A comprehensive review of synthesized derivatives of methotrexate in relation to their anticancer potential.

Comp	CCRF-CEM ID50  mol/L
MTX	0.006 × 10-6
MTX- γ-monomethyl ester	0.43 × 10-6
MTX-dimethyl ester	0.40 × 10-6
MTX- α-monoethyl ester	6.2 × 10-6
MTX- γ-monoethyl ester	0.58 × 10-6
MTX-diethyl ester	0.012 × 10-6
MTX- α-monobµtyl ester	2.0 × 10-6
MTX- γ-monobµtyl ester	0.76 × 10-6
MTX-dibµtyl ester	0.057 × 10-6

Comp	R	X	Sµrvival in L1210
			ID50 µM
1	C2H5	H	51%
5	n-C4H9	H	25%
6	n-C4H9	Cl	66%
12	n-C8H17	H	Inactive
13	n-C8H17	Cl	Inactive

Table 10: In vitro growth inhibitory activity alkyl ester derivatives of MTX [24].

Several derivatives of MTX have been synthesized by substitution of alkyl group at 7-position. Inhibitory activity against Streptococcus faecium ATCC 8043 showed that synthesized derivatives were 1000 times less potent than the parent drug. The inhibitory action against p388 murine leukemia shows similar results. The lack of activity of both derivatives against DHFR showed that it might be due to steric effect of –CH3. Studies against L1210 leukemia in mouse showed that these derivatives were inactive (Table 11).

Table 11: Biological data for alkyl derivatives of MTX [25].

Many MTX derivatives were synthesized by MTX diethyl esters and various amines. The procedure involves the use of an excess of amines without solvent. Four of synthesized derivatives have been tested for CCRF-CEM and three for RBL (Rat basophilic leukemia) cells to make a comparison between two cell lines. Inhibitory activity against lymphoblastic leukemia CCRF-CEM showed that bis-amides derivatives were less active than MTX or MTX esters. Bis (benzylamide) showed higher activity in vivo against L1210 in mice, and this activity considered as bis (benzylamide) derivative release free MTX at site other than serum. 1h show most significant activity and IL%. Derivatives show better result for RBL than CCRF-CEM (Table 12).

Table 12: Growth inhibitory activity of selected bisamide derivatives of MTX [26].

Various derivatives of MTX were synthesized having general formula 8-alkyl-7,8-dihydromethotrexate. Synthetic pathway includes alkylation of 7,8-dihydromethotrexate. In vitro studies tested against Lactobacillus casei, thymidylate synthetase, and DHFR. All derivatives were less potent for DHFR than MTX but more potent for thymidylate synthetase. In vitro inhibitory activity tested against CCRF-CEM show all derivatives have less inhibitory activity than MTX. Derivative having H at 8-position has the same activity as MTX but it was inactive for l1210 leukemia (Table 13).

Table 13: In Vitro biological data of MTX analogµes [27].

MTX γ-L-glutamate diethyl ester, MTX α-L-glutamate esters and α- γ-bis (L-glutamate tetraethyl esters) derivatives were synthesized. Major product obtained was γ-isomer. Further esterification of both isomers gave triethyl esters. Antiproliferating activity tested against L1210 leukemia. The increase in life span due to γ and α diethyl ester is 40% and 10% respectively while MTX has +60%. Growth inhibition activity again CCRF-CEM show γ-isomer 10 times more activity but α-isomer didn’t show same result. L isomer show higher activity than D isomer (Table 14).

Table 14: Growth inhibitory activity of glµtamate ester derivatives of MTX [28].

Aza derivative of MTX has been synthesized by additional N-atom between phenyl and carbonyl of the side chains. Photochemical method was used to insert additional N-atom. Inhibitory activity was tested against DHFR and thymidylate synthetase of Lactobacillus casei and CCRF-CEM and found to be less cytotoxic. In vivo inhibition was tested against L-1210 leukemia (mice) and show significant activity than MTX in the order of 55% and 88% respectively. (Table 15) summarizes the obtained results.

Table 15: Inhibition of L. casei (ATCC 7469) growth and DHFR and thymidylate synthetase by MTX derivatives [29].

Tripepetide derivatives of methotrexate have been synthesized by reacting with four different amino acids. These synthesized derivatives have been tested for anti-proliferative activity against W256 (rat) and L 1210 (mouse) leukemia. In this work an extra amino acid added between glutamic acid and amino benzoyl portion. This insertion of amino acid made these derivatives to shows borderline activity. Generally, an increase in dose increase in life spam (%ILS) (Table 16).

Table 16: Anti proliferative activity of peptide derivatives of MTX [30].

Various diesters of MTX and DCM have been prepared by the reaction of acid catalysed esterification. Neutral esterification also carries out using Cs₂CO₃. In vitro anticancer activity was tested against L1210 in mice. Different doses injected to check the increase in median life span [31].

