-
-
edited by: LEONARDFINE Columbia University New York, NY 10027
ERICS. PROSKAUER
Antimetabolites of Coenzyme Q and Their Potential Clinical Use as Antitumor Agents A New Biochemical Approach to Cancer Chemotherapy Karl Folkers and Thomas H. Porter Institute for Biomedical Research, The University of Texas at Austin, Austin, TX 78712 Cancer includes more than 100 disease entities that present a variety of histopathology and clinical symptoms. It is the third leading cause of death in the i.'nited States after accidents and cardiovascular disease. One of every four Americans will developcancer, and most will eventuall; dieof theirdisease. It isestimated that in 1975alone there were6fi5.000 new cases and 380,000 deaths from cancer in America (I). Roughly half of all cancer deaths are caused by cancer of three organs: the lung, the large intestine (colon), and the breast. Lung cancer, a disease of the 20th century for which there has been little improvement in life expectancy, kills more Americans than any other type of cancer-an estimated 72,000 persons in 1975-and its incidence has more than doubled since 1947 in both men and women (I). Investieators ex~erienced with cancer e~idemioloev -. suggests that ahout 80% of all human cancers are causatively related to environment factors. It is estimated that sume 50°0 of all female cancers and about 3:1W of all male cancers in the Wescern world are related to nutritional factors (orimarilv high fat, low fiber diets) (Z), while some 20% of cancers in the U S . are caused by occupation-related chemical hazards (3). Many now believe that one of the most promising approaches is preventive medicine--to identify those cancer-causing factors by epidemiologicalstudies and eliminate them or lower their environmental levels. For treatment of cancer patients, there are now four major modalities: (1) surgery; (2) radiation therapy; (3) immunotherapy; and (4) chemotherapy. For a long time chemotherapy was considered as a treatment of last r e s o r t m o r e palliative than curative, often producing unpleasant and sometimes dangerous side effects (4). Recent advances in chemotherapy now offer promise of cancer cures. Appropriate combinations of new drugs and drugs following surgery now offer greater chance of success. For instance, more than 90% of patients with early Hodgkin's disease and about 70%with advanced disease are surviving five years (5). I t is firmly established that chemotherapy combined with surgery and radiotherapy is curative in 80-90% of patients with W i l d s
892
Journal of Chemical Education
tumor, a previously fatal childhood cancer of the kidney. Markedly improved survival rates of patients with Ewing's sarcoma, choriocarcinoma, various types of non-Hodgkin's lymphomas, and uterine cancer have been achieved (5).In all there are 10 human cancers which are highly responsive to chemotherapy, and 50% of these patients should achieve normal life expectancy (6).Unfortunately these successes do not include the major cancer killers such as breast, colon, or lung cancer. For example, the median survival rate of all lung cancer patients from diagnosis to death remains less than six months (7). Rational approaches t o the design of new cancer chemotherapeutic agents are needed. We believe one such approach is the development of antimetabolites of coenzyme Qlo. Discovery of Coenzyme Qlo in Cancer Tlssue and Early Research on Antlmetabolltes of Coenzyme Qro Coenzyme $10 (CoQlo) became of interest in cancer research shortly after it was isolated in pure form and structurally elucidated. In 1968, Scichiri et al. (8)showed apparent low levels of coenzyme Qlo in rat and human neoplastic tissues. The concentration of CoQlo in the mitochondria of ascites h e ~ a t o m awas one-half that of normal liver and reeeneratine" liver. In mitochondria of human cancer tissues, the concentrationsof C 4 1 nweresienificantlv lower than that of control tissues taken from the &me indiGiduals. The activity of the succinate-cytochrome C reductase was very low in rat ascites hepatoma and was related to the concentration of CoQlo. A lower concentration of CoQ7o in human neo~lastictissue .-" in comparison with normal tissue coupled with a lower compliment of mitochondria per cell seems to offer one advantage of selectivity in the potential use of antimetabolites of CoQlo for antitumor activitv. in ~atients. These data seem to sueeest . that analogs of coenzyme Qlo which are antagonists orinhibitors of either functional or biosynthetic CoQlo-enzymes in tumor tissues could become clinically useful chemotherapeutic agents for cancer that are novel and different in structure from existing agents and that are based upon a
