Indian Journal of Chemistry Vol. 41B, February 2002, pp. 388-393

Synthesis and anticancer activity evaluation of some hemin and hematoporphyrin derivatives

Sham M Sondhi*, Shefali Rajvanshi & Monika lahar

Department of Chemistry, University of Roorkee. Roorkee 247667, India and

Sunanada G Dastidar

Ranbaxy Research Laboratories, New Delhi 110 020, India

Received 3 October 20()(), accepted (revised) 19 June 200 I

Sulphamerazine, sulphadiazine, and sulphaguanidine are coupled with hemin to give bis coupled products la, Ib and Ie respectively. 3, 4-Diphenyl iminothiazoline, sulphamerazine, sulphaacetamide, sulphathiazole and sulphadiazine on cou­pling with hematoporphyrin give bis coupled products 2a, 2b, 2e, 2d and 2e respectively. Compounds la-e and 2a-e have been screened for anticancer activity against a small panel of six cancer cell lines consisting of prostate( DU 145), colon (HT29), melanoma (LOX), breast(MCF7 and MCF7/ADR) and CNS(U251) tumors. Best GI50 (concentration which inhibits the cell growth by 50%) values are shown by 2e, 2. 2~M(prostate tumor, cell line DUI45); 2e 13.0)..lM(colon tumor, cell line HT29); 2b, 3.4)..lM( melanoma tumor, cell line LOX); 2e, 9.7)..lM(breast tumor, cell line MCF7); 2a, 3. I)..lM(breast, tumor, cell line MCF7/ADR) and Ie, 3.4)..lM(CNS tumor, cell line U251) respectively . Out of all the compounds reported here GI50 value shown by 2a i.e.3. I)..lMagai nst breast, tumor (MCF7/ADR) is quite close to the GI50 value i.e. 1.8)..lM, of standard drug doxorubic in .

Hemin and metalloporphyrins can carry out numerous functions' in a free state or in association with spe­cific proteins . Lipid peroxidation can be inhibited2 by hemin and could produce partial recovery to near normal levels3 of hematopoieses caused by AZT. Hemes form the prosthetic groups of a number of pro­teins and these hematoproteins exhibit impressive range of biological functions4. Metal porphyrin oli­gO£lUcleotide conjugates have been used as chemilu­minescent DNA5 probes. Pt(II) porphyrin complexes6

derived from hemin and porphyrin derivatives7 have been used in photodynamic therapy which is based on the ability of some porphyrin and porphyrin like chromophores8 to be accumulated selectively in tumor tissues and tumor necrossis can be obtained by irra­diation of neoplastic area with light of appropriate wave length. Radiolabeled porphyrins uptake9 by tu­mor is not blocked by hemin and its preinjection in­duced higher tumor uptake in rats, upto a factor of 5 with 24 hr post injection. Pretreatment of the animals with hemin caused better therapeutic ratio of porphyrin.

Hematoporphyrin glycoside derivatives useful in photochemotheraphy of malignent tumors '0, haemin­acridines showing antileukemic properties ", metal­loporphyrins showing tumor growth inhibitory ef-

fect'2 and porphyrin (Fe) intercalator causing DNA scission'3 have been reported in literature.

In the last decade'4 importance of porphyrins and metalloporphyrins has increased signi ficantly. In the clinic most activity has focused on photodynamic therapy of cancer, porp!1yrias and hematol diseases and various forms of jaundice. Importance of propi­onic acid side chains of herrun in the induction of erythroid differentiation of K562 cells have been highlighted in a recent paper reported in literature '5. In continuation'6-20 of our studies on porphyrins and metalloporphyrins we have synthesized a number of hemin and hematoporphyrin derivati ves and evaluated for anticancer activity which we wish to report in this paper.

