of the present invention is a Hydroxybenzaldehydes selected from 2-hydroxybenzaldehyde, 5-bromo-2-hydroxybenzaldehyde, 2-hydroxy-3-methoxybenzaldehyde, 2-hydroxy-1-naphthaldehyde, 5-nitro-2-hydroxybenzaldehyde, 3-formyl-4-hydroxybenzoic acid.

According to one embodiment of the present invention is a solvent are selected from Methanol, Absolute alcohol, DCM, acetonitrile, petroleum ether, water, Ethyl acetate, isopropyl alcohol, acetone, hexane, chloroform, diethyl ether, Dimethylformamide, n-propanol.

According to one embodiment of the present invention is a acid selected from glacial Acetic acid, Con. Hydrochloric acid, acetic acid, sulphuric acid, citric acid, formic acid.

According to one embodiment of the present invention is a secondary amines selected from pyrrolidine, piperidine, morpholine, diethyl amine.

EXAMPLES:


Formula I
wherein, R1 and R3 combined with each other to form a Six-membered ring
R1 = -H, -Br, -Ar, -COOH, -NO2
R2 = -H or -OCH3
R3 = -H and
x = -CH2-, -CH2-CH2-

Formula No. Formula Structure Formula Name
Formula I (a) 5-(1, 2-dihydrocyclopenta[b]chromen-3-yl)- 5, 5a, 6, 7-tetrahydrocyclopenta[1,2-b:1,5-b']dichromene
Formula I (b) 3,10-dibromo-5-(7-bromo-1,2-dihydrocyclopenta[b]chromen-3-yl)-5,5a,6,7-tetrahydrocyclopenta[1,2-b:1,5-b']dichromene
Formula I (c) 1,12-dimethoxy-5-(5-methoxy-1,2-dihydrocyclopenta[b]chromen-3-yl)-5,5a,6,7-tetrahydrocyclopenta[1,2-b:1,5-b']dichromene
Formula I (d)
Formula I (e) 5-(2,3-dihydro-1H-xanthen-4-yl)-5a,6,7,8-tetrahydro-5H-chromeno[3,2-d]xanthene
Formula I (f) 3,11-dibromo-5-(7-bromo-2,3-dihydro-1H-xanthen-4-yl)-5a,6,7,8-tetrahydro-5H-chromeno[3,2-d]xanthene
Formula I (g) 1,13-dimethoxy-5-(5-methoxy-2,3-dihydro-1H-xanthen-4-yl)-5a,6,7,8-tetrahydro-5H-chromeno[3,2-d]xanthene
Formula I (h) 5-(10,11-dihydro-9H-benzo[a]xanthen-8-yl)-5a,6,7,8-tetrahydro-5H-benzo[a]benzo[5,6]chromeno[2,3-g]xanthene
Formula I (i) 5-(7-carboxy-2,3-dihydro-1H-xanthen-4-yl)-5a,6,7,8-tetrahydro-5H-chromeno[3,2-d]xanthene-3,11-dicarboxylic acid
Formula I (j) 3,11-dinitro-5-(7-nitro-2,3-dihydro-1H-xanthen-4-yl)-5a,6,7,8-tetrahydro-5H-chromeno[3,2-d]xanthene


Example-1: 5-(1, 2-dihydrocyclopenta[b]chromen-3-yl)- 5, 5a, 6, 7-tetrahydrocyclopenta[1,2-b:1,5-b']dichromene [Formula I (a)]

A solution of 2-hydroxybenzaldehyde (1.0 eq) and cyclopentan-1-one (0.66 eq) was dissolved in Absolute alcohol (20 V) at 25-30℃. Pyrrolidine (1.0 eq) was added to the reaction mixture. After 30 minutes gl. Acetic acid (4.0 eq) was added and the reaction was kept for 8-10 hours at 95°C -100°C. After completion the formed precipitates was filtered to give crude product which was washed with Absolute alcohol (5 V), and dried under vacuum. The crude was further purified by recrystallization from the DCM/acetonitrile or DCM/petroleum ether mixed solvents to give pure product (Formula I (a)). The filtrate was poured into ice-water (100 V), the formed precipitates were filtered and dried, followed by neutral alumina column chromatography in petroleum ether: DCM gradient elution to get second crop of product. The column chromatography resulted in compound of (Formula I (a)). The combined purified yield of Formula I (a) from solid and filtrate of second crop was found to be 46%.


