Journal of Medical Oncology and Therapeutics

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Review Article - Journal of Medical Oncology and Therapeutics (2020) Volume 5, Issue 1

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

Nemat A1, Iqbal M1*, Mehmood T2

1Department of Chemistry, School of Natural Sciences (SNS), National University of Sciences and Technology (NUST), Islamabad, Pakistan

2Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences-UVAS, Lahore, Pakistan

*Corresponding Author:
Iqbal M
Department of Chemistry
School of Natural Sciences (SNS)
National University of Sciences and Technology (NUST)
Islamabad, Pakistan
: +92 300 6005249
: [email protected]

Accepted date: February 11, 2020

Citation: Nemat A, Iqbal M, Mehmood T. A comprehensive review of synthesized derivatives of methotrexate in relation to their anticancer potential.J Med Oncl Ther. 2020;5(1):04-20.

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Methotrexate (MTX) is a chemotherapeutic agent with certain side effects. Efforts were made to synthesize analogues of MTX to enhance the activity and reduce side effects. Various derivatives were synthesized by structural modification of parent drug and mainly assayed for anticancer activity and binding affinity with dihydrofolatereductase (DHFR) and folylpolyglutamate synthetase (FPGS). In vitro study was carried out on cell lines CCRF. CEM, ATCC8, L. Casei, P388, L1210, L1210/R81, and H35. These synthesized derivatives have been assayed for human squamous cell carcinoma namely SSC25, SSC68, SSC78, SSC25/R1, SSC68/R1, and SSC78/R1. Some modification proved to enhance anticancer activity while others are detrimental. Some of derivatives were also tested for other biological activity like anti-malarial, anti-bacterial and anti-diabetic and show better activity. This article deals with a comprehensive overview of the synthesized derivatives by structural modifications and the impact of these modifications on its activity.


Methotrexate, Derivatives, Anticancer activity, Review.


Methotrexate is known as amethopterin which suppresses the immune system and used as chemotherapeutic agents. It was synthesized in 1947 and in 1956 it proved to cure as for metastatic cancer [1].


There is another analogue of folic acid and methotrexate known as aminopterin. It is also used in chemotherapy as it supresses the production of folic acid. It has comparable activity with methotrexate.


Methotrexate effects folic acid pathway [2], as it closely resembles folic acid, acting as antimetabolite and used in the treatment of various diseases like arthritis and cancer [3]. Although methotrexate has many applications but it has certain disadvantages, e.g. fast metabolism and low selectivity for tumor cell [4]. The mechanism of inhibition of cancer cells by MTX starts from inhibition of DNA and RNA synthesis, particularly in rapidly dividing cancerous cells thus acting as anticancer drug [5]. Dihydrofolatereductase (DHFR) is used to catalyse the dihydrofolate (DHF) to tetrahydrofolate (THF). THF is necessary for folate reduction and hence its needed for DNA synthesis [6]. Methotrexate is used to treat rheumatoid arthritis [7], psoriasis [8], cancer [9] and ectopic pregnancy [10]. Keeping in view all the uses of methotrexate it has certain disadvantages as it causes life threatening side effects on vital organs [11]. There are a number of derivatives synthesized by modification of MTX showing different activity and binding affinity.

Literature Review

Lysine and ornithine derivatives of MTX were synthesized and both derivatives have same binding affinity as MTX and lysine have the same potency as MTX. Reaction mechanism involved protection and de-protection of amino acid and deprotected derivative is eight times more potent. Lysyl derivative has the same potency in protected and de-protected form. Lysyl derivative was more potent than ornithine derivatives. Presence of Cbz group didn’t affect activity but removal improved activity 3 folds in ornithine. Inhibition and DHFR binding were tested in liver of chicken. Results are summarized in (Table 1).

Comp R1 R2 n I50 × 108Ma
1(MTX) H H 2 9
2 t-Bµ Cbz 3 310
3 t-Bµ Cbz 4 95
4 H Cbz 3 33
5 H Cbz 4 38
6 H H 3 25
7 H H 4 13
a=The enzyme concentration was 18.4 × 10-8 M

Table 1: Lysine and ornithine derivatives and their I50 valµes [12].


Various MTX derivatives have been synthesized having a different group by replacing γ-COOH and only a few were tested for anti-proliferative activity. These derivatives have been tested against DHFR, FPGS, L1210 and L1210/R81. Ornithine derivative was found to be more potent against DHFR and FPGS (Table 2).