Sulphamerazine, sulphadiazine, sulphaguanidine, sulphathiazole and sulphaacetamide were obtained from Aldrich or Sigma Chemical Company. 3, 4-Diphenyl-2-iminothiazoline used for synthesis was synthesized by the condensation of phenacyl thiocy­anate with aniline hydrochloride as reported in litera­ture2'. Various coupling agents i.e. 1, l' -carbonyl di­imidazole, 1, 3-dicycIohexyl carbodi imide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiirrude hydro­chloride (EDC) were used for the bis coupling of sul­phamerazine with hemin. Best results were obtained

SaNDHI et al. : SYNTHESIS & ANTICANCER ACfIVITY EV ALVA TION OF HEMIN & HEMATOPORPHYRINS 389

H:>C

DMF; EDC; RNH2

stirring at RT for 4 days

H:>C

CH2 ! CH2 ! CONHR

1

CH2 I

CH2 I

CONHR

Scheme I

by using EDC and so in the synthesis of 1a-c (Scheme I) EDC was used as a coupling agent. Thus hemin and EDC in the ratio of 1:2 mole equivalent were taken in dry DMF and stirred at room tempera­ture for 2 hr and then sulphamerazine (2 mole equiva­lent) was added and reaction mixture was stirred at room temperature for four days. Removal of solvent under reduced pressure and washing of residue with water removed excess EDC. The crude product was crystallized from methanol to give bis coupled prod­uct 1a in 50% yield (Scheme I). Similarly sulphadiaz­ine and sulphaguanidine were coupled with hemin to give 1b and 1c respectively. IR spectrum of 1a shows absorption bands at 3391, 1644 and 1590 cm' ! corre­sponding to -NH-, CONH- and aromatic groups. Due to bulky nature of hemin derivatives synthesized, low resolution electron spray (LRES) or fast atom bom­bardment technique (FAB) was used for recording mass spectrum and in case of 1a and 1b, M+ ion peak could not be observed but in case of 1c, M+ ion peak at 1008 was observed which was in agreement with the structure assigned to 1c. Compounds 1a-c gave satisfactory elemental analysis for C and H. Spectral data and physical constants of 1a-c are reported in Table I. and are in agreement with the assigned struc­tures.

3, 4-Diphenyl-2-iminothiazoline2! was bis coupled with hematoporphyrin using EDC as a coupling agent.

The crude product was washed with methanol and was recrystallized from THF to give pure bis coupled product 2a (Scheme II) in 50% yield. IR spectrum of 2a give absorption bands at 3675, 3385, 1690 and 1597 cm'; corresponding to -OH, -NH-, -CON= and aromatic groups respectively. LRES MS of 2a gave M+ ion peak at 1067.3 which was in agrrement with the structure assigned to 2a. Similarly sulpha merazine, sulphaacetamide, sulphathiazole and sul­phadiazine were coupled with hematoporphyrin to give corresponding bis coupled products i.e 2b, 2c, 2d and 2e respectively. Compounds 2a-e gave satisfac­tory elemental analysis for C and H.Yield, mp, and spectral data of 1a-c and 2a-e reported in Table I fully support the structures assigned to them.

Anticancer activity evaluation of 1a-c and 2a-e was carried out against a small panel of six cancer cell lines consisting of prostate (DU 145) colon (HT29) melanoma (LOX) breast (MCF7) breast(MCF7/ADR) and CNS (U251) tumors. The effect of various com­pounds screened is expressed in terms of 50% growth inhibitory concentration (GI50). The GI50 values in JlM concentration of 1a-c and 2a-e are reported in Table II. From Table II it is clear that best GI50 val­ues are shown by 2c, 2.2~ (prostrate tumor, cell line DUI45); 2c 13.0JlM (colon tumor, cell line HT 29); 2b, 3.4JlM (melanoma tumor, cell line LOX);2c, 9.7JlM (breast tumor, cell line MCF7); 2a, 3.1JlM

390

Compd

la

Ib

Ie

2a

2b

2c

2d

2e

INDIAN J. CHEM., SEC B, FEBRUARY 2002

Table I - Physical constants and spectral data of hemin la-e and hematoporphyrin 2a-e derivatives