Analytical Result:
FT-IR (KBr disc, cm-1): 3445.88, 3063.68, 3026.07, 2952.88, 2919.89, 2855.90, 2359.59, 2329.48, 1640.09, 1607.69, 1581.42, 1483.68, 1453.59, 1426.68, 1399.72, 1363.72, 1352.85, 1308.69, 1293.44, 1267.67, 1222.38, 1169.66, 1146.40, 1117.28, 1084.33, 1033.50, 986.49, 956.61, 921.90, 879.19, 855.18, 800.71, 758.58, 667.74, 615.57, 570.79, 459.74, 426.09. 1H NMR (400 MHz, CDCl3): δ ppm, 7.232-7.126 (m, 4H), 7.054-7.004 (m, 3H), 6.909-6.816 (m, 4H), 6.769 (dd, 1H), 6.592 (s, 1H), 5.996 (s, 1H), 4.151 (d, 1H), 2.901-2.825 (m, 1H), 2.781-2.704(m, 2H), 2.672-2.614(m, 3H), 2.483-2.420 (m, 1H), 2.202-2.131 (m, 1H), 1.922-1.848 (m, 1H). 13C NMR (400 MHz, CDCl3): δ ppm, 152.59, 151.74, 150.74, 149.48, 142.40, 137.47, 129.16, 128.17, 127.72, 127.20, 126.60, 125.44, 123.74, 123.53, 122.76, 122.12, 122.05, 121.96, 118.79, 118.49, 118.07, 116.62, 114.94, 112.09, 102.15, 46.58, 33.81, 28.32, 27.24, 26.62, 24.96. DEPT135 NMR (400 MHz, CDCl3): δ ppm, 129.16, 128.18, 127.72, 127.20, 126.61, 125.44, 122.76, 122.12, 122.05, 118.79, 118.08, 116.62, 114.94, 112.09, 46.58, 33.81, 28.32, 27.23, 26.63, 24.96. Elemental analysis calculated for C31H24O3: Experimental yield, C = 83.65%; H = 5.391%.Theoretical Calculated Yield, C = 83.76%; H = 5.44%.
SC X-ray Analysis
Crystal System monoclinic
Molecular Formula C31H24O3
Space group P21/c
a/Å 12.0845(13)
b/Å 21.091(2)
c/Å 9.2175(10)
α/° 90
β/° 95.828(3)
γ/° 90
Volume/Å3 2337.2(4)
R factor% 7.96
Z 4









Example-2: 3,10-dibromo-5-(7-bromo-1,2-dihydrocyclopenta[b]chromen-3-yl)-5,5a,6,7-tetrahydrocyclopenta[1,2-b:1,5-b']dichromene [Formula I (b)]

A solution of 5-bromo-2-hydroxybenzaldehyde (1.0 eq) and cyclopentan-1-one (0.66 eq) was dissolved in Absolute alcohol (20V) at 25-30℃. Pyrrolidine (1.0 eq) was added to the reaction mixture. After 30 minutes gl. Acetic acid (4.0 eq) was added and the reaction was kept for 7-8 hours at 90-95°C. After completion the formed precipitate was filtered to give crude product which was washed with Absolute alcohol (5 V), and dried under vacuum. The crude was further purified by recrystallization from the DCM/acetonitrile or DCM/petroleum ether mixed solvents to give pure product (Formula I (b)). The filtrate was poured into ice-water (100 V) the formed precipitates were filtered and dried followed by neutral alumina column chromatography in petroleum ether: DCM gradient elution to get second crop of product. The column chromatography resulted in (Formula I (b)). The combined purified yield of Formula I (b) from solid and filtrate of second crop was found to be 61%.


Analytical Result:
FT-IR (KBr disc, cm-1): 2963.14, 2947.94, 2840.03, 2745.26, 2352.95, 1691.77, 1659.40,1606.35, 1571.64, 1482.45, 1259.88, 1246.64, 1224.38, 1153.41, 1124.45, 1050.02, 1020.16, 944.44, 908.35, 868.54, 842.19, 794.86, 757.02, 735.56, 687.46, 658.66. 1H NMR (400 MHz, CDCl3): δ ppm, 7.529 (d, 1H), 7.330-7.193 (m, 3H), 7.117 (dd, 1H, J1= 8.4 Hz), 7.036 (s, 1H), 6.876 (d, 1H), 6.675 (d, 1H), 6.644 (d, 1H), 6.483 (s, 1H), 5.979 (s, 1H), 4.642 (d, 1H), 2.809-2.714 (m, 4H), 2.662-2.506 (m, 2H), 2.439-2.380 (m, 1H), 1.971-1.906 (m, 1H), 1.702-1.633 (m, 1H). 13C NMR (400 MHz, CDCl3): δ ppm, 151.58, 151.48, 150.40, 149.64, 142.63, 137.19, 131.30, 130.81, 129.91, 129.01, 127.94, 125.19, 123.73, 122.84, 119.23, 118.65, 118.41, 118.09, 116.60, 115.13, 114.66, 114.46, 113.82, 111.81, 101.74, 44.25, 30.93, 28.00, 25.73, 25.14, 23.59. DEPT135 NMR (400 MHz, CDCl3): δ ppm, 131.31, 130.81, 129.91, 129.01, 127.94, 119.23, 118.65, 118.41, 118.10, 116.60, 111.81, 44.25, 30.93, 28.00, 25.73, 25.14, 23.59. Elemental analysis calculated for calculated for C31H21Br3O3: Experimental yield, C = 54.65%; H = 3.033%. Theoretical Calculated Yield, C = 54.66%; H = 3.11%.

