Synthesis and Fragmentation Behavior Study of n-alkyl/benzyl Isatin Derivatives Present in Small/Complex Molecules: Precursor for the Preparation of Biological Active Heterocycles

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  • ABSTRACT

    N-Alkyl/benzyl substituted isatin derivatives are intermediates and synthetic precursors for the preparation of biological active heterocycles. N-alkyl/benzyl isatins have showed various biological activities, such as cytotoxicity, antiviral, caspase inhibition, cannabinoid receptor 2 agonists for the treatment of neuropathic pain, etc. In this study, N-alkyl/benzyl isatin derivatives were synthesized from isatin and alkyl/benzyl halides in presence of K2CO3 in DMF and excellent to quantitative yields (~95%) were obtained. Isatins and benzyl-isatins were condensed with fluorescein hydrazide to form fluorescein hydrazone. All the compounds were subjected to their fragmentation behavior study using LC/MSn. N-Alkyl substituted isatin derivatives fragmented at nitrogen-carbon (N-C) bond, hence gave daughter ion as [RN+H]+. Whereas, N-benzyl substituted isatin derivatives fragmented at carbon-carbon (C-C) bond of alkyl chain which linked with nitrogen molecules, therefore gave N-methyl fragments [RNCH2]+. This study demonstrated that, isatin moiety present in a small/large molecule or in a matrix of reaction mixture with/without N-alkyl/benzyl substituents can be identified by mass spectroscopic fragmentation behavior study.


  • KEYWORD

    Isatin , N-Alkyl/aryl isatin , Fragmentation behavior , Heterocycles

  • Introduction

    Isatin (1, 1H-indole-2,3-dione) is an indole derivative which was first obtained by oxidation of indigo dye,1,2 later on, it was synthesized by condensation of chloral hydrate, aniline and hydroxylamine in sulphuric acid which is so called Sandmeyer isonitrosoacetanilideisatin synthesis.3,4 Isatin is occurring in Isatis tinctoria,5 Calanthe discolour6 in Couroupita guianensis7 and many other sources.8-10 It has been used to test the presence of thiophene in crude benzene.11 Large number of biological active molecules which consist of isatin moiety showed various biological activities, such as anticonvulsant, antiviral, free radical scavengers, cytotoxic, antitumor, antineoplastic, antileishmanial, enhancer of ATRA-induced differentiation, ADAMTS-5 (Aggrecanase-2) inhibitors and inhibitors of NADPH oxidase (Chart 1). Thus, synthesis and study of isatin derivatives have been increasingly promoted in the scientific area. N-Alkyl/ benzyl isatins can be prepared by using NaH / K2CO3 / CaH2 / F3CCOOH / EtN(i-Pr)2 / Et3N / C5H5N / POCl3 / LiAlH4 / KOH / etc. as reagents.

    Due to the intriguing structures of isatin and its derivatives as well as their presence in the various forms in natural / synthetic biological active molecules, we focused on how to figure out the presence of isatin, N-alkyl/benzyl isatins moiety in natural / synthetic molecules before isolating from natural sources and/or purifying the reaction mixtures. In order to achieve this goal, we need to study the properties of the compounds of our interests. Since our group has been heavily involving in the field of mass spectroscopic research,12-15 we have designed and synthesized a series of N-alkyl/benzyl isatin derivatives and studied their fragmentation behavior using LC/MSn. These studies turned out to give us a solution to identify the presence of isatin moiety in natural / synthetic molecule before isolating the bioactive compounds from natural sources or before purifying the reaction mixtures. In addition, we can apply this method to indentify complex compounds containing the bioactive skeletons in natural source.