Solvent. of m.p. Yield

cryst. (0C) ( (%)

MeOH 185 50

MeOH 217 20

MeOH 200 40

THF >230 50

THF >230 90

THF >240 40

THF >240 70

THF >240 65

IR(KBr) . · 1

Vmax. m cm

3391 (-NH-)

1644 (-CON H-), 1590(Ar)

3384 (-NH-), I 633(-CON H-), 1588(Ar)

3583 & 3433 (-NHd, 3313(-NH-) 1646 (-CONH-), 1582(Ar)

3675(-OH),3385(-NH-), 1690(-CON=), 1597 (Ar)

3597 (-OH), 3430 (-NH-), 1670 (-CONH-), 159 1 (Ar)

3500 (-OH), 3400 (-NH-), 1670 (-CONH-), 1538 (Ar)

3539 (-OH), 344 8 (-NH-), 1685 (-CONH .. ), 1538 (Ar)

3500 (-OH), 3380 (-NH-), 1650 (-C ONH-), 1588(Ar)

LRES or FABMS

rnJz (relt. int. %)

1093 (M+-CH3; 0.2),844

CH3

(M+- NH-fi-SOl'lH-<~j ; 1.4)

986 (M+-· N); 0.5 NH-<, ;;

N 936 (rnJz 986-H20 ; 3)

1008 (M+, 0.1), 972 (M+ -2H20; 7)

1067.3 (M+, 6) 1049.3, (M+-H20 ; 24)1031.3, (M+ -H20; 31 )824.3

[M+-(2H2O + L~-COCH~H2 ) Ph IN-Ph

10.OJ

1001(M+, 7), 1073(M+-H20; 12), 1055 (M+-2H20 ; 13)

CH3

962 [M+-(2H2O+ N~ ; 15J

'<~J'

932.5 (M+-CH3CONH; 6), 9 14.4 (rnJz 932.5-H20;7) 1073 (M+; 7)

1037.1 (M+ -2H20; 40),

N-J 956 (M+-H20 + .)l I ; 65). HN S

[M+-( NH-QSOI'lH-{) H20); 8J ;

786.19 (M+ -CONH-fi-SO~H--<~)

* Compounds la-e and 2a-e gave satisfactory e lemental analysis for C & H.

SONDHI et ai. : SYNTHESIS & ANTICANCER ACTIVITY EVALUATION OF HEMIN & HEMATOPORPHYRINS 391

H~

H~

OH I

HC-CH3 CH3

OH I CH-CH3 H~

DMF; DCC; RNH2 or R = NH

stirring at RT for 4 days

CH3 H~

OH I

HC-CH3

CH2 CH2 CH2 I I I CH2 CH2 CH2 I I I COOH COOH CONHR

2a , R = fSY For 2a moiety -NH-R will be -N =R Ph~N-Ph

CH3

2 •• R = ----<Q>-So,NHi)

2c , R = ----<Q>-SO~H-COCH3

2d , R = ----<Q>-SO~H-tSJ 2 • • R = ----<Q>-So,NHi)

Scheme II

2

CH2 I CH2 I

CONHR

Table 11- Anticancer activity evalution of hemin Ia-e and hematoporphyrin 2a-e derivatives

Compd GIso Concentration in J.tM

Prostate' Colon' Melanoma" Breast' Breast Resistant" CNS"

DUI45b HT29b LOXb

la 17.6 26.0 >67.9

Ib 12.9 28.1 40.3

Ie 8.2 20.1 7.6

2a 8.4 24.1 >77.2

2b 13.6 14.8 3.4

2e 2.2 13.0 9.7

2d 9.9 27.0 11.0

2e 13.0 28.1

Doxorubicin 0.2 0.4

"Tumor type. b cell lines

(breast tumor, cell line MCF7/ADR) and Ic 3.4f.lM (CNS tumor, cell line U251) respectively.Out of all the compounds reported in Table II. GI50 value shown by 2a i.e. 3.1f.lM against breast, tumor (MCF7/ADR) was quite close to the GI50 value i.e. 1.8f.lM of standard drug doxorubicin.

