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Discovery of adamantane based highly potent HDAC inhibitors
Accepted Manuscript Discovery of adamantane based highly potent HDAC inhibitors Balasubramanian Gopalan, Thanasekaran Ponpandian, Virendra Kachhadia, Kuppusamy Bharathi mohan, Radhakrishnan Vignesh, Velaiah Sivasudar, Shridhar Narayanan, Bhonde Mandar, Rajendran Praveen, Nithyanandan Saranya, Sriram Rajagopal, Sridharan Rajagopal PII: DOI: Reference:
S0960-894X(13)00318-1 http://dx.doi.org/10.1016/j.bmcl.2013.03.002 BMCL 20238
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Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
21 January 2013 25 February 2013 1 March 2013
Please cite this article as: Gopalan, B., Ponpandian, T., Kachhadia, V., mohan, K.B., Vignesh, R., Sivasudar, V., Narayanan, S., Mandar, B., Praveen, R., Saranya, N., Rajagopal, S., Rajagopal, S., Discovery of adamantane based highly potent HDAC inhibitors, Bioorganic & Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/ j.bmcl.2013.03.002
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Discovery of adamantane based highly potent HDAC inhibitors
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Balasubramanian Gopalan, Thanasekaran Ponpandian, Virendra Kachhadia, Kuppusamy Bharathi Mohan, Radhakrishnan Vignesh, Velaiah Sivasudar, Shridhar Narayanan, Bhonde Mandar, Rajendran Praveen, Nithyanandan Saranya, Sriram Rajagopal, and Sridharan Rajagopal*
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com
Discovery of adamantane based highly potent HDAC inhibitors Balasubramanian Gopalan, Thanasekaran Ponpandian, Virendra Kachhadia, Kuppusamy Bharathi mohan, Radhakrishnan Vignesh, Velaiah Sivasudar, Shridhar Narayanan, Bhonde Mandar, Rajendran Praveen, Nithyanandan Saranya, Sriram Rajagopal, and Sridharan Rajagopal* Drug Discovery Research Centre, Orchid Chemicals & Pharmaceuticals Ltd, Chennai -600119, India.
Corresponding author. Tel.: +91 (44) 24503137 x 412; fax: +91 (44) 24501650. E-mail address: [email protected] (or) [email protected] (or) [email protected]
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ABSTRACT
Article history: Received Revised Accepted Available online
Herein, we report the development of highly potent HDAC inhibitors for the treatment of cancer. A series of adamantane and nor-adamantane based HDAC inhibitors were designed, synthesized and screened for the inhibitory activity of HDAC. A number of compounds exhibited GI50 of 10-100 nM in human HCT116, NCI-H460 and U251 cancer cells, invitro. Compound 32 displays efficacy in human tumour animal xenograft model.
Keywords: Adamantane HDAC inhibitors N-Hydroxy-4-aminomethyl cinnamide Nor-adamantane Tumour xenograft
2009 Elsevier Ltd. All rights reserved.
Histone deacetylases (HDAC) are a zinc metalloenzyme and they are an important target for the treatment of cancer and other diseases.1 Currently, more than ten HDAC inhibitors have entered into the clinical studies2 and two of them vorinostat and romidepsin have been approved by FDA for the treatment of cutaneous T-cell lymphoma (Figure-1). Figure-1 FDA approved and few of clinical HDAC inhibitors
The pharmacophore model of HDAC inhibitors (HDACi) contains three parts, a lipophilic cap connected to a hydrophilic Zn binding group by an alkylene or arylene linker.
