Indian Journal of ChemistryVol. 45B, November 2006, pp. 2512-2522

Synthesis of chemiluminescent biotinyl naphtho[1 ,8-de][ 1,2]diazepine-l A-diones

N V Krishna Murthy & A Ram Reddy*Department of Chemistry, Osmania University, Hyderabad 500 007, India

E-mail: a_ramreddy@yahoo.com

Received 16 September 2005; accepted (revised) 21 August 2006

A new chemiluminescent seven membered cyclic peri hydrazide, 6-amino-2,3-dihydronaphtho[ I,8-de](I,2]diazepine-IA-dione (AH, 4). and five of its biotin conjugates with various spacer groups are synthesized. The total framework of thechemiluminescent biotin conjugates is constructed taking advantage of a convergent synthesis from various stableintermediates employing DCC condensation and NHS activation. Geometry optimization studies show that the conjugatesexist in an enveloped conformation. AH exhibits chemiluminescence properties analogous to luminal. Thechemiluminescence of hydrazide is enhanced when it is directly conjugated to biotin through an amide linkage while viaspacers its efficiency decreased.

Key words: Cyclic hydrazides, chemiluminescence. luminal, naphthodiazepines, biotinIPC Code: Int. C1.8: C07D

The discovery of hydrazide chemiluminescence (CL)uncovered several chemiluminescent (CLt) probes 1.4.

The synthetic routes targeting new and efficient CLtmolecules for rapid and sensitive detection of myriadbiomolecules is hotly pursued at present times, owingto their wide application in organic and bioanalyticalapplications'. CLt conjugates of biotin withisoluminol and aminobutylethyl isoluminol have beensynthesized for the development of sensitive assays't'.

There has been a spur in the development ofvarious assay methods based on avidin-biotintechnology". Biotin and avidin can be derivatised byconjugation to reporter groups and are widely appliedin a variety of bioanalytical applications for impartingstringent specificity to detection methodologiesf':'.Though radioisotope labeled biotin derivativesprovide the highest sensitivity, rigorous search is onfor alternatives owing to inherent disadvantagesbecause of their shelf life and waste disposal'<"'.Efforts have been made to synthesize biotin-fluorescein fluorescent conjugates with varying spacerlengths to enhance the sensitivity of detection and toprovide efficient alternatives to radioisotopiclabelsI7,18.

In this paper the synthesis and CL properties of 6-amino- 2,3-dihydronaphtho[l ,8-de][ 1,2]diazepine-l,4-dione (AH, 4), a CLt seven membered cyclic perihydrazide, and five of its biotin conjugates withdifferent linker lengths between these two arereported. The conjugates synthesized are N-(1,4-

dioxo-I ,2,3,4-tetrahydro-naphtho[ 1,8-de] [1,2]diazepin-6-yI)-bioti namide (BAH -0, 8), N-(1 ,4-dioxo-l ,2,3,4-tetrahydro-naphtho[l, 8-de] [1,2]diazepin-6-yl)-4-[bio-tinylamino]-benzamide (BAH-6, 11), N-(1,4-dioxo-1,2,3,4-tetrahydro-naphtho[ 1,8-de][ 1,2]diazepin-6-yl)-W- {2-biotinylamino ]-ethyl} -rnalonamide (BAH-7,15), N-(1 ,4-dioxo-1 ,2,3,4-tetrahydro-naphtho[ 1,8-de][ I, 2]diazepin-6-yl)-N'- {[2-biotinylamino ]-ethyl }-adipamide (BAH-IO, 19) and N-(1,4-dioxo-l,2,3,4-tetrahydro-naphtho] l ,8-de][ 1,2]diazepin-6-yl)-N'- {[6-biotinylamino]-hexyl}succinamide (BAH-I2, 23) andare shown in Figure 1.

Results and DiscussionSynthesis of 6-amino-2,3-dihydronaphtho[I,8-

de][I,2]diazepine-I,4-dione (AH, 4). The synthesisof AH is carried out starting from 1,8-naphthoicanhydride 1, as shown in Scheme I. The first step isthe synthesis of 2,3-dihydronaphtho[1 ,8-de ]-[1,2]-diazepine-l,4-dione 2 following a modified procedureof White and Matsuol9

. Compound 1, a pale yellowsolid, when refluxed with slight excess of hydrazinehydrate in ethanol-acetic acid binary solvent mixtureat 70°C for about 8 hr readily converted to 2. It wasobtained as a pale yellow fibrous solid, in 70% yield.

The IR spectrum of 2 displays characteristic bandscomprising a cyclic hydrazide". There is no evidenceof a primary amino group from the IR spectrum,which rules out the possibility of the 6-memberedN-amino compound I. A similar difference in IR

MURTHY et aI.: CHEMILUMINESCENT BIOTINYL NAPHTHO[ I ,8-D£][ I,2jDIAZEPINE-1 ,4-DlONES 2513

H HN-N H HN-N H HN-No o

1 2 3 4

BAH-10,19BAH-7,15 H H

l 0J:-NyoHtlHH HI/'- ~NU)

S <,~N~~_ <;» If H

° °BAH-12,23

Figure 1 - Biotinyl conjugates synthesised

HH HH

000°° (a) °MN

.

