Fluorinated Vitamin D3 Analog with In Vivo Anticancer Activity - ACS

Chapter 16 Fluorinated Vitamin D Analog with In Vivo Anticancer Activity 3

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on May 26, 2018 | https://pubs.acs.org Publication Date: August 13, 1996 | doi: 10.1021/bk-1996-0639.ch016

Katsuhiko Iseki and Yoshiro Kobayashi MEC Laboratory, Daikin Industries, Ltd., Tsukuba, Ibaraki 305, Japan

To synthesize vitamin D3 analogs with in vivo anticancer activity but not calcemic activity, eight different fluorinated analogs were synthesized and structure-activity relationships were determined. Modifications such as 26,27-fluorination, side chain homologation and introduction of a hydroxyl group were carried out. The fluorinated vitamin D3 analog, (22S)-26,26,26,27,27,27-hexafluoro-24-homo1α,22,25-trihydroxyvitamin D3, was found to inhibit the growth of human colon cancer cells (HT-29) in culture to an extent ten times that of the active metabolite of vitamin D3, 1α,25-dihydroxyvitamin D3. The growth of human colon cancer (HT-29) implanted beneath the renal capsule of SCUD (severe combined immunodeficiency) mice was suppressed by 63% by the fluorinated analog (3 μg/kg body weight i.p. every other day, 5 times) with no increase in serum calcium.

The synthesis of fluorinated vitamin D3 analogs was initially prompted by extensive investigation of vitamin D3 metabolism (13). Vitamin D3 (1) in the skin or absorbed from the small intestine is transported to the liver to be hydroxylated at carbon 25 to yield 25-hydroxyvitamin D3 (2). This compound is the major circulating metabolite of vitamin D3 and undergoes further hydroxylation in the kidney, depending on physiological conditions. Low serum calcium stimulates hydroxylation at carbon 1 to produce la,25-dihydnoxyvitamin D3 (la,25-(OH)2D3,3). When circulating calcium is high, 25-hydroxyvitamin D3 (2) is hydroxylated at carbon 23, carbon 24 or carbon 26 to form 235,25-dihydroxyvitamin D (4), 2AR,25-dihydroxyvitamin D3 (5), or 25,26-dihydroxyvitamin D3 (6), respectively. lcc,25-(OH)2D3, (3), the most biologically active form of vitamin D3, is essential to the regulation of calcium metabolism in animals and is also of use for treating bone diseases such as osteoporosis. This active metabolite undergoes degradation by oxidation of the side chain, such as hydroxylation at carbon 26. 3

0097-6156/96/0639-0214$15.00/0 © 1996 American Chemical Society

Ojima et al.; Biomedical Frontiers of Fluorine Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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Fluorinated Vitamin D Analog

215

3


OH

HO*

HO"

HO

HO"

Scheme 1. Functional Metabolites of Vitamin D . 3

As a potential inhibitor of 25-hydroxylase which converts vitamin D3 to 25hydroxyvitamin D3 (2), a vitamin D3 analog blocked at this position with a fluorine atom, 25-fluorovitamin D3 (7), has been synthesized (4-5). For assessment of the physiological significance of metabolic 24-hydroxylation, 24,24-difluoro-25hydroxyvitamin D3 (8) (6-7) and 24/?-fluoro-lcx,25-dihydroxyvitamin (9) (8) were synthesized. For 23-hydroxylation, 23,23-difluoro-25-hydroxyvitamin D3 (10) was synthesized (9). 26,26,26,27,27,27-Hexafluoro-25-hydroxyvitamin D (11) (20), 26,26,26,27,27,27-hexafluoro-la,25-dihydroxyvitaminD3 (26,27-F -la^5-(OH) D3, 12) (11) and la-fluoro-25-hydroxyvitamin D3 (13) (12) were prepared to examine the 26- and la-hydroxylation of vitamin D3, respectively. 3

6

HO* no 7R = H, R*=F, R = H 8 R = F, R = H 10R = F, R = O H , R = H 9 R = H, R = OH 13 R = H, R = OH, R = F 14 R = F, R = OH 2

1

!

