Identification of HUHS190, a human naftopidil metabolite, as a novel anti-bladder cancer drug

Journal Pre-proofs Identification of HUHS190, a human naftopidil metabolite, as a novel antibladder cancer drug Tadashi Shimizu, Keiko Yamaguchi, Momoka Yamamoto, Rina Kurioka, Yukari Kino, Wataru Matsunaga, Syuhei Nakao, Hiroshi Fukuhara, Akito Tanaka, Akinobu Gotoh, Miyuki Mabuchi PII: DOI: Reference:

S0960-894X(19)30707-3 https://doi.org/10.1016/j.bmcl.2019.126744 BMCL 126744

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

21 August 2019 23 September 2019 8 October 2019

Please cite this article as: Shimizu, T., Yamaguchi, K., Yamamoto, M., Kurioka, R., Kino, Y., Matsunaga, W., Nakao, S., Fukuhara, H., Tanaka, A., Gotoh, A., Mabuchi, M., Identification of HUHS190, a human naftopidil metabolite, as a novel anti-bladder cancer drug, Bioorganic & Medicinal Chemistry Letters (2019), doi: https:// doi.org/10.1016/j.bmcl.2019.126744

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Identification of HUHS190, a human naftopidil metabolite, as a novel anti-bladder cancer drug Tadashi Shimizua,b, Keiko Yamaguchib, Momoka Yamamotob, Rina Kuriokab, Yukari Kinob, Wataru Matsunagac, Syuhei Nakaod, Hiroshi Fukuharae, Akito Tanakaa,b, Akinobu Gotohc*, and Miyuki Mabuchia*, a

Laboratory of Chemical Biology, Advanced Medicinal Research Center, Hyogo University of Health

Sciences, Kobe, Hyogo, Japan b

School of Pharmacy, Hyogo University of Health Sciences, Hyogo, Japan

c

Laboratory of Cell and Gene Therapy, Institute for Advanced Medical Sciences,

Hyogo College of Medicine, Nishinomiya, Hyogo, Japan d

Synthetic Organic Chemistry Laboratory, RIKEN Cluster for Pioneering Research, Wako, Saitama, Japan.

e Department

of Urology, Kyorin University, Tokyo, Japan.

*Corresponding author, Authors M.M. and A.G. contributed equally to this work. Tel: +81783043027, e-mail: [email protected], Keywords: HUHS190, repositioning, naftopidil, anti-bladder cancer drug, active metabolite, structureactivity relationship, intravesical administration, BCG, pirarubicin,

Abstract We carried out structure-activity relationship study on anti-cancer effects of naftopidil (1) and its metabolites, resulted in identification of 1-(4-hydroxy-2-methoxyphenyl)piperazin-1-yl)-3-(naphthalen1-yloxy) propan-2-ol (2, HUHS190), a major human metabolite of 1, which exhibited the most selective toxicities between against normal and cancer cells (Table 1). 2 was more hydrophilic compared to 1, was enough to be prepared in high concentration solution of more than 100 M in saline for an intravesical instillation drug. Moreover, serum concentration of 2 was comparable to that of 1, an oral preparation drug, after oral administration at 32 mg/kg (Figure 3). Both of 1 and 2 showed broadspectrum anti-cancer activities in vitro, for example, 1 and 2 showed inhibitory activity IC50= 21.1 M and 17.2 M for DU145, human prostate cancer cells, respectively, and IC50= 18.5 M and 10.5 M for T24 cells, human bladder cancer cells. In this study, we estimated anticancer effects of 2 in a bladder cancer model after intravesical administration similar to clinical cases. A single intravesical administration of 2 exhibited the most potent inhibitory activities among the clinical drugs for bladder cancers, BCG and Pirarubicin, without obvious side effects and toxicity (Figure 4). Thus, HUHS190 (2) can be effective for patients after post-TURBT therapy of bladder cancer without side effects, unlike the currently available clinical drugs.

