Structure-Activity Relationships of Isothiocyanates

[CANCER RESEARCH 54, 4327-4333, August 15, 1994] Structure-Activity Relationships of Isothiocyanates as Mechanism-based Inhibitors of 4-(Methy Initrosamino) -1-(3-py ridy 1)-1-butanone-induced Lung Tumorigenesis in A/J Mice1

Ding Jiao, Karin I. Eklind, Chang-In Choi, Dhimant H. Desai, Shantu G. Amin, and Fung-Lung Chung2

Division of Chemical Carcinogcnesis, Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, New York 10595

ABSTRACT duced tumor development in the lung and forestomach and N-nitroso- diethylamine-induced neoplasms in the forestomach in A/J mice (1, A structure-activity relationship study was carried out to identify struc 2). PEITC showed significant inhibitory effects against lung tumor tural features in arylalkyl and alkyl isothiocyanates that are associated with the inhibitory potency of these compounds against lung tumorigen- induction by NNK in both F344 rats and A/J mice (3, 4). PEITC also esis induced in A/J mice by the tobacco-specific nitrosamine 4-(methylni- effectively inhibited W-nitrosobenzylmethylamine-induced esopha- trosamino)-l-(3-pyridyl)-l-butanone (NNK). These features include the geal carcinogenesis in rats (5). However, PEITC showed no effect on alkyl chain length, phenyl substitution, and secondary isothiocyanates. lung tumorigenesis induced by B(a)P in A/J mice (6). Since both The naturally occurring ally! isothiocyanate, phenethyl Â¡sothiocyanate, NNK and B(a)P are important tobacco carcinogens and lung cancer is and the synthetic analogues such as 6-phenylhexyl isothiocyanate, 8-phe- attributed to tobacco usage (7), further development of more effective nyloctyl isothiocyanate, 10-phenyldecyl isothiocyanate, 1,2-diphenylethyl isothiocyanate, 2,2-diphenylethyl isothiocyanate, and alkyl isothiocyanates chemopreventive agents in the isothiocyanate family is warranted for (with 1-hexyl, 2-hexyl, and 1-dodecyl as alkyl moieties) were assayed in the prevention of lung cancers (8-11). mice for their tumor inhibitory potential. The isothiocyanates were given We have previously shown that synthetic analogues of PEITC with in corn oil by gavage at doses of either 0.04, 0.1, and 0.2 juniol or 1 and 5 a longer alkyl chain length, up to 6 carbons as in PHITC, possess /imol 2 h prior to a single i.p. injection of 10 jumol NNK. Mice were greater inhibitory activity against NNK-induced lung tumors in A/J sacrificed 16 weeks later and lung adenomas were counted. At 0.2 /Â¿mol, mice (12). These natural and synthetic isothiocyanates are potent 8-phenyloctyl isothiocyanate and 10-phenyldecyl isothiocyanate were competitive inhibitors of cytochrome P-450 isozymes in mouse lung stronger inhibitors than the previously tested 6-phenylhexyl isothiocya microsomes that are responsible for the metabolic activation of NNK nate, but the difference in potency was not obvious at the lower doses. At to methylating and pyridyloxobutylating species (13-15). Inhibition both 1 and 5 /xmol, ally! isothiocyanate was inactive, while the other five of the metabolic activation of NNK reduces the formation of O6- synthetic isothiocyanates were considerably more potent than phenethyl isothiocyanate. In the alkyl isothiocyanate series, 2-hexyl isothiocyanate methylguanine and consequently decreases the number of neoplasms was more potent than 1-hexyl isothiocyanate, while 1-dodecyl isothiocya in the lungs of NNK-treated A/J mice (3, 12). The greater inhibitions nate was the most potent at 1 itmol, reducing tumor multiplicity in the by arylalkyl isothiocyanates with a longer alkyl chain are qualitatively group treated with NNK alone from 11.1 to the background level. Also, correlated with the decreasing level of O6-methylguanine in the DNA 1,2-diphenylethyl isothiocyanate appeared to be a stronger inhibitor than of lung tissues (12). Consistent with these observations, in vitro 2,2-diphenylethyl isothiocyanate. In this study we have shown that the studies have shown that increasing the alkyl chain length enhanced the phenyl moiety is not essential for the inhibitory activity since alkyl iso binding affinity of isothiocyanates to cytochrome P-450 isozymes, thiocyanates exhibit strong inhibitory effects against lung tumorigenesis. We have also shown that secondary isothiocyanates possess a higher which resulted in greater inhibition of NNK metabolism (15). It has potency than their structural isomers bearing a primary isothiocyanate. been suggested that the increased lipophilicity of the longer chain From results of this study and of seven previously studied isothiocyanates, analogues enhances the binding affinity of the isothiocyanates to we conclude that the observed inhibitory potency of isothiocyanates in the cytochrome P-450 isozymes (12). Factors such as chemical stability, A/J mouse lung tumor model is correlated with their partition coefficients metabolism, absorption, and molecular geometry of these isothiocya (log /') and the pseudo first order rate constants for the reaction of nates may also play a role in the relative inhibitory potency of these isothiocyanates toward glutathione (A,,,,J. These results reveal that both compounds. Since the major metabolic pathway of isothiocyanates in high lipophilicity and low reactivity of isothiocyanates are important for rodents and humans proceeds via conjugation with GSH (16-18), inhibitory activity toward NNK-induced lung tumorigenesis. These obser vations provide a structural basis for the discovery of more effective their chemical reactivity toward GSH would seem to be important for chemopreventive agents. in vivo potency. The identification of the structural features of iso thiocyanates is important to explain their inhibitory efficacy and to guide the design of more effective chemopreventive agents. Our goals INTRODUCTION in this study were: (a) to determine the optimal chain length for Naturally occurring isothiocyanates of cruciferous origin have been maximal tumor inhibition by testing synthetic arylalkyl isothiocya shown to protect against tumorigenesis induced by environmental nates with longer alkyl chain lengths than those previously assayed; carcinogens such as polycyclic aromatic hydrocarbons and nitro- (b) to examine other structural features that are critical to the inhib samines in rodents (1-3). For example, BITC3 inhibited B(o)P-in- itory activities by replacing the aromatic moiety with allyl and alkyl groups, by increasing the number of aromatic rings, or by including