Example-3: 1,12-dimethoxy-5-(5-methoxy-1,2-dihydrocyclopenta[b]chromen-3-yl)-5,5a,6,7-tetrahydrocyclopenta[1,2-b:1,5-b']dichromene [Formula I (c)]

A solution of 2-hydroxy-3-methoxybenzaldehyde (1.0 eq) and cyclopentan-1-one (0.66 eq) was dissolved in Absolute alcohol (20V) at 25-30℃. Pyrrolidine (1.0 eq) was added to the reaction mixture. After 30 minutes gl. Acetic acid (4.0 eq) was added and the reaction was kept for 6-8 hours at 95-100°C temperature. After completion the formed precipitates was filtered to give crude product which was washed with Absolute alcohol (5 V), and dried under vacuum. The crude was further purified by neutral alumina column chromatography in petroleum ether: DCM gradient elution. The column chromatography resulted in 18% pure product (Formula I (c)).

Analytical Result:
FT-IR (KBr disc, cm-1): 3439.46, 2921.97, 2843.70, 2360.76, 1729.90, 1643.64, 1606.50, 1576.54, 1478.75, 1400.21, 1363.15, 1326.97, 1269.99, 1219.73, 1167.21, 1102.66, 1082.62, 1015.95, 986.13, 927.67, 840.98, 774.37, 732.74. 1H NMR (400 MHz, CDCl3): δ ppm, 6.945-6.887 (m, 1H), 6.868-6.792 (m, 5H), 6.749-6.686 (m, 2H), 6.559-6.537 (m, 2H), 5.983 (s, 1H), 4.845 (d, 1H), 3.768 (s, 3H), 3.736 (s, 3H), 3.650 (s, 3H), 2.919-2.768 (m, 2H), 2.669-2.531 (m, 4H), 2.441-2.373 (m, 1H), 1.978-1.909 (m, 1H), 1.844-1.757 (m, 1H). 13C NMR (400 MHz, CDCl3): δ ppm, 149.46, 149.03, 148.64, 146.84, 143.07, 142.13, 141.44, 140.80, 137.17, 124.71, 124.44, 122.92, 122.33, 121.53, 121.49, 121.30, 119.43, 119.19, 119.15, 118.25, 112.88, 112.16, 111.70, 111.17, 102.21, 56.72, 56.63, 56.34, 46.74, 33.90, 27.99, 26.99, 26.64, 25.01. DEPT135 NMR (400 MHz, CDCl3): δ ppm, 122.33, 121.53, 121.49, 121.30, 119.43, 119.15, 118.25, 112.88, 112.16, 111.70, 111.17, 56.73, 56.63, 56.34, 46.74, 33.91, 27.99, 26.99, 26.64, 25.01. Elemental analysis calculated for C34H30O6: Experimental yield, C = 76.38%; H = 5.612%. Theoretical Calculated Yield, C = 76.39%; H = 5.66%.


Example-4: [Formula I (d)]

A solution of 2-hydroxy-1-naphthaldehyde (1.0 eq) and cyclopentan-1-one (0.66 eq) was dissolved in Absolute alcohol (20V) at 25-30℃. Pyrrolidine (1.0 eq) was added to the reaction mixture. After 30 minutes gl. Acetic acid (4.0 eq) was added and the reaction was kept for 8 hours at 95°C temperature. After completion the formed precipitates was filtered to give crude product which was washed with Absolute alcohol (5 V), and dried under vacuum. The crude was further purified by neutral alumina column chromatography in petroleum ether: DCM gradient elution. The column chromatography resulted in 24% pure product (Formula I (d)).

Analytical Result:
FT-IR (KBr disc, cm-1): 3444.48, 3056.19, 2926.07, 2855.62, 1729.31, 1622.03, 1591.58, 1512.33, 1460.70, 1435.94, 1385.38, 1330.06, 1240.26, 1201.94, 1140.91, 1112.08, 1076.96, 1007.93, 943.17, 926.01, 902.31, 862.09, 812.57, 773.03, 744.81. 1H NMR (400 MHz, CDCl3): δ ppm, 8.120 (d, 1H), 7.954 (d, 1H), 7.897 (d, 1H), 7.797-7.731 (m, 3H), 7.658-7.620 (m, 2H), 7.590-7.441 (m, 4H), 7.410-7.333 (m, 3H), 7.294 (s, 1H), 7.143 (d, 1H), 7.068 (d, 1H), 6.935 (d, 1H), 6.635 (s, 1H), 4.596 (s, 1H) 3.101-3.051 (m, 1H), 3.000-2.693 (m, 4H), 2.610-2.545 (m, 1H), 2.252-2.185 (m, 1H), 1.998-1.943 (m, 1H), 1.846-1.757 (m, 1H). 13C NMR (400 MHz, CDCl3): δ ppm, 150.60, 148.73, 148.57, 148.49, 143.62, 136.07, 133.19, 130.37, 129.85, 129.83, 129.54, 128.85, 128.66, 128.51, 128.47, 128.42, 128.34, 127.21, 126.82, 126.48, 126.32, 124.04, 123.96, 123.61, 122.45, 121.95, 121.77, 119.78, 119.22, 118.15, 116.84, 116.28, 115.07, 114.85, 112.35, 106.85, 100.43, 46.37, 30.77, 28.14, 27.01, 26.07, 25.35. DEPT135 NMR (400 MHz, CDCl3): δ ppm, 128.66, 128.51, 128.47, 128.43, 128.34, 127.21, 126.82, 126.48, 126.32, 124.04, 123.96, 123.61, 122.45, 121.95, 121.77, 119.22, 118.15, 116.84, 115.07, 106.85, 46.37, 30.77, 28.14, 27.01, 26.07, 25.35. Elemental analysis calculated for C43H30O3: Experimental yield, C = 86.67%; H = 5.043%. Theoretical Calculated Yield, C = 86.84%; H = 5.08%.