In the past few years a number of articles have been published by changing the cap, linker and zinc binding portions of the HDACi.3 But recent studies have focused on varying the cap or linker portion where the metal binding portion remaining same which can be either a hydroxamic acid or an ortho-amino benzamide group which are well tolerated in all invivo studies. In an efforts to produce a highly potent HDAC inhibitors to treat cancer, we have reported a number of molecules having good HDAC inhibition as well as antiproliferative activity.4 Some of these compounds showed good PK/PD properties and tumour growth inhibition in animal xenograft model. For developing a novel HDACi, we mainly concentrated on modifying the cap portion of the HDACi pharmacophore which might determine the potency. After a thorough literature survey we found that the adamantane moiety might be more suitable in the cap portion, since the adamantane moiety is highly lipophilic, stress-free bulky cyclic hydrocarbon and incorporation of this moiety into several molecules results in compounds which modify the biological availability. Several adamantane derived drugs are available in the market (Amantadine, Memantine, Rimantadine,
Scheme-1. Reagents and condition: (a) MeOH, rt, 3h then NaBH 4 (1.6 eq), 5 oC, 1h, 90-95% (b) NH2OH.HCl (10 eq), KOH (20 eq), MeOH, rt, 30 min, 45-50%. Tromantadine, Vildagliptin, Saxagliptin and Arterolane) and also they show diverse biological properties such as antiviral,5 antibacterial,6 antifungal,7 anti-inflammatory,8 anti-diabetic,9 11β-HSD1 inhibitory activities10 and other medical properties.11 Recently Jung et al reported the therapeutic effect of adamantyl HDAC inhibitors H6CAHA in combination with radiation.12 To explore the therapeutic effect of adamantyl based HDAC inhibitors, we designed compounds having adamantane moiety as the cap structure, which was connected by different groups to N-hydroxy 4-aminomethylcinamide (1) which has been shown to be a better HDAC inhibitor in a number of HDAC inhibitors which have entered the clinic. 13
Following the known procedures 1-adamantylamine derivatives were prepared from 1-adamantylamine and homoaza-adamantylamine from 2-adamantanone.14 Synthesis of compounds 5 to 11 and 30 were carried by the reductive amination reaction between methyl-4-formyl cinnamate (3)15 and corresponding adamantylamines (2) with NaBH4 and the resulting esters (4) were treated with methanolic NH2OK at room temperature provided the corresponding hydroxamic acid analogues (Scheme-1). Syntheses of one and two carbon homologated analogues of 5 are described in Scheme-5. The 1-adamantane carboxylic acids (17a, 17b & 17c) were purchased from commercial sources. The nor-adamantane carboxylic acid 17d (Scheme-2) and 2-adamantane acetic acid 17e (Scheme-3) were synthesized from 2-adamantanone. The carboxylic acids group in 17 was converted into carboxaldehyde derivatives 18 by the step wise reduction and oxidation reactions with BH3.SMe2/THF and PCC/MDC respectively. The obtained aldehyde derivatives 18 were treated with 1a in MeOH followed by NaBH4 yielding the methylene linked derivatives 19. The hydroxamic acid analogues 22, 24, 26, 29 and 32 were obtained by the treatment of methanolic NH2OK to 19. The carbonyl linked derivatives (20a, 20b, 20c, 20d and 20e) were prepared by the reaction between adamantane carboxylic acid derivatives 17 and 1a in presence of condensing agent, EDCI,
and base (Et3N). The obtained amide linked derivatives were converted into the hydroxamic acid analogues 21, 23, 25, 28 and 31, following the above procedure.
Scheme-2. Reagents and condition: (a) MeMgCl (1.1 eq), THF, 0 oC, 30 min, 98% (b) (i) 4% aq. NaOCl, AcOH, 0 oC, 3h (ii) CCl4, reflux, 7h (iii) KOH (3 eq), MeOH, reflux, 3h (c) Br2 (5 eq), aq.NaOH (12 eq), Dioxane:H2O (1:5), 0 oC to rt, 15h, 74%.
Scheme-3. Reagents and condition: (a) (OEt)2POCH2COOEt (1.1 eq), NaH (60% dispersion in oil, 1.4 eq), THF, 0 oC, 1h, 98% (b) 10% Pd/C, 30psi H2, EtOH, rt, 1h, 99% (c) NaOH (2 eq), MeOH:H2O (9.8:0.2), 70 oC, 2h, 88%. The compounds 27 and 33 were prepared from 2adamantanone and 1-acetyl noradamantane 14 respectively by reductive amination with 1a followed by hydroxamate formation (Scheme-4).