Received 3/25/94; accepted 6/6/94. secondary isothiocyanates; (c) to correlate lipophilicity and chemical The costs of publication of this article were defrayed in part by the payment of page reactivity of isothiocyanates with their relative inhibitory potency in charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by Grant CA 46535 from the National Cancer Institute. cyanate, PHITC, 6-phenylhexyl isolhiocyanate; POITC, 8-phenyloctyl isothiocyanate; This is Paper 19 in the series. "Dietary Inhibitors of Chemical Carcinogenesis." PDITC, 10-phenyldecyl isothiocyanate; OPBITC, 4-oxo-4-(3-pyridyl)bulyl isothiocya 2 To whom requests for reprints should be addressed. nate; HITC, 1-hexyl isothiocyanate; 2HITC, 2-hexyl isothiocyanate; DDITC, 1-dodecyl ' The abbreviations used are: BITC, benzyl isothiocyanale; NNK, 4-(methylnitro- isothiocyanate; 12DPEITC, 1,2-diphenylelhyl isothiocyanate; 22DPEITC, 2,2-diphenyl samino)-l-(3-pyridyl)-l-butanone; B(a)P, benzo(a)pyrene; AITC, allyl isothiocyanate; ethyl isothiocyanate; GSH. glutathione; HPLC. high performance liquid chromalography; PITC, phenyl isothiocyanate; PEITC, phenethyl isothiocyanate; PPITC, 3-phenylpropyl DCI, desorption chemical ionization; NMR, nuclear magnetic resonance; m, multiplet; t, isothiocyanate; PBITC, 4-phenylbutyl Â¡solhiocyanate; PPelTC, 5-phenylpentyl isothio- triplet. 4327