Example-5: 5-(2, 3-dihydro-1H-xanthen-4-yl)-5a, 6, 7, 8-tetrahydro-5H-chromeno[3,2-d]xanthene [Formula I (e)]

A solution of 2-hydroxybenzaldehyde (1.0 eq) and cyclohexan-1-one (0.66 eq) was dissolved in Absolute alcohol (20 V) at 25-30℃. Pyrrolidine (1.0 eq) was added to the reaction mixture. After 30 minutes gl. Acetic acid (4.0 eq) was added and the reaction was kept for 8-9 hours at 90-95°C. After completion the formed precipitates was filtered to give crude product which was washed with Absolute alcohol (5 V), and dried under vacuum. The crude was further purified by recrystallization from the DCM/ acetonitrile or DCM/ petroleum ether mixed solvents to give 41% pure product (Formula I (e)).

Analytical Result:
FT-IR (KBr disc, cm-1): 3450.41, 3029.03, 2934.38, 2846.93, 2360.58, 1660.79, 1636.01, 1594.09, 1578.06, 1484.97, 1452.06, 1435.32, 1392.62, 1372.69, 1339.76, 1312.43, 1296.11, 1279.47, 1253.07, 1224.48, 1187.61, 1169.22, 1143.33, 1116.69, 1103.90, 1064.26, 1048.41, 1032.25, 993.82, 940.14, 914.62, 881.87, 847.14, 791.51, 750.05, 712.16, 589.26, 568.48, 461.70. 1H NMR (400 MHz, CDCl3): δ ppm, 7.161-7.123 (m, 4H), 7.073-7.035 (m, 1H), 7.005-6.956 (m, 3H), 6.908-6.882 (m, 2H), 6.862-6.802 (m, 2H), 6.491 (d, 1H), 6.127 (s, 1H), 5.379 (d, 1H), 2.706-2.442 (m, 6H), 2.033-1.901 (m, 2H), 1.818-1.763 (m, 2H), 1.716-1.663(m, 1H) 1.584-1.512 (m, 2H). 13C NMR (400 MHz, CDCl3): δ ppm, 153.23, 151.98, 151.05, 147.45, 133.70, 131.14, 129.58, 128.39, 128.06, 127.07, 125.94, 125.37, 122.71, 122.12, 121.97, 121.49, 120.89, 120.64, 120.62, 117.78, 117.35, 116.16, 114.60, 111.78, 98.61, 43.24, 33.85, 31.48, 30.86, 28.99, 25.70, 25.17, 21.89. DEPT135 NMR (400 MHz, CDCl3): δ ppm, 129.58, 128.39, 128.06, 127.07, 125.94, 125.37, 121.97, 121.49, 120.89, 120.62, 117.78, 117.35, 116.17, 114.60, 43.24, 33.85, 31.48, 30.86, 28.99, 25.70, 25.17, 21.89. Elemental analysis calculated for C33H28O3: Experimental yield, C = 83.76%; H = 5.967%. Theoretical Calculated Yield, C = 83.87%; H = 5.97%.

SC X-ray Analysis
Crystal System monoclinic
Molecular Formula C33H28O3
Space group P21/c
a/Å 10.684(2)
b/Å 12.9897(19)
c/Å 17.308(3)
α/° 90
β/° 91.847(8)
γ/° 90
Volume/Å3 2400.7(8)
R factor% 8.32
Z 4

Example-6: 3, 11-dibromo-5-(7-bromo-2, 3-dihydro-1H-xanthen-4-yl)-5a, 6, 7, 8-tetrahydro-5H-chromeno[3,2-d]xanthene [Formula I (f)]

A solution of 5-bromo-2-hydroxybenzaldehyde (1 eq) and cyclohexan-1-one (0.66 eq ) was dissolved in Absolute alcohol (20 V) at 25-30℃. Pyrrolidine (1 eq) was added to the reaction mixture. After 30 minutes gl. Acetic acid (4 eq) was added and the reaction was kept for 8-9 hours at 90-95°C. After completion the reaction mixture was poured into ice-water (100V) the formed precipitates were filtered and dried followed by neutral alumina column chromatography in petroleum ether: DCM gradient elution. The column chromatography resulted in 60% pure document (Formula I (f)).