-
As our interest in cancer chemotherapy increased, many of these new antagonists of coenzyme QIOwere tested for antitumor activity against a panel of tumors a t the National Cancer Institute. Many were active, particularly against Walker carcinosarcoma in rats. At thii stage of the investigations, the synthesis of new quinones as potential antitumor agents could he designed upon these new leads, active comIa pounds, derived from data supplied by NCI and also upon the new knowledge of the existence and role of coenzyme Q;" ..in NO.of Svvival No. wlih the metabolism of cancer cells. Test Do80 deathslNo. time IK)BIIcI~(NI/ It is important to emphasize that the coenzyme $10 of system (mglkglday) baaled (avg days) No. beeled TJC cancer cells is a lipoidal substance; this is largely because of Sarmma 180 12.5 0110 ... 6/10 O.lgb the 50 carbon atoms in its isoprenoid side chain. ConseL1210 IkukemLs 25 ... 11.0 . .. 125 quently, one may presume that effective inhibitors of coen.. . 9.7 ... 110 12.5 zyme Qlo would, of necessity, also have to be lipoidal in nature ... ... 8.8 ... ... Conbolaa for annronriate site alienment. For this reason. the svnthesis of sekirafnew categorgs of lipoidal quinones and th; expanCmaol almls-lwd 10 rnllkglday ofTwwn-BO + mllm 'TO&$ p B & d -11 MI-. sion of several previously initiated groups of analogs were undertaken as part of our research toward new and effective 20-year established essential biochemical role of coenzyme anticancer agents. Quinones were designed with long side Qlo in the electron transfer processes and coupled oxidative chains to impart lipoidal character and with various ring phosphorylation of mitochondria. substituents that could offer differences in oxidation-reducThe first series of antimetabolites of coenzyme Qlo syntion potentials, hydrogen bonding, and alkylation sites. thesized primarily as potential antitumor agents were alkA rationale based upon coenzyme Qlo for the synthesis of ylhydroxy-2,3-dimethoxy-1,4-benzoquinones (I) (9,lO). Repotential antitumor agents constitutes a new approach in the sults of exploratory testing in animal tumors-sarcoma 180 research toward chemotherapy of cancer. There are multiple ascites tumor in mice and L1210 in mice-of one such quinone, 6-phytyl-5-hydroxy-2,3-dimethoxy-1,4-benmquinone sites for potential inhibition of coenzyme Qlo-enzymes by effective analogs of CoQlo. The three quinone precursors of (Ia), were positive (Table 1)(11). This compound represented CoQloimply three enzyme sites in biosynthesis for inhibition, a lead to a new class of cancer chemotherapeutic agents. The and inhibition may occur at sites of functionality of CoQloin results with this compound were later confirmed by G. A. mitochondria1 (succinoxidase, NADH-oxidase (21-23), and LePage at The University of Alberta Cancer Research Unit, a-glycerophosphate oxidase (25))and in the Golgi apparatus. Edmonton, Alberta, Canada. Selective toxicity for tumor cells can be based on the differSynthesis of Antlmetabolltes of Coenzyme 010 ential inhibition of pathways of CoQlo functionality andlor CoQlo biosynthesis. For a number of years, antimetabolites of coenzyme Qlo were svntbesized as ootential antimalarial drum and were Alkynhio-2.3-Oimethoxy- I, 4-Benzoquinones designid using the knbwledge ahout the existen& and role of The category of 5-alkythio-2,3-dimethoxy-1,4-benzoquicoenzvme Qa in the metabolism of olasmodia (12). Manv of none compounds (VII), first synthesized by Wikbolm et al. these hew (;inone analogs were hiihly active against P.ber(18) as potential antimetaholites of coenzyme Qlo, was exehei in mice and