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1994 American Association for Cancer Research. CHEMOPREVENTIVE ACTIVITIES. LIPOPIIIIJCITY. AND REACTIVITY order to establish a convenient guide for predicting their inhibitory which is consistent with the M+ 1 of the molecular formula C|5H,|NS (cal activities against lung tumorigenesis (Figure 1). culated, 247.405). 'H-NMR (360 MHz, CDCI,, 6 in ppm, tetramethylsilane as internal reference): 0 1.30-1.45 (m, 8H), 1.55 - 1.75 (m, 4H), 2.60 (t, J = 7.6 Hz, 2H), 3.50 (t, J = 6.6 Hz, 2H), 7.18-7.32 (m, 5H). "C-NMR (92.52 MHz, MATERIALS AND METHODS CDCI,, S in ppm, tetramethylsilane as internal reference): 8 26.51, 28.70, Animals. Female A/J mice were obtained from The Jackson Laboratory 29.11, 29.22, 29.93, 31.39, 35.92, 45.03, 125.59, 128.23, 128.38, 129.61 (Bar Harbor, ME). The mice were fed the semipurified diet AIN-76A with 5% (-NCS), 142.75. The DCI-mass spectrometry of PDITC showed a base peak at corn oil (Dyets, Bethlehem, PA) and were maintained under the following mie 276, which is consistent with the M + l of the molecular formula standard conditions: 20 Â±2Â°C(SD), 50 Â±10% relative humidity, and a 12-h C^H^NS (calculated, 275.4ft). 'H-NMR: Â«1.30-1.45 (m, 12H), 1.55-1.75 light. 12-h dark cycle. Animals were used in bioassay at 7 weeks of age after (m, 8H), 2.70 (t, J = 7.6, 2H), 3.5 (t, J = 6.6, 2H), 7.18-7.32 (m, 5H). 13C-NMR: 26.54, 28.77, 29.27, 29.35, 29.40, 29.40, 29.94, 31.49, 35.96, 45.05, 2 weeks of quarantine. Instrumentation. A HPLC system equipped with an automatic gradient 125.56, 128.21, 128.38, 129.59 (-NCS), 142.87. controller, two Model 501 pumps, and a Waters 990 photodiode array detector Pulmonary Adenoma Bioassay in the A/J Mouse. The single-dose model in conjunction with a 4.6 x 250-mm Partisi! 5 ODS-3 reverse-phase C|K for NNK lung tumor induction in the A/J mouse was used in this study (21). analytical column (Whatman, Clifton, NJ) was used in the determination of the As an extension of the previous bioassay with PHITC, POITC, and PDITC capacity factor k' of isothiocyanates. In the kinetic studies, we used a Hewlett- were examined with reference to PHITC to determine the optimal chain length Packard 8452A diode array spectrophotometer (Hewlett-Packard, Palo Alto, in arylalkyl isothiocyanates for maximal potency (12). The doses chosen were CA) interfaced with a computer and the MS-DOS UV/VIS software. NMR based on the lowest dose (0.2 /unni i for PHITC (the most potent inhibitor spectra were recorded on a Bruker AM 360 WB spectrometer (Bruker, Bil- among the previously tested arylalkyl isothiocyanates) to be effective. In the lerica, MA) using CDCI, as solvent. DCI-mass spectra were obtained on a second bioassay, six other isothiocyanates of various structures were assayed Hewlett-Packard 5988A mass spectrometer. using PEITC as a reference compound at doses tested previously (3). Groups Chemicals. AITC, BITC, P1TC, and PEITC of highest purity were pur of 20 A/J mice were each given isothiocyanate by gavage at a dose of 5, 1, 0.2, chased from Aldrich Chemical Co. (Milwaukee, WI). PPITC, 8-phenyloctyl 0.1, or 0.04 fimol/mouse depending upon the isothiocyanate tested. Two h chloride, and 10-phenyldecyl chloride were obtained from Fairfield Chemical later, mice were given 10 /xmol NNK (in 0.1 ml saline) by i. p. injection. Mice Co. (Blythewood, SC). HITC, 2HITC, DDITC, 12DPEITC, and 22DPEITC in control groups were treated with either corn oil alone or isothiocyanate in with greater than 98% purity were obtained from Trans World Chemicals corn oil, followed by i.p. injection of saline. Sixteen weeks after these treat (Rockville, MD). GSH (reduced form) was purchased from Sigma (St. Louis, ments the mice were killed, and pulmonary adenomas were counted. The MO). PBITC, PPelTC, OPBITC, PHITC, and the tobacco-specific lung car statistical significance of bioassay data was determined by Student's t test. cinogen NNK were previously synthesized and characterized in our laborato Measurement of Lipophilicity by HPLC. The fully silanized Whatman ries (12, 19,20). Partisil ODS-3 C,s analytical column was used under the same HPLC condi POITC and PDITC were newly synthesized. The general procedures for tions for all isothiocyanates to minimize variability. The mobile phase consists synthesizing isothiocyanates through the reduction of azide and reacting the of a mixture of 70% acetonitrile and 30% water eluting at a flow rate of 1 resulting amine with thiophosgene were used (19). POITC and PDITC were ml/min. Sample solutions of isothiocyanates were prepared by dissolving 2 ftl both obtained with HPLC purity greater than 98%. The spectral data are as of each isothiocyanate in 1 ml of acetonitrile with 0.1% formamide. Ten fil of follows: the DCI-mass spectrometry of POITC showed a base peak at mie 248, each sample were used for each HPLC analysis. The peak of formamide (log P = - 1.69) was used as an unretained reference peak (22). The capacity factor k' was calculated as follows: (CH2)n N C K' = (tg - Â«â€ž)//,â€ž