Analytical Result:
FT-IR (KBr disc, cm-1): 3465.49, 2930.11, 2861.66, 2840.24, 2857.83, 2323.18, 1633.15, 1612.37, 1476.93, 1415.33, 1337.90, 1298.55, 1267.09, 1248.59, 1225.60, 1188.26, 1169.14, 1143.71, 1100.30, 1064.77, 1051.23, 993.63, 940.66, 912.52, 890.27, 865.31, 814.72, 774.09, 702.87, 570.54. 1H NMR (400 MHz, CDCl3): δ ppm, 7.240-7.206 (m, 4H), 7.119 (dd, 1H), 7.062 (d, 1H), 6.743 (d, 1H), 6.676 (d, 1H), 6.646 (d, 1H), 6.394 (d, 1H), 6.048 (s, 1H), 5.230 (d, 1H), 2.635-2.506 (m, 5H), 2.450-2.376 (m, 1H), 1.933-1.892 (m, 2H), 1.823-1.712 (m, 2H), 1.682-1.660 (m, 1H), 1.519-1.467 (m, 2H). 13C NMR (400 MHz, CDCl3): δ ppm, 152.07, 150.88, 149.74, 147.39, 134.74, 132.16, 132.02, 131.03, 130.56, 130.19, 128.36, 127.83, 124.76, 124.01, 122.62, 119.79, 119.26, 117.87, 117.01, 116.19, 114.17, 113.87, 113.48, 111.77, 98.64, 42.79, 33.96, 31.28, 30.66, 28.89, 25.53, 24.89, 21.62. DEPT135 NMR (400 MHz, CDCl3): δ ppm, 132.02, 131.03, 130.56, 130.20, 128.36, 127.84, 119.79, 119.26, 117.87, 117.01, 116.19, 42.79, 33.96, 31.28, 30.66, 28.89, 25.53, 24.89, 21.62. Elemental analysis calculated for C33H25Br3O3: Experimental yield, C = 55.80%; H = 3.491%. Theoretical Calculated Yield, C = 55.88%; H = 3.55%.

SC X-ray Analysis
Crystal System monoclinic
Molecular Formula C33H25Br3O3
Space group P21/c
a/Å 12.0357(8)
b/Å 10.4709(8)
c/Å 22.6914(19)
α/° 90
β/° 101.309(2)
γ/° 90
Volume/Å3 2804.2(4)
R factor% 9.39
Z 4

Example-7: 1, 13-dimethoxy-5-(5-methoxy-2, 3-dihydro-1H-xanthen-4-yl)-5a, 6, 7, 8-tetrahydro-5H-chromeno [3, 2-d] xanthene [Formula I (g)]

A solution of 2-hydroxy-3-methoxybenzaldehyde (1 eq) and cyclohexan-1-one (0.66 eq) was dissolved in Absolute alcohol (20 V) at 25-30℃. Pyrrolidine (1 eq) was added to the reaction mixture. After 30 minutes gl. Acetic acid (4 eq) was added and the reaction was kept for 7-8 hours at 95-100°C. After completion the formed precipitates was filtered to give crude product which was washed with Absolute alcohol (5 V), and dried under vacuum. The crude was further purified by neutral alumina column chromatography in petroleum ether: DCM gradient elution. The column chromatography resulted in 12% pure product (Formula I (g)).

Analytical Result:
FT-IR (KBr disc, cm-1): 3446.35, 3039.22, 2932.44, 2836.15, 1596.84, 1579.12, 1480.00, 1438.38, 1376.14, 1330.44, 1272.90, 1239.43, 1214.57, 1193.65, 1168.11, 1145.95, 1118.81, 1096.36, 1078.05, 1058.67, 1032.01, 992.91, 971.42, 929.97, 904.94, 875.75, 827.50, 786.84, 771.07, 740.35, 726.23, 569.45. 1H NMR (400 MHz, CDCl3): δ ppm, 6.878-6.692 (m, 8H), 6.593 (d, 1H), 6.444 (d, 1H), 6.078 (s, 1H), 5.445 (d, 1H), 3.719 (s, 3H), 3.684 (s, 3H), 3.538 (s, 3H), 2.763-2.735 (m, 2H), 2.556-2.431 (m, 4H), 1.978-1.882 (m, 2H), 1.806-1.518 (m, 5H). 13C NMR (400 MHz, CDCl3): δ ppm, 148.78, 147.85, 147.13, 146.79, 143.29, 141.73, 141.52, 133.13, 131.30, 123.77, 122.89, 121.99, 121.54, 121.39, 120.99, 120.94, 120.11, 119.57, 118.31, 117.62, 116.85, 114.18, 112.28, 110.39, 98.57, 58.29, 57.24, 56.40, 42.89, 34.33, 31.65, 30.75, 28.97, 25.89, 25.21, 21.92. DEPT135 NMR (400 MHz, CDCl3): δ ppm, 121.99, 121.54, 120.99, 120.94, 120.11, 119.57, 118.31, 117.62, 116.85, 114.18, 110.39, 58.29, 57.24, 56.40, 42.89, 34.33, 31.65, 30.75, 28.97, 25.89, 25.21, 21.92. Elemental analysis calculated for C36H34O6: Experimental yield, C = 76.77%; H = 6.134%. Theoretical Calculated Yield, C = 76.85%; H = 6.09%.