Scheme-4. Reagents and condition: (a) 1a, MeOH, rt, 15h then NaBH4 (1.6 eq), 5 oC, 1h, 75-87% (b) NH2OH.HCl (10 eq), KOH (20 eq), MeOH, rt, 30 min, 38-42%. The experimental procedure, analytical data and biological protocol for all the compounds are given in the earlier publication.16
Scheme-5. Reagents and condition: (a) BH3.SMe2 (1.2 eq), THF, 0 oC to rt, 3h, 70-75% (b) PCC (1.5 eq), MDC, 2h, 65-72% (c) MeOH, rt, 3h then NaBH4 (1.6 eq), 5 oC, 1h, 80-89% (d) NH2OH.HCl (10 eq), KOH (20 eq), MeOH, rt, 30 min, 41-45% (e) EDCI (1.3 eq), anhydrous. HOBt (0.5 eq), Et3N (2 eq), DMF, rt, 6h, 72-81%.
Table-1 HDAC and anti-cancer activity of adamantane derived hydroxamic acids
Cpd. No
5
6
7
Adamantane derivatives
pan HDAC IC50 nMa
NCI-H460 GI50 µMa
HCT116 GI50 µMa
U251 GI50 µMa
70
2.8
0.5
2.7
460
6
1
2.8
260
7
1.6
7
8
250
8
3
4.2
240
9.5
2.8
6
440
5
2.2
3.2
11
40
4
11
14
21
67
0.1
0.1
0.17
22
1.1
0.3
0.5
0.8
23
77
0.5
0.5
1.9
24
6
0.03
0.03
0.18
25
1.8
1.3
1.1
1.6
26
0.9
1.1
0.45
1.3
27
60
0.07
0.08
0.17
28
8
0.4
0.5
0.26
29
0.28
0.01
0.06
0.05
SAHA
110
1
1
2
9
10
a
Variation is within ±10%
All the synthesised adamantane analogues were compared to the approved drug Vorinostat (SAHA) for their ability to inhibit partially purified mouse liver HDAC enzymes (pan HDAC, triplicate run) and viability of three human cancer cells (HCT116, NCI-H460 and U251). The determination of HDAC and anti-cancer activity by fluorometric and MTT assay respectively. The results are presented in table-1. Initially we started our SAR studies with unsubstituted adamantane, (compound 5) which shows the HDAC inhibitory activity about 70 nM with 0.5-2.8 µM range of anti-cancer activity. Replacing hydrogen in 3rd position of 5 with ethoxy, methoxy, hydroxyl, phenyl and hydroxymethyl (compounds 6, 7, 8, 9 & 10), decreased the potency against HDAC as compared to 5 by 3 fold and the anti-cancer activity also reduced. In the case of substitution of the adamantane ring with chloro in 3rd position (11), there was 2 fold increases in HDAC inhibition, but the anti-cancer activity was reduced. The SAR of unsubstituted adamantane 5 was further modified and studied. Introduction of one carbon linker between the adamantane ring and 1 like methylene or carbonyl groups improved the anti-cancer activity whereas methylene linker 22 showed 70 fold increase in HDAC inhibition compared to 5, whereas the carbonyl linker 21 showed similar potency. Replacing hydrogen with fluorine in 21 results in similar HDAC activity but it decreases the anti-cancer effect by 5 fold (compound 23) whereas in the case of 22, 5 fold decrease in
HDAC and 10 fold increase in anti-cancer activity (compound 24) was observed. Both HDAC and anti-cancer activity were better in methylene linker as compared to carbonyl linker. When the carbon linker was increased by one more carbon from 22, the compound 25 and 26 showed good HDAC and anticancer activity. In all these compounds the linker portion was attached to the C1 of the adamantane ring. When the linker position was changed to 2nd position the compound 27 and 28 were showed better anticancer-activity as compared to the corresponding 1-adamantane analogue 5 and 25 whereas the HDAC activity was 4 fold decrease in 28 and almost similar in 27. The compounds 29 with a two carbon linker showed 3 fold increase in HDAC activity compared to 26 and good anticancer activity. The compound 29 showed the HDAC IC50 0.28 nM and anti-cancer activity 10 nM, 60 nM and 50 nM in HCT116, NCI-H460 and U251 respectively. After establishing good HDAC and anticancer activity in this series, we focused our efforts in exploring the other structural analogue of adamantane ring. The homoazaadamantane compound 30 showed moderate HDAC and anticancer activity compared to adamantane partner. In the case of noradamantane analogue 32, there was 10 fold increases in HDAC and anticancer activity compared to the corresponding adamantane analogue. It was interesting to note that compound 32 showed picomolar HDAC activity (0.02 nM) and low nanomolar anticancer activity (Table 2).