Various γ-monoamides of the AMT and MTX have been synthesized by modification of coupling method of mixed carboxylic- anhydride method. All derivatives have been tested in vitro against L1210 leukemia, wild type L1210 and sublime (CEM/MTX). In vitro γ-N-aryl alkyl and γ-N-aryl derivatives show higher potency than γ-N-tert-alkyl analogues. Results show that AMT derivatives have more potency than MTX derivatives and all MTX derivatives show more potency against sublime L1210/R81 than MTX. It was also found that all derivatives show more activity against cell lines than human cells. In vivo studies showed γ-N-tert-alkyl analogues inactive however significant activity was shown by γ-N-aryl alkyl and γ-N-aryl derivatives. The results are shown in (Table 17).

Table 17: In Vitro biological data for monoamides derivatives of MTX [32].

Various derivatives of MTX and AMT have been synthesized by replacing glutamate moiety with DL-2-aminoalkanedioic acid having up to 10 CH₂ alkyl group. All derivatives have been tested against L1210 leukemia, CEM leukemia (human), CEM/ MTX, and L1210/R81. Derivative with 9 CH₂ alkyl group show higher activity with CEM, and with 6 CH₂ alkyl group against L1210 cell lines (Table 18).

Table 18: Biological data for aminoalkanedioic acid derivatives of MTX [33].

γ-tert-butyl esters of the AMT and MXT have been synthesized with the new synthetic scheme. Affinity for DHFR and inhibitory activities tested against L1210, CEM, and several other cell lines of human carcinoma (Tables 19a-b).

Table 19a: Ester derivatives of MTX [34].

In this article antibodies coupled with MTX by two different methods. First one is water soluble carbiimide coupling and other is a modification of anhydride coupling and later method was found to be more effective. In vivo studies of antibody MTX conjugate show that antibodies associated with cytotoxic the drug were more potent than drug alone. Drug, mixture, and γ-globulin-MTX conjugate were used as a control group [35].

A poly (γ-L-glutamate) derivative of methotrexate has been synthesized in 4 steps, and this derivative contains 2-3 glutamate units more than MTX. Synthetic rout involved peptide coupling, blocking groups removed by catalytic hydrogenolysis. A coupling reagent used was diphenylphosphoryl azide [36].

5,6,7,8-tetrahydromethotrexate (3) and di-hydro-methotrexate (2) were synthesized and affinity was checked against DHFR. It was found that tetra hydro-MTX shows more potency than dihydro-MTX for mice, S. faecalis, P. cerevisiae, dogs and chicks. It was also found that both reduced derivatives were less potent against DHFR and more potent than MTX against thymidylate synthetase. Results show dihydro-MTX is more potent than 5,6,7,8-tetrahydromethotrexate (Table 20).

Table 19b: DHFR inhibition and cell growth of MTX derivatives.

Table 20: DHFR affinity dihydro and tetra hydro derivatives of MTX [37].

Anilides of MTX and AMT have been synthesized and their anti-proliferative activity and binding affinity were tested against DHFR, L1210, and W1-L2 and it was found that the presence of a hydrophobic ring with an acid group enhances potency. All the anilides found to be potent, however γ-amide containing -(BOH₂) found to be most potent. It was proposed that CONH group of these derivatives involved in hydrogen bonding and enhances affinity for DHFR (Table 21).

Table 21: DHFR affinity and anti-proliferative activity of anilides of MTX [38].

Several derivatives of methotrexate have been synthesized by modifying glutamyl moiety along with two oxide analogues. These derivatives have been tested for DHFR affinity against L. Casei, chicken liver, L 1210- FR8, inhibitory activity tested against S. facium. All derivatives show inhibitory activity except oxide derivatives, rather these showed deleterious effect. This detrimental effect may be due to decrease in basicity of pteridine ring (Table 22) [39].

Table 22: Inhibitory activity of derivatives of MTX.

MTX bisamide derivatives have been synthesized with different aryl, alkyl aryl, and alkyl amines. MTX-dianilide was also prepared. These derivatives have been tested for DHFR inhibit and anti-proliferative for L1210 leukemia. Results showed that methyl group on benzylic carbon decrease activity. Most active compounds was that having R group aryl amine having no methyl group (Table 23).

Table 23: Biological activity of anilides and dianilide of MTX [40].

α and γ-esters of MTX have been synthesized having 8, 12 and 16 Carbon chain length. These synthesized derivatives have been tested for DHFR affinity and inhibitory activity against CEM. It was found that all derivatives have less inhibitory activity than MTX and γ esters have more inhibitory activity than α esters. It was found that by increasing chain length there is an increase in cytotoxicity but decrease in DHFR affinity (Table 24).

Table 24: DHFR affinity and inhibitory activity against CEM of esters of MTX [41].

Stretched MTX derivatives having different numbers of (Gab) as a spacer between glutamate and MeAPA moiety have been synthesized. The DHFR affinity and inhibitory activity have been evaluated. DHFR inhibition was directly related to number of Gab spacers, but growth inhibitory potency lost slightly (Table 25).

Table 25: DHFR affinity and inhibitory activity of derivatives of MTX [42].

Dihydro-2H-1,4-benzothiazine and dihydro-2H-1,4- benzoxazine MTX derivatives have been synthesized and tested for anti-proliferative activity against hSC and hPBMC in vitro. In vivo activity checked against rat arthritis. 3c was found to be more potent than MTX (Table 26).