2

2

1

1

2

2

1

2

Scheme 2. Fluorinated Vitamin D Analogs. 3


2

216

BIOMEDICAL FRONTIERS OF FLUORINE CHEMISTRY

Many fluorinated vitamin D3 analogs have been found quite useful for determining the significance of hydroxylation in vitamin D metabolism, as well as proving augmented biological activity in certain cases of importance. 24,24-Difluorola,25-dihydroxyvitamin D (14) (13) and 26,27-F -la,25-(OH) D3 (12) (14) have been shown 5-10 times more potent than loc,25-(OH)2D3 (3) in in vivo vitamin D responsive systems such as increasing intestinal calcium transport and bone calcium mobilization in vitamin D-deficient rats. Of great importance is the fact that the action of these fluoro analogs persists for longer times than that of la,25-(OH)2D (3). 26,27-F6-la,25-(OH)2D3 (12) is presently being studied for application to the treatment of bone diseases such as osteoporosis. In 1981, Suda et ah discovered new functions of la,25-(OH)2D3 (3), such as inhibition of cell growth and the induction of cell differentiation in human leukemia cells (25-76). This active metabolite induces the differentiation of human colon cancer cells (27). In an in vivo study, la,25-(OH)2D (3) was found to inhibit the growth of human colon cancer xenografts in mice (18). Theriskof colon cancer has been shown inversely correlated with the dietary intake of vitamin D3 (29). la,25-(OH)2D3 (3) would thus appear useful for the prevention and treatment of human cancer including colon cancer. The high calcemic activity of this drug, however, would limit such potential application. Problems may be encountered at hypercalcemia at concentrations required for inhibiting cell growth and the inducement of cell differentiation. Thus, many analogs of la,25-(OH)2D3 (3) with in vitro anticancer activity but not calcemic activity have been synthesized as clinically useful anticancer agents (20-21). However, few low calcemic analogs with in vitro anticancer activity have in vivo anticancer effect (22-24), possibly owing to their rapid clearance in vivo. To obtain therapeutic agents for colon cancer, eightfluorinatedanalogs of la,25(OH)2D (3) were synthesized in this study and their structure-activity relationships were determined based on calcemic activity in rats and antiproliferative effect on HT29 human colon cancer cells in culture. The analog DD-003, (22S)-26,26,26,27,27,27hexafluoro-24-homo-la,22,25-trihydroxyvitamin D3 (25), expresses high anticancer activity toward colon cancer cells in culture with no hypercalcemia in rats. In vivo activity of DD-003 toward human colon cancer was determined. The growth of HT-29 colon cancer implanted beneath the renal capsule of SCID (severe combined immunodeficiency) mice was effectively suppressed by DD-003 (26).


3

6

2

3

3

3

Design of Fluorinated Vitamin D Analogs 3

The design was such as would permit the separation of activities in tumor prevention and calcium regulation and provide in vivo anticancer activity. 26,27-Hexafluoro analog 12 is about 100 and 10 times more potent than loc,25-(OH)2D3 (3) for inhibiting the growth of human cancer cells in culture and elevating serum calcium, respectively. The action of the fluoro analog 12 persists longer than that of la,25(OH)2D (3) for augmenting serum phosphorus following oral administration (14). la,22,25-Trihydroxyvitamin D3 (15) (2 7-2 8) and 24-homo-la,25dihydroxyvitamin D3 (16) (29-30) induce the differentiation of human leukemia cells (HL-60) but express no calcemic activity. Homologation of the side chain and introduction of a hydroxyl group into this chain lessens calcium-regulating capacity. 3


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3

The introduction of a hydroxyl group into the side chain and homologation of this side chain of 26,27-F6-la,25-(OH)2D3 (12) were the objectives of the present research. OH


OH

HO*

OH

High calcemic activity Anticancer (in vitro) Long lasting

15 Low calcemic activity Anticancer (in vitro)

HO*

OH 16 Low calcemic activity Anticancer (in vitro)

