Discovery of a Novel Oral Antithrombotic Drug Apixaban

Discovery of a Novel Oral Antithrombotic Drug Apixaban Shuangyu Ma Background Thrombosis, which is generally defined as the formation of blood clot inside blood vessels, is a major health issue in western world. Thrombosis associated disease, such as ischemic stroke and high blood pressure, remain to be the leading causes of morbidity and mortality each year in developed countries [1]. To make things worse, thrombosis is also known as a chronic disease with episodic recurrence [2]. According the American Heart Association, thirty percent of the patients diagnosed with venous thromboembolism develop recurrence within the next 10 years [2]. The balance between thrombosis formation and bleeding is prudently regulated by the blood coagulation cascade, one of the most delicate systems in human body (figure 1, [3]). The signal transmission and amplification along the cascade was conducted by a series of trypsin-like serine proteases with high specificity, often referred as zymogens [4]. The zymogens are cleaved by the previous coagulation factor to become activated proteases, which can thereby cleave the next zymogen along the cascade and achieve the signal amplification. Blood coagulation process can be triggered by intrinsic pathway or extrinsic pathway; both are followed by the activation of common pathway. The prothrombin is then cleaved by activated factor X to generate thrombin, eventually lead to the formation of stable fibrin clot. Fig 1. Blood coagulation cascade: (figure extracted from Haematology (2nd edition) by C. J. Pallister and M. S. Watson [4]) Until now, the most well-known oral anticoagulant is warfarin, which is the current standard therapy for clinic thrombosis treatment and prevention. Despite its widely application, warfarin has a huge draw back on its dosing issue. Many of the coagulation factors, including factors II, VII, IX and X are Vitamin K dependent; the ÃŽ ³-carboxylation by Vitamin K is essential for their biological activity. Targeting on vitamin-K conversion cycle, warfarin produces an anticoagulation effect by reducing activities of the four vitamin K-dependent coagulation factors. However, the responses to warfarin can be interfered dramatically by genetic and environment factors, such as the common mutation on cytochrome P450 encoding gene, disease states variation, drug-drug interaction or even diet change [5]. Hence, warfarin treated patients need blood test regularly (often once a week or even worse) to determine a safe dosage case by case. Despite handling with great cautious, the anticoagulant-asso ciated intracerebral hemorrhage keeps increasing [2]. Thus, many efforts have been put on developing a novel antithrombotic drug with low risk and high potency. From lead to drug: the development of apixaban The drug Apixaban, approved by FDA in 2012 for thromboembolism treatment and prevention, is developed by Pfizer and Bristol-Myers Squibb to serve as an optimized risk-benefit oral anticoagulant. The mechanism of apixaban is entirely different with warfarin it specifically targets on activated coagulation factor ten (FXa), rather than vitamin K. Several clinical trial has proved its higher efficacy compared to asparin and lower risk compared to warfarin treatment [6]. Given the central position in the blood coagulation cascade, FXa evolved as an attractive drug target for developing anticoagulants. The DuPont Merck Pharmaceutical Company first launched a high throughput screening on FXa inhibitor in 1997 [7]. Because of the substrate similarity between FXa and the GPIIb/IIa receptor, they screened the compound library which was originally designed to be GPIIb/IIa receptor antagonist. A hit was found with low micromolar FXa affinity (coumpound 1, Ki= 38.5 ÃŽ ¼M). However, most of the reported FXa inhibitor in literature at that time posse the bisamindinoaryl structure, such as 2 and 3 (figure 2a). Inspired by these compounds, they modified the hit to be bisamidines and obtained the first list of leads (including compound 4), as shown in figure 2b. Aiming at a higher potency, molecular modeling studies were conducted to further optimize the lead