Table 26: In Vitro anti-proliferative activity against hSC and hPBMC of derivatives of MTX [43].

Various derivatives of MTX have been synthesized and few were studied for anti-proliferative activity against L1210 was tested. 9Aa and 9Ab showed higher activity than MTX (Table 27).

Table 27: Anti-proliferative activity against L1210 of MTX derivatives [44].

Derivatives of MTX have been synthesized by using a different number of Carbon chain length and N-halo acetylation. These derivatives have been tested for DHFR affinity and antiproliferative against L1210 and L1210/R81. N-bromoacetyl- L-ornithine found to be more potent than other synthesized activity (Table 28).

Table 28: Anti-proliferative activity against L1210 of Halo derivatives of MTX [45].

Aminoalkanephosphonic, aminophosphonoalkanoic and aminoalkanesulfonic MTX derivative have been synthesized instead of glutamate moiety. All derivatives have been tested for enzyme affinity against FPGS and inhibitory activity was tested against MTX, MTX resistant cells. The maximum number of CH2 for optimal activity was found to be two and less than this was detrimental. Removal of α- COOH was found to lose the anti-proliferative activity, and reason behind is α-COOH involved in binding activity (Table 29).

Table 29: Inhibitory activity of derivatives of MTX [46].

In this article synthesis approach were made to derivatives of methotrexate or aminopterin which are basically folic acid antagonists. The synthesis scheme involved protection, de-protection and various steps. Synthesis begins from potassium phthalimide and end product is p-[(2,4-Diamino-6-pteridinyl) methyl]amino]-benzoic acid which involves a number of intermediate [47].

Ortho and Meta isomers of AMT and MTX have been synthesized and were assayed for biological activities. General structures of these isomers are as follows. Besides these few derivatives were synthesized having CH2CH=CHCOOH instead of saturated side chain. These derivatives were being assayed for binding affinity with DHFR and FPGS from mouse liver. None of the derivative was found to be more potent than the parent drug [48].

Various methotrexate derivatives synthesized by replacement of glutamic acid with amines and amino acid esters. These derivatives were tested for antibacterial and antitumor activity against L1210 in mice. Many derivatives showed significant activity and some show better than MTX. ID₅₀ value of MTX is 0.002 (Table 30).

Table 30: Antitµmor activity against L1210 of amine and ester derivatives of MTX [49].

In this research work, glutamate moiety changed to amino acids having longer carbon chain. It was found that cytotoxicity increased by increasing carbon number from 2-5. Anticancer activity was tested on L1210 leukemia and results are comparable to parent drug (Table 31).

Table 31: Anticancer activity of amino acid derivatives of MTX [50].

γ–Phosphonates and γ–sulphonate derivatives of MTX have been synthesized and tested for DHFR inhibition and L1210 carcinoma. Results showed that γ–sulphonates are more active than γ– phosphonates and both are less active than the parent drug. but γ–phosphonates have more potency in MTX resistant line L1210/R81 (Table 32).

Table 32: DHFR inhibition of derivatives of MTX [51].

A novel drug delivery system was formed using peptide labile spacer attached to MTX and tuftsin which is peptide carrier. These drug delivery systems were tested for cell lines MonoMac 6. It was found that all conjugates trigger toxic effect greater than MTX [52].

In this article 3’ Halo, and 3’, 5’-dihalo derivatives have been synthesized and synthesis was done in several steps. All derivatives showed a good growth inhibitory effect against lymphosarcoma cell lines (Table 33).

Table 33: di-Halo derivatives of MTX [53].

In this article halogenated derivatives were synthesized, but after modification in pteridine ring (Table 34).

Table 34: Halo derivatives of MTX [54].

Derivatives have been synthesized by replacing glutamate moiety with histidine and other related groups. Biological assay data of a few were given yet various derivatives have been synthesized. These derivatives have significant inhibition activity against DHFR but less for FPGS (Table 35).

Table 35: Histidine Derivatives of MTX [55].

Conclusion

Changes in Structure of MTX were made to enhance efficacy and find a relation of structural changes and activity. Those derivatives, which are structurally related to MTX show good activity. Increase in carbon chain length between –COOH silibinin conjugate, SO₂H at gamma position, Aza derivatives, aminoalkanedioic acid derivatives, associated with antibodies, Anilides, dihydro-2H,1,4 benzothiazine, thiophene-2-carboxy group instead of benzyl show higher activity and proved to be more potent.

Substitution of alkyl group at 7-position, bisamides, alkylation at 8-position, lysyl derivative of gamma position, methyl group at benzylic carbon, removal of alpha COOH decreases activity as compared to MTX. Gamma substituted binds more effectively than alpha substituted so alpha COOH is more important in activity. Gamma isomers are more actives than alpha isomer. Diesters, monoamides showed varying degree of activity. Less than two CH₂ between COOH proved to be detrimental. So, new researchers can focus on those modifications which show better anticancer activity and can synthesize even better derivatives.

Conflict of Interest

The authors declare that they have no conflict of interests.

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