N°(b) °MN

-

N°(c) 0Cr5~-~ °~ ~ . ~ ~... .I I ~ ~ ~ ~..0- ..0- ..0-...0- I I

..0- ..0- ..0- ..0-'~ 2y3 NO, 4 NH,

0050 ° (a) 00&0 °.:(e) •••

Y' I ~ Y' I ~~..0- ~..0-

5 N02 NH26

a) NH2-NH2.xHp; CH3COOH;C2HsOH;Reflux70°CBhr.b) HNO:/H2S04; heat70°C 1hr.c) Pd/C,H!CH30H; SnCI2·2H20/CHPH;Pd/C/NH2-NH2·2H20d) HN03, H2S04

Scheme I - Synthesis of AH from I,8-naphthoic anhydride

2514 INDIAN J CHEM, SEC B, NOVEMBER 2006

spectra is displayed by the isomeric compoundsphthalhydrazide U and N-aminophthalimide IU21

,

The IH NMR spectrum of 2 exhibits a broad signalat 85.7 ppm assigned to the protons attached to thetwo nitrogens of hydrazide, and are exchanged withdeuterium, on addition of D20. The NMR spectrumshows three signals for the six aromatic protons ofnaphthalene moiety, which confirms that theannulated benzene rings in naphthalene are in plane.In addition, the electron impact and chemicalionization mode mass spectra and the fragmentationpattern further confirm the structure of 2. Theelimination of a fragment of molecular weight 15rather than 16 indicates the formation of a cyclichydrazide 2 rather than I.The cyclic hydrazide 2 was converted to 6-nitro-

2,3-dihydronaphtho[ 1,8-de][ 1,2] diazepine-l,4-dione3 by the addition of pre-cooled mixture of fumingHN03 and cone. H2S04 and subsequently heating at60°C for 1 hr. The compound 3 was obtained as abright yellow powder with 60% yield. The IRspectrum shows the nitro groups characteristic intenseasymmetric and symmetric stretching bands at 1595and 1537 ern" in addition to the amide I and II bands,indicating the introduction of the nitro group in thenaphthalene ring, without affecting the hydrazidemoiety.Unlike in 2 the IH NMR spectrum of 3 shows all

the aromatic protons as chemically non-equivalent,indicating a loss of symmetry in the naphthalenemoiety of the molecule. The coupling constant of thetwo downfield signals at 88.65 and 8.70 ppm is about2.5 Hz suggesting meta coupling, i.e. substitution at6-position. The regio selective nitro substitution atC-6, rather than at 5- or 7-position, is probablybecause of two factors. The stability of C-6 attackedintermediates and the bond localization in thenaphthalene ring favor the nitronium ion substitutionat 6-position. It is interesting to note that thehydrazide protons resonated as a distinct doublet fardownfield, at 8 12.1 ppm. Similar shift is observed incase of 5-nitro-2,3-dihydrophthalazine-1 ,4-dione. Theformation of the doublet is because of the

cqo C¢0 NHI N-NH2 I I,0- ,0- NH

° °II III

anisotropism caused by the geometry of the sevenmembered ring wherein the two hydrazide nitrogensof 3 are not equivalent. This is also supported by thegeometry optimized model of 3. The new sevenmembered ring formed is not a planar one and the twonitrogen atoms are slightly puckered deviating fromthe planarity. The molecular ion peak in the massspectrum of 3 is intense and occurs at 257, which is inconsonance with the odd number of nitrogen's rule. Inaddition there is a fragment corresponding to a loss of-N02 which further confirms the nitration.The compound 3 was reduced in a Paar shaker with

H2 (1 atm) and in presence of Pd/C (ref. 22), whichreadily yielded 6-amino-2,3-dihydronaphtho[ I ,8-de]-[1,2]diazepine-l ,4-dione (AH, 4), a bright fluorescentcompound. AH was also obtained on reduction of 3with Pd/C: hydrazine hydrate+' or SnCh.2H20 inmethanol." or Pd/C and ammonium formate". Aspecial distinguishing feature in the electronicabsorption spectrum of AH is the appearance of a newbroad absorption maximum at 432 nm. The IRspectrum of AH shows peaks corresponding to theprimary amino group and to the associated -NHgroup of the hydrazide. In the IH NMR spectrum ofAH the two amino protons resonate as a broad signalat 8 5.99 ppm. This broadening is due to thequadrapole resonance effect of the nitrogen atom towhich the protons are attached. In addition, asignificant diatropic shift was noticed in the chemicalshifts of two hydrazide protons when compared to 3.In compound 3 they resonated at 8 12.1 ppm while inAH they occurred at 8 5.75 ppm. This observation issimilar to the chemical shifts on reduction of 5-nitro-2,3-dihydrophthalazine-1,4-dione to luminol.Structure of amino compound AH, deduced from

IH NMR was further established from the l3C NMRspectrum. Eleven signals were obtained for the twelvecarbons present, indicating that two of the carbons arechemically equivalent. The two chemical shiftequivalent carbons are the amide carbonyl carbons i.e.C-l and C-4. It is interesting to observe that there issignificant variance in the chemical shifts of thealternate aromatic carbons, arising from substitutionon the naphthalene ring. All the ten aromatic carbonsin naphthalene ring gave different signals in l3CNMR. This may be probably due to the distortion inthe planarity of the bicyclic ring. The molecular ionpeak obtained by .electro~impact is at m/z 227, whileit is at 228 in chemical ionization mode of massspectrometry, further confirming the formation ofA.H. The fragmentation pattern resembles that of the

MURTHY et al.: CHEMILUMINESCENT BlOTINYL NAPHTHO[I,8-DE][1,2]DIAZEPINE-l,4-DIONES 2515

parent hydrazide with a sequential loss of both the-NH moieties rather than the -NH2 group initially.The overall yield of AH obtained from the aboveprocess is 42%.Synthesis of AH was also attempted by alternate

routes, involving 1 as starting material and the samereagents, but by manipulating the order of appearanceof the intermediates. The routes are summarized inScheme I. However, both these routes were found tobe inferior, as the anhydride is not as stable as thehydrazide in acidic medium. Nitration of 1 not onlyprovides the desired nitro compound 5 in low yields,but also other regio isomeric nitro compounds and thehydrolyzed diacid as the side products. Moreover,attempts to convert 5 to 3 by hydrazinolysis or 3-aminoanhydride 6 by reduction were not met withconsiderable success. The overall yields obtained bythese methods, i.e., 1 to AH via 5 and 3 is 10%, whilethrough 5 and 6 is 3%. The marginalized yields in theabove two conversions can be probably due to thenegative electronic and steric effects of the nitrogroup.Synthesis of Biotin-AH conjugates. Five

conjugates of biotin with AH were synthesized usingdifferent linkers to separate them spatially and theirstructures were established by chemical and spectralmethods. The synthesis of all the conjugates wascarried out using biotin-N-hydroxysuccinimide 7 asthe biotinylating agent. Compound 7 was preparedfollowing the procedure of Becker et a126

.