    Experimental

      >  General

    Chemicals and solvents were commercial reagent grade and were used without further purifications. Melting points were determined on a Barnstead electrothermal digital melting point apparatus model 9100 and are uncorrected. NMR spectra were obtained using a Bruker-500 spectrometer and Agilent technologies 400MR spectrometer. Electrospray ionization (ESI) mass spectrometry (MS) experiments were performed using an Agilent high performance liquid chromatography (HPLC) 1200 connected to an Agilent 6320 ion trap mass spectrometer fitted with an electrospray ionization (ESI) ion source (Agilent Technologies, Palo Alto, CA, USA). Agilent 6300 series ion trap LC/MS system software, Version 6.2, was used for data acquisition and analysis. Direct injection was performed using a connector. The mobile phase was composed of two solvents, methanol and water with 1:1 ratio. Flow rate was 0.3 mL/min, run time was 3 min. The ESI ion source capillary temperature was set at 325 ℃; the dry gas flow rate was set to 10.0 L/min; nebulizer pressure was set to 40.00 psi; maximum accumulation time was 300,000 µs; spectra were taken in positive/negative mode and scan began at 50 m/z and ended at 500 m/z; the injection volume was 2 µL.

      >  General procedure for the synthesis of N-substituted isatin (2a-i).

    A flask equipped with a magnetic stirring bar was charged with DMF (100 mL) and potassium carbonate (13 mmol). The mixture was stirred at room temperature for 5 min., isatin (1,10 mmol) was then added and the stirring was continued for 45 min. Alkyl/benzyl halide (11 mmol, chloride or bromide) was added to the reaction mixture and the stirring was continued at 80 ℃ for 12 h. The mixture was then diluted with water (200 mL), extracted with ethyl acetate and dried over anhydrous sodium sulfate. The solvent was removed under vacuum, and the residue was purified by flash column chromatography on silica gel using ethyl acetate and hexane (1:9) solvent system. Analytical pure N-substituted isatin was obtained in quantitative yields. 1-Methyl-1H-indole-2,3-dione (2a). Orange solid (90%). Mp 133-134 ℃ [lit.16 mp. 132-134 ℃; lit.17 mp. 133 ℃]. MS (ESI): Calculated 161, found 162 [M+H]+.; 1-Ethyl-1H-indole-2,3-dione (2b). Yellowish-orange solid (96%). Mp. 161-162 ℃ [lit.18 mp. 162-163 ℃]. MS (ESI): Calculated 175, found 176 [M+H]+ ; 198 [M+Na]+ ; 215 [M+K]+.; 1-Propyl-1H-Indole-2,3-dione (2c). Orange solid (92%). Mp. 61-62 ℃ [lit.16 mp. 62 -62 ℃]. MS (ESI): Calculated 189, found 212 [M+Na]+.; 1-Butyl-1H-Indole-2,3-dione (2d). Red thick oil (85%) [lit.19 mp. 36 ℃]. MS (ESI): Calculated 203, found 204 [M+H]+ ; 226 [M+Na]+.; 1-Hexyl-1H-Indole-2,3-dione (2e). Yellow low melting solid (85%) [lit.16 mp. 39 -40 ℃]. MS (ESI): Calculated 231, Found 254 [M+Na]+.; 1-(iso-Butyl)-1H-Indole-2,3-dione (2f).19 Red solid (89%). Mp. 90-91.C [lit.20 mp. 88-89 ℃]. MS (ESI): Calculated 203, found 204 [M+H]+.; 1-(sec-Butyl)-1H-Indole-2,3-dione (2g).21 Red low melting solid (87%). MS (ESI): Calculated 203, found 204 [M+H]+.; 1-Benzyl-1H-Indole-2,3-dione (2h). Orange slid (95%).22,23 Mp 130-131 ℃ [lit.17 mp. 122-123 ℃; lit.24 mp. 129-131 ℃]. MS (ESI): Calculated 237, Found 238 [M+H]+ ; 260 [M+Na]+.; 1-(3-Methoxybenzyl)-1H-Indole-2,3-dione (2i).9,10 Orange solid (82%). Mp 109-110 ℃. MS (ESI): Calculated 267, Found 268 [M+H]+ ; 290 [M+Na]+.