Comp R n X DHFR IC50 µM FPGS Ki µM L1210 Ki µM L1210/R81 IC50 µM
MTX Me 2 COOH 0.035 0.002 220
AMT H 2 COOH 0.035  - 0.002 84
2 Me 4 NH2 0.065  - 0.4 220
3 Me 3 NH2 0.16 20.4 1.3 86
4 Me 2 NH2 0.12  - 2.42 290
5 Me 1 NH2 0.18  - 0.44 405
6 H 3 NH2 0.072 0.15 1.3 32

Table 2: Analogµes of MTX and their IC50 valµes [13].


Various Derivatives of MTX have been synthesized by modification of γ-COOH like MTX-N (idoacetyl) L-lysine, another with Cbz, and NH2 Group. Bonding Affinity for DHFR tested against L-Casei and L1210. N (idoacetyl) L-lysine shows the best activity for L. Casei than MTX, but less activity for L1210. All other derivatives exhibit less activity and possible reason is the presence of charged groups at the end of chain lessens binding affinity results mentioned in (Table 3).

Comp R L. casei (ID 50)b L1210 (ID 50)b
MTX image 6.2 2.7
1 image 4.5 31
4 image 14 11
5 image 23 11
b=Competitive [3H] MTX binding assay affinity for DHFR

Table 3: Analogμes of MTX and their binding affinity [14].


Lysyl derivatives of methotrexate have been synthesized by reaction of γ-COOH with the amine group of lysine. These derivatives vary due to the number of Lysyl groups attached. It was found that synthesized derivatives show less affinity to DHFR from 2-3 folds as well as decrease in cytotoxic activity by 30-120 folds. Minimum activity was shown by derivative with three Lysyl groups; moreover, an increase in the number of Lysyl derivative does not affect activity. Activity was tested against L1210 and H35 cell line and these showed same result (Table 4).

Comp L1210 DHFR IC50 nM L1210 cells IC50 µM H35 Cells IC50 µM
MTX 50 0.024 0.01
1 87 0.76 0.4
2 86 1.8 0.5
3 140 2.9 0.56

Table 4: Dihydrofolate redµctase inhibitory activity and cytotoxicity of lysyl derivatives of MTX [15].


3 [MTX (γ-e)-(Lys)3],R= NH2CH[(CH2)4NH] CONHCH[(CH2)4NH2]CONHCH[(CH2)4 NH2]


A fluorescent derivative of MTX has been synthesized by the lysine derivative of MTX to dansyl analogue and this analogue show higher activity against DHFR of L. Casei [16].

Protected dilysine and trislysine analogues of methotrexate have been synthesized. Two schemes were used to synthesize lysine derivatives. In Scheme (I) the substituted L-glutamyl-L-lysine intermediate 6 was formed stepwise from dilysine, while in the scheme (II) synthesis was done from two N-terminal lysine joined in 6 steps [17].

Shams A. Nadhum et al 2015 synthesized a conjugate of silibinin and MTX in order to enhance efficacy than parent drugs and to minimize their side effects. Scheme of reaction consists of 6 steps in which firstly imine derivative formed. This imine derivative is converted into imine-MTX-cysteine and after 2-3 steps imine conjugate of MTX and silibinin. Imine conjugate hydrolyzed to get conjugate of MTX and silibinin. Anticancer activity was tested against HEP-2 cell lines from human epidermoid larynx carcinoma for 24 hr and 48 hr. General trend shows compound 5 and 6 have a high inhibition rate on high concentration dose and decreased at low concentration. Silibinin shows 33.1 % inhibition MTX-silibinin conjugate shows 41.2% [17].

Andre et al 1984 synthesized a polygultamate derivative bind as well as MTX and leave mammalian cell more slowly. This analogue contained gama-SO3H instead of COOH and was synthesized in 78% yield. Binding affinity is measure of interaction by which a molecule (protein) binds with drug and in turn potency of drug can be found which actually drugs activity, for anticancer activity cell lines of humane lymphoblastic leukemia cells were used for measuring binding and anticancer efficacy, MTX used as positive control. The binding affinity of analogue was same as of MTX. Three experiments were conducted using same quantity and different doses and it revealed that by increasing frequency increase in molar potency of the drug shown in (Table 5) [18].

Comp X L1210DHFR IC50 (nM) L. Casei DHFR IC50 (nM) L1210 cells IC50 (nM)
MTX CO2H 1 17 0.3
mAPA-HCysA SO2H 0.95 10 0.01

Table 5: Dihydrofolate redµctase (DHFR) binding and cytotoxicity of glµtamate derivatives of MTX [19].