n=0 . P lTC where Â¡Kisthe retention time of the isothiocyanate and /â€žisthe retention time of the unrctaincd peak. For isothiocyanates with known log P values, a linear 1 , BITC correlation between log P and log k' was obtained with a correlation coeffi cient of 0.97 (Fig. 2), log P = 2.322 + 3.675 log k'. With this linear equation, H lTC 2 , PE ITC the log P values of all isothiocyanates were calculated from their k' values. 3, PPITC Measurement of the First Order Reaction Rate Constants of Isothio cyanates. A stock solution of each isothiocyanate in acctonitrile (10 mM) was 4 , PBITC prepared. GSH solution (10 mM) was freshly prepared by dissolving 30.7 mg 2H ITC 5, PPelTC of GSH (reduced form) in a 10-ml mixture of 0.2 Mphosphate buffer (pH 7.4) and methanol (1:1). Both solvents were degassed under vacuum before use. 6 , PH ITC N=C=:=S This high concentration of methanol is necessary to ensure miscibility for those DDITC highly lipophilic isothiocyanates. In each UV cell, 1 ml of GSH solution was 8 POI TC added and a UV spectrum was recorded as a background. After addition of 10 10, PDITC fil of isothiocyanate solution to the UV cell, the product formation was monitored by the programmed UV scans with selected time intervals. The O change of the absorbance at wavelength 270 nm, an absorption wavelength for the conjugate at which there is essentially no absorption for isothiocyanates, except for PITC, was used for calculations of the observed first order reaction rate constants Â¿obb.Inthe case of PITC, absorbance at 300 nm was used. The OPB ITC following equation was fitted with observed data by using the built-in regres N ~O sion method. i N CHj 12DPEITC

NNK where A(>,A,, and Aa are the absorbance at time zero, during the run, and at the end of the run, respectively. Three separate measurements were made for each Fig. 1. Structures of isothiocyanates and NNK. isothiocyanate. 4328

RESULTS Table 2 Effects of synthetic and naturally occurring i.mthiocyanates an NNK-induced lung lumorigenicity" Inhibition of NNK-induced Pulmonary Adenoma by Long Dose Tumor Chain Arylalkyl Isothiocyanates. POITCand PDITCwere tested in (/xmol/mouse)CornTreatment No. of(%)'40202019"2(12(\2(1 animals multiplicity'' incidence this assay at 0.2, 0.1, and 0.04 /nmol/mouse. PHITC was included as oil/NNKPEITC/NNK a positive control because our previous studies had shown that 0.2 1AITC/NNK'' jLimolof PHITC was inhibitory (12). The tumor multiplicities and 51HITC/NNK Â±1.211.6Â±1.0i.sÂ±o.y4.6Â±OV0.3 incidences are summarized in Table 1. During the bioassay, all groups showed normal body weight gains. The mice in the control groups 512HITC/NNK pretreated with 0.2 /xmol of isothiocyanates or with corn oil prior to saline administration developed only background tumors normally 5 Â±O.I7 found in the A/J mouse; in some cases, no tumor was found. Mice 1DDITC/NNK 19"202(12020202010101.6Â±0.4/0.3Â±0.1/ pretreated with corn oil, prior to NNK administration, developed 10.2 Â±0.6tumors/mouse with an incidence of 100%. These responses 5 122DPEITC/NNK 0.6Â±0.2/0.6Â±0.2/'i.iÂ±o.y0.535*45*