compound, which resulted in the removal of methylene unit between the isoxazoline ring and the amidine carbonyl together with the à ¯Ã¢â‚¬Å¾Ã†â€™ substitution to the carbonyl. After the comparison of different substitution groups, they obtained compound 5 with Ki of 94 nM (Figure 3) [7]. Fig 2 a. The structure of hit compound and several known FXa inhibitor b. The bisamidine lead compounds (figure extracted from [7]). Fig 3. Effects of the amidine group position and à ¯Ã¢â‚¬Å¾Ã†â€™ substitution (figure extracted from [7]) However, as an oral drug candidate, this dibasic lead compound might not have good oral absorption due to its hydrophilicity. Therefore, the next goal is to reduce its basicity by replacing one of the amidine groups with a neutral species. The molecular modeling of compound 5 bound to FXa indicated that the p-amidine group fits in an hydrophobic aryl-binding pocket of FXa, which is consist of three aromatic residues (Trp215, Tyr99 and Phe174, figure 4a) [8]. Therefore, it is possible to replace the p-amidine with aromatic functional groups without largely interrupt the affinity. They employed a biaryl group and obtained compound 6 with only 2-fold decrease in potency. Further investigation on different substitution at the terminal phenyl ring resulted in large enhancement on potency, such as compound 7 (Ki = 6.3 nM). (Figure 4b) Fig 4 a. Effects of substitutions on terminal phenyl ring (figures extracted from [8]). b. Chemical structure of compound 6 and 7 Nevertheless, another concern emerged to be the metabolic instability, as compound 7 bears an ester side chain, which is labile to esterase once it gets inside human body. Since the corresponding acid metabolite is three-fold less potent than the original ester [9], replacing the ester side chain appears becomes the imperative next step. Again, a list of compound 7 analogs bearing different side chains were synthesized and tested. Among all the functional groups, tetrazole substitution was found to be most potent, and compound 8 (Ki= 0.52 nM) was developed with additional modification on the biaryl ring motif (figure 5a). Variation on the position of amide moiety attachment and isoxazoline aromatization resulted in compound 9 (Ki= 0.15 nM, figure 5b) [10], while further investigation on a series of five-membered heterocyclic ring gives compound 10 with equal potency (Ki= 0.15 nM, figure 5c) and lower basicity [11] . Fig 5. Chemical structure of compound 8, 9 and 10 Despite the high potency and selectivity, compound 10 is still far from perfect due to potential mutagenicity of the biarylanilines motif [12]. Therefore, the cleavage of amide bond must be prevented by either ligating or eliminating the nitrogen (figure 6). Compound 11 was developed to have the optimized pyrazole with an even higher FXa affinity (Ki = 0.03 nM). After the scaffold was settled, more compound 11 analogs with various substitution groups and terminal aromatic rings were synthesized and compared to afford the final drug candidate as compound 12 (apixaban, Ki= 0.08, figure 7a) [1]. The optimized drug selectivity towards FXa was shown in figure 7b. The therapeutic index of the final drug compared to warfarin was demonstrated in rabbit thrombosis models and shown figure 7c) [6]. The drug candidate was sent for preclinical studies followed with several clinic trials, and eventually approved by FDA for venous thromboembolism prevention after hip/knee replacement and artrial fibrillation treatment. Another clinical trial for acute venous thromboembolism is still in progress. Fig 6. Compound 11 bearing the optimized pyrazole. Fig 7 a. Apixaban (compound 12) b. In vitro Ki values of apixaban for FXa and other human enzymes with structural or functional similarities (figure extracted from [6]). c. Therapeutic index of apixaban and warfarin in rabbit models (figure extracted from [6]). Conclusion Comparing to the initial lead compound, the eventual drug changed dramatically in chemical structure, while its FXa inhibiting potency was enhanced by 107. Tracing back to each modification step, the comprehensive consideration in drug potency, selectivity, absorption, metabolism and toxicity is truly