HO-N~

o

O.1NNaHC03

Synthesis of BAH-O (8). BAH-O was obtained bythe coupling the activated ester 7 with AH in DMF inpresence of NaHC03 as shown in Scheme II. The ESImass spectrum of BAH-O shows the molecular ion at492 (M+K+). The IR spectrum of BAH-O ischaracterized by broad peaks at 3420 and 3242 andmultiple peaks at 1747, 1731 and 1704 ern",indicating the presence of hydrazide and biotinmoieties. The peaks corresponding to the NHS ester at1819 and 1788 cm' are missing. The IH NMRspectrum shows that chemical shifts of most of theprotons are not greatly affected in comparison to itsprecursor biotin and AH. When the amino group isconverted to an amide, a broad peak at 8 5.97 ppm isobtained and the intensity counts conformed to asingle proton confirming the coupling between biotinand AH. There is no marked difference in thechemical shifts of C-7, C-8 and C-9 protons of thenaphthalene ring moiety in the conjugate whencompared to AH. However, the protons attached to C-10 and C-5 of naphthalene ring show a very smalldownfield shift. The protons at bridge of biotin areshifted up field to 8 4.22 and 4.34 ppm in theconjugate, in comparison to 8 4.28 and 4.47 ppm infree biotin.Synthesis of BAH-6 (11). p-Aminobenzoic acid

(PABA) and 7 are condensed in DMF in basicconditions to yield the biotinyl-amide 9. Thecompound was characterized by IR. Compound 9 wasconverted to its NHS ester 10 by reaction with NHS

o

HNANH

H-+-t-H °(J,~~,(t)7 0

DCC•.DMF

DMF ...°A

HN NH

HTIH

«, "~ns ""~ Y' """"o ~ I ~

BAH-O 8 0 0, N-NH H

Scheme II - Synthesis of BAH-O (8)

2516 INDIAN J CHEM, SEC B, NOVEMBER 2006

and DCC in DMF. In the IR spectrum of 10, peakscharacteristic of the NHS ester moiety are observed.The condensation of 10 and AH in DMF andNaHC03 yielded BAH-6 11. The ESI mass spectrumconfirmed the molecular weight as 572. In addition tothe IR peaks of AH and biotin, a peak correspondingto the aromatic stretching of PABA is observed. IHNMR spectrum of BAH-6 clearly indicates an overalldeshielding effect on the naphthalene ring protons dueto the conjugation with PABA.

Synthesis of BAH-7, BAH-I0 and BAH-12. Thesynthesis of BAH-7 15, BAH-lO 19 and BAH-12 23were carried out in conditions similar to BAH-6 andtheir synthetic path is shown in Scheme III. Theconjugates were characterized by their electronicabsorption, IR, IH NMR and mass spectra andelemental analysis.

Synthesis of spacers. The three bifunctionalspacers 12, 16, 20 for compounds BAH-7, BAH-IOand BAH-12 were synthesized and characterized byspectral techniques. The compound 12 wassynthesized by the condensation of malonic acid andethylene diamine in l,4-dioxane medium. Compound16 was prepared by the condensation of adipic acidand ethylenediamine and 20 by the condensation ofhexamethylenediarnine and succinic acid in 1,4-dioxane".

The compound 12 was coupled to biotin in DMFand NaHC03 to generate 13. The IR spectrum of 13shows peaks corresponding to the amide NHstretching and another broad peak at 2930 cm'corresponding to the carboxylic acid. 13 was thenconverted to its NHS ester 14, by reaction with DCCand NHS, in DMF. IR spectrum of 14 gives a clearindication of the introduction of the NHS ester group,in addition to the other absorptions of biotin. Theeffect of conjugation on the proton resonances issimilar to BAH-6. The NMR spectrum of theconjugate shows that the protons from the spacer arenot affected largely. BAH-IO and BAH-12 wereprepared employing a similar strategy. The IH NMRspectrum of BAH-12 is slightly different from theremaining conjugates. The hydrazide NH protons-inBAH-12 occur as a distinct doublet at 0 7.5 ppm akintoAH.

The chemical shift values of the affected protons ofall the conjugates are shown in Table I. From thetable it can be noticed that the protons attached to thehydrazide nitrogen atoms occurred as a distinctdoublet at 0 5.75 ppm in AH, but are merged into abroad signal resonating at down field in BAH-O, BAH-6, BAH-7 and BAH-lO, while their chemical shift isunaffected in BAH-12. The paratropic shift in theseprotons may be due to the intra-molecular H-bond

oH ......J SpacerL-COOH.....2....... . . )l --1 Spacert--COOH

H2,,. IL.........:......_--,r Biotin NH

113,17,21

NHS12,16,20

AH 0 0

)l ..A spacer~o <,OBiotin N N

Ho

14,18,22 0

BAH-7, BAH-10, BAH-12 ••••

o

H2N~~~OH

o 16

oH2N~~yJOH

o20

Scheme III - Synthesis of BAH-7, BAH-lO and BAH-12

-

MURTHY et al.: CHEMILUMINESCENT BIOTINYL NAPHTHO[1,8-DE][ I,2]DIAZEPINE-l A-DIONES 2517

Table I - 'H NMR chemical shifts (in ppm) of AH. biotin and their conjugateso

HN)lNHHtt

sAH 5.75 (d) 7.27 (d)

Biotin 5.59 (s), 5.68 (s) 4.28 (rn), 4.47 (rn)

BAH-O 5.79 (b) 7.29 (d) 6.36 (s), 6.50 (s) 4.22 (m), 4.34 (m)BAH-6 5.80 (b) 7.85 (d) 6.35 (s), 6.45 (s) 4.15 (m), 4.25 (rn)