5 60*45*25*10*30* 5.0 112DPEITC/NNK 51AITC/saline'' Â±O.I70.4Â±0./0.2Â±0.2/0.2Â±0.1/

4.5 PPelTC HITC 5HITC/saline 5 2HITC/saline 5 10 20* PBITC DDITC/saline 5 10 0.0Â±0.0^o.oÂ±o.o''0.6Â±0.270.3Â±0./10010010010075"10030*70s25*1 4.0 22DPEITC/saline512DPEITC/saline 10III10ll.lÂ±0.710.2Â±0.99.5 150*20* PPITC 5Corn oil/salineTumor

O) Q r a Tested in a single-dose model; experimental details given in the text. O 3.5 PE ITC ' Mean Â±SE; those significantly different from corn oil/NNK control according to Student's t tests are denoted by superscripts. ' Tumor incidence was compared between each treatment group versus the corn oil/NNK group using a procedure analogous to the DunneÂ»testfor multiple comparisons. 3.0 Tested with the same protocol in a separate bioassay. Positive control (corn oil/ NNK): 9.2 Â±0.6 nodules/mouse, 100% incidence. Negative control (corn oil/saline): 1.0 Â±0.4 nodules/mouse, 50% incidence. " One accidental death occurred during the experiment. 2.5 1'P < 0.001. x P <0.05.

2.0 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 are similar to those observed previously (19). All three isothiocyanates Log k' caused significant reduction of this tumor multiplicity, except for PHITC and Fig. 2. Plot of log P against log k' for isothiocyanates. A linear correlation between the POITC at the dose level of 0.04 p,mol/mouse. Previously we have shown that two parameters was obtained: log P = 2.322 + 3.675 log k' (r = 0.97). PHJTC was the most potent inhibitor among all the arylalkyl isothiocyanates tested (12) and that it is about 50-100 times more potent than the naturally occurring PEITC (23). However, at 0.2 /unol/mouse in the current experi Table 1 Effects of synthetic long chain arylalkyl isothiocyanates on NNK-induced lung ment, longer chain homologues POITC and PDITC appear to be more potent tumorigenicity" than PHITC (P < 0.05). Such a trend is not obvious at the lower doses. At the Dose Tumor incidence lowest dose, only PDITC showed significant reduction of tumor multiplicity. Treatment(^mol/mouse)Corn No. of(%)r4020 animals multiplicity* Both POITC and PDITC caused a clear dose-dependent inhibition, but they oil/NNKPHITC/NNKPOITC/NNKPDITC/NNKPOITC/salinePDITC/salineCorn are not significantly different in their potencies. Inhibition of NNK-induced Pulmonary Adenoma in A/J Mice 0.10.040.2 20202019"2020 100100<*/1001007(/by Isothiocyanates of Other Structural Features. Six isothiocya nates were tested together with PEITC in this bioassay at two different dose levels, 5 and 1 /nmol/mouse. The tumor multiplicity 0.10.040.2 7.9Â±0./9.0Â±0.62.8Â±0.6'/ and tumor incidence in all treated groups are summarized in Table 2. In these experiments, all animal groups showed body weight 6.3Â±0.81' 0.10.040.20.2Tumor20 7.9Â±0.6/'Q.2Â±Q.ld0.0Â±0.0rfOÃŸÂ±0.(fl100100100 gains comparable to those of the control groups. The control 201(1101010.2Â±0.65.2Â±oy5.5Â±0.6''10.9Â±0.93.3Â±0.5d100Iff< 0.001. mustard, was inactive at both doses. In agreement with previous '' One accidental death occurred during the experiment. results, PEITC was shown to be effective at 5 jxmol/mouse or !P < 0.05. higher doses, but not at 1 jxmol/mouse (12). 4329