4.3

0.1

MCF7b MCF7/ADRb U251 b

>67.9 >67.9 18.3

>69.1 22.1 13.6

29.2 21.0 3.4

>77.2 3.1 10.4

10.3 10.8 11.0

9.7 11.3 11.3

>83.9 9.8 13.8

12.3 4.2 9.8

0.1 1.8 0.2

Experimental Section

Melting points were determined on lSGW appara­tus and are uncorrected. IR spectra were recorded on a Perkin Elemer 1600 Ff spectrophotometer (principal sharply defined IR peaks are reported) and mass spec­tra using lEOL SX-102 and AEI M9-9 double focus-

392 INDIAN J. CHEM., SEC B, FEBRUARY 2002

ing high resolution mass spectrometer at a resolving power of 15.000. TLC was performed by using silica gel G (Merck) and spots were visualized by iodine vapour or by irradiation with UV light (254 nm).

Condensation of sulphamerazine with hemin 1a. Hemin (652 mg; 1 m mole) was taken is dry DMF (20 mL) and to it was added EDC (384 mg: 2 m moles). The reaction contents were stirred at room temp for 2 hr and then to it was added sulphamerazine (528 mg; 2 m moles). The reaction contents were stirred for 72 hr at room temperature and then solvent was removed under reduced pressure. The solid residue left behind was washed with water and air dried to give crude bis coupled product which was purified by crystallization from methanol to give pure product 1a.

Similarly sulphadiazine and sulphaguanidine were coupled with hemin to give bis coupled products 1b and Ic respectively. Yield, m.p., solvent of crystalli­zation and spectral data of 1a-c are reported in Table I.

Condensation of 3, 4-diphenyl-2-iminothiazoline with hematoporphyrin 2a. Hematoporphyrin (600 mg; 1 m mole) was dissolved in dry DMF (20 mL) and to it was added EDC (384 mg; 2 m moles). The reaction contents were stirred at room temperature for 2 hr and then 3, 4-diphenyl-2-iminothiazoline (504 mg; 2 m moles) was added to it. The reaction contents were stirred at room temperature for 72 hr and then solvent was removed in vaccuo. The solid residue left behind was washed with water and air dried to give crude bis coupled product. This crude product was washed thoroughly with methanol and methanol in­soluble product was crystallized from THF to give bis coupled product 2a.

Similarly sulphamerazine, sulphaacetamide, sul­phathiazole and sulphadiazine were coupled with he­matoporphyrin to give 2b, 2c, 2d, and 2e respectively. Yield m.p., solvent of crystalization and spectral data of 2a-e are reported in Table I.

Anticancer activity screening. Compounds 1a-c and 2a-e were tested over a broad concentration range (ten told dilutions starting from > 100~M to 10 nM) against six human cancer cell lines comprised of dif­ferent tumor types maintained in growing condition in RPMI 1640 medium containing 10% fetal calf serum and incubated at 37°C under 5% CO2 atmosphere. All cell lines were inoculated on to a series of standard 96 well microtitre plate on day zero, followed by 24 hr incubation in the absence of test compound. The in­oculation density used currently in the screen was as per Monk et. a/22

• All the test compounds i.e. 1a-c and

2a-e were dissolved in DMSO and diluted further in culture medium. An aliquot of each dilution was added to the growing cells in 96 well plates and incu­bated for 48 hr. After incubation, the assay was termi­nated by adding 50 ~L of trichloroacetic acid (TCA) and incubating at 4°C for 30 min. The precipitated cells were washed and stained with sulphorhodamine B dye for 30 min and the excess dye was washed off with acetic acid. Adsorbed dye is solubilized in tris base (alkaline pH) and quantitated by measuring the OD at 490 nm in an ELISA reader. GI50 values were calculated according to Boyd & Pau1l23 and are re­ported in Table II.