Naftopidil (1, Figure 1) is an 1-adrenergic receptor blocker1, and it is widely used for the treatment of benign prostatic hyperplasia to reduce prostatic smooth muscle tone and to exert an immediate effect on urinary flow. Interestingly, Honma and Yamada et al. reported2 that prostate cancer incidence was significantly lower in men treated with 1 for three months or longer, for treatment of prostatic hypertrophy, compared to tamulosin, another 1-adrenergic receptor blocker. Nakagawa et al. 3 exhibited the anti-cancer effects of 1 was 1-adrenergic-independent and observed even for bladder

cancer cells, not only for prostate cancer cells. Identification of 1-adrenergic and organ independent anti-cancer effects of 1 was attractive for novel drug discovery in anti-cancer because of its safety in long-term clinical usage4 while its mechanism is still unclear. 5-6 Chen et al. 7 have paid attention to anticancer effects of 1 and synthesized new naftopidil-based arylpiperazine derivatives because anti-cancer effects of 1 is not satisfied. In this study, we focused on anti-cancer effects of clinical metabolites from 1 and its derivarives8-9 because some major metabolites were clinically identified in patients (Figure S1). Bladder cancer is one of the most common urothelial carcinomas worldwide, and still the leading cause of cancer death. 10-12 Almost 75% of patients have bladder cancer with non-muscle invasive bladder cancers. 10, 13 Treatment for these patients usually begins with transurethral resection of the bladder tumour (TURBT) followed by risk level-appropriate post-TURBT adjuvant therapy. 13 Removal of superficial tumours by TURBT treatment is an effective method; however, patients often suffer from relapses.13, 14 Intravesical administrations of Bacillus Calmette-Guerin (BCG) and /or traditional anticancer drug such as pirarubicin are now widely recommended to suppress these relapses. However, insufficient anti-cancer effects and/or serious adverse side effects often restrict the clinical usages, which often resulted in advance-stage tumours, organ failure or death.15-18 Thus, the development of novel intravesical anti-bladder cancer drug without serious side effects, which is effective and hydrophilic enough for intravesical administration to avoid systemic disadvantages, is strongly required for post-TURBT therapy.13 The Naftopidil derivatives studied in this work were synthesized as shown in Figure 2. 8-9 The 1-naphtol derivatives (3, 4) reacted with epichlorohydrin in the presence of K2CO3 and potassium iodide to afford compounds 5 or 6. Treatment of 5 or 6 with 1-substituted phenylpiperazine derivatives gave 7a-7c, or 7d-7g, respectively. Deprotection of 2-methoxymethyl ether (MOM) of 7a, 7b, or 7c by hydrochloric acid afforded biologically estimated compounds 8, 2, or 9, respectively. Deprotection of benzyl group of 7d by Pd catalytic reduction under H2 atmosphere gave 10. Sequential deprotection of MOM group

and benzyl group of 7e, 7f, or 7g resulted in the synthetic compounds 11-13, respectively. We excluded glucuronidated metabolites from our study while some have also been identified in clinical study because they were too hydrophilic to be cell-permeable and because they do not have a drug-like structure. Growth inhibition effects in vitro on both of normal human cell lines, HEK293 (embryonic kidney) and PNT1A (prostatic epithelial cells) and a broad range of human cancer cells such as PC3 and DU145 cells (prostate cancer), KK47 and T24 cells (bladder cancer) and 786-O cells (kidney carcinoma), were examined to estimate their inhibitory activities (Table 1, Figure S2 and S3). Naftopidil (1) showed weak and non-selective inhibitory activities, with IC50 values for growth inhibitions on normal cells of 27 - 47 M and with those on cancer cells of 13-31 M. Demethylation of methoxy moiety (8), a reported metabolite in humans (Figure S1), 8-9 showed almost the same inhibitory activities as those of 1. Interestingly, introduction of hydroxy at the 4-position of phenyl of 1 (2, HUHS190), a major metabolite in humans, exhibited more potent inhibition for the growth of cancer cells, values of IC50 were 11-24 M, while it exhibited little inhibition for normal cells, indicating better selective inhibition compared with 1 to avoid toxicity. The introduction of two hydroxy groups on the phenyl and naphthyl rings (12) resulted in a decrease in the inhibitory activities. Other related derivatives (10, 11) also showed weak inhibitions. The introduction of two hydroxy groups on the phenyl and naphthyl rings (13) showed almost the same inhibitory activities as those of 2 against cancer cells, but it also had inhibitory activities on normal cells. Therefore, we selected 2 (HUHS190) as the compound to study further because it showed potent inhibitory activities against cancer cells with weak toxicity on normal cells (Table 1). 2 was more hydrophilic than 1 and could be prepared in high concentration solution of more than 100 M in saline, which is adequate for an intravesical instillation drug. We also compared serum concentrations of 2 with 1, a widely used oral medicine, in rats after oral administrations at 32 mg/kg, respectively (Figure 3). Both of 1 and 2 were quickly absorbed and