Table 3 Partition coefficients (log P) and pseudo first order rate constants thiol groups 103-104 times faster than with amino or hydroxyl groups (27). Among the isothiocyanates examined, PITC and AITC were the most reactive. In the arylalkyl isothiocyanate series, the rate constants " Mean Â±SE, based on three separate measurements. decreased with increasing alkyl chain length. The rate constants " From Ref. 26. cFrom Ref. 12. dropped significantly when the alkyl chain length reaches 3 carbons or longer; thus, POITC and PDITC appeared to be the least reactive In the aliphatic isothiocyanate series, HITC at 5 Â¿Â¿mol/mousewas ones. Similarly, alkyl isothiocyanates with longer alkyl chains showed very effective in reducing both tumor multiplicity and incidence. At lower fcabs.The Â£obsof the longer chain isothiocyanate DDITC is the lower dose, where PEITC is not effective, HITC still showed a about one-fourth of that of its shorter chain homologue HITC. Inter strong inhibition with an approximately 58% reduction of tumor estingly, the structural isomers HITC and 2HITC have an alkyl chain multiplicity. As predicted on the basis of the trend observed in the with same number of carbons but HITC reacted with GSH twice as arylalkyl isothiocyanate series, its longer chain homologue DDITC fast as its isomer, 2HITC, which bears a secondary isothiocyanate showed a much stronger inhibition, reducing both tumor multiplicity group. The diphenylethyl isothiocyanates, 12DDPEITC and and incidence essentially to the background levels. Interestingly, 22DPEITC, had rate constants similar to those of PEITC and BITC. 2HITC, a branched structural isomer of HITC, caused greater inhibi The second phenyl group at either position 1 or position 2 did not tion than HITC at both dose levels (P < 0.001). This provides an significantly alter the chemical reactivity. In summary, both the longer example of a secondary isothiocyanate that has an improved efficacy alkyl chain and the secondary alkyl isothiocyanates examined by us over the primary isothiocyanate as an inhibitor of NNK-induced lung had lower reactivity toward the thiol group of GSH. Their rate tumorigenesis in A/J mice. These results also show for the first time constants may differ by as much as one order of magnitude due to that the phenyl group in the arylalkyl isothiocyanate series is not steric and electronic influences. critical for tumor inhibition. Correlation of Lung Tumor-inhibitory Activities with Lipophi In a comparison of 12DPEITC and 22DPEITC with PEITC, both licity and Reactivity of Isothiocyanates. To assist structure-activity analogues showed significantly greater inhibitory potency than PEITC relationship study, the relative potency of all 16 isothiocyanates in the (P < 0.001). 12DPEITC was more inhibitory than 22DPEITC at 1 lung tumorigenesis assays in A/J mice was rated on the basis of their /xmol/mouse (P < 0.05). However, at 5 /j.mol, the difference in their percentile inhibition of NNK-induced tumors at 3 doses tested, potency is not obvious. Lipophilicity of Isothiocyanates. The capacity factor k' for 16 namely 5, 1, and 0.2 /xmol/mouse (Table 4). To further delineate the isothiocyanates studied in the present and previous works was ob relationships of inhibitory potency with the physical and chemical tained using a single HPLC system (details in "Materials and Meth properties of isothiocyanates, the relative inhibitory potencies are ods"). A list of measured k' values and log P (partition coefficients plotted against log P and Â£obsobtained in this study (Fig. 3). The plot between 1-octanol and aqueous phases) calculated on the basis of k' can be divided into three regions. The first region is the one located values is presented in Table 3. Since HPLC retention time and k' at the bottom of the right side of the plot and is defined by low log P values have been used to obtain log P and, in some cases, the retention values, as seen for compounds such as AITC, PITC, BITC, PEITC, times of drugs are directly correlated with biological activity (22, 24, and OPBITC. Some of these isothiocyanates such as AITC, PITC, and 25), it seems reasonable to use the log P values as a measure of BITC are inactive even at the highest doses, 5 or 25 jxmol/mouse (27). relative lipophilicity of isothiocyanates. The lipophilicity scale meas Most of these compounds are characterized by high reactivity and, in ured by these log P values for isothiocyanates is consistent with that particular, low lipophilicity. For example, OPBITC has the lowest log predicted on the basis of chemical structures. For instance, the in P value among these 16 isothiocyanates. The second region, in the crease of lipophilic property of arylalkyl isothiocyanates with alkyl middle area of the plot, encircles compounds with modest log P and chain length is well reflected by log P values. The change Alog P/CH2 ^obs- Most isothiocyanates with modest to strong tumor inhibitor is about 0.5 for longer chain analogues [(CH2)4 to (CH2)10], which is potency reside in this region, such as arylalkyl isothiocyanates with in agreement with the trend observed for fatty acids and alcohol medium alkyl chain length or diphenylethyl isothiocyanates and hexyl homologues with Alog P/CH2, being 0.53 and 0.52, respectively (26). For HITC and DDITC, the Alog P/CH2 = (7.13 - 3.98)/6 = 0.52, isothiocyanates. The front left corner of the plot, where compounds are associated with large log P and small Â£obsisthe third region and also falls into the same range. The log P values for 2HITC and is occupied by highly potent inhibitors such as PHITC, POITC, 12DPEITC are relatively smaller than those for HITC and 22DPEITC, PDITC, and DDITC. respectively. This also agrees with the trend that the branched isomers have smaller log P values than their unbranched homologues (26). Chemical Reactivity of Isothiocyanates toward GSH. Since the concentration of GSH used in these reactions was 10 rriMwhich is 100 4 D. Jiao and F-L. Chung, unpublished data. 4330