Table-2 HDAC and anti-cancer activity of homo and nor-adamantane series
Cpd. No
HDAC IC50 nMa
NCI-H461 GI50 µMa
HCT116 GI50 µMa
U251 GI50 µMa
30
110
4.9
1.8
1.7
31
25
0.35
0.38
0.6
32
0.02
0.01
0.01
0.03
33
0.03
0.03
0.03
0.05
a
Structure
Variation is within ±10%
On the basis of above in-vitro results compound 32 was chosen for animal xenograft model. SCID mice were implanted subcutaneously with a suspension of NCI-H460 human lung carcinoma cells, tumours were permitted to grow approximately 100 mm3. The compound 32 was administrated intraperitoneally with a dose of 5 mg/kg (since MTD is 7.5 mg/kg) once daily for 21 days and it significantly inhibited the tumour growth by 45% with acceptable body weight loss (Figure 2) (<10% relative to the mean starting weight of the mice).
In summary, a systematic study of adamantane based HDACi was explored. Adamantane with two alkylene linker showed good HDAC inhibition and anticancer activity. Replacing adamantane with nor-adamantane group resulted in more potent compounds having sub-nano molar HDAC activity and nano molar anticancer activity. In vivo efficacy of compound 32 was studied in animal xenograft model at 5 mg/kg IP in NCI-H460, which showed significant tumour growth inhibition of with acceptable body weight reduction (<10 %). Further profiling of these compounds is in progress.
Figure-2 Anti-tumour effect of 32 on NCI-H460 (Lung) tumour xenograft in SCID (Severe combined immunodeficiency) mice
*p<0.05,**p<0.01,***p<0.001 as compared to Vehicle control Note: Statistical analysis carried out by Two-way ANOVA followed by Bonferroni post tests using Graph pad prism, (Version 4).
4.
Acknowledgements: The authors thank Orchid Chemicals & Pharmaceuticals Ltd, for the support and Dr. C. V. Srinivasan for his technical comments. References and notes: 1.
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S0960-894X(13)00318-1 http://dx.doi.org/10.1016/j.bmcl.2013.03.002 BMCL 20238
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
21 January 2013 25 February 2013 1 March 2013
Please cite this article as: Gopalan, B., Ponpandian, T., Kachhadia, V., mohan, K.B., Vignesh, R., Sivasudar, V., Narayanan, S., Mandar, B., Praveen, R., Saranya, N., Rajagopal, S., Rajagopal, S., Discovery of adamantane based highly potent HDAC inhibitors, Bioorganic & Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/ j.bmcl.2013.03.002
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Discovery of adamantane based highly potent HDAC inhibitors
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Balasubramanian Gopalan, Thanasekaran Ponpandian, Virendra Kachhadia, Kuppusamy Bharathi Mohan, Radhakrishnan Vignesh, Velaiah Sivasudar, Shridhar Narayanan, Bhonde Mandar, Rajendran Praveen, Nithyanandan Saranya, Sriram Rajagopal, and Sridharan Rajagopal*
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com
Discovery of adamantane based highly potent HDAC inhibitors Balasubramanian Gopalan, Thanasekaran Ponpandian, Virendra Kachhadia, Kuppusamy Bharathi mohan, Radhakrishnan Vignesh, Velaiah Sivasudar, Shridhar Narayanan, Bhonde Mandar, Rajendran Praveen, Nithyanandan Saranya, Sriram Rajagopal, and Sridharan Rajagopal* Drug Discovery Research Centre, Orchid Chemicals & Pharmaceuticals Ltd, Chennai -600119, India.