included the following structural twes: al.. panded by Porter et al. (25) and VorkapiE et al. (26). kylhydroxyquinolinequinones (11) (18,alkylarninoquinolineauinonesand naohthoauinones (111 and IVJ,(14.15) alkylthiohydroxquinol&equi~ones(V) (16), benzodioxinquinones (VI) (17),alkylthio-2,3-dimethoxy-1.4-benzoquinones (VII) (18), alkylquinoxalinequinones (VIII) (19), and benzothiaxolequinones (IX) (20). As a class of compounds, the 5.8-quinolinequinones were most active as antimalarials; for instance, 7-n-octadecylmercapto-6-hydroxy-53-quinolinequinone gave 515 cure8 and (T- C ) = 7.9 at 320 mg/kg. These auinone analogs demonstrated the effectiveness both curaThe 35 new alkylthio-1.4-benzoquinones prepared in these Gvely and pro~hylacticallyof antimetaholites of coenzyme studies orovided wide variation in alkvl side-chain lendh and Q," in the treatment of malaria in mice with Little or no taxicity ring substituents with potentially significant variation in redox ootential of the new quinones. Many side chains contained-aromatic and aromatic-substituted-moieties. For the most &, these sulfur-containinr!quinone analogs had little or no sidehain unsaturation. The sdfur.atom adjacent to the nucleus could be expected to influence the oxidation-reduction potentials. e he-shorter, saturated, and unbranched side chains which were incorporated extensively into these analogr. would significantly diffe; from the nature and stereochemistry of the 6-decaprenyl side chain of CoQlo and would influence the partitioning of the analog in the "lipid milieu" of the mitochondria. It is known that CoQlo is in a "lipid milieu" of the mitochondria, and cardiolipin of the mitochondria has often been thought to be associated with CoQlo. In addition, new alkylsulfonyl (X) and methylamino (XI) derivatives were prepared. Treatment of 5-alkylmercapto2.3-dimetboxy-1.4-benzoquinone with excess methylamine in MeOH yielded bis-2,5-(methylamino)-3-methoxy-6-alkylmercapto-1,4-benzoquinones,benzoquinones containing two methylamino groups as determined by PMR spectroscopy
Table 1. lnhlbltlon of Sarcoma 180 and LIZ10 Leukemia by an Analog of Coenzyme 0,0
Volume 61 Number 10 October 1984
893
Table 2. Best Ten AMHumor AlkyHhlo-1,4-~nzOquIno~~ Agalnst Walker Carclnosarcma 256 n
No.
N S C ~
Structure
Toxicity Day Survivws
Dose m g k g
via
X = CI. R = S(CH2hCH3
Vllb
X = CI. R = S(CHehCH3
Vlk
X = CI. R = S(CH2InCHs
Vlld
x = cI, R = s cH2cn ncn,
Cures
% TIC
8"
Vlle
Vllf
vllg
Vllh
Vlll
Vlll
and elemental analysis. The structure of these new diadducta were assigned on the basis of a very low carbonyl-stretching
frequent;. The antitumor activities against Walker carcinosareoma of 35 alkyl-thio-1,4-benzoquinonesof this series was determined a t the National Cancer Institute. All of these quinones except two were active (% TIC 2 125). The Walker survival system is a sensitive test system for % TIC values, but substantiallv lone-term survivors or cures are not verv commonlv observed, so ;hat this parameter is of great impor-tance in t h i search for effective antitumor agents. The 10 best antitumor 894
Journal of Chemical Education
analogs of coenzyme $10 (Table 2) all showed long-term curea. 5-Phytylthio-2,3-dimethoxy-1,4-benzoquinone (NSC 276371, VIIg) was the most potent quinone with % TIC = 789 and 316 cures a t 0.78 mgkg. Also particularly impressive were the 6-n-odylthio-5-chloro-2,3-dimethoxy-1,4-benzoquhone (NSC 252188, IIIa) which cured 616 rats with % TIC = 584 a t 3.13 mgkg and 5-8-naphthylthio-2,3-dimethoxy-1,4-benzoquinone (NSC 234214, VIIj) which gave 416 cures with % TIC = 789 a t 40 meke. In " eeneral. a 5-chloro on the auinone nucleus or a side chain with unsaturation or branching, such as the phytyl side chain of NSC 276371, sewed u, increase an-
- -
TaMe 3. Antitumor ActlvHy d 8-n-OclylthloS-Chl0r0-Z,3-Dlmethoxy-l,4-Benzulna (Vlla) and 5o-PhenylpropyHhlo-2,3-Dimethoxy-1,4-~ulnone (Vllh) against Sarcoma 180 AacHas Tumor and Taper Llver Tumora Turn
Sbum
S180
Dose, mglkglday
0 25 50
ATFCV
Deaths
1.10 0 0
015 115 115
VC 1.00 0 0
Resuiia
+b +b
w%CH3m n40 NSC 252188. Vlla
+ + + toxic
NSC 258835. Vllh Vlla
TLT
+' mx1c4
VII~
n T
-
+
Tabla 4.