      >  General procedure for the synthesis of isatin derivates (3a-b)

    A mixture of fluorescein hydrazide (2, 0.3 mmol) and substituted isatins (0.3 mmol) were reacted according to the reported method.25 (E)-3',6'-dihydroxy-2-(2-oxoindolin-3-ylid-eneamino)spiro[isoindoline-1,9'-xanthen]-3-one (3a). Orange solid; (98%); mp. 261 ℃ [lit.25 mp. 261 ℃]. MS (ESI): Calculated 475, Found 476 [M+H]+ ; 498 [M+Na]+.; (E)-2-((1-benzyl-2-oxoindolin-3-ylidene)amino)-3',6'-dihydroxyspiro[iso indoline-1,9'-xanthen]-3-one (3b). Orange solid; (99%); mp. 269 ℃. 1H NMR (DMSO-d6, 400 MHz): δ, 9.93 (s, 2H), 7.98 (d, J = 6.8 Hz, 1H), 7.68 (td, J = 7.8, 1.2 Hz, 1H), 7.63 (td, J = 7.8, 1.2 Hz, 1H), 7.36 (td, J = 7.8, 1.2 Hz, 1H), 7.34-7.28 (m, 4H), 7.28-7.24 (m, 1H), 7.21 (d, J = 7.8 Hz, 1H), 7.17 (d, J = 7.8 Hz, 1H), 7.04 (t, J = 7.8 Hz, 1H), 6.98 (d, J = 7.8 Hz, 1H), 6.60 (d, J = 2.4 Hz, 2H), 6.58 (d, J = 8.8 Hz, 2H), 6.44 (dd, J = 8.4Hz, 2H), 4.85 (s, 2H) ppm. 13 C NMR (DMSO-d6, 100 MHz): d 163.01, 160.50, 159.05, 152.60, 151.80, 151.08, 136.40, 134.90, 133.85, 129.80, 129.20, 128.91, 128.48, 128.42, 128.06, 127.85, 124.54, 124.47, 123.58, 117.13, 112.90, 110.28, 110.21, 102.84, 67.65 and 43.28 ppm. MS (ESI): Calculated 565, Found 566 [M+H]+ ; 588 [M+Na]+.

    Results and Discussion

    N-Substituted alkyl/benzyl isatin derivatives (2a-i) were synthesized from commercially available isatin (1) in presence of potassium carbonate (K2CO3) in DMF at 80 ℃. Condensation of isatin (1) / N-benzylisatin (2h) with FL-Hydrazide using literature procedure16 resulted in compounds 3a-b. (Scheme 1).

    Isatin (1) was first introduced into the ESI ion source of Ion trap mass spectrometer by direct injection using methanol and water solvent system. Molecular ion peak were obtained m/z at 148 in positive mode as [M+H]+ (100%) (Figure 1A) and m/z at 146 in negative mode as [M-H]- (100%) (Figure 1B). Fragmentation (MS2) of 1 gave the product ion at m/z 120 with the loss of carbon monoxide (CO) (Figure 1C).

    For the fragmentation behavior study of N-alkyl/benzyl isatin derivatives (2a-i), first, multistage mass spectral data (MS2 and MS3) were taken for N-substituted alkyl isatin derivatives (2a-g). It elucidates that, MS2 of compounds 2a-g gave m/z at 148 (100%) with the loss of alkyl group attached with nitrogen atom of isatin skeleton.

    Further fragmentation (MS3) of m/z 148 ion gave the product ion at m/z 120. Details of fragmentation behavior study are depicted in Scheme 2.

    Scheme 2. Proposed MSn (MS2 and MS3) mechanism pattern of N-alkyl substituted isatin (2a-g).

    It should be noted that, all the compounds 2a-g gave fragments at m/z 148 with the loss of substituted alkyl group and the alkyl chain did not affect the fragmentation behavior in compounds 2a-g (Figure 2).

    MS2 fragment at m/z 148 obtained from parent compounds (2a-g) were extensively studied for their MS3 fragmentation behavior. All the fragments had m/z at 120 (100%) with the loss of one molecule of carbon monoxide (CO) while some of them gave m/z at 92 (0-60%) with the loss of two molecule of carbon monoxide (CO) along with m/z at 120 (100%). Figure 3 shows a short chain substituent N-alkyl isatin, namely N-ethyl isatin (2b, Figure 3A), long chain substituent N-alkyl isatin, N-hexyl isatin (2e, Figure 3B) and branched chain substituent N-alkyl isatin, N-iso-butyl isatin (2f, Figure 3C).