reached the maximum concentration (Cmax), 110 ng/mL, and 397 ng/m at 15 minutes, respectively after oral administration. The AUC values for 6 h (AUC0-6) were almost the same, which exhibited that 2 was available for oral drug as well as intravesical instillation drug. We have finally constructed an allogenetic bladder cancer model, in which a small number of MB49 cells, derived from C57BL/6 mice, were injected into C57BL/6 mice’s smooth muscle layer of bladder using a needle after laparotomy. The test compounds, BCG, pirarubicin, or HUHS190, were intravesically administered in clinical situations and were maintained for 2 h. Pirarubicin exhibited potent inhibitory activity in vitro; its IC50 was 2.2 x 10-8 M (Figure S4), which was the same value as that against mouse bladder carcinoma, MBT-2 cells derived from C3H mice (IC50=2.2 x 10-8 M) and mouse lymphoblastoma L5178Y cells (IC50=2.3 x 10-8 M).19 Compounds 1 and 2 showed weak inhibitory activities, IC50=2.3 and 1.2 x 10-5 M, respectively. As expected, little inhibitory activity was observed for BCG in this cell assay (IC50 >4 mg/mL, Figure S4). As shown in Figure 4, mice began to die nine days after the implantation and administration day (day 0), and the exact causes of death were not clear. Mice treated with saline or pirarubicin incrementally died, and 70-80 % of them died after 28 days. Mice treated with BCG had a higher viability within 25 days than the control mice, but after that, the BCG-treated mice died almost at the same rate as the control mice. On the other hand, mice treated with 2 had a more gradual mortality rate, and only 30 % of them died on the last day (28 days), indicating that 2 had a more potent anticancer effect than BCG or pirarubicin. In conclusion, we examined structure-activity relationships of naftopidil metabolites on their anticancer effects, resulted in the identification of 1-(4-hydroxy-2-methoxyphenyl)piperazin-1-yl)-3(naphthalen-1-yloxy) propan-2-ol (2, HUHS190), a major human metabolites from 1. 2 demonstrated the most excellent selective toxicities between normal and cancer cells in vitro (Table 1). Moreover, 2 was hydrophilic enough to be prepared in high concentration solution of more than 100 M in saline for an intravesical instillation drug, while serum concentrations of 2 was higher than that of 1 as well

(Figure 3). A single intravesical administration of 2 prolonged the life of model mice in vivo bearing MB49 cells under the outside surface of the bladder compared to clinical drugs, such as BCG and pirarubicin, for 28 days without obvious side effects and toxicity (Figure 4). Combination of intravesical administration of 2 and continuously oral administrations after the operation is anticipated to be more effective for the suppression of relapse, while BCG and pirarubicin, drugs for bladder cancer, are not available for oral administration. Thus, HUHS190 (2) can be effective for patients after post-TURBT therapy of bladder cancer without side effects, unlike the currently available clinical drugs.

Acknowledgments

This work was supported by JSPS KAKENHI Grant Number 15K10613, 16K07186.

Supplementary data The supplementary data (experimental procedures, Figures S1-S4) are available.

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captions Table 1

Growth inhibitory activities (IC50 M) of 1, its metabolites and their derivatives

Figure 1. Structure of Naftopidil (1) and HUHS190 (2)

Figure 2. Synthesis of Naftopidil derivatives studied in this study.

Figure 3. Comparison of oral administrations of 1 and 2 at 32 mg/kg in rats. Compounds were suspended in 0.5 % methyl cellulose (0.5 % MC). Male SD rats were fasted for 18 hours before the p.o. test. After the compounds were administered of compounds, 0.25. 0.5, 1, 2, or 6 hours later, the blood was collected from the abdominal vein under anaesthesia. The concentration of each sample was determined using HPLC, and Cmax and AUC0-6 values were obtained.

Figure 4. Comparison of the anti-anticarcinogenic activities of HUHS190 (50 mg/mL), BCG (2 mg/mL), and Pirarubicin (1 mg/mL) in C57BL/6 mice bearing MB49 cells under the surface of the bladder. MB49 cells were implanted to the bladder muscle of C57BL/6 mice under anesthesia by needle after laparotomy; following that, each compound was instilled to the bladder, and sustained for 2 hours. The Mice were observed for 4 weeks.