γ-Hydrazide, γ-n-butylamide, γ-benzylamide and γ-tert-butyl ester analogues of MTX have been synthesized. The binding affinity of synthesized derivatives was tested for DHFR from L1210 mouse leukemia cells and Lactobacillus casei. It has been found that γ-terminal region of MTX is site for modifications. Binding affinity has been tested to DHFR of L. Casei and L1210 by spectrophotometric assay and radio-ligand assay respectively. It was found that gamma substituted compound binds effectively than α-Substituted. γ-tert-butyl ester shows 1.9 times higher binding affinity than MTX. γ-tert-butyl ester and γ hydrazide showed same affinity as MTX. γ-n-butyl amide showed slightly same activity as MTX. γ-benzyl amide showed less activity (Table 6).

Comp R1 R2 L. Casei ID50 µm L. Casei (Binding affinity) ID50 µm L1210 (Binding Assay) ID50 µm L1210 ID50 µm
1 H O-t-Bµ 0.025 0.012 0.0029 0.0029
2 H NHNH2 0.0021 0.013 0.012 0.012
3 H NH-n-Bµ 0.053 0.036 0.0033 0.0033
4 H NHCH2C6H5 0.055 0.022 0.003 0.003
6 CH3 -t-Bµ 0.33 3.9 0.011 0.011
7 -t-Bµ CH3 0.2 2.2 0.019 0.019
8 -t-Bµ NHNH2 0.17 2.2 ND ND
9 -t-Bµ H 0.036 0.21 0.035 0.035
10 CH2Ph NH-nC4H9 ND 0.25 ND ND

Table 6: Binding affinity of side chain modified MTX derivatives for analogµes of methotrexate [20].


Several MTX derivatives have been synthesized by modification of the glutamyl moiety with peptide side chain. Nine different amino acids were used for modification of the side chain. Various intermediate peptides were also separated. Biological activity was tested for L1210 leukemia and W25 carcinoma in mouse and rat respectively. It has been found that α-COOH is more important for activity than gamma(γ)-COOH. All derivatives were inactive, which show glutamyl moiety is necessary for activity. If α-COOH is present, an increase in length of the alkyl chain restores activity (Table 7).

Comp           R L1210 Dose (mg/kg) × no. of administration L1210 %ILS 
1a Glycine 23 × 7 0
1b DL-alanine 33 × 6 0
1c Β-alanine 25 × 6 19
1d Sarcosine 50 × 6 0
1e DL-α-aminobµtyric acid 78 × 9 16
1f γ- aminobµtyric acid 10 × 8 0
1g DL-Valine 20 × 7 0
1h L-leµcine 90 × 9 24
1i L-phenylalanine 45 × 10 14

Table 7: Biological data of analogµes of methotrexate [21].


Different derivatives of MTX have been synthesized by alkylation of the side chain. DHFR affinity and antiproliferating activity was tested against L1210 in mice. It was found that those derivatives have a high inhibition rate, which was structurally closed to MTX. Minute changes in Structure such as derivatization of pyridine ring, chlorination gave good results. But the substitution of aliphatic group decreased the activity greatly. Introducing the carbon around the benzene ring decreased binding affinity, but between –COOH enhances the binding affinity, but none of the synthesized derivatives show significant anti-proliferative activity (Tables 8a-8e).

No      R Inhibition of DHFR I50 µm Cytotoxicity to KB cells ED50 µg/µL Inhibition of leµkemia cells L1210
1 -NMe- (MTX) 0.026 0.004 1.5
2 CH(CO2H)(CH2)2CO2H 0.026 0.003 0.67
26 CH(CO2H)(CH2)4CO2H 0.013 0.025 20
27 (CH2)3CO2H 0.38 >100 25
28 CH2CO2H 1.1 88 80

Table 8a: Biological data for derivatives of MTX [22].

No      R Inhibition of DHFR I50 µm Cytotoxicity to KB cells ED50 µg/µL Inhibition of leµkemia cells L1210
29 CH2COGlµ 0.38 41 500
30 CH2COAsp 1 >100 200
31 (CH2)2COGlµ 0.46 37 500
32 SO2Glµ 1.1 45 100

Table 8b: Biological data for derivatives of MTX [22].

No      R Inhibition of DHFR I50 µm Cytotoxicity to KB cells ED50 µg/µL Inhibition of leµkemia cells L1210
33 Et   0.050 0.022
34 H   0.030 0.006
35 Me   20 >100

Table 8c: Biological data for derivatives of MTX [22].