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ARTICLE INFO
ABSTRACT
Article history: Received Revised Accepted Available online
Herein, we report the development of highly potent HDAC inhibitors for the treatment of cancer. A series of adamantane and nor-adamantane based HDAC inhibitors were designed, synthesized and screened for the inhibitory activity of HDAC. A number of compounds exhibited GI50 of 10-100 nM in human HCT116, NCI-H460 and U251 cancer cells, invitro. Compound 32 displays efficacy in human tumour animal xenograft model.
Keywords: Adamantane HDAC inhibitors N-Hydroxy-4-aminomethyl cinnamide Nor-adamantane Tumour xenograft
2009 Elsevier Ltd. All rights reserved.
Histone deacetylases (HDAC) are a zinc metalloenzyme and they are an important target for the treatment of cancer and other diseases.1 Currently, more than ten HDAC inhibitors have entered into the clinical studies2 and two of them vorinostat and romidepsin have been approved by FDA for the treatment of cutaneous T-cell lymphoma (Figure-1). Figure-1 FDA approved and few of clinical HDAC inhibitors
The pharmacophore model of HDAC inhibitors (HDACi) contains three parts, a lipophilic cap connected to a hydrophilic Zn binding group by an alkylene or arylene linker.
In the past few years a number of articles have been published by changing the cap, linker and zinc binding portions of the HDACi.3 But recent studies have focused on varying the cap or linker portion where the metal binding portion remaining same which can be either a hydroxamic acid or an ortho-amino benzamide group which are well tolerated in all invivo studies. In an efforts to produce a highly potent HDAC inhibitors to treat cancer, we have reported a number of molecules having good HDAC inhibition as well as antiproliferative activity.4 Some of these compounds showed good PK/PD properties and tumour growth inhibition in animal xenograft model. For developing a novel HDACi, we mainly concentrated on modifying the cap portion of the HDACi pharmacophore which might determine the potency. After a thorough literature survey we found that the adamantane moiety might be more suitable in the cap portion, since the adamantane moiety is highly lipophilic, stress-free bulky cyclic hydrocarbon and incorporation of this moiety into several molecules results in compounds which modify the biological availability. Several adamantane derived drugs are available in the market (Amantadine, Memantine, Rimantadine,
Scheme-1. Reagents and condition: (a) MeOH, rt, 3h then NaBH 4 (1.6 eq), 5 oC, 1h, 90-95% (b) NH2OH.HCl (10 eq), KOH (20 eq), MeOH, rt, 30 min, 45-50%. Tromantadine, Vildagliptin, Saxagliptin and Arterolane) and also they show diverse biological properties such as antiviral,5 antibacterial,6 antifungal,7 anti-inflammatory,8 anti-diabetic,9 11β-HSD1 inhibitory activities10 and other medical properties.11 Recently Jung et al reported the therapeutic effect of adamantyl HDAC inhibitors H6CAHA in combination with radiation.12 To explore the therapeutic effect of adamantyl based HDAC inhibitors, we designed compounds having adamantane moiety as the cap structure, which was connected by different groups to N-hydroxy 4-aminomethylcinamide (1) which has been shown to be a better HDAC inhibitor in a number of HDAC inhibitors which have entered the clinic. 13
Following the known procedures 1-adamantylamine derivatives were prepared from 1-adamantylamine and homoaza-adamantylamine from 2-adamantanone.14 Synthesis of compounds 5 to 11 and 30 were carried by the reductive amination reaction between methyl-4-formyl cinnamate (3)15 and corresponding adamantylamines (2) with NaBH4 and the resulting esters (4) were treated with methanolic NH2OK at room temperature provided the corresponding hydroxamic acid analogues (Scheme-1). Syntheses of one and two carbon homologated analogues of 5 are described in Scheme-5. The 1-adamantane carboxylic acids (17a, 17b & 17c) were purchased from commercial sources. The nor-adamantane carboxylic acid 17d (Scheme-2) and 2-adamantane acetic acid 17e (Scheme-3) were synthesized from 2-adamantanone. The carboxylic acids group in 17 was converted into carboxaldehyde derivatives 18 by the step wise reduction and oxidation reactions with BH3.SMe2/THF and PCC/MDC respectively. The obtained aldehyde derivatives 18 were treated with 1a in MeOH followed by NaBH4 yielding the methylene linked derivatives 19. The hydroxamic acid analogues 22, 24, 26, 29 and 32 were obtained by the treatment of methanolic NH2OK to 19. The carbonyl linked derivatives (20a, 20b, 20c, 20d and 20e) were prepared by the reaction between adamantane carboxylic acid derivatives 17 and 1a in presence of condensing agent, EDCI,
and base (Et3N). The obtained amide linked derivatives were converted into the hydroxamic acid analogues 21, 23, 25, 28 and 31, following the above procedure.