impressive and reveals the tremendous efforts embedded in a single drug. It is worth noting that molecular modeling with FXa structure played a critical role during the rational drug modification process. The discovery of apixaban also demonstrated the importance of defining new drug target in modern drug development. The success of apixaban is largely attributed to the better drug target FXa. However, the clinical trial of apixaban on acute coronary syndrome was discontinued due to increased bleeding events [6], which suggests that other anticoagulants are still in demand for treating various type of thrombosis. Reference [1] Pinto, D. J.; Orwat, M. J.; Koch, S.; Rossi, K. A.; Alexander, R. S.; Smallwood, A.; Wong, P. C.; Rendina, A. R.; Luettgen, J. M.; Knabb, R. M.; He, K.; Xin, B.; Wexler, R. R; Lam, P. Y. Discovery of 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl) phenyl)-4, 5, 6, 7-tetrahydro-1 H-pyrazolo [3, 4-c] pyridine-3-carboxamide (Apixaban, BMS-562247), a highly potent, selective, efficacious, and orally bioavailable inhibitor of blood coagulation factor Xa.Journal of medicinal chemistry. 2007,50, 5339-5356. [2] Go, A. S., Mozaffarian, D., Roger, V. L., Benjamin, E. J., Berry, J. D., Borden, W. B., Turner, M. B et al. Heart Disease and Stroke Statistics: 2013 Update: A Report From the American Heart Association. Circulation. 2013, 127, 143-146. [3] Pallister, C. J.; Watson, M. S. Haematology, 2nd ed. Scion Publishing: Banbury, 2010; pp 336–347. [4] Krishnaswamy, S. Exositeà ¢Ã¢â€š ¬Ã‚ driven substrate specificity and function in coagulation. Journal of thrombosis and haemostasis. 2005, 3, 54-67. [5] Hirsh, J., Fuster, V., Ansell, J., Halperin, J. L. Foundation guide to warfarin therapy. Journal of the American College of Cardiology. 2003, 41, 1633-1652. [6] Wong, P. C.; Pinto, D. J.; Zhang, D. Preclinical discovery of apixaban, a direct and orally bioavailable factor Xa inhibitor.Journal of thrombosis and thrombolysis. 2011,31, 478-492. [7] Quan, M. L.; Pruitt, J. R.; Ellis, C. D.; Liauw, A. Y.; Galemmo, R. A., Jr.; Stouten, P. F. W.; Wityak, J.; Knabb, R. M.; Thoolen, M. J.; Wong, P. C.; Wexler, R. R. Bisbenzamidine isoxazoline derivatives as factor Xa inhibitors.Bioorganic Medicinal Chemistry Letters. 1997, 7, 2813-2818. [8] Quan, M. L.; Liauw, A. Y.; Ellis, C. D.; Pruitt, J. R.; Carini, D. J.; Bostrom, L. L.; Huang, P. P.; Harrison, K.; Knabb, R. M.; Thoolen, M. J.; Wong, P. C.; Wexler, R. R. Design and Synthesis of Isoxazoline Derivatives as Factor Xa Inhibitors 1. Journal of medicinal chemistry. 1999,42, 2752-2759. [9] Quan, M. L.; Ellis, C. D.; Liauw, A. Y.; Alexander, R. S.; Knabb, R. M.; Lam, G.; Wright, M. R.; Wong, P. C.; Wexler, R. R. Design and Synthesis of Isoxazoline Derivatives as Factor Xa Inhibitors 2. Journal of medicinal chemistry. 1999,42, 2760-2773. [10] Pruitt, J. R., Pinto, D. J., Estrella, M. J., Bostrom, L. L., Knabb, R. M., Wong, P. C. Wright, M. R., Wexler, R. R. Isoxazolines and isoxazoles as factor Xa inhibitors.Bioorganic medicinal chemistry letters,2000, 10, 685-689. [11] Pinto, D. J.; Orwat, M. J.; Wang, S.; Fevig, J. M.; Quan, M. L.; Amparo, E.; Cacciola, J.; Rossi, K. A.; Alexander, R. S.; Smallwood, A. M.; Luettgen, J. M.; Liang, L.; Aungst, B. J.; Wright, M. R.; Knabb, R. M.; Wong, P. C.; Wexler, R. R.; Lam, P. Y. Discovery of 1-[3-(Aminomethyl) phenyl]-N-[3-fluoro-2-(methylsulfonyl)-[1, 1-biphenyl]-4-yl]-3-(trifluoromethyl) -1H-pyrazole-5- carboxamide (DPC423), a Highly Potent, Selective, and Orally Bioavailable Inhibitor of Blood Coagulation Factor Xa 1.Journal of medicinal chemistry. 2001, 44, 566-578. [12] Pinto, D. J.; Orwat, M. J.; Quan, M. L.; Han, Q.; Galemmo, R. A., Jr.; Amparo, E.; Wwllsg, B.; Ellisj, C.; Hek, M. Y.; Alexanderf, R. S.; Knabbb, R. M.; Mersingerg, L.; Kettnera, C.; Baih, S.; Hed, K. Wexlera, R. R.; Lam, P. 1-[3-Aminobenzisoxazol-5†²-yl]-3-trifluoromethyl -6-[2†²-(3-(R) -hydroxy-N-pyrrolidinyl) methyl-[1, 1†²]-biphen-4-yl]-1, 4, 5, 6-tetrahydropyrazolo-[3, 4-c]-pyridin -7-one (BMS-740808) a highly potent, selective, efficacious, and orally bioavailable inhibitor of blood coagulation factor Xa.Bioorganic medicinal chemistry letters. 2006,16, 4141-4147.