BAH-7 5.76 (b) 7.70 (b, m) 6.60 (s), 6.73 (s) 4.09 (rn), 4.27 (rn)BAH-IO 5.76 (b) 7.83 (rn) 6.36 (s), 6.72 (s) 4.13 (rn), 4.27 (rn)

BAH-12 5.75 (d)' 7.80 (rn) 6.35 (s), 6.50 (s) 4.10 (t), 4.30 (t)b, broad; s, singlet; d, doublet; t, triplet; rn, multiplet

NaphthaleneC-5

Compd

formed with the carbonyl oxygen of ureido ring ofbiotin moiety. The proton attached to C-5 ofnaphthalene ring of the hydrazide experiences adownfield shift in all the conjugates. The paratropicshift of C-5 proton of the naphthalene ring uponconjugation may be due to the regiospecificinteraction with the valeric acid side chain of thebiotin in BAH-O or with the spacer group chain incase of BAH-6, BAH-7, BAH-IO and BAH-12.Similar effects were observed by Nakashima et al. inthe naphthoamide moiety of amino-f-cyclodextrinderivatives. The associative hydrophobic interactionbetween the ~-cyclodextrin and the naphthoamide,leading to the formation of intra-molecular inclusioncomplex, caused an up field shift in the naphthalenering protons at lower "temperature, while at highertemperature a downfield shift due to the repulsiveinteractions". The NH protons of the ureido amidemoiety of biotin, which were observed at <5 5.59 and5.68 ppm in free biotin, appeared around <5 6.36 and6.50 ppm in all the conjugates. The down field shift inthese protons is probably due to the intra-molecularH-bonding with the hydrazide carbonyl oxygen.Similarly the protons at bridge carbons of biotin,which were observed at <5 4.28 and 4.47 ppm in freebiotin, are now shifted up field to <5 4.22 and 4.34ppm. This may be due to the folding up of the valericacid side chain of biotin to give an envelopedconformation to the conjugate which is alsoresponsible for the little but uniform up field shiftshown by the side chain protons' of biotin. This with thebroadening and downfield shift of the hydrazidesignal in the conjugates indicates the existence of theconjugate in an enclosed conformation, represented inFigure 2.

Figure 2 - Schematic representation of enclosed structure ofBAH-O

In case of BAH-12 however, the hydrazide protonsare distinct and resonated at <5 5.75 ppm as observedin case of free AH. The ureido amide protons ofbiotin in 21 as well as in BAH-12 underwent adownfield shift, while the hydrazide protons of AHmoiety experienced no change in chemical shift uponconjugation. It is quite interesting and is differentfrom the earlier observations.Alternate Synthetic Methods. The preparation of

the conjugate compounds was also attempted usingalternate synthetic routes. Such a method for thesynthesis of BAH-6 is summarized in Scheme IV.Though this is a realized route for the synthesis of thefinal conjugate it was not preferred. AH is alkalilabile and since most reactions described above werecarried out in basic conditions it led to the formationof a mixture of products thus reducing the yield of theproduct and therefore Scheme IV is not followed forthe synthesis of the conjugates.

2518 INDIAN J CHEM, SEC B, NOVEMBER 2006

+HO-~~

o

BAH-6

Scheme IV.~ Alternate procedure for the synthesis of BAH-6

Chemiluminescence. The compounds 2, 3 and AHare angularly annulated peri diazepinones and areanalogs of luminol. Therefore, these compounds areexpected - to exhibit CL similar to luminol. Asanticipated these compounds are CLt. The CLreaction of these hydrazides can be initiated only-withthe addition of alkali like NaOH. Similar results wereobtained in case of. the conjugates also. The CLt lightoutput of 2, 3, AH and the conjugates is given inTable n. Among the three hydrazides, All is themostCl.t, This is brought about by the presence of theelectron donating "amino group. Electron donatinggroups enhance CL, whereas electron withdrawinggroups quench CL, akin to fluorescence. In case of thebiotin conjugates the CL is quenched in general. Norelationship between the length of the spacers and theCL intensity is observed. No immediate explanationcan be provided for such a behavior. However, it ispossible that the non-radiative decay pathway may bemore active in presence of the spacers, which leads tolower CL light output. In case of BAH-O, where AHis directly coupled to biotin, there is an enhancementin the light output.

Experimental SectionAll starting chemicals were obtained from

commercial sources and were of analytical grade orbetter and were used without further purification.Melting points were determined using a TempoMelting Point Apparatus, India and are reporteduncorrected. IR spectra were recorded using JASCO

Table 11- CL light output (in arbitrary units) of 2, 3, AH, BAH-0, BAH-6, BAH-7, BAH-lO and BAH-12 (60 J.lM) inDMSOlNaOH (62.5 mM)

Compd CL light output

655668512721372201762358200

23AH

BAH-OBAH-6BAH-7BAH-lOBAH-12Luminol

Ff/IR-420 instrument. NMR spectra were recordedusing Bruker (300 MHz) and Gemini (300 MHz)instruments. The chemical shifts are expressed as ppm

. using tetramethylsilane as an internal standard(0= 0.0). Mass spectra of compounds 2, 3 and AHwere recorded using Shrader laboratories VG 7070 Hmass spectrometer. Mass spectra of the five biotinconjugates were recorded using Micromass Q-TOFmass spectrometer in the electron spray ionizationmode. Elemental analysis was carried out usingPerkin-Elmer 250°C analyzer. Chemiluminescencemeasurements were carried out using a TurnerDesigns TD-20/20 luminometer.2, 3-Dihydronaphtho[l, 8-de][l, 2]diazepine-l, 4-