Scheme-2. Reagents and condition: (a) MeMgCl (1.1 eq), THF, 0 oC, 30 min, 98% (b) (i) 4% aq. NaOCl, AcOH, 0 oC, 3h (ii) CCl4, reflux, 7h (iii) KOH (3 eq), MeOH, reflux, 3h (c) Br2 (5 eq), aq.NaOH (12 eq), Dioxane:H2O (1:5), 0 oC to rt, 15h, 74%.
Scheme-3. Reagents and condition: (a) (OEt)2POCH2COOEt (1.1 eq), NaH (60% dispersion in oil, 1.4 eq), THF, 0 oC, 1h, 98% (b) 10% Pd/C, 30psi H2, EtOH, rt, 1h, 99% (c) NaOH (2 eq), MeOH:H2O (9.8:0.2), 70 oC, 2h, 88%. The compounds 27 and 33 were prepared from 2adamantanone and 1-acetyl noradamantane 14 respectively by reductive amination with 1a followed by hydroxamate formation (Scheme-4).
Scheme-4. Reagents and condition: (a) 1a, MeOH, rt, 15h then NaBH4 (1.6 eq), 5 oC, 1h, 75-87% (b) NH2OH.HCl (10 eq), KOH (20 eq), MeOH, rt, 30 min, 38-42%. The experimental procedure, analytical data and biological protocol for all the compounds are given in the earlier publication.16
Scheme-5. Reagents and condition: (a) BH3.SMe2 (1.2 eq), THF, 0 oC to rt, 3h, 70-75% (b) PCC (1.5 eq), MDC, 2h, 65-72% (c) MeOH, rt, 3h then NaBH4 (1.6 eq), 5 oC, 1h, 80-89% (d) NH2OH.HCl (10 eq), KOH (20 eq), MeOH, rt, 30 min, 41-45% (e) EDCI (1.3 eq), anhydrous. HOBt (0.5 eq), Et3N (2 eq), DMF, rt, 6h, 72-81%.
Table-1 HDAC and anti-cancer activity of adamantane derived hydroxamic acids
Cpd. No
5
6
7
Adamantane derivatives
pan HDAC IC50 nMa
NCI-H460 GI50 µMa
HCT116 GI50 µMa
U251 GI50 µMa
70
2.8
0.5
2.7
460
6
1
2.8
260
7
1.6
7
8
250
8
3
4.2
240
9.5
2.8
6
440
5
2.2
3.2
11
40
4
11
14
21
67
0.1
0.1
0.17
22
1.1
0.3
0.5
0.8
23
77
0.5
0.5
1.9
24
6
0.03
0.03
0.18
25
1.8
1.3
1.1
1.6
26
0.9
1.1
0.45
1.3
27
60
0.07
0.08
0.17
28
8
0.4
0.5
0.26
29
0.28
0.01
0.06
0.05
SAHA
110
1
1
2
9
10
a
Variation is within ±10%
All the synthesised adamantane analogues were compared to the approved drug Vorinostat (SAHA) for their ability to inhibit partially purified mouse liver HDAC enzymes (pan HDAC, triplicate run) and viability of three human cancer cells (HCT116, NCI-H460 and U251). The determination of HDAC and anti-cancer activity by fluorometric and MTT assay respectively. The results are presented in table-1. Initially we started our SAR studies with unsubstituted adamantane, (compound 5) which shows the HDAC inhibitory activity about 70 nM with 0.5-2.8 µM range of anti-cancer activity. Replacing hydrogen in 3rd position of 5 with ethoxy, methoxy, hydroxyl, phenyl and hydroxymethyl (compounds 6, 7, 8, 9 & 10), decreased the potency against HDAC as compared to 5 by 3 fold and the anti-cancer activity also reduced. In the case of substitution of the adamantane ring with chloro in 3rd position (11), there was 2 fold increases in HDAC inhibition, but the anti-cancer activity was reduced. The SAR of unsubstituted adamantane 5 was further modified and studied. Introduction of one carbon linker between the adamantane ring and 1 like methylene or carbonyl groups improved the anti-cancer activity whereas methylene linker 22 showed 70 fold increase in HDAC inhibition compared to 5, whereas the carbonyl linker 21 showed similar potency. Replacing hydrogen with fluorine in 21 results in similar HDAC activity but it decreases the anti-cancer effect by 5 fold (compound 23) whereas in the case of 22, 5 fold decrease in