SC X-ray Analysis
Crystal System monoclinic
Molecular Formula C36H34O6
Space group P21/c
a/Å 11.5501(15)
b/Å 18.962(2)
c/Å 14.0652(17)
α/° 90
β/° 110.951(3)
γ/° 90
Volume/Å3 2876.8(6)
R factor% 10.11
Z 4

Example-8: 5-(10,11-dihydro-9H-benzo[a]xanthen-8-yl)-5a,6,7,8-tetrahydro-5H-benzo[a]benzo[5,6]chromeno[2,3-g]xanthene [Formula I (h)]

A solution of 2-hydroxy-1-naphthaldehyde (1 eq) and cyclohexane-1-one (0.66 eq) was dissolved in Absolute alcohol (20 V) at 25-30℃. Pyrrolidine (1 eq) was added to the reaction mixture. After 30 minutes gl. Acetic acid (4 eq) was added and the reaction was kept for 6-8 hours at 85-95°C temperature. After completion the formed precipitates was filtered to give crude product which was washed with Absolute alcohol (5 V) and dried under vacuum. The crude was further purified by neutral alumina column chromatography in petroleum ether: DCM gradient elution. The column chromatography resulted in 26% pure product (Formula I (h)).



Analytical Result:
FT-IR (KBr disc, cm-1): 3430.77, 3062.04, 2927.82, 2854.85, 1737.31, 1654.22, 1621.49, 1591.50, 1512.58, 1461.20, 1434.50, 1386.70, 1327.32, 1281.90, 1228.07, 1200.20, 1141.94, 1111.62, 1078.10, 1007.69, 944.30, 903.79, 863.86, 812.99, 774.56, 745.77, 659.51, 540.93, 470.55. 1H NMR (400 MHz, CDCl3): δ ppm, 8.255 (d, 1H), 8.136 (d, 1H), 8.037 (d, 1H), 7.805-7.755 (m, 3H), 7.674-7.468 (m, 6H), 7.404-7.367 (m, 3H, ), 7.285 (s, 1H), 7.222 (d, 1H), 6.984 (d, 1H), 6.953 (d, 1H), 6.827 (s, 1H), 5.577 (d, 1H), 3.110-3.049 (m, 1H), 2.929-2.859 (m, 1H), 2.764-2.728 (m, 2H), 2.662-2.539 (m, 2H), 2.081-2.005 (m, 2H), 1.827-1.738 (m, 3H), 1.682-1.576 (m, 2H). 13C NMR (400 MHz, CDCl3): δ ppm, 151.67, 149.47, 148.81, 145.82, 133.38, 132.72, 131.51, 129.84, 129.76, 129.60, 129.34, 128.90, 128.61, 128.52, 128.50, 128.33, 128.26, 128.17, 126.57, 126.40, 125.81, 124.18, 123.88, 123.71, 123.27, 121.26, 121.24, 119.61, 118.02, 116.78, 116.48, 115.82, 114.67, 113.60, 113.28, 112.94, 98.80, 43.54, 33.41, 31.98, 30.91, 28.56, 26.55, 25.42, 21.37. DEPT135 NMR (400 MHz, CDCl3): δ ppm, 128.61, 128.52, 128.49, 128.32, 128.26, 128.17, 126.57, 126.40, 125.81, 124.18, 123.88, 123.71, 123.27, 121.26, 121.23, 119.61, 118.02, 116.78, 116.48, 112.94, 43.53, 33.41, 31.98, 30.91, 28.56, 26.55, 25.41, 21.37. Elemental analysis calculated for C45H34O3: Experimental yield, C = 86.76%; H = 5.481%. Theoretical Calculated Yield, C = 86.79%; H = 5.50%.

Example-9: 5-(7-carboxy-2,3-dihydro-1H-xanthen-4-yl)-5a,6,7,8-tetrahydro-5H-chromeno[3,2-d]xanthene-3,11-dicarboxylic acid [Formula I (i)]

A solution of 3-formyl-4-hydroxybenzoic acid (1 eq) and cyclohexan-1-one (0.66 eq) was dissolved in Absolute alcohol (20 V) at 25-30℃. Pyrrolidine (1 eq) was added to the reaction mixture. After 30 minutes gl. Acetic acid (4 eq) was added and the reaction was kept for 8-9 hours at 90-95°C. After completion the reaction mixture was poured into ice-water (100 V), the formed precipitates were filtered to give crude product and dried followed by neutral alumina column chromatography in DCM: Methanol gradient elution. The column chromatography resulted in 22% pure product (Formula I (i)).