dione 2. To a suspension of 1,8-naphthoic anhydride(1, 1.98 g, 10 mmoles) in ethanol (40 mL), wereadded acetic acid (5.7 mL, 10 mmoles) and hydrazine.hydrate (2.43 mL, 50 mmoles). The reaction mixturewas refluxed at 70°C for 8 hr. The hot reactionmixture was then poured onto 20 g of crushed ice andallowed to stand at ice-bath temperature for one hourtill the entire solid precipitates out. The yellow fibroussolid was filtered at pump. The compound was dried.The dried compound was shaken with 200 mL of 2%NaOH to remove any acid formed. The crudecompound was recrystallized in ethanol to give 2 in79% yield; m.p. fuses above 240°C; IR (KBr): 3270,1665, 1633 cm': IH NMR: (DMSO-d6) 0 5.7 (d, 2),7.8 (dd;, 15,6=7.2Hz & 16,7=7,2 Hz, 2), 8.32 (d, 1=7.2 Hz. 2), 8.49 (d, 1=7.2 Hz, 2); rnIz 212 (M+); Anal.Found: C, 68.02; H, 3.90; N, 13.05; 0, 15.03.C12H8N202 requires C, 67.92; H, 3.80; N, 13.20; 0,15.08%.6-Nitro-2, 3-dihydronaphtho[l, 8-de ][1, 2]diaze-

pine-I,4-dione 3. Cone. H2S04 (1.43 mL) was addedto 2 (2.12 g, 10 mmoles) and kept in ice. A mixture offuming HN03 (0.66 mL, 10.5 mmoles) and H2S04(1.43 mL, 26.25 mmoles) was made and stored in ice.

MURTHY et al.: CHEMILUMINESCENT BIOTINYL NAPHTHO[I,8-DE][1,2jDIAZEPINE-l,4-DIONES 2519

The cooled nitrating mixture was slowly added to thecompound taking care not to allow the temperature torise above 30°C. An oily liquid was initially formed.The reaction was carried out by heating at 60 °C forl hr. The reaction mixture was then poured on ice. Theyellow solid was separated and was allowed to settledown and filtered, washed thoroughly with cold waterand dried. The crude product was extracted in hotchloroform and was purified by silica gel column,using chloroform as eluent. The chloroform fractionswere pooled and dried in a rotary evaporator to obtainTLC pure bright yellow compound 3. Yield 63%;m.p. fuses above 260°C; IR (KBr): 1537 (VNO), 1595em"; IH NMR: (DMSO-d6) 8 8.2 (m, C-9, JS•9 = 7.9Hz & hlO = 7.4 Hz, 1),8.45 (d, C-8, JS•9 = 7.9 Hz, I),8.55 (d, C-IO, J9,I0 = 7.4 Hz, 1), 8.65 (d, C-7, JS•7 =2.5 Hz, 1),8.7 (d, C-5, JS.7 = 2.5 Hz, I), 12.1 (d, NH-NH, 2); MS: rn/z 257 (M+).

6-Amino-2, 3-dihydronaphtho[1,8-de ][1,2]diaze-pine-l,4-dione 4, AH.(a) Hy-Pd/Charcoal reduction. A suspension of 3

(2.57 g, 10 mmoles) in HPLC grade methanol (200mL) was reduced with Pd/charcoal, under H2atmosphere for 4 hr, in a Paar shaker. This was thenfiltered off and the orange red compound wasrecovered (1.9 g, 84%) by evaporating methanol.(b) Reduction with SnCh.2H20. A suspension of

3 (I g, 3.9 mmoles) in methanol (35 mL) was reducedwith SnCI2.2H20 (0.9 g, 3.75 mmoles) under refluxfor 8 hr. The product was filtered and dried in a hotair oven at 90°C fOT 30 min. The product wasrecovered (0.57g, 65%).(c) Reduction with hydrazine hydrate. A

suspension of 3 (1.29 g, 5 mmoles) in methanol (50mL) was reduced with hydrazine hydrate (25 mmoles)and Pd/charcoal under stirring for 6 hr. The volumewas reduced in vacuum and was poured into ice-watermixture. The precipitate was filtered, dried and theproduct recovered (0.8 g, 70% ).Though the above three methods afforded AH in

good yields, the method described using Pd/C and H2gas was preferred for the ease of processing andhigher yield. m.p. fused above 240°C; IR (KBr): 3445,3316,3235 (VNH), 1644, 1600, 1582, 1550 ern"; IHNMR: (DMSO-d6) 8 5.75 (d, NH-NH, 2), 5.99 (broad,-NH2, 2), 7.27 (d, C-5, JS•7 = 2.2 Hz, 1),7.6 (m, C-7,hs = 7.3 Hz & JS•7 = 2.1 Hz, 1), 7.88 (rn, C-8, hs =7.3 Hz & JS•9 = 8.1 Hz, 1),8.05 (m, C-9, J8.9 = 8.1 Hz& hlO = 7.6 Hz, 1),8.49 (m, C-IO, J9.10 = 7.6 Hz, 1);I3C NMR: 9117 (C5), 120 (C7), 123 (IIa), 128 (C8),

129 (C7a), 130 (CII), 130.5 (C9), 131 (CI2), 133(CIO), 146 (C6), 160 (CI & C4); MS: rn/z 227 (M+);Anal. Found: C, 63.35; H, 4.01; N, 18.57; 0, 14.07.CI2H9N302 requires C, 63.43; H, 3.99; N, 18.49; 0,14.08%.Synthesis ofBAH-O (8). Compound 7 (409 mg, 1.2

mmoles) was dissolved in hot DMF (3 mL) and to thisAH (227 mg, 1.0 mmole), dissolved in hot DMF(1 ml.), was added with stirring. The reaction wascarried out by adding 2 mL of O.IN NaHC03, with2-3 drops of pyridine, by stirring overnight. Thecompound BAH-Owas precipitated out in ether. Theprecipitate was washed repeatedly with ether anddried in vacuum to obtain 8. Yield 75%; m.p.decomposes above 250°C; IR (KBr): 3420, 3242,2941, 2360, 1747, 1731, 1704,1469, 1369 em"; IHNMR: ~)7.29 (d, 1), 7.63 (d, 1),7.88 (m, 1), 8.05 (m,1),8.53 (m, 1),5.79 (d, broad, 2), 5.97 (s, I), 1.38 (m,broad, 2), 1.50 (2), 1.80 (d, 2), 2.02 (d, 2), 2.79 (d, I),2.83 (m, I), 3.11 (m, I), 4.22 (m, broad, I), 4.34 (m,1), 6.36 (s, 1), 6.50 (s, 1); LRMS: 476 (M+Nat; 495(M+Kt; Anal. Found: C, 58.14; H, 5.23; N, 15.12; 0,14.86; S, 6.65. C22H23Ns04S requires C, 58.26; H,5.1l;N, 15.44;0, 14.11;S, 7.07%.