No      R Inhibition of DHFR I50 µm Cytotoxicity to KB cells ED50 µg/µL Inhibition of leµkemia cells L1210
2 -NH- 0.026 0.003 0.67
1 -NMe- 0.026 0.004 1.5
36 -NEt- 0.045 0.005 10
37 -NBn- 14 >100 80
38 -NPhe- 0.28 26 80
39 -NMeCH2- 0.75 0.043 50
40 -O- 0.51 3.5 1.3

Table 8d: Biological data for derivatives of MTX [22].

No      R Inhibition of DHFR I50 µm Cytotoxicity to KB cells ED50 µg/µL Inhibition of leµkemia cells L1210
41 H 0.63 0.05 100
42 Br 2.5 76 200
43 NO2 38 >100 200

Table 8e: Biological data for derivatives of MTX [22].


In a study [23] α and γ-monoesters of MTX were formed and anticancer activity checked against lymphoblastic leukemia (CCRF-CEM) cell lines. γ–monoesters were inhibitory than α-isomer and difference was about 10 folds, but with increase in chain length this difference reduced 2 to 2.5 folds. Monoesters showed a less inhibitory effect than diesters. Diethyl ester was 516 times more activity than α-monoethyl ester and 48 times than γ-monoester. Dibutyl esters show the same result as diethyl esters (Table 9).

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

Table 9: In vitro biological data for ester derivatives of MTX [23].

In this work numbers of alkyl esters derivatives of MTX were prepared by the direct esterification method. But with 2º alcohols or 1º alcohols, reaction at room temperature gives a poor yield. So, reaction mixture heated at 55-60ºC. In vitro growth inhibitory activity was also studied with two murine leukemia L1210 in hybrid mice and p1534 leukemia in mice. Dibutyl ester showed a 25% increase in life span. Binding affinity was tested with DHFR of Lactobacillus casei ATCC 7469 and it shows 1000 times less tightly than MTX (Table 10).

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).

R X S. faeciµm L1210-FR8 L. case P388 CCRF-CEM
ATCC8043 ID50 ID50 mol/l ID50 mol/l ID50 mol/l ID50 mol/l
H(MTX) H 0.002 1.5×10-9 3×10-9 0.01 0.018
CH3 H 0.17 7×10-6 1× 10-5 0.1 0.1
H Cl 0.01 1×10-9 3×10-9 0.007 0.009
CH3 Cl 1 4×10-6 9×10-6 0.1 0.1

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).

Comp            R CCRF-CEM RBL
ID50 ID50
MTX H 0.003 0.003
1a NH2  -  -
1b NH-n-C3H7 7  -
1c NH-n-C4H9  -
1d NH-s-C4H9  -  -
1e NH-s-C6H13 10
1f NH-c-C6H11 1 0.22
1g c-NC4H8  -  -
1h NHCH2C6H5 7.6 2.5
1i NH CH2CH2C6H5  -
1j NHCH2CH2C6H3-3,4 (OMe)2  -  -
1k NHCH2CH2CH2OH  -  -
1l NHCH2CH2NMe2  -
1m NHCH2CH2CH2NMe2  -

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).

Comp   R L. casei (×10-11)a DHFR (×10-11)a Thymidylate Synthetase (X 10-6)a CCRF-CEM ID50 µg/mL
 - MTX 4 0.3 >100 0.018
7 H 2 2 1 0.021
8 Methyl 10 14 4 0.23
9 Ethyl 35 90 20 0.27
10 n-Bµtyl 120 38 19 0.23
11 n-hexyl 27 180 43 0.58
12 Cyclohexylmethyl 350 87 17 0.14
13 Benzyl 35 74 8 0.78
14 3,4 dichlorobenzyl 1940 360 13 >1.0
15 1-Naphthylmethyl 47 450 16 >1.0
a=Molar conc. for 50% inhibition

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).

Comp Hµman Lymphoblastic Leµkemia Cells (CCRF-CEM)ID50 µg/mL
MTX 0.003
1 0.88
2 10+
3 (LLL) 3.8
3(DLL) 9
4 1.8
5 0.85

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.

No. Comp L. casei
M X (10-8)
Thymidylate synthetase
M X (10-6)
MTX - 0.01 0.3 100
5 image 6 60 -
6 image 6 30 -
7 image 150 5 190
8 image 8 8 250
10 image 104 400 -

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).