Antitumor ActlvHy d 5w-Phenylpropylthlo-2,3-Dlmathoxy-1,4-Benqu (NSC 258835, Vllh) and 8-n-OctyHhlo-5-Chloro2.3-Dlmethoxy-l,&Benrwlnone (NSC 252188, Vlla) against HI34 and 8C3HED Tumor Systems a
No.
SbllOture
Vlla Vlla Vllh Vllh
X = CI, R = (CHrhCHo X = CI, R = (CH2hCH3 X = H. R = (CHr)& X = H. R = (CHr)$-@
Vlla Vllh Vlla Vllh
X X X X
= Cl. R = (CHzhCHs = H. R = (We)& = CI. R = (CH&C& = H, R = (CH*)&
@up
Tumor
I II II IV V I II Ill V VII Vlll
H134b H134 Hi34 HI34 Hi34 6C3HEDC 6C3HW 6C3HED 6C3HED 6C3HED 6C3HED
~esuhs~ (suwlval days or estimate)
Treatment CanboCbiton X 100. 1:10000 Bmglkg. daily X 5 12mglkg. dally X 5 6mgIkg. daily X 5 12mgIkg. dally X 5 Connol-Uitan X 100, 1:10000 Adriamycin, 10rnglkglday 1 3mglkg, daily X 5 3mglkg. daily X 5 Adnarnycln, day 1 7a, daily X 5 Adrbamycin. day 1 7h, dally X 5
+ +
titumor activity in nearly all cases. A methyl group in position 5 instead of hydrogen gave little, if any, increase in % T/C. In addition, selected quinone analogs of this series showed activity against other tumor systems-sarcoma 180, Taper liver tumor, lymphosarcoma (6C3HED), and H13Pindieating a broad spectrum of antitumor activities for these compounds. Data from the Memorial Sloan-KetteringCancer Center showed that NSC 258835 (VIIh) and NSC 252188 (VIIa) had significant activity against sarcoma 180and Taper liver tumor (Table 3). NSC 258835 (VIIh) reduced the average total packed cell volume to zero at 12.5 mgkg against S-180. In addition these two quinones, VIIa and VIIh, showed notable activity against 6C3HED and HI34 when compared to the chemotherapeutic drug, adriamycin, as a standard (Table 4). NSC 258835 (VIIh) resulted in two tumor-free survivors a t 50 days when treated with 3 mgkg daily X 5 against 6C3HED. Significnntly, adriamycin and NSC 252188 (VIIa) when given together result in a synergistic antitumor effect (36.8 10 survival days and 3 tumor-free survivors at 50 days; control
*
+
6.0 1.0 11.0 i 2.0 (1 w i v o r ) 13.0 1.5 (3 suvlvors) 12.0 i 3.6 15.0 i 2.5 14.8 i 1.0 29.2 i 10 (1 tumor-freeswivor at 50 days) 18.5 i 3.6 (1 turn-Wee suwlvw st 50 days) 24 4 i 5.0 (2 I--Wee suwlvm at 50 days) 36.8 i 10 (3 tumor-Wee s w v l v m at 50 days) 26.6 2.3 (2 tumor-Wee suwlvm at 50 days)
*
= 14.8 f 1survival davs (Tabie 4).
were tested for Nineteen of 38alkylihio-1,4-bekoquinones their inhibitorv effects on mitochondria1 succinoxidase and N A D H - O Xactivities ~ ~ ~ ~ (Table ~ 5). The inhibitory activities of these analogs were measured by their effects on the oxygen uptake of the beef heart mitoehondrial coenzyme Qlo enzymes as determined by a Gilson respirometer. For most analogs, inhibition of the CoQ-enzymes was determined at several concentrations so that the concentration at which 50%inhibition was effected could be established by extrapolation. In general, the quinoue analogs were stronger inhibitors of NADH-oxidase than of succinoxidase activity. The 5-chloro-1,4-benzoquinoneswere far stronger inhibitors in both enzyme systems than the corresponding "deschloro"-quinones. Many of the most potent antitumor compounds [6-n-octylmercapto-5-chloro-2,3-dimethoxy1,4-benzoquinone(NSC 252188, VIIa) and B-n-dodecylmercapto-5-chloro-2,3-dimethoxy-1,4-benzoquinone(NSC 220334, VIIb) were also among the best in vitro inhibitors of coenzyme Qlo enzyme systems. Although a perfed correlation Volume 61 Number 10 October 1984
895
Table 5.
lnhlbltory E f f d ol Banzoqulnm Analogs ol Coenzyme (I10 on Mltomondrlal Succlnoxldase and NADHhidase Actlvlly
NO.