Acknowledgement We are thankful to Head, RSIC, CDRI, Lucknow,

Prof. J W Lown, Department of Chemistry, Univer­sity of Albenta, Canada, and technical staff of Chem­istry Department, University of Roorkee, Roorkee for FABMS, elemental analysis, LRES and IR spectra. Financial help from UGC, New Delhi (Monika Johar) is greatfully acknowledged.

References I Fiorucci G, Percario Z A, Coccia E M, Battistini A, Rossi G

B, Romeo G & Affabaris E, J Interferon Cytokine Res, IS , 1995,39S.

2 Kimie I, Tachio A, Masaki S & Ryohei K, Chem Pharm Bull, 38, 1990,2S8.

3 Lutton J D, Mathew A, Levere R D & Abraham N G, Am J Hematol, 3S, 1990, I.

4 Chapman S K, Daff S & Munro A W, Struct Bonding (Ber· lin), 88, 1997,39.

S Kluetsch T, Schubert F & Cech D, Collect Czech Chem Com, 61,1996 (Spec Issue), S294

6 Brunner H, Obermeier H & Szeimies R, Chem Ber, 128, 1995,173.

7 Nakajima S, Sakata I & Tokemura T, Drug Delivery Syst, II , 1996, lOS .

8 Vicente Maria Da Graca H, Rev Port Quim, 3, 1996,47. 9 Huenerbein M, Sinn H, Schrenk H, Maier-Borst W & Frie­

drich E A, Proc SPIE-int Soc Opt Eng, 1993, 1616. (Interna­tional conference on photodynamic therapy and laser medi­cine), 1991, 199.

10 Bourhim A, Czernecki S, Krausz P, Viari A & Vigny P, J Carbohydr Chem, 9,1990,761 .

II Lown J W & Joshua A V, J Chem Soc Chem Commun, 1982, 1298.

12 Sakata [, Nakajima S, Koshimizu K, Takada H & Inui H, Eur Pat Appl EP 3S0948, Chem Abstr, 114,1991, 6ISSy.

13 Hashimoto Y, Lee C S, Shudo K & Okamoto T, Tetrahedron Lett, 24, 1983, IS23.

14 Cannon J B, J Pharm Sci, 82,1993, 43S. IS Nakajima 0, Iwasaki S & Hashimoto y , Cell Mol Biol(Paris),

43, 1997, liS. 16 Lawn J W, SQndhi S M, Ong C W, Skorobogaty A, kishikawa

H & Dabrowiak J C, Biochemistry, 2S, 1986, Sill.

SONDHI et al. : SYNTHESIS & ANTICANCER ACTIVITY EVALUATION OF HEMIN & HEMATOPORPHYRINS 393

17 Sondhi S M, Magan A & Lown 1 W, Bull Chern Soc Jpn, 65, 1992,579.

18 Sondhi S M & Magan A, Indian J.Het Chem, 4, 1994, 155.

19 Sondhi S M, Verma R P, Sharma V K, Singhal N, Kraus 1 L, Camplo M & Cherman 1 C, Phosphorus Sulfur and Silicon, 122,1997,215.

20 Sondhi S M, Singhal N, Verma R P, Arora S K & Dastidar S

G, lndian J.Chem, 40 B, 2001, 113. 21 Mahajan M P, Vasudeva S K & Ralhan N K, Indian J.Chem

10,1972,318. 22 Monks A, Scudiero D, Skehan P, Shoemaker R, Paull K, Vis­

tica D, Hose C, Languely 1, Cronise P & Vaigrowolf A, J Natl Cancer blStt, 83, 1991,757.

23 .Boyd M R & Paull K D, Drug Dev Res, 34, 1995, 91.