Scheme 3. Design of Fluorinated Vitamin D Analogs. 3

Synthesis of Fluorinated Vitamin D3 Analogs 26,26 26,27 27,27-Hexafluoro-24-homo-la,22,25-trihydroxyvitamin D3. 22Hydroxylated anlogs (DD-003 and DD-004) were synthesized using the ene reaction of hexafluoroacetone for side chain construction (25). The results of retrosynthetic analysis indicated that aldehyde 17, readily available from vitamin D via the oxidation procedure of Toh and Okamura (57), should be used as starting material. This aldehyde was reacted with allylmagnesium chloride in ether-THF at 0 C to give alcohol 18 (47%) along with isomer 19 (39%). Alcohol 18 was acetylated and the acetate thus obtained was treated with hexafluoroacetone in a sealed tube at 150*C for 35 h to afford allyl acetate 20 in 73% yield in two steps. The deacetylation of 20, followed by hydrogenation of the resulting alcohol in the presence of Wilkinson's catalyst gave alcohol 21. The silylation of 21, hydrogenolysis and PCC oxidation furnished ketone 22 in 84% overall yield. According to the general approach of Lythgoe et al (32-33), the Horner-Wittig reaction of ketone 22 with the phosphinoyl 9

9

2

#


218



carbanion derived from 23 and n-BuLi afforded the bis(TBS) ether which was desilylated with cation exchange resin (50W-X4) in MeOH to give DD-003 in 86% yield. In the same manner, DD-004 was prepared from allyl alcohol 19.

a) CH =CHCH MgCl; b) AcÔ, Py; c) CF3COCF3,150 C; d) K C 0 , MeOH; e) H , RhCl(PPh ) ; f) TBSOTf, 2,6-lutidine; g) H , Pd-C; h) PCC; i) *-BuLi, 23; j) 50W-X8, MeOH. #

2

2

2

2

3

3

3

2

Scheme 4. Synthesis of 26,26,26,27,27,27-Hexafluoro-24-homola,22,25-trihydroxyvitamin D . 3

26 26 26,27^7,27-Hexafluoro-24-homo-la,23,25-trihydroxyvitamin D3. 23Hydroxylated analogs (DD-014 and DD-015) were synthesized using the coupling reaction of aldehyde 24 with sulfone 25 to construct the side chain (Iseki, K; Takahashi, M.; Kobayashi, Y., Daikin Industries Ltd., unpublished data.). The Wittig reaction of aldehyde 17 with (methoxymethyl)triphenylphosphonium chloride, followed by treatment of the enol ether thus obtained with mercuric acetate gave aldehyde 24 in 89% yield. Reaction of 24 with the carbanion derived from sulfone 25 and Swem oxidation furnished sulfonyl ketone 26 in 67% overall yield. The desulfonylation of 26 with samarium iodide afforded ketone 27 in 94% yield. Reduction of 27 with sodium borohydride gave alcohol 28 (69%) along with isomer 29 (31%). The demethoxymethylation of 28, acetylation, hydrogenolysis and PCC oxidation provided ketone 30 in 46% overall yield. Ketone 30 was converted to DD014 by the Wittig reaction with the phosphinoyl carbanion derived from 23 and 9

t


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219

3

dcprotection with cation exchange resin (50W-X4) in MeOH in 82% yield. In the same manner, DD-015 was prepared from alcohol 29. v^CHO

_V~CHO


a),b) OBn 24

OBn 17

J' if.

PhSO, C F V s A > O M O M 3

HO*^*OH DD-014 R = OH,R = H DD-015 R = H, R = OH 1

2

e) 94%

2

1

CF xhdMOM CF f)

(3mom

2CF3

3

OBn

g),h),i),j) 46%

OBn 28 R = OH, R = H (69%) 29 R = H , R = OH(31%)

27

1

2

1

2

CF >OH 3

oac O

C F

k),l) 82%

3

DD-014 25

30

3

v

TBSO ^ OTBS « 23 TO

+

a) (MeOCH )Ph P Cr, r-BuOK; b) Hg(OAc) ; c) 25, n-BuLi; d) Swem oxidation; e) Sml ; f) NaBH^; g) cone. HCl-dioxane, 65 C; h) Ac 0, Py; i) H , Pd-C; j) PCC; k) H-BuLi, 23; 1) 50W-X8, MeOH. 2

3

2

-

2

2

2

Scheme 5. Synthesis of 26,26,26,27,27,27-Hexafluoro-24-homolot,23,25-trihydroxyvitamin D . 3