Earthquake in Pakistan Essay

Earthquake activity in Pakistan is mainly concentrated in the north and western sections of the country, along the boundary of the Indian plate and the Iranian and Afghan micro-plates. The Chaman Fault runs along Pakistan’s western frontier with Afghanistan from Kalat, in the northern Makran range, past Quetta and then on to Kabul, Afghanistan. A fault also runs along the Makran coast and is believed to be of the same nature as the West Coast fault along the coast of Maharashtra, India. An active subduction zone exists off the Makran coast. The great 1945 earthquake was centred in this region. This zone forms the boundary between the Arabian and the Iranian micro-plate, where the former subducts or dives beneath the latter. Thrust zones run along the Kirthar, Sulaiman and Salt ranges. There are four faults in and around Karachi and other parts of deltaic Indus, and Makran coast. The first is the Allah Bund fault that passes through Shahbundar, Jah, Pakistan Steel Mills, and runs through eastern parts of the city and ends near Cape Monz. This fault, in fact, has caused extensive damage in the past many centuries in the deltaic areas. The destruction of Bhanbhor in the 13th century and damage to Shahbundar in 1896 were caused by this fault. The other one emanates from the Rann of Kutchh. The third one is the Pubb fault which ends into Arabian sea near Makran coast and the last one is located in the lower Dadu district near Surajani and falls in the vicinity of Karachi. Tsunamis or tidal waves have also affected the coast of Pakistan. The worst case was in 1945 when an earthquake of magnitude 7. 9 struck the Makran coast, waves as high as 12 meters were reported.

Lord Byron

In `Excerpt from Don Juan`, answer the following: Select 3 stanzas from Canto I that you can explain. Do not give plot summary, so carefully choose a stanza that lends itself to analysis or some research. Write about three to four sentences. In Subject Line, identify your stanza, e.g., 44 (Canto I, Stanza 44). Stanza 5 Brave men were living before Agamemnon And since, exceeding various and Sage, A good deal like him too, though quite the same none;  Ã‚   35 But then they shone not on the Poet’s page, And so have been forgotten: – I condemn none, But can’t find any in present age Fit for my poem (that is, for my New One) So, as I said, I’ll take my friend Don Juan. –  Ã‚  Ã‚   40 In analyzing Stanza 5, it’s interesting to read as we wonder who else Byron may have considered in this poem he sat down to write. Was he considering other brave men, poets, heroes before finalizing it with Don Juan? Who were the others â€Å"a great deal like him† (Canto 1, Stanza 5, Line 35). This stanza can be more interesting to readers who examine the history of what was viewed as the scandalous controversial nature of his writing of â€Å"Don Juan† and what concessions did Byron have to end up making before it was published. Stanza 5 sums up what he was saying about heroes in the previous stanzas. Stanza 6 Most epic poets plunge â€Å"in medias res† (Horace makes this the heroic turnpike road), And then your hero tells, whene’er you please, What went before—by the way of episode, While seated after dinner at his ease,  Ã‚   45 Beside his mistress in some soft abode, Palace, or garden, paradise, or cavern, Which serves the happy couple for a tavern. The analysis of Stanza 6 lets the reader in that Byron is intentionally not following Horace’s recommendation of when to start an epic. Byron is (intentionally?) not following the rules of what at the time was being seen by other writers as the better way of starting an epic, which was in the middle. This stanza proves to us the writer is choosing not to write using the examples of Homer or Virgil but writing this epic his own way (Canto 1, Stanza 6, Lines 41-44). Stanza 7 That is the usual method, but not mine— My way is to begin with the beginning;  Ã‚   50 The regularity of my design Forbids all wandering as the worst of sinning, And therefore I shall open with a line (Although it cost me half an hour in spinning) Narrating somewhat of Don Juan’s father,  Ã‚   55 And also of his mother, if you’d rather. Although the reader isn’t aware of it until later, Byron reveals to us that he knew in advance he would digress in the poem (Canto 1, Stanza 7, Line 54). The reader now learns that was the writer Byron’s intention from the start. The reader can wonder if Byron is even conscious of how he changes some of the â€Å"traditional† epic writing in writing this work. In `Excerpt from Childe Harold`s Pilgrimage`, answer the following: 1. Does the Byronic hero know any form of Keatsian love? Yes, in that Keatsian love is often associated with â€Å"beauty-as-truth.† In Canto 2, Stanza 9 we read as Byron writes of having loved and it was still in his thoughts although he is now alone with those thoughts. We also read of this â€Å"beauty-as-truth† love in Canto III Stanza I when he relates of the love for his daughter. 