Comp       R        X n L 1210 mg/kg %ILS
Dose (mg/kg) × no. of admin
1a H Gly 1 40×10 69
1b H Gly 2 33×6 14
1c CH3 Gly 1 100×8 40
1d CH3 Gly 2 100×8 0
1e CH3 DL-ala 2 50×6 19
1f CH3 Sar 2 100×6 0
1g CH3 L-Leµ 2 50×8 0
1h CH3 L-phe 1 100×7 0
1i CH3 L-phe 2 50×10 25

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).

Comp R2 R4 R5 DHFR L1210 L1210/R81
IC50 µm IC50 µm IC50 µm
MTX Me H H 0.02 0.01-0.03 220
3a H H t-BµNH 0.044 0.12 22
3b H H (1-admantyl)NH 0.1 0.68 25
3c H H C6H5CH2NH 0.087 0.005 17
3d H H 3,4-Cl2 C6H3CH2NH 0.13 0.046 32
3e H H 2,6-Cl2 C6H3 CH2NH 0.08 0.0037 61
3f H H C6H5NH 0.06 0.0035 10
3g H H 3,4-(OCH2O) C6H3NH 0.045 0.0032 28
3h Me H 3,4-(OCH2O) C6H3NH 0.042 nd 25
3i H H 3,4-(OH)2 C6H3NH 0.057 0.037 23
3j Me H 3,4-(OH)2 C6H3NH 0.031 0.03 38
AMT H H H 0.02 0.003 84

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).

Comp R1 n R2 DHFR CEM L1210 IC50 µM L1210/R81 IC50 µM
IC50 µm IC50 µM
MTX Me 2 H 0.025 0.032 0.0046 197
AMT H 2 H 0.025 0.001 0.002 84
2 Me 6 H 0.023 0.15 0.0012 68
3 Me 7 H 0.032 0.062 0.0042 73
4 Me 8 H 0.029 0.056 0.0031 78
5 Me 9 H 0.034 0.016 0.0071 58
6 Me 10 H 0.026 0.64 0.026 56
7 H 6 H 0.54 nd 0.02 >218
8 H 9 H 0.081 nd 0.00065 110
9 H 10 H 0.067 nd 0.0011 215

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).

Comp R3 R2 R1
1(γ-tBMTX) H t-Bµ Me
2(γ-tBAMT) H t-Bµ H

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).

Hµman Sqµamoµs Cell Carcinoma
Comp CEM L1210 L1210/R71 L1210/R81 SSC25 IC50 µm SSC68 IC50 µm SSC78 IC50 µm SSC25/R1 IC50 µm SSC68/R1 IC50 µm SSC78/R1 IC50 µm
IC50 µm IC50 µm IC50 µm IC50 µm
MTX 0.62 0.056 40 25 0.4 0.37 0.48 0.78 1.4 1.4
AMT 0.45 0.023 3.5 6.5 0.066 0.19 0.08 1.8 3.5 0.43
1(γ-tBMTX) 0.032 0.002 19 220 0.014 0.032 0.013 0.15 0.25 0.071
2(γ-tBAMT) 0.001 0.002 7.9 84 0.0016 0.0037 0.0025 0.043 0.29 0.014

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

Comp DHFR mg/mla Thyimdylate synthetase mg/ml S. faecalis mg/ml L. casei mg/ml
MTX 9 45000 0.15 0.01
-3 16 1125 0.011 0.008
-2 46 2250 0.047 0.056
a=These are the concentration for 50% inhibition.

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).

Comp R X Y DHFR, L1210 W1-L2 IC50, nM
IC50, nM IC50, nM
1 H (CH2)3NHCO o-COOH 52 0.75 48
2 H (CH2)2CONH m-COOH 32 25 6.1
3 Me (CH2)2CONH m-COOH 35 1.6 2.2
4 Me (CH2)2CONH m-(BOH2) 30 0.7 nd

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].

Comp S. faciµm ID 50, µg/ml
6a 0.017
6b 0.37
6c 0.002
6d 0.003
6e 0.002
6f 0.001
7a 1
7d 1

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).

Comp R CEM ID50 µM L1210 ID50 µM
- MTX 0.003 0.01
1 image >10 3.4
2 n-C5H10N - >10
3 4-ClC6H4CH2N >10 6.6
4 4-MeOC6H4CH2N >10 8.2
5 C6H5CH2N(CH3) 6.4 9.4
6 (C6H5CH2 )2N 7.6 0.69
7 C6H5 CH (CH3)NH >10 >10
8 C6H5NH 3.3 0.41
9 H2 NNH 7.5 0.95

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).