Compounds (NSC NO.) None Standsrd Inhlbltwc
Vllm
Vlln
VllO vllp vllq Vll,
Vllb
896
Journal of Chemical Education
Conc.'
SucclnOxldBse RBI. Enz. nmol. for ~ c t i v n y ~ 50% lnh.
Conc:
NADKOxldme ReI. Enz. nmol. tw ~ctlvlty' 50% lnh.
was not expected, a reasonable and satisfactory correlation was achieved, particularly within the same category of quinones. The effectiveness of these alkylthio-1,4-benzoquinones,in vitro and in uivo, may be due to their having a combination of molecular characteristics for inhibition, including electron transoort. relative affinities for bindine to functional andlor hiosinth&ic sites of CoQlo in cancer c h i , and lipoidal character. Another new category of ethoxy-1,4-benzoquinones, compounds XII, XIII, and XIV, has been recently synthesized by Porter et al. (27) as potential antimetabolitesof coenzyme Qlo and antitumor agents.
Six analogs have, as a primary variation, one ethoxy group in place of one of the two methoxy groups of CoQlo. Four of these six analoes have two alkvlamino moum attached in positions 2 and 5 of the benziquinone nucieus furnishing coenzyme Qlo analogs with increasing lipoidal cbarader. The fifth compound contains the isoprenoid farnesyl as a side chain, and the sixth is an alkylmercapto analog. Five of these six analogs showed some degree of antitumor activity against Walker carcinosarcoma 256, and two of these five showed
Table 7.
Table 6. Anlllumor ActlvHy ot Substituted 61s-(1.4Benz0qulnOyl)-Alkyldlthlolsagalnrt Walker Carclnollarcoma In Rats
No.
NSC NO.
XVla
300239
R = H. x = 3
XVlb
299911
R = H, x = 4
XVld
302310
R = CH3. x = 3
XVlf
302968
R = CHs. x = 9
........, Day Survivors Cures
% TIC
50 25 12.5 6.25 3.13 50 25 12.5
6.25 3.13 50 25 12.5 6.25 3.13
50 25
lnhlbnory EHect ot Bls-(1,4-Benzoqulno~)-Alkyldllhiol Analogs on MHochondrlal CoOID Enzymes In Beef Heart r-Succinoxidase~-~
NO.
StruChlre
Dose. mglkg
Inhibitor Structure
,-NADKOxidasea-
Rel. Act.
Conc. nmollfl.
I%)
50% lnhibilion nmollfl.
Conc. nmollfl.
?
Rel. Act. (%)
50 % Inhibition nmollfl.
None Standard Inhibitors XVld
x=3,R=CHs
XVlf
x=6.R=CHI
XVlg
x=9,R=CHs
XVla
x=3.R=H
XVlb
x=4,R=H
Comem of mitochordrialprotein was 0.593 mg per fiark lor svccimxidaseand 0.508 mg per flash InNADKaxiblse. speclflc activity in microatomoxygen uptake per min per mg protein. 6o-CyclohexylpentyI-5h~roxy-2,MImemxy-l1Cbeeeoquinone.
w.
Volume 61 Number 10 October 1964
897
limited cures a t % TIC values above 125,and baseline defmed for positive activity. The "ethoxy analog" of CO& showed inhibition of NADH-oxidase a t a concentration of 10-3 M. 2,5-Bisi l-Alky1amino)-I,4genzoquinones and Bisi2,3Dimethoxy-5-Methyl- 1,4-Benzoquinonyfi-Alkyldithiols
xvl
XVIaR=Hx-3 XV&R=H;Z=~
Twelve compounds of another new class of quinone analogs, his-2,5-(l-alkyl-amino)-1,4-henzoquinones (XV), were synthesized by Porter et al. (28)by the addition of the appropriate alkylamine t o 5-methyl-2,3-dimethoxy-1,4-henzoquinone.