HDAC and 10 fold increase in anti-cancer activity (compound 24) was observed. Both HDAC and anti-cancer activity were better in methylene linker as compared to carbonyl linker. When the carbon linker was increased by one more carbon from 22, the compound 25 and 26 showed good HDAC and anticancer activity. In all these compounds the linker portion was attached to the C1 of the adamantane ring. When the linker position was changed to 2nd position the compound 27 and 28 were showed better anticancer-activity as compared to the corresponding 1-adamantane analogue 5 and 25 whereas the HDAC activity was 4 fold decrease in 28 and almost similar in 27. The compounds 29 with a two carbon linker showed 3 fold increase in HDAC activity compared to 26 and good anticancer activity. The compound 29 showed the HDAC IC50 0.28 nM and anti-cancer activity 10 nM, 60 nM and 50 nM in HCT116, NCI-H460 and U251 respectively. After establishing good HDAC and anticancer activity in this series, we focused our efforts in exploring the other structural analogue of adamantane ring. The homoazaadamantane compound 30 showed moderate HDAC and anticancer activity compared to adamantane partner. In the case of noradamantane analogue 32, there was 10 fold increases in HDAC and anticancer activity compared to the corresponding adamantane analogue. It was interesting to note that compound 32 showed picomolar HDAC activity (0.02 nM) and low nanomolar anticancer activity (Table 2).
Table-2 HDAC and anti-cancer activity of homo and nor-adamantane series
Cpd. No
HDAC IC50 nMa
NCI-H461 GI50 µMa
HCT116 GI50 µMa
U251 GI50 µMa
30
110
4.9
1.8
1.7
31
25
0.35
0.38
0.6
32
0.02
0.01
0.01
0.03
33
0.03
0.03
0.03
0.05
a
Structure
Variation is within ±10%
On the basis of above in-vitro results compound 32 was chosen for animal xenograft model. SCID mice were implanted subcutaneously with a suspension of NCI-H460 human lung carcinoma cells, tumours were permitted to grow approximately 100 mm3. The compound 32 was administrated intraperitoneally with a dose of 5 mg/kg (since MTD is 7.5 mg/kg) once daily for 21 days and it significantly inhibited the tumour growth by 45% with acceptable body weight loss (Figure 2) (<10% relative to the mean starting weight of the mice).
In summary, a systematic study of adamantane based HDACi was explored. Adamantane with two alkylene linker showed good HDAC inhibition and anticancer activity. Replacing adamantane with nor-adamantane group resulted in more potent compounds having sub-nano molar HDAC activity and nano molar anticancer activity. In vivo efficacy of compound 32 was studied in animal xenograft model at 5 mg/kg IP in NCI-H460, which showed significant tumour growth inhibition of with acceptable body weight reduction (<10 %). Further profiling of these compounds is in progress.
Figure-2 Anti-tumour effect of 32 on NCI-H460 (Lung) tumour xenograft in SCID (Severe combined immunodeficiency) mice
*p<0.05,**p<0.01,***p<0.001 as compared to Vehicle control Note: Statistical analysis carried out by Two-way ANOVA followed by Bonferroni post tests using Graph pad prism, (Version 4).
4.
Acknowledgements: The authors thank Orchid Chemicals & Pharmaceuticals Ltd, for the support and Dr. C. V. Srinivasan for his technical comments. References and notes: 1.
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