Synthesis of BAH-6 (11).(a) Synthesis of 4-(biotinylamino) benzoic acid 9.

Compound 7 (341 mg, 1.0 mmole) was dissolved in 3mL of hot DMF and cooled. To this, a solution ofPABA (178 mg, 1.3 mmoles) in 2 mL O.IN NaHCOjwas added drop by drop with stirring and the stirringwas continued for 4 hr. The precipitate wasneutralized with IN HCI, filtered and washed with 5mL of cold O.IN HCI and dried with ether. The solidwas dried over NaOH in vacuum. Yield 75%, m.p.fused above 240°C; IR (KBr): 3389, 3249, 1760,1731,1702,1631,1595,1349 cm'.(b) Synthesis of 4-(biotinylamino)benzoic acid -

NHS ester 10. Compound 9 (372 mg, 1.0 mmole)was dissolved in 7 mL of N-methyl pyrrolidone. Tothis was added NHS (140 mg, 1.2 mmoles) and DCC(230 mg, 1.1 mmoles) and the reaction mixture stirredgently overnight at room temp. The dicyclohexyl ureawas filtered off and the filtrate precipitated with ether.The precipitate was filtered and dried in vacuum. TheTLC pure compound was obtained in 45% yield. m.p.decomposed above 260°C; IR (KBr): 3521, 1818,1788,1746,1731,1702,1631,1597,1468 ern".(c) Synthesis of BAH-6 (11). Compound 10 (322

mg, 0.7 mmole) was dissolved in 6 mL of hot DMFand to this AH (113.5 mg, 0.5 mmole), dissolved in 1

2520 INDIAN J CHEM. SEC B. NOVEMBER 2006

mL of hot DMF was added. The reaction was carriedout by the addition of 2 mL O.IN NaHC03, with 2-3drops of pyridine, by stirring overnight. Thecompound was precipitated out in ether. Theprecipitate was washed repeatedly with ether anddried in vacuum to obtain BAH-6. Yield 45%; m.p.decomposes around 240°C; IR (KBr): 3423, 1700(sh.), 1631, 1599, 1393 ern"; IH NMR: (DMSO-d6)

~1.4 (m, broad, 2), 1.6 (m, broad, 3), 1.8 (t, 1),2.25 (t,2),2.5-2.6 (m, 2), 2.7-2.8 (m, 1),4.15 (m, broad, I),4.25 (m, broad, 1),6.35 (1), 6.45 (1),5.95 (broad, I),5.8 (broad, 2), 8.5 (m, 1),8.2 (I), 8(1), 7.85 (1), 6.9(I), 6.85 (2), 7.55 (d, 2), 9.9 (I); LRMS: 595(M+Nat; 611 (M+Kt; Anal. Found: C, 60.93; H,5.18; N, 14.58; 0, 13.76; S, 5.55. C29H2SN60SSrequires C, 60.83; H, 4.93; N, 14.68; 0, 13.97; S,5.60%.

Synthesis of BAH -7 (15)(a) Synthesis of N-(2-aminoethyl)-malanomic

acid 12. To a solution of malonic acid (1.04 g, 10mmoles) in 50 mL of 1,4-dioxane, anhydrousethylenediamine (3 g, 50 mmoles) was added drop bydrop with stirring and subsequently the stirring wascontinued overnight. The product was filtered,washed twice with 1,4-dioxane and recrystallizedtwice in ethanol. The TLC pure compound gave apositive test with ninhydrin and was obtained in 70%yield. m.p. 200°C; IR (KBr): 3425, 3102, 2992, 1654,1567, 1435 em"; IH NMR: ~1.2 (s, 1), 2.5 (s,2), 2.7-2.8 (broad, 4),3.55 (2), 8.25 (1).

(b) Synthesis of N-{2-biotinylamino]-ethyl}-malonamie acid 13. Compound 12 was dissolved inminimum of O.IN NaHC03 and diluted in 2 mL ofabsolute alcohol and to this, a solution of 7 (341 mg,1.0 mmole), dissolved in 3 mL of hot DMF, wasadded drop by drop with stirring. The stirring wascontinued for 4 hr. The reaction was diluted withabsolute ethanol and was precipitated with ether. Theprecipitate was filtered off, washed with 5 mL of coldO.IN HCI and dried over NaOH in vacuum to obtain13 in 79% yield. The compound did not give a pinkspot with ninhydrin, but on exposure to HCI fumesthe pink spot was obtained. IR (KBr): 3426, 1732,1715, 1698, 1683, 1670, 1648, 1636, 1625, 1558,1541 cm'.(e) Synthesis of N-{2-biotinylamino]-ethyl}-

malonamic acid NHS ester 14. Compound 13 (372mg. 1.0 mmole) was dissolved in 5 mL N-methylpyrrolidone and to this NHS (200 mg, 1.75 mmoles)and DCC (330 mg, 1.6 mmoles) were added. The

reaction was stirred gently overnight at room temp.The dicyclohexyl urea was filtered off and the filtrateprecipitated with ether. The precipitate was filteredand dried in vacuum. The TLC pure compound wasobtained in 59% yield. IR (KBr): 3440, 3288, 1819,1788 (shoulder), 1750,1699,1682,1631,1600,1474cm',