26,2626,27^27,27-Hexafluoro-24-homo-la,24,25-trihydroxyvitamin D3. 24Hydroxylated analogs (DD-019 and DD-020) were synthesized using the regio- and stereoselective opening of epoxides to introduce the hydroxyl group into the side chain (Iseki, K; Oishi, S.; Kobayashi, Y., Daikin Industries Ltd., unpublished data.). Allyl acetate 20, prepared from 17, was converted to epoxy alcohol 31 by methoxymethylation, deacetylation and epoxidation in 38% overall yield. The acetylation of 31, hydrogenolysis, methoxymethylation and deacetylation gave epoxy alcohol 32 in 83% yield in four steps. The mesylation of 32 and reduction with dissolving sodium followed by hydrogenation of the resulting allyl alcohol in the presence of platinum oxide furnished alcohol 33 in 17% overall yield. The acetylation of 33, demethoxymethylation and PCC oxidation gave ketone 34 in 68% yield in three steps. Ketone 34 was converted to DD-020 by the Wittig reaction with the phosphinoyl carbanion derived from 23 and deprotection with cation exchange resin t


220



(50W-X4) in MeOH in 20% yield. In the same manner, DD-019 was prepared from the 22R isomer of 20.

a) MOMC1, /-Pr NEt; b) K C 0 ; c) MCPBA, Na HP0 ; d) AcÔ, Et N, DMAP; e) H , Pd-C; 0 MOMC1, z-Pr NEt; g) K C 0 , MeOH; h) MsCl, Py; i) Na, NH ; j) H , Pt0 ; k) AcÔ, Et N, DMAP; 1) cone. HCl-dioxane, 60°C; m) PCC; n) *-BuLi, 23; o) 50W-X8, MeOH. 2

2

2

2

3

2

2

2

2

4

3

3

3

3

Scheme 6. Synthesis of 26,26,26,27,27,27-Hexafluoro-24-homola,24 25-trihydroxyvitamin D . 9

3

,

26,26,26,27^7,27-Hexafluoro-24-homo-la,24 ,25-trihydroxyvitamin I>j (Iseki, K ; Nagai, T.; Kobayashi, Y., Daikin Industries Ltd., unpublished data.). Alcohol 36 (34), easily prepared from propargyl alcohol and hexafluoroacetone, was reduced with lithium aluminum hydride followed by demethoxymethylation of the (E)-alcohol thus obtained to give the corresponding diol which was converted to epoxide 37 using f-butyl hydroperoxide and vanadyl acetylacetonate. The reduction of 37 and f-butyldiphenysilylation furnished silyl ether 38 in 86% yield in two steps. The diesterification of 38, separation of the resulting diastereomers, treatment with lithium aluminum hydride and silylation with f-butyldiphenylsilyl chloride gave (/?)-alcohol 39 (28%) along with (S)-alcohol 40 (30%). The acetonization of 40 and removal of the silyl group afforded alcohol 41 in 84% yield in two steps. The mesylation of 41 and treatment with potassium thiophenoxide, followed by oxidation of the resulting thioether with m-chloroperbenzoic acid provided sulfone 42 in 94% yield in three steps. The coupling reaction of 42 with aldehyde 17, acetylation and reduction with


16. ISEKI & KOBAYASHI


221

3

sodium amalgam gave 43 in 41% overall yield. The hydrogenation of 43 catalyzed with platinum oxide, deketalization, acetylation, hydrogenolysis and PCC oxidation furnished ketone 44 in 39% yield in five steps. The Wittig reaction of 44 with the phosphinoyl carbanion derived from 23 and deprotection with cation exchange resin (50W-X4) in MeOH gave DD-011 in 33% yield. In the same manner, DD-010 was obtained from alcohol 39. CF >£)MOM


HO'

3

a),b),c) 38%

CF

36

3

37 f),g),h)

16%^

TBDPSO^^n 38 r« ! ~r V CF i).J)

H

C F 3

2

v

HO ^"OH DD-011 R = OH, R = H 84% ' DD-010 R = H, R = OH T B D P S O - ^ V ^ h 39 R = O H , R = H (28%) 40 R = H , R = O H (30%) 1

2

1

1

2

1

k),l),m)