2. Beginning with stanza 17, the narrator talks about Waterloo. Why? Waterloo is current to at this time to Byron. Just a few months before this, the fate of Europe had been decided because of that Battle. So it is important that the reader is aware that it is sacred ground to him. The battle was fought on June 18th, 1815 which makes this a very relevant event during his lifetime of 1788-1824. 3. In what ways is this poem about mid-life crises? Childe in this epic refers to a â€Å"knight† and we read as this knight is gloomily wandering as a vicious world-worn man. In his thoughts throughout the â€Å"pilgramage† it relates closely to a man who is going through similar thoughts a man in mid-life crises might go through as though he has already fully lived. 4. How does the Byronic hero relate to nature? Byron relates better to nature than he does to humans. In Canto 4, Stanza 178, Byron states this: There is a pleasure in the pathless woods, There is a rapture on the lonely shore. There is society where none intrudes, By the deep Sea, and music in its roar: I love not Man the less, but Nature more, From these our interviews, in which I steal From all I may be, or have been before, To mingle with the Universe, and feel What I can ne'er express, yet cannot all conceal However, in reading this piece, I feel its obvious throughout to the reader that the writer can connect more easily with nature than humans. Because most of Byron’s work is autographical in nature, this is easy to understand if the reader about Byron’personal life. References â€Å"Characteristics of the Byronic Hero.† University of Michigan. Online. Internet. 17 May 2003. (2002, February 11). Childe Harold's Pilgrimage. Retrieved May 17, 2007, from The Project Gutenberg Web site: http://www.gutenberg.org/dirs/etext04/chp110h.htm (2007). George Gordon, Lord Byron. Retrieved May 17, 2007, from Bob's Byway Web site: http://www.poeticbyway.com/xbyron.html ; ;

Examples Demonstrate Use of Subjunctive Mood in Spanish

The subjunctive mood can be daunting for English speakers who are learning Spanish. This is mostly because even though English has its own subjunctive mood,  we dont use its distinctive forms very often. Therefore, the subjunctive can often most easily be learned by studying examples of its use. What Is the Subjunctive Mood? Lets start with the basics: The mood (sometimes called the mode) of the verb either expresses the speakers attitude toward the verb or describes how the verb is used in a sentence. The most common mood—the indicative mood—is used to refer to what is real, to state facts, to make declarations. For example, the verb in Leo el libro (I am reading the book) is in the indicative mood. In contrast, the subjunctive mood is typically used in such a way that the verbs meaning relates to how the speaker feels about it. In the sentence Espero que està © feliz (I hope that she is happy), the second verb, està © (is), may or may not be reality; what is important here is the speakers attitude toward the second  half of the sentence. Examples of the Subjunctive Mood Proper uses of the subjunctive mood can best be seen through examples. In these sample sentences, the Spanish verbs are all in the subjunctive mood (even if the English verbs are not). The explanations can help you understand why the verbs are in the subjunctive mood in the first place. Quiero que no tengas frà ­o. (I want you to not be cold.)Its irrelevant whether or not the person is cold or not. The sentence expresses a wish, not necessarily reality.Siento que tengas frà ­o. (Im sorry you are cold.)The sentence expresses the speakers emotions about a perceived reality. What is important in this sentence is the speakers feelings, not if the other person is actually cold.Te doy mi chaqueta para que no tengas frà ­o. (Im giving you my coat so you wont be cold.)The sentence expresses the speakers intent, not necessarily reality.Se permite que lleven chaquetas allà ­. (People are allowed to wear jackets there.)The phrase expresses permission for an action to take place.Dile a ella que lleve una chaqueta. (Tell her to wear her jacket.)This expresses a command or wish of the speaker.Es preferible que ustedes no viajen maà ±ana a Londres. (It is preferable that you dont travel to London tomorrow.)The subjunctive is often used in giving advice.No hay nadie que tenga frà ­o. (Nobody is cold.)This is an expression of a negation of the action in a subordinate clause.Tal vez tenga frà ­o. (Perhaps he is cold.)This is an expression of doubt.Si yo fuera rico, tocarà ­a el violà ­n. (If I were a rich man, I would play the fiddle.)This is an expression of a statement contrary to fact. Note that in this English translation, were is also in the subjunctive mood. Comparing the Subjunctive and Indicitive Moods These sentence pairs show differences