    Multistage mass spectral data (MS2 and MS3) were also taken for N-benzyl isatin derivatives (2h-i) and different fragmentation behavior was observed than the N-alkyl isatin derivatives (2a-g) (Scheme 3).

    MS2 fragmentation of compound 2h gave three fragments,m/z 160: with the loss of benzyl group attached with nitrogen atom of isatin skeleton,m/z 132: might be attributed to the loss of carbon monoxide generated from fragment ionm/z 160, andm/z 91: most likely generated from phenyl ring attached with nitrogen atom by methylene (-CH2) chain. To our notice, the fragment atm/z 91 is a common tropylium ion or phenylmethylium ion generated from parent compound 2h (Figure 4A).

    Similarly MS2 fragmentation of compound 2i having 3-methoxy group attached with phenyl ring gave three fragments,m/z 160: with the loss of benzyl group attached with nitrogen atom of isatin skeleton,m/z 132: might be attributed to the loss of carbon monoxide generated from fragment ionm/z 160, andm/z 121: most likely generated from 3-methoxy phenyl ring attached with nitrogen atom by methylene (-CH2) chain. The fragment atm/z 121 is also a common tropylium ion or phenylmethylium ion generated from parent compound 2i (Figure 4B).

    MS2 fragment atm/z 160 obtained from parent compounds 2h-i were extensively studied for their MS3 fragmentation behavior in a similar fashion as for 2a-g. Both fragments atm/z 160 gavem/z 132 (100%) with the loss of one molecule of carbon monoxide (CO) along withm/z 148 (20-30%) with the loss of methane. Figure 5 shows N-benzyl isatin (2h, Figure 5A) and N-(3-methoxy)benzyl isatin (2i, Figure 5B), respectively.

    Since one of the main objectives of our study is to identify if the isatin moiety presents in a complex molecules or in a fraction of natural extracts by using this fragmentation behavior method, we therefore applied this method to indentify isatin moiety present in a complex fluorescein hydrazone isatin/benzylisatin derivatives, namely (E)-3',6'-dihydroxy-2-((2-oxoindolin-3-ylidene)amino)spiro-[isoindoline-1,9'-xanthen]-3-onen (3a)16 and (E)-2-((1-be-nzyl-2-oxoindolin-3-ylidene)amino)-3',6'-dihydroxyspiro[isoindoline-1,9'-xanthen]-3-one (3b), respectively.

    As shown in Scheme 4, compounds 3a-b gave same pattern as we expected. From Figure 6A, we can see that, compound 3a gavem/z 476 [M+H]+ and sodium adduct atm/z 498 [M+Na]+ . MS2 fragmentation ofm/z 476 gave fragment atm/z 448 with the loss of one molecule of carbon monoxide from the parent (Figure 6B). As for benzyl isatin moiety containing compound 3b, the exactly same fragmentation pattern as for small molecules like 2h-i were observed. It showedm/z at 566, MS2 fragmentation ofm/z 566 gavem/z at 488 with the loss of benzyl group attached with nitrogen atom of isatin skeleton, and in MS3 fragmentation of daughter fragmentm/z 488gavem/z at 460 with the loss of carbon monoxide. Details of mass spectra are shown in Figure 6.

    From the Figure 6, conclusion cab be drown that, isatin moiety (with / without substituent) presents in small / large or complex mixtures its gives similar fragmentation patterns.

    Conclusions

    A series N-alkyl/benzyl isatin derivatives (2a-i) and two fluorescein Isatin hydrazone (3a-b) were synthesized and subjected to their mass spectroscopic fragmentation behavior study using Ion Trap LC/MS. Fragmentation of N-alkyl substituted isatin derivatives gave daughter ion as [RN+H]+ hence the breakage occurred nitrogen-carbon (NC) bond, whereas fragmentation of N-benzyl substituted isatin derivatives gave N-methyl fragments [RNCH2]+, hence the breakage occurred in carbon-carbon (C-C) bond of alkyl chain. Same fragmentation behavior were observed for the complex molecule fluorescein hydrazone (3a-b). Based on this fragmentation behavior pattern, whether isatin moiety present or not in a small/large molecule or in a matrix of reaction mixture with or without substituents can be easily identified. Further studies about this method for identification of basic skeleton in natural mixture are in progress.