Particularly potent were compounds XVIa, NSC 300239, and XVIc, NSC 292681, which showed 3/6 cures with % TIC = 821 a t 3.13 m g k g and 316 cures with % T/C = 789 a t 50 mglkg, respectively. Data in Table 7 show that XVIa is one of the two compounds which also gave relatively strong inhibitory activity in succinoxidase and NADH-oxidase. These his-2,5-(alkylamin0)-1,4-henzoqninonesand his(1,4-henzoquinonyl)-alkyldithiols represent the "traditional surprise" in medicinal chemistry because these quinones were submitted with a relatively low expectancy of antitumor activity. The analogs with the nitrogen-containing side chains possess little or no side-chain unsaturation, or branching; several contain groups in their side chains which could reasonably be expected to prevent appropriate stereochemical fit at receptor sites. The nitrogen atom adjacent to the quinone ring of the first group of analogsprohahly influences the oxidation-reduction potentials in a way different from that of the analogs with sulfur-containing side chains. The short saturated unbranched side chains would significantly differ from the stereochemistry of the 50-carbon side chains of CoQlo, which in turn may influence unfavorahly the behavior of the analog in the "lipid milieu" of the mitochondria. The his-(1,4-henzoquinonyl)-alkyldithiolsare indeed snrprising quinone analogs. Two compounds, XVIa and XVIc, gave potent activity. These derivatives have two sites for ring alkylation-the rings being located a t the ends of an alkyl chain of varying lengths.
One analog of 11 tested was weakly inhibitory to NADHoxidase of beef heart mitochondria. Three of seven compounds tested for antitumor activity were marginally active against Walker carcinosarcoma 256. Bis-2,5-(l-decylamin0)-3methoxy-6-methyl-1.4-henzoquinone(NSC 268687, XVa) showed 2/6 cures with % T/C = 438 a t 25 mgkg. In a similar manner 1,4-addition was carried out with appropriately substituted quinones (29) and alkyldithiols. Six new his-(1,4-henzoquinonyl)-alkyldithiolswere synthesized and tested hv NCI against Walker carcinosarcoma in rats (Tahle 6).~ u ; ~ r i s i n ~all l ; nix quinones showed cures and activity defined as Db TIC 2 125. Table 8. Anthumor Acllvny 016-Phytyl-5-Hydroxy-2,3Df~hoxy-l,4-BenzcquInone(la) agalnst Walker Carclnosarcoma 256 NO. NSC NO.
la
277618
Dose mglkg
Toxicity Cay SUrVivwS
50 25 12.5 6.25
414 414 414 414 414
3.13
Table 9.
CWBS % TIC 416
923
... ... ...
815 143 123 109
...
2-Alkyithio- 1,4-Naphthoquinone
A new series of hicyclic quinones, the 2-alkylthio-1,4-naphthoquinone (XVII) have heen recently synthesized by
""*
Effects of 5-Hydmxy-l,4-Benzoqulnme Analogs on Succlnoxldase and NADH-Oxldase Systems from Beef Hearl Mnochondrla n
CHO ,
-Succinoxidase-,
C~ncenmtion
No.
Analogs Comrol
ld
898
R = ~ c H J , ~ ~ J
Journal of Chemical Education
(M)
Relative Activity (%)
...
100
...
51
11 (1)
4X
lo-'
50% Inhibition (nmolelflask)
r-NADH-Oxidsse-,
(M)
Relative Activity (%)
...
100
...
52
611)
Concentration
2X
lo@
50% Inhibition (nmolelflask)
Porter et al. (30) as potential antitumor agents. Fourteen new members of this series-four new 2-alkylthio-1,4-naphthoquinones (XVIIa), four new 2-amino-3-alkyltbio-1,4-naphthoquinones (XVIIb), four 2-hydroxy-3-alkyltbio-1,4naphthoquinones (XVIIc), and two 3-alkylthio-5-hydroxy1,4-naphtho-quinones (XVI1d)-have been prepared by 1.4-addition of the annronriate alkvl mercantan to suitablv s;hstirud 1,4-naph;hW;inones. ~ i e v e nof kese compounds were tested for inhibition of mitochondrial coenzvme Onn enzymes, succinoxidase, NADH-oxidase, and 10 of the ne& synthesized quinones plus one previously prepared quinone, which bad shown exemplary in vitro enzymatic inhibition, were assayed for antitumor activity against Walker carcinosarcoma 256. The alkylated hydroxy-1,4-naphthoquinones were the best inhibitors of succinoxidase and NADH-oxidase and also the best antitumor agents in this tumor system.