Analytical Result:
FT-IR (KBr disc, cm-1): 3432.35, 3073.94, 2930.34, 2859.26, 1694.63, 1611.28, 1492.47, 1443.39, 1385.81, 1262.62, 1231.74, 1192.80, 1147.96, 1119.46, 1054.16, 996.14, 944.01, 917.53, 898.14, 838.04, 773.65, 731.08, 688.64, 640.13, 556.44, 536.98, 463.51. 1H NMR (400 MHz, DMSO-d6): 12.803 (s, 3H), 7.863 (s, 1H), 7.778-7.653 (m, 5H), 7.028 (d, 1H), 6.914 (d, 1H), 6.840 (d, 1H), 6.787 (s, 1H), 6.464 (s, 1H), 5.274 (d, 1H), 2.327-2.296 (m, 2H), 2.113-1.701 (m, 7H), 1.561-1.247 (m, 4H). Elemental analysis calculated for C36H28O9: Experimental yield, C = 71.40%; H = 4.666%. Theoretical Calculated Yield, C = 71.52%; H = 4.67%.

Example-10: 3, 11-dinitro-5-(7-nitro-2, 3-dihydro-1H-xanthen-4-yl)-5a, 6, 7, 8-tetrahydro-5H-chromeno[3,2-d]xanthene [Formula I (j)]

A solution of 2-hydroxy-5-nitrobenzaldehyde (1 eq) and cyclohexan-1-one (0.66 eq) was dissolved in gl. Acetic acid solution (30 V). After dissolving addition of drop wise a mixture of 2.5 ml Con. H2SO4 and 2.5 ml gl. CH3COOH solution. The reaction was stirred for 5-6 hours at 25-30℃. After completion the reaction mixture was poured into ice-water (100 V). The formed precipitates were filtered to give crude product and dried followed by neutral alumina column chromatography in Petroleum ether: Ethyl acetate gradient elution. The column chromatography resulted in 12% pure product (Formula I (j)).

Analytical Result:
FT-IR (KBr disc, cm-1): 3438.80, 3088.87, 2925.27, 2854.90, 2675.36, 1731.87, 1709.37, 1654.31, 1615.10, 1582.97, 1520.07, 1481.23, 1439.71, 1338.67, 1263.85, 1125.01, 1088.70, 1000.55, 942.66, 898.80, 827.80, 748.14. 1H NMR (400 MHz, CDCl3): 8.107-8.061 (m, 4H), 7.973 (dd, 1H), 7.883 (d, 1H), 6.967 (d, 1H), 6.916 (d, 1H), 6.819 (d, 1H), 6.598 (s, 1H), 6.241 (s, 1H), 5.321(d, 1H), 2.722-2.658 (m, 5H), 2.400-2.499(m, 1H), 1.996-1.805 (m, 4H), 1.696 (m, 1H), 1.554-1.492 (m, 2H). Elemental analysis calculated for C33H25N3O9: Experimental yield, C = 65.25%; H = 4.172%.; N = 6.95%. Theoretical Calculated Yield, C = 65.24%; H = 4.15%; N = 6.92.

Table 1: MTT Assay study:
IC50 (µM) IC50 (µM) IC50 (µM)
Formula I A549 MDA-MB-231 L132
Formula I (a) 25.81±5.00 19.23±2.04 25.04±5.00
Formula I (b) 11.70±1.42 12.29±2.03 30.47±2.6
Formula I (c) 10.44±3.441 20.45±6.75 34.19±3.79
Formula I (d) 11.13±2.71 16.47±5.57 43.54±4.64
Formula I (e) 23±4.58 15.48±2.79 33.22±5.71
Formula I (f) 13.74±1.92 11.26±1.7 24.80±3.72
Formula I (g) 20.04±6.79 25.50±9.92 32.20±3.46
Formula I (h) 13.77±3.35 9.700±2.29 24.60±3.83
Formula I (i) 9.296±2.484 18.55±4.75 35.53±4.85
Formula I (j) 14.12±4.54 15.68±4.13 31.73±5.52
5-Fluorouracil - 70.07±8.96 96.33±25.37
Crizotinib(lung) 15.90±3.24 - 12.26±1.89

Where,
A549 is Lung cancer cell line.
MDA-MB-231 is Triple negative breast cancer cell line.
L132 is Normal cell line.

The series of Oxa-spiro compounds were screened for their cytotoxicity against A549 cells and MDA-MB-231 cells by MTT (3-(4, 5- dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) assay.

All synthesized compounds show good anticancer activity on lung cancer cell line A549 and triple negative breast cancer cell line MDA-MB-231 as revealed by MTT assay. All compounds show enhanced cytotoxicity on breast cancer cell line as compared to standard drug 5-Fluorouracil. The un substituted conjugated oxa-spiro derivative, compound of formula-I (a), was found to have IC50 value 19.23±2.04 µM on MDA-MB-231 cells. Substitution by electron releasing -OCH3 (compound of formula-I (c)) did not exhibit any enhancement in cytotoxicity on cancerous cell line, rather little increase was observed in IC50 value (20.45±6.75 µM). When bulky substituent was introduced to a spiro molecule to result in compound of formula-I (d) by using -naphthaldehyde as starting material, little improvement was obtained in cytotoxicity. When substitution was carried out by -Br, which is an electron withdrawing group but also releases electrons by mesomeric effect, (compound of formula-I (b)), a marked increase in cytotoxicity was observed (IC50 value 12.29±2.03 µM). When cyclopentane ring was replaced by cyclohexane ring to generate compound of formula-I (e), the cytotoxicity was improved showing IC50 value 15.48±2.79 µM. When formula-I (e) was substituted by electron releasing group, -OCH3, the potency was reduced (25.50±9.92 µM). Substitution by -COOH group also reduced the efficacy exhibiting IC50 value 18.55±4.75 µM. Substitution by electron withdrawing -NO2 group (Compound of formula-I (j)) showed comparable activity as that of compound of formula-I (e). When -Br group was introduced, the activity was much improved (11.26±1.7 µM, compound of formula-I (f)). When bulky aromatic ring was introduced to compound of formula-I (e), by using -naphthaldehyde as starting material to generate compound of formula-I (h), excellent cytotoxicity was observed showing IC50 value of 9.700±2.29 µM. All synthesized molecules were quite non-toxic in a desired range of concentration over normal cell line L132.