Comp R1 R2 DHFR, IC50, µM CEM, IC50, µM
MTX H H 0.0033 0.025
1 (α) n-C8H7 H 0.36 3
2(γ) H n-C8H7 0.0054 0.92
3(α) n-C12H25 H 0.44 2.1
4(γ) H n-C12H25 0.034 0.37
5(α) n-C16H33 H 1.2 0.25
6(γ) H n-C16H33 0.037 0.11

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).

Comp n L1210 DHFR IC50 µm L1210 cells IC50 µm L. casei TS IC50 µm
mAPA-Gab1-Glµ (1a) 1 0.082 0.82 0.53
mAPA-Gab2-Glµ (1b) 2 0.09 1.3 5.6
mAPA-Gab3-Glµ (1c) 3 0.31 4.4 29
mAPA-Gab4-Glµ (1d) 4 0.54 7.7 >100
mAPA-Gab5-Glµ (1e) 5 0.84 12 >100
mAPAGlµ (MTX)  - 0.035 0.5 0.02

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).

Comp A n hSC, IC50 nM hPBMC, IC50 nM
MTX  -  - 1 1
3a O 1 0.52 1
3b O 2 1.4 1.9
3c S 1 0.3 0.5
3d S 2 0.77 1.3
MTX-33 C 1 3.8 0.83

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).

Comp R1 Z L1210 IC50 nM Chang Liver IC50 nM
MTX H - 20 14
9Aa H 8.1 3.1
9Ab Et image 3.7 1.6
9Ba H image 88 36
9Bb Et image 268 35

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).

Comp n X DHFR L1210 Cells IC50, µM L1210/R81
IC50, nM
MTX  -  - 25 0.005 200
1 4 I  -
2 4 Br 72 0.096 240
3 4 Cl 32 0.033 93
4 3 Br 46 0.126 155
5 3 Cl 32 0.062 105

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).

Comp R X n Y % inhibition
3 Me COOH 2 SO2H 59
4 H COOH 2 SO2H 77
5 Me COOH 2 PO(OH)2 53
6 H COOH 2 PO(OH)2 100
7 H COOH 1 PO(OH)2 22
8 H COOH 3 PO(OH)2 52
9 H COOH 4 PO(OH)2 44
10 Me H 0 SO2H 19
11 Me H 1 SO2H 22
12 Me H 2 SO2H 18
13 Me H 3 SO2H 26
14 Me H 1 PO(OH)2 22
15 Me H 2 PO(OH)2 16
16 Me H 3 PO(OH)(OEt) 9

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).

Comp R ID50µg/ml
2 -oc (=O)OCH2 CH(CH3)2 0.0001
3 Ethyl 4 - amino bµtyrate 0.003
4 Methyl DL-2-amino-bµtyrate 0.02
5 Ethyl L -1eµcinate 0.003
6 Methyl DL-norleµcinate 0.010
7 Ethyl L- methihioninate 0.001
8 Ethyl L-tryptophanate 0.006
9 Methyl L-tyrosinate 0.010
10 Methyl c-carbobenzyloxy-L - lysinate 0.035
11 Dimethyl DL-asparate 0.005
12 Methyl L-prolinate 0.030
13 C yclohexanamine 0.002
14 Morpholine 0.018
15 image 0.350
16 image 0.010
17 image 0.005
18 image 0.010
19 -N3 (azide) 0.005

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).

Comp n L1210 Binding assay ID50, nM L1210spectrophoto-metric assay ID50, nM L1210/R71 ID50, nM
MTX(1) 2 11 9 32
2 3 12 6.7 39
3 4 18 6.9 39
4 5  - 35

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).

Comp R X DHFR inhibition L1210 IC50 µM
IC50 µM
1 MTX Me CO2H 0.5 0.012
2 AMP H CO2H 0.5 0.0031
3 Me SO3H 0.55 0.3
4 H SO3H 0.62 0.037
5 Me CO2PO3H2  -
6 H CO2PO3H2  -  -
7 Me PO3H2 1.34 0.19
8 H PO3H2 1.26 0.035

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).

Comp X X'
V Br H
VI Cl Cl

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


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

Comp R R’ X X’
V NH2 CH3 Cl Cl

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).

Comp R L1210
IC50, µM
IC50, µM
IC50, µM
MTX -COOH 0.007 0.073 3.2
4 image 0.091 1.3 >200
5 image 0.15 0.37 >200

Table 35: Histidine Derivatives of MTX [55].



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.