Benzothiazolequinones In the future, dioxobenzothiazolequinones will have high priority in the continuing program to explore new heterobicyclic quinone systems for potential antitumor activity. Two new 5-alkyl-6-hydroxybenzothiazole-4,7-quiuones synthesized during the malarial program by Friedman et al. (20) containing n-undecyl (IXa) and n-pentadecyl (IXb) side chains are some of our most potent inhibitors of mitochondrial succinoxidase and NADH-oxidase (Table 10).
In addition, in prophylactic tests against sporozoite-induced
P.gallinaceurn malaria in chicks, 5-n-undecyl-6-hydroxybenzothiazole-4,7-quinone(Na) was effective without toxicity a t dose levels of 30 mgkg (115 cures) and 120 mgkg (415 cures). A new step to the deamination of 2-amino-6-methoxy-
Antitumor Activity 01 Alkylqulnoxallnequlnones agalnst Walker Carclnosarcoma 256
Table 11.
A reasonable correlation of activity exists hetween in viva antitumor activity in the Walker system and in vitro enzyme inhibitory activity for these compounds. Two Prornislng Classes of Coenzyme Ole Analogs as Potential Antitumor Agents
This series should he expanded hecause of the exemplary activities of 6-~hvtvl-5-hvdroxv-2.3-dimethoxv-1.4-benzo. - quinone (la) both as an adtitugor agent (25) ( ~ a b l 8) e and as an inhibitor in vitro of mitochondrial succinoxidase and NADH-oxidase activities (Table 9). Unpublished data of the effects of two other 5-hydroxy-1,4-benzoquinoneson NADH-oxidase and succinoxidase systems of beef heart mitochondria show the excellent in uitro activities of these compounds (Table 9). 6-(8,ll-Heptadecyl-dienyl)-5-bydroxy-2,3-dimethoxy-1.4-benzoquinone(Ic) is even more active than the 6-phenyl analog (Ia) in the NADH-oxidase system.
Table 10.
No.
IXa
Structure
Xllla
266930
R = (CH2)&H3
Vlllb
268482
R = (CH2hCHs
Dose. mglkg
Toxicity Day Survivors cures
% TIC
... ...
50 25 12.5 6.25 3.13
016 016 416 616 616
016 016 016 116 416
278 449 504
50 25 12.5 6.25 3.13
616 616 616 616 616
416 316 016 116 116
598 596 116 117 110
lnhlbitlon by 6-Hydroxy-4.7-Dlxobenzothiazolequlnones of Succlnoxldase and NADH-Oxldase Actlvltles I n Beef Heart Mltochondrla
C0Q Analogs Control 6-o-cycIohexylpenlyl-5hydroxy-2.3dimethoxy 1.4-benzoquinone (standard) R = +C4,H2,
Control
NO.
NSC NO.
c ~ S u c c i n o x i d a s e 7a N ~A D H - O x i d a s e a Concentration Speclflc Relative Antimetabolie Concsnlration Specific Relative Antimetabolite @ moles) Activity Activity COOIndexb @moles) Activity Activity CoQ l?dexb
... 0.016
0.334 0.150
100% 45%
0.004 0.008 0.016 0.032
0.241 0.129 0.073 0.057
72% 39% 22% 17%
...
0.342 0.213
100% 62%
...
...
5
0.016
0.427 0.202
100% 47%
2.3
0.004 0.006 0.032
0.210 0.126 0.082
49% 30% 19%
...
0.432 0.241
100% 56%
... 5
0.016
... 9.5
1.6
... 11
'2.4 nmoies of COO,. iscontained in 0.716 mg of mi~adandrialp r a m per each flask. 4 Definedas ms ratio d nmoles of analcg per n m l w of Co(llo in me saual mitOchaMial peparation whlch causes approximately 50% inhibhionof enryme activity.
Volume 61 Number 10 October 1984
899
Table 12. lnhlbnory Elfect of Coenzyme Q10Analogs, XlVa and XIVb, on Mltochondrlal Succlnoxldase and NADH-Oxldase Actlvlly +uccinoxidasNo.