Table 4 Relative inhibitory potencies of isothiocyanates against NNK-induced lung tumorigenicity in A/J mouse % of inhibition"

5 /Â¿mol 1 /Ã•IlHll 0.2 /Â¿mol

CompoundAITCPITCB1TCOPBITCPEITCPPITCPBITCPPelTCPHITCPOITCPDITC12DPEITC22DPEITCHITC2HITCDDITCMultiplicity00014769898%99NDND9594859797Incidence00II411m897595NDND5555257(175Multiplicity0NDNDND085911mi100NDNDÂ«X,mi51Xh95IncidenceÃœNDNDND0254253100NDND754003065MultiplicityND1NDNDND05347fi2856X73NDNDNDNDNDIncidencepotency*NDNDNDND0

+10 +0 + + +0 + + +30 + + +10 + + + +30 + + + +ND + + + +ND + + + +ND + + +ND + +ND + + + + + + " Data are compiled from the present and the previous studies (Refs. 12, 19, and 28). Data for PITC, BITC, OPBITC, PEITC, PPITC, PBITC. PPelTC, and PHITC are from previous studies. 6 The rating of the inhibitory potency is based on the percentage of inhibition. - and +, negative and positive inhibitory activity, respectively. -, inhibition of tumor multiplicity <20% at 5 /i nini +, inhibition of tumor multiplicity <80% at 5 /Â«noibut no inhibition at the lower doses; + +, inhibition of tumor multiplicity <90% at 5 /Â¿moland <60% at 1 /Â¿mol; + + +, inhibition of tumor multiplicity >90% at 5 /Â¿moland <9()% at 1 /Â¿mol;+ + + +, inhibition of tumor multiplicity >9()% at 5 /Â¿molor >65% at 0.2 /Â¿mol.In some cases, the inhibition of tumor incidence is also taken into consideration in the rating. ' ND. not determined.