(d) Synthesis of BAH-7 (15). Compound 14 (329mg, 0.7 mmole) was dissolved in 6 mL of hot DMFand to this AH (113.5 mg, 0.5 mmole), dissolved in ImL of hot DMF, was added. The reaction was carriedout in 0.5 mL of O.IN NaHC03 with 2 drops ofpyridine by stirring overnight. The compound wasprecipitated out in ether, washed repeatedly with etherand dried in vacuum. The TLC pure compound wasobtained in 40% yield. m.p. fused above 240°C; IR(KBr): 3412, 3288,1700 (sh), 1631, 1595, 1383, 1350cm': IH NMR: (DMSO-d6) 0 7.97 (2),7.7-7.8 (broad,m, 3), 6.8 (I), 5.76 (b, 2), 5.95 (1),6.6 (I), 6.73 (I),4.09 (I), 4.27 (I), 3.05 (I), 2.81 (1), 2.76 (1),2.17 (2),1.75 (1),1.57 (1),1.51 (2),1.45 (2), 2.68 (2),2.54 (2),1.37 (I), 1.260), 2.05-2.0 (2); LRMS: 604 (M+Nat;620 (M+Kt; Anal. Found: C, 55.89; H, 5.43; N,16.73; 0, 16.64; S, 5.31. C27H .•IN706S requires C,55.75; H, 5.37; N, 16.86; 0, 16.50; S, 5.51 %.

Synthesis of BAH-I0 (19)(a) Synthesis of N-(2-aminoethyl)adipamic acid

16. To a solution of adipic acid (1.46 g, 10 mmoles)in 50 mL of 1,4-dioxane, anhydrous ethylenediamine(3 g, 50 mmoles) was added drop by drop withstirring and subsequently the stirring was continuedovernight. The product was filtered washed twicewith 1,4-dioxane and recrystallized twice in ethanol.The melting point of the product was 200°C. Thecompound gave a positive test with ninhydrin andobtained in 75% yield. IR (KBr): 3505, 2580, 2203.1653,1564,1397 cm': IH NMR: ~1-1.2 (1),1.5 (t, 2),1.85 (d, 2), 2.15 (t, 2), 2.25 (t, 2), 2.45-2.5 (2),2.7 (t.r:2), 3.2 (2).

(b) Synthesis of N-(2-biotinylamino)ethyl)adipamic acid 17. The compound 16 was dissolvedin minimum of O.IN NaHC03 and diluted in 2 mL ofabsolute alcohol and to this, 7 (341 mg, 1.0 rnrnole),dissolved in 3 mL of hot DMF, was added. The DMFsolution was added drop by drop with stirring and thestirring was continued for 4 hr. The reaction wasdiluted with absolute ethanol and was precipitatedwith ether. The precipitate was filtered off washedwith 5 mL of cold O.IN HCI and dried over NaOH invacuum. The compound gave a single spot with

MURTHY et al : CHEMILUMINESCENT I3IOTINYL NAPHTHO[I,8-DE][l,2]D1AZEPINE-I,4-D10NES 2521

DACA but gave a negative test with ninhydrin. Ithowever gave a pink spot after exposure to HCIfumes. The compound 17 was obtained in 78% yield.JR (KBr): 3531, 3311, 2935, 1748, 1699, 1639, 1557,1417,1242 cm'

(c) Synthesis of N-(2-biotinylamino)ethyl)adipamic acid NHS ester 18. The compound 17 (990mg, 2.4 rnmoles) was dissolved in 7 ml. of N-methylpyrrolidone and to this, NHS (335 mg, 2.91 mmoles)and DCC (492 mg, 2.39 rnmoles) were added. Thereaction was stirred gently overnight at room temp.The dicyclohexyl urea was filtered off and the filtrateprecipitated with ether, filtered and dried in vacuum.The TLC pure compound gave a single spot withDACA and was obtained in 60% yield. IR (KBr):3464, 3328, 2930, 2852, 1819, 1788, 1699 (shoulder),1628 crn'.

(d) Synthesis of BAH-I0 (19). The compound 18(318 mg, 0.7 mmole) was dissolved in 3 mL of hotDMF and to this, AH (113.5 mg, 0.5 rnrnole),dissolved in 1 mL of hot DMF was added, Thereaction was carried out in 2 mL of 0.1 N aHCO,with a 2-3 drops of pyridine, by stirring overnight.The compound was precipitated out in ether, washedrepeatedly with ether and dried in vacuum. The TLCpure compound was obtained in 51 % yield. m.p. fusedabove 270°C; IR (KBr): 3532, 1748, 1733, 1716,1699,1650, 1361, 1631, 1597, 1559 ern"; IH NMR:(DMSO-d6) 8 7.83 (2),7.97 (2), 8.41 (1),5.76 (broad,2), 6.0 (broad, 1), 6.72 (1), 6.36 (1), 4.27 (1),4.13 (1),3.13 (1),2.84 (I), 2.78 (1), 2.27 (2),1.87 (2),1.65 (2),1.43 (2), 4.32 (1), 1.32 (1) 2.59 (2), 2.48-2.51 (2), 2-2.05 (2), 1.51 (2), 3.67 (2), 3.05 (d, 2); Anal. Found:C, 57.81; H, 5.87; N, 15.68; 0, 15.46; S, 5.18.C-'OH:17N70r,Srequires C, 57.77; H, 5.98; N, 15.72; 0,15.39; S, 5.14%.

Synthesis of BAH-12 (23)(a) Synthesis of N-(6-aminohexyl)succinamic

acid 20. To a solution of succinic anhydride (J g, 10mmoles) in 20 mL of 1A-dioxane, hexamethylene-diamine (5.8 g, 50 mmoles), dissolved in 15 mL oflA-dioxane, was added with stirring. The stirring wascontinued overnight. The product was filtered,washed twice with 1A-dioxane and recrystallizedtwice in ethanol. The TLC pure compound obtained in60% and gave a pink spot with ninhydrin. rn.p. 200°C;IR (KBr): 3402, 3304, 2936, 1693, 1636, 1541, 1402ern"; IH NMR: ~1.9 (s, 2), 2.3 (2),2.6 (t, 2), 2.5 (t, 2),2.8 (r, 2).