Similar trend was observed for lung cancer cell line A549. All molecules showed enhanced cytotoxicity as compared to standard drug crizotinib except un-substituted spiro derivative compounds of formula-I (a), formula-I (e), and methoxy substituted compound of formula I (g). When bulky aromatic ring was introduced, by using -naphthaldehyde as starting material to generate compound of formula-I (d) and formula-I (h), improved cytotoxicity was observed showing IC50 value of 11.13±2.71 µM and 13.77±3.35 µM respectively. Attachment of the electron withdrawing groups like -Br and -NO2 increased cytotoxicity as evident by IC50 values of compounds of formula-I (b), formula-I (f), and formula-I (j). When -COOH group was introduced (compound of formula-I (i)), excellent cytotoxicity was observed.

Table 2: Quantum Yield:
Compound No. Quantum Yield (%)
Formula I (a) 65.98
Formula I (b) 16.42
Formula I (c) 57.70
Formula I (e) 47.21
Formula I (f) 58.81
Formula I (g) 34.30
Formula I (i) 01.38
Quinine Sulphate 54.60

Fluorescence quantum yield was determined on the basis of the absorption and fluorescence spectra, using Quinine sulphate as reference. Quantum yields are calculated by using following formula:

ɸX = ɸSt (Ix / ISt) (ASt/ Ax) (ηX / ηSt)2

Where
ɸ = the quantum yield,
I = fluorescence intensity,
A = absorbance at excitation wavelength
η = solvent's refractive index,
The subscript 'St' denotes standard quinine sulphate, whose quantum yield is known, while 'x' denotes the sample.

Calculated relative quantum yield for Spiro compounds is shown in table 2.

All molecules show good quantum yields except compound of formula-I (i). Compound of formula-I (a) and formula-I (f) shows much better quantum yield than quinine sulphate.
, Claims:CLAIMS

We claim:

1. A novel conjugated oxa-spiro derivative compound formula I:

wherein, R1 and R3 combined with each other to form a six-membered ring
R1 = -H, -Br, -Ar, -COOH, -NO2
R2 = -H or -OCH3
R3 = -H and
x = -CH2-, -CH2-CH2-

2. A process for preparation of a novel conjugated oxa-spiro derivative compound of formula I comprises: reaction of Hydroxybenzaldehydes and cycloalkanone in presence of solvent with secondary amine and acid.
3. The novel conjugated oxa-spiro derivative compound of formula I as claimed in claim 2, wherein Hydroxybenzaldehydes selected from 2-hydroxybenzaldehyde, 5-bromo-2-hydroxybenzaldehyde, 2-hydroxy-3-methoxybenzaldehyde, 2-hydroxy-1-naphthaldehyde, 5-nitro-2-hydroxybenzaldehyde, 3-formyl-4-hydroxybenzoic acid.

4. The novel conjugated oxa-spiro derivative compound of formula I as claimed in claim 2, wherein solvent are selected from Methanol, Absolute alcohol, DCM, acetonitrile, petroleum ether, water, Ethyl acetate, isopropyl alcohol, acetone, hexane, chloroform, diethyl ether, Dimethylformamide, n-propanol.

5. The novel conjugated oxa-spiro derivative compound of formula I as claimed in claim 2, wherein acid selected from glacial Acetic acid, Con. Hydrochloric acid, acetic acid, sulphuric acid, citric acid, formic acid.

6. The novel conjugated oxa-spiro derivative compound of formula I as claimed in claim 2, wherein secondary amines selected from pyrrolidine, piperidine, morpholine, diethyl amine.

7. The novel conjugated oxa-spiro derivative compound of formula I as claimed in claim 2, wherein reaction is carried out at 25 °C to 100 °C for 30-540 min.

8. The novel conjugated oxa-spiro derivative compound of formula I as claimed in claim 2, wherein reaction is carried out time 30-540 min.

9. The novel conjugated oxa-spiro derivative compound of formula I as claimed in claim 2, wherein yield of the reaction is 12-67%.


Dated this on 07th day of October, 2024

Signature:
Name: Bhavik Patel
Applicant's Agent: IN/PA-1379
INFINVENT IP

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