O

N OH

Naftopidil (1)

N

OCH3

O

N OH

N

OCH3

OH A major metabolite from 1 in human (HUHS190, 2)

Figure 1. Structure of Naftopidil (1) and HUHS190 (2)

O

OH Cl

O

O

a

7a (R1=OMOM, R2=H, R3=H) K2CO3 KI

3

R

3 (R3=H) 4 (R3=OBn)

R3

5 6 (R3=OBn) O

R1

HN

7b

(R3=H)

R1

N N

R2

2

R R3

3,

R2=OMOM,

7a - 7g

7d

(R1=OCH

7e

(R1=OMOM,

3,

R2=H,

R3=OBn)

R2=H,

R3=OBn)

400 HUHS190

Cmax (ng/mL)

300

397.1

204.4

Naftopidil (1)

110.1

201.5

0 1

2

3

4

5

6

7

Time (h)

60

～ ～

～ ～

Figure 3. Comparison of oral administrations of 1 and 2 at 32 mg/kg in rats. Compounds were suspended in 0.5 % methyl cellulose (0.5 % MC). Male SD rats were fasted for 18 hours before the p.o. test. After the compounds were administered of compounds, 0.25. 0.5, 1, 2, or 6 hours later, the blood was collected from the abdominal vein under anaesthesia. The concentration of each sample was determined using HPLC, 100 and Cmax and AUC0-6 values were obtained. 80

AUC0-6 (ng・ h/mL)

HUHS190 (2)

Naftopidil

rvival (%)

Seum concentration (ng/mL)

500

200

b a, b

7g (R1=OMOM, R2=OMOM, R3=OBn)

Figure 2. Synthesis of Naftopidil derivatives studied in this study.

0

a

7f (R1=OCH3, R2=OMOM, R3=OBn)

Conditions; a) H2, Pd-C, b) HCl

100

a

R3=H)

7c (R1=OMOM, R2=OMOM, R3=H)

OH

N

(R1=OCH

a, b a, b

8 2 9 10 11 12 13

Table 1.

Growth inhibitory activities (IC50 M) of 1, its metabolites and their derivatives1) O

R1

N OH

N R2

R3

NO.

R

1 OMe (Naftopidil) 8

Normal cells

Structure 1

OH

2 OMe (HUHS190)

3

2)

Cancer cells

2

R

R

HEK293

KK475)

T245)

786-O6)

H

H

47.3

27.0

31.2

21.1

19.0

18.5

13.3

H

H

>50

22.2

21.7

18.0

18.9

18.0

18.7

OH

H

>50

>50

24.4

17.2

11.8

10.5

11.4

PNT1A

3)

PC3

4)

DU145

4)

9

OH

OH

H

>50

29.9

30.3

11.6

9.6

13.4

17.5

10

OMe

H

OH

>50

34.9

28.0

35.1

17.6

16.1

46.9

11

OH

H

OH

>50

37.4

26.1

>50

33.0

14.8

9.7

12

OMe

OH

OH

>50

37.2

45.7

>50

>50

37.4

38.7

13

OH

OH

OH

40.4

17.1

17.7

20.3

17.9

15.6

18.5

1) Dose-dependency on each cells were shown in Figure S2 (normal cells) and Figure S3 (cancer cells).

2) Human Embryonic Kidney cells

293. 3) Human normal prostate epithelium immortalized with SV40. 4) Human prostate cancer cells. 5) Human bladder carcinoma. 6) Human kidney carcinoma.

Table 1. Growth inhibitory activities (IC50 M) of 1, its metabolites and their derivatives1) O OH

NO.

Structure R1

1 OMe (Naftopidil) 8

OH

2 OMe (HUHS190) 9

OH

　

R1

N

Normal Rcells 3

N

　

R2

R2

R3

　 HEK2932) PNT1A3) 　 PC34)

H

H

　

47.3

27.0

H

H

　

>50

OH

H

　

OH

H

　

Cancer cells DU1454)

KK475)

T245)

78

　 31.2

21.1

19.0

18.5

1

22.2

　 21.7

18.0

18.9

18.0

1

>50

>50

　 24.4

17.2

11.8

10.5

1

>50

29.9

　 30.3

11.6

9.6

13.4

1

10 11 12 13

OMe OH OMe OH

H H OH OH

OH OH OH OH

　 　 　 　

>50 >50 >50 40.4

34.9 37.4 37.2 17.1

　 　 　 　

28.0 26.1 45.7 17.7

35.1 >50 >50 20.3

17.6 33.0 >50 17.9

1) Dose-dependency on each cells were shown in Figure S2 (normal cells) and Figure S3 (cancer cells). 2) Human Embryonic Kidney cells 293. 3) Human normal prostate epithelium immortalized with SV40. 4) Human prostate cancer cells. 5) Human bladder carcinoma. 6) Human kidney carcinoma.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☒The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

16.1 14.8 37.4 15.6

4

3 1