(b) Synthesis of N-{6-biotinylamino]-hexyl}-succinamic acid 21. The compound 20 (244 mg, 1.3mmoles) was dissolved in minimum of O.IN NaHCO,and diluted in absolute ethanol and to this, 7 (341 mg,I mrnole), dissolved in 3 mL of hot DMF, was addeddrop by drop with stirring. The stirring was continuedfor 4 hr. The reaction was precipitated with ether. Theprecipitate was filtered washed with 5 mL of coldO.IN HCl and dried over NaOH in vacuum. Thecompound gave a positive test with DACA but did notgi ve a color with ninhydrin and obtained in 80%yield. IR (KBr): 3458, 3302, 2934, 1700, 1638, 1542,1418 ern"; IH NMR: 8 1.2-1.5 (rn, 12), 1.6 (d, 2), 2-2.2 (rn, 4), 2.3 (2), 2.4 (2), 2.6 (2), 2.8 (J), 3.1 (2),3.2(1),4.1 (1),4.3 (J), 6.4 (d, 2), 7.6-7.8 (1).

(c) Synthesis of N-{ 6-[biotinylamino]-hexyl}-succinamic acid NHS ester 22. The compound 21(330 mg, 0.75 mmole) was dissolved in 7 mL of N-methyl pyrrolidone and to this, NHS (115 rng, 1.0mmole) and DCC (170 mg, 0.825 rnrnole) wereadded. The reaction was stirred gently overnight atroom temp. The dicyclohexyl urea was filtered offand the filtrate precipitated with ether. The precipitatewas filtered and dried in vacuum. The compound gavea single spot with DACA and was obtained in 85%yield. lR (KBr): 3446, 3298,2936, 1818, 1780, 1699,1635, 1542, 1349 cm'.

(d) Synthesis of BAH-12 (23). The compound 22(323 mg, 0.6 mmole) was dissolved in 6 mL of hotDMF and AH (113.5 rng, 0.5 mrnole), dissolved in1 mL of hot DMF, was added to it. The reaction wascarried out in 2 mL of O.lN NaHCO-, with 2-3 dropsof pyridine by stirring overnight. The compound wasprecipitated out in ether. The precipitate was washedrepeatedly with ether and dried in vacuum. The TLCpure compound obtained in 56% yield. m.p. fusedabove 250°C; TR (KBr): 3483, 3300, 2937, 1698,1631 1351 crn': IH NMR: (DMSO-dr,) ~8.72 (broad,1),8.5 (d, 1),8.4-8.5 (m, 1),8.0 (m, 1),7.85 (m, I),5.75 (d, 2), 6 (broad, 1),6.35 (1), 6.5 (1),4.1 (r, 1),4.3 (t, I), 3.07 (1), 2.83 (1), 2.78 (l), 2.13 (2), 2.2 (2),2.0 I (2), 1.48 (2), 2.95 (2H), 2.49 (t, 2), 6.4 (1), 7.7(rn, 1), 1.22 (J), 1.57 (2), 2.06 (2), 1.34 (2), 1.29 (2)2.99 (2), 2.56 (2); LRMS: 674 (M+Nat; 690 (M+Kt;Anal. Found: C, 58.83; H, 6.41; N, 14.93; 0, 14.84; S,4.99; CJ2H41N706S requires C, 58.97; H, 6.34; N,15.04; 0, 14.73; S, 4.92%.

ConclusionA new CLt 7-membered peri cyclic hydrazide, AH,

and five of its biotin conjugates with various spacer

10 Wilbur D S, Path are P M, Hamlin D K, Frownfelter M B,Kegley B B, Leung W Y & Gee K R, Bioconj Chem, II,2000,584.

II Langer P R, Waldrop A A & Ward D C, Proc Nat Acad SciUSA, 78, 1981, 6633.

12 Wilchek M & Bayer E A, Methods Enzymol, 138,1987,429.13 Forster C, McInnes J L, Skingle D C & Symons R H, Nuc

Acids Res, 13,1985,745.14 Yankofsky S A, Gurevitch R, Niv A, Cohen G & Goldstein

L, Anal Biochem, 118,1981, 307.15 Thuy L P, Sweetman L & Nyhan W L, C/in Chim Acta, 202,

1991,191.16 Mayer A & Neuenhofer S, Angew Chell! Jilt Edn Engl, 33,

1994,1044.17 Gruber H J, Kada G, Marek M & Kaiser K, Biochim Biopliys

Acta, 1381,1998,203.18 Gruber H J, Marek M, Schindler H & Kaiser K, Bioconj

Chern, 8, 1997, 552.19 White E H & Matsuo K. J Org Chern, 32,1967,1921.20 Caswell L R & Cavasos G, J Heterocycl Chern, 32, 1995,

907.21 The Aldrich Library of Infra Red Spectra, Edn. Ill, 1101E &

1087C.22 Wehner V & Jager V, Allgew Chem Jilt Edn Engl, 29, 1990,

1169.23 Furst, Berlo R C & Hooton S, Chem Rev, 65, 1965, 51.24 Bellamy F D & Ou K, Tetrahedron Lett, 25, 1984, 839.25 Barrett G M & Spilling C D, Tetrahedron Left, 29, 1988,

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1961, 13.28 Nakashima H, Takenaka Y, Higashi M & Yoshida N, J

Client Soc Perkin Trails 2, 2002. 2096.

2522INDIAN J CHEM, SEC B, NOVEMBER 2006

groups were synthesized. The compounds werecharacterized by IR, NMR, Mass spectra andelemental analysis. The compounds exhibit CLanalogous to luminol. The CL of these hydrazides wasenhanced when directly connected to the biotinthrough an amide linkage while via spacer itsefficiency decreased. No linear relationship existedbetween the spacer length and CLt light output.

AcknowledgementOne of the authors (NVK) thanks the Chief

Executive, BRIT and officer-in-charge, JONAKI,BRIT for their kind encouragement.

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