DISCUSSION itory activity. These evaluations are expected to provide alternate structure leads for future studies. While previously tested isothiocya The selection of isothiocyanates for the bioassays in this study was nates are primary isothiocyanates, a secondary isothiocyanate, 2HITC based on the following rationales. The evaluation of arylalkyl isothio (a structural isomer of HITC), was tested for the first time. Since a cyanates with longer alkyl chains (C8 and C,,,), together with results major route of metabolism for isothiocyanates is via conjugation with from previously tested isothiocyanates, may provide information GSH, the chemical reactivity of isothiocyanates toward GSH would about the optimal chain length required for maximal inhibition. Since conceivably be an important factor in determining their potency (16, lipophilicity appears to be an important factor in the inhibition of NNK-induced lung tumorigenesis by arylalkyl isothiocyanates, sev 17, 29, 30). The secondary isothiocyanate group is expected to be less amenable to conjugation because of greater steric hindrance and eral lipophilic isothiocyanates without aryl moiety, such as HITC, electron-donating effect due to the a-substitution. For similar reasons, 2HITC, and DDITC, were also tested in this study. Thus, we were able to examine whether the phenyl group is essential for the inhib- the biphenyl analogues of PEITC, 12DPEITC and 22DPEITC, were tested in this bioassay and were also found to be more lipophilic than PEITC. Finally, AITC occurs naturally in many of the vegetables, food additives and flavoring agents consumed by humans, and it is a strong phase II enzyme inducer (31-33). Our earlier data had suggested that the tumor-inhibitory potency of isothiocyanates appears to be associated with increased lipophilicity and chemical stability (12). Now we have obtained further evidence that an increase of the alkyl chain length diminishes the reaction rates of isothiocyanates toward GSH. The reactivity of the NCS functional group toward nucleophiles declines with an increase of electron density around the carbon atom of the NCS group. The observed low reactivity of long chain arylalkyl isothiocyanates may be explained by the reduced electron-withdrawing effect of the phenyl ring along the alkyl chain. In some instances, the high lipophilicity or low reactivity alone may enhance the inhibitory activity of the isothiocyanate. For example, HITC and 2HITC have similar log P values, 2HITC being slightly less lipophilic (Table 3). However, the reaction rate constant of 2HITC is only one-half that of HITC, which is likely related to the significantly stronger inhibitory activity of 2HITC. In another case, PEITC, 22DPEITC, and 12DPEITC all have similar reaction rate constants (Table 3), but their tumor-inhibitory activity is in the order: PEITC < 22DPEITC < 12DPEITC. This trend is consistent with the order of their log P values. However, with low lipophilicity and low reactivity, OPBITC is yet an inactive compound. Evidence obtained Fig. 3. Plot of relative inhibitory potency of isothiocyanates against log P and *l)b,. For thus far strongly supports the notion that the chemopreventive effi convenience, the relative inhibitory potency is expressed as 0 (â€”), 1 (+), 2 (++), 3 cacy of isothiocyanates is mainly determined by structural character ( + + +), and 4 ( + + + + ). Three regions of the plot, upper front left corner, the middle and the lower right region, are associated with high, intermediate, and low potency of istics associated with high lipophilicity and low reactivity. Thus, isothiocyanates. See text for detailed discussion. future studies of isothiocyanates as inhibitors of NNK-induced lung 4331

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1994 American Association for Cancer Research. CHEMOPREVENTIVE ACTIVITIES, LIPOPH1LICTTY, AND REACTIVITY tumorigenesis should be focused on various lipophilic isothiocyanates plicated by the fact that there are multiple isozymes involved in NNK with low reactivity. For example, it has been reported that the rate metabolism. For instance, at least cytochrome P-450 2A1 and 2B1 constants of the reaction of ^-substituted aromatic isothiocyanates have been shown to be partially responsible for NNK metabolism in with thioglycolate are correlated with the Hammett constant cr of the the lung of the A/J mouse (13). The binding specificity of isothiocya substituents (34). It would be interesting to test compounds with nates toward cytochrome P-450 isozymes must be examined in order electron-donating groups, such as a methyl or methoxy group at the p to assess the potential of isothiocyanates to inhibit bioactivation of position of the phenyl moiety which will result in low reactivity of other carcinogens. In this context, the isothiocyanates of various isothiocyanates. structural features will be useful probes for studying cytochrome In the tumor bioassay, the reactive isothiocyanates with low li- P-450 binding site structures and their substrate recognition. pophilicity, such as AITC, BITC and PITC, were poor inhibitors. These compounds interact readily with nuleophiles in vivo; conse ACKNOWLEDGMENTS quently little of them reaches the target cytochrome P-450s. As shown in this study, these isothiocyanates react with GSH at a substantially We thank C. S. Yang for helpful discussions. high rate even without glutathione S-transferases. 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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1994 American Association for Cancer Research. Structure-Activity Relationships of Isothiocyanates as Mechanism-based Inhibitors of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced Lung Tumorigenesis in A/J Mice

Ding Jiao, Karin I. Eklind, Chang-In Choi, et al.

Cancer Res 1994;54:4327-4333.

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