XlVa
Rel. C O ~ C . ~A c l
Compound
-NADH-Oxidase-
50% inhibitionr
Conc:
Rel. Actb
50% Inhibitions
EsDodecylmercapto-5-memy1 2-methoxy-1.4-benzoquinone
nmoies in a flask Coment of me mmcnondial protein was 0 593 mp per ikrh Percenta8~at spec lic actwdr in me presence oi tnnbitm lo met ol me mnnol a d 0 832m~croalomsorroenoer mm om ma f a NAOMxlaase
"
me specific activity of he comrol wao 0.506 miC(OB1Omaoxygen per min. per mg f a wccinoxidsse
benzothiazole (XVIII) t o 6-methoxy-benzothiazole (XIX) was worked out recently, and it is hoped this new step will allow adequate numbers and quantities of analogs of this series (eqn. (1)).
Two such compounds have been recently prepared in these laboratories - 6-n-dodecylthio-5-methyl-2-methoxy-1,4benzoquinone, NSC 285693, XXIVa, and 6-0-naphthylthio-5-methyl-2-methoxy-l,4-henzoquinone, XXIVb (29).
cm
6-Al~lamino-5,s-ouinoxalinequfnones
This series of analogs of coenzyme Qlo should be extended because of the unexpectedly potent activity of two exploratory members of this group-6-n-propylamino and 6-n-dodecylamino-5,8-quinoxlainequinones(VIIIa and VIIIb, respectively)-against Walker carcinosarcoma 256 (Table 11) (29). Design and Synthesis of lnhlbltors of Blosynthesis of Coenzyme Olo as Potential Antitumor Agents
T o date, most of our analogs of coenzyme Qlo could be considered more antagonists of functionality of coenzyme $10 in succinoxidase, NADH-oxidase, andfor a-glycerophosphate oxidase than as inhibitors of biosynthesis of coenzyme Qlo. Equation (2), which shows the last three steps in the biosyuthesis of coenzyme Qlo, would indicate that structures of potential antagonists of hiosynthesis could be designed with only one methoxy group or a hydroxy group in place of one methoxy group.
Neither XXIVa nor XXIVh demonstrated significant inhibitorv activitv in mitochondrial succinoxidase and NADHoxidase, even a t high levels (Table 11); however, XXIVa, the only compound of this series so far tested against Walker carcinosarcoma 256, gave 116 cures with % TIC = 229modest but promising activity for the first member of a new series. In an assay for mitochondria1 hiosynthesis recently developed by Folkers e t al. (31),XXIVa showed 83% inhibition of the conversion of W-p-hydroxyhenzoic acid to 1%coenzyme $10-a significant result because this analog has only one methoxy group as do the intermediates XX, XXI, and XXII. lnhlbltlon of Two Human Cell Llnes by Antimetabolite
Joseph R. Bertino and Barbara Moroson of the School of Medicine, Yale University, tested a few selected analogs of coenzyme Qlo from this research and found that two inparticular, NSC 262188 (VIIa) and NSC 277818 (la), inhihiced the human cell lines 4265 and K562 and the experimental animal tumor line W2.X (Table 12) (32). Line 4265 is a human lvm~hohlastoidline and K562 is believed to he an acuce mv~loblasticleukemia line. The same NSC 252188 (Ia) showed 616 cures. % TIC = 584.. aeainst Walker carcinosarcoma 256 " in rats ( ~ a h l 2). e
lnhlbltlon by Antimetabolites of Coenzyme Qlo of Nuclelc Acid Synthesis In Leukemia L1210 Cells ,41' 1,, 0
900
Journal of Chemical Education
I
C&
Jl
(21
Thirteen diversified antimetabolites of CoQio having antitumor activitv in vivo were tested for inhibition of u ~ t a k e of tritiated thymidine and uridine into DNA and RNA, re-
Table 14. InhlbRlon of Nuclelc Acid Synthesis In LIZ10 Cells by Selected 1,4-Benzoqulnone Analogs of Coenzyme Qro
Table 13. lnhlbltlon of Two Human Tumor and One Experlmental Animal Tumor Cell Llnes by Two Antlmetabolltes ol Coenzyme (1.,