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  • [Chart 1.] Biological active compounds containing isatin skeleton.
    Biological active compounds containing isatin skeleton.
  • [Scheme 1.] Synthesis of N-substituted isatin derivatives (2a-i, 3a-b)
    Synthesis of N-substituted isatin derivatives (2a-i, 3a-b)
  • [Figure 1.] Mass spectra of Isatin (MS2 scan and MS2 fragmentation): A) in positive modem/z = 148; B) in negative modem/z = 146; C) fragmentation (MS2 ) gavem/z = 120.
    Mass spectra of Isatin (MS2 scan and MS2 fragmentation): A) in positive modem/z = 148; B) in negative modem/z = 146; C) fragmentation (MS2 ) gavem/z = 120.
  • [Scheme 2.] proposed MSn (MS2 and MS3) mechanism pattern of N-alkyl substituted isatin (2a-g).
    proposed MSn (MS2 and MS3) mechanism pattern of N-alkyl substituted isatin (2a-g).
  • [Figure 2.] MS2 Fragmentation behavior of substituted N-alkyl isatin (2a and 2f): A) N-methyl isatin (2a); B) N-iso-butyl isatin (2f).
    MS2 Fragmentation behavior of substituted N-alkyl isatin (2a and 2f): A) N-methyl isatin (2a); B) N-iso-butyl isatin (2f).
  • [Figure 3.] MS3 Fragmentation behavior of substituted N-alkyl isatin (2b, 2e and 2f): A) N-ethyl isatin (2b); B) N-n-hexyl isatin (2e); C) N-iso-butyl isatin (2f).
    MS3 Fragmentation behavior of substituted N-alkyl isatin (2b, 2e and 2f): A) N-ethyl isatin (2b); B) N-n-hexyl isatin (2e); C) N-iso-butyl isatin (2f).
  • [Scheme 3.] Proposed MSn (MS2 and MS3) mechanism behavior of N-benzyl substituted isatin (2h-i)
    Proposed MSn (MS2 and MS3) mechanism behavior of N-benzyl substituted isatin (2h-i)
  • [Figure 4.] MS2 Fragmentation behavior of substituted N-benzyl isatin (2h-i): A) N-phenyl isatin (2h); B) N-(3-methoxy)phenyl isatin (2i).
    MS2 Fragmentation behavior of substituted N-benzyl isatin (2h-i): A) N-phenyl isatin (2h); B) N-(3-methoxy)phenyl isatin (2i).
  • [Figure 5.] MS3 Fragmentation behavior of substituted N-benzyl isatin (2h-i): A) N-phenyl isatin (2h); B) N-(3-methoxy)phenyl isatin (2i).
    MS3 Fragmentation behavior of substituted N-benzyl isatin (2h-i): A) N-phenyl isatin (2h); B) N-(3-methoxy)phenyl isatin (2i).
  • [Scheme 4.] Proposed MSn mechanism behavior of fluorescein hydrazone isatin/benzyl isatin derivatives (3a-b)
    Proposed MSn mechanism behavior of fluorescein hydrazone isatin/benzyl isatin derivatives (3a-b)
  • [Figure 6.] Mass spectra of compounds 3a-b: A)m/z 476 [M+H]+ andm/z 498 [M+Na]+ of 3a; B) MS2 ofm/z 476 gave fragment atm/z 448; C)m/z 566 [M+H]+ andm/z 588 [M+Na]+ of 3b; D) MS2 ofm/z 566 gave fragment atm/z 488; E) MS3 ofm/z 488 gave fragment atm/z 460.
    Mass spectra of compounds 3a-b: A)m/z 476 [M+H]+ andm/z 498 [M+Na]+ of 3a; B) MS2 ofm/z 476 gave fragment atm/z 448; C)m/z 566 [M+H]+ andm/z 588 [M+Na]+ of 3b; D) MS2 ofm/z 566 gave fragment atm/z 488; E) MS3 ofm/z 488 gave fragment atm/z 460.