H

2

F

o V * O

PhSO/ 42

FC 3

C F

2

C

-

C

3

F

O q),r),s),t),u) ° r 39%

n),o),p) 41%

3

3

°

o V * n ^ r gv cCF, 41

OBn '*..^CHO

jTP(0)Ph

2

DD-011

33%

OTBS TBSO OBn 17 O 44 23 a) LiAlH ; b) cone. HCl-MeOH; c) f-BuOOH, VO(acac) ; d) LiAlH ; e) TBDPSC1, imidazole; f) i) (-)-camphanic chloride, ii) resolution; g) LiAlH ; h) TBDPSQ, imidazole; i) Me C(OMe) , TsOH; j) TBAF; k) MsCl, Et N; 1) PhSH, KOH; m) MCPBA; n) w-BuLi, then 17; o) Ac 0; p) Na-Hg; q) H , Pt0 ; r) cone. HC1MeOH; s) Ac 0, DMAP, Py; t) H , Pd-C; u) PCC; v) w-BuLi, 23; w) 50W-X8, MeOH. Scheme 7. Synthesis of 26,26,26,27,27,27-Hexafluoro-24-homola,24 ,25-trihydroxyvitamin D . 4

2

4

4

2

2

3

2

2

2

2

2

,

3

In Vitro Biological Activity of Fluorinated Vitamin D Analogs 3

Growth Inhibition of Human Colon Cancer Cells (HT-29) in Culture. In vitro assessment for inhibition activity by the fluorinated vitamin D analogs toward the growth of human cancer cells, HT-29, was carried out by measuring mitochondrial dehydrogenase of viable cells with MTT (26). Activity values relative to la,25(OH) D (3) are shown in Table I. Based on half-maximum concentrations, the potency of the 22(5)-hydroxylated analog (DD-003) in inhibiting HT-29 cell growth was shown to be 10 times that of la,25-(OH) D (3). DD-003 was 10 times less 3

2

3

2

3


222


effective than 26^7-F -la,25-(OH) D3 (12). The configuration of the 22-hydroxyl group was essential for growth inhibition activity. The 22(5)-isomer (DD-003) was 100 times more active than the 22(R) isomer (DD-004) and the 23(rt)-isomer (DD014), 10timesmore than la,25-(OH)2D3 (3). The effect of configuration of hydroxyl group on growth inhibition was much less in the case of 23-isomers (DD-014 vs. DD015), and that of 24'(S)-isomer (DD-011) was basically the same as that of the 24(/?)isomer (DD-010). To induce the differentiation of human leukemia cells, HL-60, DD003 was found 6 times more potent than la,25-(OH)2D3 (3). 6

2


,

Table I. Inhibition of Colon Cancer Cell Proliferation (HT-29) -Activity Relative to la,25-Dihydroxyvitamin D 3

la,25-(OH) D (3) 2

3

DD-003

DD-014

DD-020

DD-019

DD-010

DD-004 All values >1 indicate the analog more potent than la,25-(OH) D3 (3) 2

In Vitro Binding Assays. The fluorinated vitamin D3 analogs were examined for binding activity to vitamin D3 receptor (VDR) and vitamin D binding protein (DBP). Table II shows relative activities toward la,25-(OH) D (3). The 22(5)hydroxylated analog (DD-003) was 100 times less effective than la,25-(OH) D (3) for binding to the chick embryonic intestinal la,25-(OH) D3 receptor (VDR). The configuration of the 22-hydroxyl group was essential for VDR binding activity. The 22(5)-isomer (DD-003) was 125timesmore active than the 22(R) isomer (DD-004) and the 23(/?)-isomer (DD-014), 17timesmore potent than DD-003. The effect of the configuration of hydroxyl group on binding to VDR was much less for the 23-isomers (DD-014 vs. DD-015), and that of 24(S)-isomer (DD-011) was basically the same as that of the 24'(/?)-isomer (DD-010). As for the binding affinity toward vitamin D binding protein (DBP) from vitamin D deficient rats, the 22(S)-hydroxylated analog (DD-003) was 250timesless effective than lot,25-(OH) D (3). Allfluorinatedanalogs except 26,27-F -la,25-(OH) D3 (12) were much less active than la,25-(OH) D3 (3). Binding affinity toward VDR and DBP showed no correlation with inhibitory activity toward human cancer cell growth, HT-29. 2

3

2

2

,

2

3

6

2


2

3

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Fluorinated Vitamin D Analog 3

223

Table II. In Vitro Binding Activity -Activity Relative to la^5-Dihydroxyvitamin D3Compound

la,25-(OH)2D3 Receptor

la,25-(OH) D (3) 26,27-F6-la,25-(OH) D3 (12) DD-003 DD-004 DD-014 2

3


2

DD-015 DD-011 DD-010

Vitamin D Binding Protein 1 0.25 0.004

1 0.5 0.01 0.00008 0.17 0.06 0.5 0.5

0.003 0.02 0.003 0.005 0.006

All values >1 indicate the analog more potent than la,25-(OH)2D3. In Vivo Calcemic Activity of Fluorinated Vitamin D Analogs 3