between the indicative and subjunctive. Note how in most of the examples, the English verb form is the same in translating the two Spanish moods. Example 1 Indicative: Es cierto que sale tarde. (It is definite that she is leaving late.)Subjunctive: Es imposible que salga tarde. Es probable que salga tarde. (It is impossible that she is leaving late. It is likely that she will leave late.)Explanation: In the indicative sentence, the early departure is presented as fact. In the others, it is not. Example 2 Indicative: Busco el carro barato que funciona. (Im looking for the cheap car that works.)Subjunctive: Busco un carro barato que funcione. (Im looking for a cheap car that works.)Explanation: In the first example, the speaker knows that there is a car that matches the description, so the indicative is used as an expression of reality. In the second example, there is doubt that such a car exists, so the subjunctive is used. Example 3 Indicative: Creo que la visitante es Ana. (I believe the visitor is Ana.)Subjunctive: No creo que la visitante sea Ana. (I dont believe the visitor is Ana.)Explanation: The subjunctive is used in the second example because the subordinate clause is negated by the main clause. Generally, the indicative is used with creer que or pensar que, while the subjunctive is used with no creer que or no pensar que. Example 4 Indicative: Es obvio que tienes dinero. (It is obvious you have money.)Subjunctive: Es bueno que tengas dinero. (It is good you have money.)Explanation: The indicative is used in the first example because it expresses reality or apparent reality. The subjunctive is used in the other example because the sentence is a reaction to the statement in the subordinate clause. Example 5 Indicative: Habla bien porque es experto. (He speaks well because hes an expert.)Subjunctive: Habla bien como si fuera experto. (He speaks well as if he were an expert.)Explanation: The subjunctive is used in the second example because its irrelevant to the sentence whether hes an expert, although the sentence suggests that he isnt. Example 6 Indicative: Quizà ¡s lo pueden hacer. (Perhaps they can do it [and Im sure of it].)Subjunctive: Quizà ¡s lo puedan hacer. (Perhaps they can do it [but I doubt it].)Explanation: In a sentence such as this, the subjunctive is used to emphasize uncertainty or doubt, while the indicative is used to emphasize certainty. Note how the Spanish verb form is used to indicate an attitude that might need further explanation in English. Example 7 Indicative: Hay polà ­ticos que tienen coraje. (There are politicians who have courage.)Subjunctive:  ¿Hay polà ­ticos que tengan coraje? (Are there politicians with courage?)Explanation: The subjunctive is used in the second example to express doubt, and it is not clear that the subject of the sentence  exists in reality. Example 8 Indicative: Llegarà © aunque mi carro no funciona. (I will arrive even though my car isnt running.)Subjunctive: Llegarà © aunque mi carro no funcione. (I will arrive even if my car isnt running.)Explanation: The indicative is used in the first sentence because the speaker knows their car isnt working. In the second sentence, the speaker doesnt know whether or not it is running, so the subjunctive is used. Example 9 Indicative: La pirà ¡mide ha sido reconstruida por el gobierno provincial. (The pyramid has been restored by the provincial government.)Subjunctive: Estoy feliz que la pirà ¡mide se haya reconstruido. (I am happy that the pyramid has been restored.)Explanation: The indicative is used in the first sentence because it is a direct statement of fact. The main point of the second example is the speakers reaction to the event, so the subjunctive is used. Example 10 Indicative: Cuando està ¡s conmigo se llena mi corazà ³n.  (When you are with me my heart is full.)Subjunctive: Cuando està ©s conmigo iremos por un helado. (When you are with me we will go for an ice cream.)Explanation: When the indicative is used with cuando in a sentence such as the first example, it refers to a recurring action. The use of the subjunctive in the second example indicates that the event has yet to take place. Finding the Subjunctive in English The subjunctive was once used in English more than it is today—it is now employed most in formal speech, not everyday conversation. Still, cases where it is still used in English might help you remember some of the instances where it is used in Spanish. Contrary-to-fact condition: If I were the president, Id keep us out of war.Expression of a desire: I would like it if he were my father.Expressions of request or advice: I insist that he go. We recommended that he fill out the form. In all the above cases, a straightforward translation to Spanish would likely use the subjunctive mood. But remember that there are numerous instances where the subjunctive is used in Spanish where we make no distinction in English.