Male Wistar rats (3 weeks old) were kept on a low calcium and vitamin D-deficient diet. When serum calcium decreased to less than 6 mg/dl, each compound was injected on five consecutive days intraperitoneally (7.2 nmol/kg/day). Rat serum was collected from the tail vein to measure serum calcium colorimetrically at 575 nm using 0-cresolphthalein. As shown in Figure 1, la,25-(OH)2D3 (3) considerably increased serum calcium while the 22(S)-hydroxylated analog (DD-003) had much less effect. The 22(/?> isomer (DD-004) was less potent than the 22(S)-isomer (DD-003). Hydroxylation at carbon 22 or carbon 23 greatly reduced calcemic activity compared to la,25-(OH)2D3 (3). The extent of serum calcium increase by 24'hydroxylated isomers (DD-010 and DD-011) was virtually the same as that by loc,25(OH)2D3 (3). The calcemic activity of fluorinated vitamin D3 analogs showed close correlation with VDR binding affinity but not with DBP binding affinity. Inhibition of HT-29 Human Colon Cancer Growth beneath the Renal Capsule of SCID (Severe Combined Immunodeficiency) Mice by DD-003 In Vivo Growth Inhibition of HT-29 Tumor Cells. Fibrin clots of colon cancer cells (HT-29) were implanted beneath the renal capsule of 5 week-old SCID mice. At 7 days following implantation, the mice were administered 3 |ig/kg body weight DD003 or the vehicle i.p. every other day 5 times. Eleven days after the initial administration, tumor growth and serum calcium were determined. In both groups, mice appeared healthy and essentially the same in final body weight. Tumors in DD003-treated mice were smaller, invading to a lesser extent surrounding tissue. Central necrosis was greater compared to the control. DD-003 greatly suppressed HT-29 tumor growth. As shown in Table IE, serum calcium of the mice treated with DD-003 was basically the same as that of the control mice and malignant growth of colon cancer cells was inhibited by 63%.


224


«3 ^ 8 B

/la,25-(OH) D 2

3

1 _ Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on May 26, 2018 | https://pubs.acs.org Publication Date: August 13, 1996 | doi: 10.1021/bk-1996-0639.ch016

3

1

7

6

§ 5

0

t

1

t

2

3

t

4

t t

Time (day) INJECTION Figure 1. Effects of Vitamin D Analogs on Serum Calcium. 3

Table III. Inhibition of Human Colon Cancer Growth in SCID Mice by DD-003 Initial Tumor diameter (mm)

Increase in Tumor diameter (mm)

Serum Calcium (mg/dl)

Control

1.61 ±0.15

2.29 ±0.39

7.9 ±0.6

DD-003

1.40 ±0.38

0.85 ± 0.26*

8.4 ±0.7

^Significantly different from the control; P < 0.001. Reproduced with permission fromreference26. Time- and Dose-dependent Growth Inhibition of HT-29 Tumor Cells. As shown in Figure 2, at 2 days following implantation of colon cancer cells (HT-29), the mice were given 1 ^g/kg body weight of DD-003 or the vehicle i.p. three times a week for a specified period of time. Tumors in the control mice grew greatly, reaching a plateau in 19 days. Inhibition of cell growth was not significant after 1 week of DD003 treatment but became progressively significant with prolonged time. The tumors remained small throughout the experimental period. Since 3 weeks of treatment were sufficient to detect drug effect on tumor growth, as shown in Figure 2, dose-dependent studies were conducted in which various amounts of DD-003 were injected i.p. three times a week for three weeks. As shown in Table IV, DD-003 considerably inhibited the growth of colon cancer cells dosedependently. Body weight and serum calcium of the mice treated with DD-003 at all concentrations were essentially the same as for the control mice.


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2.5r

0

5

10 15 20 25 Time (day) Figure 2. Time-dependent Growth Inhibition of HT-29 in SCID Mice by DD-003: * P

Fluorinated Vitamin D3 Analog with In Vivo Anticancer Activity - ACS

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